{"paper_id":"b59fe90c-3e9f-417b-97f0-39180f3318f4","body_text":"Bioengineered 3D microvessels reveal novel determinants of Trypanosoma congolense sequestration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bioengineered 3D microvessels reveal novel determinants of Trypanosoma congolense sequestration Teresa Porqueddu, Mariana De Niz, Aitor Casas-Sánchez, Viola Introini, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5104603/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Feb, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract In the mammalian host, Trypanosoma congolense cytoadheres to the vascular endothelium in a process known as sequestration. Although sequestration influences clinical outcome, disease severity and organ pathology, its determinants and mediators remain unknown. Challenges such as the variability of animal models, the only-recently developed tools to genetically manipulate the parasite, and the lack of physiologically relevant in vitro models have hindered progress. Here, we engineered brain and cardiac 3D bovine endothelial microvessel models that mimic the bovine brain microvasculature and the bovine aorta, respectively. By perfusing these models with two T. congolense strains, we simulated physiologically relevant conditions and investigated the roles of flow for parasite sequestration and tropism for different endothelial beds. We discovered that sequestration is dependent on cyclic AMP signalling, closely linked to parasite proliferation, but not associated with parasite transmission to the tsetse fly vector. Finally, by comparing the expression profiles of sequestered and non-sequestered parasites collected from a rodent model, we showed gene expression changes in sequestered parasites, including of the surface variant antigens. This work presents a physiologically-relevant platform to study trypanosome interactions with the vasculature and provides a deeper understanding of the molecular and biophysical mechanisms underlying T. congolense sequestration. Biological sciences/Microbiology/Parasitology/Parasite physiology Biological sciences/Microbiology/Parasitology/Parasite host response sequestration Trypanosoma congolense animal African trypanosomiasis vascular bioengineering Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Main Trypanosoma congolense is a unicellular, intravascular parasite that causes animal African trypanosomiasis, or nagana, in several mammals, and particularly pathogenic for livestock and dogs in Africa. The parasite replicates in the blood, where it binds to the vascular endothelium, in a process known as sequestration. Whilst most infections result in a chronic disease, a small proportion of animals develop an acute, rapidly fatal illness. In rodent models, acute cerebral disease is determined by increased T. congolense sequestration in the brain and is characterized by immune cell recruitment and early death 1 . In other parasitic diseases, such as malaria and babesiosis 2 , 3 , sequestration also determines clinical course, disease severity, and organ pathology. Sequestration in Trypanosomes has recently been suggested to play a role in transmission, since silencing of a gene orthologous to a T. brucei negative regulator of differentiation to the insect-transmissible form resulted in reduction of attachment to a plastic substrate and an increase in peripheral parasitaemia in vivo 4 . Despite its importance for disease pathogenesis, the determinants of sequestration, be it molecular, biophysical, or biochemical, remain unknown. Many challenges have precluded the study of sequestration determinants, namely the large phenotypic variability of animal models, the short timespan in the development of acute cerebral trypanosomiasis, and the absence of physiologically-relevant in vitro models that could reproduce the complexity of the parasite-endothelial cell interaction. In cerebral malaria, the development of bioengineered in vitro human 3D brain microvessels 5 has allowed for the recent development of many advances in severe and cerebral malaria research. In this study, we adapted this microvessel system to the study of trypanosomiasis by engineering two 3D bovine endothelial microvessel models that mimic the bovine brain microvasculature and the aorta. Strains from T. congolense that cause acute and chronic trypanosomiasis showed different tropism for cardiac and brain 3D microvessels under different flow mechanical conditions. Using a combination of the microvessels systems, simpler cytoadhesion assays, and mouse and tsetse fly experimental infections, we found that sequestration is dependent on cyclic adenosine monophosphate (cAMP), closely associated to parasite proliferation, but not associated with transmission. Furthermore, we assessed the T. congolense gene expression remodelling that takes place in sequestered parasites. Results The use of acute and chronic T. congolense rodent models has shed light on the importance of parasite sequestration in vascular pathogenesis and inflammation 1 . Yet, uncovering the determinants of parasite sequestration through the exclusive use of animal models is challenging, given the difficulties to disentangle the cellular and biophysical components of the whole organism. To overcome this challenge, we developed a 3D endothelialised bovine microvessel system. The devices consist of a 3D microfluidic network with a pre-defined geometry on a collagen scaffold fabricated by soft lithography and injection moulding (Fig. 1 A). The system supports the growth of primary microvascular endothelial cells in lumenised microvessels, perfusable with T. congolense , which allows for the study of parasite sequestration under controlled conditions, including flow and endothelial cell type, independently of other host factors. Our previous results showed that different parasite strains accumulate in various organotypic beds in mouse models, leading to distinct clinical presentations. Whilst T. congolense 1/148 parasites accumulate highly in the brain microvasculature, causing acute cerebral trypanosomiasis, IL3000 parasites present a tropism for the heart, and cause chronic, wasting disease 1 . To better understand this organ preference in vitro , we developed two microvessel systems: one mimicking the heart vasculature with bovine aorta endothelial cells (BAOEC) and one mimicking the brain with bovine brain microvascular endothelial cells (BBMVEC). Endothelial cell identity of both cell types was assessed by immunostaining on 2D monolayers. Both BBMVEC and BAOEC expressed adherens (VE-cadherin, ß-catenin) and tight junction markers (ZO-1), as well as the pan-endothelial cell marker, Von Willebrand factor, (Supplementary Fig. 1). We then seeded both endothelial cell types in a 13 x 13 microfluidic branched network of 120 µm microvessels. This geometry recapitulates a large range of flow velocities (42.5-fold range) and generates microvessels analogous to post-capillary venules in terms of surface-to-volume ratio (20 mm 2 /mm 3 ). After 3 days in culture, BAOEC and BBMVEC form a 3D tubular geometry with empty lumens. Immunofluorescence labelling with junctional markers revealed that both cell types align with flow when grown in 3D. However, they are morphologically distinct: BAOEC are smaller and more rounded, whilst BBMVEC are bigger and more elongated with a higher aspect ratio (unpaired t-test, p < 0.001) (Fig. 1 B). Nonetheless, they both express markers of adherens (VE-cadherin, ß-catenin) and tight junctions (ZO-1) (Fig. 1 C and D). The actin cytoskeleton appears to be cortical on BAOEC, while more stress fibres are present on BBMVEC, probably due to the endothelial stretching found in brain microvascular models. In conclusion, we developed two in vitro microvessel systems mimicking heart and brain vasculature, with expected endothelial and junctional marker expression, to study organ-specific trypanosome sequestration. T. congolense sequestration to 3D microvessels is dependent on wall shear stress, parasite strain and endothelial cell type Blood flow velocity and the associated wall shear stress (WSS) varies along the hierarchical vascular bed. In healthy conditions, in the arteriovenous microcirculation, WSS ranges range between 5 and 40 dyn/cm 2 in arterioles and capillaries, and 1 and 5 dyn/cm 2 in venules 6 – 8 . However, in pathological conditions, such as when there is vascular obstruction, flow and WSS may reduce 1 , 6 . To assess the role of WSS and organotypic endothelial cell types in T. congolense sequestration, we first determined the flow mechanical stress that T. congolense parasites can withstand. To this end, we used a much simpler setup. BAOEC or BBMVEC were seeded into 6-channel µ-slides overnight at maximum confluence. Fluorescently-labelled T. congolense parasites were introduced and allowed to cytoadhere for 30 minutes, followed by perfusion with increasing flow rates to measure parasite binding strength against detachment (Fig. 2 A). We observed that both 1/148 and IL3000 parasites remained bound to BAOEC, withstanding high WSS values. In fact, at WSS of 11.97 dyn/cm 2 , 25%±11 and 43%±21 of 1/148 and IL3000 parasites, respectively, remained sequestered (Fig. 2 B). IL3000 presented similar binding kinetics to BBMVEC (51%±16 at maximum WSS) (Fig. 2 C). Conversely, 1/148 presented a different behaviour on BBMVEC, with parasites being significantly released back to circulation when exposed to WSS forces higher than 0.6 dyn/cm 2 ( p -value < 0.001, 2-way ANOVA with Sidak’s correction for multiple comparisons). This shows that different parasite strains display heterogeneous binding behaviour to different endothelial beds and suggests that 1/148 presents lower sequestration strength to brain microvessels at physiological WSS. Having shown that at least a proportion of both T. congolense 1/148 and IL3000 can withstand these forces, we tested the role of WSS in parasite sequestration. When perfused at a constant flow rate of 10µl/min, endothelial cells in the outer edge of the 13 x 13 grid are exposed to flow velocities that range from 0.4 to 15.2 mm/s, which translates into WSS values between 0.08 and 3.4 dyn/cm 2 . We perfused 3D aorta or brain microvessels with 1.5 million fluorescently-labelled parasites of either IL3000 or 1/148 strains (Fig. 2 D and Video 1). Following a 15-minute perfusion, unbound parasites were washed under flow for 10 minutes and microvessels were fixed and stained for subsequent microscopical analysis and binding quantification. Parasites presented widespread cytoadhesion to 3D microvessels by live microscopy (video 1). Scanning electron microscopy showed that parasites sequester to the luminal side of the endothelial cells lining the 3D vessels on top of the collagen matrix (Fig. 2 E). Within the 3D microvessel, a smooth, empty lumen was surrounded by tightly clustered endothelial cells, to which the parasites sequestered (Fig. 2 E). In agreement with previous literature 9 , close interaction between the flagellum and the endothelial cell was observed (Fig. 2 F). Several microfilaments, which have been previously reported, coming out of the endothelial cell and surrounding the parasite cell body could also be observed (Fig. 2 F). Having confirmed that parasites sequester to the 3D microvessels, we proceeded with binding quantifications. Since we were using fluorescently-labelled parasites, we calculated the area occupied by parasite binding (obtained from the total fluorescence area) as a proxy for the number of sequestered parasites because these two variables (number of sequestered parasites and fluorescence area) correlate highly to each other (R 2 = 0.91, Pearson’s correlation, p-value < 0.001) (Fig. 2 G). Overall, IL3000 parasites presented higher sequestration to bovine aorta microvessels than 1/148 ( p -value < 0.0001, ordinary one-way ANOVA with Tukey’s multiple comparisons test), whilst both strains showed similar binding levels to bovine brain microvessels (Fig. 2 H). Despite these variations, T. congolense sequestration, regardless of the strain and endothelial bed, is significantly higher than sequestration of Plasmodium falciparum HB3var03 to human 3D brain microvessels, a malaria parasite line associated to severe and cerebral malaria in humans 5 , 10 , 11 . Even though P. falciparum was perfused at higher concentrations, T, congolense 1/148 presented a 5-fold higher binding, and IL3000 a 15-fold. These results highlight the affinity and extend of binding of T. congolense compared to other parasitic disease that affect brain microvessels. Sequestration of both strains to aorta 3D microvessels negatively correlated with WSS (Fig. 2 I). More specifically, IL3000 presented high binding level at WSS of 0.5 dyn/cm 2 and below, then it slowly decreased when it reached a WSS of 1 dyn/cm 2 , after which it plateaued. In contrast, 1/148 displayed lower binding levels than those of IL3000 across all WSS regions, being significantly lower in the range between 0.25 and 0.8 dyn/cm 2 ( p -value = 0.02 for WSS values of 0.5–0.8 and p -value = 0.001 for WSS values 0.25 to 0.45, 2-way ANOVA with Sidak’s correction for multiple comparisons) (Fig. 2 I). These observations agree with findings in mouse models, where T. congolense IL3000 shows higher sequestration to the heart microvasculature than 1/148 1 . When exposed to bovine brain microvessels, IL3000 sequestration followed the same binding pattern than in aorta microvessels and reached similar sequestration levels (Fig. 2 J). However, 1/148 presented a different sequestration pattern with two independent sequestration peaks. First, sequestration was dramatically high at 0.08 dyn/cm 2 , representing accumulations in 40% of the total vessel area 12867µm 2 ± 4483 of sequestration area, which is 3 times what was observed for IL3000 (i.e. 4365µm 2 ± 719) ( p -value = 0.03, 2-way ANOVA with Sidak’s correction for multiple comparisons). Then, there was also non-significant sequestration bump at 1.4 dyn/cm 2 . When comparing 1/148 sequestration across different vascular beds, we observed that 1/148 parasites bound similarly to aorta and brain at WSS higher than 0.25 dyn/cm 2 but presented significantly more binding to brain at pathological WSS levels of less than 0.25 (Fig. 2 K) ( p -value = 0.03, 2-way ANOVA with Sidak’s correction for multiple comparisons). Interestingly, in the mouse model of acute cerebral trypanosomiasis, T. congolense 1/148 preferentially sequesters in the small capillaries of the brain, often leading to vascular occlusion and vessel blockage 1 , which greatly reduce blood flow and the associated WSS. We conclude that T. congolense sequestration is dependent on flow mechanical properties, irrespective of the parasite strain and endothelial cell organotype. Nevertheless, parasite strains display distinct sequestration behaviours, which may be important for the clinical outcome. T. congolense sequestration can be prevented by interfering with cAMP homeostasis Having established the role of WSS in sequestration, we asked how we could interfere with sequestration in vitro . It has been previously suggested that cAMP phosphodiesterase inhibition results in lower T. congolense sequestration 4 . Therefore, we attempted to reproduce that phenotype by treating IL3000 parasites with 10µM or 20µM of NPD-1015, an inhibitor of cAMP phosphodiesterases PDEB1 and PDEB2 12 ) that interferes with cAMP homeostasis, increasing the cAMP intracellular levels in the parasite and resulting in growth arrest 13 . After 24 hours, the drug was removed, and parasites were added to a 2D monolayer of BAOEC and allowed to sequester. Subsequently, we washed unbound parasites and quantified the number of sequestered parasites by microscopy (Fig. 3 A and B). Treatment did not affect the health of parasites; we observed vigorous parasite motility and did not detect abnormal cell debris in the wells. However, there was a significant growth arrest upon NPD-1015 treatment: whilst untreated parasites proliferated 82%±14 over 24 hours, parasites treated with 10µM NPD-1015 grew only 42%±4 and those treated with 20µM NPD-1015 did not proliferate at all (-7%±10) ( p -value = 0.0026, one-way ANOVA with Tukey’s correction for multiple comparisons) (Fig. 3 C). We observed that parasite exposed to 10µM of NPD-1015 presented a 5-fold lower binding ( p -value < 0.0001, one-way ANOVA with Tukey’s correction for multiple comparisons), whilst exposure to 20µM of NPD-1015 decreased binding levels 20 times ( p -value < 0.0001, one-way ANOVA with Tukey’s correction for multiple comparisons) (Fig. 3 B and D). As we washed the drug before adding the parasites to the endothelial cell monolayers, we ensured that any effect observed is parasite-derived. We then asked if NPD-1015 could revert binding of sequestered parasites. Therefore, we co-cultured T. congolense IL3000 parasites in a 2D BAOEC monolayer for 24 hours. Subsequently, we removed non-sequestered parasites by washing and added 20µM of NPD-1015 for 24 hours longer. We observed a reduction of sequestration, indicating that NPD-1015 induced detachment of parasites. This suggests that increasing intracellular cAMP not only prevents sequestration, but also reverts it (Fig. 3 E). We then used the 3D microvessels system to test whether a similar binding reduction occurred under flow. For that, we incubated parasites with 20µM NPD-1015 and after 24 hours perfused the microvessels with treated parasites (Fig. 3 F). Overall, there was a 25% reduction of sequestration ( p -value = 0.03, unpaired t-test) (Fig. 3 G), consistent with our previous observation in the 2D assay, but with a lower effect than in EC monolayers, suggesting that the tridimensional architecture of the microvessel or the presence of flow might increase cytoadhesion efficiency. Although NPD-1015 treatment did not affect parasite sequestration dependence on WSS (Fig. 3 H), an increase in parasitic cAMP significantly reduced parasite binding in regions exposed to a range of WSS between 0.25 and 1.4 dyn/cm 2 . No differences in binding were observed when the parasite was exposed to a WSS below 0.25 dyn/cm 2 . In conclusion, we showed that interfering with cAMP homeostasis is sufficient to both prevent and revert sequestration, and this effect is more pronounced under specific flow mechanical cues. Sequestered parasites proliferate more in the mammalian host and have similar transmission ability to non-sequestered parasites Next, we hypothesised that sequestration provides an adaptive advantage for T. congolense , so that sequestered parasites proliferate faster than non-sequestered. To test that, we examined the cell cycle distribution of sequestered and non-sequestered IL3000 parasites, grown in vitro , on plastic, without endothelial cells, by quantifying the kinetoplast and nuclei number in each trypanosome cell. (Fig. 4 A). We noticed that the population of sequestered parasites contained a higher proportion of proliferating parasites, which typically can be distinguished by having with two kinetoplasts and one nucleus (2K1N, 20%±8 vs. 5%±4) relative to non-sequestered population ( p -value < 0.0001, one-way ANOVA with Tukey’s correction for multiple comparisons) (Fig. 4 A), suggesting that sequestered parasites divide more frequently than non-sequestered parasites. Parasites in S phase (i.e. with kinetoplast butterfly-shaped) were considered as 2K1N. We did not observe statistically significant differences in the number of mitotic parasites (2K2N configuration). To test whether sequestered parasites divide more frequently in vivo , we used intravital microscopy data previously collected from mice infected with either T. congolense 1/148 or IL3000 1 . In 1/148 infections, we analysed video recordings from 8 major organs (i.e. adipose tissue, brain, heart, liver, lungs, kidneys, spleen) at days 1–6 post-infection, corresponding to the timepoint after which infected animals start developing acute cerebral trypanosomiasis. In IL3000 infections, we analysed data from the same organs, but at the first peak of parasitaemia, the interval between the first and the second peaks of parasitaemia, where peripheral parasitaemia is barely detected, and the second peak of parasitaemia (Fig. 4 B). Before image acquisition, Hoechst and FITC-Dextran were injected intravenously into the mice, to allow detection of intravascular parasites and their DNA using intravital microscopy (Fig. 4 C). We differentiated between sequestered and non-sequestered parasites based on their displacement during the video as previously described 1 . We observed that, overall, sequestered parasites were more often found replicating and dividing (i.e. 2K1N or 2K2N) than non-sequestered parasites (p-value < 0.0001, one-way ANOVA with Tukey’s correction for multiple comparisons) in both 1/148 and IL3000 infections (Fig. 4 D). This shows that sequestered parasites divide more than non-sequestered parasites, irrespective of the parasite strain. Finally, we asked whether the tissue microenvironment might affect parasite division (Supplementary Fig. 2). In both IL3000 and 1/148 mouse infections, we observed an enrichment of dividing parasites (2K1N and 2K2N) in all organs, regardless of the parasite strain, except on lung and kidney for 1/148. Therefore, we conclude that the association between sequestration and T. congolense cell division is neither strain- nor endothelial cell organotype-dependent. Given our results suggesting that sequestration facilitates parasite proliferation (or vice-versa) and recent work suggesting that non-sequestered parasites may be growth-arrested, insect-transmissible forms 4 , we tested the ability of sequestered and non-sequestered parasites to differentiate into procyclic (insect) forms (Fig. 4 E). We separated in vitro sequestered from non-sequestered parasites, incubated them in differentiation trypanosome media (DTM), at 27ºC, without CO 2 , and followed parasite differentiation and growth for 5 days (Fig. 4 E). We observed that both sequestered and non-sequestered parasites could successfully differentiate into procyclic forms with similar dynamics, reaching similar procyclic parasite number within 5 days (Fig. 4 F). Procyclic parasites were identified by their morphology (pointy and elongated cells, with the flagellum starting from the mid body) and motility (not sequestering, fast swimmers). We also quantified their proliferation rate over 24 hours after procyclic differentiation and did not observe any difference (Fig. 4 G). Since cell cycle arrest precedes procyclic differentiation in the related organism T. brucei 14 , we forced T. congolense cell cycle arrest with the administration of NPD-1015 24 hours before induction of differentiation. Again, we did not observe any difference in the ability of each parasite population to differentiate into procyclic forms (Fig. 4 F) or of differentiated procyclic forms to grow (Fig. 4 G). Finally, we assessed the ability of sequestered, non-sequestered, and NPD-1015-treated bloodstream form parasites to infect tsetse flies. As T. brucei PDEB1 gene deletion was shown to disrupt social motility 15 and pH taxis 16 of procyclic parasites, we thoroughly washed the parasites to before feeding them to the flies, removing any traces of NPD-1015. Furthermore, we fed the tsetse flies with a low inoculum (10 5 parasites/ mL blood) to increase the probability of observing differences in fly-infectivity. The lower inoculum, the higher the proportion of parasites that must be competent to achieve infection. In contrast, with a higher inoculum, even a small proportion of fly-infective trypanosomes could represent enough parssites to saturate fly infection rates and mask potential differences between groups. We observed that sequestered and non-sequestered parasites infected similar proportions of flies (37 ± 3% and 26 ± 9%, respectively), whereas NPD-1015-treated parasites infected significantly fewer flies (12 ± 4%) ( p -value = 0.0043, one-way ANOVA with Tukey’s correction for multiple comparisons) (Fig. 4 H). Moreover, despite similar overall infection rates between sequestered and non-sequestered parasites, we observed that the former resulted in heavier infections (higher parasite load in the midguts) than both non-sequestered and NPD-1015-treated parasites ( q -value = 0.0002, < 0.0001, = 0.021, respectively, 2-way ANOVA with Benjamin, Krieger and Yekutieli method correction for false discovery rate) (Fig. 4 I). In summary, our data suggest that sequestration is associated to higher proliferation rates in the mammalian host and heavier infections in the vector, which might affect transmission potential, even though both sequestered and non-sequestered parasites are fly-transmissible. T. congolense sequestered parasites show distinct transcriptomes to non-sequestered The striking differences in cell cycle stage between sequestered and non-sequestered parasites both in vitro and in vivo , suggest that these two parasite forms are intrinsically distinct. Therefore, we compared their gene expression profiles during acute cerebral trypanosomiasis in vivo . We infected C57BL/6J mice with 2000 T. congolense 1/148 parasites 17 and, at the first peak of parasitaemia (day 6 post-infection), we collected systemic blood and three organs: the brain, the adipose tissue, and the kidney. From these samples, we extracted total RNA and performed multiplexed trypanosome targeted RNA sequencing based on the spliced-leader 18 enrichment (SL-seq) 19 , 20 . These organs were chosen because the parasite population in their vasculature is predominantly in its sequestered form 1 . Therefore, we obtained transcriptomes of non-sequestered parasites from systemic blood samples, whereas sequestered parasites enriched transcriptomes were obtained from the tissues. First, we removed the sequencing reads that mapped to the mouse genome. Then, we mapped the remaining reads to the annotated T. congolense IL3000 genome 21 (Supplementary file 1), given that genome homology analysis shows that IL3000 and 1/148 strains have 94.8%±2.8 nucleotide sequence identity (Supplementary Fig. 3). We compared the transcriptomes from parasites of each tissue and blood to see if we were able to detect significant tissue-specific differences. We observed that the transcriptomes of non-sequestered parasites (from the systemic blood) clustered together (Fig. 5 A). Correlation analysis further showed that transcriptomes of sequestered parasites were more different from non-sequestered parasites, than sequestered parasites collected from different tissues ( R 2 = 0.58–0.61 vs. 0.91–0.96, Pearson’s correlation). Therefore, in subsequent analyses, we compared the transcriptomes of sequestered parasites irrespective of the tissue they derived from to the group of non-sequestered parasites. We detected 523 differentially expressed genes, of which 323 were upregulated in sequestered parasites (Fig. 5 B). Upregulated genes included phosphatidic acid phosphatase, DNA repair protein, sister chromatid cohesion C-terminus, UDP-Gal/UDP-GlcNAc-dependent glycosyltransferase (UGT), transferrin receptor-like proteins (both Fam14 and 15) and the orthologue to flagellum attachment zone protein (FAZP). Downregulated genes included those encoding for ALBA and other RNA-binding proteins, PAD-like genes (protein associated with differentiation), amastin, cAMP phosphodiesterase A, and PLAC8 family (Fig. 5 B). We asked if genes previously identified as upregulated in T. congolense parasites upon silencing of a negative regulator of differentiation to insect-transmissible forms (TcoREG9.1) 4 , and that therefore could be assumed to be characteristic of insect-transmissible parasites, were enriched within our dataset. We did not find compelling evidence of enrichment (Fig. 5 C). Based on what we know from T. brucei , at the peak of infection, the population of parasites is expected to contain more insect-transmissible forms than in the ascending phase of infection 22 . Therefore, we also tested a gene set comprising genes upregulated in the first peak infection compared to the ascending phase of infection 22 , but also did not find any evidence of enrichment. Together, these results corroborate our previous observation that T. congolense sequestration (or their lack of) is not associated with transmission ability. Given that sequestration is a physical interaction between the parasite and the endothelial cell and considering that the T. congolense cell surface is tightly packed with the major antigen, variant surface glycoprotein (VSG), we specifically looked for changes in their expression. VSGs cannot be accurately characterised using standard differential expression tools, so we used the software VAPPER 23 to profile them in sequestered and non-sequestered parasites. T. congolense VSGs cluster into 15 phylogenetically-distinct lineages (or phylotypes), between which genetic recombination is rare 24 . We found genes from all phylotypes being expressed at the mRNA level, consistent with previous observations in insect forms (Fig. 5 D). However, we found that genes belonging to VSG phylotype 8 were predominantly expressed in sequestered parasites, irrespective of the mouse organ, whereas genes from phylotypes 11 and 14 were more abundant in non-sequestered parasites ( p -value < 0.0001, 2-way ANOVA with Sidak’s multiple comparisons test) (Fig. 5 D and 5 E). These results suggest functional differentiation amongst the VSG repertoire and a role of phylotype 8 genes in sequestration. Our results support the conclusions that sequestered and non-sequestered T. congolense bloodstream forms present different transcriptomes, and that sequestration might be directly linked to VSG expression. Discussion Sequestration is emerging as an essential process of T. congolense interaction with the mammalian host, although its mechanisms remain unknown. In this work, we have significantly improved our understanding of trypanosome sequestration through the development of physiologically-relevant in vitro system and their use in combination with in vivo animal models. We have discovered the importance of flow mechanical cues as an important determinant of sequestration, revealed that cAMP intracellular levels modulate sequestration, found a link between sequestration and cell cycle that is independent of transmission ability, and characterised the gene expression profiles of sequestered parasites. Novel bioengineering tools are gaining relevance in the infection biology field. One of their main advantages is the possibility to generate animal species-specific tissues in vitro to study zoonoses or veterinary infections. To the best of our knowledge, we have generated for the first time a non-human microvessel model and showed their potential to study animal pathogens. By modelling bovine small arterioles or postcapillary venules, this system mimics the natural host’s endothelium and the preferred environment for trypanosome sequestration. Despite low throughput and technical complexity of microfabrication, our versatile bioengineered method supports the generation of organ-specific vasculature and approximates in vitro settings to natural conditions, by modelling a wide range of physiological WSS and flow velocities within a single device. Future studies could explore these models as platforms to test sequestration mediators or study endothelial cell responses to trypanosomes. In the malaria field, similar approaches have led to a better understanding of cerebral malaria, such as the identification of polyclonal 25 or monoclonal antibodies 26 that inhibit parasite binding, or the discovery of new mechanisms of brain microvessel dysfunction 27 , 28 . In this study, we have exploited them to reveal the biophysical and molecular determinants of T. congolense sequestration as well as suggest a link between parasite binding and proliferation. Furthermore, microvessels might help us to characterise mechanisms of vascular transmigration of tissue-invading related trypanosome species, such as T. brucei 29 – 33 , or assess the role of haematocrit levels in sequestration and/or extracellular matrix invasion. T. congolense can withstand high WSS both in 2D and 3D vascular models, corroborating previous observations of parasites sequestering in large arteries of the mouse 1 , where WSS is approximately around 10 dyn/cm 2 . However, based on our microvessels models, sequestration gradually increases as WSS decreases. Indeed, maximum sequestration is achieved in pathologically low WSS values, when combining T. congolense 1/148 and brain endothelium. This is reminiscent of acute cerebral trypanosomiasis, where brain pathology is associated with high parasite accumulation in the microvasculature and vascular occlusion, which ultimately culminates in ischaemic or haemorrhagic stroke-like events 1 . The subtle but significant differences in the effect of WSS in sequestration between parasite strains and host endothelial cell types suggest that there may be more than one host ligand and/or parasite receptor of sequestration. Indeed, in P. falciparum , it has been well described that binding heterogeneity arises from the combination of different host receptors and parasite ligands from the PfEMP1 family 5 . Here, we found several surface-expressed proteins upregulated in sequestered parasites, including VSGs, invariant surface glycoproteins, and flagellum attachment zone proteins. Future research could investigate further if these candidates are sequestration mediators. Previous studies suggested that sequestration to live, but not fixed, bovine aorta endothelial cell monolayers increased T. congolense proliferation 34 . Here, we corroborated these results by showing that, in vivo , T. congolense parasites divide more when sequestered. These data suggest that sequestration might bring a metabolic advantage for parasites, i.e. the physical contact between the parasite and the endothelial cell may facilitate hijacking of host cellular functions and/or nutrients. However, here we uncovered an additional layer of complexity: faster cell division is observed even when the parasite is attached to a plastic substrate although at a lower rate than in the presence of endothelial cells. This indicates that the link between sequestration and proliferation is at least partly intrinsic to the parasite and not just a consequence of a potential metabolic benefit. It has been previously suggested that sequestered and non-sequestered parasites could be two distinct life forms: the first adapted for proliferation in the mammalian host (analogous to the T. brucei slender form), and the latter adapted for fly transmissibility (analogous to the T. brucei stumpy form) 4 . However, our results suggest that both sequestered and non-sequestered parasites can differentiate to the insect stage in vitro and successfully infect tsetse flies in similar timeframes. This could be because the number of parasites in G0/G1 (1K1N) present in the sequestered parasite population are sufficient to establish a successful procyclic form population, masking the phenotype of the remaining proliferating population. However, in these conditions, we would have expected faster differentiation of non-sequestered populations (as the number of parasites in G0/G1 is larger) and of NPD-1015-treated parasites (because this drug induces growth arrest and detachment). Instead, our results strongly suggest otherwise: NPD-1015-treated parasites infect flies at lower rates (perhaps because cAMP is important for successful infection, as reported for T. brucei 35 ), and sequestered parasites result in heavier fly infections, which might result in higher transmission risk if transposed to heavier mouthpart infections. An alternative is that cell-cycle arrested forms are not necessary for fly transmission, which would explain why differentiation to procyclic forms is not more efficient if the starting population has more cells in G0/G1. A similar hypothesis, suggesting that proliferative T. brucei parasites are capable of infecting flies has been recently proposed 36 . While it remains unclear why sequestration promotes cell division, or vice-versa, cAMP homeostasis appears central to this question. When we inhibited cAMP phosphodiesterases with NPD-1015, we observed growth arrest, but also a drastic decrease in sequestration. NPD-1015 treatment not only prevented attachment to endothelial cells, but also caused detachment of already sequestered cells. It has been thoroughly described that cAMP phosphodiesterase inhibition induces growth arrest in mammalian cells 37 , 38 , and alters endothelial cell permeability and barrier properties 39 – 42 , which may result in altered expression of surface molecules. As we washed the drug away before adding the parasites to the endothelial cells, we show that NPD-1015 prevents T. congolense sequestration independently of endothelial cells, but parasite detachment could be partly result from changes in endothelial cell biology. Although, previously, it was shown that a similar inhibitor was able to prevent adhesion of Crithidia fasciculata , this is the first time that detachment of already attached cells is observed 43 . Therefore, whilst cAMP signalling might play an evolutionarily-conserved role in kinetoplastid parasite attachment, the specific effects in sequestration seem distinct in T. congolense bloodstream forms. To conclude, we present a new experimental model for assessing trypanosome interactions with the vascular endothelium and add new insights into the roles and characteristics of T. congolense sequestration. Our work lays the ground for additional mechanistic examinations of sequestration, including receptor-ligand discovery. Methods Animal Experiments This study was conducted in accordance with EU regulations and ethical approval was obtained from the Animal Ethics Committee of Instituto de Medicina Molecular (AWB_2021_11_LF_TrypColonization), the Animal Ethics Committee of the European Molecular Biology Laboratory, and the Animal Ethics Committee of Instituto Gulbenkian de Ciência (A003.2023). Infections were performed either at the rodent facility at the Parc de Recerca Biomedica de Barcelona (PRBB), where EMBL Barcelona is located, at iMM’s rodent facility, or at IGC’s mouse facility, in 6–10 weeks old, wild-type, male C57BL/6J mice. Mice were infected by intraperitoneal injection of 10 6 [ T. congolense savannah 1/148 (MBOI/NG/60/1-148) 44 ]. Blood for perfusion of 3D microvessels was obtained by cardiac puncture. Mice were sacrificed by CO 2 narcosis. Cell culture Bovine brain microvasculature endothelial cells (BBMVEC) (Cell Applications #B840-05), Bovine aorta endothelial cells (BAOEC) (Cell Applications #B304-05) and Human brain microvascular endothelial cells (HBMEC) (Cell Systems #ACBRI 376) were cultured as per supplier’s instructions respectively in bovine brain endothelial cell growth media (Cell Applications, USA), bovine endothelial cell growth media (Cell Applications, USA), or in complete endothelial growth media-2MV (Lonza) containing 5% foestal bovine serum. T. congolense savannah 1/148 (MBOI/NG/60/1–148) were expanded in 7–10 weeks-old, male C57BL/6 J mice (Charles River, France), harvested from blood by cardiac puncture and purified by anion exchange chromatography. T. congolense savannah IL3000 SM parasites were cultured in HMI-93 medium supplemented with 10% goat serum on 10mm-diameter Petri dishes, at 34ºC, until 80–100% confluency. When necessary, parasite cells were stained with fluorescent dye 5(6)- Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), at a 1:1000 dilution, as per manufacturer’s instructions. Plasmodium falciparum clone HB3 was selected for expression of PfEMP1 variants HB3VAR03 as previously described 45 , and cultured in human O + erythrocytes in RPMI-1640 medium containing 25 mM HEPES, 4 mM L-glutamine, 0.04 mM hypoxanthine, 5 mM glucose, and 10% human type B + serum at 37°C and 90% N 2 , 5% CO 2 , and 5% O 2 . Late-stage P. falciparum infected erythrocytes were enriched using a MACS cell separator with LD columns (Miltenyi Biotec #130-042-901) and fluorescently labelled (PKH26 Red Fluorescent Cell Linker Midi Kit (Sigma #MIDI26-1KT) before perfusion in human brain microvessels. Microvessel Fabrication and immunofluorescence analysis Microvessels were prepared by soft lithography and injection moulding of a collagen hydrogel in between polymethylmethacrylate (PMMA) jigs and polydimethylsiloxane (PDMS) stamps, as previously described for human brain microvessels 5 . BBMVEC, BAOEC or HBMVEC were seeded into the collagen at a concentration of 7x10 6 cells/mL Devices were kept for 3 days to allow for vessel forming, replacing medium approximately every 12 hours under gravity-driven flow. 1.5x10 6 fluorescently-labelled T. congolense parasites and 10x10 6 P. falciparum -infected erythrocytes were perfused at a defined flow rate of 10µL/min for 15 minutes. Microvessels were washed with 150µL of PBS for 10 minutes, at the same flow rate. Vessels were fixed with 3.7% paraformaldehyde and washed twice with PBS. Cells were permeabilized using a 2% Bovine Serum Albumin 0.1% Triton-X100 in PBS and stained with 4µg/mL dihydrochloride (DAPI) or specific antibodies. Parasite binding quantification was performed by confocal microscopy (Zeiss LSM 980 confocal microscope), 10X magnification, with a total scanning with 30–50µm of depth and analysed using ImageJ. Detachment Assay Parasites were either isolated from mouse blood by anion exchange chromatography (1/148) (Lanham and Godfrey, 1970) or harvested from culture (IL3000), stained with 5 mM Vybrant CFDA SE Cell Tracer dye (#V12883, Invitrogen) diluted 1000 times in trypanosome dilution buffer (TDB) (5 mM KCl, 80 mM NaCl, 1 mM MgSO 4 , 20 mM Na 2 HPO 4 , 2 mM NaH 2 PO 4 , 20 mM glucose, pH 7.4), and incubated for 25 min at 34ºC, 5% CO 2 . At the end of the incubation period, parasites were washed and resuspended in TDB, added to the endothelial cell monolayers, and incubated for 1 hour at 34ºC, 5% CO 2 . Flow was applied with a perfusion syringe pump containing PBS at defined flow rates, for 1 min each. Parasites were imaged live on a Zeiss LSM 980 (Carl Zeiss Microimaging) with a 20X water- immersion objective (0.8 numerical aperture and 0.55 mm working distance) before and after each flow session. We acquired 10 fields of view per condition (each WSS value), per replicate (3 replicates), with green laser (488nm, maximum power of 13mW). For all acquisitions, the software used was ZEN blue edition v.2.6, allowing export of images in czi format. Scanning Electron Microscopy Microvessels were fixed in Karnovsky solution (2% PFA, 2.5% glutaraldehyde in 0.1M cacodylate buffer, pH 7.4) at least overnight at 4ºC. First, 200 µl of fixative were added to the inlet and incubated for 15 minutes. After that, additional 300µl were added to the device. After fixation, the devices were washed with 0.1M cacodylate, at 4ºC, opened and the collagen containing the microvessels was removed from the jigs and post-fixed with 2.5% Glutaraldehyde in 0.1M Cacodylate buffer pH 7.4, for 1 hour at 4ºC. After washing twice with 0.1M cacodylate, samples were incubated with 2% tannic acid and 4.2% sucrose for 1 hour, at 4ºC. Samples were subsequently washed twice with distilled water, stained with 1% methylene blue, embedded in 2% low-melt agarose, and sectioned as 150µm sections in the vibratome. Sections were kept in water until further processing. Samples were dehydrated in an ascending acetone sequence, critical point dried and sputter coated with platinum (60s, diffuse coating). Images were acquired with a FEI Quanta 650 FEG scanning electron microscope using the detector LEI for secondary electrons at 10 kV, spot 3, high vacuum and dwell time of 3µs. Cytological analysis To assess cell cycle status, parasites were grown in MATEK glass-bottom dishes overnight. Non-sequestered parasites were removed by aspiration, fixed with 2% formaldehyde, and airdried on to glass slides for at least 4 hours. Then, parasites were rehydrated with PBS, permeabilized with 2% Bovine Serum Albumin − 0.1% Triton- X100 in PBS for 10 minutes, incubated with 4µg/ml of 4’,6-diamidino-2-phenylindole (DAPI) in PBS for 10 minutes and then washed twice for 10 min in PBS. Slides were then mounted with fluoromount-G (ThermoFisher Scientific) and sealed with a glass coverslip secured with nail polish. Parasites remaining sequestered to the MATEK glass-bottom dish following aspiration were fixed with 2% formaldehyde, permeabilised with 2% Bovine Serum Albumin − 0.1% Triton- X100 in PBS for 10 minutes, stained with DAPI for 10 minutes, and washed twice with PBS. Cell cycle status was assessed under a Zeiss LSM980 using a bright field 63X objective and a 561nm laser. Intravital Imaging Intravital microscopy involved separate surgeries targeting specific organs, as previously outlined for the brain 46 , lungs and heart, liver, pancreas, spleen, kidneys 47 , 48 , and adipose tissue. Briefly, mice were anesthetized with a ketamine (120mg/kg) and xylazine (16mg/kg) mixture via intra-peritoneal injection. Reflexes were checked, and upon their absence, mice received intravenous injections into the retro-orbital sinus of three markers: Hoechst 33,342 for nucleic acid labelling (stock diluted in dH2O at 100mg/ml, injection of 40µg/kg mouse) and 70 kDa FITC-Dextran for intravascular space labelling (stock diluted in 1 x PBS at 100mg/ml, injection of 500mg/kg mouse). Temporary glass windows (Merk rectangular cover glass, 100mm x 60mm) or circular cover glasses (12mm) of 0.17mm thickness were implanted in each organ. These windows were secured with stitches or surgical glue. For heart and lung imaging, vacuum immobilization was used to prevent thoracic cavity collapse. Brain imaging utilized semi-closed or open cranial windows, reaching depths of around 190µm or up to 300µm into the tissue, respectively. Imaging sessions were conducted on spinning disc microscopes: Zeiss Cell Observer SD (Carl Zeiss Microimaging, equipped with a Yokogawa CSU-X1 confocal scanner, and an Evolve 512 EMCCD camera and a Hamamatsu ORCA-Flash 4.0 VS camera) or a 3i Marianas SDC (spinning disc confocal) microscope (Intelligent Imaging Innovations, equipped with a Yokogawa CSU-X1 confocal scanner and a Photo-metrics Evolve 512 EMCCD camera). Laser units 405, 488, and 647 were utilized for imaging Hoechst, FITC-Dextran, and AF67-CD31 respectively. Imaging was performed using either an oil-immersion plan apochromat 63 x objective with 1.4 Numerical Aperture (NA) and 0.17mm working distance (WD), or a 40 x LD C-Apochromat corrected, water immersion objective with 1.1 NA and 0.62 WD. Images were acquired for 20 seconds at a rate of 20 frames per second. ZEN blue edition v.2.6., or 3i Slidebook reader v.6.0.22 software was used for all acquisitions. Differentiation assays For the differentiation assays, T. congolense IL3000 SM cells were incubated overnight with DMSO or 20µM NPD-1015 in 5mL of TcBSF1 media on T25 culture flasks. Non-sequestered parasites were removed from the flasks by pipetting, and remaining sequestered parasites were added additional 5ml of media. Then, detachment was forced by vortexing for a few seconds. Parasites were centrifuged at 1200xg for 10 minutes and 3x10 6 parasites per condition were incubated in DTM medium at 27˚C, without CO 2 . The number of live cells were counted at 3 and 5 days after on a haemocytometer. At day 5 post differentiation induction, 10 6 procyclic cells were passaged into a new T25 flask with 5mL DTM and counted 24 hours after to estimate parasite population growth. Tsetse Fly infections T. congolense IL3000-SM bloodstream forms cryopreserved parasites were cultured in TcBSF1 media on T25 culture flasks, at 34ºC, 5% CO 2 . Twenty-four hours before fly infection, 5x10 6 parasites were supplemented with 20µM NPD-1015 or the same volume of DMSO. On the experiment day, non-sequestered parasites supplemented with DMSO were collected by pipetting and undisturbed sequestered parasites were washed and collected after vortexing for a few seconds to force detachment. These and drug-treated (non-sequestered) parasites were washed twice by centrifugation at 1200xg for 10 minutes to remove traces of DMSO and NPD-1015. Parasites were counted with a haemocytometer and fed to experimental teneral (unfed, 0–48 hours post-eclosion) male and female tsetse flies ( Glossina morsitans morsitans ) at a concentration of 10 5 trypanosomes per mL of sterile defibrinated horse blood (TCS Biosciences) via a silicone membrane as previously described 49 . Flies were maintained by feeding on non-infected sterile horse defibrinated blood, and killed by decapitation and midguts were dissected out at days 10 post-infection. Midgut infections were scored after breaking down the entire tissue in a PBS drop, and were classified as heavy, medium or mild, based on the number of parasites observed under the microscope. Transcriptomics analysis Four mice were infected with 2000 T. congolense 1/148 parasites and euthanized at days 6 post-infection. Blood was collected by cardiac puncture and mice were perfused with 50ml heparinized PBS. Brain, gonadal adipose tissue, and kidney were dissected and flash-frozen in liquid nitrogen. Organs were homogenized in Qiazol (Qiagen, UK) with silica beads on a bead beater for 2 rounds of 45 seconds. RNA was extracted using the RNeasy Universal Plus kit (Qiagen, UK) according to the manufacturer’s protocol and RNA concentration and integrity were checked by fluorometry (Qubit DNA HS, Thermo Fisher Scientific) and parallel capillary electrophoresis (TapeStation, Agilent), respectively. Trypanosome-specific cDNA libraries were prepared using custom primers targeting the spliced leader sequence as previously described 19 , and sequenced as 75bp single-end reads on the NextSeq 550 platform (Illumina, USA). Reads were aligned to the T. congolense IL3000 2018 genome available from tritrypDB version 51 using STAR 50 . The output from read alignment was processed with SAMtools 51 , and transcript abundances were estimated using stringtie 52 . Differential expression between sequestered (blood) and non-sequestered (tissues) samples was performed in R, using edgeR 53 and limma-voom 54 . Log 2 Fold change of 1 and p -value < 0.05 was considered significant. VSG profiling was conducted with VAPPER 55 . Declarations Data availability Sequencing reads are available from NCBI under BioProject accession number PRJNA1159173. Acknowledgments We thank Dr Álvaro Acosta-Serrano (The University of Notre Dame du Lac) and Dr Catarina Gadelha (University of Nottingham) for providing T. congolense 1/148 and IL3000-SM parasites, respectively. We acknowledge the support of the Rodent and Bioimaging facilities at Instituto de Medicina Molecular, the Mouse and Advanced Imaging facilities at Instituto Gulbenkian de Ciência, and the animal facility at PRBB. The SEM work was carried out in part by INL User Facilities, with support from the Electron Microscopy Facility at Instituto Gulbenkian de Ciência. Microvessels masters were fabricated by MicroFabSpace and Microscopy Characterization Facility, Unit 7 of ICTS “NANBIOSIS” from CIBER-BBN at IBEC. NPD-1015 was kindly provided by Dr Harry De Koning (University of Glasgow). We also would like to thank the efforts of Leonor Pinho and Cristina Bancells for project support, as well as Ana Nascimento, Antonio Temudo, Jose Rino, Livia Piatti and Matt Govendir for technical support. We thank Jonathan Thornton for providing and maintaining the experimental tsetse flies, and the Liverpool School of Tropical Medicine for their support in maintaining the tsetse colony, managed by Dr Aitor Casas-Sanchez. This work was funded by the Marie Skłodowska-Curie Actions post-doctoral fellowship FEBRIS [101026717]) to VI, EMBO postdoctoral fellowship (ALTF 1048-2016) and HSFP (LT000047/2019-L) long-term postdoctoral fellowship awarded to MDN; EMBO Scientific Exchange Grant #9333 to SSP; Prémio Maria de Sousa, from Fundação Bial and Ordem dos Médicos, project 7/2021 to SSP, Fundação para a Ciência e a Tecnologia (PeX/2022.02187.PTDC) to SSP, “la Caixa” Foundation (ID 10001043) through a Junior Leader Postdoctoral Fellowship to SSP (LCF/BQ/PR23/11980034), the European Research Council (ERC) (FatTryp, ref. 771714) to LMF and EMBL core funding to MB. Declaration of Interests The authors declare no competing interests. References Silva Pereira S et al (2022) Immunopathology and Trypanosoma congolense parasite sequestration cause acute cerebral trypanosomiasis. Elife 11 Ghazanfari N, Mueller SN, Heath WR (2018) Cerebral Malaria in Mouse and Man. 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Proceedings of the National Academy of Sciences 102, 15545–15550 Wang J, Vasaikar S, Shi Z, Greer M, Zhang B (2017) WebGestalt 2017: a more comprehensive, powerful, flexible and interactive gene set enrichment analysis toolkit. Nucleic Acids Res 45:W130–W137 Jain C, Koren S, Dilthey A, Phillippy AM, Aluru S (2018) A fast adaptive algorithm for computing whole-genome homology maps. in Bioinformatics vol. 34 i748–i756Oxford University Press Additional Declarations There is NO Competing Interest. Supplementary Files SF1.xlsx Supplementary File 1 Sequencing statistics and differential expression results of sequestered vs. non-sequestered T. congolense parasites. SF1.png Supplementary Figure 1 Immunofluorescence analysis of bovine aorta endothelial cells and bovine brain endothelial cells grown on a cell monolayer under static conditions. Adherens junction markers (ß-catenin in blue, VE cadherin in yellow), tight junctions markers (ZO-1 in red), actin cytoskeleton staining (phalloidin in cyan), pan-endothelial cell marker (Von Willebrand factor in green), and nuclei (4′,6-diamidino-2-phenylindole (DAPI) in magenta). Scale bar = 100µm. SF2.png Supplementary Figure 2 Parasite cytological analysis within individual organs of the mouse. Quantification of kinetoplast-nuclei counts of parasites imaged by intravital microscopy. Mice were infected with T. congolense IL3000 (blue) or 1/148 (pink), by organ. Nuclei were stained with Hoechst and intravascular environment was stained with 70kDa FITC-dextran. Error bars show standard error of the mean. SF3.png Supplementary Figure 3 Genome homology between T. congolense strains IL3000 and 1/148, estimated with mashmap, an approximate aligner for long DNA sequences. Each colour dot/line indicates a match between IL3000 and 1/148. Colours indicate different DNA strands; inverted lines indicate inversions. 58 Video1.avi Video 1 Sequestration of fluorescently-labelled Trypanosoma congolense (in green) to bovine brain microvessels during perfusion, acquired by live imaging. Scale bar = 50µm. Cite Share Download PDF Status: Published Journal Publication published 27 Feb, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-5104603\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":358711750,\"identity\":\"7493300a-a52b-47c2-8fde-0d6da895a2f1\",\"order_by\":0,\"name\":\"Teresa Porqueddu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Instituto Superior Técnico\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Teresa\",\"middleName\":\"\",\"lastName\":\"Porqueddu\",\"suffix\":\"\"},{\"id\":358711751,\"identity\":\"f1c9f251-f338-4946-9f55-c90ae991ab79\",\"order_by\":1,\"name\":\"Mariana De 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Barcelona\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Viola\",\"middleName\":\"\",\"lastName\":\"Introini\",\"suffix\":\"\"},{\"id\":358711754,\"identity\":\"af19ac10-d32d-434d-8678-785f2e7e23c0\",\"order_by\":4,\"name\":\"Maria Zorrinho-Almeida\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Universidade Católica Portuguesa\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Maria\",\"middleName\":\"\",\"lastName\":\"Zorrinho-Almeida\",\"suffix\":\"\"},{\"id\":358711755,\"identity\":\"3964ef64-519b-440f-b022-a7cdd592df60\",\"order_by\":5,\"name\":\"Silvia Sanz Sender\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"EMBL Barcelona\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Silvia\",\"middleName\":\"Sanz\",\"lastName\":\"Sender\",\"suffix\":\"\"},{\"id\":358711756,\"identity\":\"aeb21e3c-c054-450e-96d1-0a50d813ed81\",\"order_by\":6,\"name\":\"Diana Carrasqueira\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Universidade Católica Portuguesa\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Diana\",\"middleName\":\"\",\"lastName\":\"Carrasqueira\",\"suffix\":\"\"},{\"id\":358711757,\"identity\":\"a730b7fd-5c0c-4506-815b-1154ffb35d86\",\"order_by\":7,\"name\":\"Luisa Figueiredo\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0002-5752-6586\",\"institution\":\"Universidade de Lisboa\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Luisa\",\"middleName\":\"\",\"lastName\":\"Figueiredo\",\"suffix\":\"\"},{\"id\":358711758,\"identity\":\"362e7f54-49a7-4714-8970-a8bc3011365d\",\"order_by\":8,\"name\":\"Maria Bernabeu\",\"email\":\"\",\"orcid\":\"https://orcid.org/0000-0001-7212-6209\",\"institution\":\"EMBL Barcelona\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Maria\",\"middleName\":\"\",\"lastName\":\"Bernabeu\",\"suffix\":\"\"},{\"id\":358711749,\"identity\":\"3ea5ff4e-6b7c-4349-93a2-1b1209c70c1c\",\"order_by\":9,\"name\":\"Sara Silva Pereira\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDADNgYGxgcka2E2gLGI1yVBlBb59rMPPxdU3JPjkz78rJqn5g6DuXwDfi0GZ9KNpWecKTZm40szu81z7BmDZRsBWwwY0hikedsSEtt4GMxu8zYcZjA4Rshh/c+Yf/P+S6hv42H/VkyUFoYbaWzSvA0JCWw8PGbMRGkxuPGMzXrGsQTDNh6eYsk5x57xWLYlEHJYGvPtgpoEefke9o0f3tTckTNnPkDIZcAoRGIf4DEgrAFNCwNRWkbBKBgFo2BEAQAAZzpuUP19RQAAAABJRU5ErkJggg==\",\"orcid\":\"https://orcid.org/0000-0002-6590-6626\",\"institution\":\"Universidade Católica Portuguesa\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Sara\",\"middleName\":\"Silva\",\"lastName\":\"Pereira\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-09-17 16:06:34\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-5104603/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-5104603/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1038/s42003-025-07739-z\",\"type\":\"published\",\"date\":\"2025-02-27T05:00:00+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":65284698,\"identity\":\"9f3ed23e-059a-4c0c-a2f1-c1d8cfaa5a37\",\"added_by\":\"auto\",\"created_at\":\"2024-09-25 15:35:32\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1923656,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003e3D bovine microvessels.\\u003c/strong\\u003e A. Representative images of an assembled 3D microvessel device, showing collagen previously injected in between PDMS micro-patterned stamps and PMMA jig, next to a 5 cents coin (21.25mm). B. Roundness (4 x [Area / (π x major axis\\u003csup\\u003e2\\u003c/sup\\u003e)] and aspect ratio (height/width) of bovine brain microvascular endothelial cells (BBMVEC) and bovine aorta endothelial cells (BAOEC) after vessel formation. C. Immunofluorescence z-projections of multiple bovine aorta microvessels showing adherens junctions markers (ß-catenin in blue, VE cadherin in yellow), tight junction markers (ZO-1 in red), actin cytoskeleton staining (phalloidin in cyan), and nuclei (4′,6-diamidino-2-phenylindole (DAPI) in magenta). D. As C, but for bovine brain microvessels.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage12.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5104603/v1/82cafe2422d3a0cf48189206.png\"},{\"id\":65284700,\"identity\":\"45e183f5-49c5-4c5a-9e4f-8747d9d33743\",\"added_by\":\"auto\",\"created_at\":\"2024-09-25 15:35:32\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":901157,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003e3D bovine microvessels show that \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eTrypanosoma congolense\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e sequestration depends on flow.\\u003c/strong\\u003e A. Diagram showing experimental procedures of 2D detachment assay. B. Number of \\u003cem\\u003eT. congolense\\u003c/em\\u003e parasites (1/148 in pink and IL3000 SM in blue) remaining sequestered to bovine aorta endothelial cells (BAOEC) after increasing flow rates (mean ± standard deviation). C. As B, but for bovine brain microvascular endothelial cells (BBMVEC). D. Schematic of 3D microvessel perfusion with \\u003cem\\u003eT. congolense\\u003c/em\\u003e. E. Scanning electron micrograph of bovine aorta microvessel, showing an empty lumen surrounded by a layer of bovine aorta endothelial cells (EC) to which IL3000 Single Marker parasites (white arrows) have sequestered, onto a layer of collagen (COL). Bottom bar indicates magnification (958X), working distance (10.6mm) and scale (100µm). F. Zoomed-in section of a scanning electron micrograph of bovine aorta microvessel, showing part of a parasite (P) closely interacting with the endothelial cell (EC) via the flagellum tip (FLA). Bottom bar indicates magnification (50000X), working distance (10.6mm) and scale (3µm). G. Correlation of number of sequestered parasites manually quantified from micrographs and fluorescence area (µm2) estimated from Fiji. Pearson’s correlation R\\u003csup\\u003e2\\u003c/sup\\u003e=0.91, p-value\\u0026lt;0.0001. H. Quantification of the total area of sequestration across individual edges of the microvessel device between IL3000 SM and 1/148, in bovine aorta microvessels and bovine brain microvessels, and with \\u003cem\\u003ePlasmodium falciparum\\u003c/em\\u003e strain HB3var03 in human brain microvessels (median ± range). Stars indicate statistically-significant results (**** \\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt;0.0001, one-way ANOVA with Tukey’s multiple comparisons test). I. Area of sequestration occupied in the 3D microvessels at regions exposed to different wall shear stress rates. Dots indicate the mean values, shaded regions show the standard error of the mean, from a total of 4 biological replicates. Statistical analyses were performed for individual wall shear stress values and for binned regions (dotted vertical lines) using a 2-way ANOVA with Sidak’s multiple comparisons test. * \\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026lt; 0.05, **\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026lt; 0.01. J. As in I, but for bovine brain microvessels. K. Quantification of 1/148 sequestration to brain and aorta microvessels throughout different regions of the device (mean ± range) (2-way ANOVA with Sidak’s multiple comparisons test, * \\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026lt; 0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage21.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5104603/v1/dfb4989618ab1a28c1a141c1.png\"},{\"id\":65285282,\"identity\":\"17d51279-32ef-46a4-956f-1e1780bdadac\",\"added_by\":\"auto\",\"created_at\":\"2024-09-25 15:43:32\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":910443,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCyclic AMP homeostasis is essential for \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eTrypanosoma congolense\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e sequestration. \\u003c/strong\\u003eA. Diagram showing experimental procedure for testing the effect of NPD-1015 on \\u003cem\\u003eT. congolense\\u003c/em\\u003e sequestration ability. B. Representative widefield micrographs of \\u003cem\\u003eT. congolense\\u003c/em\\u003e IL3000 SM parasites sequestered to a bovine aorta endothelial cell (BAOEC) monolayer. Parasites were incubated with DMSO (control), 10µM or 20µM of NPD-1015 for 24 hours before co-culture with endothelial cells. White arrows indicate parasites. Scale bar = 50µm C. Quantification of parasite proliferation during 24 hours of BAOEC co-culture, in the presence or absence of a previous treatment with NPD-1015. D. Quantification of parasites sequestered to BAOEC with and without previous NPD-1015 treatment (as described in A and B). E. Quantification of parasites remaining sequestered to BAOEC after 24-hour treatment with 2µM 0NPD-1015. F. Diagram showing experimental procedure for testing the effect of NPD-1015 on \\u003cem\\u003eT. congolense\\u003c/em\\u003e sequestration to 3D bovine aorta microvessels. G. Total area of sequestration between DMSO-treated and NPD-1015-treated parasites across the3D microvessels. Unpaired t-test, ***\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026lt; 0.001. H. Area of sequestration occupied in the 3D microvessels at regions exposed to different wall shear stress rates. Dots indicate the mean values, shaded regions show the standard error of the mean, from a total of 3 biological replicates. Statistical analyses were performed for individual wall shear stress values for individual wall shear stress values and for binned regions (dotted vertical lines) using a mixed-effects analysis with Tukey’s multiple comparisons test. * \\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026lt; 0.05, **\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026lt; 0.01.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage33.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5104603/v1/f451ab3a6d6d7708557f2b73.png\"},{\"id\":65284703,\"identity\":\"ad5694c9-2125-405e-8158-c0fd6532a35b\",\"added_by\":\"auto\",\"created_at\":\"2024-09-25 15:35:32\",\"extension\":\"jpeg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":881870,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSequestration is associated with cell cycle but does not affect transmission. \\u003c/strong\\u003eA. Representative image and respective quantification of kinetoplast-nuclei counts of \\u003cem\\u003eT. congolense\\u003c/em\\u003e IL3000 SM parasites.\\u0026nbsp; Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) in magenta. Scale bar = 10µm. Parasite bodies are delineated in white. B. Parasitaemia throughout infection with 1/148 and IL3000 parasites estimated by hemocytometry. Reproduced from Silva Pereira \\u003cem\\u003eet al.\\u003c/em\\u003e 2022 (\\u003cem\\u003eeLife\\u003c/em\\u003e). C. Representative image of kinetoplast-nuclei counts of \\u003cem\\u003eT. congolense\\u003c/em\\u003e 1/148 parasites imaged by intravital microscopy. Nuclei are stained with Hoechst (in cyan) and intravascular space is stained with 70kDa FITC-Dextran (in yellow). Scale bar = 20µm. D. Quantification of kinetoplast-nuclei counts of \\u003cem\\u003eT. congolense\\u003c/em\\u003e 1/148 and IL3000 parasites during mouse infections. Data was collected from 8 major organs (adipose tissue, brain, spleen, liver, kidneys, lungs, heart, and pancreas) throughout the infection course. Stars indicate statistically-significant results (unpaired t-test). **** \\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026lt; 0.0001. E. Simplified representation of the \\u003cem\\u003eT. congolense\\u003c/em\\u003e life cycle, highlighting the differentiation from the mammalian-stage bloodstream form to the insect-stage procyclic form. During\\u003cem\\u003e in vitro \\u003c/em\\u003edifferentiation, \\u003cem\\u003eT. congolense \\u003c/em\\u003eIL3000 SM parasites were incubated at 27ºC without CO\\u003csub\\u003e2\\u003c/sub\\u003e for 5 days to differentiate into procyclic forms. F. Number of parasites during and after induction of differentiation by temperature and pH alteration, for sequestered and non-sequestered parasites untreated, treated with NPD-1015, or treated with DMSO. Starting parasite populations of 3x10\\u003csup\\u003e6\\u003c/sup\\u003e cells. G. Growth of procyclic parasite population over 24h starting 5 days after induction of differentiation, when all parasites are procyclic forms. Starting parasite populations of 10\\u003csup\\u003e6\\u003c/sup\\u003e cells. H. Percentage of tsetse flies successfully infected with sequestered, non-sequestered or NPD-1015-treated \\u003cem\\u003eT. congolense\\u003c/em\\u003e IL3000 SM parasites. Stars indicate statistically-significant results (one-way ANOVA with Tukey’s multiple comparisons test). **\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026lt; 0.01. I. Tsetse fly midgut infection load at day 10 post-infection. Infections were scored as heavy, mild or low according to the number of parasites observed in dissected midguts. Individual \\u003cem\\u003ep\\u003c/em\\u003e-values and \\u003cem\\u003eq\\u003c/em\\u003e-values of false discovery rates are shown when significant. 2-way ANOVA with Benjamini, Krieger and Yekutieli multiple comparison correction.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5104603/v1/5b6171136c13d6fa6555f9f1.jpeg\"},{\"id\":65284705,\"identity\":\"352b0774-cf88-4a89-b768-454ea4bee127\",\"added_by\":\"auto\",\"created_at\":\"2024-09-25 15:35:32\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":358809,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSequestered parasites display differential transcriptomes and changes in VSG expression. \\u003c/strong\\u003eA. Multidimensional scaling plot showing distribution of sequestered and non-sequestered parasite samples and the tissues they derive from. B. MA plot showing differentially expressed genes (Log\\u003csub\\u003e2\\u003c/sub\\u003e Fold Change) and their abundance (Log\\u003csub\\u003e2\\u003c/sub\\u003e counts per million transcripts (CPM)). Genes mentioned in the text are labelled and represented by a triangle. C. Gene set enrichment analysis using genes upregulated upon silencing of gene TcoREG9.1 as query. Genes were pre-ranked by their log\\u003csub\\u003e2\\u003c/sub\\u003e fold change value upon sequestration. Gene distribution is shown by black arrows along the full ranked transcriptome. Green line indicates distribution of enrichment scores. D. Expressed variant antigen profiles of sequestered and non-sequestered parasites per sample and per organ of origin, compared to the\\u003cem\\u003e T. congolense \\u003c/em\\u003e1/148 genomic variant antigen profile. Phylotypes are colour-coded according to key. E. Expressed variant antigen profiles of sequestered and non-sequestered parasites. ****=\\u003cem\\u003ep\\u003c/em\\u003e-value \\u0026lt;0.0001, independent t-test. Bars are colour-coded according to key.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage51.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5104603/v1/312c3442d97b06a4ceaab6cd.png\"},{\"id\":77299875,\"identity\":\"6a36df51-e57b-4d6f-a827-f8d4baf65493\",\"added_by\":\"auto\",\"created_at\":\"2025-02-27 08:09:01\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":5793501,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5104603/v1/efad2281-b03a-4b2d-9c49-be5294061ac0.pdf\"},{\"id\":65285281,\"identity\":\"38f2bdd3-4396-4d99-be13-1d05f2d469ef\",\"added_by\":\"auto\",\"created_at\":\"2024-09-25 15:43:32\",\"extension\":\"xlsx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":108330,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSupplementary File 1 \\u003c/strong\\u003eSequencing statistics and differential expression results of sequestered \\u003cem\\u003evs.\\u003c/em\\u003e non-sequestered \\u003cem\\u003eT. congolense\\u003c/em\\u003eparasites.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"SF1.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5104603/v1/ee740f878a52cea909128304.xlsx\"},{\"id\":65284708,\"identity\":\"996e5ae7-bccc-4718-b5d4-156f5616313b\",\"added_by\":\"auto\",\"created_at\":\"2024-09-25 15:35:33\",\"extension\":\"png\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":62802263,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSupplementary Figure 1 Immunofluorescence analysis of bovine aorta endothelial cells\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eand bovine brain endothelial cells grown on a cell monolayer under static conditions\\u003c/strong\\u003e. \\u0026nbsp;Adherens junction markers (ß-catenin in blue, VE cadherin in yellow), tight junctions markers (ZO-1 in red), actin cytoskeleton staining (phalloidin in cyan), pan-endothelial cell marker (Von Willebrand factor in green), and nuclei (4′,6-diamidino-2-phenylindole (DAPI) in magenta). Scale bar = 100µm.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"SF1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5104603/v1/b950afaf6ad5d9637365a797.png\"},{\"id\":65285283,\"identity\":\"885bd629-63aa-44c5-8dca-6dea3bba1e20\",\"added_by\":\"auto\",\"created_at\":\"2024-09-25 15:43:32\",\"extension\":\"png\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":564443,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSupplementary Figure 2 Parasite cytological analysis within individual organs of the mouse. \\u003c/strong\\u003eQuantification of kinetoplast-nuclei counts of parasites imaged by intravital microscopy. Mice were infected with \\u003cem\\u003eT. congolense\\u003c/em\\u003e IL3000 (blue) or 1/148 (pink), by organ. Nuclei were stained with Hoechst and intravascular environment was stained with 70kDa FITC-dextran. Error bars show standard error of the mean.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"SF2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5104603/v1/f9536c0194b653583dbb0d0a.png\"},{\"id\":65284707,\"identity\":\"30c5156c-064d-4a1b-bc17-b3d77726a7cb\",\"added_by\":\"auto\",\"created_at\":\"2024-09-25 15:35:32\",\"extension\":\"png\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":2743027,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSupplementary Figure 3 Genome homology between \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eT. congolense\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e strains IL3000 and 1/148, estimated with mashmap, an approximate aligner for long DNA sequences. \\u003c/strong\\u003eEach colour dot/line indicates a match between IL3000 and 1/148. Colours indicate different DNA strands; inverted lines indicate inversions.\\u003c/p\\u003e\\n\\u003cp\\u003e58\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"SF3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5104603/v1/36adb95e05f88b6aa0a89dd6.png\"},{\"id\":65284706,\"identity\":\"682cdc86-aa1f-4b84-aade-1be3f8366b98\",\"added_by\":\"auto\",\"created_at\":\"2024-09-25 15:35:32\",\"extension\":\"avi\",\"order_by\":5,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":5110416,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eVideo 1\\u003c/strong\\u003e Sequestration of fluorescently-labelled \\u003cem\\u003eTrypanosoma congolense\\u003c/em\\u003e (in green) to bovine brain microvessels during perfusion, acquired by live imaging. Scale bar = 50µm.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Video1.avi\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5104603/v1/3956c047cda5caac2e2f3e98.avi\"}],\"financialInterests\":\"There is \\u003cb\\u003eNO\\u003c/b\\u003e Competing Interest.\",\"formattedTitle\":\"Bioengineered 3D microvessels reveal novel determinants of Trypanosoma congolense sequestration\",\"fulltext\":[{\"header\":\"Main\",\"content\":\"\\u003cp\\u003e \\u003cem\\u003eTrypanosoma congolense\\u003c/em\\u003e is a unicellular, intravascular parasite that causes animal African trypanosomiasis, or nagana, in several mammals, and particularly pathogenic for livestock and dogs in Africa. The parasite replicates in the blood, where it binds to the vascular endothelium, in a process known as sequestration. Whilst most infections result in a chronic disease, a small proportion of animals develop an acute, rapidly fatal illness. In rodent models, acute cerebral disease is determined by increased \\u003cem\\u003eT. congolense\\u003c/em\\u003e sequestration in the brain and is characterized by immune cell recruitment and early death\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. In other parasitic diseases, such as malaria and babesiosis\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e, sequestration also determines clinical course, disease severity, and organ pathology. Sequestration in Trypanosomes has recently been suggested to play a role in transmission, since silencing of a gene orthologous to a \\u003cem\\u003eT. brucei\\u003c/em\\u003e negative regulator of differentiation to the insect-transmissible form resulted in reduction of attachment to a plastic substrate and an increase in peripheral parasitaemia \\u003cem\\u003ein vivo\\u003c/em\\u003e\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eDespite its importance for disease pathogenesis, the determinants of sequestration, be it molecular, biophysical, or biochemical, remain unknown. Many challenges have precluded the study of sequestration determinants, namely the large phenotypic variability of animal models, the short timespan in the development of acute cerebral trypanosomiasis, and the absence of physiologically-relevant \\u003cem\\u003ein vitro\\u003c/em\\u003e models that could reproduce the complexity of the parasite-endothelial cell interaction. In cerebral malaria, the development of bioengineered \\u003cem\\u003ein vitro\\u003c/em\\u003e human 3D brain microvessels\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e has allowed for the recent development of many advances in severe and cerebral malaria research.\\u003c/p\\u003e \\u003cp\\u003eIn this study, we adapted this microvessel system to the study of trypanosomiasis by engineering two 3D bovine endothelial microvessel models that mimic the bovine brain microvasculature and the aorta. Strains from \\u003cem\\u003eT. congolense\\u003c/em\\u003e that cause acute and chronic trypanosomiasis showed different tropism for cardiac and brain 3D microvessels under different flow mechanical conditions. Using a combination of the microvessels systems, simpler cytoadhesion assays, and mouse and tsetse fly experimental infections, we found that sequestration is dependent on cyclic adenosine monophosphate (cAMP), closely associated to parasite proliferation, but not associated with transmission. Furthermore, we assessed the \\u003cem\\u003eT. congolense\\u003c/em\\u003e gene expression remodelling that takes place in sequestered parasites.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003eThe use of acute and chronic \\u003cem\\u003eT. congolense\\u003c/em\\u003e rodent models has shed light on the importance of parasite sequestration in vascular pathogenesis and inflammation\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. Yet, uncovering the determinants of parasite sequestration through the exclusive use of animal models is challenging, given the difficulties to disentangle the cellular and biophysical components of the whole organism. To overcome this challenge, we developed a 3D endothelialised bovine microvessel system. The devices consist of a 3D microfluidic network with a pre-defined geometry on a collagen scaffold fabricated by soft lithography and injection moulding (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). The system supports the growth of primary microvascular endothelial cells in lumenised microvessels, perfusable with \\u003cem\\u003eT. congolense\\u003c/em\\u003e, which allows for the study of parasite sequestration under controlled conditions, including flow and endothelial cell type, independently of other host factors.\\u003c/p\\u003e \\u003cp\\u003eOur previous results showed that different parasite strains accumulate in various organotypic beds in mouse models, leading to distinct clinical presentations. Whilst \\u003cem\\u003eT. congolense\\u003c/em\\u003e 1/148 parasites accumulate highly in the brain microvasculature, causing acute cerebral trypanosomiasis, IL3000 parasites present a tropism for the heart, and cause chronic, wasting disease\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. To better understand this organ preference \\u003cem\\u003ein vitro\\u003c/em\\u003e, we developed two microvessel systems: one mimicking the heart vasculature with bovine aorta endothelial cells (BAOEC) and one mimicking the brain with bovine brain microvascular endothelial cells (BBMVEC). Endothelial cell identity of both cell types was assessed by immunostaining on 2D monolayers. Both BBMVEC and BAOEC expressed adherens (VE-cadherin, \\u0026szlig;-catenin) and tight junction markers (ZO-1), as well as the pan-endothelial cell marker, Von Willebrand factor, (Supplementary Fig.\\u0026nbsp;1). We then seeded both endothelial cell types in a 13 x 13 microfluidic branched network of 120 \\u0026micro;m microvessels. This geometry recapitulates a large range of flow velocities (42.5-fold range) and generates microvessels analogous to post-capillary venules in terms of surface-to-volume ratio (20 mm\\u003csup\\u003e2\\u003c/sup\\u003e/mm\\u003csup\\u003e3\\u003c/sup\\u003e). After 3 days in culture, BAOEC and BBMVEC form a 3D tubular geometry with empty lumens. Immunofluorescence labelling with junctional markers revealed that both cell types align with flow when grown in 3D. However, they are morphologically distinct: BAOEC are smaller and more rounded, whilst BBMVEC are bigger and more elongated with a higher aspect ratio (unpaired t-test, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB). Nonetheless, they both express markers of adherens (VE-cadherin, \\u0026szlig;-catenin) and tight junctions (ZO-1) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC and D). The actin cytoskeleton appears to be cortical on BAOEC, while more stress fibres are present on BBMVEC, probably due to the endothelial stretching found in brain microvascular models. In conclusion, we developed two \\u003cem\\u003ein vitro\\u003c/em\\u003e microvessel systems mimicking heart and brain vasculature, with expected endothelial and junctional marker expression, to study organ-specific trypanosome sequestration.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eT. congolense sequestration to 3D microvessels is dependent on wall shear stress, parasite strain and endothelial cell type\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eBlood flow velocity and the associated wall shear stress (WSS) varies along the hierarchical vascular bed. In healthy conditions, in the arteriovenous microcirculation, WSS ranges range between 5 and 40 dyn/cm\\u003csup\\u003e2\\u003c/sup\\u003e in arterioles and capillaries, and 1 and 5 dyn/cm\\u003csup\\u003e2\\u003c/sup\\u003e in venules\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR7\\\" citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e. However, in pathological conditions, such as when there is vascular obstruction, flow and WSS may reduce\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. To assess the role of WSS and organotypic endothelial cell types in \\u003cem\\u003eT. congolense\\u003c/em\\u003e sequestration, we first determined the flow mechanical stress that \\u003cem\\u003eT. congolense\\u003c/em\\u003e parasites can withstand. To this end, we used a much simpler setup. BAOEC or BBMVEC were seeded into 6-channel \\u0026micro;-slides overnight at maximum confluence. Fluorescently-labelled \\u003cem\\u003eT. congolense\\u003c/em\\u003e parasites were introduced and allowed to cytoadhere for 30 minutes, followed by perfusion with increasing flow rates to measure parasite binding strength against detachment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). We observed that both 1/148 and IL3000 parasites remained bound to BAOEC, withstanding high WSS values. In fact, at WSS of 11.97 dyn/cm\\u003csup\\u003e2\\u003c/sup\\u003e, 25%\\u0026plusmn;11 and 43%\\u0026plusmn;21 of 1/148 and IL3000 parasites, respectively, remained sequestered (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). IL3000 presented similar binding kinetics to BBMVEC (51%\\u0026plusmn;16 at maximum WSS) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). Conversely, 1/148 presented a different behaviour on BBMVEC, with parasites being significantly released back to circulation when exposed to WSS forces higher than 0.6 dyn/cm\\u003csup\\u003e2\\u003c/sup\\u003e (\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001, 2-way ANOVA with Sidak\\u0026rsquo;s correction for multiple comparisons). This shows that different parasite strains display heterogeneous binding behaviour to different endothelial beds and suggests that 1/148 presents lower sequestration strength to brain microvessels at physiological WSS.\\u003c/p\\u003e \\u003cp\\u003eHaving shown that at least a proportion of both \\u003cem\\u003eT. congolense\\u003c/em\\u003e 1/148 and IL3000 can withstand these forces, we tested the role of WSS in parasite sequestration. When perfused at a constant flow rate of 10\\u0026micro;l/min, endothelial cells in the outer edge of the 13 x 13 grid are exposed to flow velocities that range from 0.4 to 15.2 mm/s, which translates into WSS values between 0.08 and 3.4 dyn/cm\\u003csup\\u003e2\\u003c/sup\\u003e. We perfused 3D aorta or brain microvessels with 1.5\\u0026nbsp;million fluorescently-labelled parasites of either IL3000 or 1/148 strains (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD and Video 1). Following a 15-minute perfusion, unbound parasites were washed under flow for 10 minutes and microvessels were fixed and stained for subsequent microscopical analysis and binding quantification.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eParasites presented widespread cytoadhesion to 3D microvessels by live microscopy (video 1). Scanning electron microscopy showed that parasites sequester to the luminal side of the endothelial cells lining the 3D vessels on top of the collagen matrix (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE). Within the 3D microvessel, a smooth, empty lumen was surrounded by tightly clustered endothelial cells, to which the parasites sequestered (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE). In agreement with previous literature\\u003csup\\u003e\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e, close interaction between the flagellum and the endothelial cell was observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eF). Several microfilaments, which have been previously reported, coming out of the endothelial cell and surrounding the parasite cell body could also be observed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eF).\\u003c/p\\u003e \\u003cp\\u003eHaving confirmed that parasites sequester to the 3D microvessels, we proceeded with binding quantifications. Since we were using fluorescently-labelled parasites, we calculated the area occupied by parasite binding (obtained from the total fluorescence area) as a proxy for the number of sequestered parasites because these two variables (number of sequestered parasites and fluorescence area) correlate highly to each other (R\\u003csup\\u003e2\\u003c/sup\\u003e\\u0026thinsp;=\\u0026thinsp;0.91, Pearson\\u0026rsquo;s correlation, p-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eG). Overall, IL3000 parasites presented higher sequestration to bovine aorta microvessels than 1/148 (\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001, ordinary one-way ANOVA with Tukey\\u0026rsquo;s multiple comparisons test), whilst both strains showed similar binding levels to bovine brain microvessels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eH). Despite these variations, \\u003cem\\u003eT. congolense\\u003c/em\\u003e sequestration, regardless of the strain and endothelial bed, is significantly higher than sequestration of \\u003cem\\u003ePlasmodium falciparum\\u003c/em\\u003e HB3var03 to human 3D brain microvessels, a malaria parasite line associated to severe and cerebral malaria in humans\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. Even though \\u003cem\\u003eP. falciparum\\u003c/em\\u003e was perfused at higher concentrations, \\u003cem\\u003eT, congolense\\u003c/em\\u003e 1/148 presented a 5-fold higher binding, and IL3000 a 15-fold. These results highlight the affinity and extend of binding of \\u003cem\\u003eT. congolense\\u003c/em\\u003e compared to other parasitic disease that affect brain microvessels.\\u003c/p\\u003e \\u003cp\\u003eSequestration of both strains to aorta 3D microvessels negatively correlated with WSS (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eI). More specifically, IL3000 presented high binding level at WSS of 0.5 dyn/cm\\u003csup\\u003e2\\u003c/sup\\u003e and below, then it slowly decreased when it reached a WSS of 1 dyn/cm\\u003csup\\u003e2\\u003c/sup\\u003e, after which it plateaued. In contrast, 1/148 displayed lower binding levels than those of IL3000 across all WSS regions, being significantly lower in the range between 0.25 and 0.8 dyn/cm\\u003csup\\u003e2\\u003c/sup\\u003e (\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;=\\u0026thinsp;0.02 for WSS values of 0.5\\u0026ndash;0.8 and \\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;=\\u0026thinsp;0.001 for WSS values 0.25 to 0.45, 2-way ANOVA with Sidak\\u0026rsquo;s correction for multiple comparisons) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eI). These observations agree with findings in mouse models, where \\u003cem\\u003eT. congolense\\u003c/em\\u003e IL3000 shows higher sequestration to the heart microvasculature than 1/148\\u003csup\\u003e1\\u003c/sup\\u003e. When exposed to bovine brain microvessels, IL3000 sequestration followed the same binding pattern than in aorta microvessels and reached similar sequestration levels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eJ). However, 1/148 presented a different sequestration pattern with two independent sequestration peaks. First, sequestration was dramatically high at 0.08 dyn/cm\\u003csup\\u003e2\\u003c/sup\\u003e, representing accumulations in 40% of the total vessel area 12867\\u0026micro;m\\u003csup\\u003e2\\u003c/sup\\u003e\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4483 of sequestration area, which is 3 times what was observed for IL3000 (i.e. 4365\\u0026micro;m\\u003csup\\u003e2\\u003c/sup\\u003e\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;719) (\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;=\\u0026thinsp;0.03, 2-way ANOVA with Sidak\\u0026rsquo;s correction for multiple comparisons). Then, there was also non-significant sequestration bump at 1.4 dyn/cm\\u003csup\\u003e2\\u003c/sup\\u003e. When comparing 1/148 sequestration across different vascular beds, we observed that 1/148 parasites bound similarly to aorta and brain at WSS higher than 0.25 dyn/cm\\u003csup\\u003e2\\u003c/sup\\u003e but presented significantly more binding to brain at pathological WSS levels of less than 0.25 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eK) (\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;=\\u0026thinsp;0.03, 2-way ANOVA with Sidak\\u0026rsquo;s correction for multiple comparisons). Interestingly, in the mouse model of acute cerebral trypanosomiasis, \\u003cem\\u003eT. congolense\\u003c/em\\u003e 1/148 preferentially sequesters in the small capillaries of the brain, often leading to vascular occlusion and vessel blockage\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e, which greatly reduce blood flow and the associated WSS. We conclude that \\u003cem\\u003eT. congolense\\u003c/em\\u003e sequestration is dependent on flow mechanical properties, irrespective of the parasite strain and endothelial cell organotype. Nevertheless, parasite strains display distinct sequestration behaviours, which may be important for the clinical outcome.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eT. congolense sequestration can be prevented by interfering with cAMP homeostasis\\u003c/h2\\u003e \\u003cp\\u003eHaving established the role of WSS in sequestration, we asked how we could interfere with sequestration \\u003cem\\u003ein vitro\\u003c/em\\u003e. It has been previously suggested that cAMP phosphodiesterase inhibition results in lower \\u003cem\\u003eT. congolense\\u003c/em\\u003e sequestration\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. Therefore, we attempted to reproduce that phenotype by treating IL3000 parasites with 10\\u0026micro;M or 20\\u0026micro;M of NPD-1015, an inhibitor of cAMP phosphodiesterases PDEB1 and PDEB2\\u003csup\\u003e12\\u003c/sup\\u003e) that interferes with cAMP homeostasis, increasing the cAMP intracellular levels in the parasite and resulting in growth arrest\\u003csup\\u003e\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e. After 24 hours, the drug was removed, and parasites were added to a 2D monolayer of BAOEC and allowed to sequester. Subsequently, we washed unbound parasites and quantified the number of sequestered parasites by microscopy (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA and B). Treatment did not affect the health of parasites; we observed vigorous parasite motility and did not detect abnormal cell debris in the wells. However, there was a significant growth arrest upon NPD-1015 treatment: whilst untreated parasites proliferated 82%\\u0026plusmn;14 over 24 hours, parasites treated with 10\\u0026micro;M NPD-1015 grew only 42%\\u0026plusmn;4 and those treated with 20\\u0026micro;M NPD-1015 did not proliferate at all (-7%\\u0026plusmn;10) (\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;=\\u0026thinsp;0.0026, one-way ANOVA with Tukey\\u0026rsquo;s correction for multiple comparisons) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC).\\u003c/p\\u003e \\u003cp\\u003eWe observed that parasite exposed to 10\\u0026micro;M of NPD-1015 presented a 5-fold lower binding (\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001, one-way ANOVA with Tukey\\u0026rsquo;s correction for multiple comparisons), whilst exposure to 20\\u0026micro;M of NPD-1015 decreased binding levels 20 times (\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001, one-way ANOVA with Tukey\\u0026rsquo;s correction for multiple comparisons) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB and D). As we washed the drug before adding the parasites to the endothelial cell monolayers, we ensured that any effect observed is parasite-derived. We then asked if NPD-1015 could revert binding of sequestered parasites. Therefore, we co-cultured \\u003cem\\u003eT. congolense\\u003c/em\\u003e IL3000 parasites in a 2D BAOEC monolayer for 24 hours. Subsequently, we removed non-sequestered parasites by washing and added 20\\u0026micro;M of NPD-1015 for 24 hours longer. We observed a reduction of sequestration, indicating that NPD-1015 induced detachment of parasites. This suggests that increasing intracellular cAMP not only prevents sequestration, but also reverts it (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE).\\u003c/p\\u003e \\u003cp\\u003eWe then used the 3D microvessels system to test whether a similar binding reduction occurred under flow. For that, we incubated parasites with 20\\u0026micro;M NPD-1015 and after 24 hours perfused the microvessels with treated parasites (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF). Overall, there was a 25% reduction of sequestration (\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;=\\u0026thinsp;0.03, unpaired t-test) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eG), consistent with our previous observation in the 2D assay, but with a lower effect than in EC monolayers, suggesting that the tridimensional architecture of the microvessel or the presence of flow might increase cytoadhesion efficiency. Although NPD-1015 treatment did not affect parasite sequestration dependence on WSS (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eH), an increase in parasitic cAMP significantly reduced parasite binding in regions exposed to a range of WSS between 0.25 and 1.4 dyn/cm\\u003csup\\u003e2\\u003c/sup\\u003e. No differences in binding were observed when the parasite was exposed to a WSS below 0.25 dyn/cm\\u003csup\\u003e2\\u003c/sup\\u003e. In conclusion, we showed that interfering with cAMP homeostasis is sufficient to both prevent and revert sequestration, and this effect is more pronounced under specific flow mechanical cues.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eSequestered parasites proliferate more in the mammalian host and have similar transmission ability to non-sequestered parasites\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eNext, we hypothesised that sequestration provides an adaptive advantage for \\u003cem\\u003eT. congolense\\u003c/em\\u003e, so that sequestered parasites proliferate faster than non-sequestered. To test that, we examined the cell cycle distribution of sequestered and non-sequestered IL3000 parasites, grown \\u003cem\\u003ein vitro\\u003c/em\\u003e, on plastic, without endothelial cells, by quantifying the kinetoplast and nuclei number in each trypanosome cell. (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). We noticed that the population of sequestered parasites contained a higher proportion of proliferating parasites, which typically can be distinguished by having with two kinetoplasts and one nucleus (2K1N, 20%\\u0026plusmn;8 \\u003cem\\u003evs.\\u003c/em\\u003e 5%\\u0026plusmn;4) relative to non-sequestered population (\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001, one-way ANOVA with Tukey\\u0026rsquo;s correction for multiple comparisons) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA), suggesting that sequestered parasites divide more frequently than non-sequestered parasites. Parasites in S phase (i.e. with kinetoplast butterfly-shaped) were considered as 2K1N. We did not observe statistically significant differences in the number of mitotic parasites (2K2N configuration).\\u003c/p\\u003e \\u003cp\\u003eTo test whether sequestered parasites divide more frequently \\u003cem\\u003ein vivo\\u003c/em\\u003e, we used intravital microscopy data previously collected from mice infected with either \\u003cem\\u003eT. congolense\\u003c/em\\u003e 1/148 or IL3000\\u003csup\\u003e1\\u003c/sup\\u003e. In 1/148 infections, we analysed video recordings from 8 major organs (i.e. adipose tissue, brain, heart, liver, lungs, kidneys, spleen) at days 1\\u0026ndash;6 post-infection, corresponding to the timepoint after which infected animals start developing acute cerebral trypanosomiasis. In IL3000 infections, we analysed data from the same organs, but at the first peak of parasitaemia, the interval between the first and the second peaks of parasitaemia, where peripheral parasitaemia is barely detected, and the second peak of parasitaemia (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB). Before image acquisition, Hoechst and FITC-Dextran were injected intravenously into the mice, to allow detection of intravascular parasites and their DNA using intravital microscopy (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC). We differentiated between sequestered and non-sequestered parasites based on their displacement during the video as previously described\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. We observed that, overall, sequestered parasites were more often found replicating and dividing (i.e. 2K1N or 2K2N) than non-sequestered parasites (p-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001, one-way ANOVA with Tukey\\u0026rsquo;s correction for multiple comparisons) in both 1/148 and IL3000 infections (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD). This shows that sequestered parasites divide more than non-sequestered parasites, irrespective of the parasite strain.\\u003c/p\\u003e \\u003cp\\u003eFinally, we asked whether the tissue microenvironment might affect parasite division (Supplementary Fig.\\u0026nbsp;2). In both IL3000 and 1/148 mouse infections, we observed an enrichment of dividing parasites (2K1N and 2K2N) in all organs, regardless of the parasite strain, except on lung and kidney for 1/148. Therefore, we conclude that the association between sequestration and \\u003cem\\u003eT. congolense\\u003c/em\\u003e cell division is neither strain- nor endothelial cell organotype-dependent.\\u003c/p\\u003e \\u003cp\\u003eGiven our results suggesting that sequestration facilitates parasite proliferation (or vice-versa) and recent work suggesting that non-sequestered parasites may be growth-arrested, insect-transmissible forms\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e, we tested the ability of sequestered and non-sequestered parasites to differentiate into procyclic (insect) forms (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eE). We separated \\u003cem\\u003ein vitro\\u003c/em\\u003e sequestered from non-sequestered parasites, incubated them in differentiation trypanosome media (DTM), at 27\\u0026ordm;C, without CO\\u003csub\\u003e2\\u003c/sub\\u003e, and followed parasite differentiation and growth for 5 days (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eE). We observed that both sequestered and non-sequestered parasites could successfully differentiate into procyclic forms with similar dynamics, reaching similar procyclic parasite number within 5 days (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eF). Procyclic parasites were identified by their morphology (pointy and elongated cells, with the flagellum starting from the mid body) and motility (not sequestering, fast swimmers). We also quantified their proliferation rate over 24 hours after procyclic differentiation and did not observe any difference (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eG). Since cell cycle arrest precedes procyclic differentiation in the related organism \\u003cem\\u003eT. brucei\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/em\\u003e\\u003c/sup\\u003e, we forced \\u003cem\\u003eT. congolense\\u003c/em\\u003e cell cycle arrest with the administration of NPD-1015 24 hours before induction of differentiation. Again, we did not observe any difference in the ability of each parasite population to differentiate into procyclic forms (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eF) or of differentiated procyclic forms to grow (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eG). Finally, we assessed the ability of sequestered, non-sequestered, and NPD-1015-treated bloodstream form parasites to infect tsetse flies. As \\u003cem\\u003eT. brucei\\u003c/em\\u003e PDEB1 gene deletion was shown to disrupt social motility\\u003csup\\u003e\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u003c/sup\\u003e and pH taxis\\u003csup\\u003e\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u003c/sup\\u003e of procyclic parasites, we thoroughly washed the parasites to before feeding them to the flies, removing any traces of NPD-1015. Furthermore, we fed the tsetse flies with a low inoculum (10\\u003csup\\u003e5\\u003c/sup\\u003e parasites/ mL blood) to increase the probability of observing differences in fly-infectivity. The lower inoculum, the higher the proportion of parasites that must be competent to achieve infection. In contrast, with a higher inoculum, even a small proportion of fly-infective trypanosomes could represent enough parssites to saturate fly infection rates and mask potential differences between groups. We observed that sequestered and non-sequestered parasites infected similar proportions of flies (37\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3% and 26\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;9%, respectively), whereas NPD-1015-treated parasites infected significantly fewer flies (12\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4%) (\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;=\\u0026thinsp;0.0043, one-way ANOVA with Tukey\\u0026rsquo;s correction for multiple comparisons) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eH). Moreover, despite similar overall infection rates between sequestered and non-sequestered parasites, we observed that the former resulted in heavier infections (higher parasite load in the midguts) than both non-sequestered and NPD-1015-treated parasites (\\u003cem\\u003eq\\u003c/em\\u003e-value\\u0026thinsp;=\\u0026thinsp;0.0002, \\u0026lt;\\u0026thinsp;0.0001, =\\u0026thinsp;0.021, respectively, 2-way ANOVA with Benjamin, Krieger and Yekutieli method correction for false discovery rate) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eI).\\u003c/p\\u003e \\u003cp\\u003eIn summary, our data suggest that sequestration is associated to higher proliferation rates in the mammalian host and heavier infections in the vector, which might affect transmission potential, even though both sequestered and non-sequestered parasites are fly-transmissible.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eT. congolense\\u003c/b\\u003e \\u003cb\\u003esequestered parasites show distinct transcriptomes to non-sequestered\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe striking differences in cell cycle stage between sequestered and non-sequestered parasites both \\u003cem\\u003ein vitro\\u003c/em\\u003e and \\u003cem\\u003ein vivo\\u003c/em\\u003e, suggest that these two parasite forms are intrinsically distinct. Therefore, we compared their gene expression profiles during acute cerebral trypanosomiasis \\u003cem\\u003ein vivo\\u003c/em\\u003e. We infected C57BL/6J mice with 2000 \\u003cem\\u003eT. congolense\\u003c/em\\u003e 1/148 parasites\\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e and, at the first peak of parasitaemia (day 6 post-infection), we collected systemic blood and three organs: the brain, the adipose tissue, and the kidney. From these samples, we extracted total RNA and performed multiplexed trypanosome targeted RNA sequencing based on the spliced-leader\\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e enrichment (SL-seq)\\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. These organs were chosen because the parasite population in their vasculature is predominantly in its sequestered form\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. Therefore, we obtained transcriptomes of non-sequestered parasites from systemic blood samples, whereas sequestered parasites enriched transcriptomes were obtained from the tissues.\\u003c/p\\u003e \\u003cp\\u003eFirst, we removed the sequencing reads that mapped to the mouse genome. Then, we mapped the remaining reads to the annotated \\u003cem\\u003eT. congolense\\u003c/em\\u003e IL3000 genome\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e (Supplementary file 1), given that genome homology analysis shows that IL3000 and 1/148 strains have 94.8%\\u0026plusmn;2.8 nucleotide sequence identity (Supplementary Fig.\\u0026nbsp;3). We compared the transcriptomes from parasites of each tissue and blood to see if we were able to detect significant tissue-specific differences. We observed that the transcriptomes of non-sequestered parasites (from the systemic blood) clustered together (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA). Correlation analysis further showed that transcriptomes of sequestered parasites were more different from non-sequestered parasites, than sequestered parasites collected from different tissues (\\u003cem\\u003eR\\u003c/em\\u003e\\u003csup\\u003e2\\u003c/sup\\u003e\\u0026thinsp;=\\u0026thinsp;0.58\\u0026ndash;0.61 \\u003cem\\u003evs.\\u003c/em\\u003e 0.91\\u0026ndash;0.96, Pearson\\u0026rsquo;s correlation). Therefore, in subsequent analyses, we compared the transcriptomes of sequestered parasites irrespective of the tissue they derived from to the group of non-sequestered parasites. We detected 523 differentially expressed genes, of which 323 were upregulated in sequestered parasites (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB). Upregulated genes included phosphatidic acid phosphatase, DNA repair protein, sister chromatid cohesion C-terminus, UDP-Gal/UDP-GlcNAc-dependent glycosyltransferase (UGT), transferrin receptor-like proteins (both Fam14 and 15) and the orthologue to flagellum attachment zone protein (FAZP). Downregulated genes included those encoding for ALBA and other RNA-binding proteins, PAD-like genes (protein associated with differentiation), amastin, cAMP phosphodiesterase A, and PLAC8 family (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003eWe asked if genes previously identified as upregulated in \\u003cem\\u003eT. congolense\\u003c/em\\u003e parasites upon silencing of a negative regulator of differentiation to insect-transmissible forms (TcoREG9.1)\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e, and that therefore could be assumed to be characteristic of insect-transmissible parasites, were enriched within our dataset. We did not find compelling evidence of enrichment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC). Based on what we know from \\u003cem\\u003eT. brucei\\u003c/em\\u003e, at the peak of infection, the population of parasites is expected to contain more insect-transmissible forms than in the ascending phase of infection\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u003c/sup\\u003e. Therefore, we also tested a gene set comprising genes upregulated in the first peak infection compared to the ascending phase of infection\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u003c/sup\\u003e, but also did not find any evidence of enrichment. Together, these results corroborate our previous observation that \\u003cem\\u003eT. congolense\\u003c/em\\u003e sequestration (or their lack of) is not associated with transmission ability.\\u003c/p\\u003e \\u003cp\\u003eGiven that sequestration is a physical interaction between the parasite and the endothelial cell and considering that the \\u003cem\\u003eT. congolense\\u003c/em\\u003e cell surface is tightly packed with the major antigen, variant surface glycoprotein (VSG), we specifically looked for changes in their expression. VSGs cannot be accurately characterised using standard differential expression tools, so we used the software VAPPER\\u003csup\\u003e\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e to profile them in sequestered and non-sequestered parasites. \\u003cem\\u003eT. congolense\\u003c/em\\u003e VSGs cluster into 15 phylogenetically-distinct lineages (or phylotypes), between which genetic recombination is rare\\u003csup\\u003e\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e. We found genes from all phylotypes being expressed at the mRNA level, consistent with previous observations in insect forms (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD). However, we found that genes belonging to VSG phylotype 8 were predominantly expressed in sequestered parasites, irrespective of the mouse organ, whereas genes from phylotypes 11 and 14 were more abundant in non-sequestered parasites (\\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001, 2-way ANOVA with Sidak\\u0026rsquo;s multiple comparisons test) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD and \\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE). These results suggest functional differentiation amongst the VSG repertoire and a role of phylotype 8 genes in sequestration.\\u003c/p\\u003e \\u003cp\\u003eOur results support the conclusions that sequestered and non-sequestered \\u003cem\\u003eT. congolense\\u003c/em\\u003e bloodstream forms present different transcriptomes, and that sequestration might be directly linked to VSG expression.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eSequestration is emerging as an essential process of \\u003cem\\u003eT. congolense\\u003c/em\\u003e interaction with the mammalian host, although its mechanisms remain unknown. In this work, we have significantly improved our understanding of trypanosome sequestration through the development of physiologically-relevant \\u003cem\\u003ein vitro\\u003c/em\\u003e system and their use in combination with \\u003cem\\u003ein vivo\\u003c/em\\u003e animal models. We have discovered the importance of flow mechanical cues as an important determinant of sequestration, revealed that cAMP intracellular levels modulate sequestration, found a link between sequestration and cell cycle that is independent of transmission ability, and characterised the gene expression profiles of sequestered parasites.\\u003c/p\\u003e \\u003cp\\u003eNovel bioengineering tools are gaining relevance in the infection biology field. One of their main advantages is the possibility to generate animal species-specific tissues \\u003cem\\u003ein vitro\\u003c/em\\u003e to study zoonoses or veterinary infections. To the best of our knowledge, we have generated for the first time a non-human microvessel model and showed their potential to study animal pathogens. By modelling bovine small arterioles or postcapillary venules, this system mimics the natural host\\u0026rsquo;s endothelium and the preferred environment for trypanosome sequestration. Despite low throughput and technical complexity of microfabrication, our versatile bioengineered method supports the generation of organ-specific vasculature and approximates \\u003cem\\u003ein vitro\\u003c/em\\u003e settings to natural conditions, by modelling a wide range of physiological WSS and flow velocities within a single device. Future studies could explore these models as platforms to test sequestration mediators or study endothelial cell responses to trypanosomes. In the malaria field, similar approaches have led to a better understanding of cerebral malaria, such as the identification of polyclonal\\u003csup\\u003e\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e or monoclonal antibodies\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e that inhibit parasite binding, or the discovery of new mechanisms of brain microvessel dysfunction\\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e. In this study, we have exploited them to reveal the biophysical and molecular determinants of \\u003cem\\u003eT. congolense\\u003c/em\\u003e sequestration as well as suggest a link between parasite binding and proliferation. Furthermore, microvessels might help us to characterise mechanisms of vascular transmigration of tissue-invading related trypanosome species, such as \\u003cem\\u003eT. brucei\\u003c/em\\u003e\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR30 CR31 CR32\\\" citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e, or assess the role of haematocrit levels in sequestration and/or extracellular matrix invasion.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eT. congolense\\u003c/em\\u003e can withstand high WSS both in 2D and 3D vascular models, corroborating previous observations of parasites sequestering in large arteries of the mouse\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e, where WSS is approximately around 10 dyn/cm\\u003csup\\u003e2\\u003c/sup\\u003e. However, based on our microvessels models, sequestration gradually increases as WSS decreases. Indeed, maximum sequestration is achieved in pathologically low WSS values, when combining \\u003cem\\u003eT. congolense\\u003c/em\\u003e 1/148 and brain endothelium. This is reminiscent of acute cerebral trypanosomiasis, where brain pathology is associated with high parasite accumulation in the microvasculature and vascular occlusion, which ultimately culminates in ischaemic or haemorrhagic stroke-like events\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. The subtle but significant differences in the effect of WSS in sequestration between parasite strains and host endothelial cell types suggest that there may be more than one host ligand and/or parasite receptor of sequestration. Indeed, in \\u003cem\\u003eP. falciparum\\u003c/em\\u003e, it has been well described that binding heterogeneity arises from the combination of different host receptors and parasite ligands from the PfEMP1 family\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e. Here, we found several surface-expressed proteins upregulated in sequestered parasites, including VSGs, invariant surface glycoproteins, and flagellum attachment zone proteins. Future research could investigate further if these candidates are sequestration mediators.\\u003c/p\\u003e \\u003cp\\u003ePrevious studies suggested that sequestration to live, but not fixed, bovine aorta endothelial cell monolayers increased \\u003cem\\u003eT. congolense\\u003c/em\\u003e proliferation\\u003csup\\u003e\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e. Here, we corroborated these results by showing that, \\u003cem\\u003ein vivo\\u003c/em\\u003e, \\u003cem\\u003eT. congolense\\u003c/em\\u003e parasites divide more when sequestered. These data suggest that sequestration might bring a metabolic advantage for parasites, i.e. the physical contact between the parasite and the endothelial cell may facilitate hijacking of host cellular functions and/or nutrients. However, here we uncovered an additional layer of complexity: faster cell division is observed even when the parasite is attached to a plastic substrate although at a lower rate than in the presence of endothelial cells. This indicates that the link between sequestration and proliferation is at least partly intrinsic to the parasite and not just a consequence of a potential metabolic benefit.\\u003c/p\\u003e \\u003cp\\u003eIt has been previously suggested that sequestered and non-sequestered parasites could be two distinct life forms: the first adapted for proliferation in the mammalian host (analogous to the \\u003cem\\u003eT. brucei\\u003c/em\\u003e slender form), and the latter adapted for fly transmissibility (analogous to the \\u003cem\\u003eT. brucei\\u003c/em\\u003e stumpy form)\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. However, our results suggest that both sequestered and non-sequestered parasites can differentiate to the insect stage \\u003cem\\u003ein vitro\\u003c/em\\u003e and successfully infect tsetse flies in similar timeframes. This could be because the number of parasites in G0/G1 (1K1N) present in the sequestered parasite population are sufficient to establish a successful procyclic form population, masking the phenotype of the remaining proliferating population. However, in these conditions, we would have expected faster differentiation of non-sequestered populations (as the number of parasites in G0/G1 is larger) and of NPD-1015-treated parasites (because this drug induces growth arrest and detachment). Instead, our results strongly suggest otherwise: NPD-1015-treated parasites infect flies at lower rates (perhaps because cAMP is important for successful infection, as reported for \\u003cem\\u003eT. brucei\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e\\u003c/em\\u003e\\u003c/sup\\u003e), and sequestered parasites result in heavier fly infections, which might result in higher transmission risk if transposed to heavier mouthpart infections. An alternative is that cell-cycle arrested forms are not necessary for fly transmission, which would explain why differentiation to procyclic forms is not more efficient if the starting population has more cells in G0/G1. A similar hypothesis, suggesting that proliferative \\u003cem\\u003eT. brucei\\u003c/em\\u003e parasites are capable of infecting flies has been recently proposed\\u003csup\\u003e\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eWhile it remains unclear why sequestration promotes cell division, or vice-versa, cAMP homeostasis appears central to this question. When we inhibited cAMP phosphodiesterases with NPD-1015, we observed growth arrest, but also a drastic decrease in sequestration. NPD-1015 treatment not only prevented attachment to endothelial cells, but also caused detachment of already sequestered cells. It has been thoroughly described that cAMP phosphodiesterase inhibition induces growth arrest in mammalian cells\\u003csup\\u003e\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u003c/sup\\u003e, and alters endothelial cell permeability and barrier properties\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR40 CR41\\\" citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e\\u003c/sup\\u003e, which may result in altered expression of surface molecules. As we washed the drug away before adding the parasites to the endothelial cells, we show that NPD-1015 prevents \\u003cem\\u003eT. congolense\\u003c/em\\u003e sequestration independently of endothelial cells, but parasite detachment could be partly result from changes in endothelial cell biology. Although, previously, it was shown that a similar inhibitor was able to prevent adhesion of \\u003cem\\u003eCrithidia fasciculata\\u003c/em\\u003e, this is the first time that detachment of already attached cells is observed\\u003csup\\u003e\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e\\u003c/sup\\u003e. Therefore, whilst cAMP signalling might play an evolutionarily-conserved role in kinetoplastid parasite attachment, the specific effects in sequestration seem distinct in \\u003cem\\u003eT. congolense\\u003c/em\\u003e bloodstream forms.\\u003c/p\\u003e \\u003cp\\u003eTo conclude, we present a new experimental model for assessing trypanosome interactions with the vascular endothelium and add new insights into the roles and characteristics of \\u003cem\\u003eT. congolense\\u003c/em\\u003e sequestration. Our work lays the ground for additional mechanistic examinations of sequestration, including receptor-ligand discovery.\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAnimal Experiments\\u003c/h2\\u003e \\u003cp\\u003e This study was conducted in accordance with EU regulations and ethical approval was obtained from the Animal Ethics Committee of Instituto de Medicina Molecular (AWB_2021_11_LF_TrypColonization), the Animal Ethics Committee of the European Molecular Biology Laboratory, and the Animal Ethics Committee of Instituto Gulbenkian de Ci\\u0026ecirc;ncia (A003.2023). Infections were performed either at the rodent facility at the Parc de Recerca Biomedica de Barcelona (PRBB), where EMBL Barcelona is located, at iMM\\u0026rsquo;s rodent facility, or at IGC\\u0026rsquo;s mouse facility, in 6\\u0026ndash;10 weeks old, wild-type, male C57BL/6J mice. Mice were infected by intraperitoneal injection of 10\\u003csup\\u003e6\\u003c/sup\\u003e [\\u003cem\\u003eT. congolense\\u003c/em\\u003e savannah 1/148 (MBOI/NG/60/1-148)\\u003csup\\u003e44\\u003c/sup\\u003e]. Blood for perfusion of 3D microvessels was obtained by cardiac puncture. Mice were sacrificed by CO\\u003csub\\u003e2\\u003c/sub\\u003e narcosis.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eCell culture\\u003c/h3\\u003e\\n\\u003cp\\u003eBovine brain microvasculature endothelial cells (BBMVEC) (Cell Applications #B840-05), Bovine aorta endothelial cells (BAOEC) (Cell Applications #B304-05) and Human brain microvascular endothelial cells (HBMEC) (Cell Systems #ACBRI 376) were cultured as per supplier\\u0026rsquo;s instructions respectively in bovine brain endothelial cell growth media (Cell Applications, USA), bovine endothelial cell growth media (Cell Applications, USA), or in complete endothelial growth media-2MV (Lonza) containing 5% foestal bovine serum. \\u003cem\\u003eT. congolense\\u003c/em\\u003e savannah 1/148 (MBOI/NG/60/1\\u0026ndash;148) were expanded in 7\\u0026ndash;10 weeks-old, male C57BL/6 J mice (Charles River, France), harvested from blood by cardiac puncture and purified by anion exchange chromatography. \\u003cem\\u003eT. congolense\\u003c/em\\u003e savannah IL3000 SM parasites were cultured in HMI-93 medium supplemented with 10% goat serum on 10mm-diameter Petri dishes, at 34\\u0026ordm;C, until 80\\u0026ndash;100% confluency. When necessary, parasite cells were stained with fluorescent dye 5(6)- Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), at a 1:1000 dilution, as per manufacturer\\u0026rsquo;s instructions. \\u003cem\\u003ePlasmodium falciparum\\u003c/em\\u003e clone HB3 was selected for expression of PfEMP1 variants HB3VAR03 as previously described\\u003csup\\u003e\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e\\u003c/sup\\u003e, and cultured in human O\\u0026thinsp;+\\u0026thinsp;erythrocytes in RPMI-1640 medium containing 25 mM HEPES, 4 mM L-glutamine, 0.04 mM hypoxanthine, 5 mM glucose, and 10% human type B\\u0026thinsp;+\\u0026thinsp;serum at 37\\u0026deg;C and 90% N\\u003csub\\u003e2\\u003c/sub\\u003e, 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e, and 5% O\\u003csub\\u003e2\\u003c/sub\\u003e. Late-stage \\u003cem\\u003eP. falciparum\\u003c/em\\u003e infected erythrocytes were enriched using a MACS cell separator with LD columns (Miltenyi Biotec #130-042-901) and fluorescently labelled (PKH26 Red Fluorescent Cell Linker Midi Kit (Sigma #MIDI26-1KT) before perfusion in human brain microvessels.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMicrovessel Fabrication and immunofluorescence analysis\\u003c/h2\\u003e \\u003cp\\u003eMicrovessels were prepared by soft lithography and injection moulding of a collagen hydrogel in between polymethylmethacrylate (PMMA) jigs and polydimethylsiloxane (PDMS) stamps, as previously described for human brain microvessels\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e. BBMVEC, BAOEC or HBMVEC were seeded into the collagen at a concentration of 7x10\\u003csup\\u003e6\\u003c/sup\\u003e cells/mL Devices were kept for 3 days to allow for vessel forming, replacing medium approximately every 12 hours under gravity-driven flow. 1.5x10\\u003csup\\u003e6\\u003c/sup\\u003e fluorescently-labelled \\u003cem\\u003eT. congolense\\u003c/em\\u003e parasites and 10x10\\u003csup\\u003e6\\u003c/sup\\u003e \\u003cem\\u003eP. falciparum\\u003c/em\\u003e-infected erythrocytes were perfused at a defined flow rate of 10\\u0026micro;L/min for 15 minutes. Microvessels were washed with 150\\u0026micro;L of PBS for 10 minutes, at the same flow rate. Vessels were fixed with 3.7% paraformaldehyde and washed twice with PBS. Cells were permeabilized using a 2% Bovine Serum Albumin 0.1% Triton-X100 in PBS and stained with 4\\u0026micro;g/mL dihydrochloride (DAPI) or specific antibodies. Parasite binding quantification was performed by confocal microscopy (Zeiss LSM 980 confocal microscope), 10X magnification, with a total scanning with 30\\u0026ndash;50\\u0026micro;m of depth and analysed using ImageJ.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eDetachment Assay\\u003c/h3\\u003e\\n\\u003cp\\u003eParasites were either isolated from mouse blood by anion exchange chromatography (1/148) (Lanham and Godfrey, 1970) or harvested from culture (IL3000), stained with 5 mM Vybrant CFDA SE Cell Tracer dye (#V12883, Invitrogen) diluted 1000 times in trypanosome dilution buffer (TDB) (5 mM KCl, 80 mM NaCl, 1 mM MgSO\\u003csub\\u003e4\\u003c/sub\\u003e, 20 mM Na\\u003csub\\u003e2\\u003c/sub\\u003eHPO\\u003csub\\u003e4\\u003c/sub\\u003e, 2 mM NaH\\u003csub\\u003e2\\u003c/sub\\u003ePO\\u003csub\\u003e4\\u003c/sub\\u003e, 20 mM glucose, pH 7.4), and incubated for 25 min at 34\\u0026ordm;C, 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e. At the end of the incubation period, parasites were washed and resuspended in TDB, added to the endothelial cell monolayers, and incubated for 1 hour at 34\\u0026ordm;C, 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e. Flow was applied with a perfusion syringe pump containing PBS at defined flow rates, for 1 min each. Parasites were imaged live on a Zeiss LSM 980 (Carl Zeiss Microimaging) with a 20X water- immersion objective (0.8 numerical aperture and 0.55 mm working distance) before and after each flow session. We acquired 10 fields of view per condition (each WSS value), per replicate (3 replicates), with green laser (488nm, maximum power of 13mW). For all acquisitions, the software used was ZEN blue edition v.2.6, allowing export of images in czi format.\\u003c/p\\u003e\\n\\u003ch3\\u003eScanning Electron Microscopy\\u003c/h3\\u003e\\n\\u003cp\\u003eMicrovessels were fixed in Karnovsky solution (2% PFA, 2.5% glutaraldehyde in 0.1M cacodylate buffer, pH 7.4) at least overnight at 4\\u0026ordm;C. First, 200 \\u0026micro;l of fixative were added to the inlet and incubated for 15 minutes. After that, additional 300\\u0026micro;l were added to the device. After fixation, the devices were washed with 0.1M cacodylate, at 4\\u0026ordm;C, opened and the collagen containing the microvessels was removed from the jigs and post-fixed with 2.5% Glutaraldehyde in 0.1M Cacodylate buffer pH 7.4, for 1 hour at 4\\u0026ordm;C. After washing twice with 0.1M cacodylate, samples were incubated with 2% tannic acid and 4.2% sucrose for 1 hour, at 4\\u0026ordm;C. Samples were subsequently washed twice with distilled water, stained with 1% methylene blue, embedded in 2% low-melt agarose, and sectioned as 150\\u0026micro;m sections in the vibratome. Sections were kept in water until further processing. Samples were dehydrated in an ascending acetone sequence, critical point dried and sputter coated with platinum (60s, diffuse coating). Images were acquired with a FEI Quanta 650 FEG scanning electron microscope using the detector LEI for secondary electrons at 10 kV, spot 3, high vacuum and dwell time of 3\\u0026micro;s.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCytological analysis\\u003c/h2\\u003e \\u003cp\\u003eTo assess cell cycle status, parasites were grown in MATEK glass-bottom dishes overnight. Non-sequestered parasites were removed by aspiration, fixed with 2% formaldehyde, and airdried on to glass slides for at least 4 hours. Then, parasites were rehydrated with PBS, permeabilized with 2% Bovine Serum Albumin \\u0026minus;\\u0026thinsp;0.1% Triton- X100 in PBS for 10 minutes, incubated with 4\\u0026micro;g/ml of 4\\u0026rsquo;,6-diamidino-2-phenylindole (DAPI) in PBS for 10 minutes and then washed twice for 10 min in PBS. Slides were then mounted with fluoromount-G (ThermoFisher Scientific) and sealed with a glass coverslip secured with nail polish. Parasites remaining sequestered to the MATEK glass-bottom dish following aspiration were fixed with 2% formaldehyde, permeabilised with 2% Bovine Serum Albumin \\u0026minus;\\u0026thinsp;0.1% Triton- X100 in PBS for 10 minutes, stained with DAPI for 10 minutes, and washed twice with PBS. Cell cycle status was assessed under a Zeiss LSM980 using a bright field 63X objective and a 561nm laser.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIntravital Imaging\\u003c/h2\\u003e \\u003cp\\u003eIntravital microscopy involved separate surgeries targeting specific organs, as previously outlined for the brain\\u003csup\\u003e\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e\\u003c/sup\\u003e, lungs and heart, liver, pancreas, spleen, kidneys\\u003csup\\u003e\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e\\u003c/sup\\u003e, and adipose tissue. Briefly, mice were anesthetized with a ketamine (120mg/kg) and xylazine (16mg/kg) mixture via intra-peritoneal injection. Reflexes were checked, and upon their absence, mice received intravenous injections into the retro-orbital sinus of three markers: Hoechst 33,342 for nucleic acid labelling (stock diluted in dH2O at 100mg/ml, injection of 40\\u0026micro;g/kg mouse) and 70 kDa FITC-Dextran for intravascular space labelling (stock diluted in 1 x PBS at 100mg/ml, injection of 500mg/kg mouse). Temporary glass windows (Merk rectangular cover glass, 100mm x 60mm) or circular cover glasses (12mm) of 0.17mm thickness were implanted in each organ. These windows were secured with stitches or surgical glue. For heart and lung imaging, vacuum immobilization was used to prevent thoracic cavity collapse. Brain imaging utilized semi-closed or open cranial windows, reaching depths of around 190\\u0026micro;m or up to 300\\u0026micro;m into the tissue, respectively.\\u003c/p\\u003e \\u003cp\\u003eImaging sessions were conducted on spinning disc microscopes: Zeiss Cell Observer SD (Carl Zeiss Microimaging, equipped with a Yokogawa CSU-X1 confocal scanner, and an Evolve 512 EMCCD camera and a Hamamatsu ORCA-Flash 4.0 VS camera) or a 3i Marianas SDC (spinning disc confocal) microscope (Intelligent Imaging Innovations, equipped with a Yokogawa CSU-X1 confocal scanner and a Photo-metrics Evolve 512 EMCCD camera). Laser units 405, 488, and 647 were utilized for imaging Hoechst, FITC-Dextran, and AF67-CD31 respectively. Imaging was performed using either an oil-immersion plan apochromat 63 x objective with 1.4 Numerical Aperture (NA) and 0.17mm working distance (WD), or a 40 x LD C-Apochromat corrected, water immersion objective with 1.1 NA and 0.62 WD. Images were acquired for 20 seconds at a rate of 20 frames per second. ZEN blue edition v.2.6., or 3i Slidebook reader v.6.0.22 software was used for all acquisitions.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDifferentiation assays\\u003c/h2\\u003e \\u003cp\\u003eFor the differentiation assays, \\u003cem\\u003eT. congolense\\u003c/em\\u003e IL3000 SM cells were incubated overnight with DMSO or 20\\u0026micro;M NPD-1015 in 5mL of TcBSF1 media on T25 culture flasks. Non-sequestered parasites were removed from the flasks by pipetting, and remaining sequestered parasites were added additional 5ml of media. Then, detachment was forced by vortexing for a few seconds. Parasites were centrifuged at 1200xg for 10 minutes and 3x10\\u003csup\\u003e6\\u003c/sup\\u003e parasites per condition were incubated in DTM medium at 27˚C, without CO\\u003csub\\u003e2\\u003c/sub\\u003e. The number of live cells were counted at 3 and 5 days after on a haemocytometer. At day 5 post differentiation induction, 10\\u003csup\\u003e6\\u003c/sup\\u003e procyclic cells were passaged into a new T25 flask with 5mL DTM and counted 24 hours after to estimate parasite population growth.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTsetse Fly infections\\u003c/h2\\u003e \\u003cp\\u003e \\u003cem\\u003eT. congolense\\u003c/em\\u003e IL3000-SM bloodstream forms cryopreserved parasites were cultured in TcBSF1 media on T25 culture flasks, at 34\\u0026ordm;C, 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e. Twenty-four hours before fly infection, 5x10\\u003csup\\u003e6\\u003c/sup\\u003e parasites were supplemented with 20\\u0026micro;M NPD-1015 or the same volume of DMSO. On the experiment day, non-sequestered parasites supplemented with DMSO were collected by pipetting and undisturbed sequestered parasites were washed and collected after vortexing for a few seconds to force detachment. These and drug-treated (non-sequestered) parasites were washed twice by centrifugation at 1200xg for 10 minutes to remove traces of DMSO and NPD-1015. Parasites were counted with a haemocytometer and fed to experimental teneral (unfed, 0\\u0026ndash;48 hours post-eclosion) male and female tsetse flies (\\u003cem\\u003eGlossina morsitans morsitans\\u003c/em\\u003e) at a concentration of 10\\u003csup\\u003e5\\u003c/sup\\u003e trypanosomes per mL of sterile defibrinated horse blood (TCS Biosciences) via a silicone membrane as previously described \\u003csup\\u003e\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e\\u003c/sup\\u003e. Flies were maintained by feeding on non-infected sterile horse defibrinated blood, and killed by decapitation and midguts were dissected out at days 10 post-infection. Midgut infections were scored after breaking down the entire tissue in a PBS drop, and were classified as heavy, medium or mild, based on the number of parasites observed under the microscope.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTranscriptomics analysis\\u003c/h2\\u003e \\u003cp\\u003eFour mice were infected with 2000 \\u003cem\\u003eT. congolense\\u003c/em\\u003e 1/148 parasites and euthanized at days 6 post-infection. Blood was collected by cardiac puncture and mice were perfused with 50ml heparinized PBS. Brain, gonadal adipose tissue, and kidney were dissected and flash-frozen in liquid nitrogen. Organs were homogenized in Qiazol (Qiagen, UK) with silica beads on a bead beater for 2 rounds of 45 seconds. RNA was extracted using the RNeasy Universal Plus kit (Qiagen, UK) according to the manufacturer\\u0026rsquo;s protocol and RNA concentration and integrity were checked by fluorometry (Qubit DNA HS, Thermo Fisher Scientific) and parallel capillary electrophoresis (TapeStation, Agilent), respectively. Trypanosome-specific cDNA libraries were prepared using custom primers targeting the spliced leader sequence as previously described\\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e, and sequenced as 75bp single-end reads on the NextSeq 550 platform (Illumina, USA). Reads were aligned to the \\u003cem\\u003eT. congolense\\u003c/em\\u003e IL3000 2018 genome available from tritrypDB version 51 using STAR\\u003csup\\u003e\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e\\u003c/sup\\u003e. The output from read alignment was processed with SAMtools\\u003csup\\u003e\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e\\u003c/sup\\u003e, and transcript abundances were estimated using stringtie\\u003csup\\u003e\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e\\u003c/sup\\u003e. Differential expression between sequestered (blood) and non-sequestered (tissues) samples was performed in R, using edgeR\\u003csup\\u003e\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e\\u003c/sup\\u003e and limma-voom\\u003csup\\u003e\\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e\\u003c/sup\\u003e. Log\\u003csub\\u003e2\\u003c/sub\\u003e Fold change of 1 and \\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 was considered significant. VSG profiling was conducted with VAPPER\\u003csup\\u003e\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSequencing reads are available from NCBI under BioProject accession number PRJNA1159173.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank Dr \\u0026Aacute;lvaro Acosta-Serrano (The University of Notre Dame du Lac) and Dr Catarina Gadelha (University of Nottingham) for providing \\u003cem\\u003eT. congolense\\u0026nbsp;\\u003c/em\\u003e1/148 and IL3000-SM parasites, respectively.\\u0026nbsp;We acknowledge the support of the Rodent and Bioimaging facilities at Instituto de Medicina Molecular, the Mouse and Advanced Imaging facilities at Instituto Gulbenkian de Ci\\u0026ecirc;ncia, and the animal facility at PRBB. The SEM work was carried out in part by INL User Facilities, with support from the Electron Microscopy Facility at Instituto Gulbenkian de Ci\\u0026ecirc;ncia. Microvessels masters were fabricated by MicroFabSpace and Microscopy Characterization Facility, Unit 7 of ICTS \\u0026ldquo;NANBIOSIS\\u0026rdquo; from CIBER-BBN at IBEC. NPD-1015 was kindly provided by Dr Harry De Koning (University of Glasgow). We also would like to thank the efforts of Leonor Pinho and Cristina Bancells for project support, as well as Ana Nascimento, Antonio Temudo, Jose Rino, Livia Piatti and Matt Govendir for technical support. We thank Jonathan Thornton for providing and maintaining the experimental tsetse flies, and the Liverpool School of Tropical Medicine for their support in maintaining the tsetse colony, managed by Dr Aitor Casas-Sanchez.\\u0026nbsp;This work was funded by\\u0026nbsp;the Marie Skłodowska-Curie Actions post-doctoral fellowship FEBRIS [101026717]) to VI,\\u0026nbsp;EMBO postdoctoral fellowship (ALTF 1048-2016) and HSFP (LT000047/2019-L) long-term postdoctoral fellowship awarded to MDN; EMBO Scientific Exchange Grant #9333 to SSP; Pr\\u0026eacute;mio Maria de Sousa, from Funda\\u0026ccedil;\\u0026atilde;o Bial and Ordem dos M\\u0026eacute;dicos, project 7/2021 to SSP,\\u0026nbsp;Funda\\u0026ccedil;\\u0026atilde;o para a Ci\\u0026ecirc;ncia e a Tecnologia (PeX/2022.02187.PTDC) to SSP,\\u0026nbsp;\\u0026ldquo;la Caixa\\u0026rdquo; Foundation (ID 10001043) through a Junior Leader Postdoctoral Fellowship to SSP (LCF/BQ/PR23/11980034), the European Research Council (ERC) (FatTryp, ref. 771714) to LMF and EMBL core funding to MB.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDeclaration of Interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eSilva Pereira S et al (2022) Immunopathology and \\u003cem\\u003eTrypanosoma congolense\\u003c/em\\u003e parasite sequestration cause acute cerebral trypanosomiasis. Elife 11\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGhazanfari N, Mueller SN, Heath WR (2018) Cerebral Malaria in Mouse and Man. Front Immunol 9:2016\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eGallego-Lopez GM, Cooke BM, Suarez CE (2019) Interplay between attenuation-and virulence-factors of \\u003cem\\u003eBabesia B\\u003c/em\\u003eovis and their contribution to the establishment of persistent infections in cattle. Pathogens 8:1\\u0026ndash;13\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSilvester E et al (2024) A conserved trypanosomatid differentiation regulator controls substrate attachment and morphological development in \\u003cem\\u003eTrypanosoma congolense\\u003c/em\\u003e. PLoS Pathog 20:e1011889\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBernabeu M et al (2019) Binding Heterogeneity of \\u003cem\\u003ePlasmodium falciparum\\u003c/em\\u003e to Engineered 3D Brain Microvessels Is Mediated by EPCR and ICAM-1. \\u003cem\\u003emBio\\u003c/em\\u003e 10, e00420-19\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHudetz AG (1997) Blood flow in the cerebral capillary network: A review emphasizing observations with intravital microscopy. Microcirculation 4:233\\u0026ndash;252\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLipowsky HH (2005) Microvascular rheology and hemodynamics. \\u003cem\\u003eMicrocirculation\\u003c/em\\u003e vol. 12 5\\u0026ndash;15 10739680590894966\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eItoh Y, Suzuki N (2012) Control of brain capillary blood flow. J Cereb Blood Flow Metab 32:1167\\u0026ndash;1176\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHemphill A, Ross CA (1995) Flagellum-mediated adhesion of \\u003cem\\u003eTrypanosoma congolense\\u003c/em\\u003e to bovine aorta endothelial cells. Parasitol Res 81:412\\u0026ndash;420\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eClaessens A et al (2012) A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells. 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Nucleic Acids Res 45:W130\\u0026ndash;W137\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eJain C, Koren S, Dilthey A, Phillippy AM, Aluru S (2018) A fast adaptive algorithm for computing whole-genome homology maps. in \\u003cem\\u003eBioinformatics\\u003c/em\\u003e vol. 34 i748\\u0026ndash;i756Oxford University Press\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"nature-portfolio\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Nature Portfolio\",\"twitterHandle\":\"\",\"acdcEnabled\":false,\"dfaEnabled\":false,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"sequestration, Trypanosoma congolense, animal African trypanosomiasis, vascular bioengineering\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5104603/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5104603/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eIn the mammalian host, \\u003cem\\u003eTrypanosoma congolense\\u003c/em\\u003e cytoadheres to the vascular endothelium in a process known as sequestration. Although sequestration influences clinical outcome, disease severity and organ pathology, its determinants and mediators remain unknown. Challenges such as the variability of animal models, the only-recently developed tools to genetically manipulate the parasite, and the lack of physiologically relevant \\u003cem\\u003ein vitro\\u003c/em\\u003e models have hindered progress. Here, we engineered brain and cardiac 3D bovine endothelial microvessel models that mimic the bovine brain microvasculature and the bovine aorta, respectively. By perfusing these models with two \\u003cem\\u003eT. congolense\\u003c/em\\u003e strains, we simulated physiologically relevant conditions and investigated the roles of flow for parasite sequestration and tropism for different endothelial beds. We discovered that sequestration is dependent on cyclic AMP signalling, closely linked to parasite proliferation, but not associated with parasite transmission to the tsetse fly vector. Finally, by comparing the expression profiles of sequestered and non-sequestered parasites collected from a rodent model, we showed gene expression changes in sequestered parasites, including of the surface variant antigens. This work presents a physiologically-relevant platform to study trypanosome interactions with the vasculature and provides a deeper understanding of the molecular and biophysical mechanisms underlying \\u003cem\\u003eT. congolense\\u003c/em\\u003e sequestration.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Bioengineered 3D microvessels reveal novel determinants of Trypanosoma congolense sequestration\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-09-25 15:35:27\",\"doi\":\"10.21203/rs.3.rs-5104603/v1\",\"editorialEvents\":[],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"communications-biology\",\"isNatureJournal\":true,\"hasQc\":false,\"allowDirectSubmit\":false,\"externalIdentity\":\"commsbio\",\"sideBox\":\"Learn more about [Communications Biology](http://www.nature.com/commsbio/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Communications Biology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"ejp\",\"reportingPortfolio\":\"Communications Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"70659b67-5956-482e-83d4-266bac36c4e3\",\"owner\":[],\"postedDate\":\"September 25th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":38165171,\"name\":\"Biological sciences/Microbiology/Parasitology/Parasite physiology\"},{\"id\":38165172,\"name\":\"Biological sciences/Microbiology/Parasitology/Parasite host response\"}],\"tags\":[],\"updatedAt\":\"2025-02-27T08:08:49+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-5104603\",\"link\":\"https://doi.org/10.1038/s42003-025-07739-z\",\"journal\":{\"identity\":\"communications-biology\",\"isVorOnly\":false,\"title\":\"Communications Biology\"},\"publishedOn\":\"2025-02-27 05:00:00\",\"publishedOnDateReadable\":\"February 27th, 2025\"},\"versionCreatedAt\":\"2024-09-25 15:35:27\",\"video\":\"\",\"vorDoi\":\"10.1038/s42003-025-07739-z\",\"vorDoiUrl\":\"https://doi.org/10.1038/s42003-025-07739-z\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5104603\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5104603\",\"identity\":\"rs-5104603\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}