Different isolates of Leishmania aethiopica elicit distinct effector functions in primary human monocytes

Monocytes; Leishmania aethiopica; association; phagocytosis; ROS; chemokines; cytokines. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint

In Ethiopia, cutaneous leishmaniasis (CL) is caused by Leishmania (L.) aethiopica and presents as a spectrum of clinical forms ranging from self-healing to persistent, disfiguring lesions. Monocytes are some of the first cells to encounter Leishmania parasites in the host. Despite this, their role in L. aethiopica infection has not been investigated. In this study, primary human monocytes were co-incubated with different L. aethiopica isolates – three isolates recently collected from CL patients and one long-term cultured isolate – and their main effector functions were evaluated. After 2 hours of co-incubation, over 30% of monocytes associated with all four L. aethiopica isolates, with significantly higher association with recently isolated L. aethiopica than with the long-term cultured parasites. Phagocytosis of L. aethiopica parasites by monocytes was confirmed by confocal microscopy. Co-incubation of monocytes with all L. aethiopica isolates resulted in upregulation of reactive oxygen species in monocytes. Following incubation of monocytes with all L. aethiopica, five chemokines – monocyte chemoattractant protein (MCP)-1, MCP-4, macrophage inflammatory protein (MIP)-1α, MIP-1β, and interleukin (IL-8) – and four cytokines – tumour necrosis factor (TNF)-α, IL- 1β, IL-6, and IL-10 – were detected. Our results show that the interaction of monocytes with the long-term cultured L. aethiopica differ as compared to those recently collected from CL patients. Furthermore, we define an in vitro model for the investigation of monocyte effector functions that may be useful to elucidate the role that parasites and their interactions with monocytes play in the different presentations of lesions caused by L. aethiopica. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint

The leishmaniases are a group of neglected tropical diseases caused by protozoan parasites of the genus Leishmania. These diseases present as a wide spectrum of clinical manifestations, ranging from self-healing skin lesions to potentially fatal visceral infections. They are primarily classified into two main forms: visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). VL is characterised by the dissemination of Leishmania parasites to internal organs such as the liver, spleen and bone marrow [1, 2]. CL includes a variety of presentations where the parasite primarily infects the skin and mucosal tissue. CL is endemic in 90 countries, with 205,986 new cases reported by the WHO in 2022 [3]. Eight countries – Afghanistan, Algeria, Brazil, Colombia, Iran, Iraq, Peru and Syria – accounted for 85% of the global cases, and children under 15 years old represented 37% of CL cases worldwide [3]. The burden of CL includes permanent scarring and potential disfigurement, resulting in important psychological effects and social stigmatisation [4-6]. In Ethiopia, CL present mainly as three clinical forms [7, 8]: - Localised cutaneous leishmaniasis (LCL), which is the most common form of CL, presents as one or more ulcerative skin lesions that develop at or near the site of the sandfly bite [7]. While LCL is typically self-healing, treatment is sometimes provided for persistent lesions [7]. - Mucocutaneous leishmaniasis (MCL) is a severe and debilitating form of CL that predominantly affects the mucosal tissues of the nose, mouth, and pharynx [7]. - Diffuse cutaneous leishmaniasis (DCL) is a chronic form of CL characterised by the widespread dissemination of non-ulcerating lesions [7, 8]. Both DCL and MCL require treatment [9]. According to the most recent WHO data, Ethiopia reported 1505 CL cases in 2023 [10]; however, this is likely to be an underestimate of the actual burden due to underreporting, as a result of limited resources, remoteness of endemic areas, and the lack of a system to report cases [11, 12]. The Ethiopian Ministry of Health reported an estimate of 20,000 to 30,000 yearly CL cases in 2013 [7]. The number of people at risk of CL in Ethiopia was estimated at 29 million [13]. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Leishmania parasites are transmitted during the blood meal of female sandflies [14]. L. aethiopica causes most CL cases in Ethiopia [8]. Three Phlebotomus (P.) species have been identified as vectors for L. aethiopica: P. pedifer and P. longipes are considered to be the main vectors, with P. sergenti only identified once as naturally infected with L. aethiopica [15-17]. Since sandflies feed by lacerating capillaries to create a blood pool, monocytes are likely to be among the first cells to come into contact with Leishmania parasites [18]. Monocytes are circulating immune cells that make up ~10% of peripheral blood cells in humans [19]. In the response to infection, monocytes are rapidly recruited and can perform a range of antimicrobial functions, including phagocytosis, production of reactive oxygen species (ROS), and the release of a range of cytokines and chemokines [19]. Furthermore, monocytes are able to differentiate into dendritic cells and macrophages [20]. In humans, monocytes are divided into at least three subsets, based on the expression of CD14 and CD16 [21]. The subsets have distinct functions that have been characterised by both functional and ribonucleic acid sequencing (RNA- seq) studies: classical monocytes (CD14++ CD16-) mainly play a role in phagocytosis, adhesion and migration; intermediate monocytes predominantly carry out antigen presentation (CD14++ CD16+); while non-classical monocytes (CD14+ CD16+) are principally responsible for inflammatory cytokine production, and complement- and Fc receptor-mediated phagocytosis [22-24]. However, RNA-seq studies have proposed different ways of classifying monocytes, often into more subsets, noting the heterogeneity within each of the three conventional subsets [25, 26]. Human monocytes, isolated from peripheral blood, have been shown to become infected by several species of Leishmania in vitro [27] and produce reactive oxygen species (ROS). Human monocytes were shown to kill L. donovani promastigotes in a hydrogen peroxide-dependent manner [28]. Hoover et al. showed monocyte killing of L. donovani amastigotes when interferon (IFN)-γ was added to cultures, although the mechanism of killing was not determined [29]. Another study showed IFN-γ induced killing of L. donovani by human monocytes, which correlated with increased hydrogen peroxide production [30]. Ritter and Moll demonstrated that the chemokine monocyte chemoattractant protein (MCP)-1 acted synergistically with IFN-γ to induce human (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint monocyte killing of L. major, in a process correlating with increased superoxide production [31]. A more recent study showed that classical, but not intermediate and non-classical, monocytes killed L. braziliensis amastigotes in a ROS-dependent manner [32]. Most of the work on monocyte effector functions in response to Leishmania parasites has been done with parasite isolates that had been maintained in culture for an extended period of time and/or passaged in vivo in animals, but rarely with parasites recently isolated from patients. The only study assessing the interaction between blood immune cells and L. aethiopica was done with a parasite that was kept in culture for decades. Here, the main aim was to investigate different effector functions of human monocytes to three recently isolated L. aethiopica and compare those to long-term cultured L. aethiopica. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint

Leishmania aethiopica parasites Four isolates of L. aethiopica were used in this study: one long-term cultured isolate, (MHOM/ET/72/L100) [33, 34] referred to as L. aethiopica lab, and three isolates recently obtained from LCL patients in Lay Gayint, Amhara region, north-west Ethiopia, as described in [35, 36], referred to as clinical isolates L. aethiopica 1, 2 and 3. These parasites were grown from skin scrapings and were frozen as soon as they reached stationary phase. They were sent to the UK, where large stocks of frozen parasites were prepared for further use. Once thawed, the parasites were used for a maximum of 3 weeks. Parasites were grown in M199 medium supplemented with 10 μM hemin, 1x Eagle’s minimum essential medium (MEM) vitamin solution, 25 mM HEPES, 0.2 μM folic acid, 100 µM adenine, 10% heat-inactivated fetal bovine serum (HI FBS), 8 μM 6-biopterin (Sigma-Aldrich, USA), 100 units/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific, USA) and were incubated at 26°C. Stationary phase L. aethiopica were enriched for metacyclic parasites by eliminating non-metacyclic parasites by peanut agglutination, as described in [37]. Of note, the percentage of metacyclic parasites was significantly lower with L. aethiopica lab than each of the three clinical isolates (Table S1). Parasites were stained using 1μM CellTrace™ Far Red dye (Thermo Fisher Scientific, USA). Over 98% of parasites were stained with the Far Red dye. Monocytes Primary human monocytes were purified from the peripheral blood of healthy volunteers. 100-120 mL of blood was collected in heparin tubes (BD, USA) and monocytes were isolated by double gradient centrifugation [38]. The blood was first overlayed onto an equal volume of Histopaque®-1077 Hybri-Max™ (Sigma-Aldrich, USA) to collect the peripheral blood mononuclear cells (PBMCs), that were then overlayed onto an equal volume of 46% Percoll® solution. Monocytes were obtained from the interphase ring. All steps were performed in polypropylene tubes to avoid monocyte adhesion. Monocyte purity following purification was >80%. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint 2x105 monocytes were incubated alone or in the presence of 2x106 L. aethiopica parasites stained with Far Red dye (L. aethiopicaFR). Incubations were performed in RPMI medium (Thermo Fisher Scientific, USA) supplemented with 5% HI FBS (Sigma- Aldrich, USA), 100 units/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific, USA) in 5 mL round-bottom polypropylene tubes at 37°C, 5% CO2, for 2 hours. The supernatants were collected and frozen at -20oC until further use. Flow cytometry: monocyte association with L. aethiopica and ROS production After 1 hour 45 minutes of incubation, cells were stained with anti-CD14PE (clone M5E2; BioLegend, USA) and anti-CD16eFluor™ 450 (clone CB16; Thermo Fisher Scientific, USA) for 15 minutes. The different monocytes subsets were defined as follows: classical monocytes as CD14++ CD16-, intermediate monocytes as CD14++ CD16+ and non- classical monocytes as CD14+ CD16+ (Figure S1). Cells were then washed twice in PBS and were resuspended in 500 µL of ROS detection solution from the ROS-IDTM Total ROS detection kit (Enzo Life Sciences, USA). Cells were incubated in ROS detection solution for a further 30 minutes at 37°C with 5% CO2, after which tubes were placed on ice and immediately processed. The percentage of monocyte subsets associated with each L. aethiopicaFR isolate and the ROS production were assessed by flow cytometry. The percentages of live monocytes after the 2-hour incubation were measured by exclusion of Zombie Violet (BioLegend, USA) and were >98%. Flow cytometry acquisition was performed using an LSR II (BD, USA) and data were analysed on FlowJo™ v10.10 Software (BD, USA). Confocal microscopy 3x105 purified monocytes were incubated alone or in the presence of 3x106 L. aethiopicaFR parasites for 2 hours, before being washed in PBS and transferred onto 24 mm x 24 mm glass coverslips (Thermo Fisher Scientific, USA) precoated with an excess of 0.01% poly-L-lysine. After 30 minutes at room temperature, the coverslips were washed with PBS and fixed with 2% paraformaldehyde (Sigma-Aldrich, USA). Twenty minutes later, the coverslips were washed twice in PBS and incubated with a (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint rabbit anti-human CD14 IgG polyclonal antibody (PA5-13305, Thermo Fisher Scientific, USA) and incubated overnight at 4°C. The coverslips were washed with PBS and Alexa Fluor™ 488 conjugated goat anti-rabbit IgG antibody (A27034, Thermo Fisher Scientific, USA) was added and incubated for 1 hour at room temperature. Coverslips were then transferred to a 6-well plate and washed with PBS. The coverslips were then placed over 10 µL of VECTASHIELD mounting medium with DAPI (Vector Laboratories, USA) on a glass microscope slide. Slides were visualised under an SP8 LIGHTNIGHT confocal microscope (Leica Microsystems, Germany) with a 100x objective and images were acquired using the LAS X software (Leica Microsystems, Germany). 3-colour imaging was performed to assess staining with DAPI, CD14 staining of monocytes and the Far Red label of L. aethiopica. Images were acquired as z-stacks of at least 20 images to enable 3- dimensional visualisation. Images were analysed using Fiji software [39]. Chemokine and cytokine measurements The levels of the following chemokines: IL-8, eotaxin, eotaxin-3, interferon-γ-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, MCP-4, macrophage- derived chemokines (MDC), macrophage inflammatory protein (MIP)-1α, MIP-1β and thymus- and activation regulated chemokine (TARC) and cytokines: IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-13, TNF-α were measured by multiplex assay using V- PLEX Proinflammatory Panel 1 and Chemokine Panel 1 Kits (Meso Scale Diagnostics, Rockville, USA). Statistics Data were evaluated for statistical differences using Mann-Whitney and Kruskal-Wallis tests (GraphPad Prism 10). Differences were considered statistically significant at p<0.05. Unless otherwise specified, results are expressed as mean ± standard deviation (SD). (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint

Association of monocytes with L. aethiopica The ability of the different monocyte subsets to associate with L. aethiopica was determined. Results presented in Figure 1A-D and Table S2 show that all three subsets had the ability to associate with monocytes, with the classical monocytes displaying the highest mean association. Of note, not all monocytes associated with L. aethiopica (hereafter referred to as unassociated monocytes). We also compared the association between each monocyte subset and the different clinical L. aethiopica isolates and show that there was no significant difference in association (Figure 1E-G and Table S3). However, the percentages of monocytes associated with L. aethiopica lab were significantly lower than with the clinical isolates, except for the intermediate monocytes co-incubated with L. aethiopica 3 (p=0.1049) (Figure 1E-G and Table S4). The flow cytometry experiments described above showed that primary human monocytes have the ability to associate with all L. aethiopica parasites. However, the co-localisation of flow cytometry signals (monocytes and L. aethiopica) does not necessarily mean internalisation of the parasite. Therefore, confocal microscopy was used to demonstrate that monocytes have the ability to phagocytose L. aethiopica.

presented in Figure 2A-E and Figure S2 confirm that L. aethiopica are internalised by monocytes. ROS production Next, we investigated whether the different monocyte subsets produced an oxidative burst in response to co-incubation with the parasites. Results presented in Table S5 show that the mean MFIs of ROS in the unassociated and the associated monocyte subsets were always higher as compared to baseline, however it was not always significant. It was also higher in the associated monocytes than in the unassociated monocytes, although it was not significant for the classical subset incubated with L. aethiopica 1 (p=0.0584, Table S5). All three different monocyte subsets associated with L. aethiopica produced similar levels of ROS (Figure 3A-D, Table S6). Of note, unassociated classical monocytes (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint produced higher levels of ROS than unassociated non-classical monocytes with L. aethiopica lab (Table S6). For each monocyte subset, there was no difference in ROS production in response to all parasite clinical isolates (Table S7). When compared to L. aethiopica lab, unassociated classical and non-classical monocytes produced significantly higher levels of ROS following co-incubation with L. aethiopica 3 (Table S8). Chemokine and cytokine production Chemokines The levels of eotaxin, eotaxin-3, IP-10, MDC and TARC were below the detection limit. In contrast, IL-8, MCP-1, MCP4, MIP-1α and MIP-1β were significantly upregulated in response to all parasite isolates (Table S9). No significant differences were observed in the production of chemokines following co-incubation with the three clinical isolates (Figure 4 and Table S10). Notably, the levels of MIP-1α and MIP-1β were significantly lower in the supernatants of monocytes co-incubated with L. aethiopica lab as compared to L. aethiopica 2 and 3 (Figure 4 and Table S11). Cytokines IFN-γ, IL-2, IL-4, IL-12p70, and IL-13 were undetectable. By contrast, the levels of IL- 1β, IL-6, IL-10 and TNF-α were significantly increased in response to all parasite isolates, except IL-6 with L. aethiopica 1 (p=0.0982, Table S12). There were no significant differences in cytokine production between the three clinical isolates (Figure 5 and Table S13). However, the levels of IL-1β and TNF-α were significantly lower following co-incubation with L. aethiopica lab compared to L. aethiopica 2 (Figure 5 and Table S14). (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint

When feeding, sand flies lacerate the skin of the host to form a pool of blood, into which Leishmania parasites are deposited [18, 40]. Monocytes are therefore likely to be one of the first cells to encounter parasites. However, little is known about the interactions of monocytes with L. aethiopica. Unlike many other CL-causing Leishmania species, there are no established animal models for L. aethiopica infection [41, 42]. Therefore, here we set up an in vitro model, using primary human monocytes. We measured phagocytosis, ROS production and release of chemokines and cytokines in response to co-incubation with L. aethiopica and compared those responses to three different recently isolated L. aethiopica and a long-term cultured laboratory isolate. Our results show that all four parasite isolates associated with primary human monocytes. However, some monocytes remained unassociated; this might be due to the short incubation time. Interestingly, while there was no difference between the clinical isolates, association with each of the three clinical isolates was significantly higher than with L. aethiopica lab. Phagocytosis of L. aethiopica parasites and their entry into monocytes was confirmed using confocal microscopy. The ability of primary human monocytes to associate with Leishmania parasites has already been shown, using different approaches, and using different incubation times as well as different multiplicities of infection (MOIs). This includes whole blood [43, 44], PBMCs [27, 45, 46] and purified human peripheral blood monocytes [32, 47]. Some of the studies specify the parasite stage, e.g. metacyclic parasites; however, an approach to enrich for metacyclic promastigotes is not described [43, 44, 46, 47]. To ensure consistency in the parasite stages that we used, promastigotes were grown to stationary phase and PNA was used in order enrich for metacyclic parasites. Additionally, L. aethiopica parasites were kept in culture for a maximum of three weeks. The time parasites are kept in culture is of crucial importance. Indeed, several studies have shown that after in vitro culture, parasites lose their virulence as shown by decreased parasites loads in vivo and in vitro [48-50]. Altered gene and protein expression profiles, as well as chemokine and cytokine production, were reported following extensive in vitro passages [51]. In particular, glycoprotein (GP)63 and lipophosphoglycan (LPG)2 expression were shown to be reduced [52, 53]. Both GP63 and LPG are two of the main (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint surface molecules on Leishmania parasites that play a crucial role in phagocytosis [54- 56]. Therefore, it is likely that prolonged culture of L. aethiopica lab may have resulted in changes in LPG and GP63, leading to the observed differences in association, compared to the clinical L. aethiopica isolates. A recent study assessed the ability of immune cells to associate with GFP-transfected L. aethiopica lab [57]. The authors used an MOI of 10:1 (parasites to leukocytes) and reported that an estimated 20% of associated cells were CD14+ cells, as gated in the whole population of GFP+ cells in whole blood, at both 4- and 24-hour timepoints. Since the percentage of monocytes varies greatly between individuals [58], this method does not allow for consistency in MOI between experiments. However, no study has previously shown the association of purified monocytes with L. aethiopica. Our results show that monocytes associated with L. aethiopica promastigotes upregulated ROS, as compared to monocytes cultured in the absence of parasites. Incubation with the three clinical isolates, but not L. aethiopica lab, also resulted in increased ROS in unassociated monocytes as compared to baseline. ROS MFI in associated monocytes was higher than in unassociated monocytes with all four parasite isolates. Human monocytes have been shown to produce ROS in response to multiple Leishmania species [28, 31, 32, 47, 59]. However, the set-up of these experiments varied greatly, from the incubation time, the parasite species, their life cycle stages, to the ways ROS was measured. Some of the studies have shown that ROS production contributes to killing of the parasite [28, 31, 32]. Yet, others have identified defence mechanisms used by Leishmania to inhibit ROS production by phagocytes [60-62]. Of note, our results showed that unassociated monocytes produced an oxidative burst with the clinical isolates of L. aethiopica, but not with L. aethiopica lab. This could be caused by differences in the surface molecules of L. aethiopica lab, especially in LPG and GP63, as discussed above. Our results showed that five different chemokines were detectable in the supernatants of monocytes incubated with L. aethiopica – MCP-1, MCP-4, MIP-1α, MIP-1β, and IL-8. There were higher levels of MIP-1α and MIP-1β following incubation with the clinical isolates than L. aethiopica lab. The lower levels of these chemokines in response to L. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint aethiopica lab could result from altered cell surface molecules due to the prolonged time of the parasite isolate in culture. Little is known about the role of MCP-1 and MCP-4 in leishmaniasis. Several in vitro studies using murine and human monocytes and macrophages have shown that the addition of MCP-1 results in increased parasite killing [31, 63, 64]. The in vitro production of MIP-1α and MIP-1β by monocytes in response to Leishmania parasites has not been previously demonstrated. MIP-1α and MIP-1β were also elevated in the supernatants of PBMCs incubated with L. amazonensis in both mouse and human macrophages [63, 64]. In addition to activating the monocytes to kill the parasites, MCP- 1, MCP-4, MIP-1α, MIP-1β and IL-8 may contribute to the recruitment of more immune cells and therefore impact on disease development. Our results also show that production of TNF-α, IL-1β, IL-6 and IL-10 were increased in response to L. aethiopica. In our study, IL-6 and IL-10 were similar between all four L. aethiopica isolates. Little is known about the production of both cytokines by monocytes co-incubated with Leishmania parasites. Two studies have documented the production of IL-6 in response to L. aethiopica; however, it was by total PBMCs in response to soluble Leishmania antigen (SLA) [65, 66]. Two studies have shown the production of IL-10 by monocytes in response to L. braziliensis [46, 67]. TNF-α and IL-1β were increased in response to L. aethiopica 2, compared to L. aethiopica lab. The production of TNF-α by monocytes in response to pathogens has been extensively documented [68-70], but not to L. aethiopica. There are conflicting reports on the production of TNF- α by monocytes in response to Leishmania parasites, showing parasite killing or contribution to pathology [71, 72]. Specifically in CL caused by L. aethiopica, TNF-α has been shown to be produced by the PBMCs of CL patients in response to incubation with L. aethiopica SLA, although the cellular source was not identified [73, 74]. Little is known about the role of IL-1β produced by monocytes in response to Leishmania parasites. Santos et al. identified intermediate monocytes as the main producers of IL- 1β. The authors showed that IL-1β did not contribute to L. braziliensis killing by human monocyte-derived macrophages, and suggested that instead, the IL-1β produced contributed to the pathology of CL [75]. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint It has to be noted that the purity of the monocyte population used in our study was >80% and we can therefore not exclude that other cells contributed to the levels of chemokines and cytokines detected. Eotaxin, eotaxin-3, IP-10, MDC, TARC, IFN-γ, IL-2, IL-4, IL-8, IL-12p70, and IL-13 were not detectable in the supernatants of activated monocytes. It is possible that these chemokines and cytokines were still produced but were captured by cell surface receptors, or that they were produced at levels below the detection limit. Although there were no significant differences between clinical isolates in the effector functions measured in this study, there were some differences between L. aethiopica lab and some of the clinical L. aethiopica; such as the production of TNF-α between L. aethiopica lab and L. aethiopica 2, but not L. aethiopica 1 and L. aethiopica 3; this suggests that there may still be differences between the clinical isolates. And indeed, while the three clinical L. aethiopica were isolated from LCL patients who had similar lesions, these patients were of different ages, at different times after the onset of symptomatic disease, and had different numbers and sizes of lesions. One of the main limitations was that we did not measure survival or killing of the parasites. This is usually done by counting intracellular parasites inside the monocytes. However, it is not possible to assess whether these parasites will survive or be killed. Another method that is commonly used is the transformation assay, where parasites are washed away after co-incubation with the monocytes and the released intracellular parasites are allowed to grow in culture and counted [76]. However, in our experience it has proven impossible to completely eliminate extracellular parasites after the 2-hour incubation, whether by extensive washing or by disrupting the parasite cell membrane using sodium dodecyl sulfate. In summary, our study describes an in vitro model to investigate monocyte effector functions in response to L. aethiopica. Our results emphasise that care should be taken when using parasites that have been passaged in vitro for a long period of time. This model could be applied to L. aethiopica isolates from patients with LCL, DCL or MCL and might contribute to elucidating why L. aethiopica causes different clinical presentations. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint

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J Microbiol Methods. 2023;215:106847. Epub 2023/10/24. doi: 10.1016/j.mimet.2023.106847. PubMed PMID: 37871728. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint

The authors are thankful to the staff of Nefas Mewcha Hospital for their enthusiastic collaboration during the data collection of this study. ECC is funded by a Wellcome Trust Studentship (PS3750). This research is jointly funded by the UK Medical Research Council (MRC) and the Foreign Commonwealth and Development Office (FCDO) under the MRC/FCDO Concordat agreement (MR/R021600/1) (EY, JAC, PK). JAC is funded by Wellcome via core funding of the Wellcome Sanger Institute (grant 206194). (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint FIGURE LEGENDS Figure 1 – Association of monocyte subsets with L. aethiopica. Primary human monocytes were co-incubated for 2 hours with L. aethiopicaFR at an MOI of 1:10. The association of each monocyte subset with parasites was determined by flow cytometry. For each L. aethiopica isolate, association was compared between the three monocyte subsets (A- D), and for each monocyte subset, association with the different L. aethiopica isolates was compared (E-G). L. aethiopica lab: n=8, L. aethiopica 1: n=7, L. aethiopica 2: n=9, L. aethiopica 3: n=8. Statistical differences between monocyte subsets were determined using a Kruskal-Wallis test (A-D), between the clinical isolates using a Kruskal-Wallis test (E-G), and between L. aethiopica lab and each clinical isolate using a Mann-Whitney test (E-G). Data summaries and statistical analyses are shown in Tables S2-S4. Figure 2 – Phagocytosis of L. aethiopica by monocytes. Primary human monocytes were co-incubated with L. aethiopicaFR for 2 hours at an MOI of 1:10. Following staining with primary and secondary antibodies, monocytes were visualised using confocal microscopy. Images are shown for monocytes incubated without parasites (A), and for monocytes incubated with L. aethiopica lab (B), L. aethiopica 1 (C), L. aethiopica 2 (D) and L. aethiopica 3 (E). One representative image is shown for each parasite, out of three experimental repeats. Figure 3 – Comparing ROS production by different monocyte subsets. Primary human monocytes were co-incubated with L. aethiopicaFR for 2 hours at an MOI of 1:10. ROS production by monocytes was measured by flow cytometry using a ROS-IDTM Total ROS detection kit. ROS production (as a percentage of baseline ROS production) was compared between the three monocyte subsets for L. aethiopica lab (A), L. aethiopica 1 (B), L. aethiopica 2 (C) and L. aethiopica 3 (D). L. aethiopica lab: n=8, L. aethiopica 1: n=6, L. aethiopica 2: n=9, L. aethiopica 3: n=8. Statistical differences between monocyte subsets were determined using a Kruskal-Wallis test. Data summaries and statistical analyses are shown in Table S6. Figure 4 – Chemokine production in response to co-incubation with the four L. aethiopica isolates. Primary human monocytes were co-incubated with L. aethiopica at an MOI of 1:10. After 2 hours, supernatants from each tube were collected. Supernatants were analysed with a V-PLEX Chemokine Panel 1 Human Kit (Meso Scale Diagnostics) to determine the concentration of 10 different chemokines. The concentrations of five chemokines were compared in response to the different parasite isolates: IL-8 (A), MCP-1 (B), MCP-4 (C), MIP-1α (D), and MIP-1β (E). n=10 for all parasite isolates. Statistical differences between L. aethiopica lab and each clinical isolate were determined using a Mann-Whitney test, and between the three clinical isolates using a Kruskal-Wallis test. Data summaries and statistical analyses are shown in Tables S10 and S11. Figure 5 – Cytokine production in response to co-incubation with the four L. aethiopica isolates. Primary human monocytes were co-incubated with L. aethiopica at an MOI of 1:10. After 2 hours, supernatants from each tube were collected. Supernatants were analysed with a V-PLEX Proinflammatory Panel 1 Human Kit (Meso Scale Diagnostics) to determine the concentration of 10 different cytokines. The concentrations of four cytokines were compared in response to the different parasite isolates: IL-1β (A), IL-6 (B), IL-10 (C), TNF-α (D). n=10 for all parasite isolates. Statistical differences between L. aethiopica lab and each clinical isolate were determined using a Mann-Whitney test, and between the three clinical isolates using a Kruskal-Wallis test. Data summaries and statistical analyses are shown in Tables S13 and S14. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Figure S1 – Gating for monocyte subsets. Monocytes were stained with anti-CD14PE and anti-CD16eFluor™ 450 and divided into subsets according to the following definitions: classical monocytes as CD14++ CD16-, intermediate monocytes as CD14++ CD16+ and non-classical monocytes as CD14+ CD16+. Figure S2 – Phagocytosis of L. aethiopica promastigotes by primary human monocytes. Primary human monocytes were co-incubated for 2 hours with L. aethiopicaFR at an MOI of 1:10. Following the incubation, monocytes were washed and transferred onto a microscope slide, where they were fixed and stained using anti-CD14 and DAPI. The images show staining for three markers: monocyte surface (anti-CD14FITC; green), nuclei (DAPI; blue) and L. aethiopica (Far Red dye; red). A. The three different channels separately (i-iii) and combined (iv), for a monocyte infected with L. aethiopica 1. B. A representative series of 24 z-stack images for a monocyte infected with L. aethiopica 1. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint TABLES Table S1: PNA- recovery in stationary phase population: comparison of L. aethiopica lab with each clinical isolate. L. aethiopica PNA- recovery PNA- recovery with L. aethiopica lab p value* 1 8.64±2.50 3.48±0.72 0.0079 2 8.82±1.11 0.0079 3 10.30±1.52 0.0079 Stationary phase L. aethiopica promastigotes were enriched for metacyclic parasites using PNA agglutination as described in Materials and Methods. PNA- recovery (%) is shown as shown as mean ± standard deviation (SD). n=5 for all parasite isolates. Statistical differences between L. aethiopica lab and each of the clinical isolates were determined using a Mann-Whitney test*. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Table S2: Comparing association between monocyte subsets, for each L. aethiopica isolate. L. aethiopica Monocyte subset Association p value** p value*** lab Classical 31.2±9.4 0.0070 C vs I 0.1017 Intermediate 16.2±7.1 C vs NC 0.0063 Non-classical 11.5±8.2 I vs NC >0.9999 1 Classical 61.3±6.0 0.9999 2 Classical 64.0±9.4 0.0006 C vs I 0.0089 Intermediate 41.6±10.9 C vs NC 0.0009 Non-classical 34.0±16.8 I vs NC >0.9999 3 Classical 54.9±10.6 0.0388 C vs I 0.2313 Intermediate 30.3±18.5 C vs NC 0.0400 Non-classical 26.3±17.1 I vs NC >0.9999 Primary human monocytes were co-incubated for 2 hours with L. aethiopicaFR promastigotes at an MOI of 1:10. The association (%) of each subset with parasites was determined by flow cytometry, and subsets were compared for each parasite isolate. L. aethiopica lab: n=8, L. aethiopica 1: n=7, L. aethiopica 2: n=9, and L. aethiopica 3: n=8. Statistical differences were determined using a Kruskal-Wallis test** and Dunn’s multiple comparisons test***. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Table S3: Comparing association with the three clinical isolates of L. aethiopica, for each of the monocyte subsets. Monocyte subset L. aethiopica Association p value** Classical 1 61.3±6.0 0.0986 2 64.0±9.4 3 54.9±10.6 Intermediate 1 36.8±8.2 0.1209 2 41.6±10.9 3 30.3±18.5 Non-classical 1 31.8±7.9 0.4202 2 34.0±16.8 3 26.3±17.1 Primary human monocytes were co-incubated for 2 hours with L. aethiopicaFR promastigotes at an MOI of 1:10. Association (%) was determined by flow cytometry and is shown as mean ± SD. L. aethiopica 1: n=7, L. aethiopica 2: n=9, and L. aethiopica 3: n=8. Statistical differences between the three clinical isolates were determined using a Kruskal-Wallis test**. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Table S4 – Comparing monocyte association with L. aethiopica lab and three clinical isolates of L. aethiopica. Primary human monocytes were co-incubated for 2 hours with L. aethiopicaFR promastigotes at an MOI of 1:10. Association (%) was determined by flow cytometry and shown as mean ± SD. L. aethiopica lab: n=8, L. aethiopica 1: n=7, L. aethiopica 2: n=9, and L. aethiopica 3: n=8. Statistical differences between L.a. lab and each of the clinical isolates were determined a Mann-Whitney test*. Monocyte subset L. aethiopica Association (%) Association with L. aethiopica lab (%) p value* Classical 1 61.3±6.0 31.2±9.4 0.0003 2 64.0±9.4 <0.0001 3 54.9±10.6 0.0002 Intermediate 1 36.8±8.2 16.2±7.1 0.0003 2 41.6±10.9 <0.0001 3 30.3±18.5 0.1049 Non- classical 1 31.8±7.9 11.5±8.2 0.0012 2 34.0±16.8 0.0055 3 26.3±17.1 0.0281 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Table S5: ROS production by monocyte subsets following co-incubation with the different L. aethiopica isolates. L. aethiopica Monocyte subset Monocyte ROS production (MFI) p value* lab Classical Baseline: 270.5±53.0 Associated: 369.6±48.0 0.0020 Unassociated: 286.4±31.9 0.4227 p value* 0.0065 Intermediate Baseline: 304.2±67.5 Associated: 391.9±64.2 0.0141 Unassociated: 313.6±37.1 0.4556 p value 0.0131 Non-classical Baseline: 335.7±92.4 Associated: 408.4±78.0 0.3625 Unassociated: 321.2±68.8 0.8932 p value 0.0490 1 Classical Baseline: 245.2±14.3 Associated: 344.7±36.4 0.0022 Unassociated: 296.7±33.9 0.0065 p value 0.0584 Intermediate Baseline: 278.8±24.6 Associated: 419.4±60.4 0.0022 Unassociated: 326.1±29.9 0.0173 p value 0.0043 Non-classical Baseline: 318.7±65.3 Associated: 423.4±87.5 0.0238 Unassociated: 328.5±68.5 0.8160 p value 0.0411 2 Classical Baseline: 262.1±55.6 Associated: 378.0±49.7 0.0003 Unassociated: 316.7±40.2 0.0582 p value 0.0082 Intermediate Baseline: 294.6±69.5 Associated: 429.2±64.3 0.0016 Unassociated: 345.5±46.5 0.0659 p value 0.0174 Non-classical Baseline: 327.1±87.8 Associated: 469.1±103.8 0.0028 Unassociated: 360.2±68.0 0.3943 p value 0.0334 3 Classical Baseline: 264.1±35.6 Associated: 392.3±46.2 0.0002 Unassociated: 324.5±36.1 0.0040 p value 0.0186 Intermediate Baseline: 294.2±49.1 Associated: 457.3±48.8 0.0003 Unassociated: 352.3±56.7 0.0190 p value 0.0042 Non-classical Baseline: 320.5±77.7 Associated: 460.7±81.1 0.0196 Unassociated: 346.3±68.5 0.2216 p value 0.0194 Primary human monocytes were co-incubated for 2 hours with L. aethiopicaFR promastigotes at an MOI of 1:10. Monocyte ROS production was measured by flow cytometry using a ROS- IDTM Total ROS detection kit and is shown as mean MFI ± SD. L. aethiopica lab: n=8, L. aethiopica 1: n=6, L. aethiopica 2: n=9, and L. aethiopica 3: n=8. Statistical differences were determined using a Mann-Whitney test*. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Table S6: Comparing ROS production by monocyte subsets in response to L. aethiopica. L. aethiopica Association Subsets ROS p value** Groups p value*** lab Associated Classical 138.6±22.4 0.3045 NA Intermediate 131.6±26.1 Non-classical 131.2±26.8 Unassociated Classical 110.4±17.8 0.0187 C v I 0.7618 Intermediate 107.4±19.5 C v NC 0.0215 Non-classical 98.2±12.8 I v NC >3646 1 Associated Classical 140.4±10.3 0.5063 NA Intermediate 150.6±26.9 Non-classical 136.4±20.9 Unassociated Classical 120.8±8.3 0.5063 NA Intermediate 115.1±8.6 Non-classical 105.8±8.2 2 Associated Classical 146.6±25.2 0.6517 NA Intermediate 152.9±27.6 Non-classical 143.2±30.4 Unassociated Classical 122.8±17.3 0.0810 NA Intermediate 125.3±20.2 Non-classical 115.5±18.8 3 Associated Classical 147.4±18.1 0.5691 NA Intermediate 156.6±23.5 Non-classical 148.7±16.0 Unassociated Classical 123.5±10.7 0.6838 NA Intermediate 121.2±9.5 Non-classical 112.7±8.0 Primary human monocytes were co-incubated for 2 hours with L. aethiopicaFR promastigotes at an MOI of 1:10. Monocyte ROS production was measured by flow cytometry using a ROS- IDTM Total ROS detection kit, was calculated as % of baseline ROS production, and is shown as mean ± SD. L. aethiopica lab: n=8, L. aethiopica 1: n=6, L. aethiopica 2: n=9, and L. aethiopica 3: n=8. Statistical differences between monocyte subsets were determined using a Kruskal-Wallis test**. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Table S7: Comparing ROS production by monocyte subsets co-incubated with the clinical isolates. Primary human monocytes were co-incubated for 2 hours with L. aethiopicaFR promastigotes at an MOI of 1:10. Monocyte ROS production was measured by flow cytometry using a ROS- IDTM Total ROS detection kit, was calculated as % of baseline ROS production, and is shown as mean ± SD. L. aethiopica 1: n=6, L. aethiopica 2: n=9, and L. aethiopica 3: n=8. Statistical differences between the three clinical isolates were determined using a Kruskal- Wallis test**. Monocyte subset Association L. aethiopica ROS p value** Classical Associated 1 140.4±10.3 0.4585 2 147.8±25.4 3 149.7±18.1 Unassociated 1 120.8±8.3 0.9373 2 123.2±16.9 3 123.5±10.7 Intermediate Associated 1 151.4±25.8 0.6040 2 149.6±26.2 3 157.8±22.4 Unassociated 1 117.3±10.4 0.5280 2 123.6±18.7 3 120.1±9.2 Non-classical Associated 1 133.6±17.5 0.4812 2 147.1±29.5 3 146.2±16.1 Unassociated 1 103.4±8.5 0.4432 2 113.2±17.9 3 109.1±6.0 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Table S8 – Comparison of ROS production by monocyte subsets between L. aethiopica lab with each clinical isolate. Monocyte subset Association L. aethiopica ROS ROS with L. aethiopica lab p value* Classical Associated 1 140.4±10.3 139.4±22.3 0.8032 2 147.8±25.4 0.4954 3 149.7±18.1 0.5936 Unassociated 1 120.8±8.3 108.0±15.7 0.0982 2 123.2±16.9 0.1191 3 123.5±10.7 0.0465 Intermediate Associated 1 151.4±25.8 132.1±25.7 0.2128 2 149.6±26.2 0.2076 3 157.8±22.4 0.0690 Unassociated 1 117.3±10.4 105.7±16.4 0.1695 2 123.6±18.7 0.0607 3 120.1±9.2 0.0716 Non-classical Associated 1 133.6±17.5 125.9±26.6 0.6509 2 147.1±29.5 0.6096 3 146.2±16.1 0.8275 Unassociated 1 103.4±8.5 97.4±10.4 0.3287 2 113.2±17.9 0.0778 3 109.1±6.0 0.0298 Primary human monocytes were co-incubated for 2 hours with L. aethiopicaFR promastigotes at an MOI of 1:10. P Monocyte ROS production was measured by flow cytometry using a ROS- IDTM Total ROS detection kit, was calculated as % of baseline ROS production, and is shown as mean ± SD. L. aethiopica lab: n=8, L. aethiopica 1: n=6, L. aethiopica 2: n=9, and L. aethiopica 3: n=8. Statistical differences between L. aethiopica lab and each clinical isolate were determined using a Mann-Whitney test*. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Table S9 – Chemokine production in response to co-incubation with the four L. aethiopica isolates. Chemokine L. aethiopica Concentration Baseline concentration p value* MCP-1 lab 169.8±145.0 58.32±63.41 0.0185 1 172.6±140.3 0.0288 2 177.4±139.5 0.0115 3 175.0±136.5 0.0115 MCP-4 lab 15.59±12.36 4.337±2.064 0.0002 1 19.65±18.02 0.0068 2 24.28±13.77 <0.0001 3 19.38±8.883 <0.0001 MIP-1α lab 352.0±374.6 22.34±27.03 <0.0001 1 1271±1596 <0.0001 2 1692±1419 <0.0001 3 799.0±1044 <0.0001 MIP-1β lab 430.9±412.2 81.66±118.6 0.0011 1 1187±1844 0.0005 2 2134±3655 <0.0001 3 1013±1743 0.0002 IL-8 lab 785.3±500.3 371.3±447.8 0.0232 1 798.8±453.5 0.0232 2 983.0±314.7 0.0029 3 876.4±373.3 0.0147 Primary human monocytes were co-incubated for 2 hours with L. aethiopica promastigotes at an MOI of 1:10. Supernatants were collected and analysed on a V-PLEX Chemokine Panel 1 Human Kit to determine the concentration of 10 different chemokines. Chemokine concentration (pg/mL) is shown as mean ± SD. n=10 for all parasite isolates. Statistical differences were determined using a Mann-Whitney test*. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Table S10 – Comparing chemokine production in response to co-incubation with clinical isolates. Chemokine L. aethiopica Concentration p value** MCP-1 1 494.9±310.3 0.9683 2 535.5±340.5 3 517.9±310.8 MCP-4 1 474.6±344.2 0.5661 2 656.1±423.3 3 521.5±299.6 MIP-1α 1 5172±4493 0.0964 2 11508±11824 3 4971±4077 MIP-1β 1 1459±932.7 0.1289 2 2941±2593 3 1610±1426 IL-8 1 635.6±667.5 0.6069 2 980.8±848.4 3 784.3±704.2 Primary human monocytes were co-incubated for 2 hours with L. aethiopica promastigotes at an MOI of 1:10. Supernatants were collected and analysed on a V-PLEX Chemokine Panel 1 Human Kit to determine the concentration of 10 different chemokines. Chemokine concentration was calculated as % of baseline production (%) is shown as mean ± SD. n=10 for all parasite isolates. Statistical differences between the three clinical isolates were determined using a Kruskal-Wallis test**. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Table S11 – Comparing chemokine production in response to co-incubation with L.a. lab and clinical isolates. Chemokine L. aethiopica Concentration Concentration with L. aethiopica lab p value* MCP-1 1 494.9±310.3 491.5±364.4 0.9118 2 535.5±340.5 0.7394 3 517.9±310.8 0.7959 MCP-4 1 474.6±344.2 397.2±269.4 0.7394 2 656.1±423.3 0.0892 3 521.5±299.6 0.2176 MIP-1α 1 5172±4493 2364±2248 0.1051 2 11508±11824 0.0021 3 4971±4077 0.0433 MIP-1β 1 1459±932.7 1018±1110 0.1655 2 2941±2593 0.0039 3 1610±1426 0.0433 IL-8 1 635.6±667.5 577.0±642.5 0.9705 2 980.8±848.4 0.4359 3 784.3±704.2 0.6305 Primary human monocytes were co-incubated for 2 hours with L. aethiopica promastigotes at an MOI of 1:10. Supernatants were collected and analysed on a V-PLEX Chemokine Panel 1 Human Kit to determine the concentration of 10 different chemokines. Chemokine concentration was calculated as % of baseline production (%) is shown as mean ± SD. n=10 for all parasite isolates. Statistical differences between L. aethiopica lab and each clinical isolate were determined using a Mann-Whitney test*. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Table S12 – Cytokine production in response to co-incubation with the four L. aethiopica isolates. Cytokine L. aethiopica Concentration Baseline concentration p value** TNF-α lab 134.9±98.14 6.137±6.628 0.0001 1 443.9±458.2 0.0001 2 597.5±395.8 <0.0001 3 299.7±221.2 <0.0001 IL-1β lab 6.984±8.142 0.9005±0.9754 0.0185 1 20.94±28.01 0.0147 2 22.22±23.20 0.0005 3 9.353±10.82 0.0052 IL-6 lab 29.10±43.89 6.586±10.40 0.0433 1 88.40±137.6 0.0982 2 64.95±101.3 0.0147 3 48.22±96.99 0.0433 IL-10 lab 0.9682±0.9523 0.2390±0.2181 0.0147 1 1.448±1.693 0.0355 2 1.567±1.343 0.0068 3 1.209±1.070 0.0185 Primary human monocytes were co-incubated for 2 hours with L. aethiopica promastigotes at an MOI of 1:10. Primary human monocytes were co-incubated for 2 hours with L. aethiopica promastigotes at an MOI of 1:10. Supernatants were collected and analysed on a V-PLEX Proinflammatory Panel 1 Human Kit to determine the concentration of 10 different cytokines. Cytokine concentration (pg/mL) is shown as mean ± SD. n=10 for all parasite isolates. Statistical differences were determined using a Mann-Whitney test*. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Table S13 – Comparing cytokine production in response to co-incubation with clinical isolates. Cytokine L. aethiopica Concentration p value** TNF-α 1 8838±9970 0.2842 2 19028±26340 3 11782±18872 IL-1β 1 1625±1435 0.4137 2 3468±4069 3 1603±1901 IL-6 1 981.0±858.2 0.5764 2 1138±1359 3 708.0±817.3 IL-10 1 519.8±455.6 0.7159 2 683.1±482.4 3 531.6±372.5 Primary human monocytes were co-incubated for 2 hours with L. aethiopica promastigotes at an MOI of 1:10. Supernatants were collected and analysed on a V-PLEX Proinflammatory Panel 1 Human Kit to determine the concentration of 10 different cytokines. Cytokine concentration was calculated as % of baseline production (%) is shown as mean ± SD. n=10 for all parasite isolates. Statistical differences between the three clinical isolates were determined using a Kruskal-Wallis test**. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Table S14 – Comparing cytokine production in response to co-incubation with L.a. lab and clinical isolates. Cytokine L. aethiopica Concentration Concentration with L. aethiopica lab p value* TNF-α 1 8838±9970 4417±5926 0.2176 2 19028±26340 0.0185 3 11782±18872 0.1431 IL-1β 1 1625±1435 787.6±651.1 0.1655 2 3468±4069 0.0355 3 1603±1901 0.6305 IL-6 1 981.0±858.2 568.7±675.5 0.5288 2 1138±1359 0.1051 3 708.0±817.3 0.7959 IL-10 1 519.8±455.6 388.2±259.0 0.5288 2 683.1±482.4 0.2799 3 531.6±372.5 0.4359 Primary human monocytes were co-incubated for 2 hours with L. aethiopica promastigotes at an MOI of 1:10. Supernatants were collected and analysed on a V-PLEX Proinflammatory Panel 1 Human Kit to determine the concentration of 10 different cytokines. Cytokine concentration was calculated as % of baseline production (%) is shown as mean ± SD. n=10 for all parasite isolates. Statistical differences between L. aethiopica lab and each clinical isolate were determined using a Mann-Whitney test*. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Figure 1 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Figure 2 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Figure 3 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Figure 4 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Figure 5 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Figure S1 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint Figure S2 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 2, 2025. ; https://doi.org/10.1101/2025.02.26.640320doi: bioRxiv preprint