Unravelling the cellular sources and location of IL-17A production during a Giardia infection

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Van Crombrugge, Bregt Decorte, Leen J.M. Seys, Peter Geldhof This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6836025/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract IL-17A plays a crucial role in the immune defense against Giardia infection, yet its cellular sources remain incompletely defined. In this study, the site-specific expression and cellular origin of IL-17A were investigated, focusing on both adaptive and innate immune cells in the small intestine, Peyer’s patches and mesenteric lymph nodes of Giardia muris infected C57BL/6 mice. RT-qPCR analyses showed that IL-17A mRNA expression was significantly upregulated in the small intestine, slightly elevated in the Peyer’s patches and unchanged in the mesenteric lymph nodes. Flow cytometry revealed that CD4⁺ T helper cells in the lamina propria of the small intestine are the predominant source of anti-giardial IL-17A. No increase in IL-17A was detected by γδT cells, Tc cells, NK(T) cells, B cells, neutrophils, dendritic cells and innate lymphoid cells. Within the CD3- innate cell population, increased IL-17A production was observed in MHC-II+CD11c+CD11b+/- cells, including a subset of cells expressing typical macrophage markers, namely MHC-II⁺CD11c⁺CD11b⁺CD64⁺F4/80⁺ cells. In T cell-deficient mice, both IL-17A expression and parasite clearance were severely impaired. Our findings demonstrate the importance of the adaptive immunity and simultaneously identify Th cells in the lamina propria as the main source of anti-giardial IL-17A, with a possible supporting role from macrophage-like antigen-presenting cells. Biological sciences/Immunology/Adaptive immunity/Cellular immunity Biological sciences/Immunology/Cytokines/Interleukins Biological sciences/Immunology/Infectious diseases/Parasitic infection Giardia IL-17A T cells ILCs MHC-II macrophages Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Giardia duodenalis , also referred to as Giardia lamblia or Giardia intestinalis , is a flagellated unicellular protozoan parasite with a broad host tropism, infecting both animals and humans. G. duodenalis has a high global incidence, with over 200 million human cases reported annually 1 . Giardia infection can lead to a spectrum of clinical signs that extends from acute to chronic 2 . Acute giardiasis generally lasts one to three weeks and symptoms disappear quickly. If a chronic situation develops, infection can last up to several months. Symptom severity ranges from subclinical to severe malabsorption, diarrhea, abdominal pain and weight loss 3 . These variations are due to different factors, such as the state of the immune system, age and diet of the host, the genotype of the Giardia species and complicating co-infections 4 . In recent years, interleukin 17A (IL-17A) has gained recognition as a central cytokine in the immune response against Giardia infection. Dreesen et al. 5 reported a significant increase in IL-17A mRNA expression in the small intestine after G. muris infection in C57BL/6 mice. Additionally, the use of IL-17A receptor (IL-17RA) knock-out mice underscored the importance of this cytokine during Giardia infections, as mice with a defect in their IL-17A/IL-17RA axis were unable to clear a Giardia infection 5,6 . Subsequent research has predominantly focused on the downstream effector mechanisms of IL-17A, elucidating the mechanisms that lead to clearance of the parasite. Specifically, IL-17A activates the complement system by stimulating the production of mannose-binding lectin 2 (Mbl2) and triggers the secretion of Giardia -specific IgA by B cells 7 . Less research has focused on the mechanisms leading to the production of IL-17A. IL-17A production can occur across various immune sites in the body. The immune architecture of the intestine consists of inductive sites, where adaptive immune cells are primed; and effector sites, where these activated immune cells preserve barrier function and regulate protective immunity. Intestinal inductive sites encompass the mesenteric lymph nodes (MLNs) and gut-associated lymphoid tissues, such as Peyer’s patches (PP), whereas intestinal effector sites include the lamina propria (LP) and the epithelial layer of the small intestine (SI), housing intra-epithelial lymphocytes (IEL) 8 . The PP have already been investigated as an important site of immune activity during Giardia infection. In the 1990s, Hill 9 observed a blastogenic response in PP leukocytes of G. muris infected mice, correlating with the time of peak-infection. Additionally, Djamatun and Faubert 10 observed increased in vitro IL-4 and IL-5 cytokine production by PP cells isolated from G. muris infected mice. Notably, these studies predate the identification of IL-17A as a central player during anti-giardial immunity. Varying reports have been made on the MLN during Giardia infection. A previous study observed a slight increase in IL-17A production by extracted MLN cells of G. duodenalis infected mice after ex vivo stimulation with Giardia extracts 11 . In contrast, Dann et al. 6 reported no increase in IL-17A mRNA production in the MLN of G. muris infected mice. Generally, T helper 17 (Th17) cells are known to be the classical IL-17A producers, although several innate immune cells have also been reported to produce IL-17A under certain conditions. These innate IL-17A cell sources include ɣδT cells, innate lymphoid cells type 3 (ILC3), natural killer (NK) cells, NKT cells, Paneth cells, neutrophils and dendritic cells (DCs) 12-17 . In the context of a Giardia infection, the exact cellular sources of IL-17A remain unclear. Dann et al. 6 reported increased IL-17A production in CD4 + T cells, most likely Th17 cells, isolated from the SI of G. muris infected mice. Interestingly, CD4 deficient mice, which lack all Th cell subsets; and recombination activating gene 2 (Rag2) deficient mice, which lack both B and T cells, still exhibited enhanced expression of IL-17A mRNA in small intestinal tissue following a G. muris infection 6 . This suggests that innate immune cells could be potential additional sources of IL-17A in the defense against a Giardia infection. As innate counterparts of Th17 cells, ILC3s have been speculated to be potential producers of anti-giardial IL-17A 6,18-20 . Supporting this hypothesis, Lee et al. 18 reported increased IL-17A production in ILC3s isolated from the intestine of C57BL/6 mice after stimulation with G. duodenalis trophozoites. In contrast, Yordanova et al. 20 reported no IL-17A production in ILC3s of G. muris infected mice. At this time, IL-17A production by innate cell sources following Giardia infection remains to be clarified. The present study investigates IL-17A production in response to G. muris infection across the different inductive and effector sites in the intestine. It further aims to clarify the cellular origins of anti-giardial IL-17A and simultaneously determine the relative contribution of Th17 cells compared to innate cell sources regarding IL-17A expression. Methods Ethical statement All animal experiments were conducted in accordance with the European Union (E.U.) Animal Welfare Directives, the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) and the ARRIVE guidelines for reporting animal research. Guidelines for Good Clinical Practice and ethical approval to conduct the studies were obtained from the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University (ethical committee approval numbers EC2023-085 and EC2024-033). Murine infection studies Six independent infection studies were performed in either C57BL/6J mice or B6.129P2-Tcrb tm1Mom Tcrd tm1Mom /J mice (T cell knock-out (TKO) mice), obtained from Jackson Laboratories. An overview of all infection studies is provided in Table 1 . In each experiment, 6-week old mice were either orally infected with 10 3 G. muris cysts suspended in 0.1 mL phosphate-buffered saline (PBS) (n = 4-5) or used as uninfected control mice (n = 4-5). Individual fecal cyst counts were performed every 2-3 days as previously described 5 . The presence of trophozoites in the SI was quantified for each animal at the time of euthanasia according to Paerewijck et al. 7 . All mice were euthanized by cervical dislocation. RT-qPCR Tissue samples of SI, PP and MLN of C57BL/6 and TKO mice were collected for real-time quantitative polymerase chain reaction (RT-qPCR) analysis. A 1 cm long segment of small intestinal tissue was collected 4 cm distal of the gastroduodenal junction. All PP along the small intestinal tract were pooled together and the MLN were dissected from the mesentery. To allow further RNA extraction, all samples were immediately snap-frozen in liquid nitrogen and stored at -80°C until further processing. RNA isolation and RT-qPCR analysis were performed according to Dreesen et al. 5 . In short, RNA isolation was performed using the RNeasy minikit (Qiagen). An Agilent 2100 Bioanalyzer (Agilent Technologies) was used to verify the quality of the isolated RNA and total RNA concentration was determined using a Nano-Drop ND-1000 spectrophotometer (NanoDrop Technologies). The iScript cDNA synthesis kit (Bio-Rad) was used to acquire cDNA for further RT-qPCR analysis. Gene expression of IL-17A and Mbl2 was measured using primer sequences obtained from Paerewijck et al. 21 and all analyses were run on a StepOnePlus real-time PCR system (Applied Biosciences). The data were analyzed using the ΔC T method and normalized based on the housekeeping genes Hprt1 and Tbp. Isolation of intestinal cells Single cell suspensions of IEL and LP cells from the SI of C57BL/6 and TKO mice were prepared following a protocol adapted from Lefrançois and Lycke 22 . After carefully excising the SI, the IEL fraction was isolated by incubating the intestine at 37°C for 20 minutes in RPMI medium containing 5% FCS, 2mM EDTA and 1mM DTT. The IEL cell suspension was filtered through a 40µM cell strainer and kept on ice until further processing. The remaining small intestinal tissue was incubated for 15 minutes at 37°C in a digestion buffer containing Dispase I (50U/mL), DNAse I (0,05mg/mL) and collagenase VIII (0,6mg/mL) in RPMI medium. The LP suspension was then filtered through a 100µM cell strainer. Both IEL and LP fractions were centrifuged at 400 g. IEL cell pellets were resuspended in 44% Percoll, overlaid on 67% Percoll and centrifuged at 1800 rpm for 20 minutes. Similarly, LP pellets were resuspended in 40% Percoll and overlaid on 80% Percoll before centrifugation. Buffy coats containing lymphocytes were collected and washed in PBS before antibody staining for flow cytometry. Flow cytometry Cells from the IEL fraction of C57BL/6 mice in study 2 were stained with an antibody panel targeting T cell subsets (panel 1). After a blocking step with Fc-Block, the cells were stained for 30 minutes at 4°C with CD45.2-PerCP-Cy5.5 (BD) (clone 104), TCRβ-BV421 (Biolegend) (clone H57-597), TCRɣδ-PE-Cy7 (Biolegend) (clone GL3), CD4-AF700 (eBioscience) (clone GK1.5) and CD8-APC-Cy7 (BD) (clone 53-6.7). eF506 (eBioscience) was used as a live/dead marker to exclude non-viable cells. Next, the cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD) to allow for intracellular staining with IL-17A-AF647 (BD) (clone TC11-18H10). Fluorescence-minus-one (FMO) controls were included for the IL-17A staining. For the LP cells of C57BL/6 mice in study 2, the same T cell panel was used. In a follow-up infection study (study 3), two additional panels were incorporated alongside panel 1 in order to investigate a broader range of immune cell types within the LP compartment. The first additional panel (panel 2) was directed against ILCs, specifically ILC3s, and consisted of the following antibodies: CD45.2-AF700 (Biolegend) (clone 30-F11), CD90.2-PerCP-Cy5.5 (Biolegend) (clone 30-H12), CD127-FITC (Biolegend) (clone SB/199), CD117-APC-Cy7 (Invitrogen) (clone 2BB) and a negative lineage consisting of CD3-BV421 (BD) (145-2C11), CD5-BV421 (Biolegend) (clone 53-7.3), TCRβ-BV421 (Biolegend) (clone H57-597), TCRɣδ-BV421 (Biolegend) (GL3), CD19-BV421 (BD) (clone 1D3), F4/80 (BD) (clone T45-2342), Ly6C/G-BV421 (BD) (clone RB6-8C5) and CD11b-V450 (BD) (clone M1/70). eF506 (eBioscience) was used as a live/dead marker to exclude non-viable cells. An intracellular staining was performed for IL-17A-AF647 (BD) (clone TC11-18H10) and RORɣt-PE (Invitrogen) (clone AFKJS-9) after fixation and permeabilization of the cells. The third panel was designed to target NKT cells, NK cells, T cells, neutrophils and DCs, using the following antibodies: CD3-PerCP-Cy5.5 (Biolegend) (clone 17A2), NK1.1-BV421 (Invitrogen) (clone PK136), CD11b-APC-Cy7 (BD) (clone M1/70), CD11c-PE-Cy7 (BD) (clone HL3), Ly6G-AF700 (Biolegend) (clone 1A8) and MHC-II-AF488 (eBioscience) (clone M5/114.15.2). eF506 (eBioscience) was used as a live/dead marker to exclude non-viable cells. An intracellular staining was carried out for IL-17A-AF647 (BD) (clone TC11-18H10) following cell fixation and permeabilization. For both panels, FMO controls were included for the IL-17A staining. As TKO mice are deficient in T cell populations, staining of LP cells was limited to panel 2 and panel 3. In panel 3, a modification was made by substituting the staining for CD3 by CD19-PerCP-Cy5.5 (Biolegend) (clone 6D5) to include the identification of B cells. In the final study, a flow cytometry panel was designed specifically to target antigen-presenting cells. The LP cells were stained for 30 minutes at 4°C with CD45.2-FITC (Biolegend) (clone 30-F11), a co-staining for CD19-PerCP-Cy5.5 (Biolegend) (clone 6D5) and CD3-PerCP-Cy5.5 (Biolegend) (clone 17A2), MHC-II-AF700 (Biolegend) (clone M5/114.15.2), CD11c-PE-eF610 (Invitrogen) (clone N418), CD11b-BUV395 (BD) (clone M1/70), CD26-BUV737 (BD) (clone H194-112), CD64-BV711 (Biolegend) (clone X54-5/7.1), F4/80-BV785 (Biolegend) (clone BM8) and Ly6C-eFluor450 (Invitrogen) (clone HK1.4). An intracellular staining was performed for IL-17A-AF647 (BD) (clone TC11-18H10) after fixation and permeabilization of the cells. An FMO control was included for the IL-17A staining. The samples from study 2, 3 and 5 were passed on a CytoFlex flow cytometer (Beckman Coulter) and the data were analyzed using the CytExpert Software Version 2.5 (Beckman Coulter Inc.). The samples from study 6 were run on a LSRFortessa 5-laser (BD Biosciences) and analyzed using the FlowJo v10.7 software (BD Biosciences). Statistical analysis All quantitative data are reported as the mean and standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 10. A one-tailed Mann-Whitney test was used for comparisons between two groups and a non-parametric Kruskal-Wallis test followed by a Dunn’s multiple comparison test was applied to compare differences among more than two groups. A P-value of < 0.05 was considered statistically significant. Results T helper cells located in the lamina propria of the small intestine are the main cellular source of IL-17A in response to a Giardia infection In a first experiment, IL-17A expression levels were monitored in the PP, MLN and SI of G. muris infected mice to determine the intestinal location of the anti-giardial IL-17A response. C57BL/6 mice were monitored for 21 days after G. muris infection. The fecal cyst excretion pattern was in line with previous observations 5,23 , reaching a peak around day 7 after infection and declining progressively, with minimal excretion by day 21 (Fig. 1a). Small intestinal trophozoite counts at day 7, 14 and 21 p.i. mirrored this pattern, with the highest number of trophozoites present at day 7 p.i., followed by a steady reduction. At day 21 p.i., only a minor amount of trophozoites was left in the SI (Fig. 1b). Additionally, IL-17A and Mbl2 gene expression levels in the SI, PP and MLN were analyzed by RT-qPCR at day 7, 14 and 21 p.i.. In the SI, there was a significant upregulation of IL-17A expression at day 7 and day 14 after infection compared to uninfected control mice. An increase in IL-17A expression was also noted in the PP on day 7 p.i., although it did not reach statistical significance. In the MLN, no differences in IL-17A mRNA production could be detected at any of the analyzed time points (Fig. 1c). Expression of Mbl2, an important downstream regulated gene of IL-17A 7 , showed the same expression pattern as IL-17A, with significant upregulation in the SI at all analyzed time points. No differences in Mbl2 expression could be detected in the PP and MLN (Supplementary Fig. S1). To identify which immune cells are responsible for this IL-17A production in the small intestine during a Giardia infection, a second experiment was set up in C57BL/6 mice. The parasitological parameters (cyst and trophozoite counts) for this study are shown in Supplementary Fig. S2. At day 14 p.i., IL-17A production by IEL and by immune cells in the LP of infected and uninfected control mice was analyzed using flow cytometry. In this experiment, leukocytes (CD45 + cells), Th cells (CD45 + TCRβ + CD4 + ), cytotoxic T (Tc) cells (CD45 + TCRβ + CD8 + ), ɣδT cells (CD45 + TCRɣδ + ) and ‘non-T’ cells (CD45 + TCRβ - TCRɣδ - ) were investigated. The gating strategy is documented in Supplementary Fig. S3. No IL-17A production could be detected in the IEL compartment by any cell type (Supplementary Fig. S4). In contrast, there was significantly elevated production of IL-17A by leukocytes in the LP at day 14 p.i. compared to uninfected control mice. Within this leukocyte population, Th cells demonstrated a clear and significant upregulation of IL-17A production. ɣδT cells and Tc cells showed no IL-17A production after infection. Notably, a small increase in IL-17A production following Giardia infection was also observed in a cell population negative for both T cell receptors (Fig. 2). To further characterize the IL-17A producing cells, a similar infection experiment was designed, this time including additional time points (day 4 and day 7 p.i.) and a broader range of immune cell types was investigated. This study focused exclusively on LP cells, as the previous study revealed no IL-17A production in the IEL. Along with leukocytes, Th cells, Tc cells, ɣδT cells and ‘non-T' cells, the following cell types were also included: all T cells, NKT cells, NK cells, neutrophils, DCs and ILCs, including ILC3s. These specific cell types were selected based on previous reports indicating their ability to produce IL-17A under certain conditions 12-17 . ILC3s specifically have been suggested multiple times as a possible innate source of anti-giardial IL17A 6,18-20 . The gating strategy for the additional cell types can be found in Supplementary Fig. S5, 6. Parasitological data of this infection experiment are presented in Supplementary Fig. S7. At day 14 p.i., a significant increase in IL-17A production was detected in the leukocyte population. Within the leukocytes this increase was present in the T cells and more precisely in the Th cell subset, confirming the observations made in study 2 (Fig. 3). Within the T cell population, again no IL-17A production could be detected in the Tc cells or the ɣδT cells at any time point during Giardia infection (Supplementary Fig. S8). In the population negative for TCRβ and TCRɣδ, a small but significant level of IL-17A production was detected at day 14 after infection, consistent with the prior experiment (Fig. 3). However, when looking further into multiple non-T cell populations, specifically NK(T) cells, neutrophils, DCs, ILCs and ILC3s, no IL-17A production could be detected at any time point by any of these cell types (Supplementary Fig. S8, 9). Only at day 7 p.i., a minor amount of IL-17A production could be detected from a population of MHC-II + cells, more specifically MHC-II + CD11c + CD11b - cells (Fig 3). Mice deficient in T cells lose the majority of their IL-17A production and lack the ability to overcome a G. muris infection To confirm that Th cells are the primary source of IL-17A in the defense against a Giardia infection, a new infection experiment was designed using TKO mice. The aim of this study was to compare C57BL/6 and TKO mice regarding their IL-17A production and their ability to overcome a Giardia infection. Infected C57BL/6 and TKO mice were monitored for 21 days after infection, a timeframe during which C57BL/6 mice typically clear the infection. Cyst shedding by C57BL/6 mice had almost entirely ceased by day 21 p.i.., whereas TKO mice maintained a high level of cyst excretion throughout the whole study period (Fig. 4a). Similarly, intestinal trophozoite counts at the end of the study revealed that C57BL/6 mice had almost completely eliminated the parasite, while TKO mice still carried up to 60 million trophozoites in their SI (Fig. 4b). These observations indicate that mice deficient in T cells are not able to overcome a Giardia infection. Relative IL-17A and Mbl2 expression levels were measured in the SI, PP and MLN of both mouse strains using RT-qPCR. Within C57BL/6 mice, a significant IL-17A upregulation was detected in the SI and an upwards trend was again visible in the PP, confirming the results of the first study. In all intestinal compartments and at every time point after infection, the magnitude of the IL-17A response was considerably lower in TKO mice compared to the expression levels in C57BL/6 mice. This observation highlights the predominant role of T cells in anti-giardial IL-17A production. However, within the TKO mice, IL-17A mRNA expression was still significantly elevated at day 21 p.i. compared to control mice (Fig. 4c). The expression of Mbl2 was significantly elevated after Giardia infection in C57BL/6 mice, but not in TKO mice (Supplementary Fig. S10). In the following phase, the aim was to investigate whether the minor amount of IL-17A mRNA upregulation observed in TKO mice subsequently translates into IL-17A production and which immune cells are responsible for this production. Therefore, IL-17A production by LP cells of TKO mice was assessed using flow cytometry. As IL-17A mRNA production only started at day 21 p.i. in TKO mice, flow cytometry experiments were conducted on day 21 and at an additional later time point, i.e. 2 months p.i.. Cyst and trophozoite counts remained persistently high until the end of the observation period (Fig. 5a,b). In the CD45 + population of TKO mice, no IL-17A upregulation could be detected at either time point after infection (Fig. 5c). Within the CD45 + population, no IL-17A production was observed by ILCs, ILC3s, NK cells, B cells, DCs or neutrophils (Supplementary Fig. S11). An upwards trend was visible in the MHC-II + cell population, although not statistically significant. The only cell type that was found to significantly produce IL-17A in TKO mice following a Giardia infection were MHC-II + CD11c + CD11b - cells, consistent with the previous observation in C57BL/6 mice (Fig. 5c). Subsets of innate MHC-II+ cells produce IL-17A following a G. muris infection In a final experiment, we aimed to further characterize the MHC-II + cells that produced IL-17A after Giardia infection through more detailed phenotypic analysis. Since MHC-II + IL-17A-producing cells were identified at day 7 p.i. in C57BL/6 mice, this time point was revisited using a panel targeting distinct MHC-II + antigen-presenting cell subsets. The gating strategy for this panel can be found in Supplementary Fig. S12. As a first step, IL-17A upregulation in CD3 + T cells, CD3 - non-T cells and the MHC-II + population was confirmed (Supplementary Fig. S13). Secondly, different MHC-II + subpopulations were analysed. Elevated IL-17A levels were detected in the MHC-II + CD11c + CD11b - population, as seen previously, and additionally in the MHC-II + CD11c + CD11b + subset. These MHCII + CD11c + populations might comprise of conventional DCs (cDC), monocyte-derived cells and tissue macrophages. To distinguish between these populations, the markers CD26 (lineage marker for cDCs) and CD64 (marker for monocyte-derived cells and macrophages) were used. We could not detect an increase in IL-17A production in the MHCII + CD11c + CD26 + population, regardless of the expression of CD11b. In contrast, when looking at macrophage associated markers, an increase in IL-17A production was found in the MHC-II + CD11c + CD11b + CD64 + F4/80 + population (Fig. 6a, b). Further analysis revealed these cells to be negative for Ly6C expression (Supplementary Fig. S14). Discussion Interleukin-17A is a pro-inflammatory cytokine primarily associated with mucosal defense against extracellular pathogens. IL-17A promotes the recruitment of neutrophils, strengthens epithelial barrier integrity and induces the expression of antimicrobial peptides. Its function is crucial in initiating effective immune responses against pathogens, while maintaining homeostasis at mucosal surfaces 12,24,25 . The involvement of IL-17A in the immune response against Giardia infection was first highlighted by Dreesen et al. 5 , who showed that mice deficient in the IL-17A receptor exhibit increased parasite burdens and impaired clearance of infection. IL-17A contributes to host protection by inducing Mbl2 expression and stimulating the production of Giardia -specific IgA antibodies 6,7 . These observations establish IL-17A as a central molecule in the host defense against Giardia . Despite its recognized importance, the cellular pathways leading to anti-giardial IL-17A production, specifically its cellular sources and their exact location, remain poorly defined. Potential sites of anti-giardial IL-17A production include secondary lymphoid organs, such as the MLN and the PP. In the present study, IL-17A mRNA expression levels were measured in the PP and MLN of G. muris infected mice. A slight elevation in IL-17A production could be detected at day 7 after infection in the PP. While not significantly increased, this trend suggests a potential involvement of the PP in early immune responses against Giardia . This is consistent with prior observations that have pointed to immune activation in the PP during infection, including cytokine responses and cellular proliferation 9,10 . Further research focused specifically on the PP could help clarify their role during Giardia infection, particularly in the early stages of infection when antigen-presentation and priming of naive T cells takes place. While increased IL-17A production has been reported in MLN cells of G. duodenalis infected mice, this observation was made following ex vivo stimulation of these cells with parasite extract, a method that may not accurately reflect in vivo conditions 11 . In our study, we observed no increase in IL-17A mRNA expression in the MLN after G. muris infection, in line with previous observations by Dann et al. 6 . Taken together, our results from the PP and MLN highlight the local nature of the anti-giardial immune response, with IL-17A production confined to intestinal tissues in close proximity to the site of infection. IL-17A production in small intestinal tissue in response to Giardia infection has been widely documented 5-7,26 . In agreement with these findings, we observed significantly increased IL-17A mRNA expression at days 7 and 14 p.i.. Our study further expands these data by examining the IL-17A response in the IEL and the LP fraction of the SI. This approach provides further insight into both the location of the anti-giardial IL-17A response and the cells that are responsible for its production. Th17 cells are known as the classical producers of IL-17A. Consistently, we found CD4 + T cells residing in the LP to be the major source of IL-17A production in the case of a Giardia infection. These results are in line with Dann et al. 6 , who reported a significant increase in IL-17A production by CD45 + CD4 + cells in the LP of G. muris infected C57BL/6 mice. They also detected IL-17A production by CD45 + cells in the IEL, while this was not observed in the current study. As Th17 cells are less common in the IEL, they speculated that this production may originate from innate immune cell types. To investigate this hypothesis, they infected CD4 -/- and Rag2 -/- mice with G. muris , which respectively lack Th cells and all T and B cells. Interestingly, they still observed a significant IL-17A mRNA increase following infection in these mutant mice. Our flow cytometry experiments in C57BL/6 mice also revealed a population of IL-17A-producing cells that were both TCRβ - and TCRɣδ - , raising the possibility of an innate IL-17A source. Therefore, we aimed to elucidate the contribution of innate immune cells to anti-giardial IL-17A production by comparing the IL-17A response between TKO and C57BL/6 mice. This comparison revealed that even though there is still a significant IL-17A upregulation after infection within the TKO mice, the magnitude of this response is markedly reduced relative to wild-type mice. To further investigate the level of IL-17A protein production in C57BL/6 and TKO mice and simultaneously identify their cellular sources, a broad panel of immune cell types capable of producing IL-17A was explored. Using flow cytometry, IL-17A production by Tc cells, ɣδT cells, B cells, NK cells, NKT cells, neutrophils, DCs and ILCs was evaluated. Specifically in the case of Giardia infection, ILC3s have been speculated to be potential producers of IL-17A, as they are seen as the innate counterpart of Th17 cells 6,18-20 . Furthermore, ILC3s are known to be important in the defense against extracellular pathogens and they are responsive to IL-1β and IL-23, which are both upregulated during Giardia infection 5,27,28 . In the present study, no infection-related IL-17A induction could be detected in any of the investigated cell types. However, when examining specific subsets of MHC-II + cells, an IL-17A increase could be observed in both C57BL/6 and TKO mice. This result aligns with findings by Lee et al. 18 , who reported an IL-17A increase in LP MHC-II + cells from C57BL/6 mice upon stimulation with G. duodenalis trophozoites. These cells were addressed as ILC3s. However, their identification relied solely on MHC-II expression, which is a marker shared by various antigen-presenting cells such as dendritic cells, macrophages, and B cells. Without a broader panel of innate markers or clear lineage exclusion strategies, identifying these cells as ILC3s remains speculative. Our results also corroborate those of Yordanova et al. 20 , who found no evidence of IL-17A production in ILC3s of G. muris infected mice. Given the MHC-II + CD11c + CD11b +/- phenotype of the IL-17A producing cells we observed, they most likely represent a certain subset of DCs or macrophages 29 . Further phenotypic analysis revealed these cells to be CD26 - , which rules out cDCs as a likely source 30 . Alternatively, these cells could represent an intermediate migratory stage of DCs or plasmacytoid DCs (pDCs) 31,32 . pDCs are typically known for shaping the innate and adaptive immunity in the gastrointestinal tract and participate in immunological responses through the presentation of antigens 33 . The IL-17A producing subset of MHC-II + CD11c + CD11b + CD64 + F4/80 + cells we observed matches the classical phenotype of tissue-resident macrophages 29,34 . In the context of Giardia infection, while research on the role of macrophages in protective immunity is limited, studies have demonstrated their ability to phagocytose trophozoites in both mice and humans 35-39 . Notably, two reports from the same research group have identified an accumulation of macrophages in the lamina propria of G. duodenalis infected mice 40,41 . Further characterization revealed these cells to be F4/80 + , CD11b + , CD11c int , CX3CR1 + , MHCII + , Ly6C - , very similar to the population detected in the current study and also consistent with the tissue-resident macrophage phenotype. Interestingly, when macrophages were depleted using an anti-CSF-1R antibody, this did not impair the clearance of Giardia or affect Th cell numbers in the LP, suggesting that macrophages are dispensable for protective immunity. However, they speculated that during Giardia infection, macrophages may play a role in maintaining mucosal homeostasis and epithelial barrier function through the clearance of apoptotic cells. Additionally, while macrophages did not seem to be important for the differentiation of naïve T cells into Th cells, they speculated that these cells might support effector T cell proliferation through MHC-II-dependent antigen presentation. Given this context, the detection of IL-17A production by these macrophages could indicate a previously unrecognized protective role in anti- Giardia immunity. Alternatively, our observed phenotype might also correspond to monocyte-derived DCs (moDCs), which share overlapping surface markers with tissue-resident macrophages 42 . Overall, MHC-II + innate cell types are not typically known as IL-17A producing cells. However, a recent study identified elevated IL-17A expression levels in moDCs in humans with asthma and COPD 17 . In the context of Giardia infection, the functional relevance of these innate IL-17A producing cells remains unclear. While their contribution is minor, it is possible that they play a niche role in early parasite sensing or facilitating crosstalk between innate and adaptive immunity. Conclusion This study provides critical insights into the cellular sources of IL-17A during Giardia infection, highlighting the predominant role of Th17 cells in mounting a protective IL-17A response. Our results demonstrate that IL-17A production is largely restricted to Th17 cells within the LP of the SI, with a minimal contribution from innate immune cells. Moreover, the inability of TKO mice to elicit an effective IL-17A response and clear the infection underscores the critical role of the adaptive immune response in controlling Giardia infection. Further research into the upstream signals that drive Th17 cell differentiation will be essential to gain a deeper understanding on the mechanisms underlying the protective immune response against Giardia . Declarations Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Acknowledgements This study was fnancially supported by an FWO PhD-fellowship attributed to C.V.C.. Author contributions C.V.C., L.S. and P.G. conceived and designed the studies. C.V.C. performed the experiments with contributions of B.D. and L.S.. Data analysis was performed by C.V.C. and L.S.. The manuscript was written by C.V.C., L.S. and P.G.. All authors read and approved the final manuscript. Additional information Competing interests . The authors declare no competing interests. Supplementary material . The online version contains supplementary material. References Lane, S. & Lloyd, D. Current Trends in Research into the Waterborne Parasite Giardia . Crit. Rev. Immunol. 28 , 123-147 (2002). Zhou, P. et al. Role of Interleukin-6 in the Control of Acute and Chronic Giardia lamblia Infections in Mice. Infect. Immun. 71 , 1566-1568 (2003). Adam, R. D. The biology of Giardia spp. Microbiol. Rev. 55 , 706-732 (1991). Halliez, M. C. & Buret, A. G. Extra-intestinal and long term consequences of Giardia duodenalis infections. World J. Gastroenterol. 19 , 8974-8985 (2013). Dreesen, L. et al. Giardia muris infection in mice is associated with a protective interleukin 17A response and induction of peroxisome proliferator-activated receptor alpha. Infect. Immun. 82 , 3333-3340 (2014). Dann, S. M. et al. IL-17A promotes protective IgA responses and expression of other potential effectors against the lumen-dwelling enteric parasite Giardia . Exp. Parasitol. 156 , 68-78 (2015). Paerewijck, O. et al. Interleukin-17 receptor A (IL-17RA) as a central regulator of the protective immune response against Giardia . Sci. Rep. 7 , 8520 (2017). Brandtzaeg, P., Kiyono, H., Pabst, R. & Russell, M. W. Terminology: nomenclature of mucosa-associated lymphoid tissue. Mucosal Immunol. 1 , 31-37 (2008). Hill, D. R. Lymphocyte proliferation in Peyer's patches of Giardia muris -infected mice. Infect. Immun. 58 , 2683-2685 (1990). Djamiatun, K. & Faubert, G. M. Exogenous cytokines released by spleen and Peyer's patch cells removed from mice infected with Giardia muris . Parasite immunol. 20 , 1365-3024 (1998). Solaymani-Mohammadi, S. & Singer, S. M. Giardia duodenalis : The double-edged sword of immune responses in giardiasis. Exp. Parasitol. 126 , 292-297 (2010). Cua, D. J. & Tato, C. M. Innate IL-17-producing cells: the sentinels of the immune system. Nat. Rev. Immunol. 10 , 479-489 (2010). Haas, J. D. et al. CCR6 and NK1.1 distinguish between IL-17A and IFN-gamma-producing gammadelta effector T cells. Eur. J. Immunol. 39 , 3488-3497 (2009). Passos, S. T. et al. IL-6 promotes NK cell production of IL-17 during toxoplasmosis. J. Immunol. 184 , 1776-1783 (2010). Li, L. et al. IL-17 produced by neutrophils regulates IFN-gamma-mediated neutrophil migration in mouse kidney ischemia-reperfusion injury. J. Clin. Invest. 120 , 331-342 (2010). Coury, F. et al. Langerhans cell histiocytosis reveals a new IL-17A-dependent pathway of dendritic cell fusion. Nat. Med. 14 , 81-87 (2008). Paplinska-Goryca, M. et al. The Expressions of TSLP, IL-33, and IL-17A in Monocyte Derived Dendritic Cells from Asthma and COPD Patients are Related to Epithelial–Macrophage Interactions. Cells 9 , 1944 (2020). Lee, H.-Y., Park, E.-A., Lee, K.-J., Lee, K.-H. & Park, S.-J. Increased Innate Lymphoid Cell 3 and IL-17 Production in Mouse Lamina Propria Stimulated with Giardia lamblia . Korean J. Parasitol. 57 , 225-232 (2019). Yordanova, I. A. et al. RORγt+ Treg to Th17 ratios correlate with susceptibility to Giardia infection. Sci. Rep. 9 , 20328 (2019). Yordanova, I. A., Lamatsch, M., Kühl, A. A., Hartmann, S. & Rausch, S. Eosinophils are dispensable for the regulation of IgA and Th17 responses in Giardia muris infection. Parasite Immunol. 43 (2021). Paerewijck, O., Maertens, B., Gagnaire, A., De Bosscher, K. & Geldhof, P. Delayed development of the protective IL-17A response following a Giardia muris infection in neonatal mice. Sci. Rep. 9 , 8959 (2019). Lefrançois, L. & Lycke, N. Isolation of mouse small intestinal intraepithelial lymphocytes, Peyer's patch, and lamina propria cells. Curr. Prot. Immunol. 17 , 3.19.11-13.19.16 (2001). Heyworth, M. F., Carlson, J. R. & Ermak, T. H. Clearance of Giardia muris infection requires helper/inducer T lymphocytes. J. Exp. Med. 165 , 1743-1748 (1987). Ouyang, W., Kolls, J. K. & Zheng, Y. The Biological Functions of T Helper 17 Cell Effector Cytokines in Inflammation. Immun. 28 , 454-467 (2008). Ishigame, H. et al. Differential Roles of Interleukin-17A and -17F in Host Defense against Mucoepithelial Bacterial Infection and Allergic Responses. Immun. 30 , 108-119 (2009). Maertens, B., Gagnaire, A., Paerewijck, O., De Bosscher, K. & Geldhof, P. Regulatory role of the intestinal microbiota in the immune response against Giardia . Sci. Rep. 11 , 10601 (2021). Zeng, B. et al. ILC3 function as a double-edged sword in inflammatory bowel diseases. Cell Death Dis. 10 , 315 (2019). Obendorf, J. et al. Increased expression of CD25, CD83, and CD86, and secretion of IL-12, IL-23, and IL-10 by human dendritic cells incubated in the presence of Toll-like receptor 2 ligands and Giardia duodenalis . Parasit. Vectors 6 , 317 (2013). Cerovic, V., Bain, C. C., Mowat, A. M. & Milling, S. W. Intestinal macrophages and dendritic cells: what's the difference? Trends Immunol. 35 , 270-277 (2014). Guilliams, M. et al. Unsupervised High-Dimensional Analysis Aligns Dendritic Cells across Tissues and Species. Immun. 45 , 669-684 (2016). Naik, S. H. et al. Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo. Nat. Immunol. 8 , 1217-1226 (2007). Swiecki, M., Colonna, M., Swiecki, M. & Colonna, M. The multifaceted biology of plasmacytoid dendritic cells. Nat. Rev. Immunol. 15 , 471-485 (2015). Arimura, K. et al. Crucial role of plasmacytoid dendritic cells in the development of acute colitis through the regulation of intestinal inflammation. Mucosal Immunol. 10 , 957-970 (2016). Bain, C. C. & Mowat, A. M. The monocyte-macrophage axis in the intestine. Cell. Immunol. 291 , 41-48 (2014). Li, L. et al. Mouse macrophages capture and kill Giardia lamblia by means of releasing extracellular trap. DCI 88 , 205-212 (2018). Hill, D. R. & Pearson, R. D. Ingestion of Giardia lamblia trophozoites by human mononuclear phagocytes. Infect. Immun. 55 , 3155-3161 (1987 Dec). Hill, D. R. & Pohl, R. Ingestion of Giardia lamblia trophozoites by murine Peyer's patch macrophages. Infect. Immun. 58 , 3202-3207 (1990). Belosevic, M. & Daniels, C. W. Phagocytosis of Giardia lamblia trophozoites by cytokine-activated macrophages. Clin. Exp. Immunol. 87 , 304-309 (1992). Owen, R. L., Allen, C. L. & Stevens, D. P. Phagocytosis of Giardia muris by macrophages in Peyer's patch epithelium in mice. Infect. Immun. 33 , 591-601 (1981). Fink, M. Y. et al. Proliferation of resident macrophages is dispensable for protection during Giardia duodenalis infections. ImmunoHorizons 3 , 412-421 (2019). Maloney, J., Keselman, A., Li, E. & Singer, S. M. Macrophages expressing arginase 1 and nitric oxide synthase 2 accumulate in the small intestine during Giardia lamblia infection. Microbes Infect. 17 , 462-467 (2015). Guilliams, M. et al. Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. Nat. Rev. Immunol. 14 , 571-578 (2014). Tables Table 1. Summary of G. muris infection studies. All experiments were performed with 6-week old mice. Individual fecal cyst counts were monitored every 2-3 days and intestinal trophozoite counts were performed at the time of euthanasia for each animal. Abbreviations: SI, small intestine; PP, Peyer’s patches; MLN, mesenteric lymph nodes; IEL, intra-epithelial lymphocytes; LP, lamina propria; TKO, T cell knock-out. Study Objective Mouse Strain Number of animals Sampling time Samples Data analyzed 1 Intestinal location: SI/PP/MLN C57BL/6 5 controls + 15 infected Day 7, 14, 21 (n=5) SI, PP and MLN IL-17A gene expression (RT-qPCR) 2 Intestinal location: IEL/LP C57BL/6 4 controls + 4 infected Day 14 (n=4) IEL and LP cells IL-17A producing cells (flow cytometry) 3 IL-17A producing cells in the LP C57BL/6 12 controls + 12 infected Day 4, 7, 14 (n=4) LP cells IL-17A producing cells (flow cytometry) 4 Involvement of T cells in IL-17A production C57BL/6 and TKO For each strain: 4 controls + 12 infected Day 7, 14, 21 (n=4) SI, PP and MLN IL-17A gene expression (RT-qPCR) 5 IL-17A producing non-T cells in the LP TKO 8 controls + 8 infected Day 21, 2 months (n=4) LP cells IL-17A producing cells (flow cytometry) 6 IL-17A producing non-T cells in the LP C57BL/6 4 controls + 4 infected Day 7 (n=4) LP cells IL-17A producing cells (flow cytometry) Additional Declarations No competing interests reported. Supplementary Files SupplementaryFileVanCrombruggeUnravellingthecellularsourcesandlocationofIL17AproductionduringaGiardiainfection.pdf Cite Share Download PDF Status: Published Journal Publication published 05 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 07 Jul, 2025 Reviews received at journal 06 Jul, 2025 Reviews received at journal 23 Jun, 2025 Reviewers agreed at journal 17 Jun, 2025 Reviewers agreed at journal 17 Jun, 2025 Reviewers invited by journal 17 Jun, 2025 Editor assigned by journal 17 Jun, 2025 Editor invited by journal 11 Jun, 2025 Submission checks completed at journal 10 Jun, 2025 First submitted to journal 06 Jun, 2025 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6836025","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":473037504,"identity":"2721e09c-ba24-454f-978e-3e129439dc3b","order_by":0,"name":"Charlotte E. 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(\u003cstrong\u003ea\u003c/strong\u003e) Fecal cyst counts monitored every 2-3 days until day 21 p.i.. Each point on the graph represents the mean cyst output per gram of feces, with SEM as error bars (n=15 for day 4-6; n=10 for day 8-13; n=5 for day 15-21). (\u003cstrong\u003eb\u003c/strong\u003e) Small intestinal trophozoite counts at day 7, 14 and 21 p.i. (n=5). (\u003cstrong\u003ec\u003c/strong\u003e) Relative IL-17A mRNA expression levels in the SI, PP and MLN of C57BL/6 mice at day 7, 14 and 21 p.i. as measured by RT-qPCR (n=5). Differences were analyzed using a Kruskal-Wallis test (*P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6836025/v1/3ea7334a2f66c6b98bd654a1.png"},{"id":85194540,"identity":"a70fdf36-33e9-4221-b829-3810a394d507","added_by":"auto","created_at":"2025-06-23 09:13:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":60003,"visible":true,"origin":"","legend":"\u003cp\u003eIL-17A production by different cell types in the LP compartment of \u003cem\u003eG. muris\u003c/em\u003e infected C57BL/6 mice at day 14 p.i. as measured by flow cytometry, expressed as %IL-17A+ cells of parent population. Differences were analyzed using a Mann-Whitney test (*P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6836025/v1/76c189b1085c95abbda79b4e.png"},{"id":85194542,"identity":"fdd7f17e-7602-43be-96b6-37aa4ee5bdd7","added_by":"auto","created_at":"2025-06-23 09:13:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1661137,"visible":true,"origin":"","legend":"\u003cp\u003eIL-17A production by different cell types in in the LP compartment of \u003cem\u003eG. muris\u003c/em\u003e infected C57BL/6 mice at day 4, 7 and 14 p.i. as measured by flow cytometry, expressed as %IL-17A+ cells of parent population. Differences were analyzed using a Mann-Whitney test (*P \u0026lt; 0.05, **P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6836025/v1/50fa602b318222571ee6ac98.png"},{"id":85194543,"identity":"3bea6236-4329-4a92-a87f-34be3b229706","added_by":"auto","created_at":"2025-06-23 09:13:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":434895,"visible":true,"origin":"","legend":"\u003cp\u003eMonitoring IL-17A expression in three different intestinal compartments of \u003cem\u003eG. muris\u003c/em\u003e infected C57BL/6 and TKO mice. (\u003cstrong\u003ea\u003c/strong\u003e) Fecal cyst counts monitored every 2-3 days until day 21 p.i.. for C57BL/6 and TKO mice. Each point on the graph represents the mean cyst output per gram of feces, with SEM as error bars (n=12 for day 5-7; n=8 for day 10-14; n=4 for day 17-21). (\u003cstrong\u003eb\u003c/strong\u003e) Small intestinal trophozoite counts at day 21 p.i. for C57BL/6 and TKO mice (n=4). (\u003cstrong\u003ec\u003c/strong\u003e) Relative IL-17A mRNA expression levels in the SI, PP and MLN of C57BL/6 and TKO mice at day 7, 14 and 21 p.i. as measured by RT-qPCR (n=4). Differences were analyzed using a Kruskal-Wallis test (*P \u0026lt; 0.05, **P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6836025/v1/f4d96375638a7621cd4c1586.png"},{"id":85194546,"identity":"17ec2b8c-5883-4ce5-b357-8f3285ff610e","added_by":"auto","created_at":"2025-06-23 09:13:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1621068,"visible":true,"origin":"","legend":"\u003cp\u003eIL-17A production in the LP of \u003cem\u003eG. muris\u003c/em\u003einfected TKO mice. (\u003cstrong\u003ea\u003c/strong\u003e) Fecal cyst counts monitored every 2-3 days until day 21 p.i. and until 2 months p.i.. Each point on the graph represents the mean cyst output per gram of feces, with SEM as error bars (n=4). (\u003cstrong\u003eb\u003c/strong\u003e) Small intestinal trophozoite counts at day 21 and 2 months p.i. (n=4). (\u003cstrong\u003ec\u003c/strong\u003e) IL-17A production by different cell types in in the LP compartment of \u003cem\u003eG. muris\u003c/em\u003e infected TKO mice at day 21 and 2 months p.i. as measured by flow cytometry, expressed as %IL-17A+ cells of parent population. Differences were analyzed using a Kruskal-Wallis test (*P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6836025/v1/61252ddd4282b80d3b5f2615.png"},{"id":85194551,"identity":"959041c0-363e-4f7d-851e-9716e3fda8a4","added_by":"auto","created_at":"2025-06-23 09:13:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":42571,"visible":true,"origin":"","legend":"\u003cp\u003eIL-17A production by different MHC-II+CD11c+CD11b- cell subsets (\u003cstrong\u003ea\u003c/strong\u003e) and MHC-II+CD11c+CD11b+ cell subsets (\u003cstrong\u003eb\u003c/strong\u003e) in the LP compartment of \u003cem\u003eG. muris\u003c/em\u003e infected C57BL/6 mice at day 7 p.i. as measured by flow cytometry, expressed as %IL-17A+ cells of parent population. Differences were analyzed using a Mann-Whitney test (*P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6836025/v1/5b20d531a23b005b2b38fdc8.png"},{"id":95564170,"identity":"d71a87bb-40f6-4684-8ef3-dd9bf62528ca","added_by":"auto","created_at":"2025-11-10 16:08:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3855958,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6836025/v1/86d1b684-fc6d-48da-84e5-4e8c59668ab4.pdf"},{"id":85194545,"identity":"cee73fbb-c398-411b-8edc-c96b6e0e6d9a","added_by":"auto","created_at":"2025-06-23 09:13:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2452746,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileVanCrombruggeUnravellingthecellularsourcesandlocationofIL17AproductionduringaGiardiainfection.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6836025/v1/ac93258c701fafce407c5339.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eUnravelling the cellular sources and location of IL-17A production during a \u003cem\u003eGiardia\u003c/em\u003e infection\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eGiardia\u003c/em\u003e \u003cem\u003eduodenalis\u003c/em\u003e, also referred to as \u003cem\u003eGiardia\u003c/em\u003e \u003cem\u003elamblia\u003c/em\u003e or \u003cem\u003eGiardia\u003c/em\u003e \u003cem\u003eintestinalis\u003c/em\u003e, is a flagellated unicellular protozoan parasite with a broad host tropism, infecting both animals and humans. \u003cem\u003eG. duodenalis\u003c/em\u003e has a high global incidence, with over 200 million human cases reported annually\u003csup\u003e1\u003c/sup\u003e. \u003cem\u003eGiardia\u003c/em\u003e infection can lead to a spectrum of clinical signs that extends from acute to chronic\u003csup\u003e2\u003c/sup\u003e. Acute giardiasis generally lasts one to three weeks and symptoms disappear quickly. If a chronic situation develops, infection can last up to several months. Symptom severity ranges from subclinical to severe malabsorption, diarrhea, abdominal pain and weight loss\u003csup\u003e3\u003c/sup\u003e. These variations are due to different factors, such as the state of the immune system, age and diet of the host, the genotype of the \u003cem\u003eGiardia\u003c/em\u003e species and complicating co-infections\u003csup\u003e4\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn recent years, interleukin 17A (IL-17A) has gained recognition as a central cytokine in the immune response against \u003cem\u003eGiardia\u003c/em\u003e infection. Dreesen et al.\u003csup\u003e5\u003c/sup\u003e reported a significant increase in IL-17A mRNA expression in the small intestine after \u003cem\u003eG. muris\u003c/em\u003e infection in C57BL/6 mice. Additionally, the use of IL-17A receptor (IL-17RA) knock-out mice underscored the importance of this cytokine during \u003cem\u003eGiardia\u003c/em\u003e infections, as mice with a defect in their IL-17A/IL-17RA axis were unable to clear a \u003cem\u003eGiardia\u003c/em\u003e infection\u003csup\u003e5,6\u003c/sup\u003e. Subsequent research has predominantly focused on the downstream effector mechanisms of IL-17A, elucidating the mechanisms that lead to clearance of the parasite. Specifically, IL-17A activates the complement system by stimulating the production of mannose-binding lectin 2 (Mbl2) and triggers the secretion of \u003cem\u003eGiardia\u003c/em\u003e-specific IgA by B cells\u003csup\u003e7\u003c/sup\u003e. Less research has focused on the mechanisms leading to the production of IL-17A.\u003c/p\u003e\n\u003cp\u003eIL-17A production can occur across various immune sites in the body. The immune architecture of the intestine consists of inductive sites, where adaptive immune cells are primed; and effector sites, where these activated immune cells preserve barrier function and regulate protective immunity. Intestinal inductive sites encompass the mesenteric lymph nodes (MLNs) and gut-associated lymphoid tissues, such as Peyer’s patches (PP), whereas intestinal effector sites include the lamina propria (LP) and the epithelial layer of the small intestine (SI), housing intra-epithelial lymphocytes (IEL)\u003csup\u003e8\u003c/sup\u003e. The PP have already been investigated as an important site of immune activity during \u003cem\u003eGiardia\u003c/em\u003e infection. In the 1990s, Hill\u003csup\u003e9\u003c/sup\u003e observed a blastogenic response in PP leukocytes of \u003cem\u003eG.\u003c/em\u003e \u003cem\u003emuris\u003c/em\u003e infected mice, correlating with the time of peak-infection. Additionally, Djamatun and Faubert\u003csup\u003e10\u003c/sup\u003e observed increased \u003cem\u003ein vitro\u003c/em\u003e IL-4 and IL-5 cytokine production by PP cells isolated from \u003cem\u003eG.\u003c/em\u003e \u003cem\u003emuris\u003c/em\u003e infected mice. Notably, these studies predate the identification of IL-17A as a central player during anti-giardial immunity. Varying reports have been made on the MLN during \u003cem\u003eGiardia\u003c/em\u003e infection. A previous study observed a slight increase in IL-17A production by extracted MLN cells of \u003cem\u003eG. duodenalis\u003c/em\u003e infected mice after \u003cem\u003eex vivo\u003c/em\u003e stimulation with \u003cem\u003eGiardia\u003c/em\u003e extracts\u003csup\u003e11\u003c/sup\u003e. In contrast, Dann et al.\u003csup\u003e6\u003c/sup\u003e reported no increase in IL-17A mRNA production in the MLN of \u003cem\u003eG. muris\u003c/em\u003e infected mice.\u003c/p\u003e\n\u003cp\u003eGenerally, T helper 17 (Th17) cells are known to be the classical IL-17A producers, although several innate immune cells have also been reported to produce IL-17A under certain conditions. These innate IL-17A cell sources include ɣδT cells, innate lymphoid cells type 3 (ILC3), natural killer (NK) cells, NKT cells, Paneth cells, neutrophils and dendritic cells (DCs)\u003csup\u003e12-17\u003c/sup\u003e. In the context of a \u003cem\u003eGiardia\u003c/em\u003e infection, the exact cellular sources of IL-17A remain unclear. Dann et al.\u003csup\u003e6\u003c/sup\u003e reported increased IL-17A production in CD4\u003csup\u003e+\u003c/sup\u003e T cells, most likely Th17 cells, isolated from the SI of \u003cem\u003eG.\u003c/em\u003e \u003cem\u003emuris\u003c/em\u003e infected mice. Interestingly, CD4 deficient mice, which lack all Th cell subsets; and recombination activating gene 2 (Rag2) deficient mice, which lack both B and T cells, still exhibited enhanced expression of IL-17A mRNA in small intestinal tissue following a \u003cem\u003eG. muris\u0026nbsp;\u003c/em\u003einfection\u003csup\u003e6\u003c/sup\u003e. This suggests that innate immune cells could be potential additional sources of IL-17A in the defense against a \u003cem\u003eGiardia\u003c/em\u003e infection. As innate counterparts of Th17 cells, ILC3s have been speculated to be potential producers of anti-giardial IL-17A\u003csup\u003e6,18-20\u003c/sup\u003e. Supporting this hypothesis,\u0026nbsp;Lee et al.\u003csup\u003e18\u003c/sup\u003e reported increased IL-17A production in ILC3s isolated from the intestine of C57BL/6 mice after stimulation with \u003cem\u003eG. duodenalis\u003c/em\u003e trophozoites. In contrast, Yordanova et al.\u003csup\u003e20\u003c/sup\u003e\u0026nbsp; reported no IL-17A production in ILC3s of \u003cem\u003eG.\u003c/em\u003e \u003cem\u003emuris\u003c/em\u003e infected mice. At this time, IL-17A production by innate cell sources following \u003cem\u003eGiardia\u003c/em\u003e infection remains to be clarified.\u003c/p\u003e\n\u003cp\u003eThe present study investigates IL-17A production in response to \u003cem\u003eG.\u003c/em\u003e \u003cem\u003emuris\u003c/em\u003e infection across the different inductive and effector sites in the intestine. It further aims to clarify the cellular origins of anti-giardial IL-17A and simultaneously determine the relative contribution of Th17 cells compared to innate cell sources regarding IL-17A expression.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the European Union (E.U.) Animal Welfare Directives, the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) and the ARRIVE guidelines for reporting animal research. Guidelines for Good Clinical Practice and ethical approval to conduct the studies were obtained from the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University (ethical committee approval numbers EC2023-085 and EC2024-033).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMurine infection studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix independent infection studies were performed in either C57BL/6J mice or B6.129P2-Tcrb\u003cem\u003e\u003csup\u003etm1Mom\u003c/sup\u003e Tcrd\u003csup\u003etm1Mom\u003c/sup\u003e\u003c/em\u003e/J mice (T cell knock-out (TKO) mice), obtained from Jackson Laboratories. An overview of all infection studies is provided in \u003cstrong\u003eTable 1\u003c/strong\u003e. In each experiment, 6-week old mice were either orally infected with 10\u003csup\u003e3\u003c/sup\u003e \u003cem\u003eG.\u003c/em\u003e \u003cem\u003emuris\u003c/em\u003e cysts suspended in 0.1 mL phosphate-buffered saline (PBS) (n = 4-5) or used as uninfected control mice (n = 4-5). Individual fecal cyst counts were performed every 2-3 days as previously described\u003csup\u003e5\u003c/sup\u003e. The presence of trophozoites in the SI was quantified for each animal at the time of euthanasia according to Paerewijck et al.\u003csup\u003e7\u003c/sup\u003e. All mice were euthanized by cervical dislocation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue samples of SI, PP and MLN of C57BL/6 and TKO mice were collected for real-time quantitative polymerase chain reaction (RT-qPCR) analysis. A 1 cm long segment of small intestinal tissue was collected 4 cm distal of the gastroduodenal junction. All PP along the small intestinal tract were pooled together and the MLN were dissected from the mesentery. To allow further RNA extraction, all samples were immediately snap-frozen in liquid nitrogen and stored at -80°C until further processing.\u003c/p\u003e\n\u003cp\u003eRNA isolation and RT-qPCR analysis were performed according to Dreesen et al.\u003csup\u003e5\u003c/sup\u003e. In short, RNA isolation was performed using the RNeasy minikit (Qiagen). An Agilent 2100 Bioanalyzer (Agilent Technologies) was used to verify the quality of the isolated RNA and total RNA concentration was determined using a Nano-Drop ND-1000 spectrophotometer (NanoDrop Technologies). The iScript cDNA synthesis kit (Bio-Rad) was used to acquire cDNA for further RT-qPCR analysis. Gene expression of IL-17A and Mbl2 was measured using primer sequences obtained from Paerewijck et al.\u003csup\u003e21\u003c/sup\u003e and all analyses were run on a StepOnePlus real-time PCR system (Applied Biosciences). The data were analyzed using the ΔC\u003csub\u003eT\u003c/sub\u003e method and normalized based on the housekeeping genes Hprt1 and Tbp.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of intestinal cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle cell suspensions of IEL and LP cells from the SI of C57BL/6 and TKO mice were prepared following a protocol adapted from Lefrançois and Lycke\u003csup\u003e22\u003c/sup\u003e. After carefully excising the SI, the IEL fraction was isolated by incubating the intestine at 37°C for 20 minutes in RPMI medium containing 5% FCS, 2mM EDTA and 1mM DTT. The IEL cell suspension was filtered through a 40µM cell strainer and kept on ice until further processing. The remaining small intestinal tissue was incubated for 15 minutes at 37°C in a digestion buffer containing Dispase I (50U/mL), DNAse I (0,05mg/mL) and collagenase VIII (0,6mg/mL) in RPMI medium. The LP suspension was then filtered through a 100µM cell strainer. Both IEL and LP fractions were centrifuged at 400 g. IEL cell pellets were resuspended in 44% Percoll, overlaid on 67% Percoll and centrifuged at 1800 rpm for 20 minutes. Similarly, LP pellets were resuspended in 40% Percoll and overlaid on 80% Percoll before centrifugation. Buffy coats containing lymphocytes were collected and washed in PBS before antibody staining for flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells from the IEL fraction of C57BL/6 mice in study 2 were stained with an antibody panel targeting T cell subsets (panel 1). After a blocking step with Fc-Block, the cells were stained for 30 minutes at 4°C with CD45.2-PerCP-Cy5.5 (BD) (clone 104), TCRβ-BV421 (Biolegend) (clone H57-597), TCRɣδ-PE-Cy7 (Biolegend) (clone GL3), CD4-AF700 (eBioscience) (clone GK1.5) and CD8-APC-Cy7 (BD) (clone 53-6.7). eF506 (eBioscience) was used as a live/dead marker to exclude non-viable cells. Next, the cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD) to allow for intracellular staining with IL-17A-AF647 (BD) (clone TC11-18H10). Fluorescence-minus-one (FMO) controls were included for the IL-17A staining.\u003c/p\u003e\n\u003cp\u003eFor the LP cells of C57BL/6 mice in study 2, the same T cell panel was used. In a follow-up infection study (study 3), two additional panels were incorporated alongside panel 1 in order to investigate a broader range of immune cell types within the LP compartment. The first additional panel (panel 2) was directed against ILCs, specifically ILC3s, and consisted of the following antibodies: CD45.2-AF700 (Biolegend) (clone 30-F11), CD90.2-PerCP-Cy5.5 (Biolegend) (clone 30-H12), CD127-FITC (Biolegend) (clone SB/199), CD117-APC-Cy7 (Invitrogen) (clone 2BB) and a negative lineage consisting of CD3-BV421 (BD) (145-2C11), CD5-BV421 (Biolegend) (clone 53-7.3), TCRβ-BV421 (Biolegend) (clone H57-597), TCRɣδ-BV421 (Biolegend) (GL3), CD19-BV421 (BD) (clone 1D3), F4/80 (BD) (clone T45-2342), Ly6C/G-BV421 (BD) (clone RB6-8C5) and CD11b-V450 (BD) (clone M1/70). eF506 (eBioscience) was used as a live/dead marker to exclude non-viable cells. An intracellular staining was performed for IL-17A-AF647 (BD) (clone TC11-18H10) and RORɣt-PE (Invitrogen) (clone AFKJS-9) after fixation and permeabilization of the cells. The third panel was designed to target NKT cells, NK cells, T cells, neutrophils and DCs, using the following antibodies: CD3-PerCP-Cy5.5 (Biolegend) (clone 17A2), NK1.1-BV421 (Invitrogen) (clone PK136), CD11b-APC-Cy7 (BD) (clone M1/70), CD11c-PE-Cy7 (BD) (clone HL3), Ly6G-AF700 (Biolegend) (clone 1A8) and MHC-II-AF488 (eBioscience) (clone M5/114.15.2). eF506 (eBioscience) was used as a live/dead marker to exclude non-viable cells. An intracellular staining was carried out for IL-17A-AF647 (BD) (clone TC11-18H10) following cell fixation and permeabilization. For both panels, FMO controls were included for the IL-17A staining.\u003c/p\u003e\n\u003cp\u003eAs TKO mice are deficient in T cell populations, staining of LP cells was limited to panel 2 and panel 3. In panel 3, a modification was made by substituting the staining for CD3 by CD19-PerCP-Cy5.5 (Biolegend) (clone 6D5) to include the identification of B cells.\u003c/p\u003e\n\u003cp\u003eIn the final study, a flow cytometry panel was designed specifically to target antigen-presenting cells. The LP cells were stained for 30 minutes at 4°C with CD45.2-FITC (Biolegend) (clone 30-F11), a co-staining for CD19-PerCP-Cy5.5 (Biolegend) (clone 6D5) and CD3-PerCP-Cy5.5 (Biolegend) (clone 17A2), MHC-II-AF700 (Biolegend) (clone M5/114.15.2), CD11c-PE-eF610 (Invitrogen) (clone N418), CD11b-BUV395 (BD) (clone M1/70), CD26-BUV737 (BD) (clone H194-112), CD64-BV711 (Biolegend) (clone X54-5/7.1), F4/80-BV785 (Biolegend) (clone BM8) and Ly6C-eFluor450 (Invitrogen) (clone HK1.4). An intracellular staining was performed for IL-17A-AF647 (BD) (clone TC11-18H10) after fixation and permeabilization of the cells. An FMO control was included for the IL-17A staining.\u003c/p\u003e\n\u003cp\u003eThe samples from study 2, 3 and 5 were passed on a CytoFlex flow cytometer (Beckman Coulter) and the data were analyzed using the CytExpert Software Version 2.5 (Beckman Coulter Inc.). The samples from study 6 were run on a \u0026nbsp;LSRFortessa 5-laser (BD Biosciences) and analyzed using the FlowJo v10.7 software (BD Biosciences).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll quantitative data are reported as the mean and standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 10. A one-tailed Mann-Whitney test was used for comparisons between two groups and a non-parametric Kruskal-Wallis test followed by a Dunn’s multiple comparison test was applied to compare differences among more than two groups. A P-value of \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eT helper cells located in the lamina propria of the small intestine are the main cellular source of IL-17A in response to a \u003cem\u003eGiardia\u003c/em\u003e infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a first experiment, IL-17A expression levels were monitored in the PP, MLN and SI of\u003cem\u003e\u0026nbsp;G. muris\u0026nbsp;\u003c/em\u003einfected mice to determine the intestinal location of the anti-giardial IL-17A response.\u0026nbsp;C57BL/6 mice were monitored for 21 days after \u003cem\u003eG. muris\u003c/em\u003e infection. The fecal cyst excretion pattern was in line with previous observations\u003csup\u003e5,23\u003c/sup\u003e, reaching a peak around day 7 after infection and declining progressively, with minimal excretion by day 21 (Fig. 1a). Small intestinal trophozoite counts at day 7, 14 and 21 p.i. mirrored this pattern, with the highest number of trophozoites present at day 7 p.i., followed by a steady reduction. At day 21 p.i., only a minor amount of trophozoites was left in the SI (Fig. 1b). Additionally, IL-17A and Mbl2 gene expression levels in the SI, PP and MLN were analyzed by RT-qPCR at day 7, 14 and 21 p.i.. In the SI, there was a significant upregulation of IL-17A expression at day 7 and day 14 after infection compared to uninfected control mice. An increase in IL-17A expression was also noted in the PP on day 7 p.i., although it did not reach statistical significance. In the MLN, no differences in IL-17A mRNA production could be detected at any of the analyzed time points (Fig. 1c). Expression of Mbl2, an important downstream regulated gene of IL-17A\u003csup\u003e7\u003c/sup\u003e, showed the same expression pattern as IL-17A, with significant upregulation in the SI at all analyzed time points. No differences in Mbl2 expression could be detected in the PP and MLN (Supplementary Fig. S1).\u003c/p\u003e\n\u003cp\u003eTo identify which immune cells are responsible for this IL-17A production in the small intestine during a \u003cem\u003eGiardia\u003c/em\u003e infection, a second experiment was set up in C57BL/6 mice. The parasitological parameters (cyst and trophozoite counts) for this study are shown in Supplementary Fig. S2. At day 14 p.i., IL-17A production by IEL and by immune cells in the LP of infected and uninfected control mice was analyzed using flow cytometry. In this experiment, leukocytes (CD45\u003csup\u003e+\u003c/sup\u003e cells), Th cells (CD45\u003csup\u003e+\u003c/sup\u003eTCRβ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e), cytotoxic T (Tc) cells (CD45\u003csup\u003e+\u003c/sup\u003eTCRβ\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e), ɣδT cells (CD45\u003csup\u003e+\u003c/sup\u003eTCRɣδ\u003csup\u003e+\u003c/sup\u003e) and ‘non-T’ cells (CD45\u003csup\u003e+\u003c/sup\u003eTCRβ\u003csup\u003e-\u003c/sup\u003eTCRɣδ\u003csup\u003e-\u003c/sup\u003e) were investigated. The gating strategy is documented in Supplementary Fig. S3. No IL-17A production could be detected in the IEL compartment by any cell type (Supplementary Fig. S4). In contrast, there was significantly elevated production of IL-17A by leukocytes in the LP at day 14 p.i. compared to uninfected control mice. Within this leukocyte population, Th cells demonstrated a clear and significant upregulation of IL-17A production. ɣδT cells and Tc cells showed no IL-17A production after infection. Notably, a small increase in IL-17A production following \u003cem\u003eGiardia\u003c/em\u003e infection was also observed in a cell population negative for both T cell receptors (Fig. 2).\u003c/p\u003e\n\u003cp\u003eTo further characterize the IL-17A producing cells, a similar infection experiment was designed, this time including additional time points (day 4 and day 7 p.i.) and a broader range of immune cell types was investigated. This study focused exclusively on LP cells, as the previous study revealed no IL-17A production in the IEL. Along with leukocytes, Th cells, Tc cells, ɣδT cells and ‘non-T' cells, the following cell types were also included: all T cells, NKT cells, NK cells, neutrophils, DCs and ILCs, including ILC3s. These specific cell types were selected based on previous reports indicating their ability to produce IL-17A under certain conditions\u003csup\u003e12-17\u003c/sup\u003e. ILC3s specifically have been suggested multiple times as a possible innate source of anti-giardial IL17A\u003csup\u003e6,18-20\u003c/sup\u003e. The gating strategy for the additional cell types can be found in Supplementary Fig. S5, 6. Parasitological data of this infection experiment are presented in Supplementary Fig. S7. At day 14 p.i., a significant increase in IL-17A production was detected in the leukocyte population. Within the leukocytes this increase was present in the T cells and more precisely in the Th cell subset, confirming the observations made in study 2 (Fig. 3). Within the T cell population, again no IL-17A production could be detected in the Tc cells or the ɣδT cells at any time point during \u003cem\u003eGiardia\u003c/em\u003e infection (Supplementary Fig. S8). In the population negative for TCRβ and TCRɣδ, a small but significant level of IL-17A production was detected at day 14 after infection, consistent with the prior experiment (Fig. 3). However, when looking further into multiple non-T cell populations, specifically NK(T) cells, neutrophils, DCs, ILCs and ILC3s, no IL-17A production could be detected at any time point by any of these cell types (Supplementary Fig. S8, 9). Only at day 7 p.i., a minor amount of IL-17A production could be detected from a population of MHC-II\u003csup\u003e+\u003c/sup\u003e cells, more specifically MHC-II\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e-\u003c/sup\u003e cells (Fig 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMice deficient in T cells lose the majority of their IL-17A production and lack the ability to overcome a \u003cem\u003eG.\u003c/em\u003e \u003cem\u003emuris\u003c/em\u003e infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm that Th cells are the primary source of IL-17A in the defense against a \u003cem\u003eGiardia\u003c/em\u003e infection, a new infection experiment was designed using TKO mice. The aim of this study was to compare C57BL/6 and TKO mice regarding their IL-17A production and their ability to overcome a \u003cem\u003eGiardia\u003c/em\u003e infection. Infected C57BL/6 and TKO mice were monitored for 21 days after infection, a timeframe during which C57BL/6 mice typically clear the infection. Cyst shedding by C57BL/6 mice had almost entirely ceased by day 21 p.i.., whereas TKO mice maintained a high level of cyst excretion throughout the whole study period (Fig. 4a). Similarly, intestinal trophozoite counts at the end of the study revealed that C57BL/6 mice had almost completely eliminated the parasite, while TKO mice still carried up to 60 million trophozoites in their SI (Fig. 4b). These observations indicate that mice deficient in T cells are not able to overcome a \u003cem\u003eGiardia\u003c/em\u003e infection. Relative IL-17A and Mbl2 expression levels were measured in the SI, PP and MLN of both mouse strains using RT-qPCR. Within C57BL/6 mice, a significant IL-17A upregulation was detected in the SI and an upwards trend was again visible in the PP, confirming the results of the first study. In all intestinal compartments and at every time point after infection, the magnitude of the IL-17A response was considerably lower in TKO mice compared to the expression levels in C57BL/6 mice. This observation highlights the predominant role of T cells in anti-giardial IL-17A production. However, within the TKO mice, IL-17A mRNA expression was still significantly elevated at day 21 p.i. compared to control mice (Fig. 4c). The expression of Mbl2 was significantly elevated after \u003cem\u003eGiardia\u003c/em\u003e infection in C57BL/6 mice, but not in TKO mice (Supplementary Fig. S10).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;In the following phase, the aim was to investigate whether the minor amount of IL-17A mRNA upregulation observed in TKO mice subsequently translates into IL-17A production and which immune cells are responsible for this production. Therefore, IL-17A production by LP cells of TKO mice was assessed using flow cytometry. As IL-17A mRNA production only started at day 21 p.i. in TKO mice, flow cytometry experiments were conducted on day 21 and at an additional later time point, i.e. 2 months p.i.. Cyst and trophozoite counts remained persistently high until the end of the observation period (Fig. 5a,b). In the CD45\u003csup\u003e+\u003c/sup\u003e population of TKO mice, no IL-17A upregulation could be detected at either time point after infection (Fig. 5c). Within the CD45\u003csup\u003e+\u003c/sup\u003e population, no IL-17A production was observed by ILCs, ILC3s, NK cells, B cells, DCs or neutrophils (Supplementary Fig. S11). An upwards trend was visible in the MHC-II\u003csup\u003e+\u003c/sup\u003e cell population, although not statistically significant. The only cell type that was found to significantly produce IL-17A in TKO mice following a \u003cem\u003eGiardia\u003c/em\u003e infection were MHC-II\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e-\u003c/sup\u003e cells, consistent with the previous observation in C57BL/6 mice (Fig. 5c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubsets of innate MHC-II+ cells produce IL-17A following a \u003cem\u003eG. muris\u003c/em\u003e infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a final experiment, we aimed to further characterize the MHC-II\u003csup\u003e+\u003c/sup\u003e cells that produced IL-17A after \u003cem\u003eGiardia\u003c/em\u003e infection through more detailed phenotypic analysis. Since MHC-II\u003csup\u003e+\u003c/sup\u003e IL-17A-producing cells were identified at day 7 p.i. in C57BL/6 mice, this time point was revisited using a panel targeting distinct MHC-II\u003csup\u003e+\u003c/sup\u003e antigen-presenting cell subsets. The gating strategy for this panel can be found in Supplementary Fig. S12. As a first step, IL-17A upregulation in CD3\u003csup\u003e+\u003c/sup\u003e T cells, CD3\u003csup\u003e-\u003c/sup\u003e non-T cells and the MHC-II\u003csup\u003e+\u003c/sup\u003e population was confirmed (Supplementary Fig. S13). Secondly, different MHC-II\u003csup\u003e+\u003c/sup\u003e subpopulations were analysed. Elevated IL-17A levels were detected in the MHC-II\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e-\u003c/sup\u003e population, as seen previously, and additionally in the MHC-II\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e subset. These MHCII\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e populations might comprise of conventional DCs (cDC), monocyte-derived cells and tissue macrophages. To distinguish between these populations, the markers CD26 (lineage marker for cDCs) and CD64 (marker for monocyte-derived cells and macrophages) were used. We could not detect an increase in IL-17A production in the MHCII\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD26\u003csup\u003e+\u003c/sup\u003e population, regardless of the expression of CD11b. In contrast, when looking at macrophage associated markers, an increase in IL-17A production was found in the MHC-II\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eCD64\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e population (Fig. 6a, b). Further analysis revealed these cells to be negative for Ly6C expression (Supplementary Fig. S14).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eInterleukin-17A is a pro-inflammatory cytokine primarily associated with mucosal defense against extracellular pathogens. IL-17A promotes the recruitment of neutrophils, strengthens epithelial barrier integrity and induces the expression of antimicrobial peptides. Its function is crucial in initiating effective immune responses against pathogens, while maintaining homeostasis at mucosal surfaces\u003csup\u003e12,24,25\u003c/sup\u003e. The involvement of IL-17A in the immune response against \u003cem\u003eGiardia\u003c/em\u003e infection was first highlighted by Dreesen et al.\u003csup\u003e5\u003c/sup\u003e, who showed that mice deficient in the IL-17A receptor exhibit increased parasite burdens and impaired clearance of infection. IL-17A contributes to host protection by inducing Mbl2 expression and stimulating the production of \u003cem\u003eGiardia\u003c/em\u003e-specific IgA antibodies\u003csup\u003e6,7\u003c/sup\u003e. These observations establish IL-17A as a central molecule in the host defense against \u003cem\u003eGiardia\u003c/em\u003e. Despite its recognized importance, the cellular pathways leading to anti-giardial IL-17A production, specifically its cellular sources and their exact location, remain poorly defined.\u003c/p\u003e\n\u003cp\u003ePotential sites of anti-giardial IL-17A production include secondary lymphoid organs, such as the MLN and the PP. In the present study, IL-17A mRNA expression levels were measured in the PP and MLN of \u003cem\u003eG.\u003c/em\u003e \u003cem\u003emuris\u003c/em\u003e infected mice. A slight elevation in IL-17A production could be detected at day 7 after infection in the PP. While not significantly increased, this trend suggests a potential involvement of the PP in early immune responses against \u003cem\u003eGiardia\u003c/em\u003e. This is consistent with prior observations that have pointed to immune activation in the PP during infection, including cytokine responses and cellular proliferation\u003csup\u003e9,10\u003c/sup\u003e. Further\u0026nbsp;research focused specifically on the PP could help clarify their role during \u003cem\u003eGiardia\u003c/em\u003e infection, particularly in the early stages of infection when antigen-presentation and priming of naive T cells takes place. While increased IL-17A production has been reported in MLN cells of \u003cem\u003eG. duodenalis\u003c/em\u003e infected mice, this observation was made following \u003cem\u003eex vivo\u003c/em\u003e stimulation of these cells with parasite extract, a method that may not accurately reflect\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e conditions\u003csup\u003e11\u003c/sup\u003e. In our study, we observed no increase in IL-17A mRNA expression in the MLN after \u003cem\u003eG. muris\u003c/em\u003e infection, in line with previous observations by Dann et al.\u003csup\u003e6\u003c/sup\u003e. Taken together, our results from the PP and MLN highlight the local nature of the anti-giardial immune response, with\u0026nbsp;IL-17A production confined to intestinal tissues in close proximity to the site of infection.\u003c/p\u003e\n\u003cp\u003eIL-17A production in small intestinal tissue in response to \u003cem\u003eGiardia\u003c/em\u003e infection has been widely documented\u003csup\u003e5-7,26\u003c/sup\u003e. In agreement with these findings, we observed significantly increased IL-17A mRNA expression at days 7 and 14 p.i.. Our study further expands these data by examining the IL-17A response in the IEL and the LP fraction of the SI. This approach provides further insight into both the location of the anti-giardial IL-17A response and the cells that are responsible for its production. Th17 cells are known as the classical producers of IL-17A. Consistently, we found CD4\u003csup\u003e+\u003c/sup\u003e T cells residing in the LP to be the major source of IL-17A production in the case of a \u003cem\u003eGiardia\u003c/em\u003e infection. These results are in line with Dann et al.\u003csup\u003e6\u003c/sup\u003e, who reported a significant increase in IL-17A production by CD45\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e cells in the LP of \u003cem\u003eG. muris\u003c/em\u003e infected C57BL/6 mice. They also detected IL-17A production by CD45\u003csup\u003e+\u003c/sup\u003e cells in the IEL, while this was not observed in the current study. As Th17 cells are less common in the IEL, they speculated that this production may originate from innate immune cell types. To investigate this hypothesis, they infected CD4\u003csup\u003e-/-\u003c/sup\u003e and Rag2\u003csup\u003e-/-\u003c/sup\u003e mice with \u003cem\u003eG.\u003c/em\u003e \u003cem\u003emuris\u003c/em\u003e, which respectively lack Th cells and all T and B cells. Interestingly, they still observed a significant IL-17A mRNA increase following infection in these mutant mice. Our flow cytometry experiments in C57BL/6 mice also revealed a population of IL-17A-producing cells that were both TCRβ\u003csup\u003e-\u003c/sup\u003e and TCRɣδ\u003csup\u003e-\u003c/sup\u003e, raising the possibility of an innate IL-17A source. Therefore, we aimed to elucidate the contribution of innate immune cells to anti-giardial IL-17A production by comparing the IL-17A response between TKO and C57BL/6 mice. This comparison revealed that even though there is still a significant IL-17A upregulation after infection within the TKO mice, the magnitude of this response is markedly reduced relative to wild-type mice.\u003c/p\u003e\n\u003cp\u003eTo further investigate the level of IL-17A protein production in C57BL/6 and TKO mice and simultaneously identify their cellular sources, a broad panel of immune cell types capable of producing IL-17A was explored. Using flow cytometry, IL-17A production by Tc cells, ɣδT cells, B cells, NK cells, NKT cells, neutrophils, DCs and ILCs was evaluated. Specifically in the case of \u003cem\u003eGiardia\u003c/em\u003e infection, ILC3s have been speculated to be potential producers of IL-17A, as they are seen as the innate counterpart of Th17 cells\u003csup\u003e6,18-20\u003c/sup\u003e. Furthermore, ILC3s are known to be important in the defense against extracellular pathogens and they are responsive to IL-1β and IL-23, which are both upregulated during \u003cem\u003eGiardia\u003c/em\u003e infection\u003csup\u003e5,27,28\u003c/sup\u003e. In the present study, no infection-related IL-17A induction could be detected in any of the investigated cell types. However, when examining specific subsets of MHC-II\u003csup\u003e+\u003c/sup\u003e cells, an IL-17A increase could be observed in both C57BL/6 and TKO mice. This result aligns with findings by Lee et al.\u003csup\u003e18\u003c/sup\u003e, who reported an IL-17A increase in LP MHC-II\u003csup\u003e+\u003c/sup\u003e cells from C57BL/6 mice upon stimulation with \u003cem\u003eG.\u003c/em\u003e \u003cem\u003eduodenalis\u003c/em\u003e trophozoites. These cells were addressed as ILC3s. However, their identification relied solely on MHC-II expression, which is a marker shared by various antigen-presenting cells such as dendritic cells, macrophages, and B cells. Without a broader panel of innate markers or clear lineage exclusion strategies, identifying these cells as ILC3s remains speculative. Our results\u0026nbsp;also corroborate those of Yordanova et al.\u003csup\u003e20\u003c/sup\u003e, who found no evidence of IL-17A production in ILC3s of \u003cem\u003eG. muris\u003c/em\u003e infected mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven the MHC-II\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+/-\u003c/sup\u003e phenotype of the IL-17A producing cells we observed, they most likely represent a certain subset of DCs or macrophages\u003csup\u003e29\u003c/sup\u003e. Further phenotypic analysis revealed these cells to be CD26\u003csup\u003e-\u003c/sup\u003e, which rules out cDCs as a likely source\u003csup\u003e30\u003c/sup\u003e. Alternatively, these cells could represent an intermediate migratory stage of DCs or plasmacytoid DCs (pDCs)\u003csup\u003e31,32\u003c/sup\u003e. pDCs are typically known for shaping the innate and adaptive immunity in the gastrointestinal tract and participate in immunological responses through the presentation of antigens\u003csup\u003e33\u003c/sup\u003e. The IL-17A producing subset of MHC-II\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eCD64\u003csup\u003e+\u003c/sup\u003eF4/80\u003csup\u003e+\u003c/sup\u003e cells we observed matches the classical phenotype of tissue-resident macrophages\u003csup\u003e29,34\u003c/sup\u003e. In the context of \u003cem\u003eGiardia\u003c/em\u003e infection, while research on the role of macrophages in protective immunity is limited, studies have demonstrated their ability to phagocytose trophozoites in both mice and humans\u003csup\u003e35-39\u003c/sup\u003e. Notably, two reports from the same research group have identified an accumulation of macrophages in the lamina propria of \u003cem\u003eG. duodenalis\u0026nbsp;\u003c/em\u003einfected mice\u003csup\u003e40,41\u003c/sup\u003e. Further characterization revealed these cells to be F4/80\u003csup\u003e+\u003c/sup\u003e, CD11b\u003csup\u003e+\u003c/sup\u003e, CD11c\u003csup\u003eint\u003c/sup\u003e, CX3CR1\u003csup\u003e+\u003c/sup\u003e, MHCII\u003csup\u003e+\u003c/sup\u003e, Ly6C\u003csup\u003e-\u003c/sup\u003e, very similar to the population detected in the current study and also consistent with the tissue-resident macrophage phenotype. Interestingly, when macrophages were depleted using an anti-CSF-1R antibody, this did not impair the clearance of \u003cem\u003eGiardia\u003c/em\u003e or affect Th cell numbers in the LP, suggesting that macrophages are dispensable for protective immunity. However, they speculated that during \u003cem\u003eGiardia\u003c/em\u003e infection, macrophages may play a role in maintaining mucosal homeostasis and epithelial barrier function through the clearance of apoptotic cells. Additionally, while macrophages did not seem to be important for the differentiation of naïve T cells into Th cells, they speculated that these cells might support effector T cell proliferation through MHC-II-dependent antigen presentation. Given this context, the detection of IL-17A production by these macrophages could indicate a previously unrecognized protective role in anti-\u003cem\u003eGiardia\u003c/em\u003e immunity.\u0026nbsp;Alternatively, our observed phenotype might also correspond to monocyte-derived DCs (moDCs), which share overlapping surface markers with tissue-resident macrophages\u003csup\u003e42\u003c/sup\u003e. Overall, MHC-II\u003csup\u003e+\u003c/sup\u003e innate cell types are not typically known as IL-17A producing cells. However, a recent study identified elevated IL-17A expression levels in moDCs in humans with asthma and COPD\u003csup\u003e17\u003c/sup\u003e. In the context of \u003cem\u003eGiardia\u003c/em\u003e infection, the functional relevance of these innate IL-17A producing cells remains unclear. While their contribution is minor, it is possible that they play a niche role in early parasite sensing or facilitating crosstalk between innate and adaptive immunity.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides critical insights into the cellular sources of IL-17A during \u003cem\u003eGiardia\u0026nbsp;\u003c/em\u003einfection, highlighting the predominant role of Th17 cells in mounting a protective IL-17A response. Our results demonstrate that IL-17A production is largely restricted to Th17 cells within the LP of the SI, with a minimal contribution from innate immune cells. Moreover, the inability of TKO mice to elicit an effective IL-17A response and clear the infection underscores the critical role of the adaptive immune response in controlling \u003cem\u003eGiardia\u003c/em\u003e infection. Further research into the upstream signals that drive Th17 cell differentiation will be essential to gain a deeper understanding on the mechanisms underlying the protective immune response against \u003cem\u003eGiardia\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was fnancially supported by an FWO PhD-fellowship attributed to C.V.C..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.V.C., L.S. and P.G. conceived and designed the studies. C.V.C. performed the experiments with contributions of B.D. and L.S.. Data analysis was performed by C.V.C. and L.S.. The manuscript was written by C.V.C., L.S. and P.G.. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e. The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary material\u003c/strong\u003e. The online version contains supplementary material.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLane, S. \u0026amp; Lloyd, D. Current Trends in Research into the Waterborne Parasite \u003cem\u003eGiardia\u003c/em\u003e. \u003cem\u003eCrit. Rev. Immunol.\u003c/em\u003e\u003cstrong\u003e28\u003c/strong\u003e, 123-147 (2002).\u003c/li\u003e\n\u003cli\u003eZhou, P.\u003cem\u003e et al.\u003c/em\u003e Role of Interleukin-6 in the Control of Acute and Chronic \u003cem\u003eGiardia lamblia \u003c/em\u003eInfections in Mice. \u003cem\u003eInfect. Immun.\u003c/em\u003e\u003cstrong\u003e71\u003c/strong\u003e, 1566-1568 (2003).\u003c/li\u003e\n\u003cli\u003eAdam, R. D. The biology of \u003cem\u003eGiardia\u003c/em\u003e spp. \u003cem\u003eMicrobiol. Rev.\u003c/em\u003e\u003cstrong\u003e55\u003c/strong\u003e, 706-732 (1991).\u003c/li\u003e\n\u003cli\u003eHalliez, M. C. \u0026amp; Buret, A. G. Extra-intestinal and long term consequences of \u003cem\u003eGiardia duodenalis\u003c/em\u003e infections. \u003cem\u003eWorld J. Gastroenterol.\u003c/em\u003e\u003cstrong\u003e19\u003c/strong\u003e, 8974-8985 (2013).\u003c/li\u003e\n\u003cli\u003eDreesen, L.\u003cem\u003e et al.\u003c/em\u003e\u003cem\u003eGiardia muris\u003c/em\u003e infection in mice is associated with a protective interleukin 17A response and induction of peroxisome proliferator-activated receptor alpha. \u003cem\u003eInfect. Immun.\u003c/em\u003e\u003cstrong\u003e82\u003c/strong\u003e, 3333-3340 (2014).\u003c/li\u003e\n\u003cli\u003eDann, S. M.\u003cem\u003e et al.\u003c/em\u003e IL-17A promotes protective IgA responses and expression of other potential effectors against the lumen-dwelling enteric parasite \u003cem\u003eGiardia\u003c/em\u003e. \u003cem\u003eExp. Parasitol.\u003c/em\u003e\u003cstrong\u003e156\u003c/strong\u003e, 68-78 (2015).\u003c/li\u003e\n\u003cli\u003ePaerewijck, O.\u003cem\u003e et al.\u003c/em\u003e Interleukin-17 receptor A (IL-17RA) as a central regulator of the protective immune response against \u003cem\u003eGiardia\u003c/em\u003e. \u003cem\u003eSci. Rep.\u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, 8520 (2017).\u003c/li\u003e\n\u003cli\u003eBrandtzaeg, P., Kiyono, H., Pabst, R. \u0026amp; Russell, M. W. Terminology: nomenclature of mucosa-associated lymphoid tissue. \u003cem\u003eMucosal Immunol.\u003c/em\u003e\u003cstrong\u003e1\u003c/strong\u003e, 31-37 (2008).\u003c/li\u003e\n\u003cli\u003eHill, D. R. Lymphocyte proliferation in Peyer's patches of \u003cem\u003eGiardia muris\u003c/em\u003e-infected mice. \u003cem\u003eInfect. Immun.\u003c/em\u003e\u003cstrong\u003e58\u003c/strong\u003e, 2683-2685 (1990).\u003c/li\u003e\n\u003cli\u003e Djamiatun, K. \u0026amp; Faubert, G. M. Exogenous cytokines released by spleen and Peyer's patch cells removed from mice infected with \u003cem\u003eGiardia muris\u003c/em\u003e. \u003cem\u003eParasite immunol.\u003c/em\u003e\u003cstrong\u003e20\u003c/strong\u003e, 1365-3024 (1998).\u003c/li\u003e\n\u003cli\u003e Solaymani-Mohammadi, S. \u0026amp; Singer, S. M. \u003cem\u003eGiardia duodenalis\u003c/em\u003e: The double-edged sword of immune responses in giardiasis. \u003cem\u003eExp. Parasitol.\u003c/em\u003e\u003cstrong\u003e126\u003c/strong\u003e, 292-297 (2010).\u003c/li\u003e\n\u003cli\u003e Cua, D. J. \u0026amp; Tato, C. M. Innate IL-17-producing cells: the sentinels of the immune system. \u003cem\u003eNat. Rev. Immunol.\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, 479-489 (2010).\u003c/li\u003e\n\u003cli\u003e Haas, J. D.\u003cem\u003e et al.\u003c/em\u003e CCR6 and NK1.1 distinguish between IL-17A and IFN-gamma-producing gammadelta effector T cells. \u003cem\u003eEur. J. Immunol.\u003c/em\u003e\u003cstrong\u003e39\u003c/strong\u003e, 3488-3497 (2009).\u003c/li\u003e\n\u003cli\u003e Passos, S. T.\u003cem\u003e et al.\u003c/em\u003e IL-6 promotes NK cell production of IL-17 during toxoplasmosis. \u003cem\u003eJ. Immunol.\u003c/em\u003e\u003cstrong\u003e184\u003c/strong\u003e, 1776-1783 (2010).\u003c/li\u003e\n\u003cli\u003e Li, L.\u003cem\u003e et al.\u003c/em\u003e IL-17 produced by neutrophils regulates IFN-gamma-mediated neutrophil migration in mouse kidney ischemia-reperfusion injury. \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e\u003cstrong\u003e120\u003c/strong\u003e, 331-342 (2010).\u003c/li\u003e\n\u003cli\u003e Coury, F.\u003cem\u003e et al.\u003c/em\u003e Langerhans cell histiocytosis reveals a new IL-17A-dependent pathway of dendritic cell fusion. \u003cem\u003eNat. Med.\u003c/em\u003e\u003cstrong\u003e14\u003c/strong\u003e, 81-87 (2008).\u003c/li\u003e\n\u003cli\u003e Paplinska-Goryca, M.\u003cem\u003e et al.\u003c/em\u003e The Expressions of TSLP, IL-33, and IL-17A in Monocyte Derived Dendritic Cells from Asthma and COPD Patients are Related to Epithelial\u0026ndash;Macrophage Interactions. \u003cem\u003eCells\u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, 1944 (2020).\u003c/li\u003e\n\u003cli\u003e Lee, H.-Y., Park, E.-A., Lee, K.-J., Lee, K.-H. \u0026amp; Park, S.-J. Increased Innate Lymphoid Cell 3 and IL-17 Production in Mouse Lamina Propria Stimulated with \u003cem\u003eGiardia lamblia\u003c/em\u003e. \u003cem\u003eKorean J. Parasitol.\u003c/em\u003e\u003cstrong\u003e57\u003c/strong\u003e, 225-232 (2019).\u003c/li\u003e\n\u003cli\u003e Yordanova, I. A.\u003cem\u003e et al.\u003c/em\u003e ROR\u0026gamma;t+ Treg to Th17 ratios correlate with susceptibility to \u003cem\u003eGiardia \u003c/em\u003einfection. \u003cem\u003eSci. Rep.\u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, 20328 (2019).\u003c/li\u003e\n\u003cli\u003e Yordanova, I. A., Lamatsch, M., K\u0026uuml;hl, A. A., Hartmann, S. \u0026amp; Rausch, S. Eosinophils are dispensable for the regulation of IgA and Th17 responses in \u003cem\u003eGiardia muris\u003c/em\u003e infection. \u003cem\u003eParasite Immunol.\u003c/em\u003e\u003cstrong\u003e43\u003c/strong\u003e (2021).\u003c/li\u003e\n\u003cli\u003e Paerewijck, O., Maertens, B., Gagnaire, A., De Bosscher, K. \u0026amp; Geldhof, P. Delayed development of the protective IL-17A response following a \u003cem\u003eGiardia muris\u003c/em\u003e infection in neonatal mice. \u003cem\u003eSci. Rep.\u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, 8959 (2019).\u003c/li\u003e\n\u003cli\u003e Lefran\u0026ccedil;ois, L. \u0026amp; Lycke, N. Isolation of mouse small intestinal intraepithelial lymphocytes, Peyer's patch, and lamina propria cells. \u003cem\u003eCurr. Prot. Immunol.\u003c/em\u003e\u003cstrong\u003e17\u003c/strong\u003e, 3.19.11-13.19.16 (2001).\u003c/li\u003e\n\u003cli\u003e Heyworth, M. F., Carlson, J. R. \u0026amp; Ermak, T. H. Clearance of \u003cem\u003eGiardia muris\u003c/em\u003e infection requires helper/inducer T lymphocytes. \u003cem\u003eJ. Exp. Med.\u003c/em\u003e\u003cstrong\u003e165\u003c/strong\u003e, 1743-1748 (1987).\u003c/li\u003e\n\u003cli\u003e Ouyang, W., Kolls, J. K. \u0026amp; Zheng, Y. The Biological Functions of T Helper 17 Cell Effector Cytokines in Inflammation. \u003cem\u003eImmun.\u003c/em\u003e\u003cstrong\u003e28\u003c/strong\u003e, 454-467 (2008).\u003c/li\u003e\n\u003cli\u003e Ishigame, H.\u003cem\u003e et al.\u003c/em\u003e Differential Roles of Interleukin-17A and -17F in Host Defense against Mucoepithelial Bacterial Infection and Allergic Responses. \u003cem\u003eImmun.\u003c/em\u003e\u003cstrong\u003e30\u003c/strong\u003e, 108-119 (2009).\u003c/li\u003e\n\u003cli\u003e Maertens, B., Gagnaire, A., Paerewijck, O., De Bosscher, K. \u0026amp; Geldhof, P. Regulatory role of the intestinal microbiota in the immune response against \u003cem\u003eGiardia\u003c/em\u003e. \u003cem\u003eSci. Rep.\u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e, 10601 (2021).\u003c/li\u003e\n\u003cli\u003e Zeng, B.\u003cem\u003e et al.\u003c/em\u003e ILC3 function as a double-edged sword in inflammatory bowel diseases. \u003cem\u003eCell Death Dis.\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, 315 (2019).\u003c/li\u003e\n\u003cli\u003e Obendorf, J.\u003cem\u003e et al.\u003c/em\u003e Increased expression of CD25, CD83, and CD86, and secretion of IL-12, IL-23, and IL-10 by human dendritic cells incubated in the presence of Toll-like receptor 2 ligands and \u003cem\u003eGiardia duodenalis\u003c/em\u003e. \u003cem\u003eParasit. Vectors\u003c/em\u003e\u003cstrong\u003e6\u003c/strong\u003e, 317 (2013).\u003c/li\u003e\n\u003cli\u003e Cerovic, V., Bain, C. C., Mowat, A. M. \u0026amp; Milling, S. W. Intestinal macrophages and dendritic cells: what's the difference? \u003cem\u003eTrends Immunol.\u003c/em\u003e\u003cstrong\u003e35\u003c/strong\u003e, 270-277 (2014).\u003c/li\u003e\n\u003cli\u003e Guilliams, M.\u003cem\u003e et al.\u003c/em\u003e Unsupervised High-Dimensional Analysis Aligns Dendritic Cells across Tissues and Species. \u003cem\u003eImmun.\u003c/em\u003e\u003cstrong\u003e45\u003c/strong\u003e, 669-684 (2016).\u003c/li\u003e\n\u003cli\u003e Naik, S. H.\u003cem\u003e et al.\u003c/em\u003e Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo. \u003cem\u003eNat. Immunol.\u003c/em\u003e\u003cstrong\u003e8\u003c/strong\u003e, 1217-1226 (2007).\u003c/li\u003e\n\u003cli\u003e Swiecki, M., Colonna, M., Swiecki, M. \u0026amp; Colonna, M. The multifaceted biology of plasmacytoid dendritic cells. \u003cem\u003eNat. Rev. Immunol.\u003c/em\u003e\u003cstrong\u003e15\u003c/strong\u003e, 471-485 (2015).\u003c/li\u003e\n\u003cli\u003e Arimura, K.\u003cem\u003e et al.\u003c/em\u003e Crucial role of plasmacytoid dendritic cells in the development of acute colitis through the regulation of intestinal inflammation. \u003cem\u003eMucosal Immunol.\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, 957-970 (2016).\u003c/li\u003e\n\u003cli\u003e Bain, C. C. \u0026amp; Mowat, A. M. The monocyte-macrophage axis in the intestine. \u003cem\u003eCell. Immunol.\u003c/em\u003e\u003cstrong\u003e291\u003c/strong\u003e, 41-48 (2014).\u003c/li\u003e\n\u003cli\u003e Li, L.\u003cem\u003e et al.\u003c/em\u003e Mouse macrophages capture and kill \u003cem\u003eGiardia lamblia\u003c/em\u003e by means of releasing extracellular trap. \u003cem\u003eDCI\u003c/em\u003e\u003cstrong\u003e88\u003c/strong\u003e, 205-212 (2018).\u003c/li\u003e\n\u003cli\u003e Hill, D. R. \u0026amp; Pearson, R. D. Ingestion of \u003cem\u003eGiardia lamblia \u003c/em\u003etrophozoites by human mononuclear phagocytes. \u003cem\u003eInfect. Immun.\u003c/em\u003e\u003cstrong\u003e55\u003c/strong\u003e, 3155-3161 (1987 Dec).\u003c/li\u003e\n\u003cli\u003e Hill, D. R. \u0026amp; Pohl, R. Ingestion of \u003cem\u003eGiardia lamblia \u003c/em\u003etrophozoites by murine Peyer's patch macrophages. \u003cem\u003eInfect. Immun.\u003c/em\u003e\u003cstrong\u003e58\u003c/strong\u003e, 3202-3207 (1990).\u003c/li\u003e\n\u003cli\u003e Belosevic, M. \u0026amp; Daniels, C. W. Phagocytosis of \u003cem\u003eGiardia lamblia \u003c/em\u003etrophozoites by cytokine-activated macrophages. \u003cem\u003eClin. Exp. Immunol.\u003c/em\u003e\u003cstrong\u003e87\u003c/strong\u003e, 304-309 (1992).\u003c/li\u003e\n\u003cli\u003e Owen, R. L., Allen, C. L. \u0026amp; Stevens, D. P. Phagocytosis of \u003cem\u003eGiardia muris\u003c/em\u003e by macrophages in Peyer's patch epithelium in mice. \u003cem\u003eInfect. Immun.\u003c/em\u003e\u003cstrong\u003e33\u003c/strong\u003e, 591-601 (1981).\u003c/li\u003e\n\u003cli\u003e Fink, M. Y.\u003cem\u003e et al.\u003c/em\u003e Proliferation of resident macrophages is dispensable for protection during \u003cem\u003eGiardia duodenalis\u003c/em\u003e infections. \u003cem\u003eImmunoHorizons\u003c/em\u003e\u003cstrong\u003e3\u003c/strong\u003e, 412-421 (2019).\u003c/li\u003e\n\u003cli\u003e Maloney, J., Keselman, A., Li, E. \u0026amp; Singer, S. M. Macrophages expressing arginase 1 and nitric oxide synthase 2 accumulate in the small intestine during \u003cem\u003eGiardia lamblia\u003c/em\u003e infection. \u003cem\u003eMicrobes Infect.\u003c/em\u003e\u003cstrong\u003e17\u003c/strong\u003e, 462-467 (2015).\u003c/li\u003e\n\u003cli\u003e Guilliams, M.\u003cem\u003e et al.\u003c/em\u003e Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. \u003cem\u003eNat. Rev. Immunol.\u003c/em\u003e\u003cstrong\u003e14\u003c/strong\u003e, 571-578 (2014).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Summary of \u003cem\u003eG. muris\u003c/em\u003e infection studies. All experiments were performed with 6-week old mice. Individual fecal cyst counts were monitored every 2-3 days and intestinal trophozoite counts were performed at the time of euthanasia for each animal. Abbreviations: SI, small intestine; PP, Peyer\u0026rsquo;s patches; MLN, mesenteric lymph nodes; IEL, intra-epithelial lymphocytes; LP, lamina propria; TKO, T cell knock-out.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"624\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStudy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMouse Strain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of animals\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSampling time\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSamples\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eData analyzed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eIntestinal location: SI/PP/MLN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eC57BL/6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e5 controls + 15 infected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eDay 7, 14, 21 (n=5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003eSI, PP and MLN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003eIL-17A gene expression (RT-qPCR)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eIntestinal location: IEL/LP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eC57BL/6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e4 controls + 4 infected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eDay 14 (n=4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003eIEL and LP cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003eIL-17A producing cells (flow cytometry)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eIL-17A producing cells in the LP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eC57BL/6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e12 controls + 12 infected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eDay 4, 7, 14 (n=4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003eLP cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003eIL-17A producing cells (flow cytometry)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eInvolvement of T cells in IL-17A production\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eC57BL/6 and TKO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003eFor each strain: 4 controls + 12 infected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eDay 7, 14, 21 (n=4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003eSI, PP and MLN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003eIL-17A gene expression (RT-qPCR)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eIL-17A producing non-T cells in the LP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eTKO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e8 controls + 8 infected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eDay 21, 2 months (n=4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003eLP cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003eIL-17A producing cells (flow cytometry)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 108px;\"\u003e\n \u003cp\u003eIL-17A producing non-T cells in the LP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003eC57BL/6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e4 controls + 4 infected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eDay 7 (n=4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003eLP cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121px;\"\u003e\n \u003cp\u003eIL-17A producing cells (flow cytometry)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Giardia, IL-17A, T cells, ILCs, MHC-II, macrophages","lastPublishedDoi":"10.21203/rs.3.rs-6836025/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6836025/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"IL-17A plays a crucial role in the immune defense against Giardia infection, yet its cellular sources remain incompletely defined. In this study, the site-specific expression and cellular origin of IL-17A were investigated, focusing on both adaptive and innate immune cells in the small intestine, Peyer’s patches and mesenteric lymph nodes of Giardia muris infected C57BL/6 mice. RT-qPCR analyses showed that IL-17A mRNA expression was significantly upregulated in the small intestine, slightly elevated in the Peyer’s patches and unchanged in the mesenteric lymph nodes. Flow cytometry revealed that CD4⁺ T helper cells in the lamina propria of the small intestine are the predominant source of anti-giardial IL-17A. No increase in IL-17A was detected by γδT cells, Tc cells, NK(T) cells, B cells, neutrophils, dendritic cells and innate lymphoid cells. Within the CD3- innate cell population, increased IL-17A production was observed in MHC-II+CD11c+CD11b+/- cells, including a subset of cells expressing typical macrophage markers, namely MHC-II⁺CD11c⁺CD11b⁺CD64⁺F4/80⁺ cells. In T cell-deficient mice, both IL-17A expression and parasite clearance were severely impaired. Our findings demonstrate the importance of the adaptive immunity and simultaneously identify Th cells in the lamina propria as the main source of anti-giardial IL-17A, with a possible supporting role from macrophage-like antigen-presenting cells.","manuscriptTitle":"Unravelling the cellular sources and location of IL-17A production during a Giardia infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-23 09:13:40","doi":"10.21203/rs.3.rs-6836025/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-07T19:46:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-06T22:49:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-23T13:45:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"10278321465339677577956403977944058819","date":"2025-06-17T10:24:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152931506358912130051447460946517989044","date":"2025-06-17T10:04:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-17T08:23:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-17T07:30:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-11T16:50:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-10T12:42:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-06T10:03:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"52b3981e-f1eb-4080-a9b4-e6f41e512eb9","owner":[],"postedDate":"June 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50439940,"name":"Biological sciences/Immunology/Adaptive immunity/Cellular immunity"},{"id":50439941,"name":"Biological sciences/Immunology/Cytokines/Interleukins"},{"id":50439942,"name":"Biological sciences/Immunology/Infectious diseases/Parasitic infection"}],"tags":[],"updatedAt":"2025-11-10T16:03:49+00:00","versionOfRecord":{"articleIdentity":"rs-6836025","link":"https://doi.org/10.1038/s41598-025-22662-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-05 15:58:14","publishedOnDateReadable":"November 5th, 2025"},"versionCreatedAt":"2025-06-23 09:13:40","video":"","vorDoi":"10.1038/s41598-025-22662-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-22662-3","workflowStages":[]},"version":"v1","identity":"rs-6836025","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6836025","identity":"rs-6836025","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}