Single-cell transcriptomics reveals altered myeloid cell profiles associated with the early establishment of leishmania reservoirs

doi:10.21203/rs.3.rs-3931457/v1

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Single-cell transcriptomics reveals altered myeloid cell profiles associated with the early establishment of leishmania reservoirs

https://doi.org/10.21203/rs.3.rs-3931457/v1

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Current drug regimens to treat visceral leishmaniasis (VL) are associated with a significant frequency of infection relapses, particularly in immunosuppressed patients. Understanding the cellular and tissue-specific persistence of Leishmania infantum post-treatment is crucial for improving therapeutic outcomes. Using a rhesus macaque model of VL, despite the administration of miltefosine (HePC) shortly after infection, L. infantum was detected in various tissues, including the spleen, bone marrow, and peripheral and mesenteric lymph nodes (LNs). Notably, lower HePC penetration in pLNs correlated with persistent parasites, culminating in mLNs relapse three months post-treatment. Our analysis of splenic neutrophils, monocytes/macrophages, and dendritic cells post-HePC treatment revealed parasite reservoirs. Single-cell transcriptomic analysis unveiled myeloid cell heterogeneity and indicated a correlation between the failure to eradicate parasites and incomplete immune cell restoration in the spleen. This study provides valuable insights for developing more effective treatments targeting parasite reservoirs that potentially may reduce relapses.

Health sciences/Diseases/Infectious diseases/Parasitic infection

Health sciences/Medical research/Experimental models of disease

Leishmaniasis, a group of neglected tropical diseases (NTD) caused by protozoan parasites of the genus Leishmania, manifests in three primary forms: cutaneous, mucocutaneous, and visceral leishmaniasis (VL). VL, the most severe form, accounts for nearly 20,000 fatalities annually, predominantly in India, Sudan, Ethiopia, Somalia, and Brazil. In Southern Europe, about 700 new cases are reported each year ^1–3, reflecting an expansion of endemic leishmaniasis, driven by the presence of the sand fly vector and increased numbers of migrants and travelers ^2,4.

Several anti-leishmanial drugs are available to effectively treat VL, Amphotericin B, Pentamidine, and Miltefosine (HePC), are among the most widely used. Despite their efficacy in reducing the Leishmania burden, persistence and relapse have been reported in humans ^5,6. Thus, HePC, although showing an initial cure rate of over 90%, results in relapses in approximately 20% of the patients within 12 months post-treatment ⁷, with lower efficacy in children compared to adults ⁸. The increasing relapse rate, particularly in HIV-Leishmania co-infected patients in Asia and Africa ^7,9, and the onset of Post Kala Azar Dermal Leishmaniasis (PKDL) post-treatment ¹⁰ demonstrate that despite efficient anti-leishmanial therapies, relapsing disease emerges due to failure to completely eradicate the parasite after treatment and a lack of immune competence that leads to a loss of control of parasite reservoirs. The failure to eradicate parasites may favor the emergence of drug-resistant strains ^11–13. Thus, identify the cellular and anatomical parasite reservoirs that persist after treatment is of crucial importance.

Leishmania predominantly infects myeloid cells, including monocytes, macrophages, dendritic cells, and polymorphonuclear neutrophils (PMNs) ¹⁴. PMNs are the first line of phagocytic defense at infection sites ¹⁵, playing a dual role in controlling and disseminating the parasite ^16–19, by reducing the incoming parasite burden and subsequently facilitating the safe passage of surviving parasites to naive host cells ^20,21. Monocytes in the blood are vital for replenishing macrophages in various tissues, including the intestines, and the spleen serves as a reservoir for these undifferentiated monocytes ^22–24. Therefore, understanding the role of these cell populations in maintaining parasite reservoirs under treatment is of interest in VL management.

Because such analyses are impractical because of the challenges in sampling relevant tissue sites in humans, non-human primates (NHP) constitute powerful experimental models for understanding host-pathogen interactions due to their close phylogenetic relationship and similar physiological and anatomical systems to humans ²⁵. We have previously reported that rhesus macaque (RM) infected with L. infantum manifested hypergammaglobulinemia, anemia, splenomegaly, and disorganization of the white pulp similar to that observed in VL patients ^26,27. Thus, RMs offer the opportunity to decipher the early events associated with L. infantum dissemination, particularly to identify parasite reservoirs that persist after HePC. RMs were treated with a daily dose of HePC for 21 days immediately following infection to maximize therapeutic impact and to understand early parasite dissemination and persistence.

Herein, we demonstrate that despite early HePC administration, parasites persist in different anatomical tissues, with an inverse relationship between drug concentration and parasite burden. We identified the myeloid cell subsets that represent the main reservoirs post-HePC interruption. Furthermore, single-cell RNA sequencing reveals the heterogeneity of splenic myeloid cells in treated RMs, highlighting the impact of L. infantum on innate immunity. Together, our findings provide significant advancements in understanding the establishment and persistence of parasite reservoirs, despite aggressive early HePC therapy, and the associated persistent immune alterations in treated RMs.

HePC treatment in L. infantum-infected RMs

To assess the impact of early HePC therapy on parasite seeding, RMs were intravenously infected with L. infantum promastigotes and received early treatment post-infection (on day one). A daily dose of HePC (5 mg/kg) was administrated by oral gavage (Fig. 1A). This dose was adapted to clinical dose and tolerability used in patients ^28,29. RMs were sacrificed at various time-points following the 21-day HePC treatment to assess L. infantum persistence relative to HePC’s pharmacodynamics in tissues. Consequently, the animals developed transient anemia during the first weeks after inoculation, marked by reduced erythrocyte counts (Fig. 1B) and blood hemoglobin levels (Fig. 1C). This state of anemia was not improved by the administration of HePC (Fig. 1B and C). Additionally, we observed an early and transient neutrophilia in infected and HePC-treated RMs but not in infected and untreated RM (Fig. 1D). We also noticed a transient depletion of monocytes at week 4 post-infection in untreated RMs, which was prevented by HePC treatment (Fig. 1E).

Parasite burdens at weeks 2 and 3 post-infection were assessed using a quantitative PCR (qPCR) assay. Parasite kinetoplast DNA was assessed in blood, BM aspirates, and PLNs (Fig. 1F-H). At week 3, the parasite loads were reduced by 99% in HePC-treated RMs both in the blood (32.8 ± 34 vs HePC, 0.02 ± 0.03 copies/10⁶ cells) and BM aspirates (7.1 ± 12 vs HePC, 0.08 ± 0.05 copies/10⁶ cells) (Fig. 1F-G), whereas no significant reduction was observed in PLNs (0.9 ± 0.5 vs HePC, 0.8 ± 0.3 copies/10⁶ cells) (Fig. 1H).

At the peak of infection (week 2), we assessed the levels of IL-1Ra, an inhibitor of inflammation ³⁰ that has been associated with pathogenesis ³¹, and CXCL13, a marker of germinal center activity ³², reported to be lower in the spleen of dogs with canine leishmaniasis ³³. Our results revealed lower levels of CXCL13 and higher levels of IL-1Ra early post-infection in L. infantum-infected RMs compared to uninfected RMs irrespectively of HePC treatment (Fig. I-J). The decrease CXCL13 levels during infection could be consistent with an early abortive Tfh differentiation ²⁷. We also observed a tendency of increased IFN-inducible CXC chemokine (CXCL10/IP-10) during the acute phase in both HePC-treated and non-treated RMs (Fig. 1K). Thus, despite early HePC administration, parasites persist in PLNs, which may alter immunity.

L. infantum persists after HePC treatment

Having observed that parasites are still present in PLNs despite early HePC treatment, we quantified the parasite loads in tissues following HePC therapy interruption (HTi). RMs were sacrificed at 1, 4, and 9 weeks post-HTi. Parasite burden in the spleen, PLNs, and BM aspirates was quantified by qPCR. We also determined the presence of parasites in MLNs, which drain the small and large intestines ³⁴, a known anatomical site of parasite presence in dogs ³⁵. In untreated RMs, the spleen was the most heavily parasitized organ (87.3 ± 100 copies/10⁶ cells) with a parasite load 50- to 100-fold higher than MLNs (2.3 ± 1.2 copies/10⁶ cells) and PLNs (0.75 ± 0.6 copies/10⁶ cells) (Fig. 2A-C). Significant reductions in parasite burden were observed in the spleen and PLNs of HePC-treated animals after HTi, with a similar trend in BM samples (Fig. 2A-B and 2D), however, parasites were still detected despite early HePC therapy. The parasite burdens for treated RM were 5.5 ± 18.2 copies/10⁶ cells in MLNs, 0.4 ± 0.3 copies/10⁶ cells in the spleen, and 0.2 ± 0.1 copies/10⁶ cells in PLNs (Fig. 2A-C). Since we cannot rule out the amplification of dead parasite DNA ³⁶, we performed a parasite outgrowth assay to assess the viability of parasites detected by qPCR. Thus, splenic and PLN cell suspensions of HTi- RMs were serial-diluted and incubated for 21 days, to allow promastigote conversion and multiplication. Our data revealed an outgrowth of L. infantum from both the spleen and PLNs of HePC-treated RMs (Fig. 2E). Therefore, early HePC treatment did not fully eradicate parasites in different anatomical lymphoid organs, providing tissue reserves for L. infantum.

HePC Pharmacodynamics

Given parasite persistence despite HePC administration, we quantified the remaining antileishmanial drug at the time of RM sacrifice. HePC levels were quantified in the spleen, PLN, and MLN cell pellets using UPLC-MS (Fig. 3A). One-week post-treatment interruption, the levels of HePC remained elevated, decreasing thereafter at week 4 post-HTi, but were still detectable after 9 weeks (Fig. 3A). After one week of treatment interruption, splenic HePC levels (1308 ± 1824 pg/10⁶ cells) were two-fold higher than in MLNs (563 ± 769 pg/10⁶ cells) and 10-fold higher than in PLNs (156 ± 214 pg/10⁶ cells) (Fig. 3B-D). We calculated the ratio of HePC amount to parasite burden in these organs at this time point (Fig. 3E). These ratios (1000-fold) were consistent across organs, indicating that despite lower HePC levels in PLNs and MLNs compared to the spleen, the impact on parasite reduction at week 1 post-HTi was similar (Fig. 3E). Interestingly, at week 9 post-HTi, MLN parasite burden was higher than in earlier-sacrificed RMs, suggesting a parasite relapse in the gut (Fig. 3D). Although HePC accumulation differs among lymphoid tissues, it would not seem sufficient to complete parasite eradication, leading to persistent anatomical parasite reservoirs.

Identification of cellular reservoirs that persist in HePC-treated RMs

We then assessed the nature of infected cells in L. infantum infected RMs as well as in RMs having received HePC. Given the scarce number of myeloid cells in LNs for performing cell sorting, we only analyzed the spleen. Myeloid cell subsets were identified from total splenic cell suspension including neutrophils and sorted by flow cytometry following the gating strategy shown in Fig. 4A. After exclusion of lymphoid cells (CD3⁺CD20⁻ and CD3⁻CD20⁺), neutrophils are mostly contained in the HLA-DR⁻ population (Fig. 4A). From the HLA-DR⁺ population, we identified monocytes/macrophages (CD11b^highCD14^high) and a subset including dendritic cells (DCs, CD11b^lowCD14^low) (Fig. 4A). The numbers of splenic HLA-DR⁺CD14^highCD11b^high and HLA-DR⁺CD14^lowCD11b^low increased in L. infantum-infected RMs, whereas no difference was observed for neutrophils (Fig. 4B), corroborating the myeloid infiltration observed in mice models ³⁷. HePC treatment decreased HLA-DR⁺CD14^highCD11b^high and HLA-DR⁺CD14^lowCD11b^low cell numbers, although they remained higher compared to non-infected RMs (Fig. 4B). We calculated the total number of parasites considering both the number of cell subsets per spleen and the number of parasites in sorted cells. Our results indicated that both CD11b^highCD14^high and CD11b^lowCD14^low subsets contained 2- to 10-fold more parasites than neutrophils in untreated animals (Fig. 4C). Although HePC reduced the amounts of infected neutrophils, the differences were not statistically significant (Fig. 4C). The same tendency occurred for both HLA-DR⁺CD14^highCD11b^high and HLA-DR⁺CD14^lowCD11^low cell subsets (Fig. 4C). In summary, we demonstrated the persistence of infected myeloid cells despite the early administration of HePC.

Persistent heterogeneity of splenic myeloid cell populations despite HePC

We next evaluated several myeloid cell biomarkers that can be impacted by parasite persistence in the spleen (Fig. 5A-C). First, we evaluated the expression of CD36, a class B scavenger receptor reported to be internalized upon amastigote contact ³⁸. We observed lower percentage of HLA-DR⁺CD14^highCD11b^high expressing CD36 in L. infantum-infected RMs that is recovered after HePC treatment (Fig. 5A). CD354, a potent amplifier of immune reactions ³⁹, already described during VL ⁴⁰, was enhanced in L. infantum-infected RMs but the percentage remains higher in HTi RMs (Fig. 5B). Finally, we assessed the surface expression of CD183 (CXCR3), a chemokine receptor that binds CXCL10/IP-10 and contributes to the recruitment of inflammatory myeloid cells to damaged tissues ^41,42. Herein, we found higher levels of CD183 on HLA-DR⁺CD14^lowCD11^low in L. infantum-infected RMs (Fig. 5C). These results suggested a heterogeneity of myeloid cells identified in the spleen and a partial restoration associated with HePC treatment.

Single-cell transcriptomic analysis of splenic cells

To gain deeper insight into splenic cells in L. infantum and HePC-treated RMs, we utilized single-cell transcriptomic analysis (scRNA). This approach helps to understand cell heterogeneity based on gene expression ^43,44. After eliminating erythrocytes and dead cells using ficoll density gradient centrifugation, we processed the cells with the BD Rhapsody Single-Cell Analysis System. Post quality control, cells were clustered by their differential gene expression (DEG) profiles. Figure 6A shows the Uniform Manifold Approximation and Projection (UMAP) visualization of splenic cell populations derived from both naïve, L. infantum-infected, and HTi-treated RMs. Whereas the expression of CD3E and MS4A1 transcripts defined T and B cell populations, respectively, the expression of MAMU-DRA, CD163, CD68, FCGR3, and CLEC9A transcripts defined myeloid cells (Fig. 6 panels B-H). The heatmap shows the hierarchical clustering of DEG with the top 10 genes (Fig. 6I). The relative average gene expression against the percentage of expressed cells related to the clustering is showed in Fig. 6J. Thus, genes associated with myeloid cells are primarily expressed in clusters 9 and 12.

To better assess myeloid cell heterogeneity, we reruned these two clusters (clusters 9 and 12), allowing us to identify eight clusters (Fig. 7A, B, and Tables S1-8). These included genes related to dendritic cells (cluster 3), in which MAMU-DRA, MAMU-DRB1, IFI30, MS4A6A, CLEC9A, SLAMF7, and BHLEH40 transcripts are enriched (Fig. 7C). A second group is characterized by an enrichment of IRF8, GZMB, SELL, LILRA3, and PTPRS (cluster 4), indicating plasmacytoid dendritic cells (pDC). A third group of transcripts defining macrophage populations was observed (cluster 5) that comprise transcriptional signatures in which CD68, C1QC, C1QB, MAF, HMOX1, APOE, and CD209 transcripts are generally associated with tissue-resident macrophage signature, whereas CD163, MRC1 (encoding for CD206), and SLC40A1 transcripts are reminiscent to M2 macrophage signature (Fig. 7C) ^45–48. Clustering analysis also revealed that cluster 6 comprises transcriptionally signatures associated with non-classical monocytes (FCGR3 (CD16), CX3CR1, KLF4, ITGAX, and PILRA transcripts) and tumor-associated macrophages (S100A4, S100A6, C5AR1, and TCF7L2). Finally, cluster 7 featured genes linked to both myeloid (C1QC, C1QB, HMOX1, and MAF, cluster 5) and B cells (FCRL1, BANK1, EBF1, and KLHL14) (Fig. 7C); this latter gene being generally associated with Follicular B cells. This observation suggested the presence of tangible macrophages having the capacity to phagocyte and eliminate dying cells within splenic B cell follicles ^49–51. Three additional clusters named 0,1 and 2 were less defined regarding gene signature. Cluster 0 expressed higher level of DAPK1 (Death Associated Protein Kinase 1) and NLRP1 (NLR family pyrin domain containing 1) genes, which are associated with inflammation (Fig. 7C).

The UMAP profiles shows the distribution of myeloid cell clusters in naïve, Leishmania-infected, and HePC treated RMs (Fig. 7D-F). Notably, cluster 5 disappeared (Fig. 7D) and cluster 6 emerged in the spleen of L. infantum-infected RMs (Fig. 7E), indicating a profound alteration in the myeloid cell subsets. The proportion of pDC (cluster 4) increased in L. infantum-infected RM (Fig. 7D vs E and 7H, supplemental Fig. 1), whereas DCs (cluster 3) remained relatively unchanged (Fig. 7G; supplemental Fig. 1). The scRNA transcriptomics analysis highlighted an intermediate profile of splenic myeloid cell populations isolated from HTi RM, compared to naïve and L. infantum-infected RMs (Fig. 7E-F). Thus, similarly to L. infantum-infected RM, cluster 6 is still present in HTi RMs (Fig. 7E vs 7F), as is cluster 5 compared to uninfected RM (Fig. 7D vs 7E).

Venn diagrams illustrated the numbers of common and discriminative gene transcripts in clusters 3 to 6 (Fig. 7G-J), with DEG listed in supplementary tables (Tables S9-12). We noticed a persistent immune activation (Gene ontology [GO]:0001775, p value = 8.153E-10) in cluster 3 (DC) despite early treatment (supplemental Fig. 2). Cluster 4 (pDC) also demonstrated a significant association with positive regulation of signal transduction (GO:0009967, p value = 1.568E-5). Most importantly, in cluster 6, genes related to external stimulus (GO:0043207, p value = 2.253E-25), cellular development (GO:2000026, p value = 3.912E-16), and Golgi and reticulum endoplasmic component (GO:0005764, p value = 1.438E-10) were prevalent in treated L. infantum-infected RMs (supplemental Fig. 2). The myeloid cell population in cluster 5, though reemerged in early treated RM exhibited an altered gene profile compared to naïve RM.

Altogether, these results demonstrated that despite early therapy, the lack of complete parasite eradication is associated to the absence of full restoration of splenic myeloid cell population and genes profile.

It is generally accepted that current treatment options for VL usually achieve long-term clinical success in most immunocompetent patients. However, the risk of VL treatment failure and relapse remains and, worryingly, seems to be increasing ⁵². Even with the first line HePC or liposomal amphotericin B treatments, the highest cure rate at 12 months post-treatment never exceeds 94% in the best-case scenarios ⁷. Nevertheless, the cellular pathophysiological events behind parasite persistence after treatment and even clinical cure remain elusive. A key question is identifying the specific cells and tissues where Leishmania parasites persist upon treatment.

Here, analyses of L. infantum-infected RMs treated with HePC revealed evidence of parasite persistence mainly in monocytes/macrophages and DCs. Parasites were still detected in the spleen, PLNs, and MLNs after three weeks of HePC therapy, demonstrating the wide dissemination and persistence of L. infantum despite early treatment. Of interest, parasite burden was inversely proportional to HePC concentration and varied depending on the tissue, with this being particularly relevant in MLNs. These results provide major advances regarding Leishmania therapy and cure, marking the first known identification of parasite reservoirs in RM model closely resembling humans. Thus, even at an early stage of infection, a dose of 5 mg/kg of HePC was insufficient to completely eradicate L. infantum in RMs, leading to the formation of parasite reservoirs. The effectiveness of drug-induced parasite killing is influenced by pharmacokinetics and pharmacodynamics as well as host-factors. VL treatment failure and the risk of relapse are still present and, worryingly, seem to be increasing ⁵². A high occurrence of relapses, have been proposed to be associated to insufficient drug exposure ⁵³, and questioned the long-term efficacy of anti-leishmanial drugs. Of interest, we observed that PLNs are less sensitive to HePC-mediated parasite killing during the early phase of the treatment. Furthermore, our observation that several anatomical lymphoid sites contain parasites after HePC treatment pave the way of our understanding of VL cure, requiring particular attention to such reserves. However, we cannot exclude that other reservoirs persist under HePC in particular in the genital tract ⁵⁴. This parasite persistence could contribute to future relapses and emergence of resistant strains ^11–13.

Earlier reports in rodents demonstrated the importance of an effective host immune response during chemotherapy against Leishmania. Furthermore, defects or alterations in immune cells may compromise the efficacy of drugs ^55–57, a concern especially relevant in patients undergoing immunosuppressive therapies or those co-infected with HIV. Thus, the emergence of co-infection with HIV is one of the major challenges in VL, which makes therapy more complex and increases the chances of drug resistance and relapse ^58–61. The detection of parasites in the MLNs of L. infantum-infected RMs is certainly not anecdotic for humans and differ from murine VL model in which no intestinal infection is observed in chronically infected mice ⁶². MLNs, a specialized lymphoid organ crucial for intestinal immune responses, are located along the intestine and drain the small and the large intestine ^34,63,64. In VL patients, elevated levels of LPS from microbial translocation, as indicated by I-FABP levels, has been reported ⁶⁵ and are more pronounced in HIV co-infected individuals ^66,67. The gut, a predominant site for HIV replication and a viral reservoir, is of particular importance in potential VL cure strategies ^68–70. Therefore, our observation that L. infantum parasites persist in MLNs, with a high parasite burden observed 3 months after HePC interruption, may be crucial in understanding VL relapse, especially in people living with HIV.

Our results further revealed that IL-1Ra and CXCL13 levels do not return to pre-infection levels. CXCL13 is a marker of germinal center activity ³² and it is essential in homing of B and Tfh cells. Lowered levels of CXCL13 are consistent with our previous report showing early abortive Tfh differentiation and abnormal splenic architecture in RMs ²⁷. The inability of HePC to restore CXCL13 levels suggests persistence alteration of lymphoid follicles, and incomplete recovery of the splenic cell subsets despite early treatment. Infection of monocytes and neutrophils by Leishmania is well documented ¹⁸. Splenic monocytes represent a large reservoir of undifferentiated cells that can be mobilized in response to injury ²⁴. Therefore, Leishmania infection of splenic monocytes may contribute to parasite dissemination in distal inflamed tissues contributing to parasite relapse. Whereas neutrophils are considered short-lived cells, our data intriguingly indicate they sustain infection in the spleen. This may reflect that either infected cells in the tissues persist longer or become reinfected after HePC interruption due to the release of amastigote from ruptured splenic monocytes/DCs. Given that a certain plasticity of neutrophils has been previously described in cancer in which environmental factors prolong their survival ^71–73, it cannot be excluded that infected PMNs survive longer, as we and others have previously reported for myeloid cells ^74–77. Thus, the infection of granulocytes in tissues may act as Trojan horses for parasites reducing the efficacy of treatment. Indeed, whereas most of the drugs kill extracellular parasites, just a few of them have the capacity to kill parasites inside host cells representing a caveat in Leishmania drug discovery. This persistence of parasite burden in tissues may also contribute to induce distinct myeloid polarization ^78–84, affecting DC maturation ^85,86, and polarizing immune cells towards non-protective cells ⁸⁷. We found that splenic DC expresses CXCR3 (CD183), a marker of cDC1 associated with tissue inflammation ⁸⁸ and parasite elimination ⁴¹. We also observed higher expression of CD354, which not only mediates innate immune response ³⁹ but also promotes macrophage survival ⁸⁹.

Transcriptomics analyses clearly demonstrated the presence of different myeloid cell populations as previously described ^45–48. This unsupervised approach provides a comprehensive atlas of splenic myeloid populations that are reprogrammed by L. infantum infection. This led to the identification of new cell subsets that persist after HePC treatment. Collectively, these analyses indicate incomplete immune restoration and continued altered myeloid cell populations, consistent with the detection of parasite in the spleen despite 21 days of an early HePC treatment. Unlike a recent report in a mouse infected with L. donovani ⁹⁰, the alignment of L. infantum genome concomitantly with RM genome did not confirmed parasite presence in these myeloid cell subsets in comparison to uninfected RMs. While 61,484 reads aligned with the parasite genome, > 99% of these alignments were either in intergenic or intronic regions. Furthermore, all the reads that mapped on the L. infantum genome are heavily clipped leading us to consider them as background noise. Thus, a possible limitation of this study could relate to the number isolated cells or to the difference in visceral VL models. Cell enrichment might enhance the sensitivity of Leishmania detection through scRNA analysis.

In conclusion, early therapy can reduce parasite burden in lymphoid organs but not effectively enough to prevent the establishment of tissue reservoirs. Importantly, these anatomical and cellular reservoirs may reactivate, mainly but not only, in immunocompromised patients which will impact on patient’s quality of life. Therefore, enhancing current therapies to achieve a sterilizing cure, capable of eradicating the formation of competent reservoirs associated with altered immune profile of myeloid cells, is of great importance.

Animal, parasites, and infections. Ten female rhesus macaques (Macaca mulatta) of Chinese origin were used in this study. Animals were seronegative for STLV-1 (Simian T Leukemia Virus type-1), SRV-1 (type D retrovirus), herpes-B viruses, and SIVmac. All animals were housed in compliance with French regulations for animal care and use (Cynbiose SAS, VetAgro-Sup, Marcy l’Étoile, France). They were inoculated intravenously via the saphenous vein with 2x10⁷ stationary-phase L. infantum promastigotes (clone MHOM/MA/67/ITMAP-263) per kg of body weight. At day one post-infection RMs were treated for 21 days by nasogastric gavage with 5 mg/kg of HePC (Sigma-Aldrich). RMs were sacrificed at three time points (1, 4, and 9 weeks post-HePC interruption). Tissues were collected, including axillary and inguinal peripheral LNs (PLNs), mesenteric LNs (MLNs), spleen, bone marrow (BM) aspirates, and peripheral blood. A hemogram was performed by Biovelys (Vet Agro Sup, Lyon).

Ethics statement. All the animal experiments described in the present study were conducted at Cynbiose SAS (VetAgro-Sup, Marcy l’Étoile, France). The study was reviewed by the Animal Welfare Body of Cynbiose and the Ethics Committee of VetAgro-Sup approved under the number 4305-2016020215429763. All experiments were conducted in accordance with the European Directive 2010/63/UE as published in the French Official Journal of February 7th, 2013.

Parasite quantification. DNA was extracted from cell pellets of blood or tissues using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s protocol. A TaqMan-based qPCR assay was used to detect and quantify L. infantum kinetoplastid DNA minicircles as described elsewhere ^27,91. Briefly, mixtures were composed of TaqMan Universal Master mix II, no UNG (2X, Applied Biosystems), 400 nM of forward primer (5’CTTTTCTGGTCCTCCGGGTAGG-3’), 400 nM of reverse primer (5’-CCACCCGGCCCTATTTTACACCAA-3’) and 250 nM of TaqMan probe (5’-FAM-TTTTCGCAGAACGCCCCTACCCGC-TAMRA-3’). Thermocycling settings consisted of one hold of 15 min at 95°C followed by a two-step temperature (95°C for 15 s and 60°C for 60 s) over 40 cycles in an ABI Prism 7900 HT (Applied Biosystems). A standard curve was established, and sample normalization was performed by quantifying the macaque albumin host gene. Thus, parasite load was expressed as kinetoplastid copy number/10⁶ nucleated cells.

Quantification of cytokines/chemokines by ELISA. ELISA was used to quantify IL-1Ra (Human IL-1ra/IL-1F3, R&D Systems) and CXCL13 (Human CXCL13/BLC/BCA-1, R&D Systems), while CXCL10/IP-10 was quantified by flow cytometry using a LEGENDplex assay (Biolegend). Experiments were performed according to the manufacturer’s instructions.

Parasite outgrowth assay. Parasites were quantified in the spleen and peripheral LN (PLN) using culture microtitration. Briefly, cell suspension was resuspended in Schneider’s drosophila medium (Gibco) supplemented with 20% heat-inactivated FCS (Gibco), penicillin/streptomycin (100 U/mL, Life technologies), glutamine (2 mM, Life technologies) and sodium pyruvate (1 mM, Life technologies). Serial dilutions were performed in a 96-well plate ranging from 1x10⁶ to 5 cells. Plates were sealed hermetically and incubated at 26°C for 21 days to allow promastigote conversion and multiplication. After that, wells were incubated with Alamar blue reagent (ThermoFisher) at 37°C, and after overnight incubation, fluorescence was monitored (λex = 560 nm and λem = 590 nm) on an EnSpire plate reader (Perkin Emer) ⁹². Parasite density was calculated through a standard curve ranging from 1x10⁷ to 1 L. infantum promastigotes.

HePC quantification. Pharmacokinetics of HePC were performed in the sera and PLN and MLN splenic cells. After protein precipitation with acetonitrile, dried cell pellet and sera were analyzed using a Waters ACQUITY ultra-performance liquid chromatography (UPLC) system with a 2.1x50-mm, 1.7-m ACQUITY UPLC BEH RP18 shield column coupled to a Waters Qua9ro Premier TQ mass spectrometer operated in positive ion electrospray and multiple reaction monitoring mode (MRM).

Cell sorting and immunophenotyping. Cells from the spleen were stained with a cocktail of antibodies, including anti-CD3 (clone SP34-2), anti-CD11b (clone ICRF44), and anti-CD20 (clone 2H7) from BD biosciences and anti-CD14 (clone TÜK-4) and anti-HLA-DR (clone AC122) from Miltenyi Biotec. Cells were then fixed with paraformaldehyde and sorted (FACS ARIA III cytometer, BD Biosciences) for DNA parasite quantification by qPCR. In parallel, splenocytes were also stained for immunophenotyping with anti-CD36 (clone FA6.152) and anti-CD16 (clone 3G8) from Beckman Coulter, anti-CD14 (clone TÜK-4) and anti-HLA-DR (clone AC122) from Miltenyi Biotec, anti-CD354 (clone TREM-26) from Biolegend and anti-CD183 (clone 1C6/CXCR3) from BD biosciences. The purity of each cell type was higher than 97%.

Single-cell RNA-seq. To conduct single-cell analysis, the genomic sequence for Macaca mulatta was obtained from the Ensembl database (accession number GCA_003339765.3). The corresponding sequence from Leishmania Infantum was obtained from NCBI with accession number GCA_900500625.2. The genomes and their annotations were integrated using the "Reference files generator for BD Rhapsody sequencing" pipeline, resulting in the creation of a consolidated reference tailored for single-cell analysis. The raw sequencing reads underwent processing through the BD Rhapsody analysis pipeline, encompassing tasks such as read quality filtering, read annotation, putative cell determination, and the generation of a single-cell expression matrix. To organize cell clusters based on gene expression profiles, we employed a clustering algorithm utilizing the Seurat package (version 4.3.0.1). Initially, a standard linear transformation was applied as a preprocessing step using the "Scaledata" function before conducting principal component analysis (PCA) on each gene. Cell clustering was achieved through the "FindNeighbors" function on the first 20 dimensions, followed by the "FindClusters" function with a resolution of 0.3–0.5. Visualization was performed using Uniform Manifold Approximation and Projection (UMAP) with specific parameters, including "dims" set to 1:20, "n.neighbors" set to 50, "min.dist" set to 0.3, and "spread" set to 1.

For the generation of the unsupervised heatmap, the "FindAllMarkers" function was employed with specific settings, including "only.pos = TRUE," "min.pct = 0.25," and "logfc.threshold = 0.58." Thus, we identified markers in each cluster through differential expression gene (DEG) based on average log2 fold change (avg_log2FC) and p-value criteria, utilizing the "DoHeatmap" function for visualization. The Venn diagrams were constructed using the VennDiagram package (version 1.7.3) and gene tables were generated using the "FindAllMarkers" function. Upon identifying common gene signatures from the Venn diagrams, we characterized genes of interest within clusters. Subsequently, a supervised heatmap was generated using the "DotPlot" function. Gene enrichment analysis was performed using ToppFun enrichment analysis to identify biological pathways and processes.

Statistical analyses. Statistics were performed with the GraphPad Prism 6 software. Data are presented as means ± SEM. Statistical differences were assessed using the Mann-Whitney U test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Acknowledgments

We acknowledge Stéphanie Blanc and Hugues Contamin (Cynbiose, Marcy l’Etoile, France) for the non-human primate housing and their support. We also thank Stéphanie Dupuy from BioMedTech Facilities (INSERM US36, CNRS UMS2009, Université Paris Cité, France) for the cytometry platform.

This work was supported by the European Community’s Seventh Framework Programme under grant agreement No.602773 (Project KINDRED) and grant agreement N°603240 (Project NMTryp) and Infect-ERA ERA-NET (Project InLeish) to JE. RS This work has also been funded by National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020.RS was supported by the Fundação para a Ciência e Tecnologia (FCT) (contract CEECIND/00185/2020). JE also thanks the Canada Research Chair program for financial assistance. SA was supported by a post-doctoral fellowship granted by Fédération pour la Recherche Médicale (FRM: SPF20160936115) and Infect-Era (ANR-16-IFEC-0002). MP by a doctoral fellowship granted by Infect-Era (ANR-16-IFEC-0002) and SB by a fellowship support from Formation Desjardins pour la Recherche et l'Innovation (CHU de Québec). VR by a post-doctoral fellowship granted by KINDRED.

Author contributions:

MP, SB, VR, YF, CB, CS, JC, CJB, GR, OZA and SA performed the experiments and analyses under the supervision of AD, AP, SA and JE. MPC, RS, JMD and JE acquired the fundings. MP, MPC, RS, ACS, JMD, SA and JE wrote and edit the manuscript.

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Single-cell transcriptomics reveals altered myeloid cell profiles associated with the early establishment of leishmania reservoirs

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Abstract

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INTRODUCTION

RESULTS

HePC Pharmacodynamics

Identification of cellular reservoirs that persist in HePC-treated RMs

Persistent heterogeneity of splenic myeloid cell populations despite HePC

Single-cell transcriptomic analysis of splenic cells

DISCUSSION

METHODS

Declarations

References

Additional Declarations

Supplementary Files

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