Platelet DKK1 promotes tolerogenic dendritic cells and non-healing responses in cutaneous leishmaniasis
Olivia C. Ihedioha, Anutr Sivakoses, Haley Q. Marcarian, Malini Sajeev, Diane McMahon-Pratt, Alfred L.M. Bothwell
TL;DR
Platelet DKK1 promotes immune tolerance and worsens infection outcomes in cutaneous leishmaniasis by influencing specific immune cell responses.
Contribution
This study reveals that platelet DKK1 regulates Th2 and IL-10-Th1 responses by inducing tolerogenic DC-10 cells during Leishmania infection.
Findings
Mice deficient in platelet DKK1 show reduced neutrophilic responses and contain Leishmania infection better.
DKK1 promotes M2 macrophages that support parasite proliferation.
DKK1 induces tolerogenic DC-10 cells, regulating Th2 and IL-10-Th1 immune responses.
Abstract
Activation of platelet TLR1/2 at the initiation of Leishmania major infection results in the release of the Wnt antagonist Dickkopf-1 (DKK1). To examine the role of platelet MyD88 and/or DKK1 in regulating the immune response, genetic approaches using BALB/c mice deficient in platelet MyD88 (MyD88PKO) or DKK1 (DKK1PKO) were used. Genetically deficient mice exhibit significant changes in the immune response throughout infection. Initially, the levels of activated neutrophils and neutrophil-platelet aggregates were reduced at the site of infection. Subsequently, an anti-leishmanial Th1-response was observed in these mice, which in contrast to their WT counterparts, fail to develop progressive lesions. Further, WT mice developed comparatively elevated levels of CD206+ M2 macrophages and tolerogenic DC-10 cells. Previously, Chae et al. showed that DKK1 promoted type 2 immunity during…
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Taxonomy
TopicsResearch on Leishmaniasis Studies · Phagocytosis and Immune Regulation · Mast cells and histamine
Introduction
Members of the genus of parasite protozoa, Leishmania, cause a range of clinical manifestations, from limited cutaneous and mucocutaneous disease to potentially fatal visceral disease.1 Within cutaneous leishmaniasis, clinical manifestations can present from self-healing lesions to chronic debilitating infection.2 The control or progression of disease is complex and is determined by an interplay between the Leishmania parasite and host factors, involving a joint effort of the innate and adaptive immune responses.3 Thus, the innate immune cells (neutrophils, dendritic cells [DCs], macrophages), in addition to CD8^+^ and CD4^+^ T cells, participate in regulating disease outcome.4^,^5 Dendritic cells are critical in determining disease outcome; further, dendritic cell subsets (dendritic cell type 1 [cDC1] and type 2 [cDC2]) regulate disease outcome by preferentially driving Th1 and Th2 responses, respectively.6 Dendritic cDC1 cells direct the differentiation of naive T cells to host-protective Th1 cells through the production of IL-12.7^,^8 An IL-12 response promotes macrophage activation through cognate interactions and/or the release of cytokines, including interferon (IFN-γ) and tumor necrosis factor (TNF-α), which control infection.9^,^10^,^11 Conversely, the sustained release of IL-4 and IL-10 in the draining lymph nodes of Leishmania major (L. major)-infected BALB/c mice andthe differentiation and expansion of Th2 cells12^,^13^,^ result in M2 macrophage development, impaired intracellular killing of Leishmania, and disease progression.14^,^15 Furthermore, studies have identified a subset of CD4^+^ Th1 cells that produce IL-10 in response to Leishmania infection.16^,^17^,^18 Th1-IL-10 cells develop in response to an early, strong inflammatory setting and can be important mediators of immune suppression in chronic cutaneous leishmaniasis.16 IL-10-producing CD4 Th1 cells are T-bet^+^, Foxp3^−^, and CD25^−^ and maintain an effector phenotype.16 A subset of tolerogenic, IL-10-producing dendritic cells (DC-10) is capable of polarizing CD4^+^ T cells toward an IL-10-producing Th1 phenotype. Phenotypically, tolerogenic DC-10 cells display MHC II^low^, CD80^low^, CD86^low^, and CD40 ^low^ markers.19 Further, monocyte-derived tolerogenic DC-10 cells fail to produce IL-12, and important driver of a Th1 healing response.20
The innate immune response (involving neutrophils, dendritic cells, platelets, and macrophages) drives the development of acquired immune response (healing/non-healing) in murine leishmaniasis and acts within the first few hours post-infection.3 This early response also detects invading pathogens through specific receptors that recognize the pathogen-associated molecular patterns (PAMPs) conserved on invading microbes.21 Specifically, the family of Toll-like receptors (TLRs) recognizes a variety of PAMPs (lipids, proteins, peptidoglycan, DNA, and RNA) and triggers innate immune responses, subsequently shaping the adaptive immune response.22 The myeloid differentiation protein 88 (MyD88) has been demonstrated to be a critical downstream protein in initiating TLR signaling.23 Platelets are highly abundant and ubiquitously well-positioned to act as innate/first responders in the detection of a pathogen challenge, triggering the host’s immune response to fight the developing infection.24 Previous studies have confirmed the expression of TLR1-7 and MyD88 in human and mouse platelets.25 Activation via TLR1, TLR2, and TLR4 leads to platelet granule exocytosis (release of mediators and cytokines) and occurs via the MyD88-dependent pathway.26^,^27 Further, activation through platelet TLR causes the modulation of leukocyte inflammatory responses and heterotypic cell aggregation.28
Our previous studies showed that leukocyte-platelet aggregates (LPAs) are required for leukocyte migration to the infection site and induction of Th2 immune response during Leishmania infection.29^,^30 This aberrantly high LPA formation is driven by DKK1 released by activated platelets following recognition of L. major-derived lipophosphoglycan (LPG) via TLR1/2.29 Since MyD88 is a downstream adapter of the TLR1/2,28 in the current study, we utilized mice with selective deletion of MyD88 or DKK1 from platelets to establish that DKK1 released via the MyD88-dependent pathway determines disease outcome. These studies further show that DKK1 critically promotes the shift of dendritic cells toward the DC-10 phenotype, leading to inhibition of dendritic cell maturation and the development of a chronic Th2 immune response, which is critical for disease progression in L. major infection. Together with the regulation of polymorphonuclear leukocyte (PMN)/LPA aggregation, which selectively draws host cells to the site of infection, DKK1 controls the developing milieu promoting susceptibility to infection.
Results
Minimal P-selectin expression in activated platelets from infected MyD88(PKO) mice on days 3 and 14 PI
P-selectin is an established marker for detecting activated platelets.31 Studies have shown that LPG derived from L. major engages TLR2 expressed on immune cells and modulates anti-leishmanial immune responses.32^,^33^,^34^,^35 Our previous study showed that L. major-derived LPG acts as a primary virulence factor that triggers platelet activation through TLR1/2, leading to the production of DKK1.29 Based on the knowledge that the TLR1/2 protein complex initiates its signaling pathway via the adapter molecule MyD88, this study examined whether LPG and MyD88 influence platelet activation by modulating platelet TLR2-mediated signaling.
Thus, P-selectin expression was assessed at varying time points in platelets obtained from infected BALB/c, DKK1^(PKO)^, MyD88^(PKO)^, and non-infected BALB/c mice. Compared to the infected BALB/c and DKK1^(PKO)^ mice, the expression of P-selectin was significantly reduced in platelets obtained from infected MyD88^(PKO)^ mice on days 3 and 14 PI (Figures S1B and S1C). Whereas DKK1^(PKO)^ mice demonstrated no significant impairment in P-selectin expression. Additionally, the level of P-selectin expression was suppressed on day 14 PI (compared to day 3 PI) in all experimentally infected mice (Figures S1B and S1C). Our previous study showed that platelet activation and P-selectin expression are mainly controlled by LPG.28 Since the LPG effect diminishes within 72 h. This indicates that, as LPG expression decreases in the mammalian host, it would play little to no important role as the disease progresses. Thus, the reduced level of P-selectin expression on day 14 PI (compared to day 3 PI) might be due to the decline in LPG expression. To exclude the possibility of off-target effects, the baseline platelet characterization was performed. Data showed comparable platelet counts, activation and aggregation in naive wild-type BALB/c, DKK1^(PKO)^, and MyD88^(PKO)^ mice (Figures S2B–S2F). Since the MyD88 adapter protein is fully functional in DKK1^(PKO)^-infected mice, these findings established that DKK1 signaling is indispensable for the platelet activation process in the early stages of L. major infection.
Reduced DKK1 production in infected MyD88(PKO) and DKK1(PKO) mice on day 3 PI
MyD88 is considered the primary downstream signaling protein of TLR1/2,26 and DKK1 production during Leishmania infection is primarily dependent on TLR1/2.29 Thus, to investigate whether the deficiency of MyD88 blocks DKK1 production, the plasma DKK1 levels obtained from infected BALB/c, DKK1^(PKO)^, MyD88^(PKO)^, and non-infected BALB/c mice were compared on day 3 PI. We confirmed significantly decreased production of plasma DKK1 in DKK1^(PKO)^- and MyD88^(PKO)^-infected mice compared to infected BALB/c mice (Figure S3A). Considering the artificial nature of the infection model involving the injection of 2x10^6^ promastigotes into a subcutaneous site, we explored the level of DKK1 production in mice inoculated with fewer parasites (1x10^5^ and 1x10^4^). At day 3 PI, DKK1 responses were observed in BALB/c mice at all parasite doses. Consistent with mice inoculated with 2x10^6^ parasite, there was a significant decrease in plasma DKK1 production in DKK1^(PKO)^ and MyD88^(PKO)^ 1x10^5^ and 1x10^4^ parasite-infected mice compared to infected WT BALB/c mice (Figures S3B–S3D). Relative to day 3 PI, the level of DKK1 production decreased by day 14 PI in BALB/c mice that received 1x10^5^ parasites (Figures S3B–S3C). In addition, the level of DKK1 production in mice that received a low dose of parasite was less than mice that received a higher parasitic dose (Figures S3A–S3D). This finding suggests that fewer promastigotes can still activate the platelet MyD88 signaling pathway to release DKK1.
Impaired LPA and NPA formation in infected MyD88(PKO) and DKK1(PKO) mice on days 3 and 14 PI
Under inflammatory conditions, P-selectin (CD62P) and P-selectin glycoprotein ligand-1 (PSGL-1) mediate leukocyte platelet aggregates formed by adhesion between mature leukocytes and activated platelets.30^,^36^,^37 Our previous work demonstrated that LPG-activated platelets induce P-selectin expression, leading to platelet-leukocyte aggregate formation, which is vital for the initial recruitment of leukocytes to inflammatory sites.29^,^38^,^39 Furthermore, the elevated formation of LPAs, usually seen in the blood of L. major-infected mice, was reduced by pre-treating mice with a DKK1 inhibitor prior to infection with L. major.30 These findings suggest that the leukocyte platelet aggregate is driven by DKK1 production. Since LPG activates the TLR1/2-MyD88 pathway to induce DKK1 production, we initially examined the possibility that the absence of MyD88 protein or failure of DKK1 production could reduce LPA formation in the blood of DKK1^(PKO)^- and MyD88^(PKO)^-infected mice. Relative to the BALB/c-infected mice, LPA formation was significantly reduced in DKK1^(PKO)^- and MyD88^(PKO)^-infected mice on day 3 PI (Figures S4C and S4D). Given these results, we then characterized the levels of neutrophil platelet aggregates (NPAs) within infected tissue using infected BALB/c, DKK1^(PKO)^, MyD88^(PKO)^,and non-infected mice. NPA are essential for the infiltration of activated neutrophils to the infection site. Interestingly, both DKK1^(PKO)^ and MyD88^(PKO)^ mice displayed lower levels of NPA at the site of infection compared to the higher levels observed in infected BALB/c mice on day 3 PI (Figures S4E and S4F). On day 14 PI, all infected mice showed a decrease in LPA and NPA formation compared to day 3 PI (Figures S4C–S4F). Additionally, on day 3PI, the percentage and number of neutrophils at the infection site in DKK1^(PKO)^- and MyD88^(PKO)^-infected mice were significantly decreased (Figures S4G and S4H). Although on day 14 PI, the LPA and NPA levels in DKK1^(PKO)^ and MyD88^(PKO)^ were somewhat lower in comparison to BALB/c mice, this difference was not statistically significant; the LPA and NPA formation were similar in all the infected mouse groups (Figures S4D and S4F). These findings imply that DKK1 produced via the TLR1/2-MyD88 pathway might be crucial for the early, elevated formation of LPA and NPA at the site of infection.
Decreased MPO+, CD11b+ and MHC class II+ neutrophils in MyD88(PKO)- and DKK1(PKO)-infected mice on days 3 and 14 PI
Myeloperoxidase is a primary enzyme abundantly produced by PMN and serves as a marker of PMN maturation and activation.40 Previously, we have demonstrated that DKK1 signaling via PMN-LRP6 elevated neutrophil MPO expression in the infection site of L. major-infected mice.38 To examine whether failure to transmit activation signal due to deficiency of MyD88 or impaired DKK1 production regulates recruitment of activated neutrophils to the infection site, MPO^+^ neutrophils were assessed in cells generated from the footpad of infected BALB/c, DKK1^(PKO)^, MyD88^(PKO)^, and non-infected mice. DKK1^(PKO)^- and MyD88^(PKO)^-infected mice had significantly fewer MPO^+^ neutrophils compared to infected BALB/c mice (Figures 1A and 1B).Figure 1. Decreased MPO^+^, CD11b^+^, and MHC class II^+^ neutrophils in MyD88^(PKO)^- and DKK1^(PKO)^-infected mice(A–G) Six-week-old female WT-BALB/c, MyD88^(PKO)^ and DKK1^(PKO)^ mice were challenged with infective metacyclic promastigote (2 ×10^6^ parasites, n = 5 per group) of L. major via the footpad. Non-infected BALB/c mice (n = 10/2 feet per mouse) were given 0.9% NaCl saline. Cells were isolated from the footpads of all infected and non-infected mice at days 3 and 14 PI. Samples were analyzed by flow cytometry for MPO^+^, CD11b^+^, and MHC class II^+^ neutrophils. A representative flow cytometry dot plot of MPO^+^, CD11b^+^, and MHC class II^+^ neutrophils on day 3 is presented in Figure S5. The percentage of MPO^+^, CD11b^+^, and MHC class II^+^ cells in the different experimental groups is shown in column graphs (A, B, C, D, E, and F), while the absolute number of neutrophils obtained on day 3 PI is indicated (G). In all experiments, infected and non-infected BALB/c mice served as positive and negative controls, respectively. Results are presented as mean (±SEM) and are representative of two independent experiments. One-way ANOVA followed by Bonferroni’s post hoc test was used to analyze the data ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, non-significant (p > 0.05).
Elevated expression of CD11b and MHC class II in neutrophils is another way to identify activated neutrophils.41^,^42 CD11b^+^ and MHC class II^+^ neutrophils obtained from the footpads of infected BALB/c, DKK1^(PKO)^, MyD88^(PKO)^, and non-infected BALB/c mice were evaluated to further determine whether MyD88 activation signals and subsequent DKK1 production are essential in the migration of activated neutrophils to the infection site. We found that DKK1^(PKO)^- and MyD88^(PKO)^-infected animals had significantly fewer CD11b^+^ and MHC II^+^ neutrophils than the infected BALB/c mice (Figures 1C–1F). Additionally, compared to day 3 PI, all infected mice had fewer MPO^+^, CD11b^+^, and MHC II^+^ neutrophils on day 14 PI (Figures 1A–1F). Furthermore, the absolute number of neutrophils obtained on day 3 PI were significantly reduced in DKK1^(PKO)^- and MyD88^(PKO)^-infected mice (Figure 1G). Considering that reduced MPO^+^, CD11b^+^, and MHC class II^+^ neutrophils were found in DKK1^(PKO)^- and MyD88^(PKO)^-infected mice, these findings imply that DKK1 release via the MyD88 signaling pathway promotes sustained neutrophil activation and infiltration to the infection site in BALB/c mice.
Increased CD38+CD206- macrophages and CD8α+CD11b− dendritic cells in infected DKK1(PKO) and MyD88 (PKO) mice at the site of infection
We previously showed that systemic treatment with a DKK1 inhibitor reduced leukocyte accumulation at the inflammatory site following parasite infection.30 Although the role of PMN recruitment and LPA formation clearly depends on DKK1, we considered the possibility that deleting MyD88 or the absence of DKK1 in platelets might also affect the infiltration of macrophages and dendritic cells into the infection site. By day 14 PI, migration of dendritic cells and macrophages to the infection site increased relative to day 7 PI (Figures 2A–2D). In contrast, DKK1^(PKO)^ and MyD88^(PKO)^ mice exhibited significantly reduced infiltration of these immune cells on both days 7 and 14 PI compared to infected BALB/c mice (Figures 2A–2D). Notably, although LPA formation was similar across all groups of mice by day 14 PI, dendritic cell and macrophage levels remained lower in DKK1^(PKO)^ and MyD88^(PKO)^ mice than in WT BALB/c mice.Figure 2. Increased CD38^+^ macrophages and CD8α^+^ dendritic cells in MyD88^(PKO)^- and DKK1^(PKO)^-infected mice(A–D) Six-week-old female WT-BALB/c, MyD88^(PKO)^ and DKK1^(PKO)^ mice were challenged with infective metacyclic promastigote (2 ×10^6^ parasites, n = 5 per group) of L. major via the footpad. One week and two weeks post-infection, cells from the infected foot of each mouse in BALB/c, MyD88^(PKO)^, DKK1^(PKO)^, and non-infected BALB/c mice (n = 10/2 feet per mouse) were harvested and counted for macrophages (A and B) and dendritic cells (C and D) by flow cytometry.(E–H) Two weeks post-infection, the infected foot from each mouse in BALB/c, MyD88^(PKO)^, DKK1^(PKO)^ (n = 5 per group), and non-infected BALB/c mice (n = 10/2 feet per mouse) were examined for CD206^+^ and CD38^+^ macrophages (E and F), as well as CD11b^+^ and CD8α^+^ dendritic cells (G and H) by flow cytometry. Representative flow cytometry dot plots showing the analysis of CD206^+^, CD38^+^ macrophages and CD11b^+^, CD8α^+^ dendritic cells and a dot plot of each sample in all the experimental groups performed on day 14 PI are presented in Figure S6. Results are presented as mean ± SEM and are representative of two independent experiments. Data analysis was done using one-way ANOVA followed by Bonferroni’s post hoc test ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and “ns” indicates non-significant.
Having observed a significant decrease in the frequency of macrophages and dendritic cells in infected DKK1^(PKO)^ and MyD88^(PKO)^ mice, we investigated whether DKK1 influences the M1-M2 balance, thereby shaping the disease outcome. Accordingly, we compared the abundance of M1 (F4/80^+^CD38^+^CD206^-^) and M2 (F4/80^+^CD38^−^CD206^+^) subsets in dermal macrophages obtained from infected BALB/c, DKK1^(PKO)^, MyD88^(PKO)^, and non-infected BALB/c mice. Relative to infected BALB/c mice, DKK1^(PKO)^- and MyD88^(PKO)^-infected mice showed a reduced F4/80^+^CD38^−^CD206^+^ cell population and an increased F4/80^+^CD38^+^CD206^-^ cell population. Notably, the F4/80^+^CD38^−^CD206^+^ cell population dominates in the infection site in BALB/c mice (Figures 2E and 2F).
The cDC1 and cDC2 subsets of conventional dendritic cells at the infection site were distinguished based on the surface expression of CD11c, CD11b, and CD8α molecules, as previously reported.42 In general, the percentage of cDC1 (CD11c^+^CD11b^−^CD8α^+^) and cDC2 (CD11c^+^CD11b^+^CD8α^−^) cells were compared in the experimental and control mice. We observed a significant decrease in CD11c^+^CD11b^+^ cells in infected DKK1^(PKO)^ and MyD88^(PKO)^ mice compared to infected BALB/c mice. However, the portion of CD11c^+^CD8α^+^ cells was significantly higher in infected DKK1^(PKO)^ and MyD88^(PKO)^ mice (Figures 2G and 2H). These data suggest that DKK1 released via the MyD88-dependent pathway promotes the infiltration of M2 macrophages and cDC2 cells at the infection site in BALB/c mice.
Impaired Th2 cytokine production and upregulated IFN-γ in MyD88(PKO)- and DKK1(PKO)-infected mice on weeks 2 and 15 PI
Previously, we have shown using in vitro studies that DKK1 preferentially induces Th2 cytokines.30 To confirm whether failure to transmit activation signals due to a deficiency of MyD88 or a defect in DKK1 production promotes a Th1 response, cytokine production from SLAG-activated lymph node cells obtained from infected BALB/c, DKK1^(PKO)^, MyD88^(PKO)^, and non-infected BALB/c mice was assessed. Compared to infected BALB/c, the production of IL-10 and IL-4 was significantly inhibited in infected DKK1^(PKO)^ and MyD88^(PKO)^ mice on weeks 2 and 15 PI. However, IFN-γ production was elevated in the mutant mice (Figures 3A–3F). These data confirmed that the Th2 response in BALB/c infected mice is dependent on DKK1 produced via the TLR1/2-MyD88-dependent pathway.Figure 3DKK1 promotes IL-10 induction in BALB/c-infected mice(A–F) Six-week-old female BALB/c, MyD88^(PKO)^ and DKK1^(PKO)^ mice were challenged with infective metacyclic promastigote (2 ×10^6^ parasites, n = 5 per group) of L. major via the footpad. Non-infected BALB/c mice (n = 5) were given 0.9% NaCl saline. Two or fifteen weeks post-infection, cells from draining lymph nodes were isolated. Lymph node cells were incubated with SLAG (50 μg/mL derived from WT parasites). Cell culture supernatant samples obtained were analyzed by ELISA for cytokine production, as shown in the column graphs (A–F). In all the experiments, BALB/c infected and non-infected mice served as positive and negative controls, respectively. Results are presented as mean ± SEM and are representative of two independent experiments. One-way ANOVA followed by Bonferroni’s post hoc test was used to analyze the data ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, “ns” indicates not significant (p > 0.05).
Reduced IL-10-Th1 cells, CD4+IL-10+ and CD8+IL-10+ T cells in the draining lymph nodes of MyD88(PKO)- and DKK1(PKO)-infected mice
Since MyD88^(PKO)^- and DKK1^(PKO)^-infected mice produce high levels of IFN-γ and low levels of IL-10 cytokines, we further evaluated the relative contribution of T cell subsets in producing these cytokines. The results showed that the percentage and number of CD4^+^ T cells were comparable in the draining lymph nodes of infected BALB/c, MyD88^(PKO)^ and DKK1^(PKO)^ mice (Figures 4A and 4B). However, the percentage and number of CD8^+^T cells were significantly lower in MyD88^(PKO)^- and DKK1^(PKO)^-infected mice compared to infected BALB/c mice (Figures 4C and 4D). The total number of CD3^+^T cells was significantly reduced in MyD88^(PKO)^- and DKK1^(PKO)^-infected mice compared to infected BALB/c mice. (Figure S7C). In addition, infected MyD88^(PKO)^ and DKK1^(PKO)^ mice showed a higher percentage of CD4^+^ and CD8^+^ T cell-producing IFN-γ. Similarly, the MFI of IFN-γ-producing CD4^+^ and CD8^+^ T cells was significantly higher in these mutant mice. However, there was an increased MFI and percentage of IL-10-producing CD4^+^ and CD8^+^ T cells, while IFN-γ-producing CD4^+^ and CD8^+^ T cells were low in the infected BALB/mice (Figures 4E–4L) Interestingly, the percentage of IL-10-Th1 cells was elevated in BALB/c-infected mice compared to MyD88^(PKO)^- and DKK1^(PKO)^-infected mice (Figure 4M). This suggests that DKK1 generated via the MyD88-dependent pathway promotes polarization of immune suppressive T cells in BALB/c-infected mice.Figure 4. Impaired CD8^+^IL-10^+^IFN-γ^−^, CD4^+^IL-10^+^IFN-γ^−^, and CD4^+^IL-10^+^IFNg^+^ T cells in MyD88^(PKO)^- and DKK1^(PKO)^-infected mice on day 14 PI(A–M) Six-week-old female BALB/c, MyD88^(PKO)^, and DKK1^(PKO)^ mice were challenged with infective metacyclic promastigote (2 ×10^6^ parasites, n = 5 per group) of L. major via the footpad. Non-infected BALB/c mice (n = 5) were given 0.9% NaCl saline. Two weeks post-infection, the draining and non-draining lymph node cells were isolated. Lymph node cells were incubated with a cell stimulation cocktail for 5 h. After a further 3 h in BFA, cells were stained for intracellular IL-10 and IFN-γ. The stained lymph node cells from each mouse in BALB/c, MyD88^(PKO)^- and DKK1^(PKO)^-infected mice were determined for the percentage and frequency of CD4^+^ and CD8^+^ T cells (A, B, C, and B), percentage and MFI of CD8^+^IFN-γ^+^ or CD8^+^IL-10^+^ T cells (E, F, G, and H), percentage and MFI of CD4^+^IFN-γ^+^ or CD4^+^IL-10^+^ T cells (I, J, K, and L), and percentage of CD4^+^ IFN-γ^+^ IL-10^+^ T cells (M) by flow cytometry. Representative flow cytometry dot plots showing the analyses CD4^+^ and CD8^+^ T cells, CD8^+^IFN-γ^+^ or CD8^+^IL-10^+^ T cells, CD4^+^IFN-γ^+^ or CD4^+^IL-10^+^ T cells, and CD4^+^ IFN-γ^+^ IL-10^+^ T cells performed on day 14 PI is indicated (Figure S7A). Results are presented as mean ± SEM. and are representative of triplicate experiments. Data analysis was done using one-way ANOVA followed by Bonferroni’s post hoc test ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and “ns” indicates non-significant.
rDKK1-treated dendritic cells display tolerogenic DC-10 phenotype
Tolerogenic DC-10 cells are potent inducers of IL-10-producing Th1 cells, well-known to block Leishmania resolution by suppressing Th1 cell-mediated immunity.15^,^19 Tolerogenic DC-10 are usually detected using reduced expression of antigen-presenting molecules and elevated IL-10 production.19 Given that IL-10-producing Th1 cells are elevated in BALB/c-infected mice, we consider the possibility that DKK1 increases IL-10-producing Th1 cells by inducing tolerogenic DC-10. Thus, naive bone marrow-derived dendritic cells were activated in vitro with r-TNF-α, r-DKK1, r-IL-10, or (r-TNF-α + rDKK1). Compared to r-TNF-activated dendritic cells, the expression and percentage of MHC II^+^, CD86^+^, and CD80^+^ molecules were impaired in r-DKK1-treated DCs (Figures 5B–5G). Interestingly, r-DKK1 blocked TNF-α-induced expression and percentage of MHC II^+^, CD-80^+^, and CD86^+^ molecules on dendritic cells (Figures 6B and 6C). Additionally, IL-10 production was elevated in r-DKK1-treated dendritic cells, while production of IL-12 by r-TNF-α treated dendritic cells was inhibited by r-DKK1 (Figures 7A and 7B). The tolerogenic effect of rDKK1-treated dendritic cells was comparable to rIL-10-treated dendritic cells. To directly link the DKK-1-mediated induction of DC-10 and increased CD4 IL-10-producing cells, dendritic cells pulsed with soluble Leishmania antigen (SLAG) were co-cultured with T cells from infected WT-BALB/c mice, and intracellular IL-10 staining of CD4-T cells was performed. Relative to r-TNF-α-treated dendritic cells, r-DKK1-treated dendritic cells significantly increased IL-10-producing CD4-T cells (Figure 8B). These data suggest that DKK1 may regulate IL-10-Th1 cells by activation of a tolerogenic DC-10 phenotype.Figure 5. Minimal expression and percentage of MHC II^+^, CD86^+^, and CD80^+^ dendritic cells in the presence of recombinant DKK1(A) Monocyte-derived dendritic cells were incubated in rDKK1(100 ng/mL), rIL-10 (20 ng/mL) and rTNF-α (10 ng/mL). Cells harvested at 24 and 48 h post-incubation were used to determine the expression and percentage of MHC II^+^, CD86^+^, and CD80^+^ cells by flow cytometry. Representative flow cytometry dot plots generated 24 h post-incubation showed the analysis of MHC II^+^, CD86^+^ and CD80^+^ as indicated (A).(B–G) The percentage (B, D, and F) and MFI (C, E, and G) of MHC II^+^, CD86^+^ and CD80^+^ in the different experimental conditions are shown in the bar graphs. Results are presented as mean (±SEM) and are representative of triplicate experiments. One-way ANOVA followed by Bonferroni’s post hoc test was used to analyze the data (∗p < 0.05, ∗∗p < 0.01; ∗∗∗p < 0.001; and “ns” indicates non-significant.Figure 6r-DKK1 blocked TNF-α-induced MHC II^+^ and CD86^+^ expression of dendritic cells(A) Monocyte-derived dendritic cells were incubated in rDKK1(100 ng/mL), rTNF-α (10 ng/mL), and (rDKK1 + rTNF-α). Cells harvested at 48 h post-incubation were used to determine the percentage of MHC II^+^ and CD86^+^ cells by flow cytometry. Representative flow cytometry dot plots generated 48 h post-incubation showed the analysis of MHC II^+^ and CD86^+^ as indicated (A).(B and C) The percentage (B and C) of MHC II^+^ and CD86^+^ in the different experimental conditions are shown in the bar graphs. Results are presented as mean (±SEM) and are representative of triplicate experiments. One-way ANOVA followed by Bonferroni’s post hoc test was used to analyze the data (∗p < 0.05, ∗∗p < 0.01; and “ns” indicates non-significant.Figure 7. Elevated IL-10 and reduced IL-12 production in rDKK1-treated dendritic cells(A and B) Monocyte-derived dendritic cells were incubated in r-DKK1(100 ng/mL), r-IL-10 (20 ng/mL), r-TNF-α (10 ng/mL) or r-TNF-α + r-DKK1. Cell culture supernatants harvested 24 and 48 h post-incubation were used to determine IL-10 and IL-12 production by ELISA, as shown in the bar graphs (A and B). Results are presented as mean ± SEM and are representative of triplicate experiments. One-way ANOVA followed by Bonferroni’s post hoc test was used to analyze the data (∗p < 0.05, ∗∗p < 0.01). “ns” indicates not significant.Figure 8rDKK1-treated dendritic cells significantly increased the percentage of IL-10-producing CD4 T cells(A and B) Rested immature dendritic cells were stimulated with 100 ng/mL of recombinant DKK1 or 10 ng/mL of recombinant TNF-α for 24 h. The rDKK1 and r-TNF-α-treated dendritic cells were incubated for 4 h in a 24-well plate (2 × 10^5^ per well) in 200 μL of medium in the presence of SLAG (100 ng/ml). SLAG-loaded rDKK1 and r-TNF-α-treated dendritic cells (2 ×10^5^) were then washed and co-cultured with infected WT-BALB/c lymph node T cells (1 ×10^6^) in complete RPMI 1640 medium in a 1:5 ratio for 72 h. The cells were surface-stained with appropriate antibodies before intracellular IL-10 staining with a cell stimulation cocktail and BD Golgi plug. The stained lymph node cells from the experimental condition were determined for the percentage of CD4^+^IL-10^+^ T cells by flow cytometry. Representative flow cytometry dot plots showed the analysis of CD4^+^IL-10^+^ T cells (A). The percentage of CD4^+^IL-10^+^ T cells in the different experimental conditions is indicated (B). The non-treated and r-TNF-α-treated dendritic cells serve as controls. Results are presented as mean ± SEM and are representative of three replicate experiments. One-way ANOVA followed by Bonferroni’s post hoc test was used to analyze the data ∗∗∗p < 0.001; ns indicates not significant (p > 0.05).
Significant decrease in lesion size and parasite load in MyD88(PKO)- and DKK1(PKO)-infected mice
Lesion size was significantly reduced in MyD88^(PKO)^- and DKK1^(PKO)^-infected mice compared to infected BALB/c mice (Figure 9A). In weeks 6 and 15 PI, the parasitic load was significantly decreased in MyD88^(PKO)^- and DKK1^(PKO)^-infected mice relative to infected BALB/c mice (Figures 7B and 7C) and appeared to plateau, consistent with observed lesion development. Although previous studies have shown that administration of a DKK1 inhibitor can partially ameliorate disease, the disease still progressed.30 These data clearly demonstrate that platelet DKK1 released through MyD88 signaling is critical for disease progression, and the release of DKK1 from platelets promotes parasite survival and proliferation in the infection site of BALB/c mice.Figure 9. Lesion size and parasite burden decreased in MyD88^(PKO)^- and DKK1^(PKO)^-infected mice(A–C) Six-week-old female WT-BALB/c, MyD88^(PKO)^ and DKK1^(PKO)^ mice were challenged with infective metacyclic promastigote (2 ×10^6^ parasites) of L. major via the footpad. The infected foot from each mouse in BALB/c, MyD88^(PKO)^- and DKK1^(PKO)^-infected mice (n = 5 per group) were measured for lesion size weekly using a vernier caliper (A), and parasite burden (at day week 6 and 15PI) was determined by limiting dilution assay (B and C). Results are presented as mean ± SEM. For Figure (A), mice in each infected group were compared with the non-infected group and data analysis was done using one-way ANOVA followed by Bonferroni’s post hoc test ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001.
Discussion
Platelets are established to aggregate in the periphery with leukocytes upon TLR1/2 engagement during Leishmania infection.30^,^39 Aside from platelets, Leishmania species and their LPGs have been shown to interact with TLR2 expressed on macrophages, neutrophils, dendritic cells, or natural killer cells, modulating the immunological response to the parasite.31^,^33^,^43^,^44^,^45 The interaction between LPG and TLR2 produces different outcomes depending on the specific TLR2 co-receptor that is up-regulated; the TLR2-TLR6 complex induces pro-inflammatory cytokines, while the TLR1-TLR2 heterodimer induces anti-inflammatory cytokines.35^,^46^,^47 In addition, silencing TLR6 exacerbates L. major infection, while silencing TLR1 improves the protective response.48 Immune pressure during Leishmania infection is maintained by IFN-γ-producing CD4 and CD8 T cells, IL-12-producing cells, and inducible nitric oxide synthase (iNOS).49^,^50 Impairment of these responses during latency has been shown in each case to promote parasite proliferation and the reappearance of lesions. However, the explanation as to why these control mechanisms fail to eliminate the parasite is not well understood. Our recent in vitro study showed that TLR1/2 plays a role in recognizing Leishmania-derived LPG and induction of DKK1 in activated platelets.29 In turn, DKK1 potentiates Th2 cytokine expression and parasite survival.30 However, the mechanisms regulating how DKK1 is produced and modulates Th2 polarized responses are incompletely understood. To gain a more complete understanding of this process, mice with conditional deletion of MyD88 or DKK1 in platelets were used to evaluate whether the MyD88 activation signal and subsequent release of DKK1 from platelets regulate disease outcome by promoting a Th2 response in Leishmania infection. First, we demonstrated that the transmission of activation signals through MyD88 is crucial for platelet activation and early DKK1 production. Lack of platelet MyD88 or DKK1 resulted in impaired P-selectin expression. These findings highlighted MyD88 as the downstream protein regulating platelet activation and DKK1 production following interaction between LPG and platelet TLR1/2.
In this study, we have demonstrated that DKK1 produced via the platelet TLR1/2-MyD88 dependent pathway elevates LPA and NPA formation, as well as the infiltration of activated neutrophils into the infection site in BALB/c mice. These findings align with our previous studies, which demonstrated that DKK1 increases LPA and leukocytes in blood harvested from infected BALB/c mice.30 Also, our recent work has established that DKK1 signaling through its receptor (LRP6) elevates NPA formation and the migration of activated neutrophils to the site of infection.37 Further, these findings are consistent with previous studies, which demonstrated that activated platelets release platelet-derived growth factor, which is required for the infiltration of a subpopulation of effector monocytes to the site of Leishmania infection.51 These findings provide direct evidence that platelet DKK1 produced via the MyD88-dependent pathway plays a pivotal role in modulating the local inflammatory responses through the Wnt3a pathway. The comparable LPA and NPA formation at the infection site in all infected mice on day 14 PI is consistent with the fact that the expression level of LPG required for DKK1 production wanes in the mammalian host as the parasite transforms into the amastigote stage.
The impact of Th1 versus Th2 immunity on intracellular infections is attributed to classical versus alternative activation of macrophages leading to resistance or susceptibility to infection.52 Our previous study demonstrated that DKK1 serves as a key regulator of leukocyte infiltration and polarization of immune responses in pathological type 2 cell-mediated inflammation.30 Because DKK1 produced via the MyD88 activation signal promotes the migration of leukocytes to the infection site, we tested the hypothesis that the Th2 response in infected BALB/c mice may be mediated by DKK1 produced via a MyD88-dependent pathway. The elevated production of IL-10 and IL-4 in BALB/c-infected mice, in contrast to the mice with platelets deficient in MyD88 or DKK1, suggests that DKK1 released following activation of MyD88 promotes Th2 cytokine production. This is consistent with our previous study, which established that r-DKK-1 induced Th2 responses (IL-4, IL-10, and IL-13) in naive CD4^+^ T cells stimulated with anti-CD3 and anti-CD28 antibodies. Further, Gata-3 and c-Maf expression, which are known as the transcription factors essential for the expression of IL-4, IL-10, and IL-13, were elevated in r-DKK1-treated CD4T cells. This evidence suggests the ability of DKK1 to drive Th2 cell polarization.30 This effect is associated with elevated IL-10-producing CD4 and CD8T cells, cDC2 dendritic cells and M2 macrophages. In the context of Leishmania infection, a Th2 response is considered detrimental, as it hampers responses controlling parasite survival/growth and promotes disease progression.14 Also, M2 macrophages and cDC2 dendritic cells have been linked to the development of pathology; parasites survive and multiply within M2 macrophages.51 The impaired production of Th2 cytokines (specifically IL-10 and IL-4) in DKK1^(PKO)^- and MyD88^(PKO)^-infected mice confirms that the Th2 response in BALB/c infected mice may be mediated by DKK1.
Although Th2 and M2-like activation profiles induced by DKK1 promote disease progression, these responses appear to be secondary during the initial phase of infection to the neutrophilic regulation of disease progression. Tacchini-Cottier et al. demonstrated the role of an early wave of PMN in the development of the Th2 response characteristic of mice susceptible to L. major infection.53 Our previous study explored the potential role of DKK1-LRP6 signaling in the migration and longevity of activated neutrophils in the infection site using BALB/c mice with PMNs deficient in LRP6 (LRP6^NKO^) or BALB/c mice deficient in both PMN LRP6 and platelet DKK1 (LRP6^NKO^ DKK1^PKO^). Mouse PMNs were found to express LRP6, and the interaction between DKK1 and LRP6 facilitates early migration of activated PMN in the infection site of BALB/c mice.29 This finding indicates that DKK1-LRP6 signaling influences early infiltration and the effector function of PMNs. Leishmania prolongs neutrophil lifespan by inhibiting spontaneous apoptosis, thereby facilitating intracellular proliferation. Our mouse model showed that DKK1 activity influences PMN apoptosis.29 Relative to infected BALB/c mice, the percentage of apoptotic PMNs significantly increased in infected LRP6^NKO^ and LRP6^NKO^DKK1^PKO^ mice. The elevated viable activated PMNs obtained from infected BALB/c mice indicate that DKK1-LRP6 signaling influences the longevity of PMNs. Further, the elevated parasite load observed in infected BALB/c mice indicates that increased PMN activation and the inhibition of PMN apoptosis, mediated by DKK1-LRP6 signaling play critical roles in parasite survival and disease progression.29 Since DKK1 is a strong inducer of type 2 cell-mediated immune responses,30 blocking of DKK1-signaling in PMN generated from BALB/c mice might further lessen the Th2 responses and have a protective effect. Thus, DKK1 signaling in PMNs is likely to drive Th2 and M2-like activation profiles in BALB/c mice infected with L. major.
While BALB/c mice are inherently susceptible to L. major due to Th2-polarizing conditions, effective healing immune responses in both mouse and human models depend on sustained Th1 immunity. Dendritic cells are central to this process, acting as host antigen-presenting cells and cytokine sources that direct a Th1 or Th2 response. The interactions between Leishmania and dendritic cells are complex and involve paradoxical functions that can activate or block T cell responses, leading to a protective response or facilitate disease progression.54 The magnitude and profile of dendritic cell activation differ significantly depending on the cytokines released early in the microenvironment and on dendritic cell subsets.54 Although BALB/c mice typically produce early IL-4 that drives susceptibility to Leishmania,13^,^55 previous studies show that early recombinant IL-4 administration (within 8 h post infection with L. major) can paradoxically render mice resistant by promoting dendritic cell IL-12 mRNA expression.56 Also, IL-4 has been shown to block dendritic cell-derived IL-10.57 In L. major-infected BALB/c mice, deletion of IL-4 receptor alpha on dendritic cells increases susceptibility to infection, as these results in reduced IL-12 and higher IL-10 levels.58 Further studies identified CD11b^+^Ly6C^+^ inflammatory dendritic cells as the most highly infected dendritic cell subset harboring and spreading L. major parasites in CD11c^cre^IL-4Rα^−/lox^ mice.59 Notably, in the current study, the level of CD11c^+^CD11b^+^ cells is selectively diminished in DKK1-deficient MyD88^(PKO)^- and DKK1^(PKO)^-infected mice.
In contrast, IL-10 compromises healing responses in intensity or function, acting through effects on T cells, macrophages and dendritic cells.15^,^60^,^61^,^62^,^63 Adoptive transfer studies have shown that IL-10 produced by the CD4^+^ CD25^−^ Foxp3^−^ T cells suppressed the healing response in cutaneous leishmaniasis.15 In addition, it was demonstrated that a significant portion of the CD4 T cells in the dermis and a smaller subset of CD4 T cells localized in the draining node were CD4 T cells that produce both IL-10 and IFN-γ.15 It has been found that DC-10 cells direct the development of IL-10 producing Th1 cells.19 In particular, IL-10 inhibits the up-regulation of antigen-presenting molecules and IL-12 production, thus inhibiting the capacity of dendritic cells to generate Th1 responses.64 IL-10 exerts its actions through interaction with the IL-10 receptor, resulting in the activation of a series of intracellular signaling molecules, including STAT proteins.65^,^66 In the current study, we have demonstrated that the presence of platelet-derived DKK1 in vivo promotes the development of tolerogenic DC-10 dendritic cells and M2 macrophages. This effect on dendritic cells can be direct, as demonstrated in vitro that rDKK1 induces tolerogenic DC-10 cells and blocks activation by TNFα. IL-10 has been identified as a primary factor that can block dendritic cell maturation induced by various stimuli.67^,^68^,^69^,^70 Notably, the tolerogenic effect of r-DKK1 and r-IL-10 treated dendritic cells in vitro were comparable. As DC-10 cells are important for Th1-IL-10 cell differentiation, this suggests that both IL-10 and DKK1 can contribute to the development of Th1-IL-10 cells by limiting dendritic cell maturation and their capacity to initiate Th1 responses, resulting in parasite proliferation in BALB/c infected mice. We previously showed that the marked potentiation of IL-10 secretion by DKK1 in Th2 cell polarization conditions was blocked either by SGK-1 or p38 MAPK inhibition, confirming that both kinases are required for DKK1-mediated Th2 cytokine secretion.30 We speculate that DKK1-induced tolerogenic dendritic cells may be Wnt/β-catenin independent.
Previous studies have shown lpg1^-^ mutant parasite (deficient in LPG) can cause a slow onset of infection/disease.71 Also, amastigotes isolated from mice infected with lpg1^-^ mutant parasite are still infective, despite the initial attenuation of the promastigote infection.72 In our previous study, lpg1^−^mutant parasite (analogous to amastigotes in terms of LPG) exhibits delayed induction of DKK1 production as the disease progresses.29 The increase in DKK1 production in lpg1^-^ mutant parasite was observed on day 42 post-infection.29 Since LPG levels decline during transformation into the amastigote stage (within 72 h), this suggests that LPG-independent or alternate mechanisms exist in the amastigote stage of infection that trigger the production of DKK1, as evidenced by the infections with lpg1^−^mutant parasite. Thus, MyD88 ^PKO^ and DKK1 ^PKO^ mice may still display their resistance phenotypes when inoculated with amastigotes; however, this remains to be demonstrated. Overall, our study has highlighted the importance of DKK1 produced through the platelet-MyD88 signaling pathway in dendritic cell polarization. This has been shown to be critical for regulating IL-10 levels and ultimately influencing the differentiation of Th2 and Th1-IL-10 cells. Consequently, DKK1 promotes disease progression and parasite survival in M2 macrophages by inducing Th2 and Th1-IL-10 responses. Thus, inhibiting the TLR1/2-MyD88 pathway or DKK1 production in platelets may be an attractive approach to limit parasite proliferation in Leishmania infection.
Limitations of the study
Our results highlighted that platelet-DKK1 promotes disease progression through the induction of tolerogenic DCs and subsequent pathological Th2 and IL-10-Th1 T cell responses in WT-BALB/c mice. However, this study was limited by the lack of DKK1 reconstitution in mutant mice. It is possible that the reduced neutrophil-platelet aggregates, tolerogenic DC-10 cells, and lesion progression and induction of anti-leishmanial Th1-responses in MyD88^(PKO)^- and DKK1^(PKO)^-infected mice could stem from off-target knock-out effects or compensatory mechanisms. In addition, the in vitro data showed that recombinant DKK1-treated dendritic cells display a tolerogenic DC-10 phenotype. However, a mechanistic experiment to dissect the signaling pathway was not addressed. Finally, failure in developing progressive lesions in MyD88^(PKO)^- and DKK1^(PKO)^-infected mice and reduced inflammation suggest that blocking DKK1 signaling might promote lesion healing by activating the Wnt signaling pathway. Thus, this study is weakened by the inability to measure Wnt target gene expression (Axin, Tcf, and Lef) in the infected site with qPCR.
Resource availability
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Alfred L.M. Bothwell ([email protected]).
Materials availability
All data associated with this study are available in the article or the online supplemental information.
Data and code availability
Data requests should be directed to and will be fulfilled by the lead contact. The study in this article does not report original code. Additional information will be made available by the lead contact upon request.
Acknowledgments
We thank INFRAFRONTIER/EMMA (www.infrafrontier.eu, INFRAFRONTIER Consortium73) for providing the fertilized embryos (DKK1^fl/fl−/−^). We also appreciate the UNMC Flow Cytometry Research Facility (The UNMC Flow Cytometry Research Facility is administrated through the Office of the Vice Chancellor for Research and supported by state funds from the Nebraska Research Initiative [10.13039/100012050NRI] and The Fred and Pamela Buffett Cancer Center’s National Cancer Institute Cancer Support Grant [P30 CA036727]. Major instrumentation has been provided by the office of the Vice Chancellor for research, The University of Nebraska Foundation, the Nebraska Banker’s Fund, and the 10.13039/100000002NIH-10.13039/100000097NCRR Shared Instrument Program). In addition, we acknowledge Dr. Joann B Sweasy, Dr. Stephen M Beverley, Dr. Jennifer M Lundberg, and Dr. Wook-Jin Chae for their excellent suggestions and helpful advice. This work was supported by AI-137060 (awarded to A.L.M.B.).
Author contributions
O.C.I.: writing the original draft, acquisition and analysis of the data, visualization, validation, project administration, resources, investigation, methodology, data curation, formal analysis, conceptualization. A.S., H.Q.M., and M.S.: review and editing, validation, visualization, conceptualization, and resources. D.M.-P.: review and editing, visualization, validation, supervision, resources, methodology, investigation, and conceptualization. A.L.M.B.: review and editing, visualization, validation, supervision, project administration, resources, methodology, funding acquisition, investigation, and conceptualization. All authors read and approved the final manuscript.
Declaration of interests
The authors declare no conflicts of interest.
STAR★Methods
Key resources table
REAGENT or RESOURCESOURCEIDENTIFIERAntibodiesPE/Cyanine7 rat monoclonal anti-mouse CD206 (MMR) AntibodyBiolegendCat#141720; RRID: AB_2562248APC mouse monoclonal anti-mouse/rat CD62P antibodyBiolegendCat#148304; RRID: AB_2565273PE rat monoclonal anti-mouse CD38 AntibodyBiolegendCat#102708; RRID: AB_312929APC rat monoclonal anti-mouse F4/80 antibodyeBioscienceCat#17480182; RRID: AB_2784648V450 rat monoclonal anti-mouse CD86 antibodyBD BiosciencesCat#15850209APC rat monoclonal anti-mouse CD80 antibodyeBioscienceCat#17080182; RRID: AB_469417PE rat monoclonal anti-mouse CD86 antibodyBiolegendCat#105105; RRID: AB_313158PE-Cyanine5 rat monoclonal anti-mouse MHC class II antibodyeBioscienceCat#15532182; RRID: AB_468800PE rat monoclonal anti-mouse MHC II antibodyeBioscienceCat#12598082; RRID: AB_466083PECy5 rat monoclonal anti-mouse MHC II antibodyBiolegendCat#107612; RRID: AB_313327PE hamster monoclonal anti-mouse CD11c antibodyBD BiosciencesCat#553802; RRID: AB_395061FITC Armenian hamster monoclonal anti-mouse CD11c antibodyeBioscienceCat#11011482; RRID: AB_464940PE rat monoclonal anti-mouse CD41 antibodyBiolegendCat#133906; RRID: AB_2129745Alexa Fluor 700 monoclonal anti-mouse CD41 antibodyBiolegendCat#133926; RRID: AB_2572130Alexa Fluor 647 mouse monoclonal anti-mouse myeloperoxidase antibodyBD BiosciencesCat#570233; RRID: AB_3662122PE rat monoclonal anti-mouse IL-10 antibodyeBioscienceCat#12710182; RRID: AB_466176APC rat monoclonal anti-mouse IFN gamma antibodyeBioscienceCat#17731182; RRID: AB_469504FITC rat monoclonal anti-mouse CD4 antibodyeBioscienceCat#11004282; RRID: AB_464896FITC rat monoclonal anti-mouse CD11b antibodyeBioscienceCat#11011282; RRID: AB_464935PECy5 rat monoclonal anti-mouse CD11b antibodyBiolegendCat#101210; RRID: AB_312793PerCP-Cy5.5 rat monoclonal anti-mouse CD45 antibodyeBioscienceCat#45045182; RRID: AB_1107002Pacific Blue rat monoclonal anti-mouse Ly-6G antibodyBiolegendCat#127612; RRID: AB_2251161FITC rat monoclonal anti-mouse Ly-6G antibodyBiolegendCat#127605; RRID: AB_1236488Pacific blue rat monoclonal anti-mouse CD3 antibodyBiolegendCat#100214Alexa Fluor 700 rat monoclonal anti-mouse CD8 antibodyBiolegendCat#100730; RRID: AB_493703PE Mouse IgG1, κ Isotype Ctrl AntibodyBiolegendCat#400111APC Mouse IgG2a, κ Isotype Ctrl AntibodyBiolegendCat#400219; RRID: AB_326468Alexa Fluor® 647 Mouse IgG1, κ Isotype ControlBD BiosciencesCat#566011; RRID: AB_2869735PE rat monoclonal anti-mouse CD4 antibodyeBioscienceCat#12004182; RRID: AB_465506APC-Cy7 rat monoclonal anti- mouse CD3 antibodyBiolegendCat#100222; RRID: AB_2242784FITC rat monoclonal anti-mouse CD8 antibodyeBioscienceCat#11008182; RRID: AB_464915APC rat monoclonal anti-mouse IL-10 antibodyBD BiosciencesCat#17710182; RRID: AB_469502APC Mouse IgG2b, κ Isotype Ctrl AntibodyBiolegendCat#400320; RRID: AB_241949Chemicals, peptides, and recombinant proteinsMouse GM-CSF recombinant Protein, PeproTech®Thermo Fisher ScientificCat#3150320UGMouse IL-4 Recombinant Protein, PeproTechThermo Fisher ScientificCat#2141420UGMouse TNF-alpha Recombinant Protein, PeproTech®Thermo Fisher ScientificCat#31501A20UGMouse IL-10 Recombinant ProteinNACat: 2101010UGRecombinant Mouse DKK1 ProteinResearch and Development (R&D)Cat#5897DK010/CFAdenosine 5′-diphosphate sodium saltSigma AldrichCat#20398349Critical commercial assaysMouse Th1/Th2 Uncoated ELISA KitThermo Fisher ScientificCat#88771144Mouse IL-12 p40/70 (IL-12B) ELISA KitThermo Fisher ScientificCat#EMIL12BDKK1 Mouse ELISA KitThermo Fisher ScientificCat#EMDKK1Experimental models: Organisms/strainsParasite: Wild-type Leishmania major LV 39 clone 5Richard Titus (https://doi.org/10.1111/j.1365-3024.1987.tb00490.x)LV39 clone 5 (Rho/SU/59/P)Mouse: DKK1^fl/fl−/−^INFRAFRONTIER/EMMAwww.infrafrontier.eu, INFRAFRONTIER Consortium73Mouse: MyD88^fl/fl−/−^Ruslan Medzhitov’ labN/AMouse: WT BALB/c mouseJackson laboratoryN/AOligonucleotidesPrimers for mDKK1 Flox1 (5^’^-AGAACTAACCC CGGCCCCACAGCAGA-3′)This paperN/APrimers for mDKK1 Flox2 (5^’^-CTCCTCAGGGAAGACAACAAAGCCG-3^’^)This paperN/APrimers for MyD88-O1MR9481 Forward (5^’^-GTTGTGTGTGTCCGACCGT-3^’^)This paperN/APrimers for MyD88-01MR9482 Reverse (5^’^-GTCAGAAACAACCACCACCATGC-3^’^)This paperN/APrimer for PF4-Cre Forward (5^’^-CCCATACAGCACACCTTTTG-3^’^)This paperN/APrimer for PF4-Cre Reverse (5^’^-TGCACAGTCAGCAGG TT -3^’^This paperN/ASoftware and algorithmsGraphPad Prism 10 softwareGraphPad softwarehttps://www.graphpad.com/FlowJo (version 10.8.1)BD Bioscienceshttps://www.flowjo.com/BiorenderBiorenderhttps://Biorender.com.Adobe illustratorAdobehttps://www.adobe.com/products/ilustrator/free-trial-download.htmlOthereBioscience™ Cell Stimulation Cocktail (500XThermo Fisher ScientificCat#00497093BD GolgiPlug™ Protein Transport Inhibitor (Containing Brefeldin A)Fisher ScientificCat#BDB555029CD11b MicroBeads UltraPure, mouseMiltenyi BiotecCat#130126725Modified Tyrodes bufferThermo Fisher ScientificCat#J67607.K2Intracellular staining perm wash bufferBD BiosciencesCat#558050Medium 199Thermo Fisher ScientificCat#12350039Percoll®Sigma AldrichCat#GE17-0891-02Prostaglandin E_1_Sigma AldrichNAIndomethacinThermo Fisher ScientificCat#A19910.22Tyrode’s Solution, HEPES-bufferedThermo Fisher ScientificCat#J67607.APFetal Bovine SerumThermo Fisher ScientificCat#16000044HEPESThermo Fisher ScientificCat#15630106GentamycinThermo Fisher ScientificCat#15750060Acid Citrate Dextrose Solution AG-BiosciencesCat#786-493
Experimental model and study participant details
Mice
In vivo mice studies, Female WT BALB/c mice (4 weeks old) were purchased from the Jackson Laboratory. Sperm from mice heterozygous for MyD88 flox and PF4-Cre recombinase was used for in vitro fertilization, and pups carrying a heterozygous floxed allele of MyD88 and PF4-Cre were backcrossed to female BALB/c mice for eight generations. Thereafter, the heterozygous MyD88 floxed mice were intercrossed with mice expressing Cre recombinase under the control of the PF4 promoter to generate mice in which MyD88 was selectively deficient in platelets (MyD88^PKO^). PF4-Cre-DKK1 deficient mice (DKK1^PKO^) were generated by specific deletion of DKK1 in megakaryocytes and platelets. All knockout mice were genotyped using standard polymerase chain reaction (PCR) procedures.74 Genomic DNA from Proteinase K-digested tail tissue was extracted using a tail lysis buffer consisting of 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1% SDS, and 100 mM EDTA (pH 8.0). The primers used for the PCR reactions are the following: mDKK1 Flox1 (5^’^-AGAACTAACCC CGGCCCCACAGCAGA-3^’^); mDKK1 Flox2 (5^’^-CTCCTCAGGGAAGACAACAAAGCCG-3^’^); MyD88-O1MR9481 F (5^’^-GTTGTGTGTGTCCGACCGT-3^’^); MyD88-01MR9482 R (5^’^-GTCAGAAACAACCACCACCATGC-3^’^); PF4-Cre F (5^’^-CCCATACAGCACACCTTTTG-3^’^); PF4-Cre R (5^’^-TGCACAGTCAGCAGG TT -3^’^). All mice were maintained under specific-pathogen-free conditions with a 12-h light/dark cycle at 22 ± 1°C, appropriate bedding, cage enrichment, and ad libitum access to food and water. The hygiene management protocol mandates a strict separation between the central breeding facility and the experimental units. When the same mouse strain was assigned to multiple experimental groups, littermates were randomly assigned to different groups. All experiments utilized age-matched littermates (maximum of 5 mice per cage), unless otherwise specified, and all mice were housed at the University of Arizona Animal or University of Nebraska Medical Center Care Facilities. The mouse protocols were approved by the University of Arizona in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Mice strains were transferred to the University of Nebraska Medical Center (UNMC) and utilized under approved protocols. The code/number for the ethical approval of this study is IACUC #23-079-02-FC.
Primary cell culture
CD11b^+^ monocytes were isolated and purified from female mouse bone marrow cells using CD11b MicroBeads UltraPure mouse magnetic bead (Miltenyi Biotec) according to the manufacturer’s protocol. Isolated monocytes (1 × 10^6^ cells/mL) were differentiated into dendritic cells by 6 days of culturing with RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 10 mM HEPES, 50 μM 2-mercaptoethanol (Thermo Fisher Scientific), 20 ng/mL of IL-4 (Thermo Fisher Scientific) and 100 ng/mL of GM-CSF (Thermo Fisher Scientific) in 6-well tissue culture plates (Corning Costar) at 37 °C under 5% CO_2_. Cells were confirmed dendritic cells by flow cytometric analyses for CD11c^+^ expression.
Parasite strain
Metacyclic promastigotes of the wild-type L. major LV39 clone 5 were used for in vivo murine infections. For soluble Leishmania antigen (SLAG) preparation, L. major parasites (5 × 10^8^ parasites/ml grown in M199 medium without FCS) were subjected to five repeated freeze-thaw cycles at −80°C and room temperature, respectively.
Method details
L. major in vivo infections in mice
The wild-type L. major LV39 clone 5 was cultured at 26°C in M199 culture medium (Thermo Fisher Scientific) supplemented with 20% heat-inactivated FBS (Thermo Fisher Scientific), 20 mM HEPES (Thermo Fisher Scientific) and 50 μg/mL gentamycin (Thermo Fisher Scientific). Prior to using parasites for infection, metacyclic promastigotes were isolated from stationary-phase cultures by centrifugation through a 45% and 90% Percoll gradient (Sigma Aldrich) as previously described.75^,^76 Live parasites were isolated at the 90%–45% interface. Metacyclic promastigotes were washed three times in cold phosphate-buffered saline by centrifugation, resuspended in PBS at 2X10^8^/mL and 10 μL containing 2x10^6^, 1x10^5^ or 1x 10^4^ metacyclic promastigotes was injected into the top of the right hind footpad.
Platelet preparation and P-selectin expression
P-selectin expression was assessed in platelets isolated from infected female BALB/c, DKK1^(PKO)^, MyD88^(PKO)^, and non-infected mice, as previously described with minor modifications.29 Briefly, blood was collected via maxillary bleeding into tubes containing 3.2% citrate buffer (G-Biosciences) from infected BALB/c, DKK1^(PKO)^, MyD88^(PKO)^ and non-infected mice at days 3 and 14 PI. Blood was diluted with 250 μL modified Tyrode’s-HEPES buffer and centrifuged at 250 x g for 15 min at room temperature. Platelet-rich plasma (PRP) was discarded, and platelets were then isolated by centrifugation at 900 × g for 30 min. Isolated platelets (10x10^6^/mL) were washed at 550 x g for 10 min in the presence of indomethacin (10 mM; Thermo Fisher Scientific) and PGE1 (140 nM; Sigma Aldrich). Platelets were resuspended to the required density in modified Tyrode’s HEPES buffer (Thermo Fisher Scientific) and rested for 30 min at 37°C in the presence of 10 mM indomethacin before staining. Staining for P-selectin expression was done using PE-conjugated CD41 and APC-conjugated CD62P. Stained platelets (1 × 10^6^/mL in Tyrode’s-HEPES buffer; 100 μL) were acquired with an LSR II flow cytometer, and data were processed using FlowJo software. The mean fluorescent intensity of P-selectin was compared across the experimental groups.
Flow cytometry-based platelet aggregation assay
Platelet aggregation was assessed in platelets obtained from naive female BALB/c, DKK1^(PKO)^, MyD88^(PKO)^ as previously described with minor modifications.77 Briefly, Platelet-rich plasma was obtained by centrifugation of citrate-anticoagulated blood for 10 min at 200 g. Isolated platelets were washed at 550 x g for 10 min with PGE1 (140 nM) and indomethacin (10 mM) to prevent platelet activation. Samples were centrifuged for 10 min at 1000 x g, resulting in platelet pelleting. The pellet was subsequently washed and resuspended in Tyrodes buffer. The resting state of platelets was verified by flow cytometry following PE-conjugated CD41(Biolegend) and APC-conjugated CD62-P (Biolegend) antibody labeling. In addition, the absolute number and percentage of CD41^+^ cells in naive female BALB/c, DKK1^(PKO)^, MyD88^(PKO)^ were determined within the CD41^+^ gated population. Two fractions of the isolated platelets were differently labeled with PE-conjugated CD41 or Alexa Fluor 700-conjugated CD41 (BioLegend). Fractions were incubated in the dark for 15 min at 37°C. Differently labeled platelets were mixed in a 1:1 ratio, and 244 μM ADP (Sigma Aldrich) was added. Non-ADP-stimulated platelet mix was analyzed in parallel (serves as a control). To induce aggregation, tubes were shaken at 1000 rpm for 5 min in an Eppendorf Thermomixer. Samples were fixed and acquired with an LSR II flow, and data were analyzed with FlowJo software. Dot plot quadrants (Q1–Q4) were set by using the non-stimulated platelet mix. Platelet aggregation was quantified as the percentage of CD41^+^ double-positive events.
Cell isolation from infected footpad and lymph node
Neutrophils, dendritic cells, and macrophages were isolated from the footpad according to an established protocol.78 Infected footpads were excised above the ankle, deskinned, and cut crosswise in small increments from the bottom toward the ankle, then minced with toothed forceps. For non-infected mice, phosphate-buffered saline (PBS) was injected into both feet to increase cell yield, and cells from both feet were collected. The number of mice in the non-infected group increased from five to ten, and cells were pooled for analysis. Cells were also collected from both draining and non-draining lymph nodes, and single-cell suspensions were prepared. Tissue-derived cells were filtered through a 70 μm cell strainer (Thermo Fisher Scientific), spun down at 300 x g for 7 min, and resuspended in FACS buffer for staining.
Analysis of dendritic and macrophage subtypes
The abundance of cDC1 and cDC2 subtypes in dermal dendritic cells obtained from the footpads of infected female BALB/c, DKK1^(PKO),^ MyD88^(PKO),^ and non-infected BALB/c mice was assessed on day 14 post-infection using PE-conjugated CD11c (BD Bioscience), PECy5-conjugated CD11b (Biolegend), and Alexa fluor 700-conjugated CD8α antibodies (Biolegend). The cDC1 and cDC2 subtypes were identified by measuring the percentage of CD11b^−^CD8α^+^ and CD11b^+^CD8α^−^ cells within the CD11c^+^ gated population, respectively. In addition, the frequency of cDC1 and cDC2 was assessed using flow cytometry. The abundance of M1 and M2 subtypes in dermal macrophages obtained from the footpads of infected BALB/c, DKK1^(PKO)^, MyD88^(PKO)^, and non-infected BALB/c mice was assessed on day 14 post-infection using APC-conjugated F4/80 (eBioscience), PE-conjugated CD38 (Biolegend), and PE-Cy7-conjugated CD206 (Biolegend). The M1 and M2 subtypes were determined by measuring the percentage of CD38^+^CD206^-^ and CD38^−^CD206^+^ cells within the F4/80^+^gated population, respectively.
Neutrophil-platelet aggregate (NPA) and leukocyte-platelet aggregate (LPA) formation
NPA assessment was conducted as previously described, with minor modifications.29^,^39 Cells (1 x 10^6^/mL) were isolated from the footpads of infected female BALB/c, MyD88^(PKO)^, DKK1^(PKO)^, and non-infected BALB/c mice on days 3 and 14 PI, and stained with Pacific Blue-conjugated Ly6G (BioLegend) and PE-conjugated CD41 (BioLegend) antibodies for 15 min at room temperature. To assess LPA, a similar staining protocol was applied to cells (1 x 10^6^/mL) isolated from blood. These cells were stained with PerCP-Cy5.5-conjugated CD45 (eBioscience) and PE-conjugated CD41 (BioLegend). Stained samples were acquired using LSR II flow cytometry within 4–6 h. Data analysis was performed using FlowJo software. Live gating was applied to leukocyte-sized events to exclude single platelets. Leukocytes were identified based on forward and side scatter characteristics in conjunction with CD45 expression. Neutrophils were distinguished based on the forward and side scatter characteristics as well as Ly6G expression. The Ly6G^+^ and CD41^+^ subpopulations defined NPA, while the CD45^+^ and Ly6G^+^ subpopulations defined LPA.
CD11b+ and MHC class II+ neutrophils
Neutrophil activation, defined by CD11b^+^ and MHC^+^ cells, was assessed using a minor modification of established protocols.38 Cells were isolated from the footpads of infected and non-infected BALB/c mice, as well as from infected MyD88^(PKO)^ and DKK1^(PKO)^ mice on days 7 and 14 PI. These cells were stained with Pacific-blue conjugated Ly6G (BioLegend), FITC conjugated CD11b (eBioscience), and PE-conjugated MHC class II (eBioscience) antibodies for 15 min at room temperature. Stained cells were evaluated by LSR II flow cytometry and analyzed using FlowJo software. Neutrophils were characterized by forward and side scatter properties and Ly6G expression. Activated neutrophils were defined as the CD11b^+^ and MHC class II^+^ subpopulation.
Myeloperoxidase positive (MPO+) neutrophils
Activated neutrophils were also identified by quantifying myeloperoxidase (MPO^+^) neutrophils using a standardized flow cytometry intracellular staining protocol with minor modifications.39 In brief, cells (1x10^6^/mL) isolated from the footpad of infected female BALB/c, MyD88^(PKO),^ DKK1^(PKO)^ as well as from non-infected BALB/c mice on days 3 and 14 PI were fixed at room temperature in tubes containing BD Cytofix Fixation Buffer (BD Biosciences) that has been prewarmed to 37°C. Cells were subsequently washed and permeabilized using BD Perm/Wash Buffer (BD Biosciences). Following permeabilization, cells were stained at room temperature with FITC-conjugated Ly6G and either Alexa Fluor 647 mouse IgG1 isotype control or Alexa Fluor 647-conjugated myeloperoxidase antibody (BD Biosciences) in BD Perm/Wash Buffer. LSR II flow cytometry was used for data acquisition, and FlowJo software was employed for data analysis.
Plasma DKK1 ELISA
Plasma was collected from blood drawn from infected female BALB/c, DKK1^(PKO)^, MyD88^(PKO)^ and non-infected mice as previously described.28 Plasma was isolated from blood by centrifugation at 900 x g for 30 min. DKK1 concentration in the plasma was determined by Enzyme-linked immunosorbent assay (ELISA) using a mouse DKK1 ELISA kit (Thermo Fisher Scientific) according to the manufacturer’s guidelines.
Ex-vivo lymph node cell stimulation and cytokine determination using ELISA
Single-cell suspensions from draining and non-draining lymph nodes of infected female BALB/c, MyD88^(PKO)^, DKK1^(PKO)^ and non-infected BALB/c mice were activated with SLAG at a concentration of 50 μg/mL. Cells (1 × 10^6^ cells/mL) were cultured in a complete RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Gibco) and 10 mM HEPES at 37 °C under 5% CO_2._ The supernatant was collected 72 h post-incubation with SLAG and stored at −80°C for cytokine analysis. The concentrations of IL-4, IL-10, and IFN−γ in the supernatant were determined using mouse Th1 and Th2 cytokine panel ELISA kit (eBioscience) according to the manufacturer’s protocol.
Intracellular staining for IFN-γ and IL-10 in CD4+ and CD8+T cell
On day 14 PI, single-cell suspension obtained from draining and non-draining lymph node cells of non-infected BALB/c, infected BALB/c, MyD88^(PKO)^ and DKK1^(PKO)^ mice were stimulated with a cell stimulation cocktail (Thermo Fisher Scientific; 2 μl/ml) for 5 h, and BD Golgi Plug (BD Thermo Fisher Scientific; 1 μL/mL) added for the final 3 h. Cell stimulation was performed in complete RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin and 10 mM HEPES. The cells were surface-stained with the following antibodies: PacBlue-conjugated anti-CD3 (Biolegend), FITC-conjugated anti-CD4 (eBioscience), and Alexa Fluor-conjugated anti-CD8 antibodies (Biolegend). Intracellular staining was performed following the manufacturer’s guidelines (eBioscience) using APC-conjugated anti-IFN-γ (eBioscience) or PE-conjugated anti-IL-10 antibodies (BD Biosciences). PE mouse IgG1(BD Biosciences) and APC mouse IgG2a antibodies (eBioscience) served as isotype controls. Data acquisition and analysis were done using LSR II flow cytometry and FlowJo software, respectively.
In vitro cell stimulation of monocyte-derived dendritic cells and functional analysis
Immature dendritic cells were allowed to rest for 24 h in complete RPM1 1640 media without cytokines to remove the residual effect of GM-CSF. Rested immature dendritic cells were stimulated with 100 ng/mL of recombinant DKK1 (R & D Systems), 20 ng/mL of recombinant IL-10 (Thermo Fisher Scientific), or 10 ng/mL of recombinant TNF-α (Thermo Fisher Scientific). Cell supernatant was collected at 24 and 48 h post-incubation and stored at −80°C for cytokine analysis. To prevent measuring recombinant IL-10, cells were washed 6 h post-activation with recombinant IL-10, and supernatant was collected 24- and 48-h post-washing of the dendritic cells. used for cell activation, The concentration of IL-10 and IL-12p40 in the supernatant was determined using a mouse IL-12p40 as well as a Th1 and Th2 cytokine panel ELISA kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. For the detection of MHC II and co-stimulatory molecules, dendritic cells were stained with the following antibodies: FITC-conjugated CD11c (eBioscience), PE-conjugated MHC II (eBioscience), APC-conjugated CD80 (eBioscience) and V450-conjugated CD86 (eBioscience). To determine whether DKK-1 was able to induce a direct tolerogenic dendritic cell response or block the effects of inflammatory cytokines on dendritic cells, washed monocyte-derived dendritic cells were incubated in rDKK1(100 ng/mL), rTNF-α (10 ng/mL) or (rDKK1 + rTNF-α). Cells harvested at 48 h post-incubation were used to determine the percentage of MHC II^+^ and CD86^+^ cells by flow cytometry. For the detection of MHC II and CD86 molecules, dendritic cells were stained with FITC-conjugated CD11c (eBioscience), PECy5-conjugated MHC II (eBioscience) and PE-conjugated CD86 (Biolegend). Data acquisition and analysis were done using LSR II flow cytometry and FlowJo software, respectively.
Co-culture of T-cells and DCs pulsed with SLAG
Rested immature dendritic cells were stimulated with 100 ng/mL of recombinant DKK1 and/or 10 ng/mL of recombinant TNF-α for 24 h. The rDKK1 and r-TNF-α-treated dendritic cells were incubated for 4 h in a 24-well plate (2 × 10^5^ per well) in 200 μL of medium in the presence of SLAG (50 μg/mL). Meanwhile, T-cells isolated from the lymph nodes of infected WT-BALB/c mice were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 10 mM HEPES, 50 μM 2-mercaptoethanol. SLAG-loaded rDKK1 and/or r-TNF-α-treated dendritic cells (2 x 10^5^) were then washed and co-cultured with T-cells (1 x 10^6^) in complete RPMI 1640 medium in a 1:5 ratio for 3 days. The cells were surface-stained with the following antibodies: APC-Cy7-conjugated anti-CD3 (Biolegend), FITC-conjugated anti-CD8 (eBiosciences), and PE-conjugated anti-CD4 antibodies (eBiosciences). Intracellular CD4^+^IL-10^+^ cells were detected by intracellular staining of cells with APC-conjugated anti-IL-10 antibodies (BD Biosciences) using a cell stimulation cocktail and BD Golgi Plug. APC mouse IgG2a antibodies served as an isotype control. Stained cells were analyzed by flow cytometry, and FACS analyses are shown after gating on the CD4^+^ lymphocyte population.
Assessment of parasite load and lesion size
Parasite load was estimated by limiting dilution analysis at weeks 6 and 15 PI as previously described.79 Briefly, cells obtained from the infection site were suspended in Schneider medium supplemented with 20% heat-inactivated fetal bovine serum and 1% Penicillin-streptomycin. After cell count, ten serial dilutions were prepared; for each dilution, eight wells (100 μL) were set up in 96-well microtiter plates. Cell incubation was performed at 26 °C for 10 days. The total number of negative wells (absence of motile promastigotes) and positive wells (presence of motile promastigotes) were identified by an inverted light microscope. The number of viable parasites in the infection site of each mouse was determined from the highest dilution at which promastigotes could be grown.
Lesion size was monitored weekly by using a Vernier caliper to determine the thickness of the infected hind footpad and compare it with that of the non-infected hind footpad. Lesion size = Size of infected footpad - Contralateral uninfected footpad (mm).
Quantification and statistical analysis
Statistical tests and the exact n values were noted in the corresponding figure legends. For all experiments, n represents the number of mice per group. All data were shown as means ± SEM. Analyses were performed using GraphPad Prism 10 software. Flow cytometry data were analyzed using FlowJo software. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. Statistical significance was identified with ∗p < 0.05; ∗p < 0.01; ∗∗p < 0.001; and ns, not significant. The graphical abstract was created with https://Biorender.com.
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