The Long Pentraxin 3 (PTX3) Suppresses Immunity to Cutaneous Leishmaniasis by Negatively Regulating Th17 Response

The long Pentraxin 3 (PTX3), a soluble pattern recognition molecule, plays a critical role in inflammation, tissue repair and wound healing. Here, we show that PTX3 regulates disease pathogenesis in cutaneous leishmaniasis (CL). PTX3 expression is increased in active skin lesions in patients and mice during CL, with higher levels being expressed in individuals with severe disease. PTX3 deficient (PTX3-/-) mice were highly resistant to L. major infection and the enhanced resistance was associated with increased IL-17 response. Neutralization of IL-17A abolished this enhanced resistance while treatment with recombinant PTX3 resulted in reduced IL-17A response and increased susceptibility to L. major infection. Naïve CD4+ T cells from PTX3-/- mice displayed increased differentiation into Th17 cells, which was reversed in the presence of recombinant PTX3. The enhanced Th17 response observed in PTX3-/- cells was associated with increased Leishmania specific IL-6 production from dendritic cells along with enhanced expression of Th17-specific transcription factors including RORγt, AhR and STAT3. Addition of recombinant PTX3 significantly inhibited the expression of Th17-specific transcription factors and dramatically reduced the frequency of Th17 cells in Th17-polarizing cultures of PTX3-/- CD4+ T cells. Collectively, our results show that PTX3 contributes to the pathogenesis of CL by suppressing Th17 differentiation and IL-17A production. Author Summary Cutaneous leishmaniasis (CL) is caused by several species of Leishmania. Currently, there is no approved vaccine against human CL because of the poor understanding of the mechanisms that regulate disease pathogenesis and correlates of protective immunity. Because the long pentraxin 3 (PTX3, a soluble pattern recognition molecule that forms an integral part of the host innate immunity), regulates inflammation and tissue repair, which are critical physiological events associated with resolution of skin lesions during CL, we investigated its role in disease pathogenesis. Here, we show that PTX3 levels were elevated in skin-lesions in patients and mice during CL. Using a loss of function approach, we showed that PTX3 contributes to pathogenesis, and this was associated with increased IL-17A responses. Neutralization and recombinant cytokine treatment studies showed that the increased resistance of PTX3 deficient mice to L. major is due to enhanced Th17 response in these mice. We further show that PTX3 negatively regulates IL-6 production by dendritic cells and the expression of IL-17A-specific transcription factors (including RORγT, STAT3, IRF4, BATF and AhR) in CD4+ T cells. Collectively, these findings show that PTX3 is a negative regulator of Th17 response and protective immunity during L. major infection.

148 splenocytes compared to their WT counterparts. In contrast and consistent with our 149 mouse infection studies, the frequency of CFSE lo CD4 + IFN- + T cells in both WT and 150 PTX3 -/splenocytes under Th1 polarizing conditions were comparable (Figs 2B and E).

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We confirmed these findings by ELISA, which showed higher levels of IL-17A in cell 152 culture supernatants of PTX3 -/splenocytes under Th17 polarization condition ( Fig 2D) 153 but comparable levels of IFN- in both WT and PTX3 -/splenocytes under Th1 154 polarization condition (Fig 2E). We also observed similar increased frequencies of Th17 155 cells using purified CD4 + T cells from PTX3 -/spleens under Th17 polarizing conditions 156 ( Supplementary Fig 4). These results show that deficiency of PTX3 potentiates Th17 198 these mice. To confirm this, we co-cultured DCs from WT and PTX3 -/mice with 199 Leishmania-PEPCK TCR-transgenic CD4 + T cells (1:10) in presence of PECPK peptide.

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Although some reports have suggested that IL-17 plays a pathogenic role in 208 leishmaniasis (11-13), others showed that they play a protective role (7, 9, 10). Because 209 we found that enhanced resistance of PTX3 -/mice to L. major was not associated with 210 superior IFN- response, we postulated that the enhanced resistance was mediated by

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Next, we evaluated if administration of rPTX3 to WT mice could lead to increased 217 susceptibility to L. major infection. We infected WT mice with L. major and administered 218 rPTX3 intralesionally once a week for 3 weeks. WT mice treated with rPTX3 had 219 increased lesion size (Fig 5C) that corresponded with significantly increased parasite 220 burden ( Fig 5D) compared to PBS treated controls. The enhanced susceptibility 221 following rPTX3 treatment was accompanied by significant (p < 0.05) reduction in the 222 frequency of CD4 + IL-17A + T cells in the dLNs and spleen compared to PBS treated F), there was no difference in the frequency of CD4 + IFN- + T cells in dLNs and spleen of Supplementary Fig 6) and unchanged levels of IFN- (Fig 5H and Supplementary Fig 6) 228 in cell culture supernatant fluids of SLA-stimulated dLN and spleen cells from rPTX3-229 treated mice. Collectively, these findings confirm that PTX3 enhances susceptibility to L.  E show that IL-17 cooperates with suboptimal dose of IFN- to mediate increased NO   250   production and more effective parasite killing (compared to treatment with IFN- or IL-17   251   alone). Similarly, we observed increased iNOS2 mRNA expression at the site of infection 252 in PTX3 -/mice compared to WT counterparts (Fig 6F), which confirmed our in vitro 253 findings. Collectively, our results show that the enhanced resistance of PTX3 -/mice to L. 254 major is due to enhanced IL-17A production, which synergizes with IFN- to enhance NO 255 production, leading to more effective killing of parasites in infected macrophages.

Discussion:
Herein we showed that PTX3 levels were elevated in skin lesions from patients and mice 260 suffering from CL, suggesting that this innate pattern recognition molecule may play a 261 critical role in disease pathogenesis. Using a loss of function approach, we showed that 262 deficiency of PTX3 resulted in enhanced resistance to L. major, and this was associated 263 with increased IL-17 (but not IFN- response. Neutralization studies showed that the 264 enhanced resistance of PTX3 -/mice to L. major is due to enhanced Th17 responses in 265 these mice. In contrast, administration of rPTX3 led to increased susceptibility to L.

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All human subjects that were part of this study were adults, and informed written consent 410 was obtained from them.

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Antibody treatment was continued once weekly at 0.5 mg/mouse for additional 4 weeks.

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The lesion thickness was monitored weekly and mice were sacrificed after 4 weeks post