Neutrophil-dendritic cell interaction plays an important role in live attenuated Leishmania vaccine induced immunity

Background Neutrophils are involved in the initial host responses to pathogens. Neutrophils can activate T cell responses either independently or through indirect involvement of Dendritic cells (DCs). Recently we have demonstrated direct neutrophil-T cell interactions that initiate adaptive immune responses following immunization with live attenuated Leishmania donovani centrin deleted parasite vaccine (LdCen(-/-)). However, neutrophil-DC interactions in T cell priming in vaccine immunity in general are not known. In this study we evaluated the interaction between neutrophils and DCs during LdCen(-/-) infection and compared with wild type parasite (LdWT) both in vitro and in vivo. Methodology/findings LdCen(-/-) parasite induced increased expression of CCL3 in neutrophils caused higher recruitment of DCs capable of inducing a strong proinflammatory response and elevated co-stimulatory molecule expression compared to LdWT infection. To further illustrate neutrophil-DCs interactions in vivo, we infected LYS-eGFP mice with red fluorescent LdWT/LdCen(-/-) parasites and sort selected DCs that engulfed the neutrophil containing parasites or DCs that acquired the parasites directly in the ear draining lymph nodes (dLN) 5d post infection. The DCs predominantly acquired the parasites by phagocytosing infected neutrophils. Specifically, DCs containing LdCen(-/-) parasitized neutrophils exhibited a proinflammatory phenotype, increased expression of costimulatory molecules and initiated higher CD4(+)T cell priming ex-vivo. Notably, potent DC activation occurred when LdCen(-/-) parasites were acquired indirectly via engulfment of parasitized neutrophils compared to direct engulfment of LdCen(-/-) parasites by DCs. Neutrophil depletion in LdCen(-/-) infected mice significantly abrogated expression of CCL3 resulting in decreased DC recruitment in ear dLN. This event led to poor CD4(+)Th1 cell priming ex vivo that correlated with attenuated Tbet expression in ear dLN derived CD4(+) T cells in vivo. Conclusions Collectively, LdCen(-/-) containing neutrophils phagocytized by DC markedly influence the phenotype and antigen presenting capacity of DCs early on and thus play an immune-regulatory role in shaping vaccine induced host protective response. Author summary Visceral Leishmaniasis (VL), caused by the protozoan parasites of the genus Leishmania is a neglected tropical disease. Leishmania donovani is the principal causative agent of VL in East Africa and the Indian subcontinent whereas in Europe, North Africa, and Latin America VL is mainly caused by Leishmania infantum. No licensed vaccine exists against VL. We have reported previously that live attenuated centrin gene-deleted L. donovani (LdCen(-/-)) parasite vaccine induced strong innate immunity which leads to a protective Th1 response in animal models. We recently demonstrated that neutrophils play an indispensable role following immunization with LdCen(-/-) parasites in inducing protective Th1 immune response. However, neutrophils also secrete chemokines that attract other innate cells such as dendritic cells and regulate their activities. In the current study we analyzed the interplay between neutrophils and DCs, and its effects on T cell activation during LdCen(-/-) infection and compared with wild type parasite (LdWT) infection. We observed that higher recruitment of DCs occurred in LdCen(-/-) infected mice ear draining lymph nodes compared to LdWT. This recruitment is facilitated by increased secretion of the chemokine CCL3 by neutrophils. A markedly decreased DC recruitment was observed in LdCen(-/-) infected mice following CCL3 neutralization indicating the key role of neutrophils in DC recruitment. Further, we demonstrated that DCs that ingest LdCen(-/-) infected neutrophils are better activated than those that acquire the parasites independent of neutrophils. Notably neutrophil depletion in LdCen(-/-) infected mice also attenuated activation of DCs in the ear dLN that resulted in poor CD4(+)T cell priming. Our results reveal that interaction between neutrophils and DCs play an important role in shaping proinflammatory immune response induced by a live attenuated Leishmania vaccine.