Clearance of apoptotic exudate neutrophils (efferocytosis) induces either pro- or anti-inflammatory responses in mouse macrophages depending on host genetic background. In this study, we investigated whether neutrophil efferocytosis induces a stable macrophage phenotype that could be recalled by late restimulation with LPS. Bone marrow-derived macrophages previously stimulated by pro- but not anti-inflammatory neutrophil efferocytosis expressed a regulatory/M2b phenotype characterized by low IL-12 and high IL-10 production following restimulation, increased expression of LIGHT/TNF superfamily 14, Th2-biased T cell responses, and permissive replication of Leishmania major. Induction of regulatory/M2b macrophages required neutrophil elastase activity and was partially dependent on TLR4 signaling. These results suggested that macrophage differentiation to a regulatory phenotype plays a role in resolution of inflammation but could contribute to increased humoral Ab responses and parasite persistence in the infected host.

Neutrophils and dendritic cells (DCs) converge at localized sites of acute inflammation in the skin following pathogen deposition by the bites of arthropod vectors or by needle injection. Prior studies in mice have shown that neutrophils are the predominant recruited and infected cells during the earliest stage of Leishmania major infection in the skin, and that neutrophil depletion promotes host resistance to sand fly transmitted infection. How the massive influx of neutrophils aimed at wound repair and sterilization might modulate the function of DCs in the skin has not been previously addressed. The infected neutrophils recovered from the skin expressed elevated apoptotic markers compared to uninfected neutrophils, and were preferentially captured by dermal DCs when injected back into the mouse ear dermis. Following challenge with L. major directly, the majority of the infected DCs recovered from the skin at 24 hr stained positive for neutrophil markers, indicating that they acquired their parasites via uptake of infected neutrophils. When infected, dermal DCs were recovered from neutrophil depleted mice, their expression of activation markers was markedly enhanced, as was their capacity to present Leishmania antigens ex vivo. Neutrophil depletion also enhanced the priming of L. major specific CD4⁺ T cells in vivo. The findings suggest that following their rapid uptake by neutrophils in the skin, L. major exploits the immunosuppressive effects associated with the apoptotic cell clearance function of DCs to inhibit the development of acquired resistance until the acute neutrophilic response is resolved. Prior studies in mice have shown that the inoculation of Leishmania major into the skin by sand fly bite or by needle provokes a massive recruitment of neutrophils that take up the parasite, and that this response somehow suppresses immunity since neutrophil depletion results in better control of the infection. We investigated how neutrophils recruited to the injection site might interact with and suppress the function of dendritic cells (DCs) in the skin. Infected neutrophils recovered from the skin expressed increased levels of apoptotic markers compared to uninfected neutrophils, and were efficiently taken up by dermal DCs when injected back into the skin. When dermal DCs were permitted to take up parasites in the absence of neutrophils, their expression of activation markers and their ability to present Leishmania antigens were enhanced. Neutrophil depletion also enhanced the activation of Leishmania specific CD4⁺ T cells in vivo. The results suggest that for insect borne pathogens like Leishmania that provoke a strong inflammatory response at the site of infection, the immunosuppressive effects associated with the apoptotic cell clearance function of DCs will inhibit the early development of immunity.

Infection of C57BL/6 mice with most Leishmania major strains results in a healing lesion and clearance of parasites from the skin. Infection of C57BL/6 mice with the L. major Seidman strain (LmSd), isolated from a patient with chronic lesions, despite eliciting a strong Th1 response, results in a nonhealing lesion, poor parasite clearance, and complete destruction of the ear dermis. We show here that in comparison to a healing strain, LmSd elicited early upregulation of IL‐1β mRNA and IL‐1β‐producing dermal cells and prominent neutrophil recruitment to the infected skin. Mice deficient in Nlrp3, apoptosis‐associated speck‐like protein containing a caspase recruitment domain, or caspase‐1/11, or lacking IL‐1β or IL‐1 receptor signaling, developed healing lesions and cleared LmSd from the infection site. Mice resistant to LmSd had a stronger antigen‐specific Th1 response. The possibility that IL‐1β might act through neutrophil recruitment to locally suppress immunity was supported by the healing observed in neutropenic Genista mice. Secretion of mature IL‐1β by LmSd‐infected macrophages in vitro was dependent on activation of the Nlrp3 inflammasome and caspase‐1. These data reveal that Nlrp3 inflammasome‐dependent IL‐1β, associated with localized neutrophil recruitment, plays a crucial role in the development of a nonhealing form of cutaneous leishmaniasis in conventionally resistant mice.

Neutrophils are the first cells recruited to the dermal site of Leishmania infection following injection by needle or sand fly bite. The role of neutrophils in either promoting or suppressing host immunity remains controversial. We discuss the events driving neutrophil recruitment, their interaction with the parasite and apoptotic fate, and the nature of their encounters with other innate cells. We suggest that the influence of the neutrophil response on infection outcome critically depends on the timing of their recruitment and the tissue environment in which it occurs.

Significance: Protozoan parasites of the genus Leishmania are transmitted by sand flies and produce diseases in humans ranging from localized cutaneous lesions to fatal visceral infection. Although these clinical outcomes have clear parasite species associations, the genes controlling these differences are not known. We provide, to our knowledge, the first experimental demonstration of genetic exchange in the sand fly vector between different Leishmania species: a cutaneous strain of Leishmania major and a visceral strain of Leishmania infantum. Eleven full genomic hybrids were generated that displayed differences in their ability to grow in the skin or viscera of mice, indicating that the genes controlling these traits may be polymorphic within the parental species and are potentially amenable to identification by classical linkage analysis.

As key cells, able to host and kill Leishmania parasites, inflammatory monocytes/macrophages are potential vaccine and therapeutic targets to improve immune responses in Leishmaniasis. Macrophage phenotypes range from M1, which express NO-mediated microbial killing, to M2 macrophages that might help infection. Resistance to Leishmaniasis depends on Leishmania species, mouse strain, and both innate and adaptive immunity. C57BL/6 (B6) mice are resistant and control infection, whereas Leishmania parasites thrive in BALB/c mice, which are susceptible to develop cutaneous lesions in the course of infection with Leishmania major, but not upon infection with Leishmania braziliensis. Here, we investigated whether a deficit in early maturation of inflammatory monocytes into macrophages in BALB/c mice underlies increased susceptibility to L. major versus L. braziliensis parasites. We show that, after infection with L. braziliensis, monocytes are recruited to peritoneum, differentiate into macrophages, and develop an M1 phenotype able to produce proinflammatory cytokines in both B6 and BALB/c mice. Nonetheless, more mature macrophages from B6 mice expressed inducible NO synthase (iNOS) and higher NO production in response to L. braziliensis parasites, whereas BALB/c mice developed macrophages expressing an incomplete M1 phenotype. By contrast, monocytes recruited upon L. major infection gave rise to immature macrophages that failed to induce an M1 response in BALB/c mice. Overall, these results are consistent with the idea that resistance to Leishmania infection correlates with improved maturation of macrophages in a mouse-strain and Leishmania-species dependent manner. All-trans retinoic acid (ATRA) has been proposed as a therapy to differentiate immature myeloid cells into macrophages and help immunity to tumors. To prompt monocyte to macrophage maturation upon L. major infection, we treated B6 and BALB/c mice with ATRA. Unexpectedly, treatment with ATRA reduced proinflammatory cytokines, iNOS expression, and parasite killing by macrophages. Moreover, ATRA promoted an M1 to M2 transition in bone marrow-derived macrophages from both strains. Therefore, ATRA uncouples macrophage maturation and development of M1 phenotype and downmodulates macrophage-mediated immunity to L. major parasites. Cautions should be taken for the therapeutic use of ATRA, by considering direct effects on innate immunity to intracellular pathogens.