Apoptotic cell clearance of Leishmania major-infected neutrophils by dendritic cells inhibits CD8+ T-cell priming in vitro by Mer tyrosine kinase-dependent signaling

Neutrophils are the predominant recruited and infected cells during the early stages of Leishmania major infection in the skin, and depletion of neutrophils promotes immunity to infection transmitted by sand fly bite. In order to better understand how the acute neutrophilic response suppresses immunity, we assessed the consequences of the interaction between neutrophils recovered from the skin-inoculation site and bone marrow-derived dendritic cells (DCs) in vitro. The capture of infected, apoptotic neutrophils by the DCs completely inhibited their cross-presentation function that was dependent on engagement of the receptor tyrosine kinase Mer on the DCs. The capture of uninfected neutrophils, or neutrophils infected with Toxoplasma gondii, had only slight immunomodulatory effects. These studies define the clearance of infected, apoptotic neutrophils by DCs and Mer receptor signaling as central to the early immune evasion strategies of L. major, with relevance to other vector-borne pathogens delivered by bite to the skin.The phagocytosis of dying cells in the absence of inflammation was described over 100 years ago by Metchnikoff in the context of the removal of regressing tissue during amphibian morphogenesis.^{1, 2} Many studies have since addressed how dying cells that are continuously generated as a consequence of normal tissue turnover signal for their clearance by dendritic cells (DCs) or macrophages in a manner that maintains homeostasis. Phagocytosis of apoptotic cells by macrophages and DCs can inhibit their production of proinflammatory mediators while leading to the production of TGF-β and to the generation of regulatory T cells.^{3, 4, 5} Under steady-state conditions, the capture of apoptotic cells by DCs is thought to contribute to the maintenance of peripheral tolerance.⁶ In infection-driven inflammatory settings, the ingestion of apoptotic cells by macrophages and DCs may be exploited by pathogens to promote infection by inhibiting components of the antimicrobial response. For example, Trypanosoma cruzi grows better in macrophages that have ingested apoptotic lymphocytes;⁷ the killing of Streptococcus pneumonia by alveolar macrophages is suppressed following their uptake of apoptotic cells;⁸ and Mycobacterium tuberculosis-induced activation of human DCs for T-cell priming is inhibited by their co-culture with apoptotic neutrophils.⁹During infection, PMNs and DCs, which are normally located in distinct anatomical compartments, may colocalize at sites of inflammation. As neutrophils are short-lived cells that must be targeted for orderly removal, the function of DCs as antigen-presenting cells (APCs) in these infectious foci may become subordinate to their role in the clearance of dying neutrophils. For microbes that gain entry into the skin via the bite of an arthropod vector, the inflammatory signals that direct the recruitment and colocalization of neutrophils and DCs can be especially pronounced as they are driven not only by the microbial stimuli, but also by salivary constituents and by the tissue injury associated with the bite itself. In this context, we have previously reported that the inoculation of Leishmania major into the skin by sand fly bite or by needle results in the rapid recruitment of large numbers of neutrophils that phagocytose the parasite and constitute the predominant initial infected cell in the site.^{10, 11, 12} Although the majority of the infected neutrophils die and release viable parasites that are taken up by inflammatory monocytes in the skin, a significant proportion appear to be engulfed by DCs, with most infected DCs acquiring their early infections via this process.¹¹ As neutrophil depletion just before infection augments the development of immunity to sand fly-transmitted infection and of a Leishmania-specific T-cell response, the capture of infected neutrophils by DCs in the skin was suggested as a key mechanism to inhibit their APC function and to delay the development of acquired resistance. The current studies were designed to model these cellular interactions in vitro, and to directly explore the immunologic consequences of the capture of L. major-infected neutrophils by DCs. The findings reveal a profound impairment of the capacity of DCs for T-cell priming following their uptake of infected neutrophils that is mediated by Mer tyrosine kinase-dependent signaling.