Bacterial coinfections during murine cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is a parasitic disease causing chronic, ulcerating skin lesions. Most humans infected with the causative Leishmania protozoa are asymptomatic. Leishmania spp. are usually introduced by sand flies into the dermis of mammalian hosts in the presence of bacteria from either the host skin, sand fly gut, or both. We hypothesized that bacteria at the dermal inoculation site of Leishmania major will influence the severity of infection that ensues. A C57BL/6 mouse ear model of infection with Leishmania major, Staphylococcus aureus, or both showed that single pathogen infections caused localized lesions that peaked after 2-3 days for S. aureus and 3 weeks for L. major infection, but that coinfection produced lesions that were two-fold larger throughout 4 weeks after coinfection. Coinfection increased S. aureus burdens over 7 days, whereas L. major burdens (3, 7, 28 days) were the same in singly and coinfected ears. Inflammatory lesions throughout the first 4 weeks of coinfection had more neutrophils than did singly infected lesions, and the recruited neutrophils from early (day 1) lesions had similar phagocytic and NADPH oxidase capacities. However, most neutrophils were apoptotic, and transcription of immunomodulatory genes that promote efferocytosis were not upregulated, suggesting that the increased numbers of neutrophils may, in part, reflect defective clearance and resolution of the inflammatory response. In addition, the presence of more IL-17A-producing γδ and non-γδ T cells in early lesions (1-7 days), and L. major antigen-responsive Th17 cells after 28 days of coinfection, with a corresponding increase in IL-1beta, may recruit more naïve neutrophils into the inflammatory site. Neutralization studies suggest that IL-17A contributed to an enhanced inflammatory response, whereas IL-1beta instead had a net proinflammatory effect. Taken together, these data suggest that coinfection of L. major infection with S. aureus exacerbates disease, both by promoting more inflammation and neutrophil recruitment and by increasing neutrophil apoptosis and delaying resolution of the inflammatory response. We also investigated the role of inflammasomes in bacterial coinfection using mice deficient in ASC, an adapter protein necessary for efficient inflammasome assembly. Inflammasomes are multi-protein complexes that form in response to danger signals, leading to the production of active IL-1beta. L. major-S. aureus coinfection in ASC knockout mice mirrored the results of IL-1beta neutralization, leading to further lesion exacerbation. We also coinfected wild-type mice with L. major and P. aeruginosa strains that were deficient in flagellin, a potent activator of the NLRC4 inflammasome, or ExoS, ExoT, and ExoY (ExoSTY), exotoxins that compete with flagellin for injection into eukaryotic host cells via the type III secretion system (T3SS). Wild-type P. aeruginosa and flagellin mutants exacerbated lesions in coinfection with L. major, but ExoSTY mutants did not. This suggests that NLRC4 inflammasome activation does not affect lesion exacerbation in L. major-P. aeruginosa coinfection, but exotoxins are required. Based on this observation, we explored the role of bacterial exotoxins and leishmanial factors by performing co-inoculations of live L. major or S. aureus with heat-killed bacteria or parasites, respectively. We also performed L. major coinfections with S. aureus strains expressing different exotoxins. We found that co-inoculated heat-killed S. aureus, but not heat-killed L. major, recapitulated coinfected lesion exacerbation, suggesting that S, aureus antigens or heat-stable virulence factors can influence the immune response to L. major similarly to live bacteria. S. aureus strains that do not highly express alpha-toxin, or with Staphylococcus epidermidis, a human skin commensal, recapitulated lesion exacerbation in coinfection with L. major. S. aureus toxic shock syndrome toxin-1 was not required for coinfection lesion exacerbation, but worsened lesion pathology. Thus, enhanced lesion pathology in L. major-S. aureus coinfection can occur without the action of several S. aureus virulence factors, but live L. major or a heat-labile factor produced by L. major, may be required. These data illustrate the profound impact that coinfecting microorganisms can exert on inflammatory lesion pathology and host adaptive immune responses. In particular, our study provides new insights into how bacterial coinfection affects the host immune response to Leishmania, which may contribute to novel approaches for reducing the severity of CL disease.