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Inflammatory monocytes can be manipulated by environmental cues to perform multiple functions. To define the role of monocytes during primary or secondary infection with an intra-phagosomal pathogen we employed Leishmania major-red fluorescent protein (RFP) parasites and multi-color flow cytometry to define and enumerate infected and uninfected inflammatory cells in the skin. During primary infection, infected monocytes had altered maturation and were the initial mononuclear host cell for parasite replication. In contrast, at a distal site of secondary infection in mice with a healed but persistent primary infection, this same population rapidly produced inducible nitric oxide synthase (iNOS) in an IFN-γ dependent manner and was critical for parasite killing. Maturation to a dendritic cell-like phenotype was not required for monocyte iNOS-production, and enhanced monocyte recruitment correlated with IFN-γ dependent cxcl10 expression. In contrast, neutrophils appeared to be a safe haven for parasites in both primary and secondary sites. Thus, inflammatory monocytes play divergent roles during primary versus secondary infection with an intra-phagosomal pathogen. [ABSTRACT FROM AUTHOR]

Primary Leishmania major infection typically produces cutaneous lesions that not only heal but also harbor persistent parasites. While the opposing roles of CD4+ T-cell-derived IFN-γ and IL-10 in promoting parasite killing and persistence have been well established, how these responses develop from naïve precursors has not been directly monitored throughout the course of infection. We used peptide:Major Histocompatibility Complex class II (pMHCII) tetramers to investigate the endogenous, parasite-specific primary CD4+ T-cell response to L. major in mice resistant to infection. Maximal frequencies of IFN-γ+ CD4+ T cells were observed in the spleen and infected ears within a month after infection and were maintained into the chronic phase. In contrast, peak frequencies of IL-10+ CD4+ T cells emerged within 2 weeks of infection, persisted into the chronic phase, and accumulated in the infected ears but not the spleen, via a process that depended on local antigen presentation. T helper type-1 (Th1) cells, not Foxp3+ regulatory T cells, were the chief producers of IL-10 and were not exhausted. Therefore, tracking antigen -specific CD4+ T cells revealed that IL-10 production by Th1 cells is not due to persistent T-cell antigen receptor stimulation, but rather driven by early antigen encounter at the site of infection. [ABSTRACT FROM AUTHOR]

Ecotin is a potent inhibitor of family S1A serine peptidases, enzymes lacking in the protozoan parasite Leishmania major. Nevertheless, L. major has three ecotin-like genes, termed inhibitor of serine peptidase (ISP). ISP1 is expressed in vector-borne procyclic and metacyclic promastigotes, whereas ISP2 is also expressed in the mammalian amastigote stage. Recombinant ISP2 inhibited neutrophil elastase, trypsin and chymotrypsin with Kis between 7.7 and 83 nM. L. major ISP2–ISP3 double null mutants (Δ isp2/3) were created. These grew normally as promastigotes, but were internalized by macrophages more efficiently than wild-type parasites due to the upregulation of phagocytosis by a mechanism dependent on serine peptidase activity. Δ isp2/3 promastigotes transformed to amastigotes, but failed to divide for 48 h. Intracellular multiplication of Δ isp2/3 was similar to wild-type parasites when serine peptidase inhibitors were present, suggesting that defective intracellular growth results from the lack of serine peptidase inhibition during promastigote uptake. Δ isp2/3 mutants were more infective than wild-type parasites to BALB/c mice at the early stages of infection, but became equivalent as the infection progressed. These data support the hypothesis that ISPs of L. major target host serine peptidases and influence the early stages of infection of the mammalian host. [ABSTRACT FROM AUTHOR]

Primary Leishmania major infection typically produces cutaneous lesions that not only heal but also harbor persistent parasites. While the opposing roles of CD4(+) T-cell-derived IFN-γ and IL-10 in promoting parasite killing and persistence have been well established, how these responses develop from naïve precursors has not been directly monitored throughout the course of infection. We used peptide:Major Histocompatibility Complex class II (pMHCII) tetramers to investigate the endogenous, parasite-specific primary CD4(+) T-cell response to L. major in mice resistant to infection. Maximal frequencies of IFN-γ(+) CD4(+) T cells were observed in the spleen and infected ears within a month after infection and were maintained into the chronic phase. In contrast, peak frequencies of IL-10(+) CD4(+) T cells emerged within 2 weeks of infection, persisted into the chronic phase, and accumulated in the infected ears but not the spleen, via a process that depended on local antigen presentation. T helper type-1 (Th1) cells, not Foxp3(+) regulatory T cells, were the chief producers of IL-10 and were not exhausted. Therefore, tracking antigen-specific CD4(+) T cells revealed that IL-10 production by Th1 cells is not due to persistent T-cell antigen receptor stimulation, but rather driven by early antigen encounter at the site of infection., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)