TLR2 signaling in skin non-hematopoietic cells induces early neutrophil recruitment in response to Leishmania major infection

Neutrophils are rapidly recruited to the mammalian skin in response to infection with the cutaneous Leishmania pathogen. The parasites use neutrophils to establish the disease, however, the signals driving early neutrophil recruitment are poorly known. Here, we identified the functional importance of TLR2 signaling in this process. Using bone-marrow chimeras and immunohistology we identified the TLR2-expressing cells involved in this early neutrophil recruitment to be of non-hematopoietic origin. Keratinocytes are damaged and briefly in contact with the parasites during infection. We show that TLR2 triggering by L. major is required for their secretion of neutrophil-attracting chemokines. Furthermore, TLR2 triggering by L. major phosphoglycans is critical for neutrophil recruitment impacting negatively on disease development, as shown by better control of lesion size and parasite load in Tlr2-/- compared to wild type infected mice. Conversely, restoring early neutrophil presence in Tlr2-/- mice through injection of wild type neutrophils or CXCL1 at the onset of infection resulted in delayed disease resolution comparable to that observed in wild type mice. Taken together, our data demonstrate a new role for TLR2-expressing non-hematopoietic skin cells in the recruitment of the first wave of neutrophils following L. major infection, a process delaying disease control

Regulatory B cells shape the development of Th2 immune responses in BALB/c mice infected with Leishmania major through IL-10 production.

Recent evidence indicates that B cells are required for susceptibility to infection with Leishmania major in BALB/c mice. In this study, we analyzed the role of the IL-10 produced by B cells in this process. We showed that B cells purified from the spleen of BALB/c mice produced IL-10 in response to stimulation with L. major in vitro. In vivo, early IL-10 mRNA expression is detected after L. major infection in B cells from draining lymph nodes of susceptible BALB/c, but not of resistant C57BL/6 mice. Although adoptive transfer of naive wild-type B cells prior to infection in B cell-deficient BALB/c mice restored Th2 cell development and susceptibility to infection with L. major of these otherwise resistant mice, adoptive transfer of IL-10(-/-) B cells mice did not. B cells stimulated by L. major, following in vitro or in vivo encounter, express the CD1d and CD5 molecules and the IL-10 produced by these cells downregulate IL-12 production by L. major-stimulated dendritic cells. These observations indicate that IL-10 secreting B cells are phenotypically and functionally regulatory B cells. Altogether these results demonstrate that the IL-10 produced by regulatory CD1d+ CD5+ B cells in response to L. major is critical for Th2 cell development in BALB/c mice

Infection and Immunity

Texto completo: acesso restrito. p. 1575–1584Infection with Leishmania braziliensis causes cutaneous or mucocutaneous leismaniasis in humans. Toll-like receptor 9 (TLR9) expression has been found in granulomas of lesions in L. braziliensis-infected individuals. L. braziliensis inoculation in mice induces very small lesions that are self-healing, whereas deficiency in the TLR adaptor molecule, MyD88, renders mice susceptible to infection. The TLR involved has not been identified, prompting us to investigate if TLR9 triggering by the parasite contributes to the strong resistance to infection observed in L. braziliensis-inoculated mice. The parasites activated wild-type (WT) dendritic cells (DCs) in vitro but not DCs derived from TLR9−/− mice. TLR9−/− mice inoculated with L. braziliensis exhibited a transient susceptibility characterized by increased lesion size and parasite burden compared to those of WT mice. Surprisingly, elevated levels of gamma interferon (IFN-γ) were measured at the site of infection and in draining lymph node T cells of TLR9−/− mice at the peak of susceptibility, suggesting that unlike observations in vitro, the parasite could induce DC activation leading to the development of Th1 cells in the absence of TLR9 expression. Taken together, these data show that TLR9 signaling is important for the early control of lesion development and parasite burden but is dispensable for the differentiation of Th1 cells secreting IFN-γ, and the high levels of this cytokine are not sufficient to control early parasite replication following L. braziliensis infection

<i>L. braziliensis</i> infection prevents the upregulation of IFN-inducible genes due to SGS pre-immunization.

<p>BALB/c mice were inoculated 3 times in the right ear pinna every 2 wks with <i>Lu. intermedia</i> SGS and then challenged in the left ear 2 wks later with <i>Lu. intermedia</i> SGS plus 1×10⁶<i>L. braziliensis</i> stationary phase promastigotes. The left ears were collected 2 wks after infection, homogenized and the expression of IFN-inducible genes such as (A) Ifit1, Irgm1 and Irgm2, and the expression of IFN-related genes like (B) Stat1 and CXCL9 was determined by real-time quantitative PCR. Relative mRNA expression normalized to the housekeeping gene HPRT is presented as the mean +SEM (n = 5 mice per group); * p<0.05 by Student's <i>t</i>-test.</p

Genes differentially expressed in response to SGS pre-immunization.

<p>Genes differentially expressed in response to SGS pre-immunization.</p

IFN-inducible genes are upregulated in human PBMCs by individuals exposed to sand fly bites.

<p>PBMCs from people exposed to <i>Lu. intermedia</i> bites living in an endemic area who expressed high anti-SGS antibody were isolated and stimulated <i>in vitro</i> with SGS (equivalent to 1.5 pairs of salivary glands) for 72 hours. For the control group, PBMCs were isolated from individuals living in a non-endemic area of Salvador, Bahia, Brazil and stimulated with SGS for 72 hours. The expression of (A) Ifit, (B) Irgm, (C) Stat1, and (D) CXCL9 was determined by real-time quantitative PCR. *** p<0.0001.</p