Macrophages play host to Leishmania major, a parasite that causes leishmaniasis in 500,000 people annually. Macrophage-expressed CD40, a costimulatory molecule, induces interleukin-12 (IL-12)-dependent and interferon-gamma (IFN-gamma)-dependent host-protective immune responses to Leishmania and other intracellular pathogens. Paradoxically, IL-10, another CD40-induced cytokine in macrophages, promotes Leishmania infection. How CD40 signaling regulates the secretion of these two counteractive cytokines remains unknown. Here we show that weak CD40 signals induce extracellular stress-related kinase-1/2 (ERK-1/2)-dependent IL-10 expression, whereas stronger signals induce p38 mitogen-activated protein kinase (p38MAPK)-dependent IL-12 production. p38MAPK and ERK-1/2 therefore have counter-regulatory actions. Leishmania skews CD40 signaling toward ERK-1/2, inducing IL-10, which inhibits activation of CD40-induced p38MAPK and expression of inducible nitric oxide synthase-2 (iNOS-2) and IL-12. ERK-1/2 inhibition or IL-10 neutralization restores CD40-induced p38MAPK activation and parasite killing in macrophages and the BALB/c mouse, a susceptible host. These data uncover a new immune evasion strategy, whereby Leishmania differentially modulates CD40-engaged, reciprocally functioning signaling modules, and provide a new conceptual framework for immune homeostasis.

Leishmania donovani, a protozoan parasite, resides and replicates as amastigotes within macrophages. The parasite inflicts the disease visceral leishmaniasis by suppressing host cell function. Neither a therapeutic vaccine nor an effective anti-leishmanial drug to reverse the immunosuppression is available. Although miltefosine (hexadecylphosphocholine or HPC) is a promising orally bioavailable anti-leishmanial drug, its efficacy is seriously compromised by contra-indications in pregnant women. Further rational redesigning of the drug requires studies on its mechanism of action, which is unknown at present. Because miltefosine is proposed to have immunomodulatory functions, we examined whether miltefosine exerts its anti-leishmanial functions by activating macrophages. We observed that miltefosine’s anti-leishmanial function was significantly compromised in IFN-γ-deficient macrophages suggesting the importance of endogenous IFN-γ in miltefosine-induced anti-leishmanial functions of macrophages. Miltefosine induced IFN-γ, neutralization of which reduced the anti-leishmanial functions of macrophages. IFN-γ responsiveness is reduced in L. donovani-infected macrophages but is significantly restored by miltefosine, as it enhances IFN-γ receptors and IFN-γ induced STAT-1 phosphorylation but reduced activation of SHP-1, the phosphatase implicated in the down-regulation of STAT-1 phosphorylation. Miltefosine induced protein kinase C-dependent and PI3K-dependent p38MAP kinase phosphorylation and anti-leishmanial function. Miltefosine promotes p38MAP kinase-dependent anti-leishmanial functions and IL-12-dependent Th1 response. Leishmania donovani-infected macrophages induced Th2 response but miltefosine treatment reversed the response to Th1-type. Thus, our data define for the first time the mechanistic basis of host cell-dependent anti-leishmanial function of miltefosine.

No abstract

Anergy and apoptosis are major pathways of T cell tolerance. Impairments in these mechanisms are believed to contribute to the development of autoimmune diseases such as SLE and arthritis. Here we analyzed features of T cell anergy, activation and activation-induced cell death in lupus-prone and MHC-matched healthy mice. Unexpectedly, lupus mice exhibit reduced calcium flux, IL-2 production and proliferation in spleen cells upon anti-CD3 stimulation. Such T cell hyporesponsiveness is abrogated upon ionomycin stimulation, suggesting a specific impairment in TCR signaling pathway. This suggests that autoimmune T cells exhibit features of anergy, rather than resistance to it. In contrast, autoimmune T cells produce higher levels of pro-inflammatory cytokine interferon-gamma, and exhibit features of activation, i.e., increased activation markers. Consistently, quantitative gene expression analyses show lower Il2, but higher Ifn-g, mRNA in lupus T cells than in controls. Further, lupus T cells exhibit increased constitutive phosphorylation of ZAP-70 and other T cell signaling molecules, which do not increase upon anti-CD3 stimulation. Finally, lupus T cells resist apoptosis upon specific TCR signaling. Thus, autoimmune T cells display features of activation as well as anergy. Such split anergic T cells display resistance to activation-induced cell death. These data lead us to posit that persistent in vivo T cell activation results in subsequent induction of anergy that manifests as impaired responsiveness to TCR mediated signaling and prevents T cells from undergoing activation-induced cell death. Thus, induction of anergy may actually prevent autoreactive T cells from being eliminated. Ongoing experiments will delineate mechanisms underlying this novel mechanism of autoimmune T cell persistence.