CD40 Signaling Is Impaired in L. major–infected Macrophages and Is Rescued by a p38MAPK Activator Establishing a Host-protective Memory T Cell Response

Abstract

Leishmania, a protozoan parasite, lives and multiplies as amastigote within macrophages. It is proposed that the macrophage expressed CD40 interacts with CD40 ligand on T cells to induce IFN-γ, a Th1-type cytokine that restricts the amastigote growth. Here, we demonstrate that CD40 cross-linking early after infection resulted in inducible nitric oxide synthetase type-2 (iNOS2) induction and iNOS2-dependent amastigote elimination. Although CD40 expression remained unaltered on L. major–infected macrophages, delay in the treatment of macrophages or of mice with anti-CD40 antibody resulted in significant reduction in iNOS2 expression and leishmanicidal function suggesting impaired CD40 signaling in Leishmania infection. The inhibition of CD40-induced iNOS2 expression by SB203580, a p38-mitogen activated protein kinase (p38MAPK)-specific inhibitor, and the reversal of the inhibition by anisomycin, a p38MAPK activator, suggested a crucial role of p38MAPK in CD40 signaling. Indeed, the CD40-induced p38MAPK phosphorylation, iNOS2 expression and anti-leishmanial function were impaired in Leishmania-infected macrophages but were restored by anisomycin. Anisomycin's effects were reversed by SB203580 emphasizing the role of p38MAPK in CD40-induced iNOS2-dependent leishmanicidal function. Anisomycin administration in L. major–infected BALB/c mice resulted in significant reduction in the parasite load and established a host-protective Th1-type memory response. Also implicated in these findings is a scientific rationale to define novel anti-parasite drug targets and to bypass the problem of drug resistance.

Reciprocal Regulation of Protein Kinase C Isoforms Results in Differential Cellular Responsiveness

“…Induction of IL-12 and iNOS2 in leishmaniasis is associated with host-protective function, whereas induction of IL-10 is associated with disease-exacerbating function (21,22,39). We have shown in this study that inhibition of PKCd and z/l enhanced the antileishmanial effect of CD40, whereas inhibition of PKCa, b, and ε abrogated the anti-leishmanial effect of CD40.…”

“…Because host protection is associated with reduced amastigote number in macrophages and enhanced iNOS2 expression (11,12,22), we tested the effect of PKCa/b, b, d, ε, and z/l inhibitors on amastigote number and iNOS2 expression in thioglycolate-elicited BALB/c-derived peritoneal macrophages. We observed that the inhibition of PKCd and z/l isoforms enhanced the anti-leishmanial activity of CD40, as inhibition of PKCd and z/l along with anti-CD40 treatment resulted in reduced amastigote count (Fig.…”

Mechanisms of Immune Evasion in Leishmaniasis

“…This is further supported by studies with L. major infected B7-1 knockout, B7-2 knockout, and B7-1/B7-2 double knockout BALB/c mice (Brown et al, 2002). Signaling via CD40-CD40L interactions, critical for the induction of anti-leishmanial responses (Campbell et al, 1996; Kamanaka et al, 1996), is also impaired by L. major (Awasthi et al, 2003). …”

SHP-1 Plays a Crucial Role in CD40 Signaling Reciprocity

“…for three alternate days beginning from the fourth day after infection. Disease severity was assessed by measurement of footpad swelling using a digital micrometer (Mitituyo, Kawasaki, Japan) and parasite burden as described earlier (26). Mice were used per the animal use protocol approved by the Institutional Animal Care and Use Committee.…”