SummaryTwo different Toll-like receptors (TLRs) have been shown to play a role in host responses to Leishmania infection. TLR-2 is involved in parasite survival in macrophages upon activation by lipophosphoglycan (LPG), a virulence factor expressed by Leishmania. In contrast, activation of TLR-9 has been shown to promote a host-protective response. However, whether there is a relationship between the interaction of LPG and TLR-2, on one hand, with the effect of TLR-9, on the other hand, remains unknown. In this study, we report that in-vitro infection of macrophages with a L. major parasite with high expression levels of LPG results in decreased TLR-9 expression compared to infection with a L. major parasite with lower expression levels of LPG. Addition of anti-LPG as well as anti-TLR-2 antibodies prevents this reduction of TLR-9 expression. Also, the addition of purified LPG to macrophages results in a decrease of TLR-9 expression, which is shown to be mediated by transforming growth factor (

TLRs recognize pathogen-expressed Ags and elicit host-protective immune response. Although TLR2 forms heterodimers with TLR1 or TLR6, recognizing different ligands, differences in the functions of these heterodimers remain unknown. In this study, we report that in Leishmania major-infected macrophages, the expression of TLR1 and TLR2, but not TLR6, increased; TLR2–TLR2 association increased, but TLR2–TLR6 association diminished. Lentivirus-expressed TLR1–short hairpin RNA (shRNA) or TLR2–shRNA administration reduced, but TLR6–shRNA increased L. major infection in BALB/c mice. Corroboratively, Pam3CSK4 (TLR1–TLR2 ligand) and peptidoglycan (TLR2 ligand) increased L. major infection but reduced TLR9 expression, whereas pegylated bisacycloxypropylcysteine (BPPcysMPEG; TLR2–TLR6 ligand) reduced L. major number in L. major-infected macrophages, accompanied by increased TLR9 expression, higher IL-12 production, and inducible NO synthase expression. Whereas MyD88, Toll/IL-1R adaptor protein, and TNFR-α–associated factor 6 recruitments to TLR2 were not different in Pam3CSK4-, peptidoglycan-, or BPPcysMPEG-treated macrophages, only BPPcysMPEG enhanced p38MAPK and activating transcription factor 2 activation. BPPcysMPEG conferred antileishmanial functions to L. major-infected BALB/c-derived T cells in a macrophage–T cell coculture and in BALB/c mice; the protection was TLR6 dependent and IL-12 dependent, and it was accompanied by reduced regulatory T cell number. BPPcysMPEG administration during the priming with fixed L. major protected BALB/c mice against challenge L. major infection; the protection was accompanied by low IL-4 and IL-10, but high IFN-γ productions and reduced regulatory T cells. Thus, BPPcysMPEG, a novel diacylated lipopeptide ligand for TLR2–TLR6 heterodimer, induces IL-12–dependent, inducible NO synthase–dependent, T-reg–sensitive antileishmanial protection. The data reveal a novel dimerization partner-dependent duality in TLR2 function.

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Leishmania major is a parasite that resides and replicates in macrophages. We previously showed that the parasite enhanced CD40-induced Raf-MEK-ERK signaling but inhibited PI3K-MKK-p38MAPK signaling to proleishmanial effects. As Raf and PI3K have a Ras-binding domain but exert opposite effects on Leishmania infection, we examined whether Ras isoforms had differential roles in Leishmania infection. We observed that L. major enhanced N-Ras and H-Ras expression but inhibited K-Ras expression in macrophages. L. major infection enhanced N-Ras activity but inhibited H-Ras and K-Ras activity. TLR2 short hairpin RNA or anti-TLR2 or anti-lipophosphoglycan Abs reversed the L. major–altered N-Ras and K-Ras expressions. Pam3CSK4, a TLR2 ligand, enhanced N-Ras expression but reduced K-Ras expression, indicating TLR2-regulated Ras expression in L. major infection. Whereas N-Ras silencing reduced L. major infection, K-Ras and H-Ras silencing enhanced the infection both in macrophages in vitro and in C57BL/6 mice. BALB/c-derived macrophages transduced with lentivirally expressed N-Ras short hairpin RNA and pulsed with L. major–expressed MAPK10 enhanced MAPK10-specific Th1-type response. CD40-deficient mice primed with these macrophages had reduced L. major infection, accompanied by higher IFN-γ but less IL-4 production. As N-Ras is activated by Sos, a guanine nucleotide exchange factor, we modeled the N-Ras–Sos interaction and designed two peptides from their interface. Both the cell-permeable peptides reduced L. major infection in BALB/c mice but not in CD40-deficient mice. These data reveal the L. major–enhanced CD40-induced N-Ras activation as a novel immune evasion strategy and the potential for Ras isoform–targeted antileishmanial immunotherapy and immunoprophylaxis.

SummaryToll-like receptors (TLRs) recognize pathogen-associated molecular patterns and results in innate immune system activation that results in elicitation of the adaptive immune response. One crucial modulator of the adaptive immune response is CD40. However, whether these molecules influence each other's expression and functions is not known. Therefore, we examined the effects of TLRs on CD40 expression on macrophages, the host cell for the protozoan parasite Leishmania major. While polyinosinic-polycytidylic acid [poly (I:C)], a TLR-3 ligand, lipopolysaccharide (LPS), a TLR-4 ligand, imiquimod, a TLR-7/8 ligand and cytosine-phosphate-guanosine (CpG), a TLR-9 ligand, were shown to enhance CD40 expression, CD40 stimulation enhanced only TLR-9 expression. Therefore, we tested the synergism between CD40 and CpG in anti-leishmanial immune response. In Leishmaniainfected macrophages, CpG was found to reduce CD40-induced extracellular stress-regulated kinase (ERK)1/2 activation; with the exception of interleukin (IL)-10, these ligands had differential effects on CD40-induced IL-1α, IL-6 and IL-12 production. CpG significantly enhanced the anti-leishmanial function of CD40 with differential effects on IL-4, IL-10 and interferon (IFN)-γ production in susceptible BALB/c mice. Thus, we report the first systematic study on CD40-TLR cross-talk that regulated the experimental L. major infection.