Interdependencies between Toll-like receptors in Leishmania infection.

Multiple pathogen-associated molecular patterns (PAMPs) on a pathogen's surface imply their simultaneous recognition by the host cell membrane-located multiple PAMP-specific Toll-like receptors (TLRs). The TLRs on endosomes recognize internalized pathogen-derived nucleic acids and trigger anti-pathogen immune responses aimed at eliminating the intracellular pathogen. Whether the TLRs influence each other's expression and effector responses-termed TLR interdependency-remains unknown. Herein, we first probed the existence of TLR interdependencies and next determined how targeting TLR interdependencies might determine the outcome of Leishmania infection. We observed that TLRs selectively altered expression of their own and of other TLRs revealing novel TLR interdependencies. Leishmania major-an intra-macrophage parasite inflicting the disease cutaneous leishmaniasis in 88 countries-altered this TLR interdependency unfolding a unique immune evasion mechanism. We targeted this TLR interdependency by selective silencing of rationally chosen TLRs and by stimulation with selective TLR ligands working out a novel phase-specific treatment regimen. Targeting the TLR interdependency elicited a host-protective anti-leishmanial immune response and reduced parasite burden. To test whether this observation could be used as a scientific rationale for treating a potentially fatal L. donovani infection, which causes visceral leishmaniasis, we targeted the inter-TLR dependency adopting the same treatment regimen. We observed reduced splenic Leishman-Donovan units accompanied by host-protective immune response in susceptible BALB/c mice. The TLR interdependency optimizes TLR-induced immune response by a novel immunoregulatory framework and scientifically rationalizes targeting TLRs in tandem and in sequence for redirecting immune responses against an intracellular pathogen.

TLR11 or TLR12 silencing reduces Leishmania major infection.

Toll-like receptors (TLRs) recognize the pathogen-associated molecular patterns (PAMPs) and induce host-protective immune response. The role of the profilin-recognizing TLR11/TLR12 in Leishmania infection is unknown. Herein, we report that TLR11/ TLR12 expression increases in virulent L. major-infected macrophages but is prevented by miltefosine, an anti-leishmanial drug. While lipohosphoglycan (LPG) increases, LPG or TLR2 blockade prevents, the heightened TLR11/TLR12 expression. LPG-TLR2 interaction triggers MyD88- and TIRAP-mediated signaling enhancing ERK-1/2 activation and increased production of IL-10 that promotes TLR11/TLR12 expression. Profilin expression was higher in the virulent L. major and L. donovani parasites than that observed in the avirulent parasites. TLR11 or TLR12 silencing reduces parasite burden and increases IFN-γ, but reduces IL-4, production indicating that TLR11 and TLR12 play a pro-leishmanial role.

Counteractive functions are encrypted in the residues of CD154.

CD40, as a single receptor that binds CD154 (CD40-ligand or CD40L), regulates counteractive effector functions such as production of pro- and anti-inflammatory cytokines. Therefore, we examined whether such dual messages are encrypted in CD40L. As such message encryption was never investigated, we hypothesized that mutation of certain amino acid residues should in principle enhance pro-inflammatory cytokine production whereas mutation of some others would enhance anti-inflammatory cytokine secretion. We mutated six such residues, which were previously showed to participate in CD40L function. Here, we report that the mutant CD154 129E→V was superior to the wild-type CD154 in killing of Leishmania donovani, induction of inducible nitric oxide synthase (iNOS) and production of IL-12 and relative phosphorylation of p38MAPK and ERK-1/2 in PBMC-derived macrophages. By contrast, 128S→V promoted L. donovani survival, reducing iNOS, but increasing IL-10 expression and predominant ERK-1/2 phosphorylation. The mutant 144G→V did not have significant effects. Other mutants (142E→V, 143K→A, 145Y→F) mimicked the wild-type CD154. Molecular dynamics simulation suggested that these mutations induced differential conformational changes in the CD40-CD154 complex. Therefore, assortment of the contrasting messages encrypted in a given ligand performing counteractive functions presents a novel fundamental biological principle that can be used for devising various therapies.

TLR9-deficiency reduces TLR1, TLR2 and TLR3 expressions in Leishmania major-infected macrophages.

The parasite Leishmania major counteractively modulates TLR2 and TLR9 expression and their functions. Although TLR1, TLR3, TLR4, and TLR7 are also implicated in Leishmania infection, whether their expression was altered in TLR2 or TLR9 deficiency remained unknown. Therefore, we examined TLR1, TLR3, TLR4 and TLR7 expression in L. major infection in TLR2-deficient or TLR9-deficient macrophages. We observed that TLR9-deficiency reduced TLR1, TLR2 and TLR3 but not TLR7 expression in the macrophages treated with live or killed L. major promastigotes. TLR2-deficiency had little effects by comparison. TLR9-deficient macrophages had reduced CD40 expression and less IL-12 and TNF-α expression. Thus, we report that TLR9 modulates TLR1, TLR2 and TLR3, but not TLR7, expression in L. major-infected macrophages.

Pegylated bisacycloxypropylcysteine, a diacylated lipopeptide ligand of TLR6, plays a host-protective role against experimental Leishmania major infection.

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.

Anti-herpes virus activities of Achyranthes aspera: an indian ethnomedicine, and its triterpene acid.

The aim of this study was to evaluate the antiviral potential of methanolic extract (ME) of Achyranthes aspera, an Indian folk medicine and one of its pure compound oleanolic acid (OA) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The ME possessed weak anti-herpes virus activity (EC50 64.4µg/ml for HSV-1 and 72.8µg/ml for HSV-2). While OA exhibited potent antiherpesvirus activity against both HSV-1 (EC50 6.8µg/ml) and HSV-2 (EC50 7.8µg/ml). The time response study revealed that the antiviral activity of ME and OA is highest at 2-6h post infection. The infected and drug-treated peritoneal macrophage at specific time showed increased level of pro-inflammatory cytokines (IL6 and IL12). Further, the PCR of DNA from infected cultures treated with ME and OA, at various time intervals, failed to show amplification at 48-72h, similar to that of HSV infected cells treated with acyclovir, indicating that the ME and OA probably inhibit the early stage of multiplication (post infection of 2-6h). Thus, our study demonstrated that ME and OA have good anti-HSV activity, with SI values of 12, suggesting the potential use of this plant.