Surya Prakash Pandey

Microbial Sensing by Toll-Like Receptors and Intracellular Nucleic Acid Sensors

Recognition of an invading pathogen is critical to elicit protective responses. Certain microbial structures and molecules, which are crucial for their survival and virulence, are recognized by different families of evolutionarily conserved pattern recognition receptors (PRRs). This recognition initiates a signaling cascade that leads to the transcription of inflammatory cytokines and chemokines to eliminate pathogens and attract immune cells, thereby perpetuating further adaptive immune responses. Considerable research on the molecular mechanisms underlying host-pathogen interactions has resulted in the discovery of multifarious PRRs. In this review, we discuss the recent developments in microbial recognition by Toll-like receptors (TLRs) and intracellular nucleic acid sensors and the signaling pathways initiated by them.

NLRC5 Deficiency Does Not Influence Cytokine Induction by Virus and Bacteria Infections

Nucleotide-binding domain and leucine rich repeat containing gene family receptors (NLRs) are cytosolic proteins that respond to a variety of pathogen and host components to induce inflammatory cytokines. NLRC5 is a recently identified member of the NLR family that has been implicated in positive and negative regulation of antiviral innate immune responses. To clarify whether NLRC5 controls antiviral innate immunity in vivo, we generated NLRC5-deficient mice. Macrophages and dendritic cells derived from NLRC5-deficient mice induced relatively normal levels of IFN-β, IL-6, and TNF-α after treatment with RNA viruses, DNA viruses, and bacteria. The serum cytokine levels after polyinosinic-polycytidylic acid infection were also comparable between control and NLRC5-deficient mice. NLRC5 overexpression promoted IL-1β production via caspase-1, suggesting that NLRC5 constitutes an inflammasome. However, there was no reduction of IL-1β in NLRC5-deficient cells in response to known inflammasome activators, suggesting that NLRC5 controls IL-1β production through an unidentified pathway. These findings indicate that NLRC5 is dispensable for cytokine induction in virus and bacterial infections under physiologic conditions.

Pivotal role of RNA-binding E3 ubiquitin ligase MEX3C in RIG-I-mediated antiviral innate immunity.

Significance Upon virus infection, host cells detect viral nucleic acids and induce antiviral innate immune responses by producing cytokines. The retinoic acid inducible gene-1 (RIG-I)-like receptors are cytoplasmic sensors for RNA viruses that mediate antiviral innate immunity. In this study, we identify K homology (KH) domain- and really interesting new gene (RING)-finger domain-containing MEX3C as a molecule that regulates RIG-I function. MEX3C preferentially colocalizes with viral RNA and RIG-I in cytoplasmic granules in infected cells and mediates K63-linked ubiquitination of RIG-I, which is important for activation of downstream signaling. We show that MEX3C is required for cytokine production after infection with RNA viruses that are detected by RIG-I by generating MEXC-deficient mice. Our study demonstrates a critical role of MEX3C in induction of RIGI-mediated antiviral innate immune responses. The RIG-I–like receptors, retinoic acid inducible gene-1 (RIG-I), melanoma differentiation-associated protein 5, and laboratory of genetics and physiology-2, are cytoplasmic sensors for RNA viruses that mediate the antiviral innate immune responses. We demonstrate that really interesting new gene-finger domain- and K homology domain-containing MEX3C regulates RIG-I function. MEX3C colocalizes with RIG-I in the stress granules of virally infected cells, and its overexpression causes the lysine-63–linked ubiquitination of RIG-I and activates IFN-β promoter. Embryonic fibroblast cells, macrophages, and conventional dendritic cells derived from Mex3c-deficient mice showed defective production of type I IFN after infection with RNA viruses that are recognized by RIG-I. These results demonstrate that MEX3C is an E3 ubiquitin ligase that modifies RIG-I in stress granules and plays a critical role in eliciting antiviral immune responses.

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

Immunological homeostasis is often maintained by counteractive functions of two different cell types or two different receptors signaling through different intermediates in the same cell. One of these signaling intermediates is protein kinase C (PKC). Ten differentially regulated PKC isoforms are integral to receptor-triggered responses in different cells. So far, eight PKC isoforms are reported to be expressed in macrophages. Whether a single receptor differentially uses PKC isoforms to regulate counteractive effector functions has never been addressed. As CD40 is the only receptor characterized to trigger counteractive functions, we examined the relative role of PKC isoforms in the CD40-induced macrophage functions. We report that in BALB/c mouse macrophages, higher doses of CD40 stimulation induce optimum phosphorylation and translocation of PKCα, βI, βII, and ε whereas lower doses of CD40 stimulation activates PKCδ, ζ, and λ. Infection of macrophages with the protozoan parasite Leishmania major impairs PKCα, βI, βII, and ε isoforms but enhances PKCδ, ζ, and λ isoforms, suggesting a reciprocity among these PKC isoforms. Indeed, PKCα, βI, βII, and ε isoforms mediate CD40-induced p38MAPK phosphorylation, IL-12 expression, and Leishmania killing; PKCδ and ζ/λ mediate ERK1/2 phosphorylation, IL-10 production, and parasite growth. Treatment of the susceptible BALB/c mice with the lentivirally expressed PKCδ- or ζ-specific short hairpin RNA significantly reduces the infection and reinstates host-protective IFN-γ–dominated T cell response, defining the differential roles for PKC isoforms in immune homeostasis and novel PKC-targeted immunotherapeutic and parasite-derived immune evasion strategies.

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.

TLR–CD40 Cross-Talk in Anti-Leishmanial Immune Response

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) and activate innate immune cells to induce cytokines and co-stimulatory molecules such as CD40 and to enhance antigen presentation to T cells (1) that, upon activation, can either eliminate or support the pathogen (2). Herein, we propose that this duality in TLR functions results from their cross-talk with CD40. While all TLRs enhance CD40 expression, CD40 augments the expression of only TLR9 (3). As both CD40 and TLR9 induce expression of IL-12, a cytokine that induces the IFN-γ secreting Th1 cell differentiation (4), the CD40–TLR9 cross-regulation implies a positive feedback loop. By contrast, TLR1–TLR2 heterodimer down-regulates TLR9 expression (5) and antagonizes the development of Th1 response but favors the differentiation of regulatory T (T-reg) cells (Pandey et al., unpublished observation). Low CD40 expression levels in dendritic cells also promote T-reg cell differentiation (6). This duality can emerge from the sharing of signaling molecules. CD40 induces TRAF6-mediated, ERK-1/2-dependent IL-10 (7), which can inhibit the TLR-induced p38-MAPK activation and IL-12 production, antagonizing Th1 development. CD40-induced TRAF3-dependent p38-MAPK activation (7) can synergize with the TLR-activated p38-MAPK-dependent IL-12 production and Th1 differentiation. Using Leishmania infection, we show that the TLR–CD40 cross-talk can induce contrasting anti-leishmanial immune responses. Leishmania, a protozoan parasite, lives in macrophages. Leishmania expresses lipophosphoglycan (LPG), proteoglycans, flagellin, and profilin for possible recognition by the host cell-expressed TLRs. Recognition of the Leishmania-expressed PAMPs results in differential immune responses, which can either reduce or exacerbate Leishmania infection. As TLRs modulate the expression of CD40, a co-stimulatory molecule whose expression levels modulate anti-leishmanial T cell responses, we propose that TLR–CD40 cross-talk significantly regulate the outcome of an anti-leishmanial immune response.

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.

Inhibition of CD40-Induced N-Ras Activation Reduces Leishmania major Infection

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.

TLR11 or TLR12 silencing reduces Leishmania major infection

HIGHLIGHTSTLR11 and TLR12 expression is increased by virulent Leishmania major parasites.TLR2‐LPG interaction augments TLR11 and TLR12 expression.IL‐10 increases TLR11 and TLR12 expression.TLR11 and TLR12 silencing reduces parasite growth.The anti‐leishmanial effect is accompanied by Th1 response. ABSTRACT 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‐&ggr;, but reduces IL‐4, production indicating that TLR11 and TLR12 play a pro‐leishmanial role.

Host DNA Induced Inflammation and Autoimmune Diseases

DNA sensors initiate innate immune responses upon recognition of microbial and self-derived DNA in the intracellular compartments or cytoplasm. These sensors include TLR9, AIM2 like receptors and many other recently identified cytosolic DNA sensors. The otherwise protective nature of host defense by these receptors can turn hostile when they recognize self-DNA through various mechanisms and aberrantly activate DNA sensing pathways leading to unregulated or inappropriate type I IFN production and consequent autoimmune and autoinflammatory diseases. In this chapter, we highlight the current findings that shed light on the complex initiator and effector mechanisms that contribute to autoimmune disease pathology, including DNA sensing receptors, self and non-self discrimination, type I IFN system, mechanisms of enhanced self-DNA access to TLR9 and defective host DNA clearance.