Karishma Kamdar

A specific role for TLR1 in protective T H 17 immunity during mucosal infection

TLR1/TLR2 complexes are required for induction of IL-6 and IL-23 to generate protective TH17-mediated immunity and IgA production after oral but not systemic Yersinia enterocolitica infection.

Genetic and Metabolic Signals during Acute Enteric Bacterial Infection Alter the Microbiota and Drive Progression to Chronic Inflammatory Disease

Chronic inflammatory disorders are thought to arise due to an interplay between predisposing host genetics and environmental factors. For example, the onset of inflammatory bowel disease is associated with enteric proteobacterial infection, yet the mechanistic basis for this association is unclear. We have shown previously that genetic defiency in TLR1 promotes acute enteric infection by the proteobacteria Yersinia enterocolitica. Examining that model further, we uncovered an altered cellular immune response that promotes the recruitment of neutrophils which in turn increases metabolism of the respiratory electron acceptor tetrathionate by Yersinia. These events drive permanent alterations in anti-commensal immunity, microbiota composition, and chronic inflammation, which persist long after Yersinia clearence. Deletion of the bacterial genes involved in tetrathionate respiration or treatment using targeted probiotics could prevent microbiota alterations and inflammation. Thus, acute infection can drive long term immune and microbiota alterations leading to chronic inflammatory disease in genetically predisposed individuals.

Toll-like receptor signaling and regulation of intestinal immunity.

The intestine is a complex organ that must maintain tolerance to innocuous food antigens and commensal microbiota while being also able to mount inflammatory responses against invading pathogenic microorganisms. The ability to restrain tolerogenic responses while permitting inflammatory responses requires communication between commensal bacteria, intestinal epithelial cells and immune cells. Disruption or improper signaling between any of these factors may lead to uncontrolled inflammation and the development of inflammatory diseases. Toll-like receptors (TLR) recognize conserved molecular motifs of microorganisms and, not surprisingly, are important for maintaining tolerance to commensal microbiota, as well as inducing inflammation against pathogens. Perturbations in individual TLR signaling can lead to a number of different outcomes and illustrate a system of regulation within the intestine in which each TLR plays a largely non-redundant role in mucosal immunity. This review will discuss recent findings on the roles of individual TLRs and intestinal homeostasis.

Arginine in α-defensins: differential effects on bactericidal activity correspond to geometry of membrane curvature generation and peptide-lipid phase behavior

Background: Complete Lys-for-Arg substitutions in α-defensins Crp4 and RMAD4 affect microbicidal activities very differently. Results: The peptide-lipid phase behavior of Crp4, RMAD4, and associated mutants correlated with differential biological activities. Conclusion: A stringent agreement exists between α-defensin bactericidal effects and an induced negative Gaussian curvature model of membrane disruption. Significance: These findings provide a basis for molecular engineering of novel peptide mimetic microbicides. The conserved tridisulfide array of the α-defensin family imposes a common triple-stranded β-sheet topology on peptides that may have highly diverse primary structures, resulting in differential outcomes after targeted mutagenesis. In mouse cryptdin-4 (Crp4) and rhesus myeloid α-defensin-4 (RMAD4), complete substitutions of Arg with Lys affect bactericidal peptide activity very differently. Lys-for-Arg mutagenesis attenuates Crp4, but RMAD4 activity remains mostly unchanged. Here, we show that the differential biological effect of Lys-for-Arg replacements can be understood by the distinct phase behavior of the experimental peptide-lipid system. In Crp4, small-angle x-ray scattering analyses showed that Arg-to-Lys replacements shifted the induced nanoporous phases to a different range of lipid compositions compared with the Arg-rich native peptide, consistent with the attenuation of bactericidal activity by Lys-for-Arg mutations. In contrast, such phases generated by RMAD4 were largely unchanged. The concordance between small-angle x-ray scattering measurements and biological activity provides evidence that specific types of α-defensin-induced membrane curvature-generating tendencies correspond directly to bactericidal activity via membrane destabilization.

In Vitro Activation of the Rhesus Macaque Myeloid α-Defensin Precursor proRMAD-4 by Neutrophil Serine Proteinases

α-Defensins are mammalian antimicrobial peptides expressed mainly by cells of myeloid lineage or small intestinal Paneth cells. The peptides are converted from inactive 8.5-kDa precursors to membrane-disruptive forms by post-translational proteolytic events. Because rhesus myeloid pro-α-defensin-4 (proRMAD-4(20–94)) lacks bactericidal peptide activity in vitro, we tested whether neutrophil azurophil granule serine proteinases, human neutrophil elastase (NE), cathepsin G (CG), and proteinase-3 (P3) have in vitro convertase activity. Only NE cleaved proRMAD-4(20–94) at the native RMAD-4 N terminus to produce fully processed, bactericidal RMAD-4(62–94). The final CG cleavage product was RMAD-4(55–94), and P3 produced both RMAD-4(55–94) and RMAD-4(57–94). Nevertheless, NE, CG, and P3 digests of proRMAD4 and purified RMAD-4(62–94), RMAD-4(55–94), and RMAD-4(57–94) peptides had equivalent in vitro bactericidal activities. Bactericidal peptide activity assays of proRMAD-4(20–94) variants containing complete charge-neutralizing D/E to N/Q or D/E to A substitutions showed that (DE/NQ)-proRMAD-4(20–94) and (DE/A)-proRMAD-4(20–94) were as active as mature RMAD-4(62–94). Therefore, proregion Asp and Glu side chains inhibit the RMAD-4 component of full-length proRMAD-4(20–94), perhaps by a combination of charge-neutralizing and hydrogen-bonding interactions. Although native RMAD-4(62–94) resists NE, CG, and P3 proteolysis completely, RMAD-4(62–94) variants with disulfide pairing disruptions or lacking disulfide bonds were degraded extensively, evidence that the disulfide array protects the α-defensin moiety from degradation by the myeloid converting enzymes. These in vitro analyses support the conclusion that rhesus macaque myeloid pro-α-defensins are converted to active forms by serine proteinases that co-localize in azurophil granules.

TLR1-induced chemokine production is critical for mucosal immunity against Yersinia enterocolitica

Our gastrointestinal tract is a portal of entry for a number of bacteria and viruses. Thus, this tissue must develop ways to induce antigen-specific T cell and antibody responses quickly. Intestinal epithelial cells are a central player in barrier function and also in communicating signals from invading pathogens to the underlying immune tissue. Here we demonstrate that activation of Toll-like receptor 1 (TLR1) in the epithelium leads to the upregulation of the chemokine CCL20 during oral infection with Yersinia enterocolitica. Further, both neutralization of CCL20 using polyclonal antibody treatment and deletion of TLR1 resulted in a defect in CCR6+ dendritic cells (DCs), which produce innate cytokines that help to induce anti-Yersinia-specific T helper 17 (TH17) cells and IgA production. These data demonstrate a novel role for TLR1 signaling in the intestinal epithelium and demonstrate that together TLR1 and CCL20 are critical mediators of TH17 immunity through the activation and recruitment of DCs.

Microbicidal effects of α- and θ-defensins against antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa

Antibiotic-resistant bacterial pathogens threaten public health. Because many antibiotics target specific bacterial enzymes or reactions, corresponding genes may mutate under selection and lead to antibiotic resistance. Accordingly, antimicrobials that selectively target overall microbial cell integrity may offer alternative approaches to therapeutic design. Naturally occurring mammalian α- and θ-defensins are potent, non-toxic microbicides that may be useful for treating infections by antibiotic-resistant pathogens because certain defensin peptides disrupt bacterial, but not mammalian, cell membranes. To test this concept, clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), including vancomycin heteroresistant strains, and ciprofloxacin-resistant Pseudomonas aeruginosa (CipR-PA) were tested for sensitivity to α-defensins Crp-4, RMAD-4 and HNPs 1-3, and to RTD-1, macaque θ-defensin-1. In vitro, 3 μM Crp-4, RMAD-4 and RTD-1 reduced MRSA cell survival by 99%, regardless of vancomycin susceptibility. For PA clinical isolates that differ in fluoroquinolone resistance and virulence phenotype, peptide efficacy was independent of strain ciprofloxacin resistance, site of isolation or virulence factor expression. Thus, Crp-4, RMAD-4 and RTD-1 are effective in vitro antimicrobials against clinical isolates of MRSA and CipR-PA, perhaps providing templates for development of α- and θ-defensin-based microbicides against antibiotic resistant or virulent infectious agents.

Retinoic Acid Can Exacerbate T Cell Intrinsic TLR2 Activation to Promote Tolerance

The contribution of vitamin A to immune health has been well established. However, recent evidence indicates that its active metabolite, retinoic acid (RA), has the ability to promote both tolerogenic and inflammatory responses. While the outcome of RA-mediated immunity is dependent upon the immunological status of the tissue, the contribution of specific innate signals influencing this response have yet to be delineated. Here, we found that treatment with RA can dampen inflammation during intestinal injury. Importantly, we report a novel and unexpected requirement for TLR2 in RA-mediated suppression. Our data demonstrate that RA treatment enhances TLR2-dependent IL-10 production from T cells and this, in turn, potentiates T regulatory cell (TREG) generation without the need for activation of antigen presenting cells. These data also suggest that combinatorial therapy using RA and TLR2 ligands may be advantageous in the design of therapies to treat autoimmune or inflammatory disease.

Innate Recognition of the Microbiota by TLR1 Promotes Epithelial Homeostasis and Prevents Chronic Inflammation

There is cross-talk between the intestinal epithelium and the microbiota that functions to maintain a tightly regulated microenvironment and prevent chronic inflammation. This communication is partly mediated through the recognition of bacterial proteins by host-encoded innate receptors, such as TLRs. However, studies examining the role of TLR signaling on colonic homeostasis have given variable and conflicting results. Despite its critical role in mediating immunity during enteric infection of the small intestine, TLR1-mediated recognition of microbiota-derived ligands and their influence on colonic homeostasis has not been well studied. In this study, we demonstrate that defective TLR1 recognition of the microbiome by epithelial cells results in disruption of crypt homeostasis specifically within the secretory cell compartment, including a defect in the mucus layer, ectopic Paneth cells in the colon, and an increase in the number of rapidly dividing cells at the base of the crypt. As a consequence of the perturbed epithelial barrier, we found an increase in mucosal-associated and translocated commensal bacteria and chronic low-grade inflammation characterized by an increase in lineage-negative Sca1+Thy1hi innate lymphoid-like cells that exacerbate inflammation and worsen outcomes in a model of colonic injury and repair. Our findings demonstrate that sensing of the microbiota by TLR1 may provide key signals that regulate the colonic epithelium, thereby limiting inflammation through the prevention of bacterial attachment to the mucosa and exposure to the underlying immune system.