Amrita Kabi

A dual role for receptor interacting protein kinase 2 (RIP2) kinase activity in nucleotide-binding oligomerization domain 2 (NOD2)-dependent autophagy

Background: Autophagy is triggered by NOD2 as an anti-bacterial response. Results: NOD2-stimulated autophagy requires RIP2-dependent activation of p38 MAPK and repression of the PP2A phosphatase in intestinal epithelial cell lines. Conclusion: RIP2 kinase activity is necessary for anti-bacterial autophagy induction by NOD2. Significance: These findings provide novel molecular targets for modulation of autophagy as an anti-bacterial response. Autophagy is triggered by the intracellular bacterial sensor NOD2 (nucleotide-binding, oligomerization domain 2) as an anti-bacterial response. Defects in autophagy have been implicated in Crohns disease susceptibility. The molecular mechanisms of activation and regulation of this process by NOD2 are not well understood, with recent studies reporting conflicting requirements for RIP2 (receptor-interacting protein kinase 2) in autophagy induction. We examined the requirement of NOD2 signaling mediated by RIP2 for anti-bacterial autophagy induction and clearance of Salmonella typhimurium in the intestinal epithelial cell line HCT116. Our data demonstrate that NOD2 stimulates autophagy in a process dependent on RIP2 tyrosine kinase activity. Autophagy induction requires the activity of the mitogen-activated protein kinases MEKK4 and p38 but is independent of NFκB signaling. Activation of autophagy was inhibited by a PP2A phosphatase complex, which interacts with both NOD2 and RIP2. PP2A phosphatase activity inhibited NOD2-dependent autophagy but not activation of NFκB or p38. Upon stimulation of NOD2, the phosphatase activity of the PP2A complex is inhibited through tyrosine phosphorylation of the catalytic subunit in a process dependent on RIP2 activity. These findings demonstrate that RIP2 tyrosine kinase activity is not only required for NOD2-dependent autophagy but plays a dual role in this process. RIP2 both sends a positive autophagy signal through activation of p38 MAPK and relieves repression of autophagy mediated by the phosphatase PP2A.

Digesting the genetics of inflammatory bowel disease: insights from studies of autophagy risk genes.

The success of genetic analyses identifying multiple loci associated with inflammatory bowel disease (IBD) susceptibility has resulted in the identification of several risk genes linked to a common cellular process called autophagy. Autophagy is a process involving the encapsulation of cytosolic cellular components in double-membrane vesicles, their subsequent lysosomal degradation, and recycling of the degraded components for use by the cell. It plays an important part in the innate immune response to a variety of intracellular pathogens, and it is this component of autophagy that appears to be defective in IBD. This has lead to the hypothesis that Crohns disease may result from an impaired antibacterial response, which leads to ineffective control of bacterial infection, dysbiosis of the intestinal microbiota, and chronic inflammation. Several recurrent themes have surfaced from studies examining the function of autophagy-related genes in the context of IBD, with cellular context, disease status, risk variant effect, and risk gene interplay all affecting the interpretation of these studies. The identification of autophagy as a major risk pathway in IBD is a significant step forward and may lead to pathway-focused therapy in the future; however, there is more to understand in order to unravel the complexity of this disease.

Combinatorial Effects of Diet and Genetics on Inflammatory Bowel Disease Pathogenesis

Abstract:Inflammatory bowel disease (IBD) encompasses a group of disorders affecting the gastrointestinal tract characterized by acute and chronic inflammation. These are complex and multifactorial disorders that arise in part from a genetic predisposition. However, the increasing incidence of IBD in developing countries suggests that environmental factors, such as diet, are also critical components of disease susceptibility. Evidence suggests that consumption of a Western diet, enriched with saturated fat, refined carbohydrates, and food additives, is associated with increased IBD risk. Dietary components, such as omega-6 fatty acids, long-chain fatty acids, protein, and digestible carbohydrates, may contribute to IBD pathogenesis through altering intestinal microbiota, increasing intestinal permeability, and promoting inflammation; whereas omega-3 fatty acids, medium chain triglycerides, and nondigestible carbohydrates improve these parameters and intestinal health. However, the limited amount of prospective studies, small sample sizes, and the heterogeneity of disease subtype result in inconsistencies between studies and difficulty in conclusively determining the specific effects of diet on intestinal homeostasis. There are no standard clinical dietary recommendations for patients with IBD. However, exclusionary diet interventions have shown some efficacy in relieving symptoms or inducing remission, suggesting more research is needed to fully understand how diet influences disease behavior or combines with other IBD risk factors to promote disease. This review focuses on the associations of various dietary components and IBD risk in clinical studies and genetically susceptible IBD models.

The Nucleotide Synthesis Enzyme CAD Inhibits NOD2 Antibacterial Function in Human Intestinal Epithelial Cells

BACKGROUND & AIMS Polymorphisms that reduce the function of nucleotide-binding oligomerization domain (NOD)2, a bacterial sensor, have been associated with Crohns disease (CD). No proteins that regulate NOD2 activity have been identified as selective pharmacologic targets. We sought to discover regulators of NOD2 that might be pharmacologic targets for CD therapies. METHODS Carbamoyl phosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD) is an enzyme required for de novo pyrimidine nucleotide synthesis; it was identified as a NOD2-interacting protein by immunoprecipitation-coupled mass spectrometry. CAD expression was assessed in colon tissues from individuals with and without inflammatory bowel disease by immunohistochemistry. The interaction between CAD and NOD2 was assessed in human HCT116 intestinal epithelial cells by immunoprecipitation, immunoblot, reporter gene, and gentamicin protection assays. We also analyzed human cell lines that express variants of NOD2 and the effects of RNA interference, overexpression and CAD inhibitors. RESULTS CAD was identified as a NOD2-interacting protein expressed at increased levels in the intestinal epithelium of patients with CD compared with controls. Overexpression of CAD inhibited NOD2-dependent activation of nuclear factor κB and p38 mitogen-activated protein kinase, as well as intracellular killing of Salmonella. Reduction of CAD expression or administration of CAD inhibitors increased NOD2-dependent signaling and antibacterial functions of NOD2 variants that are and are not associated with CD. CONCLUSIONS The nucleotide synthesis enzyme CAD is a negative regulator of NOD2. The antibacterial function of NOD2 variants that have been associated with CD increased in response to pharmacologic inhibition of CAD. CAD is a potential therapeutic target for CD.

The dietary polysaccharide maltodextrin promotes Salmonella survival and mucosal colonization in mice.

In the latter half of the 20th century, societal and technological changes led to a shift in the composition of the American diet to include a greater proportion of processed, pre-packaged foods high in fat and carbohydrates, and low in dietary fiber (a “Western diet”). Over the same time period, there have been parallel increases in Salmonella gastroenteritis cases and a broad range of chronic inflammatory diseases associated with intestinal dysbiosis. Several polysaccharide food additives are linked to bacterially-driven intestinal inflammation and may contribute to the pathogenic effects of a Western diet. Therefore, we examined the effect of a ubiquitous polysaccharide food additive, maltodextrin (MDX), on clearance of the enteric pathogen Salmonella using both in vitro and in vivo infection models. When examined in vitro, murine bone marrow-derived macrophages exposed to MDX had altered vesicular trafficking, suppressed NAPDH oxidase expression, and reduced recruitment of NADPH oxidase to Salmonella-containing vesicles, which resulted in persistence of Salmonella in enlarged Rab7+ late endosomal vesicles. In vivo, mice consuming MDX-supplemented water had a breakdown of the anti-microbial mucous layer separating gut bacteria from the intestinal epithelium surface. Additionally, oral infection of these mice with Salmonella resulted in increased cecal bacterial loads and enrichment of lamina propria cells harboring large Rab7+ vesicles. These findings indicate that consumption of processed foods containing the polysaccharide MDX contributes to suppression of intestinal anti-microbial defense mechanisms and may be an environmental priming factor for the development of chronic inflammatory disease.

Alterations in nucleotide-binding oligomerization domain-2 expression, pathway activation, and cytokine production in Yao syndrome

Abstract Yao syndrome (YAOS) is a systemic autoinflammatory disease (SAID), formerly termed nucleotide-binding oligomerization domain-2 (NOD2)-associated autoinflammatory disease. Due to the recent identification of YAOS, the molecular mechanisms underlying its disease pathogenesis are unclear. With specific NOD2 variants as characteristic genotypic features of YAOS, our study examined NOD2 expression, transcript splicing, signaling pathway activation, and cytokine profiles in peripheral blood mononuclear cells (PBMCs) from 10 YAOS patients and six healthy individuals. All participants were genotyped for NOD2 variants; all YAOS patients were heterozygous for the NOD2 IVS8+158 variant (IVS8+158) and four patients also carried a concurrent NOD2 R702W variant (IVS8+158/R702W haplotype). Resembling other SAIDs, plasma levels of TNFα, IL-1β, IL-6, IFNγ, and S100A12 were unaltered in YAOS patients. Intron-8 splicing of NOD2 transcripts was unaffected by carriage of NOD2 IVS8+158. However, NOD2 transcript level and basal p38 mitogen-activated protein kinase (MAPK) activity were significantly elevated in PBMCs from IVS8+158 YAOS patients. Moreover, these patients’ cells had elevated basal IL-6 secretion that was enhanced by muramyl dipeptide (MDP) stimulation. Tocilizumab treatment of a YAOS IVS8+158 patient resulted in marked clinical improvement. In contrast, MDP-stimulated NF-κB activity was uniquely suppressed in haplotype IVS8+158/R702W patients, as was TNFα secretion. Our study demonstrates for the first time that NOD2 expression and pathway activation are aberrant in YAOS, and specific NOD2 genotypes result in distinct NOD2 expression and cytokine profiles. These findings may also help select therapeutic strategies in the future.

FRMBP2 directs NOD2 to the membrane

The immune system acts as a shield against harmful microbes and requires exquisite control of the activation and resolution of multiple signaling pathways. Tight regulation of these processes is required not only to produce an effective response, but also to maintain immune homeostasis and prevent autoinflammatory disease. In the intestine, this process is further complicated by the residence of a beneficial community of microbes. One major question is how the immune system determines “friend from foe,” restricting antimicrobial responses to the intestinal microbiota but retaining protective antimicrobial responses against pathogens. Answering this question is critical to uncovering mechanisms that go awry in autoinflammatory disease. In PNAS, Lipinsky et al. (1) provide mechanistic insights into how polarized intestinal epithelial cells restrict antibacterial responses to the basolateral compartment through directing the subcellular localization the bacterial sensor NOD2 (nucleotide-binding oligomerization domain-2).

p28 dynein light chains and ciliary motility in Tetrahymena thermophila

Dynein light chains are required for the assembly of axonemal dyneins into cilia and flagella. Most organisms express a single p28 dynein light chain and four to nine one‐headed inner arm dynein heavy chains. In contrast, Tetrahymena encodes three p28 dynein light chain genes (p28A, p28B, and p28C) and 18 one‐headed inner arm dynein heavy chains. In this article it is shown that mutations in p28A and p28B affected both beat frequency and waveform of cilia, while mutations in p28C affected only ciliary beat frequency. A similar set of dynein heavy chains were affected in both p28AKO and p28BKO, but a distinct set of heavy chains was affected in p28CKO. The results suggested that the p28s have non‐redundant functions in Tetrahymena and that p28C was associated with a different set of dynein heavy chains than were p28A and p28B.

Protein Phosphatase 2A Has Positive and Negative Roles in Autophagy

Autophagy is a ubiquitous, homeostatic process involving lysosomal-dependent recycling of intracellular macromolecules and organelles targeted by multi-lamellar vesicles. This multistep process involves a large network of molecules tightly regulated by post-translational mechanisms, including phosphorylation. One critical regulator of this pathway is protein phosphatase 2A (PP2A). Although referred to as a single enzymatic activity, PP2A encompasses multiple serine/threonine phosphatase enzyme complexes formed through the combination of a family of regulatory and scaffolding subunits with a common catalytic subunit. PP2A activity regulates autophagy at multiple checkpoints and can have both stimulatory and inhibitory effects on this process. The final outcome of PP2A activity on autophagic responses is dependent on multiple factors, which include the specific PP2A enzyme activated, cell type, and the nature of autophagic trigger. Alterations in autophagy are increasingly associated with chronic diseases. Therefore, understanding the molecular mechanisms controlling this process may have important implications for designing therapies targeting autophagy.

Pyrimidine synthesis inhibition enhances cutaneous defenses against antibiotic resistant bacteria through activation of NOD2 signaling

Multidrug-resistant bacterial strains are a rapidly emerging healthcare threat; therefore it is critical to develop new therapies to combat these organisms. Prior antibacterial strategies directly target pathogen growth or viability. Host-directed strategies to increase antimicrobial defenses may be an effective alternative to antibiotics and reduce development of resistant strains. In this study, we demonstrated the efficacy of a pyrimidine synthesis inhibitor, N-phosphonacetyl-l-aspartate (PALA), to enhance clearance of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Acinetobacter baumannii strains by primary human dermal fibroblasts in vitro. PALA did not have a direct bactericidal effect, but enhanced cellular secretion of the antimicrobial peptides human β-defensin 2 (HBD2) and HBD3 from fibroblasts. When tested in porcine and human skin explant models, a topical PALA formulation was efficacious to enhance MRSA, P. aeruginosa, and A. baumannii clearance. Topical PALA treatment of human skin explants also resulted in increased HBD2 and cathelicidin (LL-37) production. The antimicrobial actions of PALA required expression of nucleotide-binding, oligomerization domain 2 (NOD2), receptor-interacting serine/threonine-protein kinase 2 (RIP2), and carbamoyl phosphatase synthase II/aspartate transcarbamylase/dihydroorotase (CAD). Our results indicate that PALA may be a new option to combat multidrug-resistant bacterial infections of the skin through enhancement of an integral pathway of the cutaneous innate immune defense system.