5S rRNA pseudogene transcripts are associated with interferon production and inflammatory responses in alcohol-associated hepatitis.

BACKGROUND AND AIMS: Interferon (IFN) signaling is critical to the pathogenesis of alcohol-associated hepatitis (AH), yet the mechanisms for activation of this system are elusive. We hypothesize that host-derived 5S rRNA pseudogene (RNA5SP) transcripts regulate IFN production and modify immunity in AH. APPROACH AND RESULTS: Mining of transcriptomic datasets revealed that in patients with severe alcohol-associated hepatitis (sAH), hepatic expression of genes regulated by IFNs was perturbed and gene sets involved in IFN production were enriched. RNA5SP transcripts were also increased and correlated with expression of type I IFNs. Interestingly, inflammatory mediators upregulated in sAH, but not in other liver diseases, were positively correlated with certain RNA5SP transcripts. Real-time quantitative PCR demonstrated that RNA5SP transcripts were upregulated in peripheral blood mononuclear cells (PBMCs) from patients with sAH. In sAH livers, increased 5S rRNA and reduced nuclear MAF1 (MAF1 homolog, negative regulator of RNA polymerase III) protein suggested a higher activity of RNA polymerase III (Pol III); inhibition of Pol III reduced RNA5SP expression in monocytic THP-1 cells. Expression of several RNA5SP transcript-interacting proteins was downregulated in sAH, potentially unmasking transcripts to immunosensors. Indeed, siRNA knockdown of interacting proteins potentiated the immunostimulatory activity of RNA5SP transcripts. Molecular interaction and cell viability assays demonstrated that RNA5SP transcripts adopted Z-conformation and contributed to ZBP1-mediated caspase-independent cell death. CONCLUSIONS: Increased expression and binding availability of RNA5SP transcripts was associated with hepatic IFN production and inflammation in sAH. These data identify RNA5SP transcripts as a potential target to mitigate inflammation and hepatocellular injury in AH.

35kDa hyaluronan ameliorates ethanol driven loss of anti-microbial defense and intestinal barrier integrity in a TLR4-dependent manner.

Acute and chronic alcohol exposure compromise intestinal epithelial integrity, due to reduced expression of anti-microbial peptides (AMP) and loss of tight junction integrity. Ameliorating gut damage is beneficial in preventing associated distant organ pathologies. Orally administered purified hyaluronan (HA) polymers with an average size of 35 kDa have multiple protective effects in the gut and are well-tolerated in humans. Therefore, we tested the hypothesis that HA35 ameliorates ethanol-induced gut damage. Specifically, mechanisms that restore epithelial barrier integrity and normalize expression of the Reg3 class of C-type lectin AMPs (i.e. Reg3ß and Reg3γ) were investigated. Chronic ethanol feeding to mice reduced expression of C-type lectin AMPs in the proximal small intestine (jejunum), reduced expression of tight junction proteins and increased bacterial translocation to the mesenteric lymph node. Oral consumption of HA35 during the last 6 days of ethanol exposure ameliorated the effects of chronic ethanol. Similarly, in vitro challenge of isolated intestinal organoids from murine jejunum with ethanol reduced the expression of C-type lectin AMPs and impaired barrier integrity; these ethanol-induced responses were prevented by pre-treatment with HA35. Importantly, HA receptor null jejunum-derived organoids demonstrated that the HA receptor Tlr4, but not Cd44 nor Tlr2, was required for the protective effect of HA35. Consistent with the data from organoids, HA35 did not protect Tlr4-deficient mice from chronic ethanol-induced intestinal injury. Together, these data suggest therapeutic administration of HA35 is beneficial in restoring gut epithelial integrity and defense during the early stages of ethanol-driven intestinal damage.

Recent Advances in Understanding of Pathogenesis of Alcohol-Associated Liver Disease.

Alcohol-associated liver disease (ALD) is one of the major diseases arising from chronic alcohol consumption and is one of the most common causes of liver-related morbidity and mortality. ALD includes asymptomatic liver steatosis, fibrosis, cirrhosis, and alcohol-associated hepatitis and its complications. The progression of ALD involves complex cell-cell and organ-organ interactions. We focus on the impact of alcohol on dysregulation of homeostatic mechanisms and regulation of injury and repair in the liver. In particular, we discuss recent advances in understanding the disruption of balance between programmed cell death and prosurvival pathways, such as autophagy and membrane trafficking, in the pathogenesis of ALD. We also summarize current understanding of innate immune responses, liver sinusoidal endothelial cell dysfunction and hepatic stellate cell activation, and gut-liver and adipose-liver cross talk in response to ethanol. In addition,we describe the current potential therapeutic targets and clinical trials aimed at alleviating hepatocyte injury, reducing inflammatory responses, and targeting gut microbiota, for the treatment of ALD.

Multicellular String-Like Structure Formation by Salmonella Typhimurium Depends on Cellulose Production: Roles of Diguanylate Cyclases, YedQ and YfiN.

Bacteria face diverse stresses in the environment and, sometimes, respond by forming multi-cellular structures, e.g., biofilms. Here, we report a novel macroscopic and multi-cellular structure formed by Salmonella Typhimurium, which resembles small strings. These string-like structures, ∼1 cm long, are induced under some stress conditions: iron deprivation by 2,2-Bipyridyl or low amounts of antibiotics or ethanol in minimal media. However, cells in strings revert back to planktonic growth upon return to nutrient rich media. Compared to planktonic cells, strings are more resistant to antibiotics and oxidative stress. Also, strains lacking csgD or rpoS, which are defective in the classical rdar biofilm formation, form strings. Furthermore, some biofilm inducing conditions do not result in strings and vice-versa, demonstrating that strings are not related to classical CsgD-dependent biofilms. Cells in a string are held together by cellulose and a strain lacking bcsA, which is defective in cellulose production, does not form strings. In addition, reductive stress conditions such as dithiothreitol (DTT) or mutations in the Disulfide bonding system (DSB) also give rise to strings. The amounts of c-di-GMP are increased upon string formation and studies with single and double deletion strains of the diguanylate cyclases, yedQ (STM1987) primarily and yfiN (STM2672) partly, revealed their importance for string formation. This is the first study showcasing the ability of Salmonella to produce high amounts of cellulose in liquid culture, instead of an interface, in a CsgD-independent manner. The relevance and possible applications of strings in the production of bacterial cellulose and bioremediation are discussed.

Insights into coumarin-mediated inhibition of biofilm formation in Salmonella Typhimurium.

Coumarins have been shown to possess antimicrobial, anti-quorum sensing and anti-biofilm properties against a wide range of pathogenic bacteria. This study aimed to shed light on the effects of non-substituted coumarin on biofilm formation by the foodborne pathogen Salmonella Typhimurium. Additionally, its efficacy was tested in combination with another potent anti-biofilm agent, resveratrol. Coumarin inhibited biofilm formation for prolonged periods in millimolar concentrations with marginal effects on planktonic growth. It attenuated curli and cellulose production, likely by downregulating the transcript levels of major biofilm formation genes csgD, csgA and adrA. Coumarin further restricted motility in a dose-dependent manner. In addition, coumarin with resveratrol exhibited improved anti-biofilm properties compared with the individual compounds alone. Thus, coumarin alone or with resveratrol can be employed for inhibiting biofilms in food storage and processing units.

Salmonella Typhimurium encoded cold shock protein E is essential for motility and biofilm formation.

The ability of bacteria to form biofilms increases their survival under adverse environmental conditions. Biofilms have enormous medical and environmental impact; consequently, the factors that influence biofilm formation are an important area of study. In this investigation, the roles of two cold shock proteins (CSP) during biofilm formation were investigated in Salmonella Typhimurium, which is a major foodborne pathogen. Among all CSP transcripts studied, the expression of cspE (STM14_0732) was higher during biofilm growth. The cspE deletion strain (ΔcspE) did not form biofilms on a cholesterol coated glass surface; however, complementation with WT cspE, but not the F30V mutant, was able to rescue this phenotype. Transcript levels of other CSPs demonstrated up-regulation of cspA (STM14_4399) in ΔcspE. The cspA deletion strain (ΔcspA) did not affect biofilm formation; however, ΔcspEΔcspA exhibited higher biofilm formation compared to ΔcspE. Most likely, the higher cspA amounts in ΔcspE reduced biofilm formation, which was corroborated using cspA over-expression studies. Further functional studies revealed that ΔcspE and ΔcspEΔcspA exhibited slow swimming but no swarming motility. Although cspA over-expression did not affect motility, cspE complementation restored the swarming motility of ΔcspE. The transcript levels of the major genes involved in motility in ΔcspE demonstrated lower expression of the class III (fliC, motA, cheY), but not class I (flhD) or class II (fliA, fliL), flagellar regulon genes. Overall, this study has identified the interplay of two CSPs in regulating two biological processes: CspE is essential for motility in a CspA-independent manner whereas biofilm formation is CspA-dependent.

Interplay of cold shock protein E with an uncharacterized protein, YciF, lowers porin expression and enhances bile resistance in Salmonella Typhimurium.

Bacterial cold shock proteins (CSPs) function as RNA chaperones. To assess CSP's roles in the intracellular human pathogen Salmonella Typhimurium, we analyzed their expression in varied stress conditions. We found that cold shock protein E (cspE or STM14_0732) is up-regulated during bile salt-induced stress and that an S. Typhimurium strain lacking cspE (ΔcspE) displays dose-dependent sensitivity to bile salts, specifically to deoxycholate. We also found that an uncharacterized gene, yciF (STM14_2092), is up-regulated in response to bile stress in WT but not in the ΔcspE strain. Complementation with WT CspE, but not with a F30V CspE variant, abrogated the bile sensitivity of ΔcspE as did multicopy overexpression of yciF. Northern blotting experiments with rifampicin disclosed that the regulation of yciF expression is, most likely, due to the RNA-stabilizing activity of CspE. Importantly, electrophoretic mobility shift assays indicated that purified CspE, but not the F30V variant, directly binds yciF mRNA. We also observed that the extra-cytoplasmic stress-response (ESR) pathway is augmented in the bile-treated ΔcspE strain, as judged by induction of RpoE regulon genes (rpoE, degP, and rybB) and downstream ESR genes (hfq, rne, and PNPase). Moreover, the transcript levels of the porin genes, ompD, ompF, and ompC, were higher in bile salts-stressed ΔcspE and correlated with higher intracellular accumulation of the fluorescent DNA stain bisBenzimide H 33258, indicating greater cell permeability. In conclusion, our study has identified YciF, a CspE target involved in the regulation of porins and in countering bile stress in S. Typhimurium.

Nitric oxide synthase 2 enhances the survival of mice during Salmonella Typhimurium infection-induced sepsis by increasing reactive oxygen species, inflammatory cytokines and recruitment of neutrophils to the peritoneal cavity.

Sepsis, a leading cause of death in intensive care units, is primarily caused due to an exaggerated immune response. The hyperactive inflammatory response mediated by immune cells against infectious organisms and their toxins results in host cell death and tissue damage, the hallmarks of septic shock. Therefore, molecules that modulate inflammatory responses are attractive therapeutic targets for sepsis. Nitric oxide (NO) is a signaling molecule, which is implicated in regulating diverse immune functions. Although, the protective roles of NO in infectious diseases are well documented, its importance in sepsis is controversial. In the present study, the effects of intra-peritoneal injection of mice with Salmonella Typhimurium, a Gram-negative intracellular pathogen, were studied which leads to a rapid upregulation of serum cytokines and infiltration of neutrophils to the peritoneal cavity. Surprisingly, the induction of inflammatory cytokines and chemokines, e.g. IL6 and CCL2, and the infiltration of neutrophils into the peritoneal cavity are mitigated in mice lacking Nitric oxide synthase 2 (NOS2). The reduced inflammatory response in Nos2-/- mice is accompanied by greater bacterial burden in the peritoneal cavity, lower thymic atrophy, higher liver damage and cardiovascular dysfunction followed by decreased survival. However, no significant differences are observed in other responses between C57BL/6 wild type (WT) and Nos2-/- mice: induction of glucocorticoids, phagocytic ability and apoptosis of peritoneal cells. This study clearly highlights the NOS2-dependent and -independent responses in this mouse model of peritonitis induced sepsis. Importantly, pre-treatment of Nos2-/- mice with DETA-NO, a NO donor, upon infection, restores neutrophil recruitment, reduces bacterial numbers in the peritoneal cavity, improves liver and cardio-vascular function and enhances survival. Interestingly, DETA-NO treatment does not significantly increase the survival of infected WT mice. The implications of these results and the complex roles of NO as a target molecule during sepsis are discussed.