Trimethylamine N-oxide (TMAO), a product of gut microbiota metabolism, has previously been shown to be implicated in chronic kidney disease. A high TMAO-containing diet has been found to cause tubulointerstitial renal fibrosis in mice. However, today there are no data linking specific molecular pathways with the effect of TMAO on human renal fibrosis. The aim of this study was to investigate the fibrotic effects of TMAO on renal fibroblasts and to elucidate the molecular pathways involved. We found that TMAO promoted renal fibroblast activation and fibroblast proliferation via the PERK/Akt/mTOR pathway, NLRP3, and caspase-1 signaling. We also found that TMAO increased the total collagen production from renal fibroblasts via the PERK/Akt/mTOR pathway. However, TMAO did not induce fibronectin or TGF-β1 release from renal fibroblasts. We have unraveled that the PERK/Akt/mTOR pathway, NLRP3, and caspase-1 mediates TMAO’s fibrotic effect on human renal fibroblasts. Our results can pave the way for future research to further clarify the molecular mechanism behind TMAO’s effects and to identify novel therapeutic targets in the context of chronic kidney disease.

The NLRP3 inflammasome and IL-1β release have recently been suggested to be important for the progression of urinary tract infection (UTI). However, much is still unknown regarding the interaction of UPEC and the NLRP3 inflammasome. The purpose of this study was to elucidate what virulence factors uropathogenic Escherichia coli (UPEC) use to modulate NLRP3 inflammasome activation and subsequent IL-1β release and the role of NLRP3 for UPEC colonization of bladder epithelial cells. The bladder epithelial cell line 5637, CRISPR/Cas9 generated NLRP3, caspase-1 and mesotrypsin deficient cell lines and transformed primary bladder epithelial cells (HBLAK) were stimulated with UPEC isolates and the non-pathogenic MG1655 strain. We found that the UPEC strain CFT073, but not MG1655, induced an increased caspase-1 activity and IL-1β release from bladder epithelial cells. The increase was shown to be mediated by α-hemolysin activation of the NLRP3 inflammasome in an NF-κB-independent manner. The effect of α-hemolysin on IL-1β release was biphasic, initially suppressive, later inductive. Furthermore, the phase-locked type-1-fimbrial ON variant of CFT073 inhibited caspase-1 activation and IL-1β release. In addition, the ability of CFT073 to adhere to and invade NLRP3 deficient cells was significantly reduced compare to wild-type cells. The reduced colonization of NLRP3-deficient cells was type-1 fimbriae dependent. In conclusion, we found that the NLRP3 inflammasome was important for type-1 fimbriae-dependent colonization of bladder epithelial cells and that both type-1 fimbriae and α-hemolysin can modulate the activity of the NLRP3 inflammasome.

The NLRP3 inflammasome and IL-1β have recently been linked to the severity of uropathogenic Escherichia coli (UPEC)-mediated urinary tract infection (UTI). However, not much is known about the contribution of NLRP3 to the antimicrobial properties of neutrophils and the release of IL-1β during UPEC infection. The purpose of this study was to elucidate the mechanisms behind UPEC-induced IL-1β release from human neutrophils, and to investigate the contribution of the NLRP3 inflammasome in neutrophil-mediated inhibition of UPEC growth. We found that the UPEC strain CFT073 increased the expression of NLRP3 and increased caspase-1 activation and IL-1β release from human neutrophils. The IL-1β release was mediated by the NLRP3 inflammasome and by serine proteases in an NF-κB-and cathepsin B-dependent manner. The UPEC virulence factors α-hemolysin, type-1 fimbriae and p-fimbriae were all shown to contribute to UPEC mediated IL-1β release from neutrophils. Furthermore, inhibition of caspase-1 and NLRP3 activation increased neutrophil ROS-production, phagocytosis and the ability of neutrophils to suppress UPEC growth. In conclusion, this study demonstrates that UPEC can induce NLRP3 and serine protease-dependent release of IL-1β from human neutrophils and that NLRP3 and caspase-1 can regulate the antimicrobial activity of human neutrophils against UPEC.

The effect of a urinary tract infection on the host is a well-studied research field. However, how the host immune response affects uropathogenic Escherichia coli (CFT073) virulence is less studied. The aim of the present study was to investigate the impact of proinflammatory cytokine exposure on the virulence of uropathogenic Escherichia coli. We found that all tested proinflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8 and IFN-γ) induced an increased CFT073 growth. We also found that biofilm formation and hemolytic activity was reduced in the presence of all proinflammatory cytokines. However, a reduction in siderophore release was only observed in the presence of IL-1β, IL-6 and IL-8. Real time-qPCR showed that all proinflammatory cytokines except TNF-α significantly increased genes associated with the iron acquisition system in CFT073. We also found that the proinflammatory cytokines induced significant changes in type-1 fimbriae, P-fimbriae and gluconeogenetic genes. Furthermore, we also showed, using a Caenorhabditis elegans (C. elegans) killing assay that all cytokines decreased the survival of C. elegans worms significantly. Taken together, our findings show that proinflammatory cytokines have the ability to alter the virulence traits of UPEC.

Uropathogenic Escherichia coli (UPEC) is the most common bacteria to cause urinary tract infection (UTI). Postmenopausal women have an increased risk of recurrent UTI. This is partly explained by estrogenic effects on host defenses against UTI. Current research is mostly focused on how UPEC affects host factors, but not so much is known about how host factors like hormones affect UPEC virulence. The aim of the present study was to investigate the impact of estradiol exposure on the virulence of UPEC. We found that a postmenopausal concentration of estradiol increased CFT073 growth and biofilm formation, but not the premenopausal concentrations. Real-time qPCR showed that estradiol altered the expression of genes associated with the iron acquisition system and metabolic pathways in CFT073. We also found that estradiol in a dose-dependent manner increased the expression of fimH and papC adhesins and increased colonization and invasion of bladder epithelial cells. The premenopausal concentration of estradiol also suppressed cytokine release from bladder epithelial cells. Additionally, we also showed using a Caenorhabditis elegans killing assay that estradiol increased the survival of CFT073-infected C. elegans worms. Taken together, our findings show that estradiol has the ability to alter the virulence traits of UPEC.