Vascular smooth muscle cells in response to cholesterol crystals modulates inflammatory cytokines release and promotes neutrophil extracellular trap formation.

BACKGROUND: The formation and accumulation of cholesterol crystals (CC) at the lesion site is a hallmark of atherosclerosis. Although studies have shown the importance of vascular smooth muscle cells (VSMCs) in the disease atherosclerosis, little is known about the molecular mechanism behind the uptake of CC in VSMCs and their role in modulating immune response. METHODS: Human aortic smooth muscle cells were cultured and treated with CC. CC uptake and CC mediated signaling pathway and protein induction were studied using flow cytometry, confocal microscopy, western blot and Olink proteomics. Conditioned medium from CC treated VSMCs was used to study neutrophil adhesion, ROS production and phagocytosis. Neutrophil extracellular traps (NETs) formations were visualized using confocal microscopy. RESULTS: VSMCs and macrophages were found around CC clefts in human carotid plaques. CC uptake in VSMCs are largely through micropinocytosis and phagocytosis via PI3K-AkT dependent pathway. The uptake of CC in VSMCs induce the release inflammatory proteins, including IL-33, an alarming cytokine. Conditioned medium from CC treated VSMCs can induce neutrophil adhesion, neutrophil reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. IL-33 neutralization in conditioned medium from CC treated VSMCs inhibited neutrophil ROS production and NETs formation. CONCLUSION: We demonstrate that VSMCs due to its vicinity to CC clefts in human atherosclerotic lesion can modulate local immune response and we further reveal that the interaction between CC and VSMCs impart an inflammatory milieu in the atherosclerotic microenvironment by promoting IL-33 dependent neutrophil influx and NETs formation.

The Role of NLRP3 in Regulation of Antimicrobial Peptides and Estrogen Signaling in UPEC-Infected Bladder Epithelial Cells.

The NLRP3 inflammasome, estrogen and antimicrobial peptides have all been found to have a vital role in the protection of the bladder urothelium. However, the interdependence between these protective factors during a bladder infection is currently unknown. Our aim was to investigate the role of NLRP3 in the regulation of antimicrobial peptides and estrogen signaling in bladder epithelial cells during a UPEC infection. Human bladder epithelial cells and CRISPR/Cas9-generated NLRP3-deficient cells were stimulated with the UPEC strain CFT073 and estradiol. The gene and protein expression were evaluated with microarray, qRT-PCR, western blot and ELISA. Microarray results showed that the expression of most antimicrobial peptides was reduced in CFT073-infected NLRP3-deficient cells compared to Cas9 control cells. Conditioned medium from NLRP3-deficient cells also lost the ability to suppress CFT073 growth. Moreover, NLRP3-deficient cells had lower basal release of Beta-defensin-1, Beta-defensin-2 and RNase7. The ability of estradiol to induce an increased expression of antimicrobial peptides was also abrogated in NLRP3-deficient cells. The decreased antimicrobial peptide expression might be linked to the observed reduced expression and activity of estradiol receptor beta in NLRP3-deficient cells. This study suggests that NLRP3 may regulate the release and expression of antimicrobial peptides and affect estrogen signaling in bladder epithelial cells.

Inhibition of COX-2 signaling favors E. coli during urinary tract infection.

BACKGROUND: To avoid the overuse of antibiotics, non-steroidal anti-inflammatory drugs (NSAIDs), acting via cyclooxygenase (COX) inhibition, have been used to reduce pain and as an alternative treatment for uncomplicated urinary tract infections (UTIs). However, clinical studies evaluating NSAIDs versus antibiotics have reported an increased risk of acute pyelonephritis. Therefore, we hypothesized that COX inhibition could compromise the innate immune response and contribute to complications in patients with uncomplicated UTI. RESULTS: We here demonstrate that in particular COX-2 inhibition led to decreased expression of the antimicrobial peptides psoriasin and human ß-defensin-2 in human uroepithelial cells. Psoriasin expression was altered in neutrophils and macrophages. COX-2 inhibition also had impact on the inflammasome mediated IL-1ß expression in response to uroepithelial E. coli infection. Further, COX-2 inhibition downregulated free radicals and the epithelial barrier protein claudin 1, favoring infectivity. In addition, conditioned media from COX-2 inhibited uroepithelial cells infected with E. coli failed to activate macrophages. CONCLUSIONS: Taken together, our data suggests an adverse innate immune effect of COX-2 inhibition on uroepithelial cells during UTI.

The role of NLRP3 in regulating gingival epithelial cell responses evoked by Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) has myriads of virulence factors among which leukotoxin provides A. actinomycetemcomitans with the advantage to thrive in the surrounding hostile environment and evade host immune defences. The NLRP3 inflammasome has been associated with periodontal disease development. However, our understanding of the involvement of caspase-1, caspase-4, and NLRP3 in the release of IL-1ß and other inflammatory mediators from gingival epithelial cells during a A. actinomycetemcomitans infection is limited. The aim of this study was to investigate how the inflammasome-associated proteins caspase-1, caspase-4 and NLRP3 regulate the immune response of gingival epithelial cells during a A. actinomycetemcomitans infection. Human gingival epithelial cells (Ca9-22) deficient in NLRP3, caspase-1 or caspase-4 were created using CRISPR/Cas9. Gingival epithelial cells were stimulated with the A. actinomycetemcomitans low-leukotoxic strain NCTC9710 or the highly leukotoxic JP2 strain HK 165 for 6, 12 and 24 h. The results showed that the JP2 strain HK1651 induced higher IL-1ß and IL-1RA release and mediated more epithelial cell death compared to the NCTC9710 strain. These findings were found to be capsase-1, caspase-4 and NLRP3-dependant. A targeted protein analysis of inflammation-related proteins showed that the expression of 37 proteins were identified as being significantly altered after HK1651 infection compared to unstimulated Cas9 and NLRP3-deficient cells. Of the 37 proteins, 23 of these inflammation-related proteins released by NLRP3-deficient cells differed significantly compared to Cas9 cells after infection. This suggests that NLRP3 has a broad effect on the inflammatory response in gingival epithelial cells.

Trimethylamine N-Oxide (TMAO) Mediates Increased Inflammation and Colonization of Bladder Epithelial Cells during a Uropathogenic E. coli Infection In Vitro.

Urinary tract infections (UTIs) are among the most common infections in humans and are often caused by uropathogenic E. coli (UPEC). Trimethylamine N-oxide (TMAO) is a proinflammatory metabolite that has been linked to vascular inflammation, atherosclerosis, and chronic kidney disease. As of today, no studies have investigated the effects of TMAO on infectious diseases like UTIs. The aim of this study was to investigate whether TMAO can aggravate bacterial colonization and the release of inflammatory mediators from bladder epithelial cells during a UPEC infection. We found that TMAO aggravated the release of several key cytokines (IL-1ß and IL-6) and chemokines (IL-8, CXCL1 and CXCL6) from bladder epithelial cells during a CFT073 infection. We also found that CFT073 and TMAO mediate increased release of IL-8 from bladder epithelial cells via ERK 1/2 signaling and not bacterial growth. Furthermore, we showed that TMAO enhances UPEC colonization of bladder epithelial cells. The data suggest that TMAO may also play a role in infectious diseases. Our results can be the basis of further research to investigate the link between diet, gut microbiota, and urinary tract infection.

Targeting serotonin receptor 2B inhibits TGFß induced differentiation of human vascular smooth muscle cells.

Vascular Smooth Muscle Cells (VSMCs) are known to be the key drivers of intimal thickening which contribute to early progression of atherosclerosis. VSMCs are the major producers of extracellular matrix within the vessel wall and in response to atherogenic stimuli they could modify the type of matrix proteins produced. Serotonin receptor 2B (5-HT2B receptor/HTR2B) has been implicated in several chronic fibrotic and vascular diseases. Although studies have successfully demonstrated the efficacy of HTR2B blockade in attenuating fibrotic disease, the role of 5-HT2B receptor in TGFß mediated VSMC differentiation remain largely unknown. In the present study, we investigated the potential of targeting the 5-HT2B receptor to prevent TGFß induced VSMCs differentiation. Our results showed that 5-HT2B receptors are expressed in human atherosclerotic lesion and HTR2B expression positively correlated to the VSMCs markers. We show that AM1125, a selective 5-HT2B receptor inhibitor, significantly inhibits TGFß1 induced production of collagen and CTGF. The investigation of underlying mechanisms indicated that 5-HT2B receptor antagonism blocks phospho-Smad2 mediated downstream signaling of TGFß1 in vascular smooth muscle cells. Collectively, the HTR2B/TGF-ß1/Phospho-Smad2 pathway plays a critical role in the regulation of VSMCs differentiation. Our findings might serve 5-HT2B receptor as a therapeutic target to limit TGF-ß1 induced VSMC differentiation.

Blocking connexin 43 hemichannel-mediated ATP release reduces communication within and between tubular epithelial cells and medullary fibroblasts in a model of diabetic nephropathy.

INTRODUCTION: Fibrosis of renal tubules is the final common pathway in diabetic nephropathy and develops in the face of tubular injury and fibroblast activation. Aberrant connexin 43 (Cx43) hemichannel activity has been linked to this damage under euglycaemic conditions, however, its role in glycaemic injury is unknown. This study investigated the effect of a Cx43 blocker (Tonabersat) on hemichannel activity and cell-cell interactions within and between tubular epithelial cells and fibroblasts in an in vitro model of diabetic nephropathy. METHODS: Human kidney (HK2) proximal tubule epithelial cells and medullary fibroblasts (TK173) were treated in low (5 mM) or high (25 mM) glucose ± transforming growth factor beta-1 (TGFß1) ± Tonabersat in high glucose. Carboxyfluorescein dye uptake and ATPlite luminescence assessed changes in hemichannel-mediated ATP release, while immunoblotting determined protein expression. Co-incubation with the ATP-diphosphohydrolase apyrase or a P2X7R inhibitor (A438079) assessed ATP-P2X7R signalling. Indirect co-culture with conditioned media from the alternate cell type evaluated paracrine-mediated heterotypic interactions. RESULTS: Tonabersat partially negated glucose/TGFß1-induced increases in Cx43 hemichannel-mediated ATP release and downstream changes in adherens junction and extracellular matrix (ECM) protein expression in HK2 and TK173 cells. Apyrase and A438079 highlighted the role for ATP-P2X7R in driving changes in protein expression in TK173 fibroblasts. Indirect co-culture studies suggest that epithelial cell secretome increases Tonabersat-sensitive hemichannel-mediated dye uptake in fibroblasts and downstream protein expression. CONCLUSION: Tonabersat-sensitive hemichannel-mediated ATP release enhances TGFß1-driven heterotypic cell-cell interaction and favours myofibroblast activation. The data supports the potential benefit of Cx43 inhibition in reducing tubulointerstitial fibrosis in late-stage diabetic nephropathy.

TMAO Suppresses Megalin Expression and Albumin Uptake in Human Proximal Tubular Cells Via PI3K and ERK Signaling.

Trimethylamine-N-oxide (TMAO) is a uremic toxin, which has been associated with chronic kidney disease (CKD). Renal tubular epithelial cells play a central role in the pathophysiology of CKD. Megalin is an albumin-binding surface receptor on tubular epithelial cells, which is indispensable for urine protein reabsorption. To date, no studies have investigated the effect of TMAO on megalin expression and the functional properties of human tubular epithelial cells. The aim of this study was first to identify the functional effect of TMAO on human renal proximal tubular cells and second, to unravel the effects of TMAO on megalin-cubilin receptor expression. We found through global gene expression analysis that TMAO was associated with kidney disease. The microarray analysis also showed that megalin expression was suppressed by TMAO, which was also validated at the gene and protein level. High glucose and TMAO was shown to downregulate megalin expression and albumin uptake similarly. We also found that TMAO suppressed megalin expression via PI3K and ERK signaling. Furthermore, we showed that candesartan, dapagliflozin and enalaprilat counteracted the suppressive effect of TMAO on megalin expression. Our results may further help us unravel the role of TMAO in CKD development and to identify new therapeutic targets to counteract TMAOs effects.

Effects of estradiol on the virulence traits of Porphyromonas gingivalis.

Porphyromonas gingivalis has been strongly associated to active periodontitis sites. A number of studies have tried to elucidate the association between female steroid sex hormones and gingival health. However, until now, there is limited knowledge on estradiol effects on the virulence traits of P. gingivalis. The aim of the study was to investigate the impact of estradiol exposure on the virulence characteristics of P. gingivalis strain W50. We found that a pre- and postmenopausal concentration of estradiol increased the growth and biofilm formation of P. gingivalis W50. We also found that estradiol increased the release of lysine and arginine gingipains from W50. We then showed that IL-1ß, CXCL10 and TGF-ß1 release from gingival epithelial cells was significantly lowered by W50 pre-exposed to estradiol compared to W50 alone. Real time-qPCR showed that the gene expression of IL-18, IL-6, IL-8, TGF-ß1 and NLRP3 in gingival epithelial cells was significantly lowered by W50 pre-exposed to estradiol compared to W50 alone. We also found that estradiol in a dose-dependent manner increased P. gingivalis colonization and invasion of gingival epithelial cells. Taken together, our findings show that estradiol has the ability to alter the virulence traits of P. gingivalis.

The role of caspase-1, caspase-4 and NLRP3 in regulating the host cell response evoked by uropathogenic Escherichia coli.

The inflammasome-associated proteins caspase-1, caspase-4 and NLRP3 have been emphasised to be essential in the host cell response during urinary tract infection (UTI) by regulating IL-1ß release. Our aim was to investigate how the inflammasome-associated proteins regulate the cell response of bladder epithelial cells during infection with uropathogenic Escherichia coli (UPEC). Human bladder epithelial cells (5637) and CRISPR/Cas9 generated caspase-1, caspase-4 and NLRP3 knockdown cells were stimulated with the UPEC strain CFT073. Using Olink proteomics and real time RT-PCR, we showed that caspase-1, caspase-4 and NLRP3 are vital for the expression of many inflammatory genes and proteins from bladder epithelial cells. When investigating the effect of inflammasome-associated proteins on neutrophils, we found that conditioned medium from UPEC-infected caspase-4 knockdown cells significantly increased phagocytosis of CFT073 and significantly decreased ROS production from neutrophils. In contrast, conditioned medium from UPEC-infected NLRP3 knockdown cells significantly decreased the phagocytosis of CFT073 and significantly increased the ROS production from neutrophils. In conclusion, we showed that the inflammasome-associated proteins contribute to the host cell response during UPEC infection.

Transcriptional alterations in bladder epithelial cells in response to infection with different morphological states of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) may undergo a cyclic cascade of morphological alterations that are believed to enhance the potential of UPEC to evade host responses and re-infect host cell. However, knowledge on the pathogenic potential and host activation properties of UPEC during the morphological switch is limited. Microarray analysis was performed on mRNA isolated from human bladder epithelial cells (HBEP) after exposure to three different morphological states of UPEC (normal coliform, filamentous form and reverted form). Cells stimulated with filamentous bacteria showed the lowest number of significant gene alterations, although the number of enriched gene ontology classes was high suggesting diverse effects on many different classes of host genes. The normal coliform was in general superior in stimulating transcriptional activity in HBEP cells compared to the filamentous and reverted form. Top-scored gene entities activated by all three morphological states included IL17C, TNFAIP6, TNF, IL20, CXCL2, CXCL3, IL6 and CXCL8. The number of significantly changed canonical pathways was lower in HBEP cells stimulated with the reverted form (32 pathways), than in cells stimulated with the coliform (83 pathways) or filamentous bacteria (138 pathways). A host cell invasion assay showed that filamentous bacteria were unable to invade bladder cells, and that the number of intracellular bacteria was markedly lower in cells infected with the reverted form compared to the coliform. In conclusion, the morphological state of UPEC has major impact on the host bladder response both when evaluating the number and the identity of altered host genes and pathways.

Human Renal Fibroblasts, but Not Renal Epithelial Cells, Induce IL-1ß Release during a Uropathogenic Escherichia coli Infection In Vitro.

Understanding how uropathogenic Escherichia coli (UPEC) modulates the immune response in the kidney is essential to prevent UPEC from reaching the bloodstream and causing urosepsis. The purpose of this study was to elucidate if renal fibroblasts can release IL-1ß during a UPEC infection and to investigate the mechanism behind the IL-1ß release. We found that the UPEC strain CFT073 induced an increased IL-1ß and LDH release from renal fibroblasts, but not from renal epithelial cells. The UPEC-induced IL-1ß release was found to be NLRP3, caspase-1, caspase-4, ERK 1/2, cathepsin B and serine protease dependent in renal fibroblasts. We also found that the UPEC virulence factor α-hemolysin was necessary for IL-1ß release. Conditioned medium from caspase-1, caspase-4 and NLRP3-deficient renal fibroblasts mediated an increased reactive oxygen species production from neutrophils, but reduced UPEC phagocytosis. Taken together, our study demonstrates that renal fibroblasts, but not renal epithelial cells, release IL-1ß during a UPEC infection. This suggest that renal fibroblasts are vital immunoreactive cells and not only structural cells that produce and regulate the extracellular matrix.

The Fibrotic Effects of TMAO on Human Renal Fibroblasts Is Mediated by NLRP3, Caspase-1 and the PERK/Akt/mTOR Pathway.

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-ß1 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.

Estradiol Alters the Virulence Traits of Uropathogenic Escherichia coli.

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.

Activation of NLRP3 by uropathogenic Escherichia coli is associated with IL-1ß release and regulation of antimicrobial properties in human neutrophils.

The NLRP3 inflammasome and IL-1ß 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-1ß during UPEC infection. The purpose of this study was to elucidate the mechanisms behind UPEC-induced IL-1ß 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-1ß release from human neutrophils. The IL-1ß 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-1ß 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-1ß from human neutrophils and that NLRP3 and caspase-1 can regulate the antimicrobial activity of human neutrophils against UPEC.

IL-1RA is part of the inflammasome-regulated immune response in bladder epithelial cells and influences colonization of uropathogenic E. coli.

The NLRP3 inflammasome, IL-1ß release and pyroptosis (cell lysis) have recently been proposed to be essential for the progression of urinary tract infection (UTI) and elimination of intracellular bacterial niches. However, the effects of IL-1R antagonist (IL-1RA) on immune responses during UTI, except for its ability to disrupt IL-1ß signalling, are not well understood. The aim of this study was to investigate the role of IL-1RA in UPEC colonization of bladder epithelial cells and the subsequent host inflammatory response. Human bladder epithelial cells (5637) and CRISPR/Cas9 generated NLRP3 and caspase-1 knockdown cells and IL-1RA knockout cells were stimulated with the UPEC isolate CFT073. The results showed that the UPEC virulence factor α-hemolysin is essential for IL-1RA release, and that the inflammasome-associated proteins caspase-1 and NLRP3 affect the release of IL-1RA. IL-1RA deficient cells showed a reduced adherence and invasion by CFT073 compared to wild-type cells, suggesting that IL-1RA may oppose mechanisms that protects against bacterial colonization. A targeted protein analysis of inflammation-related proteins showed that the basal expression of 23 proteins and the UPEC-induced expression of 10 proteins were significantly altered in IL-1RA deficient bladder epithelial cells compared to Cas9 control cells. This suggests that IL-1RA has a broad effect on the inflammatory response in bladder epithelial cells.

Neisseria meningitidis-Induced Caspase-1 Activation in Human Innate Immune Cells Is LOS-Dependent.

Meningococcal disease such as sepsis and meningitidis is hallmarked by an excessive inflammatory response. The causative agent, Neisseria meningitidis, expresses the endotoxin lipooligosaccharide (LOS) that is responsible for activation of immune cells and the release of proinflammatory cytokines. One of the most potent proinflammatory cytokines, interleukin-1ß (IL-1ß), is activated following caspase-1 activity in the intracellular multiprotein complex called inflammasome. Inflammasomes are activated by a number of microbial factors as well as danger molecules by a two-step mechanism-priming and licensing of inflammasome activation-but there are no data available regarding a role for inflammasome activation in meningococcal disease. The aim of this study was to investigate if N. meningitidis activates the inflammasome and, if so, the role of bacterial LOS in this activation. Cells were subjected to N. meningitidis, both wild-type (FAM20) and its LOS-deficient mutant (lpxA), and priming as well as licensing of inflammasome activation was investigated. The wild-type LOS-expressing parental FAM20 serogroup C N. meningitidis (FAM20) strain significantly enhanced the caspase-1 activity in human neutrophils and monocytes, whereas lpxA was unable to induce caspase-1 activity as well as to induce IL-1ß release. While the lpxA mutant induced a priming response, measured as increased expression of NLRP3 and IL1B, the LOS-expressing FAM20 further increased this priming. We conclude that although non-LOS components of N. meningitidis contribute to the priming of the inflammasome activity, LOS per se is to be considered as the central component of N. meningitidis virulence, responsible for both priming and licensing of inflammasome activation.

Impact of Proinflammatory Cytokines on the Virulence of Uropathogenic Escherichia coli.

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-1ß, 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-1ß, 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.

Human renal fibroblasts are strong immunomobilizers during a urinary tract infection mediated by uropathogenic Escherichia coli.

To prevent the onset of urosepsis and reduce mortality, a better understanding of how uropathogenic Escherichia coli (UPEC) manages to infiltrate the bloodstream through the kidneys is needed. The present study elucidates if human renal interstitial fibroblasts are part of the immune response limiting a UPEC infection, or if UPEC has the ability to modulate the fibroblasts for their own gain. Microarray results showed that upregulated genes were associated with an activated immune response. We also found that chemokines released from renal fibroblasts upon a UPEC infection could be mediated by LPS and triacylated lipoproteins activating the TLR2/1, TLR4, MAPK, NF-κB and PKC signaling pathways. Furthermore, UPEC was also shown to be able to adhere and invade renal fibroblasts, mediated by the P-fimbriae. Furthermore, it was found that renal fibroblasts were more immunoreactive than renal epithelial cells upon a UPEC infection. However, both renal fibroblasts and epithelial cells were equally efficient at inducing neutrophil migration. In conclusion, we have found that human renal fibroblasts can sense UPEC and mobilize a host response with neutrophil migration. This suggests that renal fibroblasts are not only structural cells that produce and regulate the extracellular matrix, but also highly immunoreactive cells.

Staphylococcus epidermidis from prosthetic joint infections induces lower IL-1ß release from human neutrophils than isolates from normal flora.

The aim of this study was to test the hypothesis that Staphylococcus epidermidis isolated from prosthetic joint infections (PJIs) differs from S. epidermidis isolated from normal flora in terms of its capacity to induce activation of caspase-1 and release of IL-1ß in human neutrophils. The amount of active caspase-1 was determined over 6 h by detecting Ac-YVAD-AMC fluorescence in human neutrophils incubated with S. epidermidis isolates from PJIs (ST2) or normal flora. The amount of IL-1ß was detected by ELISA in neutrophil supernatants after 6 h of incubation. Mean IL-1ß release was lower after incubation with S. epidermidis from PJIs compared to isolates from normal flora, but no statistically significant difference was found in active caspase-1. Substantial inter-individual differences in both active caspase-1 and IL-1ß were noted. These results suggest that evasion of innate immune response, measured as reduced capacity to induce release of IL-1ß from human neutrophils, might be involved in the predominance of ST2 in S. epidermidis PJIs, but that other microbe-related factors are probably also important.