To Örebro University

Endothelial dysfunction is an early driver of atherosclerosis, yet the direct impact of endogenous crystals such as cholesterol crystals and monosodium urate on endothelial activation remains incompletely understood. In this study, we examine how crystalline stimuli modulate human umbilical vein endothelial cells by assessing inflammatory signaling, mitochondrial respiration, and neutrophil recruitment. Using dose- and time-controlled experiments, we show that CC and MSU are internalized by endothelial cells, activating NF-κB and STAT3 signaling pathways and inducing a robust pro-inflammatory cytokine profile. Notably, CC caused marked mitochondrial dysfunction, evidenced by impaired respiratory capacity and loss of membrane potential, revealing a novel bioenergetic vulnerability in endothelial cells. Both direct crystal stimulation and exposure to crystal-primed conditioned media triggered endothelial adhesion molecule expression and promoted neutrophil adhesion, indicating that soluble mediators released upon crystal stimulation can propagate vascular inflammation. These findings demonstrate that crystalline stimuli are potent vascular danger signals capable of driving endothelial inflammation, mitochondrial impairment, and immune cell engagement, which are hallmarks of early atherogenesis. By elucidating these multifaceted endothelial responses, this study provides important mechanistic insights into how crystal-induced signals may contribute to vascular dysfunction and the early stages of atherogenesis.

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infection (UTI). While the role of UPEC in triggering host immune responses is well studied, less is known about how host-derived cytokines influence UPEC virulence. The aim of this study was to investigate how the pro-inflammatory cytokine IL-1β affects UPEC virulence, metabolism, and host-pathogen interactions. Using the CFT073 strain, we found that IL-1β exposure induced a rapid metabolic shift characterized by decreased oxygen consumption rate (OCR) and increased extracellular acidification rate (ECAR), indicating a transition from respiration to fermentative metabolism. Microarray analysis confirmed the upregulation of fermentative (hyc) and antioxidant (katG, ahpF, grxA) genes, along with increased purine biosynthesis, supporting a metabolic state favouring stress resistance and proliferation. IL-1β also increased fimH and papC gene expression, leading to increased adhesion and invasion of bladder epithelial cells. Furthermore, IL-1β-stimulated CFT073 suppressed the gene expression of several innate immune related genes in a C. elegans infection model. Taken together, our findings suggest that IL-1β induces a virulence-enhancing metabolic change in UPEC, which could enhance its persistence and colonization of the urinary tract. This cross-kingdom signaling may have implications for infection dynamics during a UTI.

Urinary tract infection (UTI) is one of the most common bacterial infections worldwide and the most common cause is uropathogenic Escherichia coli (UPEC). Current research is mostly focused on how UPEC affects host factors, whereas the effect of host factors on UPEC is less studied. Our previous studies have shown that estrogen alters UPEC virulence. However, the effect of this altered UPEC virulence on neutrophils is unknown. The aim of the present study was to investigate how the altered UPEC virulence mediated by estrogen modulates neutrophil responses. We found that estradiol-stimulated CFT073 increased neutrophil phagocytosis, NETs formation and intracellular ROS production. We observed that the total ROS production from neutrophils was reduced by estradiol-stimulated CFT073. We also found that estradiol-stimulated CFT073 induced less cytotoxicity in neutrophils. Additionally, we found that several cytokines and chemokines like IL-8, IL-1β, CXCL6, MCP-1 and MCP-4 were increased upon estradiol-stimulated CFT073 infection. In conclusion, this study demonstrates that the estrogen-mediated alterations to UPEC virulence modulates neutrophil responses, most likely in a host-beneficial manner.

Periodontitis and infections with periodontal bacteria have been highlighted as risk factors for dementia. In recent years, attention has been drawn to the role of microglia cells in neurodegenerative diseases. However, there is limited knowledge of the influence of periodontal bacteria on microglia cells. The aim of the present study was to investigate the interactions between the periodontal bacteria Porphyromonas gingivalis and microglia cells and to unravel whether these interactions could contribute to the pathology of Alzheimer's disease. We found, through microarray analysis, that stimulation of microglia cells with P. gingivalis resulted in the upregulation of several Alzheimer's disease-associated genes, including NOX4. We also showed that P. gingivalis lipopolysaccharides (LPS) mediated reactive oxygen species (ROS) production and interleukin 6 (IL-6) and interleukin 8 (IL-8) induction via NOX4 in microglia. The viability of neurons was shown to be reduced by conditioned media from microglia cells stimulated with P. gingivalis LPS and the reduction was NOX4 dependent. The levels of total and phosphorylated tau in neurons were increased by conditioned media from microglia cells stimulated with P. gingivalis or LPS. This increase was NOX4-dependent. In summary, our findings provide us with a potential mechanistic explanation of how the periodontal pathogen P. gingivalis could trigger or exacerbate AD pathogenesis.

Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infections (UTIs) in humans. Testosterone negatively impacts UTIs by affecting the immune response, leading to higher susceptibility of chronic cystitis in individuals with elevated testosterone levels, regardless of gender. Current research is mostly focused on how testosterone affects the host response to UPEC, but not so much is known about how testosterone directly affect UPEC virulence. The aim of the present study was to investigate the impact of testosterone exposure on the virulence of UPEC. We found that testosterone directly increases UPEC growth, endotoxin release and biofilm formation. We also found that testosterone-stimulated CFT073 increased colonization and invasion of bladder epithelial cells. Testosterone-stimulated CFT073 also increased the release of IL-1β and LDH from bladder epithelial cells. Additionally, by using a Caenorhabditis elegans survival assay we also showed that testosterone decreased the survival of CFT073 infected C. elegans worms. Taken together, our findings show that testosterone directly increases the virulence traits of UPEC.

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-1β 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-1β 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.

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.

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-1β 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.