To Örebro University

Trimethylamine-N-oxide (TMAO), a gut microbiota-derived dietary metabolite, is linked to progression of chronic kidney disease (CKD). Megalin, a renal proximal tubule receptor crucial for albumin reabsorption, also plays a role in CKD. However, the relationship between them is not well explored. The aim of this study was to investigate if there are any correlations between the levels of TMAO, megalin, lysine and markers of tubular damage in CKD. Urinary metabolites (TMAO, choline, L-carnitine, betaine, lysine) and tubular markers (megalin, albumin, EGF, MCP-1) were quantified by LC-MS/MS and ELISA. Associations were evaluated using analysis of covariance (ANCOVA) adjusted for age and diabetes, with false discovery rate correction. Compared with controls, CKD patients showed higher urinary choline (FDR < 0.001), betaine (FDR = 0.007), lysine (FDR = 0.005), and soluble megalin (FDR < 0.001) but lower EGF and EGF/MCP-1 ratio (both FDR < 0.001). Correlation analyses revealed that serum TMAO was positively associated with soluble megalin and negatively with EGF/MCP-1 ratio. Choline, L-carnitine, and betaine were positively correlated with megalin. This cross-sectional study identifies associations between urinary metabolites, megalin, and tubular injury markers in advanced CKD. Although causality cannot be inferred, the results point to a potential metabolic-tubular link that should be explored in future longitudinal and mechanistic studies.

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.

With antibiotic resistance being a major global concern, there is a huge need of new treatment options to fight bacterial infections. In this study, we highlight the antibacterial and host-protective roles of a novel synthetic antimicrobial peptide in uropathogenic Escherichia coli–infected uroepithelial cells. This peptide, designed from a fragment of human cathelicidin LL-37 and named LD4-PP, was found to be highly potent against clinical isolates of E. coli as well as ESBL-producing and multi-drug resistant E. coli. Additionally, LD4-PP inhibited the formation of new biofilm, damaging both the bacterial surface and the bacterial genome. LD4-PP also modulated the host cell lipid vacuole, caveolin-1, and Rho GTPase B affecting bacterial survival. Furthermore, LD4-PP exerts immunomodulatory effects by modulating free radical formation, expression of antioxidants, and inflammasome-mediated cell death. Pronounced uroepithelial cell death was observed after E. coli infection which was significantly inhibited by LD4-PP without affecting the cellular toxicity. Overall, the peptide LD4-PP is shown to be a strong candidate for future clinical applications, particularly to prevent and treat urinary tract infections.

Periodontitis is a chronic inflammatory disease characterized by bacterial infection and immune dysregulation. Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is a key pathogen linked to disease progression. Caspase-1 and caspase-4 regulate inflammasome activation and cytokine release, yet their roles in gingival epithelial immunity remain unclear. The aim of this study was to elucidate the involvement of caspase-1 and caspase-4 in regulating the immune response to A. actinomycetemcomitans infection in gingival epithelial cells. Human gingival epithelial cells (Ca9-22) and caspase-1- and caspase-4-deficient cells were infected with A. actinomycetemcomitans for 24 h. Inflammatory mediator release was analyzed using Olink proteomics. Bacterial colonization and invasion were assessed using fluorescence-based assays and gentamicin protection assays. Caspase-1- and caspase-4-deficient cells showed significantly altered cytokine and chemokine profiles after infection with A. actinomycetemcomitans, showing reduced IL-17C and IL-18 release. We also found an increased release of TGF-α and LIF from caspase-4-deficient cells, along with elevated levels of the chemokines IL-8, CXCL9, and CXCL10. Additionally, both caspase-1- and caspase-4-deficient cells showed increased bacterial colonization and invasion, particularly in caspase-4-deficient cells. These findings suggest that caspase-1 and caspase-4 play distinct yet essential roles in gingival epithelial immunity, regulating cytokine release, barrier integrity, and defense against A. actinomycetemcomitans colonization.

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.

Trimethylamine-N-oxide (TMAO) is a gut microbiota-derived metabolite and TNF-α is proinflammatory cytokine, both known to be associated with renal inflammation, fibrosis and chronic kidney disease. However, today there are no data showing the combined effect of TMAO and TNF-α on renal fibrosis-and inflammation. The aim of this study was to investigate whether TMAO can enhance the inflammatory and fibrotic effects of TNF-α on renal fibroblasts. We found that the combination of TNF-α and TMAO synergistically increased fibronectin release and total collagen production from renal fibroblasts. The combination of TMAO and TNF-α also promoted increased cell proliferation. Both renal proliferation and collagen production were mediated through Akt/mTOR/ERK signaling. We also found that TMAO enhanced TNF-α mediated renal inflammation by inducing the release of several cytokines (IL-6, LAP TGF-beta-1), chemokines (CXCL-6, MCP-3), inflammatory-and growth mediators (VEGFA, CD40, HGF) from renal fibroblasts. In conclusion, we showed that TMAO can enhance TNF-α mediated renal fibrosis and release of inflammatory mediators from renal fibroblasts in vitro. Our results can promote further research evaluating the combined effect of TMAO and inflammatory mediators on the development of kidney disease.

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.