Bacteria and Candidaalbicans are prominent gut microbiota, and the translocation of these organisms into blood circulation might induce mixed-organism biofilms, which warrants the exploration of mixed- versus single-organism biofilms in vitro and in vivo. In single-organism biofilms, Acinetobacter baumannii and Pseudomonas aeruginosa (PA) produced the least and the most prominent biofilms, respectively. C. albicans with P. aeruginosa (PA+CA) induced the highest biofilms among mixed-organism groups as determined by crystal violet straining. The sessile form of PA+CA induced higher macrophage responses than sessile PA, which supports enhanced immune activation toward mixed-organism biofilms. In addition, Candida incubated in pre-formed Pseudomonas biofilms (PA>CA) produced even higher biofilms than PA+CA (simultaneous incubation of both organisms) as determined by fluorescent staining on biofilm matrix (AF647 color). Despite the initially lower bacteria during preparation, bacterial burdens by culture in mixed-organism biofilms (PA+CA and PA>CA) were not different from biofilms of PA alone, supporting Candida-enhanced Pseudomonas growth. Moreover, proteomic analysis in PA>CA biofilms demonstrated high AlgU and mucA with low mucB when compared with PA alone or PA+CA, implying an alginate-related mucoid phenotype in PA>CA biofilms. Furthermore, mice with PA>CA biofilms demonstrated higher bacteremia with more severe sepsis compared with mice with PA+CA biofilms. This is possibly due to the different structures. Interestingly, l-cysteine, a biofilm matrix inhibitor, attenuated mixed-organism biofilms both in vitro and in mice. In conclusion, Candida enhanced Pseudomonas alginate–related biofilm production, and Candida presentation in pre-formed Pseudomonas biofilms might alter biofilm structures that affect clinical manifestations but was attenuated by l-cysteine.
Renal ischemia is the most common cause of acute kidney injury (AKI) that might be exacerbate lupus activity through neutrophil extracellular traps (NETs) and apoptosis. Here, the renal ischemia reperfusion injury (I/R) was performed in Fc gamma receptor 2b deficient (Fcgr2b-/-) lupus mice and the in vitro experiments. At 24 h post-renal I/R injury, NETs in peripheral blood neutrophils and in kidneys were detected using myeloperoxidase (MPO), neutrophil elastase (NE) and citrullinated histone H3 (CitH3), as well as kidney apoptosis (activating caspase-3), which were prominent in Fcgr2b-/- mice more compared to wild-type (WT). After 120 h renal-I/R injury, renal NETs (using MPO and NE) were non-detectable, whereas glomerular immunoglobulin (Ig) deposition and serum anti-dsDNA were increased in Fcgr2b-/- mice. These results imply that renal NETs at 24 h post-renal I/R exacerbated the lupus nephritis at 120 h post-renal I/R injury in Fcgr2b-/- lupus mice. Furthermore, a Syk inhibitor attenuated NETs, that activated by phorbol myristate acetate (PMA; a NETs activator) or lipopolysaccharide (LPS; a potent inflammatory stimulator), more prominently in Fcgr2b-/- neutrophils than the WT cells as determined by dsDNA, PAD4 and MPO. In addition, the inhibitors against Syk and PAD4 attenuated lupus characteristics (serum creatinine, proteinuria, and anti-dsDNA) in Fcgr2b-/- mice at 120 h post-renal I/R injury. In conclusion, renal I/R in Fcgr2b-/- mice induced lupus exacerbation at 120 h post-I/R injury partly because Syk-enhanced renal NETs led to apoptosis-induced anti-dsDNA, which was attenuated by a Syk inhibitor.
Klebsiella pneumoniae is an opportunistic pathogen and a commensal organism that is possibly enhanced in several conditions with gut dysbiosis, and frequently detectable together with Candida overgrowth. Here, K. pneumoniae with or without Candida albicans was daily orally administered for 3 months in 0.8% dextran sulfate solution-induced mucositis mice and also tested in vitro. As such, Candida worsened Klebsiella-DSS-colitis as demonstrated by mortality, leaky gut (FITC-dextran assay, bacteremia, endotoxemia, and serum beta-glucan), gut dysbiosis (increased Deferribacteres from fecal microbiome analysis), liver pathology (histopathology), liver apoptosis (activated caspase 3), and cytokines (in serum and in the internal organs) when compared with Klebsiella-administered DSS mice. The combination of heat-killed Candida plus Klebsiella mildly facilitated inflammation in enterocytes (Caco-2), hepatocytes (HepG2), and THP-1-derived macrophages as indicated by supernatant cytokines or the gene expression. The addition of heat-killed Candida into Klebsiella preparations upregulated TLR-2, reduced Occludin (an intestinal tight junction molecule), and worsened enterocyte integrity (transepithelial electrical resistance) in Caco-2 and enhanced casp8 and casp9 (apoptosis genes) in HepG2 when compared with heat-killed Klebsiella alone. In conclusion, Candida enhanced enterocyte inflammation (partly through TLR-2 upregulation and gut dysbiosis) that induced gut translocation of endotoxin and beta-glucan causing hyper-inflammatory responses, especially in hepatocytes and macrophages.
The responses of macrophages to lipopolysaccharide (LPS) might determine the direction of clinical manifestations of sepsis, which is the immune response against severe infection. Meanwhile, the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, might interfere with LPS response. Transcriptomic analysis on LPS-activated wild-type macrophages demonstrated an alteration of several epigenetic enzymes. Although the Ezh2-silencing macrophages (RAW264.7), using small interfering RNA (siRNA), indicated a non-different response to the control cells after a single LPS stimulation, the Ezh2-reducing cells demonstrated a less severe LPS tolerance, after two LPS stimulations, as determined by the higher supernatant TNF-α. With a single LPS stimulation, Ezh2 null (Ezh2flox/flox; LysM-Crecre/−) macrophages demonstrated lower supernatant TNF-α than Ezh2 control (Ezh2fl/fl; LysM-Cre−/−), perhaps due to an upregulation of Socs3, which is a suppressor of cytokine signaling 3, due to the loss of the Ezh2 gene. In LPS tolerance, Ezh2 null macrophages indicated higher supernatant TNF-α and IL-6 than the control, supporting an impact of the loss of the Ezh2 inhibitory gene. In parallel, Ezh2 null mice demonstrated lower serum TNF-α and IL-6 than the control mice after an LPS injection, indicating a less severe LPS-induced hyper-inflammation in Ezh2 null mice. On the other hand, there were similar serum cytokines after LPS tolerance and the non-reduction of serum cytokines after the second dose of LPS, indicating less severe LPS tolerance in Ezh2 null mice compared with control mice. In conclusion, an absence of Ezh2 in macrophages resulted in less severe LPS-induced inflammation, as indicated by low serum cytokines, with less severe LPS tolerance, as demonstrated by higher cytokine production, partly through the upregulated Socs3.
The prevalence of obesity is increasing, and the coexistence of obesity and systemic lupus erythematosus (lupus) is possible. A high-fat diet (HFD) was orally administered for 6 months in female 8-week-old Fc gamma receptor IIb deficient (FcgRIIb−/−) lupus or age and gender-matched wild-type (WT) mice. Lupus nephritis (anti-dsDNA, proteinuria, and increased creatinine), gut barrier defect (fluorescein isothiocyanate dextran), serum lipopolysaccharide (LPS), serum interleukin (IL)-6, liver injury (alanine transaminase), organ fibrosis (liver and kidney pathology), spleen apoptosis (activated caspase 3), and aorta thickness (but not weight gain and lipid profiles) were more prominent in HFD-administered FcgRIIb−/− mice than the obese WT, without injury in regular diet-administered mice (both FcgRIIb−/− and WT). In parallel, combined palmitic acid (PA; a saturated fatty acid) with LPS (PA + LPS) induced higher tumor necrotic factor-α, IL-6, and IL-10 in the supernatant, inflammatory genes (inducible nitric oxide synthase and IL-1β), reactive oxygen species (dihydroethidium), and glycolysis with reduced mitochondrial activity (extracellular flux analysis) when compared with the activation by each molecule alone in both FcgRIIb−/− and WT macrophages. However, the alterations of these parameters were more prominent in PA + LPS-administered FcgRIIb−/− than in the WT cells. In conclusion, obesity accelerated inflammation in FcgRIIb−/− mice, partly due to the more potent responses from the loss of inhibitory FcgRIIb against PA + LPS with obesity-induced gut barrier defect.
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