Enhanced lupus progression in alcohol-administered Fc gamma receptor-IIb-deficiency lupus mice, partly through leaky gut-induced inflammation.

Alcohol can induce a leaky gut, with translocation of microbial molecules from the gut into the blood circulation. Although the contribution of inflammation to organ-mediated damage in lupus has been previously demonstrated, the mechanistic roles of alcohol consumption in lupus activation are not known. Herein, we tested the effects of 10-week lasting alcohol administration on organ damages and immune responses in 8-week-old lupus-prone Fc gamma receptor IIb-deficient (FcγRIIb-/- ) mice. Our study endpoints were evaluation of systemic inflammation and assessment of fecal dysbiosis along with endotoxemia. In comparison with alcohol-administered wild-type mice, FcγRIIb-/- mice demonstrated more prominent liver damage (enzyme, histological score, apoptosis, malondialdehyde oxidant) and serum interleukin(IL)-6 levels, despite a similarity in leaky gut (fluorescein isothiocyanate-dextran assay, endotoxemia and gut occludin-1 immunofluorescence), fecal dysbiosis (microbiome analysis) and endotoxemia. All alcohol-administered FcγRIIb-/- mice developed lupus-like characteristics (serum anti-dsDNA, proteinuria, serum creatinine and kidney injury score) with spleen apoptosis, whereas control FcγRIIb-/- mice showed only a subtle anti-dsDNA. Both alcohol and lipopolysaccharide (LPS) similarly impaired enterocyte integrity (transepithelial electrical resistance), and only LPS, but not alcohol, upregulated the IL-8 gene in Caco-2 cells. In macrophages, alcohol mildly activated supernatant cytokines (tumor necrosis factor-α and IL-6), but not M1 polarization-associated genes (IL-1ß and iNOS), whereas LPS prominently induced both parameters (more prominent in FcγRIIb-/- macrophages than wild type). There was no synergy in LPS plus alcohol compared with LPS alone in both enterocytes and macrophages. In conclusion, alcohol might exacerbate lupus-like activity partly through a profound inflammation from the leaky gut in FcγRIIb-/- mice.

Lacticaseibacillus rhamnosus dfa1 Attenuate Cecal Ligation-Induced Systemic Inflammation through the Interference in Gut Dysbiosis, Leaky Gut, and Enterocytic Cell Energy.

Despite an uncommon condition, the clinical management of phlegmon appendicitis (retention of the intra-abdominal appendiceal abscess) is still controversial, and probiotics might be partly helpful. Then, the retained ligated cecal appendage (without gut obstruction) with or without oral Lacticaseibacillus rhamnosus dfa1 (started at 4 days prior to the surgery) was used as a representative model. At 5 days post-surgery, the cecal-ligated mice demonstrated weight loss, soft stool, gut barrier defect (leaky gut using FITC-dextran assay), fecal dysbiosis (increased Proteobacteria with reduced bacterial diversity), bacteremia, elevated serum cytokines, and spleen apoptosis without kidney and liver damage. Interestingly, the probiotics attenuated disease severity as indicated by stool consistency index, FITC-dextran assay, serum cytokines, spleen apoptosis, fecal microbiota analysis (reduced Proteobacteria), and mortality. Additionally, impacts of anti-inflammatory substances from culture media of the probiotics were demonstrated by attenuation of starvation injury in the Caco-2 enterocyte cell line as indicated by transepithelial electrical resistance (TEER), inflammatory markers (supernatant IL-8 with gene expression of TLR4 and NF-κB), cell energy status (extracellular flux analysis), and the reactive oxygen species (malondialdehyde). In conclusion, gut dysbiosis and leaky-gut-induced systemic inflammation might be helpful clinical parameters for patients with phlegmon appendicitis. Additionally, the leaky gut might be attenuated by some beneficial molecules from probiotics.

Lipopolysaccharide Tolerance Enhances Murine Norovirus Reactivation: An Impact of Macrophages Mainly Evaluated by Proteomic Analysis.

Because of endotoxemia during sepsis (a severe life-threatening infection), lipopolysaccharide (LPS) tolerance (the reduced responses to the repeated LPS stimulation) might be one of the causes of sepsis-induced immune exhaustion (the increased susceptibility to secondary infection and/or viral reactivation). In LPS tolerance macrophage (twice-stimulated LPS, LPS/LPS) compared with a single LPS stimulation (N/LPS), there was (i) reduced energy of the cell in both glycolysis and mitochondrial activities (extracellular flux analysis), (ii) decreased abundance of the following proteins (proteomic analysis): (a) complex I and II of the mitochondrial electron transport chain, (b) most of the glycolysis enzymes, (c) anti-viral responses with Myxovirus resistance protein 1 (Mx1) and Ubiquitin-like protein ISG15 (Isg15), (d) antigen presentation pathways, and (iii) the down-regulated anti-viral genes, such as Mx1 and Isg15 (polymerase chain reaction). To test the correlation between LPS tolerance and viral reactivation, asymptomatic mice with and without murine norovirus (MNV) infection as determined in feces were tested. In MNV-positive mice, MNV abundance in the cecum, but not in feces, of LPS/LPS mice was higher than that in N/LPS and control groups, while MNV abundance of N/LPS and control were similar. Additionally, the down-regulated Mx1 and Isg15 were also demonstrated in the cecum, liver, and spleen in LPS/LPS-activated mice, regardless of MNV infection, while N/LPS more prominently upregulated these genes in the cecum of MNV-positive mice compared with the MNV-negative group. In conclusion, defects in anti-viral responses after LPS tolerance, perhaps through the reduced energy status of macrophages, might partly be responsible for the viral reactivation. More studies on patients are of interest.

Less Severe Sepsis in Cecal Ligation and Puncture Models with and without Lipopolysaccharide in Mice with Conditional Ezh2-Deleted Macrophages (LysM-Cre System).

Despite a previous report on less inflammatory responses in mice with an absence of the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, using a lipopolysaccharide (LPS) injection model, proteomic analysis and cecal ligation and puncture (CLP), a sepsis model that more resembles human conditions was devised. As such, analysis of cellular and secreted protein (proteome and secretome) after a single LPS activation and LPS tolerance in macrophages from Ezh2 null (Ezh2flox/flox; LysM-Crecre/-) mice (Ezh2 null) and the littermate control mice (Ezh2fl/fl; LysM-Cre-/-) (Ezh2 control) compared with the unstimulated cells from each group indicated fewer activities in Ezh2 null macrophages, especially by the volcano plot analysis. Indeed, supernatant IL-1ß and expression of genes in pro-inflammatory M1 macrophage polarization (IL-1ß and iNOS), TNF-α, and NF-κB (a transcription factor) were lower in Ezh2 null macrophages compared with the control. In LPS tolerance, downregulated NF-κB compared with the control was also demonstrated in Ezh2 null cells. In CLP sepsis mice, those with CLP alone and CLP at 2 days after twice receiving LPS injection, representing sepsis and sepsis after endotoxemia, respectively, symptoms were less severe in Ezh2 null mice, as indicated by survival analysis and other biomarkers. However, the Ezh2 inhibitor improved survival only in CLP, but not LPS with CLP. In conclusion, an absence of Ezh2 in macrophages resulted in less severe sepsis, and the use of an Ezh2 inhibitor might be beneficial in sepsis.

Less Severe Polymicrobial Sepsis in Conditional mgmt-Deleted Mice Using LysM-Cre System, Impacts of DNA Methylation and MGMT Inhibitor in Sepsis.

The O6-methylguanine-DNA methyltransferase (MGMT) is a DNA suicide repair enzyme that might be important during sepsis but has never been explored. Then, the proteomic analysis of lipopolysaccharide (LPS)-stimulated wild-type (WT) macrophages increased proteasome proteins and reduced oxidative phosphorylation proteins compared with control, possibly related to cell injury. With LPS stimulation, mgmt null (mgmtflox/flox; LysM-Crecre/-) macrophages demonstrated less profound inflammation; supernatant cytokines (TNF-α, IL-6, and IL-10) and pro-inflammatory genes (iNOS and IL-1ß), with higher DNA break (phosphohistone H2AX) and cell-free DNA, but not malondialdehyde (the oxidative stress), compared with the littermate control (mgmtflox/flox; LysM-Cre-/-). In parallel, mgmt null mice (MGMT loss only in the myeloid cells) demonstrated less severe sepsis in the cecal ligation and puncture (CLP) model (with antibiotics), as indicated by survival and other parameters compared with sepsis in the littermate control. The mgmt null protective effect was lost in CLP mice without antibiotics, highlighting the importance of microbial control during sepsis immune modulation. However, an MGMT inhibitor in CLP with antibiotics in WT mice attenuated serum cytokines but not mortality, requiring further studies. In conclusion, an absence of mgmt in macrophages resulted in less severe CLP sepsis, implying a possible influence of guanine DNA methylation and repair in macrophages during sepsis.

The Regulatory Roles of Ezh2 in Response to Lipopolysaccharide (LPS) in Macrophages and Mice with Conditional Ezh2 Deletion with LysM-Cre System.

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.

Less Severe Lipopolysaccharide-Induced Inflammation in Conditional mgmt-Deleted Mice with LysM-Cre System: The Loss of DNA Repair in Macrophages.

Despite the known influence of DNA methylation from lipopolysaccharide (LPS) activation, data on the O6-methylguanine-DNA methyltransferase (MGMT, a DNA suicide repair enzyme) in macrophages is still lacking. The transcriptomic profiling of epigenetic enzymes from wild-type macrophages after single and double LPS stimulation, representing acute inflammation and LPS tolerance, respectively, was performed. Small interfering RNA (siRNA) silencing of mgmt in the macrophage cell line (RAW264.7) and mgmt null (mgmtflox/flox; LysM-Crecre/-) macrophages demonstrated lower secretion of TNF-α and IL-6 and lower expression of pro-inflammatory genes (iNOS and IL-1ß) compared with the control. Macrophage injury after a single LPS dose and LPS tolerance was demonstrated by reduced cell viability and increased oxidative stress (dihydroethidium) compared with the activated macrophages from littermate control mice (mgmtflox/flox; LysM-Cre-/-). Additionally, a single LPS dose and LPS tolerance also caused mitochondrial toxicity, as indicated by reduced maximal respiratory capacity (extracellular flux analysis) in the macrophages of both mgmt null and control mice. However, LPS upregulated mgmt only in LPS-tolerant macrophages but not after the single LPS stimulation. In mice, the mgmt null group demonstrated lower serum TNF-α, IL-6, and IL-10 than control mice after either single or double LPS stimulation. Suppressed cytokine production resulting from an absence of mgmt in macrophages caused less severe LPS-induced inflammation but might worsen LPS tolerance.

Rapid Synergistic Biofilm Production of Pseudomonas and Candida on the Pulmonary Cell Surface and in Mice, a Possible Cause of Chronic Mixed Organismal Lung Lesions.

Due to the possible co-presence of Pseudomonas aeruginosa and Candida albicans (the most common nosocomial pathogens) in lungs, rapid interkingdom biofilm production is possible. As such, PA+CA produced more dominant biofilms on the pulmonary epithelial surface (NCI-H292) (confocal fluorescent extracellular matrix staining) with dominant psl upregulation, as demonstrated by polymerase chain reaction (PCR), after 8 h of experiments than PA alone. With a proteomic analysis, rhamnosyltransferase RhlB protein (Psl-associated quorum-sensing protein) was found to be among the high-abundance proteins in PA+CA than in PA biofilms, supporting psl-mediated biofilms in PA+CA on the cell surface. Additionally, PA+CA increased supernatant cytokines (IL-8 and IL-13, but not TNF-α, IL-6, and IL-10) with a similar upregulation of TLR-4, TLR-5, and TLR-9 (by PCR) compared with PA-stimulated cells. The intratracheal administration of PA+CA induced a greater severity of sepsis (serum creatinine, alanine transaminase, serum cytokines, and histology score) and prominent biofilms (fluorescent staining) with psl upregulation (PCR). In comparison with PA+CA biofilms on glass slides, PA+CA biofilms on biotic surfaces were more prominent (fluorescent staining). In conclusion, PA+CA induced Psl-predominant biofilms on the pulmonary cell surface and in mice with acute pneumonia, and these biofilms were more prominent than those induced by PA alone, highlighting the impact of Candida on rapid interkingdom biofilm production.

Candida Worsens Klebsiella pneumoniae Induced-Sepsis in a Mouse Model with Low Dose Dextran Sulfate Solution through Gut Dysbiosis and Enhanced Inflammation.

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.

Chlorhexidine Promotes Psl Expression in Pseudomonas aeruginosa That Enhances Cell Aggregation with Preserved Pathogenicity Demonstrates an Adaptation against Antiseptic.

Because Pseudomonas aeruginosa is frequently in contact with Chlorhexidine (a regular antiseptic), bacterial adaptations are possible. In comparison with the parent strain, the Chlorhexidine-adapted strain formed smaller colonies with metabolic downregulation (proteomic analysis) with the cross-resistance against colistin (an antibiotic for several antibiotic-resistant bacteria), partly through the modification of L-Ara4N in the lipopolysaccharide at the outer membrane. Chlorhexidine-adapted strain formed dense liquid-solid interface biofilms with enhanced cell aggregation partly due to the Chlorhexidine-induced overexpression of psl (exopolysaccharide-encoded gene) through the LadS/GacSA pathway (c-di-GMP-independence) in 12 h biofilms and maintained the aggregation with SiaD-mediated c-di-GMP dependence in 24 h biofilms as evaluated by polymerase chain reaction (PCR). The addition of Ca2+ in the Chlorhexidine-adapted strain facilitated several Psl-associated genes, indicating an impact of Ca2+ in Psl production. The activation by Chlorhexidine-treated sessile bacteria demonstrated a lower expression of IL-6 and IL-8 on fibroblasts and macrophages than the activation by the parent strain, indicating the less inflammatory reactions from Chlorhexidine-exposed bacteria. However, the 14-day severity of the wounds in mouse caused by Chlorhexidine-treated bacteria versus the parent strain was similar, as indicated by wound diameters and bacterial burdens. In conclusion, Chlorhexidine induced psl over-expression and colistin cross-resistance that might be clinically important.

Cyclic GMP-AMP Synthase (cGAS) Deletion Reduces Severity in Bilateral Nephrectomy Mice through Changes in Neutrophil Extracellular Traps and Mitochondrial Respiration.

Uremia-induced systemic inflammation is partly caused by the dissemination of microbial molecules such as lipopolysaccharide and bacterial double-stranded DNA from leaked gut damaged by immune cells in response to the microbial molecules. Cyclic GMP-AMP synthase (cGAS) can recognize fragmented DNA and induce cGAMP synthesis for the activation of the stimulator of interferon genes (STING) pathway. To study the effect of cGAS in uremia-induced systemic inflammation, we performed bilateral nephrectomy (BNx) in wild-type and cGAS knock-out mice and found that the gut leakage and blood uremia from both groups were similar. However, serum cytokines (TNF-α and IL-6) and neutrophil extracellular traps (NETs) decreased significantly in cGAS-/- neutrophils after stimulation with LPS or bacterial cell-free DNA. Transcriptomic analysis of LPS-stimulated cGAS-/- neutrophils also confirmed the down-regulation of neutrophil effector functions. The extracellular flux analysis showed that cGAS-/- neutrophils exhibited a higher respiratory rate than wild-type neutrophils despite having similar mitochondrial abundance and function. Our results suggest that cGAS may control effector functions and the mitochondrial respiration of neutrophils in response to LPS or bacterial DNA.

Obesity Exacerbates Lupus Activity in Fc Gamma Receptor IIb Deficient Lupus Mice Partly through Saturated Fatty Acid-Induced Gut Barrier Defect and Systemic Inflammation.

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-1ß), 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.

Presence of Candida tropicalis on Staphylococcus epidermidis Biofilms Facilitated Biofilm Production and Candida Dissemination: An Impact of Fungi on Bacterial Biofilms.

While Staphylococcus epidermidis (SE) is a common cause of infections in implanted prostheses and other indwelling devices, partly due to the biofilm formation, Candida tropicalis (CT) is an emerging Candida spp. with a potent biofilm-producing property. Due to the possible coexistence between SE and CT infection in the same patient, characteristics of the polymicrobial biofilms from both organisms might be different from those of the biofilms of each organism. Then, the exploration on biofilms, from SE with or without CT, and an evaluation on l-cysteine (an antibiofilm against both bacteria and fungi) were performed. As such, Candida incubation in preformed SE biofilms (SE > CT) produced higher biofilms than the single- (SE or CT) or mixed-organism (SE + CT) biofilms as determined by crystal violet staining and fluorescent confocal images with z-stack thickness analysis. In parallel, SE > CT biofilms demonstrated higher expression of icaB and icaC than other groups at 20 and 24 h of incubation, suggesting an enhanced matrix polymerization and transportation, respectively. Although organism burdens (culture method) from single-microbial biofilms (SE or CT) were higher than multi-organism biofilms (SE + CT and SE > CT), macrophage cytokine responses (TNF-α and IL-6) against SE > CT biofilms were higher than those in other groups in parallel to the profound biofilms in SE > CT. Additionally, sepsis severity in mice with subcutaneously implanted SE > CT catheters was more severe than in other groups as indicated by mortality rate, fungemia, serum cytokines (TNF-α and IL-6), and kidney and liver injury. Although CT grows upon preformed SE-biofilm production, the biofilm structures interfered during CT morphogenesis leading to the frailty of biofilm structure and resulting in the prominent candidemia. However, l-cysteine incubation together with the organisms in catheters reduced biofilms, microbial burdens, macrophage responses, and sepsis severity. In conclusion, SE > CT biofilms prominently induced biofilm matrix, fungemia, macrophage responses, and sepsis severity, whereas the microbial burdens were lower than in the single-organism biofilms. All biofilms were attenuated by l-cysteine.

Lacticaseibacillus casei Strain T21 Attenuates Clostridioides difficile Infection in a Murine Model Through Reduction of Inflammation and Gut Dysbiosis With Decreased Toxin Lethality and Enhanced Mucin Production.

Clostridioides difficile is a major cause of diarrhea in patients with antibiotic administration. Lacticaseibacillus casei T21, isolated from a human gastric biopsy, was tested in a murine C. difficile infection (CDI) model and colonic epithelial cells (Caco-2 and HT-29). Daily administration of L. casei T21 [1 × 108 colony forming units (CFU)/dose] for 4 days starting at 1 day before C. difficile challenge attenuated CDI as demonstrated by a reduction in mortality rate, weight loss, diarrhea, gut leakage, gut dysbiosis, intestinal pathology changes, and levels of pro-inflammatory cytokines [interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, macrophage inflammatory protein 2 (MIP-2), and keratinocyte chemoattractant (KC)] in the intestinal tissue and serum. Conditioned media from L. casei T21 exerted biological activities that fight against C. difficile as demonstrated in colonic epithelial cells by the following: (i) suppression of gene expression and production of IL-8, an important chemokine involved in C. difficile pathogenesis, (ii) reduction in the expression of SLC11A1 (solute carrier family 11 member 1) and HuR (human antigen R), important genes for the lethality of C. difficile toxin B, (iii) augmentation of intestinal integrity, and (iv) up-regulation of MUC2, a mucosal protective gene. These results supported the therapeutic potential of L. casei T21 for CDI and the need for further study on the intervention capabilities of CDI.

Acute Kidney Injury Induced Lupus Exacerbation Through the Enhanced Neutrophil Extracellular Traps (and Apoptosis) in Fcgr2b Deficient Lupus Mice With Renal Ischemia Reperfusion Injury.

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.

Coexistence of Pseudomonas aeruginosa With Candida albicans Enhances Biofilm Thickness Through Alginate-Related Extracellular Matrix but Is Attenuated by N-acetyl-l-cysteine.

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.

Identification and characterization of ferulic acid esterase from Penicillium chrysogenum 31B: de-esterification of ferulic acid decorated with l-arabinofuranoses and d-galactopyranoses in sugar beet pectin.

We previously described the fungus Penicillium chrysogenum 31B, which has high performance to produce the ferulic acid esterase (FAE) for de-esterifying ferulic acids (FAs) from sugar beet pulp. However, the characteristics of this fungus have not yet been determined. Therefore, in this study, we evaluated the molecular characteristics and natural substrate specificity of the Pcfae1 gene from Penicillium chrysogenum and examined its synergistic effects on sugar beet pectin. The Pcfae1 gene was cloned and overexpressed in Pichia pastoris KM71H, and the recombinant enzyme, named PcFAE1, was characterized. The 505 amino acids of PcFAE1 possessed a GCSTG motif (Gly164 to Gly168), characteristic of the serine esterase family. By comparing the amino acid sequence of PcFAE1 with that of the FAE (AoFaeB) of Aspergillus oryzae, Ser166, Asp379, and His419 were identified as the catalytic triad. PcFAE1 was purified through two steps using anion-exchange column chromatography. Its molecular mass without the signal peptide was 75 kDa. Maximum PcFAE1 activity was achieved at pH 6.0-7.0 and 50 °C. The enzyme was stable up to 37 °C and at a pH range of 3-8. PcFAE1 activity was only inhibited by Hg2+, and the enzyme had activity toward methyl FA, methyl caffeic acid, and methyl p-coumaric acid, with specific activities of 6.97, 4.65, and 9.32 U/mg, respectively, but not on methyl sinapinic acid. These results indicated that PcFAE1 acted similar to FaeB type according the Crepin classification. PcFAE1 de-esterified O-[6-O-feruloyl-ß-d-galactopyranosyl-(1→4)]-d-galactopyranose, O-[2-O-feruloyl-α-l-arabinofuranosyl-(1→5)]-l-arabinofuranose, and O-[5-O-feruloyl-α-l-arabinofuranosyl-(1→3)]-O-ß-d-xylopyranosyl-(1→4)-d-xylopyranose, indicating that the enzyme could de-esterify FAs decorated with both ß-d-galactopyranosidic and α-l-arabinofuranosidic residues in pectin and xylan. PcFAE1 acted in synergy with endo-α-1,5-arabinanase and α-l-arabinofuranosidase, which releases FA linked to arabinan, to digest the sugar beet pectin. Moreover, when PcFAE1 was allowed to act on sugar beet pectin together with Driselase, approximately 90% of total FA in the substrate was released. Therefore, PcFAE1 may be an interesting candidate for hydrolysis of lignocellulosic materials and could have applications as a tool for production of FA from natural substrates.

Identification and characterization of GH62 bacterial α-l-arabinofuranosidase from thermotolerant Streptomyces sp. SWU10 that preferentially degrades branched l-arabinofuranoses in wheat arabinoxylan.

We previously described thermotolerant Streptomyces sp. SWU10, which produced four endo-xylanases and one xylosidase able to digest xylan backbones. To achieve arabinoxylan degradation, the swu62A gene was cloned and overexpressed in Escherichia coli, and the recombinant enzyme, termed SWUAbf62A, was characterized. The 438 amino acids of SWUAbf62A revealed Glyco_hydro_62 and closely related with putative α-l-arabinofuranosidases belonging to glycoside hydrolase family 62. SWUAbf62A was purified in two steps, Ni-affinity and size-exclusion column chromatographies, and its molecular mass without signal peptide was determined to be 49 kDa. SWUAbf62A showed optimum activity at pH 5.0 and 50 °C, and more than 70% of its initial enzymatic activity remained after incubation at pH 4.1-10.5, while SWUAbf62A lost all activity after 1 h at 60 °C. SWUAbf62A activity was stimulated by Ba2+, Ca2+, and Mn2+ and decreased by Ag+, Cu2+, Fe2+, and EDTA. SWUAbf62A had no activity towards p-nitrophenyl-α-l-arabinofuranoside or p-nitrophenyl-ß-d-xylopyranoside synthetic substrates. On the other hand, SWUAbf62A had the highest activity against wheat arabinoxylan, with a specific activity of 1.29 U/mg, and was also active against sugar beet arabinan, with a specific activity of 0.14 U/mg; these results indicated that SWUAbf62A is an arabinoxylan arabinofuranohydrolase. Using 1H-NMR analysis, SWUAbf62A was found to release l-arabinofuranoses singly linked to O-3 of wheat arabinoxylan. In addition, SWUAbf62A acted synergistically with endo-xylanase (XynSW3) and α-l-arabinofuranosidase, which releases arabinose linked to O-3 of double-substituted xylose residues on arabinoxylan, to digest the wheat arabinoxylan. SWUAbf62A is an important debranching enzyme for hydrolysis of hemicelluloses to monosaccharides and can be applied in various industrial biotechnologies.

Identification and characterization of the first ß-1,3-d-xylosidase from a gram-positive bacterium, Streptomyces sp. SWU10.

In previous reports, we characterized four endo-xylanases produced by Streptomyces sp. strain SWU10 that degrade xylans to several xylooligosaccharides. To obtain a set of enzymes to achieve complete xylan degradation, a ß-d-xylosidase gene was cloned and expressed in Escherichia coli, and the recombinant protein, named rSWU43A, was characterized. SWU43A is composed of 522 amino acids and does not contain a signal peptide, indicating that the enzyme is an intracellular protein. SWU43A was revealed to contain a Glyco_hydro_43 domain and possess the three conserved amino acid residues of the glycoside hydrolase family 43 proteins. The molecular mass of rSWU43A purified by Ni-affinity column chromatography was estimated to be 60kDa. The optimum reaction conditions of rSWU43A were pH 6.5 and 40°C. The enzyme was stable up to 40°C over a wide pH range (3.1-8.9). rSWU43A activity was enhanced by Fe2+ and Mn2+ and inhibited by various metals (Ag+, Cd2+, Co2+, Cu2+, Hg2+, Ni2+, and Zn2+), d-xylose, and l-arabinose. rSWU43A showed activity on p-nitrophenyl-ß-d-xylopyranoside and p-nitrophenyl-α-l-arabinofuranoside substrates, with specific activities of 0.09 and 0.06U/mg, respectively, but not on any xylosidic or arabinosidic polymers. rSWU43A efficiently degraded ß-1,3-xylooligosaccharides to produce xylose but showed little activity towards ß-1,4-xylobiose, with specific activities of 1.33 and 0.003U/mg, respectively. These results demonstrate that SWU43A is a ß-1,3-d-xylosidase (EC 3.2.1.72), which to date has only been described in the marine bacterium Vibrio sp. Therefore, rSWU43A of Streptomyces sp. is the first ß-1,3-xylosidase found in gram-positive bacteria. SWU43A could be useful as a specific tool for the structural elucidation and production of xylose from ß-1,3-xylan in seaweed cell walls.