Bacteroidetes Species Are Correlated with Disease Activity in Ulcerative Colitis.

Fecal microbiota transplantation following triple-antibiotic therapy (amoxicillin/fosfomycin/metronidazole) improves dysbiosis caused by reduced Bacteroidetes diversity in patients with ulcerative colitis (UC). We investigated the correlation between Bacteroidetes species abundance and UC activity. Fecal samples from 34 healthy controls and 52 patients with active UC (Lichtiger's clinical activity index ≥5 or Mayo endoscopic subscore ≥1) were subjected to next-generation sequencing with HSP60 as a target in bacterial metagenome analysis. A multiplex gene expression assay using colonoscopy-harvested mucosal tissues determined the involvement of Bacteroidetes species in the mucosal immune response. In patients with UC, six Bacteroides species exhibited significantly lower relative abundance, and twelve Bacteroidetes species were found significantly correlated with at least one metric of disease activity. The abundance of five Bacteroidetes species (Alistipes putredinis, Bacteroides stercoris, Bacteroides uniformis, Bacteroides rodentium, and Parabacteroides merdae) was correlated with three metrics, and their cumulative relative abundance was strongly correlated with the sum of Mayo endoscopic subscore (R = -0.71, p = 2 × 10-9). Five genes (TARP, C10ORF54, ITGAE, TNFSF9, and LCN2) associated with UC pathogenesis were expressed by the 12 key species. The loss of key species may exacerbate UC activity, serving as potential biomarkers.

Emergence and Spread of Carbapenem-Resistant and Aminoglycoside-Panresistant Enterobacter cloacae Complex Isolates Coproducing NDM-Type Metallo-ß-Lactamase and 16S rRNA Methylase in Myanmar.

Surveillance of 10 hospitals and a regional public health laboratory in Myanmar identified 31 isolates of carbapenem-resistant Enterobacter cloacae complex harboring blaNDM-type Of these isolates, 19 were highly resistant to aminoglycosides and harbored one or more genes encoding 16S rRNA methylases, including armA, rmtB, rmtC, and/or rmtE Of the 19 isolates, 16 were Enterobacter xiangfangensis ST200, with armA on the chromosome and a plasmid harboring blaNDM-1 and rmtC, indicating that these isolates were clonally disseminated nationwide in Myanmar.IMPORTANCE The emergence of multidrug-resistant E. cloacae complex has become a public health threat worldwide. E. xiangfangensis is a recently classified species belonging to E. cloacae complex. Here, we report a clonal dissemination of multidrug-resistant E. xiangfangensis ST200 producing two types of New Delhi metallo-ß-lactamase (NDM-type MBL), NDM-1 and -4, and three types of 16S rRNA methylases, ArmA, RmtC, and RmtE, in hospitals in Myanmar. The observation of these multidrug-resistant E. xiangfangensis ST200 isolates stresses the urgency to continue molecular epidemiological surveillance of these pathogens in Myanmar and in South Asian countries.

Rifaximin prevents ethanol-induced liver injury in obese KK-Ay mice through modulation of small intestinal microbiota signature.

Exacerbation of alcoholic hepatitis (AH) with comorbid metabolic syndrome is an emerging clinical problem, where microbiota plays a profound role in the pathogenesis. Here, we investigated the effect of rifaximin (RFX) on liver injury following chronic-binge ethanol (EtOH) administration in KK-Ay mice, a rodent model of metabolic syndrome. Female, 8-wk-old KK-Ay mice were fed Lieber-DeCarli diet (5% EtOH) for 10 days, following a single EtOH gavage (4 g/kg body wt). Some mice were given RFX (0.1 g/L, in liquid diet) orally. Small intestinal contents were collected from mice without binge. Intestinal microbiota was quantified using aerobic and anaerobic culturing techniques and further analyzed by 16S rRNA sequencing in detail. EtOH feeding/binge caused hepatic steatosis, oxidative stress, and induction of inflammatory cytokines in KK-Ay mice, which were markedly prevented by RFX treatment. Hepatic mRNA levels for cluster of differentiation 14, Toll-like receptor (TLR) 4, TLR2, and NADPH oxidase 2 were increased following EtOH feeding/binge, and administration of RFX completely suppressed their increase. The net amount of small intestinal bacteria was increased over threefold after chronic EtOH feeding as expected; however, RFX did not prevent this net increase. Intriguingly, the profile of small intestinal microbiota was dramatically changed following EtOH feeding in the order level, where the Erysipelotrichales predominated in the relative abundance. In sharp contrast, RFX drastically blunted the EtOH-induced increases in the Erysipelotrichales almost completely, with increased proportion of the Bacteroidales. In conclusion, RFX prevents AH through modulation of small intestinal microbiota/innate immune responses in obese KK-Ay mice.NEW & NOTEWORTHY Here we demonstrated that rifaximin (RFX) prevents chronic-binge ethanol (EtOH)-induced steatohepatitis in KK-Ay mice. Chronic EtOH feeding caused small intestinal bacterial overgrowth, with drastic alteration in the microbiota profile predominating the order Erysipelotrichales. RFX minimized this EtOH induction in Erysipelotrichales with substitutive increases in Bacteroidales. RFX also prevented EtOH-induced increases in portal lipopolysaccharide, and hepatic cluster of differentiation 14, toll-like receptor (TLR) 2, and TLR4 mRNA levels, suggesting the potential involvement of microbiota-related innate immune responses.

Characterization of novel anti-IL-26 neutralizing monoclonal antibodies for the treatment of inflammatory diseases including psoriasis.

Interleukin (IL)-26, known as a Th17 cytokine, acts on various cell types and has multiple biological functions. Although its precise role still remains to be elucidated, IL-26 is suggested to be associated with the pathology of diverse chronic inflammatory diseases such as psoriasis, inflammatory bowel diseases and rheumatoid arthritis. To develop novel neutralizing anti-human IL-26 monoclonal antibodies (mAbs) for therapeutic use in the clinical setting, we immunized mice with human IL-26 protein. Hybridomas producing anti-IL-26 mAbs were screened for various in vitro functional assays, STAT3 phosphorylation and antibiotic assays. Although the IL-20RA/IL-10RB heterodimer is generally believed to be the IL-26 receptor, our data strongly suggest that both IL-20RA-dependent and -independent pathways are involved in IL-26-mediated stimulation. We also investigated the potential therapeutic effect of anti-IL-26 mAbs in the imiquimod-induced psoriasis-like murine model using human IL-26 transgenic mice. These screening methods enabled us to develop novel neutralizing anti-human IL-26 mAbs. Importantly, administration of IL-26-neutralizing mAb did not have an effect on the antimicrobial activity of IL-26. Taken together, our data strongly suggest that our newly developed anti-human IL-26 mAb is a potential therapeutic agent for the treatment of diverse chronic inflammatory diseases including psoriasis.

Antimicrobial cathelicidin peptide LL­37 induces NET formation and suppresses the inflammatory response in a mouse septic model.

LL­37 is the only known member of the cathelicidin family of antimicrobial peptides in humans. In addition to its broad spectrum of antimicrobial activities, LL­37 may modulate various inflammatory reactions. The authors previously revealed that LL­37 improves the survival of a murine cecal ligation and puncture (CLP) sepsis model. In the present study, the mechanism for the protective action of LL­37 was elucidated using the CLP model, focusing on the effect of LL­37 on the release of neutrophil extracellular traps (NETs). The results indicated that the intravenous administration of LL­37 suppressed the increase of damage-associated molecular patterns (DAMPs), including histone­DNA complex and high­mobility group protein 1, in addition to interleukin­1ß, tumor necrosis­α and soluble triggering receptor expressed on myeloid cells (TREM)­1 in plasma and peritoneal fluids. Notably, LL­37 significantly suppressed the decrease of mononuclear cell number in blood, and the increase of polymorphonuclear cell (neutrophil) number in the peritoneal cavity during sepsis. Furthermore, LL­37 reduced the bacterial burden in blood and peritoneal fluids. Notably, LL­37 increased the level of NETs (myeloperoxidase­DNA complex) in plasma and peritoneal fluids. In addition, it was verified that LL­37 induces the release of NETs from neutrophils, and NETs possess the bactericidal activity. Overall, these observations suggest that LL­37 improves the survival of CLP septic mice by possibly suppressing the inflammatory responses as evidenced by the inhibition of the increase of cytokines, soluble TREM­1 and DAMPs (host cell death) and the alteration of inflammatory cell numbers, and bacterial growth via the release of NETs with bactericidal activity.

Evaluation of the effect of recombinant thrombomodulin on a lipopolysaccharide-induced murine sepsis model.

To evaluate the effect of recombinant human thrombomodulin (rTM) on sepsis, the levels of nucleosome as well as high-mobility group box 1 (HMGB1) and cytokines in sera and peritoneal fluids were measured in a mouse model of lipopolysaccharide (LPS)-induced sepsis after administration of rTM. C57BL/6 mice were intraperitoneally injected with LPS (15 mg/kg; Escherichia coli O111:B4) with or without the intravenous administration of rTM (3 mg/kg; 30 min prior to or 2 h after LPS injection). The survival rates were evaluated and levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, HMGB1 and nucleosome in sera and peritoneal fluids were analyzed by ELISA. Administration of rTM prior to or after LPS improved the survival rate of septic mice. In addition, rTM administered prior to or after LPS suppressed the level of pro-inflammatory cytokine TNF-α in sera at 1-3 h after LPS injection, whereas only the administration of rTM after LPS suppressed the levels of HMGB1 and nucleosome (late-phase mediators of sepsis) (9-12 h) in sera after the LPS injection. Furthermore, administration of rTM prior to or after LPS suppressed the level of TNF-α in the peritoneal fluids at 1-3 h after LPS injection, whereas only the administration of rTM after LPS suppressed the levels of IL-6 and MCP-1 in the peritoneal fluids at 6-9 h after LPS injection. These observations indicated that administration of rTM significantly improves the survival rate and suppresses the increased levels of TNF-α, IL-6, MCP-1, HMGB1 and nucleosome in the LPS-induced septic shock model. Thus, rTM may exert a protective action on sepsis and reduce mortality, possibly by reducing not only the levels of cytokines and chemokine but also the levels of late-phase mediators of sepsis.

Spleen-derived lipocalin-2 in the portal vein regulates Kupffer cells activation and attenuates the development of liver fibrosis in mice.

The liver has an immune tolerance against gut-derived products from the portal vein (PV). A disruption of the gut-liver axis leads to liver injury and fibrosis. The spleen is connected to the PV and regulates immune functions. However, possible splenic effects on liver fibrosis development are unclear. Lipocalin-2 (Lcn2) is an antimicrobial protein that regulates macrophage activation. To clarify the role of the spleen in liver fibrosis development, we induced liver fibrosis in mice after splenectomy, and investigated liver fibrosis development. Liver fibrosis resulted in significantly increased splenic Lcn2 levels, but all other measured cytokine levels were unchanged. Splenectomized mice showed enhanced liver fibrosis and inflammation accompanied by significantly decreased Lcn2 levels in PV. Lipopolysaccharide-stimulated primary Kupffer cells, resident liver macrophages, which were treated with recombinant Lcn2 (rLcn2) produced less tumor necrosis factor-α and Ccl2 and the activation of hepatic stellate cells, the effector cells for collagen production in the liver, was suppressed by co-culture with rLcn2-treated Kupffer cells. In addition, the involvement of gut-derived products in splenectomized mice was evaluated by gut sterilization. Interestingly, gut sterilization blocked the effect of splenectomy on liver fibrosis development. In conclusion, spleen deficiency accelerated liver fibrosis development and decreased PV Lcn2 levels. The mechanism of splenic protection against liver fibrosis development may involve the splenic Lcn2, triggered by gut-derived products that enter the liver through the PV, regulates Kupffer cells activated by the gut-liver axis. Thus, the splenic Lcn2 may have an important role in regulating the immune tolerance of the liver in liver fibrosis development.

Neutrophil extracellular traps induce IL-1ß production by macrophages in combination with lipopolysaccharide.

Upon exposure to invading microorganisms, neutrophils undergo NETosis, a recently identified type of programmed cell death, and release neutrophil extracellular traps (NETs). NETs are described as an antimicrobial mechanism, based on the fact that NETs can trap microorganisms and exhibit bactericidal activity through the action of NET­associated components. In contrast, the components of NETs have been recognized as damage­associated molecular pattern molecules (DAMPs), which trigger inflammatory signals to induce cell death, inflammation and organ failure. In the present study, to clarify the effect of NETs on cytokine production by macrophages, mouse macrophage­like J774 cells were treated with NETs in combination with lipopolysaccharide (LPS) as a constituent of pathogen­associated molecular patterns. The results revealed that NETs significantly induced the production of interleukin (IL)­1ß by J774 cells in the presence of LPS. Notably, the NET/LPS­induced IL­1ß production was inhibited by both caspase­1 and caspase­8 inhibitors. Furthermore, nucleases and serine protease inhibitors but not anti­histone antibodies significantly inhibited the NET/LPS­induced IL­1ß production. Moreover, we confirmed that caspase­1 and caspase­8 were activated by NETs/LPS, and the combination of LPS, DNA and neutrophil elastase induced IL­1ß production in reconstitution experiments. These observations indicate that NETs induce the production of IL­1ß by J774 macrophages in combination with LPS via the caspase­1 and caspase­8 pathways, and NET­associated DNA and serine proteases are involved in NET/LPS­induced IL­1ß production as essential components.

Changes in Intestinal Microbiota Following Combination Therapy with Fecal Microbial Transplantation and Antibiotics for Ulcerative Colitis.

BACKGROUND: Fecal microbiota transplantation (FMT) is a potential therapeutic approach to restore normal intestinal microbiota in patients with ulcerative colitis (UC), which is associated with dysbiosis; however, treatment efficacy remains unclear. Hence, we studied the impact of antibiotic pretreatment with amoxicillin, fosfomycin, and metronidazole (AFM therapy) and FMT versus AFM alone. METHODS: AFM therapy was administered to patients for 2 weeks until 2 days before FMT. Patients' spouses or relatives were selected as donor candidates. Donor fecal samples were collected on the day of administration and transferred into the patient's colon by colonoscopy within 6 hours. Microbiome analysis was performed by 16S rRNA next-generation sequencing. RESULTS: Patients with mild-to-severe active UC (combination-therapy group, n = 21; AFM monotherapy group, n = 20) were included. Thirty-six patients completed this assessment (combination-therapy group, n = 17; AFM monotherapy group, n = 19). A higher clinical response was observed after combination therapy compared with AFM monotherapy at 4 weeks after treatment. After the 2-week AFM therapy, the Bacteroidetes composition was nearly abolished. The Bacteroidetes proportion recovered in clinical responders at 4 weeks after FMT was not observed in the AFM monotherapy group. Persistent antimicrobial-associated dysbiosis found in the AFM monotherapy group was reversed by FMT. The recovery rate of Bacteroidetes at 4 weeks after FMT correlated with endoscopic severity. CONCLUSIONS: FMT following antimicrobial bowel cleansing synergistically contributes to the recovery of the Bacteroidetes composition, which is associated with clinical response and UC severity. Thus, this therapeutic protocol may be useful for managing UC.

Antimicrobial cathelicidin peptide LL-37 inhibits the pyroptosis of macrophages and improves the survival of polybacterial septic mice.

LL-37 is the only known member of the cathelicidin family of antimicrobial peptides in humans. In addition to its broad spectrum of antimicrobial activities, LL-37 can modulate various inflammatory reactions. We previously revealed that LL-37 suppresses the LPS/ATP-induced pyroptosis of macrophages in vitro by both neutralizing the action of LPS and inhibiting the response of P2X7 (a nucleotide receptor) to ATP. Thus, in this study, we further evaluated the effect of LL-37 on pyroptosis in vivo using a cecal ligation and puncture (CLP) sepsis model. As a result, the intravenous administration of LL-37 improved the survival of the CLP septic mice. Interestingly, LL-37 inhibited the CLP-induced caspase-1 activation and pyroptosis of peritoneal macrophages. Moreover, LL-37 modulated the levels of inflammatory cytokines (IL-1ß, IL-6 and TNF-α) in both peritoneal fluids and sera, and suppressed the activation of peritoneal macrophages (as evidenced by the increase in the intracellular levels of IL-1ß, IL-6 and TNF-α). Finally, LL-37 reduced the bacterial burdens in both peritoneal fluids and blood samples. Together, these observations suggest that LL-37 improves the survival of CLP septic mice by possibly suppressing the pyroptosis of macrophages, and inflammatory cytokine production by activated macrophages and bacterial growth. Thus, the present findings imply that LL-37 can be a promising candidate for sepsis because of its many functions, such as the inhibition of pyroptosis, modulation of inflammatory cytokine production and antimicrobial activity.

Antimicrobial cathelicidin peptide LL-37 inhibits the LPS/ATP-induced pyroptosis of macrophages by dual mechanism.

Pyroptosis is a caspase-1 dependent cell death, associated with proinflammatory cytokine production, and is considered to play a crucial role in sepsis. Pyroptosis is induced by the two distinct stimuli, microbial PAMPs (pathogen associated molecular patterns) and endogenous DAMPs (damage associated molecular patterns). Importantly, cathelicidin-related AMPs (antimicrobial peptides) have a role in innate immune defense. Notably, human cathelicidin LL-37 exhibits the protective effect on the septic animal models. Thus, in this study, to elucidate the mechanism for the protective action of LL-37 on sepsis, we utilized LPS (lipopolysaccharide) and ATP (adenosine triphosphate) as a PAMP and a DAMP, respectively, and examined the effect of LL-37 on the LPS/ATP-induced pyroptosis of macrophage-like J774 cells. The data indicated that the stimulation of J774 cells with LPS and ATP induces the features of pyroptosis, including the expression of IL-1ß mRNA and protein, activation of caspase-1, inflammasome formation and cell death. Moreover, LL-37 inhibits the LPS/ATP-induced IL-1ß expression, caspase-1 activation, inflammasome formation, as well as cell death. Notably, LL-37 suppressed the LPS binding to target cells and ATP-induced/P2X7-mediated caspase-1 activation. Together these observations suggest that LL-37 potently inhibits the LPS/ATP-induced pyroptosis by both neutralizing the action of LPS and inhibiting the response of P2X7 to ATP. Thus, the present finding may provide a novel insight into the modulation of sepsis utilizing LL-37 with a dual action on the LPS binding and P2X7 activation.

Antimicrobial cathelicidin polypeptide CAP11 suppresses the production and release of septic mediators in D-galactosamine-sensitized endotoxin shock mice.

Endotoxin shock is a severe systemic inflammatory response that is caused by the augmented production and release of septic mediators. Among them, inflammatory cytokines such as tumor necrosis factor-alpha, IL-1beta and IL-6 play a pivotal role. In addition, anandamide, an endogenous cannabinoid and high-mobility group box-1 (HMGB1), a non-histone chromosomal protein has recently been recognized as members of septic mediators. We previously reported that cationic antibacterial polypeptide of 11-kDa (CAP11), an antimicrobial cathelicidin peptide (originally isolated from guinea pig neutrophils), potently neutralizes the biological activity of LPS and protects mice from lethal endotoxin shock. In this study, to clarify the protective mechanism of CAP11 against endotoxin shock, we evaluated the effects of CAP11 on the production and release of septic mediators in vitro and in vivo using a murine macrophage cell line RAW264.7 and a D-galactosamine-sensitized murine endotoxin shock model. LPS stimulation induced the production of inflammatory cytokines and anandamide and release of HMGB1 from RAW264.7 cells. Importantly, CAP11 suppressed the LPS-induced production and release of these mediators by RAW264.7 cells. Moreover, LPS administration enhanced the serum levels of HMGB1, anandamide and inflammatory cytokines in the endotoxin shock model. Of note, CAP11 suppressed the LPS-induced increase of these mediators in sera, and LPS binding to CD14-positive cells (peritoneal macrophages), accompanied with the increase of survival rates. Together these observations suggest that the protective action of CAP11 on endotoxin shock may be explained by its suppressive effect on the production and release of septic mediators by CD14-positive cells possibly via the inhibition of LPS binding to the targets.

Augmentation of the antimicrobial activities of guinea pig cathelicidin CAP11-derived peptides by amino acid substitutions.

Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensin and cathelicidin) that contribute to the innate host defense against invading microorganisms. Among these, guinea pig cathelicidin CAP11 (G1-I43) possesses potent antibacterial activities against Gram-positive and -negative bacteria, and also lipopolysaccharide-neutralizing activity. We previously revealed that the active region with antibacterial activity is localized at G1 to R18 of CAP11. In this study, to develop peptide derivatives with enhanced antimicrobial actions, we utilized the amphipathic 18-mer peptide (G1-R18) as a template. Anti-microbial activities of the peptides were assessed by alamarBlue assay (Escherichia coli, Staphylococcus aureus and Candida albicans) and colony formation assay (Porphyromonas gingivalis). Furthermore, the membrane-permeabilization activities were determined by using E. coli ML-35p as a target. By substituting K5, T9, R10, R12, and G17 with five L residues, the hydrophobicity of the peptide (1-18m1) was increased, and by substituting G1, and Q14 with K and R residues, respectively, the hydrophilicity (positive charge) of the peptide (1-18m2) was enhanced. Among the peptides, 1-18m2 exhibits the most potent antimicrobial and membrane-permeabilizing activities against the microorganisms examined. Thus, the antimicrobial activities of the amphipathic CAP11-derived 18-mer peptide can be augmented by modifying its hydrophobicity and hydrophilicity (positive charge), and 1-18m2 is the most potent among the peptide derivatives with therapeutic potential for Gram-positive and -negative bacterial, and fungal infections.

Antibacterial cathelicidin peptide CAP11 suppresses the anandamide production from lipopolysaccharide-stimulated mononuclear phagocytes.

The action of antibacterial cathelicidin peptide CAP11 on the anandamide production from mononuclear phagocytes was examined. Lipopolysaccharide (LPS)-stimulation induced the anandamide production from macrophage-like RAW264.7, accompanied with the enhanced anandamide-synthesizing enzyme activity; however, the anandamide-degrading enzyme activity was not changed by LPS-stimulation. Importantly, CAP11 suppressed the LPS-induced anandamide production and the increase of anandamide-synthesizing enzyme activity. Furthermore, CAP11 abrogated the LPS-binding to CD14-positive RAW264.7. These observations indicate that CAP11 inhibits the binding of LPS to CD14-positive mononuclear phagocytes, thereby suppressing the anandamide synthesizing enzyme activity and the anandamide production from the cells.