Within-host evolution of a gut pathobiont facilitates liver translocation.

Gut commensal bacteria with the ability to translocate across the intestinal barrier can drive the development of diverse immune-mediated diseases1-4. However, the key factors that dictate bacterial translocation remain unclear. Recent studies have revealed that gut microbiota strains can adapt and evolve throughout the lifetime of the host5-9, raising the possibility that changes in individual commensal bacteria themselves over time may affect their propensity to elicit inflammatory disease. Here we show that within-host evolution of the model gut pathobiont Enterococcus gallinarum facilitates bacterial translocation and initiation of inflammation. Using a combination of in vivo experimental evolution and comparative genomics, we found that E. gallinarum diverges into independent lineages adapted to colonize either luminal or mucosal niches in the gut. Compared with ancestral and luminal E. gallinarum, mucosally adapted strains evade detection and clearance by the immune system, exhibit increased translocation to and survival within the mesenteric lymph nodes and liver, and induce increased intestinal and hepatic inflammation. Mechanistically, these changes in bacterial behaviour are associated with non-synonymous mutations or insertion-deletions in defined regulatory genes in E. gallinarum, altered microbial gene expression programs and remodelled cell wall structures. Lactobacillus reuteri also exhibited broadly similar patterns of divergent evolution and enhanced immune evasion in a monocolonization-based model of within-host evolution. Overall, these studies define within-host evolution as a critical regulator of commensal pathogenicity that provides a unique source of stochasticity in the development and progression of microbiota-driven disease.

Lactobacillus rhamnosus Affects Rat Peritoneal Cavity Cell Response to Stimulation with Gut Microbiota: Focus on the Host Innate Immunity.

Gut microbiota contribute to shaping the immune repertoire of the host, whereas probiotics may exert beneficial effects by modulating immune responses. Having in mind the differences in both the composition of gut microbiota and the immune response between rats of Albino Oxford (AO) and Dark Agouti (DA) rat strains, we investigated if intraperitoneal (i.p.) injection of live Lactobacillus rhamnosus (LB) may influence peritoneal cavity cell response to in vitro treatments with selected microbiota in the rat strain-dependent manner. Peritoneal cavity cells from AO and DA rats were lavaged two (d2) and seven days (d7) following i.p. injection with LB and tested for NO, urea, and H2O2 release basally, or upon in vitro stimulation with autologous E.coli and Enterococcus spp. Whereas the single i.p. injection of LB nearly depleted resident macrophages and increased the proportion of small inflammatory macrophages and monocytes on d2 in both rat strains, greater proportion of MHCIIhiCD163- and CCR7+ cells and increased NO/diminished H2O2 release in DA compared with AO rats suggest a more intense inflammatory priming by LB in this rat strain. Even though E.coli- and/or Enterococcus spp.-induced rise in H2O2 release in vitro was abrogated by LB in cells from both rat strains, LB prevented microbiota-induced increase in NO/urea ratio only in cells from AO and augmented it in cells from DA rats. Thus, the immunomodulatory properties may not be constant for particular probiotic bacteria, but shaped by innate immunity of the host.

Immune Response to Enterococcus gallinarum in Lupus Patients Is Associated With a Subset of Lupus-Associated Autoantibodies.

Interactions between gut microbes and the immune system influence autoimmune disorders like systemic lupus erythematosus (SLE). Recently, Enterococcus gallinarum, a gram-positive commensal gut bacterium, was implicated as a candidate pathobiont in SLE. The present study was undertaken to evaluate the influence of E. gallinarum exposure on clinical parameters of SLE. Since circulating IgG antibodies to whole bacteria have been established as a surrogate marker for bacterial exposure, anti-E. gallinarum IgG antibodies were measured in banked serum samples from SLE patients and healthy controls in the Oklahoma Cohort for Rheumatic Diseases. The associations between anti-E. gallinarum antibody titers and clinical indicators of lupus were studied. Antibodies to human RNA were studied in a subset of patients. Our results show that sera from both patients and healthy controls had IgG and IgA antibodies reactive with E. gallinarum. The antibody titers between the two groups were not different. However, SLE patients with Ribosomal P autoantibodies had higher anti-E. gallinarum IgG titers compared to healthy controls. In addition to anti-Ribosomal P, higher anti-E. gallinarum titers were also significantly associated with the presence of anti-dsDNA and anti-Sm autoantibodies. In the subset of patients with anti-Ribosomal P and anti-dsDNA, the anti-E. gallinarum titers correlated significantly with antibodies to human RNA. Our data show that both healthy individuals and SLE patients were sero-reactive to E. gallinarum. In SLE patients, the immune response to E. gallinarum was associated with antibody response to a specific subset of lupus autoantigens. These findings provide additional evidence that E. gallinarum may be a pathobiont for SLE in susceptible individuals.

Advances and Prospects in Vaccine Development against Enterococci.

Enterococci are the second most common Gram-positive pathogen responsible for nosocomial infections. Due to the limited number of new antibiotics that reach the medical practice and the resistance of enterococci to the current antibiotic options, passive and active immunotherapies have emerged as a potential prevention and/or treatment strategy against this opportunistic pathogen. In this review, we explore the pathogenicity of these bacteria and their interaction with the host immune response. We provide an overview of the capsular polysaccharides and surface-associated proteins that have been described as potential antigens in anti-enterococcal vaccine formulations. In addition, we describe the current status in vaccine development against enterococci and address the importance and the current advances toward the development of well-defined vaccines with broad coverage against enterococci.

Development of an in-house ELISA for detection of antibodies against Enterococcus cecorum in Pekin ducks.

Enterococcus cecorum (EC) is known to cause skeletal lesions in broiler chickens and also systemic infections in Pekin ducks. Despite the importance of the pathogen, there is still a lack of serological diagnostic tools for the detection of EC infections. Here we describe the development of an in-house indirect enzyme-linked immunosorbent assay (ELISA) for the detection of EC-specific antibodies and its application by examination of 67 sera from experimentally infected Pekin ducks, 710 field samples from four Pekin duck breeder flocks previously vaccinated with inactivated vaccines, and 80 samples from commercial Pekin ducks coming from vaccinated parent flocks. All groups that had been experimentally inoculated via the air sac route were positive in the new ELISA, with significantly (P ≤ 0.05) increased mean sample/positive (S/P) ratios of 0.71-2.70 at days 7, 14 and 21 post-infection, while orally inoculated ducks and the EC-free control group remained negative with mean S/P ratios of 0.0-0.15. Antibodies were also detected in each of four vaccinated Pekin duck breeder flocks; 67.8% of the samples were antibody positive. The highest S/P ratios were found between 16 and 26 weeks (median S/P ratios from 0.15 to 1.03), but antibodies were still detected in some serum samples in weeks 61-67 post-hatch. No antibodies were detected in the commercial Pekin ducks. Antibody development in the ducks may be influenced by the composition of the inactivated vaccine. The new ELISA provides a useful tool for investigations of response to EC infections and vaccinations.

Mechanisms and consequences of gut commensal translocation in chronic diseases.

Humans and other mammalian hosts have evolved mechanisms to control the bacteria colonizing their mucosal barriers to prevent invasion. While the breach of barriers by bacteria typically leads to overt infection, increasing evidence supports a role for translocation of commensal bacteria across an impaired gut barrier to extraintestinal sites in the pathogenesis of autoimmune and other chronic, non-infectious diseases. Whether gut commensal translocation is a cause or consequence of the disease is incompletely defined. Here we discuss factors that lead to translocation of live bacteria across the gut barrier. We expand upon our recently published demonstration that translocation of the gut pathobiont Enterococcus gallinarum can induce autoimmunity in susceptible hosts and postulate on the role of Enterococcus species as instigators of chronic, non-infectious diseases.

Impact of enterococcal urinary tract infections in immunocompromised - neoplastic patients.

Infections in immunocompromised-neoplastic patients represent a severe complication. Among bacteria, Enterococcus species constitute a common causative pathogen of urinary tract infections (UTIs), especially among hospitalized patients with or without urinary tract carcinoma, related commonly to urinary tract abnormalities, urinary catheters or prolonged antibiotic treatment. Although enterococci have been considered more commonly as colonization bacteria in the intestine than virulent agents, they are frequently implicated in UTIs. The high incidence of enterococcal UTIs is associated with several risk factors including age, female gender, previous UTI, diabetes, pregnancy, immunosuppression due to cancer development and progression, renal transplantation and spinal cord injury. Clinical manifestations are usually absent or mild in enterococcal UTIs, which may also become an important source for both bacteremia and endocarditis. Over the last years, the prevalence of multidrug resistant enterococci, particularly vancomycin-resistant E. faecium and E. faecalis has significantly risen worldwide, associated with increased morbidity, limited treatment options and increased health-care costs. In this review, the current knowledge on enterococcal UTIs epidemiology and influence in the corresponding immunocompromised patients is highlighted.

The flagellin of candidate live biotherapeutic Enterococcus gallinarum MRx0518 is a potent immunostimulant.

Many links between gut microbiota and disease development have been established in recent years, with particular bacterial strains emerging as potential therapeutics rather than causative agents. In this study we describe the immunostimulatory properties of Enterococcus gallinarum MRx0518, a candidate live biotherapeutic with proven anti-tumorigenic efficacy. Here we demonstrate that strain MRx0518 elicits a strong pro-inflammatory response in key components of the innate immune system but also in intestinal epithelial cells. Using a flagellin knock-out derivative and purified recombinant protein, MRx0518 flagellin was shown to be a TLR5 and NF-κB activator in reporter cells and an inducer of IL-8 production by HT29-MTX cells. E. gallinarum flagellin proteins display a high level of sequence diversity and the flagellin produced by MRx0518 was shown to be more potent than flagellin from E. gallinarum DSM100110. Collectively, these data infer that flagellin may play a role in the therapeutic properties of E. gallinarum MRx0518.

Vaccination of breeder hens with a polyvalent killed vaccine for pathogenic Enterococcus cecorum does not protect offspring from enterococcal spondylitis.

Pathogenic strains of Enterococcus cecorum cause symmetrical paralysis in broilers due to infection of the free thoracic vertebra. The disease caused by pathogenic E. cecorum, known as enterococcal spondylitis or "kinky-back" continues to be responsible for significant losses to the broiler industry worldwide. In outbreaks of pathogenic E. cecorum, gut colonization and sepsis occur in the first three weeks-of-life. Since maternal antibodies are present during this period, we postulated that vaccination of breeders with a polyvalent killed vaccine would protect chicks from challenge. To test this hypothesis, representative isolates from seven genotype groups of pathogenic E. cecorum circulating in the US were chosen to produce adjuvanted killed vaccines (bacterins) and given to broiler-breeder hens. No single strain produced high titres of antibodies to all other strains; however, the combination of serologic reactivity of pathogenic isolates (designated SA3 and SA7) was sufficient to react with all genotypes. Vaccination of commercial broiler-breeder hens with a bacterin composed of SA3 and SA7 did not have any adverse effects. Vaccinated hens developed E. cecorum specific antibodies; however, no significant difference in survival was observed in infected embryos from hens in vaccine or adjuvant only groups. Chicks from vaccinated hens also failed to resist homologous or heterologous challenge during experimental infection. In a macrophage killing assay, pathogenic E. cecorum were found to evade opsinophagocytosis with elicited antibodies. These data suggest that pathogenic strains of E. cecorum possess virulence mechanisms that confound antibody-mediated opsinophagocytosis, complicating vaccine development for this pathogen of broilers.

Possible involvement of Enterococcus infection in the pathogenesis of chronic pancreatitis and cancer.

(Aim) Bacterial infection underlies the pathogenesis of many human diseases, including acute and chronic inflammation. Here, we investigated a possible role for bacterial infection in the progression of chronic pancreatitis. (Materials and Methods) Pancreatic juice was obtained from patients with pancreatic cancer (n = 20) or duodenal cancer/bile duct cancer (n = 16) and subjected to PCR using universal primers for the bacterial 16S ribosomal RNA gene. Bacterial species were identified by PCR using bile samples from four pancreatic cancer patients. PCR products were subcloned into T-vectors, and the sequences were then analyzed. Immunohistochemical and serologic analyses for Enterococcus faecalis infection were performed on a large cohort of healthy volunteers and patients with chronic pancreatitis or pancreatic cancer and on mice with caerulein-induced chronic pancreatitis. The effect of E. faecalis antigens on cytokine secretion by pancreatic cancer cells was also investigated. (Results) We found that 29 of 36 pancreatic juice samples were positive for bacterial DNA. Enterococcus and Enterobacter species were detected primarily in bile, which is thought to be a pathway for bacterial infection of the pancreas. Enterococcus faecalis was also detected in pancreatic tissue from chronic pancreatitis and pancreatic cancer patients; antibodies to E. faecalis capsular polysaccharide were elevated in serum from chronic pancreatitis patients. Enterococcus-specific antibodies and pancreatic tissue-associated E. faecalis were detected in mice with caerulein-induced chronic pancreatitis. Addition of Enterococcus lipoteichoic acid and heat-killed bacteria induced expression of pro-fibrotic cytokines by pancreatic cancer cells in vitro. (Conclusion) Infection with E. faecalis may be involved in chronic pancreatitis progression, ultimately leading to development of pancreatic cancer.

Translocation of a gut pathobiont drives autoimmunity in mice and humans.

Despite multiple associations between the microbiota and immune diseases, their role in autoimmunity is poorly understood. We found that translocation of a gut pathobiont, Enterococcus gallinarum, to the liver and other systemic tissues triggers autoimmune responses in a genetic background predisposing to autoimmunity. Antibiotic treatment prevented mortality in this model, suppressed growth of E. gallinarum in tissues, and eliminated pathogenic autoantibodies and T cells. Hepatocyte-E. gallinarum cocultures induced autoimmune-promoting factors. Pathobiont translocation in monocolonized and autoimmune-prone mice induced autoantibodies and caused mortality, which could be prevented by an intramuscular vaccine targeting the pathobiont. E. gallinarum-specific DNA was recovered from liver biopsies of autoimmune patients, and cocultures with human hepatocytes replicated the murine findings; hence, similar processes apparently occur in susceptible humans. These discoveries show that a gut pathobiont can translocate and promote autoimmunity in genetically predisposed hosts.

Rat strain differences in peritoneal immune cell response to selected gut microbiota: A crossroad between tolerance and autoimmunity?

AIMS: Some gut commensals can be protective, whereas others are implicated as necessary for development of inflammatory/autoimmune diseases. Peritoneal immune cells may play an important role in promoting autoimmunity in response to gut microbiota. This study investigated the phenotype and the function of peritoneal immune cells in the autoimmunity-resistant Albino Oxford (AO), and the autoimmunity-prone Dark Agouti (DA) rat strains upon stimulation with their own colonic E. coli or Enterococcus. MAIN METHODS: Rats were intraperitoneally injected with their own E. coli or Enterococcus. Peritoneal cells isolated two days later were tested for nitric oxide (NO) and cytokine production, and for arginase and myeloperoxidase (MPO) activity. The phenotype of cells was determined using flow cytometry. KEY FINDINGS: While the Enterococcus injection did not affect the composition of peritoneal cells in AO rats, the E. coli treatment increased the percentages of activated CD11bintHIS48hi neutrophils, and decreased the proportion of resident (CD11bhiHIS48int/low, CD163 + CD86+) and anti-inflammatory CD68 + CD206+ macrophages. E. coli increased the production of NO and urea, but preserved their ratio in cells from AO rats. Conversely, both E. coli and Enterococcus diminished the proportion of resident and anti-inflammatory macrophages, increased the proportion of activated neutrophils, and induced inflammatory polarization of peritoneal cells in DA rats. However, injection of E. coli maintained the ratio of typical CD11bintHIS48int neutrophils in DA rats, which correlated with the sustained MPO activity. SIGNIFICANCE: The rat strain differences in peritoneal cell response to own commensal microbiota may contribute to differential susceptibility to inflammatory/autoimmune diseases.

Differential sensitivity to infections and antimicrobial peptide-mediated immune response in four silkworm strains with different geographical origin.

The domesticated silkworm Bombyx mori has an innate immune system, whose main effectors are the antimicrobial peptides (AMPs). Silkworm strains are commonly grouped into four geographical types (Japanese, Chinese, European and Tropical) and are generally characterised by a variable susceptibility to infections. To clarify the genetic and molecular mechanisms on which the different responses to infections are based, we exposed one silkworm strain for each geographical area to oral infections with the silkworm pathogens Enterococcus mundtii or Serratia marcescens. We detected a differential susceptibility to both bacteria, with the European strain displaying the lowest sensitivity to E. mundtii and the Indian one to S. marcescens. We found that all the strains were able to activate the AMP response against E. mundtii. However, the highest tolerance of the European strain appeared to be related to the specific composition of its AMP cocktail, containing more effective variants such as a peculiar Cecropin B6 isoform. The resistance of the Indian strain to S. marcescens seemed to be associated with its prompt capability to activate the systemic transcription of AMPs. These data suggest that B. mori strains with distinct genetic backgrounds employ different strategies to counteract bacterial infections, whose efficacy appears to be pathogen-dependent.

Antibody-Based Therapy for Enterococcal Catheter-Associated Urinary Tract Infections.

Gram-positive bacteria in the genus Enterococcus are a frequent cause of catheter-associated urinary tract infection (CAUTI), a disease whose treatment is increasingly challenged by multiantibiotic-resistant strains. We have recently shown that E. faecalis uses the Ebp pilus, a heteropolymeric surface fiber, to bind the host protein fibrinogen as a critical step in CAUTI pathogenesis. Fibrinogen is deposited on catheters due to catheter-induced inflammation and is recognized by the N-terminal domain of EbpA (EbpANTD), the Ebp pilus's adhesin. In a murine model, vaccination with EbpANTD confers significant protection against CAUTI. Here, we explored the mechanism of protection using passive transfer of immune sera to show that antisera blocking EbpANTD-fibrinogen interactions not only is prophylactic but also can act therapeutically to reduce bacterial titers of an existing infection. Analysis of 55 clinical CAUTI, bloodstream, and gastrointestinal isolates, including E. faecalis, E. faecium, and vancomycin-resistant enterococci (VRE), revealed a diversity of levels of EbpA expression and fibrinogen-binding efficiency in vitro Strikingly, analysis of 10 strains representative of fibrinogen-binding diversity demonstrated that, irrespective of EbpA levels, EbpANTD antibodies were universally protective. The results indicate that, despite diversity in levels of fibrinogen binding, strategies that target the disruption of EbpANTD-fibrinogen interactions have considerable promise for treatment of CAUTI. IMPORTANCE: Urinary catheterization is a routine medical procedure, and it has been estimated that 30 million Foley catheters are used annually in the United States. Importantly, placement of a urinary catheter renders the patient susceptible to developing a catheter-associated urinary tract infection, accounting for 1 million cases per year. Additionally, these infections can lead to serious complications, including bloodstream infection and death. Enterococcus strains are a common cause of these infections, and management of enterococcal infections has been more difficult in recent years due to the development of antibiotic resistance and the ability of strains to disseminate, resulting in a major threat in hospital settings. In this study, we developed an antibiotic-sparing treatment that is effective against diverse enterococcal isolates, including vancomycin-resistant enterococci, during catheter-associated urinary tract infections.

In vitro model of production of antibodies; a new approach to reveal the presence of key bacteria in polymicrobial environments.

BACKGROUND: There is a rapid emergence of multiple resistant gram-negative bacteria due to overuse of antibiotics in the treatment of infections. Biofilms consist of polymicrobial communities that survive the host's defense system. The key bacteria in biofilms are slow growing and support an attachment and rapid growth of other microorganisms. Current antimicrobial strategies often fail due to poor diagnosis of key pathogens in biofilms. The study aims to develop anti-bacterial human antibodies in vitro from patients who had recently undergone a systemic infection by pathogenic bacteria and to use these antibodies as a tool for detecting bacteria in biofilms. METHODS: Lymphocytes were separated from whole blood of patients (n = 10) and stimulated with heat-killed bacteria to produce antibodies in vitro. The specificity of antibodies in recognizing the bacteria against which they were directed was evaluated by surface plasmon resonance system (SPR) and electron microscopy. The ulcer secretions from patients with chronic and acute leg ulcers and healthy controls were analyzed by the SPR system and the results were compared with culture studies. RESULTS: The produced antibodies recognized bacteria with high sensitivity (SPR). The antibodies against Enterococcus fecalis bound specifically to the microorganism in a bacterial co-culture that was visualized by electron microscopy. CONCLUSION: In the present work, a method for producing specific antibodies against bacteria is introduced to recognize bacterial components in body fluids of patients suffering from pathogenic biofilms. This diagnostic technique may be most useful in clinical microbiology and in the choice of antibiotics in the treatment of serious infections.

Host-derived probiotics Enterococcus casseliflavus improves resistance against Streptococcus iniae infection in rainbow trout (Oncorhynchus mykiss) via immunomodulation.

The present study evaluated the benefits of dietary administration of host-derived candidate probiotics Enterococcus casseliflavus in juvenile rainbow trout Oncorhynchus mykiss. Experimental diets were prepared by incorporating the microorganisms in the basal feed at 3 inclusion levels (i.e. 10(7) CFU g(-1) of feed [T1], 10(8) CFU g(-1) of feed [T2], 10(9) CFU g(-1) of feed [T3]). The probiotic feeds were administered for 8 weeks, with a group fed with the basal diet serving as control. The effects on growth performance, gut health, innate immunity and disease resistance were evaluated. Results showed that growth performance parameters were significantly improved in T2 and T3 groups. Activities of digestive enzymes such as trypsin and lipase were significantly higher in these two groups as well. Gut micro-ecology was influenced by probiotic feeding as shown by the significant increase in intestinal lactic acid bacteria and total viable aerobic counts in T2 and T3. Humoral immunity was impacted by dietary probiotics as total serum protein and albumin were significantly elevated in T3. The levels of serum IgM significantly increased in all probiotic fed groups at week 8; with the T3 group registering the highest increment. Respiratory burst activity of blood leukocytes were significantly improved in T2 and T3. Hematological profiling further revealed that neutrophil counts significantly increased in all probiotic fed groups. Challenge test showed that probiotic feeding significantly improved host resistance to Streptococcus iniae infection, specifically in T2 and T3 where a considerable modulation of immune responses was observed. Taken together, this study demonstrated E. casseliflavus as a potential probiotics for rainbow trout with the capability of improving growth performance and enhancing disease resistance by immunomodulation.

M2b macrophage elimination and improved resistance of mice with chronic alcohol consumption to opportunistic infections.

Alcohol abuse was found to predispose persons to opportunistic infections. In this study, we tried to improve the host antibacterial resistance of chronic alcohol-consuming (CAC) mice to opportunistic infections. Bactericidal macrophages with functions to produce IL-12 and to express mRNAs for CXCL9 and inducible nitric oxide synthase (M1 macrophages) were characterized as the main effector cells in host antibacterial innate immunities against infections with opportunistic pathogens. However, CAC mice were found to be carriers of M2b macrophages [macrophages with functions to produce IL-10 and to express mRNAs for CD163, chemokine ligand (CCL)1, and LIGHT (homologous to lymphotoxin, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for high-voltage electron microscopy on T cells)], which were inhibitory on macrophage conversion from resident macrophages to M1 macrophages. Under treatment with CCL1 antisense oligodeoxynucleotides, a specific inhibitor of M2b macrophages, CAC mouse macrophages reverted to resident macrophages, and M1 macrophages were induced by a bacterial antigen from macrophages of CAC mice that were previously treated with the oligodeoxynucleotides. Opportunistic infections (enterococcal translocation and Klebsiella pneumonia) in CAC mice were completely controlled by CCL1 antisense oligodeoxynucleotides. These results indicate that certain opportunistic infections in alcoholics are controllable through the modulation of M2b macrophages.

Differential interleukin-10 (IL-10) and IL-23 production by human blood monocytes and dendritic cells in response to commensal enteric bacteria.

Human peripheral blood contains antigen-presenting cells (APC), including dendritic cells (DC) and monocytes, that may encounter microbes that have translocated from the intestine to the periphery in disease states like HIV-1 infection and inflammatory bowel disease. We investigated the response of DC and monocytes in peripheral blood mononuclear cells (PBMC) to a panel of representative commensal enteric bacteria, including Escherichia coli, Enterococcus sp., and Bacteroides fragilis. All three bacteria induced significant upregulation of the maturation and activation markers CD40 and CD83 on myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC). However, only mDC produced cytokines, including interleukin-10 (IL-10), IL-12p40/70, and tumor necrosis factor alpha (TNF-α), in response to bacterial stimulation. Cytokine profiles in whole PBMC differed depending on the stimulating bacterial species: B. fragilis induced production of IL-23, IL-12p70, and IL-10, whereas E. coli and Enterococcus induced an IL-10-predominant response. mDC and monocyte depletion experiments indicated that these cell types differentially produced IL-10 and IL-23 in response to E. coli and B. fragilis. Bacteroides thetaiotaomicron did not induce levels of IL-23 similar to those of B. fragilis, suggesting that B. fragilis may have unique proinflammatory properties among Bacteroides species. The addition of recombinant human IL-10 to PBMC cultures stimulated with commensal bacteria abrogated the IL-23 response, whereas blocking IL-10 significantly enhanced IL-23 production, suggesting that IL-10 controls the levels of IL-23 produced. These results indicate that blood mDC and monocytes respond differentially to innate stimulation with whole commensal bacteria and that IL-10 may play a role in controlling the proinflammatory response to translocated microbes.

Tolerance to lipopolysaccharide promotes an enhanced neutrophil extracellular traps formation leading to a more efficient bacterial clearance in mice.

Tolerance to lipopolysaccharide (LPS) constitutes a stress adaptation, in which a primary contact with LPS results in a minimal response when a second exposure with the same stimulus occurs. However, active important defence mechanisms are mounted during the tolerant state. Our aim was to assess the contribution of polymorphonuclear neutrophils (PMN) in the clearance of bacterial infection in a mouse model of tolerance to LPS. After tolerance was developed, we investigated in vivo different mechanisms of bacterial clearance. The elimination of a locally induced polymicrobial challenge was more efficient in tolerant mice both in the presence or absence of local macrophages. This was related to a higher number of PMN migrating to the infectious site as a result of an increased number of PMN from the marginal pool with higher chemotactic capacity, not because of differences in their phagocytic activity or reactive species production. In vivo, neutrophils extracellular trap (NET) destruction by nuclease treatment abolished the observed increased clearance in tolerant but not in control mice. In line with this finding, in vitro NETs formation was higher in PMN from tolerant animals. These results indicate that the higher chemotactic response from an increased PMN marginal pool and the NETs enhanced forming capacity are the main mechanisms mediating bacterial clearance in tolerant mice. To sum up, far from being a lack of response, tolerance to LPS causes PMN priming effects which favour distant and local anti-infectious responses.