Intestinal Microbiota Shifts towards Elevated Commensal Escherichia coli Loads Abrogate Colonization Resistance against Campylobacter jejuni in Mice

Background: The zoonotic pathogen Campylobacter jejuni is a leading cause of bacterial foodborne enterocolitis in humans worldwide. The understanding of immunopathology underlying human campylobacteriosis is hampered by the fact that mice display strong colonization resistance against the pathogen due to their host specific gut microbiota composition. Methodology/Principal Findings: Since the microbiota composition changes significantly during intestinal inflammation we dissected factors contributing to colonization resistance against C. jejuni in murine ileitis, colitis and in infant mice. In contrast to healthy animals C. jejuni could stably colonize mice suffering from intestinal inflammation. Strikingly, in mice with Toxoplasma gondii-induced acute ileitis, C. jejuni disseminated to mesenteric lymphnodes, spleen, liver, kidney, and blood. In infant mice C. jejuni infection induced enterocolitis. Mice suffering from intestinal inflammation and C. jejuni susceptible infant mice displayed characteristical microbiota shifts dominated by increased numbers of commensal Escherichia coli. To further dissect the pivotal role of those distinct microbiota shifts in abrogating colonization resistance, we investigated C. jejuni infection in healthy adult mice in which the microbiota was artificially modified by feeding live commensal E. coli. Strikingly, in animals harboring supra-physiological intestinal E. coli loads, colonization resistance was significantly diminished and C. jejuni infection induced enterocolitis mimicking key features of human campylobacteriosis. Conclusion/Significance: Murine colonization resistance against C. jejuni is abrogated by changes in the microbiot

Novel Murine Infection Models Provide Deep Insights into the “Ménage à Trois” of Campylobacter jejuni, Microbiota and Host Innate Immunity

BACKGROUND: Although Campylobacter jejuni-infections have a high prevalence worldwide and represent a significant socioeconomic burden, it is still not well understood how C. jejuni causes intestinal inflammation. Detailed investigation of C. jejuni-mediated intestinal immunopathology is hampered by the lack of appropriate vertebrate models. In particular, mice display colonization resistance against this pathogen. METHODOLOGY/PRINCIPAL FINDINGS: To overcome these limitations we developed a novel C. jejuni-infection model using gnotobiotic mice in which the intestinal flora was eradicated by antibiotic treatment. These animals could then be permanently associated with a complete human (hfa) or murine (mfa) microbiota. After peroral infection C. jejuni colonized the gastrointestinal tract of gnotobiotic and hfa mice for six weeks, whereas mfa mice cleared the pathogen within two days. Strikingly, stable C. jejuni colonization was accompanied by a pro-inflammatory immune response indicated by increased numbers of T- and B-lymphocytes, regulatory T-cells, neutrophils and apoptotic cells, as well as increased concentrations of TNF-α, IL-6, and MCP-1 in the colon mucosa of hfa mice. Analysis of MyD88(-/-), TRIF(-/-), TLR4(-/-), and TLR9(-/-) mice revealed that TLR4- and TLR9-signaling was essential for immunopathology following C. jejuni-infection. Interestingly, C. jejuni-mutant strains deficient in formic acid metabolism and perception induced less intestinal immunopathology compared to the parental strain infection. In summary, the murine gut flora is essential for colonization resistance against C. jejuni and can be overcome by reconstitution of gnotobiotic mice with human flora. Detection of C. jejuni-LPS and -CpG-DNA by host TLR4 and TLR9, respectively, plays a key role in immunopathology. Finally, the host immune response is tightly coupled to bacterial formic acid metabolism and invasion fitness. CONCLUSION/SIGNIFICANCE: We conclude that gnotobiotic and "humanized" mice represent excellent novel C. jejuni-infection and -inflammation models and provide deep insights into the immunological and molecular interplays between C. jejuni, microbiota and innate immunity in human campylobacteriosis

Anti-Inflammatory Effects of Resveratrol, Curcumin and Simvastatin in Acute Small Intestinal Inflammation

BACKGROUND: The health beneficial effects of Resveratrol, Curcumin and Simvastatin have been demonstrated in various experimental models of inflammation. We investigated the potential anti-inflammatory and immunomodulatory mechanisms of the above mentioned compounds in a murine model of hyper-acute Th1-type ileitis following peroral infection with Toxoplasma gondii. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that after peroral administration of Resveratrol, Curcumin or Simvastatin, mice were protected from ileitis development and survived the acute phase of inflammation whereas all Placebo treated controls died. In particular, Resveratrol treatment resulted in longer-term survival. Resveratrol, Curcumin or Simvastatin treated animals displayed significantly increased numbers of regulatory T cells and augmented intestinal epithelial cell proliferation/regeneration in the ileum mucosa compared to placebo control animals. In contrast, mucosal T lymphocyte and neutrophilic granulocyte numbers in treated mice were reduced. In addition, levels of the anti-inflammatory cytokine IL-10 in ileum, mesenteric lymph nodes and spleen were increased whereas pro-inflammatory cytokine expression (IL-23p19, IFN-γ, TNF-α, IL-6, MCP-1) was found to be significantly lower in the ileum of treated animals as compared to Placebo controls. Furthermore, treated animals displayed not only fewer pro-inflammatory enterobacteria and enterococci but also higher anti-inflammatory lactobacilli and bifidobacteria loads. Most importantly, treatment with all three compounds preserved intestinal barrier functions as indicated by reduced bacterial translocation rates into spleen, liver, kidney and blood. CONCLUSION/SIGNIFICANCE: Oral treatment with Resveratrol, Curcumin or Simvastatin ameliorates acute small intestinal inflammation by down-regulating Th1-type immune responses and prevents bacterial translocation by maintaining gut barrier function. These findings provide novel and potential prophylaxis and treatment options of patients with inflammatory bowel diseases

The role of the intestinal bacterial microflora and the receptors of the innate immune system in a new murine model of acute graft-versus-host-disease

Akute Graft-versus-Host-Erkrankung (GvHD) ist eine Hauptursache für Morbidität und Mortalität nach allogener Stammzelltransplantation. TLR- und NOD- Rezeptoren sind Komponenten des angeborenen Immunsystems, welche unvariable Merkmale mikrobieller Organismen erkennen. Der Detektion der Intestinalflora durch TLR- und NOD-Rezeptoren wird eine bedeutende Rolle bei der Induktion akuter GvHD beigemessen. Die Anwendung alternativer Konditionierungsansätze mit geringerer Wirkungsintensität (RIC – Reduced-Intensity-Conditioning) vor allogener Stammzelltransplantation konnte positive Auswirkungen auf das Auftreten und die Ausprägung akuter GvHD aufzeigen, dennoch sind die Hintergründe dieser veränderten Immunbiologie größtenteils unbekannt. In einem neu etablierten murinen „Reduced-Intensity“ Konditionierungsmodell haben wir die Intestinalflora durch kulturelle und molekulare Methoden untersucht und weiterhin histologische und in situ histochemische Analysen angefertigt. Die Transplantation im allogenen „Mismatch“ führte zum Versterben der Tiere ab Tag sechs nach Transplantation, zu einer intestinalen Immunreaktion und zum Auftreten pathohistologischer Läsionen im Gastrointestinaltrakt. Im Colon waren mehr Apoptosen, T-Zellen, proliferierenden Zellen und Neutrophile nachzuweisen. Gleichzeitig kam es durch die Akkumulation Enterobacteriaceae, gramnegativer anaerober Stäbchen, grampositiver aerober Kokken und einem Verlust bakterieller Diversität zu einer proinflammatorischen Veränderung der Colonflora. Entsprechende Florenverschiebungen konnten auch im Ileum und Jejunum von Wildtyptieren beobachtet werden. Die Analyse der TLR- und NOD2-defizienten Tiere erbrachte ein signifikant besseres Überleben der TLR9- und NOD2-defizienten Tiere. Diese Tiere hatten im Vergleich zu Wildtyptieren eine mildere Entzündungsreaktion im Magen und Colon, gekennzeichnet durch den geringeren Anstieg von T-Zellen und apoptotischen Zellen bei gleichzeitig verstärkter Regeneration im Colon. Die Analysen bei MyD88-/--Tieren bestätigten weitesgehend unserer Befunde bei TLR9-defizienten Tieren. Eine veränderte Immunreaktion auf die Transplantation war ebenfalls im Colon TLR2-, TLR4-, TLR2/4-, und TRIF-defizienter Tieren zu beobachten, führte aber nicht zu verbesserten Überlebensraten dieser Tiere. Die Verabreichung von MDP und einem TLR9-Antagonisten bei akuter GvHD resultierte im Colon in einem geringeren Anstieg von T-Zellen, neutrophilen und apoptotischen Zellen. Schlussfolgernd erlauben die vorliegenden Ergebnisse wertvolle Einblicke in die Immunbiologie akuter GvHD nach „Reduced-Intensity“ Konditionierung und bilden möglicherweise Grundlage für eine Erweiterung der therapeutischen und prophylaktischen Interventionsstrategien bei Patienten mit akuter GvHD.Acute graft-versus-host-disease (GvHD) is one of the major complications of allogeneic bone marrow transplantations. TL- and NOD Receptors are mediators of the innate immune system, which recognise molecular characteristics of the bacterial flora. The detection of the intestinal flora is considered to be one of the key factors in inducing intestinal GvHD. In the past, new conditioning regimens have been explored, trying to reduce the toxicity of the conventional conditioning regimen. However, the immunopathology of these new conditioning regimens remain poorly understood. In a new established Reduced-Intensity- Conditioning regimen we analysed changes in intestinal microbial flora by using cultural and molecular methods. Furthermore, we analysed intestinal inflammation by using hematoxylin / eosin and immunhistochemical staining. In acute intestinal GvHD, we found increasing numbers of apoptotic cells, proliferating cells, T cells and neutrophils in the colon. During inflammation we found a gut flora shift towards increased numbers of enterobacteria, enterococci and Bacteroides/ Prevotella spp. Similar changes within the intestinal flora were observed in ileum and jejunum of wildtype animals. After transplantation TLR9-/- and NOD2-/- mice displayed a reduced mortality rate. In addition, these animals had less intestinal inflammation in the stomach and colon, compared to wildtype controls. The reduction of the intestinal inflammation was accompanied by less infiltration of T cells and less apoptotic cells within the colon of these animals. TLR2-, TLR4-, TLR2/4- and TRIF- knockout mice also displayed some changes in the immunopathology. However, these animals were not protected from mortality. The application of MDP and a TLR9-antagonist during conditioning or after transplantation resulted in a reduction of T cells, neutrophils and apoptotic cells in the colon. The results give new insights into the immunopathology of acute GvHD after Reduced-Intensity-Conditioning and might be the basis for new therapeutic and prophylactic options for patients suffering from GvHD

Helicobacter pylori induced gastric immunopathology is associated with distinct microbiota changes in the large intestines of long-term infected Mongolian gerbils.

BACKGROUND: Gastrointestinal (GI) inflammation in mice and men are frequently accompanied by distinct changes of the GI microbiota composition at sites of inflammation. Helicobacter (H.) pylori infection results in gastric immunopathology accompanied by colonization of stomachs with bacterial species, which are usually restricted to the lower intestine. Potential microbiota shifts distal to the inflammatory process following long-term H. pylori infection, however, have not been studied so far. METHODOLOGY/PRINCIPAL FINDINGS: For the first time, we investigated microbiota changes along the entire GI tract of Mongolian gerbils after 14 months of infection with H. pylori B8 wildtype (WT) or its isogenic ΔcagY mutant (MUT) strain which is defective in the type IV secretion system and thus unable to modulate specific host pathways. Comprehensive cultural analyses revealed that severe gastric diseases such as atrophic pangastritis and precancerous transformations were accompanied by elevated luminal loads of E. coli and enterococci in the caecum and together with Bacteroides/Prevotella spp. in the colon of H. pylori WT, but not MUT infected gerbils as compared to naïve animals. Strikingly, molecular analyses revealed that Akkermansia, an uncultivable species involved in mucus degradation, was exclusively abundant in large intestines of H. pylori WT, but not MUT infected nor naïve gerbils. CONCLUSION/SIGNIFICANCE: Taken together, long-term infection of Mongolian gerbils with a H. pylori WT strain displaying an intact type IV secretion system leads to distinct shifts of the microbiota composition in the distal uninflamed, but not proximal inflamed GI tract. Hence, H. pylori induced immunopathogenesis of the stomach, including hypochlorhydria and hypergastrinemia, might trigger large intestinal microbiota changes whereas the exact underlying mechanisms need to be further unraveled

Colonic microbiota composition in Mongolian gerbils 14 months following <i>H. pylori</i> infection by culture.

<p>Fourteen months following oral infection of Mongolian gerbils with <i>H. pylori</i> wildtype strain B8 (B8; black circles) or <i>H. pylori</i> mutant strain lacking <i>cagY</i> (B8Δ<i>cagY</i>; grey circles), the microbiota composition of luminal <b>colon</b> contents were quantitatively analyzed by culture as described in Methods. Uninfected age-matched animals served as negative controls (Naïve; white circles). Numbers of live (<b>A</b>) <i>E. coli</i>, (<b>B</b>) <i>Proteus</i> sp., (<b>C</b>) Enterococci, (<b>D</b>) Lactobacilli, (<b>E</b>) <i>Bacteroides/Prevotella</i> spp. as well as the (<b>F</b>) total bacterial load are indicated as colony forming units (CFU) per g luminal content. Numbers of animals harboring the respective bacterial species are given in parentheses. Medians and significance levels (p<i>-</i>values) determined by Mann-Whitney-U test are indicated. Data shown were pooled from three independent experiments.</p

Jejunal microbiota composition in Mongolian gerbils 14 months following <i>H. pylori</i> infection.

<p>Fourteen months following oral infection of Mongolian gerbils with <i>H. pylori</i> wildtype strain B8 (B8; black circles) or <i>H. pylori</i> mutant strain lacking <i>cagY</i> (B8Δ<i>cagY</i>; grey circles), the microbiota composition of luminal <b>jejunum</b> contents were quantitatively analyzed by culture as described in Methods. Uninfected age-matched animals served as negative controls (Naïve; white circles). Numbers of live (<b>A</b>) <i>E. coli</i>, (<b>B</b>) <i>Proteus</i> sp., (<b>C</b>) Enterococci, (<b>D</b>) Lactobacilli, (<b>E</b>) <i>Bacteroides/Prevotella</i> spp. as well as the (<b>F</b>) total bacterial load are indicated as colony forming units (CFU) per g luminal content. Numbers of animals harboring the respective bacterial species are given in parentheses and medians indicated. Data shown were pooled from three independent experiments.</p

Stomach microbiota composition in Mongolian gerbils 14 months following <i>H. pylori</i> infection.

<p>Fourteen months following oral infection of Mongolian gerbils with <i>H. pylori</i> wildtype strain B8 (B8; black circles) or <i>H. pylori</i> mutant strain lacking <i>cagY</i> (B8Δ<i>cagY</i>; grey circles), the microbiota composition of luminal <b>stomach</b> contents were quantitatively analyzed by culture as described in Methods. Uninfected age-matched animals served as negative controls (Naïve; white circles). Numbers of live (<b>A</b>) <i>E. coli</i>, (<b>B</b>) <i>Proteus</i> sp., (<b>C</b>) Enterococci, (<b>D</b>) Lactobacilli, (<b>E</b>) <i>Bacteroides/Prevotella</i> spp. as well as the (<b>F</b>) total bacterial load are indicated as colony forming units (CFU) per g luminal content. Numbers of animals harboring the respective bacterial species are given in parentheses. Medians and significance levels (p<i>-</i>values) determined by Mann-Whitney-U test are indicated. Data shown were pooled from three independent experiments.</p