Microbial signaling in the intestinal ischemia/reperfusion injury response

The intestine is home to one of the most diverse and populous ecosystems on earth. The intestinal epithelium the single layer sheet of columnar cells that provides key functions to maintain homeostasis including nutrient absorption and metabolism, innate immune system toning and the sampling of luminal contents. One of the most important roles of the epithelium is to provide a physical barrier between the host and gut microbes. Intestinal epithelial cells (IECs) are stitched together by tight junctions that maintain the architecture of the epithelial sheet and prevent uncontrolled access of luminal content to the underlying immune system compartments and the blood stream. Intestinal ischemia/reperfusion (I/R) compromises the integrity of this structure potentially leading to sepsis and multi-organ dysfunction. The epithelium experiences steady-state regeneration through migration and proliferation of IECs from the base of the intestinal crypts to tips of the villi. Microbial signals through pattern-recognition receptors foster this enterprise. Herein, I discuss my insights into the contributions of microbial signals to the host response to intestinal I/R-induced injury. I demonstrate the deleterious role of TLR adapter, MyD88, and protective role of the intracellular microbial receptor, NOD2. Then, I discuss plausible mechanisms by which these two signaling proteins yield such contrasting effects on injury.Doctor of Philosoph

From promotion to management: The wide impact of bacteria on cancer and its treatment: Prospects & Overviews

In humans, the intestine is the major reservoir of microbes. Although the intestinal microbial community exists in a state of homeostasis called eubiosis, environmental and genetics factors can lead to microbial perturbation or dysbiosis, a state associated with various pathologies including inflammatory bowel diseases (IBD) and colorectal cancer (CRC). Dysbiotic microbiota is thought to contribute to the initiation and progression of CRC. At the opposite end of the spectrum, two recently published studies in Science reveal that the microbiota is essential for chemotherapeutic drug efficacy, suggesting a beneficial microbial function in cancer management. The dichotomy between the beneficial and detrimental roles of the microbiota during cancer initiation, progression and treatment emphasize the interwoven relationship between bacteria and cancer. Moreover, these findings suggest that the microbiota could be considered as a therapeutic target, not only at the level of cancer prevention, but also during management, i.e. by enhancing the efficacy of chemotherapeutics

Epithelial Cell-Specific MyD88 Signaling Mediates Ischemia/Reperfusion-induced Intestinal Injury Independent of Microbial Status:

The Toll-like receptor/MyD88 signaling pathway has been shown to mediate protective functions during intestinal exposure to various noxious events. The goal of this study was to define the role of bacteria and MyD88 signaling in intestinal response to damage using an ischemia–reperfusion (I/R)-induced injury model. We showed that conventionalized mice displayed a better outcome to I/R-induced injury than germ-free mice (3.8 ± 1.98 vs. 11.8 ± 1.83, P < 0.05). However, mice with intestinal epithelial cell (IEC)-specific deletion of Myd88 (Myd88IEC−/−) were protected from I/R-induced injury compared with Myd88f/f control mice. Myd88IEC−/− mice also displayed a significantly reduced bacterial translocation (~85%) into lymph nodes compared with Myd88f/f mice. Expression of ccl2 and cxcl1 mRNA was significantly reduced (85% and 62%, respectively) in intestinal tissue of Myd88IEC−/− mice compared with Myd88f/f mice, which associated with a reduced number of myeloperoxidase-positive cells in intestinal tissues of I/R-exposed Myd88IEC−/− mice. Immunohistochemistry analysis showed a reduced IgA deposition and complement staining in ischemic tissue of Myd88IEC−/− mice compared with Myd88f/f mice. These findings suggest that I/R-induced intestinal injury involves IEC-derived MyD88 signaling leading to increased IgA deposition/degradation, and complement activation in conjunction with an influx of neutrophils mediated by chemokine production

The Microbiota Protects against Ischemia/Reperfusion-Induced Intestinal Injury through Nucleotide-Binding Oligomerization Domain-Containing Protein 2 (NOD2) Signaling

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), an intracellular pattern recognition receptor, induces autophagy on detection of muramyl dipeptide (MDP), a component of microbial cell walls. The role of bacteria and NOD2 signaling toward ischemia/reperfusion (I/R)–induced intestinal injury response is unknown. Herein, we report that I/R-induced intestinal injury in germ-free (GF) C57BL/6 wild-type (WT) mice is worse than in conventionally derived mice. More important, microbiota-mediated protection against I/R-induced intestinal injury is abrogated in conventionally derived Nod2−/− mice and GF Nod2−/− mice. Also, WT mice raised in specific pathogen-free (SPF) conditions fared better against I/R-induced injury than SPF Nod2−/− mice. Moreover, SPF WT mice i.p. administered 10 mg/kg MDP were protected against injury compared with mice administered the inactive enantiomer, l-MDP, an effect lost in Nod2−/− mice. However, MDP administration failed to protect GF mice from I/R-induced intestinal injury compared with control, a phenomenon correlating with undetectable Nod2 mRNA level in the epithelium of GF mice. More important, the autophagy-inducer rapamycin protected Nod2−/− mice against I/R-induced injury and increased the levels of LC3+ puncta in injured tissue of Nod2−/− mice. These findings demonstrate that NOD2 protects against I/R and promotes wound healing, likely through the induction of the autophagy response

Intestinal Epithelial Cell–Derived μ-Opioid Signaling Protects against Ischemia Reperfusion Injury through PI3K Signaling

Intestinal ischemia has a wide variety of causes, including, but not limited to, atherosclerosis, thrombosis, hypotension, and chronic inflammation. In severe cases, ischemic injury can result in death. μ-Opioid receptor (MOR) signaling has previously been shown to protect against chemically induced colitis, but the cellular origin of this effect remains unknown. Herein, we evaluated the role of intestinal epithelial cell (IEC)–derived MOR signaling in host responses to ischemia/reperfusion-induced injury. Ileal ischemia was accomplished through obstruction of the distal branches of the superior mesenteric artery (60 minutes) and reperfusion for 90 minutes (ischemia-reperfusion). Floxed-MOR mice were crossed to Villin-cre transgenic mice to selectively delete the MOR gene in IECs (MORIEC−/−). Radio-ligand binding assays demonstrated selective loss of MOR signaling in IECs of MORIEC−/− mice. The s.c. administration of the MOR agonist, [D-Arg2, Lys4] dermorphin (1–4) amide (DALDA), 10 minutes before surgery protected against both ischemic and reperfusion phases of intestinal injury, an effect abolished in MORIEC−/− mice. This cytoprotective effect was associated with enterocyte-mediated phosphoinositide 3-kinase (PI3K)/glycogen synthase kinase 3β signaling and decreased apoptosis, as determined by IHC and caspase-3 processing. PI3K blockade with Ly294002 resulted in loss of MOR-mediated cytoprotective function. Together, these data show that IEC-derived μ-opioid signaling uses the PI3K pathway to protect cells against the damaging effect of ischemia-reperfusion. Targeting MOR signaling may represent a novel mean to alleviate intestinal injury and promote the wound-healing response

Gut Commensal Bacteria and Regional Wnt Gene Expression in the Proximal Versus Distal Colon

Regional expression of Wingless/Int (Wnt) genes plays a central role in regulating intestinal development and homeostasis. However, our knowledge of such regional Wnt proteins in the colon remains limited. To understand further the effect of Wnt signaling components in controlling intestinal epithelial homeostasis, we investigated whether the physiological heterogeneity of the proximal and distal colon can be explained by differential Wnt signaling. With the use of a Wnt signaling-specific PCR array, expression of 84 Wnt-mediated signal transduction genes was analyzed, and a differential signature of Wnt-related genes in the proximal versus distal murine colon was identified. Several Wnt agonists (Wnt5a, Wnt8b, and Wnt11), the Wnt receptor frizzled family receptor 3, and the Wnt inhibitory factor 1 were differentially expressed along the colon length. These Wnt signatures were associated with differential epithelial cell proliferation and migration in the proximal versus distal colon. Furthermore, reduced Wnt/β-catenin activity and decreased Wnt5a and Wnt11 expression were observed in mice lacking commensal bacteria, an effect that was reversed by conventionalization of germ-free mice. Interestingly, myeloid differentiation primary response gene 88 knockout mice showed decreased Wnt5a levels, indicating a role for Toll-like receptor signaling in regulating Wnt5a expression. Our results suggest that the morphological and physiological heterogeneity within the colon is in part facilitated by the differential expression of Wnt signaling components and influenced by colonization with bacteria

Stochastic changes over time and not founder effects drive cage effects in microbial community assembly in a mouse model

Maternal transmission and cage effects are powerful confounding factors in microbiome studies. To assess the consequences of cage microenvironment on the mouse gut microbiome, two groups of germ-free (GF) wild-type (WT) mice, one gavaged with a microbiota harvested from adult WT mice and another allowed to acquire the microbiome from the cage microenvironment, were monitored using Illumina 16S rRNA sequencing over a period of 8 weeks. Our results revealed that cage effects in WT mice moved from GF to specific pathogen free (SPF) conditions take several weeks to develop and are not eliminated by the initial gavage treatment. Initial gavage influenced, but did not eliminate a successional pattern in which Proteobacteria became less abundant over time. An analysis in which 16S rRNA sequences are mapped to the closest sequenced whole genome suggests that the functional potential of microbial genomes changes significantly over time shifting from an emphasis on pathogenesis and motility early in community assembly to metabolic processes at later time points. Functionally, mice allowed to naturally acquire a microbial community from their cage, but not mice gavaged with a common biome, exhibit a cage effect in Dextran Sulfate Sodium-induced inflammation. Our results argue that while there are long-term effects of the founding community, these effects are mitigated by cage microenvironment and successional community assembly over time, which must both be explicitly considered in the interpretation of microbiome mouse experiments

VSL#3 probiotic modifies mucosal microbial composition but does not reduce colitis-associated colorectal cancer

Although probiotics have shown success in preventing the development of experimental colitis-associated colorectal cancer (CRC), beneficial effects of interventional treatment are relatively unknown. Here we show that interventional treatment with VSL#3 probiotic alters the luminal and mucosally-adherent microbiota, but does not protect against inflammation or tumorigenesis in the azoxymethane (AOM)/Il10−/− mouse model of colitis-associated CRC. VSL#3 (109 CFU/animal/day) significantly enhanced tumor penetrance, multiplicity, histologic dysplasia scores, and adenocarcinoma invasion relative to VSL#3-untreated mice. Illumina 16S sequencing demonstrated that VSL#3 significantly decreased (16-fold) the abundance of a bacterial taxon assigned to genus Clostridium in the mucosally-adherent microbiota. Mediation analysis by linear models suggested that this taxon was a contributing factor to increased tumorigenesis in VSL#3-fed mice. We conclude that VSL#3 interventional therapy can alter microbial community composition and enhance tumorigenesis in the AOM/Il10−/− model