Dietary iron variably modulates assembly of the intestinal microbiota in colitis-resistant and colitis-susceptible mice.

Iron deficiency, a common comorbidity of gastrointestinal inflammatory disorders such as inflammatory bowel diseases (IBD), is often treated with oral iron supplementation. However, the safety of oral iron supplementation remains controversial because of its association with exacerbated disease activity in a subset of IBD patients. Because iron modulates bacterial growth and function, one possible mechanism by which iron may exacerbate inflammation in susceptible hosts is by modulating the intestinal microbiota. We, therefore, investigated the impact of dietary iron on the intestinal microbiota, utilizing the conventionalization of germ-free mice as a model of a microbial community in compositional flux to recapitulate the instability of the IBD-associated intestinal microbiota. Our findings demonstrate that altering intestinal iron availability during community assembly modulated the microbiota in non-inflamed wild type (WT) and colitis-susceptible interleukin-10-deficient (Il10-/-) mice. Depletion of luminal iron availability promoted luminal compositional changes associated with dysbiotic states irrespective of host genotype, including an expansion of Enterobacteriaceae such as Escherichia coli. Mechanistic in vitro growth competitions confirmed that high-affinity iron acquisition systems in E. coli enhance its abundance over other bacteria in iron-restricted conditions, thereby enabling pathobiont iron scavenging during dietary iron restriction. In contrast, distinct luminal community assembly was observed with dietary iron supplementation in WT versus Il10-/- mice, suggesting that the effects of increased iron on the microbiota differ with host inflammation status. Taken together, shifts in dietary iron intake during community assembly modulate the ecological structure of the intestinal microbiota and is dependent on host genotype and inflammation status.

Human colon mucosal biofilms from healthy or colon cancer hosts are carcinogenic.

Mucus-invasive bacterial biofilms are identified on the colon mucosa of approximately 50% of colorectal cancer (CRC) patients and approximately 13% of healthy subjects. Here, we test the hypothesis that human colon biofilms comprise microbial communities that are carcinogenic in CRC mouse models. Homogenates of human biofilm-positive colon mucosa were prepared from tumor patients (tumor and paired normal tissues from surgical resections) or biofilm-positive biopsies from healthy individuals undergoing screening colonoscopy; homogenates of biofilm-negative colon biopsies from healthy individuals undergoing screening colonoscopy served as controls. After 12 weeks, biofilm-positive, but not biofilm-negative, human colon mucosal homogenates induced colon tumor formation in 3 mouse colon tumor models (germ-free ApcMinΔ850/+;Il10-/- or ApcMinΔ850/+ and specific pathogen-free ApcMinΔ716/+ mice). Remarkably, biofilm-positive communities from healthy colonoscopy biopsies induced colon inflammation and tumors similarly to biofilm-positive tumor tissues. By 1 week, biofilm-positive human tumor homogenates, but not healthy biopsies, displayed consistent bacterial mucus invasion and biofilm formation in mouse colons. 16S rRNA gene sequencing and RNA-Seq analyses identified compositional and functional microbiota differences between mice colonized with biofilm-positive and biofilm-negative communities. These results suggest human colon mucosal biofilms, whether from tumor hosts or healthy individuals undergoing screening colonoscopy, are carcinogenic in murine models of CRC.

Microbes and Cancer.

Commensal microorganisms (the microbiota) live on all the surface barriers of our body and are particularly abundant and diverse in the distal gut. The microbiota and its larger host represent a metaorganism in which the cross talk between microbes and host cells is necessary for health, survival, and regulation of physiological functions locally, at the barrier level, and systemically. The ancestral molecular and cellular mechanisms stemming from the earliest interactions between prokaryotes and eukaryotes have evolved to mediate microbe-dependent host physiology and tissue homeostasis, including innate and adaptive resistance to infections and tissue repair. Mostly because of its effects on metabolism, cellular proliferation, inflammation, and immunity, the microbiota regulates cancer at the level of predisposing conditions, initiation, genetic instability, susceptibility to host immune response, progression, comorbidity, and response to therapy. Here, we review the mechanisms underlying the interaction of the microbiota with cancer and the evidence suggesting that the microbiota could be targeted to improve therapy while attenuating adverse reactions.

The role of microbiota in cancer therapy.

The relationship between the host and the commensal microbiota regulates physiological functions including inflammation and immunity and it has been scrutinized in the context of cancer. While viruses and bacterial species have been implicated in oncogenesis, commensal microbes also have a beneficial role in the fight against cancer. Therapy efficacy, including adoptive T cell transfer, alkylating agents and immune checkpoint blockers, relies on immunity that receives its education from the gut microbiota. In cancer therapy with immunostimulating oligonucleotides and platinum salts, the microbiota also modulates the response by priming for the release of pro-inflammatory factors and reactive oxygen species, respectively. This new information offers promising clinical possibilities of modulating cancer therapy and its toxic side effects by targeting the microbiota.

Microbial genomic analysis reveals the essential role of inflammation in bacteria-induced colorectal cancer.

Enterobacteria, especially Escherichia coli, are abundant in patients with inflammatory bowel disease or colorectal cancer (CRC). However, it is unclear whether cancer is promoted by inflammation-induced expansion of E. coli and/or changes in expression of specific microbial genes. Here we use longitudinal (2, 12 and 20 weeks) 16S rRNA sequencing of luminal microbiota from ex-germ-free mice to show that inflamed Il10(-/-) mice maintain a higher abundance of Enterobacteriaceae than healthy wild-type mice. Experiments with mono-colonized Il10(-/-) mice reveal that host inflammation is necessary for E. coli cancer-promoting activity. RNA-sequence analysis indicates significant changes in E. coli gene catalogue in Il10(-/-) mice, with changes mostly driven by adaptation to the intestinal environment. Expression of specific genes present in the tumour-promoting E. coli pks island are modulated by inflammation/CRC development. Thus, progression of inflammation in Il10(-/-) mice supports Enterobacteriaceae and alters a small subset of microbial genes important for tumour development.

From promotion to management: the wide impact of bacteria on cancer and its treatment.

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 (Myd88) were protected from I/R-induced injury compared with Myd88 control mice. Myd88 mice also displayed a significantly reduced bacterial translocation (∼85%) into lymph nodes compared with Myd88 mice. Expression of ccl2 and cxcl1 mRNA was significantly reduced (85% and 62%, respectively) in intestinal tissue of Myd88 mice compared with Myd88 mice, which associated with a reduced number of myeloperoxidase-positive cells in intestinal tissues of I/R-exposed Myd88 mice. Immunohistochemistry analysis showed a reduced IgA deposition and complement staining in ischemic tissue of Myd88 mice compared with Myd88 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.

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.

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 (MOR(IEC-/-)). Radio-ligand binding assays demonstrated selective loss of MOR signaling in IECs of MOR(IEC-/-) 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 MOR(IEC-/-) mice. This cytoprotective effect was associated with enterocyte-mediated phosphoinositide 3-kinase (PI3K)/glycogen synthase kinase 3ß 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.

Intestinal inflammation targets cancer-inducing activity of the microbiota.

Inflammation alters host physiology to promote cancer, as seen in colitis-associated colorectal cancer (CRC). Here, we identify the intestinal microbiota as a target of inflammation that affects the progression of CRC. High-throughput sequencing revealed that inflammation modifies gut microbial composition in colitis-susceptible interleukin-10-deficient (Il10(-/-)) mice. Monocolonization with the commensal Escherichia coli NC101 promoted invasive carcinoma in azoxymethane (AOM)-treated Il10(-/-) mice. Deletion of the polyketide synthase (pks) genotoxic island from E. coli NC101 decreased tumor multiplicity and invasion in AOM/Il10(-/-) mice, without altering intestinal inflammation. Mucosa-associated pks(+) E. coli were found in a significantly high percentage of inflammatory bowel disease and CRC patients. This suggests that in mice, colitis can promote tumorigenesis by altering microbial composition and inducing the expansion of microorganisms with genotoxic capabilities.