Grace Y. Chen

Sterile inflammation: sensing and reacting to damage

Over the past several decades, much has been revealed about the nature of the host innate immune response to microorganisms, with the identification of pattern recognition receptors (PRRs) and pathogen-associated molecular patterns, which are the conserved microbial motifs sensed by these receptors. It is now apparent that these same PRRs can also be activated by non-microbial signals, many of which are considered as damage-associated molecular patterns. The sterile inflammation that ensues either resolves the initial insult or leads to disease. Here, we review the triggers and receptor pathways that result in sterile inflammation and its impact on human health.

Role of the gut microbiota in immunity and inflammatory disease

The mammalian intestine is colonized by trillions of microorganisms, most of which are bacteria that have co-evolved with the host in a symbiotic relationship. The collection of microbial populations that reside on and in the host is commonly referred to as the microbiota. A principal function of the microbiota is to protect the intestine against colonization by exogenous pathogens and potentially harmful indigenous microorganisms via several mechanisms, which include direct competition for limited nutrients and the modulation of host immune responses. Conversely, pathogens have developed strategies to promote their replication in the presence of competing microbiota. Breakdown of the normal microbial community increases the risk of pathogen infection, the overgrowth of harmful pathobionts and inflammatory disease. Understanding the interaction of the microbiota with pathogens and the host might provide new insights into the pathogenesis of disease, as well as novel avenues for preventing and treating intestinal and systemic disorders.

NOD-Like Receptors: Role in Innate Immunity and Inflammatory Disease

The NOD-like receptors (NLRs) are a specialized group of intracellular receptors that represent a key component of the host innate immune system. Since the discovery of the first NLR almost 10 years ago, the study of this special class of microbial sensors has burgeoned; consequently, a better understanding of the mechanism by which these receptors recognize microbes and other danger signals and of how they activate inflammatory signaling pathways has emerged. Moreover, in addition to their primary role in host defense against invading pathogens, their ability to regulate nuclear factor-kappa B (NF-kappaB) signaling, interleukin-1-beta (IL-1beta) production, and cell death indicates that they are crucial to the pathogenesis of a variety of inflammatory human diseases.

Control of pathogens and pathobionts by the gut microbiota

A dense resident microbial community in the gut, referred as the commensal microbiota, coevolved with the host and is essential for many host physiological processes that include enhancement of the intestinal epithelial barrier, development of the immune system and acquisition of nutrients. A major function of the microbiota is protection against colonization by pathogens and overgrowth of indigenous pathobionts that can result from the disruption of the healthy microbial community. The mechanisms that regulate the ability of the microbiota to restrain pathogen growth are complex and include competitive metabolic interactions, localization to intestinal niches and induction of host immune responses. Pathogens, in turn, have evolved strategies to escape from commensal-mediated resistance to colonization. Thus, the interplay between commensals and pathogens or indigenous pathobionts is critical for controlling infection and disease. Understanding pathogen-commensal interactions may lead to new therapeutic approaches to treating infectious diseases.

NOD2-mediated dysbiosis predisposes mice to transmissible colitis and colorectal cancer

Instability in the composition of gut bacterial communities (dysbiosis) has been linked to common human intestinal disorders, such as Crohns disease and colorectal cancer. Here, we show that dysbiosis caused by Nod2 deficiency gives rise to a reversible, communicable risk of colitis and colitis-associated carcinogenesis in mice. Loss of either Nod2 or RIP2 resulted in a proinflammatory microenvironment that enhanced epithelial dysplasia following chemically induced injury. The condition could be improved by treatment with antibiotics or an anti-interleukin-6 receptor-neutralizing antibody. Genotype-dependent disease risk was communicable via maternally transmitted microbiota in both Nod2-deficient and WT hosts. Furthermore, reciprocal microbiota transplantation reduced disease risk in Nod2-deficient mice and led to long-term changes in intestinal microbial communities. Conversely, disease risk was enhanced in WT hosts that were recolonized with dysbiotic fecal microbiota from Nod2-deficient mice. Thus, we demonstrated that licensing of dysbiotic microbiota is a critical component of disease risk. Our results demonstrate that NOD2 has an unexpected role in shaping a protective assembly of gut bacterial communities and suggest that manipulation of dysbiosis is a potential therapeutic approach in the treatment of human intestinal disorders.

Isolation and Molecular Characterization of Cancer Stem Cells in MMTV‐Wnt‐1 Murine Breast Tumors

In human breast cancers, a phenotypically distinct minority population of tumorigenic (TG) cancer cells (sometimes referred to as cancer stem cells) drives tumor growth when transplanted into immunodeficient mice. Our objective was to identify a mouse model of breast cancer stem cells that could have relevance to the study of human breast cancer. To do so, we used breast tumors of the mouse mammary tumor virus (MMTV)‐Wnt‐1 mice. MMTV‐Wnt‐1 breast tumors were harvested, dissociated into single‐cell suspensions, and sorted by flow cytometry on Thy1, CD24, and CD45. Sorted cells were then injected into recipient background FVB/NJ female syngeneic mice. In six of seven tumors examined, Thy1+CD24+ cancer cells, which constituted approximately 1%–4% of tumor cells, were highly enriched for cells capable of regenerating new tumors compared with cells of the tumor that did not fit this profile (“not‐Thy1+CD24+”). Resultant tumors had a phenotypic diversity similar to that of the original tumor and behaved in a similar manner when passaged. Microarray analysis comparing Thy1+CD24+ tumor cells to not‐Thy1+CD24+ cells identified a list of differentially expressed genes. Orthologs of these differentially expressed genes predicted survival of human breast cancer patients from two different study groups. These studies suggest that there is a cancer stem cell compartment in the MMTV‐Wnt‐1 murine breast tumor and that there is a clinical utility of this model for the study of cancer stem cells.

A Functional Role for Nlrp6 in Intestinal Inflammation and Tumorigenesis

The nucleotide-binding oligomerization domain-like receptor (NLR) family member, Nlrp6, has been implicated in inflammasome signaling to activate caspase-1, which is essential for the production of mature IL-1β and IL-18. However, a function for Nlrp6 in vivo has never been demonstrated. Due to the relative high expression of Nlrp6 in intestinal tissue, we hypothesized that Nlrp6 has a role in intestinal homeostasis. Indeed, Nlrp6-deficient mice are more susceptible to chemically induced colitis as well as colitis-induced tumorigenesis than wild-type (WT) mice. Nlrp6-deficient mice exhibited significantly more inflammation within the colon than WT mice after dextran sulfate sodium treatment. Their inability to resolve inflammation and repair damaged epithelium as efficiently as WT mice resulted in prolonged increases in epithelial proliferative activity that likely underlie the increased propensity for tumors in these mice during chronic inflammation. We further show that the activity of Nlrp6 in hematopoietic cells is critical for protection against inflammation-related colon tumorigenesis. This study highlights the importance of NLR function in maintaining intestinal homeostasis to prevent the development of aberrant inflammation and tumor development within the colon.

The Gut Microbiome Modulates Colon Tumorigenesis

ABSTRACT Recent studies have shown that individuals with colorectal cancer have an altered gut microbiome compared to healthy controls. It remains unclear whether these differences are a response to tumorigenesis or actively drive tumorigenesis. To determine the role of the gut microbiome in the development of colorectal cancer, we characterized the gut microbiome in a murine model of inflammation-associated colorectal cancer that mirrors what is seen in humans. We followed the development of an abnormal microbial community structure associated with inflammation and tumorigenesis in the colon. Tumor-bearing mice showed enrichment in operational taxonomic units (OTUs) affiliated with members of the Bacteroides, Odoribacter, and Akkermansia genera and decreases in OTUs affiliated with members of the Prevotellaceae and Porphyromonadaceae families. Conventionalization of germfree mice with microbiota from tumor-bearing mice significantly increased tumorigenesis in the colon compared to that for animals colonized with a healthy gut microbiome from untreated mice. Furthermore, at the end of the model, germfree mice colonized with microbiota from tumor-bearing mice harbored a higher relative abundance of populations associated with tumor formation in conventional animals. Manipulation of the gut microbiome with antibiotics resulted in a dramatic decrease in both the number and size of tumors. Our results demonstrate that changes in the gut microbiome associated with inflammation and tumorigenesis directly contribute to tumorigenesis and suggest that interventions affecting the composition of the microbiome may be a strategy to prevent the development of colon cancer. IMPORTANCE The trillions of bacteria that live in the gut, known collectively as the gut microbiome, are important for normal functioning of the intestine. There is now growing evidence that disruptive changes in the gut microbiome are strongly associated with the development colorectal cancer. However, how the gut microbiome changes with time during tumorigenesis and whether these changes directly contribute to disease have not been determined. We demonstrate using a mouse model of inflammation-driven colon cancer that there are dramatic, continual alterations in the microbiome during the development of tumors, which are directly responsible for tumor development. Our results suggest that interventions that target these changes in the microbiome may be an effective strategy for preventing the development of colorectal cancer. The trillions of bacteria that live in the gut, known collectively as the gut microbiome, are important for normal functioning of the intestine. There is now growing evidence that disruptive changes in the gut microbiome are strongly associated with the development colorectal cancer. However, how the gut microbiome changes with time during tumorigenesis and whether these changes directly contribute to disease have not been determined. We demonstrate using a mouse model of inflammation-driven colon cancer that there are dramatic, continual alterations in the microbiome during the development of tumors, which are directly responsible for tumor development. Our results suggest that interventions that target these changes in the microbiome may be an effective strategy for preventing the development of colorectal cancer.

Intracellular NOD-like receptors in innate immunity, infection and disease

The innate immune system comprises several classes of pattern‐recognition receptors, including Toll‐like receptors (TLRs) and nucleotide binding and oligomerization domain‐like receptors (NLRs). TLRs recognize microbes on the cell surface and in endosomes, whereas NLRs sense microbial molecules in the cytosol. In this review, we focus on the role of NLRs in host defence against bacterial pathogens. Nod1 and Nod2 sense the cytosolic presence of molecules containing meso‐diaminopimelic acid and muramyl dipeptide respectively, and drive the activation of mitogen‐activated protein kinase and NF‐κB. In contrast, Ipaf, Nalp1b and Cryopyrin/Nalp3 promote the assembly of inflammasomes that are required for the activation of caspase‐1. Mutation in several NLR members, including NOD2 and Cryopyrin, is associated with the development of inflammatory disorders. Further understanding of NLRs should provide new insights into the mechanisms of host defence and the pathogenesis of inflammatory diseases.

The Nod2 Sensor Promotes Intestinal Pathogen Eradication via the Chemokine CCL2-Dependent Recruitment of Inflammatory Monocytes

The intracellular sensor Nod2 is activated in response to bacteria, and the impairment of this response is linked to Crohns disease. However, the function of Nod2 in host defense remains poorly understood. We found that Nod2-/- mice exhibited impaired intestinal clearance of Citrobacter rodentium, an enteric bacterium that models human infection by pathogenic Escherichia coli. The increased bacterial burden was preceded by reduced CCL2 chemokine production, inflammatory monocyte recruitment, and Th1 cell responses in the intestine. Colonic stromal cells, but not epithelial cells or resident CD11b+ phagocytic cells, produced CCL2 in response to C. rodentium in a Nod2-dependent manner. Unlike resident phagocytic cells, inflammatory monocytes produced IL-12, a cytokine that induces adaptive immunity required for pathogen clearance. Adoptive transfer of Ly6C(hi) monocytes restored the clearance of the pathogen in infected Ccr2-/- mice. Thus, Nod2 mediates CCL2-CCR2-dependent recruitment of inflammatory monocytes, which is important in promoting bacterial eradication in the intestine.