Daniel Mucida

IL-6 and Stat3 Are Required for Survival of Intestinal Epithelial Cells and Development of Colitis-Associated Cancer

Colitis-associated cancer (CAC) is the most serious complication of inflammatory bowel disease. Proinflammatory cytokines have been suggested to regulate preneoplastic growth during CAC tumorigenesis. Interleukin 6 (IL-6) is a multifunctional NF-kappaB-regulated cytokine that acts on epithelial and immune cells. Using genetic tools, we now demonstrate that IL-6 is a critical tumor promoter during early CAC tumorigenesis. In addition to enhancing proliferation of tumor-initiating cells, IL-6 produced by lamina propria myeloid cells protects normal and premalignant intestinal epithelial cells (IECs) from apoptosis. The proliferative and survival effects of IL-6 are largely mediated by the transcription factor Stat3, whose IEC-specific ablation has profound impact on CAC tumorigenesis. Thus, the NF-kappaB-IL-6-Stat3 cascade is an important regulator of the proliferation and survival of tumor-initiating IECs.

Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth.

Approximately 2% of colorectal cancer is linked to pre-existing inflammation known as colitis-associated cancer, but most develops in patients without underlying inflammatory bowel disease. Colorectal cancer often follows a genetic pathway whereby loss of the adenomatous polyposis coli (APC) tumour suppressor and activation of β-catenin are followed by mutations in K-Ras, PIK3CA and TP53, as the tumour emerges and progresses. Curiously, however, ‘inflammatory signature’ genes characteristic of colitis-associated cancer are also upregulated in colorectal cancer. Further, like most solid tumours, colorectal cancer exhibits immune/inflammatory infiltrates, referred to as ‘tumour-elicited inflammation’. Although infiltrating CD4+ TH1 cells and CD8+ cytotoxic T cells constitute a positive prognostic sign in colorectal cancer, myeloid cells and T-helper interleukin (IL)-17-producing (TH17) cells promote tumorigenesis, and a ‘TH17 expression signature’ in stage I/II colorectal cancer is associated with a drastic decrease in disease-free survival. Despite its pathogenic importance, the mechanisms responsible for the appearance of tumour-elicited inflammation are poorly understood. Many epithelial cancers develop proximally to microbial communities, which are physically separated from immune cells by an epithelial barrier. We investigated mechanisms responsible for tumour-elicited inflammation in a mouse model of colorectal tumorigenesis, which, like human colorectal cancer, exhibits upregulation of IL-23 and IL-17. Here we show that IL-23 signalling promotes tumour growth and progression, and development of a tumoural IL-17 response. IL-23 is mainly produced by tumour-associated myeloid cells that are likely to be activated by microbial products, which penetrate the tumours but not adjacent tissue. Both early and late colorectal neoplasms exhibit defective expression of several barrier proteins. We propose that barrier deterioration induced by colorectal-cancer-initiating genetic lesions results in adenoma invasion by microbial products that trigger tumour-elicited inflammation, which in turn drives tumour growth.

Oral tolerance in the absence of naturally occurring Tregs

Mucosal tolerance prevents pathological reactions against environmental and food antigens, and its failure results in exacerbated inflammation typical of allergies and asthma. One of the proposed mechanisms of oral tolerance is the induction of Tregs. Using a mouse model of hyper-IgE and asthma, we found that oral tolerance could be effectively induced in the absence of naturally occurring thymus-derived Tregs. Oral antigen administration prior to i.p. immunization prevented effector/memory Th2 cell development, germinal center formation, class switching to IgE, and lung inflammation. Oral exposure to antigen induced development of antigen-specific CD4CD25Foxp3CD45RB cells that were anergic and displayed suppressive activity in vivo and in vitro. Oral tolerance to the Th2 allergic response was in large part dependent on TGF-beta and independent of IL-10. Interestingly, Tregs were also induced by single i.p. immunization with antigen and adjuvant. However, unlike oral administration of antigen, which induced Tregs but not effector T cells, i.p. immunization led to the simultaneous induction of Tregs and effector Th2 cells displaying the same antigen specificity.

The light and dark sides of intestinal intraepithelial lymphocytes.

The intraepithelial lymphocytes (IELs) that reside within the epithelium of the intestine form one of the main branches of the immune system. As IELs are located at this critical interface between the core of the body and the outside environment, they must balance protective immunity with an ability to safeguard the integrity of the epithelial barrier: failure to do so would compromise homeostasis of the organism. In this Review, we address how the unique development and functions of intestinal IELs allow them to achieve this balance.

Crosstalk between Muscularis Macrophages and Enteric Neurons Regulates Gastrointestinal Motility

Intestinal peristalsis is a dynamic physiologic process influenced by dietary and microbial changes. It is tightly regulated by complex cellular interactions; however, our understanding of these controls is incomplete. A distinct population of macrophages is distributed in the intestinal muscularis externa. We demonstrate that, in the steady state, muscularis macrophages regulate peristaltic activity of the colon. They change the pattern of smooth muscle contractions by secreting bone morphogenetic protein 2 (BMP2), which activates BMP receptor (BMPR) expressed by enteric neurons. Enteric neurons, in turn, secrete colony stimulatory factor 1 (CSF1), a growth factor required for macrophage development. Finally, stimuli from microbial commensals regulate BMP2 expression by macrophages and CSF1 expression by enteric neurons. Our findings identify a plastic, microbiota-driven crosstalk between muscularis macrophages and enteric neurons that controls gastrointestinal motility. PAPERFLICK:

CRISPR Interference Can Prevent Natural Transformation and Virulence Acquisition during In Vivo Bacterial Infection

Pathogenic bacterial strains emerge largely due to transfer of virulence and antimicrobial resistance genes between bacteria, a process known as horizontal gene transfer (HGT). Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci of bacteria and archaea encode a sequence-specific defense mechanism against bacteriophages and constitute a programmable barrier to HGT. However, the impact of CRISPRs on the emergence of virulence is unknown. We programmed the human pathogen Streptococcus pneumoniae with CRISPR sequences that target capsule genes, an essential pneumococcal virulence factor, and show that CRISPR interference can prevent transformation of nonencapsulated, avirulent pneumococci into capsulated, virulent strains during infection in mice. Further, at low frequencies bacteria can lose CRISPR function, acquire capsule genes, and mount a successful infection. These results demonstrate that CRISPR interference can prevent the emergence of virulence in vivo and that strong selective pressure for virulence or antibiotic resistance can lead to CRISPR loss in bacterial pathogens.

Transcriptional reprogramming of mature CD4 + helper T cells generates distinct MHC class II-restricted cytotoxic T lymphocytes

TCRαβ thymocytes differentiate into either CD8αβ+ cytotoxic T lymphocytes or CD4+ helper T cells. This functional dichotomy is controlled by key transcription factors, including the helper T cell master regulator ThPOK, which suppresses the cytolytic program in major histocompatibility complex (MHC) class II–restricted CD4+ thymocytes. ThPOK continues to repress genes of the CD8 lineage in mature CD4+ T cells, even as they differentiate into effector helper T cell subsets. Here we found that the helper T cell fate was not fixed and that mature, antigen-stimulated CD4+ T cells terminated expression of the gene encoding ThPOK and reactivated genes of the CD8 lineage. This unexpected plasticity resulted in the post-thymic termination of the helper T cell program and the functional differentiation of distinct MHC class II–restricted CD4+ cytotoxic T lymphocytes.

Specialized role of migratory dendritic cells in peripheral tolerance induction

Harnessing DCs for immunotherapies in vivo requires the elucidation of the physiological role of distinct DC populations. Migratory DCs traffic from peripheral tissues to draining lymph nodes charged with tissue self antigens. We hypothesized that these DC populations have a specialized role in the maintenance of peripheral tolerance, specifically, to generate suppressive Foxp3+ Tregs. To examine the differential capacity of migratory DCs versus blood-derived lymphoid-resident DCs for Treg generation in vivo, we targeted a self antigen, myelin oligodendrocyte glycoprotein, using antibodies against cell surface receptors differentially expressed in these DC populations. Using this approach together with mouse models that lack specific DC populations, we found that migratory DCs have a superior ability to generate Tregs in vivo, which in turn drastically improve the outcome of experimental autoimmune encephalomyelitis. These results provide a rationale for the development of novel therapies targeting migratory DCs for the treatment of autoimmune diseases.

Retinoic Acid Can Directly Promote TGF-β-Mediated Foxp3+ Treg Cell Conversion of Naive T Cells

The article by Hill et al. (2008), published in the November 14, 2008 issue of Immunity, describes a mechanism by which retinoic acid (RA) enhances TGF-β-induced Foxp3 expression. The authors propose that RA does not act directly on naive T cells during activation in culture but rather indirectly via negative regulation of an accompanying population of effector or memory CD4+ CD44hi cells. They reasoned that the increased generation of Foxp3+ cells in response to RA in culture, as described previously (Coombes et al., 2007; Elias et al., 2008; Mucida et al., 2007; Sun et al., 2007; Xiao et al., 2008), represented the lifting by RA of inhibition imparted by accompanying CD4+CD44hi T cells, rather than by direct or indirect effects of RA on the Foxp3 expression of the primed naive T cells themselves. In order to assess the effects of RA on naive T cells in the absence of accompanying CD4+CD44hi T cells, we sorted (CD4+CD25−CD44low CD62L+) GFP− T cells (more than 99.7% purity) from Foxp3-eGFP reporter mice (Figure S1A available online) by flow cytometry. After 4 days of stimulation with anti-CD3 and anti-CD28, we stained CD4 cells with 7AAD to exclude dead cells; additionally, forward and side scatter (area versus width) was used to exclude doublets, and we evaluated Foxp3 expression via GFP staining. Addition of RA enhanced Foxp3 induction more than 50% by use of 1 or 10 ng/ml doses of TGF-β (Figure S1B). Because the sorting efficiency is not 100%, it is possible that extremely low numbers of “accompanying” memory or effector cells could still influence these results. To exclude this possibility, we used FACS-sorted CD4+CD25−CD44lo CD62L+ T cells, isolated from B7-1 and B7-2 double-deficient mice (Cd80−/− Cd86−/−), which even before sorting already contain less than 5% of memory or effector CD44hi cells (data not shown). RA also greatly enhanced Foxp3 induced by TGF-β in CD4+CD25−CD44lo CD62L+ naive T cells isolated from Cd80−/− Cd86−/− mice (Figure S1C). Moreover, we showed previously that RA is able to counterbalance the inhibitory effects of costimulation on TGF-β-mediated Foxp3 induction, with either CD4+CD25− or CD4+Foxp3− T cells (Benson et al., 2007). To confirm these results, we used OTII TCR transgenic CD4+CD25−CD44lo CD62L+ cells sorted by flow cytometry and tested the effects of RA by using increasing doses of anti-CD28 stimulation. We found that RA markedly enhanced TGF-β-mediated Foxp3 induction on pure naive CD4+ T cells that were stimulated with anti-CD3 and various doses of anti-CD28 (Figure S1D). The enhanced Foxp3 expression mediated by RA is more pronounced on naive monoclonal OTII TCR transgenic T cells as compared to polyclonal T cells, consistent with a lesser frequency of “contaminating” memory T cells. Finally, because we showed previously that RA-mediated enhanced expression of Foxp3 is greatly reduced in the absence of IL-2 (Mucida et al., 2007), we investigated the effects of RA on naive T cells with various doses of exogenous IL-2. Although IL-2-deficient mice develop inflammatory disorders, Il2−/−Cd80−/−Cd86−/− mice are healthy and, more importantly, they do not contain T regulatory cells. At steady state, ~99% of all CD4+ T cells isolated from Il2−/−Cd80−/−Cd86−/− mice are naive (data not shown). The CD4+ T cells were further sorted by flow cytometry so that highly purified naive CD4+CD25−CD44lo CD62L+ cells (more than 99.9% purity) were obtained. The sorted naive CD4+ Il2−/−Cd80−/−Cd86−/− T cells were tested for TGF-β-induced Foxp3 expression in the presence of increasing doses of IL-2 and anti-CD3 and anti-CD28 coated beads, with or without RA. The data showed that 1 nM RA distinctly enhanced TGF-β (1 ng/ml)-mediated Foxp3 induction in pure naive CD4+ T cells at all doses of IL-2 examined (Figures S1E and S1F). Strikingly, although the expression of Foxp3 was much reduced, RA enhanced TGF-β-mediated Foxp3 induction not only in the absence of memory or effector T cells but also in the absence of IL-2. These data demonstrate that RA mediates enhanced TGF-β-induced Foxp3 expression upon activation of pure naive T cells in the absence of accompanying CD4+CD44hi T cells. In addition, we confirmed, as Hill et al. (2008) proposed, that RA also efficiently counteracts inhibitory effects of CD44hi T cells on Foxp3 induction (data not shown), which indicates that RA is able to enhance Foxp3 expression both, via effects directly on the primed naive T cells as well as indirectly via inhibitory effects on accompanying CD4+CD44hi T cells. There is no doubt that the new findings by Hill et al. (2008) add an important new pathway by which RA can enhance Foxp3 induction, which had been suggested previously (Elias et al., 2008; Mucida et al., 2007; Xiao et al., 2008).Nevertheless, published data, together with the data presented here, disagree with the central statement proposed by Hill et al. (2008) that the enhanced expression of TGF-β driven Foxp3 mediated by RA is an indirect effect that requires suppression of accompanying CD4+CD44hi T cells rather than via direct or indirect effects on the primed T cells themselves. Under physiological conditions, naive T cells may be exposed to cytokines and effector or memory cells, and hence it is likely that during priming of naive T cells, both mechanisms of RA-mediated enhanced TGF-β driven Foxp3 expression in the primed T cells will synergize in vivo. Therefore, elucidating and understanding both processes by which RA affects naive and already differentiated T cells is important and may lead to the identification of possible targets for therapeutic interventions to treat various inflammatory and autoimmune diseases.

Neuro-immune Interactions Drive Tissue Programming in Intestinal Macrophages

Proper adaptation to environmental perturbations is essential for tissue homeostasis. In the intestine, diverse environmental cues can be sensed by immune cells, which must balance resistance to microorganisms with tolerance, avoiding excess tissue damage. By applying imaging and transcriptional profiling tools, we interrogated how distinct microenvironments in the gut regulate resident macrophages. We discovered that macrophages exhibit a high degree of gene-expression specialization dependent on their proximity to the gut lumen. Lamina propria macrophages (LpMs) preferentially expressed a pro-inflammatory phenotype when compared to muscularis macrophages (MMs), which displayed a tissue-protective phenotype. Upon luminal bacterial infection, MMs further enhanced tissue-protective programs, and this was attributed to swift activation of extrinsic sympathetic neurons innervating the gut muscularis and norepinephrine signaling to β2 adrenergic receptors on MMs. Our results reveal unique intra-tissue macrophage specialization and identify neuro-immune communication between enteric neurons and macrophages that induces rapid tissue-protective responses to distal perturbations.