Alexei Nikolaev

Epithelial NEMO links innate immunity to chronic intestinal inflammation

Deregulation of intestinal immune responses seems to have a principal function in the pathogenesis of inflammatory bowel disease. The gut epithelium is critically involved in the maintenance of intestinal immune homeostasis—acting as a physical barrier separating luminal bacteria and immune cells, and also expressing antimicrobial peptides. However, the molecular mechanisms that control this function of gut epithelial cells are poorly understood. Here we show that the transcription factor NF-κB, a master regulator of pro-inflammatory responses, functions in gut epithelial cells to control epithelial integrity and the interaction between the mucosal immune system and gut microflora. Intestinal epithelial-cell-specific inhibition of NF-κB through conditional ablation of NEMO (also called IκB kinase-γ (IKKγ)) or both IKK1 (IKKα) and IKK2 (IKKβ)—IKK subunits essential for NF-κB activation—spontaneously caused severe chronic intestinal inflammation in mice. NF-κB deficiency led to apoptosis of colonic epithelial cells, impaired expression of antimicrobial peptides and translocation of bacteria into the mucosa. Concurrently, this epithelial defect triggered a chronic inflammatory response in the colon, initially dominated by innate immune cells but later also involving T lymphocytes. Deficiency of the gene encoding the adaptor protein MyD88 prevented the development of intestinal inflammation, demonstrating that Toll-like receptor activation by intestinal bacteria is essential for disease pathogenesis in this mouse model. Furthermore, NEMO deficiency sensitized epithelial cells to tumour-necrosis factor (TNF)-induced apoptosis, whereas TNF receptor-1 inactivation inhibited intestinal inflammation, demonstrating that TNF receptor-1 signalling is crucial for disease induction. These findings demonstrate that a primary NF-κB signalling defect in intestinal epithelial cells disrupts immune homeostasis in the gastrointestinal tract, causing an inflammatory-bowel-disease-like phenotype. Our results identify NF-κB signalling in the gut epithelium as a critical regulator of epithelial integrity and intestinal immune homeostasis, and have important implications for understanding the mechanisms controlling the pathogenesis of human inflammatory bowel disease.

Transforming growth factor beta induced FoxP3+ regulatory T cells suppress Th1 mediated experimental colitis.

Background and aims: The imbalance between effector and regulatory T cells plays a central role in the pathogenesis of inflammatory bowel diseases. In addition to the thymus, CD4+CD25+ regulatory T cells can be induced in the periphery from a population of CD25− T cells by treatment with transforming growth factor β (TGF-β). Here, we analysed the in vivo function of TGF-β induced regulatory T (Ti-Treg) cells in experimental colitis. Methods: Ti-Treg cells were generated in cell culture in the presence or absence of TGF-β and tested for their regulatory potential in experimental colitis using the CD4+CD62L+ T cell transfer model. Results: Ti-Treg cells significantly suppressed Th1 mediated colitis on CD4+CD62L+ T cell transfer in vivo, as shown by high resolution endoscopy, histology, immunohistochemistry, and cytokine analysis. Further analysis of in vivo and in vitro expanded Ti-Treg cells showed that exogenous interleukin 2 (IL-2) was crucial for survival and expansion of these cells. Conclusion: Our data suggest that regulatory Ti-Treg cells expand by TGF-β and exogenous IL-2 derived from effector T cells at the site of inflammation. In addition to Tr1 and thymic CD4+CD25+ T cells, peripheral Ti-Treg cells emerge as a class of regulatory T cells with therapeutic potential in T cell mediated chronic intestinal inflammation.

Cutting Edge: IL-23 Cross-Regulates IL-12 Production in T Cell-Dependent Experimental Colitis

Although IL-12 and IL-23 share the common p40 subunit, IL-23, rather than IL-12, seems to drive the pathogenesis of experimental autoimmune encephalomyelitis and arthritis, because IL-23/p19 knockout mice are protected from disease. In contrast, we describe in this study that newly created LacZ knockin mice deficient for IL-23 p19 were highly susceptible for the development of experimental T cell-mediated TNBS colitis and showed even more severe colitis than wild-type mice by endoscopic and histologic criteria. Subsequent studies revealed that dendritic cells from p19-deficient mice produce elevated levels of IL-12, and that IL-23 down-regulates IL-12 expression upon TLR ligation. Finally, in vivo blockade of IL-12 p40 in IL-23-deficient mice rescued mice from lethal colitis. Taken together, our data identify cross-regulation of IL-12 expression by IL-23 as novel key regulatory pathway during initiation of T cell dependent colitis.

Cutting Edge: Trans-Signaling via the Soluble IL-6R Abrogates the Induction of FoxP3 in Naive CD4+CD25− T Cells

Chronic inflammatory diseases may develop when regulatory T cells (Tregs) fail to control the balance between tolerance and immunity. Alternatively, activated immune cells might prevent the induction or activation of Tregs in such diseases. In this study, we demonstrate that trans-signaling into T cells via the soluble IL-6 receptor completely abrogates the de novo induction of adaptive Tregs. Mechanistically, IL-6 trans-signaling augmented the expression of the TGF-β signaling inhibitor SMAD7. Consequently, SMAD7 overexpression in T cells using newly created transgenic mice rendered CD4+CD25− T cells resistant to the induction of FoxP3. Finally, IL-6 trans-signaling inhibited Treg-mediated suppression in a murine model of colitis. In summary, IL-6 trans-signaling into T cells emerges as a key pathway for blockade of the development of adaptive Tregs and thus may play a pivotal role in shifting the balance between effector and regulatory T cell numbers in chronic inflammatory and autoimmune diseases.

In vivo imaging of colitis and colon cancer development in mice using high resolution chromoendoscopy

Background: Mouse models of colitis and cancer are indispensable for our understanding of the pathogenesis of these diseases. In the past, mice had to be sacrificed in order to analyse colitis activity and tumour development. We have developed a safe method for high resolution endoscopic monitoring of living mice. Methods: Mice developing colitis or colonic tumours were anaesthetised using avertine and repeatedly examined by endoscopy. A novel miniendoscope (1.9 mm outer diameter), denoted Coloview, was introduced via the anus and the colon was carefully insufflated with an air pump before analysis of the colonic mucosa. An extra working channel allowed the introduction of biopsy forceps or injection needles as well as surface staining with methylene blue in order to visualise the surface of the crypts and the pit pattern architecture. Results: Endoscopic pictures obtained were of high quality and allowed monitoring and grading of disease. Scoring of colitis activity as well as tumour size and growth was possible. In addition, pit pattern analysis using chromoendoscopy permitted discrimination between inflammatory and neoplastic changes. Biopsies yielded enough tissue for molecular and histopathological analyses. Conclusions: In summary, chromoendoscopy in mice allows monitoring of the development of colitis and colon cancer with high resolution. Manipulations such as local injection of reagents or taking biopsies can be performed easily.

IL-6 Signaling Promotes Tumor Growth in Colorectal Cancer

Recent investigations support an important role for TGF-? in the development of colorectal cancer. However, the molecular consequences ofTGF-? signaling in the colon remains incompletely understood. In a recent study in Immunity, we analyzed the role of TGF-? in a murine model of colon cancer. Using transgenic mice overexpressing TGF-? or a dominant negative TGF-? receptor II under control of the CD2 minigene, we show that TGF-? signaling in tumor infiltrating T lymphocytes regulates the growth of dysplastic colon epithelial cells, as determined by histology and a novel system for high resolution chromoendoscopy in vivo. At the molecular level, TGF-? signaling in T cells regulated STAT-3 activation in tumor cells via IL-6. IL-6 signaling required tumor cell derived soluble IL-6R rather than membrane bound IL-6R and suppression of such TGF-?-dependent IL-6 trans-signaling prevented tumor progression in vivo. Similar to these observations in mice, here we show that human colon cancer tissue expressed only low amounts of membrane bound IL-6R. In contrast, expression and activity of the matrix metalloproteinase TACE were increased. In summary, our data provide novel insights into the role of TGF-? signaling in colorectal cancer and suggest novel therapeutic approaches for colorectal cancer based on an inhibition of TGF-?-dependent IL-6 trans-signaling.

A20 deficiency in B cells enhances B-cell proliferation and results in the development of autoantibodies

A20/TNFAIP3 is an ubiquitin‐editing enzyme, important for the regulation of the NF‐κB pathway. Mutations in the TNFAIP3 gene have been linked to different human autoimmune disorders. In human B‐cell lymphomas, the inactivation of A20 results in constitutive NF‐κB activation. Recent studies demonstrate that in mice the germline inactivation of A20 leads to early lethality, due to inflammation in multiple organs of the body. In this report, we describe a new mouse strain allowing for the tissue‐specific deletion of A20. We show that B‐cell‐specific deletion of A20 results in a dramatic reduction in marginal zone B cells. Furthermore, A20‐deficient B cells display a hyperactive phenotype represented by enhanced proliferation upon activation. Finally, these mice develop higher levels of serum immunoglobulins, resulting in an excessive production of self‐reactive autoantibodies.

Interleukin 17 Drives Vascular Inflammation, Endothelial Dysfunction, and Arterial Hypertension in Psoriasis-Like Skin Disease

Objective— Interleukin (IL)-17A is regarded as an important cytokine to drive psoriasis, an inflammatory skin disease marked by increased cardiovascular mortality. We aimed to test the hypothesis that overproduction of IL-17A in the skin leading to dermal inflammation may systemically cause vascular dysfunction in psoriasis-like skin disease. Approach and Results— Conditional overexpression of IL-17A in keratinocytes caused severe psoriasis-like skin inflammation in mice (K14-IL-17Aind/+ mice), associated with increased reactive oxygen species formation and circulating CD11b+ inflammatory leukocytes in blood, with endothelial dysfunction, increased systolic blood pressure, left ventricular hypertrophy, and reduced survival compared with controls. In K14-IL-17Aind/+ mice, immunohistochemistry and flow cytometry revealed increased vascular production of the nitric oxide/superoxide reaction product peroxynitrite and infiltration of the vasculature with myeloperoxidase+CD11b+GR1+F4/80− cells accompanied by increased expression of the inducible nitric oxide synthase and the nicotinamide dinucleotide phosphate (NADPH) oxidase, nox2. Neutrophil depletion by anti-GR-1 antibody injections reduced oxidative stress in blood and vessels. Neutralization of tumor necrosis factor-&agr; and IL-6 (both downstream of IL-17A) reduced skin lesions, attenuated oxidative stress in heart and blood, and partially improved endothelial dysfunction in K14-IL-17Aind/+ mice. Conclusions— Dermal overexpression of IL-17A induces systemic endothelial dysfunction, vascular oxidative stress, arterial hypertension, and increases mortality mainly driven by myeloperoxidase+CD11b+GR1+F4/80− inflammatory cells. Depletion of the GR-1+ immune cells or neutralization of IL-17A downstream cytokines by biologicals attenuates the vascular phenotype in K14-IL-17Aind/+ mice.

Activation of epithelial STAT3 regulates intestinal homeostasis

The intestinal epithelium that lines the mucosal surface along the GI-tract is a key player for the intestinal homeostasis of the healthy individual. In case of a mucosal damage or a barrier defect as seen in patients with inflammatory bowel disease, the balance is disturbed, and translocation of intestinal microbes to the submucosa is facilitated. We recently demonstrated a pivotal role of STAT3 activation in intestinal epithelial cells (IEC) for the restoration of the balance at the mucosal surface of the gut in an experimental colitis model. STAT3 was rapidly induced in intestinal epithelial cells upon challenge of mice in both experimental colitis and intestinal wound healing models. STAT3 activation was found to be dispensable in the steady-state conditions but was important for efficient regeneration of the epithelium in response to injury. Here, we extend our previous findings by showing epithelial STAT3 activation in human patients suffering from IBD and provide additional insights how the activation of epithelial STAT3 by IL-22 regulates intestinal homeostasis and mucosal wound healing. We also demonstrate that antibody-mediated neutralization of IL-22 has little impact on the development of experimental colitis in mice, but significantly delays recovery from colitis. Thus, our data suggest that targeting the STAT3 signalling pathway in IEC is a promising therapeutic approach in situations when the intestinal homeostasis is disturbed, e.g. as seen in Crohn´s disease or Ulcerative colitis.

Transcription factor NFATc2 controls the emergence of colon cancer associated with IL-6-dependent colitis

NFAT transcription factors control T-cell activation and function. Specifically, the transcription factor NFATc2 affects the regulation of cell differentiation and growth and plays a critical role in the development of colonic inflammation. Here, we used an experimental model of colitis-associated colorectal carcinoma to investigate the contribution of NFATc2 to the promotion of colonic tumors. Compared with wild-type animals that readily presented with multiple colon tumors, NFATc2-deficient mice were protected from tumor development. This observed decrease in colonic tumor progression was associated with reduced endoscopic inflammation, increased apoptosis of lamina propria T lymphocytes, and significantly reduced levels of the critical proinflammatory cytokines interleukin (IL)-21 and IL-6. Administration of hyper IL-6 abrogated protection from tumor progression in NFATc2-knockout mice and restored tumor incidence to control levels. Taken together, our findings highlight a pivotal role for NFATc2 in the establishment of inflammation-associated colorectal tumors mediated by control of IL-6 expression.