Faculty Bibliography

BACKGROUND & AIMS: Although T-helper (Th) 17 and Th1 cells are involved in pathogenesis of intestinal inflammation, their developmental pathways and sufficiency to promote disease are not known; nor are the roles of CD4(+)CD25(+) regulatory T (T(R)) cells in their development. METHODS: We performed adoptive transfer experiments to investigate the induction and suppression of colitis using naive CD4(+)CD45RB(high) T cells and/or CD4(+)CD25(+) T(R) cells that were obtained from retinoid-related orphan receptor gamma t (RORgammat) gfp/(+) or Ly5.1/Ly5.2 congenic mice. RESULTS: We observed 3 types of colitogenic CD4(+) Th1 cells (interleukin [IL]-17A(-)interferon [IFN]-gamma(+)): RORgammat(-) classical Th1 cells that differentiated directly from naive T cells; RORgammat(+) Th1-like cells; and RORgammat(-) alternative Th1 cells that were terminally differentiated from RORgammat(+) cells via Th17 (IL-17A(+)IFN-gamma(-)), Th17/Th1 (IL-17A(+)IFN-gamma(+)), or Th1-like (IL-17A(-)IFN-gamma(+)) cells. In this pathway, CD4(+)CD25(+) T(R) cells suppress the development of not only classical Th1 cells, but also alternative Th1 cells at the transition of Th17/Th1 into alternative Th1 cells, resulting in accumulation of Th17 and Th17/Th1 cells in mice in which the development of colitis was suppressed. Furthermore, T(R) cells regulated the established balance of Th17 and Th1 cells under colitic conditions to yield a high ratio of Th17 and Th17/Th1 cells to Th1 cells in noncolitic conditions. CONCLUSIONS: Th17 and Th17/Th1 cells become colitogenic alternative Th1 cells via Th17, Th17/Th1, and Th1-like cells, independently of classical Th1 cells. T(R) cells suppress this pathway, resulting in accumulation of Th17 and Th17/Th1 cells

CD4(+) T helper lymphocytes that express interleukin-17 (T(H)17 cells) have critical roles in mouse models of autoimmunity, and there is mounting evidence that they also influence inflammatory processes in humans. Genome-wide association studies in humans have linked genes involved in T(H)17 cell differentiation and function with susceptibility to Crohn's disease, rheumatoid arthritis and psoriasis. Thus, the pathway towards differentiation of T(H)17 cells and, perhaps, of related innate lymphoid cells with similar effector functions, is an attractive target for therapeutic applications. Mouse and human T(H)17 cells are distinguished by expression of the retinoic acid receptor-related orphan nuclear receptor RORgammat, which is required for induction of IL-17 transcription and for the manifestation of T(H)17-dependent autoimmune disease in mice. By performing a chemical screen with an insect cell-based reporter system, we identified the cardiac glycoside digoxin as a specific inhibitor of RORgammat transcriptional activity. Digoxin inhibited murine T(H)17 cell differentiation without affecting differentiation of other T cell lineages and was effective in delaying the onset and reducing the severity of autoimmune disease in mice. At high concentrations, digoxin is toxic for human cells, but non-toxic synthetic derivatives 20,22-dihydrodigoxin-21,23-diol and digoxin-21-salicylidene specifically inhibited induction of IL-17 in human CD4(+) T cells. Using these small-molecule compounds, we demonstrate that RORgammat is important for the maintenance of IL-17 expression in mouse and human effector T cells. These data indicate that derivatives of digoxin can be used as chemical templates for the development of RORgammat-targeted therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease

T cell fate is associated with mutually exclusive expression of CD4 or CD8 in helper and cytotoxic T cells, respectively. How expression of one locus is temporally coordinated with repression of the other has been a long-standing enigma, though we know RUNX transcription factors activate the Cd8 locus, silence the Cd4 locus, and repress the Zbtb7b locus (encoding the transcription factor ThPOK), which is required for CD4 expression. Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart. Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8. Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells

The commensal microbiota that inhabit different parts of the gastrointestinal (GI) tract have been shaped by coevolution with the host species. The symbiotic relationship of the hundreds of microbial species with the host requires a tuned response that prevents host damage, e.g., inflammation, while tolerating the presence of the potentially beneficial microbes. Recent studies have begun to shed light on immunological processes that participate in maintenance of homeostasis with the microbiota and on how disturbance of host immunity or the microbial ecosystem can result in disease-provoking dysbiosis. Our growing appreciation of this delicate host-microbe relationship promises to influence our understanding of inflammatory diseases and infection by microbial pathogens and to provide new therapeutic opportunities

CD4⁺ T helper (Th) cells play a critical role in the development of Graft-versus-Host Disease (GvHD). The relative contributions of particular Th subsets to GVHD pathogenesis, however, are incompletely understood. In order to clarify the contribution of the Th17 subset to GVHD induction, we made use of mice knocked out at the RORgt locus (RORgt-/-), a transcription factor crucial for Th17 polarization. Methods: Haplotype matched and complete MHC mismatched murine HSCT models were used. For the haploidentical model C57BL/6 (H-2b, B6) mice served as donors while C57BL/6 x

Commensal bacteria are crucial for maturation and function of the mucosal immune system. However, the mechanisms of these interactions are poorly understood. In addition, the role of the composition of the microbiota and the importance of individual species in this community in stimulating different types of immunity are major unanswered questions. We recently showed that the balance between two major effector T cell populations in the intestine, IL-17(+) Th17 cells and Foxp3(+) Tregs, requires signals from commensal bacteria and is dependent on the composition of the intestinal microbiota. Comparison of microbiota from Th17 cell-deficient and Th17 cell-sufficient mice identified segmented filamentous bacteria (SFB) as capable of specifically inducing Th17 cells in the gut. SFB represent the first example of a commensal species that can skew the mucosal effector T cell balance and thus affect the immune fitness of the individual

The canonical microRNA (miRNA) biogenesis pathway requires two RNaseIII enzymes: Drosha and Dicer. To understand their functions in mammals in vivo, we engineered mice with germline or tissue-specific inactivation of the genes encoding these two proteins. Changes in proteomic and transcriptional profiles that were shared in Dicer- and Drosha-deficient mice confirmed the requirement for both enzymes in canonical miRNA biogenesis. However, deficiency in Drosha or Dicer did not always result in identical phenotypes, suggesting additional functions. We found that, in early-stage thymocytes, Drosha recognizes and directly cleaves many protein-coding messenger RNAs (mRNAs) with secondary stem-loop structures. In addition, we identified a subset of miRNAs generated by a Dicer-dependent but Drosha-independent mechanism. These were distinct from previously described mirtrons. Thus, in mammalian cells, Dicer is required for the biogenesis of multiple classes of miRNAs. Together, these findings extend the range of function of RNaseIII enzymes beyond canonical miRNA biogenesis, and help explain the nonoverlapping phenotypes caused by Drosha and Dicer deficiency

The stability of a lineage program (cellular memory) is dependent on mechanisms that epigenetically maintain active or repressed states of gene expression (transcriptional memory). Although epigenetic silencing of genes has been clearly demonstrated from yeast to mammals, heritable maintenance of active transcription has been less clearly defined. To investigate the potential role of active transcriptional memory during lineage diversification, we employed targeted mutation of a positive-acting cis element in the Cd4 locus to determine the impact on CD4 expression and the differentiation of CD4(+) helper T cells in mice. We show that the proximal enhancer (E4(P)) of Cd4 is essential for CD4 expression in immature CD4(+)8(+) thymocytes. Furthermore, its loss resulted in reduced and unstable expression of CD4 in mature T cells. However, if the enhancer was deleted after cells had already committed to the helper T-cell lineage, CD4 expression remained high and was stable upon cell division. 'Active' histone modifications, once initiated by E4(P), were also propagated independently of the enhancer. Thus, E4(P) is responsible for establishing an epigenetically inherited active Cd4 locus in the helper T-cell lineage. To our knowledge, this is the first genetic demonstration of active transcriptional memory in mammalian cells

The key role of interleukin (IL)-23 in the pathogenesis of autoimmune and chronic inflammatory disorders is supported by the identification of IL-23 receptor (IL-23R) susceptibility alleles associated with inflammatory bowel disease, psoriasis and ankylosing spondylitis. IL-23-driven inflammation has primarily been linked to the actions of T-helper type 17 (TH17) cells. Somewhat overlooked, IL-23 also has inflammatory effects on innate immune cells and can drive T-cell-independent colitis. However, the downstream cellular and molecular pathways involved in this innate intestinal inflammatory response are poorly characterized. Here we show that bacteria-driven innate colitis is associated with an increased production of IL-17 and interferon-gamma in the colon. Stimulation of colonic leukocytes with IL-23 induced the production of IL-17 and interferon-gamma exclusively by innate lymphoid cells expressing Thy1, stem cell antigen 1 (SCA-1), retinoic-acid-related orphan receptor (ROR)-gammat and IL-23R, and these cells markedly accumulated in the inflamed colon. IL-23-responsive innate intestinal cells are also a feature of T-cell-dependent models of colitis. The transcription factor ROR-gammat, which controls IL-23R expression, has a functional role, because Rag-/-Rorc-/- mice failed to develop innate colitis. Last, depletion of Thy1+ innate lymphoid cells completely abrogated acute and chronic innate colitis. These results identify a previously unrecognized IL-23-responsive innate lymphoid population that mediates intestinal immune pathology and may therefore represent a target in inflammatory bowel disease

The RUNX1/AML1 gene is the most frequently mutated gene in human leukemia. Conditional deletion of Runx1 in adult mice results in an increase of hematopoietic stem cells (HSCs), which serve as target cells for leukemia; however, Runx1(-/-) mice do not develop spontaneous leukemia. Here we show that maintenance of Runx1(-/-) HSCs is compromised, progressively resulting in HSC exhaustion. In leukemia development, the stem cell exhaustion was rescued by additional genetic changes. Retroviral insertional mutagenesis revealed Evi5 activation as a cooperating genetic alteration and EVI5 overexpression indeed prevented Runx1(-/-) HSC exhaustion in mice. Moreover, EVI5 was frequently overexpressed in human RUNX1-related leukemias. These results provide insights into the mechanism for maintenance of pre-leukemic stem cells and may provide a novel direction for therapeutic applications