Faculty Bibliography

Two groups have identified SAMHD1, a protein encoded by an Aicardi-Goutieres Syndrome susceptibility gene, as the factor that restricts infection of macrophages and dendritic cells with HIV-1. Here we discuss implications of this discovery for induction of antiviral protective immunity.

Micro-RNAs (miRNAs) are short (average 22 nucleotides) noncoding regulatory RNAs that inhibit gene expression by targeting complementary 3'-untranslated regions of protein-encoding mRNAs for translational repression or degradation. miRNAs play key roles in both the function and differentiation of many cell types. Drosha and Dicer, two RNAase III enzymes, function in a stepwise manner to generate a mature miRNA. Previous studies have shown that podocyte-specific deletion of Dicer during development results in proteinuric renal disease and collapsing glomerulopathy (CG); however, Dicer has functions other than the generation of miRNAs. Here we found that the podocyte-specific deletion of Drosha results in a similar phenotype to Dicer mutants, confirming that the Dicer mutant phenotype is due to the loss of miRNAs. Moreover, the inducible deletion of Drosha in 2- to 3-month-old mice (Tet-On system) resulted in CG. Thus, continuous generation of miRNAs are required for the normal function of mature podocytes and their loss leads to CG. Identifying these miRNAs may provide new insight into disease pathogenesis and novel therapeutic targets in various podocytopathies

Perturbations of the composition of the symbiotic intestinal microbiota can have profound consequences for host metabolism and immunity. In mice, segmented filamentous bacteria (SFB) direct the accumulation of potentially proinflammatory Th17 cells in the intestinal lamina propria. We present the genome sequence of SFB isolated from monocolonized mice, which classifies SFB phylogenetically as a unique member of Clostridiales with a highly reduced genome. Annotation analysis demonstrates that SFB depend on their environment for amino acids and essential nutrients and may utilize host and dietary glycans for carbon, nitrogen, and energy. Comparative analyses reveal that SFB are functionally related to members of the genus Clostridium and several pathogenic or commensal 'minimal' genera, including Finegoldia, Mycoplasma, Borrelia, and Phytoplasma. However, SFB are functionally distinct from all 1200 examined genomes, indicating a gene complement representing biology relatively unique to their role as a gut commensal closely tied to host metabolism and immunity

CD4 coreceptor expression is negatively regulated through activity of the Cd4 silencer in CD4(-)CD8(-) double-negative (DN) thymocytes and CD8(+) cytotoxic lineage T cells. Whereas Cd4 silencing is reversed during transition from DN to CD4(+)CD8(+) double-positive stages, it is maintained through heritable epigenetic processes following its establishment in mature CD8(+) T cells. We previously demonstrated that the Runx family of transcription factors is required for Cd4 silencing both in DN thymocytes and CD8(+) T cells. However, additional factors that cooperate with Runx proteins in the process of Cd4 silencing remain unknown. To identify collaborating factors, we used microarray and RNAi-based approaches and found the basic helix-loop-helix ZIP transcription factor AP4 to have an important role in Cd4 regulation. AP4 interacts with Runx1 in cells in which Cd4 is silenced, and is required for Cd4 silencing in immature DN thymocytes through binding to the proximal enhancer. Furthermore, although AP4-deficient CD8(+) T cells appeared to normally down-regulate CD4 expression, AP4 deficiency significantly increased the frequency of CD4-expressing effector/memory CD8(+) T cells in mice harboring point mutations in the Cd4 silencer. Our results suggest that AP4 contributes to Cd4 silencing both in DN and CD8(+) T cells by enforcing checkpoints for appropriate timing of CD4 expression and its epigenetic silencing

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

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness

Loss of CXCL12, a leukocyte localizing cue, from abluminal surfaces of the blood-brain barrier occurs in multiple sclerosis (MS) lesions. However, the mechanisms and consequences of reduced abluminal CXCL12 abundance remain unclear. Here, we show that activation of CXCR7, which scavenges CXCL12, is essential for leukocyte entry via endothelial barriers into the central nervous system (CNS) parenchyma during experimental autoimmune encephalomyelitis (EAE), a model for MS. CXCR7 expression on endothelial barriers increased during EAE at sites of inflammatory infiltration. Treatment with a CXCR7 antagonist ameliorated EAE, reduced leukocyte infiltration into the CNS parenchyma and parenchymal VCAM-1 expression, and increased abluminal levels of CXCL12. Interleukin 17 and interleukin 1beta increased, whereas interferon-gamma decreased, CXCR7 expression on and CXCL12 internalization in primary brain endothelial cells in vitro. These findings identify molecular requirements for the transvascular entry of leukocytes into the CNS and suggest that CXCR7 blockade may have therapeutic utility for the treatment of MS

Commensal intestinal bacteria are indispensible for the proper functioning of the mammalian immune system. Both mucosal and systemic immune mechanisms are profoundly affected by the gut microbiota. Commensals provide not only essential immune protection against life-threatening infections, but shape pre-existing immune responses and thus modulate the ability of the host to respond to environmental challenges. These immunomodulatory functions are dependent on the composition of the commensal community and seem to be a property of individual members of this community. Investigation of the underlying mechanisms requires the identification of specific examples of such interactions. We have identified the murine commensal segmented filamentous bacteria (SFB) as capable of specifically shifting the homeostasis of effector T cell subsets in the small intestinal lamina propria. SFB induce preferentially Th17 cells and augment Th17 celldependent immune responses. As a first step toward the identification of immunomodulatory SFB products, we have sequenced the SFB genome and have performed initial analysis based on its annotation. This information will hopefully help the investigation of the mechanisms by which SFB affect intestinal immune responses and the development of new genetic tools for the study of the biology of this unique microorganism