[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Intestinal bacteria form a resident community that has co-evolved with the mammalian host. In addition to playing important roles in digestion and harvesting energy, commensal bacteria are crucial for the proper functioning of mucosal immune defenses. Most of these functions have been attributed to the presence of large numbers of 'innocuous' resident bacteria that dilute or occupy niches for intestinal pathogens or induce innate immune responses that sequester bacteria in the lumen, thus quenching excessive activation of the mucosal immune system. However it has recently become obvious that commensal bacteria are not simply beneficial bystanders, but are important modulators of intestinal immune homeostasis and that the composition of the microbiota is a major factor in pre-determining the type and robustness of mucosal immune responses. Here we review specific examples of individual members of the microbiota that modify innate and adaptive immune responses, and we focus on potential mechanisms by which such species-specific signals are generated and transmitted to the host immune system.
Preview · Article · Feb 2011 · Current opinion in microbiology
[Show abstract][Hide abstract] ABSTRACT: 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 1β increased, whereas interferon-γ 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.
Full-text · Article · Feb 2011 · Journal of Experimental Medicine
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Interleukin (IL)-17-producing CD4(+) helper T cells (Th17) mediate mucosal immunity and are involved in the pathogenesis of inflammatory bowel disease (IBD). They are believed to arise from the same precursor population as regulatory T (Treg) cells, but little is known about how these T-cell subsets interact under chronic inflammatory conditions. We studied Th17 and Treg cells isolated from intestinal lamina propria of patients with IBD to investigate their role in pathogenesis.
FoxP3 expression (a marker of Treg cells) and IL-17 production were assessed in CD4(+) lamina propria lymphocytes isolated from IBD patients and healthy subjects. IL-17(+)FoxP3(+) and IL-17(+) CD4(+) T-cell clones were generated by limiting dilution. An in vitro suppression assay was performed to assess the functional capacity of derived T-cell clones.
IL-17(+)FoxP3(+) T cells were identified in inflamed intestinal mucosa of patients with Crohn disease (CD), but not in patients with ulcerative colitis (UC) or healthy controls. These cells shared phenotypic characteristics of Th17 and Treg cells, and showed potent suppressor activity in vitro. Transforming growth factor-β was necessary and sufficient to induce development of an IL-17(+) FoxP3(+) cell population in CD4(+) lamina propria lymphocytes derived from patients with UC.
The inflammatory environment in the intestinal mucosa of patients with CD contributes to the generation of a distinct population of Treg cells that are FoxP3(+) and produce IL-17. These cells are likely to arise during differentiation of Th17 and Treg cells. Specific microenvironmental cues from tissues are likely to determine their commitment to either lineage and affect the balance between regulation and inflammation in the intestine.
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells serve a key function in host defence, linking innate detection of microbes to activation of pathogen-specific adaptive immune responses. Whether there is cell-intrinsic recognition of human immunodeficiency virus (HIV) by host innate pattern-recognition receptors and subsequent coupling to antiviral T-cell responses is not yet known. Dendritic cells are largely resistant to infection with HIV-1, but facilitate infection of co-cultured T-helper cells through a process of trans-enhancement. Here we show that, when dendritic cell resistance to infection is circumvented, HIV-1 induces dendritic cell maturation, an antiviral type I interferon response and activation of T cells. This innate response is dependent on the interaction of newly synthesized HIV-1 capsid with cellular cyclophilin A (CYPA) and the subsequent activation of the transcription factor IRF3. Because the peptidylprolyl isomerase CYPA also interacts with HIV-1 capsid to promote infectivity, our results indicate that capsid conformation has evolved under opposing selective pressures for infectivity versus furtiveness. Thus, a cell-intrinsic sensor for HIV-1 exists in dendritic cells and mediates an antiviral immune response, but it is not typically engaged owing to the absence of dendritic cell infection. The virulence of HIV-1 may be related to evasion of this response, the manipulation of which may be necessary to generate an effective HIV-1 vaccine.
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Sep 2010 · Genes & development
[Show abstract][Hide abstract] ABSTRACT: Commensal microbes can have a substantial impact on autoimmune disorders, but the underlying molecular and cellular mechanisms remain largely unexplored. We report that autoimmune arthritis was strongly attenuated in the K/BxN mouse model under germ-free (GF) conditions, accompanied by reductions in serum autoantibody titers, splenic autoantibody-secreting cells, germinal centers, and the splenic T helper 17 (Th17) cell population. Neutralization of interleukin-17 prevented arthritis development in specific-pathogen-free K/BxN mice resulting from a direct effect of this cytokine on B cells to inhibit germinal center formation. The systemic deficiencies of the GF animals reflected a loss of Th17 cells from the small intestinal lamina propria. Introduction of a single gut-residing species, segmented filamentous bacteria, into GF animals reinstated the lamina propria Th17 cell compartment and production of autoantibodies, and arthritis rapidly ensued. Thus, a single commensal microbe, via its ability to promote a specific Th cell subset, can drive an autoimmune disease.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Apr 2010 · Genes & development
[Show abstract][Hide abstract] ABSTRACT: HIV-1 replication requires transport of nascent viral DNA and associated virion proteins, the retroviral preintegration complex (PIC), into the nucleus. Too large for passive diffusion through nuclear pore complexes (NPCs), PICs use cellular nuclear transport mechanisms and nucleoporins (NUPs), the NPC components that permit selective nuclear-cytoplasmic exchange, but the details remain unclear. Here we identify a fragment of the cleavage and polyadenylation factor 6, CPSF6, as a potent inhibitor of HIV-1 infection. When enriched in the cytoplasm, CPSF6 prevents HIV-1 nuclear entry by targeting the viral capsid (CA). HIV-1 harboring the N74D mutation in CA fails to interact with CPSF6 and evades the nuclear import restriction. Interestingly, whereas wild-type HIV-1 requires NUP153, N74D HIV-1 mimics feline immunodeficiency virus nuclear import requirements and is more sensitive to NUP155 depletion. These findings reveal a remarkable flexibility in HIV-1 nuclear transport and highlight a single residue in CA as essential in regulating interactions with NUPs.
[Show abstract][Hide abstract] ABSTRACT: The vertebrate immune system is poised in a state of equilibrium that permits accurate and rapid protective responses against pathogens but curtails potential for causing harm to the host through targeting of "self" and provoking overexuberant inflammatory processes. In this Review we discuss this balance achieved in large part by interactions of different classes of T lymphocytes that have potent pro- or anti-inflammatory activity in the context of genetic and environmental factors, particularly the commensal microbiota.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Identification of the Th17 T cell subset as important mediators of host defense and pathology prompted us to determine their susceptibility to human immunodeficiency virus (HIV) infection.
We found that a sizeable portion of Th17 cells express HIV coreceptor CCR5 and produce very low levels of CCR5 ligands macrophage inflammatory protein (MIP)-1alpha and MIP-1beta. Accordingly, CCR5(+) Th17 cells were efficiently infected with CCR5-tropic HIV and were depleted during viral replication in vitro. Remarkably, HIV-infected individuals receiving treatment had significantly reduced Th17 cell counts, compared with HIV-uninfected subjects, regardless of viral load or CD4 cell count, whereas treatment-naive subjects had normal levels. However, there was a preferential reduction in CCR5(+) T cells that were also CCR6 positive, which is expressed on all Th17 cells, compared with CCR6(-)CCR5(+) cells, in both treated and untreated HIV-infected subjects. This observation suggests preferential targeting of CCR6(+)CCR5(+) Th17 cells by CCR5-tropic viruses in vivo. Th17 cell levels also inversely correlated with activated CD4(+) T cells in HIV-infected individuals who are receiving treatment.
Our findings suggest a complex perturbation of Th17 subsets during the course of HIV disease potentially through both direct viral infection and virus indirect mechanisms, such as immune activation.
Full-text · Article · Feb 2010 · The Journal of Infectious Diseases