SUMMARY The gastrointestinal tract of mammals is inhabited by hundreds of distinct species of commensal microorganisms that exist in a mutualistic relationship with the host. How commensal microbiota influence the host immune system is poorly understood. We show here that colonization of the small intestine of mice with a single commensal microbe, segmented filamentous bacterium (SFB), is sufficient to induce the appearance of CD4+ T helper cells that produce IL-17 and IL-22 (Th17 cells) in the lamina propria. SFB adhere tightly to the surface of epithelial cells in the terminal ileum of mice with Th17 cells but are absent from mice that have few Th17 cells. Colonization with SFB was correlated with increased expression of genes associated with inflammation and anti-microbial defenses, and resulted in enhanced resistance to the intestinal pathogen Citrobacter rodentium. Thus, manipulation of this commensalregulated pathway may provide new opportunities for enhancing mucosal immunity and treating autoimmune disease.

SUMMARY IL-17-producing T-helper cells (Th17) are potent effectors of inflammation, but little is known about the requirements for their differentiation in vivo at steady state. We found that specific commensal microbiota are required for Th17 cell differentiation in the lamina propria (LP) of the small intestine. Differentiation of Th17 cells correlated with presence of Cytophaga-Flavobacter-Bacteroidetes bacteria in the intestine, was independent of TLR, IL-21 or IL-23 signaling, but required appropriate activation of TGF-β. Absence of Th17 cell-inducing bacteria was accompanied by increased Foxp3+ regulatory T cells in the LP. Our results suggest that the composition of the microbiota regulates the Th17:Treg balance in the lamina propria and may thus influence intestinal immunity, tolerance, and susceptibility to inflammatory bowel diseases.

T helper 17 cells (T H -17 cells) are interleukin 17 (IL-17)-secreting CD4 + T cells involved in autoimmune disease and mucosal immunity. In naive CD4 + T cells from mice, IL-17 is expressed in response to a combination of IL-6 or IL-21 and transforming growth factor-β (TGF-β) and requires induction of the transcription factor RORγt. It has been suggested that human T H -17 cell differentiation is independent of TGF-β and thus differs fundamentally from mouse. We show here that a combination of TGF-β, IL-1β and IL-6, IL-21 or IL-23 in serum-free conditions was necessary and sufficient to induce IL-17 expression in naive human CD4 + T cells from cord blood. TGF-β upregulated RORγT expression but at the same time inhibited its ability to induce IL-17 expression. Inflammatory cytokines relieved this inhibition and increased IL-17 expression directed by RORγT, similar to what has been reported with mouse cells. Other gene products detected in T H -17 cells upon RORγT induction include CCR6, the IL-23 receptor (IL23R), IL17F and IL26. Together, these studies identify RORγT as having a central role in differentiation of human T H -17 cells from naive CD4 + T cells and suggest that similar cytokine pathways are involved in this process in mouse and human.T H -17 cells, the T helper cells that produce IL-17 and other pro-inflammatory cytokines, have been shown to have key functions in a wide variety of autoimmune disease models in mice and are thought to be similarly involved in human disease (reviewed 1-3). In healthy humans, IL-17-secreting cells are present in the CD45RO + CCR6 + populations of T cells from peripheral blood4,5 and gut5. T H -17 cells or their products have been associated with the pathology of multiple inflammatory or autoimmune disorders in humans. IL-17 protein and T H -17 CD4 + T cells are found in lesions from multiple sclerosis patients6-8 where they are thought to contribute to the disruption of the blood-brain barrier9. IL-17 is produced by CD4 + T cells of rheumatoid synovium10 and is thought to contribute to inflammation in rheumatoid arthritis11,12. In psoriasis, products associated with T H -17 cells, including IL-17, IL-17F, . IL-17 is induced in the gut mucosa from Crohn's disease and ulcerative colitis patients and T H -17 cells are detected13,16. IL-23, which is produced by dendritic cells in the intestine17, contributes significantly to T H -17 cell differentiation18. Strikingly, polymorphisms in the IL23R gene are associated with Crohn's disease, further implicating the T H -17 cell pathway in the pathogenesis19. HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptThe mechanisms leading to differentiation of T H -17 cells have been well established in mice but they are still poorly understood in humans. In mice, differentiation of T H -17 cells that secrete IL-17 (also referred to as IL-17A) and IL-17F requires the expression of the transcription factors RORγt, an orphan nuclear hormone receptor, STAT3 and IRF4 (reviewed in reference 20). RORγt is sufficien...

SummaryExosomes are extracellular vesicles (EVs) secreted upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane. The mechanisms involved in their biogenesis have not yet been fully identified although they could be used to modulate exosome formation and therefore are a promising tool in understanding exosome functions. We have performed an RNA interference screen targeting 23 components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins in MHC class II (MHC II)-expressing HeLa-CIITA cells. Silencing of HRS, STAM1 or TSG101 reduced the secretion of EV-associated CD63 and MHC II but each gene altered differently the size and/or protein composition of secreted EVs, as quantified by immuno-electron microscopy. By contrast, depletion of VPS4B augmented this secretion while not altering the features of EVs. For several other ESCRT subunits, it was not possible to draw any conclusions about their involvement in exosome biogenesis from the screen. Interestingly, silencing of ALIX increased MHC II exosomal secretion, as a result of an overall increase in intracellular MHC II protein and mRNA levels. In human dendritic cells (DCs), ALIX depletion also increased MHC II in the cells, but not in the released CD63-positive EVs. Such differences could be attributed to a greater heterogeneity in size, and higher MHC II and lower CD63 levels in vesicles recovered from DCs as compared with HeLa-CIITA. The results reveal a role for selected ESCRT components and accessory proteins in exosome secretion and composition by HeLa-CIITA. They also highlight biogenetic differences in vesicles secreted by a tumour cell line and primary DCs.

In eukaryotic cells, the nuclear envelope separates the genomic DNA from the cytoplasmic space and regulates protein trafficking between the two compartments. This barrier is only transiently dissolved during mitosis. Here, we found that it also opened at high frequency in migrating mammalian cells during interphase, which allowed nuclear proteins to leak out and cytoplasmic proteins to leak in. This transient opening was caused by nuclear deformation and was rapidly repaired in an ESCRT (endosomal sorting complexes required for transport)-dependent manner. DNA double-strand breaks coincided with nuclear envelope opening events. As a consequence, survival of cells migrating through confining environments depended on efficient nuclear envelope and DNA repair machineries. Nuclear envelope opening in migrating leukocytes could have potentially important consequences for normal and pathological immune responses.

Dissection of the genetic basis of Aicardi-Goutières syndrome has highlighted a fundamental link between nucleic acid metabolism, innate immune sensors and type I interferon induction. This had led to the concept of the human interferonopathies as a broader set of Mendelian disorders in which a constitutive upregulation of type I interferon activity directly relates to disease pathology. Here, we discuss the molecular and cellular basis of the interferonopathies, their categorization, future treatment strategies and the insights they provide into normal physiology.

Dendritic cells (DC) serve a key function in host defense, linking innate detection of microbes to the activation of pathogen-specific adaptive immune responses(1,2). Whether there is cell-intrinsic recognition of HIV-1 by host innate pattern-recognition receptors and subsequent coupling to antiviral T cell responses is not yet known(3). DC are largely resistant to infection with HIV-1(4), but facilitate infection of co-cultured T-helper cells through a process of trans-enhancement(5,6). We show here that, when DC resistance to infection is circumvented(7,8), HIV-1 induces DC 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 (CA) with cellular cyclophilin A (CypA) and the subsequent activation of the transcription factor IRF3. Because the peptidyl-prolyl isomerase CypA also interacts with CA to promote HIV-1 infectivity, our results suggest that CA conformation has evolved under opposing selective pressures for infectivity versus furtiveness. Thus, a cell intrinsic sensor for HIV-1 exists in DC and mediates an antiviral immune response, but it is not typically engaged due to absence of DC infection. The virulence of HIV-1 may be related to evasion of this response, whose manipulation may be necessary to generate an effective HIV-1 vaccine.

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