Plasmacytoid dendritic cells promote HIV-1–induced group 3 innate lymphoid cell depletion

Group 3 innate lymphoid cells (ILC3s) have demonstrated roles in promoting antibacterial immunity, maintaining epithelial barrier function, and supporting tissue repair. ILC3 alterations are associated with chronic inflammation and inflammatory disease; however, the characteristics and relevant regulatory mechanisms of this cell population in HIV-1 infection are poorly understood due in part to a lack of a robust model. Here, we determined that functional human ILC3s develop in lymphoid organs of humanized mice and that persistent HIV-1 infection in this model depletes ILC3s, as observed in chronic HIV-1–infected patients. In HIV-1–infected mice, effective antiretroviral therapy reversed the loss of ILC3s. HIV-1–dependent reduction of ILC3s required plasmacytoid dendritic cells (pDCs), IFN-I, and the CD95/FasL pathway, as targeted depletion or blockade of these prevented HIV-1–induced ILC3 depletion in vivo and in vitro, respectively. Finally, we determined that HIV-1 infection induces CD95 expression on ILC3s via a pDC- and IFN-I–dependent mechanism that sensitizes ILC3s to undergo CD95/FasL-mediated apoptosis. We conclude that chronic HIV-1 infection depletes ILC3s through pDC activation, induction of IFN-I, and CD95-mediated apoptosis.     

NOD2 drives early IL-33–dependent expansion of group 2 innate lymphoid cells during Crohn’s disease–like ileitis

Innate lymphoid cells (ILCs) are enriched at barrier surfaces, including the gastrointestinal tract. While most studies have focused on the balance between pathogenic group 1 ILCs (ILC1s) and protective ILC3s in maintaining gut homeostasis and during chronic intestinal inflammation, such as Crohn’s disease (CD), less is known regarding ILC2s. Using an established murine model of CD-like ileitis, i.e., the SAMP1/YitFc (SAMP) mouse strain, we showed that ILC2s, compared with ILC1s and ILC3s, were increased within draining mesenteric lymph nodes and ilea of SAMP versus AKR (parental control) mice early, during the onset of disease. Gut-derived ILC2s from CD patients versus healthy controls were also increased and expanded, similarly to ILC1s, in greater proportion compared with ILC3s. Importantly, we report that the intracellular bacteria–sensing protein, nucleotide-binding oligomerization domaining–containing protein 2, encoded by Nod2, the first and strongest susceptibility gene identified for CD, promoted ILC2 expansion, which was dramatically reduced in SAMP mice lacking NOD2 and in SAMP mice raised under germ-free conditions. Furthermore, these effects occurred through a mechanism involving the IL-33/ST2 ligand-receptor pair. Collectively, our results indicate a functional link between NOD2 and ILC2s, regulated by the IL-33/ST2 axis, that mechanistically may contribute to early events leading to CD pathogenesis.     

Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage

Disruption of the intestinal epithelial barrier allows bacterial translocation and predisposes to destructive inflammation. To ensure proper barrier composition, crypt-residing stem cells continuously proliferate and replenish all intestinal epithelial cells within days. As a consequence of this high mitotic activity, mucosal surfaces are frequently targeted by anticancer therapies, leading to dose-limiting side effects. The cellular mechanisms that control tissue protection and mucosal healing in response to intestinal damage remain poorly understood. Type 3 innate lymphoid cells (ILC3s) are regulators of homeostasis and tissue responses to infection at mucosal surfaces. We now demonstrate that ILC3s are required for epithelial activation and proliferation in response to small intestinal tissue damage induced by the chemotherapeutic agent methotrexate. Multiple subsets of ILC3s are activated after intestinal tissue damage, and in the absence of ILC3s, epithelial activation is lost, correlating with increased pathology and severe damage to the intestinal crypts. Using ILC3-deficient Lgr5 reporter mice, we show that maintenance of intestinal stem cells after damage is severely impaired in the absence of ILC3s or the ILC3 signature cytokine IL-22. These data unveil a novel function of ILC3s in limiting tissue damage by preserving tissue-specific stem cells.The intestinal epithelium combines efficient uptake of nutrients and water while providing a physical barrier between the intestinal microbiota and the body (Peterson and Artis, 2014). Damage sustained by intestinal epithelial cells (IECs) needs to be swiftly and efficiently repaired to prevent inappropriate immune responses to commensal bacteria. Intestinal damage is an early event in the development of both graft-versus-host disease (Reddy and Ferrara, 2003) and alimentary mucositis (Sonis, 2004) and a driver of bacterial translocation and T cell activation in inflammatory bowel disease (Salim and Söderholm, 2011).A major pathway involved in the intestinal epithelial response to damage is the activation of Stat3, which is expressed along the crypt–villus axis of the intestinal epithelium (Grivennikov et al., 2009; Heneghan et al., 2013). Phosphorylated Stat3 translocates to the nucleus and activates genes involved in proliferation, survival, and mucosal defense (Bollrath et al., 2009; Pickert et al., 2009; Ernst et al., 2014). Mutations in STAT3 have been identified as susceptibility factors for inflammatory bowel disease (Bollrath et al., 2009; Anderson et al., 2011; Demaria et al., 2012), and in mice, upon DSS-induced colitis, epithelial Stat3 is required for mucosal wound healing (Pickert et al., 2009).Intestinal regeneration depends on the continuous differentiation of epithelial cells from crypt-residing intestinal stem cells (ISCs; Potten et al., 1978; Günther et al., 2013; Ritsma et al., 2014). Even though multiple intestinal progenitor cells have been described, the best-characterized populations are the Lgr5-expressing cells that reside at the crypt bottom, interspersed with Paneth cells. These stem cells have the ability to give rise to all IECs ex vivo (Sato et al., 2009). Similar to its role in differentiated epithelial cells, Stat3 activation is also an important pathway for survival of intestinal epithelial stem cells (Matthews et al., 2011).Type 3 innate lymphoid cells (ILC3s) are innate immune cells that reside in the lamina propria of both the small and large intestines and are involved in tissue homeostasis, early defense against enteric pathogens, and containment of microbiota (Spits and Cupedo, 2012; Artis and Spits, 2015). In the intestines, multiple ILC3 subsets exist, two of which can be distinguished by mutual exclusive expression of the natural cytotoxicity receptor NKp46 and the chemokine receptor CCR6 (Sawa et al., 2010; Reynders et al., 2011). Most Nkp46⁺ ILC3s are found dispersed throughout the lamina propria, a localization that depends on the expression of CXCR6 (Satoh-Takayama et al., 2014). In contrast, the majority of CCR6⁺ ILC3s are located in close proximity to the intestinal crypts in anatomically defined sites known as cryptopatches (Kanamori et al., 1996). Recent findings indicated that under inflammatory conditions, such as experimental graft-versus-host disease, ILC3s can interact with the epithelial stem cells in the crypts, protecting them from T cell–mediated killing (Hanash et al., 2012).The well-known ability of ILC3s to condition the local microenvironment, the close proximity of ILC3s to intestinal crypts, and the ability of ILC3s to communicate with epithelial stem cells led us to hypothesize that ILC3s are involved in directing intestinal epithelial responses to tissue damage. Using the methotrexate (MTX) model of small intestinal damage, we now show that ILC3s are activated immediately after MTX administration, leading to a rapid activation of epithelial Stat3 and maintenance of ISCs. Our data reveal a novel function for ILC3s as organizers of the intestinal epithelial response to tissue damage through activation of epithelial cells and maintenance of ISCs and suggest that ILC3s might in future be therapeutically harnessed to prevent stem cell loss during chemotherapy.     

CD8+ T-cell immunity to HIV infection

Fifteen years after the first, definitive reports of HIV-1-specific, CD8+ T cells [147,148], there is ample evidence for the importance of these cells in control of HIV-1 infection. As much is known of their role in the natural history of HIV-1 infection and their cellular and molecular mechanisms of reactivity than of T-cell responses to any other human virus. Indeed, HIV-1-related research has led the scientific field in revealing many new, fundamental principles of cellular immunity in the last 15 years. From these data, there are multiple, posited mechanisms for loss of CD8+ T-cell control of HIV-1 infection. These include both intrinsic defects in T-cell function and loss of T-cell recognition of HIV-1 because of its extraordinary genetic diversity and disruption of antigen presentation. Efforts have begun on devising approaches to reverse these immune defects in infected individuals and develop vaccines that induce T-cell immunity for protection from infection. Combination antiretroviral drug regimens now provide exceptional, long-lasting control of HIV-1 infection, even though they do not restore anti-HIV-1 T-cell immunity fully in persons with chronic HIV-1 infection. Very encouraging results show that such treatment can maintain normal T-cell reactivity specific for this virus in some persons with early HIV-1 infection. Unfortunately, the antiviral treatment does not cure the host of this persistent, latent virus. This has led to new strategies for immunotherapeutic intervention to enhance the level and breadth of the T-cell repertoire specific for the host's residual virus in persons with chronic HIV-1 infection. Although the principles of immunotherapy stem from early in the last century, modern era approaches are integrating highly sophisticated, molecular and cell biology reagents and methods for control of HIV-1 infection. The most promising immunotherapies are autologous virus activated in vivo by STI or administered in autologous DC that have been engineered ex vivo. There are also compelling rationales supported by animal models and early clinical trials for use of cytokines and chemokines as recombinant proteins or DNA to augment anti-HIV-1 T-cell reactivity and trafficking of T cells and APC to tissue sites of infection. For prevention of HIV-1 infection, the discouragingly poor results of vaccine development in the late 1980s and early 1990s have led to very encouraging, recent studies in monkeys that show partially protective and possibly sterilizing immunity. Finally, clinical trials of new-generation DNA and live vector vaccines already have indications of improved induction of HIV-1-specific T-cell responses. Knowledge of HIV-1-specific T-cell immunity and its role in protection from HIV-1 infection and disease must continue to expand until the goal of complete control of HIV-1 infection is accomplished.     

CX3CR1+ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22

Interleukin (IL)-22–producing group 3 innate lymphoid cells (ILC3) promote mucosal healing and maintain barrier integrity, but how microbial signals are integrated to regulate mucosal protection offered by these cells remains unclear. Here, we show that in vivo depletion of CX₃CR1⁺ mononuclear phagocytes (MNPs) resulted in more severe colitis and death after infection with Citrobacter rodentium. This phenotype was rescued by exogenous IL-22, which was endogenously produced by ILC3 in close spatial proximity to CX₃CR1⁺ MNPs that were dependent on MyD88 signaling. CX₃CR1⁺ MNPs from both mouse and human tissue produced more IL-23 and IL-1β than conventional CD103⁺ dendritic cells (cDCs) and were more efficient than cDCs in supporting IL-22 production in ILC3 in vitro and in vivo. Further, colonic ILC3 from patients with mild to moderate ulcerative colitis or Crohn’s disease had increased IL-22 production. IBD-associated SNP gene set analysis revealed enrichment for genes selectively expressed in human intestinal MNPs. The product of one of these, TL1A, potently enhanced IL-23– and IL-1β-induced production of IL-22 and GM-CSF by ILC3. Collectively, these results reveal a critical role for CX₃CR1⁺ mononuclear phagocytes in integrating microbial signals to regulate colonic ILC3 function in IBD.Mononuclear phagocytes (MNPs) are sentinels of the intestinal lamina propria, capable of responding to microbial products, and play a crucial role in orchestrating intestinal lymphocyte homeostasis. MNPs can be subdivided based on their expression of CD103 or CX₃CR1, and each group has been ascribed critical functions in maintaining intestinal homeostasis (Bogunovic et al., 2009; Merad et al., 2013). CD103⁺ cells, which can be further subdivided based on the expression of CD11b, differentiate from a common DC precursor and are thought to be the conventional, migratory myeloid DCs (Varol et al., 2010). CD103⁺ CD11b⁻ DCs require Irf8, Id2, and Batf3 for their development and are thought to play a critical role in cross-priming virus- and tumor-specific CTLs (Hildner et al., 2008; Merad et al., 2013). Loss of these cells, however, does not alter intestinal T cell homeostasis or lead to spontaneous inflammation (Edelson et al., 2010). CD103⁺CD11b⁺ DCs, in contrast, require Notch2 signaling, produce IL-23 in response to flagellin-induced TLR5 activation, resulting in IL-22 production by ILC3, and have additionally been proposed to support Th17 polarization (Lewis et al., 2011; Kinnebrew et al., 2012). These cells can produce retinoic acid, which promotes the expression of the gut-homing receptor CCR9 and synergizes with TGFβ to induce regulatory T cells (Sun et al., 2007). One recent study suggests that Notch2-dependent CD103⁺ CD11b⁺ DCs regulate protection from C. rodentium–induced colitis (Satpathy et al., 2013). However, specific depletion of CD103⁺ CD11b⁺ intestinal DCs revealed that these cells are not the MNP subset required for protection against C. rodentium or IL-22 production (Welty et al., 2013).In contrast to CD103⁺ cDCs, CX₃CR1⁺ MNPs differentiate from monocyte precursors (Varol et al., 2010). Although these cells were previously thought to be tissue-resident and to promote local T_reg differentiation (Hadis et al., 2011), recent data from our group showed that they can up-regulate CCR7 and migrate to secondary lymphoid organs, suggesting a broader role in orchestrating immunity (Diehl et al., 2013). Notably, we observed that interaction with microbiota limits the migration of these cells to mesenteric LNs (MLNs; Diehl et al., 2013), and an increase in CX₃CR1⁺ cells has been described in the lamina propria during mouse (Zigmond et al., 2012) and human colitis (Kamada et al., 2008). A recent study reported that fractalkine receptor (CX₃CR1) expression supports innate cell–dependent clearance of C. rodentium infection (Manta et al., 2013), but a functional role for CX₃CR1⁺ MNPs in regulating colitis-associated ILC3 remains unclear. To evaluate this question, we employed novel mouse models to enable selective depletion of CX₃CR1⁺ MNPs in vivo. Our results reveal a critical role for CX₃CR1⁺ MNPs from both mouse and human tissue in supporting IL-22 induction in ILC3 in vitro and in vivo. Moreover, we identify the ability of TL1A produced by MNPs to potently enhance IL-23– and IL-1β–induced production of IL-22 and GM-CSF by ILC3.     

Plasmacytoid DCs help lymph node DCs to induce anti-HSV CTLs

Antiviral cell-mediated immunity is initiated by the dendritic cell (DC) network in lymph nodes (LNs). Plasmacytoid DCs (pDCs) are known to migrate to inflamed LNs and produce interferon (IFN)-alpha, but their other roles in antiviral T cell immunity are unclear. We report that LN-recruited pDCs are activated to create local immune fields that generate antiviral cytotoxic T lymphocytes (CTLs) in association with LNDCs, in a model of cutaneous herpes simplex virus (HSV) infection. Although pDCs alone failed to induce CTLs, in vivo depletion of pDCs impaired CTL-mediated virus eradication. LNDCs from pDC-depleted mice showed impaired cluster formation with T cells and antigen presentation to prime CTLs. Transferring circulating pDC precursors from wild-type, but not CXCR3-deficient, mice to pDC-depleted mice restored CTL induction by impaired LNDCs. In vitro co-culture experiments revealed that pDCs provided help signals that recovered impaired LNDCs in a CD2- and CD40L-dependent manner. pDC-derived IFN-alpha further stimulated the recovered LNDCs to induce CTLs. Therefore, the help provided by pDCs for LNDCs in primary immune responses seems to be pivotal to optimally inducing anti-HSV CTLs.     

Stress impairs murine intestinal barrier function: improvement by glucagon-like peptide-2

Stress-induced intestinal barrier dysfunction may be involved in chronic intestinal disorders. Glucagon-like peptide-2 (GLP-2) is an intestinotrophic growth hormone that can rapidly improve intestinal epithelial barrier function. Here, we investigated whether mouse intestine is responsive to chronic psychological stress and whether pretreatment with GLP-2 can ameliorate stress-induced changes. Mice were subjected to water avoidance stress (WAS; 1 h/day for 10 days) with GLP-2 or saline administered 4 h before each WAS session. After the final stress period, the intestine was removed for assessment of physiological/morphological changes. Compared with controls (sham-stressed mice), stressed mice demonstrated enhanced ion secretion and permeability in the jejunum, ileum, and colon. In addition, increased numbers of bacteria were observed adhering to and/or penetrating the epithelium, associated with infiltration of mononuclear cells into the mucosa. GLP-2 treatment improved intestinal barrier function in stressed mice and ameliorated other aspects of impaired host defense. Our study extends previous findings in rats of stress-induced intestinal dysfunction and provides insights into potential novel therapeutics.     

Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection

Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1–infected individuals to a mean of 49.4 ± SD 12.9% of CD8⁺ T cells expressing Tim-3 in HIV-1–infected chronic progressors versus 28.5 ± 6.8% in HIV-1–uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1–infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4⁺ T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1–specific CD8⁺ T cells. Tim-3–expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1–specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1–associated T cell dysfunction.     

Loss of HIV-1-specific CD8+ T cell proliferation after acute HIV-1 infection and restoration by vaccine-induced HIV-1-specific CD4+ T cells

Virus-specific CD8(+) T cells are associated with declining viremia in acute human immunodeficiency virus (HIV)1 infection, but do not correlate with control of viremia in chronic infection, suggesting a progressive functional defect not measured by interferon gamma assays presently used. Here, we demonstrate that HIV-1-specific CD8(+) T cells proliferate rapidly upon encounter with cognate antigen in acute infection, but lose this capacity with ongoing viral replication. This functional defect can be induced in vitro by depletion of CD4(+) T cells or addition of interleukin 2-neutralizing antibodies, and can be corrected in chronic infection in vitro by addition of autologous CD4(+) T cells isolated during acute infection and in vivo by vaccine-mediated induction of HIV-1-specific CD4(+) T helper cell responses. These data demonstrate a loss of HIV-1-specific CD8(+) T cell function that not only correlates with progressive infection, but also can be restored in chronic infection by augmentation of HIV-1-specific T helper cell function. This identification of a reversible defect in cell-mediated immunity in chronic HIV-1 infection has important implications for immunotherapeutic interventions.     

Antigen-driven clonal selection shapes the persistence of HIV-1–infected CD4+ T cells in vivo

Clonal expansion of infected CD4⁺ T cells is a major mechanism of HIV-1 persistence and a barrier to achieving a cure. Potential causes are homeostatic proliferation, effects of HIV-1 integration, and interaction with antigens. Here, we show that it is possible to link antigen responsiveness, the full proviral sequence, the integration site, and the T cell receptor β-chain (TCRβ) sequence to examine the role of recurrent antigenic exposure in maintaining the HIV-1 reservoir. We isolated CMV- and Gag-responding CD4⁺ T cells from 10 treated individuals. Proviral populations in CMV-responding cells were dominated by large clones, including clones harboring replication-competent proviruses. TCRβ repertoires showed high clonality driven by converging adaptive responses. Although some proviruses were in genes linked to HIV-1 persistence (BACH2, STAT5B, MKL1), the proliferation of infected cells under antigenic stimulation occurred regardless of the site of integration. Paired TCRβ and integration site analysis showed that infection could occur early or late in the course of a clone’s response to antigen and could generate infected cell populations too large to be explained solely by homeostatic proliferation. Together, these findings implicate antigen-driven clonal selection as a major factor in HIV-1 persistence, a finding that will be a difficult challenge to eradication efforts.     

Yersinia pseudotuberculosis disrupts intestinal barrier integrity through hematopoietic TLR-2 signaling

Intestinal barrier function requires intricate cooperation between intestinal epithelial cells and immune cells. Enteropathogens are able to invade the intestinal lymphoid tissue known as Peyer's patches (PPs) and disrupt the integrity of the intestinal barrier. However, the underlying molecular mechanisms of this process are poorly understood. In mice infected with Yersinia pseudotuberculosis, we found that PP barrier dysfunction is dependent on the Yersinia virulence plasmid and the expression of TLR-2 by hematopoietic cells, but not by intestinal epithelial cells. Upon TLR-2 stimulation, Y. pseudotuberculosis-infected monocytes activated caspase-1 and produced IL-1β. In turn, IL-1β increased NF-κB and myosin light chain kinase activation in intestinal epithelial cells, thus disrupting the intestinal barrier by opening the tight junctions. Therefore, Y. pseudotuberculosis subverts intestinal barrier function by altering the interplay between immune and epithelial cells during infection.