IL-9–mediated survival of type 2 innate lymphoid cells promotes damage control in helminth-induced lung inflammation

IL-9 fate reporter mice established type 2 innate lymphoid cells (ILC2s) as major producers of this cytokine in vivo. Here we focus on the role of IL-9 and ILC2s during the lung stage of infection with Nippostrongylus brasiliensis, which results in substantial tissue damage. IL-9 receptor (IL-9R)–deficient mice displayed reduced numbers of ILC2s in the lung after infection, resulting in impaired IL-5, IL-13, and amphiregulin levels, despite undiminished numbers of Th2 cells. As a consequence, the restoration of tissue integrity and lung function was strongly impaired in the absence of IL-9 signaling. ILC2s, in contrast to Th2 cells, expressed high levels of the IL-9R, and IL-9 signaling was crucial for the survival of activated ILC2s in vitro. Furthermore, ILC2s in the lungs of infected mice required the IL-9R to up-regulate the antiapoptotic protein BCL-3 in vivo. This highlights a unique role for IL-9 as an autocrine amplifier of ILC2 function, promoting tissue repair in the recovery phase after helminth-induced lung inflammation.The cytokine IL-9 was discovered more than 20 yr ago and described as a T cell and mast cell growth factor produced by T cell clones (Uyttenhove et al., 1988; Hültner et al., 1989; Schmitt et al., 1989). Subsequently, IL-9 was shown to promote the survival of a variety of different cell types in addition to T cells (Hültner et al., 1990; Gounni et al., 2000; Fontaine et al., 2008; Elyaman et al., 2009). Until recently, Th2 cells were thought to be the dominant source of IL-9 and the function of IL-9 was mainly studied in the context of Th2 type responses in airway inflammation and helminth infections (Godfraind et al., 1998; Townsend et al., 2000; McMillan et al., 2002; Temann et al., 2002). IL-9 blocking antibodies were shown to ameliorate lung inflammation (Cheng et al., 2002; Kearley et al., 2011) and are currently in clinical trials for the treatment of patients with asthma (Parker et al., 2011). The paradigm that Th2 cells are the dominant source of IL-9 was challenged when it became apparent that naive CD4⁺ T cells cultured in the presence of TGF-β and IL-4 initiate high IL-9 expression without coexpression of IL-4, suggesting the existence of a dedicated subset of IL-9–producing T cells (Dardalhon et al., 2008; Veldhoen et al., 2008; Angkasekwinai et al., 2010; Chang et al., 2010; Staudt et al., 2010). Subsequently, the generation of an IL-9–specific reporter mouse strain enabled the study of IL-9–producing cell types in vivo and revealed that in a model of lung inflammation IL-9 is produced by innate lymphoid cells (ILCs) and not T cells (Wilhelm et al., 2011). IL-9 production in ILCs was transient but important for the maintenance of IL-5 and IL-13 in ILCs. Such type 2 cytokine-producing ILCs (ILC2s; Spits and Di Santo, 2011) were first described as a population of IL-5– and IL-13–producing non-B/non-T cells (Fort et al., 2001; Hurst et al., 2002; Fallon et al., 2006; Voehringer et al., 2006) and later shown to play a role in helminth infection via IL-13 expression (Moro et al., 2010; Neill et al., 2010; Price et al., 2010; Saenz et al., 2010). In addition, important functions were ascribed to such cells in the context of influenza infection (Chang et al., 2011; Monticelli et al., 2011) and airway hyperactivity in mice (Barlow et al., 2012) and humans (Mjösberg et al., 2011). However, although the contribution of ILC2s to host immunity against helminths in the gut is well established (Moro et al., 2010; Neill et al., 2010; Price et al., 2010; Saenz et al., 2010), the function of ILC2s in helminth-related immune responses in the lung remains unknown. ILC2s are marked by expression of the IL-33R (Moro et al., 2010; Neill et al., 2010; Price et al., 2010), as well as the common γ chain (γ_c) cytokine receptors for IL-2 and IL-7 (Moro et al., 2010; Neill et al., 2010). Interestingly, gene expression array analyses have demonstrated that the receptor for IL-9, another member of the γ_c receptor family, is also expressed in ILC2s and differentiates them from Th2 cells (Price et al., 2010) and ROR-γt⁺ ILCs (Hoyler et al., 2012). However, the function of IL-9R expression for ILC2 biology has not been addressed so far.Here we show that the production of IL-5, IL-13, and amphiregulin during infection with Nippostrongylus brasiliensis in the lung depends on ILC2s and their expression of IL-9R. The ability to signal via the IL-9R was crucial for the survival of ILC2s, but not Th2 cells. The absence of IL-9 signaling in IL-9R–deficient mice resulted in reduced lung ILC2 numbers and, consequently, diminished repair of lung damage in the chronic phase after helminth-induced lung injury despite the presence of an intact Th2 cell response. Thus, we identify IL-9 as a crucial autocrine amplifier of ILC2 function and survival.     

Interleukin (IL)-23 mediates Toxoplasma gondii–induced immunopathology in the gut via matrixmetalloproteinase-2 and IL-22 but independent of IL-17

Peroral infection with Toxoplasma gondii leads to the development of small intestinal inflammation dependent on Th1 cytokines. The role of Th17 cells in ileitis is unknown. We report interleukin (IL)-23–mediated gelatinase A (matrixmetalloproteinase [MMP]-2) up-regulation in the ileum of infected mice. MMP-2 deficiency as well as therapeutic or prophylactic selective gelatinase blockage protected mice from the development of T. gondii–induced immunopathology. Moreover, IL-23–dependent up-regulation of IL-22 was essential for the development of ileitis, whereas IL-17 was down-regulated and dispensable. CD4⁺ T cells were the main source of IL-22 in the small intestinal lamina propria. Thus, IL-23 regulates small intestinal inflammation via IL-22 but independent of IL-17. Gelatinases may be useful targets for treatment of intestinal inflammation.Within 8 d after peroral infection with Toxoplasma gondii, susceptible C57BL/6 mice develop massive necrosis in the ileum, leading to death (Liesenfeld et al., 1996). T. gondii–induced ileitis is characterized by a CD4 T cell–dependent overproduction of proinflammatory mediators, including IFN-γ, TNF, and NO (Khan et al., 1997; Mennechet et al., 2002). Activation of CD4⁺ T cells by IL-12 and IL-18 is critical for the development of small intestinal pathology (Vossenkämper et al., 2004). Recently, we showed that LPS derived from gut flora via Toll-like receptor (TLR)–4 mediates T. gondii–induced immunopathology (Heimesaat et al., 2006). Thus, the immunopathogenesis of T. gondii–induced small intestinal pathology resembles key features of the inflammatory responses in inflammatory bowel disease (IBD) in humans and in models of experimental colitis in rodents (Liesenfeld, 2002). However, most animal models of IBD assessed inflammatory responses in the large intestine, and models of small intestinal pathology are scarce (Kosiewicz et al., 2001; Strober et al., 2002; Olson et al., 2004; Heimesaat et al., 2006).IL-12 shares the p40 subunit, IL-12Rβ1, and components of the signaling transduction pathways with IL-23 (Parham et al., 2002). There is strong evidence that IL-23, rather than IL-12, is important in the development of colitis (Yen et al., 2006). The association of IL-23R encoding variant Arg381Gln with IBD (Duerr et al., 2006) and the up-regulation of IL-23p19 in colon biopsies from patients with Crohn''s disease (Schmidt et al., 2005) underline the importance of IL-23 in intestinal inflammation. Effector mechanisms of IL-23 include the up-regulation of matrixmetalloproteinases (MMPs; Langowski et al., 2006), a large family of endopeptidases that mediate homeostasis of the extracellular matrix. MMPs were significantly up-regulated in experimental models of colitis (Tarlton et al., 2000; Medina et al., 2003) and in colonic tissues of IBD patients (von Lampe et al., 2000).Studies in mouse models of autoimmune diseases have associated the pathogenic role of IL-23 with the accumulation of CD4⁺ T cells secreting IL-17, termed Th17 cells (Aggarwal et al., 2003; Cua et al., 2003). Moreover, increased IL-17 expression was reported in the intestinal mucosa of patients with IBD (Fujino et al., 2003; Nielsen et al., 2003; Kleinschek et al., 2009).In addition to IL-17, Th17 cells also produce IL-22, a member of the IL-10 family (Dumoutier et al., 2000). IL-22, although secreted by certain immune cell populations, does not have any effects on immune cells in vitro or in vivo but regulates functions of some tissue cells (Wolk et al., 2009). Interestingly, IL-22 has been proposed to possess both protective as well as pathogenic roles. In fact, IL-22 mediated psoriasis-like skin alterations (Zheng et al., 2007; Ma et al., 2008; Wolk et al., 2009). In contrast, IL-22 played a protective role in experimental models of colitis (Satoh-Takayama et al., 2008; Sugimoto et al., 2008; Zenewicz et al., 2008; Zheng et al., 2008), in a model of Klebsiella pneumoniae infection in the lung (Aujla et al., 2007), and against liver damage caused by concanavalin A administration (Radaeva et al., 2004; Zenewicz et al., 2007). IL-22 has been reported to be produced by CD4⁺ T cells (Wolk et al., 2002; Zheng et al., 2007), γδ cells (Zheng et al., 2007), CD11c⁺ cells (Zheng et al., 2008), and natural killer cells (Cella et al., 2008; Luci et al., 2008; Sanos et al., 2009; Satoh-Takayama et al., 2008; Zheng et al., 2008). The role of IL-22 in small intestinal inflammation remains to be determined.In the present study, we investigated the role of the IL-23–IL-17 axis in T. gondii–induced small intestinal immunopathology. We show that IL-23 is essential in the development of small intestinal immunopathology by inducing local MMP-2 up-regulation that could be inhibited by prophylactic or therapeutic chemical blockage. Interestingly, IL-23–dependent IL-22 production was markedly up-regulated and essential for the development of ileal inflammation, whereas IL-17 production was down-regulated after T. gondii infection. IL-22 was mostly produced by CD4⁺ T cells in the small intestinal lamina propria.     

Inherited IL-17RC deficiency in patients with chronic mucocutaneous candidiasis

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent or persistent infections of the skin, nail, oral, and genital mucosae with Candida species, mainly C. albicans. Autosomal-recessive (AR) IL-17RA and ACT1 deficiencies and autosomal-dominant IL-17F deficiency, each reported in a single kindred, underlie CMC in otherwise healthy patients. We report three patients from unrelated kindreds, aged 8, 12, and 37 yr with isolated CMC, who display AR IL-17RC deficiency. The patients are homozygous for different nonsense alleles that prevent the expression of IL-17RC on the cell surface. The defect is complete, abolishing cellular responses to IL-17A and IL-17F homo- and heterodimers. However, in contrast to what is observed for the IL-17RA– and ACT1-deficient patients tested, the response to IL-17E (IL-25) is maintained in these IL-17RC–deficient patients. These experiments of nature indicate that human IL-17RC is essential for mucocutaneous immunity to C. albicans but is otherwise largely redundant.In humans, chronic mucocutaneous candidiasis (CMC) is characterized by infections of the skin, nail, digestive, and genital mucosae with Candida species, mainly C. albicans, a commensal of the gastrointestinal tract in healthy individuals (Puel et al., 2012). CMC is frequent in acquired or inherited disorders involving profound T cell defects (Puel et al., 2010b; Vinh, 2011; Lionakis, 2012). Human IL-17 immunity has recently been shown to be essential for mucocutaneous protection against C. albicans (Puel et al., 2010b, 2012; Cypowyj et al., 2012; Engelhardt and Grimbacher, 2012; Huppler et al., 2012; Ling and Puel, 2014). Indeed, patients with primary immunodeficiencies and syndromic CMC have been shown to display impaired IL-17 immunity (Puel et al., 2010b). Most patients with autosomal-dominant (AD) hyper-IgE syndrome (AD-HIES) and STAT3 deficiency (de Beaucoudrey et al., 2008; Ma et al., 2008; Milner et al., 2008; Renner et al., 2008; Chandesris et al., 2012) and some patients with invasive fungal infection and autosomal-recessive (AR) CARD9 deficiency (Glocker et al., 2009; Lanternier et al., 2013) or Mendelian susceptibility to mycobacterial diseases (MSMD) and AR IL-12p40 or IL-12Rβ1 deficiency (de Beaucoudrey et al., 2008, 2010; Prando et al., 2013; Ouederni et al., 2014) have low proportions of IL-17A–producing T cells and CMC (Cypowyj et al., 2012; Puel et al., 2012). Patients with AR autoimmune polyendocrine syndrome type 1 (APS-1) and AIRE deficiency display CMC and high levels of neutralizing autoantibodies against IL-17A, IL-17F, and/or IL-22 (Browne and Holland, 2010; Husebye and Anderson, 2010; Kisand et al., 2010, 2011; Puel et al., 2010a).These findings paved the way for the discovery of the first genetic etiologies of CMC disease (CMCD), an inherited condition affecting individuals with none of the aforementioned primary immunodeficiencies (Puel et al., 2011; Casanova and Abel, 2013; Casanova et al., 2013, 2014). AR IL-17RA deficiency, AR ACT1 deficiency, and AD IL-17F deficiency were described, each in a single kindred (Puel et al., 2011; Boisson et al., 2013). A fourth genetic etiology of CMCD, which currently appears to be the most frequent, has also been reported: heterozygous gain-of-function (GOF) mutations of STAT1 impairing the development of IL-17–producing T cells (Liu et al., 2011; Smeekens et al., 2011; van de Veerdonk et al., 2011; Hori et al., 2012; Takezaki et al., 2012; Tóth et al., 2012; Al Rushood et al., 2013; Aldave et al., 2013; Romberg et al., 2013; Sampaio et al., 2013; Soltész et al., 2013; Uzel et al., 2013; Wildbaum et al., 2013; Frans et al., 2014; Kilic et al., 2014; Lee et al., 2014; Mekki et al., 2014; Mizoguchi et al., 2014; Sharfe et al., 2014; Yamazaki et al., 2014). We studied three unrelated patients with CMCD without mutations of IL17F, IL17RA, ACT1, or STAT1. We used a genome-wide approach based on whole-exome sequencing (WES). We found AR complete IL-17RC deficiency in all three patients.     

Activin-A induces regulatory T cells that suppress T helper cell immune responses and protect from allergic airway disease

Activin-A is a pleiotropic cytokine that participates in developmental, inflammatory, and tissue repair processes. Still, its effects on T helper (Th) cell–mediated immunity, critical for allergic and autoimmune diseases, are elusive. We provide evidence that endogenously produced activin-A suppresses antigen-specific Th2 responses and protects against airway hyperresponsiveness and allergic airway disease in mice. Importantly, we reveal that activin-A exerts suppressive function through induction of antigen-specific regulatory T cells that suppress Th2 responses in vitro and upon transfer in vivo. In fact, activin-A also suppresses Th1-driven responses, pointing to a broader immunoregulatory function. Blockade of interleukin 10 and transforming growth factor β1 reverses activin-A–induced suppression. Remarkably, transfer of activin-A–induced antigen-specific regulatory T cells confers protection against allergic airway disease. This beneficial effect is associated with dramatically decreased maturation of draining lymph node dendritic cells. Therapeutic administration of recombinant activin-A during pulmonary allergen challenge suppresses Th2 responses and protects from allergic disease. Finally, we demonstrate that immune cells infiltrating the lungs from individuals with active allergic asthma, and thus nonregulated inflammatory response, exhibit significantly decreased expression of activin-A''s responsive elements. Our results uncover activin-A as a novel suppressive factor for Th immunity and a critical controller of allergic airway disease.Immune responses by differentiated effector Th1, Th2, and Th17 cell subsets provide protection against pathogens but can also lead to chronic inflammation, autoimmunity, or allergy if not tightly controlled (Reiner, 2007; Steinman, 2007). Critical controllers of these responses are immunosuppressive cytokines, such as IL-10 and TGF-β1, and regulatory T lymphocytes. Subsets of regulatory T cells suppress responses by other effector Th cells mainly via cell-to-cell interactions (Nakamura et al., 2001) or the release of immunosuppressive cytokines (Asseman et al., 1999; Chen et al., 2003; Hawrylowicz and O''Garra, 2005; Ostroukhova et al., 2006; Li et al., 2007). However, blockade of these cytokines does not completely inhibit immune regulation (von Boehmer, 2005; Tang and Bluestone, 2008; Vignali et al., 2008), suggesting that other, as yet unidentified, cytokines are also involved.The cytokine activin-A, a member of the TGF-β superfamily, participates in essential biological processes, such as development, hematopoiesis, wound repair, and fibrosis (Vale et al., 1988; Werner and Alzheimer, 2006). Activin-A^−/− mice are embryonic lethal (Matzuk et al., 1995b), whereas activin receptor type IIA (act-RIIA)^−/− mice reach adulthood but have major deficiencies in their reproductive systems (Matzuk et al., 1995a). Although most studies have focused on the role of activin-A in developmental and fibrotic processes, certain reports demonstrate elevated levels of this cytokine in immune-mediated diseases such as rheumatoid arthritis (Ota et al., 2003) and inflammatory bowel disease (Hubner et al., 1997; Dohi et al., 2005). Activin-A is also increased in the sera of individuals with allergic asthma (Karagiannidis et al., 2006), and in the lung and bronchoalveolar lavage (BAL) of mice during acute (Rosendahl et al., 2001; Hardy et al., 2006) and chronic allergic airway inflammation and remodeling (Le et al., 2007). In addition, activin-A is induced in human (Karagiannidis et al., 2006) and mouse effector Th2 lymphocytes (Ogawa et al., 2006), which are key players in allergic responses. However, whether activin-A has enhancing or suppressive actions during Th immune responses and subsequent disease remains unclear.Certain studies indicate that recombinant activin-A (r-activin-A) reduces in vitro nonspecific proliferation of human Th (Karagiannidis et al., 2006) and mouse B cells (Yu et al., 1998; Werner and Alzheimer, 2006), and inhibits certain functions of human natural killer cells (Robson et al., 2009). In other reports, r-activin-A attenuates in vitro endotoxin-induced maturation and phagocytosis of mouse macrophages (Wang et al., 2008; Zhou et al., 2009) and CD40 ligand–dependent cytokine and chemokine release by human monocytes and DCs (Robson et al., 2008). Nevertheless, a few reports have suggested that activin-A has proinflammatory effects, i.e., during in vivo endotoxin administration and allergen challenge in mice (Hardy et al., 2006; Jones et al., 2007). Collectively, these studies indicate a dual nature of this cytokine, a characteristic feature of certain immune mediators (Veldhoen et al., 2006; Zenewicz et al., 2008). Of note, our previous studies revealed a dual role for another cytokine, osteopontin, in allergic airway inflammation (Xanthou et al., 2007).In the present study, we have investigated the in vivo role of activin-A in Th-mediated immune responses and, more specifically, during Th2-associated allergic airway inflammation. We demonstrate that antibody-mediated depletion of activin-A during pulmonary allergen challenge resulted in significant exacerbation of Th2-mediated allergic airway disease, indicating that endogenous activin-A is suppressive. In fact, our findings reveal that activin-A induces the generation of antigen-specific regulatory T cells that suppress both primary and effector Th responses in vitro and upon adoptive transfer in vivo. Functional in vitro analysis shows that activin-A–mediated suppressive effects on Th responses are dependent on both IL-10 and TGF-β1. Importantly, activin-A–induced antigen-specific regulatory T cells transfer protection against allergic airway disease correlated with decreased DC maturation. Collectively, our data reveal that activin-A can suppress Th responses and represents a critical therapeutic target for allergic asthma.     

Identification of human CCR8 as a CCL18 receptor

The CC chemokine ligand 18 (CCL18) is one of the most highly expressed chemokines in human chronic inflammatory diseases. An appreciation of the role of CCL18 in these diseases has been hampered by the lack of an identified chemokine receptor. We report that the human chemokine receptor CCR8 is a CCL18 receptor. CCL18 induced chemotaxis and calcium flux of human CCR8-transfected cells. CCL18 bound with high affinity to CCR8 and induced its internalization. Human CCL1, the known endogenous CCR8 ligand, and CCL18 competed for binding to CCR8-transfected cells. Further, CCL1 and CCL18 induced heterologous cross-desensitization of CCR8-transfected cells and human Th2 cells. CCL18 induced chemotaxis and calcium flux of human activated highly polarized Th2 cells through CCR8. Wild-type but not Ccr8-deficient activated mouse Th2 cells migrated in response to CCL18. CCL18 and CCR8 were coexpressed in esophageal biopsy tissue from individuals with active eosinophilic esophagitis (EoE) and were present at markedly higher levels compared with esophageal tissue isolated from EoE patients whose disease was in remission or in normal controls. Identifying CCR8 as a chemokine receptor for CCL18 will help clarify the biological role of this highly expressed chemokine in human disease.Chemokines are chemotactic cytokines that guide the directed migration of leukocytes in the steady-state and in inflammation through a subfamily of seven-transmembrane spanning G protein–coupled receptors. The CC chemokine ligand (CCL) 18 was identified in the late 1990s by several groups as a gene highly expressed in the lung and induced in alternatively activated macrophages (AAMs) and thus initially given the names PARC (pulmonary and activation-regulated chemokine), AMAC-1 (alternative macrophage activation-associated CC chemokine-1), DC-CK1 (dendritic cell chemokine 1), and macrophage inflammatory protein (MIP) 4 (Adema et al., 1997; Hieshima et al., 1997; Kodelja et al., 1998). CCL18 is highly expressed in many human chronic inflammatory diseases, which include pulmonary fibrosis and certain cancers, and a wide range of allergic diseases (Pivarcsi et al., 2004; Schutyser et al., 2005; de Nadaï et al., 2006; Chen et al., 2011; Lucendo et al., 2011). In some diseases, the level of circulating CCL18 has been demonstrated to be a biomarker for disease activity and outcome (Prasse et al., 2009). In patients with systemic sclerosis, for instance, levels of CCL18 have been associated with the complication of interstitial lung disease, and in patients with idiopathic pulmonary fibrosis and certain cancers, levels of CCL18 have been correlated with poor outcomes (Prasse et al., 2007, 2009; Chen et al., 2011). Understanding the role of CCL18 in these diseases has been hampered by the lack of an identified receptor and by the lack of a known murine orthologue.CCL18 is secreted primarily by cells of the myeloid lineage and has been identified in alveolar macrophages, tolerogenic dendritic cells, AAMs, and keratinocytes (Hieshima et al., 1997; Kodelja et al., 1998; Pivarcsi et al., 2004; Bellinghausen et al., 2012). In macrophages, CCL18 is induced by the Th2 cytokines IL-4 and IL-13, which program macrophages to differentiate into AAMs (Kodelja et al., 1998). AAMs contribute to the healing phase of acute inflammatory reactions and to tissue remodeling and fibrosis in chronic inflammatory diseases. Concordant with the spectrum of AAM activity, the abundance of CCL18 has been found to correlate with disease severity in fibrotic diseases, such as pulmonary fibrosis and scleroderma, and diseases of dysregulated macrophage biology (Schutyser et al., 2005; Prasse et al., 2007, 2009). In allergic diseases, increased CCL18 is also frequently associated with eosinophil infiltration in affected tissues (Pivarcsi et al., 2004; Schutyser et al., 2005; de Nadaï et al., 2006; Lucendo et al., 2011).CCL18 activity has been detected on peripheral blood lymphocytes (Adema et al., 1997; Hieshima et al., 1997). However, studies using cells transfected with CCR1-7, CXCR1-4, and CX₃CR1 did not reveal a specific CCL18 signaling receptor but did find that CCL18 could antagonize CCR3 (Hieshima et al., 1997; Nibbs et al., 2000). Investigation into atopic dermatitis and allergic diseases enabled the first identification of a specific T cell subset that responded to CCL18, and thus provided clues to its possible chemokine receptor (Günther et al., 2005; de Nadaï et al., 2006). In atopic dermatitis, CCL18 is the most abundantly expressed chemokine in lesional skin (Pivarcsi et al., 2004; Günther et al., 2005). CCL18 bound to blood skin-tropic cutaneous leukocyte antigen expressing (CLA⁺) memory T cells from individuals with atopic dermatitis and induced migration of CD4⁺ T cell clones derived from atopic dermatitis lesional skin in vitro and into human skin transplanted in SCID mice in vivo (Günther et al., 2005). The signature homing receptors of skin-tropic T cells are CLA in combination with the chemokine receptors CCR4, CCR10, and CCR8 (Schaerli et al., 2004; Islam et al., 2011). More CCR8-expressing cells are found in inflamed skin of individuals with active atopic dermatitis compared with skin of healthy individuals (Gombert et al., 2005). In studies of human asthma, CCL18 was found to induce the migration of TCR-activated in vitro differentiated human Th2 cells (de Nadaï et al., 2006). CCL18 induced migration of human peripheral blood Th2 cells and regulatory T cells ex vivo (Bellinghausen et al., 2012; Chenivesse et al., 2012). Th2 but not Th1 cells are programmed to selectively express the chemokine receptors CCR4 and CCR8, a receptor profile shared with regulatory T cells (D’Ambrosio et al., 1998; Wei et al., 2010). Skin-homing CLA⁺ T cells, Th2 cells, and regulatory T cells thus all express CCR4 and CCR8. However, TCR activation is a prerequisite for functional CCR8 but not CCR4 expression on in vitro differentiated Th2 cells (D’Ambrosio et al., 1998). Here, we report that CCL18 is an endogenous agonist of the human CCR8 receptor.     

Small intestinal eosinophils regulate Th17 cells by producing IL-1 receptor antagonist

Eosinophils play proinflammatory roles in helminth infections and allergic diseases. Under steady-state conditions, eosinophils are abundantly found in the small intestinal lamina propria, but their physiological function is largely unexplored. In this study, we found that small intestinal eosinophils down-regulate Th17 cells. Th17 cells in the small intestine were markedly increased in the ΔdblGATA-1 mice lacking eosinophils, and an inverse correlation was observed between the number of eosinophils and that of Th17 cells in the small intestine of wild-type mice. In addition, small intestinal eosinophils suppressed the in vitro differentiation of Th17 cells, as well as IL-17 production by small intestinal CD4⁺ T cells. Unlike other small intestinal immune cells or circulating eosinophils, we found that small intestinal eosinophils have a unique ability to constitutively secrete high levels of IL-1 receptor antagonist (IL-1Ra), a natural inhibitor of IL-1β. Moreover, small intestinal eosinophils isolated from IL-1Ra−deficient mice failed to suppress Th17 cells. Collectively, our results demonstrate that small intestinal eosinophils play a pivotal role in the maintenance of intestinal homeostasis by regulating Th17 cells via production of IL-1Ra.The small intestinal lamina propria (LP) contains a variety of immune cells. These include Th17 cells, a subset of activated CD4⁺ T cells characterized by the production of IL-17A, IL-17F, IL-21, and IL-22 (Korn et al., 2009). Th17 cells have the potential to protect or damage the intestinal tissue environment, so their activity must be tightly regulated (O’Connor et al., 2009; Morrison et al., 2011). Several cytokines are known to promote the development of Th17 cells; IL-6 and TGF-β are required for the differentiation of Th17 cells from naive CD4⁺ T cells, and IL-1β and IL-23 are critical for the maintenance of Th17 cells, as well as their differentiation (Zhou et al., 2007; Chung et al., 2009). Commensal bacteria contribute to the generation of small intestinal Th17 cells in the steady state (Atarashi et al., 2008, 2015; Ivanov et al., 2009). In particular, commensal-induced IL-1β production by intestinal macrophages is required for the development of Th17 cells (Shaw et al., 2012).IL-1β is a proinflammatory cytokine primarily produced by activated macrophages and acts as a key mediator in various inflammatory diseases, including inflammatory bowel disease and rheumatoid arthritis (Sims and Smith, 2010). Consequently, mice deficient for IL-1 receptor antagonist (IL-1Ra), which competes with IL-1β for receptor binding, spontaneously develop arthritis with a marked increase in Th17 cells (Nakae et al., 2003; Koenders et al., 2008). In humans, a decrease in the IL-1Ra to IL-1 ratio has been linked to inflammatory bowel disease (Casini-Raggi et al., 1995). IL-1Ra secreted by intestinal epithelial cells upon TLR5 activation reduces tissue damage (Carvalho et al., 2011), and treatment with recombinant IL-1Ra ameliorates intestinal graft-versus-host disease by inhibiting Th17 responses (Jankovic et al., 2013). Thus, the balance between IL-1β and IL-1Ra is critical for controlling Th17 cells and maintaining intestinal immune homeostasis.Eosinophils are commonly known as proinflammatory cells, mediating the host responses against helminth infections, as well as the pathogenesis of various allergic diseases and gastrointestinal disorders (Rothenberg and Hogan, 2006). However, recent studies found that eosinophils also play various roles in maintaining homeostasis, such as supporting glucose homeostasis by sustaining alternatively activated macrophages in adipose tissue (Wu et al., 2011) and promoting the generation and survival of plasma cells (Chu et al., 2014b; Jung et al., 2015). Under steady-state conditions, eosinophils develop in the bone marrow and migrate primarily to the gastrointestinal tract (Mishra et al., 1999; Rothenberg and Hogan, 2006). Small intestinal eosinophils have unique phenotypes and extended life spans (Carlens et al., 2009; Verjan Garcia et al., 2011). However, their function under healthy homeostatic conditions remains to be fully elucidated.In this study, we show that small intestinal eosinophils down-regulate Th17 cells by constitutively secreting a large amount of IL-1Ra. We found a decrease in serum IL-1Ra and a concomitant increase in small intestinal Th17 cells in ΔdblGATA-1 mice, which lack eosinophil-lineage cells (Yu et al., 2002). In WT mice, the number of Th17 cells in the small intestine was inversely correlated with that of eosinophils. Furthermore, eosinophils isolated from the small intestine of WT mice, but not of IL-1Ra–deficient mice, inhibited the Th17 cells. Our findings demonstrate a hitherto unappreciated role of small intestinal eosinophils to regulate intestinal homeostasis by controlling Th17 cells.     

Dual-reactive B cells are autoreactive and highly enriched in the plasmablast and memory B cell subsets of autoimmune mice

Rare dual-reactive B cells expressing two types of Ig light or heavy chains have been shown to participate in immune responses and differentiate into IgG⁺ cells in healthy mice. These cells are generated more often in autoreactive mice, leading us to hypothesize they might be relevant in autoimmunity. Using mice bearing Igk allotypic markers and a wild-type Ig repertoire, we demonstrate that the generation of dual-κ B cells increases with age and disease progression in autoimmune-prone MRL and MRL/lpr mice. These dual-reactive cells express markers of activation and are more frequently autoreactive than single-reactive B cells. Moreover, dual-κ B cells represent up to half of plasmablasts and memory B cells in autoimmune mice, whereas they remain infrequent in healthy mice. Differentiation of dual-κ B cells into plasmablasts is driven by MRL genes, whereas the maintenance of IgG⁺ cells is partly dependent on Fas inactivation. Furthermore, dual-κ B cells that differentiate into plasmablasts retain the capacity to secrete autoantibodies. Overall, our study indicates that dual-reactive B cells significantly contribute to the plasmablast and memory B cell populations of autoimmune-prone mice suggesting a role in autoimmunity.While developing in the BM, B cells undergo stochastic rearrangement of Ig heavy (IgH) and Ig light (IgL) chain V(D)J gene segments resulting in the random expression of Ig H and L (κ and λ) chains in the emerging B cell population (Schlissel, 2003; Nemazee, 2006). During V(D)J recombination, allelic and isotypic exclusion at the Ig loci are also established, leading to the expression of a unique H and L chain pair and, therefore, of BCRs with unique specificity in each B cell (Langman and Cohn, 2002; Nemazee, 2006; Vettermann and Schlissel, 2010). These mechanisms ensure that developing B cells expressing BCRs reactive with self-antigens (i.e., autoreactive B cells) undergo tolerance induction, whereas those expressing BCRs specific for a foreign antigen or a peripheral self-antigen proceed in differentiation and selection into the periphery (Burnet, 1959). Autoreactive B cells are silenced by central tolerance in the BM via receptor editing and, less frequently, clonal deletion (Halverson et al., 2004; Ait-Azzouzene et al., 2005), whereas peripheral B cell tolerance proceeds via anergy and clonal deletion (Goodnow et al., 2005; Pelanda and Torres, 2006, 2012; Shlomchik, 2008). Despite these tolerance mechanisms, small numbers of autoreactive B cells are detected in peripheral tissues of healthy mice and humans (Grandien et al., 1994; Wardemann et al., 2003) and their numbers are increased in autoimmunity (Andrews et al., 1978; Izui et al., 1984; Warren et al., 1984; Samuels et al., 2005; Yurasov et al., 2005, 2006; Liang et al., 2009).A small population of dual-reactive B cells expressing two types of L chains (or more rarely H chains) has been observed both in mice and humans (Nossal and Makela, 1962; Pauza et al., 1993; Giachino et al., 1995; Gerdes and Wabl, 2004; Rezanka et al., 2005; Casellas et al., 2007; Velez et al., 2007; Kalinina et al., 2011). These allelically and isotypically (overall haplotype) included B cells are <5% of all peripheral B cells in normal mice (Barreto and Cumano, 2000; Rezanka et al., 2005; Casellas et al., 2007; Velez et al., 2007), but they are more frequent in Ig knockin mice in which newly generated B cells are autoreactive and actively undergo receptor editing (Li et al., 2002a,b; Liu et al., 2005; Huang et al., 2006; Casellas et al., 2007). B cells that coexpress autoreactive and nonautoreactive antibodies can escape at least some of the mechanisms of central and peripheral B cell tolerance and be selected into the mature peripheral B cell population (Kenny et al., 2000; Li et al., 2002a,b; Gerdes and Wabl, 2004; Liu et al., 2005; Huang et al., 2006), sometimes with a preference for the marginal zone (MZ) B cell subset (Li et al., 2002b).Furthermore, dual-reactive B cells observed within a normal polyclonal Ig repertoire exhibit characteristics of cells that develop through the receptor editing process, including delayed kinetics of differentiation and more frequent binding to self-antigens (Casellas et al., 2007). Hence, dual-reactive B cells might play a role in autoantibody generation and autoimmunity. However, the contribution of these B cells to autoimmunity has not yet been established. Our hypothesis is that haplotype-included autoreactive B cells are positively selected within the context of genetic backgrounds that manifest defects in immunological tolerance and contribute to the development of autoimmunity.Until recently, the analysis of dual-reactive B cells was impaired by the inability to detect dual-κ cells, which are the most frequent among haplotype-included B cells (Casellas et al., 2007; Velez et al., 2007). To overcome this issue, we took advantage of Igk^h mice that bear a gene-targeted human Ig Ck allele in the context of a wild-type Ig repertoire (Casellas et al., 2001) and crossed these to MRL-Fas^lpr/lpr (MRL/lpr) and MRL mice that develop an autoimmune pathology with characteristics similar to human lupus (Izui et al., 1984; Rordorf-Adam et al., 1985; Theofilopoulos and Dixon, 1985; Cohen and Eisenberg, 1991; Watanabe-Fukunaga et al., 1992). MRL/lpr mice, moreover, display defects in receptor editing (Li et al., 2002a; Lamoureux et al., 2007; Panigrahi et al., 2008) and reduced tolerance induction (Li et al., 2002a), which could potentially contribute to higher frequency of haplotype-included autoreactive B cells.We found that the frequency of dual-κ cells increased with age and progression of disease in autoimmune-prone mice and independent of the expression of Fas. Dual-κ B cells exhibited higher prevalence of autoreactivity than single-κ B cells and were frequently selected into the antigen-activated cell subsets in MRL/lpr and MRL mice where up to half of the plasmablasts and memory B cells were dual-κ B cells. Moreover, disruption of Fas expression appeared to mediate increased survival of dual-reactive memory B cells. Overall, these data indicate that dual-reactive B cells significantly contribute to the plasmablast and memory B cell populations of autoimmune-prone mice suggesting a role in the development of autoimmunity.     

B cell depletion reduces the development of atherosclerosis in mice

B cell depletion significantly reduces the burden of several immune-mediated diseases. However, B cell activation has been until now associated with a protection against atherosclerosis, suggesting that B cell–depleting therapies would enhance cardiovascular risk. We unexpectedly show that mature B cell depletion using a CD20-specific monoclonal antibody induces a significant reduction of atherosclerosis in various mouse models of the disease. This treatment preserves the production of natural and potentially protective anti–oxidized low-density lipoprotein (oxLDL) IgM autoantibodies over IgG type anti-oxLDL antibodies, and markedly reduces pathogenic T cell activation. B cell depletion diminished T cell–derived IFN-γ secretion and enhanced production of IL-17; neutralization of the latter abrogated CD20 antibody–mediated atheroprotection. These results challenge the current paradigm that B cell activation plays an overall protective role in atherogenesis and identify new antiatherogenic strategies based on B cell modulation.Atherosclerosis-related cardiovascular diseases are the leading cause of mortality worldwide. Immune-mediated reactions initiated in response to multiple potential antigens, including oxidatively modified lipoproteins and phospholipids, play prominent roles in atherosclerotic lesion development, progression, and complications (Binder et al., 2002; Hansson and Libby, 2006; Tedgui and Mallat, 2006). Besides the critical requirement for monocytes/macrophages (Smith et al., 1995), adaptive immunity substantially contributes to the perpetuation of the immunoinflammatory response, further promoting vascular inflammation and lesion development (Binder et al., 2002; Hansson and Libby, 2006; Tedgui and Mallat, 2006). Mice on a severe combined immunodeficiency or Rag-deficient background show reduced susceptibility to atherosclerosis under moderate cholesterol overload (Dansky et al., 1997; Daugherty et al., 1997; Zhou et al., 2000). Resupplementation of these mice with purified T lymphocytes accelerates lesion development (Zhou et al., 2000), even though it does not fully recapitulate lesion development of the immunocompetent mice. The proatherogenic T cells are related to the Th1 lineage (Gupta et al., 1997; Buono et al., 2005), and are counterregulated by both Th2 (Binder et al., 2004; Miller et al., 2008) and T reg cell responses (Ait-Oufella et al., 2006; Tedgui and Mallat, 2006).The development of atherosclerosis is also associated with signs of B cell activation, particularly manifested by enhanced production of natural IgM type and adaptive IgG type anti–oxidized low-density lipoprotein (oxLDL) autoantibodies (Shaw et al., 2000; Caligiuri et al., 2002). However, in contrast to other immune-mediated diseases, i.e., rheumatoid arthritis and systemic lupus erythematosus, B cells have been assigned a protective role in atherosclerosis (Caligiuri et al., 2002; Major et al., 2002; Binder et al., 2004; Miller et al., 2008). Although IgG type anti-oxLDL antibodies show variable association with vascular risk, circulating levels of IgM type anti-oxLDL antibodies have been more frequently linked with reduced vascular risk in humans (Karvonen et al., 2003; Tsimikas et al., 2007). In mice, IL-5– and IL-33–mediated atheroprotective effects have been indirectly associated with specific B1 cell activation and enhanced production of natural IgM type anti-oxLDL antibodies (Binder et al., 2004; Miller et al., 2008). On the other hand, splenectomy (Caligiuri et al., 2002) or transfer of μMT-deficient (B cell–deficient) bone marrow (Major et al., 2002) into lethally irradiated atherosclerosis-susceptible mice resulted in profound reduction of IgG (Caligiuri et al., 2002) or total (Major et al., 2002) anti-oxLDL antibody production, and was associated with acceleration of lesion development. These studies led to the current paradigm that overall B cell activation is atheroprotective. Surprisingly, however, whether mature B cell depletion accelerates atherosclerotic lesion development in immunocompetent mice, as expected from previous studies, is still unexplored. This is a critical question given the potentially important risk of cardiovascular complications that might arise from the clinical use of B cell–depleting CD20-targeted immune therapy in patients with severe rheumatoid arthritis or systemic lupus erythematosus, who are at particularly high risk of cardiovascular diseases (for review see Roman et al., 2001). We have therefore designed a series of experiment to address this important question.