Induction and stability of human Th17 cells require endogenous NOS2 and cGMP-dependent NO signaling

Nitric oxide (NO) is a ubiquitous mediator of inflammation and immunity, involved in the pathogenesis and control of infectious diseases, autoimmunity, and cancer. We observed that the expression of nitric oxide synthase-2 (NOS2/iNOS) positively correlates with Th17 responses in patients with ovarian cancer (OvCa). Although high concentrations of exogenous NO indiscriminately suppress the proliferation and differentiation of Th1, Th2, and Th17 cells, the physiological NO concentrations produced by patients’ myeloid-derived suppressor cells (MDSCs) support the development of RORγt(Rorc)⁺IL-23R⁺IL-17⁺ Th17 cells. Moreover, the development of Th17 cells from naive-, memory-, or tumor-infiltrating CD4⁺ T cells, driven by IL-1β/IL-6/IL-23/NO-producing MDSCs or by recombinant cytokines (IL-1β/IL-6/IL-23), is associated with the induction of endogenous NOS2 and NO production, and critically depends on NOS2 activity and the canonical cyclic guanosine monophosphate (cGMP)–cGMP-dependent protein kinase (cGK) pathway of NO signaling within CD4⁺ T cells. Inhibition of NOS2 or cGMP–cGK signaling abolishes the de novo induction of Th17 cells and selectively suppresses IL-17 production by established Th17 cells isolated from OvCa patients. Our data indicate that, apart from its previously recognized role as an effector mediator of Th17-associated inflammation, NO is also critically required for the induction and stability of human Th17 responses, providing new targets to manipulate Th17 responses in cancer, autoimmunity, and inflammatory diseases.Nitric oxide (NO; a product of nitrite reduction or the NO synthases NOS1, NOS2, and NOS3; Culotta and Koshland, 1992), is a pleiotropic regulator of neurotransmission, inflammation, and autoimmunity (Culotta and Koshland, 1992; Bogdan, 1998, 2001; Kolb and Kolb-Bachofen, 1998) implicated both in cancer progression and its immune-mediated elimination (Culotta and Koshland, 1992; Coussens and Werb, 2002; Hussain et al., 2003; Mantovani et al., 2008). In different mouse models, NO has been paradoxically shown to both promote inflammation (Farrell et al., 1992; Boughton-Smith et al., 1993; McCartney-Francis et al., 1993; Weinberg et al., 1994; Hooper et al., 1997) and to suppress autoimmune tissue damage through nonselective suppression of immune cell activation (Bogdan, 2001; Bogdan, 2011), especially at high concentrations (Mahidhara et al., 2003; Thomas et al., 2004; Niedbala et al., 2011). Although previous studies demonstrated a positive impact of NO on the induction of Th1 cells (Niedbala et al., 2002) and forkhead box P3–positive (FoxP3⁺) regulatory T (T reg) cells (Feng et al., 2008) in murine models, the regulation and function of the NO synthase (NOS)–NO system have shown profound differences between mice and humans (Schneemann and Schoedon, 2002, Schneemann and Schoedon, 2007; Fang, 2004), complicating the translation of these findings from mouse models to human disease.In cancer, NOS2-derived NO plays both cytotoxic and immunoregulatory functions (Bogdan, 2001). It can exert distinct effects on different subsets of tumor-infiltrating T cells (TILs), capable of blocking the development of cytotoxic T lymphocytes (CTLs; Bronte et al., 2003), suppressing Th1 and Th2 cytokine production, and modulating the development of FoxP3⁺ T reg cells (Brahmachari and Pahan, 2010; Lee et al., 2011). NOS2-driven NO production is a prominent feature of cancer-associated myeloid-derived suppressor cells (MDSCs; Mazzoni et al., 2002; Kusmartsev et al., 2004; Vuk-Pavlović et al., 2010; Bronte and Zanovello, 2005), which in the human system are characterized by a CD11b⁺CD33⁺HLA-DR^low/neg phenotype consisting of CD14⁺ monocytic (Serafini et al., 2006; Filipazzi et al., 2007; Hoechst et al., 2008; Obermajer et al., 2011) and CD15⁺ granulocytic (Zea et al., 2005; Mandruzzato et al., 2009; Rodriguez et al., 2009) subsets (Dolcetti et al., 2010; Nagaraj and Gabrilovich, 2010).Production of NO in chronic inflammation is supported by IFN-γ and IL-17 (Mazzoni et al., 2002; Miljkovic and Trajkovic, 2004), the cytokines produced by human Th17 cells (Veldhoen et al., 2006; Acosta-Rodriguez et al., 2007a,b; van Beelen et al., 2007; Wilson et al., 2007). Human Th17 cells secrete varying levels of IFN-γ (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; Kryczek et al., 2009; Miyahara et al., 2008; van Beelen et al., 2007; Wilson et al., 2007) and have been implicated both in tumor surveillance and tumor progression (Miyahara et al., 2008; Kryczek et al., 2009; Martin-Orozco and Dong, 2009). Induction of Th17 cells typically involves IL-1β, IL-6, and IL-23 (Bettelli et al., 2006; Acosta-Rodriguez et al., 2007a,b; Ivanov et al., 2006; van Beelen et al., 2007; Veldhoen et al., 2006; Wilson et al., 2007; Zhou et al., 2007), with the additional involvement of TGF-β in most mouse models (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006; Zhou et al., 2007; Ghoreschi et al., 2010), but not in the human system (Acosta-Rodriguez et al., 2007a; Wilson et al., 2007). IL-1β₁, IL-6, and IL-23 production by monocytes and DCs, and the resulting development of human Th17 cells, can be induced by bacterial products, such as LPS or peptidoglycan (Acosta-Rodriguez et al., 2007a; Acosta-Rodriguez et al., 2007b; van Beelen et al., 2007). However, the mechanisms driving Th17 responses in noninfectious settings, such as autoimmunity or cancer, remain unclear.Here, we report that the development of human Th17 cells from naive, effector, and memory CD4⁺ T cell precursors induced by the previously identified Th17-driving cytokines (IL-1β, IL-6, and IL-23) or by IL-1β/IL-6/IL-23-producing MDSCs, is promoted by exogenous NO (or NO produced by human MDSCs) and critically depends on the induction of endogenous NOS2 in differentiating CD4⁺ T 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.     

Circulating precursors of human CD1c+ and CD141+ dendritic cells

Two subsets of conventional dendritic cells (cDCs) with distinct cell surface markers and functions exist in mouse and human. The two subsets of cDCs are specialized antigen-presenting cells that initiate T cell immunity and tolerance. In the mouse, a migratory cDC precursor (pre-CDC) originates from defined progenitors in the bone marrow (BM). Small numbers of short-lived pre-CDCs travel through the blood and replace cDCs in the peripheral organs, maintaining homeostasis of the highly dynamic cDC pool. However, the identity and distribution of the immediate precursor to human cDCs has not been defined. Using a tissue culture system that supports the development of human DCs, we identify a migratory precursor (hpre-CDC) that exists in human cord blood, BM, blood, and peripheral lymphoid organs. hpre-CDCs differ from premonocytes that are restricted to the BM. In contrast to earlier progenitors with greater developmental potential, the hpre-CDC is restricted to producing CD1c⁺ and CD141⁺ Clec9a⁺ cDCs. Studies in human volunteers demonstrate that hpre-CDCs are a dynamic population that increases in response to levels of circulating Flt3L.Conventional DCs (cDCs) induce immunity or tolerance by capturing, processing, and presenting antigen to T lymphocytes (Banchereau and Steinman, 1998). In the mouse, cDCs are short-lived cells, whose homeostasis in lymphoid and nonlymphoid tissues is critically dependent on continual replenishment from circulating pre-CDC (Liu et al., 2007; Liu and Nussenzweig, 2010). Murine pre-CDCs are BM-derived cells that are present in very small numbers in the blood but increase in response to Flt3L injection (Liu et al., 2007, 2009). pre-CDCs have a very short dwell time in the blood, 65% of these cells leave the circulation within 1 min after leaving the BM (Liu et al., 2007, 2009). Upon leaving the circulation, pre-CDCs seed tissues where they differentiate to cDCs, which divide further under the control of Flt3L (Liu et al., 2007, 2009). Thus, in addition to the BM and blood, mouse pre-CDCs are also found in peripheral lymphoid organs and nonlymphoid tissues (Naik et al., 2006; Bogunovic et al., 2009; Ginhoux et al., 2009; Liu et al., 2009; Varol et al., 2009).Mouse cDCs can be divided into two major subsets, CD11b⁺ DCs and CD8⁺/CD103⁺ DCs that differ in their microanatomic localization, cell surface antigen expression, antigen-processing activity, and ability to contribute to immune responses to specific pathogens (Merad et al., 2013; Murphy, 2013). Despite these important differences, both CD11b⁺ and CD8⁺/CD103⁺ cDC subsets of mouse DCs are derived from the same immediate precursor (pre-CDC) that expresses CD135 (Flt3), the receptor for Flt3L, a cytokine that is critical to DC development in vivo (McKenna et al., 2000; Waskow et al., 2008).Similar to the mouse, humans have two major subsets of cDCs. CD141 (BDCA3)⁺Clec9a⁺ DCs (CD141⁺ cDC herein) appear to be the human counterpart of mouse CD8⁺/CD103⁺ DCs, expressing XCR1, Clec9a, IRF8, and TLR3 and producing IL-12 (Robbins et al., 2008; Bachem et al., 2010; Crozat et al., 2010; Jongbloed et al., 2010; Poulin et al., 2010; Haniffa et al., 2012). CD1c (BDCA1)⁺ cDCs appear to be more closely related to mouse CD11b⁺ DCs, expressing IRF4, inducing Th17 differentiation upon A. fumigatus challenge, and imprinting intraepithelial homing of T cells (Robbins et al., 2008; Crozat et al., 2010; Schlitzer et al., 2013; Yu et al., 2013). In the mouse, the superior ability of CD8⁺/CD103⁺ DCs to cross-present exogenous antigens to CD8⁺ T cells is attributed to both differential antigen uptake (Kamphorst et al., 2010) and to increased expression of proteins and enzymes that facilitate MHC class I presentation (Dudziak et al., 2007). Human CD141⁺ cDCs are more efficient than CD1c⁺ cDCs in cross-presentation (Bachem et al., 2010; Crozat et al., 2010; Jongbloed et al., 2010; Poulin et al., 2010), but this difference appears to result from differences in antigen uptake and cytokine activation rather than a specialized cell-intrinsic program (Segura et al., 2012; Cohn et al., 2013; Nizzoli et al., 2013).Both CD1c⁺ cDCs and CD141⁺ cDCs are present in human blood and peripheral tissues. Each subset in the blood resembles its tissue counterpart in gene expression but appears less differentiated (Haniffa et al., 2012; Segura et al., 2012; Schlitzer et al., 2013). These observations are consistent with the idea that less differentiated human cDCs travel through the blood to replenish the cDC pool in the peripheral tissues (Collin et al., 2011; Segura et al., 2012; Haniffa et al., 2013). Others have postulated the existence of a less differentiated circulating DC progenitor based on absence of CD11c, expression of CD123, and response to Flt3L (O’Doherty et al., 1994; Pulendran et al., 2000), but the progenitor potential of these putative precursors that produced large amounts of IFN-α was never tested directly and they appear to correspond at least in part to plasmacytoid DCs (Grouard et al., 1997; Siegal et al., 1999). Thus, whether there is an immediate circulating precursor restricted to human immature and mature CD1c⁺ and CD141⁺ cDCs is not known.Here, we report the existence of a migratory pre-CDC in humans (hpre-CDC) that develops from committed DC progenitors (hCDPs) in the BM (Lee et al., 2015) and is the immediate precursor of both CD1c⁺ and CD141⁺ cDCs, but not pDCs or monocytes. hpre-CDCs are present in BM, cord, and peripheral blood, as well as peripheral lymphoid tissues. In studies of human volunteers, Flt3L injection induces expansion of hpre-CDCs in the circulation. Thus, human cDC precursors constitute a dynamic circulating population whose homeostasis is regulated by Flt3L, a cytokine that is responsive to inflammatory and infectious agents.     

Activating receptors promote NK cell expansion for maintenance,IL-10 production,and CD8 T cell regulation during viral infection

Natural killer (NK) cells have the potential to deliver both direct antimicrobial effects and regulate adaptive immune responses, but NK cell yields have been reported to vary greatly during different viral infections. Activating receptors, including the Ly49H molecule recognizing mouse cytomegalovirus (MCMV), can stimulate NK cell expansion. To define Ly49H''s role in supporting NK cell proliferation and maintenance under conditions of uncontrolled viral infection, experiments were performed in Ly49h^−/−, perforin 1 (Prf1)^−/−, and wild-type (wt) B6 mice. NK cell numbers were similar in uninfected mice, but relative to responses in MCMV-infected wt mice, NK cell yields declined in the absence of Ly49h and increased in the absence of Prf1, with high rates of proliferation and Ly49H expression on nearly all cells. The expansion was abolished in mice deficient for both Ly49h and Prf1 (Ly49h^−/−Prf1^−/−), and negative consequences for survival were revealed. The Ly49H-dependent protection mechanism delivered in the absence of Prf1 was a result of interleukin 10 production, by the sustained NK cells, to regulate the magnitude of CD8 T cell responses. Thus, the studies demonstrate a previously unappreciated critical role for activating receptors in keeping NK cells present during viral infection to regulate adaptive immune responses.Classical (non–T) NK cells are generally found at low frequencies in leukocyte populations (Biron et al., 1999). They have the potential to mediate antiviral and immunoregulatory functions through a variety of mechanisms (Orange et al., 1995; Su et al., 2001; Lee et al., 2007; Robbins et al., 2007; Strowig et al., 2008). By altering cell availability, in vivo conditions changing NK cell numbers may indirectly influence all of their effects. Activating receptors on NK cells are linked to stimulatory pathways overlapping with those used by TCRs to drive cell expansions (Murali-Krishna et al., 1998; Pitcher et al., 2003; French et al., 2006; MacFarlane and Campbell, 2006; Biron and Sen, 2007; Lee et al., 2007) and can induce NK cell proliferation (Dokun et al., 2001; French et al., 2006). Although particular activating receptors have been reported to recognize microbial products (Lanier, 1998; Vidal and Lanier, 2006; Jonjic et al., 2008), dramatic NK cell expansion has not been observed during infections. Except under rare experimental conditions (Caligiuri et al., 1991; Yamada et al., 1996; Fehniger et al., 2001; Huntington et al., 2007a; Sun et al., 2009), NK cell division is generally induced for limited periods of time as a consequence of transient innate cytokine exposure (Biron et al., 1984; Biron et al., 1999; Dokun et al., 2001; Nguyen et al., 2002; Yokoyama et al., 2004). Increasing proportions of NK cell subsets with activating receptors recognizing particular viral ligands can be detected during certain infections (Dokun et al., 2001; Gumá et al., 2006), but this is observed without dramatic increases in overall NK cell numbers, and many viral infections induce striking reductions in NK cell functions, frequencies, and yields (Biron et al., 1999; Tarazona et al., 2002; Lehoux et al., 2004; Reed et al., 2004; Azzoni et al., 2005; Vossen et al., 2005; Morishima et al., 2006). Thus, particular conditions of viral challenges must result in differential regulation of NK cell proportions and numbers, with consequences for the delivery of NK cell functions.An NK cell activating receptor in the mouse is Ly49H (Lanier, 1998; Gosselin et al., 1999; Smith et al., 2000; Vidal and Lanier, 2006). This molecule recognizes a mouse CMV (MCMV) ligand (Arase et al., 2002; Smith et al., 2002), is expressed on NK cell subsets in strains of particular genetic backgrounds, including C57BL/6 (B6) mice, and is reported to be an exclusive marker for the classical NK cell subset (Smith et al., 2000). Through an associated molecule, Ly49H stimulates using signaling pathways overlapping with those used by the TCR (MacFarlane and Campbell, 2006; Biron and Sen, 2007). Additional markers for all NK cells include CD49b, expressed on other activated cell types (Arase et al., 2001); NKp46, selectively expressed on classical NK cells (Gazit et al., 2006; Walzer et al., 2007a; Walzer et al., 2007b); CD122, the IL-2Rβ chain, expressed on all NK cells and activated T cells (Huntington et al., 2007b); and NK1.1, exclusively expressed on C57BL6 (B6) NK and NKT cells (Lian and Kumar, 2002; MacDonald, 2002; Yokoyama et al., 2004; Huntington et al., 2007b). The TCR with associated CD3 molecules is not expressed on their cell surfaces (Biron et al., 1999). The mechanisms for NK cell–enhanced resistance to MCMV infection are incompletely characterized (Lee et al., 2007), but Ly49H contributes to their protective effects (Scalzo et al., 1990; Brown et al., 2001; Daniels et al., 2001; Lee et al., 2001; Lee et al., 2003). Engagement of the Ly49H receptor can lead to killing of target cells (Arase et al., 2002; Smith et al., 2002), and the correlation of increases in viral burdens resulting from the absence of Ly49H (Scalzo et al., 1990) as compared with those resulting from defects in cytotoxicity functions, such as mutation of the membrane pore-forming protein perforin 1 (Prf1; Tay and Welsh, 1997; Loh et al., 2005; van Dommelen et al., 2006), supports a role for Ly49H-dependent killing of virus-infected cells in the delivery of NK cell antiviral effects. The receptor may have other functions associated with its ability to stimulate proliferation, however, as the proportions of NK cells expressing Ly49H are increased during MCMV infection (Dokun et al., 2001).The studies presented in this paper were undertaken to dissect the proliferative from the cytotoxic functions accessed through Ly49H, and to define the contribution of the regulation of NK cell numbers to protection during infection. To carry out the work, responses were evaluated, during MCMV infections, in wild-type (wt) B6 mice and mice deficient in Ly49h (Fodil-Cornu et al., 2008), Prf1 (Kägi et al., 1994), or both. As expected, single Ly49h^−/− and Prf1^−/− mice had profoundly increased viral burdens, but significant differences in NK cell expansion were discovered. The NK cell populations were decreasing in infected Ly49h^−/− mice, whereas infected Prf1^−/− mice had an unexpected dramatic proliferation of NK cells uniformly expressing Ly49H. The expansion was proven to be dependent on Ly49H. During uncontrolled infection in the absence of Prf1, Ly49H beneficially promoted effects for survival, because the sustained NK cells produced IL-10 to control the magnitude of the CD8 T cell response and limit immunopathology. The data suggest that Ly49H-dependent cytotoxicity acts to control viral infection and NK cell expansion, but that in the absence of the killing function, Ly49H promotes a continued NK cell expansion critical for supporting life over death because the NK cells are available to regulate adaptive immune responses.     

Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo

Autoreactive B lymphocytes first encountering self-antigens in peripheral tissues are normally regulated by induction of anergy or apoptosis. According to the “two-signal” model, antigen recognition alone should render B cells tolerant unless T cell help or inflammatory signals such as lipopolysaccharide are provided. However, no such signals seem necessary for responses to T-independent type 2 (TI-2) antigens, which are multimeric antigens lacking T cell epitopes and Toll-like receptor ligands. How then do mature B cells avoid making a TI-2–like response to multimeric self-antigens? We present evidence that TI-2 antigens decorated with ligands of inhibitory sialic acid–binding Ig-like lectins (siglecs) are poorly immunogenic and can induce tolerance to subsequent challenge with immunogenic antigen. Two siglecs, CD22 and Siglec-G, contributed to tolerance induction, preventing plasma cell differentiation or survival. Although mutations in CD22 and its signaling machinery have been associated with dysregulated B cell development and autoantibody production, previous analyses failed to identify a tolerance defect in antigen-specific mutant B cells. Our results support a role for siglecs in B cell self-/nonself-discrimination, namely suppressing responses to self-associated antigens while permitting rapid “missing self”–responses to unsialylated multimeric antigens. The results suggest use of siglec ligand antigen constructs as an approach for inducing tolerance.B lymphocytes can respond rapidly to nonself-antigens, yet even at mature stages of development can be rendered tolerant if they encounter self-antigen (Goodnow et al., 2005). How B cells distinguish self from nonself has been explained in part by Bretscher and Cohn’s associative recognition (“two-signal”) hypothesis (Bretscher and Cohn, 1970), which posits that B cells can only achieve activation after a second signal is delivered, the first being recognition of antigen by the BCR. Without this second signal, tolerance is induced. In response to T-dependent antigens, activated helper T cells provide this second signal. In a T-independent type 1 response, the second signal might come from the B cells’ Toll-like receptors (TLRs) recognizing conserved microbial motifs attached to the antigen (e.g., lipopolysaccharide; Coutinho et al., 1974). This model, however, fails to explain how T-independent type 2 (TI-2) responses occur, as TI-2 antigens require neither T cells (Mond et al., 1995) nor recognition by known innate immune receptors (Gavin et al., 2006), and can elicit antibody responses in cultures of single B cells (Nossal and Pike, 1984). Although we do not dispute contributory roles of innate immune receptors, cytokines, or accessory cells in amplifying their responses (Mond et al., 1995; Vos et al., 2000; Hinton et al., 2008), TI-2 antigens appear to have only two surprisingly simple properties, high molecular weight and ≥20 closely spaced BCR epitopes (Dintzis et al., 1976), and are thus unlikely to have innate receptors specialized for their recognition.Alternatively, B cells might be capable of “missing self”–recognition (Parish, 1996; Nemazee and Gavin, 2003) similar to that originally observed in NK cells (Kärre et al., 1986). In NK cell recognition, the decision to lyse a target cell depends on integration of opposing signals from activating and inhibitory receptors (Lanier, 2008). Activating receptors trigger recruitment of tyrosine kinases to immunotyrosine activating motifs of associated adapter molecules but are kept in check by inhibitory receptors recognizing classical MHC I molecules expressed on target cells (Lanier, 2008). Inhibitory receptors carry immunotyrosine inhibitory motifs (ITIMs), which serve as docking sites for phosphatases, such as SHP-1, that counteract activation (Ravetch and Lanier, 2000). Target cells that down-regulate MHC I are lysed owing to unopposed activation, hence missing self–recognition.Extrapolating from this model, we hypothesize that besides their BCR epitopes, self-antigens carry self-markers that can engage inhibitory receptors on B cells, preventing antiself TI-2–like responses and rendering activation dependent on second signals. The concept that self-markers might facilitate self-tolerance was first suggested many years ago by Burnet and Fenner (1949) but has garnered little experimental support with respect to lymphocyte tolerance. According to our model, antigens that simultaneously cross-link the BCRs and inhibitory receptors should prevent or blunt B cell responses. Conversely, antigens that bind only the BCR and not inhibitory receptors are predicted to elicit a TI-2 response, provided that they carry the appropriate number and spacing of epitopes. This missing self–model of self-/nonself-discrimination would explain why B cells constitutively express so many inhibitory receptors that recognize ubiquitous self-components, and why null mutations in those receptors or their signaling machinery can lead to autoantibody formation (Nishimura et al., 1998; Pan et al., 1999; Ravetch and Lanier, 2000; Nemazee and Gavin, 2003).In this study, we chose to test if self-/nonself-discrimination is regulated by self-markers through the roles of the sialic acid–binding Ig-like lectins (siglecs) CD22 and Siglec-G in B cells. The siglec family consists of 9 members in mice and 13 members in humans (for review see Crocker et al., 2007). In mice, mature B cells express CD22 (Siglec 2) and Siglec-G, which bind to host sialic acids carried on glycans of glycoproteins and glycolipids and have properties of inhibitory receptors. They carry ITIMs capable of recruiting the tyrosine phosphatase SHP-1 and attenuating BCR signaling (Campbell and Klinman, 1995; Doody et al., 1995; Cornall et al., 1998). Mice carrying null mutations in either CD22 or Siglecg exhibit B cell hyperactivity, variable responses to T-independent antigens, and a tendency toward autoantibody formation (O’Keefe et al., 1996; Otipoby et al., 1996; Sato et al., 1996; Nitschke et al., 1997; Cornall et al., 1998; O’Keefe et al., 1999; Ding et al., 2007; Hoffmann et al., 2007). Mouse CD22 exhibits a strong preference for sialoside ligands with the disaccharide sequence NeuGcα2-6Gal (Collins et al., 2006a; Crocker et al., 2007), whereas Siglec-G, before this study, has had an unknown ligand specificity. Their disaccharide ligands represent terminal sugars commonly carried on N- and O-linked glycans of glycoproteins and are found on virtually all cells, including B cells (Crocker et al., 2007). It is well documented that CD22 binds to glycans on endogenous B cell glycoproteins in cis, and masks the ligand binding site from binding synthetic polymeric ligands (Hanasaki et al., 1995; Razi and Varki, 1998; Razi and Varki, 1999; Collins et al., 2002; Han et al., 2005). Yet, CD22 is able to recognize native ligands on glycoproteins of apposing cells in trans, causing it to redistribute to the site of cell contact (Lanoue et al., 2002; Collins et al., 2004). Although mutations of the ligand binding domain of CD22 (Jin et al., 2002; Poe et al., 2004) and ablation of enzymes involved in the synthesis of its glycan ligands (Hennet et al., 1998; Poe et al., 2004; Collins et al., 2006b; Ghosh et al., 2006; Grewal et al., 2006; Naito et al., 2007; Cariappa et al., 2009) document the importance of siglec ligands in the regulation of CD22 function, a unifying role for CD22 ligand interactions in B cell biology has not yet emerged (Crocker et al., 2007; Walker and Smith, 2008). Although less is known about the specificity of Siglec-G and its interaction with ligands, it is assumed that similar concepts regarding cis and trans ligands will apply to the modulation of Siglec-G function (Hoffmann et al., 2007; Chen et al., 2009).Because siglecs see sialylated glycans that are usually absent from microbes, with the notable exceptions of some pathogenic microbes (Crocker et al., 2007; Carlin et al., 2009a), one possible role of siglecs is to discriminate self from nonself. Though CD22 and Siglec-G have been implicated to play roles in B cell tolerance, evidence has been indirect, inferred from the facts that they possess ITIMs able to recruit SHP-1 and dampen Ca²⁺ signaling (Otipoby et al., 1996; Sato et al., 1996; Nitschke et al., 1997; O’Keefe et al., 1999; Ding et al., 2007; Hoffmann et al., 2007). Hypomorphic or null alleles of CD22 and SHP-1 (Ptpn6) have been correlated with anti-DNA production and development of lupus erythematosus (Shultz et al., 1993; O’Keefe et al., 1999; Mary et al., 2000). CD22 mutations also lead to increased in vivo B cell proliferation and turnover (Otipoby et al., 1996; Nitschke et al., 1997; Poe et al., 2004; Haas et al., 2006; Onodera et al., 2008). However, studies designed to directly assess tolerance induction in antigen-specific CD22^−/− or SHP-1 mutant B cells found, paradoxically, a more robust tolerance relative to unmutated controls (Cyster and Goodnow, 1995; Cornall et al., 1998; Ferry et al., 2005). This suggests that the autoimmune phenotypes of siglec and SHP-1 null mutants could be caused by abnormal B cell selection and development rather than failure of tolerance. It is generally assumed that physical association of CD22 with the BCR will allow CD22 to exert a maximal inhibitory response (Pezzutto et al., 1987; Doody et al., 1995; Lanoue et al., 2002; Courtney et al., 2009), but evidence to support this has been garnered only from in vitro experiments (Ravetch and Lanier, 2000; Lanoue et al., 2002; Tedder et al., 2005; Courtney et al., 2009). In this paper, we show in wild-type mice with unaltered B cell selection and development that decorating a TI-2 antigen with siglec ligands not only prevents its immunogenicity but can also tolerize B cells to subsequent challenges with the unsialylated, immunogenic form. The results suggest that one function of B cell inhibitory receptors like siglecs is to assist B cells in distinguishing self from nonself.     

Intestinal monocytes and macrophages are required for T cell polarization in response to Citrobacter rodentium

Dendritic cells (DCs), monocytes, and macrophages are closely related phagocytes that share many phenotypic features and, in some cases, a common developmental origin. Although the requirement for DCs in initiating adaptive immune responses is well appreciated, the role of monocytes and macrophages remains largely undefined, in part because of the lack of genetic tools enabling their specific depletion. Here, we describe a two-gene approach that requires overlapping expression of LysM and Csf1r to define and deplete monocytes and macrophages. The role of monocytes and macrophages in immunity to pathogens was tested by their selective depletion during infection with Citrobacter rodentium. Although neither cell type was required to initiate immunity, monocytes and macrophages contributed to the adaptive immune response by secreting IL-12, which induced Th1 polarization and IFN-γ secretion. Thus, whereas DCs are indispensable for priming naive CD4⁺ T cells, monocytes and macrophages participate in intestinal immunity by producing mediators that direct T cell polarization.Inducing specific immunity and maintaining tolerance requires cells of the mononuclear phagocyte lineage. This lineage is comprised of three closely related cell types: DCs, monocytes, and macrophages (Shortman and Naik, 2007; Geissmann et al., 2010a,b; Liu and Nussenzweig, 2010; Yona and Jung, 2010; Chow et al., 2011). DCs are essential to both immunity and tolerance (Steinman et al., 2003); however, the role monocytes and macrophages play in these processes is not as well defined (Geissmann et al., 2008).In mice, DCs and monocytes arise from the same hematopoietic progenitor, known as the macrophage–DC progenitor (MDP; Fogg et al., 2006). Their development diverges when MDPs become either common DC progenitors (CDPs) that are Flt3L-dependent, or monocytes, which are dependent on CSF1 (M-CSF; Witmer-Pack et al., 1993; McKenna et al., 2000; Fogg et al., 2006; Waskow et al., 2008). CDPs develop into either plasmacytoid DCs or preDCs that leave the bone marrow to seed lymphoid and nonlymphoid tissues, where they further differentiate into conventional DCs (cDCs; Liu et al., 2009). In contrast, monocytes circulate in the blood and through tissues, where they can become activated and develop into several different cell types, including some but not all tissue macrophages (Schulz et al., 2012; Serbina et al., 2008; Yona et al., 2013).Despite their common origin from the MDP, steady-state lymphoid tissue cDCs can be distinguished from monocytes or macrophages by expression of cell surface markers. For example, cDCs in lymphoid tissues express high levels of CD11c and MHCII, but lack the expression of CD115 and F4/80 found in monocytes and macrophages, respectively. However, this distinction is far more difficult in peripheral tissues, like the intestine or lung, or during inflammation when monocytes begin to express many features of DC including high levels of MHCII and CD11c (Serbina et al., 2003; León et al., 2007; Hashimoto et al., 2011).The function of cDCs in immunity and tolerance has been explored extensively using a series of different mutant mice to ablate all or only some subsets of cDCs (Jung et al., 2002; Liu and Nussenzweig, 2010; Chow et al., 2011). In contrast, the methods that are currently available to study the function of monocytes and macrophages in vivo are far more restricted and less specific (Wiktor-Jedrzejczak et al., 1990; Dai et al., 2002; MacDonald et al., 2010; Chow et al., 2011). For example, Ccr2^−/− and Ccr2^DTR mice (Boring et al., 1997; Kuziel et al., 1997; Serbina and Pamer, 2006; Tsou et al., 2007) have been used to study monocytes (Boring et al., 1997; Peters et al., 2004; Hohl et al., 2009; Nakano et al., 2009). However, CCR2 is also expressed on some subsets of cDCs, activated CD4⁺ T cells, and NK cells (Kim et al., 2001; Hohl et al., 2009; Egan et al., 2009; Zhang et al., 2010). Thus, it is challenging to dissect the precise role of monocytes as opposed to other cell types in immune responses in Ccr2^−/− or Ccr2^DTR mice. Inducible DTR expression in CD11c^Cre x CX₃CR1^LsL-DTR mice is far more specific (Diehl et al., 2013), but restricted to a small subset of mononuclear phagocytes.Here, we describe a genetic approach to targeting monocytes and macrophages that spares cDCs and lymphocytes, and we compare the effects of monocyte and macrophage ablation to cDC depletion on the adaptive immune response to intestinal infection with Citrobacter rodentium.     

The Src family kinases Hck,Fgr, and Lyn are critical for the generation of the in vivo inflammatory environment without a direct role in leukocyte recruitment

Although Src family kinases participate in leukocyte function in vitro, such as integrin signal transduction, their role in inflammation in vivo is poorly understood. We show that Src family kinases play a critical role in myeloid cell–mediated in vivo inflammatory reactions. Mice lacking the Src family kinases Hck, Fgr, and Lyn in the hematopoietic compartment were completely protected from autoantibody-induced arthritis and skin blistering disease, as well as from the reverse passive Arthus reaction, with functional overlap between the three kinases. Though the overall phenotype resembled the leukocyte recruitment defect observed in β₂ integrin–deficient (CD18^−/−) mice, Hck^−/−Fgr^−/−Lyn^−/− neutrophils and monocytes/macrophages had no cell-autonomous in vivo or in vitro migration defect. Instead, Src family kinases were required for the generation of the inflammatory environment in vivo and for the release of proinflammatory mediators from neutrophils and macrophages in vitro, likely due to their role in Fcγ receptor signal transduction. Our results suggest that infiltrating myeloid cells release proinflammatory chemokine, cytokine, and lipid mediators that attract further neutrophils and monocytes from the circulation in a CD18-dependent manner. Src family kinases are required for the generation of the inflammatory environment but not for the intrinsic migratory ability of myeloid cells.Src family kinases are best known for their role in malignant transformation and tumor progression, as well as signaling through cell surface integrins (Parsons and Parsons, 2004; Playford and Schaller, 2004). Due to their role in cancer development and progression, Src family kinases have become major targets of cancer therapy (Kim et al., 2009; Zhang and Yu, 2012). Src family kinases are also present in immune cells with dominant expression of Lck and Fyn in T cells and NK cells; Lyn, Fyn, and Blk in B cells and mast cells; and Hck, Fgr, and Lyn in myeloid cells such as neutrophils and macrophages (Lowell, 2004).The best known function of Src family kinases in the immune system is their role in integrin signal transduction. Indeed, Hck, Fgr, and Lyn mediate outside-in signaling by β₁ and β₂ integrins in neutrophils and macrophages (Lowell et al., 1996; Meng and Lowell, 1998; Mócsai et al., 1999; Suen et al., 1999; Pereira et al., 2001; Giagulli et al., 2006; Hirahashi et al., 2006), Lck participates in LFA-1–mediated T cell responses (Morgan et al., 2001; Fagerholm et al., 2002; Feigelson et al., 2001; Suzuki et al., 2007), and Src family kinases are required for LFA-1–mediated signal transduction and target cell killing by NK cells (Riteau et al., 2003; Perez et al., 2004).Src family kinases also mediate TCR signal transduction by phosphorylating the TCR-associated immunoreceptor tyrosine-based activation motifs (ITAMs), leading to recruitment and activation of ZAP-70 (van Oers et al., 1996; Zamoyska et al., 2003; Palacios and Weiss, 2004). However, their role in receptor-proximal signaling by the BCR and Fc receptors is rather controversial. Although the combined deficiency of Lyn, Fyn, and Blk results in defective BCR-induced NF-κB activation, receptor-proximal BCR signaling (ITAM phosphorylation) is not affected (Saijo et al., 2003). Genetic deficiency of Lyn, the predominant Src family kinase in B cells, even leads to enhanced BCR signaling and B cell–mediated autoimmunity (Hibbs et al., 1995; Nishizumi et al., 1995; Chan et al., 1997). Similarly, both positive (Hibbs et al., 1995; Nishizumi and Yamamoto, 1997; Parravicini et al., 2002; Gomez et al., 2005; Falanga et al., 2012) and negative (Kawakami et al., 2000; Hernandez-Hansen et al., 2004; Odom et al., 2004; Gomez et al., 2005; Falanga et al., 2012) functions for Fyn and Lyn during Fc receptor signaling in mast cells have been reported. In addition, Hck^−/−Fgr^−/− neutrophils respond normally to IgG immune complex–induced activation (Lowell et al., 1996) and Fc receptor–mediated phagocytosis of IgG-coated red blood cells is delayed but not blocked in Hck^−/−Fgr^−/−Lyn^−/− macrophages (Fitzer-Attas et al., 2000; Lowell, 2004). The differential requirement for Src family kinases in TCR, BCR, and Fc receptor signaling is thought to derive from the fact that Syk, but not ZAP-70, is itself able to phosphorylate ITAM tyrosines (Rolli et al., 2002), making Src family kinases indispensable for signaling by the ZAP-70–coupled TCR but not by the Syk-coupled BCR and Fc receptors.Autoantibody production and immune complex formation is one of the major mechanisms of autoimmunity-induced tissue damage. In vivo models of those processes include the K/B×N serum transfer arthritis (Korganow et al., 1999) and autoantibody-induced blistering skin diseases (Liu et al., 1993; Sitaru et al., 2002, 2005), which mimic important aspects of human rheumatoid arthritis, bullous pemphigoid, and epidermolysis bullosa acquisita. Activation of neutrophils or macrophages (Liu et al., 2000; Wipke and Allen, 2001; Sitaru et al., 2002, 2005; Solomon et al., 2005), recognition of immune complexes by Fcγ receptors (Ji et al., 2002; Sitaru et al., 2002, 2005), and β₂ integrin–mediated leukocyte recruitment (Watts et al., 2005; Liu et al., 2006; Chiriac et al., 2007; Monach et al., 2010; Németh et al., 2010) are indispensable for the development of those in vivo animal models.The role of Src family kinases in β₂ integrin signaling and the requirement for β₂ integrins during autoantibody-induced in vivo inflammation prompted us to test the role of Src family kinases in autoantibody-induced inflammatory disease models. We found that Hck^−/−Fgr^−/−Lyn^−/− mice were completely protected from autoantibody-induced arthritis and inflammatory blistering skin disease. Surprisingly, this was not due to a cell-autonomous defect in β₂ integrin–mediated leukocyte migration but to defective generation of an inflammatory microenvironment, likely due to the role of Src family kinases in immune complex–induced neutrophil and macrophage activation.     

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.     

Conversion of Helicobacter pylori CagA from senescence inducer to oncogenic driver through polarity-dependent regulation of p21

The Helicobacter pylori CagA bacterial oncoprotein plays a critical role in gastric carcinogenesis. Upon delivery into epithelial cells, CagA causes loss of polarity and activates aberrant Erk signaling. We show that CagA-induced Erk activation results in senescence and mitogenesis in nonpolarized and polarized epithelial cells, respectively. In nonpolarized epithelial cells, Erk activation results in oncogenic stress, up-regulation of the p21^Waf1/Cip1 cyclin-dependent kinase inhibitor, and induction of senescence. In polarized epithelial cells, CagA-driven Erk signals prevent p21^Waf1/Cip1 expression by activating a guanine nucleotide exchange factor–H1–RhoA–RhoA-associated kinase–c-Myc pathway. The microRNAs miR-17 and miR-20a, induced by c-Myc, are needed to suppress p21^Waf1/Cip1 expression. CagA also drives an epithelial-mesenchymal transition in polarized epithelial cells. These findings suggest that CagA exploits a polarity-signaling pathway to induce oncogenesis.Infection with Helicobacter pylori cagA-positive strains is the strongest risk factor for the development of gastric carcinoma, the second leading cause of cancer-related death worldwide (Peek and Blaser, 2002; Parkin, 2004; Hatakeyama, 2008). The cagA gene encodes an ∼130–145-kD CagA protein, which is delivered via a bacterial type IV secretion system into gastric epithelial cells (Segal et al., 1999; Asahi et al., 2000; Backert et al., 2000; Odenbreit et al., 2000; Stein et al., 2000). Upon delivery, CagA is localized to the inner surface of the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs by host cell kinases (Backert and Selbach, 2005). Tyrosine-phosphorylated CagA acquires the ability to specifically bind to and deregulate SH2 domain–containing proteins such as SHP-2, Csk, and Crk (Higashi et al., 2002; Tsutsumi et al., 2003; Suzuki et al., 2005). CagA also interacts with Grb2 and c-Met in a phosphorylation-independent manner (Mimuro et al., 2002; Churin et al., 2003). Accordingly, the bacterial oncoprotein mimics the function of mammalian scaffolding/adaptor proteins, such as Gab, and thereby manipulates host-signaling molecules to provoke pathogenic actions (Hatakeyama, 2008). Many, if not all, of these CagA–host protein interactions trigger a cascade of signaling events that culminate in activation of the Erk microtubule-associated protein (MAP) kinase pathway, deregulation of which generates a growth-promoting oncogenic signal, in both Ras-dependent and -independent manners (Mimuro et al., 2002; Churin et al., 2003; Higashi et al., 2004; Suzuki et al., 2005).In polarized epithelial cells, CagA disrupts the tight junctions and causes loss of apical-basal epithelial polarity (Amieva et al., 2003; Saadat et al., 2007). This CagA activity is achieved through the interaction of CagA with Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK), an evolutionally conserved serine/threonine kinase originally isolated in Caenorhabditis elegans which plays a fundamental role in the establishment and maintenance of cell polarity (Saadat et al., 2007; Zeaiter et al., 2008). In mammals, there are four PAR1 isoforms (PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4) that redundantly phosphorylate MAPs and thereby destabilize microtubules, allowing asymmetric distribution of molecules which regulate cell polarity (Suzuki and Ohno, 2006). CagA functions as a universal inhibitor of PAR1 isoforms by directly binding to their kinase catalytic domains independent of CagA tyrosine phosphorylation (Saadat et al., 2007; Lu et al., 2009). The C-terminal 16-aa sequence of CagA that is specifically required for PAR1 binding has been designated as the CagA-multimerization (CM) sequence (Ren et al., 2006; Saadat et al., 2007; Lu et al., 2008). Recent structural analysis confirmed the importance of CM, which is also termed MARK kinase inhibitor sequence (MKI), for PAR1 interaction (Nesić et al., 2010).Consistent with the tumor-relevant activities of CagA, proliferation of gastric epithelial cells in patients infected with H. pylori cagA-positive strains has been reported to be significantly higher than that in patients infected by cagA-negative strains (Peek et al., 1997; Cabral et al., 2007). Furthermore, systemic expression of CagA in mice led to the development of gastrointestinal and hematological malignancies (Ohnishi et al., 2008; Miura et al., 2009). Hence, CagA is the first bacterial oncoprotein to be discovered in the context of human malignancy. Paradoxically, however, CagA has also been reported to act as a potent inhibitor of cell proliferation in vitro, an observation which is inconsistent with the oncogenic role of CagA (Tsutsumi et al., 2003; Higashi et al., 2004; Murata-Kamiya et al., 2007).In this paper, we show that CagA-deregulated Erk signaling in nonpolarized epithelial cells induces accumulation of the p21^Waf1/Cip1 cyclin-dependent kinase (CDK) inhibitor (hereafter referred to as p21), which in turn causes senescence-like proliferation arrest. In contrast, deregulated Erk signaling caused by CagA in polarized epithelial cells induces proliferation without accumulation of p21. We then describe the mechanism that determines the fate of epithelial cells in response to CagA, either senescence or forced mitogenesis, in an epithelial polarity-dependent manner. Our work reveals that CagA exploits the guanine nucleotide exchange factor (GEF)–H1–RhoA–RhoA-associated kinase (ROCK)–c-Myc–microRNA–p21 axis, a long-sought signaling pathway which connects epithelial polarity with the cell cycle, to exert its oncogenic action.     

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.