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.     

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.     

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.     

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.     

The G protein βγ subunit mediates reannealing of adherens junctions to reverse endothelial permeability increase by thrombin

The inflammatory mediator thrombin proteolytically activates protease-activated receptor (PAR1) eliciting a transient, but reversible increase in vascular permeability. PAR1-induced dissociation of Gα subunit from heterotrimeric Gq and G12/G13 proteins is known to signal the increase in endothelial permeability. However, the role of released Gβγ is unknown. We now show that impairment of Gβγ function does not affect the permeability increase induced by PAR1, but prevents reannealing of adherens junctions (AJ), thereby persistently elevating endothelial permeability. We observed that in the naive endothelium Gβ1, the predominant Gβ isoform is sequestered by receptor for activated C kinase 1 (RACK1). Thrombin induced dissociation of Gβ1 from RACK1, resulting in Gβ1 interaction with Fyn and focal adhesion kinase (FAK) required for FAK activation. RACK1 depletion triggered Gβ1 activation of FAK and endothelial barrier recovery, whereas Fyn knockdown interrupted with Gβ1-induced barrier recovery indicating RACK1 negatively regulates Gβ1-Fyn signaling. Activated FAK associated with AJ and stimulated AJ reassembly in a Fyn-dependent manner. Fyn deletion prevented FAK activation and augmented lung vascular permeability increase induced by PAR1 agonist. Rescuing FAK activation in fyn^−/− mice attenuated the rise in lung vascular permeability. Our results demonstrate that Gβ1-mediated Fyn activation integrates FAK with AJ, preventing persistent endothelial barrier leakiness.A persistent increase in endothelial permeability during inflammatory conditions such as pneumonia, trauma, and burn leads to the life-threatening illness acute respiratory distress syndrome (Mehta and Malik, 2006; Liu and Matthay, 2008). Increased endothelial permeability occurs because of loss of cell–cell contacts and disruption of cell–extracellular matrix (ECM) adhesions (Yuan, 2002; Mehta and Malik, 2006). Focal adhesion kinase (FAK) and VE-cadherin play a fundamental role in establishing the endothelial barrier to macromolecules and liquid by maintaining intercellular adhesion and cell–ECM adhesivity (Nelson et al., 2004; van Buul et al., 2005; Wu, 2005; Mehta and Malik, 2006; Dejana et al., 2008; Rudini and Dejana, 2008). We have shown that thrombin, a serine protease generated early on during acute respiratory distress syndrome, plays a critical role in increasing endothelial permeability by inducing the loss of VE-cadherin homotypic adhesion and redistribution of focal adhesions dependent on FAK (Mehta et al., 2002; Kouklis et al., 2004; Holinstat et al., 2006). Interestingly, the thrombin-induced increase in endothelial permeability is reversed within 2–3 h, indicating activation of endogenous pathways that limit the persistent increase in endothelial permeability produced by thrombin (Kouklis et al., 2004; Holinstat et al., 2006; Kaneider et al., 2007).Thrombin binds to endothelial cell surface receptor, protease-activating receptor 1 (PAR1) and PAR4 (Coughlin, 2000, 2005; Kataoka et al., 2003). We have shown that the permeability increasing effects of thrombin in lung endothelium are predominantly mediated through PAR1 because thrombin and selective PAR1 peptide agonists failed to induce endothelial contraction and lung microvascular permeability increase in mice lacking PAR1 (Vogel et al. 2000). PAR1 is a seven-transmembrane domain receptor that couples to heterotrimeric G proteins of the Gq and G12/13 families (Hung et al., 1992; Coughlin 1999). Upon ligation by thrombin, PAR1 signals the dissociation of the α-subunits of Gq and G12/13 from the Gβγ dimer. Gαq and Gα12/13 activate myosin light chain kinase and RhoA pathways, which by inducing endothelial cell contraction increase permeability (Goeckeler and Wysolmerski, 1995; Dudek and Garcia, 2001; Holinstat et al., 2003; McLaughlin et al., 2005; Knezevic et al., 2007; Singh et al., 2007; Gavard and Gutkind, 2008; Korhonen et al., 2009). However, the role of Gβγ after its dissociation from these heterotrimeric G proteins in the mechanism of PAR1-induced alteration in endothelial barrier function is unknown.The Gβγ pathway has progressively emerged as a critical element of GPCR signaling (Clapham and Neer, 1997; Cabrera-Vera et al., 2003; Oldham and Hamm, 2008). Gβγ is known to induce cyclic AMP generation (Tang and Gilman, 1992; Taurin et al., 2007), Ca²⁺ signaling (Herlitze et al., 1996; Blackmer et al., 2001), oxidant generation (Niu et al., 2003), neurotransmission (Blackmer et al., 2005), chemotaxis (Neptune and Bourne, 1997; Jin et al., 2000), and caveolae-mediated transcytosis (Shajahan et al., 2004). The β subunit of Gβγ contains WD 40 repeats that are thought to mediate protein–protein interactions (Neer et al., 1994; Chen et al., 2004b). Studies show that Gβ interacts with receptor for activated C kinase 1 (RACK1; Dell et al., 2002; Chen et al., 2004a), p60cSrc (Luttrell et al., 1996; McGarrigle and Huang, 2007), and Fyn (Yaka et al., 2002; Thornton et al., 2004). Fyn, p60cSrc, and RACK1 are known to influence adherens junctions (AJ) and focal adhesions (Xing et al., 1994; Bockholt and Burridge, 1995; Thomas and Brugge, 1997; Roura et al., 1999; Owens et al., 2000; Mourton et al., 2001; Schaller, 2001; Piedra et al., 2003). We tested the hypothesis that, besides restraining Gα subunits, Gβγ orchestrates signaling to terminate endothelial permeability increase through its ability to coordinate intercellular and cell–matrix interactions.To explore the function of Gβγ in regulating endothelial permeability, we interfered with expression of Gβγ, RACK1, or Fyn using small interfering RNA (siRNA) or Fyn knockout mice. We show that Gβγ plays a fundamental role in signaling endothelial barrier recovery. Moreover, we identified Fyn and FAK as the key downstream effectors of Gβγ. Fyn-mediated activation of FAK facilitated the association of FAK with p120-catenin and reannealing of AJ, which reversed the increased endothelial permeability responses produced by G protein–coupled receptor agonists.     

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.     

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.     

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.     

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.     

Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML

Leukemic stem cells (LSCs) isolated from acute myeloid leukemia (AML) patients are more sensitive to nuclear factor κB (NF-κB) inhibition-induced cell death when compared with hematopoietic stem and progenitor cells (HSPCs) in in vitro culture. However, inadequate anti-leukemic activity of NF-κB inhibition in vivo suggests the presence of additional survival/proliferative signals that can compensate for NF-κB inhibition. AML subtypes M3, M4, and M5 cells produce endogenous tumor necrosis factor α (TNF). Although stimulating HSPC with TNF promotes necroptosis and apoptosis, similar treatment with AML cells (leukemic cells, LCs) results in an increase in survival and proliferation. We determined that TNF stimulation drives the JNK–AP1 pathway in a manner parallel to NF-κB, leading to the up-regulation of anti-apoptotic genes in LC. We found that we can significantly sensitize LC to NF-κB inhibitor treatment by blocking the TNF–JNK–AP1 signaling pathway. Our data suggest that co-inhibition of both TNF–JNK–AP1 and NF-κB signals may provide a more comprehensive treatment paradigm for AML patients with TNF-expressing LC.NF-κB is a major mediator of immunity, inflammation, tissue regeneration, and cancer promotion signaling. It regulates multiple cell behaviors such as proliferation, survival, differentiation, and migration (Naugler and Karin, 2008; DiDonato et al., 2012; Perkins, 2012). Leukemic cells (LCs), including leukemic stem cells (LSCs), demonstrate increased NF-κB activity, which provides a critical survival signal (Kuo et al., 2013). In vitro studies demonstrated that NF-κB inhibition can largely eliminate LSC with minimal effects on normal hematopoietic stem and progenitor cells (HSPCs), suggesting the potential for NF-κB inhibition as an anti-leukemia therapy (Guzman et al., 2001). However, the use of NF-κB inhibitors alone in vivo does not effectively eliminate the acute myeloid leukemia (AML) cells, indicating that additional survival signals might be compensating for the effects of NF-κB inhibition. In addition, the clinical use of NF-κB inhibitors is also limited by potential side effects, including compromised T/B cell immunity, inflammatory tissue damage, and skin/liver cancer development (Chen et al., 2001; Zhang et al., 2004, 2007; Maeda et al., 2005; Sakurai et al., 2006; Luedde et al., 2007; Bettermann et al., 2010; Ke et al., 2010).TNF, a pro-inflammatory cytokine, has been shown to be a key mediator of inflammatory reactions in tumor tissues and is responsible for elevated NF-κB activity in many tumors. NF-κB levels are significantly increased in most tumor tissues, being produced by tumor-infiltrating hematopoietic cells, tumor cells, and/or tumor stromal cells (Anderson et al., 2004; Balkwill, 2006; Mantovani et al., 2008; Grivennikov and Karin, 2011; Ren et al., 2012). Animal model studies demonstrate that TNF plays an essential role in the pathogenesis of many types of cancer such as skin, liver, and colon cancers by directly stimulating tumor cell proliferation/survival or by inducing a tumor-promoting environment (Moore et al., 1999; Knight et al., 2000; Sethi et al., 2008; Balkwill, 2009; Oguma et al., 2010). Also, supportive care for some cancers includes inhibition of TNF signaling through use of soluble receptors and neutralizing antibodies (Egberts et al., 2008; Popivanova et al., 2008).Elevated serum TNF levels have been identified in patients with BM failure, including aplastic anemia and myelodysplastic syndromes (MDSs), suggesting that the hematopoietic-repressive activity of TNF might contribute to the cytopenic phenotype of such patients (Molnár et al., 2000; Dybedal et al., 2001; Dufour et al., 2003; Lv et al., 2007). The observed increased levels of TNF during disease progression in MDS patients imply that TNF might also be involved in the leukemic transformation of mutant HSPC (Tsimberidou and Giles, 2002; Stifter et al., 2005; Li et al., 2007; Fleischman et al., 2011). Increased levels of TNF are detected in the peripheral blood (PB) and BM of most human leukemia patients and are correlated to higher white blood cell counts and poorer prognosis (Tsimberidou et al., 2008). In fact, the importance of TNF in leukemogenesis is further documented in Fancc knockout mice and Bcr-Abl–transduced chronic myelogenous leukemia (CML) animal models. In these animals, TNF is required for inducing the leukemic transformation of Fancc mutant cells and promotes the proliferation of CML stem cells (Gallipoli et al., 2013).TNF can stimulate both survival and death signals within the same type of cells in a context-dependent fashion. TNF-dependent survival signals are mediated primarily by canonical NF-κB signaling (Sakurai et al., 2003; Skaug et al., 2009; Vallabhapurapu and Karin, 2009), whereas the TNF-induced death signal is driven by caspase-8–dependent apoptosis or RIP1/3-dependent necroptosis (Wang et al., 2008; He et al., 2009; Zhang et al., 2009, 2011; Feoktistova et al., 2011; Günther et al., 2011; Kaiser et al., 2011; Oberst et al., 2011; Tenev et al., 2011; Xiao et al., 2011). In addition, TNF can also stimulate the activation of MKK4/7-JNK signaling, although the role of the MKK4/7-JNK signaling pathway is also cell context–dependent (Liu and Lin, 2005; Bode and Dong, 2007; Kim et al., 2007; Chen, 2012). Many studies suggest that TNF-induced MKK4/7-JNK signaling is responsible for most of the side effects associated with NF-κB signal inactivation (Chen et al., 2001; Zhang et al., 2004, 2007; Maeda et al., 2005; Sakurai et al., 2006; Luedde et al., 2007; Ke et al., 2010).The role of MKK4/7-JNK signaling in the regulation of hematopoiesis is not entirely clear. Sustained JNK activation has been reported in many types of AML cells, coordinating with AKT/FOXO signaling to maintain an undifferentiated state (Sykes et al., 2011). In Bcr/Abl-induced CML, JNK1-AP1 signaling is required for the development of leukemia by mediating key survival signals (Hess et al., 2002). In Fanconi anemia, JNK is required for the TNF-induced leukemic clonal evolution of Fancc mutant HSPC (Li et al., 2007). These studies suggest that the JNK signal promotes the development and progression of leukemia by inducing proliferative and survival activities (Chen et al., 2001; Zhang et al., 2004, 2007; Maeda et al., 2005; Sakurai et al., 2006; Luedde et al., 2007; Bettermann et al., 2010; Ke et al., 2010).In this study, we searched for the survival signals which compensate for the inhibition of NF-κB signaling in AML stem and progenitor cells. We found that TNF stimulates JNK and NF-κB, which act as parallel survival signals in LC, whereas TNF acts through JNK to induce a death signal in HSPC. Inhibition of TNF-JNK signaling not only significantly sensitizes AML stem and progenitor cells to NF-κB inhibitor treatment but also protects HSPC from the toxicity of such treatment.     

DOCK8 regulates lymphocyte shape integrity for skin antiviral immunity

DOCK8 mutations result in an inherited combined immunodeficiency characterized by increased susceptibility to skin and other infections. We show that when DOCK8-deficient T and NK cells migrate through confined spaces, they develop cell shape and nuclear deformation abnormalities that do not impair chemotaxis but contribute to a distinct form of catastrophic cell death we term cytothripsis. Such defects arise during lymphocyte migration in collagen-dense tissues when DOCK8, through CDC42 and p21-activated kinase (PAK), is unavailable to coordinate cytoskeletal structures. Cytothripsis of DOCK8-deficient cells prevents the generation of long-lived skin-resident memory CD8 T cells, which in turn impairs control of herpesvirus skin infections. Our results establish that DOCK8-regulated shape integrity of lymphocytes prevents cytothripsis and promotes antiviral immunity in the skin.DOCK8, which is highly expressed only within the immune system, functions as an atypical guanine nucleotide exchange factor (GEF) to activate small Rho GTPases (Côté and Vuori, 2002; Ruusala and Aspenström, 2004; Meller et al., 2005; Harada et al., 2012; Mou et al., 2012) and its role as an adaptor in TLR9-MYD88 signaling suggests additional functions beyond GEF activity (Jabara et al., 2012). DOCK proteins and their orthologs participate in diverse biological processes, including gonadal and epidermal cell migration during embryonic development, tumor cell invasion, and leukocyte chemotaxis and trafficking through LNs (Kunisaki et al., 2006; Côté and Vuori, 2007; Gotoh et al., 2008; Kikuchi et al., 2008; Nishikimi et al., 2009, 2013; Harada et al., 2012).For most people without any obvious immune deficiency, infections with HSV, varicella-zoster virus, or human papillomavirus cause self-limited cold sores, chickenpox, or warts. However, these viruses can reemerge from latency to cause disease in up to ∼30% of the population (Higgins et al., 1993; Kilkenny and Marks, 1996; Harpaz et al., 2008). In contrast to normal individuals, DOCK8-deficient patients with autosomal-recessive loss-of-function mutations in DOCK8 have impaired cellular and humoral immunity (Engelhardt et al., 2009; Zhang et al., 2009; Su et al., 2011; Jing et al., 2014) that manifests as extreme susceptibility to skin and other infections (Chu et al., 2012). Patients often suffer from disseminated and persistent viral skin infections including those caused by HSV, varicella-zoster virus, human papillomavirus, and molluscum contagiosum. Their chronic viral infections may reflect multiple defects that affect T cell activation, proliferation, survival, and priming by dendritic cells (Zhang et al., 2009; Lambe et al., 2011; Randall et al., 2011; Harada et al., 2012; Crawford et al., 2013), NK cell cytotoxicity (Ham et al., 2013; Mizesko et al., 2013), and antiviral cytokine production (Zhang et al., 2009).T effector cells are a critical component of immunity to the types of viral skin infections characteristically seen in DOCK8 deficiency. These cells must scan for and target pathogens within the large volume of the skin, which is organized into two layers. The epidermis is composed of interlocking arrays of keratinocytes that impede the passage of immune effector cells (Honda et al., 2014). In contrast, the dermis is composed of a dense network of packed collagen fibers, through which immune cells must navigate (Wolf et al., 2009; Honda et al., 2014). The collagen fibers make up as much as one third of the wet weight of skin, as compared with ∼10% of aorta or ∼1% or less of other organs such as spleen and brain (Lowry et al., 1941; Neuman and Logan, 1950). Thus, the extracellular environments of the epidermis and dermis are characterized by many highly confined spaces, which are likely to tax the structural integrity of cells navigating to their targets. Given the presumptive role of DOCK8 in controlling cell cytoskeletal function and migration capacity, the fact that DOCK8-deficient patients—in comparison with other combined immunodeficiency patients—seem to suffer disproportionately from a broad variety of skin infections, and the evidence for physical constraints on immune cell movement in skin, we investigated whether the skin viral susceptibility of these patients might relate to a defect in effector cell migration. Our studies revealed an unexpected, critical role for DOCK8 in maintaining lymphocyte cellular integrity during migration in dense environments that limits host resistance.     

Annular PIP3 accumulation controls actin architecture and modulates cytotoxicity at the immunological synapse

The immunological synapse formed by a T lymphocyte on the surface of a target cell contains a peripheral ring of filamentous actin (F-actin) that promotes adhesion and facilitates the directional secretion of cytokines and cytolytic factors. We show that growth and maintenance of this F-actin ring is dictated by the annular accumulation of phosphatidylinositol trisphosphate (PIP₃) in the synaptic membrane. PIP₃ functions in this context by recruiting the exchange factor Dock2 to the periphery of the synapse, where it drives actin polymerization through the Rho-family GTPase Rac. We also show that synaptic PIP₃ is generated by class IA phosphoinositide 3-kinases that associate with T cell receptor microclusters and are activated by the GTPase Ras. Perturbations that inhibit or promote PIP₃-dependent F-actin remodeling dramatically affect T cell cytotoxicity, demonstrating the functional importance of this pathway. These results reveal how T cells use lipid-based signaling to control synaptic architecture and modulate effector responses.Stimulation of the TCR induces dramatic cytoskeletal remodeling that reshapes the interface between the T cell and the APC into an immunological synapse (IS; Gomez and Billadeau, 2008; Dustin et al., 2010). First, an intense burst of actin polymerization drives radially symmetric spreading over the APC. Subsequently, the filamentous actin (F-actin) within this circular lamellipodium resolves into an annular configuration (Bunnell et al., 2001; Stinchcombe et al., 2006; Sims et al., 2007). The resulting F-actin ring regulates the trafficking and clustering of signaling complexes and integrins (Varma et al., 2006; Nguyen et al., 2008; Babich et al., 2012; Yi et al., 2012). It also provides a structural framework for specifying effector function. Clearance of F-actin from the central synaptic membrane is coupled to the polarization of the microtubule-organizing center (MTOC) toward the APC (Huse, 2012). These events together facilitate the directional release of soluble factors into the IS. This is particularly important for CD8⁺ CTLs, which kill APCs by directional secretion of cytolytic perforins and granzymes (Stinchcombe and Griffiths, 2007).The pathways regulating synaptic F-actin architecture are not well understood. Studies suggest that TCR-induced actin polymerization and cell spreading require the Rho family GTPase Rac (Ku et al., 2001; Sanui et al., 2003; Nolz et al., 2006; Zipfel et al., 2006). Like all small GTPases, Rac is activated by specific guanine nucleotide exchange factors (GEFs) that catalyze its transition from an inactive, GDP-bound form into an active, GTP-bound form that recruits downstream effectors (Jaffe and Hall, 2005). T cells express several GEFs that could potentially regulate Rac. The most prominent is Vav, which functions as a core component of the TCR signaling complex (Tybulewicz, 2005). Recent work, however, has suggested that GEFs other than Vav might control Rac-dependent F-actin remodeling at the IS (Miletic et al., 2009). T cells also express Dock2, a Rac-specific CDM family GEF that catalyzes nucleotide exchange via its conserved DHR-2 domain (Côté and Vuori, 2007). T cells lacking Dock2 display marked defects in Rac activation and TCR trafficking (Sanui et al., 2003), implying that Dock2 might be involved in shaping synaptic F-actin. The N-terminal region of Dock2 binds constitutively to the scaffolding protein Elmo, which confers stabilization and enhances GEF activity toward Rac. Dock2 also contains a so-called DHR-1 domain, which binds specifically to phosphatidylinositol 3,4,5-trisphosphate (PIP₃; Côté and Vuori, 2007). In that regard, it is intriguing that TCR signaling induces robust PIP₃ accumulation at the IS (Costello et al., 2002; Harriague and Bismuth, 2002; Huppa et al., 2003; Garçon et al., 2008). The possibility that this pool of PIP₃ might regulate F-actin organization via recruitment of Dock2, however, has not been explored.It is generally thought that synaptic PIP₃ is produced by class I phosphoinositide 3-kinases (PI3Ks). It remains controversial, however, precisely which isoforms contribute to this process (Alcázar et al., 2007; Garçon et al., 2008; Sauer et al., 2008), and it is also unclear how these proteins might be recruited and activated by TCR signaling. Previous studies in T cells have focused on the role of phosphotyrosine (pTyr)-containing signaling motifs that can bind and allosterically activate certain PI3K isoforms (Carpenter et al., 1993; Holt et al., 1994; Pagès et al., 1994; Zhang et al., 1998; Shim et al., 2004, 2011). However, class I PI3Ks also interact with the small GTPase Ras (Rodriguez-Viciana et al., 1994, 1996), which functions synergistically with pTyr peptides to induce full PI3K activity (Jimenez et al., 2002). Ras is strongly activated by TCR signaling (Genot and Cantrell, 2000), but whether it promotes synaptic PIP₃ accumulation through PI3K is not known.In the present study, we demonstrate that the size and shape of the synaptic F-actin ring is dictated by an annular accumulation of PIP₃ in the overlying plasma membrane. This PIP₃ is generated by class IA PI3K isoforms downstream of Ras, and coordinates F-actin architecture by recruiting the Dock2/Elmo complex to the periphery of the IS. Specific perturbations in PIP₃-Dock2 signaling either reduce or enhance CTL-mediated killing, indicative of an important role in T cell effector responses. These results identify a previously uncharacterized mechanism for controlling synaptic F-actin, and provide insight into how lipid second messenger signaling shapes lymphocyte structure and function.     

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.