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.     

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.     

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.     

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.     

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.     

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.     

Histone H2AX stabilizes broken DNA strands to suppress chromosome breaks and translocations during V(D)J recombination

The H2AX core histone variant is phosphorylated in chromatin around DNA double strand breaks (DSBs) and functions through unknown mechanisms to suppress antigen receptor locus translocations during V(D)J recombination. Formation of chromosomal coding joins and suppression of translocations involves the ataxia telangiectasia mutated and DNA-dependent protein kinase catalytic subunit serine/threonine kinases, each of which phosphorylates H2AX along cleaved antigen receptor loci. Using Abelson transformed pre–B cell lines, we find that H2AX is not required for coding join formation within chromosomal V(D)J recombination substrates. Yet we show that H2AX is phosphorylated along cleaved Igκ DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation. We also show that H2AX prevents chromosome breaks emanating from unrepaired RAG endonuclease-generated TCR-α/δ locus coding ends in primary thymocytes. Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation. We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs.The rapid phosphorylation of histone H2A proteins in chromatin for large distances around DNA double strand breaks (DSBs) is a conserved feature of the cellular DNA damage response. In mammalian cells, the H2AX histone variant comprises 2–25% of the H2A pool and is nonuniformly incorporated into chromatin (Rogakou et al., 1998; Bewersdorf et al., 2006). Upon DSB induction, the ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ATR (ATM and Rad3 related) protein kinases phosphorylate H2AX on a conserved carboxyl terminal serine residue to form γ-H2AX around DNA breakage sites (Rogakou et al., 1999; Paull et al., 2000; Burma et al., 2001; Ward and Chen, 2001; Stiff et al., 2004). Generation of γ-H2AX creates binding sites for repair and checkpoint proteins, some of which catalyze other covalent modifications of γ-H2AX to generate binding sites for additional repair and checkpoint proteins, all of which assemble into complexes in chromatin surrounding DNA breaks (Downs et al., 2007; Bonner et al., 2008). H2ax^−/− cells exhibit increased sensitivity to agents that cause DSBs, elevated levels of spontaneous and DSB-induced genomic instability, and defective repair of chromosomal DSBs (Bassing et al., 2002a; Celeste et al., 2002; Xie et al., 2004; Franco et al., 2006). Although H2ax^−/− cells display apparent normal activation of p53-dependent cell cycle checkpoints and apoptotic responses (Bassing et al., 2002a; Celeste et al., 2002), H2ax^−/− cells are defective in the G2/M checkpoint after induction of only a few DSBs (Fernandez-Capetillo et al., 2002). The phenotypes of H2ax^−/− cells suggest that the ability of γ-H2AX to retain repair and checkpoint proteins around DSBs may promote accessibility of DNA ends, stabilize disrupted DNA strands, and/or amplify checkpoint signals (Bassing and Alt, 2004; Stucki and Jackson, 2006; Bonner et al., 2008; Kinner et al., 2008).The health and survival of humans and mice depends on the ability of their adaptive immune systems to generate lymphocytes with receptors capable of recognizing and eliminating large varieties of pathogens. In developing lymphocytes, Ig and TCR variable region exons are assembled from germline V (variable), D (diversity), and J (joining) gene segments by the lymphoid-specific RAG1/RAG2 (RAG) endonuclease and the ubiquitously expressed nonhomologous end-joining (NHEJ) DSB repair factors (Bassing et al., 2002b). The RAG proteins catalyze the coupled cleavage of DNA strands between a pair of gene segments and their flanking recombination signal sequences to generate covalently sealed coding ends (CEs) and blunt signal ends (SEs; Fugmann et al., 2000). RAG-mediated cleavage occurs only in G1 phase because of cell cycle phase-restricted expression of RAG2 (Lee and Desiderio, 1999). The DNA-PKcs/Artemis endonuclease opens CEs (Ma et al., 2002), which are then processed by nucleases and polymerases (McElhinny and Ramsden, 2004). Core NHEJ factors join together CEs and SEs to form coding joins (CJs) and signal joins (SJs), respectively (Bassing et al., 2002b). RAG1/RAG2 can hold CEs and SEs within stable synaptic complexes (Agrawal and Schatz, 1997; Lee et al., 2004); however, ATM and, likely, the Mre11–Rad50–Nbs1 (MRN) complex maintain chromosomal CEs in proximity to facilitate end-joining in G1 phase cells (Bredemeyer et al., 2006; Deriano et al., 2009; Helmink et al., 2009). The large combination of V(D)J joining events and the imprecision in CJ formation cooperate to generate a diverse repertoire of antigen receptor specificities.Despite its benefits, V(D)J recombination poses substantial threats to the viability and genomic integrity of lymphocytes and lymphoma predisposition in host organisms. For example, DNA-PKcs–deficient mice lack mature lymphocytes as a result of inability to repair RAG-generated DSBs, but they only occasionally develop lymphoma (Bosma et al., 1983; Gao et al., 1998; Taccioli et al. 1998). However, DNA-PKcs/p53–deficient mice rapidly succumb to pro-B lymphomas with RAG-dependent IgH/c-myc translocations (Vanasse et al., 1999; Gladdy et al., 2003), demonstrating that p53 protects organisms from oncogenic translocations during V(D)J recombination. RAG-generated DSBs activate ATM (Perkins et al., 2002; Bredemeyer et al., 2008), which is required for both normal coding join formation and normal p53 activation (Perkins et al., 2002; Bredemeyer et al., 2006). Consequently, Atm^−/− mice exhibit impaired lymphocyte development, increased frequencies of antigen receptor locus translocations in nonmalignant lymphocytes, and marked predisposition to thymic lymphomas with RAG-dependent TCR-α/δ translocations (Barlow et al., 1996; Elson et al., 1996; Xu et al., 1996; Borghesani et al., 2000; Liyanage et al., 2000; Petiniot et al., 2000, 2002; Callén et al., 2007; Matei et al., 2007; Vacchio et al., 2007). The observation that RAG-dependent γ-H2AX foci colocalized with TCR-α/δ loci suggested that H2AX may coordinate DSB repair, signaling, and surveillance during V(D)J recombination (Chen et al., 2000). Consistent with this notion, αβ T cells of H2ax^−/− mice contain elevated frequencies of TCR-α/δ translocations and H2ax^−/−p53^−/− mice develop pro–B lymphomas with RAG-dependent IgH/c-myc translocations (Celeste et al., 2002, 2003; Bassing et al., 2003, 2008). ATM and DNA-PKcs generate γ-H2AX along RAG-cleaved DNA strands (Savic et al., 2009). However, H2ax^−/− mice do not exhibit impaired or blocked lymphocyte development, as observed in mice deficient for ATM or DNA-PKcs, suggesting that H2AX is not involved in the processing and/or joining of chromosomal coding ends. However, H2AX prevents the progression of IgH locus DNA breaks into chromosome breaks and translocations during class switch recombination (CSR; Franco et al., 2006; Ramiro et al., 2006), which is consistent with the notion that H2AX does function in chromosomal end joining (Reina-San-Martin et al., 2003; Bassing and Alt, 2004). Consequently, the mechanisms by which H2AX suppresses antigen receptor locus translocations during V(D)J recombination remain unknown.Based on the disparate phenotypes of Atm^−/− and H2ax^−/− mice, we hypothesized that formation of γ-H2AX for long distances along RAG-cleaved antigen receptor loci promotes chromatin changes that hold together broken DNA strands (Bassing and Alt, 2004). We proposed that this stabilization of disrupted DNA strands would not be required for coding join formation in the G1 phase of developing lymphocytes but that this H2AX-dependent function would be important for preventing the irreversible dissociation of unrepaired coding ends persisting into S phase (Bassing and Alt, 2004). Quantitative analysis of DNA end joining during V(D)J recombination of endogenous loci in developing lymphocytes is difficult because of asynchronous induction of RAG DSBs at multiple genomic locations in cycling cells and expansion of lymphocytes in which functional coding joins have been assembled. Consequently, we have used a cell line–based system that enables the controlled induction of RAG DSBs at single defined chromosomal locations in G1-arrested cells to test our hypothesized functions of H2AX during V(D)J recombination.     

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.     

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.     

MGL1 promotes adipose tissue inflammation and insulin resistance by regulating 7/4hi monocytes in obesity

Adipose tissue macrophages (ATMs) play a critical role in obesity-induced inflammation and insulin resistance. Distinct subtypes of ATMs have been identified that differentially express macrophage galactose-type C-type lectin 1 (MGL1/CD301), a marker of alternatively activated macrophages. To evaluate if MGL1 is required for the anti-inflammatory function of resident (type 2) MGL1⁺ ATMs, we examined the effects of diet-induced obesity (DIO) on inflammation and metabolism in Mgl1^−/− mice. We found that Mgl1 is not required for the trafficking of type 2 ATMs to adipose tissue. Surprisingly, obese Mgl1^−/− mice were protected from glucose intolerance, insulin resistance, and steatosis despite having more visceral fat. This protection was caused by a significant decrease in inflammatory (type 1) CD11c⁺ ATMs in the visceral adipose tissue of Mgl1^−/− mice. MGL1 was expressed specifically in 7/4^hi inflammatory monocytes in the blood and obese Mgl1^−/− mice had lower levels of 7/4^hi monocytes. Mgl1^−/− monocytes had decreased half-life after adoptive transfer and demonstrated decreased adhesion to adipocytes indicating a role for MGL1 in the regulation of monocyte function. This study identifies MGL1 as a novel regulator of inflammatory monocyte trafficking to adipose tissue in response to DIO.Chronic inflammation is an important consequence of obesity that impacts upon the development of insulin resistance, diabetes, and metabolic syndrome (Hotamisligil, 2006; Neels and Olefsky, 2006). Obesity-induced systemic inflammation is characterized by chronic elevations in circulating inflammatory cytokines (e.g., TNF and IL-6), adipokines, and monocytes (Hotamisligil et al., 1995; Ghanim et al., 2004). At the tissue level, inflammatory pathways are induced in visceral adipose tissue that lead to a striking accumulation of macrophages (Weisberg et al., 2003; Xu et al., 2003). These adipose tissue macrophages (ATMs) are now recognized to be a significant participant in the inflammatory response to obesity as they generate a wide range of inflammatory cytokines in hypertrophied adipose tissue (Coppack, 2001). Attenuation of inflammatory genes such as Jnk1 and Ikkb in macrophages has been shown to decrease inflammation and prevent the development of insulin resistance and glucose intolerance with diet-induced obesity (DIO; Arkan et al., 2005; Solinas et al., 2007). Signals between inflammatory ATMs and adipocytes impair insulin sensitivity in adipocytes and influence adipocyte cell death (Cinti et al., 2005; Lumeng et al., 2007a).The biology of ATMs in both lean and obese states is slowly being revealed. ATMs are derived from the bone marrow and differentiate in adipose tissue from circulating monocytes (Weisberg et al., 2003). Two distinct types of ATMs have been identified. ATMs in obese mice have an activation pattern similar to that seen with classical or M1 macrophage activation, with high expression of Tnfa, Il6, and Nos2 (Lumeng et al., 2007b). We will refer to these cells as type 1 ATMs and define them as F4/80⁺ CD11c⁺ macrophage galactose-type C-type lectin⁻ 1 (MGL1⁻; Lumeng et al., 2007b, 2008; Nguyen et al., 2007). Type 1 ATMs organize themselves into clusters that have been described as “crownlike structures” (CLSs) that are closely coupled to adipocyte death (Cinti et al., 2005; Murano et al., 2008). These ATMs ingest triglyceride and take on an appearance similar to foam cells (Cinti et al., 2005; Lumeng et al., 2007a).A second population of ATMs has the phenotype F4/80⁺ CD11c⁻ MGL1⁺; we will refer to these as type 2 ATMs (Lumeng et al., 2008). These ATMs are the predominant macrophage type in adipose tissue in lean mice and express genes that overlap with alternatively activated or M2 macrophages, such as Arg1, Il10, Mgl1, and Mgl2 (Lumeng et al., 2007b). Type 2 ATMs localize in interstitial spaces between adipocytes and are present in both lean and obese states. It is believed that the alternative activation state of type 2 ATMs maintains homeostasis by suppressing proinflammatory signals activated with obesity as macrophage-specific knockouts of Pparg and Ppard demonstrate worse insulin resistance and inflammation (Hevener et al., 2007; Odegaard et al., 2007; Kang et al., 2008).Type 2 ATMs predominate in lean mice, whereas obesity induces the accumulation of type 1 ATMs leading to a proinflammatory environment (Lumeng et al., 2007b). The mechanism behind this shift in ATM phenotype may relate to the differential recruitment of monocyte subtypes to adipose tissue (Weisberg et al., 2006; Gordon, 2007; Lumeng et al., 2008; Nishimura et al., 2008). In mice, a population of 7/4^hi CCR2⁺ Ly-6C^hi CX₃CR1^lo monocytes are preferentially recruited to sites of tissue inflammation and generate classically activated macrophages (Gordon and Taylor, 2005; Gordon, 2007). In contrast, 7/4^mid CCR2⁻ Ly-6C^lo CX₃CR1^hi monocytes appear to be regulated by different stimuli and may play a role in patrolling noninflamed tissues that give rise to resident tissue macrophages (Geissmann et al., 2003; Auffray et al., 2007; Bouhlel et al., 2007; Charo, 2007). Importantly, 7/4^hi monocytes are increased with obesity, suggesting that they may be a specific inflammatory mediator of obesity-induced inflammation (Tsou et al., 2007).Type 2 ATMs express high levels of macrophage galactose-type C lectin 1 (MGL1/CD301), a marker of alternatively activated macrophages (Kang et al., 2008; van Kooyk, 2008). MGL1 is type 2 transmembrane protein expressed on macrophages and DCs in multiple tissues that is part of a family of scavenger receptors including macrophage mannose receptor (CD206; van Kooyk, 2008; van Vliet et al., 2008a). Functions for mMGL1, its homologue mMGL2, and human MGL in DCs include antigen presentation, suppression of effector T cell function, and negative regulation of cell migration (van Vliet et al., 2006, 2008a, 2008b). mMGL1 has binding specificity for Lewis X and Lewis A structures, which differentiates it from mMGL2 and hMGL (Tsuiji et al., 2002; Singh et al., 2009). MGL1 ligands include sialoadhesin, apoptotic cells, and commensal bacteria such as Streptococcus sp. (Kumamoto et al., 2004; Yuita et al., 2005; Saba et al., 2009). This latter interaction triggers production of IL-10 in macrophages and explains the increased inflammation seen with experimental colitis in Mgl1^−/− mice (Saba et al., 2009).We sought to examine the role of MGL1 in obesity-induced inflammation and insulin resistance by evaluating the response of Mgl1^−/− mice to DIO. We hypothesized that the protective functions of type 2 ATMs would be attenuated in Mgl1^−/− mice, and that they would show increased inflammation and worse insulin resistance. Surprisingly, we observed the opposite result as obese Mgl1^−/− mice had improved glucose tolerance and impaired accumulation of type 1 ATMs in visceral adipose tissue. The mechanism of this effect is related to the expression of MGL1 on inflammatory 7/4^hi monocytes and the lack of maintenance of these monocytes in the circulation caused by the intrinsic properties of Mgl1^−/− monocytes. Overall, we demonstrate that MGL1 is a novel cell surface receptor involved in the regulation of monocyte/macrophage activation and trafficking to fat in obesity.     

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.     

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.     

Deficiency for endoglin in tumor vasculature weakens the endothelial barrier to metastatic dissemination

Therapy-induced resistance remains a significant hurdle to achieve long-lasting responses and cures in cancer patients. We investigated the long-term consequences of genetically impaired angiogenesis by engineering multiple tumor models deprived of endoglin, a co-receptor for TGF-β in endothelial cells actively engaged in angiogenesis. Tumors from endoglin-deficient mice adapted to the weakened angiogenic response, and refractoriness to diminished endoglin signaling was accompanied by increased metastatic capability. Mechanistic studies in multiple mouse models of cancer revealed that deficiency for endoglin resulted in a tumor vasculature that displayed hallmarks of endothelial-to-mesenchymal transition, a process of previously unknown significance in cancer biology, but shown by us to be associated with a reduced capacity of the vasculature to avert tumor cell intra- and extravasation. Nevertheless, tumors deprived of endoglin exhibited a delayed onset of resistance to anti-VEGF (vascular endothelial growth factor) agents, illustrating the therapeutic utility of combinatorial targeting of multiple angiogenic pathways for the treatment of cancer.Efforts to achieve an effective cure against malignant diseases are commonly thwarted by the rapid emergence of therapy-resistant cancer cells that serve as the basis for resurgence of the tumor despite initial shrinkage. High hopes were placed on the development of antiangiogenic drugs, as it was thought that this class of agents would be inherently impervious to mechanisms of acquired resistance by means of targeting the nonmalignant and genetically stable tumor endothelial cells (Kerbel, 1991, 1997). However, the initial clinical experience with drugs selectively targeting the tumor neovasculature, such as bevacizumab, sunitinib, and sorafenib, has been sobering. Major clinical responses to these drugs, with targeting of the prototypical proangiogenic vascular endothelial growth factor (VEGF) as a common denominator, are rare, and the median prolongation of progression-free survival is typically 2–6 mo with minimal effect on overall survival after long-term follow up (Hurwitz et al., 2004; Escudier et al., 2007; Motzer et al., 2007). Mechanistic insight into evasive or intrinsic resistance to antiangiogenic therapy comes from recent preclinical trials (Bergers and Hanahan, 2008; Ebos et al., 2009b). Specifically, pharmacological inhibition of VEGF signaling in mouse models of cancer results in up-regulation of compensatory angiogenic pathways (Casanovas et al., 2005) and enhanced protective coverage of pericytes (Pietras and Hanahan, 2005). In parallel, tumors escalate the seeding of metastases as a result of hypoxia-induced increased local invasiveness (Ebos et al., 2009a; Pàez-Ribes et al., 2009). In yet other studies, contradictory results were presented demonstrating no association between anti-VEGF therapy and metastatic behavior (Chung et al., 2012; Singh et al., 2012; Welti et al., 2012). Clearly, in depth mechanistic studies are warranted to resolve the apparent controversies.Members of the TGF-β family act pleiotropically on most, if not all, cell types in the body by engaging a heterotetrameric complex of type I and type II receptors (ten Dijke and Arthur, 2007; Massagué, 2008). Genetic targeting studies in mice provide ample evidence for a role of signaling by TGF-β ligands, receptors, and downstream mediators during developmental angiogenesis, although the precise mechanism remains unclear (David et al., 2009; Cunha and Pietras, 2011; van Meeteren et al., 2011). Moreover, pharmacological blocking of signaling by the endothelial cell–restricted type I receptor activin receptor-like kinase 1 (ALK1) inhibits tumor growth by impairing pathological angiogenesis (Cunha et al., 2010; Mitchell et al., 2010; Hu-Lowe et al., 2011). Signaling by ALK1 is complemented by the TGF-β co-receptor endoglin (ten Dijke et al., 2008; Pérez-Gómez et al., 2010; Nassiri et al., 2011). Endoglin (also known as CD105) is selectively expressed by endothelial cells actively engaged in vasculogenesis, angiogenesis, and inflammation and acts to promote endothelial cell proliferation, migration, and tube formation (Jonker and Arthur, 2002; Torsney et al., 2003; Lebrin et al., 2004; Jerkic et al., 2006). Germline mutations in the gene encoding endoglin are causative of the vascular syndrome hereditary hemorrhagic telangiectasia (HHT), characterized by arteriovenous malformations and frequent bleedings (Shovlin, 2010), a condition partially phenocopied by mice lacking a single copy of Eng (Bourdeau et al., 1999; Li et al., 1999; Arthur et al., 2000; Torsney et al., 2003) and more recently in mice with endothelial-specific endoglin depletion (Mahmoud et al., 2010). In tumors, endoglin is selectively up-regulated on endothelial cells (Westphal et al., 1993; Burrows et al., 1995; Miller et al., 1999; Bernabeu et al., 2009), and in many different tumor types, including breast, colon, and lung carcinoma, abundant expression of endoglin is a predictor of poor survival (Kumar et al., 1999; Takahashi et al., 2001b; Wikström et al., 2002; Charpin et al., 2004; Dales et al., 2004; Martone et al., 2005). Accordingly, partial genetic ablation or antibody targeting of endoglin delays tumor growth in mouse models of cancer through inhibition of angiogenesis (Seon et al., 1997; Takahashi et al., 2001a; Düwel et al., 2007; Seon et al., 2011). Collectively, endoglin appears as a valid therapeutic target for efforts to suppress tumor angiogenesis, but it is not known whether the long-term efficacy of such targeting would be limited by induction of adaptive mechanisms.Here, we have delineated a novel mode of metastatic dissemination associated with tumors refractory to attenuated expression of endoglin. Deficiency for even a single copy of endoglin was characterized by an increased seeding of metastases caused by a weakened endothelial cell barrier to tumor cell intra- and extravasation. Strikingly, endoglin-deficient endothelial cells adapted a more mesenchymal phenotype consequential to enhanced signaling through the TGF-β type I receptor ALK5; increased endothelial-to-mesenchymal transition (EndMT) in the tumor vasculature thus constitutes a novel mechanism for metastatic dissemination.     

DOCK5 functions as a key signaling adaptor that links FcεRI signals to microtubule dynamics during mast cell degranulation

Mast cells play a key role in the induction of anaphylaxis, a life-threatening IgE-dependent allergic reaction, by secreting chemical mediators that are stored in secretory granules. Degranulation of mast cells is triggered by aggregation of the high-affinity IgE receptor, FcεRI, and involves dynamic rearrangement of microtubules. Although much is known about proximal signals downstream of FcεRI, the distal signaling events controlling microtubule dynamics remain elusive. Here we report that DOCK5, an atypical guanine nucleotide exchange factor (GEF) for Rac, is essential for mast cell degranulation. As such, we found that DOCK5-deficient mice exhibit resistance to systemic and cutaneous anaphylaxis. The Rac GEF activity of DOCK5 is surprisingly not required for mast cell degranulation. Instead, DOCK5 associated with Nck2 and Akt to regulate microtubule dynamics through phosphorylation and inactivation of GSK3β. When DOCK5–Nck2–Akt interactions were disrupted, microtubule formation and degranulation response were severely impaired. Our results thus identify DOCK5 as a key signaling adaptor that orchestrates remodeling of the microtubule network essential for mast cell degranulation.Mast cells play a key role in induction of anaphylaxis, a life-threatening allergic reaction which occurs rapidly after exposure of certain antigens, such as foods, drugs, and insect venoms (Sampson et al., 2005). Mast cells express the high-affinity receptor for IgE, FcεRI, on their surface, and binding of multivalent antigens to FcεRI-bound IgE induces receptor aggregation and triggers mast cell activation (Kawakami and Galli, 2002; Kraft and Kinet, 2007). Activated mast cells secrete preformed chemical mediators, including proteases and vasoactive amines such as histamine, which are stored in cytoplasmic secretory granules (Kawakami and Galli, 2002; Lundequist and Pejler, 2011). This process involves the movement of secretory granules and their fusion with the plasma membrane followed by exocytosis to release the chemical mediators (Blott and Griffiths, 2002; Lundequist and Pejler, 2011). Degranulation of mast cells is therefore a complex and multistep process that is tightly regulated by FcεRI-mediated signals.Upon aggregation of FcεRI with IgE and antigens, two parallel signaling cascades operate. One cascade is initiated by activation of the Src family protein tyrosine kinase Lyn, which is bound to the FcεRI β subunit, and involves subsequent activation of the nonreceptor protein tyrosine kinase Syk (Kawakami and Galli, 2002; Kraft and Kinet, 2007; Alvarez-Errico et al., 2009; Gilfillan and Rivera, 2009; Kambayashi et al., 2009). The activated Syk then phosphorylates multiple substrates, including PLC-γ (Kawakami and Galli, 2002; Kraft and Kinet, 2007; Alvarez-Errico et al., 2009; Gilfillan and Rivera, 2009; Kambayashi et al., 2009). The other cascade uses Fyn, another FcεRI-associated Src family protein tyrosine kinase (Kraft and Kinet, 2007; Alvarez-Errico et al., 2009; Gilfillan and Rivera, 2009; Kambayashi et al., 2009). Fyn phosphorylates the adaptor protein Gab2, which leads to activation of phosphatidylinositol 3-kinase (PI3K) through association with the p85α regulatory subunit (Gu et al., 2001; Parravicini et al., 2002; Nishida et al., 2005, 2011). Several lines of evidence indicate that although the Lyn–Syk–PLC-γ axis regulates granule-plasma membrane fusion and exocytosis by controlling calcium response (Nishida et al., 2005; Alvarez-Errico et al., 2009; Gilfillan and Rivera, 2009; Kambayashi et al., 2009), the Fyn–Gab2 pathway plays a key role in translocation of secretory granules to the plasma membrane (Parravicini et al., 2002; Nishida et al., 2005, 2011). However, little is known about the distal events controlling mast cell degranulation. In particular, movement of secretory granules requires dynamic rearrangement of microtubules (Martin-Verdeaux et al., 2003; Smith et al., 2003; Nishida et al., 2005; Dráber et al., 2012), yet the signaling events regulating this step of mast cell activation are poorly understood.GSK3β is a serine/threonine kinase that negatively regulates microtubule dynamics (Cohen and Frame, 2001; Zhou and Snider, 2005). In resting cells, GSK3β phosphorylates many microtubule-binding proteins and inhibits their ability to interact with microtubules and to promote microtubule assembly (Zhou et al., 2004; Yoshimura et al., 2005; Kim et al., 2011). This inhibitory effect is relieved when GSK3β is phosphorylated at serine residue of position 9 (Ser9; Cohen and Frame, 2001). Although knockdown experiments revealed a role for GSK3β in cytokine production, chemotaxis, and survival of human mast cells (Rådinger et al., 2010; Rådinger et al., 2011), aggregation of FcεRI also induces GSK3β phosphorylation at Ser9 (Rådinger et al., 2010). Therefore, phosphorylation-dependent inactivation of GSK3β may be involved in FcεRI-mediated regulation of microtubule dynamics in mast cells.DOCK5 is a member of the atypical guanine nucleotide exchange factors (GEFs) for the Rho family of GTPases (Côté and Vuori, 2002). Although DOCK5 does not contain the Dbl homology domain typically found in GEFs (Schmidt and Hall, 2002), DOCK5 mediates the GTP–GDP exchange reaction for Rac through DOCK homology region 2 (DHR-2; also known as CZH2 or Docker) domain (Brugnera et al., 2002; Côté and Vuori, 2002; Meller et al., 2002). DOCK5 is widely expressed in various tissues and regulates multiple cellular functions, including myoblast fusion and bone resorption (Laurin et al., 2008; Vives et al., 2011), yet its roles in the immune system and immune responses remain unexplored. In this study, we demonstrate that DOCK5 regulates FcεRI-mediated anaphylactic responses in vivo and mast cell degranulation in vitro. Unexpectedly, this regulation by DOCK5 does not require its Rac GEF activity, but instead involves association with Nck2 and Akt. When this interaction was blocked in mast cells, FcεRI-mediated phosphorylation and inactivation of GSK3β were impaired, resulting in a marked reduction in microtubule dynamics and a severe defect in degranulation. Our results thus define a novel regulatory mechanism controlling degranulation response in mast cells.     

TIM1 is an endogenous ligand for LMIR5/CD300b: LMIR5 deficiency ameliorates mouse kidney ischemia/reperfusion injury

Leukocyte mono-immunoglobulin (Ig)–like receptor 5 (LMIR5)/CD300b is a DAP12-coupled activating receptor predominantly expressed in myeloid cells. The ligands for LMIR have not been reported. We have identified T cell Ig mucin 1 (TIM1) as a possible ligand for LMIR5 by retrovirus-mediated expression cloning. TIM1 interacted only with LMIR5 among the LMIR family, whereas LMIR5 interacted with TIM4 as well as TIM1. The Ig-like domain of LMIR5 bound to TIM1 in the vicinity of the phosphatidylserine (PS)-binding site within the Ig-like domain of TIM1. Unlike its binding to TIM1 or TIM4, LMIR5 failed to bind to PS. LMIR5 binding did not affect TIM1- or TIM4-mediated phagocytosis of apoptotic cells, and stimulation with TIM1 or TIM4 induced LMIR5-mediated activation of mast cells. Notably, LMIR5 deficiency suppressed TIM1-Fc–induced recruitment of neutrophils in the dorsal air pouch, and LMIR5 deficiency attenuated neutrophil accumulation in a model of ischemia/reperfusion injury in the kidneys in which TIM1 expression is up-regulated. In that model, LMIR5 deficiency resulted in ameliorated tubular necrosis and cast formation in the acute phase. Collectively, our results indicate that TIM1 is an endogenous ligand for LMIR5 and that the TIM1–LMIR5 interaction plays a physiological role in immune regulation by myeloid cells.A growing number of studies have characterized a variety of paired activating and inhibitory receptors (Ravetch and Lanier, 2000; Klesney-Tait et al., 2006; Lanier, 2009). We have previously identified a leukocyte mono-Ig–like receptor (LMIR) mainly expressed in myeloid cells (Kumagai et al., 2003; Izawa et al., 2007; Yamanishi et al., 2008). The mouse LMIR family is also known as the CMRF-35–like molecule/myeloid-associated Ig-like receptor/dendritic cell–derived Ig-like receptor/CD300 family (Luo et al., 2001; Chung et al., 2003; Yotsumoto et al., 2003). LMIR1 and LMIR3 are immunoreceptor tyrosine-based inhibitory motif–containing inhibitory receptors, whereas other members are activating receptors that associate with immunoreceptor tyrosine-based activation motif–containing adaptor proteins (Luo et al., 2001; Chung et al., 2003; Kumagai et al., 2003; Yotsumoto et al., 2003; Izawa et al., 2007; Yamanishi et al., 2008). LMIR5 is a DAP12-coupled activating receptor predominantly expressed in myeloid cells (Yamanishi et al., 2008). However, the ligands for LMIR remained unknown. In this study, we identified T cell Ig mucin 1 (TIM1) as a ligand for LMIR5 by retrovirus-mediated expression cloning (Kitamura et al., 2003).TIM1–4 are characterized as important regulators of immune responses associated with autoimmunity and allergic diseases (McIntire et al., 2001; Kuchroo et al., 2003, 2008). The TIM molecules are type 1 cell-surface glycoproteins, consisting of an N-terminal IgV domain and a mucin domain. TIM1/hepatitis A virus cellular receptor 1 (Kaplan et al., 1996)/kidney injury molecule–1 (KIM-1; Ichimura et al., 1998) is expressed in activated T cells and delivers a signal that enhances T cell activation and proliferation (Meyers et al., 2005; Umetsu et al., 2005). TIM1 can also interact with itself (Santiago et al., 2007b). In addition, a soluble form of KIM-1/TIM1 is released by shedding (Bailly et al., 2002). On the other hand, TIM4 is expressed in macrophages and dendritic cells and is a natural ligand for TIM1 (Meyers et al., 2005). Interestingly, TIM1 and TIM4 recognize phosphatidylserine (PS) and are critical for the efficient clearance of apoptotic cells (Kobayashi et al., 2007; Miyanishi et al., 2007; Santiago et al., 2007a; Ichimura et al., 2008). Recent reports have demonstrated that the narrow cavity built by the CC’ and FG loops of the Ig-like domain is a binding site for PS (Kobayashi et al., 2007; Santiago et al., 2007a,b). In addition, TIM1/KIM-1 expression is strongly induced in the injured kidney epithelial cells (Ichimura et al., 1998, 2008; Waanders et al., 2010), and confers a phagocytic phenotype on epithelial cells (Ichimura et al., 2008). TIM1 is also a marker for renal tubular damage (Waanders et al. 2010).In the present study, using biological and biochemical analysis, we demonstrate that TIM1 and TIM4 are endogenous ligands for LMIR5. In addition, we generated LMIR5^−/− mice and delineated the physiological significance of the LMIR5–TIM1 interaction by using an acute kidney injury model.