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

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

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

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

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.     

Broadly neutralizing antibodies that inhibit HIV-1 cell to cell transmission

The neutralizing activity of anti–HIV-1 antibodies is typically measured in assays where cell-free virions enter reporter cell lines. However, HIV-1 cell to cell transmission is a major mechanism of viral spread, and the effect of the recently described broadly neutralizing antibodies (bNAbs) on this mode of transmission remains unknown. Here we identify a subset of bNAbs that inhibit both cell-free and cell-mediated infection in primary CD4⁺ lymphocytes. These antibodies target either the CD4-binding site (NIH45-46 and 3BNC60) or the glycan/V3 loop (10-1074 and PGT121) on HIV-1 gp120 and act at low concentrations by inhibiting multiple steps of viral cell to cell transmission. These antibodies accumulate at virological synapses and impair the clustering and fusion of infected and target cells and the transfer of viral material to uninfected T cells. In addition, they block viral cell to cell transmission to plasmacytoid DCs and thereby interfere with type-I IFN production. Thus, only a subset of bNAbs can efficiently prevent HIV-1 cell to cell transmission, and this property should be considered an important characteristic defining antibody potency for therapeutic or prophylactic antiviral strategies.HIV-1–infected individuals produce high titers of antibodies against the virus, but only a small fraction of the patients develop a broadly neutralizing serologic activity, generally after 2–4 yr of infection (Sather et al., 2009; Simek et al., 2009; Stamatatos et al., 2009; Walker et al., 2011; McCoy and Weiss, 2013). The serologic anti–HIV-1 activity in some of these individuals can be accounted for by a combination of antibodies targeting different sites on the HIV-1 envelope spike (Scheid et al., 2009; Bonsignori et al., 2012; Klein et al., 2012a; Georgiev et al., 2013) and in others, by a predominant highly expanded clone (Scheid et al., 2011; Walker et al., 2011; Burton et al., 2012; McCoy and Weiss, 2013). Although the presence of broad neutralizing activity does not correlate with a better clinical outcome, passive transfer of broadly neutralizing antibodies (bNAbs) can protect against infection in macaques or in mouse models (Hessell et al., 2009; Pietzsch et al., 2012; McCoy and Weiss, 2013). In addition, bNAbs can suppress viremia in humanized mice (Klein et al., 2012b). Moreover, antibodies against the HIV-1 envelope spike appear to be the unique correlate of protection in the RV144 HIV-1 vaccine trial (Haynes et al., 2012). Therefore, it has been proposed that vaccines that would elicit such antibodies may be protective against the infection in humans.The recent development of efficient methods for cloning of human anti–HIV-1 antibodies from single cells (Scheid et al., 2009) led to the discovery of dozens of new bNAbs and new targets for neutralization (Burton et al., 2012; McCoy and Weiss, 2013). The new antibodies target at least six different sites of vulnerability on the HIV-1 spike. These include the CD4-binding site (VRC01, NIH45-46, 3BNC60/117, and CH103), the glycan-dependent V1/V2 loops (PG16 and PGT145) and V3 loop (PGT121, PGT128, and the 10-1074 family), a conformational epitope on gp120 (3BC176), a domain in the vicinity of the CD4bs (8ANC195), and the gp41 membrane-proximal external region (MPER; 2F5, 4E10, and 10E8; Scheid et al., 2009, 2011; Walker et al., 2011; Wu et al., 2011; Kwong and Mascola, 2012; Mouquet et al., 2012; West et al., 2012; Liao et al., 2013). Some of these antibodies display remarkable antiviral activity with median 50% inhibitory concentrations (IC50s) < 0.2 µg/ml for up to 95% of isolates tested (Diskin et al., 2011; Scheid et al., 2011; Walker et al., 2011; Wu et al., 2011; Burton et al., 2012; Liao et al., 2013).The antiviral activity of bNAbs is typically measured in vitro using cell-free pseudovirus particles and reporter cell lines, such as the HeLa-derived TzMbl cell (Heyndrickx et al., 2012). In these assays, neutralization is mediated by inhibition of free virus binding to cellular receptors and/or by inhibition of viral fusion. Although cell-free HIV-1 is infectious, the virus replicates more efficiently and rapidly through direct contact between cells, and this mode of transmission likely mediates a significant fraction of viral spread and immune evasion in vivo (Dimitrov et al., 1993; Sourisseau et al., 2007; Sattentau, 2011; Murooka et al., 2012; Dale et al., 2013). In addition, this form of dissemination appears to be less susceptible to inhibition by antiretroviral drugs than cell-free virus transmission (Chen et al., 2007; Sigal et al., 2011; Abela et al., 2012).Cell to cell spread of HIV-1 is in large part mediated through virological synapses, where viral particles accumulate at the interface between infected cells and targets (Sattentau, 2011; Dale et al., 2013). Synapse formation involves HIV-1 Env-CD4 coreceptor interactions and requires cytoskeletal rearrangements and adhesion molecules (Sattentau, 2011; Dale et al., 2013).Here, we examined the antiviral activity of a panel of 15 newly identified bNAbs targeting all known sites of vulnerability in conventional neutralization and cell to cell transmission assays. We show that only a subset of the bNAbs that target the CD4-binding site or the glycan/V3 loop efficiently neutralize cell to cell viral transfer in co-cultures of infected T cells with primary lymphocytes. We further characterized the antiviral mechanisms used by the effective antibodies and report that they affect multiple steps of viral cell to cell transfer.     

Spermatozoa capture HIV-1 through heparan sulfate and efficiently transmit the virus to dendritic cells

Semen is the main vector for HIV-1 dissemination worldwide. It contains three major sources of infectious virus: free virions, infected leukocytes, and spermatozoa-associated virions. We focused on the interaction of HIV-1 with human spermatozoa and dendritic cells (DCs). We report that heparan sulfate is expressed in spermatozoa and plays an important role in the capture of HIV-1. Spermatozoa-attached virus is efficiently transmitted to DCs, macrophages, and T cells. Interaction of spermatozoa with DCs not only leads to the transmission of HIV-1 and the internalization of the spermatozoa but also results in the phenotypic maturation of DCs and the production of IL-10 but not IL-12p70. At low values of extracellular pH (∼6.5 pH units), similar to those found in the vaginal mucosa after sexual intercourse, the binding of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs were strongly enhanced. Our observations support the notion that far from being a passive carrier, spermatozoa acting in concert with DCs might affect the early course of sexual transmission of HIV-1 infection.The UNAIDS/World Health Organization AIDS epidemic update estimated that 33 million people were living with HIV at the end of 2007. New infections have been occurring in the past few years at a rate of 3.2 million per year, and almost 2 million individuals succumbed to AIDS-related diseases in 2007 (UNAIDS, 2007). Most infections are acquired through sexual transmission during vaginal or anal intercourse, with semen being the major transmission vector for HIV-1 (Miller and Shattock, 2003; Pope and Haase, 2003; Haase, 2005; Lederman et al., 2006).Semen contains three major sources of infectious virus: free virions, spermatozoa-associated virions, and infected leukocytes (Miller and Shattock, 2003; Gupta and Klasse, 2006; Lederman et al., 2006; Hladik and McElrath, 2008). The role of each of these sources in sexual transmission of HIV-1 is not well defined. Few studies have addressed this important question. Free virus and seminal infected leukocytes appear to play an important role in sexual transmission of HIV-1 (Miller and Shattock, 2003; Gupta and Klasse, 2006; Lederman et al., 2006; Hladik and McElrath, 2008). The role of spermatozoa, however, has been a matter of debate (Mermin et al., 1991; Dussaix et al., 1993; Quayle et al., 1997; Pudney et al., 1998), in spite of the fact that the presence of viral particles and/or nucleic acids in spermatozoa from HIV-1–infected men has been largely demonstrated using a variety of techniques (Baccetti et al., 1994, 1998; Bagasra et al., 1994; Nuovo et al., 1994; Dulioust et al., 1998; Muciaccia et al., 1998, 2007; Barboza et al., 2004).The identity of the receptor for HIV-1 expressed in the spermatozoa remains unclear. On the basis of its ability to recognize the HIV gp120, it has been proposed that mannose receptors (MRs) might function as HIV-1 receptors on the spermatozoa (Bandivdekar et al., 2003; Cardona-Maya et al., 2006; Fanibunda et al., 2008). It has also been shown that the gp120 can be recognized by galactosyl-alkyl-acylglycerol, a glycolipid expressed on the spermatozoa, suggesting that, as described for keratinocytes and epithelial cells, this molecule also contributes to the attachment of HIV-1 to the spermatozoa (Brogi et al., 1996, 1998).After deposition of HIV-1 on the mucosa, the virus must cross the mucosal epithelium to interact with T CD4⁺ lymphocytes, macrophages, and DCs, which are the most important targets of infection (Mermin et al., 1991; Miller and Shattock, 2003; Haase, 2005; Gupta and Klasse, 2006; Lederman et al., 2006; Hladik and McElrath, 2008). These cells express the HIV-1 receptors CD4 and coreceptors CCR5 or CXCR4 which are required for infection (Haase, 2005; Gupta and Klasse, 2006; Lederman et al., 2006; Hladik and McElrath, 2008). It is now clear that DCs are able to capture HIV-1 at entry sites and transport the virus to draining lymph nodes, where HIV-1 is efficiently transmitted to T CD4⁺ cells, which become the center of viral replication (Geijtenbeek et al., 2000; Gurney et al., 2005; Wilkinson and Cunningham, 2006; Wu and KewalRamani, 2006).The pathways used by HIV-1 to cross the mucosal epithelium are not well defined. The virions may transcytose through the genital epithelium (Gupta and Klasse, 2006; Hladik and McElrath, 2008) or may pass the barrier through genital lesions, either as cell-free or cell-associated virus (Piot and Laga, 1989; Serwadda et al., 2003; Galvin and Cohen, 2004). The latter possibility could account for the association of HIV infections with sexually transmitted diseases (Miller and Shattock, 2003; Haase, 2005; Lederman et al., 2006). It is of note that epithelial microabrasions in the vagina are detected in 60% of healthy women after consensual intercourse (Norvell et al., 1984; Guimarães et al., 1997), suggesting that the presence of genital microlesions represent a frequent scenario for the transmission of HIV-1. Anal intercourse is also often associated with mucosal trauma and, because the rectal epithelium is only one cell layer thick, it provides a low degree of protection against trauma, favoring the access of virus to the underlying target cells (Shattock and Moore, 2003). Moreover, the access of HIV-1 to target cells may be facilitated by other mechanisms such as the binding of HIV-1 to DC projections that extend to the luminal surface (Pope and Haase, 2003; Haase, 2005; Wu and KewalRamani, 2006; Sharkey et al., 2007). Transmission may also be favored by the induction of a local inflammatory response triggered by semen (Pandya and Cohen 1985; Thompson et al., 1992; Berlier et al., 2006; Sharkey et al., 2007).In this paper, we analyze the interaction of HIV-1 with human spermatozoa and the ability of spermatozoa to transmit the virus to immature DCs. We found that spermatozoa capture HIV-1 and efficiently transmit the virus to DCs through a mechanism that requires cell-to-cell contacts. DC–spermatozoa contacts lead to the phenotypic maturation of the DCs and the production of IL-10 (but not IL-12). Acidic values of extracellular pH similar to those found in the vaginal mucosa after sexual intercourse markedly increased both the attachment of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs. Our results suggest that spermatozoa-associated virus may play a central role in the sexual transmission of HIV-1.     

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.     

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.     

Lung dendritic cells induce migration of protective T cells to the gastrointestinal tract

Developing efficacious vaccines against enteric diseases is a global challenge that requires a better understanding of cellular recruitment dynamics at the mucosal surfaces. The current paradigm of T cell homing to the gastrointestinal (GI) tract involves the induction of α4β7 and CCR9 by Peyer’s patch and mesenteric lymph node (MLN) dendritic cells (DCs) in a retinoic acid–dependent manner. This paradigm, however, cannot be reconciled with reports of GI T cell responses after intranasal (i.n.) delivery of antigens that do not directly target the GI lymphoid tissue. To explore alternative pathways of cellular migration, we have investigated the ability of DCs from mucosal and nonmucosal tissues to recruit lymphocytes to the GI tract. Unexpectedly, we found that lung DCs, like CD103⁺ MLN DCs, up-regulate the gut-homing integrin α4β7 in vitro and in vivo, and induce T cell migration to the GI tract in vivo. Consistent with a role for this pathway in generating mucosal immune responses, lung DC targeting by i.n. immunization induced protective immunity against enteric challenge with a highly pathogenic strain of Salmonella. The present report demonstrates novel functional evidence of mucosal cross talk mediated by DCs, which has the potential to inform the design of novel vaccines against mucosal pathogens.Because efficient trafficking of immune cells to the gastrointestinal (GI) tract is critical for host defense against pathogenic challenge, studying cellular recruitment pathways to the GI tract is the key to developing novel vaccines against mucosally transmitted diseases, including HIV-1 infection.Naive T cells acquire the capacity to migrate to extra-lymphoid tissues once activated by their cognate antigen (Butcher et al., 1999; von Andrian and Mackay, 2000). These antigen-experienced effector T cells migrate preferentially to the tissues where they first encountered the antigen (Kantele et al., 1999; Campbell and Butcher, 2002). For example, early observations demonstrated that cells activated in the GI tract home back to the intestinal effector sites (Cahill et al., 1977; Hall et al., 1977). Integrin α4β7 and chemokine receptor 9 (CCR9) are among the best studied gut-specific homing molecules (Berlin et al., 1995; Zabel et al., 1999). The α4β7 ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) mediates recruitment of T cells to the intestinal lamina propria (Berlin et al., 1995), and the CCR9 ligand TECK, expressed by small intestinal epithelial cells, recruits T cells to the small bowel (Zabel et al., 1999).DCs are well recognized as the initiators of the adaptive immune response (Steinman and Cohn, 1973), as well as mediators of tolerance to self-antigens in steady-state conditions (Hawiger et al., 2001). Additionally, there is increasing evidence regarding the role played by DCs as conductors of immunological traffic to the skin and the GI tract (Johansson-Lindbom et al., 2003, 2005; Mora et al., 2003, 2005; Sigmundsdottir et al., 2007). DCs can imprint T cells to migrate to the tissue in which the T cells were originally activated. For example, gut-associated DCs induce the gut-homing receptors α4β7 and CCR9 on T cells upon activation (Johansson-Lindbom et al., 2003; Mora et al., 2003; Stagg et al., 2002).RA is necessary and sufficient to induce gut-homing receptors on T cells (Iwata et al., 2004). The main pathway of RA biosynthesis in vivo is dependent on the intracellular oxidative metabolism of retinol (Napoli, 1999; Duester, 2000), catalyzed by a family of alcohol dehydrogenases including retinal dehydrogenase (RALDH), a class I aldehyde dehydrogenase that mediates the irreversible oxidation of retinal to RA. RA in turn is thought to induce RALDH-2 in a positive feedback loop (Yokota et al., 2009; Hammerschmidt et al., 2011; Villablanca et al., 2011) and RA levels correlate with the ability of the intestinal DCs to induce gut-tropic T cells. Vitamin A is introduced via dietary or biliary sources (Jaensson-Gyllenbäck et al., 2011). Among the cellular sources of RA in the intestinal mucosa are DCs (Iwata et al., 2004), stromal cells (Hammerschmidt et al., 2008; Molenaar et al., 2009), intestinal epithelial cells (Bhat, 1998; Lampen et al., 2000), and intestinal macrophages (Denning et al., 2007), with the DCs likely playing a key role in the induction of gut-homing phenotype on T cells. Among the intestinal DCs, the CD103⁺ DC subsets express high levels of RALDH-2 and are capable of generating high levels of RA (Johansson-Lindbom et al., 2005; Coombes et al., 2007; Sun et al., 2007; Jaensson et al., 2008). In contrast, the CD103⁻CD11b⁺CX3CR1⁺ macrophage-like population in the intestinal lamina propria expresses RALDH-1 and not RALDH-2 and exhibits a lower RA-producing capacity (Schulz et al., 2009; Denning et al., 2011), and therefore a decreased capacity to induce gut-homing potential on T cells (Jaensson et al., 2008).Collectively, a paradigm has emerged wherein only the intestinal CD103⁺ DCs, which are capable of metabolizing vitamin A, can induce GI-specific homing on T cells (Jaensson et al., 2008). This paradigm however, is difficult to reconcile with reports of GI T cell responses after i.n. delivery of antigens that do not directly target the GI lymphoid tissue. For example, mice infected i.n. with influenza virus show no activated virus-specific CD8⁺ T cells in the mesenteric LNs (MLNs) or Peyer’s patches (PPs), yet flu-specific CD8⁺ cells within the lung-associated tissues express α4β7 and memory CD8⁺ T cells are established within the small intestinal epithelium (Masopust et al., 2010). Similarly, a recent study demonstrates that i.n. challenge with H1N1 influenza results in the accumulation of T_H17 cells within the small intestinal lamina propria (SILP; Esplugues et al., 2011). Furthermore, Ciabattini et al. (2011) have demonstrated that after i.n. immunization, antigen-specific T cells are generated in the mediastinal LN and migrate to the MLN in an α4β7- and CD62L-dependent manner. Additionally, it is known that lungs harbor prominent extrahepatic stores of vitamin A (Okabe et al., 1984; Dirami et al., 2004). Its metabolite, RA, plays an important role in pulmonary alveolar development (Dirami et al., 2004) and has a putative therapeutic role in emphysema (Massaro and Massaro, 1997). Finally, although RA production has been considered to be the forte of gut-resident DCs, other DC populations also express RALDH, particularly lung-resident DCs that express RALDH-2 (Heng and Painter, 2008; Guilliams et al., 2010). However, the ability of lung DCs to induce GI-specific T cell homing has not yet been reported.All of the aforementioned factors led us to hypothesize that lung DCs would up-regulate the expression of gut-homing molecules integrin α4β7 and CCR9 on T cells, which in turn would license the migration of T cells to the GI tract. Our hypothesis was made credible by the concept of a common mucosal immunological system proposed by Bienenstock et al. (1978) more than 30 years ago. Indeed, there is increasing appreciation of the mucosal immune system as an integrated network of tissues, cells, and effector molecules, although the cellular factors that link different mucosal compartments are not well understood (Gill et al., 2010).In this study, we show that lung DCs can imprint expression of the gut-homing integrin α4β7 and CCR9 on co-cultured T cells in vitro and on adoptively transferred cells in vivo, licensing T cells to migrate to the GI lamina propria and confer protective immunity against intestinal pathogens. We define a new pathway of DC-mediated mucosal cross talk and challenge the existing dogma that only GI-resident DCs can recruit antigen-specific T cells back to the gut.     

Identification of CD112R as a novel checkpoint for human T cells

T cell immunoglobulin and ITIM domain (TIGIT) and CD226 emerge as a novel T cell cosignaling pathway in which CD226 and TIGIT serve as costimulatory and coinhibitory receptors, respectively, for the ligands CD155 and CD112. In this study, we describe CD112R, a member of poliovirus receptor–like proteins, as a new coinhibitory receptor for human T cells. CD112R is preferentially expressed on T cells and inhibits T cell receptor–mediated signals. We further identify that CD112, widely expressed on antigen-presenting cells and tumor cells, is the ligand for CD112R with high affinity. CD112R competes with CD226 to bind to CD112. Disrupting the CD112R–CD112 interaction enhances human T cell response. Our experiments identify CD112R as a novel checkpoint for human T cells via interaction with CD112.T cell activation is orchestrated by the cosignaling network, which is involved in all stages of the T cell response (Croft, 2003; Zhu et al., 2011). The B7/CD28 family of Ig superfamily (IGSF) and several members of TNF receptor superfamily are the major groups of T cell cosignaling molecules (Chen and Flies, 2013). The importance of these cosignaling pathways has been emphasized in a variety of human diseases, including graft versus host disease, autoimmunity, infection, and cancer (Rosenblum et al., 2012; Yao et al., 2013; Drake et al., 2014).Poliovirus receptor (PVR)–like proteins are a newly emerging group of IGSF with T cell cosignaling functions (Chan et al., 2012; Pauken and Wherry, 2014). This group of molecules share PVR signature motifs in the first Ig variable–like (IgV) domain and are originally known to mediate epithelial cell–cell contacts (Takai et al., 2008; Yu et al., 2009). The two ligands, CD155 (PVR/Necl-5) and CD112 (PVRL2/nectin-2), interact with CD226 (DNAM-1) to costimulate T cells, and they also inhibit T cell response through another coinhibitory receptor, T cell Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT; Yu et al., 2009). CD155 seems to be the predominant ligand in this ligand/receptor network because the interaction between CD112 and TIGIT is very weak (Yu et al., 2009). Adding to the complexity of this network, CD155, but not CD112, interacts with CD96, another PVR-like protein present on T cells and NK cells, though the function of this interaction is still unclear (Fuchs et al., 2004; Seth et al., 2007; Chan et al., 2014). In addition to its intrinsic inhibitory function, TIGIT exerts its T cell inhibitory effects through ligating CD155 on DCs to increase IL-10 secretion or competes with the costimulatory receptor CD226 for ligand interaction (Yu et al., 2009; Lozano et al., 2012; Stengel et al., 2012). Although the molecular and functional relationship between CD226 and TIGIT is still unclear, this novel cosignaling pathway represents important immunomodulators of T cell responses, as well as valuable targets for future immunotherapy (Joller et al., 2011, 2014; Levin et al., 2011; Johnston et al., 2014; Zhang et al., 2014; Chauvin et al., 2015). In this study, we identified CD112R as a new coinhibitory receptor of the PVR family for human T cells.     

Wiskott-Aldrich syndrome protein–mediated actin dynamics control type-I interferon production in plasmacytoid dendritic cells

Mutations in Wiskott-Aldrich syndrome (WAS) protein (WASp), a regulator of actin dynamics in hematopoietic cells, cause WAS, an X-linked primary immunodeficiency characterized by recurrent infections and a marked predisposition to develop autoimmune disorders. The mechanisms that link actin alterations to the autoimmune phenotype are still poorly understood. We show that chronic activation of plasmacytoid dendritic cells (pDCs) and elevated type-I interferon (IFN) levels play a role in WAS autoimmunity. WAS patients display increased expression of type-I IFN genes and their inducible targets, alteration in pDCs numbers, and hyperresponsiveness to TLR9. Importantly, ablating IFN-I signaling in WASp null mice rescued chronic activation of conventional DCs, splenomegaly, and colitis. Using WASp-deficient mice, we demonstrated that WASp null pDCs are intrinsically more responsive to multimeric agonist of TLR9 and constitutively secrete type-I IFN but become progressively tolerant to further stimulation. By acute silencing of WASp and actin inhibitors, we show that WASp-mediated actin polymerization controls intracellular trafficking and compartmentalization of TLR9 ligands in pDCs restraining exaggerated activation of the TLR9–IFN-α pathway. Together, these data highlight the role of actin dynamics in pDC innate functions and imply the pDC–IFN-α axis as a player in the onset of autoimmune phenomena in WAS disease.Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by thrombocytopenia, eczema, recurrent infections, and autoimmune phenomena. The disease is caused by mutations of the WAS gene that encodes the WAS protein (WASp) involved in controlling actin dynamics. Members of the WASp family regulate a variety of actin-dependent processes that range from cell migration to phagocytosis, endocytosis, and membrane trafficking (Thrasher and Burns, 2010). Efforts to understand the cellular basis of the disease have identified diverse and cell-specific actin-related defects in cells of the adaptive and innate immune system. In T cells, TCR engagement induces cytoskeletal rearrangement, driving assembly of signaling platforms at the synaptic region. WASp plays a crucial role in this process by controlling ex novo actin polymerization required to stabilize synapse formation and signaling (Dupré et al., 2002; Sasahara et al., 2002; Badour et al., 2003; Snapper et al., 2005; Sims et al., 2007). WASp is also required on the APC side of the immune synapse for proper transmission of activating signals (Pulecio et al., 2008; Bouma et al., 2011). Defective signaling through antigen receptors affects the function of invariant natural killer T cells (Astrakhan et al., 2009; Locci et al., 2009) and B cells (Meyer-Bahlburg et al., 2008; Westerberg et al., 2008; Becker-Herman et al., 2011). Furthermore, altered actin polymerization and integrin signaling in WASp-deficient immune cells cause defective homing and directional migration of T, B, and DCs (de Noronha et al., 2005; Westerberg et al., 2005; Gallego et al., 2006). Moreover, WASp-mediated actin polymerization controls phagocytic cup formation in monocytes, macrophages, and DCs (Leverrier et al., 2001; Tsuboi, 2007) and it is involved in polarization and secretion of cytokine/cytotoxic granules in T cells/NK cells (Orange et al., 2002; Gismondi et al., 2004; Morales-Tirado et al., 2004; Trifari et al., 2006). Together, the cellular defects identified in WASp-deficient immune cells provide clues to understand the immunodeficiency of WAS patients. However, the mechanisms by which perturbation of actin dynamics promote autoimmune phenomena are less clear. Impairment of T and B cell tolerance have been reported in WAS patients and in Was-deficient mice, but the exact cellular mechanisms that link loss of WASp function to autoimmunity have not been fully elucidated yet (Humblet-Baron et al., 2007; Maillard et al., 2007; Marangoni et al., 2007; Becker-Herman et al., 2011; Recher et al., 2012).Plasmacytoid DCs (pDCs) are the major class of type-I IFN–producing cells that react rapidly upon pathogen encounter to secrete large amount of this cytokine. Recognition of foreign nucleic acid by TLR7 and TLR9 occurs in endosomes and leads to production of type-I IFN and proinflammatory cytokines. Several studies have unveiled that activation of IFN regulatory factor 7 (IRF-7) and IFN-α production in pDCs relies upon strict spatiotemporal compartmentalization of TLR9 and its ligands within the endocytic pathway (Honda et al., 2005; Guiducci et al., 2006; Sasai et al., 2010).Besides their beneficial antiviral properties, type-I IFNs produced by pDCs contribute to breakage of tolerance in several human autoimmune diseases, including systemic lupus erythematosus (SLE), Sjogren syndrome, and psoriasis (Blanco et al., 2001; Båve et al., 2005; Nestle et al., 2005; Gottenberg et al., 2006; Becker-Herman et al., 2011). In these diseases, uncontrolled pDC activation depends on triggering of TLR7/9 by self–nucleic acid–containing immune complexes (Barrat et al., 2005), binding of self-DNA to antimicrobial peptides (Lande et al., 2007), and clustering of self-DNA within neutrophil extracellular traps (Garcia-Romo et al., 2011).The most common autoimmune features that develop in a high proportion of WAS patients include hemolytic anemia, vasculitis, renal disease, and arthritis (Humblet-Baron et al., 2007; Bosticardo et al., 2009). These symptoms partially overlap with those commonly observed in type-I IFN–driven diseases. Based on this, we hypothesized an involvement of the pDC–IFN-α axis in promoting autoimmunity in WAS. We provide evidences in patients and the demonstration in a mouse model that WASp deficiency in pDCs controls activation of IFN-I genes contributing to autoimmune phenomena in WAS.