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.     

DOCK8 regulates lymphocyte shape integrity for skin antiviral immunity

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

Self-RNA–antimicrobial peptide complexes activate human dendritic cells through TLR7 and TLR8

Dendritic cell (DC) responses to extracellular self-DNA and self-RNA are prevented by the endosomal seclusion of nucleic acid–recognizing Toll-like receptors (TLRs). In psoriasis, however, plasmacytoid DCs (pDCs) sense self-DNA that is transported to endosomal TLR9 upon forming a complex with the antimicrobial peptide LL37. Whether LL37 also interacts with extracellular self-RNA and how this may contribute to DC activation in psoriasis is not known. Here, we report that LL37 can bind self-RNA released by dying cells, protect it from extracellular degradation, and transport it into endosomal compartments of DCs. In pDC, self-RNA–LL37 complexes activate TLR7 and, like self-DNA–LL37 complexes, trigger the secretion of IFN-α without inducing maturation or the production of IL-6 and TNF-α. In contrast to self-DNA–LL37 complexes, self-RNA–LL37 complexes also trigger the activation of classical myeloid DCs (mDCs). This occurs through TLR8 and leads to the production of TNF-α and IL-6, and the differentiation of mDCs into mature DCs. We also found that self-RNA–LL37 complexes are present in psoriatic skin lesions and are associated with mature mDCs in vivo. Our results demonstrate that the cationic antimicrobial peptide LL37 converts self-RNA into a trigger of TLR7 and TLR8 in human DCs, and provide new insights into the mechanism that drives the auto-inflammatory responses in psoriasis.Dendritic cells (DCs) sense viral infections through a subset of nucleic acid–recognizing Toll-like receptors (TLRs) expressed in endosomal compartments (Akira et al., 2006). These receptors include TLR3, which detects double-stranded viral RNA (Alexopoulou et al., 2001); TLR7 and TLR8, which recognize guanosine- and uridine-rich single-stranded RNA (ssRNA) (Diebold et al., 2004; Heil et al., 2004; Lund et al., 2004); and TLR9, which recognizes the phosphodiester backbone in natural DNA or unmethylated CpG motifs (Hemmi et al., 2000; Haas et al., 2008). TLR7 and TLR9 are selectively expressed by human plasmacytoid dendritic cells (pDCs) (Jarrossay et al., 2001; Kadowaki et al., 2001; Hornung et al., 2002), a subset of dendritic cells specialized in type I IFN production (Colonna et al., 2004; Gilliet et al., 2008). In contrast, classical human myeloid DCs (mDCs), which are potent stimulators of T cell responses by virtue of their inherent capacity to present antigens and migrate from the periphery into secondary lymphoid organs, do not express TLR7 and TLR9, but instead express TLR3 and TLR8 (Jarrossay et al., 2001; Kadowaki et al., 2001; Hornung et al., 2002).Through endosomal TLRs, pDCs and mDCs efficiently sense viral nucleic acids but do not respond to self-nucleic acids released into the extracellular environment by dying host cells. Structural differences such as the high levels of unmethylated CpG motifs in viral DNA and clusters of U or GU-rich sequences in viral RNA have been considered a key factor in the discrimination between viral and self-nucleic acids (Krieg, 2002; Diebold et al., 2004; Heil et al., 2004). However, more recently, it has become clear that this discrimination is mainly achieved by the intracellular localization of these TLRs, which allows recognition of viral nucleic acids released into endosomal compartments by the endocytosed virus (Barton et al., 2006). In contrast, self-nucleic acids are rapidly degraded in the extracellular environment and fail to access endosomal compartments spontaneously (Barton et al., 2006). Self-DNA and self-RNA can, however, become a potent trigger of pDC activation when they are aberrantly transported into TLR-containing endosomes in the context of autoimmunity. In systemic lupus erythematosus (SLE), self-RNA and self-DNA are complexed with autoantibodies against the nucleic acid or nucleoproteins, which deliver the nucleic acids into endosomal compartments of pDCs via FcγRII-mediated endocytosis (Rönnblom et al., 2003; Barrat et al., 2005; Means et al., 2005). As a result, pDCs are continuously activated to produce type I IFNs, which drives the development of autoimmunity and disease formation. This has been demonstrated in mouse models of SLE using TLR7- or TLR9-deficient mice or inhibitors for TLR7 and TLR9 (Leadbetter et al., 2002; Lau et al., 2005; Christensen et al., 2006; Barrat et al., 2007), and by a study showing that TLR7 gene duplication in mice induces SLE-like disease (Deane et al., 2007).In psoriasis, a chronic autoimmune-inflammatory disease of the skin, self-DNA forms complexes with the cationic antimicrobial peptide LL37. Self-DNA–LL37 complexes gain access to endosomal TLR9, leading to an aberrant activation of pDCs to produce IFN-α (Lande et al., 2007). pDC-derived IFN-α initiates the autoimmune-inflammatory cascade in psoriasis, a process characterized by the activation of mDCs and their maturation into DCs that stimulate pathogenic autoimmune T cells (Nestle et al., 2005). However, whether LL37 also interacts with extracellular self-RNA, and whether this pathway is involved in the activation of mDCs in psoriasis, is unknown.Here, we found that LL37 also forms complexes with extracellular self-RNA. These complexes are highly protected from RNase degradation and gain access to the endosomal compartments of both pDCs and mDCs. Self-RNA–LL37 complexes induce TLR7 activation in pDCs and trigger the secretion of IFN-α. Self-RNA–LL37 complexes also trigger the direct activation of mDCs to secrete TNF-α and IL-6 and differentiate into mature DCs. This maturation of mDCs is triggered by endosomal TLR8 and is enhanced by the concomitant activation of pDCs to produce IFN-α. In-vivo, self-RNA–LL37 complexes are specifically found in psoriatic skin and the number of these complexes correlates with the presence of mature mDCs. The current findings identify self-RNA–LL37 complexes as endogenous triggers of TLR7 and TLR8 in human DCs, and provide a new link between the expression of antimicrobial peptides and DC-mediated inflammation in psoriasis.     

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.     

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

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

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

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

Identification of human CCR8 as a CCL18 receptor

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

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.     

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.     

Whole-exome sequencing-based discovery of STIM1 deficiency in a child with fatal classic Kaposi sarcoma

Classic Kaposi sarcoma (KS) is exceedingly rare in children from the Mediterranean Basin, despite the high prevalence of human herpesvirus-8 (HHV-8) infection in this region. We hypothesized that rare single-gene inborn errors of immunity to HHV-8 may underlie classic KS in childhood. We investigated a child with no other unusually severe infectious or tumoral phenotype who died from disseminated KS at two years of age. Whole-exome sequencing in the patient revealed a homozygous splice-site mutation in STIM1, the gene encoding stromal interaction molecule 1, which regulates store-operated Ca²⁺ entry. STIM1 mRNA splicing, protein production, and Ca²⁺ influx were completely abolished in EBV-transformed B cell lines from the patient, but were rescued by the expression of wild-type STIM1. Based on the previous discovery of STIM1 deficiency in a single family with a severe T cell immunodeficiency and the much higher risk of KS in individuals with acquired T cell deficiencies, we conclude that STIM1 T cell deficiency precipitated the development of lethal KS in this child upon infection with HHV-8. Our report provides the first evidence that isolated classic KS in childhood may result from single-gene defects and provides proof-of-principle that whole-exome sequencing in single patients can decipher the genetic basis of rare inborn errors.Kaposi sarcoma (KS) is an angiogenic, inflammatory neoplasm first described by Moritz Kaposi (1872). The etiology of KS was unknown until Chang et al. (1994) isolated KS-associated herpesvirus, also known as human herpesvirus (HHV)-8, from KS lesions. Cells of endothelial origin are the principal targets of HHV-8 infection and the major components proliferating in KS lesions (Ganem, 2010). It has been clearly established that all forms of KS are associated with HHV-8 (Dupin et al., 1995; Moore and Chang, 1995; Schalling et al., 1995; Chang et al., 1996). Infection with HHV-8 is necessary but not sufficient for the development of KS. Despite the high seroprevalence of HHV-8, at up to 30% in the Mediterranean Basin (classic KS) and 70% in sub-Saharan Africa (endemic KS), the incidence of KS is very low (Boshoff and Weiss, 2001). Individuals with acquired immunodeficiency caused by HIV infection (epidemic KS; Martellotta et al., 2009) or immunosuppression after organ transplantation (iatrogenic KS; Lebbé et al., 2008) are at a much higher risk of developing KS than the general population. Host factors in other patients are therefore likely to play a critical role in determining the outcome of HHV-8 infection, including the development of KS in particular.As a first approach to testing the hypothesis that KS may result from inborn errors of immunity to HHV-8 in some patients (Casanova and Abel, 2007), we focused on classic KS in childhood. Classic KS is exceedingly rare in children, with only 30 cases reported since 1960, and follows a disseminated and often lethal course (Dutz and Stout, 1960; Bisceglia et al., 1988; Akman et al., 1989; Zurrida et al., 1994; Erdem et al., 1999; Landau et al., 2001; Ferrari et al., 2002; Hussein, 2008). We previously reported two children from the Mediterranean Basin with KS caused by an underlying primary immunodeficiency, one with autosomal recessive complete IFN-γR-1 deficiency (Camcioglu et al., 2004) and the other with X-linked recessive Wiskott-Aldrich syndrome (Picard et al., 2006). These children had several other clinical phenotypes, including mycobacterial disease in the IFN-γR1–deficient child and EBV-driven lymphoma in the child with Wiskott-Aldrich syndrome. These two cases provided the first evidence that classic KS in childhood may result from inborn errors of immunity, at least in children with multiple infectious and tumoral phenotypes, including KS.More recently, we described three unrelated Turkish children with classic KS in the absence of any other infectious or tumoral phenotype, each born to consanguineous parents (Sahin et al., 2010). This suggested that classic KS in children with no overt signs of immunodeficiency might also result from single-gene defects impairing HHV-8 immunity. Because the age of onset, clinical features, and outcome of KS reported in previous studies differ from child to child (Dutz and Stout, 1960; Bisceglia et al., 1988; Akman et al., 1989; Zurrida et al., 1994; Erdem et al., 1999; Landau et al., 2001; Ferrari et al., 2002; Picard et al., 2006; Camcioglu et al., 2004; Hussein, 2008; Sahin et al., 2010), we further hypothesized that the single-gene inborn errors of immunity underlying classic KS in children are heterogeneous. Using the recently developed whole-exome sequencing approach, which has been used to determine the genetic basis of other rare Mendelian disorders in small numbers of affected individuals (Choi et al., 2009; Hoischen et al., 2010; Lalonde et al., 2010; Ng et al., 2010; Walsh et al., 2010), we focused our investigation on a single Turkish child with early-onset disseminated KS that eventually ran a lethal course.     

Circulating precursors of human CD1c+ and CD141+ dendritic cells

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

Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection

Competition for iron influences host–pathogen interactions. Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron. In response, the host secretes siderophore-binding proteins, such as lipocalin 24p3, which limit siderophore-mediated iron import into bacteria. Mammals produce 2,5-dihydroxy benzoic acid, a compound that resembles a bacterial siderophore. Our data suggest that bacteria use both mammalian and bacterial siderophores. In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro. In addition, mice lacking the mammalian siderophore resist E. coli infection. Finally, we show that the host responds to infection by suppressing siderophore synthesis while up-regulating lipocalin 24p3 expression via TLR signaling. Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.The growth and metabolism of many pathogenic microbes is exquisitely iron sensitive (Schaible and Kaufmann, 2004). Mammals have evolved a defense mechanism to sequester iron that permits host cells to maintain access to iron while preventing invading microbes from acquiring the metal (Ganz, 2009; Cassat and Skaar, 2013). Iron is predominantly intracellular or tightly bound to proteins. Invading pathogens therefore obtain iron from their host by secreting small, iron-binding siderophores, which remove iron from host protein–iron complexes due to their higher affinity for iron (Miethke and Marahiel, 2007). In parallel, mammals have evolved siderophore-binding proteins as components of the innate immune system, e.g., 24p3 (Flo et al., 2004). 24p3 is a member of the lipocalin family of proteins that transport a variety of ligands (Flower, 2000). 24p3 binds small hydrophobic molecules, the siderophores (Goetz et al., 2002). By binding iron-laden bacterial siderophores, 24p3 participates in innate host defense (Flo et al., 2004; Berger et al., 2006; Saiga et al., 2008). 24p3 expression in innate immunity is highly induced in response to stimulation by TLR2, TLR4, and TLR5 agonists (Flo et al., 2004; Saiga et al., 2008; Van Maele et al., 2010). 24p3 is also one of the secondary granule proteins of neutrophils (Kjeldsen et al., 1993). Therefore, the 24p3 released upon neutrophil degranulation, complements 24p3 secreted by TLR stimulated macrophages, epithelial cells and liver opposing salvage of host iron by bacterial siderophores (Borregaard and Cowland, 2006).24p3 specifically binds catecholate or mixed phenolate type siderophores (Holmes et al., 2005). The ability of 24p3 to curtail the growth of bacteria is limited to those species that depend on catecholes and mixed phenolates for iron acquisition (Berger et al., 2006, Flo et al., 2004; Saiga et al., 2008). Strains of E. coli and Staphylococcus aureus have modified siderophores that evade capture by 24p3 (Fischbach et al., 2006). Moreover, pathogenic strains of E. coli secrete multiple siderophores (Henderson et al., 2009). Although 24p3 is highly induced by infection with these bacteria, secreted 24p3 may not be able to sequester the multiple siderophores excreted by these bacteria because its binding pocket is selective for catecholes (Holmes et al., 2005; Reigstad et al., 2007). Interestingly, 24p3 is also highly induced by E. coli infection of C3H/HeJ mice that harbor a mutation in TLR4 (Reigstad et al., 2007). Despite high levels of 24p3, these mice succumb to E. coli infection; the effect of 24p3 on E. coli growth in C3H/HeJ mice is insignificant (Hagberg et al., 1985). As stated above, 24p3 is a secondary granule protein in neutrophils, which is critical for motility and chemotaxis (Rathore et al., 2011; Schroll et al., 2012; Liu et al., 2013). 24p3 deficiency confers enhanced sensitivity upon mice to a variety of pathogens independent of their ability to secrete siderophores (Liu et al., 2013). These studies suggest that additional mechanisms may be in place that synergize with 24p3 to counter bacterial hijacking of iron.Mammalian-derived 24p3 also captures iron indicating the existence of siderophore-like molecules (Yang et al., 2003). In addition, older studies have suggested the existence of low-molecular weight iron binding compounds or siderophore-like molecules that are capable of binding iron and that stimulate proliferation of bacteria (Fernandez-Pol, 1978; Jones et al., 1980). However, these molecules were not implicated in iron carrier function of 24p3. Using 24p3 as bait we recently identified a siderophore in mammalian cells that facilitates iron loading onto 24p3 (Devireddy et al., 2010). The mammalian siderophore, 2,5-dihydroxy benzoic acid (2,5-DHBA) is chemically similar to 2,3-DHBA, the iron-binding moiety of E. coli enterobactin (Raymond et al., 2003). One of the short chain dehydrogenases (SDR) family of reductases, 3-OH butyrate dehydrogenase-2 (Bdh2), a homologue of bacterial EntA, catalyzes a rate-limiting step in the 2,5-dihydroxy benzoic acid (DHBA) biosynthetic pathway (Devireddy et al., 2010).However, the role of the mammalian siderophore in innate immunity is not clear. Due to its similarity to bacterial siderophore, we hypothesized that the mammalian siderophore promotes bacterial growth. We now show that the mammalian siderophore augments bacterial growth and can rescue a growth deficit conferred by the absence of the E. coli siderophore, enterobactin. To counter bacterial coopting of the mammalian siderophore, the host responds by suppressing mammalian siderophore biosynthesis. Thus, negative regulation of the mammalian siderophore provides an ingenious strategy to withhold iron from invading pathogens. Of note, mammalian siderophore-deficient mice resist pathogenic E. coli. In addition to decreasing mammalian siderophore the host responds by secreting 24p3 to sequester iron-laden bacterial siderophores. Thus, reciprocal regulation of 24p3 and the mammalian siderophore restricts the growth of invading pathogens.