TNF-related apoptosis-inducing ligand (TRAIL) exerts therapeutic efficacy for the treatment of pneumococcal pneumonia in mice

Apoptotic death of alveolar macrophages observed during lung infection with Streptococcus pneumoniae is thought to limit overwhelming lung inflammation in response to bacterial challenge. However, the underlying apoptotic death mechanism has not been defined. Here, we examined the role of the TNF superfamily member TNF-related apoptosis-inducing ligand (TRAIL) in S. pneumoniae–induced macrophage apoptosis, and investigated the potential benefit of TRAIL-based therapy during pneumococcal pneumonia in mice. Compared with WT mice, Trail^−/− mice demonstrated significantly decreased lung bacterial clearance and survival in response to S. pneumoniae, which was accompanied by significantly reduced apoptosis and caspase 3 cleavage but rather increased necrosis in alveolar macrophages. In WT mice, neutrophils were identified as a major source of intraalveolar released TRAIL, and their depletion led to a shift from apoptosis toward necrosis as the dominant mechanism of alveolar macrophage cell death in pneumococcal pneumonia. Therapeutic application of TRAIL or agonistic anti-DR5 mAb (MD5-1) dramatically improved survival of S. pneumoniae–infected WT mice. Most importantly, neutropenic mice lacking neutrophil-derived TRAIL were protected from lethal pneumonia by MD5-1 therapy. We have identified a previously unrecognized mechanism by which neutrophil-derived TRAIL induces apoptosis of DR5-expressing macrophages, thus promoting early bacterial killing in pneumococcal pneumonia. TRAIL-based therapy in neutropenic hosts may represent a novel antibacterial treatment option.Streptococcus pneumoniae is the most prevalent pathogen, and is responsible for causing community-acquired pneumonia in humans. Despite the fact that all clinically relevant serotypes of S. pneumoniae are susceptible against the most common antibiotics, S. pneumoniae remains a significant cause of morbidity and lethality worldwide (Welte et al., 2012). Therefore, the development of novel antibiotic-independent therapeutic strategies is urgently needed to decrease the disease burden associated with pneumococcal infections of the lung.Because of their pivotal role in bacterial phagocytosis and orchestration of innate immune responses to bacterial infections, alveolar macrophages represent the first line of lung protective immunity against inhaled S. pneumoniae (Calbo and Garau, 2010). Recruited neutrophils support macrophages in lung bacterial clearance during established pneumonia (Knapp et al., 2003; Herbold et al., 2010; Calbo and Garau, 2010), and resident alveolar and lung macrophages, along with inflammatory recruited exudate macrophages, critically contribute to resolution of lung inflammation (Knapp et al., 2003; Winter et al., 2007).An important feature of S. pneumoniae–induced lung infection is the rapid induction of apoptosis in alveolar macrophages within 24 h, resulting in a transient depletion of alveolar macrophages from distal airways (Paton, 1996; Rubins et al., 1996; Dockrell et al., 2003; Knapp et al., 2003; Maus et al., 2004, 2007; Winter et al., 2007; Taut et al., 2008; Hahn et al., 2011b). Inhibition of S. pneumoniae–induced macrophage apoptosis decreases lung pneumococcal clearance, thereby promoting invasive pneumococcal disease progression in mice (Dockrell et al., 2003; Marriott et al., 2005). Conversely, activation of apoptotic cascades in macrophages and neutrophils limits pathogen-driven inflammatory cascades during pneumococcal disease (Marriott et al., 2004, 2006). Moreover, phagocytosis of apoptotic macrophages by lung macrophages down-regulates the overall inflammatory response and decreases invasive disease progression of pneumococcal pneumonia (Fadok et al., 1998; Marriott et al., 2006). Together, these data suggest that macrophage apoptosis is protective in terms of limiting excessive proinflammatory responses during pneumococcal lung infections.The TNF superfamily member TNF-related apoptosis-inducing ligand (TRAIL) exhibits a complex ligand/receptor cross-talk (Schneider et al., 2003). In humans, four membrane-bound TRAIL receptors have been identified, of which TRAIL-R1 (DR4) and TRAIL-R2 (DR5) are apoptosis-inducing receptors, and TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2) act as “decoy” receptors because of absent or nonfunctional death domains (Ashkenazi and Dixit, 1999). In mice, three decoy receptors, but only one death-mediating receptor for TRAIL, death receptor 5 (DR5), have been identified (Wu et al., 1999; Schneider et al., 2003). Previously, a role for caspases and TNF superfamily member Fas ligand has been established in lung infection models (Ali et al., 2003; Matute-Bello et al., 2005). More recently, there has been emerging evidence for a role of TRAIL to induce apoptosis in leukocyte subsets (Katsikis et al., 1997; Renshaw et al., 2003; Zheng et al., 2004; Lum et al., 2005; McGrath et al., 2011; Zhu et al., 2011), alveolar epithelial cells, and other host cell-types in models of LPS-induced acute lung injury, peritonitis (McGrath et al., 2011), as well as viral and bacterial infections (Zheng et al., 2004; Ishikawa et al., 2005; Hoffmann et al., 2007; Brincks et al., 2008, 2011; Stary et al., 2009; Cziupka et al., 2010; Zhu et al., 2011). These data collectively demonstrate that TRAIL plays a role in inducing apoptosis in different cell types in pulmonary inflammation and infection models.Despite the increased acknowledgment that TRAIL is a key player in several immune reactions within the lung, there are currently no data available regarding the role of TRAIL in macrophage apoptosis and disease progression in bacterial pneumonia induced by the major prototype lung-tropic pathogen, S. pneumoniae. Our data reveal a novel neutrophil-macrophage cross talk mechanism by which alveolar accumulating neutrophils responding to the infection secrete TRAIL that induces alveolar macrophage apoptosis and regulates bacterial killing subsequent to pneumococcal challenge. Importantly, we also show for the first time that treatment of neutropenic mice with agonistic anti-DR5 antibody compensates for the lack of neutrophil-derived TRAIL, and significantly improves survival of pneumococcal pneumonia. This finding may be of great interest for future antibiotic-independent immunomodulatory strategies in immunocompromised patients at risk of acquiring bacterial infections. The implications of these findings will be discussed.     

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.     

IL-27 inhibits HIV-1 infection in human macrophages by down-regulating host factor SPTBN1 during monocyte to macrophage differentiation

The susceptibility of macrophages to HIV-1 infection is modulated during monocyte differentiation. IL-27 is an anti-HIV cytokine that also modulates monocyte activation. In this study, we present new evidence that IL-27 promotes monocyte differentiation into macrophages that are nonpermissive for HIV-1 infection. Although IL-27 treatment does not affect expression of macrophage differentiation markers or macrophage biological functions, it confers HIV resistance by down-regulating spectrin β nonerythrocyte 1 (SPTBN1), a required host factor for HIV-1 infection. IL-27 down-regulates SPTBN1 through a TAK-1–mediated MAPK signaling pathway. Knockdown of SPTBN1 strongly inhibits HIV-1 infection of macrophages; conversely, overexpression of SPTBN1 markedly increases HIV susceptibility of IL-27–treated macrophages. Moreover, we demonstrate that SPTBN1 associates with HIV-1 gag proteins. Collectively, our results underscore the ability of IL-27 to protect macrophages from HIV-1 infection by down-regulating SPTBN1, thus indicating that SPTBN1 is an important host target to reduce HIV-1 replication in one major element of the viral reservoir.Macrophages, as a major target of HIV-1, play an important role in HIV-1 infection. Macrophage infection is found extensively in body tissues and contributes to HIV-1 pathogenesis (Koenig et al., 1986; Salahuddin et al., 1986; Wang et al., 2001; Smith et al., 2003). Macrophage lineage cells are among the first cells to be infected because most viruses involved in the first round of infection use CCR5 as the co-receptor to initiate HIV-1 replication in vivo (Philpott, 2003). Once infected, macrophages have been shown to promote rapid virus dissemination by transmitting virus particles to CD4⁺ T cells via a transit “virological synapse” (Groot et al., 2008). Although most CD4⁺ T cells are eventually killed by HIV-1, infected macrophages survive longer and can harbor virus particles in intracellular compartments (Raposo et al., 2002; Pelchen-Matthews et al., 2003), thus maintaining a hidden HIV-1 reservoir for ongoing infection (Wahl et al., 1997; Lambotte et al., 2000; Zhu et al., 2002; Smith et al., 2003; Sharova et al., 2005). Collectively, macrophage infection is involved throughout the progression of disease. Therefore, restriction of macrophage infection may provide a key to eradication of HIV-1 infection.HIV-1 infection is modulated by a variety of host cellular factors. HIV-1 has evolved to have specific viral proteins to counteract certain host restriction factors. Human HIV-1 restriction factors, including APOBEC3G and BST-2, have been reported (Neil et al., 2008; Sheehy et al., 2002) and models of how HIV-1 overcomes these restrictions have been described in reviews (Evans et al., 2010; Goila-Gaur and Strebel, 2008). More recently, SAMHD1, a restriction factor of myeloid cells, was found to limit HIV replication by depleting intracellular dNTPs, and it is largely opposed by Vpx (Hrecka et al., 2011; Laguette et al., 2011; Lahouassa et al., 2012). Release of these host restrictions, however, does not guarantee productive infection. HIV-1, with a limited genome of nine open reading frames, has to fully exploit an array of cellular proteins to facilitate its life cycle at almost every step (Goff, 2007). Genome-wide siRNA screens, using 293T or HeLa cells as HIV-1 targets, have revealed hundreds of potential supportive host factors (Brass et al., 2008; Zhou et al., 2008), only some of which have been validated in primary target cells. Regulation of host factors, both inhibitory and supportive, may offer great opportunities to prevent HIV-1 infection of macrophages.Cytokine-mediated immunoregulation is an effective way to inhibit HIV-1 infection in cells of myeloid lineage (Kedzierska and Crowe, 2001). Our previous studies have demonstrated that IL-27 strongly inhibits HIV-1 replication in terminally differentiated monocyte-derived macrophages (MDMs) (Fakruddin et al., 2007). IL-27 is an IL-12 family cytokine mainly produced by dendritic cells and macrophages (Kastelein et al., 2007). It was originally characterized as a proinflammatory cytokines to induce Th1 responses in T cells (Pflanz et al., 2004; Villarino et al., 2004). However, the IL-27 receptor complex, consisting of WSX-1 and glycoprotein 130 (gp130), is also expressed on monocytes (Pflanz et al., 2004) and recent evidence has supported a role for IL-27 in monocyte activation (Kalliolias and Ivashkiv, 2008; Guzzo et al., 2010a). In the current study, we aim to investigate the role of IL-27 stimulation during monocyte differentiation in modulating macrophage susceptibility to HIV-1 infection, and our study will help to evaluate whether IL-27 can be used to prevent HIV-1 infection of macrophages.     

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

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

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.     

Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension

Idiopathic pulmonary arterial hypertension (PAH [IPAH]) is an insidious and potentially fatal disease linked to a mutation or reduced expression of bone morphogenetic protein receptor 2 (BMPR2). Because intravascular inflammatory cells are recruited in IPAH pathogenesis, we hypothesized that reduced BMPR2 enhances production of the potent chemokine granulocyte macrophage colony-stimulating factor (GM-CSF) in response to an inflammatory perturbation. When human pulmonary artery (PA) endothelial cells deficient in BMPR2 were stimulated with tumor necrosis factor (TNF), a twofold increase in GM-CSF was observed and related to enhanced messenger RNA (mRNA) translation. The mechanism was associated with disruption of stress granule formation. Specifically, loss of BMPR2 induced prolonged phospho-p38 mitogen-activated protein kinase (MAPK) in response to TNF, and this increased GADD34–PP1 phosphatase activity, dephosphorylating eukaryotic translation initiation factor (eIF2α), and derepressing GM-CSF mRNA translation. Lungs from IPAH patients versus unused donor controls revealed heightened PA expression of GM-CSF co-distributing with increased TNF and expanded populations of hematopoietic and endothelial GM-CSF receptor α (GM-CSFRα)–positive cells. Moreover, a 3-wk infusion of GM-CSF in mice increased hypoxia-induced PAH, in association with increased perivascular macrophages and muscularized distal arteries, whereas blockade of GM-CSF repressed these features. Thus, reduced BMPR2 can subvert a stress granule response, heighten GM-CSF mRNA translation, increase inflammatory cell recruitment, and exacerbate PAH.Idiopathic pulmonary arterial hypertension (PAH [IPAH]) is a lethal disorder characterized by obliterative changes in small- to medium-sized pulmonary arteries (PAs; Pietra et al., 1989). Familial IPAH has been linked to heterozygous germline mutations in the bone morphogenetic protein receptor 2 (BMPR2; Lane et al., 2000), but the penetrance of disease is as low as 15–20% (Newman et al., 2004). The BMPR2 mutation is therefore thought to increase susceptibility to IPAH in the context of environmental or other genetic factors (Newman et al., 2004). It is important to note that even PAH patients without a BMPR2 mutation have reduced expression of this receptor (Atkinson et al., 2002; Alastalo et al., 2011).Inflammation is associated with IPAH; i.e., the PA lesions have elevated levels of inflammatory cytokines such as IL-1, IL-6, and TNF (Humbert et al., 1995; Itoh et al., 2006; Soon et al., 2010) and contain inflammatory cells, including macrophages, T cells, B cells, and dendritic cells (Tuder et al., 1994), and tertiary lymphoid follicles (Perros et al., 2012). Evidence for inflammation as a cause of PAH rather than a consequence has come from several sources. Recent publications show the essential role of perivascular macrophages in the pathogenesis of hypoxia-induced pulmonary hypertension (Vergadi et al., 2011), as well as interactions between activated fibroblasts and macrophages that are critical in the development of vascular pathology (Li et al., 2011). Hepatopulmonary syndrome in rats results in a proliferative pulmonary vasculopathy similar to that seen in patients that develop portopulmonary hypertension, which is completely prevented when CD68-positive macrophages are depleted (Thenappan et al., 2011). More recently, macrophages have been linked directly to endothelial apoptosis and to the development of PAH in the athymic rat in which blockade of vascular endothelial growth factor receptor (VEGFR2) is induced (Tian et al., 2013). In this model, immune reconstitution with regulatory T cells prevents the development of PAH, at least in part by enhancing the expression and activity of BMPR2 to preserve endothelial function (Tamosiuniene et al., 2011). This is consistent with experiments showing that heterozygous BMPR2 mutant mice develop PAH under inflammatory stress that does not cause PAH in control mice (Song et al., 2005). Moreover, conditional ablation of BMPR2 in PA endothelial cells (ECs [PAECs]) of transgenic mice resulted in some of the animals developing PAH, in association with perivascular infiltration of CD68-positive cells (Hong et al., 2008). Loss of function of BMPR2 in smooth muscle cells (SMCs) doubles the level of the proinflammatory cytokine IL-6 by a phospho (p)-p38–dependent signaling mechanism (Hagen et al., 2007). Moreover, mice overexpressing IL-6 develop severe PAH (Steiner et al., 2009). It was not known, however, how an increase in p38 activity could elevate levels of IL-6 or other cytokines or chemokines that recruit macrophages or other inflammatory and/or bone marrow–derived progenitor cells (Asosingh et al., 2008) that could contribute to the pathobiology of PAH (Davie et al., 2004; Frid et al., 2006).In this study, we investigate the contribution to PAH of GM-CSF because it is a potent proinflammatory chemokine, implicated in the mobilization of ECs and other progenitor cells to sites of injury (Takahashi et al., 1999), in the expansion of myeloid-derived suppressor cells in autoimmunity (Rosborough et al., 2012) and in pulmonary hypertension (Yeager et al., 2012), in survival and activation of monocytes/macrophages (Toren and Nagler, 1998) in pulmonary hypertension (Li et al., 2011), and in the regulation of leukotriene B4 in macrophages (Serezani et al., 2012), which has been implicated in PAH (Tian et al., 2013).     

Macrophages retain hematopoietic stem cells in the spleen via VCAM-1

Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)⁺ macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE^−/− mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques.Leukocytosis correlates closely with cardiovascular mortality. In the steady state, blood leukocytes derive exclusively from bone marrow hematopoietic stem cells (HSCs). Supporting cells (Sugiyama et al., 2006; Ding et al., 2012; Ding and Morrison, 2013), including macrophages (Winkler et al., 2010; Chow et al., 2011), maintain the bone marrow HSC niche and regulate hematopoietic stem and progenitor cell (HSPC) activity by supplying various cytokines and retention factors. Systemic inflammation can stimulate extramedullary hematopoiesis in adult mice and humans. Splenic myelopoiesis supplies inflammatory monocytes to atherosclerotic plaques (Robbins et al., 2012) and the ischemic myocardium (Leuschner et al., 2012). In ischemic heart disease, HSPCs emigrate from the bone marrow, seed the spleen, and amplify leukocyte production (Dutta et al., 2012). Splenic HSPCs localize in the red pulp near the sinusoids in parafollicular areas (Kiel et al., 2005). Likewise, after adoptive transfer of GFP⁺ HSPCs, GFP⁺ colonies populate the splenic red pulp of atherosclerotic ApoE^−/− mice (Robbins et al., 2012). During myocardial infarction (MI), proinflammatory monocytes derived from the spleen accelerate atherosclerotic progression (Dutta et al., 2012). Collectively, these data suggest that splenic myelopoiesis has promise as a therapeutic target; however, the components of the splenic hematopoietic niche are incompletely understood, especially compared with the well-studied bone marrow niche. Understanding HSC retention factors and their regulation in the spleen was the purpose of this study.Because the spleen harbors very few HSCs in the steady state, we investigated the splenic hematopoietic niche after injecting the Toll-like receptor ligand LPS to activate extramedullary hematopoiesis. In the bone marrow, macrophages are an integral part of the HSC niche (Winkler et al., 2010; Chow et al., 2011) and differentiation depends on the receptor for macrophage colony-stimulating factor (M-CSFR, CD115; Auffray et al., 2009). We thus hypothesized that splenic hematopoietic niche assembly also requires M-CSFR signaling. In line with knockout studies (Takahashi et al., 1994; Dai et al., 2002), in vivo knockdown of M-CSFR with nanoparticle-encapsulated siRNA reduced splenic macrophage numbers substantially. Interestingly, decreased macrophage numbers were associated with a reduction of splenic HSCs. Depleting macrophages with diphtheria toxin (DT) in CD169 iDTR mice reproduced the findings obtained with M-CSF–directed siRNA treatment, thereby indicating that macrophages have a key role in splenic HSC maintenance. To investigate how splenic macrophages retain HSCs, we measured changes in splenic expression of major bone marrow retention factors after M-CSFR silencing. Silencing M-CSFR selectively reduced splenic VCAM-1, and the adhesion molecule was primarily expressed by macrophages. Inhibiting macrophage expression of VCAM-1 with siRNA targeting this adhesion molecule reduced splenic HSPC numbers. Finally, we found that M-CSFR and macrophage-directed VCAM-1 silencing in mice with atherosclerosis mitigated blood leukocytosis and dampened inflammation in atherosclerotic plaques and the infarcted myocardium. These data reveal the importance of VCAM-1 expression by splenic macrophages for extramedullary hematopoiesis and illustrate the therapeutic potential of RNAi as an antiinflammatory that mutes emergency overproduction and provision of myeloid cells.     

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.     

Langerhans cell (LC) proliferation mediates neonatal development,homeostasis, and inflammation-associated expansion of the epidermal LC network

Most tissues develop from stem cells and precursors that undergo differentiation as their proliferative potential decreases. Mature differentiated cells rarely proliferate and are replaced at the end of their life by new cells derived from precursors. Langerhans cells (LCs) of the epidermis, although of myeloid origin, were shown to renew in tissues independently from the bone marrow, suggesting the existence of a dermal or epidermal progenitor. We investigated the mechanisms involved in LC development and homeostasis. We observed that a single wave of LC precursors was recruited in the epidermis of mice around embryonic day 18 and acquired a dendritic morphology, major histocompatibility complex II, CD11c, and langerin expression immediately after birth. Langerin⁺ cells then undergo a massive burst of proliferation between postnatal day 2 (P2) and P7, expanding their numbers by 10–20-fold. After the first week of life, we observed low-level proliferation of langerin⁺ cells within the epidermis. However, in a mouse model of atopic dermatitis (AD), a keratinocyte signal triggered increased epidermal LC proliferation. Similar findings were observed in epidermis from human patients with AD. Therefore, proliferation of differentiated resident cells represents an alternative pathway for development in the newborn, homeostasis, and expansion in adults of selected myeloid cell populations such as LCs. This mechanism may be relevant in locations where leukocyte trafficking is limited.Current data indicate that many macrophage subsets and most DCs in nonlymphoid tissues and in the secondary lymphoid organs of mice originate and are renewed from bone-marrow hematopoietic stem cell–derived progenitors with myeloid-restricted differentiation potential (Fogg et al., 2006; Liu et al., 2009). However, exceptions must exist to this major pathway of macrophage and DC generation, because Langerhans cells (LCs) and microglia remain of host origin after syngeneic bone marrow transplant (Merad et al., 2002; Ajami et al., 2007; Mildner et al., 2007), and LCs remain of donor origin after a limb graft (Kanitakis et al., 2004). Epidermal LCs have been shown to be a cycling population (Giacometti and Montagna, 1967; Czernielewski et al., 1985; Czernielewski and Demarchez, 1987). LC precursors were proposed to reside in the dermis (Larregina et al., 2001) or in the hair follicle (Gilliam et al., 1998), and cells with features of proliferating LC precursors have been found in fetal and newborn skin (Elbe et al., 1989; Chang-Rodriguez et al., 2005). On the other hand, monocytes can give rise to LC-like cells in vitro (Geissmann et al., 1998; Mohamadzadeh et al., 2001), and LCs can be replaced by bone marrow–derived cells in a selected experimental setting, i.e., after allogeneic bone marrow transplant, UV light irradiation, and conditional genetic ablation (Katz et al., 1979; Frelinger and Frelinger, 1980; Merad et al., 2002; Bennett et al., 2005). The nature of the endogenous LC precursor is thus unclear.LC development is controlled by M-CSF receptor and TGF-β1 (Borkowski et al., 1996; Ginhoux et al., 2006; Kaplan et al., 2007), but the LC precursor is particularly enigmatic because, in contrast to most organs, migration of leukocytes into the epidermis, as well as the brain, is rarely observed in a steady state; when such migration is observed, it is typically associated with inflammation. The mechanisms by which LCs develop and are renewed may differ from those involved in organs where hematopoietic cells circulate constantly, such as the spleen, liver, or lung. Although the roles of epidermal LCs remain controversial, recent evidence indicates a role as scavengers for viruses such as HIV-1 (de Witte et al., 2007) and possibly for carcinogens (Strid et al., 2008), as well as their role in promoting and regulating T cell–mediated immune responses (Bennett et al., 2007; Stoitzner et al., 2008; Elentner et al., 2009; Vesely et al., 2009). Understanding the mechanisms that control the development and homeostasis of DCs and macrophages in the skin or brain is thus of importance in understanding the pathophysiology of inflammation in these organs. In this study, we investigated the development of the LC network of the epidermis, and how it is maintained in a steady state and during epidermal inflammation.