Ignacio J. Juncadella

Apoptotic cell clearance by bronchial epithelial cells critically influences airway inflammation

Lung epithelial cells can influence immune responses to airway allergens. Airway epithelial cells also undergo apoptosis after encountering environmental allergens; yet, relatively little is known about how these are cleared, and their effect on airway inflammation. Here we show that airway epithelial cells efficiently engulf apoptotic epithelial cells and secrete anti-inflammatory cytokines, dependent upon intracellular signalling by the small GTPase Rac1. Inducible deletion of Rac1 expression specifically in airway epithelial cells in a mouse model resulted in defective engulfment by epithelial cells and aberrant anti-inflammatory cytokine production. Intranasal priming and challenge of these mice with house dust mite extract or ovalbumin as allergens led to exacerbated inflammation, augmented Th2 cytokines and airway hyper-responsiveness, with decreased interleukin (IL)-10 in bronchial lavages. Rac1-deficient epithelial cells produced much higher IL-33 upon allergen or apoptotic cell encounter, with increased numbers of nuocyte-like cells. Administration of exogenous IL-10 ‘rescued’ the airway inflammation phenotype in Rac1-deficient mice, with decreased IL-33. Collectively, these genetic and functional studies suggest a new role for Rac1-dependent engulfment by airway epithelial cells and in establishing the anti-inflammatory environment, and that defects in cell clearance in the airways could contribute to inflammatory responses towards common allergens.

Notch Signaling Regulates Mouse and Human Th17 Differentiation

Th17 cells are known to play a critical role in adaptive immune responses to several important extracellular pathogens. Additionally, Th17 cells are implicated in the pathogenesis of several autoimmune and inflammatory disorders as well as in cancer. Therefore, it is essential to understand the mechanisms that regulate Th17 differentiation. Notch signaling is known to be important at several stages of T cell development and differentiation. In this study, we report that Notch1 is activated in both mouse and human in vitro-polarized Th17 cells and that blockade of Notch signaling significantly downregulates the production of Th17-associated cytokines, suggesting an intrinsic requirement for Notch during Th17 differentiation in both species. We also present evidence, using promoter reporter assays, knockdown studies, as well as chromatin immunoprecipitation, that IL-17 and retinoic acid-related orphan receptor γt are direct transcriptional targets of Notch signaling in Th17 cells. Finally, in vivo inhibition of Notch signaling reduced IL-17 production and Th17-mediated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Thus, this study highlights the importance of Notch signaling in Th17 differentiation and indicates that selective targeted therapy against Notch may be an important tool to treat autoimmune disorders, including multiple sclerosis.

Unexpected requirement for ELMO1 in clearance of apoptotic germ cells in vivo

Apoptosis and the subsequent clearance of dying cells occurs throughout development and adult life in many tissues. Failure to promptly clear apoptotic cells has been linked to many diseases. ELMO1 is an evolutionarily conserved cytoplasmic engulfment protein that functions downstream of the phosphatidylserine receptor BAI1, and, along with DOCK1 and the GTPase RAC1, promotes internalization of the dying cells. Here we report the generation of ELMO1-deficient mice, which we found to be unexpectedly viable and grossly normal. However, they had a striking testicular pathology, with disrupted seminiferous epithelium, multinucleated giant cells, uncleared apoptotic germ cells and decreased sperm output. Subsequent in vitro and in vivo analyses revealed a crucial role for ELMO1 in the phagocytic clearance of apoptotic germ cells by Sertoli cells lining the seminiferous epithelium. The engulfment receptor BAI1 and RAC1 (upstream and downstream of ELMO1, respectively) were also important for Sertoli-cell-mediated engulfment. Collectively, these findings uncover a selective requirement for ELMO1 in Sertoli-cell-mediated removal of apoptotic germ cells and make a compelling case for a relationship between engulfment and tissue homeostasis in vivo.

Cutting Edge: CD4 Is the Receptor for the Tick Saliva Immunosuppressor, Salp15

Salp15 is an Ixodes scapularis salivary protein that inhibits CD4+ T cell activation through the repression of TCR ligation-triggered calcium fluxes and IL-2 production. We show in this study that Salp15 binds specifically to the CD4 coreceptor on mammalian host T cells. Salp15 specifically associates through its C-terminal residues with the outermost two extracellular domains of CD4. Upon binding to CD4, Salp15 inhibits the subsequent TCR ligation-induced T cell signaling at the earliest steps including tyrosine phosphorylation of the Src kinase Lck, downstream effector proteins, and lipid raft reorganization. These results provide a molecular basis to understanding the immunosuppressive activity of Salp15 and its specificity for CD4+ T cells.

Unexpected link between an antibiotic, pannexin channels and apoptosis.

Plasma membrane pannexin 1 channels (PANX1) release nucleotide find-me signals from apoptotic cells to attract phagocytes. Here we show that the quinolone antibiotic trovafloxacin is a novel PANX1 inhibitor, by using a small-molecule screen. Although quinolones are widely used to treat bacterial infections, some quinolones have unexplained side effects, including deaths among children. PANX1 is a direct target of trovafloxacin at drug concentrations seen in human plasma, and its inhibition led to dysregulated fragmentation of apoptotic cells. Genetic loss of PANX1 phenocopied trovafloxacin effects, revealing a non-redundant role for pannexin channels in regulating cellular disassembly during apoptosis. Increase in drug-resistant bacteria worldwide and the dearth of new antibiotics is a major human health challenge. Comparing different quinolone antibiotics suggests that certain structural features may contribute to PANX1 blockade. These data identify a novel linkage between an antibiotic, pannexin channels and cellular integrity, and suggest that re-engineering certain quinolones might help develop newer antibacterials.

Macrophages redirect phagocytosis by non-professional phagocytes and influence inflammation

Professional phagocytes (such as macrophages) and non-professional phagocytes (such as epithelial cells) clear billions of apoptotic cells and particles on a daily basis. Although professional and non-professional macrophages reside in proximity in most tissues, whether they communicate with each other during cell clearance, and how this might affect inflammation, is not known. Here we show that macrophages, through the release of a soluble growth factor and microvesicles, alter the type of particles engulfed by non-professional phagocytes and influence their inflammatory response. During phagocytosis of apoptotic cells or in response to inflammation-associated cytokines, macrophages released insulin-like growth factor 1 (IGF-1). The binding of IGF-1 to its receptor on non-professional phagocytes redirected their phagocytosis, such that uptake of larger apoptotic cells was reduced whereas engulfment of microvesicles was increased. IGF-1 did not alter engulfment by macrophages. Macrophages also released microvesicles, whose uptake by epithelial cells was enhanced by IGF-1 and led to decreased inflammatory responses by epithelial cells. Consistent with these observations, deletion of IGF-1 receptor in airway epithelial cells led to exacerbated lung inflammation after allergen exposure. These genetic and functional studies reveal that IGF-1- and microvesicle-dependent communication between macrophages and epithelial cells can critically influence the magnitude of tissue inflammation in vivo.

An essential role for calcium flux in phagocytes for apoptotic cell engulfment and the anti-inflammatory response

Cells undergo programmed cell death/apoptosis throughout the lifespan of an organism. The subsequent immunologically silent removal of apoptotic cells plays a role in the maintenance of tolerance; defects in corpse clearance have been associated with autoimmune disease. A number of receptors and signaling molecules involved in this process have been identified, but intracellular signaling downstream of corpse recognition is only now being defined. Calcium plays a key role as a second messenger in many cell types, leading to the activation of downstream molecules and eventual transcription of effector genes; however, the role of calcium signaling during apoptotic cell removal is unclear. Here, using studies in cell lines and in the context of a whole organism, we show that apoptotic cell recognition induces both an acute and sustained calcium flux within phagocytes and that the genes required for calcium flux are essential for engulfment. Furthermore, we provide evidence that both the release of calcium from the endoplasmic reticulum and the entry of extracellular calcium through CRAC channels into the phagocytes are important during engulfment. Moreover, knockdown in Caenorhabditis elegans of stim-1 and jph-1, two genes linked to the entry of extracellular calcium into cells, led to increased persistence of apoptotic cells in the nematode. Loss of these genes seemed to affect early signaling events, leading to a decreased enrichment of actin adjacent to the apoptotic cell during corpse removal. We also show that calcium is crucial for the secretion of TGF-β by the phagocytes during the engulfment of apoptotic cells. Taken together, these data point to an earlier unappreciated and evolutionarily conserved role for calcium flux at two distinguishable steps: the formation of the phagocytic cup and the internalization of the apoptotic cell, and the anti-inflammatory signaling induced in phagocytes by contact with apoptotic cells.

Boosting Apoptotic Cell Clearance by Colonic Epithelial Cells Attenuates Inflammation In Vivo

Few apoptotic corpses are seen even in tissues with high cellular turnover, leading to the notion that the capacity for engulfment in vivo is vast. Whether corpse clearance can be enhanced in vivo for potential benefit is not known. In a colonic inflammation model, we noted that the expression of the phagocytic receptor Bai1 was progressively downmodulated. Consistent with this, BAI1-deficient mice had more pronounced colitis and lower survival, with many uncleared apoptotic corpses and inflammatory cytokines within the colonic epithelium. When we engineered and tested transgenic mice overexpressing BAI1, these had fewer apoptotic cells, reduced inflammation, and attenuated disease. Boosting BAI1-mediated uptake by intestinal epithelial cells (rather than myeloid cells) was important in attenuating inflammation. A signaling-deficient BAI1 transgene could not provide a similar benefit. Collectively, these complementary genetic approaches showed that cell clearance could be boosted in vivo, with potential to regulate tissue inflammation in specific contexts.

Conformational Rearrangement within the Soluble Domains of the CD4 Receptor Is Ligand-specific

Ligand binding induces shape changes within the four modular ectodomains (D1–D4) of the CD4 receptor, an important receptor in immune signaling. Small angle x-ray scattering (SAXS) on both a two-domain and a four-domain construct of the soluble CD4 (sCD4) is consistent with known crystal structures demonstrating a bilobal and a semi-extended tetralobal Z conformation in solution, respectively. Detection of conformational changes within sCD4 as a result of ligand binding was followed by SAXS on sCD4 bound to two different glycoprotein ligands: the tick saliva immunosuppressor Salp15 and the HIV-1 envelope protein gp120. Ab initio modeling of these data showed that both Salp15 and gp120 bind to the D1 domain of sCD4 and yet induce drastically different structural rearrangements. Upon binding, Salp15 primarily distorts the characteristic lobal architecture of the sCD4 without significantly altering the semi-extended shape of the sCD4 receptor. In sharp contrast, the interaction of gp120 with sCD4 induces a shape change within sCD4 that can be described as a Z-to-U bi-fold closure of the four domains across its flexible D2–D3 linker. Placement of known crystal structures within the boundaries of the SAXS-derived models suggests that the ligand-induced shape changes could be a result of conformational changes within this D2–D3 linker. Functionally, the observed shape changes in CD4 receptor causes dissociation of lymphocyte kinase from the cytoplasmic domain of Salp15-bound CD4 and facilitates an interaction between the exposed V3 loops of CD4-bound gp120 molecule to the extracellular loops of its co-receptor, a step essential for HIV-1 viral entry.

IP3 Receptor-Mediated Ca2+ Release in Naive CD4 T Cells Dictates Their Cytokine Program

IP3 (inositol 1,4,5-trisphosphate) receptors (IP3Rs) regulate the release of Ca2+ from intracellular stores in response to IP3. Little is known about regulation of the expression of IP3Rs and their role during the activation of CD4 T cells. In this study we show that mouse naive CD4 T cells express IP3R1, IP3R2, and IP3R3, but that gene expression of IP3R3 primarily is down-regulated upon activation due to loss of the Ets-1 transcription factor. Down-regulation of IP3R expression in activated CD4 T cells is associated with the failure of TCR ligation to trigger Ca2+ release in these cells. We also show that down-regulation of specific IP3Rs in activated CD4 T cells correlates with the requirement of IP3R-mediated Ca2+ release only for the induction of, but not for the maintenance of, IL-2 and IFN-γ expression. Interestingly, while inhibition of IP3R function early during activation blocks IL-2 and IFN-γ production, it promotes the production of IL-17 by CD4 T cells. Thus, IP3Rs play a key role in the activation and differentiation of CD4 T cells. The immunosuppressive effect of pharmacological blockers of these receptors may be complicated by promoting the development of inflammatory CD4 T cells.