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Background Relative enlargement of the pulmonary artery (PA) on chest CT imaging is associated with respiratory exacerbations in patients with COPD or cystic fibrosis. We sought to determine whether similar findings were present in patients with asthma and whether these findings were explained by differences in ventricular size. Methods We measured the PA and aorta diameters in 233 individuals from the Severe Asthma Research Program III cohort. We also estimated right, left, and total epicardial cardiac ventricular volume indices (eERVVI, eELVVI, and eETVVI, respectively). Associations between the cardiac and PA measures (PA-to-aorta [PA/A] ratio, eERVVI-to-eELVVI [eRV/eLV] ratio, eERVVI, eELVVI, eETVVI) and clinical measures of asthma severity were assessed by Pearson correlation, and associations with asthma severity and exacerbation rate were evaluated by multivariable linear and zero-inflated negative binomial regression. Results Asthma severity was associated with smaller ventricular volumes. For example, those with severe asthma had 36.1 mL/m2 smaller eETVVI than healthy control subjects (P = .003) and 14.1 mL/m2 smaller eETVVI than those with mild/moderate disease (P = .011). Smaller ventricular volumes were also associated with a higher rate of asthma exacerbations, both retrospectively and prospectively. For example, those with an eETVVI less than the median had a 57% higher rate of exacerbations during follow-up than those with eETVVI greater than the median (P = .020). Neither PA/A nor eRV/eLV was associated with asthma severity or exacerbations. Conclusions In patients with asthma, smaller cardiac ventricular size may be associated with more severe disease and a higher rate of asthma exacerbations. Trial Registry ClinicalTrials.gov; No.: NCT01761630; URL: www.clinicaltrials.gov

BACKGROUND Pharmacogenetic studies in asthma cohorts, primarily made up of White people of European descent, have identified loci associated with response to inhaled beta agonists and corticosteroids (ICSs). Differences exist in how individuals from different ancestral backgrounds respond to long-acting beta agonist (LABA) and ICSs. Therefore, we sought to understand the pharmacogenetic mechanisms regulating therapeutic responsiveness in individuals of African descent. METHODS We did ancestry-based pharmacogenetic studies of children (aged 5-11 years) and adolescents and adults (aged 12-69 years) from the Best African Response to Drug (BARD) trials, in which participants with asthma uncontrolled with low-dose ICS (fluticasone propionate 50 μg in children, 100 μg in adolescents and adults) received different step-up combination therapies. The hierarchal composite outcome of pairwise superior responsiveness in BARD was based on asthma exacerbations, a 31-day difference in annualised asthma-control days, or a 5% difference in percentage predicted FEV1. We did whole-genome admixture mapping of 15 159 ancestral segments within 312 independent regions, stratified by the two age groups. The two co-primary outcome comparisons were the step up from low-dose ICS to the quintuple dose of ICS (5 × ICS: 250 μg twice daily in children and 500 μg twice daily in adolescents and adults) versus double dose (2-2·5 × ICS: 100 μg twice daily in children, 250 μg twice daily in adolescents and adults), and 5 × ICS versus 100 μg fluticasone plus a LABA (salmeterol 50 μg twice daily). We used a genome-wide significance threshold of p

Background Cystic fibrosis related diabetes (CFRD) is associated with pulmonary decline and compromised nutritional status. Emerging data suggest that CFTR dysfunction may play a direct role in the pathogenesis of CFRD; however, studies investigating the effect of CFTR modulators on glycemic outcomes in patients with cystic fibrosis (CF) have shown mixed results. The impact of elexacaftor-tezacaftor-ivacaftor (ETI) on glycemic control is currently unknown. Our objective was to investigate the effect of ETI initiation on glycemia in adults with CF using continuous glucose monitoring (CGM). Methods In this prospective observational study, 34 adults with CF and at least one F508del CFTR mutation wore CGM sensors for 14 days prior to starting ETI and again 3-12 months after ETI initiation. Hypoglycemia symptoms were queried at each visit, and most recent anthropometric measures and spirometry data were obtained by chart review. Results Twenty-three participants completed the study. Compared to baseline, average glucose (AG), standard deviation (SD), % time >200 mg/dL, and peak sensor glucose decreased with ETI treatment, and % time in target range 70-180 mg/dL increased. Improvements in glycemic parameters were most notable in individuals with CFRD. There was no significant change in CGM-measured or self-reported hypoglycemia before and after ETI initiation. Conclusion Initiation of ETI in adults with CF was associated with improvement CGM-derived measures of hyperglycemia and glycemic variability with no effect on hypoglycemia. Further studies are needed to investigate underlying etiology of these changes and the long-term impact of ETI on glycemic control in patients with CF.

SARS-CoV-2 in an Immunocompromised Host This letter describes an immunocompromised patient who had persistent infection with SARS-CoV-2 over a period of months, despite several courses of remdesivi...

Background Cystic fibrosis (CF) is an inherited disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that promotes persistent lung infection and inflammation and progressive loss of lung function. Patients with CF have increased lung lymphoid follicles (LFs) and B cell-activating factor of tumor necrosis factor family (BAFF) that regulates B cell survival and maturation. A direct role for CFTR in B cell activation and disease pathogenesis in CF remains unclear. Methods The number of LFs, BAFF+, TLR4+ and proliferation marker Ki67+ B cells in lung explants or resections from subjects with CF and normal controls was quantified by immunostaining. The role of CFTR in B cell activation and LF development was then examined in two independent cohorts of uninfected CFTR-deficient mice (Cftr −/− ) and wild type controls. The number of lung LFs, B cells and BAFF+, CXCR4+, immunoglobulin G+ B cells was examined by immunostaining. Lung and splenocyte B cell activation marker and major histocompatibility complex class II (MHC class II) expression was quantified by flow cytometry. Inflammatory cytokine levels were measured in supernatants from isolated B cells from Cftr −/− and wild type mice stimulated in vitro with Pseudomonas aeruginosa lipopolysaccharide (LPS). Results There was a significant increase in well-formed LFs in subjects with CF compared to normal controls. Increased B cell activation and proliferation was observed in lung LFs from CF subjects as was quantified by a significant increase in B cell BAFF, TLR4 and Ki67 expression. Uninfected Cftr −/− mice had increased lung LFs and BAFF+ and CXCR4+ B cells compared to wild type controls. Lung B cells isolated from uninfected Cftr −/− mice demonstrated increased MHC class II expression. In vitro, isolated B cells from Cftr −/− mice produced increased IL-6 when stimulated with LPS compared to wild type controls. Conclusions These data support a direct role for CFTR in B cell activation, proliferation and inflammatory cytokine production that promotes lung LF follicle development in cystic fibrosis.

The immune responses of type 2 T helper cells (Th2) play an important role in asthma and promote the differentiation of alternatively activated (M2) macrophages. M2 macrophages have been increasingly understood to contribute to Th2 immunity. We hypothesized that M2 macrophages are altered in asthma and modulate Th2 responses. The aim of this study was to characterize the phenotype and function of human monocyte-derived M2 and bronchoalveolar lavage fluid (BALF) macrophages from healthy control subjects and subjects with asthma. Phenotypic characteristics and effector function of M2 macrophages were examined using monocyte-derived and BALF macrophages obtained from subjects with asthma (n = 28) and healthy volunteers (n = 9) by flow cytometry and quantitative PCR. Resting monocyte-derived (M0) and M2 macrophages were generated by the addition of macrophage colony–stimulating factor or macrophage colony–stimulating factor plus IL-4, respectively. M2 macrophage cytokine expression and their impact on dendritic and CD4+ T cell activation were examined in vitro. High levels of CD206 and major histocompatibility complex class II expression identify macrophages with an M2 phenotype that are increased 2.9-fold in the BALF of subjects with asthma compared with control subjects. M2 macrophages have elevated IL-6, IL-10, and IL-12p40 production compared with conventional macrophages and modulate dendritic and CD4+ T cell interactions. Histamine receptor 1 and E-cadherin expression identify M2 macrophage subsets associated with increased airflow obstruction. M2 macrophages have a distinct cell surface and effector phenotype and are found in increased numbers in subjects with asthma. These findings suggest that M2 macrophages may play an important role in allergic asthma through their bidirectional interactions with immune and structural cells, and inflammatory mediators.

Fatty acid binding protein 4 (FABP4), an intracellular lipid chaperone and adipokine, is expressed by lung macrophages, but the function of macrophage–FABP4 remains elusive. We investigated the role of FABP4 in host defense in a murine model of Pseudomonas aeruginosa pneumonia. Compared with wild-type (WT) mice, FABP4-deficient (FABP4(−/−)) mice exhibited decreased bacterial clearance and increased mortality when challenged intranasally with P. aeruginosa. These findings in FABP4(−/−) mice were associated with a delayed neutrophil recruitment into the lungs and were followed by greater acute lung injury and inflammation. Among leukocytes, only macrophages expressed FABP4 in WT mice with P. aeruginosa pneumonia. Chimeric FABP4(−/−) mice with WT bone marrow were protected from increased mortality seen in chimeric WT mice with FABP4(−/−) bone marrow during P. aeruginosa pneumonia, thus confirming the role of macrophages as the main source of protective FABP4 against that infection. There was less production of C-X-C motif chemokine ligand 1 (CXCL1) in FABP4(−/−) alveolar macrophages and lower airway CXCL1 levels in FABP4(−/−) mice. Delivering recombinant CXCL1 to the airways protected FABP4(−/−) mice from increased susceptibility to P. aeruginosa pneumonia. Thus, macrophage-FABP4 has a novel role in pulmonary host defense against P. aeruginosa infection by facilitating crosstalk between macrophages and neutrophils via regulation of macrophage CXCL1 production.—Liang, X., Gupta, K., Rojas Quintero, J., Cernadas, M., Kobzik, L., Christou, H., Pier, G. B., Owen, C. A., Çataltepe, S. Macrophage FABP4 is required for neutrophil recruitment and bacterial clearance in Pseudomonas aeruginosa pneumonia.

Severe asthma is a debilitating and treatment refractory disease. As many as half of these patients have complex neutrophil-predominant lung inflammation that is distinct from milder asthma with type 2 eosinophilic inflammation. New insights into severe asthma pathogenesis are needed. Concomitant exposure of mice to an aeroallergen and endotoxin during sensitization resulted in complex neutrophilic immune responses to allergen alone during later airway challenge. Unlike allergen alone, sensitization with allergen and endotoxin led to NETosis. In addition to neutrophil extracellular traps (NETs), enucleated neutrophil cytoplasts were evident in the lungs. Surprisingly, allergen-driven airway neutrophilia was decreased in peptidyl arginine deiminase 4-deficient mice with defective NETosis but not by deoxyribonuclease treatment, implicating the cytoplasts for the non-type 2 immune responses to allergen. Neutrophil cytoplasts were also present in mediastinal lymph nodes, and the cytoplasts activated lung dendritic cells in vitro to trigger antigen-specific interleukin-17 (IL-17) production from naïve CD4+ T cells. Bronchoalveolar lavage fluid from patients with severe asthma and high neutrophil counts had detectable NETs and cytoplasts that were positively correlated with IL-17 levels. Together, these translational findings have identified neutrophil cytoplast formation in asthmatic lung inflammation and linked the cytoplasts to T helper 17-mediated neutrophilic inflammation in severe asthma. ispartof: SCIENCE IMMUNOLOGY vol:3 issue:26 ispartof: location:United States status: published

Severe asthma is a debilitating and treatment refractory disease. As many as half of these patients have complex neutrophil-predominant lung inflammation that is distinct from milder asthma with type 2 eosinophilic inflammation. New insights into severe asthma pathogenesis are needed. Concomitant exposure of mice to an aeroallergen and endotoxin during sensitization resulted in complex neutrophilic immune responses to allergen alone during later airway challenge. Unlike allergen alone, sensitization with allergen and endotoxin led to NETosis. In addition to neutrophil extracellular traps (NETs), enucleated neutrophil cytoplasts were evident in the lungs. Surprisingly, allergen-driven airway neutrophilia was decreased in peptidyl arginine deiminase 4-deficient mice with defective NETosis but not by deoxyribonuclease treatment, implicating the cytoplasts for the non-type 2 immune responses to allergen. Neutrophil cytoplasts were also present in mediastinal lymph nodes, and the cytoplasts activated lung dendritic cells in vitro to trigger antigen-specific interleukin-17 (IL-17) production from naïve CD4

Severe asthma is typically characterized by chronic airway inflammation that is refractory to corticosteroids and associated with excess morbidity. Patients were recruited into the National Heart, Lung, and Blood Institute–sponsored Severe Asthma Research Program and comprehensively phenotyped by bronchoscopy. Bronchoalveolar lavage (BAL) cells were analyzed by flow cytometry. Compared with healthy individuals (n = 21), patients with asthma (n = 53) had fewer BAL natural killer (NK) cells. Patients with severe asthma (n = 29) had a marked increase in the ratios of CD4+ T cells to NK cells and neutrophils to NK cells. BAL NK cells in severe asthma were skewed toward the cytotoxic CD56dim subset, with significantly increased BAL fluid levels of the cytotoxic mediator granzyme A. The numbers of BAL CD56dim NK cells and CCR6−CCR4− T helper 1–enriched CD4+ T cells correlated inversely with lung function [forced expiratory volume in 1 s (FEV1) % predicted] in asthma. Relative to cells from healthy controls, peripheral blood NK cells from asthmatic patients had impaired killing of K562 myeloid target cells despite releasing more cytotoxic mediators. Ex vivo exposure to dexamethasone markedly decreased blood NK cell lysis of target cells and cytotoxic mediator release. NK cells expressed airway lipoxin A4/formyl peptide receptor 2 receptors, and in contrast to dexamethasone, lipoxin A4–exposed NK cells had preserved functional responses. Together, our findings indicate that the immunology of the severe asthma airway is characterized by decreased NK cell cytotoxicity with increased numbers of target leukocytes, which is exacerbated by corticosteroids that further disable NK cell function. These failed resolution mechanisms likely contribute to persistent airway inflammation in severe asthma.

To determine whether a disintegrin and metalloproteinase-8 (Adam8) regulates allergic airway inflammation (AAI) and airway hyperresponsiveness (AHR), we compared AAI and AHR in wild-type (WT) versus Adam8−/− mice in different genetic backgrounds sensitized and challenged with OVA or house dust mite protein extract. OVA- and house dust mite–treated Adam8−/− mice had higher lung leukocyte counts, more airway mucus metaplasia, greater lung levels of some Th2 cytokines, and higher methacholine-induced increases in central airway resistance than allergen-treated WT mice. Studies of OVA-treated Adam8 bone marrow chimeric mice confirmed that leukocyte-derived Adam8 predominantly mediated Adam8’s anti-inflammatory activities in murine airways. Airway eosinophils and macrophages both expressed Adam8 in WT mice with AAI. Adam8 limited AAI and AHR in mice by reducing leukocyte survival because: 1) Adam8−/− mice with AAI had fewer apoptotic eosinophils and macrophages in their airways than WT mice with AAI; and 2) Adam8−/− macrophages and eosinophils had reduced rates of apoptosis compared with WT leukocytes when the intrinsic (but not the extrinsic) apoptosis pathway was triggered in the cells in vitro. ADAM8 was robustly expressed by airway granulocytes in lung sections from human asthma patients, but, surprisingly, airway macrophages had less ADAM8 staining than airway eosinophils. Thus, ADAM8 has anti-inflammatory activities during AAI in mice by activating the intrinsic apoptosis pathway in myeloid leukocytes. Strategies that increase ADAM8 levels in myeloid leukocytes may have therapeutic efficacy in asthma.

Little is known about the mechanisms of persistent airflow obstruction that result from chronic occupational endotoxin exposure. We sought to analyze the inflammatory response underlying persistent airflow obstruction as a result of chronic occupational endotoxin exposure. We developed a murine model of daily inhaled endotoxin for periods of 5 days to 8 weeks. We analyzed physiologic lung dysfunction, lung histology, bronchoalveolar lavage fluid and total lung homogenate inflammatory cell and cytokine profiles, and pulmonary gene expression profiles. We observed an increase in airway hyperresponsiveness as a result of chronic endotoxin exposure. After 8 weeks, the mice exhibited an increase in bronchoalveolar lavage and lung neutrophils that correlated with an increase in proinflammatory cytokines. Detailed analyses of inflammatory cell subsets revealed an expansion of dendritic cells (DCs), and in particular, proinflammatory DCs, with a reduced percentage of macrophages. Gene expression profiling revealed the up-regulation of a panel of genes that was consistent with DC recruitment, and lung histology revealed an accumulation of DCs in inflammatory aggregates around the airways in 8-week-exposed animals. Repeated, low-dose LPS inhalation, which mirrors occupational exposure, resulted in airway hyperresponsiveness, associated with a failure to resolve the proinflammatory response, an inverted macrophage to DC ratio, and a significant rise in the inflammatory DC population. These findings point to a novel underlying mechanism of airflow obstruction as a result of occupational LPS exposure, and suggest molecular and cellular targets for therapeutic development.

Fatty acid binding protein 4 (FABP4) plays an important role in regulation of glucose and lipid homeostasis as well as inflammation through its actions in adipocytes and macrophages. FABP4 is also expressed in a subset of endothelial cells, but its role in this cell type is not known. We found that FABP4-deficient human umbilical vein endothelial cells (HUVECs) demonstrate a markedly increased susceptibility to apoptosis as well as decreased migration and capillary network formation. Aortic rings from FABP4(-/-) mice demonstrated decreased angiogenic sprouting, which was recovered by reconstitution of FABP4. FABP4 was strongly regulated by mTORC1 and inhibited by Rapamycin. FABP4 modulated activation of several important signaling pathways in HUVECs, including downregulation of P38, eNOS, and stem cell factor (SCF)/c-kit signaling. Of these, the SCF/c-kit pathway was found to have a major role in attenuated angiogenic activity of FABP4-deficient ECs as provision of exogenous SCF resulted in a significant recovery in cell proliferation, survival, morphogenesis, and aortic ring sprouting. These data unravel a novel pro-angiogenic role for endothelial cell-FABP4 and suggest that it could be exploited as a potential target for diseases associated with pathological angiogenesis.

Immune responses are pathologically sustained in several common diseases, including asthma. To determine endogenous proresolving mechanisms for adaptive immune responses, we used a murine model of self-limited allergic airway inflammation. After cessation of allergen exposure, eosinophils and T cells were cleared concomitant with the appearance of increased numbers of NK cells in the lung and mediastinal lymph nodes. The mediastinal lymph node NK cells were activated, expressing CD27, CD11b, CD69, CD107a, and IFN-γ. NK cell depletion disrupted the endogenous resolution program, leading to delayed clearance of airway eosinophils and Ag-specific CD4+ T cells. NK cell trafficking to inflamed tissues for resolution was dependent upon CXCR3 and CD62L. During resolution, eosinophils and Ag-specific CD4+ T cells expressed NKG2D ligands, and a blocking Ab for the NKG2D receptor delayed clearance of these leukocytes. Of interest, NK cells expressed CMKLR1, a receptor for the proresolving mediator resolvin E1, and depletion of NK cells decreased resolvin E1-mediated resolution of allergic inflammation. Resolvin E1 regulated NK cell migration in vivo and NK cell cytotoxicity in vitro. Together, these findings indicate new functions in catabasis for NK cells that can also serve as targets for proresolving mediators in the resolution of adaptive immunity.

Objective To define the intrinsic capacity of fibroblast-like synoviocytes (FLS) to establish a 3-dimensional (3-D) complex synovial lining architecture characterized by the multicellular organization of the compacted synovial lining and the elaboration of synovial fluid constituents. Methods FLS were cultured in spherical extracellular matrix (ECM) micromasses for 3 weeks. The FLS micromass architecture was assessed histologically and compared with that of dermal fibroblast controls. Lubricin synthesis was measured via immunodetection. Basement membrane matrix and reticular fiber stains were performed to examine ECM organization. Primary human and mouse monocytes were prepared and cocultured with FLS in micromass to investigate cocompaction in the lining architecture. Cytokine stimuli were applied to determine the capacity for inflammatory architecture rearrangement. Results FLS, but not dermal fibroblasts, spontaneously formed a compacted lining architecture over 3 weeks in the 3-D ECM micromass organ cultures. These lining cells produced lubricin. FLS rearranged their surrounding ECM into a complex architecture resembling the synovial lining and supported the survival and cocompaction of monocyte/macrophages in the neo–lining structure. Furthermore, when stimulated by cytokines, FLS lining structures displayed features of the hyperplastic rheumatoid arthritis synovial lining. Conclusion This 3-D micromass organ culture method demonstrates that many of the phenotypic characteristics of the normal and the hyperplastic synovial lining in vivo are intrinsic functions of FLS. Moreover, FLS promote survival and cocompaction of primary monocytes in a manner remarkably similar to that of synovial lining macrophages. These findings provide new insight into inherent functions of the FLS lineage and establish a powerful in vitro method for further investigation of this lineage.

Interleukin 23 (IL-23) is integral to the pathogenesis of chronic inflammation. The resolution of acute inflammation is an active process mediated by specific signals and mediators such as resolvin E1 (RvE1). Here we provide evidence that RvE1, in nanogram quantities, promoted the resolution of inflammatory airway responses in part by directly suppressing the production of IL-23 and IL-6 in the lung. Also contributing to the pro-resolution effects of RvE1 treatment were higher concentrations of interferon-gamma in the lungs of RvE1-treated mice. Our findings indicate a pivotal function for IL-23 and IL-6, which promote the survival and differentiation of IL-17-producing T helper cells, in maintaining inflammation and also identify an RvE1-initiated resolution program for allergic airway responses.

Respiratory tract dendritic cells (DCs) are juxtaposed to directly sample inhaled environmental particles. Processing and presentation of these airborne Ags could result in either the development of immunity or tolerance. The purpose of this study was to determine the consequences of cigarette smoke exposure on DC function in mice. We demonstrate that while cigarette smoke exposure decreased the number of DCs in the lungs, Ag-induced DC migration to the regional thoracic lymph nodes was unaffected. However, cigarette smoking suppressed DC maturation within the lymph nodes as demonstrated by reduced cell surface expression of MHC class II and the costimulatory molecules CD80 and CD86. Consequently, DCs from cigarette smoke-exposed animals had a diminished capacity to induce IL-2 production by T cells that was associated with diminished Ag-specific T cell proliferation in vivo. Smoke-induced defects in DC function leading to impaired CD4+ T cell function could inhibit tumor surveillance and predispose patients with chronic obstructive pulmonary disease to infections and exacerbations.

Recent studies of CD1 structure and intracellular trafficking have demonstrated significant differences among the CD1 isoforms (CD1a, CD1b, CD1c and CD1d). The molecular and structural basis for the differential trafficking of CD1 molecules has also been delineated. These observations broaden our understanding of why the immune system has evolved multiple CD1 isoforms to survey different cellular compartments for lipid antigen presentation, to provide host defense against the microbial world and to offer immunoregulation with relevance to tumor immunity and autoimmunity.

Circadian rhythms are known to regulate immune responses in healthy animals, but it is unclear whether they persist during acute illnesses where clock gene expression is disrupted by systemic inflammation. Here we use a genome-wide approach to investigate circadian gene and metabolite expression in the lungs of endotoxemic mice and find that novel cellular and molecular circadian rhythms are elicited in this setting. The endotoxin-specific circadian programme exhibits unique features, including a divergent group of rhythmic genes and metabolites compared with the basal state and a distinct periodicity and phase distribution. At the cellular level, endotoxin treatment also alters circadian rhythms of leukocyte counts within the lung in a bmal1-dependent manner, such that granulocytes rather than lymphocytes become the dominant oscillating cell type. Our results show that inflammation produces a complex re-organization of cellular and molecular circadian rhythms that are relevant to early events in lung injury.

Integrins αEβ7and α4β7 are involved in localization of leukocytes at mucosal sites. Although both αEβ7 and α4β7utilize the β7 chain, they have distinct binding specificities for E-cadherin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1), respectively. We found that mutation of the metal ion-dependent adhesion site (MIDAS) in the αE A-domain (D190A) abolished E-cadherin binding, as did mutation F298A on the A-domain surface near the MIDAS cleft. A docking model of the A-domain with E-cadherin domain 1 indicates that coordination of the αE MIDAS metal ion by E-cadherin Glu31 and a novel projection of Phe298 into a hydrophobic pocket on E-cadherin provide the basis for the interaction. The location of the binding site on the αE A-domain resembles that on other integrins, but its structure appears distinctive and particularly adapted to recognize the tip of E-cadherin, a unique integrin ligand. Additionally, mutation of the β7 MIDAS motif (D140A) abolished αEβ7 binding to E-cadherin and α4β7-mediated adhesion to MAdCAM-1, and α4 chain mutations that abrogated binding of α4β1 to vascular cell adhesion molecule-1 and fibronectin similarly reduced α4β7interaction with MAdCAM-1. Thus, although specificity can be determined by the integrin α or β chain, common structural features of both subunits are required for recognition of dissimilar ligands.

CD23, a receptor for immunoglobulin E, is expressed at increased levels in asthmatic and atopic individuals and has been associated with disorders characterized by chronic inflammation. Using an established murine model, we employed several complementary strategies to investigate the role of CD23 in allergic pulmonary inflammation and airway hyperresponsiveness (AHR). Specifically, these approaches included the modulation of CD23 function in vivo by administration of anti-CD23 monoclonal antibody (mAb) or Fab fragments to wild-type mice and the analysis of CD23-deficient mice. Administration of anti-CD23 mAb, but not anti-CD23 Fab fragments, produced attenuation of pulmonary inflammation, AHR, and CD8(+) T-cell activation. On the basis of a model that the anti-CD23 mAb transduces, whereas the Fab fragment inhibits, CD23 signaling, these results suggest that CD23 negatively regulates pulmonary inflammation and AHR. This hypothesis is supported by our observation that CD23-deficient mice developed increased inflammation and AHR after sensitization and challenge with allergen. Together, these results indicate that CD23 negatively regulates pulmonary inflammation and airway hyperreactivity.

Asthma is a prevalent disease of chronic inflammation in which endogenous counterregulatory signaling pathways are dysregulated. Recent evidence suggests that innate lymphoid cells (ILCs), including natural killer (NK) cells and type 2 ILCs (ILC2s), can participate in the regulation of allergic airway responses, in particular airway mucosal inflammation. We have identified both NK cells and ILC2s in human lung and peripheral blood in healthy and asthmatic subjects. NK cells were highly activated in severe asthma, were linked to eosinophilia, and interacted with autologous eosinophils to promote their apoptosis. ILC2s generated antigen-independent interleukin-13 (IL-13) in response to the mast cell product prostaglandin D2 alone and in a synergistic manner with the airway epithelial cytokines IL-25 and IL-33. Both NK cells and ILC2s expressed the pro-resolving ALX/FPR2 receptors. Lipoxin A4, a natural pro-resolving ligand for ALX/FPR2 receptors, significantly increased NK cell-mediated eosinophil apoptosis and decreased IL-13 release by ILC2s. Together, these findings indicate that ILCs are targets for lipoxin A4 to decrease airway inflammation and mediate the catabasis of eosinophilic inflammation. Because lipoxin A4 generation is decreased in severe asthma, these findings also implicate unrestrained ILC activation in asthma pathobiology.

Rationale Endotoxin is a near ubiquitous environmental exposure that that has been associated with both asthma and chronic obstructive pulmonary disease (COPD). These obstructive lung diseases have a complex pathophysiology, making them difficult to study comprehensively in the context of endotoxin. Genome-wide gene expression studies have been used to identify a molecular snapshot of the response to environmental exposures. Identification of differentially expressed genes shared across all published murine models of chronic inhaled endotoxin will provide insight into the biology underlying endotoxin-associated lung disease. Methods We identified three published murine models with gene expression profiling after repeated low-dose inhaled endotoxin. All array data from these experiments were re-analyzed, annotated consistently, and tested for shared genes found to be differentially expressed. Additional functional comparison was conducted by testing for significant enrichment of differentially expressed genes in known pathways. The importance of this gene signature in smoking-related lung disease was assessed using hierarchical clustering in an independent experiment where mice were exposed to endotoxin, smoke, and endotoxin plus smoke. Results A 101-gene signature was detected in three murine models, more than expected by chance. The three model systems exhibit additional similarity beyond shared genes when compared at the pathway level, with increasing enrichment of inflammatory pathways associated with longer duration of endotoxin exposure. Genes and pathways important in both asthma and COPD were shared across all endotoxin models. Mice exposed to endotoxin, smoke, and smoke plus endotoxin were accurately classified with the endotoxin gene signature. Conclusions Despite the differences in laboratory, duration of exposure, and strain of mouse used in three experimental models of chronic inhaled endotoxin, surprising similarities in gene expression were observed. The endotoxin component of tobacco smoke may play an important role in disease development.

PTPN22 is a tyrosine phosphatase and functions as a damper of TCR signals. A C-to-T single nucleotide polymorphism (SNP) located at position 1858 of human PTPN22 cDNA and converting an arginine (R620) to tryptophan (W620) confers the highest risk of rheumatoid arthritis among non-HLA genetic variations that are known to be associated with this disease. The effect of the R-to-W conversion on the phosphatase activity of PTPN22 protein and the impact of the minor T allele of the C1858T SNP on the activation of T cells has remained controversial. In addition, how the overall activity of PTPN22 is regulated and how the R-to-W conversion contributes to rheumatoid arthritis is still poorly understood. Here we report the identification of an alternative splice form of human PTPN22, namely PTPN22.6. It lacks the nearly entire phosphatase domain and can function as a dominant negative isoform of the full length PTPN22. Although conversion of R620 to W620 in the context of PTPN22.1 attenuated T cell activation, expression of the tryptophan variant of PTPN22.6 reciprocally led to hyperactivation of human T cells. More importantly, the level of PTPN22.6 in peripheral blood correlates with disease activity of rheumatoid arthritis. Our data depict a model that can reconcile the conflicting observations on the functional impact of the C1858T SNP and also suggest that PTPN22.6 is a novel biomarker of rheumatoid arthritis.

Sepsis remains the leading cause of death in critically ill patients, despite modern advances in critical care. Intestinal barrier dysfunction may lead to secondary bacterial translocation and the development of the multiple organ dysfunction syndrome during sepsis. Cyclooxygenase (COX)-2 is highly upregulated in the intestine during sepsis, and we hypothesized that it may be critical in the maintenance of intestinal epithelial barrier function during peritonitis-induced polymicrobial sepsis. COX-2−/− and COX-2+/+ BALB/c mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice chimeric for COX-2 were derived by bone marrow transplantation and underwent CLP. C2BBe1 cells, an intestinal epithelial cell line, were treated with the COX-2 inhibitor NS-398, PGD2, or vehicle and stimulated with cytokines. COX-2−/− mice developed exaggerated bacteremia and increased mortality compared with COX-2+/+ mice following CLP. Mice chimeric for COX-2 exhibited the recipient phenotype, suggesting that epithelial COX-2 expression in the ileum attenuates bacteremia following CLP. Absence of COX-2 significantly increased epithelial permeability of the ileum and reduced expression of the tight junction proteins zonula occludens-1, occludin, and claudin-1 in the ileum following CLP. Furthermore, PGD2 attenuated cytokine-induced hyperpermeability and zonula occludens-1 downregulation in NS-398–treated C2BBe1 cells. Our findings reveal that absence of COX-2 is associated with enhanced intestinal epithelial permeability and leads to exaggerated bacterial translocation and increased mortality during peritonitis-induced sepsis. Taken together, our results suggest that epithelial expression of COX-2 in the ileum is a critical modulator of tight junction protein expression and intestinal barrier function during sepsis.

Autophagy, a cellular process for organelle and protein turnover, regulates innate immune responses. Here we demonstrate that depletion of the autophagic proteins LC3B and beclin 1 enhanced the activation of caspase-1 and secretion of interleukin 1β (IL-1β) and IL-18. Depletion of autophagic proteins promoted the accumulation of dysfunctional mitochondria and cytosolic translocation of mitochondrial DNA (mtDNA) in response to lipopolysaccharide (LPS) and ATP in macrophages. Release of mtDNA into the cytosol depended on the NALP3 inflammasome and mitochondrial reactive oxygen species (ROS). Cytosolic mtDNA contributed to the secretion of IL-1β and IL-18 in response to LPS and ATP. LC3B-deficient mice produced more caspase-1-dependent cytokines in two sepsis models and were susceptible to LPS-induced mortality. Our study suggests that autophagic proteins regulate NALP3-dependent inflammation by preserving mitochondrial integrity.

Myeloid antigen-presenting cells (APC) express CD1d molecules that present exogenous and endogenous lipid antigens that activate CD1d-restricted T cells, natural killer T (NKT) cells. NKT cell activation has been shown to mediate the potent downstream activation of other immune cells through cell–cell interactions and rapid, prolific cytokine production. Foreign antigens are not required for NKT cell activation. The endogenous lipids bound to CD1d are sufficient for activation of NKT cells in the setting of Toll-like receptor-induced cytokines. The most potent NKT cell antigens identified are glycosphingolipids (GSL). The GSL repertoire of endogenous ligands bound to CD1d molecules that are expressed in myeloid APC at steady state and in the setting of activation has not been delineated. This report identifies the range of GSL bound to soluble murine CD1d (mCD1d) molecules that sample the endoplasmic reticulum/secretory routes and cell surface-cleaved mCD1d that also samples the endocytic system. Specific GSL species are preferentially bound by mCD1d and do not solely reflect cellular GSL. GM1a and GD1a are prominent CD1d ligands for molecules following both the ER/secretory and lysosomal trafficking routes, whereas GM2 was eluted from soluble CD1d but not lysosomal trafficking CD1d. Further, after LPS activation, the quantities of soluble CD1d-bound GM3 and GM1a markedly increased. A unique α-galactose-terminating GSL was also found to be preferentially bound to mCD1d at steady state, and it increased with APC activation. Together, these studies identify the range of GSL presented by CD1d and how presentation varies based on CD1d intracellular trafficking and microbial activation.

A major step in understanding differences in the nature of Ag presentation was the realization that MHC class I samples peptides transported to the endoplasmic reticulum from the cytosol, whereas MHC class II samples peptides from lysosomes. In contrast to MHC class I and II molecules that present protein Ags, CD1 molecules present lipid Ags for recognition by specific T cells. Each of the five members of the CD1 family (CD1a–e) localizes to a distinct subcompartment of endosomes. Accordingly, it has been widely assumed that the distinct trafficking of CD1 isoforms must also have evolved to enable them to sample lipid Ags that traffic via different routes. Among the CD1 isoforms, CD1a is unusual because it does not have a tyrosine-based cytoplasmic sorting motif and uniquely localizes to the early endocytic recycling compartment. This led us to predict that CD1a might have evolved to focus on lipids that localize to early endocytic/recycling compartments. Strikingly, we found that the glycolipid Ag sulfatide also localized almost exclusively to early endocytic and recycling compartments. Consistent with colocalization of CD1a and sulfatide, wild-type CD1a molecules efficiently presented sulfatide to CD1a-restricted, sulfatide-specific T cells. In contrast, CD1a:CD1b tail chimeras, that retain the same Ag-binding capacity as CD1a but traffic based on the cytoplasmic tail of CD1b to lysosomes, failed to present sulfatide efficiently. Thus, the intracellular trafficking route of CD1a is essential for efficient presentation of lipid Ags that traffic through the early endocytic and recycling pathways.

SummaryHuman and murine dendritic cell (DC) subsets are often defined by phenotypic features that predict their functional characteristics. In humans and mice, DC have been shown to have the ability to polarize naive CD4 T cells to a T helper type 1 (Th1) or Th2 phenotype. However, human myeloid DC generated from monocytes (monocyte-derived DC) have often been regarded as a homogeneous population, both phenotypically and functionally. Monocytes give rise to subpopulations of DC in vitro that can be separated on the basis of their expression of CD1a, a well-described DC subset marker. Importantly, we show that the CD1a+ DC subset produces significant quantities of interleukin-12p70 (IL-12p70) upon stimulation and, similar to the murine CD8α+ DC subset, can polarize naive CD4+ T cells to a Th1 phenotype. In contrast, CD1a− DC, similar to murine CD8α− DC, do not produce significant amounts of IL-12p70 upon stimulation or polarize T cells to a Th1 phenotype. Like monocyte-derived DC, CD1a+ and CD1a− DC subsets obtained from CD34+ haematopoietic progenitors under distinct culture conditions were found to have these same features, suggesting that CD1a expression is a marker for myeloid DC that are a major source of IL-12 and Th1 CD4+ T cell polarization in man.

An Na(+)-dependent active process for myo-inositol (MI) uptake, sharing a common carrier system with glucose and sensitive to phlorizin, was previously established in primary cultures of bovine retinal pigment epithelial (RPE) cells (26, 32). The present report further examines the nature of glucose-induced inhibition of MI transport in primary cultures of RPE cells. RPE cells were grown in supplemented Dulbecco's modification of Eagle's medium (DMEM) containing 5 mM D-glucose (basic growth media) or 40 mM D-glucose or its nonmetabolizable analogue, alpha-methyl-D-glucoside (alpha MG); 1-5 mM nonradioactive MI, pyruvate, or lactate; or 0.2-20 microM phorbol 12-myristate 13-acetate (TPA) or straurosporin (modified growth media), for up to 4 weeks. The capacity of RPE cells to accumulate 3H-MI (ratios of intracellular transported radioactive MI, [MI]i, to external free MI concentration, [MI]i/[MI]o) decreased by up to 41% or 34% when cells were grown for 10 days or longer with 40 mM D-glucose or 40 mM alpha MG, respectively, compared to cells grown in basic growth media. The rate of uptake of 3H-MI also was reduced to 63 +/- 15% or 48 +/- 8% of the control values when cells were fed 1 or 5 mM nonradioactive MI, respectively. In addition, cellular capacity to bind to [3H]phlorizin was reduced to 52 +/- 7%, 61 +/- 5%, or 38 +/- 6% of the controls when RPE cells were fed 40 mM D-glucose, 40 mM alpha MG, or 5 mM nonradioactive MI, respectively. Growth media containing either pyruvate or lactate, the glucose metabolites, did not suppress the ability of RPE cells to accumulate MI. An 18 +/- 8% reduction in [3H]thymidine incorporation into DNA occurred when cells were grown in 40 mM glucose for 12-14 days, compared to cells grown with 5 mM glucose. Chronic treatment (12-14 days) of the cells with phorbol ester, an activator of protein kinase C, caused up to twofold increase in MI uptake, [3H]phlorizin binding, cell number, and DNA synthesis. However, when the rates of MI uptake into cells grown in basic growth media or TPA-treated media were normalized to cell number, no significant difference in MI uptake was found between the treated and untreated cells. Addition of staurosporin, a protein kinase C inhibitor, together with TPA, in the growth media reversed the phorbol-induced increase of MI uptake.(ABSTRACT TRUNCATED AT 400 WORDS)

The presentation of lipid and glycolipid Ags to T cells is mediated through CD1 molecules. In the mouse and rat only a single isoform, CD1d, performs these functions, while humans and all other mammals studied have members of both group I (CD1a, -b, and -c) and group II (CD1d) isoforms. Murine CD1d contains a cytoplasmic tyrosine-based sorting motif that is similar to motifs recognized by adaptor protein complexes that sort transmembrane proteins. Here we show that the adaptor protein complex, AP-3, directly interacts with murine CD1d and controls its targeting to lysosomes. AP-3 deficiency results in a redistribution of CD1d from lysosomes to the cell surface of thymocytes, B cell-depleted splenocytes, and dendritic cells. The altered trafficking of CD1d in AP-3-deficient mice results in a significant reduction of NK1.1+TCR-β+ and CD1d tetramer-positive cells, consistent with a defect in CD1d self-Ag presentation and thymocyte-positive selection. The AP-3 complex has recently been shown to associate with the human CD1b isoform, which has an intracellular distribution pattern similar to that of murine CD1d. We propose that lysosomal sampling may be so critical for efficient host defense that mice have evolved mechanisms to target their single CD1 isoform to lysosomes for sampling lipid Ags. Here we show the dominant mechanism for this trafficking is mediated by AP-3.

Mucosal tissues, such as the lung, are continually exposed to both foreign and environmental Ags. To counter the potential inflammatory tissue injury of chronic Th1-mediated responses against these Ags, mucosal sites may skew toward Th2 immune responses. However, the mechanism by which this occurs is unknown. Dendritic cells (DC), as orchestrators of the immune response, skew Th1/Th2 differentiation by cytokine secretion and expression of specific cell surface markers. We compared DC from mucosal and systemic locations. In this study, we show that the lung lacks a CD8α+ DC subpopulation and contains DC that appear less mature than splenic DC. Furthermore, we demonstrate that pulmonary DC produce significant levels of IL-6 and fail to produce the Th1-polarizing cytokine IL-12. Importantly, we demonstrate that IL-6 negatively regulates IL-12 production, as pulmonary DC from IL-6−/− mice produce significant levels of IL-12 and induce Th1 polarization of naive CD4+ T cells. Furthermore, we demonstrate that IL-6 is sufficient to explain the differential polarizing abilities of pulmonary and splenic DC, as splenic DC cocultures supplemented with IL-6 polarize naive T cells toward Th2, and pulmonary DC cultures in which IL-6 was removed with neutralizing Ab resulted in more Th1 polarization, pointing to IL-6 as the mechanism of Th2 polarization in the lung. We propose that the Th2 response seen in the lung is due to DC-mediated inhibition of Th1 responses via IL-6 production, rather than enhanced Th2 responses, and that this regulation decreases the likelihood of chronic inflammatory pathology in the lung.

Integrins are important for T cell interactions with endothelial cells. Because the integrin alpha(E)beta(7) is expressed on some circulating gut-homing T cells and as T cell numbers are reduced in the intestinal lamina propria of alpha(E)-deficient mice, we evaluated whether alpha(E)beta(7) mediates binding to intestinal endothelial cells. We found that anti-alpha(E)beta(7) mAbs partially blocked the binding of cultured intraepithelial T cells to human intestinal microvascular endothelial cells (HIMEC). Furthermore, alpha(E)beta(7)-transfected K562 cells bound more efficiently than vector-transfected K562 cells to HIMEC. Finally, HIMEC bound directly to an alpha(E)beta(7)-Fc fusion protein. These interactions were partially blocked by anti-alpha(E)beta(7) mAbs, and endothelial cell binding to the alpha(E)beta(7)-Fc was dependent upon the metal ion-dependent adhesion site within the alpha(E) A domain. Of note, the HIMEC lacked expression of E-cadherin, the only known alpha(E)beta(7) counterreceptor as assessed by functional studies, flow cytometry, and RT-PCR. Thus, HIMEC/alpha(E)beta(7) binding was independent of E-cadherin. In addition, this interaction appeared to be tissue selective, as HIMEC bound to the alpha(E)beta(7)-Fc, whereas microvascular endothelial cells from the skin did not. Finally, there was evidence for an alpha(E)beta(7) ligand on intestinal endothelial cells in vivo, as alpha(E)beta(7) expression enhanced lymphocyte binding around vessels in the lamina propria in tissue sections. Thus, we have defined a novel interaction for alpha(E)beta(7) at a nonepithelial location. These studies suggest a role for alpha(E)beta(7) in interactions with the intestinal endothelium that may have implications for intestinal T cell homing or functional responses.

We examined the roles of B7-1 (CD80) and B7-2 (CD86) in a model of allergic pulmonary inflammation and airway hyperresponsiveness (AHR) by using mice with germline deletions of the B7-1 and/or B7-2 molecules. Multiple parameters of the allergic response were affected to varying degrees by the absence of B7-1 and/or B7-2. Mice lacking both B7-1 and B7-2 had no elevation of serum immunoglobulin E, lack of airway eosinophilia, and no AHR. These same disease parameters were also reduced in mice lacking either B7-1 or B7-2. Lack of B7-1 and/or B7-2 resulted in an increase in T-helper 1 cytokine production. Our observations suggest that whereas B7-2 is quantitatively more significant in the induction of this response, B7-1 and B7-2 may have complementary roles in mediating the development of allergic pulmonary inflammation.

Background. The impaired renal function and vasodilatation that accompany age need to be re-addressed based upon the new knowledge concerning vascular nitric oxide (NO)-dependent systems. The present study examined the effects of age on the NO-related renal response. Methods. The study was performed in euvolaemic, conscious Wistar rats, aged 5 and 18 months. Renal function and haemodynamic measurements with fluorescent microspheres were employed to assess differences between groups. Results. A first set of experiments showed that ageing rats had a reduced natriuretic and diuretic response to acetylcholine, whereas the response to sodium nitroprusside was preserved. In the same regard, a reduction of the renal functional effects of L-arginine (L-Arg) and L-glycine (L-Gly) was found in the older rats. In the ageing rats, these responses were accompanied by an enhanced effect of the L-Arg competitive analogue, NwNLA, which provoked a marked reduction of renal function. This effect of NwNLA was blocked by the simultaneous administration of a small dose of L-Arg in the ageing but not in the young rats. Systemic haemodynamic studies revealed that in ageing rats, NwNLA reduced renal blood flow and increased renal vascular resistances in a significantly higher proportion than in younger animals. However, flow to other organs, namely, brain, spleen or liver, was affected in a similar manner in both young and old rats. Ultrastructural alterations were found in endothelial cells, which might constitute the anatomical basis for the observed functional derangements. Conclusions. The present experiments reveal that ageing is accompanied by significant differences in NO-related responses in the kidney which do not appear to affect blood flow to other organs. The response to L-Arg and L-Arg competitive analogues supports the existence of a marked dependency on NO-related mechanisms in the ageing rats, but not of a decreased baseline activity of the NO-dependent pathways.

To determine the mechanisms by which inhaled antigens produce pulmonary inflammation and bronchial hyperreactivity, we have developed a murine model of asthma. BALB/c mice are sensitized and challenged with ovalbumin (OVA). Compared with mice treated with phosphate-buffered saline (PBS), OVA-treated mice developed increased lung resistance, decreased dynamic compliance, and greater methacholine reactivity. Bronchoalveolar lavage fluid revealed significant increases in the proportion of neutrophils and eosinophils. Tissue sections of OVA-treated mice demonstrated goblet cell metaplasia and focal perivascular and peribronchial infiltrates composed of lymphocytes, neutrophils, and eosinophils. Analysis of thoracic lymphocytes via flow cytometry revealed an expansion of both CD4+ and B cell populations, with increased expression of interleukin-2 receptor on CD4+ T cells, indicated increased activation. There was also increased expression of CD44 on CD4+ and CD8+ lymphocytes, suggesting an expansion of the local memory cell population. These findings support the hypothesis that activation of T lymphocytes mediates allergic pulmonary inflammation and bronchial reactivity in asthma.

BACKGROUND Based on recent evidence showing that endothelin-1 stimulates several activation mechanisms on neutrophils, the aim of the present study was to analyze the effects of endothelin-1 on neutrophil adhesion to endothelial cells and neutrophil accumulation in the heart. METHODS AND RESULTS The experiments included (1) adhesion of 51Cr-labeled human neutrophils to bovine endothelial cells in culture both in the presence and absence of monoclonal antibodies against the alpha- and beta-subunits of integrins; (2) surface expression of the alpha- and beta-integrin antigens; (3) accumulation of 51Cr-labeled neutrophils on the isolated perfused rabbit heart; (4) in vivo accumulation of autologous neutrophils in the heart, as assessed by myeloperoxidase activity. Endothelin-1 stimulated neutrophil adhesion to endothelial cells (increase of 1 x 10(5) +/- 1 x 10(4) neutrophils per well). The endothelin-1-induced adhesion was blocked (83 +/- 6%) by the anti-CD18 antibody TS1/18 and by several anti-alpha-subunit antibodies. The expression of CD18 and CD11b on the neutrophil surface was also increased by endothelin-1. Endothelin-1 enhanced neutrophil accumulation in the isolated rabbit heart by 4.2 times throughout a TS1/18-inhibitable mechanism. Myeloperoxidase activity increased by 4.2 times in hearts infused in vivo with endothelin-1. CONCLUSIONS Endothelin-1 stimulates neutrophil adhesion to endothelial cells by an effect on the expression of adhesive molecules on the neutrophil surface. Endothelin-1 stimulates neutrophil accumulation in vivo and in vitro in the heart. Antibodies against the integrin complex block the endothelin-1-dependent neutrophil adhesion. These findings have potential importance in the pathophysiology of endothelin-1-increased states.

CD1d molecules present lipid antigens that activate CD1d-restricted natural killer T (NKT) cells. NKT cell activation leads to potent downstream activation of other immune cells through cell-cell interactions and rapid production of large quantities of cytokines. Endogenous lipids bound to CD1d are sufficient for activation of NKT cells in the setting of TLR induced cytokines. The most potent NKT cell antigens identified are glycosphingolipids (GSL). The repertoire of GSL bound to CD1d expressed in myeloid antigen presenting cells at steady state and in the setting of activation has not been delineated. We have identified the range of GSL bound to soluble CD1d molecules that sample the ER/secretory routes and cell surface cleaved CD1d that also samples the endocytic system. CD1d bound specific GSL preferentially as the CD1d GSL profile did not solely reflect cellular GSL abundance. GM1a and GD1a are prominent CD1d ligands for molecules following both the ER/secretory and lysosomal trafficking routes, while GM2 was eluted from soluble CD1d but not lysosomal trafficking CD1d. Further, after LPS activation the quantities of soluble CD1d bound GM3 and GM1a markedly increased. A unique a-galactose-terminating GSL was also found to be preferentially bound to CD1d at steady state and increased with APC activation. Our studies identify the range of GSL presented by CD1d and how presentation varies based on CD1d intracellular trafficking and microbial activation. In press PNAS