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Under steady-state conditions,aged neutrophils are removed from the circulation in bone marrow,liver,and spleen thereby maintaining myeloid cell homeostasis. The fate of these aged immune cells under inflammatory conditions,however,remains largely obscure. Here,we demonstrate that in the acute inflammatory response during endotoxemia aged neutrophils cease returning to the bone marrow and instead rapidly migrate to the site of inflammation. Having arrived in inflamed tissue,aged neutrophils were found to exhibit a higher phagocytic activity as compared to the subsequently recruited non-aged neutrophils. This distinct behavior of aged neutrophils under inflammatory conditions is dependent on specific age-related changes in their molecular repertoire that enable these 'experienced' immune cells to instantly translate inflammatory signals into immune responses. In particular,aged neutrophils engage toll-like receptor-4- and p38 mitogen-activated protein kinases-dependent pathways to induce conformational changes in β2 integrins which allow these phagocytes to effectively accomplish their mission in the front line of the inflammatory response. Hence,ageing in the circulation might represent a critical process for neutrophils that enables these immune cells to properly unfold their functional properties for host defense. View Publication

Regulation of gene expression in immune cells is known to be under genetic control,and likely contributes to susceptibility to autoimmune diseases such as multiple sclerosis (MS). How this occurs in concert across multiple immune cell types is poorly understood. Using a mouse model that harnesses the genetic diversity of wild-derived mice,more accurately reflecting genetically diverse human populations,we provide an extensive characterization of the genetic regulation of gene expression in five different naive immune cell types relevant to MS. The immune cell transcriptome is shown to be under profound genetic control,exhibiting diverse patterns: global,cell-specific and sex-specific. Bioinformatic analysis of the genetically controlled transcript networks reveals reduced cell type specificity and inflammatory activity in wild-derived PWD/PhJ mice,compared with the conventional laboratory strain C57BL/6J. Additionally,candidate MS-GWAS (genome-wide association study candidate genes for MS susceptibility) genes were significantly enriched among transcripts overrepresented in C57BL/6J cells compared with PWD. These expression level differences correlate with robust differences in susceptibility to experimental autoimmune encephalomyelitis,the principal model of MS,and skewing of the encephalitogenic T-cell responses. Taken together,our results provide functional insights into the genetic regulation of the immune transcriptome,and shed light on how this in turn contributes to susceptibility to autoimmune disease.Genes and Immunity advance online publication,22 September 2016; doi:10.1038/gene.2016.37. View Publication

IL-25,a new member of the IL-17 cytokine family,is involved in type 2 immunity initiation and has been associated with the pathogenesis of rheumatoid arthritis (RA). However,its exact role remains unclear. Here,we aimed to analyse IL-25 expression in the serum and synovial fluid of RA patients and evaluated the correlations between serum IL-25 levels,clinical and laboratory values and inflammation cytokines. Additionally,we investigated whether IL-25 can suppress Th1/Th17 responses involved in RA pathogenesis. We further determined whether IL-25 can alleviate collagen-induced arthritis (CIA) development in mice and the underlying mechanisms using in vitro and in vivo experiments. Our results showed that IL-25 was upregulated in the serum and synovial fluid of RA patients. Increased serum IL-25 levels were associated with disease severity and inflammatory response in RA patients. Furthermore,IL-25 inhibited CD4(+) T-cell activation and differentiation into Th17 cells,without affecting Th1 cells in human RA and CIA models. Administration of IL-25 could attenuate CIA development by Th17 suppression in an IL-13-dependent manner. Our findings indicate that IL-25 plays a potent immunosuppressive role in the pathogenesis of RA and CIA by downregulating Th17 cell response,and thus,may be a potential therapeutic agent for RA. View Publication

Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe,we analysed the memory humoral response against IsdB,a protein involved in iron acquisition,in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains,IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions,the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39,with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39,while part of the adaptive immune system,may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition. View Publication

The regulatory properties of B cells have been studied in autoimmune diseases; however,their role in allergic diseases is poorly understood. We demonstrate that Semaphorin 4C (Sema4C),an axonal guidance molecule,plays a crucial role in B cell regulatory function. Mice deficient in Sema4C exhibited increased airway inflammation after allergen exposure,with massive eosinophilic lung infiltrates and increased Th2 cytokines. This phenotype was reproduced by mixed bone marrow chimeric mice with Sema4C deficient only in B cells,indicating that B lymphocytes were the key cells affected by the absence of Sema4C expression in allergic inflammation. We determined that Sema4C-deficient CD19(+)CD138(+) cells exhibited decreased IL-10 and increased IL-4 expression in vivo and in vitro. Adoptive transfer of Sema4c(-/-) CD19(+)CD138(+) cells induced marked pulmonary inflammation,eosinophilia,and increased bronchoalveolar lavage fluid IL-4 and IL-5,whereas adoptive transfer of wild-type CD19(+)CD138(+)IL-10(+) cells dramatically decreased allergic airway inflammation in wild-type and Sema4c(-/-) mice. This study identifies a novel pathway by which Th2-mediated immune responses are regulated. It highlights the importance of plasma cells as regulatory cells in allergic inflammation and suggests that CD138(+) B cells contribute to cytokine balance and are important for maintenance of immune homeostasis in allergic airways disease. Furthermore,we demonstrate that Sema4C is critical for optimal regulatory cytokine production in CD138(+) B cells. View Publication

Neutrophils are recruited from the blood to sites of inflammation,where they contribute to immune defense but may also cause tissue damage. During inflammation,neutrophils roll along the microvascular endothelium before arresting and transmigrating. Arrest requires conformational activation of the integrin lymphocyte function-associated antigen 1 (LFA-1),which can be induced by selectin engagement. Here,we demonstrate that a subset of P-selectin glycoprotein ligand-1 (PSGL-1) molecules is constitutively associated with L-selectin. Although this association does not require the known lectin-like interaction between L-selectin and PSGL-1,the signaling output is dependent on this interaction and the cytoplasmic tail of L-selectin. The PSGL-1-L-selectin complex signals through Src family kinases,ITAM domain-containing adaptor proteins,and other kinases to ultimately result in LFA-1 activation. The PSGL-1-L-selectin complex-induced signaling effects on neutrophil slow rolling and recruitment in vivo demonstrate the functional importance of this pathway. We conclude that this is a signaling complex specialized for sensing adhesion under flow. View Publication

Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1,a cell surface glycoprotein expressed on MM cells. In preclinical models,elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein,we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1-CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary,human MM cells. Taken together,these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone. View Publication

Circulating levels of leptin are elevated in type-2 diabetes mellitus (T2DM) and leptin plays a role in immune responses. Elevated circulating IL-18 levels are associated with clinical complications of T2DM. IL-18 regulates cytokine secretion and the function of a number of immune cells including T-cells,neutrophils and macrophages and as such has a key role in immunity and inflammation. Pro-inflammatory monocytes exhibiting elevated cytokine secretion are closely associated with inflammation in T2DM,however,little is known about the role of leptin in modifying monocyte IL-18 secretion. We therefore aimed to investigate the effect of leptin on IL-18 secretion by monocytes. We report herein that leptin increases IL-18 secretion in THP-1 and primary human monocytes but has no effect on IL-18mRNA. Leptin and LPS signalling in monocytes occurs by overlapping but distinct pathways. Thus,in contrast to a strong stimulation by LPS,leptin has no effect on IL-1βmRNA levels or IL-1β secretion. In addition,LPS stimulates the secretion of IL-6 but leptin did not whereas both treatments up regulate IL-8 secretion from the same cells. Although leptin (and LPS) has a synergistic effect with exogenous ATP on IL-18 secretion in both THP-1 and primary monocytes,experiments involving ATP assays and pharmacological inhibition of ATP signalling failed to provide any evidence that endogenous ATP secreted by leptin-stimulated monocytes was responsible for enhancement of monocyte IL-18 secretion by leptin. Analysis of the action of caspase-1 revealed that leptin up regulates caspase-1 activity and the effect of leptin on IL-18 release is prevented by caspase-1 inhibitor (Ac-YVAD-cmk). These data suggest that leptin activates IL-18 processing rather than IL-18 transcription. In conclusion,leptin enhances IL-18 secretion via modulation of the caspase-1 inflammasome function and acts synergistically with ATP in this regard. This process may contribute to aberrant immune responses in T2DM and other conditions of hyperleptinemia. View Publication

Pathogen induced migration of neutrophils across mucosal epithelial barriers requires epithelial production of the chemotactic lipid mediator,hepoxilin A3 (HXA3). HXA3 is an eicosanoid derived from arachidonic acid. Although eosinophils are also capable of penetrating mucosal surfaces,eosinophilic infiltration occurs mainly during allergic processes whereas neutrophils dominate mucosal infection. Both neutrophils and eosinophils can respond to chemotactic gradients of certain eicosanoids,however,it is not known whether eosinophils respond to pathogen induced lipid mediators such as HXA3. In this study,neutrophils and eosinophils were isolated from human blood and placed on the basolateral side of polarized epithelial monolayers grown on permeable Transwell filters and challenged by various chemotactic gradients of distinct lipid mediators. We observed that both cell populations migrated across epithelial monolayers in response to a leukotriene B4 (LTB4) gradient,whereas only eosinophils migrated toward a prostaglandin D2 (PGD2) gradient. Interestingly,while pathogen induced neutrophil trans-epithelial migration was substantial,pathogen induced eosinophil trans-epithelial migration was not observed. Further,gradients of chemotactic lipids derived from pathogen infected epithelial cells known to be enriched for HXA3 as well as purified HXA3 drove significant numbers of neutrophils across epithelial barriers,whereas eosinophils failed to respond to these gradients. These data suggest that although the eicosanoid HXA3 serves as an important neutrophil chemo-attractant at mucosal surfaces during pathogenic infection,HXA3 does not appear to exhibit chemotactic activity toward eosinophils. ?? 2013 Elsevier Ltd. All rights reserved. View Publication

Structural bone allografts are widely used in the clinic to treat critical sized bone defects,despite lacking the osteoinductive characteristics of live autografts. To address this,we generated revitalized structural allografts wrapped with mesenchymal stem/progenitor cell (MSC) sheets,which were produced by expanding primary syngenic bone marrow derived cells on temperature-responsive plates,as a tissue-engineered periosteum. In vitro assays demonstrated maintenance of the MSC phenotype in the sheets,suggesting that short-term culturing of MSC sheets is not detrimental. To test their efficacy in vivo,allografts wrapped with MSC sheets were transplanted into 4-mm murine femoral defects and compared to allografts with direct seeding of MSCs and allografts without cells. Evaluations consisted of X-ray plain radiography,3D microCT,histology,and biomechanical testing at 4- and 6-weeks post-surgery. Our findings demonstrate that MSC sheets induce prolonged cartilage formation at the graft-host junction and enhanced bone callus formation,as well as graft-host osteointegration. Moreover,a large periosteal callus was observed spanning the allografts with MSC sheets,which partially mimics live autograft healing. Finally,biomechanical testing showed a significant increase in the structural and functional properties of MSC sheet grafted femurs. Taken together,MSC sheets exhibit enhanced osteogenicity during critical sized bone defect repair,demonstrating the feasibility of this tissue engineering solution for massive allograft healing. View Publication

Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors may provide the means for vascularization of tissue-engineered constructs and can serve as models to study vascular development and disease. Here,we report a method to efficiently produce endothelial cells from hPSCs via GSK3 inhibition and culture in defined media to direct hPSC differentiation to CD34(+)CD31(+) endothelial progenitors. Exogenous vascular endothelial growth factor (VEGF) treatment was dispensable,and endothelial progenitor differentiation was β-catenin dependent. Furthermore,by clonal analysis,we showed that CD34(+)CD31(+)CD117(+)TIE-2(+) endothelial progenitors were multipotent,capable of differentiating into calponin-expressing smooth muscle cells and CD31(+)CD144(+)vWF(+)I-CAM1(+) endothelial cells. These endothelial cells were capable of 20 population doublings,formed tube-like structures,imported acetylated low-density lipoprotein,and maintained a dynamic barrier function. This study provides a rapid and efficient method for production of hPSC-derived endothelial progenitors and endothelial cells and identifies WNT/β-catenin signaling as a primary regulator for generating vascular cells from hPSCs. View Publication

Manipulation of the CD28/CTLA-4 pathway is at the heart of a number of immunomodulatory approaches used in both autoimmunity and cancer. Although it is clear that CTLA-4 is a critical regulator of T cell responses,the immunological contexts in which CTLA-4 controls immune responses are not well defined. In this study,we show that whereas CD80/CD86-dependent activation of resting human T cells caused extensive T cell proliferation and robust CTLA-4 expression,in this context CTLA-4 blocking Abs had no impact on the response. In contrast,in settings where CTLA-4(+) cells were present as regulators� View Publication