技术资料

Biologic TNFα inhibitors are a mainstay treatment option for patients with rheumatoid arthritis (RA) refractory to other treatment options. However,many patients either do not respond or relapse after initially responding to these agents. This study was carried out to identify biomarkers that can distinguish responder from non-responder patients before the initiation of treatment. The level of cytokines in plasma and those produced by ex vivo T cells,B cells and monocytes in 97 RA patients treated with biologic TNFα inhibitors was measured before treatment and after 1 and 3 months of treatment by multiplex analyses. The frequency of T cell subsets and intracellular cytokines were determined by flow cytometry. The results reveal that pre-treatment,T cells from patients who went on to respond to treatment with biologic anti-TNFα agents produced significantly more GM-CSF than non-responder patients. Furthermore,immune cells from responder patients produced higher levels of IL-1β,TNFα and IL-6. Cytokine profiling in the blood of patients confirmed the association between high levels of GM-CSF and responsiveness to biologic anti-TNFα agents. Thus,high blood levels of GM-CSF pre-treatment had a positive predictive value of 87.5% (61.6 to 98.5% at 95% CI) in treated RA patients. The study also shows that cells from most anti-TNFα responder patients in the current cohort produced higher levels of GM-CSF and TNFα pre-treatment than non-responder patients. Findings from the current study and our previous observations that non-responsiveness to anti-TNFα is associated with high IL-17 levels suggest that the disease in responder and non-responder RA patients is likely to be driven/sustained by different inflammatory pathways. The use of biomarker signatures of distinct pro-inflammatory pathways could lead to evidence-based prescription of the most appropriate biological therapies for different RA patients. View Publication

Activin A belongs to the superfamily of transforming growth factor beta (TGF$\beta$) and is a critical regulatory cytokine in breast cancer and inflammation. However,the role of activin A in migration of breast cancer cells and immune cells was not well characterized. Here,a microfluidic device was used to examine the effect of activin A on the migration of human breast cancer cell line MDA-MB-231 cells and human blood neutrophils as well as their migratory interaction. We found that activin A promoted the basal migration but impaired epidermal growth factor (EGF)-induced migration of breast cancer cells. By contrast,activin A reduced neutrophil chemotaxis and transendothelial migration to N-Formyl-Met-Leu-Phe (fMLP). Finally,activin A promoted neutrophil chemotaxis to the supernatant from breast cancer cell culture. Collectively,our study revealed the different roles of activin A in regulating the migration of breast cancer cells and neutrophils and their migratory interaction. These findings suggested the potential of activin A as a therapeutic target for inflammation and breast cancers. View Publication

Tumor-associated macrophages (TAMs) originate as circulating monocytes,and are recruited to gliomas,where they facilitate tumor growth and migration. Understanding the interaction between TAM and cancer cells may identify therapeutic targets for glioblastoma multiforme (GBM). Vascular cell adhesion molecule-1 (VCAM-1) is a cytokine-induced adhesion molecule expressed on the surface of cancer cells,which is involved in interactions with immune cells. Analysis of the glioma patient database and tissue immunohistochemistry showed that VCAM-1 expression correlated with the clinico-pathological grade of gliomas. Here,we found that VCAM-1 expression correlated positively with monocyte adhesion to GBM,and knockdown of VCAM-1 abolished the enhancement of monocyte adhesion. Importantly,upregulation of VCAM-1 is dependent on epidermal-growth-factor-receptor (EGFR) expression,and inhibition of EGFR effectively reduced VCAM-1 expression and monocyte adhesion activity. Moreover,GBM possessing higher EGFR levels (U251 cells) had higher VCAM-1 levels compared to GBMs with lower levels of EGFR (GL261 cells). Using two- and three-dimensional cultures,we found that monocyte adhesion to GBM occurs via integrin α4β1,which promotes tumor growth and invasion activity. Increased proliferation and tumor necrosis factor-α and IFN-γ levels were also observed in the adherent monocytes. Using a genetic modification approach,we demonstrated that VCAM-1 expression and monocyte adhesion were regulated by the miR-181 family,and lower levels of miR-181b correlated with high-grade glioma patients. Our results also demonstrated that miR-181b/protein phosphatase 2A-modulated SP-1 de-phosphorylation,which mediated the EGFR-dependent VCAM-1 expression and monocyte adhesion to GBM. We also found that the EGFR-dependent VCAM-1 expression is mediated by the p38/STAT3 signaling pathway. Our study suggested that VCAM-1 is a critical modulator of EGFR-dependent interaction of monocytes with GBM,which raises the possibility of developing effective and improved therapies for GBM.Oncogene advance online publication,1 May 2017; doi:10.1038/onc.2017.129. View Publication

The high risk of neonatal death from sepsis is thought to result from impaired responses by innate immune cells; however,the clinical observation of hyperinflammatory courses of neonatal sepsis contradicts this concept. Using transcriptomic,epigenetic and immunological approaches,we demonstrated that high amounts of the perinatal alarmins S100A8 and S100A9 specifically altered MyD88-dependent proinflammatory gene programs. S100 programming prevented hyperinflammatory responses without impairing pathogen defense. TRIF-adaptor-dependent regulatory genes remained unaffected by perinatal S100 programming and responded strongly to lipopolysaccharide,but were barely expressed. Steady-state expression of TRIF-dependent genes increased only gradually during the first year of life in human neonates,shifting immune regulation toward the adult phenotype. Disruption of this critical sequence of transient alarmin programming and subsequent reprogramming of regulatory pathways increased the risk of hyperinflammation and sepsis. Collectively these data suggest that neonates are characterized by a selective,transient microbial unresponsiveness that prevents harmful hyperinflammation in the delicate neonate while allowing for sufficient immunological protection. View Publication

Recent work investigating exercise induced changes in immunocompetence suggests that some of the ambiguity in the literature is resultant from different cell isolation protocols and mitogen selection. To understand this effect,we compared post-exercise measures of T cell activation and proliferation using two different stimulation methods (costimulation through CD28 or stimulation with phytohaemagglutinin [PHA]). Further,we investigated whether exercise induced changes are maintained when T cell isolation from whole blood is delayed overnight in either a room temperature or chilled (4°C) environment. As expected,an increased proliferation response was observed post-exercise in T cells isolated from whole blood of previously trained individuals immediately after blood collection. Also,cells stimulated with PHA after resting overnight in whole blood were not adversely impacted by the storage conditions. In contrast,allowing cells to rest overnight in whole blood prior to stimulation through CD28,lessened the proliferation observed by cells following exercise rendering both the room temperature and chilled samples closer to the results seen in the control condition. Changes in early markers of activation (CD25),followed a similar pattern,with activation in PHA stimulated cells remaining fairly robust after overnight storage; whereas cell activation following stimulation through CD3+CD28 was disproportionately decreased by the influence of overnight storage. These findings indicate that decisions regarding cell stimulation methods need to be paired with the timeline for T cell isolation from whole blood. These considerations will be especially important for field based studies of immunocompetence where there is a delay in getting whole blood samples to a lab for processing as well as clinical applications where a failure to isolate T cells in a timely manner may result in loss of the response of interest. View Publication

OBJECTIVE Vitamin D deficiency has been linked to increased risk of multiple sclerosis (MS) and poor outcome. However,the specific role that vitamin D plays in MS still remains unknown. In order to identify potential mechanisms underlying vitamin D effects in MS,we profiled epigenetic changes in vitamin D receptor (VDR) gene to identify genomic regulatory elements relevant to MS pathogenesis. METHODS Human T cells derived from whole blood by negative selection were isolated in a set of 23 relapsing-remitting MS (RRMS) patients and 12 controls matched by age and gender. DNA methylation levels were assessed by bisulfite cloning sequencing in two regulatory elements of VDR. mRNA levels were measured by RT-qPCR to assess changes in VDR expression between patients and controls. RESULTS An alternative VDR promoter placed at exon 1c showed increased DNA methylation levels in RRMS patients (median 30.08%,interquartile range 19.2%) compared to controls (18.75%,9.5%),p-valuetextless0.05. Moreover,a 6.5-fold increase in VDR mRNA levels was found in RRMS patients compared to controls (p-valuetextless0.001). CONCLUSIONS An alternative promoter of the VDR gene shows altered DNA methylation levels in patients with multiple sclerosis,and it is associated with VDR mRNA upregulation. This locus may represent a candidate regulatory element in the genome relevant to MS pathogenesis. View Publication

Toxic shock syndrome (TSS) is caused by staphylococcal and streptococcal superantigens (SAgs) that provoke a swift hyperinflammatory response typified by a cytokine storm. The precipitous decline in the host's clinical status and the lack of targeted therapies for TSS emphasize the need to identify key players of the storm's initial wave. Using a humanized mouse model of TSS and human cells,we herein demonstrate that SAgs elicit in vitro and in vivo IL-17A responses within hours. SAg-triggered human IL-17A production was characterized by remarkably high mRNA stability for this cytokine. A distinct subpopulation of CD4+ effector memory T (TEM) cells that secrete IL-17A,but not IFN-$\gamma$,was responsible for early IL-17A production. We found mouse TEM-17" cells to be enriched within the intestinal epithelium and among lamina propria lymphocytes. Furthermore interfering with IL-17A receptor signaling in human PBMCs attenuated the expression of numerous inflammatory mediators implicated in the TSS-associated cytokine storm. IL-17A receptor blockade also abrogated the secondary effect of SAg-stimulated PBMCs on human dermal fibroblasts as judged by C/EBP $\delta$ expression. Finally the early IL-17A response to SAgs was pathogenic because in vivo neutralization of IL-17A in humanized mice ameliorated hepatic and intestinal damage and reduced mortality. Together our findings identify CD4+ TEM cells as a key effector of TSS and reveal a novel role for IL-17A in TSS immunopathogenesis. Our work thus elucidates a pathogenic as opposed to protective role for IL-17A during Gram-positive bacterial infections. Accordingly the IL-17-IL-17R axis may provide an attractive target for the management of SAg-mediated illnesses." View Publication

Targeting of myeloid-dendritic cell receptor DC-SIGN by numerous chronic infectious agents,including Porphyromonas gingivalis,is shown to drive-differentiation of monocytes into dysfunctional mDCs. These mDCs exhibit alterations of their fine-tuned homeostatic function and contribute to dysregulated immune-responses. Here,we utilize P. gingivalis mutant strains to show that pathogen-differentiated mDCs from primary human-monocytes display anti-apoptotic profile,exhibited by elevated phosphorylated-Foxo1,phosphorylated-Akt1,and decreased Bim-expression. This results in an overall inhibition of DC-apoptosis. Direct stimulation of complex component CD40 on DCs leads to activation of Akt1,suggesting CD40 involvement in anti-apoptotic effects observed. Further,these DCs drove dampened CD8(+) T-cell and Th1/Th17 effector-responses while inducing CD25(+)Foxp3(+)CD127(-) Tregs. In vitro Treg induction was mediated by DC expression of indoleamine 2,3-dioxygenase,and was confirmed in IDO-KO mouse model. Pathogen-infected &CMFDA-labeled MoDCs long-lasting survival was confirmed in a huMoDC reconstituted humanized mice. In conclusion,our data implicate PDDCs as an important target for resolution of chronic infection. View Publication

BACKGROUND Human rhinoviruses (HRVs) are a major trigger of asthma exacerbations,with the bronchial epithelium being the major site of HRV infection and replication. Mast cells (MCs) play a key role in asthma where their numbers are increased in the bronchial epithelium with increasing disease severity. OBJECTIVE In view of the emerging role of MCs in innate immunity and increased localization to the asthmatic bronchial epithelium,we investigated whether HRV infection of MCs generated innate immune responses which were protective against infection. METHODS The LAD2 MC line or primary human cord blood-derived MCs (CBMCs) were infected with HRV or UV-irradiated HRV at increasing multiplicities of infection (MOI) without or with IFN-β or IFN-λ. After 24 h,innate immune responses were assessed by RT-qPCR and IFN protein release by ELISA. Viral replication was determined by RT-qPCR and virion release by TCID50 assay. RESULTS HRV infection of LAD2 MCs induced expression of IFN-β,IFN-λ and IFN-stimulated genes. However,LAD2 MCs were permissive for HRV replication and release of infectious HRV particles. Similar findings were observed with CBMCs. Neutralization of the type I IFN receptor had minimal effects on viral shedding,suggesting that endogenous type I IFN signalling offered limited protection against HRV. However,augmentation of these responses by exogenous IFN-β,but not IFN-λ,protected MCs against HRV infection. CONCLUSION AND CLINICAL RELEVANCE MCs are permissive for the replication and release of HRV,which is prevented by exogenous IFN-β treatment. Taken together,these findings suggest a novel mechanism whereby MCs may contribute to HRV-induced asthma exacerbations. View Publication

SAMHD1 restricts human immunodeficiency virus type 1 (HIV-1) replication in myeloid cells and CD4(+) T cells,while Vpx can mediate SAMHD1 degradation to promote HIV-1 replication. Although the restriction mechanisms of SAMHD1 have been well-described,SAMHD1 expression and Vpx-mediated SAMHD1 degradation during chronic HIV-1 infection were poorly understood. Flow cytometric analysis was used to directly visualize ex vivo,and after in vitro SIV-Vpx treatment,SAMHD1 expression in CD4(+) T cells and monocytes. Here we report activated CD4(+) T cells without SAMHD1 expression were severely reduced,and SAMHD1 in CD4(+) T cells became susceptible to SIV-Vpx mediated degradation during chronic HIV-1 infection,which was absent from uninfected donors. These alterations were irreversible,even after long-term fully suppressive antiretroviral treatment. Although SAMHD1 expression in CD4(+) T cells and monocytes was not found to correlate with plasma viral load,Vpx-mediated SAMHD1 degradation was associated with indicators of immune activation. In vitro assays further revealed that T-cell activation and an upregulated IFN-I pathway contributed to these altered SAMHD1 properties. These findings provide insight into how immune activation during HIV-1 infection leads to irreparable aberrations in restriction factors and in subsequent viral evasion from host antiviral defenses. View Publication

The host responds to virus infection by activating type I interferon (IFN) signaling leading to expression of IFN-stimulated genes (ISGs). Dysregulation of the IFN response results in inflammatory diseases and chronic infections. In this study,we demonstrate that IFN regulatory factor 2 (IRF2),an ISG and a negative regulator of IFN signaling,influences alphavirus neuroinvasion and pathogenesis. A Sindbis virus strain that in wild-type (WT) mice only causes disease when injected into the brain leads to lethal encephalitis in Irf2(-/-) mice after peripheral inoculation. Irf2(-/-) mice fail to control virus replication and recruit immune infiltrates into the brain. Reduced B cells and virus-specific IgG are observed in the Irf2(-/-) mouse brains despite the presence of peripheral neutralizing antibodies,suggesting a defect in B cell trafficking to the central nervous system (CNS). B cell-deficient μMT mice are significantly more susceptible to viral infection,yet WT B cells and serum are unable to rescue the Irf2(-/-) mice. Collectively,our data demonstrate that proper localization of B cells and local production of antibodies in the CNS are required for protection. The work advances our understanding of host mechanisms that affect viral neuroinvasion and their contribution to immunity against CNS infections. View Publication

TRAF1 is a signaling adaptor known for its role in tumor necrosis factor receptor-induced cell survival. Here we show that monocytes from healthy human subjects with a rheumatoid arthritis-associated single-nucleotide polymorphism (SNP) in the TRAF1 gene express less TRAF1 protein but greater amounts of inflammatory cytokines in response to lipopolysaccharide (LPS). The TRAF1 MATH domain binds directly to three components of the linear ubiquitination (LUBAC) complex,SHARPIN,HOIP and HOIL-1,to interfere with the recruitment and linear ubiquitination of NEMO. This results in decreased NF-κB activation and cytokine production,independently of tumor necrosis factor. Consistent with this,Traf1(-/-) mice show increased susceptibility to LPS-induced septic shock. These findings reveal an unexpected role for TRAF1 in negatively regulating Toll-like receptor signaling,providing a mechanistic explanation for the increased inflammation seen with a disease-associated TRAF1 SNP. View Publication