技术资料

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

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

The ability of a cancer cell to migrate through the dense extracellular matrix (ECM) within and surrounding the solid tumor is a critical determinant of metastasis. Macrophages enhance invasion and metastasis in the tumor microenvironment but the basis for their effects are not fully understood. Using a microfluidic 3D cell migration assay,we found that the presence of macrophages enhanced the speed and persistence of cancer cell migration through a 3D extracellular matrix in a matrix metalloproteinases (MMP)-dependent fashion. Mechanistic investigations revealed that macrophage-released TNFα and TGFβ1 mediated the observed behaviors by two distinct pathways. These factors synergistically enhanced migration persistence through a synergistic induction of NF-κB-dependent MMP1 expression in cancer cells. In contrast,macrophage-released TGFβ1 enhanced migration speed primarily by inducing MT1-MMP expression. Taken together,our results reveal new insights into how macrophages enhance cancer cell metastasis,and they identify TNFα and TGFβ1 dual blockade as an anti-metastatic strategy in solid tumors. View Publication

Autologous chondrocyte implantation is the current gold standard cell therapy for cartilage lesions. However,in some instances,the heavily compromised health of the patient can either impair or limit the recovery of the autologous chondrocytes and a satisfactory outcome of the implant. Allogeneic human articular chondrocytes (hAC) could be a good alternative,but the possible immunological incompatibility between recipient and hAC donor should be considered. Herein,we report that allogeneic hAC inhibited T lymphocyte response to antigen-dependent and -independent proliferative stimuli. This effect was maximal when T cells and hAC were in contact and it was not relieved by the addition of exogenous lymphocyte growth factor interleukin (IL)-2. More important,hAC impaired the differentiation of peripheral blood monocytes induced with granulocyte monocyte colony-stimulating factor and IL-4 (Mo) to professional antigen-presenting cells,such as dendritic cells (DC). Indeed,a marked inhibition of the onset of the CD1a expression and an ineffective downregulation of CD14 antigens was observed in Mo-hAC co-cultures. Furthermore,compared to immature or mature DC,Mo from Mo-hAC co-cultures did not trigger an efficacious allo-response. The prostaglandin (PG) E2 present in the Mo-hAC co-culture conditioned media is a putative candidate of the hAC-mediated inhibition of Mo maturation. Altogether,these findings indicate that allogeneic hAC inhibit,rather than trigger,immune response and strongly suggest that an efficient chondrocyte implantation could be possible also in an allogeneic setting. View Publication

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses,host defense mechanisms,and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system,DCs are very rare in blood,accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore,alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity,affordability,high purity,and high yield of cells is imperative to consider. In the current study,two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability,proliferation,and phenotype were assessed using viability dyes,MTT assay,and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method,the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded textgreater 70% CD11c+ MDDCs. Therefore,our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs. View Publication

While a third of the world carries the burden of tuberculosis,disease control has been hindered by a lack of tools,including a rapid,point-of-care diagnostic and a protective vaccine. In many infectious diseases,antibodies (Abs) are powerful biomarkers and important immune mediators. However,in Mycobacterium tuberculosis (Mtb) infection,a discriminatory or protective role for humoral immunity remains unclear. Using an unbiased antibody profiling approach,we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses,such that Ltb infection is associated with unique Ab Fc functional profiles,selective binding to FcγRIII,and distinct Ab glycosylation patterns. Moreover,compared to Abs from Atb,Abs from Ltb drove enhanced phagolysosomal maturation,inflammasome activation,and,most importantly,macrophage killing of intracellular Mtb. Combined,these data point to a potential role for Fc-mediated Ab effector functions,tuned via differential glycosylation,in Mtb control. View Publication

West Nile virus (WNV) infection is a mosquito-borne zoonosis with increasing prevalence in the United States. WNV infection begins in the skin,and the virus replicates initially in keratinocytes and dendritic cells (DCs). In the skin and cutaneous lymph nodes,infected DCs are likely to interact with invariant natural killer T cells (iNKTs). Bidirectional interactions between DCs and iNKTs amplify the innate immune response to viral infections,thus controlling viral load and regulating adaptive immunity. iNKTs are stimulated by CD1d-bound lipid antigens or activated indirectly by inflammatory cytokines. We exposed human monocyte-derived DCs to WNV Kunjin and determined their ability to activate isolated blood iNKTs. DCs became infected as judged by synthesis of viral mRNA and Envelope and NS-1 proteins,but did not undergo significant apoptosis. Infected DCs up-regulated the co-stimulatory molecules CD86 and CD40,but showed decreased expression of CD1d. WNV infection induced DC secretion of type I interferon (IFN),but no or minimal interleukin (IL)-12,IL-23,IL-18 or IL-10. Unexpectedly,we found that the WNV-infected DCs stimulated human iNKTs to up-regulate CD69 and produce low amounts of IL-10,but not proinflammatory cytokines such as IFN-γ or tumour necrosis factor (TNF)-α. Both CD1d and IFNAR blockade partially abrogated this iNKT response,suggesting involvement of a T cell receptor (TCR)-CD1d interaction and type I interferon receptor (IFNAR) signalling. Thus,WNV infection interferes with DC-iNKT interactions by preventing the production of proinflammatory cytokines. iNKTs may be a source of IL-10 observed in human flavivirus infections and initiate an anti-inflammatory innate response that limits adaptive immunity and immune pathology upon WNV infection. View Publication

Lupus nephritis (LN) is a potentially dangerous end organ pathology that affects upwards of 60% of lupus patients. Bruton's tyrosine kinase (BTK) is important for B cell development,Fc receptor signaling,and macrophage polarization. In this study,we investigated the effects of a novel,highly selective and potent BTK inhibitor,BI-BTK-1,in an inducible model of LN in which mice receive nephrotoxic serum (NTS) containing anti-glomerular antibodies. Mice were treated once daily with vehicle alone or BI-BTK-1,either prophylactically or therapeutically. When compared with control treated mice,NTS-challenged mice treated prophylactically with BI-BTK-1 exhibited significantly attenuated kidney disease,which was dose dependent. BI-BTK-1 treatment resulted in decreased infiltrating IBA-1+ cells,as well as C3 deposition within the kidney. RT-PCR on whole kidney RNA and serum profiling indicated that BTK inhibition significantly decreased levels of LN-relevant inflammatory cytokines and chemokines. Renal RNA expression profiling by RNA-seq revealed that BI-BTK-1 dramatically modulated pathways related to inflammation and glomerular injury. Importantly,when administered therapeutically,BI-BTK-1 reversed established proteinuria and improved renal histopathology. Our results highlight the important role for BTK in the pathogenesis of immune complex-mediated nephritis,and BTK inhibition as a promising therapeutic target for LN. View Publication

IL-18 is a member of the IL-1 family involved in innate immunity and inflammation. Deregulated levels of IL-18 are involved in the pathogenesis of multiple disorders including inflammatory and metabolic diseases,yet relatively little is known regarding its regulation. Liver X receptors or LXRs are key modulators of macrophage cholesterol homeostasis and immune responses. Here we show that LXR ligands negatively regulate LPS-induced mRNA and protein expression of IL-18 in bone marrow-derived macrophages. Consistent with this being an LXR-mediated process,inhibition is abolished in the presence of a specific LXR antagonist and in LXR-deficient macrophages. Additionally,IL-18 processing of its precursor inactive form to its bioactive state is inhibited by LXR through negative regulation of both pro-caspase 1 expression and activation. Finally,LXR ligands further modulate IL-18 levels by inducing the expression of IL-18BP,a potent endogenous inhibitor of IL-18. This regulation occurs via the transcription factor IRF8,thus identifying IL-18BP as a novel LXR and IRF8 target gene. In conclusion,LXR activation inhibits IL-18 production through regulation of its transcription and maturation into an active pro-inflammatory cytokine. This novel regulation of IL-18 by LXR could be applied to modulate the severity of IL-18 driven metabolic and inflammatory disorders. View Publication

FNDC4 is a secreted factor sharing high homology with the exercise-associated myokine irisin (FNDC5). Here we report that Fndc4 is robustly upregulated in several mouse models of inflammation as well as in human inflammatory conditions. Specifically,FNDC4 levels are increased locally at inflamed sites of the intestine of inflammatory bowel disease patients. Interestingly,administration of recombinant FNDC4 in the mouse model of induced colitis markedly reduces disease severity compared with mice injected with a control protein. Conversely,mice lacking Fndc4 develop more severe colitis. Analysis of binding of FNDC4 to different immune cell types reveals strong and specific binding to macrophages and monocytes. FNDC4 treatment of bone marrow-derived macrophages in vitro results in reduced phagocytosis,increased cell survival and reduced proinflammatory chemokine expression. Hence,treatment with FNDC4 results in a state of dampened macrophage activity,while enhancing their survival. Thus,we have characterized FNDC4 as a factor with direct therapeutic potential in inflammatory bowel disease and possibly other inflammatory diseases. View Publication

Abnormal glucose metabolism and enhanced oxidative stress accelerate cardiovascular disease,a chronic inflammatory condition causing high morbidity and mortality. Here,we report that in monocytes and macrophages of patients with atherosclerotic coronary artery disease (CAD),overutilization of glucose promotes excessive and prolonged production of the cytokines IL-6 and IL-1β,driving systemic and tissue inflammation. In patient-derived monocytes and macrophages,increased glucose uptake and glycolytic flux fuel the generation of mitochondrial reactive oxygen species,which in turn promote dimerization of the glycolytic enzyme pyruvate kinase M2 (PKM2) and enable its nuclear translocation. Nuclear PKM2 functions as a protein kinase that phosphorylates the transcription factor STAT3,thus boosting IL-6 and IL-1β production. Reducing glycolysis,scavenging superoxide and enforcing PKM2 tetramerization correct the proinflammatory phenotype of CAD macrophages. In essence,PKM2 serves a previously unidentified role as a molecular integrator of metabolic dysfunction,oxidative stress and tissue inflammation and represents a novel therapeutic target in cardiovascular disease. View Publication

BACKGROUND: The cellular sulfonation pathway modulates key steps of virus replication. This pathway comprises two main families of sulfonate-conjugating enzymes: Golgi sulfotransferases,which sulfonate proteins,glycoproteins,glycolipids and proteoglycans; and cytosolic sulfotransferases (SULTs),which sulfonate various small molecules including hormones,neurotransmitters,and xenobiotics. Sulfonation controls the functions of numerous cellular factors such as those involved in cell-cell interactions,cell signaling,and small molecule detoxification. We previously showed that the cellular sulfonation pathway regulates HIV-1 gene expression and reactivation from latency. Here we show that a specific cellular sulfotransferase can regulate HIV-1 replication in primary human monocyte-derived macrophages (MDMs) by yet another mechanism,namely reverse transcription. METHODS: MDMs were derived from monocytes isolated from donor peripheral blood mononuclear cells (PBMCs) obtained from the San Diego Blood Bank. After one week in vitro cell culture under macrophage-polarizing conditions,MDMs were transfected with sulfotranserase-specific or control siRNAs and infected with HIV-1 or SIV constructs expressing a luciferase reporter. Infection levels were subsequently monitored by luminescence. Western blotting was used to assay siRNA knockdown and viral protein levels,and qPCR was used to measure viral RNA and DNA products. RESULTS: We demonstrate that the cytosolic sulfotransferase SULT1A1 is highly expressed in primary human MDMs,and through siRNA knockdown experiments,we show that this enzyme promotes infection of MDMs by single cycle VSV-G pseudotyped human HIV-1 and simian immunodeficiency virus vectors and by replication-competent HIV-1. Quantitative PCR analysis revealed that SULT1A1 affects HIV-1 replication in MDMs by modulating the kinetics of minus-strand DNA elongation during reverse transcription. CONCLUSIONS: These studies have identified SULT1A1 as a cellular regulator of HIV-1 reverse transcription in primary human MDMs. The normal substrates of this enzyme are small phenolic-like molecules,raising the possibility that one or more of these substrates may be involved. Targeting SULT1A1 and/or its substrate(s) may offer a novel host-directed strategy to improve HIV-1 therapeutics. View Publication