Showing papers in "BMC Immunology in 2021"

Lipopolysaccharide- TLR-4 Axis regulates Osteoclastogenesis independent of RANKL/RANK signaling.

Abstract: Lipopolysaccharide (LPS) is an endotoxin and a vital component of gram-negative bacteria’s outer membrane. During gram-negative bacterial sepsis, LPS regulates osteoclast differentiation and activity, in addition to increasing inflammation. This study aimed to investigate how LPS regulates osteoclast differentiation of RAW 264.7 cells in vitro. Herein, we revealed that RAW cells failed to differentiate into mature osteoclasts in vitro in the presence of LPS. However, differentiation occurred in cells primed with receptor activator of nuclear factor-kappa-Β ligand (RANKL) for 24 h and then treated with LPS for 48 h (henceforth, denoted as LPS-treated cells). In cells treated with either RANKL or LPS, an increase in membrane levels of toll-like receptor 4 (TLR4) receptor was observed. Mechanistically, an inhibitor of TLR4 (TAK-242) reduced the number of osteoclasts as well as the secretion of tumor necrosis factor (TNF)-α in LPS-treated cells. RANKL-induced RAW cells secreted a very basal level TNF-α. TAK-242 did not affect RANKL-induced osteoclastogenesis. Increased osteoclast differentiation in LPS-treated osteoclasts was not associated with the RANKL/RANK/OPG axis but connected with the LPS/TLR4/TNF-α tumor necrosis factor receptor (TNFR)-2 axis. We postulate that this is because TAK-242 and a TNF-α antibody suppress osteoclast differentiation. Furthermore, an antibody against TNF-α reduced membrane levels of TNFR-2. Secreted TNF-α appears to function as an autocrine/ paracrine factor in the induction of osteoclastogenesis independent of RANKL. TNF-α secreted via LPS/TLR4 signaling regulates osteoclastogenesis in macrophages primed with RANKL and then treated with LPS. Our findings suggest that TLR4/TNF-α might be a potential target to suppress bone loss associated with inflammatory bone diseases, including periodontitis, rheumatoid arthritis, and osteoporosis.

Vaccinomic approach for novel multi epitopes vaccine against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

Abstract: The spread of a novel coronavirus termed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in China and other countries is of great concern worldwide with no effective vaccine. This study aimed to design a novel vaccine construct against SARS-CoV-2 from the spike S protein and orf1ab polyprotein using immunoinformatics tools. The vaccine was designed from conserved epitopes interacted against B and T lymphocytes by the combination of highly immunogenic epitopes with suitable adjuvant and linkers. The proposed vaccine composed of 526 amino acids and was shown to be antigenic in Vaxigen server (0.6194) and nonallergenic in Allertop server. The physiochemical properties of the vaccine showed isoelectric point of 10.19. The instability index (II) was 31.25 classifying the vaccine as stable. Aliphatic index was 84.39 and the grand average of hydropathicity (GRAVY) was − 0.049 classifying the vaccine as hydrophilic. Vaccine tertiary structure was predicted, refined and validated to assess the stability of the vaccine via Ramachandran plot and ProSA-web servers. Moreover, solubility of the vaccine construct was greater than the average solubility provided by protein sol and SOLpro servers indicating the solubility of the vaccine construct. Disulfide engineering was performed to reduce the high mobile regions in the vaccine to enhance stability. Docking of the vaccine construct with TLR4 demonstrated efficient binding energy with attractive binding energy of − 338.68 kcal/mol and − 346.89 kcal/mol for TLR4 chain A and chain B respectively. Immune simulation significantly provided high levels of immunoglobulins, T-helper cells, T-cytotoxic cells and INF-γ. Upon cloning, the vaccine protein was reverse transcribed into DNA sequence and cloned into pET28a(+) vector to ensure translational potency and microbial expression. A unique vaccine construct from spike S protein and orf1ab polyprotein against B and T lymphocytes was generated with potential protection against the pandemic. The present study might assist in developing a suitable therapeutics protocol to combat SARSCoV-2 infection.

Application of imiquimod-induced murine psoriasis model in evaluating interleukin-17A antagonist

Abstract: Interleukin-17A (IL17A) is a proinflammatory cytokine critically involved in autoimmune diseases, and monoclonal antibodies of IL17A have been approved for clinical treatment of psoriasis. However, a usable psoriatic animal model has been always required for preclinical evaluation of IL17A antagonists. Imiquimod (IMQ)-induced psoriasis model is widely used in fundamental research, but it’s not able to accurately show anti-psoriatic effect of IL17A antagonists with conventional modelling condition. On female C57BL/6 mice, with optimization on the usage of IMQ, positive control reagent and anti-mIL17A antibody, a 7-day model with proper testing window, acceptable disease severity as well as high repeatability was developed, and the efficacy of IL17A antagonist can be objectively evaluated by several qualitative and quantitative indices. Meanwhile, we validated the detailed involvement of IL17A signaling in disease progression, confirmed that the expression levels of IL17A and its related cytokines were induced by IMQ application, and its downstream cytokines can be inhibited by IL17A antagonist treatment. In further study, we revealed that IL17A was transient induced by IMQ and directly caused downstream signaling activation. This finding on the kinetical change of IL17A signaling will manifest the pharmacokinetics-pharmacodynamics investigation of IL17A antagonists. Our work presents the application of a convenient psoriatic animal model in the research and development of IL17A antagonists, meanwhile providing extra evidence for understanding IL17A’s role in the progression of IMQ-induced psoriasis model, which manifest the research and development of IL17A antagonists.

Humoral and cellular response to SARS-CoV-2 BNT162b2 mRNA vaccine in hemodialysis patients.

Abstract: Background Hemodialysis (HD) patients have an increased risk of acquiring infections due to many health care contacts and may, in addition, have a suboptimal response to vaccination and a high mortality from Covid-19 infection. Methods In 50 HD patients (mean age 69.4 years, 62% men) administration of SARS-CoV-2BNT162b2 mRNA vaccine began in Dec 2020 and the immune response was evaluated 7-15 weeks after the last dose. Levels of Covid-19 (SARS-CoV-2) IgG antibody against the nucleocapsid antigen (anti-N) and the Spike antigen (anti-S) and T-cell reactivity testing against the Spike protein using ELISPOT technology were evaluated. Results Out of 50 patients, anti-S IgG antibodies indicating a vaccine effect or previous Covid-19 infection, were detected in 37 (74%), 5 (10%) had a borderline response and 8 (16%) were negative after two doses of vaccine. T-cell responses were detected in 29 (58%). Of the 37 patients with anti-S antibodies, 25 (68%) had a measurable T-cell response. 2 (40%) out of 5 patients with borderline anti-S and 2 (25%) without anti-S had a concomitant T-cell response. Twenty-seven (54%) had both an antibody and T-cell response. IgG antibodies to anti-N indicating a previous Covid-19 disease were detected in 7 (14%) patients. Conclusions Most HD patients develop a B- and/or T-cell response after vaccination against Covid-19 but approx. 20% had a limited immunological response. T-cell reactivity against Covid-19 was only present in a few of the anti-S antibody negative patients.

Enhancement of Zika virus infection by antibodies from West Nile virus seropositive individuals with no history of clinical infection

Abstract: Recent outbreaks of Zika Virus (ZIKV) infection and associated microcephaly has raised multiple scientific questions. The close antigenic relatedness between flaviviruses makes diagnosis of specific infection difficult. This relatedness also raises the potential of Antibody Dependent Enhancement (ADE) via cross reactive antibodies to flaviviruses like West Nile Virus (WNV) and Dengue Virus (DENV). Asymptomatic WNV infections are endemic throughout the US creating a large proportion of the population that is seropositive for WNV antibodies. Whether these sero-positive individuals potentially carry ZIKV enhancing antibodies remains unknown. Serum samples obtained from human subjects with symptomatic or asymptomatic WNV infection from a WNV endemic region in Texas were tested for their ability to enhance or neutralize ZIKV infection. Sero-surveillance data demonstrated a ~ 7% prevalence for WNV antibodies in the population. Sera from both symptomatic and asymptomatic WNV seropositive donors effectively neutralized WNV and to some extent DENV infection. Interestingly, WNV+ sera failed to inhibit ZIKV while significantly enhancing infection. Conversely, ZIKV specific sera effectively neutralized ZIKV, with ADE only evident at lower concentrations. The enhancement of ZIKV via WNV antibody positive sera was likely due to non-neutralizing Envelope (E) antibodies as seen with monoclonal ZIKV E antibodies. Overall, our findings suggest that WNV antibodies in the sera significantly enhance ZIKV infection in Fc receptor positive cells with limited neutralization activity. Further studies in more relevant models of ADE will be needed to confirm the relevance of these findings in vivo.

Neutrophil extracellular traps are present in the airways of ENaC-overexpressing mice with cystic fibrosis-like lung disease.

Abstract: Neutrophils are key components of the exacerbated inflammation and tissue damage in cystic fibrosis (CF) airways. Neutrophil extracellular traps (NETs) trap and kill extracellular pathogens. While NETs are abundant in the airways of CF patients and have been hypothesized to contribute to lung damage in CF, the in vivo role of NETs remains controversial, partially due to lack of appropriate animal models. The goal of this study was to detect NETs and to further characterize neutrophil-mediated inflammation in the airways of mice overexpressing the epithelial sodium channel (βENaC-Tg mice on C57BL/6 background) in their lung with CF-like airway disease, in the absence of any apparent bacterial infections. Histology scoring of lung tissues, flow cytometry, multiplex ELISA, immunohistochemistry and immunofluorescence were used to characterize NETs and the airway environment in uninfected, βENaC-Tg mice at 6 and 8 weeks of age, the most chronic time points so far studied in this model. Excessive neutrophilic infiltration characterized the lungs of uninfected, βENaC-Tg mice at 6 and 8 weeks of age. The bronchoalveolar lavage fluid (BALF) of βENaC-Tg mice contains increased levels of CF-associated cytokines and chemokines: KC, MIP-1α/β, MCP-1, G-CSF, IL-5, and IL-6. The BALF of βENaC-Tg mice contain MPO-DNA complexes, indicative of the presence of NETs. Immunofluorescence and flow cytometry of BALF neutrophils and lung tissues demonstrated increased histone citrullination, a NET-specific marker, in βENaC-Tg mice. NETs are detected in the airways of βENaC-Tg mice, in the absence of bacterial infections. These data demonstrate the usefulness of the βENaC-Tg mouse to serve as a model for studying the role of NETs in chronic CF airway inflammation.

Circular RNA circFADS2 is overexpressed in sepsis and suppresses LPS-induced lung cell apoptosis by inhibiting the maturation of miR-15a-5p.

Abstract: Circular RNA circFADS2 plays protective roles in LPS-induced inflammation, which promotes sepsis, suggesting its involvement in sepsis. Expression of circFADS2, mature miR-15a-5p, and miR-15a-5p precursor in plasma samples from sepsis patients and healthy controls was determined by RT-qPCR. The circFADS2 expression vector was transfected in lung cells, followed by the measurement of the expression levels of mature miR-15a-5p and miR-15a-5p precursor to study the role of circFADS2 in miR-15a-5p maturation. Cell apoptosis was analyzed by cell apoptosis assay. CircFADS2 was upregulated in sepsis and inversely correlated with mature miR-15a-5p, but not miR-15a-5p precursor. In lung cells, circFADS2 overexpression decreased the level of mature miR-15a-5p, but not miR-15a-5p precursor. LPS treatment decreased miR-15a-5p expression and increased circFADS2 level. Cell apoptosis analysis showed that circFADS2 overexpression reduced miR-15a-5p overexpression-induced apoptosis of LPS-treated lung cells. CircFADS2 is upregulated in sepsis to suppress LPS-induced lung cell apoptosis by inhibiting miR-15a-5p maturation.

The seroprevalence and kinetics of IgM and IgG in the progression of COVID-19.

Abstract: SARS-CoV-2 is a novel coronavirus first recognized in late December 2019 that causes coronavirus disease 19 (COVID-19). Due to the highly contagious nature of SARS-CoV-2, it has developed into a global pandemic in just a few months. Antibody testing is an effective method to supplement the diagnosis of COVID-19. However, multicentre studies are lacking to support the understanding of the seroprevalence and kinetics of SARS-CoV-2 antibodies in COVID-19 epidemic regions. A multicentre cross-sectional study of suspected and confirmed patients from 4 epidemic cities in China and a cohort study of consecutive follow-up patients were conducted from 29/01/2020 to 12/03/2020. IgM and IgG antibodies elicited by SARS-CoV-2 were tested by a chemiluminescence assay. Clinical information, including basic demographic data, clinical classification, and time interval from onset to sampling, was collected from each centre. A total of 571 patients were enrolled in the cross-sectional study, including 235 COVID-19 patients and 336 suspected patients, each with 91.9%:2.1% seroprevalence of SARS-CoV-2 IgG and 92.3%:5.4% seroprevalence of SARS-CoV-2 IgM. The seroprevalence of SARS-CoV-2 IgM and IgG in COVID-19 patients was over 70% less than 7 days after symptom onset. Thirty COVID-19 patients were enrolled in the cohort study and followed up for 20 days. The peak concentrations of IgM and IgG were reached on the 10th and 20th days, respectively, after symptom onset. The seroprevalence of COVID-19 IgG and IgM increased along with the clinical classification and treatment time delay. We demonstrated the kinetics of IgM and IgG SARS-CoV-2 antibodies in COVID-19 patients and the association between clinical classification and antibodies, which will contribute to the interpretation of IgM and IgG SARS-CoV-2 antibody tests and in predicting the outcomes of patients with COVID-19.

D-mannose ameliorates autoimmune phenotypes in mouse models of lupus

Abstract: Systemic lupus erythematosus is an autoimmune disease characterized by an overproduction of autoantibodies resulting from dysregulation in multiple immune cell types. D-mannose is a C− 2 epimer of glucose that exhibits immunoregulatory effects in models of autoimmune diseases, such as type 1 diabetes, induced rheumatoid arthritis, and airway inflammation. This study was conducted to evaluate the efficacy of D-mannose treatment in mouse models of lupus. Firstly, the effect of D-Mannose was evaluated by flow cytometry on the in vitro activation of non-autoimmune C57BL/6 (B6) bone marrow-derived dendritic cells (BMDCs) and their ability to induce antigen-specific CD4+ T cell proliferation and activation. D-mannose inhibited the maturation of BMDCs and their induction of antigen-specific T cell proliferation and activation. In vivo, D-mannose increased the frequency of Foxp3+ regulatory T cells in unmanipulated B6 mice. To assess the effect of D-mannose in mouse models of lupus, we used the graft-versus-host disease (cGVHD) induced model and the B6.lpr spontaneous model. In the cGVHD model, D-mannose treatment decreased autoantibody production, with a concomitant reduction of the frequency of effector memory and follicular helper T cells as well as germinal center B cells and plasma cells. These results were partially validated in the B6.lpr model of spontaneous lupus. Overall, our results suggest that D-mannose ameliorates autoimmune activation in models of lupus, at least partially due to its expansion of Treg cells, the induction of immature conventional dendritic cells and the downregulation of effector T cells activation. D-Mannose showed however a weaker immunomodulatory effect in lupus than in other autoimmune diseases.

Transcriptomics of type 2 diabetic and healthy human neutrophils.

Abstract: OBJECTIVES Chronic inflammatory diseases, including diabetes and cardiovascular disease, are heterogeneous and often co-morbid, with increasing global prevalence. Uncontrolled type 2 diabetes (T2D) can result in severe inflammatory complications. As neutrophils are essential to normal and aberrant inflammation, we conducted RNA-seq transcriptomic analyses to investigate the association between neutrophil gene expression and T2D phenotype. As specialized pro-resolving lipid mediators (SPM) act to resolve inflammation, we further surveyed the impact of neutrophil receptor binding SPM resolvin E1 (RvE1) on isolated diabetic and healthy neutrophils. METHODS Cell isolation and RNA-seq analysis of neutrophils from N = 11 T2D and N = 7 healthy individuals with available clinical data was conducted. Additionally, cultured neutrophils (N = 3 T2D, N = 3 healthy) were perturbed with increasing RvE1 doses (0 nM, 1 nM, 10 nM, or 100 nM) prior to RNA-seq. Data was evaluated through a bioinformatics pipeline including pathway analysis and post hoc false discovery rate (FDR)-correction. RESULTS We observed significant differential expression of 50 genes between T2D and healthy neutrophils (p < 0.05), including decreased T2D gene expression in inflammatory- and lipid-related genes SLC9A4, NECTIN2, and PLPP3 (p < 0.003). RvE1 treatment induced dose-dependent differential gene expression (uncorrected p < 0.05) across groups, including 59 healthy and 216 T2D neutrophil genes. Comparing T2D to healthy neutrophils, 1097 genes were differentially expressed across RvE1 doses, including two significant genes, LILRB5 and AKR1C1, involved in inflammation (p < 0.05). CONCLUSIONS The neutrophil transcriptomic database revealed novel chronic inflammatory- and lipid-related genes that were differentially expressed between T2D cells when compared to controls, and cells responded to RvE1 dose-dependently by gene expression changes. Unraveling the mechanisms regulating abnormalities in diabetic neutrophil responses could lead to better diagnostics and therapeutics targeting inflammation and inflammation resolution.

The role of β2 integrin in dendritic cell migration during infection.

Abstract: Dendritic cells (DCs) play a key role in shaping T cell responses. To do this, DCs must be able to migrate to the site of the infection and the lymph nodes to prime T cells and initiate the appropriate immune response. Integrins such as β2 integrin play a key role in leukocyte adhesion, migration, and cell activation. However, the role of β2 integrin in DC migration and function in the context of infection-induced inflammation in the gut is not well understood. This study looked at the role of β2 integrin in DC migration and function during infection with the nematode worm Trichuris muris. Itgb2tm1Bay mice lacking functional β2 integrin and WT littermate controls were infected with T. muris and the response to infection and kinetics of the DC response was assessed. In infection, the lack of functional β2 integrin significantly reduced DC migration to the site of infection but not the lymph nodes. The lack of functional β2 integrin did not negatively impact T cell activation in response to T. muris infection. This data suggests that β2 integrins are important in DC recruitment to the infection site potentially impacting the initiation of innate immunity but is dispensible for DC migration to lymph nodes and T cell priming in the context of T. muris infection.

Pharmacological activation of rev-erbα suppresses LPS-induced macrophage M1 polarization and prevents pregnancy loss.

Abstract: Background Circadian rhythm is an important player for reproduction. Rev-erbα, a significant clock gene, is involved in regulating cell differentiation, inflammation and metabolism. Macrophage polarization plays crucial roles in immune tolerance at the maternal-fetus interface, which also modulates the initiation and resolution of inflammation. Alteration of macrophage polarization induces adverse pregnancy outcomes such as infertility, recurrent spontaneous abortion and preterm labor. Results Decidual macrophages from LPS-induced mice abortion model displayed M1-like bias, accompanied by decreased expression of Rev-erbα. SR9009, an agonist of Rev-erbα, may reduce lipopolysaccharide (LPS)-induced M1 polarization of macrophages via activation of PI3K but not NF-κB signaling pathway. Furthermore, SR9009 could reduce M1-like polarization of decidual macrophages induced by LPS and attenuate LPS-induced resorption rates in mice model. Conclusions Both in vivo and in vitro experiments demonstrated that the pharmacological activation of Rev-erbα using SR9009 could attenuate the effect of LPS on macrophage polarization and protect pregnancy. This study may provide a potential therapeutic strategy for miscarriage induced by inflammation.

Optimizing interleukin-2 concentration, seeding density and bead-to-cell ratio of T-cell expansion for adoptive immunotherapy

Abstract: The successful ex vivo expansion of T-cells in great numbers is the cornerstone of adoptive cell therapy. We aimed to achieve the most optimal T-cell expansion condition by comparing the expansion of T-cells at various seeding densities, IL-2 concentrations, and bead-to-cell ratios. we first expanded the peripheral blood mononuclear cells (PBMCs) of a healthy donor at a range of 20 to 500 IU/mL IL-2 concentrations, 125 × 103 to 1.5 × 106 cell/mL, and 1:10 to 10:1 B:C (Bead-to-cell) ratios and compared the results. We then expanded the PBMC of three healthy donors using the optimized conditions and examined the growth kinetics. On day 28, CD3, CD4, and CD8 expression of the cell populations were analyzed by flow cytometry. T-cells of the first donor showed greater expansion results in IL-2 concentrations higher than 50 IU/mL compared to 20 IU/mL (P = 0.02). A seeding density of 250 × 103 cell/mL was superior to higher or lower densities in expanding T-cells (P = 0.025). Also, we witnessed a direct correlation between the B:C ratio and T-cell expansion, in which, in 5:1 and 10:1 B:C ratios T-cell significantly expanded more than lower B:C ratios. The results of PBMC expansions of three healthy donors were similar in growth kinetics. In the optimized condition, 96–98% of the lymphocyte population expressed CD3. While the majority of these cells expressed CD8, the mean expression of CD4 in the donors was 19.3, 16.5, and 20.4%. Our methodology demonstrates an optimized culture condition for the production of large quantities of polyclonal T-cells, which could be useful for future clinical and research studies.

CXCL9 secreted by tumor-associated dendritic cells up-regulates PD-L1 expression in bladder cancer cells by activating the CXCR3 signaling.

Abstract: Tumor-associated dendritic cells (TADCs) can interact with tumor cells to suppress anti-tumor T cell immunity. However, there is no information on whether and how TADCs can modulate programmed death-ligand 1 (PD-L1) expression by cancer cells. Human peripheral blood monocytes were induced for DCs and immature DCs were cultured alone, or co-cultured with bladder cancer T24 or control SV-HUC-1 cells, followed by stimulating with LPS for DC activation. The activation status of DCs was characterized by flow cytometry and allogenic T cell proliferation. The levels of chemokines in the supernatants of co-cultured DCs were measured by CBA-based flow cytometry. The impacts of CXCL9 on PD-L1, STAT3 and Akt expression and STAT3 and Akt phosphorylation in T24 cells were determined by flow cytometry and Western blot. Compared with the control DCs, TADCs exhibited immature phenotype and had significantly lower capacity to stimulate allogenic T cell proliferation, particularly in the presence of recombinant CXCL9. TADCs produced significantly higher levels of CXCL9, which enhanced PD-L1 expression in T24 cells. Pre-treatment with AMG487 abrogated the CXCL9-increased PD-L1 expression in T24 cells. Treatment with CXCL9 significantly enhanced STAT3 and Akt activation in T24 cells. TADCs produced high levels of CXCL9 that increased PD-L1 expression in bladder cancer T24 cells by activating the CXCR3-related signaling. Our findings may shed new lights in understanding the regulatory roles of TADCs in inhibiting antitumor T cell responses and promoting tumor growth.

Intratumoral regulatory T cells from colon cancer patients comprise several activated effector populations.

Abstract: Background Intratumoral regulatory T cells (Treg) in colon cancer are a heterogeneous cell population, with potential impact on patient outcome. Generally, a high Treg infiltration has been correlated to a worse patient outcome, but it is still unclear how the composition of different Treg subsets affects patient relapse and survival. In this study, we used mass and flow cytometry to characterize Treg in colon tumors and corresponding unaffected tissue, followed by a correlation to clinical parameters and patient outcome. Results Using mass cytometry, we defined 13 clusters of intestinal Treg, three of which were enriched in the tumors. The two most enriched clusters were defined by their expression of the proliferation marker Ki67 and CD56, respectively. The Treg accumulating in the tumors expressed inducible T-cell co-stimulator (ICOS), OX-40, and CD39, indicating that they were effector Treg (eTreg). Intratumoral CD39+ Treg also had a higher expression of Foxp3, suggesting a higher suppressive activity, and we subsequently used CD39 as a marker for eTreg. Our further studies showed that colon tumors can be divided into two tumor groups, based on the proportion of CD39+ putative eTreg in the tumors. This property was independent of both tumor microsatellite status and tumor stage, which are important factors in predicting cancer disease progression. In a prospective study of forty-four colon cancer patients, we also showed that patients with a high CD39 expression on tumor-infiltrating Treg have a tendency towards a less favorable patient outcome in terms of cumulative cancer-specific survival. Conclusions This study uncovers novel subsets of tumor-infiltrating Treg in colon cancer, and suggests that CD39 may be a potential therapeutic target in patients with microsatellite stable colon tumors, which are usually refractory to checkpoint blockade therapy.

Direct effects of mast cell proteases, tryptase and chymase, on bronchial epithelial integrity proteins and anti-viral responses.

Abstract: Mast cells (MCs) are known to contribute to both acute and chronic inflammation. Bronchial epithelial cells are the first line of defence against pathogens and a deficient anti-viral response has been suggested to play a role in the pathogenesis of asthma exacerbations. However, effects of MC mediators on bronchial epithelial immune response have been less studied. The aim of this study is to investigate the direct effects of stimulation with MC proteases, tryptase and chymase, on inflammatory and anti-viral responses in human bronchial epithelial cells (HBECs). Cultured BEAS-2b cells and primary HBECs from 3 asthmatic patients were stimulated with tryptase or chymase (0.1 to 0.5 μg/ml) for 1, 3, 6 and 24 h. To study the effects of MC mediators on the anti-viral response, cells were stimulated with 10 μg/ml of viral mimic Poly (I:C) for 3 and 24 h following pre-treatment with 0.5 μg/ml tryptase or chymase for 3 h. Samples were analysed for changes in pro-inflammatory and anti-viral mediators and receptors using RT-qPCR, western blot and Luminex. Tryptase and chymase induced release of the alarmin ATP and pro-inflammatory mediators IL-8, IL-6, IL-22 and MCP-1 from HBECs. Moreover, tryptase and chymase decreased the expression of E-cadherin and zonula occludens-1 expression from HBECs. Pre-treatment of HBECs with tryptase and chymase further increased Poly (I:C) induced IL-8 release at 3 h. Furthermore, tryptase significantly reduced type-I and III interferons (IFNs) and pattern recognition receptor (PRR) expression in HBECs. Tryptase impaired Poly (I:C) induced IFN and PRR expression which was restored by treatment of a serine protease inhibitor. Similar effects of tryptase on inflammation and anti-viral responses were also confirmed in primary HBECs from asthmatic patients. MC localization within the epithelium and the release of their proteases may play a critical role in asthma pathology by provoking pro-inflammatory and alarmin responses and downregulating IFNs. Furthermore, MC proteases induce downregulation of epithelial junction proteins which may lead to barrier dysfunction. In summary, our data suggests that mast cells may contribute towards impaired anti-viral epithelial responses during asthma exacerbations mediated by the protease activity of tryptase.

Differential polarization and the expression of efferocytosis receptor MerTK on M1 and M2 macrophages isolated from coronary artery disease patients.

Abstract: Differential polarization of macrophage into M1 and M2 mediates atherosclerotic plaque clearance through efferocytosis. Higher expression of Mer proto-oncogene tyrosine kinase (MerTK) on M2 macrophage helps in maintaining macrophage efferocytic efficiency. In healthy individuals, macrophage polarization into M1 and M2 occurs in tissues in concomitance with the acquisition of functional phenotypes depending on specific microenvironment stimuli. However, whether the macrophage differential polarization and MerTK expression vary in coronary artery disease (CAD) patients remain unknown. This study aimed to elucidate the polarization of M1 and M2 macrophage from CAD patients as well as to investigate the expression of MerTK in these macrophage phenotypes. A total of 14 (n) CAD patients were recruited and subsequently grouped into “no apparent CAD”, “non-obstructive CAD” and “obstructive CAD” according to the degree of stenosis. Thirty ml of venous blood was withdrawn to obtain monocyte from the patients. The M1 macrophage was generated by treating the monocyte with GMCSF, LPS and IFN-γ while MCSF, IL-4 and IL-13 were employed to differentiate monocyte into M2 macrophage. After 7 days of polarization, analysis of cell surface differentiation markers (CD86+/CD80+ for M1 and CD206+/CD200R+ for M2) and measurement of MerTK expression were performed using flow cytometry. Both M1 and M2 macrophage expressed similar level of CD86, CD80 and CD206 in all groups of CAD patients. MerTK expression in no apparent CAD patients was significantly higher in M2 macrophage compared to M1 macrophage [12.58 ± 4.40 vs. 6.58 ± 1.37, p = 0.040]. Differential polarization of macrophage into M1 and M2 was highly dynamic and can be varied due to the microenvironment stimuli in atherosclerotic plaque. Besides, higher expression of MerTK in patients with the least coronary obstructive suggest its vital involvement in efferocytosis.

Association between serum markers of the humoral immune system and inflammation in the Swedish AMORIS study.

Abstract: BACKGROUND Although the onset of inflammatory cascades may profoundly influence the nature of antibody responses, the interplay between inflammatory and humoral (antibody) immune markers remains unclear. Thus, we explored the reciprocity between the humoral immune system and inflammation and assessed how external socio-demographic factors may influence these interactions. From the AMORIS cohort, 5513 individuals were identified with baseline measurements of serum humoral immune [immunoglobulin G, A & M (IgG, IgA, IgM)] and inflammation (C-reactive protein (CRP), albumin, haptoglobin, white blood cells (WBC), iron and total iron-binding capacity) markers measured on the same day. Correlation analysis, principal component analysis and hierarchical clustering were used to evaluate biomarkers correlation, variation and associations. Multivariate analysis of variance was used to assess associations between biomarkers and educational level, socio-economic status, sex and age. RESULTS Frequently used serum markers for inflammation, CRP, haptoglobin and white blood cells, correlated together. Hierarchical clustering and principal component analysis confirmed the interaction between these main biological responses, showing an acute response component (CRP, Haptoglobin, WBC, IgM) and adaptive response component (Albumin, Iron, TIBC, IgA, IgG). A socioeconomic gradient associated with worse health outcomes was observed, specifically low educational level, older age and male sex were associated with serum levels that indicated infection and inflammation. CONCLUSIONS These findings indicate that serum markers of the humoral immune system and inflammation closely interact in response to infection or inflammation. Clustering analysis presented two main immune response components: an acute and an adaptive response, comprising markers of both biological pathways. Future studies should shift from single internal marker assessment to multiple humoral and inflammation serum markers combined, when assessing risk of clinical outcomes such as cancer.

Soluble cytotoxic T-lymphocyte-associated antigen 4 (sCTLA-4) as a potential biomarker for diagnosis and evaluation of the prognosis in Glioma.

Abstract: The cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is widely considered as a pivotal immune checkpoint molecule to suppress antitumor immunity. However, the significance of soluble CTLA-4 (sCTLA-4) remains unclear in the patients with brain glioma. Here we aimed to investigate the significance of serum sCTLA-4 levels as a noninvasive biomarker for diagnosis and evaluation of the prognosis in glioma patients. In this study, the levels of sCTLA-4 in serum from 50 patients diagnosed with different grade gliomas including preoperative and postoperative, and 50 healthy individuals were measured by an enzyme-linked immunosorbent assay (ELISA). And then ROC curve analysis and survival analyses were performed to explore the clinical significance of sCTLA-4. Serum sCTLA-4 levels were significantly increased in patients with glioma compared to that of healthy individuals, and which was also positively correlated with the tumor grade. ROC curve analysis showed that the best cutoff value for sCTLA-4 for glioma is 112.1 pg/ml, as well as the sensitivity and specificity with 82.0 and 78.0%, respectively, and a cut-off value of 220.43 pg/ml was best distinguished in patients between low-grade glioma group and high-grade glioma group with sensitivity 73.1% and specificity 79.2%. Survival analysis revealed that the patients with high sCTLA-4 levels (> 189.64 pg/ml) had shorter progression-free survival (PFS) compared to those with low sCTLA-4 levels (≤189.64 pg/ml). In the univariate analysis, elder, high-grade tumor, high sCTLA-4 levels and high Ki-67 index were significantly associated with shorter PFS. In the multivariate analysis, sCTLA-4 levels and tumor grade remained an independent prognostic factor. These findings indicated that serum sCTLA-4 levels play a critical role in the pathogenesis and development of glioma, which might become a valuable predictive biomarker for supplementary diagnosis and evaluation of the progress and prognosis in glioma.

TREM2 promotes natural killer cell development in CD3-CD122+NK1.1+ pNK cells.

Abstract: Triggering receptor expressed on myeloid cells 2 (TREM2) signaling is considered to regulate anti-inflammatory responses in macrophages, dendritic cell maturation, osteoclast development, induction of obesity, and Alzheimer’s disease pathogenesis. However, little is known regarding the effect of TREM2 on natural killer (NK) cells. Here, we demonstrated for the first time that CD3−CD122+NK1.1+ precursor NK (pNK) cells expressed TREM2 and their population increased in TREM2-overexpressing transgenic (TREM2-TG) mice compared with that in female C57BL/6 J wild type (WT) mice. Both NK cell-activating receptors and NK cell-associated genes were expressed at higher levels in various tissues of TREM2-TG mice than in WT mice. In addition, bone marrow-derived hematopoietic stem cells (HSCs) of TREM2-TG mice (TG-HSCs) successfully differentiated into NK cells in vitro, with a higher yield from TG-HSCs than from WT-HSCs. In contrast, TREM2 signaling inhibition by TREM2-Ig or a phosphatidylinositol 3-kinase (PI3K) inhibitor affected the expression of the NK cell receptor repertoire and decreased the expression levels of NK cell-associated genes, resulting in significant impairment of NK cell differentiation. Moreover, in melanoma-bearing WT mice, injection of bone marrow cells from TREM2-TG mice exerted greater antitumor effects than that with cells from WT control mice. Collectively, our data clearly showed that TREM2 promoted NK cell development and tumor regression, suggesting TREM2 as a new candidate for cancer immunotherapy.

Downregulation of NKG2DLs by TGF-β in human lung cancer cells

Abstract: Transforming growth factor beta (TGF-β) is a typical immuno-inhibitory cytokine and highly secreted by lung cancer cells. It was supposed that its immunosuppressive effects to NK cell might be related with the altered expression of activating and inhibitory molecules in lung cancer cells. In this study, we examined the expression of NKG2DLs, PD-L1 and PD-L2 in lung cancer cells after treatment of TGF-β and a TGF-β inhibitor, Galunisertib (LY2157299). TGF-β reduced the level of surface proteins of five NKG2DLs without altered transcription levels in lung cancer cells. Galunisertib reversed the effect of TGF-β on the expression of NKG2DLs. Since MMP inhibitors, MMPi III and MMP2 inhibitor I, restored the reduced expression of NKG2DLs after treatment of TGF-β, it was thought that TGF-β induced the expression of MMP2 which facilitated the shedding of the NKG2DLs in cancer cells. However, the expression of PD-L1, L2 were not changed by treatment with TGF-β or Galunisertib. Therefore, inhibition of TGF-β might reverse the immunosuppressive status on immune cells and restore NK cell mediated anticancer immune responses by upregulation of NKG2DLs in cancer cells.

Identification of active small-molecule modulators targeting the novel immune checkpoint VISTA

Abstract: Cancer immunotherapy has gained increasing popularity as a novel approach to treat cancer. A member of the B7 family, V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a novel immune checkpoint that regulates a broad spectrum of immune responses. VISTA is an acidic pH-selective ligand for P-selectin glycoprotein ligand-1(PSGL-1). CA-170, a first-in-class small-molecule dual antagonist of VISTA/PD-L1, was collaboratively developed by Aurigene Discovery Technologies Limited and Curis, Inc. It is currently in Phase I clinical trial. In this study, we develop homology modeling for the VISTA 3D structure and subsequent virtual screening for VISTA small-molecule hit ligands. Visualization of the binding postures of docked ligands with the VISTA protein indicates that some small molecular compounds target VISTA. The ability of antagonist to disrupt immune checkpoint VISTA pathways was investigated though functional studies in vitro. Affinity active molecule for VISTA was obtained through virtual screening, and the antagonist compound activity to VISTA was assayed in cellular level. We reported a small molecule with high VISTA affinity as antagonist, providing ideas for development VISTA-targeted small molecule compound in cancer immunotherapy.

Neutrophil to lymphocyte ratio, platelet to lymphocyte ratio, and other hematological parameters in psoriasis patients.

Abstract: Background Psoriasis is a chronic immune-mediated skin disorder. Systemic inflammation plays an important role in the pathogenesis of psoriasis. Methods A total of 477 patients with psoriasis vulgaris (PsV, n = 347), generalized pustular psoriasis (GPP, n = 37), erythrodermic psoriasis (PsE, n = 45), arthritic psoriasis (PsA, n = 25) and mixed psoriasis (n = 23), and 954 healthy control subjects were included in the study. Demographic, clinical, and laboratory information were collected and compared between subgroups. Results Compared with the healthy control group, patients with psoriasis had higher total white blood cell (WBC), neutrophil, platelet counts, neutrophil to lymphocyte ratio (NLR), and platelet to lymphocyte ratio (PLR), but lower hemoglobin (Hb) levels, lymphocyte and red blood cell (RBC) counts. NLR values in the PsV group were significantly lower than those in the GPP, PsE, and PsA groups, with GPP group being the highest. PLR values in the PsV group were significantly lower than those in the GPP, PsE, and PsA groups. There was no significant correlation between the psoriasis area severity index (PASI) score and either the NLR or PLR in the PsV group. Conclusions Elevated NLR and PLR were associated with psoriasis and differed between subtypes, suggesting that they could be used as markers of systemic inflammation in psoriasis patients.

Differential long non-coding RNA expression profile and function analysis in primary Sjogren's syndrome.

Abstract: Background Primary Sjogren's syndrome (pSS) is a chronic autoimmune disease characterized by abnormal immune cell activation. This study aimed to investigate differentially expressed long non-coding RNA (lncRNA) in peripheral blood mononuclear cells (PBMCs) in patients with pSS to identify lncRNAs that affect pSS pathogenesis. Methods Total RNA was extrated from PBMCs of 30 patients with pSS and 15 healthy persons. Transcriptome sequencing was used to screen differentially expressed lncRNAs and mRNAs in 8 RNA samples from the discovery cohort. The differentially expressed mRNAs underwent functional enrichment analysis. A protein interaction relationship (PPI) and competitive endogenous RNA (ceRNA) network was constructed. Real-time PCR was used to validate screened lncRNAs in all 45 RNA samples.. Results 1180 lncRNAs and 640 mRNAs were differentially expressed in pSS patients (fold change > 2 in healthy persons). The PPI network was constructed with 640 mRNAs and a ceRNA network with four key lncRNAs (GABPB1-AS1, PSMA3-AS1, LINC00847 and SNHG1). Real-time PCR revealed that GABPB1-AS1 and PSMA3-AS1 were significantly up-regulated 3.0- and 1.4-fold in the pSS group, respectively. The GABPB1-AS1 expression level was positively correlated with the percentage of B cells and IgG levels. Conclusions GABPB1-AS1 was significently up-regulated in pSS patients, and its expression level is positively correlated with the percentage of B cells and IgG levels. GABPB1-AS1 may be involved in the pathogenesis of pSS and may be a promising biological marker.

Mutant glucocorticoid receptor binding elements on the interleukin-6 promoter regulate dexamethasone effects.

Abstract: Glucocorticoids (GCs) have been extensively used as essential modulators in clinical infectious and inflammatory diseases. The GC receptor (GR) is a transcription factor belonging to the nuclear receptor family that regulates anti-inflammatory processes and releases pro-inflammatory cytokines, such as interleukin (IL)-6. Five putative GR binding sites and other transcriptional factor binding sites were identified on theIL-6 promoter, and dexamethasone (DEX) was noted to reduce the lipopolysaccharide (LPS)-induced IL-6 production. Among mutant transcriptional factor binding sites, nuclear factor-kappa B (NF-κB), activator protein (AP)-1, and specificity protein (Sp)1–2 sites reduced basal and LPS-induced IL-6 promoter activities through various responses. The second GR binding site (GR2) was noted to play a crucial role in both basal and inducible promoter activities in LPS-induced inflammation. We concluded that selective GR2 modulator might exert agonistic and antagonistic effects and could activate crucial signaling pathways during the LPS-stimulated inflammatory process.

Assessment of CD27 expression on T-cells as a diagnostic and therapeutic tool for patients with smear-negative pulmonary tuberculosis.

Abstract: There is a global focus on illness diagnosis in smear-negative and latent tuberculosis infectious populations (SN-TB and LTBI). CD27 has been suggested to play a direct role in active TB. Little is known about smear-negative individuals. Here, we tried to investigate whether it has a role in smear-negative populations. The expression of CD27 and MTB-specific CD27 in CD4+ T cells (“CD27−CD4+” and “CD27−IFN-γ+CD4+”) was evaluated in MTB-unexposed controls (HC), TB contacts (TB-C) and SN-TB individuals by flow cytometry. The sensitivity, specificity and AUC (area under curve) of “CD27−IFN-γ+CD4+” cells to distinguish SN-TBs from HCs and TB-Cs were determined by receiver operating characteristic (ROC) curve analysis. The clinical index was selected from the clinical laboratory and evaluated for correlation with “CD27−IFN-γ+CD4+” cells by Spearman statistical analysis. We observed that the percentages of “CD27−IFN-γ+CD4+” cells were significantly increased in the SN-TB group compared with the HC and TB-C groups (AUC was 0.88, sensitivity was 82.14%, specificity was 80.00%, and P 50 years group, after clinical treatment. The present results demonstrated that the percentage of “CD27−IFN-γ+CD4+” cells might be a conceivable molecular indicator in the diagnosis of SN-TB and was influenced by its outcome of therapy.

Macrophage mediated recognition and clearance of Borrelia burgdorferi elicits MyD88-dependent and -independent phagosomal signals that contribute to phagocytosis and inflammation

Abstract: Macrophages play prominent roles in bacteria recognition and clearance, including Borrelia burgdorferi (Bb), the Lyme disease spirochete. To elucidate mechanisms by which MyD88/TLR signaling enhances clearance of Bb by macrophages, we studied wildtype (WT) and MyD88−/− Bb-stimulated bone marrow-derived macrophages (BMDMs). MyD88−/− BMDMs exhibit impaired uptake of spirochetes but comparable maturation of phagosomes following internalization of spirochetes. RNA-sequencing of infected WT and MyD88−/− BMDMs identified a large cohort of differentially expressed MyD88-dependent genes associated with re-organization of actin and cytoskeleton during phagocytosis along with several MyD88-independent chemokines involved in inflammatory cell recruitment. We computationally generated networks which identified several MyD88-dependent intermediate proteins (Rhoq and Cyfip1) that are known to mediate inflammation and phagocytosis respectively. Our findings show that MyD88 signaling enhances, but is not required, for bacterial uptake or phagosomal maturation and provide mechanistic insights into how MyD88-mediated phagosomal signaling enhances Bb uptake and clearance.

Activation of Toll-like receptor 2 enhances peripheral and tumor-infiltrating CD8+ T cell cytotoxicity in patients with gastric cancer.

Abstract: Toll-like receptors (TLRs) play central roles in the initiation of innate immune response, and also control adaptive immunity activation. Thus, the aim of the study was to investigate the regulation of TLR activation to CD8+ T cells has not been fully elucidated in gastric cancer (GC). Thirty-two GC patients and twenty-three healthy controls were enrolled. Expression profile of TLRs in peripheral and tumor-infiltrating CD8+ T cells was investigated. Purified CD8+ T cells were stimulated with Pam3Csk4, an agonist of TLR2, and cytotoxic and co-inhibitory molecules in CD8+ T cells was measured. Direct and indirect contact coculture system between CD8+ T cells and AGS cells was set up. Modulation of TLR2 activation to CD8+ T cells was assessed by measuring lactate dehydrogenase release and cytokine secretion. TLR2 mRNA and TLR2+ cell percentage was down-regulated in GC derived peripheral and tumor-infiltrating CD8+ T cells. CD8+ T cells from GC patients showed exhausted phenotype, which presented as decreased perforin/granzyme B, increased programmed death-1, and reduced cytotoxicity to AGS cells. TLR2 activation by Pam3Csk4 enhanced perforin and granzyme B expression in CD8+ T cells, however, did not affect either proinflammatory cytokine production or co-inhibitory molecules expression. Pam3Csk4 stimulation enhanced cytolytic activation of peripheral and tumor-infiltrating CD8+ T cells from GC, but not those from healthy individuals. The present data revealed an important immunomodulatory activity of TLR2 to CD8+ T cells in GC patients.

Type 2 and type 3 innate lymphoid cells at the maternal-fetal interface: implications in preterm birth

Abstract: Preterm birth (PTB) is one of the major causes of neonatal morbidity and mortality worldwide. It is commonly accepted that the act of giving birth is the final step in a proinflammatory signaling cascade, orchestrated by an intrauterine milieu coupled to hormonal cues. Consequently, the inflammatory process plays a pivotal role during the pathogenesis of human labor, both in term and preterm deliveries. The ability of innate lymphoid cells (ILCs) to act as pro-inflammatory mediators arose the interest to study their role in normal and pathological pregnancies. The aim of this work was to analyze the relative frequencies of ILCs subsets in pregnancy and the levels of IL-4, IL-17, IL-22, and IFN-γ as inflammatory mediators. Accordingly, we hypothesized that changes in the proportions of ILCs subpopulations could be related to preterm birth. We analyzed 15 full-term delivery samples and six preterm delivery samples. In the full-term group (FTB) peripheral blood was taken during routine blood analysis, on 3 occasions: 1st, 2nd and 3rd trimester. After delivery, peripheral blood, cord blood and placenta were collected. In PTB group, peripheral blood samples were obtained on two occasions: before and 24 h after treatment with progesterone. We used flow cytometry to analyze ILCs in maternal peripheral blood, placenta, and cord blood samples. Maternal peripheral blood and cord blood samples were analyzed by enzyme-linked immunosorbent assay for IL-4, IL-17, IL-22, and IFN-γ plasma levels at the time of labor. We observed significantly increased relative frequencies of ILC2 and ILC3 in the decidua, as well as an increase of ILC2 in cord blood samples in PTB group, compared to FTB samples. We also found a decrease in IFN-γ in peripheral blood samples of the PTB group, suggesting a functional withdrawal. Additionally, IL-4, IL-17, IL-22 levels were similar in PTB and FTB groups, denoting a relevant role in mediating labor. Our results suggest that ILC2 and ILC3 play a role in PTB by mediating an inflammatory response. Further work is necessary to evaluate the importance of ILCs in the regulation of labor.

Innate lymphoid cell dysfunction during long-term suppressive antiretroviral therapy in an African cohort.

Abstract: Background Innate lymphoid cells (ILC) are lymphoid lineage innate immune cells that do not mount antigen-specific responses due to their lack of B and T-cell receptors. ILCs are predominantly found at mucosal surfaces, as gatekeepers against invading infectious agents through rapid secretion of immune regulatory cytokines. HIV associated destruction of mucosal lymphoid tissue depletes ILCs, among other immune dysfunctions. Studies have described limited restoration of ILCs during the first three years of combined antiretroviral therapy (cART). Little is known about restoration of ILCs during long-term cART, particularly in sub-Saharan Africa which hosts increasing numbers of adults with at least a decade of cART. Results We examined phenotypes and function of ILCs from peripheral blood mononuclear cells after 12 years of suppressive cART. We report that ILC1 frequencies (T-BET + CD127 + and CD161 +) were higher in cART-treated HIV-infected relative to age-matched health HIV-negative adults; P = 0.04 whereas ILC precursors (ILCP) were comparable in the two groups (P = 0.56). Interferon gamma (IFN-γ) secretion by ILC1 was higher among cART-treated HIV-infected relative to HIV-negative adults (P = 0.03). Conclusion HIV associated alteration of ILC persisted during cART and may likely affect the quality of host innate and adaptive immune responses during long-term cART.