Chronic psychological stress impairs germinal center response by repressing miR-155.

Germinal centers (GC) are vital to adaptive immunity. BCL6 and miR-155 are implicated in control of GC reaction and lymphomagenesis. FBXO11 causes BCL6 degradation through ubiquitination in B-cell lymphomas. Chronic psychological stress is known to drive immunosuppression. Corticosterone (CORT) is an adrenal hormone expressed in response to stress and can similarly impair immune functions. However, whether GC formation is disrupted by chronic psychological stress and its molecular mechanism remain to be elucidated. To address this issue, we established a GC formation model in vivo, and a GC B cell differentiation model in vitro. Comparing Naive B cells to GC B cells in vivo and in vitro, the differences of BCL6 and FBXO11 mRNA do not match the changes at the protein level and miR-155 levels that were observed. Next we demonstrated that CORT increase, induced by chronic psychological stress, reduced GC response, IgG1 antibody production and miR-155 level in vivo. The effect of chronic psychological stress can be blocked by a glucocorticoid receptor (GR) antagonist. Similarly, impaired GC B cell generation and isotope class switching were observed. Furthermore, we found that miR-155 and BCL6 expression were downregulated, but FBXO11 expression was upregulated in GC B cells treated with CORT in vitro. In addition, we demonstrated that miR-155 directly down-regulated FBXO11 expression by binding to its 3́-untranslated region. The subsequent overexpression of miR-155 significantly blocked the stress-induced impairment of GC response, due to changes in FBXO11 and BCL6 expression, as well as increased apoptosis in B cells both in vivo and in vitro. Our findings suggest perturbation of GC reaction may play a role in chronic psychological stress-induced immunosuppression through a glucocorticoid pathway, and miR-155-mediated post-transcriptional regulation of FBXO11 and BCL6 expression may contribute to the impaired GC response.

[The expression of T-bet is negatively correlated with the expressions of IFN-γ and TNF-α in CD4+ T cells of patients with active pulmonary tuberculosis].

Objective To investigate the relationship between T box expressed in T cells (T-bet) and the production of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α) and interleukin 2 (IL-2) in CD4+T cells of patients with active pulmonary tuberculosis. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by density gradient centrifugation. Individuals with latent Mycobacterium tuberculosis (MTB) infection were screened by enzyme-linked immunospot assay (ELISPOT). The expressions of T-bet, IFN-γ, TNF-α and IL-2 in CD4+T cells were detected by flow cytometry. Results The expression of IFN-γ significantly increased in PBMCs from the individuals with latent tuberculosis infection when stimulated with MTB H37Rv strain lysates. T-bet expression in CD4+IFN-γ+ cells from the patients with active pulmonary tuberculosis was significantly higher than that from the individuals with latent tuberculosis infection when stimulated with MTB H37Rv strain lysates. The expressions of IFN-γ and TNF-α in T-bet- MTB antigen-specific CD4+T cells were obviously higher than those in T-bet+ cells; however, the expression of IL-2 showed no significant difference between T-bet- cells and T-bet+ cells. Conclusion The expression of T-bet in MTB antigen-specific CD4+T cells from patients with active pulmonary tuberculosis is negatively correlated with IFN-γ and TNF-α.

Mucosal-associated invariant T cells from patients with tuberculosis exhibit impaired immune response.

OBJECTIVES: To identify factors which regulate MAIT cell response to Mycobacterium tuberculosis antigens, and to investigate the role of MAIT cells in patients with active tuberculosis. METHODS: Immune response of MAIT cells to M. tuberculosis antigens were compared between patients with active TB and healthy controls by flow cytometry and RNA sequencing. RESULTS: IFN-γ response of MAIT cells to M. tuberculosis lysates was dramatically improved by signal 3 cytokine IL-15 (p = 0.0002). Patients with active TB exhibited highly reduced IFN-γ production in MAIT cells stimulated with M. tuberculosis lysates/IL-15 compared with healthy controls (p < 0.0001) and individuals with latent TB infection (p = 0.0008). RNA sequencing of flow-sorted MAIT cells from patients with TB and healthy controls identified numerous differentially expressed genes, and the expression of genes that encode IFN-γ, TNF-α, IL-17F, granulysin and granzyme B were all down-regulated in patients with TB. MAIT cells from patients with TB has significantly lower expression of γc receptor than those from healthy controls under condition of Mtb lysates/IL-15 stimulation (p = 0.0028). Blockade of both γc and IL-2Rß receptors resulted in highly reduced frequency of IFN-γ-producing MAIT cells (79.4%) (p = 0.0011). CONCLUSIONS: MAIT cells from patients with active TB exhibited impaired cytokine and cytotoxic response to M. tuberculosis antigens.

Elevated expression of T-bet in mycobacterial antigen-specific CD4(+) T cells from patients with tuberculosis.

T-bet is a T-box transcriptional factor that controls the differentiation and effector functions of CD4 T cells. In this study, we studied the role of T-bet in regulating CD4(+) T cell immunity against tuberculosis (TB). T-bet expression in Mycobacterium tuberculosis antigen-specific CD4(+) T cells was significantly higher in patients with active TB than in individuals with latent TB infection (p<0.0001). Comparison of T-bet expression in TCM and TEM subsets showed that CD4(+)T-bet(+)M. tuberculosis antigen-specific CD4(+) T cells had significantly lower frequency of TCM (p=0.003) and higher frequency of TEM (p=0.003) than CD4(+)T-bet(-) cells. The expression of PD-1 in antigen-specific CD4(+) T cells was significantly higher in patients with TB than in individuals with latent TB infection (p=0.006). CD4(+)CD154(+)T-bet(+) T cells had significantly higher expression of PD-1 than CD4(+)CD154(+)T-bet(-) T cells (p=0.0028). It is concluded that T-bet expression might be associated with differentiation into effector memory cells and PD-1 expression in mycobacterial antigen-specific CD4(+) T cells.

miR-223 is upregulated in monocytes from patients with tuberculosis and regulates function of monocyte-derived macrophages.

Tuberculosis (TB) is a serious infectious disease that most commonly affects the lungs. Macrophages are among the first line defenders against establishment of Mycobacterium tuberculosis infection in the lungs. In this study, we found that activation and cytokine production in monocyte-derived macrophages (MDM) from patients with active TB was impaired. miR-223 expression was significantly elevated in monocytes and MDM from patients with TB compared with healthy controls. To determine the functional role of miR-223 in macrophages, stable miR-223-expressing and miR-223 antisense-expressing U937 cells were established. Compared with empty vector controls, expression of IL-1ß, IL-6, TNF-α and IL-12p40 genes was significantly higher in miR-223 antisense-expressing U937 cells, but lower in miR-223-expressing U937 cells. miR-223 can negatively regulate activation of NF-κB by inhibition of p65 phosphorylation and nuclear translocation. It is concluded that miR-223 can regulate macrophage function by inhibition of cytokine production and NF-κB activation.

Mucosal-associated invariant T-cell function is modulated by programmed death-1 signaling in patients with active tuberculosis.

RATIONALE: Mucosal-associated invariant T (MAIT) cells have been proven to play an important role in host defense against mycobacterial infection in animal infection models; however, the functional role of MAIT cells in patients with active tuberculosis (TB) is still largely unknown. OBJECTIVES: To understand the clinical features and functions of MAIT cells in patients with active TB. METHODS: MAIT cells were analyzed in patients with pulmonary TB, tuberculous pleurisy, and tuberculous peritonitis by flow cytometry. The functions of MAIT cells were compared between patients with active TB and healthy control subjects. MEASUREMENTS AND MAIN RESULTS: The frequency of MAIT cells was significantly reduced both in peripheral blood from patients with active pulmonary TB (P < 0.0001) and in tuberculous pleural effusions compared with healthy control subjects but not in ascitic fluids from patients with tuberculous peritonitis. A comparison of bacillus Calmette-Guérin (BCG)-stimulated cytokine production showed that patients with active TB had significantly higher production of IFN-γ (P = 0.0034) and tumor necrosis factor (TNF)-α (P = 0.0399) compared with healthy control subjects. In contrast, when MAIT cells were stimulated with Escherichia coli, patients with active TB had significantly lower production of IFN-γ (P = 0.0007) and TNF-α (P = 0.0032). MAIT cells in patients with active TB exhibited elevated expression of programmed death-1 (PD-1) (P = 0.0015), and blockade of PD-1 signaling resulted in a significantly higher frequency of BCG-stimulated IFN-γ production in MAIT cells (P = 0.0178). CONCLUSIONS: MAIT-cell immune response to antigen stimulation in patients with active TB is regulated by PD-1, which could be a potential target for TB immunotherapy.

[Gene expression profiling and verification for severe secondary pulmonary tuberculosis patients].

OBJECTIVE: To explore the gene expression profiles of severe secondary pulmonary tuberculosis patients. METHODS: From May 2012 to October 2013, a total of 103 eligible patients with secondary pulmonary tuberculosis were recruited from Institution of Tuberculosis Research of PLA Hospital No. 309. They were divided into severe secondary pulmonary tuberculosis (severe group) (n = 57) and mild secondary pulmonary tuberculosis (mild group) (n = 46) by the severity of disease . At the same time age-matched healthy controls (n = 45) were selected from healthy subjects undergoing physical examination. Whole genome expression profiling was performed with Affymetrix Gene expression chips for 4 cases in severe group, 3 in mild group and 5 in healthy group. Cluster and bioinformatics analysies were performed on differentially expressed genes in severe versus mild group. The remainders of three groups were 53, 43 and 40 cases respectively used for verify the results of gene chip by real-time fluorescence quantitative PCR (RT-PCR). And 20 cases in severe group, 20 in mild group and 8 in control group were used to verify the expression level of jun oncogene (JUN) on behalf of differential expressed genes. Analysis of variance and non-parametric tests were used for statistic difference analysis among three groups. RESULTS: There were 406 differentially expressed genes for severe and mild groups. There were 264 down-regulated gene and 142 up-regulated ones. The down-regulated genes were predominant. Cluster analysis show the similarity of gene expression profile in the same group . The result confirmed that the gene chip experiments were both repeatable and reliable. According to gene ontology, the differentially expressed genes were mainly involved in such biological processes as immune response, signal transduction, regulation of transcription (DNA-dependent), inflammatory response, antigen processing and presentation and chemotaxis, etc. Pathway analysis showed differentially expressed genes were involved in 22 pathways of immune response and inflammation. The major pathways included B cell receptor signaling, antigen processing and presentation, Toll-like receptor signaling, MAPK signaling and transforming growth factor-beta (TGF-ß) signaling.Real-time fluorescence quantitative PCR (RT-PCR) analysis showed that the statistics of optical density for JUN was P < 0.001 in severe versus mild group. It was down-regulated in severe group. And the expression of JUN was conformed with the result of gene expression chip. CONCLUSIONS: The patients of severe group have a larger number of differential expressed genes versus those of mild group. And severe lung tissue damage in severe group may be correlated with differences in gene expression.

Diagnostic performance of multiplex cytokine and chemokine assay for tuberculosis.

Simultaneous detection of multiple biomarkers might lead to improved diagnostic performance for Mycobacterium tuberculosis infection. In this study, we screened soluble biomarkers that had significant differences in patients with active tuberculosis and healthy controls and evaluated the diagnostic performance of the multiplex cytokine/chemokine assay. Overall, 178 patients with active pulmonary tuberculosis, 156 healthy individuals and 35 patients with bacterial pneumonia or lung cancer were evaluated. Among the 16 soluble biomarkers screened by the microbead-based multiplex assay, five cytokines/chemokines including IFN-γ, IP-10, MIG, TNF-α and IL-2 that showed most significant differences between active pulmonary tuberculosis patients and healthy controls were selected for further analysis. When analyzed individually, both IP-10 and MIG had sensitivity and specificity comparable to IFN-γ in detection of active TB. Combined detection of IFN-γ, IP-10 and MIG had significantly improved sensitivity and specificity as compared with individual cytokine and chemokine detection. The responsive levels of IFN-γ, IP-10, MIG, TNF-α and IL-2 were significantly lower in re-treatment pulmonary tuberculosis patients than in new tuberculosis patients. It is concluded that combined IFN-γ, IP-10, MIG multiplex detection had better diagnostic performance for tuberculosis than the individual cytokine/chemokine assays. The re-treatment pulmonary tuberculosis patients had poor responses to ESAT-6/CFP-10 peptides stimulation.

[Prepared the monoclonal antibody associated with hepatocellular carcinoma and analyzed its epitope].

AIM: To prepare the monoclonal antibodies associated with hepatocellular carcinoma (HCC) for diagnosis. METHODS: 3 BALB/c mice were immunized with high metastasis characteristic HCC cells (HCCLM-6). The splenocytes from the immunized mice were fused with murine myeloma cells (Sp2/0). The hybridoma cells were screened by ELISA after coating with the total protein and the culture supernatant of HCCLM-6 cells. The mAbs were characterized by immunohistochemical staining in the HCC tissues, and by indirect immunofIuorescent staining in different cell lines. The antigen and epitope recognized by the mAbs were identified by the screening premade Uni-ZAP human liver cDNA expression library. RESULTS: Twenty-eight hybridoma cells secreting mAbs were established. One clone of the hybridomas, QGA062, secreted specific mAb associated with HCC. The antigen recognized by the mAb QGA062 was identified as fibronectin (FN), and the epitope was localized among the peptide YTVSLVAIKGNQESPK. CONCLUSION: The mAb against a HCC-associated epitope in FN is established and characterized, will be a very useful reagent for diagnosis of HCC.

Recombinant heat shock protein 65 carrying hepatitis B core antigen induces HBcAg-specific CTL response.

Many studies have provided evidence that heat shock protein 65 (Hsp65) can elicit potent specific cellular adaptive immune responses (e.g. CD8(+) cytotoxic T-cell effectors or classic CTLs) based on their ability to chaperone antigenic peptides. Hsp65 is thus an effective carrier for heterologous peptide epitopes for therapeutic vaccines against cancer or chronic infectious diseases. The core antigen of hepatitis B virus (HBcAg) is extremely immunogenic, and functions as both a T-cell-dependent and a T-cell-independent antigen. Therefore, HBcAg may be a promising candidate target for therapeutic vaccine control of chronic HBV infection. Here, a chimeric protein, Hsp65Bc, was created by fusing the HBcAg sequence to the carboxyl terminus of the Hsp65 sequence in E. coli. Analysis of its antigenicity and immunogenicity revealed that HBc epitopes are surface accessible. Hsp65Bc induced moderate anti-HBc immune responses as well as a strong specific T-cell response in BALB/c mice. These results indicate that Hsp65Bc may have potential as a vaccine for treatment of HBV chronic infection.

Construction and evaluation of anti-gastrin immunogen based on P64K protein.

AIM: To construct two kinds of anti-gastrin immunogen based on P64K protein from Neisseria meningitids and to compare their immunogenic effect. METHODS: G17P64K gene was cloned and ligated into pET28a plasmid, then transformed into BL21(DE3). After inoculation of LB medium and IPTG induction, the recombinant protein was solubly expressed at a high level. The purification of G17P64K fusion protein was similar to that of P64K. An initial step of purification consisting of 30% saturated ammonium sulfate precipitation was done. Additional fine optimizations included phenyl-sepharose, G200 Sephadex gel filtration and Q-sepharose anion exchanger chromatography. Highly purified protein was obtained and sequenced at the N-terminal amino acid residues. Polypeptide was synthesized by Fmoc solid phase chemical method and cross-linked to carrier protein P64K and DT mutant by MBS method and then the rabbit anti-gastrin 17 antibody was prepared by immunizing rabbit with cross-linked and fused protein. The titer and the activity in vitro of antibody were assessed. RESULTS: G17P64K gene and the recombinant bacteria were obtained. After four steps purification, protein sample that has the purity above 90% was achieved. At the 84(th) day after the first immunization, the titer of antibody against cross-linked protein reached 51,200. Evaluation of the antibody in vitro manifested that it had a high inhibitory activity on the growth of tumor cell SW480. CONCLUSION: The P64K-polypeptide cross-linked immunogen immunized rabbit and achieved a higher titer antibody against gastrin 17 than the G17P64K fusion protein immunogen, which could inhibit the growth of the tumor cell SW480.