Loss of Bcl-6-Expressing T Follicular Helper Cells and Germinal Centers in COVID-19.

Humoral responses in coronavirus disease 2019 (COVID-19) are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined post mortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers and a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ TFH cell differentiation together with an increase in T-bet+ TH1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+ TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections, and suggest that achieving herd immunity through natural infection may be difficult.

IL-27 Gene Therapy Ameliorates IPEX Syndrome Caused by Germline Mutation of Foxp3 Gene: A Major Role for Induction of IL-10.

Inactivating mutations of Foxp3, the master regulator of regulatory T cell development and function, lead to immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome in mice and humans. IPEX is a fatal autoimmune disease, with allogeneic stem cell transplant being the only available therapy. In this study, we report that a single dose of adeno-associated virus (AAV)-IL-27 to young mice with naturally occurring Foxp3 mutation (Scurfy mice) substantially ameliorates clinical symptoms, including growth retardation and early fatality. Correspondingly, AAV-IL-27 gene therapy significantly prevented naive T cell activation, as manifested by downregulation of CD62L and upregulation of CD44, and immunopathology typical of IPEX. Because IL-27 is known to induce IL-10, a key effector molecule of regulatory T cells, we evaluated the contribution of IL-10 induction by crossing IL-10-null allele to Scurfy mice. Although IL-10 deficiency does not affect the survival of Scurfy mice, it largely abrogated the therapeutic effect of AAV-IL-27. Our study revealed a major role for IL-10 in AAV-IL-27 gene therapy and demonstrated that IPEX is amenable to gene therapy.

IL-27 Gene Therapy Induces Stat3-Mediated Expansion of CD11b+Gr1+ Myeloid Cells and Promotes Accumulation of M1 Macrophages in the Tumor Microenvironment.

IL-27 is a pleiotropic cytokine that exhibits stimulatory/regulatory functions on multiple lineages of immune cells and has a potential to be used as a therapeutic for cancer. We have recently demonstrated that administration of IL-27 producing adeno-associated virus (AAV-IL-27) exhibits potent inhibition of tumor growth in mouse models. In this study, we demonstrate that AAV-IL-27 treatment leads to significant expansion of CD11b+Gr1+ myeloid cells. AAV-IL-27-induced expansion of CD11b+Gr1+ cells is IL-27R-dependent and requires Stat3 signaling, but it is inhibited by Stat1 signaling. AAV-IL-27 treatment does not increase the self-renewal capacity of CD11b+Gr1+ cells but induces significant expansion of Lin-Sca1+c-Kit+ (LSK) and granulocyte-monocyte progenitor cells. Despite exhibiting significant suppression of T cells in vitro, IL-27-induced CD11b+Gr1+ cells lost the tumor-promoting activity in vivo and overall play an antitumor role. In tumors from AAV-IL-27-treated mice, CD11b+Gr1+ cells are largely F4/80+ and express high levels of MHC class I/II and M1 macrophage markers. Thus, IL-27 gene therapy induces Stat3-mediated expansion of CD11b+Gr1+ myeloid cells and promotes accumulation of M1 macrophages in the tumor microenvironment.

ANXA1 is identified as a key gene associated with high risk and T cell infiltration in primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease, with unclear pathogenesis. Although immune disorders, especially T cell infiltration, are thought to play a vital role in PSC, the specific pathogenesis mechanisms remain incompletely understood. This study evaluated the potential key gene associated with the PSC pathogenesis and analyzed the associations of the key gene with prognosis and immune cell infiltration by combining bioinformatics analysis and experimental verification. METHODS: Transcriptome data of PSC and normal human liver tissues (GSE159676) were obtained from the gene expression omnibus database. Differentially expressed genes (DEGs) were identified, and differences in biological states were analyzed. A protein-protein interaction (PPI) network was constructed. Hub genes were identified, and their expression was verified using transcriptome data of mice fed 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and Mdr2-/- mice (GSE179993, GSE80776), as well as by immunohistochemistry staining on clinical samples. The correlations between the key gene and other factors were evaluated by Pearson's correlation coefficient. Immune cell infiltration into human liver (GSE159676) was analyzed by xCell and verified by immunofluorescence staining on PSC liver samples. RESULTS: Of the 185 DEGs identified, 113 were upregulated and 72 were downregulated genes in PSC. Genes associated with immune cell infiltration and fibrosis were significantly enriched in PSC. PPI network showed close interactions among DEGs. A module strongly associated with immune infiltration was identified, with annexin A1 (ANXA1) being the core gene. High expression of ANXA1 in PSC was confirmed in two public datasets and by immunohistochemistry staining on clinical samples. High ANXA1 expression was strongly associated with high-risk score for PSC. Also, ANXA1 expression was positively associated with chemokines and chemokine receptors and with the infiltration of immune cells, especially T cells, into liver with PSC. Immune infiltration, fibrosis, and cancer-related processes were markedly enriched in PSC with high expression of ANXA1. CONCLUSION: ANXA1 is a key gene associated with high risk and infiltration of immune cells, especially T cells, in PSC. These findings provide new insight into the key biomarker of PSC and suggest that targeting ANXA1 may be a valuable strategy for the treatment of PSC.

IL-27 Induces CCL5 Production by T Lymphocytes, Which Contributes to Antitumor Activity.

IL-27 is a pleiotropic cytokine that exhibits stimulatory/regulatory functions on multiple lineages of immune cells including T lymphocytes. In this study, we demonstrate that IL-27 directly induces CCL5 production by T lymphocytes, particularly CD8+ T cells in vitro and in vivo. IL-27-induced CCL5 production is IL-27R-dependent. In CD4+ T cells, IL-27-induced CCL5 production was primarily dependent on Stat1 activation, whereas in CD8+ T cells, Stat1 deficiency does not abrogate CCL5 induction. A chromatin immunoprecipitation assay revealed that in the CCL5 promoter region, both putative Stat3 binding sites exhibit significant binding to Stat3, whereas only one out of four Stat1 binding sites displays moderate binding to Stat1. In tumor-bearing mice, IL-27 induced dramatic production of CCL5 in tumor-infiltrating T cells. IL-27-induced CCL5 appears to contribute to an IL-27-mediated antitumor effect. This is signified by diminished tumor inhibition in anti-CCL5- and IL-27-treated mice. Additionally, intratumor delivery of CCL5 mRNA using lipid nanoparticles significantly inhibited tumor growth. Thus, IL-27 induces robust CCL5 production by T cells, which contributes to antitumor activity.

Role of miR-219a-5p in regulating NMDAR in nonylphenol-induced synaptic plasticity damage.

Nonylphenol (NP) is a typical environmental endocrine disruptor with estrogenic effects. It serves as an emulsifier and as the main ingredient of detergents and has become an increasingly common pollutant in both fresh and salt water, vegetables, and fruits. This study aimed to clarify whether NP exposure could lead to cognitive dysfunction and synaptic plasticity impairment, and also explore the mechanism of microRNA (miR)- 219a-5p regulation of N-methyl-D-aspartate receptor (NMDAR) in NP-induced synaptic plasticity impairment in vivo and in vitro. In vivo, 30 male Sprague-Dawley rats were randomly divided into 2 groups: blank control group (pure corn oil) and NP-exposed group [NP 80 mg/(kg·d)], with 15 rats in each group. In vitro, the extracted hippocampal neurons were divided into 6 groups: blank control group, mimics NC group, miR-219 mimics group, NP group (70 µmol/L NP), NP + mimics NC group, and NP + miR-219 mimics group. In vivo, the content of NP in hippocampal tissues after 90 days of NP exposure was significantly higher in the NP-stained group than in the blank control group. NP exposure could lead to a decrease in the ability to learn and memory, ability to remember, and space spatial memory ability in rats. The dendrites in the NP-stained group were disordered, with few dendritic spines and significantly decreased dendritic spine density. The postsynaptic densities were loosely arranged, the thickness and length of the postsynaptic densities shortened, and the length and width of the synaptic gap increased. Glutamine (Glu) and γ-aminobutyric acid (GABA) contents in hippocampal tissues decreased in the NP-stained group. The expression of miR-219a-5p mRNA decreased in the NP-stained group after 3 months of NP exposure. The expression of NMDAR1, NMDAR2A, NMDAR2B, nerve growth-associated protein (GAP-43), and Ca/calmodulin-dependent kinase II (CaMKII) mRNA/proteins decreased in the NP-stained group. In vitro, NMDAR protein expression decreased, while GAP-43 and CaMKII protein expression increased in the miR-219 mimics group compared with the control group. The expression levels of NMDAR and GAP-43 and CaMKII proteins were higher in the NP + miR-219 mimics group compared with the NP group. The levels of neurotransmitters Glu and GABA decreased in the NP and NP + mimics NC groups compared with the blank group. Shortened synaptic active band length, decreased thickness of postsynaptic densities, and shortened length of postsynaptic densities were observed in the NP, NP + mimics NC, and NP + miR-219 mimics groups compared with the blank control group. In vivo, NP exposure reduced learning memory capacity and neurotransmitter content in rats and caused a decrease in dendritic spine density and synaptic number density and a decrease in miR-219a-5p expression. In vitro, high expression of miR-219a-5p inhibited the expression of NMDAR, thus reducing the effect of NP on synaptic plasticity impairment in hippocampal neurons. Our study provided a scientific basis for the prevention of cognitive impairment owing to NP exposure and the development of targeted drug treatment strategies.

Clinicopathologic characteristics of liver inflammation and fibrosis in 310 patients with chronic hepatitis B. / 310ä¾‹æ ¢æ€§ä¹™åž‹è‚ç‚Žæ‚£è€ è‚è„ç‚Žç—‡åŠçº¤ç»´åŒ–çš„ä¸´åºŠç— ç†ç‰¹å¾.

OBJECTIVES: Long-term hepatitis B virus (HBV) infection can cause recurrent inflammation in the liver, and then develop into liver fibrosis, cirrhosis, and liver cancer. The hepatic pathological change is one of the important criteria for guiding antiviral therapy in patients with chronic hepatitis B (CHB). Due to the limitations of liver biopsy, it is necessary to find valuable non-invasive indicators to evaluate the hepatic pathological changes in CHB patients and guide the antiviral therapy. This study aims to analyze the clinical characteristics of different pathological changes in CHB patients, and to explore the factors influnencing the degree of liver inflammation and fibrosis in CHB patients with normal alanine aminotransferase (ALT). METHODS: This retrospective study was conducted on 310 CHB patients. Liver biopsy was performed in all these patients. The clinical data of the patients were collected. The liver biopsy pathological results were used as the gold standard to analyze the relationship between clinical indicators and liver pathological changes. Then CHB patients with normal ALT were screened, and the independent factors influencing the degree of liver inflammation and fibrosis were explored. RESULTS: Among the 310 patients with CHB, there were 249 (80.3%) patients with significant liver inflammation [liver inflammation grade (G) ≥2] and 119 (38.4%) patients with significant liver fibrosis [liver fibrosis stage (S) ≥2]. The results of univariate analysis of total samples showed that the ALT, γ-glutamyl transferase, alkaline phosphatase, and HBV DNA were related to the significant liver pathological changes. Among the 132 CHB patients with normal ALT, the patients with liver pathology G/S≥2, G≥2, and S≥2 were 80.3% (106/132), 68.2% (90/132), and 43.2% (57/132), respectively. The results showed that the independent influencing factor of significant liver inflammation was HBV DNA>2 000 U/mL (OR=3.592, 95% CI 1.534 to 8.409), and the independent influencing factors of significant liver fibrosis were elevated alkaline phosphatase level (OR=1.022, 95% CI 1.002 to 1.043), decreased platelet count (OR=0.990, 95% CI 0.982 to 0.998), and positive in hepatitis B e antigen (HBeAg) (OR=14.845, 95% CI 4.898 to 44.995). According to the multivariate analysis, a diagnostic model for significant liver fibrosis in CHB patients with normal ALT was established, and the area under the receiver operating characteristic curve was 0.844 (95% CI 0.779 to 0.910). CONCLUSIONS: The liver pathological changes should be evaluated in combination with different clinical indicators. A considerable number of CHB patients with normal ALT still have significant liver pathological changes, which need to be identified and treated with antiviral therapy in time. Among them, HBV DNA>2 000 U/mL suggests the significant liver inflammation, and the diagnostic model for significant liver fibrosis based on alkaline phosphatase, platelet count, and HBeAg can help to evaluate the degree of liver fibrosis.

Novel CRISPR/Cas9-mediated knockout of LIG4 increases efficiency of site-specific integration in Chinese hamster ovary cell line.

AIM: To investigate the impact of deficiency of LIG4 gene on site-specific integration in CHO cells. RESULTS: CHO cells are considered the most valuable mammalian cells in the manufacture of biological medicines, and genetic engineering of CHO cells can improve product yield and stability. The traditional method of inserting foreign genes by random integration (RI) requires multiple rounds of screening and selection, which may lead to location effects and gene silencing, making it difficult to obtain stable, high-yielding cell lines. Although site-specific integration (SSI) techniques may overcome the challenges with RI, its feasibility is limited by the very low efficiency of the technique. Recently, SSI efficiency has been enhanced in other mammalian cell types by inhibiting DNA ligase IV (Lig4) activity, which is indispensable in DNA double-strand break repair by NHEJ. However, this approach has not been evaluated in CHO cells. In this study, the LIG4 gene was knocked out of CHO cells using CRISPR/Cas9-mediated genome editing. Efficiency of gene targeting in LIG4-/--CHO cell lines was estimated by a green fluorescence protein promoterless reporter system. Notably, the RI efficiency, most likely mediated by NHEJ in CHO, was inhibited by LIG4 knockout, whereas SSI efficiency strongly increased 9.2-fold under the precise control of the promoter in the ROSA26 site in LIG4-/--CHO cells. Moreover, deletion of LIG4 had no obvious side effects on CHO cell proliferation. CONCLUSIONS: Deficiency of LIG4 represents a feasible strategy to improve SSI efficiency and suggests it can be applied to develop and engineer CHO cell lines in the future.

Comparison of tenofovir alafenamide and entecavir for hepatitis B virus-related acute-on-chronic liver failure. / å¯Œé©¬é ¸ä¸™é šæ›¿è¯ºç¦éŸ¦ä¸Žæ©æ›¿å¡éŸ¦æ²»ç–—ä¹™åž‹è‚ç‚Žç— æ¯’ç›¸å ³æ ¢åŠ æ€¥æ€§è‚è¡°ç«­çš„æ•ˆæžœå¯¹æ¯”.

OBJECTIVES: Hepatitis B virus related acute-on-chronic liver failure (HBV-ACLF) is the most common type of liver failure in China, with a high mortality. Early rapid reduction of HBV-DNA load can improve the survival rate of HBV-ACLF patients. At present, the commonly used drugs are nucleoside (acid) analogues, such as entecavir (ETV), tenofovir, and so on. The newly listed tenofovir alafenamide fumarate (TAF) has attracted great attention of clinicians because of its stronger antiviral effect, higher transaminase normalization rate, better bone and kidney safety, and zero drug resistance. However, there are few clinical research data on the efficacy and safety of TAF in the treatment of Chinese HBV-ACLF patients, and there is a lack of pharmacoeconomic evaluation. This study aims to compare the efficacy, safety, and cost-effectiveness between TAF and ETV in patients with HBV-ACLF. METHODS: The data were collected from 196 HBV-ACLF patients (80 patients in the TAF group and 116 patients in the ETV group) who were hospitalized in Xiangya Hospital, Central South University from May 2020 to March 2021. Biochemistry and virology were detected before and after treatment (at baseline, Week 2, 4, and 12). Clinical features, disease prognosis, and cost-effectiveness were compared between the 2 groups. According to the baseline, HBV-ACLF patients were divided into 4 stages including pre-liver failure stage, early stage, medium stage, and end stage. And the liver transplantation rate and mortality was also compared. Pharmacoeconomic evaluation was taken using cost-effectiveness analysis and cost minimization analysis．. RESULTS: After 4 weeks of treatment, there were no significant differences in the efficacy (liver function, viral load) between the 2 groups (all P>0.05). The TAF group showed lower creatinine [(80.35±18.77) µmol/L vs (105.59±82.32) µmol/L, P<0.05] and higher estimated glomerular filtration rate (eGFR) levels [(95.65±23.21) mL/(min·1.73 m2) vs (82.68±26.32) mL/(min·1.73 m2), P<0.05] than the ETV group. After 12 weeks of treatment, the analysis of overall the liver transplantation rate and mortality between the 2 groups showed similar conclusion. However, the TAF group had a lower the liver transplantation rate and mortality than the ETV group in patients with pre-liver failure (0vs13.89%, P<0.05). No evident distinction was found in the liver transplantation rate and mortality during the early, medium, or end stages of liver failure (13.04% vs 17.65%, 37.50% vs 37.04%, and 54.55% vs 68.42%, respectively). Ratio of cost to effectiveness in the ETV group was higher than that in the TAF group. CONCLUSIONS: TAF is not more efficient than ETV group in improving liver function and reducing viral load for HBV-ACLF patients and they also show similar safety. However, TAF has a greater advantage over ETV not only in preserving renal function, but also in reducing the liver transplantation rate and mortality in patients with pre-liver failure. TAF can provide economic benefit to patients with HBV-ACLF.

Clinical features and etiological analysis for 157 cases of pyogenic liver abscess. / 157ä¾‹ç»†èŒæ€§è‚è„“è‚¿çš„ä¸´åºŠç‰¹ç‚¹åŠç— åŽŸå­¦åˆ†æž.

OBJECTIVES: To analyze the clinical features and the pathogenic constituent of patients with pyogenic liver abscess (PLA), and to find the risk factors of the multidrug resistance (MDR) pyogenic liver abscess (MDR-PLA) for effective therapy in the PLA patients. METHODS: We reviewed the PLA patients with antibiotics susceptibility test, who admitted to Xiangya Hospital of Central South University from Jan. 2010 to Dec. 2019. A total of 157 cases were divided into 2 groups: an MDR-PLA group (n=52) and a non-MDR-PLA group (n=107). The clinical data such as age, symptoms, laboratory and imaging data, and etiological test especially drug sensitivity test of the 2 groups were collected. Logistic regression analysis was performed for the risk factors of MDR-PLA. RESULTS: Anorexia (90.38%) and abdominal pain (63.46%) were more common in the MDR-PLA group than those in the non-MDR-PLA group (P<0.05). The proportions of patients with hepatolithiasis (34.62%) and biliary tract operation (38.62%) were higher in the MDR-PLA group than those in the non-MDR-PLA group (P<0.05). Klebsiellapneumoniae (59.94%) was the most common pathogen, which was sensitive to most of the antibiotics, while Escherichia coli and Enterococcus had lower drug sensitivity than Klebsiella lebsiella pneumoniae. The MDR-Enterococcus was 6.5 times of non-MDR-Enterococcus. And the multidrug resistance Klebsiella pneumoniae was 3.4 times of non-MDR-Klebsiella pneumoniae. Logistic regression analysis showed that hepatolithiasis (OR=4.895, 95% CI 1.455 to 16.463, P=0.001) and biliary tract operation (OR=3.860, 95% CI 1.156 to 12.889, P=0.004) were the risk factors for the MDR-PLA patients. CONCLUSIONS: The PLA patients with hepatolithiasis and billary tract operation history and the pathogenic bacterium of Escherichia coli and Enterococcus bacteria should be alerted for MDR bacterial infection.

CD200-CD200R Pathway in the Regulation of Tumor Immune Microenvironment and Immunotherapy.

Tumor-associated inflammation and immune responses are key components in the tumor microenvironment (TME) which regulate tumor growth, progression, and metastasis. Tumor-associated myeloid cells (TAMCs) are a group of cells that play multiple key roles including induction of tumor-associated inflammation/angiogenesis and regulation of tumor-specific T-cell responses. Thus, identification and characterization of key pathways that can regulate TAMCs are of critical importance for developing cancer immunotherapy. Recent studies suggest that CD200-CD200 receptor (CD200R) interaction may be important in regulating the TME via affecting TAMCs. In this chapter, we will give a brief overview of the CD200-CD200R axis, including the biology behind CD200-CD200R interaction and the role(s) it plays in tumor microenvironment and tumor growth, and activation/effector functions of T cells. We will also discuss CD200-CD200R's role as potential checkpoint molecules for cancer immunotherapy. Further investigation of the CD200-CD200R pathway will not only advance our understanding of tumor pathogenesis and immunity but also provide the rationale for CD200-CD200R-targeted immunotherapy of human cancer.

A Critical Role for CD200R Signaling in Limiting the Growth and Metastasis of CD200+ Melanoma.

CD200 is a cell surface glycoprotein that functions through engaging CD200R on cells of the myeloid lineage and inhibits their functions. Expression of CD200 was implicated in a variety of human cancer cells, including melanoma cells; however, its roles in tumor growth and immunity are not clearly understood. In this study, we used CD200R-deficient mice and the B16 tumor model to evaluate this issue. We found that CD200R-deficient mice exhibited accelerated growth of CD200(+), but not CD200(-), B16 tumors. Strikingly, CD200R-deficient mice receiving CD200(+) B16 cells i.v. exhibited massive tumor growth in multiple organs, including liver, lung, kidney, and peritoneal cavity, whereas the growth of the same tumors in wild-type mice was limited. CD200(+) tumors grown in CD200R-deficient mice contained higher numbers of CD11b(+)Ly6C(+) myeloid cells, exhibited increased expression of VEGF and HIF1α genes with increased angiogenesis, and showed significantly reduced infiltration of CD4(+) and CD8(+) T cells, presumably as the result of reduced expression of T cell chemokines, such as CXCL9 and CXCL16. The liver from CD200R-deficient mice, under metastatic growth of CD200(+) tumors, contained significantly increased numbers of CD11b(+)Gr1(-) myeloid cells and Foxp3(+) regulatory T cells and reduced numbers of NK cells. Liver T cells also had a reduced capacity to produce IFN-γ or TNF-α. Taken together, we revealed a critical role for CD200R signaling in limiting the growth and metastasis of CD200(+) tumors. Thus, targeting CD200R signaling may potentially interfere with the metastatic growth of CD200(+) tumors, like melanoma.

Interleukin-27 signalling induces stem cell antigen-1 expression in T lymphocytes in vivo.

Stem cell antigen-1 (Sca-1/Ly6A/E) is a cell surface glycoprotein that is often used as a biomarker for stem cells and cell stemness. However, it is not clear what factors can directly induce the expression of Sca-1/Ly6A/E in T lymphocytes in vivo, and if induction of Sca-1 is associated with T cell stemness. In this study, we show that interleukin-27 (IL-27), a member of the IL-12 family of cytokines, directly induces Sca-1 expression in T cells in vivo. We found that mice-deficient for IL-27 (either P28 or EBI3) or its signalling (IL-27Rα) had profound reduction of Sca-1 expression in naive (CD62L+  CD44- ), memory (CD62L+  CD44+ ) and effector (CD62L-  CD44+ ) T cells. In contrast, in vivo delivery of IL-27 using adeno-associated viral vectors strongly induced the expression of Sca-1 in naive and memory/effector T-cell populations in an IL-27 receptor- or signal transducer and activator of transcription 1-dependent manner. Interestingly, IL-27-induced Sca-1+ T cells do not express or up-regulate classic stem cell-associated genes such as Nanog, Oct4, Sox2 and Ctnnb1. However, IL-27-induced Sca-1+ T cells had increased expression of effector/memory-associated transcription factor T-bet, Eomes and Blimp1. Hence, IL-27 signalling directly induces the expression of Sca-1/Ly6A/E expression in T cells. Direct expansion of Sca-1+  CD62L+  CD44- T memory stem cells may explain why IL-27 enhances T-cell memory.

Tumor-derived IL-35 promotes tumor growth by enhancing myeloid cell accumulation and angiogenesis.

IL-35 is a member of the IL-12 family of cytokines that is comprised of an IL-12 p35 subunit and an IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3). IL-35 functions through IL-35R and has a potent immune-suppressive activity. Although IL-35 was demonstrated to be produced by regulatory T cells, gene-expression analysis revealed that it is likely to have a wider distribution, including expression in cancer cells. In this study, we demonstrated that IL-35 is produced in human cancer tissues, such as large B cell lymphoma, nasopharyngeal carcinoma, and melanoma. To determine the roles of tumor-derived IL-35 in tumorigenesis and tumor immunity, we generated IL-35-producing plasmacytoma J558 and B16 melanoma cells and observed that the expression of IL-35 in cancer cells does not affect their growth and survival in vitro, but it stimulates tumorigenesis in both immune-competent and Rag1/2-deficient mice. Tumor-derived IL-35 increases CD11b(+)Gr1(+) myeloid cell accumulation in the tumor microenvironment and, thereby, promotes tumor angiogenesis. In immune-competent mice, spontaneous CTL responses to tumors are diminished. IL-35 does not directly inhibit tumor Ag-specific CD8(+) T cell activation, differentiation, and effector functions. However, IL-35-treated cancer cells had increased expression of gp130 and reduced sensitivity to CTL destruction. Thus, our study indicates novel functions for IL-35 in promoting tumor growth via the enhancement of myeloid cell accumulation, tumor angiogenesis, and suppression of tumor immunity.

IL-27 enhances the survival of tumor antigen-specific CD8+ T cells and programs them into IL-10-producing, memory precursor-like effector cells.

IL-27 is a member of the IL-12 family of cytokines that is comprised of an IL-12 p40-related protein subunit, EBV-induced gene 3, and a p35-related subunit, p28. IL-27 functions through IL-27R and has been shown to have potent antitumor activity via activation of a variety of cellular components, including antitumor CD8(+) T-cell responses. However, the exact mechanisms of how IL-27 enhances antitumor CD8(+) T-cell responses remain unclear. Here we show that IL-27 significantly enhances the survival of activated tumor antigen-specific CD8(+) T cells in vitro and in vivo, and programs tumor antigen-specific CD8(+) T cells into memory precursor-like effector cells, characterized by upregulation of Bcl-6, SOCS3, Sca-1, and IL-10. While STAT3 activation and the CTL survival-enhancing effects can be independent of CTL IL-10 production, we show here that IL-27-induced CTL IL-10 production contributes to memory precursor cell phenotype induction, CTL memory, and tumor rejection. Thus, IL-27 enhances antitumor CTL responses via programming tumor antigen-specific CD8(+) T cells into a unique memory precursor type of effector cells characterized by a greater survival advantage. Our results have important implications for designing immunotherapy against human cancer.

Increased Th17 and regulatory T cell responses in EBV-induced gene 3-deficient mice lead to marginally enhanced development of autoimmune encephalomyelitis.

EBV-induced gene 3 (EBI3)-encoded protein can form heterodimers with IL-27P28 and IL-12P35 to form IL-27 and IL-35. IL-27 and IL-35 may influence autoimmunity by inhibiting Th17 differentiation and facilitating the inhibitory roles of Foxp3(+) regulatory T (Treg) cells, respectively. In this study, we have evaluated the development of experimental autoimmune encephalomyelitis (EAE) in EBI3-deficient mice that lack both IL-27 and IL-35. We found that myelin oligodendrocyte glycoprotein peptide immunization resulted in marginally enhanced EAE development in EBI3-deficient C57BL6 and 2D2 TCR-transgenic mice. EBI3 deficiency resulted in significantly increased Th17 and Th1 responses in the CNS and increased T cell production of IL-2 and IL-17 in the peripheral lymphoid organs. EBI3-deficient and -sufficient 2D2 T cells had equal ability in inducing EAE in Rag1(-/-) mice; however, more severe disease was induced in EBI3(-/-)Rag1(-/-) mice than in Rag1(-/-) mice by 2D2 T cells. EBI3-deficient mice had increased numbers of CD4(+)Foxp3(+) Treg cells in peripheral lymphoid organs. More strikingly, EBI3-deficient Treg cells had more potent suppressive functions in vitro and in vivo. Thus, our data support an inhibitory role for EBI3 in Th17, Th1, IL-2, and Treg responses. Although these observations are consistent with the known functions of IL-27, the IL-35 contribution to the suppressive functions of Treg cells is not evident in this model. Increased Treg responses in EBI3(-/-) mice may explain why the EAE development is only modestly enhanced compared with wild-type mice.

Role and mechanism of MiR-542-3p in regulating TLR4 in nonylphenol-induced neuronal cell pyroptosis.

BACKGROUND: This study aimed to investigate the spatial learning/memory and motor abilities of rats and the alteration of miR-542-3p and pyroptosis in the midbrain nigrostriatal area in vivo after nonylphenol (NP) gavage and to explore the mechanism of miR-542-3p regulation of Toll-like receptor 4 (TLR4) in NP-induced pyroptosis in BV2 microglia in vitro. METHODS: In vivo: Thirty-six specific-pathogen-free-grade Sprague-Dawley rats were divided into three equal groups: blank control group (treated with pure corn oil), NP group (treated with NP, 80 mg/kg body weight per day for 90 days), and positive control group [treated with lipopolysaccharide (LPS), 2 mg/kg body weight for 7 days]. In vitro: The first part of the experiment was divided into blank group (control, saline), LPS group [1 µg/ml + 1 mM adenosine triphosphate (ATP)], and NP group (40 µmol/L). The second part was divided into mimics NC (negative control) group, miR-542-3p mimics group, mimics NC + NP group, and miR-542-3p mimics + NP group. RESULTS: In vivo: Behaviorally, the spatial learning/memory and motor abilities of rats after NP exposure declined, as detected via Y-maze, open field, and rotarod tests. Some microglia in the substantia nigra of the NP-treated rats were activated. The downregulation of miR-542-3p was observed in rat brain tissue after NP exposure. The mRNA/protein expression of pyroptosis-related indicators (TLR4), NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), gasdermin-D (GSDMD), cysteinyl aspartate-specific proteinase-1 (caspase-1), and interleukin-1ß (IL-1ß) in the substantia nigra of the midbrain increased after NP exposure. In vitro: ASC fluorescence intensity increased in BV2 cells after NP exposure. The mRNA and/or protein expression of pyroptosis-related indicators (TLR4, NLRP3, GSDMD, caspase-1, and IL-1ß) in BV2 cells was upregulated after NP exposure. The transfection of miR-542-3p mimics inhibited NP-induced ASC expression in BV2 cells. The overexpression of miR-542-3p, followed by NP exposure, significantly reduced TLR4, NLRP3, ASC, caspase-1, and IL-1ß gene and/or protein expression. CONCLUSIONS: This study suggested that NP exposure caused a decline in spatial learning memory and whole-body motor ability in rats. Our study was novel in reporting that the upregulation of miR-542-3p targeting and regulating TLR4 could inhibit NLRP3 inflammatory activation and alleviate NP-induced microglia pyroptosis.

Characteristics of Klebsiella pneumoniae pyogenic liver abscess from 2010-2021 in a tertiary teaching hospital of South China.

OBJECTIVES: Pyogenic liver abscess (PLA) is a severe and potentially fatal infectious disease. Klebsiella pneumoniae (K. pneumoniae) is the predominant pathogen responsible for PLA. This study aims to investigate the clinical characteristics and prognostic factors of K. pneumoniae-induced pyogenic liver abscess (KP-PLA), particularly those caused by carbapenem-resistant K. pneumoniae (CRKP). METHODS: Analyses were performed on PLA patients from January 2010 to December 2021, to investigate the differences of K. pneumoniae from other etiologically infected PLA patients. Univariate and multivariate logistic regression analyses were used to compare prognostic factors between patients with carbapenem-resistant K. pneumoniae PLA (CRKP-PLA) and patients with carbapenem-sensitive K. pneumoniae PLA. RESULTS: Univariate analysis demonstrated a significant association between KP-PLA and factors including diabetes mellitus (P < 0.001), cholecystitis and cholelithiasis (P = 0.032), single abscess (P = 0.016), and abscesses with a diameter over 50 mm (P = 0.004). The CRKP group exhibited a higher prevalence of therapeutic interventions before K. pneumoniae infection, including abdominal surgery, mechanical ventilation, sputum suction, tracheal cannula, routine drainage of the abdominal cavity, and peripherally inserted central venous catheters (P < 0.05). Multivariate logistic regression analysis revealed that admission to the intensive care unit was an independent risk factor associated with CRKP-PLA (odds ratio 36; 95% confidence interval 1.77-731.56; P = 0.020). CONCLUSION: The KP-PLA patients were significantly associated with diabetes and were more likely to have single abscesses larger than 50 mm. PLA patients with a history of admission to intensive care unit or invasive therapeutic procedures should be given special consideration if combined with CRKP infection.

CD24 on thymic APCs regulates negative selection of myelin antigen-specific T lymphocytes.

Negative selection plays a key role in the clonal deletion of autoreactive T cells in the thymus. However, negative selection is incomplete; as high numbers of autoreactive T cells can be detected in normal individuals, mechanisms that regulate negative selection must exist. In this regard, we previously reported that CD24, a GPI-anchored glycoprotein, is required for thymic generation of autoreactive T lymphocytes. The CD24-deficient 2D2 TCR transgenic mice (2D2(+) CD24(-/-) ), whose TCR recognizes myelin oligodendrocyte glycoprotein (MOG), fail to generate functional 2D2 T cells. However, it was unclear if CD24 regulated negative selection, and if so, what cellular mechanisms were involved. Here, we show that elimination of MOG or Aire gene expression in 2D2(+) CD24(-/-) mice - through the creation of 2D2(+) CD24(-/-) MOG(-/-) or 2D2(+) CD24(/) ∼Aire(-/-) mice - completely restores thymic cellularity and function of 2D2 T cells. Restoration of CD24 expression on DCs, but not on thymocytes also partially restores 2D2 T-cell generation in 2D2(+) CD24(-/-) mice. Taken together, we propose that CD24 expression on thymic antigen-presenting cells (mTECs, DCs) down-regulates autoantigen-mediated clonal deletion of autoreactive thymocytes.