CD8+ tissue-resident memory T cells are expanded in primary Sjögren's disease and can be therapeutically targeted by CD103 blockade.

OBJECTIVE: Tissue-resident memory cells (Trm) are a subset of T cells residing persistently and long-term within specific tissues that contribute to persistent inflammation and tissue damage. We characterised the phenotype and function of Trm and the role of CD103 in primary Sjogren's syndrome (pSS). METHODS: In both pSS and non-pSS sicca syndrome patients, we examined Trm frequency, cytokine production in salivary glands (SG) and peripheral blood (PB). We also analysed Trm-related gene expression in SG biopsies through bulk and single-cell RNA sequencing (scRNAseq). Additionally, we investigated Trm properties in an immunisation-induced animal model of pSS (experimental SS, ESS) mouse model and assessed the effects of Trm inhibition via intraglandular anti-CD103 monoclonal antibody administration. RESULTS: Transcriptomic pSS SG showed an upregulation of genes associated with tissue recruitment and long-term survival of Trm cells, confirmed by a higher frequency of CD8+CD103+CD69+ cells in pSS SG, compared with non-specific sialadenitis (nSS). In SG, CD8+ CD103+ Trm contributed to the secretion of granzyme-B and interferon-γ, CD8+ Trm cells were localised within inflammatory infiltrates, where PD1+CD8+ T cells were also increased compared with nSS and MALT lymphoma. scRNAseq of PB and pSS SG T cells confirmed expression of CD69, ITGAE, GZMB, GZMK and HLA-DRB1 among CD3+CD8+ SG T cells. In the SG of ESS, CD8+CD69+CD103+ Trm producing Granzyme B progressively expanded. However, intraglandular blockade of CD103 in ESS reduced Trm, reduced glandular damage and improved salivary flow. CONCLUSIONS: CD103+CD8+Trm cells are expanded in the SG of pSS and ESS, participate in tissue inflammation and can be therapeutically targeted.

Establishment of a Zebrafish Xenograft Model for in Vivo Investigation of Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is a unique malignant tumor of the head and neck. Despite higher survival rates by the combination of radiotherapy and chemotherapy, the recurrence or metastasis of NPC still occurs at about 10%. Therefore, there is urgent demand to develop more effective in vivo models for preclinical trials to investigate the mechanisms of NPC development and progression and to explore better treatment approaches. In this study, we transplanted human NPC CNE1 cells into zebrafish embryos to establish a xenograft model of NPC, where the proliferation and invasion behaviors of NPC cells were investigated in vivo. Combining in vitro and in vivo analyses, we found that activating transcription factor 7 (ATF7) was involved in the occurrence and development of NPC regulated by peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1). The zebrafish NPC xenograft model established here thereby provides an in vivo tool for exploring the occurrence and development of NPC, which may help to identify new tumor markers and develop new therapeutic strategies for the treatment of NPC.

The Multiple Roles of B Cells in the Pathogenesis of Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease characterized by lymphocytic infiltration and tissue destruction of exocrine glands such as salivary glands. Although the formation of ectopic lymphoid tissue in exocrine glands and overproduction of autoantibodies by autoreactive B cells highlight the critical involvement of B cells in disease development, the precise roles of various B cell subsets in pSS pathogenesis remain partially understood. Current studies have identified several novel B cell subsets with multiple functions in pSS, among which autoreactive age-associated B cells, and plasma cells with augmented autoantibody production contribute to the disease progression. In addition, tissue-resident Fc Receptor-Like 4 (FcRL4)+ B cell subset with enhanced pro-inflammatory cytokine production serves as a key driver in pSS patients with mucosa-associated lymphoid tissue (MALT)-lymphomas. Recently, regulatory B (Breg) cells with impaired immunosuppressive functions are found negatively correlated with T follicular helper (Tfh) cells in pSS patients. Further studies have revealed a pivotal role of Breg cells in constraining Tfh response in autoimmune pathogenesis. This review provides an overview of recent advances in the identification of pathogenic B cell subsets and Breg cells, as well as new development of B-cell targeted therapies in pSS patients.

IL-17 drives salivary gland dysfunction via inhibiting TRPC1-mediated calcium movement in Sjögren's syndrome.

OBJECTIVES: This study aims to determine a role of interleukin-17A (IL-17) in salivary gland (SG) dysfunction and therapeutic effects of targeting IL-17 in SG for treating autoimmune sialadenitis in primary Sjögren's syndrome (pSS). METHODS: Salivary IL-17 levels and IL-17-secreting cells in labial glands of pSS patients were examined. Kinetic changes of IL-17-producing cells in SG from mice with experimental Sjögren's syndrome (ESS) were analysed. To determine a role of IL-17 in salivary secretion, IL-17-deficient mice and constructed chimeric mice with IL-17 receptor C (IL-17RC) deficiency in non-hematopoietic and hematopoietic cells were examined for saliva flow rates during ESS development. Both human and murine primary SG epithelial cells were treated with IL-17 for measuring cholinergic activation-induced calcium movement. Moreover, SG functions were assessed in ESS mice with salivary retrograde cannulation of IL-17 neutralisation antibodies. RESULTS: Increased salivary IL-17 levels were negatively correlated with saliva flow rates in pSS patients. Both IL-17-deficient mice and chimeric mice with non-hematopoietic cell-restricted IL-17RC deficiency exhibited no obvious salivary reduction while chimeric mice with hematopoietic cell-restricted IL-17RC deficiency showed significantly decreased saliva secretion during ESS development. In SG epithelial cells, IL-17 inhibited acetylcholine-induced calcium movement and downregulated the expression of transient receptor potential canonical 1 via promoting Nfkbiz mRNA stabilisation. Moreover, local IL-17 neutralisation in SG markedly attenuated hyposalivation and ameliorated tissue inflammation in ESS mice. CONCLUSION: These findings identify a novel function of IL-17 in driving salivary dysfunction during pSS development and may provide a new therapeutic strategy for targeting SG dysfunction in pSS patients.

Inflammasome and Its Therapeutic Targeting in Rheumatoid Arthritis.

Inflammasome is a cytoplasmic multiprotein complex that facilitates the clearance of exogenous microorganisms or the recognition of endogenous danger signals, which is critically involved in innate inflammatory response. Excessive or abnormal activation of inflammasomes has been shown to contribute to the development of various diseases including autoimmune diseases, neurodegenerative changes, and cancers. Rheumatoid arthritis (RA) is a chronic and complex autoimmune disease, in which inflammasome activation plays a pivotal role in immune dysregulation and joint inflammation. This review summarizes recent findings on inflammasome activation and its effector mechanisms in the pathogenesis of RA and potential development of therapeutic targeting of inflammasome for the immunotherapy of RA.

The Roles of Immune Cells in the Pathogenesis of Fibrosis.

Tissue injury and inflammatory response trigger the development of fibrosis in various diseases. It has been recognized that both innate and adaptive immune cells are important players with multifaceted functions in fibrogenesis. The activated immune cells produce various cytokines, modulate the differentiation and functions of myofibroblasts via diverse molecular mechanisms, and regulate fibrotic development. The immune cells exhibit differential functions during different stages of fibrotic diseases. In this review, we summarized recent advances in understanding the roles of immune cells in regulating fibrotic development and immune-based therapies in different disorders and discuss the underlying molecular mechanisms with a focus on mTOR and JAK-STAT signaling pathways.

TLR1/TLR2 signaling blocks the suppression of monocytic myeloid-derived suppressor cell by promoting its differentiation into M1-type macrophage.

Myeloid derived suppressor cells (MDSCs) play a key role in tumor immunosuppressive microenvironment, which helps tumors avoid immune destruction. Blocking the suppressive activities of MDSCs could be a promising strategy to enhance the effect of anti-tumor immunotherapies. In this study, we found that TLR1/TLR2 expression predicted favorable prognosis of lung cancer patients. In the related mice tumor model, TLR1/TLR2 activation by synthetic bacterial lipoprotein (BLP), a TLR1/2 agonist, greatly inhibited tumor growth and selectively decreased monocytic MDSCs (M-MDSCs). Furthermore, BLP treatment redirected M-MDSC differentiation towards M1 macrophage through JNK pathway, and thus blocked the suppressive activity of M-MDSCs in a TLR2-dependent manner. Therefore, our data demonstrated that TLR2 could be a promising biomarker and a potential immunotherapeutic target for lung cancer.

Deceleration of glycometabolism impedes IgG-producing B-cell-mediated tumor elimination by targeting SATB1.

B lymphocytes, known as antibody producers, mediate tumor cell destruction in the manner of antibody-dependent cell-mediated cytotoxicity; however, their anti-tumor function seems to be weakened during tumorigenesis, while the underlying mechanisms remain unclear. In this study, we found that IgG mediated anti-tumor effects, but IgG-producing B cells decreased in various tumors. Considering the underlying mechanism, glycometabolism was noteworthy. We found that tumor-infiltrating B cells were glucose-starved and accompanied by a deceleration of glycometabolism. Both inhibition of glycometabolism and deprivation of glucose through tumor cells, or glucose-free treatment, reduced the differentiation of B cells into IgG-producing cells. In this process, special AT-rich sequence-binding protein-1 (SATB1) was significantly silenced in B cells. Down-regulating SATB1 by inhibiting glycometabolism or RNA interference reduced the binding of signal transducer and activator of transcription 6 (STAT6) to the promoter of germline Cγ gene, subsequently resulting in fewer B cells producing IgG. Our findings provide the first evidence that glycometabolic inhibition by tumorigenesis suppresses differentiation of B cells into IgG-producing cells, and altering glycometabolism may be promising in improving the anti-tumor effect of B cells.

MicroRNAs 15A and 16-1 Activate Signaling Pathways That Mediate Chemotaxis of Immune Regulatory B cells to Colorectal Tumors.

BACKGROUND & AIMS: B cells infiltrate tumors, but little is known about how they affect tumor growth and progression. microRNA15A (MIR15A or miRNA15A) and microRNA16-1 (MIR16-1 or miRNA16-1) regulate cell proliferation, apoptosis, and drug resistance. We investigated their involvement in B-cell-mediated immune suppression by colorectal tumors. METHODS: Mice with disruptions of the gene cluster that encodes MIR15A and MIR16-1 (knockout mice), and control (C57BL/B6) mice were given azoxymethane with dextran sodium sulfate (AD) to induce formation of colorectal tumors. Mice were given anti-CD20 to delete B cells, or injections of agomir to increase MIR15A and MIR16-1. Proliferation of CD8+T cells was measured by carboxyfluorescein-succinimidyl-ester analysis. Colon tissues were collected from mice and analyzed by flow cytometry, microRNA (miRNA) sequencing, and for cytokine production. Intestinal epithelial cells (IECs) were isolated and transfected with miRNA mimics, to identify their targets. We analyzed miRNA expression patterns and quantified B cells in colorectal cancer tissue microarrays derived from 90 patients who underwent surgical resection, from July 2006 through April 2008, in Shanghai, China; expression data were compared with clinical outcomes. RESULTS: Tumors that developed in knockout mice following administration of AD were larger and contained greater numbers of B cells than tumors that grew in control mice. Most of the B cells in the tumors were positive for immunoglobulin A (IgA+). IgA+ B cells expressed high levels of immune regulatory molecules (programmed death ligand 1, interleukin 10, and transforming growth factor beta), and repressed the proliferation and activation of CD8+ T cells. Levels of MIR15A and MIR16-1 were reduced in colon tumors from mice, compared with nontumor colon tissue. Incubation of IECs with IL17A reduced expression of MIR15A and MIR16-1. Transgenic expression of MIR15A and MIR16-1 in IECs decreased activation of NF-κB and STAT1 by reducing expression of I-kappaB kinases; this resulted in reduced production of chemokine (C-X-C motif) ligands 9 and 10 and decreased chemotaxis of IgA+ B cells. Tumors in mice injected with AD and agomir grew more slowly than tumors in mice not given in agomir and contained fewer IgA+ B cells. We found a negative correlation between levels of MIR15A and MIR16-1 and numbers of IgA+B cells in human colorectal tumor tissues; high levels of MIR15A and MIR16-1 and low numbers of IgA+B cells were associated with longer survival times of patients. CONCLUSIONS: We found increased levels of MIR15A and MIR16-1 to reduce numbers of IgA+ B cells in colorectal tumor tissues and correlate with increased survival time of patients. In mice that lack MIR15A and MIR16-1, colon tumors grow more rapidly and contain increased numbers of IgA+ B cells. MIR15A and MIR16-1 appear to activate signaling pathways required for B-cell-mediated immune suppression.

MicroRNA 15a/16-1 suppresses aryl hydrocarbon receptor-dependent interleukin-22 secretion in CD4+ T cells and contributes to immune-mediated organ injury.

Interleukin-22 (IL-22), as a link between leukocytic and nonleukocytic cells, has gained increasing attention for its pronounced tissue-protective properties. MicroRNAs, emerging as crucial immune modulators, have been reported to be involved in the production and action of various cytokines. However, the precise control of IL-22 by microRNAs and its subsequent actions remained to be elucidated. In this study, we found a negative correlation between the expression of microRNA 15a/16-1 (miR-15a/16-1) and IL-22 in the model of concanavalin A-induced, immune-mediated liver injury. Knockout of miR-15a/16-1 ameliorated liver injury in an IL-22-dependent manner. Further results revealed that cluster of differentiation 4-positive (CD4+ ) T cells were the major source of IL-22 during liver injury and that the aryl hydrocarbon receptor was the direct target of miR-15a/16-1 in CD4+ T cells. In vivo and in vitro data showed that miR-15a/16-1 knockout CD4+ T cells produced more IL-22, while overexpression of miR-15a/16-1 down-regulated the IL-22 production by inhibiting the aryl hydrocarbon receptor. Moreover, transfer of miR-15a/16-1 knockout CD4+ T cells promoted tissue repair compared to wild-type CD4+ T cells by up-regulating IL-22. In addition, as a synergistic effect, IL-22 could down-regulate miR-15a/16-1 expression by activating phosphorylated signal transducer and activator of transcription 3-c-myc signaling, and the decrease of miR-15a/16-1 in damaged hepatocytes contributed to IL-22-mediated tissue repair by reducing cell apoptosis and promoting cell proliferation. As further proof, we demonstrated the role of miR-15a/16-1 in controlling IL-22 production and IL-22-mediated reconstruction of the intestinal epithelial barrier in a dextran sodium sulfate-induced colitis model. CONCLUSION: Our results suggest that miR-15a/16-1 acts as a essential regulator of IL-22 and that the miR-15a/16-1-aryl hydrocarbon receptor-IL-22 regulatory axis plays a central role in tissue repair; modulation of miR-15a/16-1 might hold promise in developing new strategies to enhance IL-22-mediated tissue repair. (Hepatology 2018;67:1027-1040).

STAT6 deficiency ameliorates Graves' disease severity by suppressing thyroid epithelial cell hyperplasia.

Signal transducer and activator of transcription 6 (STAT6) is involved in epithelial cell growth. However, little is known regarding the STAT6 phosphorylation status in Graves' disease (GD) and its role in thyroid epithelial cells (TECs). In this study, we found that STAT6 phosphorylation (p-STAT6) was significantly increased in TECs from both GD patients and experimental autoimmune Graves' disease mice and that STAT6 deficiency ameliorated GD symptoms. Autocrine IL-4 signalling in TECs activated the phosphorylation of STAT6 via IL-4 R engagement, and the downstream targets of STAT6 were Bcl-xL and cyclin D1. Thus, the IL-4-STAT6-Bcl-xL/cyclin D1 pathway is crucial for TEC hyperplasia, which aggravates GD. More importantly, in vitro and in vivo experiments demonstrated that STAT6 phosphorylation inhibited by AS1517499 decreased TEC hyperplasia, thereby reducing serum T3 and T4 and ameliorating GD. Thus, our study reveals that in addition to the traditional pathogenesis of GD, in which autoantibody TRAb stimulates thyroid-stimulating hormone receptors and consequently produces T3, T4, TRAb could also trigger TECs producing IL-4, and IL-4 then acts in an autocrine manner to activate p-STAT6 signalling and stimulate unrestricted cell growth, thus aggravating GD. These findings suggest that STAT6 inhibitors could be potent therapeutics for treating GD.

Biological Response Modifier in Cancer Immunotherapy.

Biological response modifiers (BRMs) emerge as a lay of new compounds or approaches used in improving cancer immunotherapy. Evidences highlight that cytokines, Toll-like receptor (TLR) signaling, and noncoding RNAs are of crucial roles in modulating antitumor immune response and cancer-related chronic inflammation, and BRMs based on them have been explored. In particular, besides some cytokines like IFN-α and IL-2, several Toll-like receptor (TLR) agonists like BCG, MPL, and imiquimod are also licensed to be used in patients with several malignancies nowadays, and the first artificial small noncoding RNA (microRNA) mimic, MXR34, has entered phase I clinical study against liver cancer, implying their potential application in cancer therapy. According to amounts of original data, this chapter will review the regulatory roles of TLR signaling, some noncoding RNAs, and several key cytokines in cancer and cancer-related immune response, as well as the clinical cases in cancer therapy based on them.

Eukaryotic translation initiation factor 3B accelerates the progression of esophageal squamous cell carcinoma by activating ß-catenin signaling pathway.

INTRODUCTION: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignant tumors. Eukaryotic translation initiation factors 3B (EIF3B) is considered to influence tumor proliferation, invasion, apoptosis and cell cycle, which act together to promote the progression of tumors. However, the role of EIF3B in ESCC is unknown. This study aims to explore the clinical and biological role of EIF3B in ESCC. RESULTS: EIF3B expressions were up-regulated in both ESCC tissues and cell lines. Overexpression of EIF3B was associated with tumor depth, lymph node metastasis and advanced TNM stage. Importantly, patients with high EIF3B expression suffered shorter overall and disease-free survival. Knockdown of EIF3B could inhibit cell proliferation and invasion, promote cell apoptosis, and interfere the cell cycle in vitro. EIF3B-knockdown cells could form smaller subcutaneous tumors in vivo. Finally, we demonstrated EIF3B could activate ß-catenin signaling pathway. METHODS: Immunohistochemical staining and Western blot were performed to detect the EIF3B expression in ESCC patient tissues and cell lines. The association between EIF3B expression and patients' prognosis was analyzed by Kaplan-Meier and Cox regression. Then, CCK-8, colony-formation, Transwell and wound-healing assay were performed to compare the bio-functional change after knockdown of EIF3B. Flow cytometry was applied to analyze the change of cell apoptosis and cycle induced by EIF3B knockdown. Tumor xenograft assay was done to verify the in-vitro results. CONCLUSIONS: EIF3B might serve as a novel marker for predicting prognosis of ESCC patients and as a potential therapeutic target, individually or together with other subunits of EIF3 complex.

NKT cells mediate the recruitment of neutrophils by stimulating epithelial chemokine secretion during colitis.

Ulcerative colitis (UC) is a kind of inflammatory bowel diseases characterized by chronic inflammation and ulcer in colon, and UC patients have increased risk of getting colorectal cancer. NKT cells are cells that express both NK cell markers and semi-invariant CD1d-restricted TCRs, can regulate immune responses via secreting a variety of cytokines upon activation. In our research, we found that the NKT cell-deficient CD1d(-/-) mice had relieved colitis in the DSS-induced colitis model. Further investigations revealed that the colon of CD1d(-/-) mice expressed less neutrophil-attracting chemokine CXCL 1, 2 and 3, and had decreased neutrophil infiltration. Infiltrated neutrophils also produced less reactive oxygen species (ROS) and TNF-α, indicating they may cause less epithelial damage. In addition, colitis-associated colorectal cancer was also relieved in CD1d(-/-) mice. During colitis, NKT cells strongly expressed TNF-α, which could stimulate CXCL 1, 2, 3 expressions by the epithelium. In conclusion, NKT cells can regulate colitis via the NKT cell-epithelium-neutrophil axis. Targeting this mechanism may help to improve the therapy of UC and prevent colitis-associated colorectal cancer.

miRNA-15a/16: as tumor suppressors and more.

Since their first discovery in chronic lymphocytic leukemia, miR-15a and miR-16 have been reported to act as tumor suppressors or potential oncomiRs in different types of cancer. This review summarizes the history, biological properties and the important functions of these two miRNAs in cancer. It also introduces their roles as regulators of immune responses and angiogenesis, endogenous controls as well as potential targets and hallmarks of cancer.

DNMT1-microRNA126 epigenetic circuit contributes to esophageal squamous cell carcinoma growth via ADAM9-EGFR-AKT signaling.

PURPOSE: MicroRNAs (miRNA) are involved in and are controlled by epigenetic regulation, and thereby form a reciprocal regulatory circuit. Using next-generation sequencing (NGS)-based miRNA profiling, this study aimed to discover esophageal squamous cell carcinoma (ESCC)-specific miRNAs and miRNA-related epigenetic modulations. EXPERIMENTAL DESIGN: NGS-based miRNA profiles were generated for four pairs of ESCC tissues and adjacent normal tissues. In situ hybridization was used to assess miRNA expression and its correlation with prognosis. miRNA-related DNA methylations were identified using bisulfite genomic sequencing, and the role of DNA methyltransferase 1 (DNMT1) was investigated using RNA interference. miRNA targets were screened by mRNA sequencing, and functional validation was performed in vitro and in vivo. RESULTS: NGS-based miRNA profiling identified 78 differentially expressed miRNAs in ESCC. Among them, microRNA126-3p (miR-126) was significantly downregulated, and its downregulation correlated with poor ESCC prognosis. Downregulation of miR-126 was due to promoter hypermethylation of its host gene, Egfl7. DNMT1 was aberrantly upregulated in ESCC and responsible for the hypermethylation of Egfl7. Intriguingly, DNMT1 was suppressed by overexpression of miR-126, indicating the existence of a regulatory feedback circuit. ADAM9 was identified as a key target of miR-126. Ectopic expression of miR-126 or silencing of ADAM9 reduced ESCC cell proliferation and migration by inhibiting epidermal growth factor receptor-AKT signaling. CONCLUSIONS: Our results indicate that miR-126 is a potential prognostic indicator for ESCC and suggest that a novel "DNMT1-miR-126 epigenetic circuit" is involved in ESCC progression. Consequently, miR-126-based epigenetic modulations may provide a basic rationale for new approaches to antitumor therapeutics.

MicroRNA-7 sensitizes non-small cell lung cancer cells to paclitaxel.

Paclitaxel (PTX) is the front-line chemotherapeutic agent against human non-small cell lung cancer (NSCLC). However, its therapeutic efficacy is restricted by the increasing frequency of chemotherapeutic resistance in NSCLC. Accumulating evidence has shown the potential role of microRNAs (miRNAs) in the chemotherapeutic sensitivity of cancer cells. Previously it was reported that microRNA-7 (miR-7) acts as an important tumor suppressor in NSCLC. Therefore, the present study was conducted to determine the regulatory role of miR-7 in PTX chemotherapy for NSCLC. Four NSCLC cell lines were used to analyze the correlation of the PTX-sensitivity and endogenoaus miR-7 expression. miR-7 expression was up- and downregulated using miR-7 mimics and inhibitors respectively, and the role of miR-7 in sensitizing NSCLC cells to PTX was assessed by cell viability and apoptosis assays. The molecular mechanism of PTX sensitivity was determined by quantitative polymerase chain reaction and western blotting. It was found that the sensitivity of NSCLC cells to PTX was dependent on endogenous miR-7. Upregulation of miR-7 enhanced the PTX-sensitivity of NSCLC cells by suppressing cell proliferation and promoting cell apoptosis, while the inhibition of miR-7 abrogated the antiproliferative proapoptotic effects of PTX. Pretreatment of miR-7 mimics enhanced the PTX-mediated downregulation of epidermal growth factor receptor (EGFR) in NSCLC cells. These results have identified miR-7 as a potential EGFR-targeting sensitizer in PTX therapy. These data may facilitate the development of novel chemotherapeutic approaches for NSCLC.