Micro RNA-19a suppresses IL-10 in peripheral B cells from patients with atherosclerosis

Background and aimsThe interleukin (IL)-10-production B cells play an important role in the pathogenesis of atherosclerosis (Asro) with unknown mechanism. Micro RNA (miR)-17-92 cluster has strong immune regulatory activities. This study tests a hypothesis that miR-17-92 cluster suppresses IL-10 expression in B cells of Asro patients.MethodsPatients with Asro were recruited into this study. Peripheral blood samples were collected from the patients. B cells were isolated from the blood samples and analyzed to elucidate the role of miR-17-92 in the regulation of IL-10 expression.ResultsPeripheral B cells from patients with Asro show lower levels of IL-10 than that from healthy subjects. The IL-10 expression in the B cells is negatively correlated with the expression of miR-19a in the B cells. The serum levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-4 in Asro patients were higher than healthy subjects. Exposure to TNF-α or IFN-γ or IL-4 suppressed IL-10 expression in B cells via increasing the expression of miR-19a in B cells, which could be abolished by Inhibition of miR-19a.ConclusionsTNF-α or IFN-γ or IL-4 suppresses IL-10 in B cells via up regulating miR-19a expression.     

Micro RNA‐550a interferes with vitamin D metabolism in peripheral B cells of patients with diabetes

The pathogenesis of diabetes is to be further investigated. Vitamin D3 (VitD3) can improve diabetes. Micro RNAs (miR) are involved in regulating cell activities. This study tests a hypothesis that miR‐550a interferes with the metabolism of VitD3 in peripheral B cells. In this study, blood samples were collected from patients with diabetes and healthy persons. The B cells were isolated from the blood samples to be treated with tumor necrosis factor (TNF)‐α. The B cells were then collected and analyzed for the expression of miR‐550a and cyp27b1. The results showed that B cells from healthy subjects were capable of converting VitD metabolite calcidiol to calcitriol, which was impaired in B cells collected from diabetic patients. The diabetic patients showed lower bone mineral density than that in healthy subject. The miR‐550a was negatively correlated with bone mineral density and the Levels of cyp27b1 in peripheral B cells of patients with diabetes. In vitro study showed that TNF‐α increased miR‐550a expression and inhibited the expression of cyp27b1 in B cells. miR‐550a mediated the effects of TNF‐α on inducing chromatin remodeling at the cyp27b1 gene locus. In conclusion, miR‐550a mediates the TNF‐α‐induced suppression of cyp27b1 expression in peripheral B cells of patients with diabetes, which can be blocked by inhibition of miR‐550a.     

Cytokine and cellular responses to human herpesvirus‐6B in patients with B‐acute lymphoblastic leukemia

Primary infection with human herpesvirus‐6 (HHV‐6), is followed by its lifelong persistence in the host. Most T‐cell responses to HHV‐6 have been characterized using peripheral blood from healthy adults; however, the role of HHV‐6 infection in immune modulation has not been elucidated for some diseases. Therefore, in this study the immune response to HHV‐6 infection in patients with B‐acute lymphoblastic leukemia (B‐ALL) was analyzed. HHV‐6 load was quantified in blood samples taken at the time of diagnosis of leukemia and on remission. The same concentrations of anti‐ and pro‐inflammatory cytokines (IL‐4, IL‐1, IL‐6, IL‐8, IL‐12p70, IL‐17a, TNF‐α and IFN‐γ) were detected in plasma samples from 20 patients with and 20 without detectable HHV‐6 virus loads in blood. Characterization of T‐cell responses to HHV‐6 showed low specific T‐cells frequencies of 2.08% and 1.46% in patients with and without detectable viral loads, respectively. IFN‐γ‐producing T cells were detected in 0.03%–0.23% and in 0%–0.2% of CD4+T cells, respectively. Strong production of IL‐6 was detected in medium supernatants of challenged T‐cells whatever the HHV‐6 status of the patients (973.51 ± 210.06 versus 825.70 ± 210.81 pg/mL). However, concentrations of TNF‐α and IFN‐γ were low. Thus, no association between plasma concentrations of cytokines and detection of HHV‐6 in blood was identified, suggesting that HHV‐6 is not strongly associated with development of B‐ALL. The low viral loads detected may correspond with latently infected cells. Alternatively, HHV‐6B specific immune responses may be below the detection threshold of the assays used.     

Decreased expression of microRNA‐21 correlates with the imbalance of Th17 and Treg cells in patients with rheumatoid arthritis

The imbalance of Th17/Treg cell populations has been suggested to be involved in the regulation of rheumatoid arthritis (RA) pathogenesis; however, the mechanism behind this phenomenon remains unclear. Recent studies have shown how microRNAs (miRNAs) are important regulators of immune responses and are involved in the development of a variety of inflammatory diseases, including RA. In this study, we demonstrated that the frequencies of CD3⁺CD4⁺IL‐17⁺Th17 cells were significantly higher, and CD4⁺CD25⁺FOXP3⁺ Treg cells significantly lower in peripheral blood mononuclear cells from RA patients. Detection of cytokines from RA patients revealed an elevated panel of pro‐inflammatory cytokines, including IL‐17, IL‐6, IL‐1β, TNF‐α and IL‐22, which carry the inflammatory signature of RA and are crucial in the differentiation and maintenance of pathogenic Th17 cells and dysfunction of Treg cells. However, the level of miR‐21 was significantly lower in RA patients, accompanied by the increase in STAT3 expression and activation, and decrease in STAT5/pSTAT5 protein and Foxp3 mRNA levels. Furthermore, lipopolysaccharide stimulation up‐regulated miR‐21 expression from healthy controls, but down‐regulated miR‐21 expression from RA patients. Therefore, we speculate that miR‐21 may be part of a negative feedback loop in the normal setting. However, miR‐21 levels decrease significantly in RA patients, suggesting that this feedback loop is dysregulated and may contribute to the imbalance of Th17 and Treg cells. MiR‐21 may thus serve as a novel regulator in T‐cell differentiation and homoeostasis, and provides a new therapeutic target for the treatment of RA.     

Exosome derived from CD137‐modified endothelial cells regulates the Th17 responses in atherosclerosis

The role of exosomes derived from endothelial cells (ECs) in the progression of atherosclerosis (AS) and inflammation remains largely unexplored. We aimed to investigate whether exosome derived from CD137‐modified ECs (CD137‐Exo) played a major role in AS and to elucidate the potential mechanism underlying the inflammatory effect. Exosomes derived from mouse brain microvascular ECs treated with agonist anti‐CD137 antibody were used to explore the effect of CD137 signalling in AS and inflammation in vitro and vivo. CD137‐Exo efficiently induced the progression of AS in ApoE^?/? mice. CD137‐Exo increased the proportion of Th17 cells both in vitro and vivo. The IL‐6 contained in CD137‐Exo which is regulated by Akt and NF‐КB pathway was verified to activate Th17 cell differentiation. IL‐17 increased apoptosis, inhibited cell viability and improved lactate dehydrogenase (LDH) release in ECs subjected to inflammation induced by lipopolysaccharide (LPS). The expression of soluble intercellular adhesion molecule1 (sICAM‐1), monocyte chemoattractant protein‐1 (MCP‐1) and E‐selectin in the supernatants of ECs after IL‐17 treatment was dramatically increased. CD137‐Exo promoted the progression of AS and Th17 cell differentiation via NF‐КB pathway mediated IL‐6 expression. This finding provided a potential method to prevent local and peripheral inflammation in AS.     

Down‐regulation of circDMNT3B is conducive to intestinal mucosal permeability dysfunction of rats with sepsis via sponging miR‐20b‐5p

Sepsis is a life‐threatening syndrome with a high risk of mortality, which is caused by the dysregulated host response to infection. We examined significant roles of circDMNT3B and miR‐20b‐5p in the intestinal mucosal permeability dysfunction of rats with sepsis. SD rats were randomly divided into 6 groups (n = 10/group): sham group, sepsis group, si‐negative control group, circDNMT3B‐si1 group, circDNMT3B‐si2 group and circDNMT3B‐si1 + anti‐miR‐20b‐5p group. The level of malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, interleukin (IL)‐6 and IL‐10 levels were measured through ELISA assay kits. Cell survival rate and cell apoptosis were evaluated by Cell‐Counting Kit‐8 Assay and flow cytometry, respectively. Luciferase reporter assays were used to investigate interactions between miR‐20b‐5p circDMNT3B in HEK‐293T cells. Silencing circDNMT3B can significantly increase the level of d ‐lactic acid, FD‐40, MDA, diamine oxidase, IL‐10 and IL‐6, compared with sepsis group, while the SOD activity was lower. Silencing circDNMT3B leads to oxidative damage and influence inflammatory factors level in intestinal tissue. CircDNMT3B was identified as a target gene of miR‐20b‐5p. Silencing circDNMT3B decreased cell survival and induced apoptosis in Caco2 cells treated with LPS, which was reversed by anti‐miR‐20b‐5p. MiR‐20b‐5p inhibitor remarkably down‐regulated mentioned‐above levels, in addition to up‐regulate SOD activity, which may relieve the damage of intestinal mucosal permeability caused by silencing circDNMT3B in sepsis rats. Down‐regulation of circDMNT3B was conducive to the dysfunction of intestinal mucosal permeability via sponging miR‐20b‐5p in sepsis rats, which may provide the novel strategy for sepsis treatment in the future.     

MicroRNA‐93‐5p expression in tumor tissue and its tumor suppressor function via targeting programmed death ligand‐1 in colorectal cancer

This work aimed to investigate miR‐93‐5p expression in tumor tissue and its in vitro effects in colorectal cancer (CRC) by targeting programmed death ligand‐1 (PD‐L1). MiR‐93‐5p and PD‐L1 expression was detected in CRC and adjacent normal tissues by quantitative real‐time polymerase chain reaction and immunohistochemistry. The correlation between miR‐93‐5p and PD‐L1 was validated by a dual‐luciferase reporter assay. HCT116 and SW480 cells were divided into blank, miR‐NC, miR‐93‐5p mimics, miR‐93‐5p inhibitor, PD‐L1 small interfering RNA (siRNA) and miR‐93‐5p inhibitor + PD‐L1 siRNA groups, and wound‐healing and transwell assays were performed to detect cell migration and invasion, respectively. Protein expression was measured by western blotting. The secretion of cytokines was detected in the CRC cell/T coculture models. MiR‐93‐5p was downregulated in CRC tissues with upregulated PD‐L1. In PD‐L1‐negative patients, miR‐93‐5p expression was increased compared with that in PD‐L1‐positive patients. MiR‐93‐5p and PD‐L1 expression levels were associated with the tumor differentiation, lymphatic metastasis, TNM, Duke's stage, and prognosis of CRC. PD‐L1 siRNA weakened the migration and invasion abilities via decreased expression of matrix metalloproteinase‐1 (MMP‐1), ‐2, and ‐9, and these effects were abolished by the miR‐93‐5p inhibitor. Additionally, anti‐PD‐L1 upregulated the expressions of interleukin‐2 (IL‐2), tumor necrosis factor‐α (TNF‐α), and interferon γ (IFN‐γ) in the coculture of T cells with CRC cells, but downregulated the expressions of IL‐1β, IL‐10, and TGF‐β. However, these changes were partially reversed by miR‐93‐5p inhibition. miR‐93‐5p is expected to be a novel target for CRC treatment since it decreases the migration and invasion, as well as the immune evasion, of CRC cells via targeting PD‐L1.     

Interleukin‐17 family cytokines in protective immunity against infections: role of hematopoietic cell‐derived and non‐hematopoietic cell‐derived interleukin‐17s

Interleukin‐17 family cytokines, consisting of six members, participate in immune response in infections and autoimmune and inflammatory diseases. The prototype cytokine of the family, IL‐17A, was originally identified from CD4+ T cells which are now termed Th17 cells. Later, IL‐17A‐producing cells were expanded to include various hematopoietic cells, namely CD8+ T cells (Tc17), invariant NKT cells, γδ T cells, non‐T non‐B lymphocytes (termed type 3 innate lymphoid cells) and neutrophils. Some IL‐17 family cytokines other than IL‐17A are also expressed by CD4+ T cells: IL‐17E by Th2 cells and IL‐17F by Th17 cells. IL‐17A and IL‐17F induce expression of pro‐inflammatory cytokines to induce inflammation and anti‐microbial peptides to kill pathogens, whereas IL‐17E induces allergic inflammation. However, the functions of other IL‐17 family cytokines have been unclear. Recent studies have shown that IL‐17B and IL‐17C are expressed by epithelial rather than hematopoietic cells. Interestingly, expression of IL‐17E and IL‐17F by epithelial cells has also been reported and epithelial cell‐derived IL‐17 family cytokines shown to play important roles in immune responses to infections at epithelial sites. In this review, we summarize current information on hematopoietic cell‐derived IL‐17A and non‐hematopoietic cell‐derived IL‐17B, IL‐17C, IL‐17D, IL‐17E and IL‐17F in infections and propose functional differences between these two categories of IL‐17 family cytokines.     

Down‐regulation of miR‐10a‐5p in synoviocytes contributes to TBX5‐controlled joint inflammation

MicroRNAs are considered to play critical roles in the pathogenesis of human inflammatory arthritis, including rheumatoid arthritis (RA). The purpose of this study was to determine the relationship between miR‐10a‐5p and TBX5 in synoviocytes and evaluate their contribution to joint inflammation. The expression of miR‐10a‐5p and TBX5 in the synovium of RA and human synovial sarcoma cell line SW982 stimulated by IL‐1β was determined by RT‐qPCR and Western blotting. The direct interaction between miR‐10a‐5p and TBX5 3′UTR was determined by dual‐luciferase reporter assay in HeLa cells. Mimics and inhibitors of miR‐10a‐5p were transfected into SW982 cells. TBX5 was overexpressed by plasmid transfection or knocked down by RNAi. Proinflammatory cytokines and TLR3 and MMP13 expressions were determined by RT‐qPCR and Western blotting. Down‐regulated expression of miR‐10a‐5p and up‐regulation of TBX5 in human patients with RA were found compared to patients with OA. IL‐1β could reduce miR‐10a‐5p and increase TBX5 expression in SW982 cells in vitro. The direct target relationship between miR‐10a‐5p and 3′UTR of TBX5 was confirmed by luciferase reporter assay. Alterations of miR‐10‐5p after transfection with its mimic and inhibitor caused the related depression and re‐expression of TBX5 and inflammatory factors in SW982 cells. Overexpression of TBX5 after pCMV3‐TBX5 plasmid transfection significantly promoted the production of TLR3, MMP13 and various inflammatory cytokines, while this effect was rescued after knocking down of TBX5 with its specific siRNA. We conclude that miR‐10a‐5p in a relation with TBX5 regulates joint inflammation in arthritis, which would serve as a diagnostic and therapeutic target for RA treatment.     

Induced miR‐99a expression represses Mtor cooperatively with miR‐150 to promote regulatory T‐cell differentiation

Peripheral induction of regulatory T (Treg) cells provides essential protection from inappropriate immune responses. CD4⁺ T cells that lack endogenous miRNAs are impaired to differentiate into Treg cells, but the relevant miRNAs are unknown. We performed an overexpression screen with T‐cell‐expressed miRNAs in naive mouse CD4⁺ T cells undergoing Treg differentiation. Among 130 candidates, the screen identified 29 miRNAs with a negative and 10 miRNAs with a positive effect. Testing reciprocal Th17 differentiation revealed specific functions for miR‐100, miR‐99a and miR‐10b, since all of these promoted the Treg and inhibited the Th17 program without impacting on viability, proliferation and activation. miR‐99a cooperated with miR‐150 to repress the expression of the Th17‐promoting factor mTOR. The comparably low expression of miR‐99a was strongly increased by the Treg cell inducer “retinoic acid”, and the abundantly expressed miR‐150 could only repress Mtor in the presence of miR‐99a. Our data suggest that induction of Treg cell differentiation is regulated by a miRNA network, which involves cooperation of constitutively expressed as well as inducible miRNAs.     

miR‐124a inhibits the proliferation and inflammation in rheumatoid arthritis fibroblast‐like synoviocytes via targeting PIK3/NF‐κB pathway

Abnormal hyperplasia of fibroblast‐like synoviocytes (FLS) leads to the progression of rheumatoid arthritis (RA). This study aimed to investigate the role of miR‐124a in the pathogenesis of RA. The viability and cell cycle of FLS in rheumatoid arthritis (RAFLS) were evaluated by Cell Counting Kit 8 and flow cytometry assay. The expression of PIK3CA, Akt, and NF‐κB in RAFLS was examined by real‐time PCR and Western blot analysis. The production of tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 was detected by ELISA. The joint swelling and inflammation in collagen‐induced arthritis (CIA) mice were examined by histological and immunohistochemical analysis. We found that miR‐124a suppressed the viability and proliferation of RAFLS and increased the percentage of cells in the G1 phase. miR‐124a suppressed PIK3CA 3'UTR luciferase reporter activity and decreased the expression of PIK3CA at mRNA and protein levels. Furthermore, miR‐124a inhibited the expression of the key components of the PIK3/Akt/NF‐κB signal pathway and inhibited the expression of pro‐inflammatory factors TNF‐α and IL‐6. Local overexpression of miR‐124a in the joints of CIA mice inhibited inflammation and promoted apoptosis in FLS by decreasing PIK3CA expression. In conclusion, miR‐124a inhibits the proliferation and inflammation in RAFLS via targeting PIK3/NF‐κB pathway. miR‐124a is a promising therapeutic target for RA.     

MiR‐377 Regulates Inflammation and Angiogenesis in Rats After Cerebral Ischemic Injury

Ischemic stroke is the leading cause of disabilities worldwide. MicroRNA‐377 (miR‐377) plays important roles in ischemic injury. The present study focused on the mechanisms of miR‐377 in protecting ischemic brain injury in rats. Cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in rats. Primary rat microglial cells and brain microvascular endothelial cells (BMECs) were exposed to oxygen‐glucose deprivation (OGD). The concentrations of cytokines (TNF‐α, IL‐1β, IL‐6, IFN‐γ, TGF‐β, MMP2, COX2, and iNOS) in the culture medium were measured by specific ELISA. Tube formation assay was for the in vitro study of angiogenesis. Luciferase reporter assay was performed to confirm whether VEGF and EGR2 were direct targets of miR‐377. The MCAO rats were intracerebroventricular (ICV) injection of miR‐377 inhibitor to assess its protective effects in vivo. MiR‐377 levels were decreased in the rat brain tissues at 1, 3, and 7 d after MCAO. Both microglia cells and BMECs under OGD showed markedly lower expression levels of miR‐377 while higher expression levels of EGR2 and VEGF compared to those under normoxia conditions. Knockdown of miR‐377 inhibited microglial activation and the release of pro‐inflammatory cytokines after OGD. Suppression of miR‐377 promoted the capillary‐like tube formation and cell proliferation and migration of BMECs. The anti‐inflammation effect of EGR2 and the angiogenesis effect of VEGF were regulated by miR‐377 after OGD. Inhibition of miR‐377 decreased cerebral infarct volume and suppressed cerebral inflammation but promoted angiogenesis in MCAO rats. Knockdown of miR‐377 lessened the ischemic brain injury through promoting angiogenesis and suppressing cerebral inflammation. J. Cell. Biochem. 119: 327–337, 2018. © 2017 Wiley Periodicals, Inc.     

Role of IL‐15 in spinal cord and sciatic nerve after chronic constriction injury: regulation of macrophage and T‐cell infiltration

The release of inflammatory mediators from immune and glial cells either in the peripheral or CNS may have an important role in the development of physiopathological processes such as neuropathic pain. Microglial, then astrocytic activation in the spinal cord, lead to chronic inflammation, alteration of neuronal physiology and neuropathic pain. Standard experimental models of neuropathic pain include an important peripheral inflammatory component, which involves prominent immune cell activation and infiltration. Among potential immunomodulators, the T‐cell cytokine interleukin‐15 (IL‐15) has a key role in regulating immune cell activation and glial reactivity after CNS injury. Here we show, using the model of chronic constriction of the sciatic nerve (CCI), that IL‐15 is essential for the development of the early inflammatory events in the spinal cord after a peripheral lesion that generates neuropathic pain. IL‐15 expression in the spinal cord was identified in both astroglial and microglial cells and was present during the initial gliotic and inflammatory (NFκB) response to injury. The expression of IL‐15 was also identified as a cue for macrophage and T‐cell activation and infiltration in the sciatic nerve, as shown by intraneural injection of the cytokine and activity blockage approaches. We conclude that the regulation of IL‐15 and hence the initial events following its expression after peripheral nerve injury could have a future therapeutic potential in the reduction of neuroinflammation.     

MiR‐101 promotes pain hypersensitivity in rats with chronic constriction injury via the MKP‐1 mediated MAPK pathway

This study was performed to characterize the effect of microRNA‐101 (miR‐101) on the pain hypersensitivity in CCI rat models with the involvement of mitogen‐activated protein kinase phosphatase 1 (MKP‐1) in spinal cord microglial cells. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in the developed CCI models were determined to assess the hypersensitivity of rats to mechanical stimulation and thermal pain. To assess inflammation, the levels of interleukin (IL)‐1β, IL‐6 and tumour necrosis factor‐α (TNF‐α) in the spinal dorsal horns of CCI rats and lipopolysaccharide (LPS)‐activated microglial cells were examined. miR‐101 and MKP‐1 gain‐ and loss‐of‐function experiments were conducted in in vivo and in vitro settings to examine the roles of miR‐101 and MKP‐1 in CCI hypersensitivity and inflammation. The results showed that miR‐101 was highly expressed in the spinal dorsal horn and microglial cells of CCI rat models. Furthermore, overexpression of miR‐101 promoted the pain hypersensitivity in CCI rat models by reducing MWT and TWL. The overexpression of miR‐101 also promoted inflammation in LPS‐exposed microglial cells, as indicated by increased levels of IL‐1β, IL‐6 and TNF‐α. MiR‐101 was shown to target MKP‐1, inhibiting its expression. Moreover, miR‐101 promoted pain hypersensitivity in CCI rat models by inhibiting MKP‐1 expression and activating the mitogen‐activated protein kinase (MAPK) signalling pathway. Taken together, miR‐101 could potentially promote hypersensitivity and inflammatory response of microglial cells and aggravate neuropathic pain in CCI rat models by inhibiting MKP‐1 in the MAPK signalling pathway.