Apolipoprotein C-III itself stimulates the Syk/cPLA2-induced inflammasome activation of macrophage to boost anti-tumor activity of CD8+ T cell.

Increased prevalence of cancer in obese individuals is involved with dyslipidemia- induced chronic inflammation and immune suppression. Although apolipoprotein C-III (ApoC3)-transgenic mice (ApoC3TG mice) or poloxamer 407 (P407)-treated mice had hyperlipidemia, CD8+ T cells with upregulated antitumor activities were observed in ApoC3TG mice, and decreased CD8+ T cell activities were observed in P407-treated mice. Increased ApoC3 expression in hepatocellular carcinoma was associated with increased infiltration of CD8+ T cells and predicted survival. Recombinant ApoC3 had no direct effects on CD8+ T cells. The upregulation of CD8+ T cells in ApoC3TG mice was due to cross-talk with context cells, as indicated by metabolic changes and RNA sequencing results. In contrast to dendritic cells, the macrophages of ApoC3TG mice (macrophagesTG) displayed an activated phenotype and increased IL-1ß, TNF-α, and IL-6 production. Coculture with macrophagesTG increased CD8+ T cell function, and the adoptive transfer of macrophagesTG suppressed tumor progression in vivo. Furthermore, spleen tyrosine kinase (Syk) activation induced by TLR2/TLR4 cross-linking after ApoC3 ligation promoted cellular phospholipase A2 (cPLA2) activation, which in turn activated NADPH oxidase 2 (NOX2) to promote an alternative mode of inflammasome activation. Meanwhile, mitochondrial ROS produced by increased oxidative phosphorylation of free fatty acids facilitated the classical inflammasome activation, which exerted an auxiliary effect on inflammasome activation of macrophagesTG. Collectively, the increased antitumor activity of CD8+ T cells was mediated by the ApoC3-stimulated inflammasome activation of macrophages, and the mimetic ApoC3 peptides that can bind TLR2/4 could be a future strategy to target liver cancer.

Deficiency of angiopoietin-like 4 enhances CD8+ T cell bioactivity via metabolic reprogramming for impairing tumour progression.

Angiopoietin-like 4 (ANGPTL4) is a secreted metabolism-modulating glycoprotein involved in the progression of tumours, cardiovascular diseases, metabolic syndrome and infectious diseases. In this study, more CD8+ T cells were activated to be effector T cells in ANGPTL4-/- mice. Impaired growth of tumours implanted in 3LL, B16BL6 or MC38 cells and reduced metastasis by B16F10 cells were observed in ANGPTL4-/- mice. Bone marrow (BM) transplantation experiments displayed that deficiency of ANGPTL4 in either host or BM cells promoted CD8+ T cell activation. However, ANGPTL4 deficiency in CD8+ T cells themselves showed more efficient anti-tumour activities. Recombinant ANGPTL4 protein promoted tumour growth in vivo with the less CD8+ T cell infiltration and it directly downregulated CD8+ T cell activation ex vivo. Transcriptome sequencing and metabolism analysis identified that ANGPTL4-/- CD8+ T cells increased glycolysis and decreased oxidative phosphorylation, which was dependent on the PKCζ-LKB1-AMPK-mTOR signalling axis. Reverse correlation of elevated ANGPTL4 levels in sera and tumour tissues with activated CD8+ T cells in the peripheral blood was displayed in patients with colorectal cancer. These results demonstrated that ANGPTL4 decreased immune surveillance in tumour progression by playing an immune-modulatory role on CD8+ T cells via metabolic reprogramming. Efficient blockade of ANGPTL4 expression in tumour patients would generate an effective anti-tumour effect mediated by CD8+ T cells.

GABA signaling enforces intestinal germinal center B cell differentiation.

Recent compelling results indicate possible links between neurotransmitters, intestinal mucosal IgA+ B cell responses, and immunoglobulin A nephropathy (IgAN) pathogenesis. Here, we demonstrated that γ-amino butyric acid (GABA) transporter-2 (GAT-2) deficiency induces intestinal germinal center (GC) B cell differentiation and worsens the symptoms of IgAN in a mouse model. Mechanistically, GAT-2 deficiency enhances GC B cell differentiation through activation of GABA-mammalian target of rapamycin complex 1 (mTORC1) signaling. In addition, IgAN patients have lower GAT-2 expression but higher activation of mTORC1 in blood B cells, and both are correlated with kidney function in IgAN patients. Collectively, this study describes GABA signaling-mediated intestinal mucosal immunity as a previously unstudied pathogenesis mechanism of IgAN and challenges the current paradigms of IgAN.

Astilbin Activates the Reactive Oxidative Species/PPARγ Pathway to Suppress Effector CD4+ T Cell Activities via Direct Binding With Cytochrome P450 1B1.

Astilbin, as a compound of flavonoids, exerts anti-inflammation, antioxidation, and immune-suppression activities. Decreased activation of NF-κB and p38 MAPK and increased activation of SOCS3 and AMPK have been found in astilbin-treated cells. However, what molecules are docked by astilbin to initiate signaling cascades and result in functional changes remains unknown. In the study, we found that astilbin efficiently suppressed TNF-α production and increased CCR9 and CD36 expression of CD4+ T cells. In vivo administration of astilbin repressed the occurrence of type 1 diabetes mellitus in non-obese diabetic mice. The PPARγ/SOCS3, PPARγ/PTEN, and PPARγ/AMPK signaling pathways were substantially activated and played key roles in astilbin-induced downregulation of CD4+ T cell functions. Transcriptome sequencing results confirmed the changes of signaling molecules involved in the immune system, inflammatory responses, and indicated variations of multiple enzymes with oxidant or antioxidant activities. Astilbin directly induced cytoplasmic ROS production of CD4+ T cells ex vivo, but had no effects on mitochondrial ROS and mitochondrial weight. When cellular ROS was depleted, astilbin-treated CD4+ T cells remarkably reversed the expression of TNF-α, IFN-γ, CCR9, CD36, and signaling molecules (PPARγ, PTEN, p-AMPK, and SOCS3). Based on bioinformatics, two P450 enzymes (CYP1B1 and CYP19A1) were selected as candidate receptors for astilbin. CYP1B1 was identified as a real docking protein of astilbin in ROS production by AutoDock Vina software analysis and surface plasmon resonance assay. Collectively, astilbin downregulates effector CD4+ T cell activities via the CYP1B1/ROS/PPARγ pathway, which firmly supports its potential use in the treatment of inflammation.

Increased angiopoietin-like 4 expression ameliorates inflammatory bowel diseases via suppressing CD8+ T cell activities.

Angiopoietin-like 4 (ANGPTL4) is involved in inflammation-associated diseases, such as rheumatoid arthritis, type 2 diabetes, atherosclerosis, and chronic obstructive pulmonary disease. The role of ANGPTL4 in the pathogenesis of inflammatory bowel disease (IBD) remains unknown. Here, the plasma ANGPTL4 levels peaked on days 3 and 5, and expression of ANGPTL4 of inflamed colons peaked on days 5 and 7 in mice with dextran sulfate sodium (DSS)-induced colitis. Simultaneously, CD8+T cells in the inflamed colons peaked at day 5 but declined at day 7. However, the ANGPTL4-/- mice treated with DSS exhibited exacerbated colitis with more CD8+T cells and macrophages infiltrating the colons. The exogenous ANGPTL4 protein protected the mice against DSS-induced colitis with less CD8+T cell and macrophage recruitment in the colons. In addition, recombinant ANGPTL4 directly downregulated the IFN-γ and NKG2D expression of CD8+T cells but had no effects on the CD86 expression and TNF-α secretion of macrophages ex vivo. Adding ANGPTL4 protein into ANGPTL4-/- mice almost blocked the onset of DSS-induced colitis. In parallel, the plasma ANGPTL4 levels were elevated in patients with Crohn's disease and ulcerative colitis at mild/moderate stage and restored to normal levels in IBD patients at a severe stage. The higher ANGPTL4 expression in the inflamed colons of patients with IBD was correlated with lower CD8+ cell infiltration, whereas no associations with macrophages. Our results demonstrated the compensatory protective effect of ANGPTL4 on IBD development at least via the downregulation of CD8+T cell activities. Adding the ANGPTL4 protein would have beneficial effects to retard the progression of IBD.

Association of Peroxiredoxin 1 and brain-derived neurotrophic factor serum levels with depression and anxiety symptoms in patients with irritable bowel syndrome.

BACKGROUND: Oxidative stress is considered a possible mechanism of irritable bowel syndrome (IBS) and depression. This study determined the possible association of serum peroxiredoxin 1 (PRDX1; a key antioxidant enzyme) and brain-derived neurotrophic factor (BDNF) with anxiety and depression symptoms in IBS patients. METHODS: According to the Rome IV diagnostic criteria, 177 IBS patients from February 2019 to July 2019 were included. Serum levels of PRDX1, BDNF, and TNFα were detected by enzyme-linked immunosorbent assay. Levels of anxiety and depression were assessed with the Self-rating Anxiety Scale (SAS) and Self-rating Depression Scale (SDS). RESULTS: Compared with normal IBS patients, patients with anxiety and depression symptoms had significantly higher serum PRDX1 (p<0.001; p=0.002) and TNFα (p<0.001; p = 0.002) and significantly lower BDNF (p < 0.001; p = 0.002). Serum PRDX1 (r = 0.659, p < 0.001; r = 0.466, p < 0.001) and TNFα (r = 0.531, p < 0.001; r = 0.449, p < 0.001) were positively correlated with SAS and SDS, respectively, whereas BDNF was negatively correlated with SAS (r = 0.594, p < 0.001) and SDS (r = 0.534, p < 0.001). Multivariable regression analysis revealed that IBS severity, BDNF, and PRDX1 were significant predictors of anxiety. BDNF was also a significant predictor of depression. CONCLUSION: Elevated PRDX1 and decreased BDNF in serum may be closely related to psychological symptoms in IBS. Results of this study suggested that PRDX1 may be an important target for IBS treatment in fighting against intestinal and psychological symptoms.

Early growth response genes 2 and 3 induced by AP-1 and NF-κB modulate TGF-ß1 transcription in NK1.1- CD4+ NKG2D+ T cells.

NK1.1- CD4+ NKG2D+ T cells are a subpopulation of regulatory T cells that downregulate the functions of CD4+ T, CD8+ T, natural killer (NK) cells, and macrophages through TGF-ß1 production. Early growth response genes 2 (Egr2) and 3 (Egr3) maintain immune homeostasis by modulating T lymphocyte development, inhibiting effector T cell function, and promoting the induction of regulatory T cells. Whether Egr2 and Egr3 directly regulate TGF-ß1 transcription in NK1.1- CD4+ NKG2D+ T cells remains elusive. The expression levels of Egr2 and Egr3 were higher in NK1.1- CD4+ NKG2D+ T cells than in NK1.1- CD4+ NKG2D- T cells. Egr2 and Egr3 expression were remarkably increased after stimulating NK1.1- CD4+ NKG2D+ T cells with sRAE or α-CD3/sRAE. The ectopic expression of Egr2 or Egr3 resulted in the enhancement of TGF-ß1 expression, while knockdown of Egr2 or Egr3 led to the decreased expression of TGF-ß1 in NK1.1- CD4+ NKG2D+ T cells. Egr2 and Egr3 directly bound with the TGF-ß1 promoter as demonstrated by the electrophoretic mobility shift assay and dual-luciferase gene reporter assay. Furthermore, the Egr2 and Egr3 expression of NK1.1- CD4+ NKG2D+ T cells could be induced by the AP-1 and NF-κB transcriptional factors, but had no involvement with the activation of NF-AT and STAT3. In conclusion, Egr2 and Egr3 induced by AP-1 and NF-κB directly initiate TGF-ß1 transcription in NK1.1- CD4+ NKG2D+ T cells. This study indicates that manipulating Egr2 and Egr3 expression would potentiate or alleviate the regulatory function of NK1.1- CD4+ NKG2D+ T cells and this strategy could be used in the therapy for patients with autoimmune diseases or tumor.

Astilbin combined with lipopolysaccharide induces IL-10-producing regulatory B cells via the STAT3 signalling pathway.

OBJECTIVES: Astilbin exerts immunoregulatory activities and plays anti-inflammatory effects in inflammation-associated diseases. IL-10-producing B cells are the major subset of regulatory B cells (Bregs) and inhibit inflammation and autoimmune diseases. This study aimed to analyse the inducing effect of astilbin on Bregs and investigate the involved molecular mechanisms. METHODS: The frequencies and activities of IL-10-producing Bregs were observed using the co-treatment of astilbin and lipopolysaccharide (LPS) ex vivo. The protective effect of astilbin/LPS-induced Bregs on dextran sulphate sodium (DSS)-induced colitis was confirmed in vivo. The molecular signalling events of Breg induction were checked via Western blot. CD40-/- and toll-like receptor (TLR) 4-/- B cells were treated with astilbin/LPS to determine the modulatory role of CD40 or TLR4 on astilbin/LPS-induced Bregs. RESULTS: Although astilbin alone could not affect Bregs, the co-treatment of astilbin and LPS remarkably induced CD19+ CD1dhi and CD19+ TIM-1+ cells which produced IL-10 ex vivo. Colonic CD19+ CD1dhi and CD19+ TIM-1+ cells were also increased in astilbin-treated mice with DSS-induced colitis. The adoptive transfer of CD19+ TIM-1+ cells pre-induced by astilbin/LPS directly suppressed the progression of DSS-induced colitis. Combined astilbin and LPS stimulated the STAT3 activation of CD19+ TIM-1+ cells but had no effects on SOCS3, AKT, NF-κB, Erk, JNK nor P38. Inhibiting the STAT3 phosphorylation of CD19+ TIM-1+ cells abolished Breg induction by astilbin/LPS. Furthermore, Breg induction was weakened in CD40-/- B cells with the decrease in STAT3 activation, but had disappeared in TLR4-/- B cells with no STAT3 activation, thereby confirming the indispensable role of TLR4 signalling in the induction of IL-10-producing Bregs. CONCLUSIONS: This study reports the new immunoregulatory role of astilbin for promoting IL-10-producing B cells and suggests the possible use of astilbin in the therapy of inflammatory diseases.

Erratum: Angiopoietin-like 4 deficiency upregulates macrophage function through the dysregulation of cell-intrinsic fatty acid metabolism.

[This corrects the article on p. 595 in vol. 10, PMID: 32195030.].

Angiopoietin-like 4 deficiency upregulates macrophage function through the dysregulation of cell-intrinsic fatty acid metabolism.

Angiopoietin-like 4 (ANGPLT4) regulates lipid metabolism by inhibiting lipoprotein lipase. Abnormal ANGTPL4 levels are associated with metabolic syndrome, atherosclerosis, inflammation, and cancer. We show here that ANGPTL4-deficient mice have abnormally large numbers of macrophages in the spleen, and that these macrophages produce large amounts of TNF-α, CD86, and inducible nitric oxide synthase. However, recombinant ANGPTL4 protein did not inhibit macrophage function ex vivo. Glycolysis and fatty-acid synthesis were upregulated in ANGPTL4-/- macrophages, whereas fatty-acid oxidation was decreased. Elevated levels of free fatty acids in the cytoplasm of ANGPTL4-/- macrophages were confirmed. ANGPTL4-/- macrophages also displayed endoplasmic reticulum (ER) stress after stimulation with LPS. Protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling was activated, but no major change in liver kinase B1 (LKB1)/adenosine 5'-monophosphate (AMP)- activated protein kinase (AMPK) phosphorylation was observed in ANGPTL4-/- macrophages. The modulation of fatty-acid metabolism prevented ER stress and the expression of inflammatory molecules, but the activation of ANGPTL4-/- macrophages was not restored by the inhibition of glycolysis. Thus, ANGPTL4 deficiency in macrophages results in ER stress due to the cell-intrinsic reprogramming of fatty-acid metabolism. Intracellular ANGPLT4 expression could thus be manipulated to modulate macrophage function.

High Expression of Angiopoietin-like Protein 4 in Advanced Colorectal Cancer and its Association with Regulatory T Cells and M2 Macrophages.

Colorectal cancer (CRC) is one of the most aggressive tumours in the human digestive system. Most CRC patients have poor prognosis due to metastasis and recurrence. Angiopoietin-like 4 (ANGPTL4) is involved in tumour development. Regulatory T (Treg) cells and M2 macrophages promote tumour growth and metastasis. Herein, we explored the changes of ANGPTL4 expression in CRC patients at different stages and observed whether in situ tumour-Treg and -M2 macrophages are correlated with ANGPTL4 expression. Serum ANGPTL4 (sANGPTL4) levels of 70 CRC patients and 10 healthy controls were detected by ELISA. ANGPTL4, Foxp3 and CD163 expression levels in CRC tissues were measured by immunohistochemistry. Recombinant ANGPTL4 (rANGPTL4) proteins were further added into cell-culture systems for induction of Treg cells and M2 macrophages. The results showed both sANGPTL4 and in situ tumour-ANGPTL4 expression levels increased in Dukes C-D stage CRC patients. Foxp3+ and CD163+ cells in tumour tissue sections were also more intensive in Dukes C-D stage patients than in Dukes A-B stage patients. Foxp3+ and CD163+ cells in tumour tissues were positively correlated with both tissue and sANGPTL4 expression (P < 0.01). Recombinant ANGPTL4 promoted the induction of murine Treg cells and M2 macrophages ex vivo. Therefore, elevated ANGPTL4 expression could be a marker for advanced CRC. Treg cell and M2 macrophage induction could be one of the mechanisms of tumour promotion mediated by ANGPTL4.

Peroxiredoxin 1 as an inflammatory marker in diarrhea-predominant and postinfectious irritable bowel syndrome.

BACKGROUND: Low-grade inflammation occurs in some patients with irritable bowel syndrome (IBS). However, the exact inflammatory markers of IBS and the relationship of these markers with IBS subtypes and symptoms are poorly defined. Peroxiredoxin 1 (PRDX1) plays an important role in inflammatory responses, including intestinal inflammation. We investigated whether PRDX1 is associated with the diagnosis, subtypes, and symptom severity of IBS. METHODS: A total of 177 IBS patients and 174 sex- and age-matched healthy controls (HCs) were recruited. The PRDX1 levels in the sera and colonic mucosa of the participants were detected by enzyme-linked immunosorbent assays and immunohistochemistry. The severity of IBS symptoms was assessed using the IBS Severity Scoring System (SSS) questionnaire. RESULTS: The PRDX1 levels in the sera (F = 71.81, P < .001) and colonic mucosa (F = 5.359, P < .001) of postinfectious (PI-IBS) and diarrhea-predominant IBS (IBS-D) groups were significantly higher than those of the other three IBS subtypes and HC group. The PRDX1 level in the serum and colonic mucosa of IBS-D (serum, P < .01, mucosa, P < .001) and PI-IBS (serum, P < .05, mucosa, P < .001) groups with the most severe symptoms was significantly higher than that in the groups with mild and moderate symptoms. Correlation analysis revealed that in patients with IBS-D (P < .001) and PI-IBS (P < .05), the levels of PRDX1 and TNF-α in sera had a significant positive correlation with IBS-SSS. CONCLUSION: Elevated PRDX1 in the serum and colon mucosa may be closely related to the progression of IBS (especially IBS-D and PI-IBS) and the expression of gastrointestinal symptoms.

Decreased expression of microRNA-510 in intestinal tissue contributes to post-infectious irritable bowel syndrome via targeting PRDX1.

OBJECTIVE: Post-infectious irritable bowel syndrome (PI-IBS) is a common functional gastrointestinal (GI) disorder that occurs after acute GI infection. Recent studies showed that microRNAs were involved in the occurrence and development of IBS. Here, we elaborated the role of miR-510 in the occurrence of PI-IBS and analyzed its mechanism. METHODS: We detected the expressions of miR-510 and PRDX1 in colonic mucosal tissues by qRT-PCR, Western blot and immunohistochemistry. Furthermore, we transfected Caco-2 cells with miR-510 mimic, anti-miR-510, si-PRDX1, and control, then evaluated the cell viability and apoptosis by CCK8 assay and flow cytometry, assessed expression levels of PRDX1 by qRT-PCR and Western blot analysis, and pro-inflammatory cytokines by qRT-PCR and ELISA. RESULTS: MiR-510 expression was downregulated and negatively correlated with TNF-α, whereas PRDX1 expression was upregulated in PI-IBS colonic mucosal tissues. LPS at concentrations of 5 and 10 µg/ml can significantly induce inflammatory injury in Caco-2 cells. MiR-510 overexpression aggravated the injury induced by LPS, as reflected by increased cell viability, decreased apoptosis, and less production of pro-inflammatory cytokines. miR-510 mimic transfection in cells significantly suppressed the mRNA and protein expression levels of PRDX1. Furthermore, the inflammatory injury induced by LPS was exacerbated by upregulating PRDX1 expression when miR-510 was knocked down. CONCLUSION: MiR-510 downregulation in intestinal tissue might contribute to PI-IBS via targeting PRDX1. The results of this study will not only enrich the pathogenesis of PI-IBS but also make us understand the biological activity of miR-510 and provide important experimental basis for PI-IBS clinical treatment targeting miR-510.

TGF-ß1 expression in regulatory NK1.1-CD4+NKG2D+ T cells dependents on the PI3K-p85α/JNK, NF-κB and STAT3 pathways.

NK1.1-CD4+NKG2D+ cells exert their immune-regulatory function in tumor as an unconventional regulatory T cell subset through the production of TGF-ß1; however, the molecular mechanisms involving with the activation of nuclear factors for TGF-ß1 transcription remain unclear. Here we determined that the PI3K-p85α subunit was specifically activated in NK1.1-CD4+NKG2D+ cells following an 8-hour stimulation by sRAE-1 or α-CD3/sRAE-1, subsequently leading to the activation of PI3K-p110, Akt, and JNK. On the contrary, α-CD3/α-CD28 stimulation did not induce the activation of PI3K-p85 and JNK. Consequently, activation of the nuclear transcription factor AP-1 as a consequence of JNK activation regulated TGF-ß1 expression in NK1.1-CD4+NKG2D+ cells. Furthermore, activation of NF-κB in NK1.1-CD4+NKG2D+ cells resulted from both protein kinase C activation downstream of TCR/CD3 signaling and PI3K activation induced by NKG2D engagement. The STAT3-Y705 phosphorylation, as activated by PI3K, under stimulations of the sRAE-1 or α-CD3/sRAE-1 also contributed to the TGF-ß1 expression in NK1.1-CD4+NKG2D+ cells. Moreover, ChIP assay confirmed that STAT3 was capable of binding with the promoter regions of TGF-ß1. In conclusion, our data showed that the TGF-ß1 transcription in NK1.1-CD4+NKG2D+ cells induced by sRAE-1 or α-CD3/sRAE-1 was involved with the AP-1, NF-κB, and STAT3 signaling pathways; therefore, regulation of AP-1, NF-κB, and STAT3 activation may play important roles in the development and function of NK1.1-CD4+NKG2D+ cells.

Chitosan nanoparticle-based delivery of fused NKG2D-IL-21 gene suppresses colon cancer growth in mice.

Nanoparticles can be loaded with exogenous DNA for the potential expression of cytokines with immune-stimulatory function. NKG2D identifies major histocompatibility complex class I chain-related protein in human and retinoic acid early induced transcript-1 in mouse, which acts as tumor-associated antigens. Biologic agents based on interleukin 21 (IL-21) have displayed antitumor activities through lymphocyte activation. The NKG2D-IL-21 fusion protein theoretically identifies tumor cells through NKG2D moiety and activates T cells through IL-21 moiety. In this study, double-gene fragments that encode the extracellular domains of NKG2D and IL-21 genes were connected and then inserted into the pcDNA3.1(-) plasmid. PcDNA3.1-dsNKG2D-IL-21 plasmid nanoparticles based on chitosan were generated. Tumor cells pretransfected with dsNKG2D-IL-21 gene nanoparticles can activate natural killer (NK) and CD8+ T cells in vitro. Serum IL-21 levels were enhanced in mice intramuscularly injected with the gene nanoparticles. DsNKG2D-IL-21 gene nanoparticles accumulated in tumor tissues after being intravenously injected for ~4-24 h. Treatment of dsNKG2D-IL-21 gene nanoparticles also retarded tumor growth and elongated the life span of tumor-bearing mice by activating NK and T cells in vivo. Thus, the dsNKG2D-IL-21 gene nanoparticles exerted efficient antitumor activities and would be potentially used for tumor therapy.