Interleukin 35 contributes to immunosuppression by regulating inflammatory cytokines and T cell populations in the acute phase of sepsis.

Cytokines interact closely with each other and play a crucial role in the progression of sepsis. We focused on the associations of a cytokine network with IL-35 in sepsis. First, the retrospective study included 42 patients with sepsis and 23 healthy controls. Blood samples were collected from patients on days 1, 2, 4. Levels of IL-35, IL-1ß, IL-4, IL-6, IL-10, IL-17A, TNF-α and IFN-γ were measured. They all increased to various extend on days 1, 2, 4, and strongly associated with markers of disease severity. Network analysis revealed a network formed by IL-35, with IL-6, IL-10, IL-17A, TNF-α and IFN-γ throughout the acute phase of sepsis(days 1, 2 and4). Then, the CLP-induced septic rats were used. The recombinant human IL-35(rIL-35) upregulated the levels of IL-10, but downregulated IL-4, IL-6, IL-17A, TNF-α and IFN-γ, while it had no significant effect on IL-1ß, and upregulated the percentages of CD4+CD25+Tregs, and iTR35, but downregulated Teff cells in the peripheral blood. The rIL-35 reduced inflammation damage and improved prognosis of the septic rats. IL-35 forms a network with other cytokines and plays a major role in the immunopathogenesis of sepsis.

miR-34b-5p promotes renal cell inflammation and apoptosis by inhibiting aquaporin-2 in sepsis-induced acute kidney injury.

OBJECTIVE: This study was designed to uncover the mechanism of miR-34b-5p-mediated aquaporin-2 (AQP2) in sepsis-induced injury using human renal tubular epithelial cells (HK-2). METHODS: Serum levels of miR-34b-5p, TNF-α, IL-1ß, IL-6, serum creatinine (SCr), and blood urea nitrogen (BUN) in septic patients with acute kidney injury (AKI) and healthy controls were detected. Lipopolysaccharide (LPS) was used to induce sepsis in HK-2 cells. LPS-induced HK-2 cells were transfected with miR-34b-5p inhibitor, miR-34b-5p mimic, pcDNA3.1-AQP2, si-AQP2, miR-34b-5p inhibitor + si-NC, or miR-34b-5p inhibitor + si-AQP2. The expressions of miR-34b-5p, AQP2, Bax, Bcl-2, cleaved caspase-3, TNF-α, IL-1ß, and IL-6 in HK-2 cells were detected. TUNEL staining revealed the apoptosis of HK-2 cells. Dual-luciferase reporter assay verified the binding between miR-34b-5p and AQP2. RESULTS: The expression of miR-34b-5p and the inflammatory responses were augmented in septic AKI patients. miR-34b-5p was up-regulated and AQP2 was down-regulated in LPS-induced HK-2 cells. miR-34b-5p inhibition or AQP2 overexpression ameliorated apoptosis and inflammation in LPS-induced HK-2 cells. In contrast, overexpressing miR-34b-5p deteriorated LPS-induced injury in HK-2 cells. AQP2 was a downstream target of miR-34b-5p. AQP2 silencing abolished the suppressive effects of miR-34b-5p inhibition on LPS-induced apoptosis and inflammatory response in HK-2 cells. CONCLUSION: miR-34b-5p inhibits AQP2 to promote LPS-induced injury in HK-2 cells.

[Hsa-let-7b-5p Inhibits Proliferation of Human Leukemia THP-1 Cells via FTO/m6A/MYC Signaling Pathway].

OBJECTIVE: To investigate the down-regulation effect of let-7b-5p on the expression of FTO in acute myeloid leukemia cell line THP-1 and inhibitory effect on THP-1 proliferation via m6A/MYC signaling pathway. METHODS: The acute myeloid leukemia cell line THP-1 and the normal human peripheral blood mononuclear cells (PBMNC) were selected as subjects. The expression of let-7b-5p and FTO mRNA in those cells was detected by qPCR, further the expression of FTO protein in those cells was detected by Western blot. And, the luciferase reporter gene assay was used to verify the targeting effect of let-7b-5p on FTO. Finally, THP-1 cells were transfected respectively with let-7b-5p mimic, and PBMNC with let-7b-5p inhibitor, there after the C-MYC mRNA m6A enrichment level in transfected cells was analyzed by dot blot, the expression levels of let-7b-5p, FTO and c-MYC were assayed by RT-PCR, the expressions of FTO and c-MYC protein were verified by Western blot, and the proliferation level of cells after transfection was detected by MTT assay. RESULTS: Compared with PBMNC, the expression of let-7b-5p in THP-1 significantly decreased, while the expression of FTO was significantly increased (P<0.05). After transfection with let-7b-5p mimic combined with FTO 3'-UTR, the luciferase activity of transfected THP-1 cells significantly decreased, but the luciferase activity significantly increased after transfection with mutant 3'-UTR, which was significantly different from the negative control group(blank vector) (P<0.05). Let-7b-5p inhibitor down-regulated c-MYC mRNA m6A enrichment, and then up-regulated the expression of FTO in transfected PBMNC cells, the effects of which were significant (P<0.05). However, let-7b-5p mimic up-regulated c-MYC mRNA m6A enrichment level and down-regulated the expression of FTO in the transfected THP-1 cells, and the cell proliferation rate was significantly lower than that in the negative control group (blank vector) (P<0.05). CONCLUSION: Human acute myeloid leukemia cell line THP-1 low expresses the let-7b-5p, which regulates c-MYC expression through let-7b-5p-/FTO-/m6A axis and promotes the proliferation of leukemia cell line THP-1.