Glt25d2 Knockout Directly Increases CD25+CD69- but Decreases CD25-CD69+ Subset Proliferation and is Involved in Concanavalin-Induced Hepatitis.

BACKGROUND/AIMS: The elaborate structure of the extracellular matrix (ECM) and the appropriate surface glycoforms upon it are indispensable to CD4+ T cell regulation. METHODS: To explore the effects of Glcα1,2Galß1 glycosylation mediated by GLT25D2 (Colgalt2) for CD4+ T cell regulation, we prepared C57BL/6J Glt25d2-/- mice. In the induction of hepatitis, after concanavalin A (Con A) challenge for 6, 12, and 24 h, more extensive parenchymal injury was noted in Glt25d2-/- mice than in wild-type (WT) mice at 12 h. Immunohistochemistry and laser scanning confocal microscopy were used to detect GLT25D2 expression, and subsets of CD4+T cells was analyzed by flow cytometry. A total of 26 cytokines in serum samples were determined using Luminex technology. RESULTS: The trend in liver injury score variation was consistent with serum alanine aminotransferase and aspartate aminotransferase levels. The levels of interleukin 4 (IL-4), IL-1ß, IL-9, and several chemokines such as macrophage inflammatory protein-2, eotaxin, and growth-related oncogene α were significantly increased in Glt25d2-/- mice compared with WT mice after Con A challenge. A further phenotype analysis of primary Glt25d2-/- CD4+ T cells showed that Glt25d2 knockout increased the frequency of the CD25+CD69- subset but decreased the frequency of the CD25-CD69+ subset after Con A challenge for 6, 12, and 24 h compared with those of WT CD4+ T cells. Activation-induced apoptosis was also significantly increased in Glt25d2-/- CD4+ T cells after Con A challenge compared with WT CD4+ T cells. Lectin microarray hybridization showed that Glt25d2 knockout increased the binding activity of Narcissus pseudonarcissus lectin to CD4+ T cells but Amaranthus caudatus lectin-binding activity was lost during Con A challenge. CONCLUSION: The present results suggest that collagen glycosylation mediated by GLT25D2 may regulate a subset of CD4+ T cells and be involved in the pathogenesis of Con A-induced hepatitis.

GLT25D2 Is Critical for Inflammatory Immune Response to Promote Acetaminophen-Induced Hepatotoxicity by Autophagy Pathway.

Acetaminophen (APAP) overdose induces hepatocyte necrosis and causes liver hepatotoxicity. Currently, the role of galactosyltransferase in APAP-induced liver injury is still unclear. This study assessed the contribution of the GLT25D2 gene, a kind of collagen galactosyltransferase, to the development of APAP-induced liver injury. This study found that the expression of GLT25D2 markedly increased first and then decreased in the liver of mice treated with APAP, however, it downregulated in the liver of APAP overdose-patients compared with normal controls. Knockout of GLT25D2 significantly ameliorated the liver injury, meanwhile, it downregulated the proinflammatory cytokines (IL-6, TNF-α) and chemokines (CXCL-10, MIG and CXCL-1) levels, however, and upregulated the anti-inflammatory cytokines (IL-22, IL-10) levels. Mechanistic explorations showed that (1) GLT25D2 knockout promoted autophagy pathway; and (2) the GLT25D2 knockout-induced autophagy selected to clear damaged mitochondria in APAP-induced liver injury by mitophagy; and (3) the autophagy intervention by Atg 7 siRNA cancelled liver protection by knockout of GLT25D2 through regulating liver inflammation. In conclusion, our study proves that the upregulated expression of GLT25D2 decreased autophagy contributing to APAP-induced hepatotoxicity by mediating the inflammatory immune regulatory mechanism.

Regulation of autophagy by GLT25D2 gene in acetaminophen-induced hepatotoxicity injury / 中华危重病急救医学

Objective To investigate whether GLT25D2 gene regulates autophagy in acetaminophen (APAP)-induced hepatotoxicity injury.Methods GLT25D2+/+ wild-type C57BL/6J mice and GLT25D2-/- C57BL/6J mice were selected as subjects. ①In vivo experiment: 20 for wild-type mice and 20 for GLT25D2-/- mice were respectively divided into phosphate buffer (PBS) control group and APAP intervention group according to random number table, with 10 mice in each group. The hepatotoxicity injury model of mice was reproduced by intraperitoneal injection of 25 g/L APAP solution 500 mg/kg. The PBS control group was intraperitoneally injected with the same amount of PBS. The mice were sacrificed immediately after model reproduction, and the liver tissues were harvested. Western Blot was used to detect the expressions of autophagy-related proteins ATG5, ATG7, microtubule-associated protein 1 light chain 3 (LC3) and P62. The ultrastructural changes in liver tissue were observed under electron microscope to observe the level of autophagy. ②In vitro experiment: primary hepatocytes extracted by two-step collagen perfusion from one GLT25D2+/+wild-type mouse and one GLT25D2-/- mouse were divided into two parts respectively. One part was treated with 5 mmol/L APAP solution. The cells were harvested at 0, 8, and 12 hours, and the expressions of autophagy-related proteins ATG5, ATG7, LC3, and P62 were determined by Western Blot. The other part was transfected with the green fluorescent protein-LC3 plasmid (GFP-LC3) for 24 hours. The cells were cultured with PBS (PBS control group) or 5 mmol/L APAP (APAP intervention group) for 12 hours, and the positive expression of GFP-LC3 was observed under the fluorescence microscope, thereby reflecting the expression of autophagosomes.Results ①In vivo experiment: compared with the corresponding PBS control group, the expressions of the positive-associated proteins ATG5, ATG7 and LC3-Ⅱ in liver tissue of the APAPintervention group were down-regulated in the wild-type and GLT25D2-/- mice, while the expression of the negative correlation protein P62 was up-regulated, indicating that the overall level of autophagy decreased after treatment with APAP. Compared with wild-type mice, the expressions of autophagy positive correlation proteins ATG5 and ATG7 were up-regulated in GLT25D2-/- mice (ATG5/β-actin: 1.21±0.29 vs. 0.84±0.19, ATG7/β-actin:1.29±0.14 vs. 1.54±0.40, bothP > 0.05), LC3-Ⅱ expression was slightly down-regulated (LC3-Ⅱ/β-actin: 0.52±0.06 vs. 0.58±0.06,P > 0.05), while negative correlation protein P62 was down-regulated (P62/β-actin: 1.13±0.94 vs. 1.54±0.40,P > 0.05), indicating that the expression of autophagy in GLT25D2-/- mice was higher than that in wild-type mice. Ultrastructural observation under electron microscope showed that the number of autophagosomes in the liver tissue of wild-type mice did not change significantly after APAP intervention as compared with that in PBS control group, but the number of autophagosomes in GLT25D2-/- mice was increased. ②In vitro experiment: with the prolongation of APAP intervention, the expressions of ATG5 and ATG7 in the primary hepatocytes of wild-type and GLT25D2-/-mice were up-regulated, LC3 was slightly fluctuated, and the expression of negative-related protein P62 was gradually down-regulated. The peak value or the trough value reached at 12 hours. It was indicated that the expression of autophagy in APAP-stimulated cells was enhanced with a time-dependent manner. Compared with wild-type mice, the expressions of autophagy correlation proteins ATG5, ATG7, LC3-Ⅱ and P62 were up-regulated in GLT25D2-/- mice at 12 hours (ATG5/β-actin: 0.93±0.09 vs. 0.74±0.06, ATG7/β-actin: 0.80±0.09 vs. 0.65±0.10, LC3-Ⅱ/β-actin:1.35±0.30 vs. 1.15±0.20, P62/β-actin: 0.36±0.02 vs. 0.31±0.03, allP > 0.05), indicating that the expression of autophagy was enhanced after gene knockout. Fluorescence microscopy showed that GFP-LC3 positive cells in both wild-type and GLT25D2-/- mice hepatocytes were significantly increased after APAP intervention as compared with those of PBS control group, and the proportion of GFP-LC3 positive cells in GLT25D2-/- mice was significantly higher than that in wild-type mice (0.64±0.08 vs. 0.36±0.05,P < 0.05).Conclusions GLT25D2 is a negative regulator of autophagy. Knockout of GLT25D2 gene can enhance the autophagy level of APAP-induced hepatotoxicity injury in mice.