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Autophagy induces apoptosis and death of T lymphocytes in the spleen of pigs infected with CSFV.

ABSTRACT: Lymphocyte depletion and immunosuppression are typical clinical characteristics of pigs infected with classical swine fever virus (CSFV). The apoptosis of virus-infected and bystander cells plays a role in the immunopathology of classical swine fever (CSF). Here, we offer the first evidence that autophagy is involved in apoptosis and death of T lymphocytes in the spleen of pigs infected with CSFV. Using immunohistochemical assays, we observed that more LC3II-positive cells appear in the T-cell zone of spleens. Spleen cell apoptosis was demonstrated using flow cytometry and TUNEL staining. Confocal immunofluorescence revealed that partial LC3II-positive cells were simultaneously TUNEL-positive. By cultivating spleen cells ex vivo, we demonstrated that the inhibition of autophagy by 3-MA treatment inhibited apoptosis and death of T lymphocytes caused by CSFV infection but did not have this effect on B lymphocytes. Further observations demonstrated that uninfected cells in the spleen were also undergoing autophagy in vivo. In summary, these results linked autophagy with the apoptosis and cell death of splenic T cells, providing a new outlook to understand the mechanism of T lymphocyte depletion and immunosuppression during CSF.

SUBMITTER: Gou H

PROVIDER: S-EPMC5648758 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Autophagy induces apoptosis and death of T lymphocytes in the spleen of pigs infected with CSFV.

Gou Hongchao H Zhao Mingqiu M Fan Shuangqi S Yuan Jin J Liao Jiedan J He Wencheng W Xu Hailuan H Chen Jinding J

Scientific reports 20171019 1

Lymphocyte depletion and immunosuppression are typical clinical characteristics of pigs infected with classical swine fever virus (CSFV). The apoptosis of virus-infected and bystander cells plays a role in the immunopathology of classical swine fever (CSF). Here, we offer the first evidence that autophagy is involved in apoptosis and death of T lymphocytes in the spleen of pigs infected with CSFV. Using immunohistochemical assays, we observed that more LC3II-positive cells appear in the T-cell z ...[more]

PMID: 29051589

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Project description:BackgroundThe immune system is one aspect of health that is affected by dietary selenium (Se) levels and selenoprotein expression. Spleen is an important immune organ of the body, which is directly involved in cellular immunity. However, there are limited reports on Se levels and spleen health. Therefore, this study established a Se-deficient pig model to investigate the mechanism of Se deficiency-induced splenic pathogenesis.MethodsTwenty-four pure line castrated male Yorkshire pigs (45 days old, 12.50 ± 1.32 kg, 12 full-sibling pairs) were divided into two equal groups and fed Se-deficient diet (0.007 mg Se/kg) or Se-adequate diet (0.3 mg Se/kg) for 16 weeks. At the end of the trial, blood and spleen were collected to assay for erythroid parameters, the osmotic fragility of erythrocytes, the spleen index, histology, terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining, Se concentrations, the selenogenome, redox status, and signaling related inflammation and apoptosis.ResultsDietary Se deficiency decreased the erythroid parameters and increased the number of osmotically fragile erythrocytes (P < 0.05). The spleen index did not change, but hematoxylin and eosin and TUNEL staining indicated that the white pulp decreased, the red pulp increased, and splenocyte apoptosis occurred in the Se deficient group. Se deficiency decreased the Se concentration and selenoprotein expression in the spleen (P < 0.05), blocked the glutathione and thioredoxin antioxidant systems, and led to redox imbalance. Se deficiency activated the NF-κB and HIF-1α transcription factors, thus increasing pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-17, and TNF-α), decreasing anti-inflammatory cytokines (IL-10, IL-13, and TGF-β) and increasing expression of the downstream genes COX-2 and iNOS (P < 0.05), which in turn induced inflammation. In addition, Se-deficiency induced apoptosis through the mitochondrial pathway, upregulated apoptotic genes (Caspase3, Caspase8, and Bak), and downregulated antiapoptotic genes (Bcl-2) (P < 0.05) at the mRNA level, thus verifying the results of TUNEL staining.ConclusionsThese results indicated that Se deficiency induces spleen injury through the regulation of selenoproteins, oxidative stress, inflammation and apoptosis.

Project description:Background:Isatin derivatives have extensive biological activities, such as antitumor. IF203, a novel isatin derivative, has not previously been reported to have antitumor activity. Methods:Acid phosphatase assays (APAs) and Ki-67 immunohistochemistry were used to detect the proliferation of HepG2 cells. Transmission electron microscope (TEM) was applied to detect ultrastructural changes. Flow cytometry (FCM) was used to detect cell cycle, apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) of HepG2 cells in vitro. TUNEL, MMP and ROS immunofluorescence assays were applied to assess apoptosis, MMP, and ROS of HepG2 cells in vivo. Western Blotting was applied to assess the levels of apoptosis- and autophagy-related proteins. Results:In this study, in vivo and in vitro experiments showed that IF203 possesses antitumor activity. The results of APAs and Ki-67 immunohistochemistry demonstrated that IF203 could inhibit the proliferation of HepG2 cells. Cell cycle assays, downregulation of Cyclin B1 and Cdc2, and upregulation of P53 suggested that IF203 could lead to G2/M cell cycle arrest. In addition, ultrastructural changes, apoptosis assays, TUNEL immunofluorescence results, upregulated expression of Bax, and downregulated expression of Bcl-2 suggest that IF203 can induce apoptosis in HepG2 cells. After IF203 treatment, intracellular ROS levels increased, MMP decreased, JC-1 green fluorescence was enhanced, and the levels of Caspase-9, Caspase-3 and Cytochrome C expression were upregulated, suggesting that IF203 could induce apoptosis of HepG2 cells through the mitochondrial apoptosis pathway. Moreover, characteristic apoptotic ultrastructural changes were accompanied by the appearance of many autophagy bubbles and upregulation of Atg5, Atg12, ULK1, Beclin-1 and LC3-II proteins, suggesting that IF203 could induce autophagy in HepG2 cells. Conclusion:This study showed that IF203 leads to the death of HepG2 cells through cell cycle arrest, apoptotic induction, and autophagy promotion.

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Autophagy induces apoptosis and death of T lymphocytes in the spleen of pigs infected with CSFV.

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Autophagy induces apoptosis and death of T lymphocytes in the spleen of pigs infected with CSFV.

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