Suppression of T Cell Autophagy Results in Decreased Viability and Function of T Cells Through Accelerated Apoptosis in a Murine Sepsis Model

Abstract

Objective: While type 1 programmed cell death (apoptosis) of T cells leads to immunosuppression in sepsis, a crosstalk between apoptosis and autophagy (type 2 programmed cell death) has not been shown. The aim of this study is to elucidate the details of the interaction between autophagy and immunosuppression.

Design: Laboratory investigation in the murine sepsis model.

Setting: University laboratory.

Subjects: Six- to 8-week-old male mice.

Interventions: We investigated the kinetics of autophagy in T cells from spleen in a cecal ligation and puncture model with green fluorescent protein-microtubule-associated protein light chain 3 transgenic mice. We analyzed apoptosis, mitochondrial homeostasis and cytokine production in T cells, and survival rate after cecal ligation and puncture using T cell-specific autophagy-deficient mice.

Measurements and main results: We observed an increase of autophagosomes, which was assessed by flow cytometry. However, an autophagy process in CD4 T cells during sepsis was insufficient including the accumulation of p62. On the other hand, a blockade of autophagy accelerated T cell apoptosis compared with the control mice, augmenting the gene expression of Bcl-2-like 11 and programmed cell death 1. Furthermore, mitochondrial accumulation in T cells occurred via a blockade of autophagy during sepsis. In addition, interleukin-10 production in CD4 T cells from the cecal ligation and puncture-operated knockout mice was markedly increased. Consequently, deficiency of autophagy in T cells significantly decreased the survival rate in the murine sepsis model.

Conclusions: We demonstrated that blocking autophagy accelerated apoptosis and increased mortality in concordance with the insufficient autophagy process in CD4 T cells in the murine sepsis model, suggesting that T cell autophagy plays a protective role against apoptosis and immunosuppression in sepsis.

Conflict of interest statement

Dr. Watanabe received support for article research from JSPS KAKENHI grant number 15K20333. The remaining authors have disclosed that they do not have any potential conflicts of interest.

Figures

Figure 1.

Although lymphocyte autophagosomes are increased by septic stimulation, the process of autophagy is insufficient in a murine sepsis model in CD4+ T cells. A, Evaluation of autophagy in green fluorescent protein (GFP)-light chain 3 (LC3) transgenic mice by flow cytometry. After incubation with anti-CD3 and anti-CD28 antibodies for 48 hr for CD4+ lymphocytes and lipopolysaccharide (LPS) for B lymphocytes, mean fluorescence intensity (MFI) of harvested splenocytes was measured by flow cytometry for 48 hr. Representative data of three independent experiments are shown. B, Quantitation of autophagic protein, LC3-B and p62, was performed by western blotting. Lymphocytes were stimulated as the above procedure. The amount of each protein level was normalized by GAPDH. Representative data of independent three experiments are shown. C, FACS profiles for harvested splenocytes of GFP-LC3 transgenic mice. Mice underwent cecal ligation and puncture (CLP) and sham procedure and were killed at the time of 24 hr after the operation. Harvested splenocytes were stained with surface antigen markers and measured by flow cytometry. Representative data of five independent experiments are shown. D, Sequential MFI of lymphocytes from GFP-LC3 transgenic mice was compared between CLP- and sham-operated mice. n = 3–6 mice in each group. Results are shown as mean ± sd in a bar graph. Data are analyzed by student t test; #p < 0.05. E, MFI of Lysotracker staining lymphocytes from sham- and CLP-operated mice are shown. Each sample was stained with surface antigen markers. Data are expressed as mean and sd, and analyzed by two-way analysis of variance (ANOVA) and student t test. n = 3–4 mice in each group; #p < 0.05. F, MFI of p62 protein conjugated with fluorescent second antibody in lymphocytes from wild-type mice. Mice underwent CLP and sham procedure, and were killed at the time of 24 hr after the operation. Harvested splenocytes were stained with p62 and surface antigen markers concomitantly, and then measured by flow cytometry. Data are expressed as mean and sd, and analyzed by two-way ANOVA and student t test. n = 4–6 mice in each group; #p < 0.05. FACS = fluorescence activated cell sorting, GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

Figure 2.

Apoptosis was accelerated by Atg5 deletion in T cells during sepsis. A, Polymerase chain reaction amplification of genomic DNA from sorted lymphocytes in Atg5f/f and CD4-Cre/Atg5f/f mice. Atg5 flox allele and CD4-Cre allele are shown in the upper and middle figure, respectively. The positive controls are tail DNA from CD4-Cre/Atg5f/f mice. In the lower figure, the deleted allele is found in CD4+ T cells and CD8+ T cells extracted from CD4-Cre/Atg5f/f mice. The positive control is liver DNA from liver specific-Cre/Atg5f/f mice. B, Representative subpopulation of splenocytes in Atg5f/f and CD4-Cre/Atg5f/f mice. Mice were performed cecal ligation and puncture (CLP) or sham procedure. Splenocytes were stained with anti-CD4+/PE and anti-CD8+/APC. Representative data of five independent experiments are shown. C, FACS profiles of splenocytes stained with Annexin V and PI in Atg5f/f and CD4-Cre/Atg5f/f mice for 24 hr postoperatively. Samples were stained with anti-CD4+/PE and anti-CD8+/APC. Representative data of five independent experiments are shown. D, Subpopulation of Annexin V positive and PI negative staining splenocytes were shown for early (postoperative period, 6 hr) and late (postoperative period, 24 hr) apoptosis. Data are expressed as mean and sd; n = 8–10 mice in each group; #p < 0.05 was significance analyzed by two-way analysis of variance (ANOVA) and student t test. E, Subpopulation of Annexin V and PI staining lymphocytes from peripheral blood were shown for early (Annexin V positive and PI negative) and late (Annexin V positive and PI positive) phase of apoptosis. Data are expressed as mean and sd; n = 6–8 mice in each group; #p < 0.05 was significance analyzed by student t test between CLP-operated Atg5f/f mice and CLP-operated CD4-Cre/Atg5f/f mice. F, Relative RNA expression for apoptotic gene in sham, CLP-operated Atg5f/f mice and sham, and CLP-operated CD4-Cre/Atg5f/f mice. Total RNA in CD4+ splenic lymphocytes was extracted from experimental mice at 24 hr after the procedure, and then relative RNA expression for the several genes was analyzed. Data are expressed as mean and sd; n = 8–10 mice in each group; #p < 0.05 was significance analyzed by two-way ANOVA and student t test. APC = allophycocyanin, Bcl-2 = B-cell leukemia/lymphoma 2, BIM = Bcl-2-like 11, FACS = fluorescence activated cell sorting, PDCD1 = programmed cell death 1, PE = phycoerythrin, PI = propidium iodide.

Figure 3.

Mitochondrial mass was increased by autophagy deficiency in T cells. FACS profiles of Mitotracker FM green staining lymphocytes in sham, cecal ligation and puncture (CLP)-operated Atg5f/f mice and sham, and CLP-operated CD4-Cre/Atg5f/f mice at 24 hr after the procedure. Representative data of five independent experiments are shown. FACS = fluorescence activated cell sorting, MFI = mean fluorescence intensity.

Figure 4.

Interleukin (IL)-10 secretion was increased by blocking autophagy in T cells, and T cell autophagy affects the late phase mortality of sepsis. A, After lymphocytes were harvested from spleen, they were stimulated with anti-CD3 and anti-CD28 antibodies for 24 hr. Then, we measured IL-2, interferon (IFN)-γ, and IL-10 concentrations in the supernatant fluid of incubated lymphocytes by enzyme-linked immunosorbent assay. Data are expressed as mean and sd; n = 8–9 mice in each group; #p < 0.05 was significance analyzed by two-way analysis of variance (ANOVA) and student t test. B, Relative RNA expression for cytokine gene in sham, cecal ligation and puncture (CLP)-operated Atg5f/f and sham, and CLP-operated CD4-Cre/Atg5f/f mice. Total RNA in CD4+ lymphocytes was extracted from experimental mice at 24 hr after the procedure, and then relative RNA expression of IL-2, interferon-γ (IFNG) and IL-10 were analyzed. Data are expressed as mean and sd; n = 8–10 mice in each group; #p < 0.05 was significance analyzed by two-way ANOVA and student t test. C, Survival rates between CLP-operated Atg5f/f mice and CLP-operated CD4-Cre/Atg5f/f mice. Survival rates were observed every 12 hr. n = 18–20 mice in each group. p < 0.05 was significance analyzed by Kaplan-Meier method and log-rank test.

Figure 5.

Interaction between autophagy and apoptosis in T cell. The summary figure illustrates the crosstalk between autophagy and apoptosis in T cells during sepsis. A, Septic stresses cause the increase of reactive oxygen species (ROS) and accumulation of damaged mitochondria. Damaged organelles are cleared by autophagic machinery and these events prevent apoptosis. B, Blocking T cell autophagy accelerates T cell apoptosis and eventually results in the immunosuppressive status. Bcl-2 = B-cell leukemia/lymphoma 2, PDCD1 = programmed cell death 1.