Splenocyte apoptosis and autophagy is mediated by interferon regulatory factor 1 during murine endotoxemia

Abstract

Sepsis-induced lymphocyte and dendritic cell apoptosis contributes to immunosuppression, which results in an inability to eradicate the primary infection as well as a propensity to acquire new, secondary infections. Another cellular process, autophagy, is also activated in immune cells and plays a protective role. In the present study, we demonstrate that interferon regulatory factor 1 (IRF-1) regulates both immune cell apoptosis and autophagy in a murine endotoxemia model. Interferon regulatory factor 1 is activated at an early phase through a Toll-like receptor 4-dependent, myeloid differentiation primary response gene 88-independent manner in splenocytes. Furthermore, IRF-1 knockout (KO) mice are protected from a lethal endotoxemia model. This protection is associated with decreased apoptosis and increased autophagy in splenocytes. Interferon regulatory factor 1 KO mice experience decreased apoptotic cell loss, especially in CD4⁺ T lymphocytes and myeloid antigen-presenting cells. Meanwhile, IRF-1 KO mice demonstrate increased autophagy and improved mitochondrial integrity. This increased autophagy in KO mice is attributable, at least in part, to deactivation of mammalian target of rapamycin/P70S6 signaling--a main negative regulator of autophagy. Therefore, we propose a novel role for IRF-1 in regulating both apoptosis and autophagy in splenocytes in the setting of endotoxemia with IRF-1 promoting apoptosis and inhibiting autophagy.

Figure 1. IRF-1 activation at early phase and is mediated through the TLR4-TRIF dependent manner in response to LPS stimulation

(A) After LPS injection at predetermined time points, splenocytes were isolated from WT mice. Nuclear proteins were isolated and IRF-1 activation was analyzed by Western blot. WT and TLR4 KO (B), TRIF KO (C), MyD88 KO (D) mice were injected with PBS or LPS for 1 h and splenocytes were isolated. Nuclear IRF-1 was analyzed by Western blot. *, P<0.05; results are representative of 3 separate independent experiments.

Figure 2. IRF-1 KO mice have decreased apoptosis in splenocytes following LPS exposure

Permanent blocks of splenic tissue obtained from WT and IRF-1 KO mice 16 h following PBS or LPS (20 mg/kg) injection were sectioned and (A) TUNEL staining or (B) cleaved caspase-3 immunohistochemistry staining was performed (magnitude X 200). Positive staining was presented as percentage of stained area over total area (% area stained). (C) Splenic tissue obtained from WT and IRF-1 KO mice 16 h following PBS or LPS (20 mg/kg) injection was imaged by transmission electron microscope (magnitude X 5000). Arrow points to apoptotic bodies. Percentage of apoptotic cells among total cells was used for apoptosis quantification. (D) WT and IRF-1 KO mice were injected with PBS or LPS (20 mg/kg) for 16 h and splenocytes were isolated. Caspase-3 cleavage was analyzed by Western blot. *, P<0.05; results are representative of 3 separate independent experiments.

Figure 3. Apoptosis is selectively reduced in splenic CD4+ T lymphocytes and myeloid APC in IRF-1 KO mice following LPS exposure

WT and IRF1-KO mice were challenged with LPS (20 mg/kg) as described and total splenocytes were harvested. Apoptotic cells (Annexin V+7-AAD+) were detected by flow cytometry. (A) Total splenocytes, (B) CD3+CD4+ T cells, (C) CD3+CD8+ T cells, (D) CD11b+NK1.1- myeloid APC including dendritic cells and macrophages, (E) CD11b+NK1.1+ NK cells, and (F) B220+ B cells were analyzed. The percent of apoptotic cells was quantified for each cell population. *, P<0.05; results are representative of 3 separate independent experiments.

Figure 4. Splenocytes from IRF-1 KO mice demonstrate increased autophagy compared to WT mice following LPS exposure

(A) Splenic tissue obtained from WT and IRF-1 KO mice 16 h following PBS or LPS (20 mg/kg) injection imaged by transmission electron microscope (magnitude X 20000). Arrows point to damaged mitochondria and autophagy. Determining the number of autophagic vacuoles per cross-sectioned cell was used for autophagy quantification. (B) WT and IRF-1 KO mice were injected with PBS or LPS (20 mg/kg) for 16 h and splenocytes were isolated. LC3B-II was analyzed by Western blot. (C) WT and IRF-1 KO mice were injected with LPS (20 mg/kg) for 16 h and splenocytes were isolated and stained with 200 nM MitoTracker Deep Red and MitoTracker Green for 45 min at 37°C. Representative flow cytometry plots are represented. *, P<0.05; results are representative of 3 separate independent experiments.

Figure 5. IRF-1 activation promotes both intrinsic and extrinsic apoptotic pathway and inhibits autophagy by modulating mTOR/P70S6 activity

(A) WT and IRF-1 KO mice were injected with PBS or LPS (20 mg/kg) for 16 h and splenocytes were isolated. Caspase-3, -8, -9 cleavage was analyzed by Western blot. (B) WT and IRF-1 KO mice were injected with PBS or LPS (20 mg/kg) for 16 h and splenocytes were isolated. mTOR and P70S6 activation were analyzed by Western blot. *, P<0.05; results are representative of 3 separate independent experiments.