TIPE2, a negative regulator of innate and adaptive immunity that maintains immune homeostasis

Abstract

Immune homeostasis is essential for the normal functioning of the immune system, and its breakdown leads to fatal inflammatory diseases. We report here the identification of a member of the tumor necrosis factor-alpha-induced protein-8 (TNFAIP8) family, designated TIPE2, that is required for maintaining immune homeostasis. TIPE2 is preferentially expressed in lymphoid tissues, and its deletion in mice leads to multiorgan inflammation, splenomegaly, and premature death. TIPE2-deficient animals are hypersensitive to septic shock, and TIPE2-deficient cells are hyper-responsive to Toll-like receptor (TLR) and T cell receptor (TCR) activation. Importantly, TIPE2 binds to caspase-8 and inhibits activating protein-1 and nuclear factor-kappaB activation while promoting Fas-induced apoptosis. Inhibiting caspase-8 significantly blocks the hyper-responsiveness of TIPE2-deficient cells. These results establish that TIPE2 is an essential negative regulator of TLR and TCR function, and its selective expression in the immune system prevents hyperresponsiveness and maintains immune homeostasis.

Figure 1. Preferential expression of TIPE2 in lymphoid tissues and inflamed spinal cord

Northern blot (A–C) and RT-PCR analyses (D–E) of TIPE2 expression in selected tissues and cell preparations. A. RNAs were extracted from freshly harvested organs of either normal C57BL/6 mice (first 13 lanes) or mice with EAE (last lane). B. RNAs were extracted from murine cell lines that were pretreated with or without 2 μg/ml of concanavalin (Con)-A, or 2 μg/ml of lipopolysacchrides (LPS) for 24 hrs. OKT, OKT-3 B cells. C. RNAs were extracted from the following cell types of the C57BL/6 mice: lane 1, total thymocytes; lane 2, enriched splenic lymphocytes; lane 3, enriched splenic macrophages. D. RT-PCR analysis of total RNA extracted from murine cell lines using specific primers for TIPE2 and GAPDH. No template DNA was added to the control lane. E. NIH3T3 fibroblasts were cultured with 0–25 ng/ml of mouse TNF-α for 4 hrs. TIPE2 and GAPDH expression was determined by RT-PCR.

Figure 2. Spontaneous development of fatal inflammatory diseases in TIPE2-deficient mice

A. The survival curves of wild type (WT) (n=36) and TIPE2^−/− littermates (n=36) over a period of 11 months. B. Increased seral cytokines in TIPE2-deficient mice. Sera were collected from 4-month-old wild type (n=6) and TIPE2^−/− littermates (n=6) and tested for cytokines by ELISA. Data shown are means and SD of all mice, and are representative of three experiments. C. Increased spleen size in TIPE2-deficient mice. Spleens of a wild type and a TIPE2-deficient littermate at 4 months of age are shown. D. Severe lnterstitial lung inflammation in TIPE2-deficient mice. Lung sections of a wild type and a TIPE2-deficient littermate at 4 months of age are shown. Sections were stained with hematoxylin and eosin, and examined by microscopy with 200x magnifications.

Figure 3. Characteristics of TIPE2-deficient mice

Wild type (n=11) and TIPE2-deficient littermates (n=11) were sacrificed at 4 months of age and tested for the following parameters. A. Body and spleen weights. B. Blood leukocyte counts and splenocyte counts. C. B220⁺, CD4⁺ and CD11b⁺ splenocyte counts by flow cytometry following staining cells with respective antibodies. D. Percentages of CD69⁺ splenocytes in B220⁺ and B220⁻ sub-populations. Data shown are means and SD, and are representative of two experiments.

Figure 4. Increased T cell immunity of TIPE2 knockout mice

A-C. Increased T cell immune responses to LCMV infection. TIPE2^−/− mice and their littermate controls (n=6), 5–6 weeks of age, were injected i.p. with 10⁶ LCMV Armstrong, and sacrificed 8 days later. Splenocytes were collected, stained with anti-CD8 and LCMV-specific D^b/gp33-tetramer, and examined by flow cytometry (A) (Ochsenbein et al., 1999). The total numbers of CD8⁺, D^b/gp33-tetramer⁺ (tet⁺) cells per spleen were presented in 4B. Splenocytes were also cultured with LCMV-gp33–41 peptide (1μM) for 48 hours, and the concentrations of IFN-γ in the supernatants were determined by ELISA (C). Results are representative of two independent experiments. D. Increased reactivity of TIPE2 knockout T cells to TCR stimulation. CD4⁺ T cells were purified from spleens of 4–5-week-old wild type (n=5) or TIPE2-deficient littermates (n=5), and stimulated with indicated amounts of plate-bound anti-CD3 and 1 μg/ml of soluble anti-CD28 mAbs. Cytokine concentrations in the culture supernatants were determined by ELISA at 24 hr, and proliferation was measured by ³H-thymidine incorporation [presented as count per minute (CPM)] at 48 hr. Data shown are means and SD, and are representative of three experiments. No significant differences were detected between knockout and control groups in terms of the number and percentage of dead cells as determined by annexin V staining.

Figure 5. Increased reactivity of TIPE2 knockout and knockdown macrophages to Toll-like receptor stimulation

A. Bone marrow-derived macrophages from wild type and TIPE2-deficient mice (n=5) were treated with or without LPS (100ng/ml) for 8 hours. IL-6, IL-12p40 and TNF-α concentrations were determined by ELISA. B. TIPE2 mRNA levels in wild type and TIPE2 knockdown (KD) RAW 264.7 macrophages as determined by RT-PCR. GAPDH was used as a loading control whereas H₂O was used as the background control. C–D. Wild type and TIPE2 knockdown RAW 264.7 cells were treated with LPS (100ng/ml) for the indicated times. IL-6 protein (C) and mRNA (D) levels were determined by ELISA and real-time PCR, respectively. E. Wild type and TIPE2 knockdown RAW 264.7 cells were treated with LPS (100ng/ml), peptidoglycans (PGN) (10μg/ml), Poly(I:C) (50μg/ml), or CpG oligodeoxynucleotides (CPG) (1μM) for 8 hours. IL-6 concentrations in the supernatants were determined by ELISA. Data shown are representative of three experiments. The p value shown is for all TLR treated WT cultures as compared to their respective TIPE2 KD groups. No significant differences were detected between the two groups in terms of the number and percentage of apoptotic cells as determined by annexin V staining.

Figure 6. Hypersensitivity of TIPE2 knockout mice to septic shock

Wild type (n=11) and TIPE2-deficient littermates (n=10), 5–6 weeks of age, were injected intravenously with a low dose LPS (15 mg/kg). A. The survival curves. B. IL-1β, IL-6, IL-12p40 and TNF-α concentrations in the sera as determined by ELISA. For the LPS treated group, mice were injected with LPS as in panel A, and sera were collected 48 hrs later. For the untreated group, sera were collected from age and sex matched naïve mice (n=6). Data shown are means and SD, and are representative of two experiments.

Figure 7. Mechanisms of TIPE2 action

A-B. Increased AP-1 and NF-κB activation in TIPE2 knockdown cells. Wild type and TIPE2 knockdown RAW 264.7 cells were stimulated with LPS (100ng/ml) for the indicated times. c-Fos and c-Jun levels in the nuclear extracts were determined by Western blot, with histone H1 serving as a loading control (A, top two panels). Total cell lysates were also blotted with antibodies to total or phosphorylated (p) IκBα, p38, ERK (A, bottom three panels) and JNK1/2 (B). C. TIPE2 binds to endogenous caspase-8. Total cell lysates of wild type and TIPE2-knockdown RAW 264.7 cells were immunoprecipitated with an anti-TIPE2 rabbit polyclonal antibody or control rabbit Ig. The immunoprecipitates and cell lysates were then blotted with anti-caspase-8 (anti-casp-8), anti-TIPE2 or anti-FLIP antibodies. The level of TIPE2 in the crude lysate was too low to be detected by immunoblot (data not shown). D. Caspase-8 blockade diminishes the hyper-reactivity of TIPE2 knockdown cells. Wild type and TIPE2 knockdown RAW 264.7 cells were stimulated with LPS (100ng/ml) for 8 hrs with or without 1μg/ml of a caspase-8 inhibitor (Calbiochem). The level of intracellular IL-6 expression was determined by flow cytometry. Data shown are representative of two experiments. E–F. TIPE2 knockout T cells are resistant to activation-induced, but not cytokine withdrawal-induced, cell death. Splenocytes from wild type and TIPE2 knockout mice (n=5) were subjected to cytokine withdrawal-induced (E) or activation-induced (F) cell death as described in Procedures. The percentage of apoptotic cells was determined by flow cytometry following staining cells with annexin V, anti-CD4 and anti-CD8 antibodies (Song et al., 2000). Data shown are means and SD, and are representative of two experiments. G.TIPE2 knockdown in EL-4 T cells renders them resistant to FasL-induced apoptosis. TIPE2 mRNA levels in non-manipulated EL-4, control RNAi (wild type) and TIPE2 RNAi (knockdown) treated EL-4 cells were determined by RT-PCR (top panel). Wild type and TIPE2 knockdown EL-4 cells were either left untreated (control), or treated with 10 ng/ml FasL for 6 hrs (bottom panel). After staining with annexin V and propidium iodide (PI), cell death was analyzed by flow cytometry. Data shown are means and SD of the percentages of annexin V⁺ PI⁺ cells, and are representative of two experiments.