Interferon-γ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway

Abstract

Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.

Keywords: BAX and BAK; COVID-19; SARS-CoV-2; TNF; Toll-like receptor; apoptosis; caspase-8; hemophagocytic lymphohistiocytosis; iNOS; interferon.

Graphical abstract

Figure 1

IFNγ primes macrophages for TLR-induced cell death (A and B) Wild-type (WT) bone-marrow-derived macrophages (BMDMs) were treated with (A) IFNγ (50 ng/mL) or (B) IFNβ (1,000 U/mL, n = 5) overnight, then with either LPS (50 ng/mL, n = 5), Pam3CSK4 (P3C, 500 ng/mL, n = 6), or PolyI:C (10 μg/mL, n = 6) for 24 h. Cell death was assessed by propidium iodide (PI) exclusion as measured by flow cytometry. (C) WT or Tnf^−/−Fasl^gld/gldTrail^−/− BMDMs were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) (left), or P3C (500 ng/mL) (right) for 24 or 48 h ± TNF (100 ng/mL). Cell death was assessed by PI exclusion as measured by flow cytometry (n = 5). (D) Immunoblot of WT BMDMs treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 8, 12, or 24 h. Treatment with the BCL-2, BCL-XL, and BCL-W inhibitor, ABT-737 (737, 1 μM) and cycloheximide (CHX, 10 μg/mL) ± Q-VD-OPh (QVD, 20 μM) for 4 h was used as a control (n = 3). (E) Immunoblot of Tnf^−/−Fasl^gld/gldTrail^−/− BMDMs that were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 12, 24, or 48 h (n = 2). Data represent the mean value ± SD, or a representative immunoblot, from n independent experiments. p > 0.05 (n.s.), p ≤ 0.001 (^∗∗∗), p ≤ 0.0001 (^∗∗∗∗). See also Figures S1 and S2 and Videos S1, S2, S3, and S4.

Figure 2

Both extrinsic caspase-8 and the mitochondrial apoptosis effector proteins BAX/BAK contribute to IFNγ/LPS-induced macrophage killing (A) WT, Mlkl^−/−, or Casp8^−/−Mlkl^−/− BMDMs were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 24 or 48 h. Treatment with LPS and Compound A (Cp. A, 1 μM) (extrinsic apoptosis) or LPS and Z-VAD-fmk (Z-VAD, 20 μM) (necroptosis) for 24 h were used as controls. Cell death was assessed by PI exclusion as measured by flow cytometry (n = 4). (B) Immunoblot analysis of WT, Mlkl^−/−, or Casp8^−/−Mlkl^−/− BMDMs that were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 8, 12 or 24 h. WT BMDMs were treated with LPS and Cp. A for 12 h as a control (n = 3). (C) WT and Bax^−/−Bak^−/− BMDMs were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 24 or 48 h. ABT-737 (1 μM) plus cycloheximide (CHX, 10 μg/mL) treatment for 6 h was used as a control. Cell death was assessed by PI exclusion as measured by flow cytometry (n = 4). (D) Immunoblot of WT and Bax^−/−Bak^−/− BMDMs that were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 12, 24, or 48 h (n = 3). Data represent the mean value ± SD, or a representative immunoblot, from n independent experiments. p > 0.05 (n.s.), p ≤ 0.05 (^∗), p ≤ 0.01 (^∗∗), p ≤ 0.001 (^∗∗∗), p ≤ 0.0001 (^∗∗∗∗). See also Figure S3.

Figure 3

Caspase-8 triggers mitochondrial cytochrome c loss and cell death through a BID-independent mechanism following IFNγ/LPS treatment (A) WT, Mlkl^−/−, or Casp8^−/−Mlkl^−/− BMDMs were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 12, 24, 36, or 48 h. Treatment with ABT-737 (1 μM) and cycloheximide (CHX, 10 μg/mL) for 5 h was used as a positive control for BAX/BAK activation (see Figure 2C). Cytochrome c retention was measured by intracellular cytochrome c staining and flow cytometric analysis (n = 3). (B) WT and Bid^−/− BMDMs were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 24 or 48 h. Cell death was assessed by PI exclusion as measured by flow cytometry (n = 6). (C) Immunoblot of WT and Bid^−/− BMDMs that were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 8 or 16 h. WT BMDMs treated with ABT-737 (1 μM) plus cycloheximide (CHX, 10 μg/mL) for 4 h were used as a positive control (n = 3). (D) WT or Bid^−/− BMDMs were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 16 or 24 h. Treatment with ABT-737 (1 μM) and cycloheximide (CHX, 10 μg/mL) for 6 h was used as a positive control for BAX/BAK activation. Cytochrome c retention was measured by intracellular cytochrome c staining and flow cytometric analysis (n = 3). (E) Immunoblot analysis of WT BMDMs that had been primed with IFNγ (50 ng/mL) overnight then stimulated with LPS (50 ng/mL) for 8, 12, or 24 h (n = 3). Data represent the mean value ± SD, or a representative immunoblot, from n independent experiments. p > 0.05 (n.s.), p ≤ 0.05 (^∗), p ≤ 0.001 (^∗∗∗), p ≤ 0.0001 (^∗∗∗∗).

Figure 4

Caspase-8 is required for transcriptional programming of macrophages upon stimulation with IFNγ/LPS, resulting in elevated NOXA and reduced BCL-2 transcripts (A–D) Mlkl^−/− and Casp8^−/−Mlkl^−/− BMDMs were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 7 h followed by RNA isolation and 3′ mRNA-sequencing. (A) Multidimensional scaling (MDS) plot and (B) differentially expressed genes (DEGs) that are up- or down-regulated in Casp8^−/−Mlkl^−/− BMDMs in comparison to Mlkl^−/− cells are shown. The effect of Casp8 deletion on genes associated with cell death was assessed by (C) a volcano plot and (D) a heatmap plot of DEGs involved in distinct cell death signaling pathways (see Table S1) that are enriched in Mlkl^−/− BMDMs versus Casp8^−/−Mlkl^−/− BMDMs . Adjusted p ≤ 0.05 and cut-off values logFC ≥ 1 or logFC ≤ −1 (n = 3). (E) Immunoblot of WT, Mlkl^−/−, or Casp8^−/−Mlkl^−/− BMDMs that were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 2, 4, 6, or 8 h (n = 2). (F) WT or Casp8^−/−Mlkl^−/− BMDMs were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) for 4, 8, or 16 h. Bcl2 expression was assessed by quantitative PCR (qPCR) using Hprt as a housekeeping gene. Baseline Bcl2 expression (dCT) is shown (right) as a control (n = 3). (G) Casp8^+/+ (WT [circles] or Ripk3^−/− [triangles]) or Casp8^−/− (Casp8^−/−Mlkl^−/− [circles] or Casp8^−/−Ripk3^−/− [triangles]) BMDMs were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) ± the BCL-2 inhibitor, ABT-199 (1 μM), or DMSO for 16 or 24 h. Treatments with LPS and Compound A (Cp. A 1 μM) (extrinsic apoptosis) or ABT-737 (1 μM) plus cycloheximide (CHX, 10 μg/mL) (intrinsic apoptosis) were used as controls. Cell death was assessed by PI exclusion as measured by flow cytometry (n = 4). (H) WT BMDMs were treated with the MCL-1 inhibitor S63845 (10 μM) ± ABT-199 (1 μM) for 12 h. Cell death was assessed by PI exclusion as measured by flow cytometry (n = 3). Data represent the mean value ± SD, or a representative immunoblot, from n independent experiments. p > 0.05 (n.s.), p ≤ 0.05 (^∗), p ≤ 0.01 (^∗∗), p ≤ 0.001 (^∗∗∗). See also Figures S4 and S5 and Tables S1–S3.

Figure 5

Caspase-8 enzymatic activity is required for IFNγ/LPS-induced killing of macrophages (A and B) WT, Ripk3^−/−, Casp8^−/−Ripk3^−/−, or Casp8^C362S/C362SRipk3^−/− (Casp8^CS/CSRipk3^−/−) BMDMs were primed with IFNγ (50 ng/mL) overnight then stimulated with LPS (50 ng/mL) for 24 or 48 h. Treatment with LPS and Compound A (Cp. A, 1 μM) for 24 h (extrinsic apoptosis), LPS and IDN-6556 (IDN, 5 μM) for 24 h (necroptosis), and ABT-737 (1 μM) and cycloheximide (CHX, 10 μg/mL) for 6 h (intrinsic apoptosis) were used as controls. (A) Cell death and (B) cytochrome c retention was measured by PI exclusion as measured by flow cytometry or intracellular cytochrome c staining and flow cytometric analysis (n = 3). (C and D) Immunoblot of WT, Ripk3^−/−, Casp8^−/−Ripk3^−/−, or Casp8^CS/CSRipk3^−/− BMDMs primed with IFNγ (50 ng/mL) overnight and stimulated with LPS (50 ng/mL) for (C) 16 and 24 h, or (D) 2–8 h. Ponceau stain is provided as a loading control (n = 2). (E) WT or Mlkl^−/− BMDMs were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) ± IDN (5 μM) or DMSO for 16, 24, or 48 h. LPS and IDN or IFNγ and IDN treatment for 24 h (necroptosis) was used as a control. Cell death was assessed by PI exclusion as measured by flow cytometry (n = 3). (F) Immunoblot of WT or Mlkl^−/− BMDMs that were treated with IFNγ (50 ng/mL) overnight then with LPS (50 ng/mL) ± IDN (5 μM) or DMSO for 8 or 16 h (n = 2). Data represent the mean value ± SD, or a representative immunoblot, from n independent experiments. p > 0.05 (n.s.), p ≤ 0.01 (^∗∗), p ≤ 0.0001 (^∗∗∗∗). See also Figure S6.

Figure 6

IFNγ/LPS-induced iNOS sensitizes macrophages to caspase-8 and BAX/BAK-mediated death (A) WT BMDMs were primed with IFNγ (50 ng/mL) overnight then stimulated with LPS (50 ng/mL) for 2–24 h. Nitrite (NO₂⁻) production and iNOS expression were measured by the Griess assay and immunoblot (n = 3). (B and C) WT BMDMs were primed with IFNγ (50 ng/mL) overnight then stimulated with LPS (50 ng/mL) ± the iNOS inhibitor 1400W (10 μM) or DMSO for 24 h. (B) Nitrite (NO₂⁻) production and cell death were measured by the Griess assay and (C) PI exclusion as measured by flow cytometry (n = 4). (D) WT BMDMs were primed with IFNγ (50 ng/mL) overnight then stimulated with LPS (50 ng/mL) ± 1400W (10 μM) for 24 h. The nitric oxide donor SNAP (200 μM) or DMSO were provided 8 h post-treatment with LPS. Cell death was assessed by PI exclusion as measured by flow cytometry (n = 5). (E) WT BMDMs were treated with 1400W (10 μM) ± IFNγ (50 ng/mL) overnight then stimulated with LPS (50 ng/mL) or TNF (100 ng/mL) for 24 h. SNAP (200 μM) or DMSO were added to cells 8 h post-treatment with LPS or TNF. Cell death was assessed by PI exclusion as measured by flow cytometry (n = 4). (F, G, and H) WT or Nos2^−/− BMDMs were primed with IFNγ (50 ng/mL) overnight then stimulated with LPS (50 ng/mL) or Pam-3-CSK4 (P3C, 500 ng/mL) for 16, 24, or 48 h. ABT-737 (1 μM) and cycloheximide (CHX, 10 μg/mL) treatment for (G) 6 h or (H) 2 h was used as a positive control. (F) Cell death and (G) cytochrome c retention was assessed by PI exclusion as measured by flow cytometry or intracellular cytochrome c staining and flow cytometric analysis (n = 6). (H) Cell death pathway activation was assessed by immunoblot of cell supernatants (S/N) and cell lysates (n = 2). Data represent the mean value ± SD, or a representative immunoblot, from n independent experiments. p ≤ 0.05 (^∗), p ≤ 0.001 (^∗∗∗), p ≤ 0.0001 (^∗∗∗∗). See also Figure S6 and S7.

Figure 7

iNOS and caspase-8 influence the host response to SARS-CoV-2, but only caspase-8 impacts hemophagocytic lymphohistiocytosis (HLH) disease severity (A and B) Rectal temperatures of (A) wild-type (WT, n = 12), Ripk3^−/− (n = 6), Casp8^−/−Ripk3^−/− (n = 10), and Tnf^−/−Fasl^gld/gldTrail^−/− (n = 6) mice, or (B) WT (n = 14) and Nos2^−/− (n = 12) mice injected with PolyI:C (10 mg/kg) for 24 h followed by LPS (5 mg/kg) to induce HLH-like disease. (C) Cleaved caspase-3 immunohistochemistry of endpoint small intestine sections taken from mice treated as described (A and B). Each image represents a separate mouse. Positive cells are indicated with red arrows. Scale bar, 100 μm. (D and E) Endpoint plasma TNF, IL-1β and IL-6 concentrations of mice treated as described in (D) A and (E) B. (F and G) (F) TCID50 infectious units per lung and (G) percentage weight loss of WT (n = 22), Nos2^+/− (n = 18) or Nos2^−/− (n = 20) mice infected with 1.5 × 10⁷ TCID50 infectious units of SARS-CoV-2 for three days. (H and I) (H) TCID50 infectious units per lung and (I) percentage weight loss of WT (n = 21), Mlkl^−/− (n = 23), Casp8^+/−Mlkl^−/− (n = 17), or Casp8^−/−Mlkl^−/− (n = 15) mice infected with 1.5 × 10⁷ TCID50 infectious units of SARS-CoV-2 for three days. Data represent the mean value ± SEM pooled from at least 2 independent experimental cohorts of mice. p > 0.05 (n.s.), p ≤ 0.05 (^∗), p ≤ 0.01 (^∗∗), p ≤ 0.001 (^∗∗∗), p ≤ 0.0001 (^∗∗∗∗).