Macrophage RIPK3 triggers inflammation and cell death via the XBP1-Foxo1 axis in liver ischaemia-reperfusion injury

Abstract

Background & aims: Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) is a central player in triggering necroptotic cell death. However, whether macrophage RIPK3 may regulate NOD1-dependent inflammation and calcineurin/transient receptor potential cation channel subfamily M member 7 (TRPM7)-induced hepatocyte death in oxidative stress-induced liver inflammatory injury remains elusive.

Methods: A mouse model of hepatic ischaemia-reperfusion (IR) injury, the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific RIPK3 knockout (RIPK3^M-KO) and RIPK3-proficient (RIPK3^FL/FL) mice.

Results: RIPK3^M-KO diminished IR stress-induced liver damage with reduced serum alanine aminotransferase/aspartate aminotransferase levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the RIPK3^FL/FL controls. IR stress activated RIPK3, inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α), x-box binding protein 1 (XBP1), nucleotide-binding oligomerisation domain-containing protein 1 (NOD1), NF-κB, forkhead box O1 (Foxo1), calcineurin A, and TRPM7 in ischaemic livers. Conversely, RIPK3^M-KO depressed IRE1α, XBP1, NOD1, calcineurin A, and TRPM7 activation with reduced serum tumour necrosis factor α (TNF-α) levels. Moreover, Foxo1^M-KO alleviated IR-induced liver injury with reduced NOD1 and TRPM7 expression. Interestingly, chromatin immunoprecipitation coupled with massively parallel sequencing revealed that macrophage Foxo1 colocalised with XBP1 and activated its target gene Zc3h15 (zinc finger CCCH domain-containing protein 15). Activating macrophage XBP1 enhanced Zc3h15, NOD1, and NF-κB activity. However, disruption of macrophage Zc3h15 inhibited NOD1 and hepatocyte calcineurin/TRPM7 activation, with reduced reactive oxygen species production and lactate dehydrogenase release after macrophage/hepatocyte coculture. Furthermore, adoptive transfer of Zc3h15-expressing macrophages in RIPK3^M-KO mice augmented IR-triggered liver inflammation and cell death.

Conclusions: Macrophage RIPK3 activates the IRE1α-XBP1 pathway and Foxo1 signalling in IR-stress livers. The XBP1-Foxo1 interaction is essential for modulating target gene Zc3h15 function, which is crucial for the control of NOD1 and calcineurin-mediated TRPM7 activation. XBP1 functions as a transcriptional coactivator of Foxo1 in regulating NOD1-driven liver inflammation and calcineurin/TRPM7-induced cell death. Our findings underscore a novel role of macrophage RIPK3 in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases.

Impact and implications: Macrophage RIPK3 promotes NOD1-dependent inflammation and calcineurin/TRPM7-induced cell death cascade by triggering the XBP1-Foxo1 axis and its target gene Zc3h15, which is crucial for activating NOD1 and calcineurin/TRPM7 function, implying the potential therapeutic targets in stress-induced liver inflammatory injury.

Keywords: ER stress; Foxo1; IRE1α; Innate immunity; Liver inflammation; Necroptosis; Reactive oxygen species; XBP1.

Graphical abstract

Fig. 1

Myeloid-specific RIPK3 deficiency alleviates IR-induced liver damage and diminishes macrophage/neutrophil infiltration and pro-inflammatory mediators in IR-stressed liver. The RIPK3^FL/FL and RIPK3^M-KO mice were subjected to 90 min of partial liver warm ischaemia, followed by 6 h of reperfusion. (A) The RIPK3 expression was detected in hepatocytes and liver macrophages from IR-stressed livers by Western blot assay. Representative of four experiments. (B) Representative histological staining (H&E) of ischaemic liver tissue (n = 6 mice/group) and Suzuki’s histological score. Scale bars, 200 and 30 μm. (C) Liver function was evaluated by sALT and sAST levels (IU/L) (n = 6 samples/group). (D) Immunofluorescence staining of CD11b⁺ macrophages in ischaemic livers (n = 6 mice/group). Quantification of CD11b⁺ macrophages. Scale bars, 100 μm. (E) Immunohistochemistry staining of Ly6G⁺ neutrophils in ischaemic livers (n = 6 mice/group). Quantification of Ly6G⁺ neutrophils. Scale bars, 100 μm. (F) qRT-PCR analysis of TNF-α, IL-6, CXCL-10, and MCP-1 mRNA levels in ischaemic livers (n = 6 samples/group). All data represent the mean ± SD. Statistical analysis was performed using a permutation t test. ∗∗∗p <0.005, ∗∗∗∗p <0.001. CXCL-10, C–X–C motif chemokine ligand 10; HPF, high-power field; IR, ischaemia and reperfusion; MCP-1, monocyte chemoattractant protein 1; qRT-PCR, quantitative reverse transcription PCR; RIPK3, receptor-interacting serine/threonine-protein kinase 3; sALT, serum alanine aminotransferase; sAST, serum aspartate aminotransferase; TNF-α, tumour necrosis factor α.

Fig. 2

Myeloid-specific RIPK3 deficiency regulates the IRE1α–XBP1 pathway and inhibits NOD1 and calcineurin/TRPM7 activation in IR-stressed liver. The WT, RIPK3^FL/FL, and RIPK3^M-KO mice were subjected to 90 min of partial liver warm ischaemia, followed by 6 h of reperfusion. (A) Western-assisted analysis and relative density ratio of RIPK3, IRE1α, XBP1s, NOD1, receptor (TNFRSF)-interacting serine-threonine kinase 2 (RIP2), p-P65, and P-65 in the WT livers after IR stress. (B) Immunofluorescence staining of RIPK3 and CD68 in ischaemic livers (n = 6 mice/group). Scale bars, 100 and 20 μm. (C) Western-assisted analysis and relative density ratio of p-JNK, JNK, and nuclear Foxo1 in the WT livers after IR stress. (D) The Kupffer cells were isolated from the WT livers after IR stress. Western-assisted analysis and relative density ratio of nuclear XBP1s and Foxo1 in Kupffer cells. (E) Western-assisted analysis and relative density ratio of IRE1α, XBP1s, NOD1, p-P65, P-65, calcineurin A, and TRPM7 in the RIPK3^FL/FL and RIPK3^M-KO livers after IR stress. (F) ELISA analysis of serum TNF-α levels in the RIPK3^FL/FL and RIPK3^M-KO mice after IR stress (n = 6 samples/group). (G) Immunofluorescence staining of TRPM7 in the RIPK3^FL/FL and RIPK3^M-KO livers after IR stress (n = 6 mice/group). Scale bars, 100 and 20 μm. All Western blots represent four experiments, and the data represent the mean ± SD. Statistical analysis was performed using a permutation t test. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.005. Foxo1, forkhead box O1; IR, ischaemia and reperfusion; IRE1α, inositol-requiring transmembrane kinase/endoribonuclease 1α; JNK, c-Jun N-terminal kinase; NOD1, nucleotide-binding oligomerisation domain-containing protein 1; RIPK3, receptor-interacting serine/threonine-protein kinase 3; TNF-α, tumour necrosis factor α; TRPM7, transient receptor potential cation channel subfamily M member 7; WT, wild-type; XBP1, x-box binding protein 1; XBP1s, spliced XBP1.

Fig. 3

Disruption of myeloid Foxo1 ameliorates liver injury and dampens NOD1 and calcineurin/TRPM7 activation in IR-stressed livers. The Foxo1^FL/FL and Foxo1^M-KO mice were subjected to 90 min of partial liver warm ischaemia, followed by 6 h of reperfusion. (A) Representative histological staining (H&E) of ischaemic liver tissue (n = 6 mice/group) and Suzuki’s histological score. Scale bars, 100 and 30 μm. (B) Liver function was evaluated by sALT and sAST levels (IU/L) (n = 6 samples/group). (C) Immunofluorescence staining of CD11b⁺ macrophages in ischaemic livers (n = 6 mice/group). Quantification of CD11b⁺ macrophages, Scale bars, 100 μm. (D) Immunohistochemistry staining of Ly6G⁺ neutrophils in ischaemic livers (n = 6 mice/group). Quantification of Ly6G⁺ neutrophils. Scale bars, 100 μm. (E) Western-assisted analysis and relative density ratio of NOD1, p-P65, P-65, calcineurin A, and TRPM7 in the Foxo1^FL/FL and Foxo1^M-KO livers after IR stress. (F) qRT-PCR analysis of TNF-α, IL-6, CXCL-10, and MCP-1 mRNA levels in ischaemic livers (n = 6 samples/group). All immunoblots represent four experiments, and data represent the mean ± SD. Statistical analysis was performed using a Permutation t-test. ∗∗p <0.01, ∗∗∗p <0.005, ∗∗∗∗p <0.001. CXCL-10, C–X–C motif chemokine ligand 10; Foxo1, forkhead box O1; IR, ischaemia and reperfusion; MCP-1, monocyte chemoattractant protein 1; NOD1, nucleotide-binding oligomerisation domain-containing protein 1; qRT-PCR, quantitative reverse transcription PCR; sALT, serum alanine aminotransferase; sAST, serum aspartate aminotransferase; TNF-α, tumour necrosis factor α; TRPM7, transient receptor potential cation channel subfamily M member 7.

Fig. 4

XBP1 interacts with Foxo1 and regulates NOD1 activation in macrophages. BMMs (1 × 10⁶) were cultured with LPS (100 ng/ml) for 6 h. (A) (B) Immunofluorescence staining for XBP1s and Foxo1 expression in macrophages after LPS stimulation (n = 4 samples/group). DAPI was used to visualise nuclei. Scale bars, 20 μm. (C) Immunofluorescence staining for macrophage XBP1s (green) and Foxo1 (red) colocalisation in LPS-stimulated macrophages. DAPI was used to visualise nuclei (blue). Scale bars, 10 μm. (D) Western blot analysis of nuclear XBP1s and Foxo1 protein expression in LPS-stimulated macrophages. (E) IP analysis of XBP1s and Foxo1 in LPS-stimulated macrophages (n = 4 samples/group). (F) BMMs from the RIPK3^FL/FL and RIPK3^M-KO mice were cultured with LPS (100 ng/ml) for 6 h. Western-assisted analysis and relative density ratio of NOD1, p-P65, and P65 in LPS-stimulated macrophages. All immunoblots represent four experiments, and the data represent the mean ± SD. Statistical analysis was performed using a permutation t test. ∗p <0.05, ∗∗p <0.01. BMM, bone marrow-derived macrophage; Foxo1, forkhead box O1; IP, immunoprecipitation; LPS, lipopolysaccharide; NOD1, nucleotide-binding oligomerisation domain-containing protein 1; XBP1, x-box binding protein 1; XBP1s, spliced XBP1.

Fig. 5

The XBP1–Foxo1 axis targets Zc3h15 and modulates NOD1-driven inflammatory response in macrophages. BMMs were collected and fixed after incubating LPS (100 ng/ml). Following chromatin shearing and Foxo1 antibody selection, the precipitated DNA fragments bound by Foxo1-containing protein complexes were used for sequencing. (A) Localisation of Foxo1-binding sites on the mouse Zc3h15 gene. The 10 exons, 9 introns, 3′ UTR, 5′ UTR, and TSSs of the mouse Zc3h15 gene on chromosome 2 are shown. (B) ChIP-PCR analysis of Foxo1 and XBP1s binding to the Zc3h15 promoter. Protein-bound chromatin was prepared from BMMs and immunoprecipitated with Foxo1 or XBP1s antibodies. For sequential ChIP, the protein-bound chromatin was first immunoprecipitated with the Foxo1 antibody, followed by elution with a second immunoprecipitation using XBP1s antibody. Then, the immunoprecipitated DNA was analysed by PCR. The normal IgG was used as a negative control. (C) Analysis of Zc3h15 mRNA levels in LPS-stimulated macrophages from the RIPK3^FL/FL and RIPK3^M-KO mice. (n = 4 samples/group). (D) Western blot analysis and relative density ratio of Zc3h15 in LPS-stimulated macrophages from the RIPK3^FL/FL and RIPK3^M-KO mice. (E) Western blot analysis and relative density ratio of Zc3h15 and NOD1 in LPS-stimulated macrophages from the Foxo1^FL/FL and Foxo1^M-KO mice. (F) qRT-PCR analysis of TNF-α, IL-1β, IL-6, CXCL-2, and CXCL-10 in LPS-stimulated macrophages from the Foxo1^FL/FL and Foxo1^M-KO mice. (n = 4 samples/group). All immunoblots represent four experiments, and the data represent the mean ± SD. Statistical analysis was performed using a permutation t test. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.001. BMM, bone marrow-derived macrophage; ChIP, chromatin immunoprecipitation; CXCL-2, C–X–C motif chemokine ligand 2; CXCL-10, C–X–C motif chemokine ligand 10; Foxo1, forkhead box O1; LPS, lipopolysaccharide; NOD1, nucleotide-binding oligomerisation domain-containing protein 1; quantitative reverse transcription PCR; RIPK3, receptor-interacting serine/threonine-protein kinase 3; TNF-α, tumour necrosis factor α; TSS, transcription start site; UTR, untranslated region; XBP1, x-box binding protein 1; XBP1s, spliced XBP1; Zc3h15, zinc finger CCCH domain-containing protein 15.

Fig. 6

XBP1 is required for the Foxo1-targeted Zc3h15 activation and NOD1 function in macrophages. BMMs from the RIPK3^FL/FL, RIPK3^M-KO, Foxo1^FL/FL, and Foxo1^M-KO mice were transfected with p-CRISPR-XBP1 KO, p-CRISPR-XBP1 KO activation or control vector followed by 6 h of LPS (100 ng/ml) stimulation. (A) Immunofluorescence staining for the Zc3h15 expression in LPS-stimulated RIPK3^FL/FL macrophages after transfecting p-CRISPR-XBP1 KO or control vector (n = 4 samples/group). DAPI was used to visualise nuclei. Scale bars, 20 μm. (B) Western blot analysis and relative density ratio of XBP1s, Zc3h15, NOD1, p-P65, and P65 in LPS-stimulated RIPK3^FL/FL macrophages. (C) Immunofluorescence staining for the Zc3h15 expression in LPS-stimulated RIPK3^M-KO macrophages after transfecting p-CRISPR-XBP1 activation or control vector (n = 4 samples/group). DAPI was used to visualise nuclei. Scale bars, 20 μm. (D) Western blot analysis and relative density ratio of XBP1s, Zc3h15, NOD1, p-P65, and P65 in LPS-stimulated RIPK3^M-KO macrophages. (E) Western blot analysis and relative density ratio of Zc3h15 in LPS-stimulated Foxo1^FL/FL macrophages. (F) Western blot analysis and relative density ratio of Zc3h15 in LPS-stimulated Foxo1^M-KO macrophages. All immunoblots represent four experiments, and the data represent the mean ± SD. Statistical analysis was performed using a permutation t test. ∗∗p <0.01, ∗∗∗p <0.005. BMM, bone marrow-derived macrophage; CRISPR, clustered regularly interspaced short palindromic repeats; Foxo1, forkhead box O1; LPS, lipopolysaccharide; NOD1, nucleotide-binding oligomerisation domain-containing protein 1; RIPK3, receptor-interacting serine/threonine-protein kinase 3; XBP1, x-box binding protein 1; Zc3h15, zinc finger CCCH domain-containing protein 15.

Fig. 7

Zc3h15 is crucial to regulate NOD1-driven inflammatory response and Calcineurin/TRPM7-induced cell death in response to oxidative stress. (A) BMMs from RIPK3^FL/FL mice were transfected with p-CRISPR-Zc3h15 KO or control vector followed by 6 h of LPS (100 ng/ml) stimulation. Immunofluorescence staining for NOD1 expression in macrophages (n = 4 samples/group). DAPI was used to visualise nuclei. Scale bars, 20 μm. (B) Western blot analysis and relative density ratio of Zc3h15, NOD1, p-P65, and P65 in LPS-stimulated RIPK3^FL/FL macrophages. (C) qRT-PCR analysis of TNF-α, IL-1β, IL-6, CXCL-2, and CXCL-10 in LPS-stimulated macrophages from the RIPK3^FL/FL mice (n = 4 samples/group). (D) BMMs from RIPK3^FL/FL mice were transfected with p-CRISPR-Zc3h15 KO or control vector followed by LPS stimulation and then cocultured with primary hepatocytes that were supplemented with H₂O₂ for 24 h. Western blot analysis and relative density ratio of calcineurin A and TRPM7 in H₂O₂-treated hepatocytes. (E) Immunofluorescence staining for TRPM7 expression in H2O2-treated hepatocytes (n = 4 samples/group). DAPI was used to visualise nuclei. Scale bars, 50 μm. (F) ELISA analysis of TNF-α levels in the coculture supernatant (n = 4 samples/group). (G) Detection of ROS production by carboxy-H2DFFDA in H₂O₂-treated hepatocytes from the RIPK3^FL/FL mice. Quantification of ROS-producing hepatocytes (green) (n = 4 samples/group). Scale bars, 100 μm. (H) LDH release from the H₂O₂-treated hepatocytes in cocultures (n = 4 samples/group). All immunoblots represent four experiments, and the data represent the mean ± SD. Statistical analysis was performed using a permutation t test. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.005. BMM, bone marrow-derived macrophage; CXCL-2, C–X–C motif chemokine ligand 2; CXCL-10, C–X–C motif chemokine ligand 10; CRISPR, clustered regularly interspaced short palindromic repeats; KO, knockout; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; NOD1, nucleotide-binding oligomerisation domain-containing protein 1; qRT-PCR, quantitative reverse transcription ROS, reactive oxygen species; PCR; RIPK3, receptor-interacting serine/threonine-protein kinase 3; TNF-α, tumour necrosis factor α; TRPM7, transient receptor potential cation channel subfamily M member 7; XBP1, x-box binding protein 1; Zc3h15, zinc finger CCCH domain-containing protein 15.

Fig. 8

Adoptive transfer of Zc3h15-expressing macrophages exacerbates IR-triggered liver inflammation and cell death. The RIPK3^M-KO mice were injected via tail vein with BMMs (1 × 10⁶ cells/mouse) transfected with lentiviral-expressing Zc3h15 (Lv-Zc3h15) or GFP control (Lv-GFP) 24 h before ischaemia. (A) Representative histological staining (H&E) of ischaemic liver tissue (n = 6 mice/group) and Suzuki’s histological score. Scale bars, 100 μm. (B) sALT levels (IU/L) (n = 6 samples/group). (C) Western-assisted analysis and relative density ratio of NOD1, p-P65, P65, calcineurin A, and TRPM7 in RIPK3^M-KO livers after adoptive transfer of Lv-Zc3h15-expressing or control cells. (D) Immunohistochemistry staining of 4-HNE⁺ cells in IR-stressed livers (n = 6 mice/group). Quantification of 4-HNE⁺ cells, Scale bars, 100 μm. (E) ELISA analysis of serum TNF-α levels (n = 6 samples/group). (F) qRT-PCR analysis of TNF-α, IL-1β, IL-6, CXCL-10, and MCP-1 in IR-stressed livers (n = 6 samples/group). (G) Immunofluorescence staining for TRPM7 expression in hepatocytes from the RIPK3^M-KO mice after adoptive transfer of Lv-Zc3h15-expressing or control cells (n = 6 samples/group). DAPI was used to visualise nuclei. Scale bars, 100 and 20 μm. All immunoblots represent four experiments, and the data represent the mean ± SD. Statistical analysis was performed using a permutation t test. ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.001. 4-HNE, 4-hydroxynonenal; BMM, bone marrow-derived macrophage; CXCL-10, C–X–C motif chemokine ligand 10; GFP, green fluorescent protein; HNF4α, hepatic nuclear factor 4α; HPF, high-power field; IR, ischaemia and reperfusion; MCP-1, monocyte chemoattractant protein 1; NOD1, nucleotide-binding oligomerisation domain-containing protein 1; qRT-PCR, quantitative reverse transcription; PCR; RIPK3, receptor-interacting serine/threonine-protein kinase 3; sALT, serum alanine aminotransferase; TNF-α, tumour necrosis factor α; TRPM7, transient receptor potential cation channel subfamily M member 7; XBP1, x-box binding protein 1; Zc3h15, zinc finger CCCH domain-containing protein 15.