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. 2024 Oct 28;11(1):71.
doi: 10.1186/s40779-024-00574-z.

Mechanism of lactic acidemia-promoted pulmonary endothelial cells death in sepsis: role for CIRP-ZBP1-PANoptosis pathway

Ting Gong  1   2 , Qing-De Wang  3 , Patricia A Loughran  3 , Yue-Hua Li  3 , Melanie J Scott  3 , Timothy R Billiar  3   4 , You-Tan Liu  5 , Jie Fan  6   7   8   9
Affiliations

Mechanism of lactic acidemia-promoted pulmonary endothelial cells death in sepsis: role for CIRP-ZBP1-PANoptosis pathway

Ting Gong et al. Mil Med Res. .

Abstract

Background: Sepsis is often accompanied by lactic acidemia and acute lung injury (ALI). Clinical studies have established that high serum lactate levels are associated with increased mortality rates in septic patients. We further observed a significant correlation between the levels of cold-inducible RNA-binding protein (CIRP) in plasma and bronchoalveolar lavage fluid (BALF), as well as lactate levels, and the severity of post-sepsis ALI. The underlying mechanism, however, remains elusive.

Methods: C57BL/6 wild type (WT), Casp8-/-, Ripk3-/-, and Zbp1-/- mice were subjected to the cecal ligation and puncture (CLP) sepsis model. In this model, we measured intra-macrophage CIRP lactylation and the subsequent release of CIRP. We also tracked the internalization of extracellular CIRP (eCIRP) in pulmonary vascular endothelial cells (PVECs) and its interaction with Z-DNA binding protein 1 (ZBP1). Furthermore, we monitored changes in ZBP1 levels in PVECs and the consequent activation of cell death pathways.

Results: In the current study, we demonstrate that lactate, accumulating during sepsis, promotes the lactylation of CIRP in macrophages, leading to the release of CIRP. Once eCIRP is internalized by PVEC through a Toll-like receptor 4 (TLR4)-mediated endocytosis pathway, it competitively binds to ZBP1 and effectively blocks the interaction between ZBP1 and tripartite motif containing 32 (TRIM32), an E3 ubiquitin ligase targeting ZBP1 for proteasomal degradation. This interference mechanism stabilizes ZBP1, thereby enhancing ZBP1-receptor-interacting protein kinase 3 (RIPK3)-dependent PVEC PANoptosis, a form of cell death involving the simultaneous activation of multiple cell death pathways, thereby exacerbating ALI.

Conclusions: These findings unveil a novel pathway by which lactic acidemia promotes macrophage-derived eCIRP release, which, in turn, mediates ZBP1-dependent PVEC PANoptosis in sepsis-induced ALI. This finding offers new insights into the molecular mechanisms driving sepsis-related pulmonary complications and provides potential new therapeutic strategies.

Keywords: Extracellular cold-inducible RNA-binding protein (eCIRP); PANoptosis; Sepsis-induced acute lung injury (ALI); Ubiquitination; ZBP1.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Elevated exosomal cold-inducible RNA-binding protein (CIRP) in plasma and bronchoalveolar lavage fluid (BALF) correlates with lactate levels and severity of sepsis-induced acute lung injury (ALI). a-f Mice were administered lactate (0.5 g/kg) intraperitoneally 6 h after cecal ligation and puncture (CLP) or sham. To inhibit lactate production, sodium oxamate (OXA, 0.5 g/kg) was injected intraperitoneally 6 h before CLP or sham. Mice were euthanized 24 h post-CLP. Mouse plasma lactate levels were measured using a lactate assay kit (a, n = 5). Plasma exosomal CIRP levels in mice were detected using an enzyme-linked immunosorbent assay (ELISA) kit across groups (b). Hematoxylin and eosin (H&E) staining of lung tissues from sham and CLP mice, with arrows indicating lung injury characterized by alveolar septal thickening and inflammatory cell infiltration (c, scale bar = 50 μm). Lung injury scores were statistically evaluated for each group (d). Significant correlations were analyzed between blood lactate levels and plasma exosomal CIRP levels across groups (e). Plasma exosomal CIRP levels were measured by Western blotting and ELISA among sham, CLP, CLP + lactate, and CLP + OXA groups (f, n = 5). g ELISA analysis of clinical samples demonstrates significantly higher levels of plasma exosomal CIRP in patients with sepsis-induced ALI compared to healthy controls. h ROC curve analysis indicates that exosomal CIRP has high diagnostic and prognostic value for sepsis-induced ALI. i ELISA detection shows that plasma exosomal CIRP levels are higher in non-survivors than survivors among patients with sepsis. j ROC curve for exosomal CIRP levels predicting patient outcomes in sepsis with AUC = 0.734. k Plasma lactate levels are significantly elevated in non-survivors compared to survivors in sepsis. l Correlation analysis reveals a significant positive relationship between plasma exosomal CIRP levels and plasma lactate levels. m Western blotting analysis indicates significantly increased exosomal CIRP levels in the BALF of patients with sepsis-induced ALI compared to controls. n ELISA analysis confirms significantly higher exosomal CIRP levels in the BALF of a larger cohort of patients with sepsis-induced ALI compared to controls. o Single-cell RNA sequencing was performed on the lungs of sham and CLP mice at 24 h (n = 3). Cell populations were identified based on copy number variations inferred from expression (annotations for each cell population are provided in the Additional file 1: Fig. S1). Cells colored by the expression levels of Cirp (p) and Ldha (q) in the lungs of mice. Data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. ROC receiver operating characteristic, AUC area under the curve, Ldha lactate dehydrogenase A
Fig. 2
Fig. 2
Lactylation of activating transcription factor 4 (ATF4)-upregulated cold-inducible RNA-binding protein (CIRP) in macrophages promotes CIRP release in sepsis. a Immunofluorescence shows significantly increased expression of CIRP (red) in F4/80 (green) macrophages in the lung tissue of cecal ligation and puncture (CLP) mice, with the right panel showing colocalization analysis (scale bar = 20 µm). The arrows from “i” to “ii” highlight the region analyzed for co-localization analysis. b Quantitative analysis indicates a higher number of CIRP+F4/80+ cells in the CLP group compared to the sham. c Western blotting analysis of bronchoalveolar lavage fluid (BALF) macrophages from CLP mice shows a significant increase in CIRP expression. d Promoter sequences of Cirp with 3 binding sites for ATF4 were predicted using databases such as University of California Santa Cruz (UCSC) and JASPAR (wild type), and mutant binding site constructs (MutA, MutB, MutC, and MutABC) were generated. e Dual-luciferase assays demonstrate ATF4 binding to the Cirp promoter region and transcriptional regulation of Cirp expression. f Western blotting analysis shows a significant increase in CIRP expression following LPS stimulation in cells, which is notably reduced after ATF4 interference. g Western blotting analysis was performed to determine cytoplasmic and nuclear CIRP levels in mouse alveolar macrophage (MH-S) cells after 6 h of stimulation with 1 µg/ml LPS. h Representative immunofluorescence images of MH-S cells treated with either vehicle or lactate (10 mmol/L) for 6 h. Lactate-treated cells exhibit increased cytoplasmic accumulation of CIRP (indicated by white arrows) (scale bar = 20 µm). i Intracellular lactate levels were measured using a kit after 6 h of treatment with LPS or LPS + OXA. j Pan-lactylation levels in macrophages following LPS stimulation (at 0, 6, 12, and 24 h) were assessed using Western blotting analysis. k The content of CIRP in the cell supernatant of alveolar macrophages stimulated with LPS or LPS + OXA was measured by enzyme-linked immunosorbent assay (ELISA), with PBS serving as the control group. l MH-S cells were treated with LPS or LPS + OXA for 24 h (scale bar = 20 µm). Confocal microscopy was used to examine the colocalization of CIRP (red) and Klac (green). DAPI (blue) staining indicates the nuclei. Below are colocalization analyses using ImageJ. The arrows from “i” to “ii” highlight the region analyzed for co-localization analysis. m A proximity ligation assay (PLA) using specific antibodies against CIRP and lactylated lysine (Klac), with nuclei stained using DAPI (scale bar = 20 μm). The right panel is a statistical graph of PLA. After 24 h of LPS stimulation in macrophages, cell lysates precipitated with agarose beads underwent a pull-down assay and were probed for CIRP and Klac protein levels using either control IgG or CIRP monoclonal antibody (n) and Klac monoclonal antibody (o). * Indicates comparison with the pGL4.1 control group, and # indicates comparison with the pGL4.1-WT group. Data are presented as the mean ± SD. ***P < 0.001; ##P < 0.01, ###P < 0.001. OXA sodium oxamate, MARCO macrophage receptor with collagenous structure, LPS lipopolysaccharide, DAPI 4',6-diamidino-2-phenylindole, IB immunoblotting
Fig. 3
Fig. 3
Toll-like receptor 4 (TLR4) mediates the endocytosis of eCIRP in mouse pulmonary vascular endothelial cells (MPVECs). a Confocal microscopy images of MPVECs isolated from C57BL/6 [wild type (WT)] mice, incubated with recombinant CIRP-GFP (10 µg/ml) or green fluorescent protein (GFP) in the presence of LPS (25 µg/ml) for 0 − 60 min (scale bar = 5 µm). b Confocal microscopy images of MPVECs isolated from WT mice, incubated with 0 − 10 µg/ml CIRP-GFP in the presence of LPS (25 µg/ml) for 30 min (scale bar = 5 µm). c Confocal microscopy images of WT MPVECs incubated with CIRP-GFP in the presence or absence of dynasore (30 µg/ml) or LPS (25 µg/ml) for 30 min (scale bar = 5 µm). d Confocal microscopy images of MPVECs isolated from WT, Tlr2−/−, or Tlr4−/− mice, incubated with CIRP-GFP (10 µg/ml) in the presence of LPS (25 µg/ml) for 30 min (scale bar = 5 µm). e Quantification of the average number of intracellular GFP-positive particles in MPVECs, calculated using a confocal microscopy program. MPVECs were isolated from WT, Tlr2−/−, or Tlr4−/− mice and incubated with CIRP-GFP (10 µg/ml) for 30 min. The first column serves as the control group, and the symbols *, **, and *** represent statistically significant differences with P-values compared to the control group. f Confocal microscopy images showing co-localization of CIRP-GFP and TLR4 in MPVECs isolated from WT mice, incubated with CIRP-GFP and LPS for 30 min (scale bar = 5 µm). Line graphs below the images display co-localization analysis of CIRP-GFP and TLR4. The arrow from “i” to “ii” highlights the region analyzed for co-localization analysis. g Immunofluorescence detection of early endosome antigen 1 (EEA1, red) in WT MPVECs treated with CIRP-GFP (green, 10 µg/ml) for 5, 15, 30 min, and 1, 3, or 6 h (scale bar = 5 µm). The bar chart shows statistical analysis of CIRP-GFP co-localization with EEA1. The first column serves as the control group, and the symbols *, **, and *** represent statistically significant differences with P-values compared to the control group. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. eCIRP extracellular cold-inducible RNA-binding protein
Fig. 4
Fig. 4
Lipopolysaccharide (LPS) and rmCIRP induce MPVEC PANoptosis. a Immunoblot analyses were conducted to evaluate the expression and activation of key cell death mediators. Pro-activated (P53), activated (P30), and inactivated (P20) GSDMD, pro-activated (P45) and activated (P20) CASP1, and activated AIM2; pro-cleaved (P35) and cleaved (P17) CASP3, pro-cleaved (P55) and cleaved (P18) CASP8; p-MLKL, MLKL, p-RIPK3, RIPK3, p-RIPK1, and RIPK1 in primary MPVECs co-treated with LPS and rmCIRP (10 µg/ml). b Immunoblot analysis of CASP1, GSDMD, and AIM2, CASP3 and CASP8, p-MLKL and MLKL in primary MPVECs treated with either LPS alone, rmCIRP alone, or co-treatment with LPS and rmCIRP for 48 h. c Immunofluorescence microscopy revealed the cellular localization of ASC specks in primary MPVECs 48 h co-treatment with LPS and rmCIRP (scale bar = 5 μm). Arrowheads indicate the PANoptosome. The bar chart presents the quantification of the percentage of cells with ASC+CASP8+RIPK3+ specks among the ASC speck+ cells. The data are expressed as the mean ± SD. ***P < 0.001. rmCIRP recombinant mouse cold-inducible RNA-binding protein, MPVEC mouse pulmonary vascular endothelial cell, GSDMD gasdermin D, CASP1 caspase-1, AIM2 absent in melanoma 2, CASP3 caspase-3, CASP8 caspase-8, p-MLKL phosphorylated mixed lineage kinase domain-like protein, MLKL total mixed lineage kinase domain-like protein, p-RIPK3 phosphorylated receptor-interacting protein kinase 3, RIPK3 receptor-interacting protein kinase 3, p-RIPK1 phosphorylated receptor-interacting protein kinase 1, RIPK1 receptor-interacting protein kinase 1, ASC apoptosis-associated speck-like protein containing a CARD
Fig. 5
Fig. 5
Z-DNA binding protein 1 (ZBP1) regulates pulmonary vascular endothelial cell (PVEC) PANoptosis in response to lipopolysaccharide (LPS) and extracellular CIRP (eCIRP). a Immunoblot analysis of ZBP1 in WT and Zbp1−/− primary MPVECs after treatment with LPS alone, rmCIRP alone, or co-treatment with LPS and rmCIRP for 48 h. b Representative images of cell death and quantification showing the percentage of cell death in WT, Zbp−/− primary MPVECs treated with LPS and rmCIRP for 48 h (scale bar = 100 μm). c Immunoblot analysis of pro-activated (P45) and activated (P20) CASP1; pro-activated (P53), activated (P30), and inactivated (P20) GSDMD; and AIM2; pro-cleaved (P35) and cleaved (P17) CASP3, pro-cleaved (P55) and cleaved (P18) CASP8; p-MLKL and MLKL in WT and Zbp1−/− MPVECs at 0 h or co-treatment with LPS and rmCIRP for 48 h. d Immunofluorescence images of WT and Zbp1−/− primary MPVECs 48 h after co-treatment with LPS and rmCIRP (scale bar = 5 μm). Arrowheads indicate the PANoptosome. The bar chart displays the quantification of the percentage of cells with ASC+CASP8+RIPK3+ specks among the ASC speck+ cells. The data are presented as mean ± SD. ***P < 0.001. rmCIRP recombinant mouse cold-inducible RNA-binding protein, MPVEC mouse pulmonary vascular endothelial cell, GSDMD gasdermin D, CASP1 caspase-1, AIM2 absent in melanoma 2, CASP3 caspase-3, CASP8 caspase-8, p-MLKL phosphorylated mixed lineage kinase domain-like protein, MLKL mixed lineage kinase domain-like protein, RIPK3 receptor-interacting protein kinase 3, ASC apoptosis-associated speck-like protein containing a CARD
Fig. 6
Fig. 6
Z-DNA binding protein 1 (ZBP1) mediates mitochondrial damage and cell death, contributing to mortality in sepsis-induced acute lung injury (ALI) in vivo. a-g Intratracheal instillation of LPS at 3 mg/kg, intravenous administration of rmCIRP at 5 mg/kg, or PBS as a control were used. TEM revealed mitochondrial structure in primary MPVECs from WT and Zbp1−/− mice at 0 h or 36 h post-treatment with lipopolysaccharide (LPS) and recombinant mouse CIRP (rmCIRP) (a, scale bar = 1 μm). Mitochondrial ROS levels were measured using MitoSOX, a fluorogenic dye specifically targeted to mitochondria to detect superoxide production. The measurements were conducted using flow cytometry (b). Analyses of Annexin V/PI staining with flow cytometry detected apoptosis (c). Histological assessment of lung tissue from WT and Zbp1−/− mice 36 h post-treatment with PBS or LPS + rmCIRP included H&E staining, arrows indicate lung injury characterized by alveolar septal thickening and inflammatory cell infiltration and a corresponding histological score (d, scale bar = 50 μm). Survival rates of male WT (n = 10) and Zbp1−/− (n = 11) mice ages 6 – 8 weeks at 96 h post intravenous CIRP injection and intratracheal LPS instillation (e). Levels of the inflammatory cytokine IL-6, TNF-α, and IL-1β in BALF were measured 36 h after intravenous CIRP injection and intratracheal LPS instillation (f). Immunofluorescence quantified ASC-positive cells within CD31-marked pulmonary vascular endothelial cells in mouse lung tissue. White arrowheads indicate ASC specks formation within CD31+ cells (g, scale bar = 20 μm). Data are presented as mean ± SD. ***P < 0.001. TEM transmission electron microscopy, ROS reactive oxygen species, MitoSOX mitochondrial superoxide indicator (fluorogenic dye), PI propidium iodide, TNF-α tumor necrosis factor-α, IL-6 interleukin 6, IL-1β interleukin 1β, BALF bronchoalveolar lavage fluid, MPVEC mouse pulmonary vascular endothelial cell, MFI mean fluorescence intensity
Fig. 7
Fig. 7
Z-DNA binding protein 1 (ZBP1)-activated receptor-interacting protein kinase 3 (RIPK3) is required for pulmonary MPVECs PANoptosome. a Immunofluorescence images of wild type (WT) primary MPVECs at 36 h post co-treatment with lipopolysaccharide (LPS) and recombinant mouse CIRP (rmCIRP) show significant co-localization of ZBP1 (green) and RIPK3 (red) (scale bar = 20 μm). The arrows from “i” to “ii” highlight the region analyzed for colocalization analysis. The images on the right show the analysis of ZBP1 and CIRP co-localization in immunofluorescence and Pearson’s correlation coefficient calculations. b Proximity ligation assay (PLA) revealed physical interactions between ZBP1 and RIPK3 as red spots in WT primary MPVECs 36 h after co-treatment with LPS and rmCIRP (scale bar = 20 μm). c Representative images of cell death and real-time analysis of cell death in WT, Zbp1−/−, and Ripk3−/− primary MPVECs co-treated with LPS and rmCIRP (scale bar = 100 μm). d Immunoblot analysis of ZBP1, RIPK3, ASC, AIM2, CASP8, and CASP3 following immunoprecipitation (IP) with anti-ZBP1 or control IgG antibodies in WT, Zbp1−/−, and Ripk3−/− primary MPVECs after 36 h of co-treatment with LPS and rmCIRP. e Hematoxylin and eosin (H&E)-stained lung tissues from WT, Zbp1−/−, and Ripk3−/− mice at 36 h post-CLP (scale bar = 50 μm), arrows indicate lung injury characterized by alveolar septal thickening and inflammatory cell infiltration. A corresponding quantification of histology scores is provided below. f Survival analysis of male WT, Zbp1−/−, and Ripk3−/− mice aged 6–8 weeks following CLP (n = 10) revealed significantly different survival rates in Zbp1−/− and Ripk3−/− mice compared to WT mice. Data are presented as mean ± SD. **P < 0.01, ***P < 0.001. MPVEC mouse pulmonary vascular endothelial cell, ASC apoptosis-associated speck-like protein containing a CARD, AIM2 absent in melanoma 2, CASP8 caspase-8, CASP3 caspase-3
Fig. 8
Fig. 8
Tripartite motif containing 32 (TRIM32) is a Z-DNA binding protein 1 (ZBP1) E3 ubiquitin (Ub) ligase destabilizing ZBP1. a Western blotting analysis showing the expression of Flag-tagged ZBP1 in endothelial cells transfected with a Flag-ZBP1 plasmid, indicating a significant overexpression of ZBP1 compared to the control group. Quantification of the ZBP1 expression level is presented in the bar graph below with a significant increase in ZBP1 expression. b Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of ZBP1-interacting proteins identified through ZBP1 immunoprecipitation followed by mass spectrometry from endothelial cells expressing ZBP1. The graph displays the enriched pathways, with Ub-mediated proteolysis being the most significant. c Co-IP assay from endothelial cell lysates using an anti-ZBP1 antibody, followed by immunoblotting for TRIM32 and ZBP1. The input and IP samples demonstrate the interaction between ZBP1 and TRIM32. d Lung vascular endothelial cells were transfected with a TRIM32 plasmid, and cell lysates were immunoprecipitated with an anti-TRIM32 antibody, followed by ZBP1 immunoblotting. e A GST-pulldown assay confirming a direct interaction between GST-tagged TRIM32 and ZBP1, as indicated by the presence of ZBP1 in the pulldown complex. f Immunofluorescence staining of TRIM32 (red) and ZBP1 (green) in endothelial cells, with nuclei counterstained with DAPI (blue). The merged image and the enlarged panel from the highlighted box show the colocalization of TRIM32 and ZBP1 (scale bar = 20 μm). Line graphs on the right display fluorescence intensity profiles for ZBP1 and TRIM32, corroborating their colocalization within the cells. The arrow from “i” to “ii” highlights the region analyzed for colocalization analysis. g Stability of ZBP1 protein was assessed over time by Western blotting in control cells, cells expressing GST-TRIM32 and TRIM32 knockdown, treated with cycloheximide (CHX) to inhibit new protein synthesis. h The plot below the blots quantifies ZBP1 levels, revealing the effects of GST-TRIM32 overexpression and siTRIM32 knockdown on ZBP1 stability. i Western blotting analysis of ZBP1 levels in the presence of CHX with and without MG132 treatment in the GST-TRIM32 overexpression group. The symbols *, **, and *** indicate statistically significant differences compared to the control group. *P < 0.05, **P < 0.01, ***P < 0.001. j Ubiquitination assay depicting the modification of ZBP1 in the presence of HA-tagged Ub and GST-TRIM32. The smear of high molecular weight bands above ZBP1 indicates polyubiquitination. The assay was conducted using an anti-Flag antibody for immunoprecipitation, and ubiquitination levels were assessed by anti-HA Western blotting. Expression levels of TRIM32 and ZBP1 were verified by analyzing 5% of the input from cell lysates. The data are presented as the mean ± SD. MG132 a proteasome inhibitor, Co-IP co-immunoprecipitation
Fig. 9
Fig. 9
eCIRP blocks TRIM32-mediated polyubiquitination and proteasomal degradation of ZBP1. a Co-IP assays demonstrate the interaction between ZBP1 and CIRP in cells treated with LPS alone or LPS with rmCIRP for 12 h. The presence of CIRP and ZBP1 in the precipitated complexes is confirmed by Western blotting. b Proximity ligation assay (PLA) reveals physical associations between ZBP1 and CIRP as red spots in WT primary MPVECs after 12 h of co-treatment with LPS alone or LPS and rmCIRP (scale bar = 20 μm). The bar chart shows the quantitative analysis of PLA signals from panel B shows a significant increase in ZBP1-CIRP association when co-treated with LPS and rmCIRP compared to LPS alone. c Immunofluorescence images display the localization of CIRP (red) and ZBP1 (green) in cells treated with LPS, with and without rmCIRP treatment. The merged and enlarged images highlight the colocalization of CIRP and ZBP1, which is enhanced with rmCIRP treatment (scale bar = 20 μm). The right images show the fluorescence intensity profiles for CIRP and ZBP1 along the lines marked “i” and “ii” that support the colocalization observation. The bar graph quantifies the colocalization coefficients, showing a significant increase with LPS and rmCIRP treatment. d Western blotting analysis monitors the stability of ZBP1 protein over time in the presence of LPS and LPS + rmCIRP, indicating that rmCIRP treatment enhances ZBP1 stability compared to LPS treatment alone. A line graph quantifying the relative ZBP1 levels suggests that rmCIRP preserves ZBP1 stability against LPS-induced degradation. The symbol *** indicates statistically significant differences compared to the control group. ***P < 0.001. e Western blotting analysis to monitor the stability of ZBP1 protein following CHX inhibition of protein synthesis in the presence of LPS, LPS + rmCIRP, and MG132. f Ubiquitination assays for ZBP1, conducted with and without rmCIRP treatment in the presence of HA-tagged ubiquitin (HA-Ub), reveal a pattern of polyubiquitination. The extent of ZBP1 ubiquitination is determined by immunoblotting using antibodies specific for HA-tagged polyubiquitin conjugates. The expression levels of TRIM32, CIRP, and ZBP1 are confirmed by analyzing 5% of the input from cell lysates. The bar chart below displays the quantification of polyubiquitinated ZBP1 levels, normalized to the control. g PLA detects physical associations between ZBP1 and TRIM32 as red spots in MPVECs after 12 h of co-treatment with LPS and rmCIRP or LPS alone (scale bar = 20 μm). The right image shows the quantitative analysis of PLA signals revealing a significant decrease in ZBP1-TRIM32 association with LPS and rmCIRP co-treatment compared to LPS alone. h Immunofluorescence images show the localization of TRIM32 (red) and ZBP1 (green) in cells treated with LPS, with and without rmCIRP treatment for 12 h. The merged and enlarged images illustrate the colocalization of TRIM32 and ZBP1 (scale bar = 20 μm). Fluorescence intensity profiles for TRIM32 and ZBP1 along the lines marked “i” and “ii” in the right mages support the colocalization data. The bar graph quantifies the colocalization coefficients, showing a significant reduction with LPS and rmCIRP treatment compared to LPS treatment alone. The data are presented as the mean ± SD. **P < 0.01, ***P < 0.001. eCIRP extracellular cold-inducible RNA-binding protein, TRIM32 tripartite motif-containing 32, ZBP1 Z-DNA binding protein 1, LPS lipopolysaccharide, rmCIRP recombinant mouse cold-inducible RNA-binding protein, PLA proximity ligation assay, WT wild type, MPVEC mouse pulmonary vascular endothelial cell, CHX cycloheximide, MG132 proteasome inhibitor, HA-tag hemagglutinin-tag, HA-Ub hemagglutinin-tagged ubiquitin, Co-IP co-immunoprecipitation
Fig. 10
Fig. 10
The current study demonstrates that ER stress, induced by sepsis or LPS stimulation in macrophages, triggers ATF4 activation, which subsequently enhances CIRP expression. The lactylation of CIRP (Lac-CIRP), a direct consequence of the septic upsurge in intracellular lactate, facilitates its migration from the nucleus to the cytoplasm and its subsequent release. The eCIRP is then internalized by PVECs through a TLR4-mediated endocytosis pathway and binds to ZBP1. This binding obstructs ZBP1’s interaction with TRIM32 and prevents TRIM32-mediated ZBP1 proteasomal degradation. This interference preserves ZBP1 stability, which in turn amplifies ZBP1-dependent PVEC PANoptosis and exacerbates ALI. ER endoplasmic reticulum, LPS lipopolysaccharide, ATF4 activating transcription factor 4, CIRP cold-inducible RNA-binding protein, eCIRP extracellular cold-inducible RNA-binding protein, PVEC pulmonary vascular endothelial cell, TLR4 Toll-like receptor 4, ZBP1 Z-DNA binding protein 1, TRIM32 tripartite motif-containing 32, PANoptosis a form of programmed cell death that combines elements of pyroptosis apoptosis and necroptosis, ALI acute lung injury
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