Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Sep 11:11:tkad006.
doi: 10.1093/burnst/tkad006. eCollection 2023.

Sustained induction of IP-10 by MRP8/14 via the IFNβ-IRF7 axis in macrophages exaggerates lung injury in endotoxemic mice

Juan Wang  1 , Guiming Chen  1 , Lei Li  1 , Sidan Luo  1 , Bingrong Hu  1 , Jia Xu  1 , Haihua Luo  1 , Shan Li  1 , Yong Jiang  1
Affiliations

Sustained induction of IP-10 by MRP8/14 via the IFNβ-IRF7 axis in macrophages exaggerates lung injury in endotoxemic mice

Juan Wang et al. Burns Trauma. .

Abstract

Background: As a damage-associated molecular pattern, the myeloid-related protein 8/14 (MRP8/14) heterodimer mediates various inflammatory diseases, such as sepsis. However, how MRP8/14 promotes lung injury by regulating the inflammatory response during endotoxemia remains largely unknown. This study aims at illuminating the pathological functions of MRP8/14 in endotoxemia.

Methods: An endotoxemic model was prepared with wild-type and myeloid cell-specific Mrp8 deletion (Mrp8ΔMC) mice for evaluating plasma cytokine levels. Lung injury was evaluated by hematoxylin and eosin (H&E) staining, injury scoring and wet-to-dry weight (W/D) ratio. The dynamic profile of interferon γ (IFNγ)-inducible protein 10 (IP-10) mRNA expression induced by macrophage MRP8/14 was determined by quantitative real-time polymerase chain reaction (qPCR). Immunoblotting was used to evaluate the increase in IP-10 level induced by activation of the JAK-STAT signaling pathway. Luciferase reporter assay was performed to detect the involvement of IRF7 in Ip-10 gene transcription. In vivo air pouch experiments were performed to determine the biological function of IP-10 induced by MRP8/14.

Results: Experiments with Mrp8ΔMC mice showed that MRP8/14 promoted the production of cytokines, including IP-10, in the bronchoalveolar lavage fluid (BALF) and lung injury in endotoxic mice. The result of qPCR showed sustained expression of Ip-10 mRNA in macrophages after treatment with MRP8/14 for 12 h. Neutralization experiments showed that the MRP8/14-induced Ip-10 expression in RAW264.7 cells was mediated by extracellular IFNβ. Western blotting with phosphorylation-specific antibodies showed that the JAK1/TYK2-STAT1 signaling pathway was activated in MRP8/14-treated RAW264.7 cells, leading to the upregulation of Ip-10 gene expression. IRF7 was further identified as a downstream regulator of the JAK-STAT pathway that mediated Ip-10 gene expression in macrophages treated with MRP8/14. In vivo air pouch experiments confirmed that the IFNβ-JAK1/TYK2-STAT1-IRF7 pathway was required for chemokine (C-X-C motif) receptor 3 (CXCR3)+ T lymphocyte migration, which promoted lung injury in the context of endotoxemia.

Conclusions: In summary, our study demonstrates that MRP8/14 induces sustained production of IP-10 via the IFNβ-JAK1/TYK2-STAT1-IRF7 pathway to attract CXCR3+ T lymphocytes into lung tissues and ultimately results in lung injury by an excessive inflammatory response in the context of endotoxemia.

Keywords: Endotoxemia; Interferon beta; Interferon regulatory factor-7; Interferon-inducible protein 10; Macrophage; Myeloid-related protein 8/14.

PubMed Disclaimer

Conflict of interest statement

None declared.

Figures

Figure 1
Figure 1
Effect of MRP8/14 on cytokine production and lung injury in endotoxic mice. (a) Mrp8 gene deficiency reduces plasma proinflammatory cytokine levels in endotoxic mice. WT mice were injected intraperitoneally (i.p.) with normal saline (NS) or LPS (20 mg/kg). In Mrp8 gene-deficient (Mrp8ΔMC) mice, MRP8/14 (4 mg/kg) or an equal volume of NS was intravenously (i.v.) injected after intraperitoneal administration of LPS (20 mg/kg) for 1 h. At 12 h after LPS administration, blood was collected, and plasma IL-1β, TNF-α, IL-6 and MCP-1 levels were measured by multiplex cytokine assays with a Luminex system. (b–d) Mrp8 gene deficiency alleviates lung injury of endotoxic mice. WT or Mrp8ΔMC mice were treated with NS, LPS (20 mg/kg), MRP8/14 (4 mg/kg) or LPS plus MRP8/14 for 12 h, followed by harvesting lung tissues from the mice and H&E staining. Representative histopathological images of lung tissues from WT mice and Mrp8ΔMC mice are shown with a scale bar of 200 μm (b). Lung injury scores were determined by semiquantitative lung injury analysis (c). The W/D ratios of lung tissues in the different groups were calculated (d). (e–g) Effect of MRP8/14 injection on lung injury in mice. WT mice were injected with NS, MRP8/14 (4 mg/kg), equal amounts of denatured MRP8/14 (dMRP8/14) or EGFP (4 mg/kg), and lung tissues were collected 12 h after intravenous injection of MRP8/14. Histopathological examination was performed by H&E staining and representative histopathological images of lung tissues from WT mice are shown with a scale bar of 200 μm (e). Lung injury scores were calculated as described above (f). W/D ratios of the lung tissues of mice in the different groups were calculated (g). The data are expressed as mean ± SD and represent three independent experiments (n = 3). *p < 0.05, **p < 0.01. WT wild-type, LPS lipopolysaccharide, MRP8/14 myeloid-related protein 8/14, IL-1β interleukin-1β, TNF-α tumor necrosis factor α, IL-6 interleukin-6, MCP-1 monocyte chemoattractant protein-1, W/D wet-to-dry, EGFP enhanced green fluorescent protein, H&E hematoxylin and eosin
Figure 2
Figure 2
IFNβ mediates MRP8/14-induced Ip-10 expression in RAW264.7 cells. (a) The dynamic profile of Ip-10 mRNA in RAW264.7 cells treated with MRP8/14 (1.5 μg/ml). Ip-10 mRNA expression levels were quantified by qPCR. (b) IP-10 protein levels in the BALF of WT or Mrp8ΔMC mice treated with LPS. WT mice were i.p. injected with LPS (20 mg/kg) or an equal volume of NS. In Mrp8ΔMC mice, MRP8/14 (4 mg/kg) or an equal volume of NS was intravenously injected after intraperitoneal LPS administration (20 mg/kg) for 1 h. BALF was collected 12 h after LPS administration, and IP-10 protein levels were quantified by a Luminex multiplex assay. (c, d) Effect of MRP8/14 on the expression of Ip-10 mRNA in RAW264.7 cells. RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml), dMRP8/14, EGFP or LPS (100 ng/ml) for 6 h (c) or 12 h (d), and Ip-10 mRNA expression levels were quantified by qPCR. (e) IP-10 protein levels in the supernatants of MRP8/14-treated RAW264.7 cells. RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for 0, 6 and 12 h, and IP-10 protein levels in the supernatants were examined by Luminex multiplex assays. (f, g) MRP8/14 induced Ifnb but not Ifna mRNA expression in RAW264.7 cells. RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for the indicated times (0, 3, 6, 9 and 12 h), followed by quantitation of Ifnb (f) and Ifna (g) mRNA expression by qPCR. (h, i) Specific induction of IFNβ protein expression in RAW264.7 cells by MRP8/14. RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml), and IFNβ (h) and IFNα (i) protein levels in the supernatants were quantified by ELISA. (j, k) IFNβ neutralizing antibody (IFNβ Ab) blocked the induction of Ip-10 mRNA expression in RAW264.7 cells induced by MRP8/14. RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for 12 h (j) or 6 h (k) in the presence or absence of the IFNβ neutralizing Ab (1 μg/ml) or isotype control IgG as a control, followed by Ip-10 mRNA analysis by qPCR. The data are expressed as mean ± SD and represent three independent experiments (n = 3). *p < 0.05, **p< 0.01, ns , not significant. MRP8/14 myeloid-related protein 8/14, IP-10 IFNγ inducible protein 10, IFNβ interferonβ, IFNα interferon α, qPCR quantitative real-time polymerase chain reaction, mRNA messenger RNA, BALF bronchoalveolar lavage fluid, WT wild type, Mrp8ΔMC  Mrp8 gene-deficient, NS normal saline, LPS lipopolysaccharide, dMRP8/14 denatured MRP8/14, EGFP enhanced green fluorescent protein, ELISA enzyme-linked immunosorbent assay
Figure 3
Figure 3
The JAK–STAT signaling pathway is involved in the regulation of Ip-10 gene expression in MRP8/14-treated RAW264.7 cells. (a–d) JAK1 and TYK2 but not JAK2 or JAK3 were phosphorylated in MRP8/14-treated RAW264.7 cells. RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for the indicated times (0, 5, 15, 30, 60, 120, 240 and 300 min), followed by total cellular protein extraction and immunoblot analysis of phosphorylated/total JAK1 (a), TYK2 (b), JAK2 (c) and JAK3 (d). The protein levels were quantified as the relative intensity of the protein bands on the blots. (e) The JAK1 inhibitor GLPG0634 blocked Ip-10 mRNA expression in RAW264.7 cells induced by MRP8/14. RAW264.7 cells were pretreated with or without GLPG0634 (500 nM) for 2 h and then stimulated with MRP8/14 (1.5 μg/ml) for 6 or 12 h. The mRNA expression levels of Ip-10 were quantified by qPCR. (f, g) IFNβ neutralizing Ab blocked the phosphorylation of JAK1 and TYK2 in RAW264.7 cells induced by MRP8/14. After pretreatment with the IFNβ neutralizing Ab (1 μg/ml) or isotype control IgG for 2 h, RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for 5 h. Phosphorylated and total JAK1 (f) and TYK2 (g) were examined by immunoblotting with specific antibodies. The protein levels were quantified as the relative intensity of the protein bands on the blots. (h) Dynamic profile of STAT1 phosphorylation in MRP8/14-treated RAW264.7 cells. RAW264.7 cells were stimulated with MRP8/14 (1.5 μg/ml) for the indicated times (0, 5, 15, 30, 60, 120, 240 and 300 min), followed by total cellular protein extraction and immunoblot analysis of phosphorylated and total STAT1. The protein levels were quantified as described above. (i) Time-dependent STAT1 nuclear translocation in MRP8/14-treated RAW264.7 cells. RAW264.7 cells were stimulated with MRP8/14 (1.5 μg/ml) for the indicated times (0, 60, 120, 240, 300 min), followed by total nuclear protein extraction and immunoblot analysis of total STAT1 and lamin as a control. The protein levels were quantified as described above. (j) The IFNβ neutralizing Ab blocked the phosphorylation of STAT1 in RAW264.7 cells treated with MRP8/14. After pretreatment with the IFNβ neutralizing Ab (1 μg/ml) or isotype control IgG for 2 h, RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for 5 h. Phosphorylated and total STAT1 were examined by immunoblotting with specific Abs. The protein levels were quantified as the relative intensity of the protein bands on the blots. (k) The STAT1 inhibitor fludarabine blocked Ip-10 mRNA expression in RAW264.7 cells induced by MRP8/14. RAW264.7 cells were pretreated with or without fludarabine (10 μM) for 2 h and then stimulated with MRP8/14 (1.5 μg/ml) for 6 or 12 h. The mRNA expression levels of Ip-10 were quantified by qPCR. The data are expressed as the mean ± SD and represent three independent experiments (n = 3). *p < 0.05, **p < 0.01. MRP8/14 myeloid-related protein 8/14, IP-10 IFNγ inducible protein 10, JAK1 Janus kinase 1, p-JAK1 phosphorylated Janus kinase 1, JAK2 Janus kinase 2, p-JAK2 phosphorylated Janus kinase 2, JAK3 Janus kinase 3, p-JAK3 phosphorylated Janus kinase 3, TYK2 tyrosine kinase 2, p-TYK2 phosphorylated tyrosine kinase 2, GLPG0634 JAK1 inhibitor, IFNβ Ab interferonβ neutralizing antibody, STAT1 signal transducer and activator of transcription 1, p-STAT1 phosphorylated signal transducer and activator of transcription 1, qPCR quantitative real-time polymerase chain reaction, mRNA messenger RNA
Figure 4
Figure 4
IRF7 functions downstream of the JAK–STAT pathway to mediate MRP8/14-induced Ip-10 gene expression in macrophages. (a) Profile of IRF7 protein expression in MRP8/14-treated RAW264.7 cells. RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for the indicated times (0, 2, 4, 6, 8, 10, 12 and 14 h), followed by total cellular protein extraction and immunoblot analysis of IRF7. The protein levels were quantified as described above. (b) Inhibition of the JAK–STAT pathway reduced IRF7 production in RAW264.7 cells induced by MRP8/14. After pretreatment with GLPG0634 (500 nM) or fludarabine (10 μM) for 2 h, RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for 12 h. IRF7 protein expression was quantified as described above. (c) Nuclear translocation of IRF7 in MRP8/14-treated RAW264.7 cells. RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for the indicated times (0, 8, 10, 12and 14 h), followed by nuclear protein extraction and immunoblot analysis of IRF7. Lamin was used as a control and the protein levels were quantified as described above. (d) Irf7 gene deficiency reduced late-stage Ip-10 mRNA expression in BMDMs induced by MRP8/14. BMDMs were isolated from WT or Irf7−/− mice and treated with or without MRP8/14 (1.5 μg/ml) for 6 or 12 h. Total RNA was extracted from BMDMs by the TRIzol method and Ip-10 mRNA expression was quantified by qPCR. (e) Irf7 gene overexpression rescued the transcriptional activity of Ip-10 that was downregulated by STAT1 inhibitor in MRP8/14-treated RAW264.7 cells. After transfection with the Ip-10 reporter (PGL3-Ip10p) with or without the irf7-expressing plasmid (pcDNA3-Irf7), RAW264.7 cells were cultured for 24 h. Then, the cells were incubated with fludarabine (10 μM) for 2 h and treated with MRP8/14 (1.5 μg/ml) for 12 h. Cell lysates were extracted for the dual-luciferase assay, and relative luciferase activities were calculated by determining the ratio of firefly and Renilla luciferase activities. The data are expressed as mean ± SD and represent three independent experiments (n = 3). *p < 0.05, **  p < 0.01. IRF7 interferon regulatory factor-7, MRP8/14 myeloid-related protein 8/14, IP-10 IFNγ inducible protein 10, JAK Janus kinase, STAT signal transducer and activator of transcription, GLPG0634 JAK1 inhibitor, WT wild-type, Irf7−/−  Irf7 gene deficiency, BMDMs bone marrow-derived macrophages, qPCR quantitative real-time polymerase chain reaction, mRNA messenger RNA
Figure 5
Figure 5
Chemotaxis of CXCR3+ lymphocytes was induced by activation of the IFNβ-JAK1/TYK2-STAT1-IRF7 pathway in vivo. (a, b) Mrp8 gene deficiency reduces the expression of Ip-10 and Ifnb mRNAs in endotoxic mice. WT mice were treated with LPS (20 mg/kg, i.p.), MRP8/14 (4 mg/kg, i.v.) or an equal volume of NS. Mrp8ΔMC mice were treated with LPS, LPS plus MRP8/14, or an equal volume of NS. After treatment with LPS or MRP8/14 for 12 h, AMs were isolated, and Ip-10 (a) and Ifnb (b) mRNA expression was quantified by qPCR. (c–e) Effect of Mrp8 gene deficiency on the phosphorylation of JAK1, TYK2 and STAT1 in AMs induced by LPS. After treatment with LPS or MRP8/14 for 12 h, AMs were isolated from mice, phosphorylated and total JAK1 (c), TYK2 (d) and STAT1 (e) levels were quantified by immunoblotting. The protein levels were quantified by determining the relative intensity of the protein bands on the blots. (f) Effect of Mrp8 gene deficiency on IRF7 expression in AMs treated with LPS. AMs were isolated from mice treated with LPS for 12 h and immunoblotting was performed to quantify IRF7 protein expression. (g–i) The IFNβ-JAK1/TYK2-STAT1-IRF7 signaling pathway was involved in the transmigration of CXCR3+ lymphocytes induced by MRP8/14 in vivo. After pretreatment with or without the IFNβ neutralizing Ab (1 μg/ml), GLPG0634 (500 nM) or fludarabine (10 μM) for 2 h, BMDMs from WT or Irf7−/− mice were treated with MRP8/14 (1.5 μg/ml) for 36 h, and cell culture supernatants were collected for the air pouch assay. After preparation of the air pouches in vivo for 5 days, the mice were i.p. administered LPS for 8 h. Then, the cell culture supernatants were injected into the air pouches and the lavage fluids were collected from the air pouches for further analysis. Wright–Giemsa staining was performed and the total number of migrated lymphocytes was counted (g). Flow cytometry was performed to analyse the proportion of CXCR3+ lymphocytes among all the migrated cells (h, i). The data are expressed as mean ± SD and represent three independent experiments (n = 3). *p < 0.05, **p < 0.01. CXCR3 chemokine (C-X-C motif) receptor 3, MRP8/14 myeloid-related protein 8/14, IP-10 IFNγ inducible protein 10, WT wild-type, Irf7−/−  Irf7 gene deficiency, Mrp8ΔMC  Mrp8 gene-deficient, LPS lipopolysaccharide, NS normal saline, IFNβ interferonβ, JAK1 Janus kinase 1, TYK2 tyrosine kinase 2, STAT1 signal transducer and activator of transcription 1, IRF7 interferon regulatory factor-7, GLPG0634 JAK1 inhibitor, IFNβ Ab interferonβ neutralizing antibody, AMs alveolar macrophages, BMDMs bone marrow-derived macrophages, qPCR quantitative real-time polymerase chain reaction, mRNA messenger RNA
Figure 6
Figure 6
IP-10 blockade and Cxcr3 gene deficiency alleviated lung injury in endotoxic mice. (a–c) Effect of IP-10 blockade and Cxcr3 gene deficiency on lung injury in endotoxic mice. WT mice were injected i.p. with NS or LPS (20 mg/kg) in the presence of IP-10 neutralizing antibody (IP-10 Ab) (2 mg/kg body weight) or equal amounts of isotype control IgG. Cxcr3−/− mice were injected i.p. with LPS (20 mg/kg). Mrp8ΔMC mice were challenged with LPS plus MRP8/14 in the presence of IP-10 Ab or equal amounts of IgG. H&E staining was performed to determine the histopathological changes in lung tissues from mice treated with LPS for 12 h and representative histopathological images are shown with a scale bar of 200 μm (a). Lung injury scores were determined by semiquantitative lung injury analysis (b). The W/D ratio of lung tissues from mice in the different groups was calculated (c). (d–f) Effect of IP-10 blockade and Cxcr3 gene deficiency on MRP8/14-induced lung injury in mice. WT mice were i.v. injected with MRP8/14 (4 mg/kg) in the presence of IP-10 Ab or equal amounts of IgG. Cxcr3−/− mice were treated with MRP8/14 alone. After MRP8/14 treatment for 12 h, lung tissues were collected from WT or Cxcr3−/− mice. H&E staining was performed to determine histopathological changes in lung tissues and representative histopathological images are shown with a scale bar of 200 μm (d). Lung injury scores were determined as described above (e). W/D ratio was calculated as described above (f). The data are expressed as mean ± SD and represent three independent experiments (n = 3). *p < 0.05, **p < 0.01. MRP8/14 myeloid-related protein 8/14, IP-10 IFNγ inducible protein 10, WT wild-type, Cxcr3−/−  Cxcr3 gene deficiency, Mrp8ΔMC  Mrp8 gene-deficient, LPS lipopolysaccharide, NS normal saline, H&E hematoxylin and eosin, W/D wet-to-dry
Figure 7
Figure 7
Signaling model for late-phase IP-10 induction by MRP8/14 in endotoxic mice. At the beginning of endotoxemia, MRP8/14 is released from activated neutrophils into the blood. Extracellular MRP8/14 binds to TLR4 on the surface of macrophages to induce IFNβ production and release. Then, IFNβ activates the JAK1/TYK2-STAT1 signaling pathway by binding with IFNAR, resulting in irf7 gene transcription and IRF7 protein synthesis. As a transcription factor, IRF7 promotes Ip-10 gene transcription, resulting in sustained production of IP-10 in the late phase of endotoxemia. Finally, the accumulated IP-10 attracts a large number of CXCR3+ lymphocytes to the site of infection, leading to an over-reactive inflammatory response and lung injury. MRP8/14 myeloid-related protein 8/14, IP-10 IFNγ inducible protein 10, IFNβ interferon β, JAK1 Janus kinase 1, TYK2 tyrosine kinase 2, STAT1 signal transducer and activator of transcription 1, IRF7 interferon regulatory factor-7, CXCR3 chemokine (C-X-C motif) receptor 3, IFNAR type I interferon receptor
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Fein AM, Calalang-Colucci MG. Acute lung injury and acute respiratory distress syndrome in sepsis and septic shock. Crit Care Clin. 2000;16:289–317. - PubMed
    1. Kumar V. Pulmonary innate immune response determines the outcome of inflammation during pneumonia and sepsis-associated acute lung injury. Front Immunol. 2020;11:1722. - PMC - PubMed
    1. Tolle LB, Standiford TJ. Danger-associated molecular patterns (DAMPs) in acute lung injury. J Pathol. 2013;229:145–56. - PubMed
    1. Xiang M, Fan J. Pattern recognition receptor-dependent mechanisms of acute lung injury. Mol Med. 2010;16:69–82. - PMC - PubMed
    1. Foell D, Wittkowski H, Vogl T, Roth J. S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules. J Leukoc Biol. 2007;81:28–37. - PubMed
-