JMJD3 Is Required for Acute Pancreatitis and Pancreatitis-Associated Lung Injury

Acute pancreatitis (AP) is an acute inflammatory disorder of the pancreas that can be complicated by the involvement of other remote organs. The current strategy of treatment in AP is limited to supportive care, adequate analgesia, and treatment of complications when they develop (1–4). The morbidity and mortality of severe AP are determined largely by the extent of the related inflammatory response (2–4). Multiple organ dysfunction syndromes will occur if the patients’ systemic inflammatory response gets worse (2–4). Severe AP with severe complications such as multiple organ failure often gives rise to high mortality (30–40%) despite intensive treatment (2, 4). Therefore, there is still an urgent need for novel approaches to better control inflammation in severe clinical conditions (2, 5). However, the molecular mechanism regulating the inflammatory response in the pancreas and other remote organs remains poorly understood.

Lysine-specific demethylase Jumonji domain-containing protein 3 (JMJD3), also known as lysine-specific demethylase 6B, is an important histone demethylase that enhances gene expression by demethylating the repressive epigenetic marker, H3K27me3 (trimethyl-lysine 27 on histone H3), in genes (6). A large number of studies have shown that JMJD3 promotes inflammatory diseases by enhancing proinflammatory gene expression (7). JMJD3 binds to Polycomb group target genes and demethylates H3K27me3 into dimethyl-lysine 27 on histone H3 (H3K27me2)/H3K27me to enable or enhance transcriptional activation of target genes (H3K27me2/3) (8). JMJD3 has been proven to promote TNF-α gene transcription by demethylating H3K27me3 from the TNF-α transcription start site to enhance the binding of RNA polymerase II (9).

AP is characterized by inflammatory disease with a significant increase in proinflammatory cytokines such as TNF-α, IL-1, and IL-6 (10). TNF-α, the cell death–inducing cytokine, is mainly produced by monocytes and macrophages. It has been proven to induce inflammatory gene upregulation, endothelial upregulation, cell death, and the recruitment and activation of immune cells in the pathogenesis of AP (11).

The NF-κB and MAPK signaling pathways are critical for TNF-α production in response to TLR and stimulator of IFN genes (STING) stimulation (12, 13). NF-κB, a nuclear transcription factor responsible for upregulating the transcription of a wide variety of inflammatory genes including TNF-α, has been shown to promote the development of AP (14). NF-κB also rapidly stimulated JMJD3 expression by binding to a cluster of three κB sites in the JMJD3 gene promoter in LPS-stimulated macrophages (15), indicating the relationship between epigenetic regulation and inflammation. In HUVECs, LPS induced the enrichment of NF-κB/p65 and JMJD3 to the promoter regions of TNF-α (15). Interestingly, JMJD3 was identified by chromatin immunoprecipitation sequencing to stimulate the genes involved in NF-κB activation (16), and TNF-α is also an activator of NF-κB (17). Thus, NF-κB, JMJD3, and TNF-α form a positive feedback loop to promote each other’s transcription. It is conceivable that the inhibition of JMJD3 expression inhibits the positive feedback loop and plays an effective therapeutic role in inflammatory diseases.

Acinar cell death is critical to AP. Dying acinar cells release damage-associated molecular patterns (DAMPs), which might contribute to pancreatic injury, proinflammatory cytokine release, and remote organ injury through specific DAMP receptors, such as TLR and STING (18). Mitochondrial DNA (mtDNA), an important source of DAMPs, was reported to induce an inflammatory response after injury through the STING and TLR9 pathways (19, 20). In addition, the oxidation of mtDNA could be induced by reactive oxygen species (ROS) generation in cell stress or infiltrating inflammatory cells, which can be recognized as a potent DAMP with a more significant potential to trigger innate immune responses through the STING and TLR9 pathways (21–23).

Considering the vital role of JMJD3 and mtDNA or oxidized-mtDNA in the regulation of the inflammatory response, we hypothesize that mtDNA and oxidized-mtDNA released from acinar cell death in AP may upregulate the expression of JMJD3, which in turn triggers the inflammatory response in pancreatic tissues and pancreatitis-associated lung injury. To test this concept, we used AP male mouse models induced by L-arginine (L-Arg) and caerulein (Cae), as well as human tissues from chronic pancreatitis (CP) patients, to investigate the possible role of JMJD3, mtDNA, and oxidized-mtDNA in the regulation of the inflammatory process of AP and the underlying mechanism.

AP patients admitted to West China Hospital of Sichuan University from January 2019 to May 2019 were enrolled. Inclusion criteria were (1) 18–45 y old and (2) met the 2012 Atlanta diagnostic revision for AP (24). Exclusion criteria were (1) gravidas and children; (2) patients with myocardial infarction, malignant diseases, end-stage liver disease, renal disease or organ failure, among others; and (3) the hospitalization admission was <24 h. In addition, physical examination individuals without any disease at the health management center were enrolled in the control group. Afterward, peripheral blood samples were acquired. All patients signed the informed consent form, which was approved by the West China Hospital Ethics Committee.

Male C57BL/6 mice, 20–25 g, were obtained from Beijing Huafukang Biotechnology Co. The experimental animal program was approved by the Committee for Animal Protection and Utilization of Sichuan University. All experiments followed the principles of laboratory animal protection. In selected experiments, C57BL/6 mice with global deletion of TLR9 (obtained from Bioindustry Division Oriental Yeast Co.) and STING (obtained from The Jackson Laboratory) expression were also used. Jmjd3-cKO mice were generated by targeting exons 15–21 (encoding the JmjCcatalytic domain) using a Cre-LoxP system. All animal experiments were approved by the Committee on Animal Protection and Utilization and the Ethics Committee.

Pancreatitis was induced by i.p. injection of the cholecystokinin analog Cae at seven doses of 50 μg/kg each, 1 h apart (25). L-Arg hydrochloride sterile solution (8%) was prepared in normal saline (NS; pH 7.0), and the nonfasted mice were i.p. injected with L-Arg hydrochloride sterile solution in two doses of 4 g/kg each, 1 h apart. The control group received only aseptic saline injections (26). For administration of GSK-J4 (Sigma), mice were divided into four groups: NS group, AP model group treated with Cae, AP model group treated with 20 mg/kg (50 mg/kg in Cae-induced AP group) GSK-J4, and AP model group treated with 50 mg/kg (100 mg/kg in Cae-induced AP group) GSK-J4. For AP-induced mice with L-Arg, GSK-J4 was injected i.p. in two doses, 20 and 50 mg/kg, at 24, 48, and 72 h after the first injection of L-Arg. For AP-induced mice with Cae, GSK-J4 was injected i.p. in two doses, 50 and 100 mg/kg at 3, 8, 24, 48, and 72 h after the first administration of Cae. The N-acetylcysteine (NAC) injection schedule was the same as GSK-J4, and both AP models were injected with 300 mg/kg NAC i.p. each time.

To detect monocytes and neutrophils infiltrating the pancreas, lung, and peripheral blood, we processed the samples as described previously (19, 26–28). Cells were dispersed in PBS at 1 × 10⁶ cells/100 μl and stained with allophycocyanin-rat anti-mouse CD45 (1:100, catalog number [Cat #] 147708; BioLegend), PerCP-Cy5.5 rat anti-mouse CD11b (1:100, Cat #101228; BioLegend), PE-rat anti-mouse Ly6G (1:100, Cat #127608; BioLegend), and FITC-rat anti-mouse Ly6C (1:100, Cat #128005; BioLegend) Abs and then measured by flow cytometry for the detection of monocytes in human peripheral blood. We used allophycocyanin-mouse anti-human HLA-DR (1:100, Cat #307610; BioLegend), PerCP-Cy5.5 mouse anti-human CD16 (1:100, Cat #302028; BioLegend), and PE-mouse anti-human CD14 (1:100, Cat #367104; BioLegend) Abs and then measured the cells by flow cytometry (29). Intracellular monocyte TNF-α was detected as previously described (30).

Monocytes and neutrophils from mouse bone marrow and peripheral blood were prepared as previously described (26). More than 85% of the separated cells were monocytes or neutrophils, as determined by flow cytometry.

Total RNA was extracted from the pancreas, lung, and isolated monocytes using an RNA Simple Total RNA Kit (TIANGEN, Beijing, China). A PrimeScript RT kit with a genomic DNA eraser (Perfect Real Time; Takara, Dalian, China) was used to synthesize the first complementary strand of DNA from the total RNA template. The resulting cDNA was added and mixed into iQ SYBR Green Supermix (perfect real-time; Bio-Rad) in a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol. The expression levels of the target genes were standardized to that of GAPDH. The mtDNA in the serum of mice and humans was concentrated and purified by a QIAamp DNA Blood Mini Kit (Qiagen). Then, mtDNA was quantified with a TaqMan probe. The PCR primers, probes, and the standard curve of mice used to amplify mouse mtDNA were designed as described previously (19). Primers were synthesized by Invitrogen. The standard samples, PCR primers, and probes of human mtDNA were designed and synthesized by Molecular Biotechnology Co. (Tianjin, China). All primer sequences quantitative PCR (qPCR) were as follows: GAPDH (mouse), forward 5′-ACCCAGAAGACTGTGGATGG-3′, reverse 5′-ACATTGGGG GTAGGAACAC-3′; JMJD3 (mouse), forward 5′-CCCCCATTTCAGCTGACTAA-3′, reverse 5′-CTGGACCAAGGGGTGTGTT-3′; homo-GAPDH, forward 5′-CTGATGCCCCCATGTTCGTC-3′, reverse 5′-CACCCTGTTGCTGATGCCAAATTC-3′; homo-JMJD3, forward 5′-TGTTCCCTGTAGCACATCAAG-3′, reverse 5′-TAGAGTGAGTGCGTTTCG-3′; mtDNA (mouse), forward 5′-ACCTACCCTATCACTCACACTAGCA-3′, reverse 5′-GAGGCTCATCCTGATCATAGAA TG-3′; FAM-labeled TAMRA-quenched probes (mouse), 5′-ATGAGTTCCCCTACCAATACCACACCC-3′; homo-mtDNA, forward 5′-CCCCACATTAGGCTTAAAAACAGAT-3′, reverse 5′-TATACCCCCGGTCGTGTAGCGGT-3′; FAM-labeled TAMRA-quenched probes (human), 5′-CAATTCCCGGACGTCTAAACCAC-3′.

Esterase staining was performed by staining with a Naphthol AS-D Chloroacetate kit (Sigma) following the instructions. Immunofluorescence of JMJD3 expression in monocytes in the pancreas and lung was detected in frozen tissue sections that were prepared as previously described (31). Frozen tissue sections were coimmunostained with rabbit anti-JMJD3 (1:400, Cat #GTX31466; GeneTex) and rat anti-mouse Ly6C (1:400, ab24973; Abcam) Abs, followed by a Cy3-conjugated goat anti-rabbit secondary Ab (1:200, ab6939; Abcam) and Alexa 488 goat anti-rat secondary Ab (1:200, ab150081; Abcam). The 8-hydroxy-2-deoxyguanosine (8-OHdG) index was stained with goat 8-OHdG (1:200, ab93295; Abcam) and rabbit anti-mouse/human TOMM 20 (1:250, ab186734; Abcam) Abs, followed by Alexa Fluor 647 donkey anti-rabbit secondary Ab (1:200, ab150075; Abcam) and Alexa 488 donkey anti-goat secondary Ab (1:400, ab150129; Abcam). The nucleus was stained with Hoechst 33342 (20 µg/ml; Sigma) for 30 min. Images were captured by using the Zeiss LSM880 confocal microscope. ROS production was measured in situ on pancreas slices as previously described (32). We used a fluorescent ROS probe, H₂DCFDA (Invitrogen), which is a substrate without fluorescence itself that converts to a green fluorescent product when it is hydrolyzed by intracellular esterase.

Pancreatic and pulmonary tissues were soaked in 4% paraformaldehyde, embedded in paraffin, and then sectioned. The tissue sections were stained with H&E to accurately assess the extent of tissue injury. The histopathological scoring analysis of the pancreas and lung was performed blindly by three independent pathologists following a corresponding scoring system based on previous studies (33, 34).

Pancreatic cells were obtained by a modified digesting and suspending technique as previously described (35). For the treatment of pancreatic cells with L-Arg in vitro, the isolated pancreatic cells were incubated with 10 mg/ml L-Arg in DMEM supplemented with 10% FBS, 1000 U/ml penicillin G, 100 μg/ml streptomycin, and 0.1 mg/ml soybean trypsin inhibitor (Sigma) for 12 h, and the necrosis rate of the separated cells was >75% (36). For the treatment of pancreatic cells with Cae in vitro, the isolated pancreatic cells were incubated with 100 μg/ml Cae for 24 h, and the cell death rate was nearly 30% (37).

mtDNA was isolated from the pancreas of C57BL/6 mice by an mtDNA isolation kit (ab65321; Abcam) under sterile conditions. The concentration of mtDNA was detected by a spectrophotometer; there was no protein contamination. The mtDNA was stored in sterile water and kept at −80°C for long-term storage. Mitochondria were isolated by a mitochondria isolation kit (MITOISO2; Sigma). The endotoxin level of mtDNA was detected at <0.25 endotoxin unit/ml. Oxidized-mtDNA was prepared by irradiating mtDNA on ice with a 254-nm germicidal UV lamp (a series of five 8-W tubes) for 0.5 h (38).

mtDNA (0.5 μg/ml) and oxidized-mtDNA (0.5 μg/ml) were used to stimulate freshly isolated monocytes at a concentration of 5 × 10⁶ cells/ml and incubated at 37°C for 0.5 h, and then monocytes were lysed to prepare protein samples. Samples containing equal amounts of protein were electrophoretically transferred onto a polyvinylidene difluoride membrane and then blocked in TBST buffer containing 5% skim milk. Abs to phospho-p38 MAPK (Thr 180/Tyr 182; #4511; Cell Signaling Technology), p38 MAPK (#8690; Cell Signaling Technology), NF-κB p65 (#8242; Cell Signaling Technology), phospho-NF-κB p65 (#3033; Cell Signaling Technology), β-actin (ab179467; Abcam), and JMJD3 (1:1000 for all; GeneTex) were used. The polyvinylidene difluoride membrane was incubated with specific primary Abs at 4°C overnight, washed with PBST, and then incubated with HRP‐conjugated secondary Abs. The target proteins on the blot were visualized by a SuperSignal West Dura Substrate (Thermo Fisher Scientific, Waltham, MA).

The 8-OHdG levels in mouse and human serum were measured using a commercial 8-OHdG ELISA kit (Abcam) following the instructions. The concentration (ng/ml) was measured at 450 nm.

Groups were compared with Prism software (GraphPad) using a 11122eq two-tailed unpaired Student t test or one-way ANOVA. Data are presented as the means ± SEM.

To study the role of JMJD3 in AP, we established a mouse model of AP by injecting L-Arg or Cae in previous reports (25, 26). As shown in (Fig. 1A, 96 h after the repetitive injection of L-Arg and 48 h after the injection of Cae in mice, significant morphological damage was observed in the pancreas and lung of AP mice by H&E histology staining. The pathomorphological changes in the pancreas of AP mice, including acinar cell necrosis, edema, and inflammatory cell infiltration, were significantly more severe than those in the control group (Fig. 1A, left, Supplemental Figs. 2A, 4A). The lungs of AP mice showed an extreme inflammatory response by an abundance of inflammatory cells (Fig. 1A, right, Supplemental Figs. 2A, 4A). The expression and distribution of JMJD3 in the AP model (Fig. 1B, Supplemental Fig. 4F) were further detected, and the expression level of JMJD3 in the pancreatic tissue (Fig. 1C) and the isolated peripheral blood monocytes (Fig. 1D) was increased in the AP mice as detected by Western blot and qPCR. In addition, the percentage of infiltrating inflammatory TNF-α⁺ monocytes was increased in the pancreas of AP mice at 24 h after the first injection of L-Arg and Cae by intracellular staining of TNF-α (26) (Fig. 1E). Eight hours after the first injection of L-Arg or Cae, JMJD3 was upregulated in the monocytes infiltrating in the pancreas in the AP group and was predominantly distributed in the nuclei of monocytes as detected by immunofluorescent staining (Fig. 1F).

Peripheral blood samples of 18 patients admitted to West China Hospital within 24 h from the onset of symptoms and diagnosed with AP and pancreatic tissue from 4 patients with CP were collected to explore the effects of JMJD3 in human pancreatitis. The control group included 18 age-matched healthy volunteers. We detected the expression of JMJD3 in the human pancreas by immunofluorescent staining and found that JMJD3 expression was upregulated (Fig. 1G). Next, we observed a significant increase in the expression of JMJD3 in the peripheral blood of the AP group by qPCR (Fig. 1H). Furthermore, the percentage of HLA-DR⁺CD16⁺CD14⁺ inflammatory monocytes in the peripheral blood of AP patients was increased (Fig. 1I). We observed the percentage of TNF-α⁺HLA-DR⁺CD16⁺CD14⁺ monocytes in the peripheral blood of the AP group by flow cytometry. The percentage of TNF-α⁺HLA-DR⁺CD16⁺CD14⁺ monocytes in the peripheral blood of the AP group was increased compared with that of the control group (Fig. 1J). These results indicated that JMJD3 might play a crucial role in the progression of AP.

Tissue damage by trauma or pathogenesis might release DAMPs from necrotic/necroptotic cells. Cell death and tissue damage were observed by H&E staining of the pancreas and lung of AP mice (Fig. 1A). Accordingly, the increased release of mtDNA was also detected in the patients with AP, as well as in the mouse model of AP (Fig. 2A, 2B). The tissue injury was also accompanied by the generation of ROS, which was confirmed in this study 48 h after the first injection of Cae (Supplemental Fig. 1A). Oxidative stress occurs in the development of AP in acinar cells, and excessive generation of ROS damages acinar cells via oxidation of mtDNA (39). Therefore, we further characterized the generation of oxidized-mtDNA using 8-OHdG as a marker. The level of oxidized-mtDNA in the pancreas of CP patients was increased as detected by immunofluorescent staining (Fig. 2C). 8-OHdG was more concentrated in the serum of patients with AP, as detected by ELISA (Fig. 2D, top). The percentage of 8-OHdG⁺HLA-DR⁺CD16⁺CD14⁺ monocytes was detected by flow cytometry in the peripheral blood of the AP group, and it was higher than that of the control group (Fig. 2D, bottom). Similar results were obtained in the mouse model of AP. In the pancreas of AP mice, strong 8-OHdG staining colocalized with TOMM20 (a mitochondrial outer membrane protein) staining (Supplemental Fig. 1B). The result was further confirmed by flow cytometry of intracellular staining for 8-OHdG in Ly6C⁺ inflammatory monocytes in the mouse pancreas and peripheral blood at 96 h after the injection of L-Arg or at 48 h after Cae in mice (Fig. 2E). The increase of oxidized-mtDNA in isolated peripheral blood monocytes was shown by immunofluorescence in AP mice (Fig. 2F). To further confirm the key roles of cell death and mtDNA release in the progression of AP, we treated mouse peripheral blood monocytes with pancreatic acinar cells incubated with L-Arg or Cae for 6 and 8 h in vitro. In the peripheral blood monocytes treated with necrotic mouse pancreatic acinar cells, the percentage of monocytes secreting TNF-α was significantly increased compared with the control mice (Fig. 2G, top). In addition, the concentration of 8-OHdG in the serum of AP mice was higher than that in control mice (Fig. 2G, bottom). The results also showed that pancreatic acinar cells isolated from the pancreas of AP mice stimulated the secretion of TNF-α in monocytes at 6 and 8 h in vitro. These findings suggested that the release of oxidized-mtDNA was detected in the mouse model/patients with AP. Next, we tried to investigate whether the oxidized-mtDNA was associated with elevated levels of JMJD3.

In the next experiment, we studied the correlation between JMJD3 elevation and mtDNA stimulation. It is interesting to find that mtDNA or oxidized-mtDNA could induce the upregulation of JMJD3 levels both in vitro and in vivo. The incubation of mtDNA or oxidized-mtDNA with the isolated bone marrow monocytes in vitro led to a quick and significant upregulation of JMJD3 mRNA levels detected by qPCR analysis. Upregulation of JMJD3 level was detected after the stimulation for 0.5 h. It was found that oxidized-mtDNA stimulated the expression of JMJD3 more efficiently than mtDNA (Fig. 3A). On stimulation with mtDNA or oxidized-mtDNA, JMJD3 was upregulated and accumulated in the nucleus, as illustrated by immunofluorescence assay (Fig. 3B). In regard to the in vivo stimulation of mtDNA/oxidized-mtDNA to JMJD3 expression, the upregulation of JMJD3 in the pancreas of the mice in the experimental group was detected 8 h after injection (Fig. 3C). JMJD3⁺Ly6C⁺ monocytes were found in the mouse lung after the injection of mtDNA and oxidized-mtDNA (Fig. 3D). Based on this observation, the pathways involved in the upregulation of JMJD3 were characterized. The elevated protein level of JMJD3 was also confirmed after the incubation of mouse bone marrow monocytes with mtDNA and oxidized-mtDNA. In addition, the upregulation of JMJD3 protein levels was accompanied by the increased phosphorylation of MAPK p38 and NF-κB p65 in the stimulated monocytes (Fig. 3E, Supplemental Fig. 4G–I). It was reported that the activation of immune cells by mtDNA and oxidized-mtDNA was closely related to the TLR9 or STING pathway. In the next experiment, we used Sting^−/− and Tlr9^−/− mice to investigate whether the upregulation of JMJD3 induced by mtDNA involves these pathways. mtDNA and oxidized-mtDNA were injected i.v. into Sting^−/− and Tlr9^−/− mice. Sting^−/− and Tlr9^−/− mice showed a reduction of JMJD3 expression in the lung tissue compared with WT mice (Fig. 3F, 3G). The monocytes isolated from Sting^−/− and Tlr9^−/− mice inhibited the activation of JMJD3 induced by mtDNA and oxidized-mtDNA (Fig. 3H, Supplemental Fig. 4J–M). Therefore, our results suggested that mtDNA and oxidized-mtDNA stimulated the expression of JMJD3 in monocytes and tissues both in vitro and in vivo, which critically involves the TLR9 and STING pathways.

Circulating mtDNA released from dead cells known as potent DAMPs is reported to stimulate immune cells and cause severe inflammatory response (40). Pancreatitis-associated lung injury is one of the most serious inflammatory complications of AP, and its exact mechanisms remain unclear (41). In this research, we postulated that pulmonary inflammation was partly induced by mtDNA and oxidized-mtDNA released by pancreatic acinar cells in mice with AP. To address these postulations, we isolated pancreatic acinar cells from normal mice and treated them with L-Arg or Cae in vitro for 12 or 24 h, respectively. Necrotic cells were also prepared through a freeze-thaw process. C57BL/6 mice were injected with NS, the pretreated pancreatic acinar cells, isolated mitochondria, mtDNA, and oxidized-mtDNA. Severe pulmonary inflammation was detected in mice 48 h after the injection of necrotic cells and all the mitochondria-derived DAMPs (Fig. 4A, Supplemental Fig. 2B). Pulmonary inflammation was further characterized by the infiltration of immune cells with esterase staining (Fig. 4B). The injection of mtDNA or oxidized-mtDNA significantly increased the percentages of TNF-α⁺CD45⁺CD11b⁺Ly6C⁺ inflammatory monocytes in the lung (Fig. 4C), as well as in the peripheral blood (Fig. 4D), compared with the control group. Because the oxidized-mtDNA stimulated the immune response more efficiently than the unoxidized mtDNA, we further investigated the role of mtDNA oxidation in the induction of inflammation in AP and lung injury. NAC, an ROS inhibitor, was administered to mice with AP via i.p. injection, and its influence on lung and pancreas pathology was evaluated. The percentage of CD45⁺CD11b⁺Ly6C⁺ inflammatory monocytes with 8-OHdG in the L-Arg/Cae–induced mouse AP model was decreased after the injection of NAC, suggesting the efficient inhibition of cell oxidation by ROS (Fig. 4E). Treatment with NAC led to a decreased level of JMJD3 in the mouse pancreas. Because 300 mg/kg NAC was administered i.p. 3 h after the first administration of L-Arg or Cae, the level of JMJD3 significantly decreased in the tissue of the AP model group as detected at 8 h (Fig. 4F), which is consistent with our hypothesis that oxidation of mtDNA contributed to the upregulation of JMJD3 expression. Accordingly, the inflammation in the pancreas and lung was partially relieved after the treatment of NAC (Fig. 4G, Supplemental Figs. 2C, 4B), indicating the indispensable role of mtDNA oxidation in the induction of severe histopathological injury. The percentages of TNF-α⁺ inflammatory monocytes were also decreased in the lung and peripheral blood of mice injected with NAC compared with that of the control (Fig. 4H, 4I). These results indicated that mitochondrial-derived DAMPs, including the oxidized form of mtDNA, play essential roles in the induction of severe pancreatic inflammation and lung injury. The inhibition of ROS by NAC in vivo decreased the oxidation of mtDNA in the circulating monocytes and resulted in the relief of pancreatic inflammation and lung injury.

To further address the role of mtDNA, TLR9, and STING pathways in the occurrence of pulmonary injury in AP, we used WT, Sting^−/−, and Tlr9^−/− mice to observe the inflammatory response in the lung in the Cae-induced disease model. As shown in (Fig. 5A and Supplemental Figs. 2D and 4C, the pathological changes in the pancreas and lung were significantly reduced in Sting^−/− and Tlr9^−/− mice. The population of circulating inflammatory cells was decreased in both peripheral blood (Supplemental Fig. 1C) and infiltrated lung tissue (Fig. 5B). In addition, the percentages of TNF-α⁺CD45⁺CD11b⁺Ly6C⁺ inflammatory monocytes in the lungs of Sting^−/− and Tlr9^−/− mice at 24 h after the administration of Cae were decreased compared with those in the lungs of WT mice (Fig. 5C). The critical role of mtDNA and oxidized-mtDNA in the induction of pulmonary inflammation was further studied by injection into WT, Sting^−/−, and Tlr9^−/− mice. Forty-eight hours after the i.v. injection, Sting^−/− and Tlr9^−/− mice showed reduced pulmonary damage compared with WT mice (Fig. 5D, Supplemental Fig. 2E). The flow cytometry results revealed decreased percentages of monocytes and neutrophils in the lungs of Tlr9^−/− and Sting^−/− mice (Fig. 5E). Furthermore, the percentage of monocytes secreting TNF-α was decreased in the lungs of both Tlr9^−/− and Sting^−/− mice (Fig. 5F). These results further confirmed that mtDNA and oxidized-mtDNA promoted the inflammatory process of AP through the Tlr9^−/− and Sting^−/− pathways.

To further study the role of JMJD3 in AP, we used the JMJD3-specific inhibitor GSK-J4 in mice with AP. As shown in (Fig. 6A and Supplemental Figs. 3A and 4D, pathological lesions were alleviated in GSK-J4–treated groups compared with the AP control groups. In addition, the percentages of monocytes and neutrophils were detected in the pancreas, lung, and peripheral blood by flow cytometry. The results showed that the percentages of monocytes and neutrophils in the pancreas (Supplemental Fig. 1D), lung (Fig. 6B, top), and peripheral blood (Fig. 6B, bottom) of the GSK-J4–treated groups were lower than those of the AP control groups. The percentage of TNF-α⁺CD45⁺CD11b⁺Ly6C⁺ inflammatory monocytes in the pancreas (Supplemental Fig. 1E), lung (Fig. 6C, left), and peripheral blood (Fig. 6C, right) of mice injected with GSK-J4 at 24 h after the administration of AP were decreased compared with the control group. These data demonstrated that JMJD3 promoted the inflammatory process of AP, and inhibition of JMJD3 activity ameliorated AP and its related lung injury with a significant reduction in monocytes expressing TNF-α.

Ιn the next experiment, AP was induced in WT and Jmjd3-cKO mice. As shown in (Fig. 6D and Supplemental Figs. 3B and 4E, the pathological alterations of the pancreas and lung were significantly reduced in Jmjd3-cKO mice. The percentages of infiltrating monocytes and neutrophils in the pancreas (Supplemental Fig. 1F), lung (Fig. 6E, top), and peripheral blood (Fig. 6E, bottom) of Jmjd3-cKO mice were decreased compared with those of WT mice, and the percentage of TNF-α⁺ inflammatory monocytes was decreased (Fig. 6F). A similar tendency was observed in the experiment by administration of mtDNA and oxidized-mtDNA. Forty-eight hours after injection, the pulmonary inflammation of Jmjd3-cKO mice was alleviated compared with that of WT mice (Fig. 6G, Supplemental Fig. 3C). The flow cytometry results suggested decreased percentages of monocytes and neutrophils (Fig. 6H), as well as TNF-α⁺CD45⁺CD11b⁺Ly6C⁺ inflammatory monocytes (Supplemental Fig. 1G), in the lungs of Jmjd3-cKO mice as compared with the control mice. These results demonstrated that JMJD3 plays an indispensable role in the induction of inflammation in AP and its lung injury.

JMJD3 is a crucial histone demethylase for the regulation of several genes involved in inflammatory signaling pathways. JMJD3 is a potent enhancer of proinflammatory genes, mainly because of its participation in the NF-κB pathway (42). The reason why NF-κB directs the transcription of JMJD3 is that there are two conserved κB sites in the promoter sequences upstream of the first coding exon of JMJD3 (26). De Santa et al. (8) demonstrated that the effect of LPS on bone marrow–derived macrophages induced significant recruitment of NF-κB/p65 and JMJD3 to TNF-α transcription initiation sites. Das et al. (42) observed that the alteration of JMJD3 expression affected the transcription of a network of NF-κB–dependent genes in human leukemia monocyte macrophages. In view of the close relationship between NF-κB and JMJD3, we hypothesized that the NF-κB-JMJD3 pathway might play a critical role in AP.

Studies have shown that ROS is generated in the early stage of acute necrotizing pancreatitis, and the main target of ROS and redox signal transduction in AP is NF-κB. It has also been shown that nearly all pathways leading to NF-κB activation can be blocked by treatment with a variety of antioxidants, including NAC and glutathione (43, 44). However, the precise molecular mechanism of ROS that activates NF-κB needs further study in AP. mtDNA is particularly vulnerable to oxidative damage because it is close to ROS production sites and lacks histone protection (45). ROS generation is an important factor for the generation of 8-OHdG, and 8-OHdG can also be abundant under UV exposure (46). Increased levels of ROS produce oxidatively modified mtDNA in newly recruited inflammatory cells, and oxidized-mtDNA induces more production of inflammatory cytokines in immune cells through the TLR9/STING-NF-κB pathways (21, 23). In vitro and in vivo experiments showed that oxidized-mtDNA has greater potential to activate dendritic cells than natural mtDNA (21). According to the observations in previous studies, purified mtDNA or even oligodeoxynucleotide (ODN) contained a single 8-OHdG residue but lacked CpG motifs, inducing arthritis in mice after intra-articular injection. In contrast, ODNs lack the oxidized base but possess identical sequences that are completely inert in vivo, except for the lack of oxidative bases (47). In another study, a significantly higher amount of TNF-α was induced from RAW264.7 macrophage-like cells by the 8-OHdG–substituted CPG ODN molecule than that of nonsubstituted control cells. In primary cultured macrophages isolated from WT mice, an increase in TNF-α production induced by DNA containing 8-OHdG was also observed, but this increase was not observed in Tlr9^−/− mice. In addition, s.c. injection of 8-OHdG containing CpG ODN resulted in increased footpad swelling compared with regular CpG ODN (48).

In our research, JMJD3 was upregulated in the early stage of AP. This discovery prompted us to further explore the cause of elevated JMJD3 and its role in the inflammatory process of AP. mtDNA activates innate immunity by the TLR9/STING-NF-κB pathways (20), and mtDNA released from necrotic pancreatic cells has been reported as a marker of AP (49). This suggests that therapeutically targeting STING and TLR9 may be a promising approach for the treatment of severe AP (50, 51). Subsequently, we found that mtDNA stimulated the expression of JMJD3 in vitro and in vivo, and oxidized-mtDNA had a more stimulatory effect. Thus, we hypothesized that mtDNA and oxidized-mtDNA triggered the STING and TLR9 pathways and activated NF-κB p65. NF-κB p65 stimulated the expression of JMJD3, which promoted TNF-α secretion from monocytes in mice. To further confirm our hypothesis, we applied the JMJD3-specific inhibitor GSK-J4 and the ROS inhibitor NAC in AP mice. Both inhibitors ameliorated AP in mice and inhibited TNF-α secretion by monocytes. In addition, the amelioration of AP and inhibition of TNF-α secretion appeared in TLR9-, STING-, and JMJD3-deficient mice with AP. Interestingly, monocytes are the major subgroup of the predominant leukocyte subset, which upregulates JMJD3 during AP. Notably, our in vitro studies showed that mtDNA released by dead acinar cells activated JMJD3 in monocytes and stimulated the secretion of TNF-α. In AP models, except for the STING and TLR9 pathways reported in this study mediating TNF-α secretion, the other TLRs, including TLR2 and TLR4, are essential for the activation of proinflammatory pathways. Stimulation of TLR4 plays a significant proinflammatory role, which subsequently induces the production of inflammatory cytokines such as IL-1β and IL-6 (52). Nevertheless, a general consensus has not been achieved because controversial outcomes were presented, and in-depth elucidation of the mechanism is warranted (53).

As with the majority of studies, the design of this study is subject to limitations. There are two major limitations in this study that could be addressed in future research. First, the study focused only on C57BL/6 male mice. It is reported that Cae is a cholecystokinin-pancreozymin analog and is used to successfully cause AP in male mice, which belongs to a hormone-induced model (54). In addition, the effects of estrogens in female mice in the setting of AP are complex and vary with sex (55). To avoid the influence of estrogens in female mice, male mice are favorably used to establish the AP model compared with the female ones (56). Although animal studies on pancreatitis typically use male animals, animal studies using both males and females, or with sex hormone treatment, also suggest that sex may play a role in the development of AP (56). Whether sex differences exist for the endogenous inhibitors of the pancreas is unknown, providing valuable insight to conduct relevant in-depth research. Second, the study focused on the crucial role of JMJD3 and related pathways in AP and pancreatitis-associated lung injury, aiming to provide therapeutic strategies for the treatment of pancreatitis. Whether the effect of JMJD3 on pancreatitis is specific is a point worthy of attention in subsequent studies.

In summary, these findings indicate that JMJD3 is crucial in the development of AP via the TLR9 and STING pathways, which are activated by mtDNA and oxidized-mtDNA released from acinar cell death. Our study suggested that targeted therapies targeting JMJD3 in monocytes might prove beneficial in AP treatment and might provide insight into the development of novel therapeutic agents for other inflammatory diseases.

All animal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee of Sichuan University (Chengdu, Sichuan, China), and the protocols were approved by the Institutional Animal Care and Use Committee of Sichuan University. Surgical incision tissue from CP patients was provided by West China Hospital of Sichuan University. Written informed consent was received from participants before inclusion in the study. This study was performed in strict accordance with recommendations from the Medical Ethics Committee of the West China Hospital of Sichuan University (2014, No. 37).

The authors have no financial conflicts of interest.