Objective Chronic pancreatitis (CP) is an inflammatory disease with progressive fibrosis leading to exocrine and endocrine dysfunction. Currently, there are no approved effective therapies for CP. Stimulator of interferon genes (STING) signalling is a key innate immune sensor of DNA. In this study, we evaluated the role of STING signalling in CP.
Design We used an experimental model of CP to test the effect of STING signalling in STING wild-type and knockout mice as well as bone marrow chimaeras (BMCs). STING was activated using a pharmacological agent. Since we found changes in Th17 cells, we used neutralising and control antibodies to determine the role of IL-17A. The effect of STING signalling was further explored in IL-17A generation and we examined the effect of IL-17A on pancreatic stellate cells (PSCs). Human pancreas from patients with CP and without CP were also stained for IL-17A.
Results STING activation decreased CP-associated pancreatic inflammation and fibrosis, whereas absence of STING led to worsening of the disease. BMCs showed that leucocytes play an important role in STING signalling–mediated amelioration of experimental CP. STING deletion was associated with increased Th17 cell infiltration in the pancreas, whereas STING agonist limited this Th17 response. Importantly, anti-IL-17A antibody treatment mitigated the severity of CP in the absence of STING signalling. STING deficiency promoted Th17 polarisation and PSCs express functional IL-17 receptor by upregulating fibrosis genes. Compared with tumour margins, pancreas from patients with CP had significant increase in IL-17A+ cells.
Conclusion Unlike acute pancreatitis, STING activation is protective in CP. STING signalling is important in regulating adaptive immune responses by diminishing generation of IL-17A during CP and presents a novel therapeutic target for CP.
- chronic pancreatitis
- experimental pancreatitis
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Significance of this study
What is already known on this subject?
Recurrent acute pancreatitis can lead to chronic pancreatitis, and there are currently no active therapies for chronic pancreatitis.
Stimulator of interferon genes (STING) activation worsens acute pancreatitis, but its role in chronic pancreatitis is not known.
Macrophages have the ability to sense pancreatic acinar cell death and produce proinflammatory cytokines.
What are the new findings?
Unlike in acute pancreatitis, we found that STING signalling is protective in chronic pancreatitis and limits fibrosis.
We found this protection to be associated with alteration in adaptive immunity with a decrease in IL-17A+ cells in the pancreas.
STING deficiency–mediated worsening of chronic pancreatitis could be reversed with IL-17A neutralisation.
STING deficiency leads to augmented Th17 polarisation, whereas STING activation restricts Th17 generation.
Pancreatic stellate cells express functional IL-17 receptor and respond to IL-17A by activating ERK1/2 and upregulating fibrosis genes.
How might it impact on clinical practice in the foreseeable future?
We found that STING signalling is important in regulating adaptive immune responses and limiting inflammation during chronic pancreatitis. IL-17A+ cells are increased in human chronic pancreatitis tissues. Activation of STING using a pharmacological agent reduces experimental chronic pancreatitis, and this provides a novel therapeutic target.
Chronic pancreatitis (CP) is described as progressive severe fibroinflammatory condition with irreversible damage to the pancreas, characterised by acinar cell death, inflammation and fibrosis.1–3 Currently, there are no FDA-approved therapies for CP. Various animal models have been developed to understand the molecular mechanism and identify potential therapeutic targets for the disease.4 Due to its chronicity and ongoing inflammation, exploring innate and adaptive immune signals during CP offers potential means of altering the natural course of the disease with hopes of rendering it from an ‘irreversible’ to a reversible disease.
Stimulator of interferon genes (STING, encoded by TMEM173) signalling can sense abnormal DNA or cyclic dinucleotides in the cytosol of cells, as well as extracellular self DNA caused by apoptosis or necrosis, and bacterial or viral DNA.5–9 STING signalling activation leads to induction of type I interferons (IFNs) and proinflammatory cytokines. Recently, we showed that innate immune cells such as macrophages have the ability to sense DNA from dying acinar cells and STING activation promotes experimental acute pancreatitis.10 Since recurrent acute pancreatitis can lead to CP,11 we sought to understand the role of STING signalling during CP.
Here, we investigated the role of STING signalling in a widely used experimental model of CP. Unlike in acute pancreatitis, we found that cGAS–STING signalling deficiency worsened CP, whereas activation of STING signalling with a pharmacological agent 5,6-dimethyllxanthenone-4-acetic acid (DMXAA) reduced severity of CP. Bone marrow chimaera studies showed that STING signalling in leucocytes plays an important role in the observed protection against CP. Interestingly, absence of STING and its activation with DMXAA were associated with an increase and a decrease in Th17 cells in the pancreas, respectively. Neutralisation of IL-17A in the presence of STING deficiency mitigated severity of CP. Importantly, our ex vivo studies show that STING activation restricts and its absence enhances Th17 polarisation, respectively. Moreover, we show that pancreatic stellate cells (PSCs) have functional IL-17 receptor and respond to IL-17A by activating ERK1/2 and upregulating fibrosis genes. In summary, our findings suggest that STING signalling plays an important role in protecting against CP by modulating Th17 response.
Materials and methods
C57BL/6J mice and C57BL/6J-Tmem173gt/J mice (STING knockout (KO) mice) were purchased from Jackson Laboratories. cGAS KO mice were gifts from Dr Lingyin Li (Stanford University, California, USA). Animal care and use was approved by Stanford University institutional animal care and use committees.
Induction of CP
CP was induced by repeated acute pancreatitis.12 In brief, sex-matched and age-matched mice (6–8 weeks old) received six hourly intraperitoneal injection of 50 µg/kg cerulein (Sigma-Aldrich, St. Louis, Missouri, USA) 3 days/week for a total of 4 weeks. Mice were sacrificed 3 days after the last cerulein injection as described.13 For STING agonist treatment, mice were intraperitoneally injected with vehicle or 10 mg/kg DMXAA (MedChem Express, Monmouth Junction, New Jersey, USA) daily during the last 5 days of cerulein injection.14 For antibody neutralising experiments, mice were treated with either isotype control or anti-mouse IL-17A (anti-IL-17A, 50 µg/mouse/day, three times/week; Bio X Cell, West Lebanon, New Hampshire, USA) antibodies during the last 2 weeks.15 To determine STING expression in leucocyte subsets (macrophages and Th17 cells) over time, mice receiving repeated cerulein or saline control (six hourly injection/day, 3 days/week) were euthanised at week 1, 2 or 3.
Human pancreas tissues
Human pancreas tissues from patients with CP and pancreatic cancer (where normal pancreas tumour margins were used) undergoing surgery were obtained from Stanford tissue bank.
Histology, immunohistochemistry (IHC) and elastase1 assay
Pancreas tissues were immediately taken from mice sacrificed by CO2 inhalation and fixed in 10% formalin. Fixed tissues were sent to Stanford Pathology Laboratory for processing of H&E and trichrome slides. Severity of fibrosis was quantified from trichrome staining and analysed by Image J software (NIH, USA) following protocols as previously described.16 17 IHC assay was performed by Stanford Pathology Laboratory. IL-17A and CD45 (Abcam, Cambridge, UK) antibodies were used for the IHC assay. Tissue blocks from human CP (n=4) and non-CP (healthy tumour margin; n=4) pancreas were stained with IL-17A using IHC. Elastase1 was detected in pancreatic lysates from control and CP mice with pancreatic elastase1 ELISA kit (LSBio, Seattle, Washington, USA).
Naive CD4+ T cells were purified from spleen of wild-type or STING KO mice using mouse CD4+CD62L+ T Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany), then cultured in 1×106 cells/mL RPMI1640 containing 2 mM L-glutamine, 50 mM 2-ME, 100 U/mL penicillin, 100 mg/mL streptomycin, 10% fetal bovine serum (FBS) and 3 mg/mL anti-CD28 (eBioscience, San Diego, California, USA) for 3 days in 96-well plates precoated with 1 µg/mL mouse anti-CD3 (eBioscience), and the polarising cytokines: 5 ng/mL human IL-12 (BioLegend, San Diego, California, USA) and 10 µg/mL anti-mouse IL-4 (BioLegend) for Th1 differentiation; 5 ng/mL TGF-β (BioLegend), 10 ng/mL IL-6 (BioLegend), 10 µg/mL anti-IL-4 (BioLegend), 10 µg/mL anti-IFNγ (BioLegend) and 20 ng/mL IL-23 (BioLegend) for Th17 differentiation.18 To test the effect of STING activation on Th17 polarisation, 50 µg/mL DMXAA was added to the culture on day 1 and the cells were collected for flow cytometry analysis on day 3.19 20
Mice PSCs were obtained from CP mice by outgrowth method and cultured in Dulbecco’s modified Eagle medium/F12 containing 10% FBS.17 To test the effect of IL-17A on PSCs, 100 ng/mL recombinant mouse IL-17A protein (R&D Systems, Minneapolis, Minnesota, USA) was added at indicated time points.21 In addition, in separate experiments, supernatant from Th17 polarised cells in culture was added to the PSCs at indicated time points.
Bone marrow (BM) chimeric mice
BM chimeric mice were prepared as previously described.22 C57BL/6J wild-type (WT) recipient mice were irradiated with a dose of 9.5 Gy. Then each mouse received 5×106 BM cells from donor WT (WT→WT) or STING KO (KO→WT) mice by retro-orbital injection. Eight weeks later, the recipient mice WT→WT or KO→WT were subjected to cerulein-induced CP. In addition, WT and STING KO mice were irradiated with a dose of 9.5 Gy. Then each mouse received 5×106 BM cells from donor WT mice by retro-orbital injection. Eight weeks later, the recipient mice WT→WT or WT→KO were subjected to cerulein-induced CP.
Immunostaining for flow cytometry
Isolation of pancreatic leucocytes was through collagenase digestion of mouse pancreas as previously described.13 Dead cells were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, Santa Clara, California, USA). For surface staining, cells were stained with antibody to the following markers: CD45, CD4, CD8, CD11b, F4/80, CD11c, CD44 and CD45RB (BioLegend). For intracellular staining, cells were stained with IFNγ, IL-4 and foxp3 (BioLegend), IL-17A and RORγt (eBioscience). Intracellular STING staining was as previously described,10 cells were stained with surface markers first, then fixed and permeabilised with kit reagents from eBioscience. Then rabbit unconjugated STING antibody or rabbit IgG isotype control (Invitrogen, Carlsbad, California, USA) was used as primary antibody and AF488 conjugated goat anti-rabbit was used as secondary antibody (Life Technologies, Carlsbad, California, USA).
Quantitative PCR (qPCR)
Mouse pancreas tissues were homogenised in TRIZOL and RNA was extracted as before.10 Then cDNA was reverse-transcribed from RNA with GoScript reverse transcription system (Promega, Madison, Wisconsin, USA). qPCR was performed on ABI-7900 sequence detection system (Applied Biosystems, Foster City, California, USA) with primers and probes as previously described.10 17 Additional primers were as follows: mouse IL-6 forward, 5′-TCGGCAAACCTAGTGCGTTA-3′; mouse IL-6 reverse, 5′-CCAGCTGAGATGCGTCTTTC-3′; mouse MMP2 forward, 5′-CCAGACAGGTGACCTTGACC-3′; mouse MMP2 reverse, 5′-AAACAAGGCTTCATGGGGGC-3′; mouse MMP3 forward, 5′-CATCCCCTGATGTCCTCGTG-3′; mouse MMP3 reverse, 5′-CTTCTTCACGGTTGCAGGGA-3′; mouse β-actin forward, 5′-CGATGCCCTGAGGCTCTTTTCC-3′; mouse β-actin reverse, 5′-CATCCTGTCAGCAATGCCTGGG-3′. The mRNA level was determined by normalising to β-actin and shown as fold change relative to control group.
Mouse pancreas tissues were homogenised in RIPA buffer (Cell Signaling, Danvers, Massachusetts, USA) with protease inhibitor cocktail (Sigma-Aldrich). Protein related to STING signalling was detected with antibodies as described previously.10 ERK1/2, p-ERK1/2 (Cell Signaling), IL-17RA and αSMA (Abcam, Cambridge, UK) and actin (Santa Cruz Biotechnology, Dallas, Texas, USA) antibodies were used for Western blot.
All statistical analyses were determined by Prism software (GraphPad Software, La Jolla, California, USA). The significance between two groups was determined by unpaired Student’s t-test. The differences among multiple groups were evaluated by one-way analysis of variance. A p value <0.05 was considered as statistically significant.
STING activation is protective in CP
Since STING senses DNA from dying acinar cells and promotes acute pancreatitis,10 we hypothesised that STING signalling plays an important role in CP. To test this, we used cerulein-induced CP murine model, dependent on recurrent acute pancreatitis and widely used in the field. We induced CP in WT and STING KO mice. CP was more severe in STING KO as compared with WT mice as shown by pancreas weight, histology, PSC activation and fibrosis-related genes, such as αSMA (αSMA) and Fn1 (fibronectin 1), respectively (figure 1A–C). At the same time, expression of STING downstream genes IFNβ, Mx1 and IRF7 was decreased (figure 1D), indicating that lack of STING signalling worsens CP. Morever, leucocyte infiltration as shown by the pan-leucocyte marker (CD45) IHC staining was also increased in the STING KO group (figure 1E). As STING deficiency worsened CP, we examined STING associated pathways in cerulein-induced CP. STING and upstream sensor cGAS mRNA were increased significantly in pancreas of cerulein-treated mice as compared with control saline-treated mice (figure 1F). In addition, STING protein and downstream STING signalling as shown by p-IRF3 increased significantly (figure 1G). These results suggest that STING signalling is activated in the pancreas and plays a protective role in CP.
In CP, STING+ CD4+ T cells are increased and STING deficiency leads to an increase in Th17 cells in the pancreas
To better understand STING’s role in CP, we first examined STING expression among pancreatic leucocytes. Consistent with figure 1E and F findings, STING expression was increased in leucocytes during CP (figure 2A). Within the STING+ leucocyte (CD45+) population, the frequency of CD4+ T cells increased (figure 2B, C), whereas no changes were observed in CD8+ T cells and macrophage frequencies (data not shown). We then went on to characterise the CD4+ T-cell subsets in CP mice. Compared with WT mice, IL-17A+ T cells (Th17) increased while IFNγ+ (Th1), IL-4+ (Th2) and Foxp3+ (Treg) cells did not increase in the pancreas of STING KO mice during CP (figure 2D, E). Based on Ki67 staining, there was no significant change in IL-17A+ T cell proliferation (figure 2E). We did not see any difference between WT and STING KO mice IL-17A+ T cells in the spleen during CP (data not shown). Moreover, an increase in IL-17A+ cells was observed by IHC staining in the STING KO group (figure 2F). These results indicate that STING deficiency promotes Th17 response in the pancreas during CP. Notably, pancreas from human CP also have significantly increased IL-17A+ cells as compared with non-CP pancreas tissues (online supplementary figure 1).
STING activation reduces CP and Th17 cells in the pancreas
Since the above studies suggested a protective role for STING, we used a STING agonist DMXAA23 24 to activate STING signalling during CP. In contrast to STING deficiency, STING activation with DMXAA reduced CP as shown by pancreas weight, histopathology, fibrosis score and fibrosis-related gene expressions (figure 3A–C). As expected, DMXAA increased STING downstream genes IFNβ, Mx1 and IRF7 expressions (figure 3D). In contrast to the findings with STING KO mice, STING activation led to a decrease in IL1-7A+ T cells in the pancreas of CP mice (figure 3E). These results suggest that STING signalling provides a protection against CP by limiting Th17 response in the pancreas.
cGAS deficiency is associated with worse CP and an increase in Th17 cells in the pancreas
Cyclic GMP-AMP synthase (cGAS) is an upstream DNA sensor of STING. DNA stimulates cGAS to generate the second messenger 2′3′-cGAMP, which then binds and activates STING downstream signalling.25 Thus, to evaluate the role of cGAS–STING signalling in CP, we examined the effect of cGAS deficiency in CP. Consistent with STING KO findings, CP was worse in cGAS KO mice as shown by pancreas weight, histopathology, fibrosis score, pancreatic elastase1 level (reflecting exocrine insufficiency) and fibrosis-related gene expression (figure 4A–C). IL-17A+ but not IFNγ+ CD4+ T cells were increased in the pancreas in cGAS-deficient mice during CP (figure 4D). Taken together, these results show that cGAS–STING pathway is protective in CP and associated with a decrease in Th17 cells in the pancreas.
Leucocyte STING plays an important role in CP
Since STING is expressed in different cells types, we sought to determine the contribution of leucocyte STING in the CP protection observed. To this end, we generated chimeric mice by engrafting irradiated WT mice with either WT (WT-WT) or STING KO (KO-WT) bone marrow (BM) and induced CP (figure 5A). Similar to STING KO mice, WT mice engrafted with STING KO BM (KO-WT) had more severe CP as compared with their WT BM recipients (WT-WT) as shown by histopathology, fibrosis score and fibrosis-related genes (figure 5B, C). Moreover, STING KO BM chimaeras (KO-WT) had a significant increase in IL-17A+ but not IFNγ+ CD4+ T cells in the pancreas (figure 5D, E). In contrast, there was no difference in pancreas pathology or CP severity between WT chimeric mice engrafted with WT BM (WT-WT) and STING KO chimeric mice engrafted with WT BM (WT-KO) (online supplementary figure 2), suggesting that leucocyte but not non-leucocyte STING signalling plays a predominant protective role in CP.
IL-17A neutralisation improves CP
The studies above show that STING deficiency worsens CP and is associated with an increase in IL-17A+CD4+ T cells in the pancreas. To determine the significance of IL-17A, we treated WT and STING KO mice with either isotype or IL-17A neutralising antibody during the last 2 weeks of the 4 weeks of repetitive cerulein administration. In WT mice, there was no statistical difference in histopathology, fibrosis score and fibrosis-related gene expression between mice treated with anti-IL-17A or isotype control antibody (figure 6A–D). Whereas in STING KO mice, IL-17A neutralisation led to significant improvement in CP severity as shown by pancreas weight, histopathology, fibrosis score and gene expression (figure 6A–D). These studies suggest that IL-17A in part mediates the CP severity observed in STING KO.
STING activation inhibits Th17 polarisation and IL-17A promotes fibrosis gene expression
To test whether STING directly impacts IL-17A generation, we set up in vitro T-cell polarisation assay using naive CD4+ T cells isolated from WT and STING KO mice spleens. Like the in vivo findings, STING deficiency promoted Th17 but not Th1 (figure 7A, B) or Th2 (not shown) polarisation. In contrast to STING deficiency, STING activation with DMXAA in WT CD4+ T cells markedly inhibited IL-17A production (figure 7C). These findings suggest that STING signalling has a T-cell intrinsic effect.
Since IL-17A neutralisation in STING KO mice improved fibrosis (figure 6), we examined whether PSCs express IL-17 receptor and respond to IL-17A. As reported for hepatic stellate cells,20 we found that PSCs express IL-17RA and respond to IL-17A by activating downstream ERK/1/2 (figure 7D) and upregulating fibrosis genes (figure 7E), IL-6, and matrix metalloproteinases (online supplementary figure 3A). Morever, PSCs respond to recombinant IL-17A and also to Th17 cell conditioned medium by activating downstream ERK/1/2 (online supplementary figure 3B). Taken together, the above results suggest that in the absence of STING signalling, Th17 polarisation is enhanced and IL-17A promotes PSC fibrosis gene expression.
STING activation or DNA sensing is important for normal host defence against pathogens, but can have deleterious effects when self DNA triggers inflammation in disease states such systemic lupus erythematosus.26 We also recently showed that STING activation worsens acute pancreatitis severity in experimental models via macrophage sensing of DNA released from dying acinar cells.10 In this study, we find that STING signalling during CP alters the adaptive immune response by regulating Th17 generation. Unlike in acute pancreatitis, STING activation plays a protective role in CP by regulating Th17 response.
Our previous study showed that STING signalling is activated in macrophages and promotes TNFα and IFNβ release and worsens acute pancreatitis.10 Here, we found that macrophages are also the main subset of STING+ leucocytes; however, their frequency (STING+ macrophages) among total CD45+ leucocytes were not different between cerulein (CP) and control saline-treated mice (data not shown). In contrast, among pancreas STING+ leucocytes, CD4+ T cells were increased in CP mice. This led us to further examine CD4+ T-cell subsets in the injured pancreas and found that STING signalling deficiency leads to an increase in pancreas Th17 cells and worsens pancreatic fibrosis. In this CP model, as STING+ macrophages start to decrease, STING+ IL-17A+CD4+ T cells start to increase midpoint (or at 2 weeks) of the repeated 3 weeks of cerulein administration, suggesting a transition period in which there is progression towards CP that is associated with rise of STING+ Th17 cells (online supplementary figure 4). Thus, STING signalling regulates different immune cell subsets under different pathological or during acute versus chronic inflammatory conditions.
STING activation inhibits T-cell proliferation in human cells and STING deficiency can promote T-cell proliferation in mice,27 but the effect of STING signalling in T-cell differentiation remains unknown. Here, we found that absence of STING promotes Th17 cell increase in the pancreas during CP, but no changes were observed in Th1, Th2 or Tregs. In vitro T-cell polarisation studies confirmed this finding where absence of STING promoted Th17 polarisation while activation of STING markedly inhibited Th17 cell generation. Thus cGAS–STING signalling can directly modulate Th17 polarisation and the specific molecular mechanism is an interest for future investigation (figure 7F).
Similar to the CP model, we found that IL-17A+ cells were increased in human CP tissues as compared with non-CP control tissues. In our cerulein-induced CP model, anti-IL-17A treatment reduced severity of CP in STING KO group, but did not have as much effect in the STING WT CP group, perhaps due to the higher extent of IL-17A overexpression observed in STING KO mice. In addition, consideration needs to be taken into account that other drivers might also be contributing that are independent of STING signalling. IL-17A was reported to activate hepatic PSCs and promote liver fibrosis.21 28 Our findings parallel these findings, and to our knowledge there are no reports whether PSCs express IL-17RA. Here, we found that PSCs express functional IL-17 receptor, and IL-17A induces PSCs to upregulate fibrosis gene expression, indicating that IL-17A may promote fibrosis in different chronic diseases. It is therefore conceivable to propose blockade of this pathway to limit and/or slow down fibrosis progression in different organs.
IL-17A overexpression in the pancreas promotes PanIN initiation and progression, while IL-17A deletion in haematopoietic cells delays this progression.29 Thus, the link between STING signalling and IL-17A we made in this study might also be relevant in pancreas cancer. In fact, STING signalling is reported to play an important role in antitumour response,19 30–32 and STING agonist treatments inhibit tumour progression in various models.33 These studies used various approaches to activate STING and enhance efficiency of STING agonist, which included nanosatellite vaccine and cationic silica nanoparticles.34 35 DMXAA can bind mouse STING,36 and functionally activate STING leading to downstream TBK1 and IRF3 signalling and induce type I IFNs.37 38 In our study, we used this widely available and well-studied STING agonist DMXAA and show that DMXAA is effective in treating established experimental CP. Thus, STING agonist developed for clinical use are promising therapeutic targets for CP. Taken together, our study reveal that cGAS–STING signalling is protective in CP via regulating Th17 response, and we propose this pathway as a novel target for the treatment of CP.
We thank Jing Guo for providing input and sharing T-cell polarisation experimental protocol.
Patient consent for publication Obtained.
QZ and MM contributed equally.
Contributors QZ, MM and AH designed the experiment, analysed the data and wrote the manuscript. QZ, MM and YW performed the research. SJP provided key input. AH provided overall guide and supervision.
Funding This work was supported by the National Institute of Health (NIH) grants DK092421 and DK105264 (to AH), P01 DK098108 and R01 AA024464 (to SJP).
Competing interests None declared.
Ethics approval Local ethics committee.
Provenance and peer review Not commissioned; externally peer reviewed.
Correction notice This article has been corrected since it published Online First. The equal contribution statement has been added.
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