Statin prevents cancer development in chronic inflammation by blocking interleukin 33 expression

Chronic inflammation is a major cause of cancer worldwide. Interleukin 33 (IL-33) is a critical initiator of cancer-prone chronic inflammation; however, its induction mechanism by the environmental causes of chronic inflammation is unknown. Herein, we demonstrate that Toll-like receptor (TLR)3/4-TBK1-IRF3 pathway activation links environmental insults to IL-33 induction in the skin and pancreas. FDA-approved drug library screen identified pitavastatin as an effective IL-33 inhibitor by blocking TBK1 membrane recruitment/activation through the mevalonate pathway inhibition. Accordingly, pitavastatin prevented chronic pancreatitis and its cancer sequela in an IL-33-dependent manner. IRF3-IL-33 axis was highly active in chronic pancreatitis and its associated pancreatic cancer in humans. Interestingly, pitavastatin use correlated with a significantly reduced risk of chronic pancreatitis and pancreatic cancer in patients. Our findings demonstrate that blocking the TBK1-IRF3 signaling pathway suppresses IL-33 expression and cancer-prone chronic inflammation. Statins present a safe and effective therapeutic strategy to prevent chronic inflammation and its cancer sequela.


Introduction
Chronic in ammation accounts for 20% of cancers worldwide [1][2][3] . Cancer-prone chronic in ammation, such as pancreatitis, in ammatory bowel disease (IBD), and hepatitis, have risen in recent decades, highlighting the urgent need for improved cancer prevention strategies in at-risk populations [4][5][6] . Several immune cells and factors, including M2 macrophages, mast cells, TGF-β, interleukin (IL)-10, and IL-13, have been identi ed to promote carcinogenesis in chronic in ammation [7][8][9][10][11] . However, inhibiting these effectors alone or combined to prevent cancer has proven challenging due to their redundant function in cancer promotion [12][13][14] . Furthermore, anti-in ammatory medications, including dexamethasone, which can reduce the risk of cancer development by broadly suppressing immune responses 15 , have severe side effects, including coagulopathy and immune hyper-activation, limiting their use as cancer-preventive agents 16,17 . To overcome these challenges, it is essential to develop safe agents that can block the development of chronic in ammation and thereby prevent its cancer sequela.
IL-33 is an epithelium-derived alarmin cytokine that is a member of the IL-1 cytokine family and drives type 2 immune responses in allergic in ammation by triggering T helper 2 (Th2) cell and type 2 innate lymphoid cell (ILC2) activation 18,19 . IL-33 is a critical initiator of chronic in ammation 20,21 . By binding to its receptor, suppressor of tumorigenesis 2 (ST2, also known as interleukin-1 receptor-like 1 (IL1RL1)), IL-33 promotes the development of a chronic in ammatory environment in damaged tissues 19 . IL-33 and ST2 are highly expressed in chronic in ammatory diseases, including colitis, pancreatitis, and chronic obstructive pulmonary disease [20][21][22][23][24] . IL-33 plays a complex role in cancer development. IL-33 induction suppresses colon tumor growth and activates CD8 + T and natural killer (NK) cells to inhibit lung metastasis in mice 25,26 . In contrast, the upregulation of IL-33 during the transition from acute to chronic in ammation initiates the development of a tumor-promoting immune environment 20,[27][28][29] . Importantly, IL-33 also acts as a nuclear protein and promotes tumorigenesis by regulating SMAD signaling in chronic in ammation 21 . Thus, blocking IL-33 expression instead of its cytokine function alone is essential to achieve cancer prevention in chronic in ammation.
However, the mechanism of IL-33 induction in epithelial cells during the development of chronic in ammation remains unknown.
Herein, we identify TLR3/4-TBK1-IRF3 signaling as the key regulator of IL-33 expression and discover statin as a novel IL-33 inhibitor by regulating TBK1 signaling in cancer-prone chronic in ammation.
TLR3/4-TBK1-IRF3 signaling was highly activated in the skin and pancreas chronic in ammation, and knockdown of IRF3 blocked Il33 expression in vitro and in vivo. Pitavastatin, an IL-33 inhibitor identi ed from the FDA-approved drug library screening, reduced membrane-bound phosphorylated TBK1 (p-TBK1) through mevalonate pathway inhibition, which resulted in the suppression of p-IRF3 and IL-33 expression.
Accordingly, pitavastatin reduced the risk of pancreatitis and PDAC in mice and humans. We conclude that blocking the TBK1-IRF3-IL-33 axis by statin represents a novel and actionable strategy to prevent chronic in ammation and its cancer sequela.

Results
Chronic in ammatory insults in the skin and pancreas activate the TLR3/4-TBK1-IRF3 signaling pathway To determine the mechanism of IL-33 induction in chronic in ammation, we subjected wild-type (WT) mice to established models of chronic in ammation in the skin and pancreas. To induce chronic dermatitis, WT mice received topical 2,4-Dinitro-1-uorobenzene (DNFB, a contact allergen) in acetone or acetone alone (control) on the back skin three times a week for 22 days 20,21 . Epidermal thickness and mast cell numbers were signi cantly increased in DNFB-treated skin (Extended Data Fig. 1a, b). To induce chronic pancreatitis, mice received intraperitoneal caerulein injections in phosphate-buffered saline (PBS) or PBS alone hourly for 6 hours per day, three days per week for three weeks 43,44 . Caerulein treatment led to in ammation and brosis in the pancreas, which was associated with a signi cant CD45 + leukocyte in ltration into the pancreas (Extended Data Fig. 1c, d). Consistent with previous reports 22,23 , IL-33 was highly expressed in the epithelial cells of DNFB-treated skin and caerulein-treated pancreas compared with acetone-treated skin and PBS-treated pancreas, respectively (Fig. 1a). IL-33 RNA and protein levels were signi cantly increased in the in amed compared with control tissues (Extended Data Fig. 1e-h). To identify the signaling pathway that induced IL-33 in chronic in ammation, we performed RNA sequencing on the epidermal keratinocytes isolated from the back skin of WT mice treated with DNFB versus acetone and the pancreas of WT mice treated with caerulein versus PBS. Among differentially expressed genes, nine common genes were increased in DNFB-treated skin and caerulein-treated pancreas (Extended Data Fig. 1i, j). Among them, S100a8 and S100a9, well-known DAMPs and TLR 3/4 ligands 45,46 , were highly enriched in chronic in ammation. Consistently, Gene Set Enrichment Analysis (GSEA) revealed the activation of the TLR3/4 signaling pathway in chronic pancreatitis (Fig. 1b, c). Thus, TLR3/4 signaling may induce IL-33 expression in chronic in ammation.
IRF3 is required for the induction of IL-33 and chronic in ammation in the skin and pancreas To determine whether Il33 expression is mediated by TBK1-IRF3 signaling in chronic in ammation, we subjected Irf3 knockout (Irf3 KO ) mice to chronic in ammatory conditions in the skin and pancreas. IL-33 RNA and protein levels were signi cantly reduced in DNFB-treated Irf3 KO compared with WT skin (Fig. 1k, l). Moreover, epidermal thickness and mast cell numbers were decreased markedly in DNFB-treated Irf3 KO compared with WT skin (Fig. 1m, n and Extended Data Fig. 3a, b). Consistent with these results, IL-33 levels were reduced in DNFB-treated skin of Trif and Myd88 double knockout (Trif,Myd88 DKO ) mice but not in Myd88 KO mice (Extended Data Fig. 3c), which indicates that TRIF adapter protein is the primary upstream activator of the IRF3 signaling pathway to induce IL-33 in chronic in ammation. IL-33 RNA and protein levels decreased in caerulein-treated Irf3 KO compared with WT pancreas (Fig. 1o, p). Likewise, caerulein-treated Irf3 KO pancreas showed less in ammation and reduced CD45 + leukocyte in ltration compared with WT pancreas (Fig. 1q and Extended Data Fig. 3d). Collectively, these ndings demonstrate that TBK1-IRF3 regulates IL-33 expression in chronic dermatitis and pancreatitis.
Pitavastatin blocks TBK1 phosphorylation and IL-33 expression via mevalonate pathway inhibition To identify a small molecule IL-33 inhibitor that can be safely used to alleviate chronic in ammation and its cancer sequela, we screened an FDA-approved drug library in a luciferase-based Il33 expression assay (Extended Data Fig. 4a, b). Among 1018 FDA-approved small molecules that were screened, we found ve candidates, which decreased Il33/control luminescence absorbance to less than 40% while having no effect on absorbance in a control luminescence assay (Extended Data Fig. 4c). Among these candidates, pitavastatin calcium (labeled as O16 in the screen) suppressed poly(I:C)-induced Il33 and endogenous Il33 levels in Pam212 and PyMt tg breast cancer cell line, respectively (Extended Data Fig. 4d, e). Pitavastatin is a lipophilic statin that inhibits β-Hydroxy β-methylglutaryl-CoA (HMG-CoA) reductase, an intermediate reaction in the mevalonate pathway 48 . Pitavastatin and zoledronic acid, another mevalonate pathway inhibitor, equally suppressed poly(I:C)-induced Il33 expression, while a TBK1 inhibitor, BX795, completely blocked poly(I:C)-induced Il33 expression in Pam212 cells (Fig. 2a). Interestingly, lipophilic statins, pitavastatin and atorvastatin, inhibited Il33 expression more potently compared with a hydrophilic statin, rosuvastatin (Extended Data Fig. 4f). Statins inhibit HMG-CoA reductase, which leads to the reduction in geranylgeranyl diphosphate (GGPP), a product of the mevalonate pathway 48,49 . GGPP plays a critical role in the membrane localization of intracellular proteins 50 . Mevalonate pathway inhibition by pitavastatin blocked poly(I:C)-induced activation of the TBK1-IRF3 signaling pathway in Pam212 cells ( Fig. 2b). Importantly, poly(I:C) treatment led to the recruitment of TBK1 to the membrane for phosphorylation (i.e., activation), and pitavastatin markedly reduced membrane-bound p-TBK1 (Fig. 2c). The addition of exogenous GGPP to Pam212 cells reversed pitavastatin effect and restored TBK1-IRF3 signaling pathway activation and membrane-bound p-TBK1 levels (Fig. 2b, c). Accordingly, pitavastatin suppression of poly(I:C)-induced Il33 expression was reversed by exogenous GGPP (Fig. 2d). Thus, pitavastatin inhibits Il33 expression by blocking GGPP-dependent membrane recruitment and activation of TBK1 (Fig. 2e).
Pitavastatin suppresses chronic in ammation and its cancer sequela in an IL-33-dependent manner Next, we investigated the impact of pitavastatin treatment on suppressing IL-33 and chronic in ammation in vivo. To test the pitavastatin effect on skin in ammation, mice were treated with topical DNFB on the back skin for 22 days together with topical pitavastatin versus carrier control (acetone).
Pitavastatin treatment dramatically blocked p-TBK1 and p-IRF3 levels compared to acetone-treated mice (Extended Data Fig. 5a). Accordingly, IL-33 RNA and protein levels were markedly decreased in pitavastatin-compared with acetone-treated mice (Extended Data Fig. 5b, c). Skin in ammation, as marked by epidermal thickness and mast cell numbers in the skin, was signi cantly reduced in pitavastatin-compared to acetone-treated mice (Extended Data Fig. 5d-f). To examine the effect of pitavastatin on chronic pancreatitis, mice were treated with caerulein for three weeks together with intraperitoneal pitavastatin versus carrier control (PBS). Pitavastatin treatment signi cantly reduced p-TBK1 and p-IRF3 levels in the pancreas with no effect on NF-kB signaling (Fig. 3a). Likewise, pitavastatin signi cantly reduced IL-33 RNA and protein levels in the caerulein-treated pancreas (Fig. 3b, c). Moreover, pitavastatin treatment preserved the normal architecture of the caerulein-treated pancreas and reduced CD45 + leukocyte in ltration into the pancreas compared with PBS-treated mice ( Fig. 3d and Extended Data Fig. 6a). Importantly, pitavastatin had no signi cant impact on the severity of pancreatitis in Il33 KO mice (Extended Data Fig. 6b, c). Thus, pitavastatin prevents chronic in ammation by suppressing the TBK1-IRF3-IL-33 signaling axis in vivo.
Chronic pancreatitis is a risk factor for the development of pancreatic cancer 51,52 . To establish a chronic pancreatitis-associated pancreatic cancer model in mice, we treated pancreas-speci c Kras and Tp53 mutant (Kras LSL-G12D , Tp53 ox/+ , p48-Cre tg or KPC) mice with hourly intraperitoneal injections of caerulein for 7 hours per day for two consecutive days (Extended Data Fig. 6d) 53,54 . This pancreatic carcinogenesis protocol led to a signi cant induction of IL-33 expression in the pancreas (Extended Data Fig. 6e). KPC mice were treated with pitavastatin versus PBS control after the caerulein injection protocol. Pitavastatin treatment signi cantly reduced pancreatic tumor weight per body weight ratio compared with PBS-treated KPC mice (Fig. 3e, f). Moreover, pitavastatin treatment blocked the progression of pancreatic tumors and retained the tumor cells in a pre-cancerous stage with high mucin production compared with PBS-treated tumors (Fig. 3e, g). In contrast, there was no signi cant difference in pancreatic tumor per body weight ratio or mucin production by tumor cells in Il33 KO KPC mice treated with pitavastatin versus PBS control (Extended Data Fig. 6f-h). These ndings demonstrate that pitavastatin blocks chronic pancreatitis-associated pancreatic cancer in an IL-33-dependent manner.

IRF3-IL-33 axis is highly active in chronic pancreatitis and pancreatic cancer in humans
To extend our ndings to cancer-prone chronic in ammation in humans, we examined IL-33 and IRF3 expression in the epithelial cells across 15 matched samples of the normal pancreas, pancreatitis, and pancreatitis-associated pancreatic ductal adenocarcinoma (PDAC). IL-33 and IRF3 were highly expressed in the nucleus of epithelial cells in pancreatitis and pancreatitis-associated PDAC samples (Fig. 4a-c).
Moreover, the number of IL-33 + epithelial cells was positively correlated with the number of IRF3 + epithelial cells across the samples (Fig. 4d). Expression of IL33 and other IRF3 target genes, TNF, IL1B, and CXCL10, were highly upregulated in pancreatic cancer compared to the normal pancreas in a large collection of samples represented in TCGA and GTEx databases ( Fig. 4e and Extended Data Fig. 7).
Pitavastatin treatment is associated with reduced risk of chronic pancreatitis and pancreatic cancer in patients Finally, we investigated the effect of pitavastatin on the risk of chronic pancreatitis and pancreatic cancer in humans using an epidemiologic approach. We compared matched cohorts of patients from the TriNetX Diamond Network, a global health network containing electronic medical record-derived data from more than 200 million patients across 92 healthcare organizations in North America and Europe (Supplementary Table S1) 55 . The risk of chronic pancreatitis was signi cantly decreased in patients treated with pitavastatin compared to those treated with ezetimibe, another cholesterol-lowering agent commonly used in the clinic, which does not affect the mevalonate pathway (control, OR 0.81; 95% CI (0.729-0.9); P<0.0001). Furthermore, the risk of pancreatic cancer was markedly decreased in the pitavastatin-treated group compared with the ezetimibe-treated control (OR 0.835; 95% CI (0.748-0.932); P=0.0013) (Fig. 4f). Collectively, these outcomes indicate that blocking TBK1-IRF3-IL-33 signaling axis by statins may prevent chronic in ammation and its cancer sequela in high-risk patients.

Discussion
Our ndings reveal that lipophilic statins suppress cancer-prone chronic in ammation by blocking the TBK1-IRF3-IL-33 signaling axis induced by chronic exposure to environmental insults. Cellular damage and release of DAMPs lead to TLR3/4-mediated activation of the TBK1-IRF3 signaling pathway. Phosphorylated IRF3 directly binds to the Il33 promoter to drive IL-33 expression during the initiation of chronic in ammation. Importantly, pitavastatin blocks Il33 expression by inhibiting the mevalonate pathway-mediated TBK1 binding to the membrane, which is required for its phosphorylation and downstream IRF3 activation. By inhibiting Il33 expression, pitavastatin blocks the cytokine and nuclear functions of IL-33 in chronic in ammation, effectively reducing the risk of chronic pancreatitis and pancreatic cancer in mice and humans. Therefore, blocking the TBK1-IRF3-IL-33 signaling axis with statins represents a safe, effective, and readily accessible strategy to prevent chronic in ammation and its cancer sequela, which can impact many individuals at high risk of developing cancer-prone chronic in ammation.
Several malignancies are associated with activated TBK1-IRF3 signaling pathway within the cancer cells, which can play a critical cell-autonomous role in cancer progression 56,57 . In particular, TBK1 activation has been linked to skin and pancreatic cancer development 58,59 . Furthermore, high TBK1 expression has been shown to induce an immunosuppressive tumor microenvironment by increasing PD-L1 expression and inhibiting CD8 + T cell in ltration in lung and liver cancer 60,61 . Accordingly, TBK1 inhibitors have tumor inhibitory effects associated with improved sensitivity to immunotherapy in several cancer types, including melanoma and liver cancer 59,60,62 . Moreover, activated TBK1-IRF3 signaling leads to the induction of angiogenesis factors, which is associated with poor prognosis in several cancers, including pancreatic cancer 57,63-65 . Our ndings demonstrate that IL-33 is a target of TBK1-IRF3 signaling in cancer-prone tissues. Likewise, IL-33's pro-tumor function can play an integral role in tumor promotion by TBK1-IRF3 signaling 27-29 . Thus, targeting the TBK1-IRF3-IL-33 signaling axis is an attractive strategy to block cancer development.
The mevalonate pathway has recently emerged as an important regulator of cancer development. Blocking the mevalonate pathway product, GGPP, inhibits cancer cells' amino acid uptake by regulating macropinocytosis 66 . Moreover, the inhibition of the mevalonate pathway by statins induces a strong antitumor T cell immunity by promoting antigen presentation 50 . Importantly, our ndings reveal a previously unknown mechanism by which the mevalonate pathway regulates TBK1-IRF3 signaling through its essential role in the membrane recruitment of TBK1, which is required for its phosphorylation/activation. Our ndings may also provide a novel explanation for how GGPP regulates PI3K/MAPK pathway activation by promoting the membrane localization of the critical signaling molecules in the pathway 67,68 . Thus, the elucidation of statins' mechanism of action in blocking the TBK1-IRF3-IL-33 signaling axis has far-reaching implications in revealing a fundamental aspect of TBK1-IRF3 and other major intracellular signaling pathways.
Our work uncovers an unprecedented role for statins as a novel class of chemopreventive agents for suppressing chronic in ammation and its cancer sequela. Statins are commonly used for long-term control of hyperlipidemia, and 56 million adults are taking statins in the United States alone 69 . Statins are well-tolerated over many years of treatment with minimal side effects 70 , which signi es their high potential as effective agents for cancer prevention. Unlike TBK1 inhibitors that are in development with a high cost and potential side effects, statins are affordable FDA-approved medications that can be safely prescribed for long-term use, which are essential requirements for an ideal chemopreventive agent. Furthermore, our ndings highlight the topical application of statins as a novel treatment strategy for cancer-prone chronic in ammation in the skin. Likewise, statins may present a breakthrough for pancreatic cancer prevention. Pancreatic cancer is an insidious cancer type known for its unresponsiveness to current treatments, including immunotherapies, due to its highly immunosuppressive tumor microenvironment with dense desmoplastic stroma 71,72 . Although statins' impact on an established tumor microenvironment is context-dependent 73-77 , our ndings strongly indicate that statin use can block the development of cancer-prone chronic in ammation and the formation of an immunosuppressive tumor microenvironment in high-risk patients. Importantly, statin use is associated with an increased survival rate in pancreatic cancer patients 78,79 . Moreover, we demonstrate that pitavastatin markedly reduces the risk of pancreatic cancer development compared with ezetimibe in a large population study. Finally, the bene cial effect of statins in blocking the TBK1-IRF3-IL-33 axis may also extend to other IL-33-dependent chronic in ammatory conditions, including chronic obstructive pulmonary disease (COPD), atopic dermatitis, and asthma [80][81][82] .

Human samples
De-identi ed formalin-xed para n-embedded human pancreas tissue sections were obtained from the Department of Pathology at Massachusetts General Hospital.

Skin chronic in ammation
Four-to six-week-old male and female mice were shaved on their abdomen and sensitized to 50 μL 0.5% 1-Fluoro-2,4-dinitrobenzene (DNFB, Millipore Sigma, St. Louis, MO, catalog no. 42085) dissolved in acetone with olive oil at 3:1 ratio (refer to as acetone). Two days after the rst sensitization, mice were sensitized to 50 μL 0.25% DNFB on their abdomen again. After ve days, mice were challenged with 100 μL 0.25% DNFB on their back skin, which was repeated three times per week for 22 days. Skin rash was monitored over the duration of the study.

Chronic pancreatitis
Mice were weighed and injected with 50 μg/kg caerulein (BACHEM, Torrance, CA, catalog no. 4030451) in 100 μL of PBS intraperitoneally every hour for 6 hours, three days per week for three weeks. Mice were harvested for analysis at the completion of the three-week treatment protocol.

Caerulein-mediated pancreatic cancer
Mice were weighed and injected with 50 μg/kg caerulein in 100 μL of PBS intraperitoneally every hour for 7 hours, for two consecutive days. Mice were harvested 30 days after the last injection.

Pitavastatin treatment
For chronic in ammation in the skin, mice were treated topically with 0.25 mM pitavastatin (Selleck Chemicals LLC, Houston, TX, catalog no. S1759) in 200 μL acetone or 200 μL acetone alone on their back skin twice a week. Pitavastatin treatments were given at the time of DNFB applications. For chronic pancreatitis and caerulein-mediated pancreatic cancer, mice were treated intraperitoneally with 2 mg/kg pitavastatin in 100 uL PBS or PBS alone. Pitavastatin was given once every three days until harvest.  -190). Then, the samples were incubated with 3% bovine serum albumin (Thermo Fisher Scienti c, catalog no. BP1600) or 5% Skim milk (BD biosciences, San Jose, CA, catalog no. 232100) in 1X Tris-Buffered Saline (Boston Bioproducts, catalog no. BM301X) containing 0.1% TWEEN, called TBS-T for 30 min. After washing with TBS-T three times, the membranes were subjected to immunoblot with proper antibodies overnight at 4 °C. The following day, the membranes were incubated with appropriate secondary antibodies after washing. Membranes were developed with Pierce ECL Western blotting substrate kit (Thermo Fisher Scienti c, catalog no. 32106). First and secondary antibodies are listed in Supplementary Table S2.
Histology, immunohistochemistry, and immuno uorescence Tissue samples were collected and xed in 4% paraformaldehyde (Millipore Sigma, catalog no. P6148) overnight at 4°C. Next, tissues were dehydrated in PBS and ethanol, processed, and embedded in para n. Five to seven μm sections of para n-embedded tissues were placed on slides, depara nized, and stained with H&E, toluidine blue (for mast cell) (Millipore Sigma, catalog no. T3260), or Alcian blue (for mucin) (VECTOR Laboratories, Burlingame, CA, catalog no. H3501). For immunohistochemistry, antigen retrieval was performed in 500 μL of antigen unmasking solution (VECTOR Laboratories, catalog no. H3300) diluted in 50 mL distilled water using a Cuisinart pressure cooker for 20 min at high pressure. Slides were washed three times for three minutes each in 1X TBS with 0.025% Triton X-100. Sections were blocked with 3% bovine serum albumin (Thermo Fisher Scienti c, catalog no. BP1600) and 5% goat serum (Millipore Sigma, catalog no. G9023) for 1 hour. Slides were incubated overnight at 4°C with a primary antibody diluted in TBS containing 3% BSA (Supplementary Table 2). The following day, slides were washed as above and incubated in 100 μL biotinylated secondary antibody (VECTOR Laboratories, catalog no. PK-6200) for 30 min. After washing, slides were stained with a 100 μL mixture of reagents A and B from VECTASTAIN Elite ABC universal kit Peroxidase (VECTOR Laboratories, catalog no. PK-6200) for 30 min. After washing again, slides were incubated with 100 μL ImmPACT DAB chromogen staining (VECTOR Laboratories, catalog no. SK-4105) for a few minutes (depending on the signal). Finally, slides were dehydrated in ethanol and xylene and mounted with a coverslip using three drops of mounting media. For immuno uorescence staining, rehydrated tissue sections were permeated with 1X PBS supplemented with 0.2% Triton X-100 for 5 min. Antigen retrieval was performed similarly to immunohistochemistry. Slides were washed three times for 3 min each in 1X PBS with 0.1% Tween-20. Sections were blocked with 3% bovine serum albumin and 5% goat serum for 1 hour. The slides were incubated overnight at 4°C with primary antibodies. The following day, slides were washed as above and stained for 2 hours at room temperature with secondary antibodies conjugated to uorochromes. Next, slides were incubated with 1:2,000 DAPI (Thermo Fisher Scienti c, catalog no. D3571) for 3 min at room temperature, then washed as above. Slides were mounted with Prolong Gold Antifade Reagent (Thermo Fisher Scienti c, catalog no. P36930). The number of positive cells was counted in randomly selected high-power eld (HPF, 200x magni cation) images in a blinded manner by a trained investigator. A pathologist reviewed clinical samples.

Quantitative PCR
Mouse dorsal skin and pancreas samples were homogenized and lysed in RLT lysis solution (QIAGEN, Hilden, Germany, catalog no. 79216)/0.1% MeOH using Mini-BeadBeater-8 (BioSpec Products, Inc., Bartlesville, OK). Trizol reagent was added to tissue samples and cell pellets to extract RNA (Thermo Fisher Scienti c, catalog no. 15-596-018). Total RNA was extracted using an RNeasy micro kit and quanti ed using NanoDrop ND-1100 (NanoDrop Technologies, Wilmington, DE). cDNA was synthesized from 1 mg of total RNA using Invitrogen SuperScripts III Reverse Transcriptase (Thermo Fisher Scienti c, catalog no. 18080085). The gene expression levels from cDNA samples were determined using QuantStudio 3 system (Thermo Fisher Scienti c) using SYBR Select Master Mix (Thermo Fisher Scienti c, catalog no. 4472908) or TaqMan Universal Master Mix II (Thermo Fisher Scienti c, catalog no. 44-400-40). Primer sequences for SYBR green and Taqman assays are listed in Supplementary Table S2. Quantitative real-time PCR for SYBR green analyses was performed in a nal reaction volume of 20 mL consisting of 5 mL of cDNA of the respective sample and 10 mL of SYBR green master mix mixed with the corresponding primers (2 mM) for each gene. TaqMan analysis was performed in a 10 mL nal reaction, including 4.5 mL cDNA and 5.5 mL TaqMan master mix and corresponding primers (20 mM). All assays were conducted in triplicate and normalized to Gapdh expression.

RNA-Seq
WT mice underwent DNFB-induced skin in ammation and caerulein-induced pancreatitis protocols. For skin RNA-seq, the epidermis was isolated from the mice's back skin. Epidermis and pancreas tissues were lysed in RLT buffer (Qiagen, catalog no. 79216) supplemented with 1% β-mercaptoethanol (Thermo Fisher Scienti c, 21-985-023). Libraries were generated and sequenced using the Smart-Seq2 protocol as previously described using Novaseq 6000 (Illumina) on the Broad Genomics Platform 83 . The raw les were mapped to the mouse genome/mm10 by STAR-2.5.3 84 . Aligned transcripts were quanti ed using RSEM-1.3.1 85 . Differentially expressed genes (DEG) were analyzed by DESEq2 86 . Original data are available in the NCBI Gene Expression Omnibus (GEO) with accession number GSE207956 (RNA-Seq).

ChIP-qPCR assay
Pam212 cells that had been transfected with poly(I:C) were xed in 1% formaldehyde (Millipore Sigma, catalog no. F8775) for 10 min and were washed with cold PBS. Cells were lysed with buffer contained with 2.5% of glycerol, 50 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, and 0.25% Triton X-100. After centrifugation at 2,000 rpm, lysates were resuspended in buffer A contained with 1 mM Tris/HCl (pH 7.9), 20 mM NaCl and 0.5 mM EDTA and incubated at room temperature for 10 min.
Following the second centrifugation at 2,000 rpm, cells were sonicated in a sonication buffer containing 10 mM HEPES, 1 mM EDTA, and 0.5% SDS for 30 minutes to achieve chromatin fragmentation.

Epidemiological analysis
A retrospective cohort analysis was performed using de-identi ed data from the TriNetX Diamond Network. A search query was used to identify the cohort of patients within the network who had received pitavastatin. Eligible patients were identi ed based on the presence of corresponding RxNorm concept unique identi ers (RXCUI) in the patients' electronic medical records. Using International Classi cation of Diseases Tenth Revision (ICD-10) codes, all patients with a history of chronic pancreatitis and pancreatic cancer prior to statin initiation were excluded from the cohorts to reduce confounding. The control cohort for each analysis included all patients within the network who had received ezetimibe but had no recorded statin use and patients with a history of any of the diagnoses mentioned above before ezetimibe initiation were also excluded.
The index event for all analyses was the initiation of pitavastatin for study cohorts and the initiation of ezetimibe for the control cohort. Cases and controls were matched using 1:1 propensity score-matching for age at index event, sex, race, and ethnicity using "greedy nearest neighbor matching" and a caliper of 0.1 pooled standard deviations. Baseline characteristics were reported by count and percentage of the total for categorical variables and means and standard deviations (SD) for continuous variables. Relative risks are presented with 95% con dence intervals. P-values are uncorrected and based on Z-tests or Fisher's exact tests. Statistical analyses were performed in real-time using the TriNetX platform.

Statistical analysis
A paired t-test was used for comparing IL-33 + and IRF3 + cell counts, and a paired t-test for the Pearson correlation coe cient was used for correlation between IL-33 + and IRF3 + counts across matched human pancreatic samples. Statistical differences between the three groups were analyzed using one-way ANOVA. Tukey multiple comparison tests were used to examine the differences in the mean ranks among all three possible pairwise comparisons. Unpaired t-test was used as the test of signi cance for tumor per body weight ratio, epidermal thickness, mast cell and leukocyte counts, RNA and protein expression levels, and other quantitative measurements. P-value < 0.05 is considered signi cant. Bar graphs show mean + SD.   o, Il33 expression levels in caerulein-treated WT versus Irf3 KO pancreas (n=6 in each group). p, IL-33 protein levels in caerulein-treated WT versus Irf3 KO pancreas (n=6 in WT and n=7 in Irf3 KO group). q, CD45 + immune cell counts in caerulein-treated WT versus Irf3 KO pancreas. Each dot represents cell counts from an HPF image. Three randomly selected HPF images are included per sample (n=3 in each group).   Graphs show mean + SD, scale bar: 100 μm.
IRF3-IL-33 signaling axis is highly active in human chronic pancreatitis-associated pancreatic cancer, and pitavastatin reduces the risk of chronic pancreatitis and pancreatic cancer in patients. a, Representative images of IL-33 and IRF3 immunostaining on adjacent sections of matched normal pancreas, chronic pancreatitis, and PDAC collected from pancreatic cancer patients. b, IL-33 + epithelial cell counts per HPF in the matched samples from human pancreatic tissues. Each dot represents the average cell counts across three randomly selected HPF images per sample (n=15 patients, paired t-test). c, IRF3 + epithelial cell counts per HPF in the matched samples from human pancreatic tissues. Each dot represents the average cell counts across three randomly selected HPF images per sample (n=15 patients, paired t-test). d, The correlation between IL-33 + and IRF3 + cell counts across normal pancreas, chronic pancreatitis, and PDAC samples (n=45 matched samples from 15 patients, t-test for the Pearson correlation coe cient). e, Box plot of IL33 expression in pancreatic cancer versus normal pancreas across TCGA/GTEx datasets (* P < 0.01, one-way ANOVA, Gene Expression Pro ling Interactive Analysis database).
f, A retrospective cohort analysis of chronic pancreatitis and pancreatic cancer risk in matched cohorts of patients treated with pitavastatin (test) versus ezetimibe (control).

Supplementary Files
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