Peroxiredoxin-I Maintains Inflammation During Pancreatitis

Acute pancreatitis is a transient and local inflammation of the pancreas characterized by immune cell infiltration, fibrosis, and edema.1Habtezion A. et al.Gastroenterology. 2019; 156: 1941-1950Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar It mainly affects acinar cells, causing acinar metaplasia, and thereby constitutes a favorable environment for the development of pancreatic cancer in human beings and mouse models.2Guerra C. et al.Cancer Cell. 2007; 11: 291-302Abstract Full Text Full Text PDF PubMed Scopus (821) Google Scholar Despite the significant involvement of redox-dependent mechanisms in pancreatitis (eg, mitogen-activated protein kinase signaling, autophagy, disulfide stress, calcium signaling), supplementation with generic antioxidants is therapeutically unsuccessful,3Perez S. et al.Redox Biol. 2015; 5: 1-14Crossref PubMed Scopus (62) Google Scholar highlighting the need to identify specific targets amenable to pharmacologic therapy. To identify redox targets relevant to pancreatitis, we first compared the transcriptional landscape of Fluorescence-activated cell sorting (FACS)-sorted acinar cells from control and cerulein-treated mice (cerulein is a pancreatitis-inducing compound). We identified an increased expression of activators of the peroxiredoxin pathway such as peroxiredoxin-1 (Prdx1), sulfiredoxin (Srxn1), and thioredoxin (Txn1) (Supplementary Figure 1A). Among the typical 2-cystein family members, mouse and human peroxiredoxin-1 protein (PRX-I), -II, -III, and -IV, only the expression of PRX-I was selectively induced in metaplastic acinar cells, at advanced stages of acute pancreatitis (Figure 1A and Supplementary Figure 1B-G). Accordingly, in primary human acinar cells cultured under conditions that mimic pancreatitis-induced metaplasia, we found substantially higher levels of PRX-I in metaplastic cells (days 3–4) compared with normal acini (day 0) (Figure 1B and Supplementary Figure 1E and F). PRX-I has been shown to interact with inflammatory factors, such as nuclear factor κB (NF-κB) and macrophage migration inhibitory factor, suggesting its involvement in the pathophysiology of pancreatitis.4Bertoldi M. Protein Pept Lett. 2016; 23: 69-77Crossref PubMed Scopus (15) Google Scholar To investigate the role of PRX-I in pancreatitis, we genetically ablated its expression using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) (Supplementary Figure 2A–D). This constitutive inactivation recapitulates the clinical context in which a drug administrated to patients would inhibit its target in all cell types. Although a previous report showed that long-term constitutive PRX-I deletion causes anemia and a shortened lifespan,5Neumann C.A. et al.Nature. 2003; 424: 561-565Crossref PubMed Scopus (612) Google Scholar we did not observe any pancreas-specific anomaly in Prdx1-/- mice (age, 3 mo). Prdx1-/- mice were born at the expected Mendelian frequency, showed normal postnatal development, and were fertile. Next, we analyzed the histology of pancreata from Prdx1+/+, Prdx1+/-, and Prdx1-/- mice treated with cerulein in early and late acute settings (Supplementary Figure 2E and F). At early acute pancreatitis time points, Prdx1+/+ and Prdx1+/- pancreata (considered together as controls [Ctrl]) showed a slight increase in PRX-I expression (Supplementary Figure 3A and B). The extent of edema and immune infiltration observed in Ctrl pancreata was not affected in Prdx1-/- mice; the low PRX-I expression, at early pancreatitis, probably explains the minimal effects observed after its genetic ablation (Supplementary Figure 3C and D). Interestingly, at late acute pancreatitis, PRX-I expression was strongly increased in metaplastic acini (Supplementary Figure 3E and F). At this time point, pancreata from Prdx1-/- mice showed a well-preserved architecture with a significantly 2-fold higher number of normal acini and a 3-fold reduction in metaplastic area compared with Ctrl (Figure 1C and D and Supplementary Figure 4A and B). CD45-positive immune cell infiltration and collagen deposit both were decreased significantly by 2-fold in Prdx1-/- compared with Ctrl mice (Figure 1C and D). PRX-I usually is described as an antioxidant enzyme with high catalytic efficiency.6Ogusucu R. et al.Free Radic Biol Med. 2007; 42: 326-334Crossref PubMed Scopus (153) Google Scholar Interestingly, the content of protein carbonyls and 4-hydroxynonenal (4-HNE)-protein adducts was comparable in pancreata from Ctrl and Prdx1-/- mice (Figure 1E). This suggested that the antioxidant function of PRX-I is not playing a predominant role in pancreatitis, which prompted us to search for additional roles of PRX-I. Previous reports have shown that PRX-I can be secreted from cultured cells in response to inflammatory stimuli and can bind to Toll-like receptor 4 to activate NF-κB–mediated production of proinflammatory cytokines.7Mullen L. et al.Mol Med. 2015; 21: 98-108Crossref PubMed Scopus (68) Google Scholar, 8Riddell J.R. et al.J Immunol. 2010; 184: 1022-1030Crossref PubMed Scopus (161) Google Scholar, 9Liu D.L. et al.Int Immunopharmacol. 2016; 41: 82-89Crossref PubMed Scopus (48) Google Scholar Accordingly, we detected PRX-I in the culture medium of primary mouse acinar cells undergoing metaplasia, highlighting their ability to release PRX-I (Figure 2A). Strikingly, primary mouse acinar cells treated with recombinant PRX-I protein released significantly more proinflammatory cytokines interleukin 6 and tumor necrosis factor-α compared with untreated cells (Figure 2B). In line with this result, Prdx1-/- pancreata showed a reduced expression of interleukin 6 and tumor necrosis factor-α, in the interstitial space between acinar cells, compared with their Ctrl counterparts (Figure 2C and D). Similarly, the expression and nuclear translocation of signal transducer and activator of transcription 3 and NF-κB (subunit p65), 2 transcriptional factors controlling the expression of proinflammatory cytokines, were decreased by 2- to 3-fold in Prdx1-/- pancreata (Figure 2C and D and Supplementary Figure 4A). Thus, our findings show that a mechanism linking the secretion of PRX-I to the production of proinflammatory cytokines may operate in vivo. In summary, we discovered that the ablation of PRX-I reduces the severity of inflammation and related acinar-to-ductal metaplasia (Supplementary Figure 4C). Our results support PRX-I as a potential therapeutic target to reduce pancreatic inflammation and related damage. The authors thank Mourad El Kaddouri, Jean-Nicolas Lodewyckx, Freddy Abrassart, and Nicolas Dauguet for technical help. Transcript profiling: GSE163254. Download .pdf (.1 MB) Help with pdf files Supplementary Data 1 Download .pdf (.81 MB) Help with pdf files Supplementary Data 2