Divergent roles of NOX1 and NOX2 in acute pancreatitis and downregulation of NOX2 in obesity

Abstract

The NADPH oxidase (NOX) enzymes are major sources of reactive oxygen species (ROS) involved in redox signaling and inflammation. However, the specific contribution of individual NOX isoforms to the pathophysiology of acute pancreatitis remains unclear. Here, we investigated the roles of NOX1 and NOX2 in ROS generation in vivo during experimental acute pancreatitis in mice as well as their impact on the regulation of the inflammatory cascade. We confirmed NOX1 expression in the exocrine compartment of the pancreas where it acts as the early and primary source of superoxide in acute pancreatitis. However, both NOX1 and DUOX were expressed in infiltrating neutrophils, suggesting disease-associated reprogramming of these cells within the inflamed pancreas. NOX1 deficiency largely restrained immune cell recruitment, whereas the absence of NOX2 resulted in marked upregulation of pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin 6, reduced protein phosphatase 2A (PP2A) levels, and enhanced MAPK signaling. In addition, pancreatitis in obese mice induced decreased NOX2 expression in pancreatic macrophages, together with an impaired phagocytic profile in both macrophages and neutrophils. In conclusion, our findings reveal opposing roles of NOX1 and NOX2 in acute pancreatitis, with NOX1 promoting neutrophil recruitment and their inflammation-dependent reprogramming, whereas NOX2 restrains the inflammatory response through PP2A.

1. Introduction

The NADPH oxidase (NOX) family of enzymes constitutes a major cellular source of reactive oxygen species (ROS), which serve as key regulators of redox signaling pathways under both physiological and pathological conditions [1]. The NOX family comprises seven isoforms: NOX1–5, DUOX1 and DUOX2, with distinct tissue distribution and regulation [2,3]. Despite this diversity, all NOX isoforms share a conserved catalytic core composed of six transmembrane domains together with cytosolic FAD- and NADPH-binding regions required for ROS generation [1]. NOX1 and NOX2 catalyze the transfer of electrons across biological membranes to reduce molecular oxygen to superoxide (O₂•^-), which is subsequently converted either spontaneously or via superoxide dismutase (SOD) activity into hydrogen peroxide (H₂O₂) [4]. NOX1 is mainly expressed in epithelial cells, whereas NOX2 is primarily expressed in phagocytes, playing a critical role in generating the respiratory burst against pathogens in the innate immune response.

ROS derived from NOXes are involved in inflammation including the activation of redox-sensitive signaling cascades and transcription factors such as NF-κB and AP-1, as well as the regulation of cytokine production and immune cell recruitment [[5], [6], [7]]. In addition, ROS generated by NOXes contribute to host defense mechanisms by supporting microbial killing and shaping innate immune responses [5,6].

Acute pancreatitis is an acute inflammatory disorder of the pancreas characterized by early acinar cell injury followed by a rapid amplification of both local and systemic inflammatory responses [8]. Similarly to other acute inflammatory diseases, in acute pancreatitis redox-dependent signaling through mitogen-activated protein kinases (MAPKs) and redox-sensitive transcription factors such as NF-κB promotes the expression of inflammatory mediators such as TNF-α, IL-1β, and IL-6, which amplify leukocyte recruitment and sustain the inflammatory cascade [[8], [9], [10], [11], [12], [13], [14], [15], [16]].While NADPH oxidase–derived ROS have been implicated in the pathogenesis of acute pancreatitis, the precise contribution of each enzyme remains controversial [17,18]. Experimental studies in pancreatic acinar cells showed that cerulein triggers the translocation of the NOX2 cytosolic subunits p47phox and p67phox to the membrane leading to NOX activation, which was linked to activation of MAPK and JAK2/STAT3 signaling and IL-6 upregulation [[19], [20], [21]]. Cerulein-induced pancreatitis in mice up-regulated NOX1 together with p22phox and the regulatory subunit p47phox [22]. Furthermore, administration of a substrate of NAD(P)H:quinone oxidoreductase 1 (NQO1) lowered the cellular NADPH/NADP⁺ ratio as well as the mRNA expression and protein levels of various NOX subunits, thereby attenuating severity of acute pancreatitis [22]. However, and strikingly, it was reported that genetic ablation of NOX1 did not impact disease severity in cerulein-induced pancreatitis [18]. In addition, although NOX1 has been localized in pancreatic epithelial cells [18], the key NOX subunits p47phox and p67phox were undetectable in isolated pancreatic acini, whereas these subunits are abundantly expressed in infiltrating neutrophils [17].

Therefore, the specific contribution of individual NOX isoforms to acute pancreatitis is still unresolved and a better understanding of the dynamic distribution of NOX isoforms during pancreatic inflammation is required, particularly in neutrophils, a cell type with transcriptional and phenotypic plasticity that can express distinct NOX isoforms within inflamed tissue environments [23,24]. In the present work, we have investigated the in vivo role of NOX1 and NOX2 in the generation of ROS during experimental acute pancreatitis and in the regulation of inflammatory cascades. We show here that NOX1 primarily contributes to ROS generation and inflammatory infiltrate in this disease, whereas NOX2 plays an unexpected opposite role in the regulation of inflammatory cascades. Furthermore, we have identified PP2A as a functionally linked downstream target to NADPH oxidase activity and demonstrate that NOX2 deficiency leads to an unexpected loss of PP2A that enhances MAPK activation and amplifies inflammation.

2. Material and methods

2.1. Animals and experimental model of acute pancreatitis

B6.129X1-Nox1^tm1Kkr/J (Nox1^-/y; NOX1 knockout (KO)) (JAX018787) and B6.129S-Cybb^tm1Din/J (Cybb^-/y; NOX2 KO) (JAX002365) strains, as well as wild-type controls, all backcrossed C57BL/6J genetic background, were used. Cyba^−/− (p22 KO) were previously described [25]. Animals were housed under standard conditions with ad libitum access to food and water. To induce an obese phenotype, mice were fed a high-fat diet (42% fat; MD.88137, Envigo) for 12 weeks, while control lean mice received a standard diet. All experiments strictly adhered to the Spanish national legislation (RD 53/2013) and the European Union Directive (2010/63/EU) on animal protection. The study protocols were approved by the Ethics Committee for Animal Experimentation of the University of Valencia (Valencia, Spain) and authorized by the Directorate General of Agriculture, Livestock and Fisheries of the Generalitat Valenciana (Approval Code: 2020/VSC/PEA/0030 Type 2).

Acute pancreatitis was induced in 3-6-month-old mice by seven intraperitoneal (i.p) injections of cerulein (Sigma-Aldrich, St. Louis, MO, USA) (50 μg/kg of body weight) at 1h intervals. Animals were sacrificed either at 1 h after the first cerulein injection, or 1h after the seventh injection, or at 18 h after the seventh injection (i.e., at 24 h after the first cerulein injection), in all cases animals were anesthetized with isoflurane 2-5%, and blood was obtained from the cava vein. The death was confirmed by cervical dislocation.

2.2. In vivo detection of ROS production

In vivo ROS production was determined using the imaging system IVIS Lumina 5X visualizing the chemiluminescent probe L-012 (Wako Chemicals USA, Inc.) as previously described (Conroy & Aviello, 2019; Aviello et al., 2019). The probe was injected i.p, 20 mg/kg diluted in distilled water in mice anesthetized with isoflurane. The images were acquired 10 min after the injection using Living Image®4.5.2 software (PerkinElmer). Different groups were administered with AZD5904 (40 mg/kg of body weight, dissolved in DMSO 1:5), an inhibitor of myeloperoxidase (MPO), 1400W (20 mg/kg of body weight), a selective irreversible inhibitor of inducible nitric oxide synthase (iNOS).

2.3. RT-PCR for gene expression

A portion of pancreas (about 30 mg) was extracted from each mouse and immediately immersed in RNA-later solution (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) to preserve RNA integrity. Thereafter, samples were homogenized in 500 μl of TRIzol™ Reagent (Thermo Fisher Scientific, Rockford, IL, USA). Then, the sample was centrifuged at 10,000 g for 10 min at 4°C. The supernatant was collected and mixed with 100 μl of chloroform. After a centrifugation at 12,000 g during 15 at 4°C, the aqueous phase was transferred to a new tube with 250 μl of isopropanol, this mix was incubated 10 min at room temperature. To precipitate RNA, the samples were centrifugated at 10,000g for 10 min at 4°C. The supernatant was discarded and the pellet was resuspended in 500 μl of cold 75% ethanol. Finally, after a centrifugation at 7,500g for 10 min at 4°C, the pellet was resuspended in 25 μl of Nuclease-Free Water (Ambion, Thermo Fisher Scientific, Rockford, IL, USA). The quantity and purity of RNA extracted were analyzed using NanoDrop ™ Lite Spectrophotometer (Thermo Fisher Scientific, Rockford, IL, USA).

Reverse transcription was performed to obtain complementary DNA (cDNA) from the isolated RNA, using PrimeScript™ RT Reagent Kit (Takara, Shiga, Japan) according to the manufacturer's instructions in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems/Thermo Fisher Scientific, Foster City, CA, USA). The expression of the genes was analyzed by RT-PCR using commercial TaqMan® probes (Applied Biosystems, Carlsbad, USA) (Table 1) and TaqMan Master Mix2X (Takara, Kusatsu, Shiga, Japan) or specifically designed primers (Table 2) using dsDNA binding dye Syber Green PCR Master mix (Takara, Kusatsu, Shiga, Japan). RT-PCR was carried out using the StepOnePlus™ Real-Time PCR System (Applied Biosystems/Thermo Fisher Scientific, Foster City, CA, USA). Each sample was analyzed in triplicate using the housekeeping gene Tbp. The threshold cycle (CT) value was determined for all samples. Relative gene expression (fold change) was calculated using the 2^−Δ(ΔCT) method. The ΔCT was determined by subtracting the CT value of the housekeeping gene from the CT of the target gene (ΔCT = CT,target−CT,housekeeping). The ΔΔCT was calculated by subtracting the average ΔCT of the control group from the ΔCT of the treated group (ΔΔCT = ΔCT,treated−ΔCT,control).

Table 1.

TaqMan probes used for RT-PCR.

Target gene	TaqMan® probe
Il-1β	Mm00434228_m1
Il-6	Mm00446190_m1
Tnf-α	Mm00443258_m1
Tbp	Mm01277042_m1

Table 2.

Specific oligos used for RT-PCR.

Target gene	Designed primers
Mpo	Forward 5′-AGGGCCGCTGATTATCTACAT- 3′
	Reverse 5’ -CTCACGTCCTGATAGGCACA- 3′
Tbp	Forward 5′-CAGCCTTCCACCTTATGCTC- 3′
	Reverse 5’ -CCGTAAGGCATCATTGGACT- 3′
Nox1	Forward 5’ -CCTTCCTGAATTATGAGAAGTC- 3′
	Reverse 5’ -CTGTTAAAATTCAAGCACAGAG- 3′

2.4. Western-blot analysis

Pancreatic tissue samples were maintained at −80°C until processing. Tissues were homogenized on ice in extraction buffer (100 mg/mL) consisting of 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Igepal CA-630, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, and 50 mM sodium orthovanadate (Sigma-Aldrich, St. Louis, MO, USA). The buffer was supplemented with protease inhibitor (8 μL/mL) and phosphatase inhibitor (1:100 dilution) cocktails (Sigma-Aldrich). Total protein lysates were harvested by mechanical lysis, clarified by centrifugation (15,000 g, 15 min at 4°C), and quantified using a BCA protein assay (Pierce, Thermo Fisher Scientific). Protein aliquots (around 40 μg) were denatured in loading buffer (69.45 mM Tris-HCl, pH 6.8, 10% v/v glycerol, 1.1% LDS, 0.005% bromophenol blue, and 50 mM DTT) at 95°C for 5 min, resolved by SDS-PAGE (120–150 V), and electrotransferred onto nitrocellulose membranes using the Trans-Blot® Turbo™ System (Bio-Rad). To prevent non-specific binding, membranes were blocked for 1 h in blocking buffer (5% bovine serum albumin in TBS-T: 20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.6) and subsequently incubated with specific primary antibodies overnight at 4°C. Following three 5-min washes in TBS-T at room temperature, membranes were incubated with HRP-conjugated secondary antibodies (Supplementary Table 2) for 1 h. Protein bands were detected using Pierce™ ECL Substrate (Thermo Fisher) and visualized with a ChemiDoc™ XRS + image system (Bio-Rad).

2.5. Histological analysis

Pancreatic tissue was excised and immediately immersed in 4% paraformaldehyde (PFA) for 24h of fixation. Then, the samples were embedded in paraffin (Sigma-Aldrich, St. Louis, MO, USA) and 4 μm sections were prepared using an automated microtome. Tissue sections were mounted on glass slides, dewaxed with xylene, rehydrated through graded ethanol series (100%, 90%, and 70%), and stained with hematoxylin and eosin (H&E; Sigma-Aldrich, St. Louis, MO, USA). All histological procedures were carried out at the Central Service for Experimental Research Support (SCSIE) of the University of Valencia.

Pancreatitis severity was assessed on tissue sections at 20X objective magnification by scoring for necrosis, edema and inflammatory infiltration across 10 non-overlapping fields. Scoring was performed according to an established procedure [26].

2.6. Immunofluorescence staining and confocal microscopy

Pancreatic tissue samples were fixed in 4% anhydrous formaldehyde, processed through routine paraffin embedding, and sectioned at 5 μm thickness. After rehydration, antigen retrieval was performed in sodium citrate buffer (pH 6) containing 0.05% Tween-20 at 95⁰C for 18 min. Sections were then equilibrated in and blocked with Tris-buffered saline supplemented with 5% donkey serum, 3% BSA and 0.4% Triton X-100 for 1h at room temperature (RT). For anti-NOS2 staining, an additional 2h incubation with a mouse-on-mouse blocking solution (Thermo Fisher Scientific; R37621) was performed. Sections were then incubated with primary antibodies (Supplementary Table 2) for NOX1 (1:1000), NOS2 (1:250), MPO (1:500), and DUOX (1:500) overnight at 4°C. The following day, slides were rinsed five times in TBS with 0.025% Triton X100 and incubated with fluorophore-conjugated secondary antibodies (Supplementary Table 2; 1:1000) for 1h at RT. After three final washes in the same buffer, nuclei were counterstained with DAPI, and the sections were mounted using ProLong™ Glass Antifade Mountant (Thermo Fisher Scientific; P36983).

The antibodies against NOX1, NOS2, MPO, and DUOX were validated for immunofluorescence in neutrophils isolated from peripheral blood and bone marrow. Peripheral blood from mice was collected by cheek bleeding in EDTA-coated tubes, pelleted, and erythrocytes were removed using ACK lysis buffer. For isolation of neutrophils from bone marrow (BM), femur and tibia bones were excised, and marrow cells were flushed with ice-cold PBS. BM neutrophils were then purified by density gradient centrifugation using a five-layer Percoll gradient, as previously described [23]. Peripheral blood cells and purified BM neutrophils were gently centrifuged onto glass coverslips (300g, 2min, 4°C) before fixation. For immunostaining of isolated blood and bone marrow cells, samples were fixed in 4% paraformaldehyde for 10 min at room temperature (RT), then blocked for 1 h at RT using Tris-buffered saline with 5% donkey serum, 3% BSA, and 0.4% TritonX100, followed by 2 h mouse-on-mouse blocking for anti-NOS2 staining. Cells were incubated with primary antibodies for 3h at RT, followed by three washes and incubation with secondary antibodies for 1h at RT. Subsequent steps were performed as outlined above. Imaging was carried out using a Zeiss LSM800 Airyscan confocal microscope, and image analysis was performed with ICY software (Institut Pasteur).

2.7. Isolation of myeloid cells from pancreas and bone marrow

Myeloid cells were isolated from the pancreas and bone marrow. For isolation from the pancreas, following the induction of acute pancreatitis, pancreatic tissues were harvested and collected in 4 mL of Hanks’ Balanced Salt Solution (HBSS) per pancreas. Subsequently, the samples were mechanically and enzymatically dissociated by incubation in a digestion buffer at 37°C for 40 min. This buffer consisted of 1 mg/mL collagenase D (Roche, reference 11088858001) dissolved in HBSS. The enzymatic digestion was stopped by the addition of 20 mL of HBSS supplemented with 10% fetal bovine serum (FBS). The cell suspension was then filtered through a 100 μm cell strainer to discard any remaining undigested tissue. Finally, the cells were harvested by centrifugation at 450 × g for 5 min, resuspended in PEB buffer (PBS supplemented with BSA and 2 mM EDTA) and counted prior to purification.

To obtain neutrophils from the bone marrow, femurs and tibias were harvested. The epiphyses were removed to access the medullary cavity, and the bone marrow was flushed out using a syringe filled with PEB buffer (PBS supplemented with 0.5% BSA and 2 mM EDTA). The extracted cells were collected in a 50 mL tube, and the bone ends were further processed to maximize cell recovery. The resulting cell suspension was then passed through a 70 μm cell strainer. After washing the filter with PEB buffer, the cell suspension was centrifuged at 2000 rpm for 5 min. To deplete erythrocytes, the cell pellet was resuspended in 5 mL1X Lysis Buffer (BD Pharm Lyse™ Lysing Buffer, Waters Biosciences) and incubated at 37°C for 5 min. The lysis reaction was halted by adding 10 mL of PEB buffer, followed by another centrifugation step. The cells were then resuspended in 4 mL of PEB buffer and counted prior to purification.

Neutrophils from the pancreas and bone marrow were isolated by magnetic-activated cell sorting (MACS). The neutrophils were enriched using the Ly6G UltraPure MicroBead Kit (Miltenyi Biotec) according to the manufacturer's protocol. Briefly, 10⁷ cells were centrifuged at 300 × g for 10 min, resuspended in 90 μL of PEB buffer, and incubated with 10 μL of microbeads for 10 min on ice. Following incubation, the cells were washed with 2 mL of PEB buffer, centrifuged again at 300 × g for 10 min, and resuspended in 500 μL of PEB. Automated magnetic separation was then performed using an autoMACS® Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany). Positive selection was carried out using the Posseld2 program, which optimizes purity by sequentially passing the sample through two magnetic columns. Purity of the isolated neutrophil populations was assessed by flow cytometry using Ly 6G-APC (130,120,803, Miltenyi Biotec). Cells from the positively selected fractions were resuspended in 100 μL of PBS and stained with the recommended antibody amounts for 15 min on ice and protected from light. After staining, cells were washed with 2 mL of PBS, centrifuged at 300 × g for 5 min, and resuspended in 500 μL of PBS. Samples were acquired on an LSR Fortessa (Waters Biosciences) equipped with 405 nm, 488 nm, 561 nm, and 640 nm lasers, and fluorescence was collected using 450/50 nm, 530/30 nm, 586/15 nm, 780/60 nm and 670/10 nm emission filters.

To isolate macrophages, single-cell suspensions from lean and obese mice were incubated for 15 min on ice, protected from light, using the following fluorescent antibodies: CD45-VioGreen (130-110-803, Miltenyi Biotec), CD11b-PE (130-113-806, Miltenyi Biotec), Ly-6G-APC (130-120-803, Miltenyi Biotec), and F4/80-BV421 (123131, BioLegend). Following incubation, the cells were washed and resuspended in PBS for analysis on a FACSAria Fusion flow cytometer (BD Biosciences) equipped with 405 nm, 488 nm, 561 nm, and 640 nm lasers. Fluorescence was detected using 450/50 nm, 530/30 nm, 586/15 nm, and 670/10 nm emission filters. Macrophages were classified as CD45⁺CD11b⁺F4/80⁺ cells.

2.8. Proteomic analysis

Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40 substitute, 0.5% sodium deoxycholate and 0.1% SDS). A total of 10 μg of protein in 20 μL was prepared for further processing. Samples were adjusted to 30 μL with 50 mM ammonium bicarbonate (ABC). Disulfide bonds were reduced by incubating samples for 20 min at 60°C in 35 μL of 2 mM DTT prepared in 50 mM ABC. After cooling to room temperature, free sulfhydryl groups were alkylated in 40 μL of 5.5 mM iodoacetamide (IAM) for 30 min at room temperature in the dark. Alkylated samples were processed using the SP3 protocol with minor modifications [27,28]. Proteins were incubated with SP3 resin. Then, 20 μL of beads were used to achieve a 1:20 protein-to-beads ratio, and acetonitrile (ACN) was added to a final concentration of 70%. After bead-based cleanup, proteins were digested with trypsin (500 ng in 100 μL of 50 mM ABC) at 37°C overnight. Digested peptides were acidified with 10% TFA to a final concentration of 1%. The final sample volume was 110 μL, corresponding to a theoretical peptide concentration of 181 ng/μL. Approximately 200 ng of each digest were diluted to 20 μL with 0.1% formic acid (FA) and loaded onto Evotip Pure tips (EvoSep) according to the manufacturer's instructions. The sample injection order was fully randomized using HyStar (Bruker).

LC–MS/MS analysis was performed on a timsTOF fleX mass spectrometer (Bruker). Peptides loaded on Evotip Pure tips were eluted onto an analytical column (Endurance 15 cm × 150 μm, 1.5 μm; Evosep) using the Evosep One system and separated using the manufacturer-defined 30 SPD chromatographic method. Eluted peptides were ionized using a CaptiveSpray source at 1600 V and 180°C and analyzed in diaPASEF mode (method: diaPASEF longGradient) with the following settings:

TIMS settings: custom mode; 1/K0 range 0.6–1.6 V s/cm²; ramp time 100 ms; duty cycle 100%; ramp rate 9.42 Hz; MS averaging 1; auto calibration off.

MS settings: scan range 100–1700 m/z; ion polarity positive; scan mode diaPASEF.

System performance was monitored using 50 ng of HeLa digest, resulting in the identification of 7417 proteins using diaPASEF and the 30 SPD gradient.

Raw diaPASEF data were processed using DIA-NN v1.8 via FragPipe 21.1. An in silico–predicted spectral library was generated from the SwissProt Human database (2024-07-23 release). Quantification results were exported as Excel files containing unique genes and protein groups filtered at FDR ≤1%.

Differential expression analysis was performed using FragPipe-Analyst (http://fragpipe-analyst.nesvilab.org/), an R Shiny web server supporting downstream quantitative proteomics analysis. The platform includes missing value imputation, quality control, unsupervised clustering, differential expression analysis using Limma, and gene ontology and pathway enrichment analysis using Enrichr. An Excel file containing the differential expression results was generated.

2.9. Cell culture

The 266-6 mouse pancreatic acinar cell line (ATCC, CRL-2151) was maintained in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.01% Fungizone. Cultures were incubated at 37°C in a humidified 5% CO₂ atmosphere and maintained in active exponential growth. To ensure experimental integrity, cells were periodically screened for mycoplasma and used within low passage numbers.

2.10. Lentiviral transduction and PP2Ac silencing

Stable knockdown of the PP2A catalytic subunit (PP2Ac) in 266-6 pancreatic acinar cells was performed using the pGIPZ shRNA lentiviral system (Thermo Scientific). Cells were seeded at 5 × 10⁴ cells/well and transduced with a 1/10 dilution of lentiviral particles in serum-free medium for 6 h. Stable transformants were selected with 4 μg/mL puromycin (Santa Cruz) for 7 days, followed by a maintenance dose of 1 μg/mL. To optimize silencing efficiency, TurboGFP-positive cells were sorted using a MoFlo™ XDP flow cytometer (Beckman Coulter), isolating the top 5% of the high-fluorescence population. High-intensity clones were expanded by single-cell seeding in 96-well plates. Successful PP2Ac silencing was confirmed by Western blot analysis using a non-silencing pGIPZ empty vector as a negative control.

2.11. Statistical analysis

Results are presented as the mean ± SEM (Standard Error of the Mean). Data were analyzed using one-way repeated measures analysis of variance (ANOVA), followed by Tukey's post hoc test for multiple group comparisons. Student's t-test was employed for comparisons involving only two independent groups. Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). A p-value of <0.05 was considered statistical significance.

3. Results

3.1. NADPH oxidase activity sustains peroxynitrite and hypochlorous acid production in pancreas during acute pancreatitis

To assess the contribution of NADPH oxidase activity to the generation of reactive oxygen species (ROS) in vivo during acute pancreatitis, we administered the chemiluminescent probe L-012 to wild type (WT) C57BL/6 mice and to Cyba-deficient mice (p22 KO mice; p22^phox is essential for NOX1-4 activity) 50 min after the last cerulein injection in order to measure ROS levels at 1 h after the last cerulein injection, when the inflammatory response is fully established [29,30]. C57BL/6 WT mice exhibited a strong luminescent signal in the upper abdominal region, indicating the high generation of ROS in vivo upon acute pancreatitis (Fig. 1A). In contrast, detectable luminescence was absent in p22 KO mice, confirming that the observed signal exclusively depended on NADPH oxidase activity (Fig. 1A).

Fig. 1.

(A) Representative in vivo bioluminescence imaging (IVIS) and chemiluminescence quantification (total photon flux, p/s) in wild-type (WT) and Cyba-deficient (p22 KO) mice with acute pancreatitis. (B) Representative images and quantification in mice treated with saline (vehicle), AZD5904 (MPO inhibitor), or 1400W (iNOS inhibitor) . In all graphs, individual data points represent single animals; bars indicate mean ± SEM; *p < 0.05 (n = 4-9 mice per group).

According to previous reports, the L-012 signal reflects in vivo mainly peroxynitrite and hypochlorous acid generated from superoxide [23,31]. To determine the contribution of each one of these highly reactive products in our model, we administered the myeloperoxidase (MPO) inhibitor AZD5904 as well as the inducible nitric oxide synthase (iNOS) inhibitor 1400W to WT mice with acute pancreatitis. Each inhibitor reduced the luminescent signal by around 50% (Fig. 1B), indicating that in acute pancreatitis both hypochlorous acid and peroxynitrite formation largely arise from NOX-derived superoxide via MPO- and iNOS-induced reactions.

3.2. NOX1, but not NOX2, is the predominant source of NOX-derived superoxide during acute pancreatitis

We next examined the specific contribution of NOX1 and NOX2 to in vivo ROS generation during acute pancreatitis by using NOX1 and NOX2-deficient mice. In NOX1 KO mice, the L-012 signal was almost absent, indicating that NOX1 is the major source of NOX-derived superoxide in acute pancreatitis (Fig. 2A). In contrast and unexpectedly, NOX2 deficiency did not significantly change luminescence intensities; instead, NOX2 KO mice displayed signals comparable to their WT counterparts or even a tendency towards a further increase (Fig. 2B). Treatment with the MPO inhibitor AZD5904 or the iNOS inhibitor 1400W during the induction of acute pancreatitis markedly reduced L-012 signal in NOX2 KO mice, confirming that both MPO and iNOS account for the formation of hypochlorous acid and peroxynitrite that arise from superoxide generated by other NOX isoenzymes (Fig. 2C).

Fig. 2.

(A) Representative in vivo bioluminescence imaging (IVIS) and chemiluminescence quantification (total photon flux, p/s) in wild-type (WT) and Nox1-deficient (NOX1 KO) mice (n = 5-9 mice per group). (B) Representative in vivo bioluminescence imaging (IVIS) and chemiluminescence quantification (total photon flux, p/s) in wild-type (WT) and Cybb-deficient (NOX2 KO) mice (n = 9-10 mice per group). (C) Representative images and quantitative analysis in NOX2 KO mice treated with saline (vehicle), AZD5904, or 1400W (n = 4-10 mice per group). In all graphs, individual data points represent single animals; bars indicate mean ± SEM; *p < 0.05.

Therefore, NOX1 is the primary contributor to NOX-dependent superoxide production during acute pancreatitis. Consistent with this interpretation, WT mice displayed a L-012 signal as early as 1 h after the first cerulein injection when leukocyte infiltration was still minimal, and this signal was quantitatively similar to that observed at later time points (Fig. 3A). The very limited inflammatory infiltration at this early stage of disease was confirmed by very low pancreatic Mpo expression, indicating minimal neutrophil recruitment (Fig. 3B). Importantly, this early luminiscence signal was fully abrogated in both p22 KO and NOX1 KO mice, confirming that NOX1-dependent superoxide production is rapidly initiated at the very beginning of acute pancreatitis (Fig. 3C).

Fig. 3.

(A) Representative in vivo bioluminescence imaging (IVIS) and chemiluminescence quantification (total photon flux, p/s) in wild-type mice at 1 h and 7h of cerulein-induced acute pancreatitis (n = 8-9 mice per group). (B) Time-course analysis of pancreatic Mpo mRNA expression in WT mice at 0, 1, and 7 h post-cerulein administration, as determined by RT-qPCR. Data are expressed as fold change relative to the 0 h control group (n = 3 mice per group). (C) Representative in vivo bioluminescence imaging (IVIS) and chemiluminescence quantification (total photon flux, p/s) in wild-type (WT), Nox1-deficient (NOX1 KO) mice and Cyba-deficient (p22 KO) mice at 1 h post-cerulein administration (n = 4-11 mice per group). In all graphs, individual data points represent single animals; bars indicate mean ± SEM; *p < 0.05.

3.3. A reprogrammed neutrophil subset expresses NOX1 and DUOX in the pancreas during acute pancreatitis

To further investigate the cellular context associated with NOX–dependent ROS generation in acute pancreatitis, we performed immunofluorescence analysis of pancreatic sections to localize NADPH oxidases, iNOS, and MPO as neutrophil marker. Immunofluorescence staining confirmed that NOX1 is expressed in pancreatic acinar cells (Supplementary Fig. S1).

Remarkably, both NOX1 and the calcium-activable oxidase DUOX were detected in a subset of infiltrating immune cells during acute pancreatitis. Co-staining revealed that NOX1 and DUOX were present in MPO⁺ neutrophils within the inflamed pancreas (Fig. 4A and B), while as expected both oxidases were not expressed in bone marrow derived neutrophils (Supplementary Fig. S2). Notably, the number of NOX1-positive and DUOX-positive neutrophils progressively increased over the course of acute pancreatitis, becoming particularly evident at 24 h after pancreatitis induction (Fig. 4B). The same time frame was observed for the expression of iNOS (NOS2) in pancreatic neutrophils. In order to further confirm the de novo expression of NOX1 in pancreatic neutrophils during acute pancreatitis, Nox1 mRNA was quantified. Neutrophils isolated from the pancreas 24h after pancreatitis induction showed marked upregulation of Nox1 mRNA when compared with neutrophils isolated from the bone marrow (Supplementary Fig. S3). Thus, expansion of the NADPH repertoire by functional reprogramming of neutrophils in inflamed environments occurs not only in the intestine and the airways (23,24), but also in acute pancreatitis.

Fig. 4.

Immunofluorescence staining of pancreatic tissue from C57BL/6 mice in MPO + neutrophils (purple) following cerulein-induced pancreatitis after (A) 7h and (B) 24h. iNOS (NOS2) orange, NOX1 green, DUOX red, DNA (DAPI blue). The upper and lower rows in A and B show different magnification of confocal microscopy images with the scale bar of 10 μm applying to all images.

We next performed an unbiased proteomic analysis of neutrophils isolated from the inflamed pancreas at 24 h after pancreatitis induction and compared their protein expression profiles with those of neutrophils obtained from bone marrow (Fig. 5A). As expected, NOX2 was detected in neutrophils from both compartments. Furthermore, NOX2 abundance remained unchanged between bone marrow and pancreatic neutrophils, confirming its stable expression as a canonical component of the neutrophil oxidative machinery (Fig. 5B).

Fig. 5.

(A) Volcano plot from proteomic analysis of neutrophils isolated from bone marrow (BM) and pancreas following cerulein-induced pancreatitis after 24h. The x-axis shows the log₂ (fold change), and the y-axis shows the -log₁₀ (adjusted p-value). Red dots indicate significantly up-regulated proteins, while blue dots indicate significantly down-regulated proteins in pancreatic neutrophils compared to BM neutrophils. Selected top differentially expressed genes (DEGs) are listed in the side panels. (B) Protein levels of NOX2 in neutrophils from bone marrow and pancreas as determined by proteomics analysis. Data are presented as mean ± SEM; each dot represents an individual biological replicate (n = 5 isolation per group) (C) Gene Set Enrichment Analysis (GSEA) displaying significantly enriched Gene Ontology (GO) terms. The dot plot illustrates GO categories that are activated or suppressed in pancreatic versus bone marrow neutrophils. Results are categorized by GeneRatio, with the dot size representing the number of proteins (count) enriched in each term and the color indicating the statistical significance (adjusted p-value).

Nevertheless, pathway enrichment analysis revealed marked reprogramming of pancreatic neutrophils during pancreatitis, exhibiting an activated and more mature phenotype. Proteins enriched in pancreatic neutrophils were associated with proteolytic activity, including serine-type endopeptidase and peptidase activity, as well as zymogen granule components. In contrast, bone marrow neutrophils were enriched in pathways related to DNA repair, glyceraldehyde-3-phosphate biosynthesis, hydrogen peroxide metabolic processes, and proteasome-associated complexes, reflecting a less activated and more immature phenotype (Fig. 5C).

3.4. NOX1 deficiency diminishes the inflammatory infiltrate, whereas NOX2 deficiency enhances the inflammatory cascade in acute pancreatitis

To evaluate the impact of NOX1 and NOX2 deficiency on the inflammatory response during acute pancreatitis, we performed histological analysis of pancreatic tissue from NOX1-and NOX2-deficient mice subjected to pancreatitis. Remarkably, the inflammatory infiltration was largely reduced in NOX1 KO mice upon pancreatitis compared with their WT counterparts (Fig. 6A and B). In contrast, NOX2 KO mice with pancreatitis showed levels of inflammatory infiltration comparable to WT animals (Fig. 6A and B).

Fig. 6.

(A) Representative H&E staining of pancreatic sections from control mice (WT Sham) and mice following cerulein-induced pancreatitis after 7h across three genotypes: wild-type (WT), Nox1-deficient (NOX1 KO), and Cybb-deficient (NOX2 KO) mice. (B) Histopathological scoring of inflammatory infiltrate in control mice (WT Sham) and mice with cerulein-induced acute pancreatitis across three genotypes: wild-type (WT), Nox1-deficient (NOX1 KO), and Cybb-deficient (NOX2 KO) mice (n = 4-8 mice per group). (C) Pancreatic Mpo mRNA expression and (D) pro-inflammatory cytokine profile (Tnf-α, Il-6, and Il-1β) in WT Sham mice and cerulein-induced acute pancreatitis groups across: wild-type (WT), Nox1-deficient (NOX1 KO), and Cybb-deficient (NOX2 KO) mice. Data were determined by RT-qPCR and are presented as fold change relative to the WT Sham group (n = 4-6 mice per group). In all graphs, individual data points represent single animals; bars indicate mean ± SEM; *p < 0.05.

Consistent with the reduced leukocyte recruitment, pancreatic Mpo expression, a gene predominantly expressed in neutrophils, was also substantially decreased in NOX1-deficient mice with acute pancreatitis. The Mpo transcript levels of WT mice and NOX2 KO mice did not significantly differ (Fig. 6C). However, NOX2-deficient mice exhibited markedly increased expression of the pro-inflammatory cytokines Tnf, Il6, and Il1ꞵ, indicating a substantial exacerbation of the inflammatory cascade in the absence of NOX2 (Fig. 6D).

3.5. NOX2 deficiency causes downregulation of PP2A that enhances MAPK activation and up-regulates proinflammatory cytokines

Considering the proposed role of protein phosphatase 2A (PP2A) as a critical redox-sensitive signaling node that restrains MAP kinase (MAPK) activation [8], we examined whether NOX2 deficiency affects PP2A abundance in the inflamed pancreas. Western blot analysis revealed that protein levels of the PP2A catalytic subunit were markedly decreased in inflamed pancreatic tissue from NOX2-deficient mice compared with WT mice subjected to pancreatitis (Fig. 7A and Supplementary Fig. S4A). Consistent with the loss of this key protein phosphatase, the deficiency in NOX2 led to increased phosphorylation of the p38 and ERK MAPK (Fig. 7B and Supplementary Fig. S4B and C) that will lead to enhanced activation of pro-inflammatory signaling cascades.

Fig. 7.

(A) Representative Western blot images of PP2Ac protein levels in pancreatic tissue from WT Sham mice and wild-type (WT), Nox1-deficient (NOX1 KO), and Cybb-deficient (NOX2 KO) mice following cerulein-induced pancreatitis after 7h. Vinculin was used as internal loading control. (B) Representative Western blot images of phosphorylated (p-) and total p38 and ERK protein levels in pancreatic tissue from WT Sham mice and cerulein-challenged wild-type (WT), Nox1-deficient (NOX1 KO), and Cybb-deficient (NOX2 KO) mice. Vinculin was used as the internal loading control. (C) Representative Western blot images of PP2Ac, phosphorylated (p-) and total p38 and ERK protein levels in pancreatic tissue from 266-6 murine acinar cells transfected with either Scrambled (Scr) or shRNA against PP2A (shPP2A). β-tubulin was used as the internal loading control. (D) Time-course analysis of pro-inflammatory cytokine (Tnf-α, Il-6, and Il-1β) mRNA expression in 266-6 murine acinar cells transfected with either Scrambled (Scr) or shRNA against PP2A (shPP2A) at 0, 1, 2 and 3 h post-transfection, as determined by RT-qPCR. Data are expressed as fold change relative to the 0 h control group (n = 3 independent experiments per group). In all graphs, individual data points represent single animals; bars indicate mean ± SEM; *p < 0.05.

To determine whether the loss of PP2A is sufficient to promote MAPK activation and up-regulation of the inflammatory cascade, we silenced the expression of the catalytic subunit of PP2A in murine pancreatic acinar cells 266-6. Indeed, knockdown of the catalytic subunit of PP2A greatly increased the phosphorylation of both p38 and ERK, confirming that PP2A restrains MAPK activation in pancreatic cells (Fig. 7C). Furthermore, PP2A-deficient 266-6 cells displayed elevated expression of the pro-inflammatory cytokines Tnf, Il6, and Il1ꞵ, demonstrating that downregulation of PP2A is sufficient to enhance the inflammatory cascade (Fig. 7D).

3.6. Obesity triggers downregulation of NOX2 and impairs the phagocytic profile of pancreatic immune cells

Our final approach was to assess whether obesity, a metabolic condition known to affect redox signaling and to aggravate inflammatory responses in acute pancreatitis [29,32,33], influences NOX2 expression and NADPH oxidase–dependent pathways in pancreatic phagocytes. To this end, neutrophils and macrophages were isolated from the pancreas of lean and obese mice with acute pancreatitis and subjected to proteomic analysis. Importantly, NOX2 levels were decreased in macrophages from obese mice with pancreatitis, with neutrophils following the same trend (Fig. 8A–C).

Fig. 8.

(A) Protein levels of NOX2 in neutrophils isolated from pancreas of lean and obese mice following cerulein-induced pancreatitis after 24h as determined by proteomics analysis. Data are presented as mean ± SEM; each dot represents an individual biological replicate (n = 4 isolation per group) (B) GSEA enrichment plots for neutrophils isolated from pancreas of lean and obese mice. Representative plots show the enrichment of the "Phagosome" and "Fc gamma R-mediated phagocytosis" pathways from KEGG database. The green line indicates the running enrichment score, while the black vertical bars represent the position of individual proteins within the ranked list. Normalized Enrichment Score (NES), nominal p-value (pval) and False Discovery Rate (FDR) are indicated for each pathway. (C) Protein levels of NOX2 in macrophages from pancreas of lean and obese mice as determined by proteomics analysis. Data are presented as mean ± SEM; each dot represents an individual biological replicate (n = 3 isolation per group), p*<0.05. (D) GSEA enrichment plot for macrophages isolated from pancreas of lean and obese mice. Representative plots show the enrichment of “Cell adhesion molecules (CAMs)” pathway from KEGG database. NES, pval and FDR are indicated.

To assess the potential functional impact of NOX2 downregulation, we performed gene set enrichment analysis (GSEA) based on proteomic data. In agreement with reduced NOX2 levels, both pancreatic macrophages and neutrophils displayed a decreased antimicrobial activity in pancreatitis upon obesity. Specifically, neutrophils showed reduced representation of pathways related to phagosome formation and Fc gamma receptor–mediated phagocytosis and macrophages exhibited decreased enrichment of cell adhesion molecules (CAMs) (Fig. 8B–D). Together, these findings indicate that obesity is associated with an impaired phagocytic phenotype of leukocytes in the inflamed pancreas.

4. Discussion

In this work we describe divergent roles for the NADPH oxidases NOX1 and NOX2 in the pathophysiology of acute pancreatitis. Our findings demonstrate that both NOX1 and NOX2 contribute to redox signaling and the inflammatory response associated with acute pancreatitis, although they exert opposite effects. While NOX1 promotes inflammation and tissue damage, surprisingly NOX2 exerts a protective role.

In agreement with a report that detected NOX1 in pancreatic acinar cells [18], our results confirm its presence in acinar cells, which is in contrast to another study that localized NOX activity in neutrophils but not in pancreatic acini [17]. Furthermore, beyond confirming its expression in the exocrine pancreas, our results provide functional insight into NOX1 activity during acute pancreatitis and challenges its previously assumed functional irrelevance. We show here that NOX1 is the primary source of ROS in the early stage of acute pancreatitis, acting as chemotactic signal to promote immune cell recruitment. Indeed, the inflammatory infiltrate was markedly reduced in NOX1-deficient mice with acute pancreatitis. Therefore, redox signals seem to predominantly originate from pancreatic epithelial cells during the early stages of acute pancreatitis.

Interestingly, while NOX1 expression was confirmed in the exocrine compartment of the pancreas, we also detected its presence in infiltrating neutrophils at later stages of acute pancreatitis. Immunofluorescence analysis identified NOX1 expression in infiltrating neutrophils within the inflamed pancreatic tissue, particularly at later stages of disease progression. We also detected the expression of the NADPH oxidase DUOX , further supporting the reprogramming of neutrophils within the inflammatory microenvironment of the pancreas.

Recent studies have described neutrophil reprogramming at mucosal barriers during inflammatory and infectious diseases, leading to tissue- and stimulus-dependent de novo expression of NOX1, DUOX2, and DUOX1 in recruited neutrophils [23,24]. While DUOX2 has been associated with enhanced inflammatory responses, myeloid NOX1 has been reported to exert protective effects [23]. In contrast, in models of colonic inflammation and pulmonary infection, epithelial NOX1 promoted neutrophil recruitment [23]. These previous findings suggest that the de novo expression of NOX1 and DUOX2 in neutrophils detected here may reflect a specialized functional adaptation to the inflammatory environment in acute pancreatitis. However, the precise impact of this reprogramming in pancreatic inflammation requires further investigation.

NOX2 represents the prototypical NOX isoform expressed in immune cells, particularly in neutrophils and macrophages [1,3]. Superoxide production by NOX2 is essential for microbial killing, and its deficiency leads to chronic granulomatous disease highlighting its critical role in immune regulation and inflammatory control [34,35]. Accordingly, NOX2-deficient mice exhibit increased susceptibility to infections, primarily due to defective neutrophil function [36,37]. Nevertheless, NOX2 deficiency has also been associated with alteration in Th17/Treg balance and increased production of pro-inflammatory cytokines, including TNF-α and IL-1β [38].

Our findings support the paradoxical contributions of NOX2 to the resolution or limitation of the inflammatory response in acute pancreatitis. In our model, the absence of NOX2 was associated with a remarkably increase in the inflammatory cascade, including marked upregulation of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the pancreas. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase highly expressed in pancreatic tissue [8] and emerges here as a central redox-sensitive node through which NOX2-derived signals may modulate the inflammatory cascade. PP2A is a heterotrimeric enzyme composed of a redox-sensitive catalytic subunit (PP2Ac), a structural scaffold subunit (PP2A-A), and a regulatory subunit (PP2A-B) that determines substrate specificity and subcellular localization [39]. The catalytic subunit contains ten cysteine residues, including a canonical CXXC motif at position 266–269, highly susceptible to reversible oxidative modifications [40,41]. Oxidation of these thiol groups alters PP2A conformation and impairs its phosphatase activity [42,43]. Accordingly, we previously reported that experimental acute pancreatitis in rats caused oxidative modification of PP2A leading to decreased phosphatase activity that coincides with sustained activation of MAPK signaling pathways and amplification of inflammatory responses [44,45].

In the present work, we found that NOX2 deficiency decreases PP2A levels, suggesting that NOX2-derived redox signals are required to maintain sufficient PP2A activity in inflamed pancreas. Remarkably, PP2A is a prominent molecular target of disulfide stress, a redox imbalance firstly described by our group that develops in the pancreas during acute pancreatitis [8,9,44]. We defined disulfide stress as a disturbance in thiol redox homeostasis characterized by oxidation of protein thiols as disulfide bonds [44]. In acute pancreatitis, disulfide stress promotes cysteinylation and disulfide bond formation in redox-sensitive proteins, contributing to sustained activation of inflammatory responses [8,30,44]. Restoration of protein thiol homeostasis depends on specific reductive systems, including thioredoxin 1 and thioredoxin-related protein of 14 kDa (TRP14), which catalyze the reduction of protein mixed disulfides and reverse protein cysteinylation [30]. Impairment of these thiol-reducing mechanisms favors accumulation of oxidized PP2A and other signaling proteins, contributing to pancreatic injury progression [44].

Our study challenges the oversimplified common assumption that NADPH oxidase derived ROS promote tissue damage by showing that NOX1-derived superoxide appears to be pathogenic, driving inflammatory cell infiltration and pancreatic injury, whereas NOX2-derived superoxide appears to be rather protective, limiting excessive inflammation. Hence, the biological effects of ROS depend on the cellular source and context, rather than simply on the total amount of ROS produced. Furthermore, our findings may have poteintial therapeutic implications since they suggest that broad inhibition of ROS production or pan-NOX inhibition could inadvertently suppress the protective effect of NOX2. Selective NOX1 inhibition might reduce pancreatic inflammation while preserving NOX2-mediated immune regulation.

Similarly, we observed here NOX2 downregulation in the pancreas of obese mice upon pancreatitis induction, a condition characterized by a more severe and exacerbated inflammatory response [8,29,46]. Diminished NOX2 levels were detected in myeloid cells, especially macrophages, together with an impaired phagocytic profile according to the proteomic analysis. Obesity is associated with an increased risk of infected necrosis, organ failure, and death in acute pancreatitis [32,33]. In this regard, reduced NOX2 levels together with impaired phagocytosis in myeloid cells that we report here may contribute to sustained inflammation and increased susceptibility to infections in obesity-associated pancreatitis.

One limitation of our study is that the sensitivity of our LC–MS/MS analysis was not sufficient to detect NOX1 and DUOX in our proteomic analysis, in contrast with the detection of these proteins when using higher sensitivity LC–MS/MS analysis and a different proteolysis protocol to confirm the de novo expression of these proteins [23,24].

In conclusion, our findings highlight the opposing roles of NOX1 and NOX2 in the pathophysiology of acute pancreatitis. NOX1 acts as a major superoxide source promoting neutrophil recruitment during acute pancreatitis and contributing to neutrophil reprogramming. In contrast, NOX2 restrains the inflammatory response through PP2A, thereby limiting pancreatic injury.

CRediT authorship contribution statement

Sergio Rius-Pérez: Data curation, Formal analysis, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing – original draft. Néstor Jiménez-Cañete: Data curation, Formal analysis, Investigation, Methodology, Software. Ashish K. Singh: Investigation, Methodology. Anabel Gil: Investigation, Methodology. Josep Benitez-Martínez: Investigation, Methodology. Inmaculada Noguera: Investigation, Methodology. Eva Blanch: Investigation, Methodology. Noemie Therry: Investigation, Methodology. Sandra Pinto: Investigation, Methodology. Ulla G. Knaus: Conceptualization, Formal analysis, Investigation, Methodology, Resources, Supervision, Writing – review & editing. Juan Sastre: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for this journal and was not involved in the editorial review or the decision to publish this article.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Fig. S1.

Immunofluorescence staining of pancreatic tissue derived from untreated C57BL/6 mice showing NOX1 (green) and DUOX (red) expression; DNA (DAPI blue). Scale bar 100 μm.

Fig. S2.

Immunofluorescence co-staining of MPO⁺ neutrophils (red) derived from bone marrow (BM) and peripheral blood of (A, B) C57BL/6 and (C) Nox1^-/y mice with anti-NOX1 and anti-DUOX antibodies. Scale bar 10 μm.

Fig. S3.

Nox1 mRNA expression in neutrophils isolated from bone marrow (BM) and pancreas following cerulein-induced pancreatitis after 24h. Data are presented as mean ± SEM; each dot represents an individual biological replicate (n = 3-4 isolation per group).

Fig. S4.

Densitometric quantification of (A) PP2Ac, (B) phospho p38 (p-p38) and (C) phospho ERK (p-ERK) protein levels in pancreatic tissue from WT Sham mice and wild-type (WT), Nox1-deficient (NOX1 KO), and Cybb-deficient (NOX2 KO) mice following cerulein-induced pancreatitis after 7h. Vinculin was used as the internal loading control. In all graphs, individual data points represent single animals; bars indicate mean ± SEM; *p < 0.05. (n = 4-5 mice per group).

Data availability

Data will be made available on request.