Recombinant human ERAP1 (E1) and ERAP2 (E2) were purchased from R&D (ERAP1-Catalog Number: 2334-ZN; ERAP2- Catalog Number: 3830-ZN) (R&D system, Minneapolis, United States). Both, ERAP1 and ERAP2 were expressed in a mouse myeloma cell line (NS0) and display enzymatic activity. The lyophilized protein (10 μg) was reconstituted in a 0.2 μm filtered solution in Tris, NaCl and Glycerol at a final concentration of 300 ng/μL 5 µL aliquots were prepared and stored at −20°C. The enzymatic activity was measured by their ability to cleave the fluorogenic peptide substrate, Arg¬7¬amido-4¬methylcoumarin (Arg¬AMC) as suggested by the datasheet. Results were analysed by Varioskan (Thermofisher) at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 min.

A limulus amebocyte lysate (LAL) chromogenic kit (# HIT302, Hycult Biotech) with a minimum detection limit of 0.04 EU/mL was used to measure endotoxin levels before treatment. Endotoxin levels were <0.1 EU/mL and the proteins were used at a final concentration of 300 ng/mL based on previous (Aldhamen et al., 2015; Saulle et al., 2021; Niu et al., 2022) and our pilot tests.

Neutrophils were obtained from the venous blood of 5 healthy volunteers. Ethical clearance was obtained from the University of Milan Ethics Committee (number 14/22). Written informed consent was obtained after receiving information about the use of their biological samples. The biological material was anonymized.

Neutrophil preparations were isolated by Ficoll-Hypaque density centrifugation followed by dextran sedimentation for 20 min (Stie and Jesaitis, 2004). After the incubation with sedimental liquid (3% Gelatin, 0.9% NaCl and 0.1% Dextrose) neutrophil containing supernatant was collect, centrifuged at 1,800 rpm for 5 min at RT and red blood cells were lysed with ammonium-potassium chloride (ACK) lysis buffer (Euroclone, Milan, Italy). Neutrophils were then washed, centrifuged and the pellet were resuspended in RPMI 1640 medium (Euroclone, Milan, Italy) (supplemented with 2% L-Glutamine 100 × 200 mM and 1% Penicillin-Streptomycin Solution 100X) with 10% FBS (fetal bovine serum) (Euroclone, Milan, Italy). Experiments were performed when cell purity was >95% as verified by flow cytometry, assessing to the following markers: CD15⁺ (Kromo Orange Bio-Legend) CD16⁺⁺(Pacific Blue Beckman Coulter) CD45^dim (ECD Bio-Legend). When >5% of contaminating eosinophils were detected in some preparations, the samples were excluded from the protocol. Since the average half-life of circulating neutrophils, in physiologic condition is about 1 day, all our experiments were performed over 24 h (Pillay et al., 2010).

Neutrophils for all experiment were treated with or without 300 ng/mL of E1 or 300 ng/mL E2 or 150 ng/mL E1 + 150 ng/mL E2 (Bio-Techne McKinley Place NE Minneapolis, United States).

For gene expression analysis and migration assay neutrophils were treated with rhERAPs for 3 h while for flow cytometry, autophagy and western blot analysis the neutrophils were challenged with rhERAPs for 24 h. Results were always compared with the untreated condition (CTR). All the experiments were performed in duplicate for each subject.

Neutrophil viability following 24 h rhERAP-treatment was determined through Trypan Blue exclusion assay. Briefly, 10 μL of cell suspension were mixed and incubated with 10 μL of 0.4% Trypan Blue (Merck-Sigma, Milan, Italy) in 96-well plates. Ten microliters of the mix were loaded on chamber slides and counted with the T20 Automated Cell Counter (Bio-Rad Laboratories, Hercules, CA, United States). Cells were routinely checked for mycoplasma contamination, and cell viability/count.

At 24 h post rhERAP administration, cells were centrifuged (1,200 rpm for 10 min at RT) for 8 min, supernatants were stored at −80°C for further analysis while cells were washed 2 times with PBS and resuspended at the concentration of 5 × 10⁵ cells/100 μL of PBS incubated with the antibody for 15 min at room temperature (RT), protected by light. Neutrophils were characterized as CD15⁺ (Kromo Orange Bio-Legend) CD16⁺⁺(Pacific Blue Beckman Coulter) CD45^dim (ECD Bio-Legend) expressing cells; neutrophil activation was assessed by the expression of MAC-1 (PC7 Bio-Legend), CD66b (APC Bio-Legend) and LAMP-1 (FITC Beckman Coulter) CCR7 (PC5.5 Bio-Legend). To verify ERAP1 and ERAP2 internalization neutrophils were incubated with anti-ERAP1 (R&D) 0.5 ug/100 uL + Goat anti-mouse Alexa Fluor and anti-ERAP2 (R&D) 0.5 ug/100 uL + Goat anti-mouse Alexa Fluor488 for 45 min. To assess ERAP internalization prior of antibody exposure neutrophils were treated for 45 min with BD’s Permeabilization buffer (cat 554715) and washed with BD’s Fix PERM buffer. After incubation with specific antibodies the cells were washed and immediately analysed by flow cytometry. The detection of intracellular granule cells during the last 6 h of stimulation, 1 μg/mL of Brefeldin A (Sigma-Aldrich) was added to block protein secretion. After 18 h neutrophils were permeabilized 30 min with the Fixation/Permeabilization Buffer (eBiosciences), stained with MPO (PE Beckman coulter), washed with PBS (Euroclone, Milan, Italy) and fixed in 1% paraformaldehyde (PFA, Sigma-Aldrich, MO, United States). Samples acquisition was performed on a CytoFLEX™ flow cytometer system equipped with CytExpert software (Beckman Coulter), and data were analyzed using Kaluza software, version 2.1.1. (Beckman Coulter).

Cellular RNA isolation, reverse transcription (RT) into cDNA, as well as amplification and quantification through real-time qPCR were performed according to a standardized protocol (Biasin et al., 2016). Briefly, 1 × 10⁶ cells were washed in PBS and collected in RNAzol^® (Duotech, Milano, Italy) and RNA extraction was performed through the phenol-chloroform method. RNA was dissolved in RNase-free water and quantified by the Nanodrop 2000 Instrument (1 μL, Thermo Scientific, Waltham, MA, United States). One μg of RNA was purified from genomic DNA with RNase-free DNase (RQ1 DNase; Promega) and reverse transcribed into first-strand cDNA with Moloney murine leukaemia virus reverse transcriptase along with random hexanucleotide primers, oligo dT, and dNTPs (Promega, Fitchburg, WI, United States). cDNA was amplified and quantified by real-time qPCR (CFX96 connect, Bio-Rad, Hercules, CA, United States) through SYBR Green PCR mix (Bio-Rad, Hercules, CA, United States), according to the following thermal profile: initial denaturation (95°C, 15 min), denaturation (15 s at 95°C × 40), annealing (1 min at 60°C) and extension (20 s at 72°C). The following genes were analysed: GAPDH, β-actin, NPRL3, GasderminD, IL-1 β, IL-8, CCL-2 and TNFα. Results for gene expression analyses were calculated by the 2^−ΔΔCT equation. Results are presented as the mean N fold (percent) ± SEM of the relative expression units to an internal reference sample and normalized to both the GAPDH and β-actin housekeeping genes.

Gene expression analyses of the main genes involved in the autophagy pathway (LAMP-1, LC3B, BCN1, TFEB, CHOP, XBP1, mTOR, SQSTM1/p62 and CASP3) were performed by droplet digital PCR (ddPCR QX200, Bio-Rad, Hercules, CA, United States). This analysis can increase the detection of rare transcripts and provide absolute quantification of RNA molecules. For all genes involved in the autophagy pathway, 3 µL of diluted cDNA (1:10.000) were mixed with specific primers and ddPCR EvaGreen SuperMix (Bio-Rad, Hercules, CA, United States). The mix was emulsified with droplet generator oil (Bio-Rad, Hercules, CA, United States) using a QX200 droplet generator, according to the manufacturer’s instructions. Droplets were transferred to a 96-well reaction plate and heat-sealed with a pierce able sealing foil sheet (PX1, PCR plate sealer, Bio-Rad, Hercules, CA, United States). PCR amplification was performed in a sealed 96-well plate using a T100 thermal cycler (Bio-Rad, Hercules, CA, United States). The thermal profile was: 3 min at 25°C, 60 min at 50°C, 10 min at 95°C, 45 cycles at 95°C for 15 s and at 60 s at 60°C, then 10 min at 98°C and finally hold at least for 30 min at 4°C. The 96-well plates were then transferred to a QX200 droplet reader (Bio-Rad, Hercules, CA, United States). Each well was queried for fluorescence to determine the number of positive events (droplets); the mRNA concentration was expressed as copies/uL. For ddPCR analysis, the QuantaSoft software version 1.7.4.0917 (Bio-Rad, Hercules, CA, United States) was used to quantify mRNA. Data are summarized as median and Interquartile (IQR) (25° and 75° percentile). All the primers were purchased as already optimized and used at 1x final dilution according to the manufacturer instructions (PrimePCR™ SYBR^® Green Assay, Bio-Rad).

A 17-cytokine multiplex assay was performed on supernatants of neutrophil cell cultures stimulated with E1, E2 or E1+E2 using magnetic bead immunoassays according to the manufacturer’s protocol (BioRad, Hercules). Arbitrary concentrations of 4,000 pg/mL and 0.1 pg/mL were assigned to values respectively over or below the detection limit.

Cell migration was assessed using 3 μm pore Transwell system (Corning). Briefly, 0.5 × 10⁶ neutrophils were either untreated (CTR) or treated with 300 ng/mL of E1, E2 or E1+E2 for 24 h and then centrifuged. The supernatants were seeded in the lower chamber and used as chemoattractant, while cells were resuspended in RPMI 1640 + 10% FBS and seeded into the upper chamber of the Transwell insert. After 3h, migration was assessed by counting the migrated cells in the lower chamber with an automatic cell counter (Bio-Rad, Hercules, CA, United States) and stained for flow cytometry analyses. As the positive control (CTR+) of the migration assay, neutrophils were resuspended in RPMI+ 2% FBS, while in the bottom chamber control (CTR+) the medium was RPMI1640 + 90%FBS.

Neutrophils (1 × 10⁶), stimulated as described above for 24 h, were cultured with latex beads coated with rabbit IgG–FITC complex (Cayman Chemical) to a final dilution of 1:100 for 1 h. Medium containing 0.05% Trypsin–EDTA (Code ECB3052D; EuroClone) was added for 10 min at 37°C in 5% CO₂. Cells were then resuspended in RPMI 1640 supplemented with 10% FBS and centrifuged at 1,500 rpm for 10 min. Pellets were fixed with 0.1% PFA for 10 min, washed and resuspended in 50 μL PBS. Samples acquisition was performed on a CytoFLEX™ flow cytometer system equipped with CytExpert software (Beckman Coulter), and data were analysed using Kaluza software, version 2.1.1. (Beckman Coulter).

To quantify the release of granule proteins upon degranulation, supernatants from 24 h rhERAP-treated neutrophils were analysed via human MPO ELISA kit (eBioscience, Vienna, Austria), and human elastase ELISA kit (R&D system Minneapolis United States) according to the manufacturers’ instructions.

For the detection of ROS, 5 × 10⁵ neutrophils pre-treated with rhERAPs for 3 h were incubated with 10 µM of 2′–7′-dichlorofluorescein diacetate (DCFDA) (Thermo Fisher Scientific, Waltham, MA, United States) solution for 30 min at 37°C. DCFDA was deacetylated by cellular esterase to a non-fluorescent compound, which was then oxidized by ROS into the fluorescent molecule 2′,7′-dichlorofluorescein (DCF) (λexc = 495 nm, λemm = 529 nm). Dye fluorescent emission was measured at different time points (30, 60, 120 min) using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific, Waltham, MA, United States). DCFDA fluorescence was normalized on nuclei after DAPI staining. Results are shown as the mean of three independent experiments performed in quintuplicate ± SD.

Oxygen consumption rate triggered by ROS production by neutrophils was evaluated by high-resolution respirometry using the O₂K oxygraph chambers (Oroboros, Instruments, Innsbruck, Austria). Granulocytes were trypsinized and 1 × 10⁶ cells were resuspended in the respiration medium MiR06 [0.5 mM EGTA, 3 mM MgCl₂, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH₂PO₄ 20 mM Hepes, 110 mM sucrose, and 1 g/L bovine serum albumin fatty acid-free, 280 U/mL catalase (pH 7.1)] and placed in the 2 mL closed oxygraphy and chamber at 37°C under continuous agitation (300 rpm). After 15 min of baseline O₂ measurement, either rhERAPs (300 ng/mL) or PMA (100 ng/mL) were added. The concentration of O₂ (µM) was monitored for 30 min and OCRs were expressed in pmol O₂/(s*Million cells).

Total proteins were extracted from 5 × 10⁶ cells using RIPA Buffer and protein concentration was measured with a BCA kit (MilliporeSigma) supplemented with 2% SDS, and a cocktail of protease inhibitors (Complete Roche Applied Science, Mannheim, Germany), sonicated, incubated on ice for 30 min on a platform rotator, and then centrifuged at maximum speed for 30 min at 4°C. Protein concentration from whole cell lysates was determined through BCA protein assay kit (Pierce, United States). Subsequently, 40ug of protein extracts were separated by 10% SDS-PAGE under reducing conditions (2.5% 2-mercaptoethanol) and were then transferred onto PVDF membranes. Membranes were blocked for 1 h with 5% bovine serum albumin (BSA) (Sigma-Aldrich, MO, United States) in Tris-buffered saline containing 0.01% Tween-20 (TBS-T), and probed overnight at 4 °C with the following primary antibodies: anti-SQSTM1 (p62) (Cell Signalling), 1:1000 in 5% BSA in TBS-T; anti-LC3B (Cell Signalling) 1:1000 in 5% BSA in TBS-T; anti-ERAP1 (R&D) 1:1000 in 5% BSA in TBS-T; anti-ERAP2 (R&D) 1:1000 in 5% BSA in TBS-T. After incubation with the appropriate HPR-conjugated secondary antibody (Cell Signalling) (1:10.000 in 5% BSA in TBS-T), immunoreactive bands were visualized by chemiluminescence (ECL, BioRad, Hercules, CA, United States). Quantification for all targets was performed through normalization to β-actin. For each experiment, a representative blot is shown, and the graphs show the mean ± SEM from n = 3 independent experiments.

24 h post-rhERAP administration, 2.5 × 10⁵ neutrophils were washed in PBS and fixed with 4% PFA for 10 min at RT, followed by permeabilization with 0.1% Triton X-100 in PBS for 10 min at RT, blocking in 5% BSA in PBS for 1 h at RT, and incubation for overnight at 4°C in an humified chamber with primary antibodies Rabbit anti-LC3B, 1:1500 (Cell Signalling); Mouse anti-SQSTM1-P62, 1:300 (Cell Signalling), prepared in 1% BSA-PBS. The next day, samples were washed three times with PBS and incubated for 45 min at RT with secondary antibodies (Goat anti-mouse Alexa Fluor 488 (ab150113) or 647 (ab150115), or Goat anti-rabbit Alexa Fluor 488,647 (ab150079), 1:500, abcam, Cambridge, United Kingdom) prepared in 1% BSA-PBS. Negative controls were performed by omitting primary antibodies. After three washes in PBS, coverslips were carefully removed from the wells and mounted on Superfrost glass slides using a mounting medium with DAPI (Enzo Life Sciences, Milan, Italy). Confocal imaging was performed with a Leica TCS SP5 AOBS microscope system using a 20×/1.30 oil immersion objective (Leica Microsystems, Wetzlar, Germany).

The Student’s t-test was done when appropriate for statistical analysis to compare continuous and categorical variables. One-way ANOVA or two-way ANOVA were applied for non-parametric comparisons. A p-value <0.05 was chosen as the cut-off for significance. Results are expressed as mean ± SEM from n = 5 independent experiments. All statistical analyses and graphs were performed with GraphPad Prism 9 (GraphPad Software, San Diego, CA, United States).

First, we confirmed the enzymatic activity of the rhERAP1 and rhERAP2 proteins (Supplementary Figure S1). To assess the internalization of these recombinant proteins, a Western blot analysis was performed on proteins extracted from neutrophils following 24 h treatment with 300 ng/mL rhERAPs. Western blot results (Figure 2A) show that neutrophils express a baseline concentration of both proteins (CTR) and effectively internalize E1 and E2 after 24-h of exposure. Indeed, increased levels of ERAP1 were detected in neutrophils treated with E1 and E1+E2 compared to CTR (p < 0.05 for both treatments) (Figure 2B). Likewise, ERAP2 levels increased in cells treated with E2 and E1+E2 compared to the control (p < 0.05 for both treatments) (Figure 2C). To further address this finding, we performed additional experiments using flow cytometry, and the results confirmed results from western blot analysis. Specifically, we marked extracellular and intracellular ERAP1 and ERAP2 separately. When cells were treated with recombinant E1 and E + E2, we observed an upregulation of ERAP1 intensity exclusively within neutrophils cells (CTR vs. E1 p < 0.05; CTRvsE1+E2 p < 0.05) (Figure 2D). Similarly, treatment with E2 and E1+E2 led to an increase of intracellular ERAP2 intensity (CTR vs. E2 p < 0.05; CTR vs. E1+E2 p < 0.05) (Figure 2E). Importantly, no fluorescence signals were detected in cells labelled with extracellular staining, indicating that the recombinant proteins were not merely bound to the cell surface but were indeed internalized (Supplementary Figure S2).

Notably, neutrophil viability assessed by an automatic cell counter, immediately and 24 h after rhERAP treatments was always over 90% in all conditions (data not shown).

To verify if rhERAPs impact neutrophil cellular homeostasis, we assessed neutrophil activation by flow cytometry and gene expression analyses. Results summarized in Figure 3A showed that the co-expression of the activation markers integrin Mac-1 (CD11b) and Carcinoembryonic antigen-related cell adhesion molecule 8 (CD66b) on neutrophils (CD15⁺ CD16^bright CD45^dim) was significantly increased following E2 and E1+E2 stimulation compared to CTR (p < 0.05 for E2 and p < 0.01 for E1+E2). E1 treatment also boosted the expression of these activation markers, as well, although differences did not reach statistical significance.

Transcriptional analyses further confirmed that the addition of rhERAPs to the culture medium triggers neutrophil activation, as the expression of several genes orchestrating the inflammasome and cytokine signalling was enhanced. In detail, gene expression of targets involved in the canonical inflammatory milieu was significantly upregulated upon E1 (IL-8: p < 0.05), E2 (NPLR3: p < 0.05; IL-1β: p < 0.05) E1+E2 stimulation (NPRL3: p < 0.05; IL-1β: p < 0.05; IL-8: p < 0.01; TNFα: p < 0.05; CCL2: p < 0.05) compared to the CTR condition and E1 vs. E1+E2 (CCL2: p < 0.05) (Figure 3B). Conversely, GSDMD expression was significantly downregulated following E1+E2 stimulation compared to CTR (p < 0.01) (Figure 3B).

To further characterize the inflammatory profile, highlighted by gene expression analysis, the secretome of rhERAP-stimulated neutrophils was analyzed as well (Figure 3C). After 24 h of rhERAP stimulation, the concentration of IL-1β, G-CSF, IFN-γ, and MIP-1β was significantly increased in E1, E2, and E1+E2 compared to CTR (p < 0.05 for all comparisons). Notably, IL-8 production, the strongest chemoattractant for neutrophils, was augmented as well in rhERAP-stimulated cells (E1 and E1+E2: p < 0.001; E2: p < 0.05) (Figure 3C). As for IL-17 and TNF-α, statistically significant differences were observed only for E1 and E1+E2 compared to CTR (p < 0.05 for both comparisons) (Figure 3C). These results are in line with the pro-inflammatory profile observed in transcriptional analyses and indicate that following rhERAP stimulation, neutrophils release several cytokines, including IL-8, IL-17, TNFα and G-CSF, which are known to drive neutrophil’s migration, activation and recruitment. In addition, these initial results suggested that rhERAPs significantly affect neutrophil balance, pinpointing the need to further delve downstream into the molecular/cellular mechanisms.

We next investigated whether rhERAPs modulate neutrophil motility. To this end, we used a transwell migration model and counted migrated neutrophils while monitoring the expression of MAC-1, the most abundant and potent neutrophil adhesive/migration marker facilitating firm adhesion and transmigration of neutrophils through endothelium to site of infection and/or inflammation (Buffone et al., 2019).

Results showed that neutrophil migration in the bottom chamber of the transwell containing conditioned medium from neutrophils that were 24 h pre-treated with E1, E2 and E1+E2, is significantly increased compared to the CTR condition (E2 and E1+E2 vs. CTR: p < 0.01; E1 vs. CTR: p < 0.05) (Figure 4A).

By using flow cytometry, we further characterized migrated neutrophils in the lower chamber of the transwell. Analysis performed on the basis of CD15⁺ CD16^bright CD45^dim revealed that most of the migrated cells are activated neutrophils, as assessed by MAC-1 and CD66b expression (E2 vs. CTR: p < 0.01) (Figure 4B). Furthermore, the expression of CCR7, a protein associated with migratory events, was significantly enhanced in the migrated neutrophils, compared to CTR (E2 and E1+E2 vs. CTR: p < 0.05) (Figure 4B). These data suggest that the extracellular milieu produced from rhERAP-exposed cells works as a chemoattractant for neutrophils.

Since neutrophil migration is usually coupled with phagocytosis activation (Mayadas et al., 2014), we investigated if this cellular function could be induced by rhERAPs. According to the gating strategy on a CTR sample (Figure 5A), (representative flow cytometry plot (A) and gaiting strategy (B) reported in Supplementary Figure S3), data demonstrate that the ability of neutrophils to phagocytize latex beads coated with fluorescently labeled rabbit IgG is significantly increased following stimulation with rhERAPs (Figure 5B). In particular, stimulation with E2, and E1+E2 induced a significant increase (nearby 2-fold) in neutrophil phagocytic activity compared to CTR (E2 vs. CTR: p < 0.01; and E1+E2 vs. CTR: p < 0.05) (Figure 5B). E1-exposure also triggered neutrophil phagocytosis though differences did not reach statistical significance. Overall, these results indicate that extracellular ERAPs may contribute to enhancing neutrophil phagocytosis.

Besides phagocytosis, antimicrobial activity of neutrophils is based on the release of granules containing myeloperoxidase (MPO) and elastase into phagosomes (Rodríguez et al., 2003) or in the extracellular milieu (Voynow and Shinbashi, 2021; Forrest et al., 2022). Thus, we verified if rhERAPs could influence enzyme-containing granule exocytosis. Results showed this to be the case. Indeed, rhERAPs led to upregulation of elastase (E2 vs. CTR: p < 0.05; E1+E2 vs. CTR: p < 0.05; E2 vs. E1: p < 0.05) (Figure 6A) and MPO concentration in neutrophils’ supernatants (E2 vs. CTR: p < 0.05; E1+E2 vs. CTR: p < 0.05) (Figure 6B).

These results were further confirmed by flow cytometry analyses, as rhERAP-activated neutrophils (CD15⁺ CD16^bright CD45^dimCD66b⁺MAC-1⁺) produced higher quantities of MPO compared to CTR (E2 vs. CTR: p < 0.01; E1+E2 vs. CTR: p < 0.01) (Figure 6C). In addition, E2 and E1+E2 treatments induced higher intracellular MPO production compared to E1 (E2 and E1+E2 vs. E1: p < 0.05). These findings suggest that rhERAPs optimize the antibacterial efficacy of neutrophils.

Given the link between neutrophil activation and reactive oxygen species (ROS) production, we investigated this pathway in rhERAP-stimulated neutrophils. Results obtained by DCFDA assay showed that E1, E2 and E1+E2 stimulations drastically reduce ROS intracellular accumulation, reaching statistical significance at 120 min post stimulation (E1 vs. CTR: p < 0.05; E2 vs. CTR: p < 0.001; E1+E2 vs. CTR: p < 0.001) (Figure 7A); these reductions are significant even when E2 and E1+E2 were compared to E1 (p < 0.05 for both treatments) (Figure 7A).

This is in line with the unchanged oxygen consumption rate (OCR) of neutrophils following rhERAP stimulation, measured by high-resolution respirometry (Figure 7B), in comparison to the ROS-releasing oxidative burst they undergo after phorbol myristate acetate (PMA) stimulation (Figure 7C).

Finally, as autophagy is implicated in the secretory pathway (Patel et al., 2013; Bhattacharya et al., 2014), we hypothesized that the upregulation of the lysosomal-mediated degranulation observed in rhERAP-stimulated cells could be linked to the modulation of autophagy by assessing mRNA expression (Figure 8A) and protein levels of autophagy-related markers (Figures 8C–F).

Results showed that rhERAPs early exposure (3 h) induce significant changes in the expression of genes that are related to the autophagy pathway either directly (BCN1, LC3B, SQSTM1/p62, TFEB, LAMP1, mTOR), or indirectly via the ER-stress-mediated Unfolded Protein Response (CHOP, XBP1, CASP3). Specifically, statistically significant differences were observed in E1 (BCN1: p < 0.05; LC3B: p < 0.05; LAMP1: p < 0.05; XBP1: p < 0.05), E2 (LAMP1: p < 0.01; TFEB: p < 0.05; XBP1: p < 0.05) and in E1+E2 stimulated cells (BCN1: p < 0.05; CHOP: p < 0.05; LC3B: p < 0.05; TFEB: p < 0.05; LAMP1: p < 0.05) compared to CTR. Finally, an increased expression of BCN1 was observed in E1 and E1+E2 vs. E2 (p < 0.05 for both treatments) and of CHOP in E1+E2 vs. E1 (p < 0.05). Notably, a drastic downregulation of CASP3, mTOR and p62 gene expression was observed in E2 compared to CTR for mTOR (p < 0.05) and in E1+E2 stimulated compared to CTR, reaching statistical significance for CASP3 (p < 0.05) and mTOR (p < 0.05) (Figure 8A). Immunofluorescence assay further confirmed autophagy activation in rhERAP-treated neutrophils. Indeed, as shown in the representative Figure 8B, E1E2E1+E2 stimulations resulted in a qualitative accumulation of LC3B along with p62 reduction (Figure 8B).

Finally, western blot analyses corroborated these results (Figure 8C). Indeed, compared to CTR, an increase in LC3B-II/I (E2: p < 0.05; E1+E2: p < 0.05) (Figure 8D), and a decrease of p62 (E1, E2 and E1+E2: p < 0.05) (Figure 8E) were observed in rhERAP-stimulated cells. These data suggest that rhERAP exposure increases the availability of the lipidated LC3 isoform for autophagosome formation and effective degradation of p62 protein, supporting an effective progression of the autophagy pathway. Notably, this was coupled with a reduction of mTOR mRNA and an increase in LAMP1 protein as assessed by flow cytometry (E1; E2; E1+E2 vs. CTR: p < 0.05 for all comparisons) (Figure 8F), consistent with the transcriptional upregulation of LAMP1 mRNA. These data further support the implication of rhERAP-induced autophagy in lysosomal-mediated neutrophil degranulation possibly via ER stress and mTOR inactivation.