The Effects of Matriptase Inhibition on the Inflammatory and Redox

biomedicines

Article The Effects of Matriptase Inhibition on the Inﬂammatory and Redox Homeostasis of Chicken Hepatic Cell Culture Models

Réka Fanni Barna 1,2,*, Máté Mackei 1 , Erzsébet Pászti-Gere 2, Zsuzsanna Neogrády 1 , Ákos Jerzsele 2 and Gábor Mátis 1

1 Division of Biochemistry, Department of Physiology and Biochemistry, University of Veterinary Medicine, 1078 Budapest, Hungary; [email protected] (M.M.); [email protected] (Z.N.); [email protected] (G.M.) 2 Department of Pharmacology and Toxicology, University of Veterinary Medicine Budapest, 1078 Budapest, Hungary; [email protected] (E.P.-G.); [email protected] (Á.J.) * Correspondence: [email protected]

Abstract: The function of the transmembrane serine protease matriptase is well described in mam- mals, but it has not been elucidated in avian species yet. Hence, the aim of the present study was to assess the effects of the 3-amidinophenylalanine (3-AphA)-type matriptase inhibitors MI432 and MI460 on the inflammatory and oxidative state of chicken primary hepatocyte mono-cultures and hepatocyte–nonparenchymal cell co-cultures, the latter serving as a proper model of hepatic inflammation in birds. Cell cultures were exposed to MI432 and MI460 for 4 and 24 h at 10, 25, and 50 µM concentrations, and thereafter the cellular metabolic activity, extracellular interleukin (IL-)6, IL-8, H2O2 and malondialdehyde concentrations were monitored. Both inhibitors caused a transient moderate reduction in the metabolic activity following 4 h exposure, which was restored after 24 h, Citation: Barna, R.F.; Mackei, M.; reflecting the fast hepatic adaptation potential to matriptase inhibitor administration. Furthermore, Pászti-Gere, E.; Neogrády, Z.; Jerzsele, Á.; Mátis, G. The Effects of Matriptase MI432 triggered an intense elevation in the cellular proinflammatory IL-6 and IL-8 production after Inhibition on the Inflammatory and both incubation times in all concentrations, which was not coupled to enhanced oxidative stress and Redox Homeostasis of Chicken lipid peroxidation based on unchanged H2O2 production, malondialdehyde levels and glutathione Hepatic Cell Culture Models. peroxidase activity. These data suggest that physiological matriptase activities might have a key Biomedicines 2021, 9, 450. https:// function in retaining the metabolic and inflammatory homeostasis of the liver in chicken, without doi.org/10.3390/biomedicines9050450 being a major modulator of the hepatocellular redox state.

Academic Editors: Fernando Capela Keywords: serine proteases; proinﬂammatory cytokines; oxidative stress; poultry; liver e Silva and Elsa Leclerc Duarte

Received: 22 March 2021 Accepted: 20 April 2021 1. Introduction Published: 21 April 2021 Matriptase belongs to the type II transmembrane serine proteases (TTSPs), which

Publisher’s Note: MDPI stays neutral have a key role in several physiological and pathological processes [1]. Concerning the with regard to jurisdictional claims in various isoenzymes, matriptase-1 is widely expressed in all types of epithelial tissues [2,3], published maps and institutional affil- while matriptase-2 is mostly localized in the liver [4]. Matriptase is required for postnatal iations. survival to establish and maintain barrier function of the epithelium [5], while improperly regulated matriptase may be involved in a number of pathological conditions, such as tumorigenesis [6], inflammation [7], virus-cell fusion [8,9], or even impaired iron homeostasis [10–12]. The inhibition of matriptase with specific inhibitors, such as the recently developed Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 3-amidinophenylalanine (3-APhA) derivatives, may represent a new, reliable therapeutic This article is an open access article approach for numerous diseases related to matriptase deregulation. For instance, based on distributed under the terms and in vitro [13,14] or in vivo [15] studies, the application of 3-APhA inhibitors can effectively conditions of the Creative Commons reduce the growth and invasion potential of tumor cells [13,14], and may possess antivi- Attribution (CC BY) license (https:// ral [8], antirheumatic [16] or hepcidin-lowering effects [17]. The key role of matriptase creativecommons.org/licenses/by/ in retaining gut-barrier integrity was confirmed on the IPEC-J2 porcine nontransformed 4.0/). small intestinal epithelial cell line, where the inhibitors MI432 and MI460 decreased the

Biomedicines 2021, 9, 450. https://doi.org/10.3390/biomedicines9050450 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 450 2 of 14

transepithelial electrical resistance and increased paracellular transport mechanisms [18]. A short-term exposure of swine-derived hepatic cell culture models to MI441 caused elevated cellular reactive oxygen species (ROS) release coupled to reduced hepcidin expression, while MI460 significantly increased hepcidin secretion [17]. Notwithstanding that matriptase can initiate the protease-activated receptor (PAR)-2-mediated inflammatory pathway [19], the hepatocellular production of the proinflammatory cytokines IL-6 and IL-8 was not affected by the applied 3-APhA type inhibitors on the same cell cultures [17]. There are only few studies investigating matriptase in avian species. Matriptase was mainly detected in mammalian cells; however, its ortholog [20] and matriptase-3 [21] were found in the chicken genome as well. The matriptase-driven proteolytic activation of H9N2 influenza virus was examined in chicken embryo kidney cells. Moreover, the cleavage of hemagglutinin was inhibited by the 3-APhA inhibitor MI462 at 50 µM concentration [8]. As the regulatory action of matriptase in birds has not been elucidated yet, the major aim of the present study was to assess the function of this membrane-anchored serine protease in the liver of chickens by applying the 3-APhA type inhibitors MI432 and MI460 on chicken primary hepatocyte mono- and hepatocyte–nonparenchymal (NP) cell co-cultures. These cell systems provide proper tools for biomedical research of the avian liver, the co-culture serving as a model of the hepatic inflammation. The inhibitors MI432 and MI460 were selected because they have already been effective to inhibit matriptase without harmful cytotoxic effects on mammalian cells [17,18,22]. To fulfill the abovementioned goals, the cellular metabolic activity and oxidative status, as well as the production of proinflammatory cytokines, were monitored, reflecting the role of matriptase in regulating redox and inflammatory homeostasis of the liver.

2. Materials and Methods 2.1. Cell Isolation and Culturing Liver cells were isolated and cell cultures were prepared as described in a previous study of our research group [23], using three-week-old male Ross-308 type broiler chickens, obtained from Gallus Ltd. (Devecser, Hungary). Cell isolation was carried out in accordance with national and EU laws and institutional guidelines, also confirmed by the Local Animal Welfare Committee of the University of Veterinary Medicine, Budapest. Experimental procedures were officially approved by the Government Office of Budapest, Food Chain Safety, Plant Protection, and Soil Conservation Directorate, Budapest, Hungary (number of permission: PEI/001/1430-4/2015; approval date: 27 April 2015). All chemicals were purchased from Merck KGaA (Darmstadt, Germany) except when otherwise specified. Briefly, a multistep in situ perfusion of the liver was performed via the gastropancreati- coduodenal vein, applying 150 mL ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA, 0.5 mM) containing Hanks’ Balanced Salt Solution (HBSS) with 0.035% NaHCO3, 150 mL EGTA-free HBSS, and finally, 100 mL HBSS buffer supplemented with 100 mg collagenase type IV (Nordmark, Uetersen, Germany), 7 mM CaCl2, and 7 mM MgCl2 to disintegrate hepatic cells. After disrupting the capsule of the liver, the gained cells were suspended in 2.5% bovine serum albumin (BSA) containing HBSS and were filtered through three layers of sterile gauze, followed by a 45 min incubation on ice. There- after, hepatocytes and NP cells were isolated by a multistep differential centrifugation as previously described [23]. Both cell fractions were resuspended in Williams’ Medium E, supplemented with 0.22% NaHCO3, 50 mg/L gentamycin, 2 mM glutamine, 4 µg/L dexam- ethasone, 20 IU/L insulin, and 5% foetal bovine serum (FBS). The viability of hepatocytes and NP cells was examined by the trypan blue exclusion test, and cell suspensions were diluted after cell counting in a Bürker chamber to the appropriate seeding concentrations (hepatocyte mono-cultures: 106 cells/mL; co-cultures: 8.5 × 105 cells/mL hepatocytes, 1.5 × 105 cells/mL NP cells). Both hepatocyte and NP cell enriched fractions have been previously characterized by flow cytometry and immunofluorescent detection of specific markers for hepatocytes and macrophages [23]. BiomedicinesBiomedicines 2021 2021, ,9 9, ,450 450 33 of of 14 14

6 5 seedingseeding concentrationsconcentrations (hepatocyte(hepatocyte mono-cultures:mono-cultures: 1010 6 cells/mL;cells/mL; co-cultures:co-cultures: 8.58.5 ×× 1010 5 5 cells/mL hepatocytes, 1.5 × 10 5cells/mL NP cells). Both hepatocyte and NP cell enriched Biomedicines 2021, 9, 450 cells/mL hepatocytes, 1.5 × 10 cells/mL NP cells). Both hepatocyte and NP cell enriched3 of 14 fractionsfractions have have been been previously previously characterized characterized by by flow flow cytometry cytometry and and immunofluorescent immunofluorescent detectiondetection of of specific specific markers markers for for hepatocytes hepatocytes and and macrophages macrophages [23]. [23]. 2 2 CellsCells were were seeded seeded on on 6-well 6-well (to (to monitor monitor extracellular extracellular H HO2O,2 ,interleukin interleukin (IL-)6, (IL-)6, IL-8, IL-8, andand malondialdehydeCellsmalondialdehyde were seeded (MDA) on(MDA) 6-well concentrationsconcentrations (to monitor extracellular andand intracellularintracellular H2O2 , glutathione interleukinglutathione (IL-)6,peroxidaseperoxidase IL-8, (GPx)and(GPx) malondialdehyde activities) activities) or or 96-well 96-well (MDA) (to (to assess assess concentrations cellular cellular metabolic metabolic and intracellular activity) activity) plates glutathioneplates (Greiner (Greiner peroxidase Bio-One, Bio-One, 2 Frickenhausen,(GPx)Frickenhausen, activities) Germany), Germany), or 96-well previously (topreviously assess cellular coated coated metabolicwith with collagen collagen activity) type type platesI I(10 (10 µg/cm (Greinerµg/cm).2). To Bio-One,To estab- estab- 2 lishFrickenhausen,lish the the co-cultures, co-cultures, Germany), NP NP cells cells previously were were seeded seeded coated firs firs witht,t, and and collagen following following type their Itheir (10 µadherence adherenceg/cm ). To in in establish 20 20 min, min, hepatocytesthehepatocytes co-cultures, werewere NP addedadded cells in werein thethe seeded cellcell ratioratio ﬁrst, ofof and 6:16:1 (hepatocyte following(hepatocyte their toto NP adherenceNP cells).cells). HepatocyteHepatocyte in 20 min, mono-cultureshepatocytesmono-cultures were were were added also also inprepared prepared the cell ratioby by seedin seedin of 6:1gg (hepatocyteonly only the the hepatocyte-enriched hepatocyte-enriched to NP cells). Hepatocyte fraction. fraction. mono- The The seedingculturesseeding volume werevolume also was was prepared 1.5 1.5 mL/well mL/well by seeding on on 6-well 6-well only pl theplatesates hepatocyte-enriched and and 100 100 µL/well µL/well on fraction.on 96-well 96-well The plates. plates. seeding All All volume was 1.5 mL/well on 6-well plates and 100 µL/well on 96-well2 plates. All cell cellcell cultures cultures were were incubated incubated at at 38.5 38.5 °C °C in in humid humid atmosphere atmosphere with with 5% 5% CO CO.2 .Culture Culture media media ◦ werecultureswere changedchanged were incubated 44 hh followingfollowing at 38.5 C seeding;seeding; in humid hepatocyte atmospherehepatocyte mono-cultures withmono-cultures 5% CO2. Culture (Figure(Figure media 1A)1A) were andand hepatocyte–NPchangedhepatocyte–NP 4 h following cell cell co-cultures co-cultures seeding; hepatocyte(Figure (Figure 1B) 1B) mono-cultures fo formedrmed confluent confluent (Figure monolayers monolayers1A) and hepatocyte–NP after after 24 24 h h cul- cul- turingcellturing co-cultures [23]. [23]. (Figure1B) formed conﬂuent monolayers after 24 h culturing [23].

Figure 1. Giemsa staining of hepatocyte mono-cultures (A) and hepatocyte–nonparenchymal cell FigureFigure 1.1. GiemsaGiemsa stainingstaining of hepatocyte mono-cultures ( (AA)) and and hepatocyte–nonparenchymal hepatocyte–nonparenchymal cell cell co-cultures (B) after 48 h culturing (20× magnification, bar = 100 µm). co-culturesco-cultures ( B(B)) after after 48 48 h h culturing culturing (20 (20×× magniﬁcation,magnification, barbar == 100 µµm).m).

2.2.2.2. Treatments TreatmentsTreatments and and Samplings Samplings AfterAfter 24 24 h h incubation,incubation, culture culture media media were were removed removed and and culturescultures werewere exposed exposed toto thethethe inhibitors inhibitors MI432 MI432MI432 and andand MI460 MI460MI460 for forfor 4 4 and and 2424 h h at at 10, 10, 25, 25, andand and 5050 50 µµM µMM concentrations.concentrations. concentrations. The The structuresstructures (Figure(Figure (Figure2 )2) 2) and and and inhibitory inhibitory inhibitory constant constant constant (Ki) (Ki) (Ki) values values values (Table (Table (Table1) of 1) the1) of of applied the the applied applied MI432 MI432 MI432 and andMI460and MI460 MI460 inhibitors inhibitors inhibitors were were alreadywere alreadyalready published publishepublishe by Hammamidd byby Hammami Hammami et al. [ et24et ]. al.al. All [24].[24]. 3-APhA AllAll 3-APhA3-APhA inhibitors inin- hibitorswerehibitors kindly were were provided kindly kindly provided provided by the Institute by by the the ofInstitute Institute Pharmaceutical of of Pharmaceutical Pharmaceutical Chemistry, Chemistry, Chemistry, Faculty of Pharmacy,FacultyFaculty of of Pharmacy,PhilippsPharmacy, University, Philipps Philipps University, Marburg,University, Germany. Marburg, Marburg, Germany. Germany.

FigureFigure 2. 2. StructuresStructures Structures of of the the used used matriptase matriptase inhibitors. inhibitors.

Table 1. The Ki (inhibitory constant) values of the applied matriptase inhibitors. TableTable 1. 1.The The K Ki i(inhibitory (inhibitory constant) constant) values values of of the the applied applied matriptase matriptase inhibitors. inhibitors.

KiKi (µM) (µM)Ki (µ M) Matriptase-1 Matriptase-1 Matriptase-1 Matriptase-2 Matriptase-2 Matriptase-2 MI432 0.002 0.11 MI432MI432 0.002 0.002 0.11 0.11 MI460 0.0018 0.18 MI460MI460 0.0018 0.0018 0.18 0.18

Culture media of 6-well plates were collected directly after the incubation time to assess extracellular H2O2, IL-6, IL-8, and MDA concentrations, while cells were lysed in Mammalian Protein Extraction Reagent (M-PER) buffer supplemented with 1% Halt Protease Inhibitor Cocktail and 1% ethylene diamine tetraacetic acid (EDTA) (Thermo Biomedicines 2021, 9, 450 4 of 14

Fisher Scientiﬁc, Waltham, MA, USA). To standardize the results, total protein concentrations of cell lysates were measured with Pierce Bicinchoninic Acid (BCA) Protein Assay (Thermo Fisher Scientiﬁc, Waltham, MA, USA), using BSA as a standard, following the manufacturer’s description. All samples were stored at −80 ◦C until further processing.

2.3. Measurements of Cellular Metabolic Activity, IL-6, IL-8, H2O2 and MDA Concentrations and GPx Activity Following 4 and 24 h inhibitor treatment, the metabolic activity of cells on 96-well plates was assessed by the Cell Counting Kit-8 (CCK-8) assay (Merck KGaA, Darmstadt, Germany) according to the manufacturer’s instructions, to detect the amount of NADH+H+ generated in the catabolic pathways. Cells were incubated with 10 µL CCK-8 reagent added to 100 µL fresh Williams’ Medium E, and the absorbance was measured at 450 nm with a Multiskan GO 3.2 reader (Thermo Fisher Scientific, Waltham, MA, USA) after 2 h incubation at 38.5 ◦C. The concentrations of IL-6 and IL-8 from cell-free supernatants were assayed by chicken-specific double-sandwich ELISA methods (MyBioSource, Inc., San Diego, CA, USA), following the manufacturer’s instructions, and measuring absorbances at 450 nm with a Multiskan GO 3.2 reader. The extracellular H2O2 concentration was monitored with the Amplex Red Hydrogen Peroxide Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), mixing 50 µL supernatant with 50 µL Amplex Red working solution (composed of 100 µM Amplex Red and 0.2 U/mL horseradish peroxidase), and detecting fluorescence after 30 min incubation at room temperature with a Victor X2 2030 fluorometer (λex = 560 nm; λem = 590 nm). The MDA as a marker of lipid peroxidation was assayed with the MDA Colorimetric Assay Kit (Merck KGaA, Darmstadt, Germany), adding 300 µL thiobarbituric acid (TBA) stock solution to 100 µL culture media. Blends were incubated at 95 ◦C for 1 h and thereafter were cooled down on ice for 10 min. Absorbance was measured at 532 nm with a Multiskan GO 3.2 reader (Thermo Fisher Scientific, Waltham, MA, USA). The GPx activity was assessed from cell lysates using a colorimetric kinetic assay kit (Merck KGaA, Darmstadt, Germany). According to the manufacturer’s instructions, 15 µL cell lysate was added to 455 µL freshly prepared GPx Assay Buffer, 25 µL NADPH Assay Reagent, and 5 µL 30 mM tert-Butyl Hyperoxide solution, the latter serving as the substrate. The absorbance was regularly detected in 10 sec intervals for 1 min at 340 nm. Enzyme activity was determined with the formula provided by the manufacturer.

2.4. Statistical Analysis All treatments were applied in triplicates (except control cells, n = 6) on both 6-well and 96-well plates. Results of the measured parameters were standardized to the total protein concentrations of cell lysates. Data were analyzed with one-way ANOVA and Tukey’s post hoc test and Spearman’s test of correlation using the R 3.6.1. software package (Vienna, Austria, 2012; accessed on 15 September 2020). Statistical signiﬁcance was set at p < 0.05; the results are expressed as mean ± SEMs.

3. Results In the case of the shorter, 4 h treatment, both inhibitors signiﬁcantly reduced the metabolic activity of the cells in mono- and co-cultures in certain cases, as indicated in Figure3A. The observed effect was similar in case of exposure to MI432 and MI460 inhibitors in each investigated cell culture model (Figure3A). In contrast, the longer, 24 h treatment with MI432 or MI460 did not signiﬁcantly affect metabolic activity of cultured cells (p > 0.05 in all cases) (Figure3B). Biomedicines 2021, 9, 450 5 of 14

Biomedicines 2021, 9, 450 5 of 14 treatment with MI432 or MI460 did not significantly affect metabolic activity of cultured cells (p > 0.05 in all cases) (Figure 3B).

A Cellular metabolic activity, 4 h 1.8 1.6 1.4 * 1.2 * 1 0.8 * * * 0.6 0.4 0.2 0 RELATIVE ABSORBANCE (MEAN ± SEM)

HEPATOCYTE MONO-CULTURE HEPATOCYTE–NP CELL CO-CULTURE

B Cellular metabolic activity, 24 h 1

0.8

0.6

0.4

0.2

0 RELATIVE ABSORBANCE (MEAN ± SEM)

HEPATOCYTE MONO-CULTURE HEPATOCYTE–NP CELL CO-CULTURE

Figure 3. The effects of MI432 and MI460 inhibitors on the metabolic activity of hepatocyte mono- Figure 3. The effects of MI432 and MI460 inhibitors on the metabolic activity of hepatocyte cultures and hepatocyte–NP cell co-cultures after 4 (A) and 24 (B) h of incubation at 10, 25, and mono-cultures and hepatocyte–NP cell co-cultures after 4 (A) and 24 (B) h of incubation at 10, 25, 50 µM concentrations, assessed by a CCK-8 test. p values belonging to signiﬁcant differences: mono- and 50 µM concentrations, assessed by a CCK-8 test. p values belonging to significant differences: cultures, 4 h: p µ = 0.0131; p µ = 0.0197; p µ = 0.0111; co-cultures, 4 h: mono-cultures, 4 MI432h: pMI432 10 10M µM = 0.0131; pMI460 25 25µM =M 0.0197; pMI460MI460 50 µM = 50 0 .0111;M co-cultures, 4 h: pMI432 25 pµMMI432 = 0.0457; 25 µM p=MI460 0.0457; 50 µM =p MI4600.0187. 50 µRelativeM = 0.0187. absorbance Relative values absorbance were calculated values were by calculatedconsidering by the consid- meanering absorbance the mean absorbanceof control hepatocyte of control mono-cultures hepatocyte mono-cultures at 4 h as 1 (mean at 4 h ± asSEM; 1 (mean nctr = ±6, SEM;nMI = nctr = 6, 3/group;nMI = 3/group; * p < 0.05). * p < 0.05).

TheThe inhibitors inhibitors MI432 MI432 and and MI460 MI460 had had different different effects effects on on the the concentration concentration of of IL-6 IL-6 in in thethe supernatants. supernatants. MI432 MI432 significantly significantly increased increased the the concentration concentration of of IL-6 IL-6 for for 4 4h hand and 24 24 h h incubationincubation times. times. This This IL-6 IL-6 elevating elevating effect effect was was more more pronounced pronounced on onmono-cultures mono-cultures for for4 h 4compared h compared to co-cultures, to co-cultures, and and the theextent extent was wassimilar similar for 24 for h 24in hboth inboth cell models. cell models. On the On otherthe otherhand, hand, MI460 MI460 did didnot notsignificantly significantly influe influencence the the IL-6 IL-6 production production of ofcultured cultured cells cells (except(except a asignificant significant reducing reducing effect effect on on co-culture co-culture at at 25 25 µMµM for for 24 24 h) h) (Figure (Figure 4A,B).4A,B). BiomedicinesBiomedicines 20212021, 9, ,9 450, 450 6 6of of 14 14

A IL-6 concentration, 4 h 5 4.5 4 3.5 3 2.5 2

(MEAN ± SEM) 1.5 1 0.5

RELATIVE IL-6 CONCENTRATION 0

HEPATOCYTE MONO-CULTURE HEPATOCYTE–NP CELL CO-CULTURE

B IL-6 concentration, 24 h 6

2 (MEAN ± SEM) 1

0 RELATIVE IL-6 CONCENTRATION

HEPATOCYTE MONO-CULTURE HEPATOCYTE–NP CELL CO-CULTURE

Figure 4. The effects of MI432 and MI460 on the IL-6 production of hepatocyte mono-cultures and Figurehepatocyte–NP 4. The effects cell of co-cultures MI432 and after MI460 4 (A on) and the 24 IL-6 (B) pr hoduction of treatment of hepatocyte at 10, 25, and mono-cultures 50 µM, assessed and by hepatocyte–NP cell co-cultures after 4 (A) and 24 (B) h of treatment at 10, 25, and 50 µM, assessed the ELISA method. p values belonging to signiﬁcant differences: mono-cultures, 4 h: pMI432 10 µM = by the ELISA method. p values belonging to significant differences: mono-cultures, 4 h: pMI432 10 µM = 0.0115; pMI432 25 µM = 0.0006; pMI432 50 µM < 0.0001; co-cultures, 4 h: pMI432 25 µM = 0.0269; pMI432 50 µM 0.0115; pMI432 25 µM = 0.0006; pMI432 50 µM < 0.0001; co-cultures, 4 h: pMI432 25 µM = 0.0269; pMI432 50 µM = 0.0421; = 0.0421; mono-cultures, 24 h: pMI432 10 µM = 0.0116; pMI432 25 µM = 0.0004; pMI432 50 µM = 0.0021; mono-cultures, 24 h: pMI432 10 µM = 0.0116; pMI432 25 µM = 0.0004; pMI432 50 µM = 0.0021; co-cultures, 24 h: co-cultures, 24 h: pMI432 10 µM = 0.0018; pMI432 25 µM < 0.0001; pMI432 50 µM = 0.0004; pMI460 25 µM = pMI432 10 µM = 0.0018; pMI432 25 µM < 0.0001; pMI432 50 µM = 0.0004; pMI460 25 µM = 0.0346. Relative concentrations were0.0346. calculated Relative by concentrations considering the were mean calculated concentration by considering of control thehepatocyte mean concentration mono-cultures of at control 4 h ashepatocyte 1 (mean ± SEM; mono-cultures nctr = 6, nMI at = 43/group; h as 1 (mean * p < 0.05;± SEM; ** p

A IL-8 concentration, 4 h 3.5 3 2.5 2 1.5

(MEAN ± SEM) 1 0.5 RELATIVE IL-8 CONCENTRATION 0

HEPAOCYTE MONO-CULTURE HEPATOCYTE NP–CELL CO-CULTURE B IL-8 concentration, 24 h 2 1.8 1.6 1.4 1.2 1 0.8 0.6 (MEAN ± SEM) 0.4 0.2 0 RELATIVE IL-8 CONCENTRATION

HEPATOCYTE MONO-CULTURE HEPATOCYTE–NP CELL CO-CULTURE

Figure 5. The effects of MI432 and MI460 on the IL-8 production of hepatocyte mono-cultures and

hepatocyte–NP cell co-cultures after 4 (A) and 24 (B) h of treatment at 10, 25, and 50 µM, assessed by Figure 5. The effects of MI432 and MI460 on the IL-8 production of hepatocyte mono-cultures and the ELISA method. p values belonging to signiﬁcant differences: mono-cultures, 4 h: pMI432 10 µM hepatocyte–NP cell co-cultures after 4 (A) and 24 (B) h of treatment at 10, 25, and 50 µM, assessed = 0.0011; pMI432 25 µM = 0.0019; pMI432 50 µM = 0.0068; co-cultures, 4 h: pMI432 10 µM = 0.0022; by the ELISA method. p values belonging to significant differences: mono-cultures, 4 h: pMI432 10 µM = pMI432 25 µM = 0.0001; pMI432 50 µM = 0.0231; mono-cultures, 24 h: pMI432 10 µM < 0.0001; pMI432 25 µM 0.0011; pMI432 25 µM = 0.0019; pMI432 50 µM = 0.0068; co-cultures, 4 h: pMI432 10 µM = 0.0022; pMI432 25 µM = 0.0001; < 0.0001; p = 0.0010; p = 0.0097; co-cultures, 24 h: p = 0.0003; pMI432 50 µM = 0.0231;MI432 50 mono-cultures,µM 24MI460 h: p 10MI432µM 10 µM < 0.0001; pMI432 25 µM < 0.0001; pMI432 50 10 µMµ =M 0.0010; ppMI460MI432 10 µM 25 µ=M 0.0097;< 0.0001; co-cultures,pMI432 50 24µ Mh: =pMI432 0.0179. 10 µM = Relative 0.0003; p concentrationsMI432 25 µM < 0.0001; were pMI432 calculated 50 µM = 0.0179. by consid-Rela- tiveering concentrations the mean concentration were calculated of control by consider hepatocyteing the mono-cultures mean concentration at 4 h as of 1(mean control± hepatocyteSEM; nctr = 6, mono-culturesnMI = 3/group; at * 4p h< as 0.05; 1 (mean ** p < ± 0.01; SEM; *** npctr< = 0.001).6, nMI = 3/group; * p < 0.05; ** p < 0.01; *** p < 0.001).

ThereThere were were no no significant significant differences differences in in extracellular extracellular H 2OO22 levelslevels in in most most cases cases when when exposingexposing the the cells cells to to MI432 MI432 or or MI460 MI460 inhibitors. inhibitors. Exceptionally, Exceptionally, MI432 MI432 at at 25 25 and and 50 50 µMµM increasedincreased the the H H22OO2 2concentrationconcentration in in culture culture media media after after 4 4 h h of of incubation incubation on on hepatocyte hepatocyte mono-cultures,mono-cultures, while while a a significant significant decrease decrease was was observed observed when when applying applying MI460 MI460 at at 10 10 µMµM forfor 4 4 and and 24 24 h h on on co-cultures co-cultures (Figure (Figure 66A,B).A,B). A significantsignificant positive correlation was found betweenbetween H H22OO2 2andand IL-8 IL-8 levels levels on on mono-cultures mono-cultures (p (p == 0.019). 0.019). BiomedicinesBiomedicines 20212021,, 99,, 450 450 88 of of 14 14

A Extracellular H2O2 production, 4 h 2.5 **

1.5 *

1 ** (MEAN ± SEM) 0.5

0 RELATIVE FLUORESCENCE INTENSITY

HEPATOCYTE MONO-CULTURE HEPATOCYTE–NP CELL CO-CULTURE

B Extracellular H2O2 production, 24 h 7 6 5 4 3 2

(MEAN ± SEM) 1 0 RELATIVE FLUORESCENCE INTENSITY

HEPATOCYTE MONO-CULTURE HEPATOCYTE–NP CELL CO-CULTURE

Figure 6. The effects of MI432 and MI460 on the extracellular H2O2 levels of hepatocyte mono- Figure 6. The effects of MI432 and MI460 on the extracellular H2O2 levels of hepatocyte cultures and hepatocyte–NP cell co-cultures after 4 (A) and 24 (B) h of treatment at 10, 25, and 50 µM, mono-cultures and hepatocyte–NP cell co-cultures after 4 (A) and 24 (B) h of treatment at 10, 25, andassessed 50 µM, by assessed the Amplex by the Red Amplex method. Redp valuesmethod. belonging p values tobelonging significant to significant differences: differences: mono-cultures, mono-cultures,4 h: pMI432 25 µM 4 =h: 0.0268;pMI432 25p µMMI432 = 0.0268; 50 µM =pMI432 0.0045; 50 µMco-cultures, = 0.0045; co-cultures, 4 h: p460 10 4µ h:M p=460 0.0048; 10 µM = co-cultures,0.0048; 24 h: co-cultures,p460 10 µM = 24 0.0199. h: p460 Relative 10 µM = 0.0199. fluorescence Relative intensities fluorescence were intens calculatedities were by considering calculated theby meanconsidering fluores- thecence mean intensity fluorescence of control intensity hepatocyte of control mono-cultures hepatocyte at mono-cultu 4 h as 1 (meanres ±at SEM;4 h asn 1ctr (mean= 6, n MI± SEM;= 3/group; nctr = 6,* pnMI< 0.05;= 3/group; ** p < * 0.01). p < 0.05; ** p < 0.01).

TheThe inhibitors inhibitors MI432 MI432 and and MI460 MI460 did did not not signifi signiﬁcantlycantly influence inﬂuence the the concentration concentration of MDAof MDA at any at concentration any concentration for any for time any in time the supernatant in the supernatant of cell cultures. of cell In cultures. one case, In there one wascase, a mild there increase was a mild by MI432 increase at 25 by µM MI432 for 4 h at on 25 hepatocyteµM for 4 h mono-cul on hepatocytetures (Figure mono-cultures 7A,B). (Figure7A,B). Biomedicines 2021, 9, 450 9 of 14 Biomedicines 2021, 9, 450 9 of 14

A MDA production, 4h 1.2

0.8

0.6

0.4

0.2

0 RELATIVE ABSORBANCE (MEAN ± SEM)

HEPATOCYTE MONO-CULTURE HEPATOCYTE–NP CELL CO-CULTURRE

B MDA production, 24 h 1.4 1.2 1 0.8 0.6 0.4 0.2 0 RELATIVE ABSORBANCE (MEAN ± SEM)

HEPATOCYTE MONO-CULTURE HEPATOCYTE–NP CELL CO-CULTURE

Figure 7. The effects of MI432 and MI460 on the malondialdehyde (MDA) concentration of hepatocyte Figuremono-cultures 7. The effects and hepatocyte–NP of MI432 and cell MI460 co-cultures on the after malondialdehyde 4 (A) and 24 (B) h (MDA) of treatment concentration at 10, 25, and of hepatocyte50 µM, assessed mono-cultures by a speciﬁc and colorimetric hepatocyte–NP assay kit. cellp value co-cultures belonging after to the4 (A signiﬁcant) and 24 ( difference:B) h of treatment

atmono-cultures, 10, 25, and 50 4 h:µM,pMI432 assessed 25 µM = by 0.0146. a specific Relative colorimetric absorbance assay values kit. were p value calculated belonging by considering to the signifi- MI432 25 µM cantthe meandifference: absorbance mono-cultures, value of control 4 h: hepatocytep = mono-cultures 0.0146. Relative at 4 absorbance h as 1 (mean values± SEM; werenctr = calculated 6, by considering the mean absorbance value of control hepatocyte mono-cultures at 4 h as 1 (mean ± nMI = 3/group; * p < 0.05). SEM; nctr = 6, nMI = 3/group; * p < 0.05). The GPx activity of cell lysates was signiﬁcantly increased by the highest concentration (50 µTheM) ofGPx MI460 activity on both of cell mono- lysates and co-cultures,was significantly while noincreased signiﬁcant by differencesthe highest were concentra- tionobserved (50 µM) in the of other MI460 cases on compared both mono- to controls and co (Figure-cultures,8). while no significant differences were observed in the other cases compared to controls (Figure 8). Biomedicines 2021, 9, 450 10 of 14 Biomedicines 2021, 9, 450 10 of 14

FigureFigure 8.8.The Theeffects effects of of MI432 MI432 and and MI460 MI460 on on the the glutathione glutathione peroxidase peroxidase (GPx) (GPx) activity activity of of hepatocyte hepatocyte mono-cultures and hepatocyte–NP cell co-cultures after 24 h of treatment at 10, 25, and 50 µM, mono-cultures and hepatocyte–NP cell co-cultures after 24 h of treatment at 10, 25, and 50 µM, assessed by a specific colorimetric assay kit. p values belonging to the significant differences: assessed by a specific colorimetric assay kit. p values belonging to the significant differences: mono- mono-cultures: pMI460 50µM = 0.0446; co-cultures: pMI460 50 µM = 0.0273. Relative activity values were cultures: p = 0.0446; co-cultures: p = 0.0273. Relative activity values were calculated MI460by considering 50µM the mean of control MI460hepatocyte 50 µM mono-cultures as 1 (mean ± SEM; nctr = 6, calculatednMI = 3/group; by considering * p < 0.05). the mean of control hepatocyte mono-cultures as 1 (mean ± SEM; nctr = 6, nMI = 3/group; * p < 0.05). 4. Discussion 4. Discussion The present study investigated the effects of matriptase inhibition on cellular met- The present study investigated the effects of matriptase inhibition on cellular metabolic, inflammatory,abolic, inflammatory, and redox and parameters redox parameters of hepatic of cellhepatic culture cell models,culture models, highlighting highlighting the po- tentialthe potential regulatory regulatory role of matriptaserole of matriptase in the chicken in the chicken liver. The liver. applied The applied hepatocyte hepatocyte mono- culturesmono-cultures and hepatocyte–NP and hepatocyte–NP cell co-cultures cell co-c wereultures previously were established previously and established characterized and bycharacterized Mackei et al. by [23 Mackei], considered et al. [23], as proper consider modelsed as forproper studying models the for avian studying hepatocellular the avian inflammatoryhepatocellular and inflammatory stress response. and Hence,stress response. they could Hence, serve asthey good could tools serve for investigations as good tools relatedfor investigations to the consequences related to of the matriptase consequences inhibition of matriptase in birds asinhibition well. in birds as well. InIn thethe presentpresent study,study, basedbased onon thethe resultsresults ofof thethe CCK-8CCK-8 assay,assay, the the aerobe aerobe catabolic catabolic activityactivity of of the the cultured cultured cells cell moderatelys moderately decreased decreased after after applying applying both both inhibitors inhibitors MI432 MI432 and MI460and MI460 in certain in certain concentrations concentrations for 4 for h. However,4 h. However, the extent the extent of the of reduction the reduction indicated indi- thatcated the that inhibitors the inhibitors were not were cytotoxic, not cytotoxic, in line in with line a with previous a previous study instudy which in which T-2 toxin T-2 causedtoxin caused a similar a similar decline decline in the metabolic in the metabolic activity ofactivity the same of the cell same culture cell models culture without models beingwithout cytotoxic being [cytotoxic25]. Following [25]. Following the longer, the 24 hlonger, incubation, 24 h noincubation, significant no differences significant were dif- observedferences were between observed control between and MI-treated control cellsand anymore,MI-treated suggesting cells anymore, the rapid suggesting metabolic the adaptationrapid metabolic of liver adaptation cells to the of applied liver cells MIs. to the applied MIs. ConcerningConcerning the the extracellular extracellular proinflammatory proinflammator cytokiney cytokine levels, levels, both both IL-6 IL-6 and IL-8and con-IL-8 centrationsconcentrations of cell-free of cell-free supernatants supernatants were were remarkably remarkably increased increased on on all all MI432-exposed MI432-exposed mono-mono- and and co-cultures, co-cultures, both both after after 4 and4 and 24 24 h incubationh incubation time time (with (with the the exception exception of IL-6 of IL-6 for thefor co-culturesthe co-cultures treated treated with with MI432 MI432 at 10at 10µM µM for for 4h). 4 h). The The MI-triggered MI-triggered elevation elevation in in the the cytokinecytokine release release was was more more pronounced pronounced on on mono-cultures mono-cultures than than on co-cultureson co-cultures after after 4 h, but4 h, thebut extent the extent was approximatelywas approximately the same the aftersame 24 af hter exposure 24 h exposure on both on cell both culture cell culture models. mod- The moreels. The notable more MI432-evoked notable MI432-evoked cytokine release cytokine of mono-culturesrelease of mono-cultures might be related might to be the related find- ingto the that finding hepatocytes that hepatocytes can predominantly can predomin produceantly a varietyproduce of a proinflammatory variety of proinflammatory cytokines, suchcytokines, as IL-6 such and as IL-8, IL-6 playing and IL-8, a key playing role in a the key inflammatory role in the inflammatory and stress response and stress [26,27 re-]. Sincesponse H 2[26,27].O2 as a Since redox H signaling2O2 as a moleculeredox signaling can induce molecule hepatocellular can induce IL-8 hepatocellular production[ 26IL-8], theproduction elevated [26], IL-8 levelsthe elevated might haveIL-8 beenlevels partly might mediated have been by partly the increased mediated extracellular by the in- ROScreased concentration extracellular in caseROS of concentration MI432 at 25 andin case 50 µ Mof forMI432 4 on at mono-cultures, 25 and 50 µM which for was4 on alsomono-cultures, reflected by thewhich observed was also positive reflected correlation by the betweenobserved H 2positiveO2 and IL-8correlation levels on between mono- cultures.H2O2 and Based IL-8 onlevels these onfindings, mono-cultures. MI432 mightBased haveon these influenced findings, the MI432 crosstalk might of different have in- Biomedicines 2021, 9, 450 11 of 14

inflammatory mediators, resulting in elevated IL-6 and IL-8 levels, and suggesting the putative role of matriptase in retaining the hepatic oxidative and inflammatory homeostasis via a multifaceted mode of action. In contrast to MI432, no proinflammatory action was exerted by MI460 as indicated by IL-6 and IL-8 concentrations (except IL-8 after 10 µM for 24 h on mono-cultures). Notwithstanding that both MI432 and MI460 are similarly structured 3-APhA based inhibitors, our results confirm that they can possess largely different actions on cell function, possibly due to their different Ki values, particularly for the inhibition of matriptase-2, highly expressed in the liver [17]. Since the inhibition of matriptase with MI432 triggered an intense surge in hepatocellular IL-6 and IL-8 release, our present results suggest that physiologically regulated matriptase may have a key role in maintaining the homeostasis in the liver of chickens, avoiding the excessive production of proinflammatory cytokines. Furthermore, as the elevated interleukin levels of MI432- treated chicken hepatic cell cultures were not alleviated after 24 h incubation, it can be suggested that the fast metabolic adaptation of liver cells to MIs was not coupled to the restoration of the physiological inflammatory homeostasis. The pleiotropic function of matriptase in the inflammatory response has been studied already in various mammalian cell types, but it was not completely assessed in avian tissues before. The effects of MI432 and MI460 on proinflammatory cytokine production were monitored by Pomothy et al. [17] on similar hepatic cell culture models originated from swine. According to their findings, the short-term (2 h) MI application did not affect the extracellular IL-6 and IL-8 levels of porcine hepatocyte–Kupffer cell co-cultures. Similarly, exposing monolayers and hydrogel-assisted 3D cultures of porcine hepatocyte mono-cultures and hepatocyte–Kupffer cell co-cultures to another 3-APhA type inhibitor, MI461, for 24 h was not capable of altering the IL-6 and IL-8 concentrations of culture media [28]. In humans, matriptase activation was coupled to several inflammatory skin disorders [29], and matriptase induced IL-6 and IL-8 production of endothelial cells via PAR- 2-mediated inflammatory signaling [19]. The activation of the PAR-2 pathway is strongly involved in the humoral and cellular inflammatory response promoted by dysregulated matriptase enzymes in context with several diseases, including epidermal tumors [7,30]. However, matriptase possessed a protective role against ulcerative colitis by restoring the gut-barrier function [31]. The observed differences between data gained from mammalian cells and those of the present study using chicken cell cultures highlight the importance of species-dependent differences in matriptase action, and also reflect the complex interplay of matriptase activity and the inflammatory response. Based on the results obtained, the redox state of the cells was mostly not influenced by the applied inhibitors, since no significant changes were found in extracellular H2O2 and MDA levels and cellular GPx activity following most of the applied MI treatments. However, in case of MI432 at 25 and 50 µM for 4 h on mono-cultures, a significantly increased ROS level was detected, combined with an elevated lipid peroxidation rate at 25 µM as indicated by the MDA measures. In contrast, 10 µM MI460 showed antioxidant action on co-cultures for both incubation times. The GPx activity of MI460-exposed cells was remarkably elevated only when applying the inhibitor in the highest concentration on both cell culture models. These data suggest that the applied 3-APhA type inhibitors may not strongly affect the hepatic cellular redox homeostasis of chickens in most concentrations; however, they might act as redox modulators under certain conditions. Similarly, the extracellular ROS levels of porcine hepatic cell culture models were not influenced by a short-term (2 h) exposure to 50 µM MI432 and MI460, but the similar 3-AphA type MI441 significantly stimulated the cellular ROS release [17]. On 3D hepatic cell culture models of pig origin, the H2O2 level was not altered after applying MI461 for 24 h [28]. The stability of the redox homeostasis of matriptase-modulated hepatocytes was also confirmed by our previous study, as sphingosine 1-phosphate as a matriptase inducer did not disturb the oxidative status of primary rat hepatocyte cultures [32]. Concerning the extrahepatic tissues, MI432 caused a transient increase in cellular ROS production of the porcine small intestinal cell line IPEC-J2 when applied for 2 h at 50 µM, but it was normalized during Biomedicines 2021, 9, 450 12 of 14

longer exposure times [22]. In contrast, the exposure of IPEC-J2 monolayers to MI439 and MI476 for 24 h elicited a remarkable antioxidative action by decreasing the extracellular H2O2 concentration on IPEC-J2 monolayers, which effect was the most pronounced at the lowest applied concentration (10 µM) [33], similarly to the action of MI460 on hepatic co-cultures in the present study. Concluding these data, matriptase inhibition might slightly influence the ROS production and the cellular antioxidative defense mechanisms in certain cases, but it cannot be considered as a major modulator of the cellular redox homeostasis. The MI460-driven activation of GPx as a major antioxidant system can be suggested to alleviate the cellular ROS release, preventing hepatic oxidative distress and contributing to the absence of increased ROS and MDA levels. Hence, addressing the results of the present study, it can be suggested that MIs do not greatly affect the oxidative state of cultured chicken liver cells, and thus they might be applied without causing oxidative distress and lipid peroxidation. Comparing the MI-driven changes on mono- and co-cultures, no considerable differences could be found, similar to the study of Pászti-Gere et al. [28] involving porcine hepatocyte mono-cultures and hepatocyte–Kupffer cell co-cultures. This finding may suggest that the presence of hepatic NP cells is not a critical factor in determining the effects of matriptase inhibition. As the presently applied co-culture with a cell ratio of 6:1 (hepatocyte to NP cells) served as a model of hepatic inflammation with moderate macrophage infiltration [23], it can be suggested that 3-AphA based MIs may act similarly under physiological and mildly inflamed conditions on the cells of the chicken liver. However, the proinflammatory action of MI432 on both cell culture models highlights that matriptase may be considered as a key regulator of hepatic inflammation in chicken, preventing excessive proinflammatory cytokine release. Notwithstanding that the underlying mechanisms of the aforementioned effects should be investigated in further studies, the present trial provided the first evidence regarding the role of matriptase in maintaining physiological homeostasis of the avian liver. If matriptase inhibitors were considered to be applied as drug candidates in the future in veterinary medicine, this newly suggested function of matriptase in avian species should be carefully addressed.

5. Conclusions In conclusion, the present study revealed that matriptase inhibition by MI432 triggered intense and substantive proinflammatory IL-6 and IL-8 production in chicken-derived hepatic cell culture models, which was not accompanied with enhanced oxidative stress and lipid peroxidation as indicated by cellular ROS production and MDA levels. The application of both inhibitors MI432 and MI460 was not cytotoxic, but caused a transient moderate metabolic depression of cultured liver cells following 4 h exposure, which was restored after 24 h, reflecting the fast hepatic adaptation potential to MI administration. These results may suggest that physiological matriptase activities should have a key function in retaining the metabolic and inflammatory homeostasis of the liver in chicken, without being a major modulator of the hepatocellular redox state.

Author Contributions: Conceptualization, G.M., Á.J., Z.N., and E.P.-G.; methodology, R.F.B., M.M., and G.M.; software, R.F.B.; validation, M.M. and R.F.B.; formal analysis, R.F.B., M.M., and G.M.; investigation, R.F.B., M.M., Z.N., and G.M.; resources, Z.N. and G.M.; data curation, R.F.B. and M.M.; Writing—Original draft preparation, R.F.B. and G.M.; Writing—Review and editing, Z.N., M.M., Á.J., and E.P.-G.; visualization, R.F.B. and M.M.; supervision, G.M. and Á.J.; project administration, Z.N. and G.M.; funding acquisition, Z.N. and G.M. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the NKFIH grants No. 124586 and No. FK 134940 of the Hungarian National Research, Development and Innovation Office. The Project was supported by the European Union and co-financed by the European Social Fund (grant agreement No. EFOP-3.6.3- VEKOP-16-2017-00005, project title: “Strengthening the scientific replacement by supporting the academic workshops and programs of students, developing a mentoring process”). Biomedicines 2021, 9, 450 13 of 14

Institutional Review Board Statement: All experimental procedures involving animals were carried out in accordance with the national and EU laws, as well as with the institutional guidelines, and were confirmed by the Local Animal Welfare Committee of the University of Veterinary Medicine, Budapest. Informed Consent Statement: Not applicable. Data Availability Statement: All raw data supporting the results of the present study can be obtained from the corresponding author upon reasonable request. Acknowledgments: The authors are especially grateful to Torsten Steinmetzer for the generous donation of the applied matriptase inhibitors. Special thanks are granted to Júlia Vörösházi and Fanni Hámor for their professional assistance. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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