animals Article The Potential of Metalloproteinase-9 Administration to Accelerate Mammary Involution and Boost the Immune System at Dry-Off Sílvia Parés 1,† , Olivia Cano-Garrido 2,3,4 , Alex Bach 1,5 , Neus Ferrer-Miralles 2,3,4 , Antonio Villaverde 2,3,4 , Elena Garcia-Fruitós 1, *,‡ and Anna Arís 1, *,‡ 1 Department of Ruminant Production, IRTA (Institut de Recerca i Tecnologia Agroalimentàries), 08140 Caldes de Montbui, Spain; silviaparesriera@gmail.com (S.P.); alex.bach@icrea.cat (A.B.) 2 Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; olivia.cano.garrido@gmail.com (O.C.-G.); neus.ferrer@uab.cat (N.F.-M.); antonio.villaverde@uab.cat (A.V.) 3 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 08193 Cerdanyola del Vallès, Spain 4 Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain 5 ICREA (Institució Catalana de Recerca i Estudis Avançats), 08010 Barcelona, Spain * Correspondence: elena.garcia@irta.cat (E.G.-F.); anna.aris@irta.cat (A.A.) † As part of the PhD Thesis at Universitat Autònoma de Barcelona, Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain. ‡ Equally contributed.   Citation: Parés, S.; Cano-Garrido, O.; Simple Summary: The cow dry period is a critical period presenting a high risk of contracting Bach, A.; Ferrer-Miralles, N.; intramammary infections. Active molecules to boost the innate immunity of the mammary gland and Villaverde, A.; Garcia-Fruitós, E.; increase infection resilience could be decisive for the milking performance of dairy cows in the next Arís, A. The Potential of lactation. Metalloproteinase-9 is a protein with a relevant role in facilitating the immune function Metalloproteinase-9 Administration and activating the regeneration of the mammary gland. The focus of this study was to test the role of to Accelerate Mammary Involution the infusion of a recombinant version of metalloproteinase 9 at cow dry off, showing, contrary to and Boost the Immune System at expectations, that it is not able to enhance the innate immunity nor to improve the involution and Dry-Off. Animals 2021, 11, 3415. regeneration of the mammary gland. https://doi.org/10.3390/ani11123415 Abstract: The dry period is decisive for the milking performance of dairy cows. The promptness of Academic Editors: Pedro mammary gland involution at dry-off affects not only the productivity in the next lactation, but also Marín Carrillo and Maya Zachut the risk of new intra-mammary infections since it is closely related with the activity of the immune Received: 5 November 2021 system. Matrix metalloproteinase-9 (MMP-9) is an enzyme present in the mammary gland and Accepted: 24 November 2021 has an active role during involution by disrupting the extracellular matrix, mediating cell survival Published: 30 November 2021 and the recruitment of immune cells. The objective of this study was to determine the potential of exogenous administration of a soluble and recombinant version of a truncated MMP-9 (rtMMP-9) Publisher’s Note: MDPI stays neutral to accelerate mammary involution and boost the immune system at dry-off, avoiding the use of with regard to jurisdictional claims in antibiotics. Twelve Holstein cows were dried abruptly, and two quarters of each cow received an published maps and institutional affil- intra-mammary infusion of either soluble rtMMP-9 or a positive control based on immunostimulant iations. inclusion bodies (IBs). The contralateral quarters were infused with saline solution as negative control. Samples of mammary secretion were collected during the week following dry-off to determine SCC, metalloproteinase activity, bovine serum albumin, lactoferrin, sodium, and potassium concentrations. The soluble form of rtMMP-9 increased endogenous metalloproteinase activity in the mammary Copyright: © 2021 by the authors. gland compared with saline quarters but did not accelerate either the immune response or involution Licensee MDPI, Basel, Switzerland. in comparison with control quarters. The results demonstrated that the strategy to increase the This article is an open access article mammary gland immunocompetence by recombinant infusion of rtMMP-9 was unsuccessful. distributed under the terms and conditions of the Creative Commons Keywords: dry period; mammary gland involution; MMP-9; immunity Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Animals 2021, 11, 3415. https://doi.org/10.3390/ani11123415 https://www.mdpi.com/journal/animals Animals 2021, 11, 3415 2 of 12 1. Introduction At the end of lactation and as calving approaches, pregnant dairy cows enter into a non-lactating (dry) period to optimize milk production in the subsequent lactation. During the dry period, the epithelial component of the mammary gland regresses, proliferates, and differentiates to allow optimal milk production in the subsequent lactation. Tissue regeneration of the mammary gland is necessary, and the omission of a dry period reduces milk production in the following lactation [1]. However, the strong presence of galactopoietic hormones due to a concomitant preg- nancy could hamper the beginning of the involution and delay the activation of the immune system [2]. Furthermore, today’s pregnant dairy cows are dried while producing ~25 kg/d of milk with some animals producing >35 kg/d [3]. These copious amounts of milk remaining in the mammary gland exert high intra-mammary pressure and may cause discomfort [4] and milk leakage [5]. In addition, high-producing dairy cows are more susceptible to intra-mammary infections during the early stages of the dry period [6]. Involution of the mammary gland starts with a complex signaling pathway of cell factors, hormonal changes, and immune stimulation. Activation of the immune system at the beginning of the dry period recruits blood leukocytes that progressively colonize the mammary gland after dry-off and phagocytize and destroy microorganisms [7]. However phagocytic activity against pathogens is diminished at dry-off as phagocytes engulf milk fat, cell debris, and other compounds derived from milk accumulation, and their activity is not fully focused in the fight against pathogenic bacteria [7]. In fact, it is assumed that the immune system does not reach effective protective levels until 8 d after dry-off [8], and thus the health of the mammary gland is compromised during this period. To reduce the risk of mastitis, antibiotics are used routinely in the mammary gland at dry-off. However, this practice has been challenged due to concerns about the potential emergence of antibiotic resistance. Matrix metalloproteinase-9 (MMP-9) is a tissue-remodeling enzyme that is physiologi- cally released by mammary epithelial cells and neutrophils entering into the mammary gland during the involution process [9,10]. Thus, MMP-9 is probably one of the enzymes involved in the breaking of the extracellular matrix of the mammary gland (ECM) [11], which could trigger a signal to the detached cells to enter into apoptosis, supporting the tissue involution [12]. Moreover, the expression of MMPs, growth factors, and cytokines is closely linked, but through a mechanism that is still widely unknown in the bovine mam- mary gland [13,14]. It has been previously demonstrated that the proteolytic degradation of ECM is a key factor through the loss of differentiated state and induction of apoptosis and involution [15]. Thus, we hypothesized that an intramammary administration of an active fragment of the MMP-9 in a recombinant soluble form (recombinant truncated MMP-9 (rtMMP-9)) could represent an effective strategy to accelerate tissue involution at dry-off. Moreover, it could boost the infiltration of immune cells into the mammary gland and stimulate other active immune factors, reducing the need to use antibiotics at dry-off [16]. A previous study of our group tried to elucidate the effect of MMP-9 at hastening mammary gland involution by administrating rtMMP-9 at dry off [17]. However, the rtMMP-9 was not administrated in a soluble format but as inclusion bodies (IBs), which are protein nanoparticles formed spontaneously during the recombinant production pro- cess [18,19]. The IBs have some advantages compared to soluble protein, such as greater stability and slow-release of active protein. On the contrary, soluble forms present the advantage of being highly active but can sometimes be unstable and have short lives because other enzymes from the tissue can degrade them. A control of inactive rtMMP-9 IBs was also included in our last study and the conclusion was that the IB format induced an unspecific immune response, so we could not determine the MMP-9 real effect [17]. In this context, the objective of the present study is to determine the net effect of soluble rtMMP-9 compared to a saline control. A positive control at stimulating inflammation is included based on rtMMP-9 IBs. Animals 2021, 11, 3415 3 of 12 2. Materials and Methods 2.1. Bacterial Strains, Plasmids, Recombinant Proteins and Growth Conditions The production of rtMMP-9 was performed using Lactococcus lactis strain NZ9000 clpP− htrA− (clpP-htrA; EmR ) [20,21] (kindly provided by INRA, Jouy-en-Josas, France; patent nº EP1141337B1). The sequence of the catalytic domain of the bovine (Bos taurus) MMP-9 (from Phe107 to Pro449 NM_174744.2) was codon optimized for the expression in L. lactis and the cloning performed adding at the C-terminal a lysine plus a histidine tag to assist protein detection and purification (GeneArt, Thermo Fisher Scientific, GmbH, Regensburg, Germany). Recombinant active fragment of MMP-9 protein (39.6 kDa) was produced by expressing the encoding gene from the CmR pNZ8148 plasmid (NIZO) under the nisA promoter control [22]. This strain was cultured in shake flasks at 30 ◦ C without shaking in M17 Broth supplemented with 0.5% glucose. Antibiotics were used at the following concentrations: chloramphenicol (5 µg/mL) and erythromycin (2.5 µg/mL). Recombinant gene expression was induced by 12.5 ng/mL nisin (Sigma-Aldrich, Barcelona, Spain) during 3 h. 2.2. Purification and Quantification of rtMMP-9 Pellets of L. lactis cells were resuspended in phosphate-buffered saline (PBS) in the presence of protease inhibitors (cOmplete™, EDTA-Free Protease Inhibitor Cocktail, Roche, Barcelona, Spain) and frozen at −80 ◦ C. After thawing, cells were disrupted at 1500 psi (4 rounds for soluble protein and 3 for protein nanoparticles) in a French press (Thermo FA-078A). For the soluble rtMMP-9, 0.05 mg/mL lysozyme was added, and the result- ing mixture was incubated at 37 ◦ C with shaking for 2 h. The lysate was centrifuged at 15,000× g for 45 min. After that, soluble rtMMP-9 was recovered from the pellet as described in [23] and filtered through a 0.22 µm filter. The protein was purified by His- tag affinity chromatography using HiTrap Chelating HP 1 mL columns (GE Healthcare, Barcelona, Spain) with an ÄKTA purifier FPLC System (GE Healthcare). The purified solu- ble rtMMP-9 was analyzed by both SDS electrophoresis/Coomassie Brilliant Blue staining and Western blotting. Concentration was determined by Bradford’s assay (BioRad, Madrid, Spain). The isolation of rtMMP-9 IBs was conducted following previous protocols [18]. The yield of rtMMP-9 IBs was determined by both SDS electrophoresis/Coomassie Bril- liant Blue staining and Western blotting, using a standard curve of known amounts of a Green Fluorescent Protein (GFP) protein. Densitometry analyses were performed with the Quantity One software (BioRad). For Western blotting, a commercial monoclonal anti- body against anti-His (#A00186-100 Genescript, Piscataway, NJ, USA) and an anti-mouse secondary antibody (#170-6516 Bio Rad) were used. 2.3. Electron Microscopy (EM) Purified IBs were checked by field emission scanning microscopy (FESEM) and trans- mission electron microscopy (TEM). In the first technique, sample microdrops were de- posited 2 min on silicon wafers (Ted Pella Inc., Redding, CA, USA) and then air-dried. Nanoparticle micrographs were acquired at a nearly native state with a high-resolution in-lens secondary electron detector in a FESEM Zeiss Merlin (Zeiss, Oberkochen, Germany) operating at 2 kV. For TEM, nanoparticles samples were fixed with aldehydes and osmium, dehydrated, and embedded in Epon resin. Ultrathin sections were deposited on copper grids, and after contrast, observed with the electron microscope TEM Jeol JEM-1400 (Jeol Ltd., Tokyo, Japan). 2.4. Analysis of Metalloproteinase Activity Metalloproteinase activity of rtMMP-9 (soluble MMP-9 and IBs) corresponding to −39.6 KDa bands and endogenous mammary MMP-9, corresponding to 92 KDa bands, were analyzed and compared by zymography as described elsewhere [23]. Animals 2021, 11, 3415 4 of 12 2.5. Intra-Mammary Infusions This experiment was performed under the evaluation and permission of the Ethical Committee of IRTA, protocol number 9705. Twelve lactating Holstein cows (210–220 d pregnant, producing >20 kg/d of milk during the last 3 d preceding dry-off, and with milk somatic cell counts <200,000 cells/mL at dry-off) were enrolled in this study at dry-off. All cows were dried abruptly with no dietary intervention before dry-off and no changes in milking frequency to decrease the amount of milk production before stop milking. At dry-off time, two quarters of each cow received an intra-mammary infusion of either 0.75 mg of soluble rMMP-9 or 12 mg of MMP-9 nanoparticles in a total volume of 10 mL of 0.9% NaCl sterile commercial saline solution. The actual enzymatic activity of the soluble and the nanostructured MMP-9 amounts used were identical, as assessed by zymography. Treatments were randomly assigned using Excel RAND() function to front or rear quarters with soluble rtMMP-9 or IBs of rtMMP-9. The respectively contralateral quarters were infused with negative control consisting of 10 mL of 0.9% NaCl sterile commercial saline solution (Braun, Barcelona, Spain). Then, broad-spectrum antibiotics (Mamyzin secado® , Boehringer Ingelheim, Barcelona, Spain) were locally administered following common production practices. No teat sealant was used. A total of 50 mL of milk or mammary secretion samples were obtained at days 0 (before last milking), 1, 2, 3, 6, and 7 post- drying at 08:00 h by manual milking. A fraction of mammary secretion samples was kept refrigerated until analyzed for somatic cell counts (SCC), and the remainder was kept frozen at −20 ◦ C until subsequent analyses. 2.6. Mammary Secretion Analyses Somatic cell counts were analyzed in fresh milk (last milking before dry-off) or mam- mary secretion samples within 4 h after extraction using a Scepter® cell counter (Merk- Millipore, Madrid, Spain) and 40 µm sensors. Briefly, 0.5 mL of mammary secretion was diluted 1:1 in PBS and cells were pelleted by centrifugation at 1000× g for 2 min. Cream and supernatant were discarded. Pellets were then washed 3 times in PBS and then re- suspended in 0.5 mL of PBS. In some cases, samples had to be diluted in PBS prior to analysis. Lactoferrin concentration in the last milking before dry-off and in mammary secre- tions was measured by ELISA, using a commercial bovine lactoferrin ELISA kit (Bethyl Laboratories Inc., Montgomery, TX, USA). The absorbance for each sample was measured at 450 nm using a Model 680 microplate reader (Bio-Rad). The concentration of bovine serum albumin (BSA) in milk and mammary secretions was analyzed by a colorimetric assay as previously described [24], with some modifica- tions. Briefly, 200 µL of skimmed milk or mammary secretion was mixed with 450 µL of distilled water and 450 µL of a solution containing 1 volume of 1.2 mM of bromocresol green dissolved in 5 mM NaOH, 3 volumes of 0.2 M succinic acid (pH 4.0), and 0.8% Brij 35 detergent. After mixing by inversion and centrifugation at 1900× g for 10 min at room temperature, 150 µL of the supernatant were added to a 96-well microplate and the optical density was read at 655 nm using a Model 680 microplate reader (Bio-Rad). Gelatinase activity was analyzed by zymography as described above. Samples were skimmed by centrifugation at 2700× g for 10 min. The fat layer was discarded with a swab and supernatant was diluted between 1:20 to 1:250 in PBS and mixed 1:1 with the loading buffer. Sodium and potassium concentrations in milk secretion were analyzed by inductively coupled plasma-Optical emission spectrometry (ICP-OES) using an ICP-OES Perkin-Elmer Optima 4300DV after dilution with Triton X-100 0.1% (v/v) instead of digested in a mi- crowave oven as described in [25]. The method was validated before analyzing all samples by evaluating the repeatability of the results from 5 samples that were analyzed after digestion with microwaves or dilution in 0.1% (v/v) Triton X-100. Animals 2021, 11, 3415 5 of 12 Animals 2021, 11, 3415 5 of 12 2.7. Statistical Analysis Each mammary quarter was the experimental unit. All data were analyzed using a mixed‐effects model that accounted for the random effects of quarter within cow, cow, 2.7. Statistical Analysis and period (enrollment week), and the fixed effects of treatment, day of sampling, and their Each 2‐way mammary quarter interaction. was thetime Sampling experimental unit. entered the All data model as a were analyzed repeated usingusing an measure a mixed-effects model that accounted for the random effects of quarter within cow, cow, autoregressive covariance matrix. Metalloproteinase activity data from zymograms were and period (enrollment week), and the fixed effects of treatment, day of sampling, and previously log transformed their 2-way interaction. to achieve Sampling a normal time entered distribution. the model Differences as a repeated wereandeclared measure using significant at p ≤ 0.05, and trends were discussed at 0.05 ≤ p ≤ 0.10. autoregressive covariance matrix. Metalloproteinase activity data from zymograms were previously log transformed to achieve a normal distribution. Differences were declared 3.significant Results at p ≤ 0.05, and trends were discussed at 0.05 ≤ p ≤ 0.10. 3.1. rMMP‐9 Production, Activity Determination and Mammary Gland Infusion 3. Results Truncated 3.1. rMMP-9 but active Production, version Activity of MMP‐9 Determination and(rtMMP‐9) was Infusion Mammary Gland successfully produced in L. lactis Truncated either as soluble or IB. but active The IBs version produced of MMP-9 were used (rtMMP-9) wasas positive control successfully producedof mammary in gland L. lactis either as soluble or IB. The IBs produced were used as positive control of mammary430 nm immunostimulation. They showed a round and compact shape with a diameter and a smooth surface gland immunostimulation. (Figurea 1). They showed When round andcomparing compact shapethe with rtMMP‐9 a 430 activity nm by diameter and athe zymography, smooth surface soluble form(Figure showed 1).about When16‐fold comparing the rtMMP-9 greater activity activity than IBsby(data not zymography, shown). the soluble Equivalent form showed about metalloproteinase 16-fold activity greatersoluble of either activityor than IB IBs (dataofnot format rtMMP‐9 shown). Equivalent metalloproteinase activity were infused in mammary gland quarters at dry‐off. of either soluble or IB format of rtMMP-9 were infused in mammary gland quarters at dry-off. 1. Field Figure 1. Figure Fieldemission emissionscanning electron scanning microscopy electron (FESEM) microscopy micrographs (FESEM) of a truncated micrographs version version of a truncated of recombinant matrix Metalloproteinase-9 (rtMMP-9) IBs (left panel) and transmission of recombinant matrix Metalloproteinase‐9 (rtMMP‐9) IBs (left panel) and transmission electronelectron microscopy (TEM) microscopy (TEM)micrographs micrographsof rtMMP-9 IBs (right of rtMMP‐9 panel). IBs (right Scale bars: panel). Scale100 nm.100 nm. bars: 3.2. Induced Metalloproteinase Activity in the Mammary Gland 3.2. Induced Metalloproteinase Activity in the Mammary Gland Metalloproteinase activity in mammary secretion was evaluated by zymography after each Metalloproteinase activity treatment (Figure 2). The in of activity mammary secretion rtMMP-9 infused, was either evaluated soluble by zymography or IBs, was easily after each treatment differentiated from the(Figure activity 2). The activitytoofthe corresponding rtMMP‐9 mammary infused, either soluble gland endogenous or IBs, was MMP-9 due to differences in the molecular weight between the wild type MMP-9 easily differentiated from the activity corresponding to the mammary gland endogenous or rt MMP- 9 (92 kDadue MMP‐9 andto39.6 kDa, respectively). differences Overall, weight in the molecular endogenous betweenmetalloproteinase the wild typeactivity MMP‐9 or rt increased as days evolved since dry-off (p < 0.001). At days 1 and 3, the activity was greater MMP‐9 (92 kDa and 39.6 kDa, respectively). Overall, endogenous metalloproteinase in quarters infused with rtMMP-9 (soluble or IBs) compared with their respective saline activity increased as days evolved since dry‐off (p < 0.001). At days 1 and 3, the activity control quarters. The activity in quarters treated with soluble rtMMP-9 was greater than was in thegreater quartersintreated quarters with infused with rtMMP‐9 IBs of rtMMP-9 (soluble atorday at day 1. However, IBs) compared 3, the with their activity was respective saline greater in the control (p IBs quarters quarters. < 0.05). The activity in quarters treated with soluble rtMMP‐9 was greater than in the quarters treated with IBs of rtMMP‐9 at day 1. However, at day 3, the activity was greater in the IBs quarters (p < 0.05). mals 2021, Animals 11, 3415 2021, 11, 3415 6 of 12 6 of 12 A. 30 Log Endogenous MMP9 activity # # 25 * * * (relative units) 20 * 15 10 5 0 0 1 3 7 Days after drying B. Figure 2.matrix Figure 2. (A) Endogenous (A) Endogenous matrix(MMP-9) metalloproteinase-9 metalloproteinase‐9 (MMP‐9) activity in mammary activityat in secretions 0, 1,mammary 3, and 7 d after dry-off secretions of pregnant dairy-cows at 0, 1,by analyzed 3, zymography. and 7 d after Continuous dry‐off of pregnant dairy‐cows lines indicate MMP-9analyzed by discontinuous treatments, zymography. lines depict Continuous controls. Filled circles lines to correspond indicate solubleMMP‐9 rtMMP-9 treatments, treatment discontinuous lines and empty circles to depict controls. its control, filled Filled squares represent circles correspond to soluble rtMMP‐9 treatment and empty circles to its control, filled IBs treatment and empty squares to its control. Asterisks indicate significant differences (p < 0.05) between treatment and squares represent IBs treatment and empty circles to its control. Asterisks indicate significant control whereas pound sign indicates differences between treatments. (B) Zymogram comparing the metalloproteinase differences (p < 0.05) between treatment and control whereas pound sign indicates differences activity (white bands) betweenobtained in mammary treatments. secretioncomparing (B) Zymogram samples (lane 1) or loading directly the metalloproteinase the rtMMP-9 activity (lane 2). The Lane (white bands) M corresponds to the molecular weight marker. obtained in mammary secretion samples (lane 1) or loading directly the rtMMP‐9 (lane 2). The Lane M corresponds to the molecular weight marker. Animals 2021, 11, 3415 7 of 12 Animals 2021, 11, 3415 7 of 12 3.3. 3.3.Mammary ImmuneResponse Mammary Immune Response andand Involution Involution Markers Markers Monitorization Monitorization after Intramammary after Intramammary Administration of rtMMP‐9 Administration of rtMMP-9 Somatic Somatic cell cell counts counts (SCC) (SCC) in inmammary mammary secretions secretions progressively progressively augmented augmented (p (p << 0.0001) as0.0001) time elapsed since dry-off as time elapsed increased since dry‐off (Figure increased 3A),3A), (Figure but but SCCSCCin milk secretion in milk from secretion fromquarters treated quarterswith soluble treated withrtMMP-9 or salineorcontrol soluble rtMMP‐9 saline did not did control differ not(Figure 3A). However, differ (Figure 3A). the However, increase inthe SCCincrease in SCC was was greater at allgreater at all times sampling sampling(p