Comparative Phenotypic and Functional Analyses of the Effects of IL-10 or TGF-β on Porcine Macrophages

Simple Summary Macrophages play a central role in innate immune response to both infectious and non-infectious stressors. They respond to different agonists modifying their phenotype and functions. In humans and mice, the regulatory cytokines IL-10 or TGF-β are both known to drive macrophage polarization into an anti-inflammatory phenotype, referred to as M2c. However, the immune systems of animal species each have their own peculiarities and the M2c subsets has never been investigated in pigs. A deep knowledge of the porcine immune system is required to design vaccines or control strategies against pathogens, which are a major constraint to pork production. Due to anatomical, physiological, and immunological similarities, swine are attracting increasing attention as a model for human diseases. To better characterize porcine macrophages, we evaluated the effects of IL-10 or TGF-β on the phenotype and function of monocyte-derived macrophages. Both cytokines downregulated the expression of MHC II DR and CD14. IL-10, but not TGF-β, statistically significantly reduced the ability of macrophages to respond to Toll-like receptor 2 (TLR2) or TLR4 agonists. Whilst these data suggest differentiation to an M2c-like immunosuppressive phenotype, the responses, and differences between IL-10 and TGF-β also reveals species-specific differences. Abstract Macrophages are phagocytic cells involved in maintaining tissue homeostasis and defense against pathogens. Macrophages may be polarized into different functionally specialized subsets. M2c macrophages arise following stimulation with IL-10 or TGF-β and mediate anti-inflammatory and tissue repair functions. M2c macrophages remain poorly characterized in the pig, thus we investigated the impact of these regulatory cytokines on porcine monocyte-derived macrophages (moMΦ). The phenotype and functionality of these cells was characterized though confocal microscopy, flow cytometry, ELISA, and RT-qPCR. Both cytokines induced CD14 and MHC II DR down-regulation and reduced IL-6, TNF-α, and CD14 expression, suggestive of an anti-inflammatory phenotype. Interestingly, neither IL-10 or TGF-β were able to trigger IL-10 induction or release by moMΦ. Differences between these cytokines were observed: stimulation with IL-10, but not TGF-β, induced up-regulation of both CD16 and CD163 on moMΦ. In addition, IL-10 down-regulated expression of IL-1β and IL-12p40 4h post-stimulation and induced a stronger impairment of moMΦ ability to respond to either TLR2 or TLR4 agonists. Overall, our results provide an overview of porcine macrophage polarization by two immunosuppressive cytokines, revealing differences between IL-10 and TGF-β, and reporting some peculiarity of swine, which should be considered in translational studies.


Introduction
Macrophages (Mϕ) are phagocytic cells, which play a central role in tissue homeostasis, by clearing cell debris, dying cells, and repairing tissues after inflammation [1,2]. They are crucial in immune response to pathogens, being able to detect foreign molecules through an array of sensing molecules, named pattern recognition receptors (PRRs) [3,4]. Macrophages also contribute to the initiation of acquired immune responses by processing and presenting antigens to naïve T lymphocytes [3]. Mϕ are a heterogenous population, with subsets in different anatomic locations presenting discrete functional characteristics (reviewed by [2]). Mϕ may also be differentially activated, resulting in their polarization into different functionally specialized subsets, referred to as 'classically' (M1) and 'alternatively' (M2) activated Mϕ [5]. Classical activation by interferon-gamma (IFN-γ) and lipopolysaccharide (LPS) produces M1 Mϕ with an increased microbicidal or tumoricidal capacity to better mediate defense to intracellular pathogens. Alternative activation induces M2 Mϕ, which are primarily associated with mechanisms of wound repair and immunosuppression [5]. M2 Mϕ were recently classified into three further subsets: M2a (activated with IL-4 or IL-13), M2b (activated by exposure of immune complexes in combination with IL-1β or LPS) and M2c (stimulated with IL-10, TGF-β or glucocorticoids) [4]. Overall, macrophages present remarkable malleability, with constant variations in their phenotype and functionality in response to subtle and continuously changing milieu of signals [1], thus, it was suggested to adopt a different nomenclature for their classification, linked to the activator/s used, e.g., M(IL-4), M(IFN-γ), M(IL-10), M(LPS), M(Ig) [6]. Despite the crucial importance of polarization on Mϕ phenotype and functionality, few studies have analyzed Mϕ polarization in pigs [7][8][9].
Pork production represents more than one-third of global meat production, and infectious diseases represent its main constraint, being responsible for major economic losses [10]. Knowledge of the porcine immune system is mandatory to understand how pigs respond to pathogens, and to rationally design vaccines or other biological interventions to control infectious diseases [11]. In addition, swine are attracting increasing attention as a biomedical model due to their anatomical, physiological, and immunological similarities to humans [12][13][14][15]. For example, pigs are used in pre-clinical toxicologic testing of pharmaceuticals [13], pre-clinical evaluation of vaccine candidates and therapeutics [16,17], and in nanomedicine studies [18,19]. A recent comparison of the porcine and human immune system showed similarities in over 80% of analyzed parameters, whereas mice and humans shared only about 10% [15]. For example, pigs and humans both have tonsils, which are absent in rodents, and the immunology of pig skin makes it an ideal model for human skin [14,15]. In addition, swine are monogastric and omnivorous, thus are more suitable than some other species to study intestinal immunology [15].
Current knowledge of the porcine immune system presents several gaps and few studies have focused on Mϕs in different polarized states. As described in humans, generation of porcine M1 resulted in release of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α), and up-regulation of the activation marker CD25 [7][8][9]. In addition, classical activation of porcine macrophages resulted in up-regulation of molecules involved in antigen presentation, such as MHC class I and II and co-stimulatory molecules CD80/CD86 and CD40 [7][8][9]. On the contrary, activation of porcine Mϕ with IL-4 induced expression of the M2a key effector arginase-1 (Arg-1) (an enzyme involved in catalyzing the hydrolysis of arginine to ornithine) and up-regulation of the surface marker CD203a (ectonucleotide pyrophosphatase/phosphodiesterase 1) [7,9]. We also reported that Mϕ in antithetic polarized status responded differently to myelotropic viruses such as porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV) [7,20,21].
However, no studies to date have deeply analyzed other M2 subtypes, like M2c, in the pig. The aim of this work was therefore to evaluate the effects of IL-10 and TGF-β (two immunosuppressive cytokines [22]) on Mϕ phenotype and functionality though an integrative analytical approach, using confocal microscopy, flow cytometry, multiplex cytokine ELISA, and RT-qPCR.

Ethical Statement
Eight healthy pigs (Sus scrofa domesticus) were utilized for blood sampling. Pigs were cross-bread, 6-24 months old, and were kept at the Experiment Station of the Istituto Zooprofilattico Sperimentale (IZS) of Sardinia. Blood sampling was approved by the local ethics committee, in accordance with the Guide of Use of Laboratory Animals issued by the Italian Ministry of Health.

Impact of IL-10 or TGF-β Activation on moMΦ Phenotype
MoMΦ were cultured in 12 well plates (to assess surface marker expression by flow cytometry) or 8-well chamber slides (for confocal microscopy). Then, 24 h postactivation with IL-10 or TGF-β, moMΦ phenotype was assessed by confocal microscopy and flow cytometry.
For confocal microscopy, moMΦ were fixed using 4% paraformaldehyde and then labelled with Alexa Fluor 488 conjugated phalloidin and Hoechst 33342 (both Molecular Probes, Thermo Fisher Scientific, Rockford, USA) to visualize actin cytoskeleton or nuclei, respectively [20]. Images were acquired on a format of 1024 × 1024 pixels, using a Leica SP5 Confocal Microscope (Leica Microsystem, Wetzlar, Germany) equipped with a 40X Plan-Apo 1.25 NA oil immersion objective. Images were then processed with LAS AF Lite software (Leica Microsystem) as previously described [20].
Flow cytometry was carried out as previously described, with slight modifications [21]. In brief, moMΦ were harvested using PBS supplemented with 10 mM EDTA and then transferred into 5 mL round bottom tubes (Corning). First, cells were stained with Zombie Aqua viability dye (BioLegend, San Diego, CA, USA) for 30 min at room temperature (RT). Then, moMΦ were washed with PBS supplemented with 0.5% bovine serum albumin (BSA) and then stained with the following murine monoclonal antibodies: anti-human CD14-PerCP-Cy5.5 (clone Tuk4; Miltenyi Biotec, Bergisch Gladbach, Germany), anti-porcine MHC II DR-FITC (clone 2E9/13, Bio-Rad Antibodies, Kidlington, United Kingdom), CD16-PE (clone G7, Thermo Scientific Pierce, Rockford, IL, USA), CD163-PE (clone 2A10/11, Bio-Rad Antibodies), CD169-FITC (clone 3B11/11, Bio-Rad Antibodies), and CD25 (clone K231.3B2, Bio-Rad Antibodies). CD25 expression was visualized by subsequent staining with BV421 rat anti-mouse IgG1 (clone A85-1, BD Horizon BD Biosciences, Franklin Lakes, NJ, USA). Incubations with either primary or secondary antibodies were carried out for 15 min at 4 • C. After washing with PBS supplemented with 2% FBS, moMΦ were resuspended in PBS supplemented with 2 mM EDTA and were analyzed with a FACS Celesta (BD Biosciences). A total 5000 live moMΦ were acquired and analysis of data was performed using BD FACS Diva Software (BD Biosciences): first doublets were excluded, then moMΦ were gated according to FSC and SSC values, dead cells excluded according to viability staining, and finally staining for surface markers were assessed.

Induction of Cytokine Release in Response to IL-10 or TGF-β Activation
MoMΦ were left untreated or stimulated with IL-10 or TGF-β and 24 h later cytokine levels in culture supernatants were evaluated. LPS was used as a positive control. Culture supernatants were harvested, cleared of debris by centrifugation (2000× g for 3 min) and stored at −80 • C until analyzed. Levels of IL-1α, IL-1β, IL-6, IL-10, IL-12, and TNF-α were determined using Porcine Cytokine/Chemokine Magnetic Bead Panel Multiplex assay (Merck Millipore, Darmstadt, Germany) and a Bioplex MAGPIX Multiplex Reader (Bio-Rad, Hercules, CA, USA), following the manufacturers' instructions, as previously described [20].

Statistical Analysis
In vitro experiments were carried out in technical duplicate and repeated with three (confocal images, multiplex ELISA) or four (flow cytometry) or five (RT-qPCR) different pigs. Baseline data distribution was determined based on Shapiro-Wilk test using R-software, version 3.6.2 (R-Foundation for Statistical Computing, Vienna, Austria), and then data were graphically and statistically analyzed with GraphPad Prism 8.01 (GraphPad Software Inc, La Jolla, USA). Data were presented as mean and standard error of the mean (SEM) and were analyzed with the parametric one-way ANOVA followed by Dunnett's multiple comparison test or the non-parametric Kruskal-Wallis test followed by a Dunn's multiple comparison test. A p value of less than 0.05 was considered statistically significant. Statistically significant differences were displayed in the figures using the following symbols: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Impact of IL-10 or TGF-β on Porcine moMΦ Phenotype
Mϕ stimulated with IL-10 and TGF-β were assessed by confocal microscopy and flow cytometry (Figure 1). moMΦ presented with a spherical shape with short hairy protrusions on their surface, as previously observed [20], and stimulation with IL-10 or TGF-β did not affect their morphology, dimension, or granularity ( Figure 1). Cytokine treatment modulated the expression of surface markers on Mϕ (Figure 2a). Both cytokines induced CD14 and MHC II DR down-regulation, with a statistically significantly (p < 0.001) reduced mean fluorescence intensity (MFI) observed in stimulated compared to untreated moMΦ. Stimulation with either cytokine also resulted in reduction of percentages of MHC II DR + cells (Figure 2b). Stimulation with IL-10, but not TGF-β, induced up-regulation of both CD16 and CD163 on moMΦ (Figure 2a), but no differences were observed between untreated and IL-10 stimulated moMΦ in terms of percentages of CD16 + or CD163 + cells (Figure 2b). IL-10 and TGF-β did not alter CD25 and CD169 expression on moMΦ, either in terms of MFI or percentages of cells expressing either of these markers (Figure 2). Values of stimulated samples were compared to the corresponding un-treated control (moMΦ) using a one-way ANOVA followed by a Dunnett's multiple comparison test. Asterisks indicate statistically significant differences: *** p < 0.001, ** p < 0.01, * p < 0.05.

Induction of Cytokine Gene Expression and Release from Porcine moMΦ in Response to IL-10 or TGF-β Stimulation
Multiplex ELISA analysis of moMΦ culture supernatants revealed no production of either pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) or IL-12 in response to both IL-10 and TGF-β (Figure 3). IL-10 was detected in the supernatants of IL-10 stimulated moMΦ, however the amount detected 24 h post-stimulation (6.66 + 1.36 ng/mL) was below the amount added to culture media at time 0 (20 ng/mL), suggesting that no de-novo synthesis of IL-10 had occurred (Figure 3). stimulation with either IL-10 or TGF-β, levels of IL-1α, IL-1β, IL-6, IL-10, IL-12, TNF-α in culture supernatants were quantified using a multiplex ELISA. Lipopolysaccharide-LPS (1 mg/mL) was used as positive control for IL-1α, IL-1β, IL-6, IL-12, and TNF-α. Mean data and SEM from three independent experiment using different animals are shown. Values for IL-10 or TGF-β stimulated samples were compared to the corresponding untreated control (moMΦ) using a one-way ANOVA followed by a Bonferroni's multiple comparison test. Asterisks indicate statistically significant differences: *** p < 0.001.
To further explore the effects of IL-10 and TGF-β on Mϕ, gene expression of several pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), IL-12p40 and IL-10 were investigated at 4, 8, and 24 h post-simulation. RT-qPCR data revealed that neither IL-10 nor TGF-β induced enhanced expression of any of the tested cytokines. On the contrary, both recombinant cytokines reduced IL-6 gene expression 24 h post-stimulation (Figure 4). Treatment with IL-10 also induced a statistically significant down-regulation of IL-1β (4 and 8 h), IL-12p40 (4 h), TNF-α (24 h) expression (Figure 4). In humans and mice, increased IL-10 gene expression is a hallmark of M2c polarization [28], but we observed that neither IL-10 nor TGF-β enhanced IL-10 expression in porcine moMΦ. On the contrary, stimulation with recombinant IL-10 resulted in statistically significant down-regulated IL-10 gene expression 4 h post-stimulation (Figure 4).

IL-10 and TGF-β Inhibit moMΦ Responses to Stimulation with TLR4 and TLR2 Agonists
The immunosuppressive ability of these two cytokines was next assessed. moMΦ were first stimulated for 24 h with Il-10 or TGF-β or left untreated, then supernatants were removed, and cells were activated with a TLR4 (LPS) or a TLR2 (Pam2Cys lipopeptide) agonist. Then, 24 h later, culture supernatants were collected, and multiplex ELISA analysis performed. IL-10 stimulation resulted in a reduced ability of moMΦ to release IL-1α, IL-1β, IL-6, IL-12, and TNF-α in response to stimulation with either LPS (Figure 5a) or Pam2Cys lipopeptide (Figure 5b). Figure 5. Impact of IL-10 or TGF-β on macrophage response to LPS stimulation. moMΦ were left untreated or stimulated with IL-10 and TGF-β. After 24 h, culture supernatants were replaced with fresh media and cells were left untreated or activated using LPS (1 mg/mL) (a) or pam2cys lipopeptide (100 ng/mL) (b). 24 h later, the amount of IL-1α, IL-1β, IL-6, IL-12, TNF-α in culture supernatants were evaluated using a multiplex ELISA. For both panels, the mean data and SEM from three independent experiments utilizing different blood donor pigs are shown. Values of IL-10 or TGF-β stimulated samples were compared to the corresponding un-treated control (moMΦ) using a Kruskal-Wallis multiple comparison test. Asterisks indicate statistically significant differences: ** p < 0.01, * p < 0.05. Stimulation with 20 ng/mL of TGF-β did not statistically significantly impair the ability of moMΦ to release IL-1α, IL-1β, IL-6, IL-12, and TNF-α in response to either LPS or Pam2Cys lipopeptide, although a decreasing trend was observed for IL-6 and IL-12 in response to both stressors and for TNF-α in response to the TLR2 agonist ( Figure 5).
3.4. CD14, TLR4, Arg-1 Expression in Macrophages Stimulated with IL-10 or TGF-β We next investigated the ability of IL-10 and TGF-β to modulate CD14 and TLR4 (two molecules involved in bacterial components recognition) gene expression over time (4,8, and 24 h post-simulation). As described in Figure 6a, both cytokines reduced CD14 gene expression, with statistical significance at 4 h (for both IL-10 and TGF-β) and at 24 h (only for IL-10). On the contrary, TLR4 expression was not altered by the two cytokines (Figure 6a). Finally, the ability of IL-10 or TGF-β to induce Arg-1 expression was assessed. Induction of this enzyme is considered a hallmark of M2a polarization in many species, including in swine [9]. IL-4 was used as positive control and as expected, a marked enhancement of Arg-1 expression was observed over time (Figure 6b). Neither IL-10 nor TGF-β triggered Arg-1 induction, except for a modest but statistically significant enhancement observed in TGF-β treated moMΦ at 4 h post-stimulation. Nevertheless, enhanced expression of Arg-1 was detected in only four out of five tested subjects.

Discussion
Mϕ are phagocytic cells which play a central role in innate immune response to both infectious and non-infectious stressors [3]. Diversity and plasticity are properties of this heterogeneous family, which allows modifications to their phenotype and functions in response to changes in the surrounding microenvironment [29]. In a spectrum of polarizing states, M1 and M2 represent two antithetic extremes [29]. Stimulation of human or murine Mϕ with either IL-10 or TGF-β or glucocorticoids polarize Mϕ toward an M2c state, which promotes anti-inflammatory activities and phagocytosis of apoptotic cells [4,28]. However, limited information is available on the existence of an M2c phenotype in pigs. Two studies focused on the effects of IL-10 and dexamethasone on porcine monocyte and moMϕ phenotypes and susceptibility to PRRSV [7,30]. In our study, for the first time, the phenotypical and functional impact of IL-10 and TGF-β on porcine Mϕ was investigated in detail.
Phenotype was first investigated. Our data revealed that stimulation with either IL-10 or TGF-β reduced MHC Class II expression on Mϕ, in accordance with what was reported in other species [2]. Stimulation with these cytokines also down-regulated surface expression of CD14, supporting the immunosuppressive effect of these two cytokines in swine. Their anti-inflammatory features were reflected also by the RT-qPCR data, which revealed that both cytokines reduced expression of pro-inflammatory cytokines (IL-6 and TNF-α) and CD14. Differences between the effects of IL-10 and TGF-β were also observed: stimulation with IL-10, but not TGF-β, resulted in enhanced expression of CD163 and CD16. M2 cells are characterized by high levels of scavenger receptors, like CD163 [28,31], and previous studies described that IL-10 induced CD163 upregulation on both porcine monocytes and Mϕ [7,30]. Similar findings were described in humans, where IL-10 stimulation enhanced both CD16 and CD163 expression on Mϕ [32], and TGF-β decreased CD163 expression [33]. Dissimilarities between the effects of the two cytokines were also revealed also by the RT-qPCR and cytokine ELISA data. Stimulation with IL-10, but not TGF-β, resulted in marked reduction of IL-1β and IL-12p40 gene expression early post-stimulation and drastically impaired the ability of moMΦ to release IL-12 and proinflammatory cytokines (IL-1α, IL-1β, IL-6, TNF-α) in response to TLR2 and TLR4 agonists. Interestingly, stimulation with TGF-β did not statistically significantly impair the ability of moMΦ to respond to these stimuli, although a clear trend was observed for IL-6 and IL-12 responses. Overall, our results highlight differences in the polarizing effects of these two cytokines, suggesting that it would be more appropriate to use a nomenclature linked to the activator(s) used, such as M(IL-10), M(TGF-β), as suggested by Murray et al. [6].
Some peculiarities in responses of porcine Mϕ were also observed. Enhanced IL-10 expression and release is regarded as a hallmark of M2c polarization in both humans and mice [28,31], but in our study, we observed that neither IL-10 or TGF-β triggered enhanced expression or release of this cytokine. In addition, a reduced IL-10 gene expression was observed in porcine moMΦ 4h post-stimulation with IL-10. Similar findings were observed in porcine M2a; with no release of IL-10 by porcine Mϕ in response to IL-4 stimulation [9,34], whereas in both humans and mice, exposure to IL-4 results in production of high levels of IL-10 and chemokines that promotes recruitment of Th2 cells [5,22]. Future studies should investigate this particularity of pigs, which should be also considered in translational studies.
We finally evaluated IL-10 and TGF-β ability to induce Arg-1 gene expression. Induction of Arg-1 is considered a hallmark of M2a polarization in humans, mice [35] and pigs [9], and we confirmed in this study enhanced expression of Arg-1 following IL-4 stimulation. Arg-1 catalyzes the hydrolysis of arginine to ornithine, resulting in increased polyamine synthesis; the latter playing an important role in tissue repair and remodeling [35]. Previous studies in rodents reported that both cytokines increased arginase activity in Mϕ [36,37] and IL-10 further enhanced Arg-1 expression in IL-4 activated M2a [38]. On the contrary, Arg-1 is not an M2c marker in human [28]. In this study, we observed that in pigs, IL-10 did not induce expression of this enzyme, instead a modest but statistically significant enhancement of Arg-1 expression was observed in moMΦ at 4 h post-stimulation with TGF-β. Nevertheless, no differences between treated and untreated cells were detected at later time points. Many differences between species exist in terms of arginine pathways in Mϕ [39]; this pathway plays an important role in immunopathology; thus, these dissimilarities should be better characterized in future studies.

Conclusions
This study has provided a detailed characterization of the polarizing effect of IL-10 and TGF-β on porcine moMΦ. Both cytokines induced CD14 and MHC II DR down-regulation, as well as reductions in IL-6 and TNF-α gene expression reflecting their immunosuppressive ability. Phenotypic and functional differences between moMΦ (IL-10) and moMΦ (TGF-β) were also observed: stimulation with IL-10, but not TGF-β, resulted in enhanced CD16 and CD163 upregulation, down-regulated expression of IL-1β and IL-12p40 and induced a stronger impairment of moMΦ ability to respond to TLR2 or TLR4 agonists. We also highlighted a peculiarity of swine MΦ: neither IL-10 or TGF-β stimulation enhanced IL-10 expression or release in porcine moMΦ. The information generated by this study helps further characterize the complex and heterogeneous Mϕ family in pigs and to highlights differences between species, that should be considered in translational studies.  Institutional Review Board Statement: The present study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the Istituto Zooprofilattico Sperimentale della Sardegna (authorization n • 1232/2020-PR).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.