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

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.

Targeting Toll-Like Receptor 2: Polarization of Porcine Macrophages by a Mycoplasma-Derived Pam2cys Lipopeptide

“…Untreated cells were used as a control. Then, 24 h post stimulation, flow cytometry was carried out as previously described [ 19 ]. In brief, cells were harvested with PBS with 10 mM EDTA and transferred into 5 mL round bottom tubes (Corning).…”

“…A total of 5000 live moMΦ were acquired. Analysis of data was performed using BD FACS Diva Software (BD Biosciences), by exclusion of doublets, gating on viable moMΦ, and then assessing the staining for surface markers [ 19 ].…”

“…Changes in the mRNA expression profile were evaluated as previously described [ 19 , 35 , 36 ]. In brief, moMΦ were seeded in 12 well plates (1 × 10 6 live cells/well) and left untreated or stimulated with Mag-Pam2Cys (10 or 100 ng/mL); at selected time points (0, 4, 8, 24 h), culture supernatants were removed, and cells were lysed with buffer RTL (Qiagen, Hilden, Germany).…”

“…We previously reported a diacylated lipopeptide, chemically synthesized based on the 14 amino acids following the cysteine immediately downstream the signal peptide of a surface protein of M. agalactiae (P48), strongly activated cells of the innate immune system including: ovine neutrophils [ 17 ] and monocytes [ 18 ], and porcine monocyte-derived macrophages (moMΦ) [ 19 , 20 ]. This diacylated lipopeptide activates cells upon recognition by TLR2, in association with TLR6 [ 3 , 17 ].…”

“…This diacylated lipopeptide activates cells upon recognition by TLR2, in association with TLR6 [ 3 , 17 ]. We most recently described that this synthetic lipopeptide induced a strong release of IL-12 and proinflammatory cytokines from porcine moMΦ [ 19 , 20 ].…”