Equine peripheral blood CD14⁺ monocyte-derived macrophage in-vitro characteristics after GM-CSF pretreatment and LPS+IFN-γ or IL-4+IL-10 differentiation

Abstract

Macrophages are a heterogeneous population of immune cells that exhibit dynamic plasticity, polarize into inflammatory or regulatory/pro-resolving macrophages, and influence the healing tissue microenvironment. This study evaluated the in-vitro morphological, proliferative, cell surface marker expression and cytokine/soluble factor secretion characteristics of control, GM-CSF pretreated and inflammatory (LPS+IFN-γ) and regulatory (IL-4 + IL-10) differentiated equine CD14⁺ monocyte-derived macrophages. Phase contrast microscopy demonstrated that LPS+IFN-γ-primed macrophages exhibited a rounded, granular morphology, whereas IL-4+IL-10-primed macrophages were elongated with a spindle-shaped morphology. GM-CSF enhanced the proliferation rate of monocytes/macrophages during adherent in-vitro culture. Flow cytometry analysis showed that GM-CSF alone and GM-CSF pretreatment with LPS+IFN-γ or IL-4+IL-10 priming increased CD86 immunopositivity by 2-fold (p=0.6); and CD206 immunopositivity remained unchanged. GM-CSF pretreatment and subsequent priming with LPS and IFN-γ yielded inflammatory macrophages that secrete significantly increased quantities of IL-1β compared to control (p=0.012) and IL-4+IL-10-primed (p=0.0047) macrophages. GM-CSF pretreatment followed by both LPS + IFN-γ and IL-4 + IL-10 priming significantly increased IL-1Ra secretion by 6-fold (p<0.05). There were no differences in TGFβ-1 secretion among control, LPS+IFN-γ or IL-4 + IL-10 primed macrophages (p=0.85). All groups contained an average of 643 ± 51.5 pg/mL of TGFβ1. Among the culture conditions evaluated, IL-4+IL-10 priming for 24 hours after 6 days of adherent culture yielded macrophages that were the least inflammatory compared to GM-CSF pretreated and LPS+IFN-γ or IL-4+IL-10-primed macrophages. These results provide a basis for subsequent in-vitro and in-vivo studies that investigate macrophage-tissue cell interactions and related biological mechanisms relevant to the field of immunomodulatory approaches for enhancing tissue healing.

Introduction

Macrophages are a heterogeneous population of innate immune cells that play a pivotal role in regulating tissue homeostasis and inflammation (Orekhov et al., 2019). Peripheral blood monocytes are recruited to distant sites of injury, subsequently differentiate into macrophages that are critical for phagocytosis, regulating inflammation, sequential interactions with other immune cells and resident tissue cells, and impact tissue regeneration and remodeling processes (Li et al., 2012; Loi et al., 2016; Neve et al., 2014; Sugg et al., 2014; Sunwoo et al., 2020). Dependent on the tissue microenvironment during injury and healing, macrophages exhibit diverse and dynamic plasticity and have been historically, albeit in-vitro, designated as classically activated, inflammatory M1 macrophages and alternatively activated, pro-resolving or regulatory M2 macrophages (Heinrich et al., 2017; Italiani and Boraschi, 2014). An imbalance in macrophage responses have been linked to dysregulated inflammation resolution in tissues, and consequently, macrophages have been implicated as the key drivers of chronic inflammation and consequent tissue fibrosis and degeneration (Ackerman et al., 2017; Nathan et al., 2019). Even though this M1/M2 dichotomy represents two extreme phenotypes/polarities that fail to recapitulate the complex biological processes impacting a dynamic spectrum in macrophage polarization in-vivo, characterizing the phenotypes of inflammatory and regulatory/pro-resolving macrophages is a necessary first step for in-vitro and in-vivo research focused on immunomodulation strategies to enhance healing outcomes.

During the remodeling phase of tissue healing, macrophages are among key immune cells that induce a catabolic/inflammatory phenotype in tissue cells, alter their bioactivities and thereby impact healing (Manning et al., 2015; Stolk et al., 2017). An initial pro-inflammatory (M1) response followed by a regulatory/pro-resolving (M2) response is thought to be essential for a regenerative tissue healing response (Howell et al., 2021; Nathan et al., 2019; Sugg et al., 2014). To this end, several recent in-vitro studies utilize macrophage-tissue cell co-cultures to investigate both cell-cell interactions controlling tissue cell bioactivity, as well as to evaluate individual therapies that target macrophage phenotype manipulation to enhance healing outcomes (Loi et al., 2016; Nathan et al., 2019; Stolk et al., 2017). Both macrophages themselves and their secreted cytokines/soluble factors are vital biological mediators that influence the healing environment (Arango Duque and Descoteaux, 2014; Iqbal and Kumar, 2015; Orekhov et al., 2019). Limited existing studies with human macrophages have suggested that following exposure to inflammatory or regulatory activation, TNF-α, IL-1β, IL-6, IL-10, TGFβ, and IL-1Ra are key sets of cytokines/soluble factors secreted by macrophages (Arango Duque and Descoteaux, 2014; Graff et al., 2012; Iqbal and Kumar, 2015; Orekhov et al., 2019).

Musculoskeletal disorders such as osteoarthritis (McIlwraith et al., 2012) and tendinopathy/desmopathy (Cadby et al., 2013), metabolic syndrome (Johnson et al., 2012; Morgan et al., 2015), asthma (Bullone and Lavoie, 2015; Turlej et al., 2001), and inflammatory bowel disease (Schumacher et al., 2000) are a few important examples of chronic inflammatory disorders in the horse. Compared to rodent (Khallou-Laschet et al., 2010; Smith et al., 2016) and human (Tarique et al., 2015) macrophage studies, the plasticity and phenotypic characteristics of equine peripheral blood CD14⁺ monocyte-derived macrophages have not been determined. This represents an important gap given the clinical significance of chronic inflammatory disorders in horses, as well as due to the high translational relevance of equine models for delineating naturally occurring diseases in human and veterinary patients. Although several factors influence macrophage plasticity/polarity, inflammatory stimuli IFN-γ and LPS, and regulatory cytokines IL-4 and IL-10 exposure are commonly used during in-vitro differentiation experiments (Couper et al., 2008; Martinez and Gordon, 2014). Further, the cytokines and soluble factors in turn secreted by these differentiated macrophages are also largely unknown. Therefore, the objective of this in-vitro study is to delineate the effects of inflammatory and regulatory in-vitro culture conditions on the proliferation, morphology, cell surface marker expression, and cytokine/soluble factor secretion of equine peripheral blood monocyte-derived macrophages. Specifically, GM-CSF pretreatment and LPS + IFN-γ inflammatory priming and IL-4 + IL–10 regulatory priming during in-vitro adherent differentiation were analyzed.

Methods

Peripheral blood mononuclear cell (PBMC) isolation, magnetic separation of CD14+ monocytes and flow cytometry analysis

All study procedures were reviewed and approved by the university’s Institutional Animal Care and Use Committee. Jugular venous blood (approximately 150 mL) was obtained from five healthy horses (3 Quarter Horses and 2 Thoroughbreds) with an average age of 13 ± 5.4 years. Peripheral blood mononuclear cells (PBMC) were isolated from individual horses separately using 1.073 Ficoll density centrifugation as previously described (Mauel et al., 2006; Moyo et al., 2013; Steinbach et al., 2009). The resultant PBMC suspension was incubated with 1:1000 CD14 antibody clone Big10 (Enzo life sciences) (Durán et al., 2013; Steinbach et al., 2009) at 4°C for 30 minutes at a concentration of 10 uL for every 1 × 10⁶ PBMCs in PBS containing 0.5% BSA and 2mM EDTA. Cell viability was determined using trypan blue dye exclusion test and >98% live cells were confirmed for subsequent experiments. Next, the cell suspension was incubated with secondary IgG1 magnetic beads (Miltenyi Biotec) and processed through an LS column using the quadroMACS^™ magnetic separator (Miltenyi Biotec) according to manufacturer’s instructions. On average, 150mL of blood yielded 20 × 10⁶ monocytes that were 95–98% immunopositive for CD14 as confirmed via flow cytometry (Supplementary Figure 1).

Macrophage generation and differentiation

All experiments were conducted with CD14⁺ monocytes isolated from each horse separately. CD14⁺ monocytes were seeded in 9.5 cm² tissue culture plates (Falcon) at 0.2 × 10⁶ cells/cm² and cultured in basal medium consisting of RPMI (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco) with and without 50ng/mL recombinant equine GM-CSF (Orekhov et al., 2019) (Kingfisher biotech) for 6 days to generate macrophages (Iqbal and Kumar, 2015; Stolk et al., 2017). A single media change was carried out on day 3. On day 6, the media were removed and the macrophages were then primed with inflammatory or regulatory stimuli for 24 hours with cytokine concentrations adapted from published studies for in-vitro canine, murine, and human macrophage differentiation (Fujiwara et al., 2016; Graff et al., 2012; Iqbal and Kumar, 2015; Menarim et al., 2019; Naskou et al., 2018; Orekhov et al., 2019). For inflammatory (M1) macrophage differentiation, basal medium containing 50 ng/mL of recombinant equine IFN-γ (Fujiwara et al., 2016; Iqbal and Kumar, 2015) (Kingfisher biotech) and 100 ng/mL of LPS (Fujiwara et al., 2016; Graff et al., 2012; Iqbal and Kumar, 2015; Menarim et al., 2019; Naskou et al., 2018) (Escherichia coli strain O55:B5; Enzo life sciences) were utilized. To promote regulatory (M2) differentiation, basal medium containing 20ng/mL of recombinant equine IL-4 (Fujiwara et al., 2016; Graff et al., 2012; Iqbal and Kumar, 2015) (Kingfisher biotech) and 20 ng/mL of recombinant equine IL-10 (Fujiwara et al., 2016; Iqbal and Kumar, 2015; Orekhov et al., 2019) (Kingfisher biotech) were used (Figure 1). For control or GM-CSF only treated macrophages, a medium change with basal medium or basal medium with GM-CSF and culture for additional 24 hours was carried out. All media were collected, centrifuged at 2000×g for 5 minutes, aliquoted and frozen at −80°C for cytokine/soluble factor analyses. The macrophages were mechanically detached using a cell scraper, collected, and processed for flow cytometry as described below.

Figure 1:

Macrophage differentiation protocol and in-vitro culture conditions evaluated in this study.

Light microscopy morphological assessment

Morphological characteristics of macrophages at day 7 of differentiation were evaluated with photomicrographs obtained in 5–8 randomly selected fields from each differentiation condition (control, GM-CSF only, LPS + IFN-γ ± GM-CSF, and IL-4 + IL-10 ± GM-CSF) using a Leica inverted light microscope (Leica Microsystems, Leica application suite LAS version 6.0) at 10x and 20x objectives (Heinrich et al., 2017; Menzyanova et al., 2019).

Proliferation assay

Monocytes were seeded onto 1.9 cm² tissue culture plates (Falcon) at 0.125 × 10⁶ cells/cm², and proliferation during differentiation was assessed via a mitochondrial metabolic assay as previously described (Durgam et al., 2019; Jayaraman and Variyar, 2021) (Cell Titer MTT 96 aqueous one solution cell proliferation assay, Promega). Cell proliferation was assessed in duplicates cultures (control, GM-CSF only, LPS + IFN-γ and IL-4 + IL-10 with and without GM-CSF pretreatment). Twenty-four hours after respective treatments were added, the old media were removed and fresh media containing tetrazolium was added in each well at a concentration of 0.5% and incubated for 3 hours. After 3 hours, 150 uL from each well was transferred to a 96-well plate and absorbance was measured at 490 nm in a microplate reader (Tecan infinite 200 pro).

Flow cytometry

Following monocyte isolation, approximately 0.5 × 10⁶ cells were stained with a secondary IgG1 antibody conjugated to PE (Miltenyi Biotec) to test the purity of a representative aliquot from each isolation via flow cytometry. Following macrophage differentiation (control, GM-CSF only, GM-CSF pretreatment and LPS + IFN-γ or IL-4 + IL-10 priming), cells were stained with CD86 clone IT 2.2 (Alexa fluor 647) as an inflammatory phenotype marker and CD206 clone 3.29B1.10 (PE) as a regulatory marker as previously described (Iqbal and Kumar, 2015; Menarim et al., 2020; Stolk et al., 2017). Autofluorescence was compensated using unstained cells, isotype controls and all antibody dilutions were determined by individual titration assays (data not shown) (Patel et al., 2022). Doublets were excluded from the FSC-A vs FSC-H plot. Cellular debris were excluded, and monocyte/macrophage populations were identified from the FSC-A vs SSC-A plot. All Flow cytometry was performed using an Attune NxT cytometer (ThermoFisher Scientific).

ELISA quantification

Cytokine and soluble factor concentrations (IL-1β, TNF-α, IL-1Ra, TGFβ-1, and IL-6) in the media of macrophages (control, LPS+IFN-γ with GM-CSF pretreatment, and IL-4+IL-10 with and without GM-CSF pretreatment) were quantified using R&D systems equine DuoSet ELISA for TNF-α (Montero et al., 2020), IL-1β (Anderson et al., 2020), IL-1Ra (Ishihara et al., 2012), and IL-6 (Anderson et al., 2020). An R&D systems human ELISA validated for equine samples for TGFβ-1 after acidification with 6N HCl and subsequent 10N NaOH neutralization (Hauschild et al., 2017) was used. One hundred uL of each sample were analyzed in duplicate following manufacturer’s instructions and absorbance was measured at 450nm using a microplate reader (BioTek ELx808).

Statistical analyses

All data were assessed for normality using Shapiro-Wilk test. Data are expressed as mean ± standard deviation or median and interquartile ranges accordingly. Comparative difference in the proliferation of macrophages with and without GM-CSF was assessed with a t-test. Comparative differences between control, inflammatory (LPS + IFN-γ with GM-CSF pretreatment) and regulatory (IL-4 + IL-10 with and without GM-CSF pretreatment) were assessed with one-way analysis of variance (CD86 and CD206 flow cytometry, TGFβ-1 and IL-1Ra ELISA) or Kruskal-Wallis test (IL-1β and TNF-α ELISA) followed by Tukey HSD or Dunn post-hoc tests, respectively for pairwise comparisons. Significance was set at p < 0.05. All statistical analyses were conducted using the R statistical program version 4.1.0. All graphs were generated using Prism GraphPad version 9.1.0.

Results

Morphology

Monocytes maintained in basal medium alone and basal medium with GM-CSF for 7 days yielded macrophages with a rounded morphology (Figure 2a). Subjectively, GM-CSF increased macrophage numbers in any given field of view. Priming with LPS + IFN-γ resulted in macrophages with a rounded, granular morphology (Figure 2b). Priming with IL-4 + IL-10 yielded macrophages with an elongated, spindle-shaped morphology (Figure 2c).

Figure 2:

Representative phase-contrast photomicrographs (n=4) of macrophages cultured in basal medium, basal medium with GM-CSF, basal medium −/+ GM-CSF followed by 24-hour priming with LPS + IFN-γ or IL-4 + IL-10. Scale bars represent 100 μm.

In-vitro macrophage proliferation

GM-CSF increased macrophage proliferation by 15-fold (p=0.03) compared to groups without GM-CSF. There were no significant differences between individual treatment groups or control.

Flow cytometry analysis of differentiated macrophages

Control macrophages were 21.1 ± 24.6 % immunopositive for CD86. GM-CSF alone and GM-CSF pre-treatment and subsequent LPS + IFN-γ and IL-4+ IL-10 priming increased CD86 expression by 2-fold (p=0.6); compared to control macrophages. Control macrophages were 73.8 ± 18.7 % immunopositive for CD206. GM-CSF alone and, GM-CSF pretreatment and subsequent LPS + IFN-γ and IL-4 + IL-10 priming did not significantly (p = 0.52) change CD206 expression (Figure 3).

Figure 3:

Flow cytometry analysis (n=4) of macrophages for CD86 (a) and CD 206 (b) maintained in basal medium designated as ‘control’, basal medium with GM-CSF designated as ‘GM-CSF’, GM-CSF pretreatment and LPS + IFN-γ priming designated as ‘LPS+IFN-γ (+)’, and GM-CSF pretreatment and IL-4 + IL-10 priming designated as ‘IL-4+IL-10 (+)’. Bars and errors represent mean ± SD percentage of macrophages immunopositive for CD86 and CD206 after 7 days of culture with differentiation treatments enlisted above. Statistical analyses were conducted within CD86 and CD206 groups between these treatments.

Cytokine analysis

Control macrophages secreted low levels of IL-1β, TNF-α and IL-1Ra. GM-CSF pre-treatment and subsequent LPS + IFN-γ significantly increased IL-1β secretion from 0 to 136.1 (100.4 – 198.3) pg/mL (p=0.012) compared to control macrophages and macrophages primed with IL-4 + IL-10 without GM-CSF pretreatment (p=0.0047). There was minimal IL-1β secretion in macrophages primed with IL-4 + IL-10 with and without GM-CSF pretreatment. (Figure 4a)

Figure 4:

ELISA quantification of cytokines in culture media during macrophage differentiation (n=5). Culture conditions evaluated included macrophages - maintained in basal medium designated as ‘control’, pretreated with GM-CSF and primed with LPS + IFN-γ designated as ‘LPS+IFN-γ (+)’, maintained in basal medium without or with GM-CSF pretreatment and primed with IL-1 + IL-10 designated as ‘IL-4+IL-10 (−)’ and ‘IL-4+IL-10 (+)’. Data points depict individual values of IL-1β (a), TNF-α (b), IL-1Ra (c) and TGF-β (d). The bars denote median for IL-1β and TNF-α, and mean for IL-1Ra and TGF-β. * represents p< 0.05 and ** represents p< 0.01

GM-CSF pretreatment followed by LPS + IFN-γ and IL-4 + IL-10 priming significantly increased TNF-α to 1285 (847–1786) pg/mL; p=0.034 and 2092 (1707–2100) pg/mL; p=0.0039, respectively, compared to control macrophages. (Figure 4b)

GM-CSF pretreatment followed by both LPS + IFN-γ and IL-4 + IL-10 priming significantly increased IL-1Ra secretion by approximately 6-fold compared to control (p=0.029; p=0.014) and macrophages primed with IL-4 + IL-10 without GM-CSF pretreatment (p=0.040; p=0.021). (Figure 4c)

Culture medium of control macrophages and all tested macrophage differentiation conditions contained 643 ± 51.5 pg/mL of TGFβ1 (p=0.85). IL-6 was not detected in the media of control or differentiated macrophages.

Discussion

This study analyzed the in-vitro differentiation and plasticity characteristics of equine peripheral blood monocyte-derived macrophages. Cell surface CD14⁺ magnetic bead-based positive selection on PBMC isolated via density gradient centrifugation was an efficient method to obtain sufficient monocytes for in-vitro macrophage studies. Recombinant equine GM-CSF enhances the proliferation rate of monocytes/macrophages during adherent in-vitro culture. GM-CSF pretreatment and subsequent inflammatory priming with LPS and IFN-γ yielded inflammatory macrophages that secrete significantly increased quantities of IL-1β and TNF-α into the culture medium. All culture conditions evaluated elicited IL-1Ra and TGFβ1 secretion from the differentiated macrophages.

Infiltrating monocytes/macrophages are key immune cells that play a crucial role in regulating acute and chronic inflammation during tissue healing, and are therefore, a potential therapeutic target to modulate regenerative tissue healing responses. In murine and human in-vitro and in-vivo studies, IFN-γ, IL-6, IL-10 have shown to be the key cytokines that impact macrophage plasticity/polarity (Couper et al., 2008; Martinez and Gordon, 2014). Due to the paucity of information on equine macrophage differentiation and plasticity characteristics, and the significance of innate immune responses during tissue healing, this study investigated the effects of inflammatory and regulatory in-vitro culture conditions on the proliferation, morphology, cell surface marker expression, and cytokine/soluble factor secretion of equine peripheral blood monocyte-derived macrophages.

In mice (Chen et al., 1988a; Chen et al., 1988b) and rabbits (Wang et al., 1994), GM-CSF exposure increased the proliferation rate of monocytes and macrophages during in-vitro culture and is consistent with the results obtained in this study. While pro-proliferative effect of GM-CSF was noted (Achuthan et al., 2021), LPS+IFN-γ or IL-4+IL-10 priming did not affect macrophage proliferation in this study and is likely reflective of the high concentrations of the stimuli used. Macrophages primed with inflammatory stimuli LPS + IFN-γ exhibited rounded granular morphology; in contrast, macrophages primed with IL-4 + IL-10 developed an elongated morphology, and these morphological differences were noted regardless of GM-CSF pretreatment. These findings are also similar in canine and murine in-vitro macrophage studies (Heinrich et al., 2017; McWhorter et al., 2013; Menzyanova et al., 2019). It is also possible that the elongate morphology noted represents overlap with dendritic cell differentiation and warrants further evaluation (Moyo et al., 2013). The functional significance of the differential macrophage morphologies and plasticity are unclear based on existing studies, and further analyses that include electron microscopy or single cell RNA sequencing may be beneficial.

Although several cell surface phenotype markers are available for distinguishing human and murine M1 and M2 macrophages in-vitro, there are limited verified antibodies that cross-react with equine antigens. In this study, flow cytometry investigation of equine macrophage differentiation after inflammatory and regulatory priming were restricted to cell surface marker expression of CD86 and CD206, respectively. CD86 expression increases in general during macrophage proliferation and activation (Raggi et al., 2017), and is reflected in the results of this study where the low CD86 expression (21%) in control macrophages was increased by approximately 2-fold irrespective of LPS+IFN-γ or IL-4+IL-10 priming. Expanding the analysis to identify combined CD34/CD86 immunopositivity may shed more light on macrophages characteristics (Ryncarz and Anasetti, 1998). CD206 maintained a moderately high expression (80%) in inflammatory, regulatory and control macrophages. CD206 is differentially expressed to a higher degree in canine (Heinrich et al., 2017) and human (Xu et al., 2019) regulatory/pro-resolving macrophages. Proinflammatory macrophages commonly express CD80, CD86, toll like receptor 2 (TLR2) and TLR4, and proresolving/regulatory macrophages commonly express CD206, CD204, and CD163 (Trombetta et al., 2018). The limited change in these markers suggests that CD86 and CD206 may not be best suited to evaluate the effects of equine macrophages with LPS+IFN-γ or IL-4+IL-10 priming. Accepting that a single marker will be insufficient to distinguish equine macrophage subtypes, incorporating gene expression/transcriptome analyses can be beneficial to establish a broader CD marker antibody panel for proinflammatory and proresolving/regulatory equine macrophages (Menarim et al., 2021).

The current study aimed to assess the impact of in-vitro GM-CSF pretreatment, and LPS+IFN-γ or IL-4+IL-10 priming of equine macrophages on subsequent cytokine and soluble factor secretion. Key cytokines and soluble factors IL-1β, TNF-α, IL-1Ra, TGFβ1, and IL-6 that impact the tissue inflammatory microenvironment were quantified (Martinez and Gordon, 2014; Orekhov et al., 2019). Their concentrations were negligible in control macrophage medium. GM-CSF pretreatment followed by LPS + IFN-γ priming significantly increased IL-1β secretion compared to control and IL-4+IL-10 primed macrophages. The finding that GM-CSF pretreatment irrespective of LPS+IFN-γ or IL-4+IL-10 priming increased TNF-α secretion suggests that GM-CSF pretreatment is responsible for the TNF-α secretion from macrophages; however, needs additional verification with GM-CSF only treatment group. The cytokine analyses results of this study indicate that GM-CSF pretreatment followed by LPS and IFN-γ priming induce an inflammatory phenotype in equine macrophages, and is consistent with canine (Heinrich et al., 2017) and murine (Holden et al., 2014) macrophage studies. IL-1β and TNF-α are potent inflammatory cytokines involved in various inflammatory diseases, and among immune cells, are produced by monocytes and macrophages (Ahn et al., 2017; Latz et al., 2013). Lack of IL-6 secretion in culture media was unexpected. Although the relative short duration of in-vitro culture may offer a potential explanation for the lack of IL-6 secretion in culture media, additional investigations are warranted.

Interleukin-1 receptor antagonist (IL-1Ra) exerts dose-dependent anti-inflammatory effects in-vitro, and therapeutic concentrations of IL-1Ra are routinely harnessed via peripheral blood monocyte activation for treating articular joint inflammatory conditions (Hale et al., 2021). In this study, IL-1Ra secretion increased with GM-CSF pretreatment and subsequent LPS+IFN-γ or IL-4+IL-10 priming suggesting that monocyte/macrophage activation during in-vitro culture and incubation as the cause, rather than the inflammatory or regulatory stimulation itself, and corroborates with other existing studies (Fjordbakk et al., 2015; Hale et al., 2021). Macrophages are involved in IL-10 secretion, an immunoregulator involved in dampening the inflammatory milieu (Couper et al., 2008; Orekhov et al., 2019), and is therefore ubiquitously utilized for proresolving/regulatory macrophage differentiation (Fujiwara et al., 2016; Iqbal and Kumar, 2015; Orekhov et al., 2019). Although a significant difference in regulatory/anti-inflammatory factors TGFβ and IL-6 were not detected in the medium of IL-4+IL-10-primed equine macrophages, the anti-inflammatory effects of net IL-1Ra increase in the regulatory primed macrophage medium warrants further evaluation. Further, an important limitation that must be considered with respect to in-vitro equine macrophage differentiation is the lack of a commercially available equine M-CSF that is typically used for M2 or alternatively activated or regulatory macrophage differentiation (Iqbal and Kumar, 2015; Orekhov et al., 2019; Stolk et al., 2017). The collective results of this study demonstrate that regulatory priming, specifically, IL-4 + IL-10 without GM-CSF pretreatment, yielded the least inflammatory equine macrophage subtype when compared with GM-CSF pretreated and LPS+IFN-γ or IL-4+IL-10-primed macrophages.

This study demonstrates that inflammatory and regulatory stimulation outlined for murine, canine, and human macrophage in-vitro differentiation are largely applicable to equine macrophages, and additionally, this study further sheds light on the biological factors secreted by equine macrophages in response to inflammatory and regulatory priming. Existing studies on in-vitro macrophage differentiation in general are focused on characterizing the differential cell surface marker expression and transcriptome profiles of proinflammatory and proresolving/regulatory macrophages. Accepting the limitations of short timeframe for LPS+IFN-γ or IL-4+IL-10 priming, and the lack of other immune cell subsets that orchestrate macrophage polarity in-vivo during in-vitro differentiation protocols used in this study, this reductionistic approach with equine macrophages provides foundational information on the biological cues secreted by macrophages.

Conclusion

This study analyzed the in-vitro differentiation and plasticity characteristics of equine peripheral blood CD14⁺ monocyte-derived macrophages. Recombinant equine GM-CSF enhances the proliferation rate of monocytes/macrophages during adherent in-vitro culture. GM-CSF pretreatment and subsequent inflammatory priming with LPS and IFN-γ yielded inflammatory macrophages that secrete significantly increased quantities of IL-1β and TNF-α into the culture medium. Among the culture conditions tested in this study, regulatory priming for 24 hours with recombinant equine IL-4 and IL-10 after 6 days of adherent culture yielded the least inflammatory macrophages among all tested conditions. All culture conditions evaluated elicited IL-1Ra and TGFβ1 secretion from the differentiated macrophages. This foundational information on the biological cues secreted by macrophage subtypes is beneficial to design and delineate subsequent in-vitro and in-vivo studies that investigate macrophage-tissue cell interactions and related biological mechanisms relevant to the field of immunomodulatory approaches to enhance tissue healing.

Supplementary Material

1

Supplementary figure 1: Flow cytometry analysis on a representative aliquot of isolated CD14+ monocytes. (a) FSC-A vs FSC-H plot with gating to exclude doublets. (b) FSC-A vs SSC-A plot with gating to exclude cellular debris and identify monocyte population. (c) Histogram of CD14+ cells and unstained control showing 98.5% immunopositive for CD14.

Highlights.

• Equine GM-CSF increases equine macrophage in vitro proliferation.

• GM-CSF pretreatment and sequential LPS+IFNγ priming induces inflammatory (M1) phenotype that secrete IL-1β.

• IL-4+IL-10 priming following adherent culture yielded regulatory (M2-like) macrophages.

• IL-1β, TNFα, IL-1Ra constitute key cytokines secreted by equine macrophages.

Abbreviations:

GM-CSF

Granulocyte Macrophage- Colony Stimulating Factor

ANOVA

Analysis of Variance

STAT

Signal Transducer and Activator of Transcription

TLR

Toll-like Receptor

M-CSF

Macrophage-Colony Stimulating Factor

Data Availability statement

The raw datasets (Proliferation, Flow cytometry, and ELISA) supporting the results of this manuscript will be made available by the authors, without any reservation, to researchers upon request.