The Human Mesenchymal Stem Cell Secretome Directs Macrophage Differentiation in an Oxygen-Dependent Manner

doi:10.21203/rs.3.rs-3006436/v1

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The Human Mesenchymal Stem Cell Secretome Directs Macrophage Differentiation in an Oxygen-Dependent Manner

https://doi.org/10.21203/rs.3.rs-3006436/v1

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Background

Chronic disease states can share inflammation as an underlying pathology. Macrophages are associated with chronic inflammation; in general, M1 phenotype macrophage activity inhibits cell proliferation and causes tissue damage whilst M2 macrophages promote proliferation and tissue repair. Human mesenchymal stem/stromal cells (hMSCs) are of particular interest for immunoregulatory diseases, through their ability to suppress T cell proliferation.

Methods

The application of a physiological oxygen environment to hMSC cultures modulates their gene expression and results in an altered secretome. Consequently, physiological, 2% O₂ hMSC SFCM was assessed in comparison to air oxygen (21% O₂) produced SFCM and applied to activated THP-1 cells in 10% and 21% O₂ to assess changes in macrophage differentiation.

Results

SFCM significantly suppressed proliferation and metabolic activity whilst promoting cell adherence compared to serum-free media (SFM) controls (p < 0.001). Terminal differentiation phenotype varied in an oxygen-dependent manner, with 21% air oxygen SFCM leading to an increased M1:M2 macrophage differentiation ratio, with cells displaying distinct pancake-like morphology, increased TNFa secretion (21% O₂ = 922 ± 5 pg/mL, and 10% O₂ = 373 ± 2 pg/mL) and CD197 expression (21% = 69.1%±6.4, and 10% = 18.6 ± 0.4%). In contrast, physiological oxygen SFCM led to the production of a greater M2:M1 macrophage differentiation ratio; displaying an elongated spindle shape, increased IL-10 secretion (21% O₂ = 3780 ± 560 pg/mL, and 10% O₂ = 9115 ± 889), and CD3 and CD14 expression. SFCM collected under 2% O₂ suppressed pro-inflammatory transcriptional profiles through downregulation of TNFa, IL-1B, and IL-12B compared to 21% O₂ collected SFCM.

Conclusion

Collectively, SFCM induced terminal macrophage differentiation in an oxygen-dependent manner. This effect was not completely replicated by the application of isolated candidate anti-inflammatory cytokines (IL-4, IL-10, IL-13 or TGFb) or a combinational cytomix, highlighting the complex effects of conditioned media products and further work needed to determine mechanism of action.

Mesenchymal Stem Cells

Secretome

Physoxia

Immunomodulation

Inflammation is critical during host infection or physical injury and is characterized by the short-term activation of immune and non-immune cells; ceasing upon pathogenic clearance or resolution of injury. Many chronic disease states share inflammation as an underlying pathology, where inflammation persists beyond the acute immune response¹. Tissue injury is associated with infiltration of immune cells to sites of injury, resulting in the release of reactive biomolecules, which in turn can contribute to further tissue damage². Neutrophils are predominantly involved in acute inflammation whilst macrophages and T cells are associated with chronic inflammation^3,4. Broadly, macrophages are classified as two types, M1 and M2, expressing distinct surface marker and secretome profiles; activating distinct subsets of T cells based on the received signal and localised tissue milieu⁵. The M1 macrophage subtype is characterised by the release of strong pro-inflammatory cytokines including, tumour necrosis factor alpha (TNFa), interleukin (IL)-12, and IL-1B and strong antimicrobial action and presentation of their antigens to Th1 subsets of T cells^6,7. Soluble factors in the localised microenvironment play a significant role in modulation of the macrophage phenotype, for instance, pro-inflammatory factors including lipopolysaccharide (LPS), interferon γ (IFNγ) and granulocyte macrophage colony-stimulating factor (GM-CSF) promote M1 phenotype polarisation and subsequently interaction with Th1 subsets of T cells^8,9. The M2 macrophage subtype is characterised by the release of strong anti-inflammatory cytokines including IL-10, and elevated expression of mannose scavenger receptor (CD206)^10,11, CD36¹² and macrophage scavenger receptor 204 (CD204)^13,14. Moreover, polarisation can be triggered by macrophage colony-stimulating factor (M-CSF), IL-4, IL-10, IL-13 and TGFb leading to stimulation of Th2 subsets of T cells^15,16. Currently, available therapeutic strategies are mainly focussed on the inhibition of inflammation through the administration of corticosteroid, non-steroidal anti-inflammatory drugs (NSAIDs) and anti-TNFa monoclonal antibodies^17–19. Recent studies have been directed towards therapeutics with immunoregulatory rather than immunosuppressive actions including, stimulation of immune cell differentiation toward regulatory T cells (Tregs) and M2 macrophages, with a view to guide the immune response toward tissue repair rather than degeneration.

Human mesenchymal stem/stromal cells (hMSCs) are a promising therapeutic tool whose scope and applications have expanded beyond tissue regeneration^20,21. hMSCs are of particular interest for immunoregulatory application in diseases displaying an inflammatory component, through their ability to suppress T cell proliferation^22–25. Proteomic profiling of the secretome composition of hMSC serum-free conditioned media (SFCM) has revealed a range of pleiotropic biomolecules including the anti-inflammatory cytokine IL-10, vascular endothelial growth factor (VEGF), GM-CSF and leptin^26–28. However, defining the SFCM components and their concentrations is complicated by secretome modification resulting from hMSC source; e.g. adipose tissue²⁹, cord blood³⁰, bone marrow³¹ or human induced pluripotent stem cell (hiPSC)-derived³²; cellular age²⁵, culture conditions³³, conditioning periods, and planar versus 3D cell culture methods³⁴.

The oxygen environment applied to in vitro hMSC cultures plays a key role in determining their biology and has been described previously^35,36. Physoxia is an inherent feature of the in vivo hMSC niche, drawing largely from the sinusoidal blood network^37,38. The physiological partial pressure of oxygen (pO₂) of in vivo bone marrow niche, has an average value of 2.7%³⁹. Physiological oxygen concentrations in tissues are significantly lower than inhaled air (21% O₂); declining gradually from entering the lungs and through the body, ranging between 0.1–9%^40–42. Consequently, the application of an increasingly in-vivo-like physiological oxygen environment to in vitro hMSC culture modulates gene expression and increasing evidence suggests this further manifests itself via an altered secretome composition^37,43. Oxygen-induced secretome profile alterations would likely impact the reparative action of hMSC SFCM and would likely better reflect the behaviour of hMSCs and/or their secretome following transplantation into in vivo tissues.

The utilisation of primary macrophages to study inflammatory processes is limited by low levels of in vitro proliferation coupled with the requirement of invasive isolation techniques, including tissue biopsy or bronchoscopy⁴⁴. To overcome these obstacles and to meet the large-scale cell requirement for in vitro experimentation, the monocyte cell line model THP-1 is routinely utilised. THP-1 cells can be activated through the addition of Phorbol 12-myristate 13-acetate (PMA) to functional macrophages through the stimulation of the protein kinase C pathway^45,46. Previous studies have demonstrated the applicability of THP-1 cells as a model for human macrophages through appropriate mRNA and protein expression of M1 and M2-specific markers following PMA polarisation^47–49.

The present study examined the efficacy of hMSC SFCM to modulate the differentiation fate of THP-1 cells towards either the M1 or M2 phenotype. Further, the mode of action of SFCM was assessed through the manipulation of selected biomolecules: IL-4, IL-10, IL-13, and TGFb, through exogenous addition to growth media, or neutralisation from SFCM. Collectively, it was demonstrated that SFCM induced terminal macrophage differentiation in an oxygen-dependent manner, where individual anti-inflammatory cytokines or a combinational cytomix had no effect on the THP-1 secretome profile and surface marker expression. Moreover, neutralisation of IL-4, IL-10, IL-13 or TGFb from SFCM resulted in the incomplete restoration of a pro-macrophagic status. This finding may be linked to growth factors present in SFCM, which represent important initial stimuli to induce the conversion of monocytic THP-1 cells to the macrophage M0 status. An initial priming step could be important for subsequent macrophage differentiation to either M1 or M2 and could be influenced by the presence of pro-inflammatory or anti-inflammatory cues, respectively^50,51. The ability of SFCM, specifically the key biomolecule mediators contained within, to modulate inflammatory processes of a myriad of disease states opens the possibility for the development of therapeutic tools and the advancement of potential cell-free approaches.

2.1 Cell Line Culture

2.1.1 hMSCs

hMSCs were isolated and expanded from human bone marrow aspirate (BMA) using an adherence-based methodology⁹¹. Three donor human BMAs (two male and one female, ages 20–36) were purchased from Lonza, USA and seeded at a density of 1×10⁵ mononuclear cells/cm² on fibronectin pre-coated culture flasks in Dulbecco’s Modified Eagle Medium (DMEM) media supplemented with 5% (v/v) fetal bovine serum (FBS), 1% (v/v) L-glutamine, 1% (v/v) non-essential amino acids (NEAA), and 1% (v/v) Penicillin-Streptomycin-Amphotericin B (PSA) (Lonza, Slough, UK). Seeded flasks were incubated in humidified incubators with distinct oxygen tensions of 21% O₂ or 2% O₂. After 7 days, half of the media volume was removed and replaced with fresh antibiotic-free growth medium followed by a complete media change after a further 7 days. Media was then changed every 3 days until confluent. Once confluent, hMSC were enzymatically passaged with 1% Trypsin/EDTA (Lonza, Slough, UK) at 1:2 split ratios. Passage 1 cells and their CM were used for all experiments.

SFCM was produced by washing 70% confluent hMSC seeded T75 flasks with phosphate-buffered saline (PBS) followed by the addition of 15 mL serum-free media (SFM) consisting of DMEM supplemented with 1% L-glutamine (v/v) and 1% NEAA (v/v). For conditioning, 20 mL of SFM were added to hMSC cultures and incubated for 24 h. Conditioned media was then collected, centrifuged for 10 minutes at 300× g, and stored at − 80°C as SFCM. Prior to use, SFCM was thawed and filtered (0.2 µm). All SFCM was produced from hMSC cultures at passage 1. Time taken for hMSC isolation from BMA plating, expansion, passaging, and confluency at passage 1 was 28–29 days (28 days for donors 1 and 2, 29 days for donor 3) and was consistent between the oxygen concentrations.

Positive expression for CD73, CD90, and CD105 and negligible expression for CD14, CD19, CD34, CD45, and HLA-DR surface molecules were confirmed, as well as trilineage differentiation potential according to ISCT guidelines, and were published previously²⁹.

2.1.2 THP-1

THP-1 cells were cultured in antibiotic-free growth media (GM) which consisted of DMEM supplemented with 10% FBS, 1% L-glutamine and 1% NEAA. THP-1 cells failed to proliferate at 2% oxygen levels and consequently, were cultured at 10% O₂, representative physiological oxygen environment, and 21% air oxygen. For sub-culturing THP-1 cell lines, confluent suspension cultures were collected and centrifuged at 180x g for 3 minutes, the supernatant removed and the cell pellet re-suspended in fresh GM. THP-1 cells were seeded at a density of 1×10⁵ cells/mL in T75 flasks. Subsequent media changes were performed twice per week based on their population doubling time (2–3 days).

THP-1 cells were seeded at 5×10⁵ cells/mL for activation through the addition of 50ng of PMA (Sigma-Aldrich, Gillingham, UK) to the cell culture media. The activation was performed for a period of 24 h in a humidified incubator at 37°C at either 21% O₂ or 10% O₂.

2.2 Cell Viability and Proliferation Assays

2.2.1. Cell Counting

Haemocytometer cell counts were performed on time-point samples over 7 days to determine the rate of cell growth from an initial seeding density of 2×10⁵ cells/mL. Cell counts were performed on the cells in different conditions, including, activated and non-activated cells in their GM, SFM, and SFCM, and under different oxygen tensions, 2% O₂ and 10% O₂. Counts were performed on triplicate flasks.

2.2.2. MTT Assay

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay (Sigma-Aldrich, Gillingham, UK) was used to determine metabolic activity. THP-1 cells were seeded in triplicate at a concentration of 2×10⁵ cells/mL before incubation in MTT for 4 h at 37°C as per manufacturer’s instructions. A total of 50 µL of DMSO were added to each well to dissolve formazan crystals, and the plate was incubated again for 45 minutes. The optical density (O.D.) of each sample was determined by reading at 570 nm on a Synergy2 plate reader (BioTek, Cheadle, UK).

2.2.3. Cytospinning

The Cytospin technique was used to assess Jurkat cell morphology. To prepare Cytospin slides for imaging, 5×10⁴ cells were centrifuged, the supernatant removed and pellets washed twice in cold PBS before resuspended in 1 mL PBS. The cells were pipetted into the Cytofunnel (Fisher Scientific, Loughborough, UK) and centrifuged by Cytospin4 centrifuge (Fisher Scientific, Loughborough, UK) at 300× g for 2 minutes. Slides were then removed, air dried for 15 minutes and fixed with 95% ethanol for 15 minutes. Following air drying, the slides were stained with May–Grünwald solution (Sigma-Aldrich, Gillingham, UK) for 5 minutes, washed, and further staining with Giemsa solution (Sigma-Aldrich, Gillingham, UK) for 15 minutes. A xylene mounting agent was placed over the slides to fix the cover slides. Images were captured, and the cell surface area was calculated for 100 cells using ImageJ software (NIH, Maryland, MD, USA).

2.3. Cytokine Challenge

To identify if the immunosuppression by SFCM was cytokine-driven, four anti-inflammatory targets were chosen: IL-4, IL-10, IL-13, and TGFb. These cytokines were individually tested on a Jurkat cell line models in SFM, and a cytomix is considered to account for the IL-4/IL-13 receptor overlapping phenomena. Concentrations were identified via dose-response curves via testing a serial dilution of an individual cytokine in SFM on the Jurkat cell line models: IL-4 (PeproTech, London, UK #200-04), IL-10 (PeproTech, London, UK #200 − 10), IL-13 (PeproTech, London, UK #200-04), and TGFb (PeproTech, London, UK #100-36E). MTT was conducted after 24 h of cell exposure to a serial dose of IL-4 (0–10,000 pg/mL), IL-13 (0–10,000 pg/mL), IL-10 (0–50 ng/mL), and TGFb (0–100 ng/mL). Toxic doses were ignored, and the highest non-toxic doses of each [(IL-4 and IL-13 (250 pg/mL), IL-10 (1 ng/mL), and TGFb (5 ng/mL)] were considered as target doses.

2.4. Cytokine Blocking

Cytokines were either individually or in combination (IL-4/IL-13) blocked from SFCM. These cytokines were blocked in SFCM using an excess amount of rabbit poly-clonal antibodies: IL-4 (PeproTech, London, UK #500-P24), IL-10 (PeproTech, London, UK #500-P20), and IL-13 (PeproTech, London, UK #500-P13). To neutralise these cytokines from SFCM, IL-10 was neutralised by 2 µg/mL of anti-human IL-10, IL-4 neutralised by 100 ng/mL anti-human IL-4, and IL-13 neutralised by 200 ng/mL anti-human IL-13.

2.5. ELISA Assay

ELISA assays were conducted for the detection of TNFa (PeproTech, London, UK # 900-K25) and IL-10 (PeproTech, London, UK #900-M21). Standard serial dilutions and culture media samples were loaded in triplicate into an overnight pre-coated surface with cytokine specific capture antibodies. Blocking was carried out for 1 hour using 1% BSA-blocking buffer, followed by a 2-hour incubation with a diluted detection antibody mixture and 30 minutes with a diluted avidin-HRP conjugate. Each step was accompanied by forcibly discarding the contents and four washing steps with diluted detergent buffer. Finally, an enzymatic reaction initiated by the addition of an ABTS-substrate (2,2′-Azino-bis (3-ethylbenzothiazoline-6- sulfonic acid)), leading to a bluish-green colour development within 5–15 minutes during which a visible signal was detected at 405 nm via a plate reader. The concentrations of unknown samples were determined by the interpolation of the standard calibration curves for each component. The differences between proliferation rates of THP-1 cells cultured in cultured under different conditions were taken into consideration. To account for this, the data was normalised and presented through determination of the secreted IL-10 and TNFa quantities per cell.

2.6 Gene Expression

2.6.1 RNA Extraction and Processing

Cell pellets were lysed and transferred to QIAshredder spin columns (Qiagen, UK). RNA extraction from cell lysates was carried out using a RNeasy Kit (Qiagen) according to manufacturer’s instructions. Purified RNA samples were quantified using a Nanodrop 2000 (Thermo Fisher, UK).

2.6.2 RT-PCR

A one-step RT-PCR reaction was designed to contain 25ng RNA per reaction tube. The gene amplification was performed on the DNA Engine thermal cycler (GENEFLOW). Primers designed using human gene sequences from NCBI Gene Viewer. Designed primers were evaluated in NCBI Primer-BLAST to check binding specificity (Table 1). Customised primer sets were purchased from Invitrogen, UK. Thermocycler cycling parameters for RT-PCR consisted of an initial denaturing step of 95ᵒC for 15 minutes followed by 40 cycles of denaturation for 1 minute at 94ᵒC, annealing for 30 seconds using primer specific temperatures and extension for 30 seconds at 72ᵒC. PCR reaction products were analysed by electrophoresis on a 2% agarose gel.

2.6. Statistical Analysis

Statistical analysis was conducted between SFCM and SFM using a two-sample t-test for most of the measured parameters. For comparison of more than 2 groups, a one-way ANOVA with Tukey’s multiple comparisons test was performed to determine pair-wise statistical significance; p ≤ 0.05 was considered significant. The analysis was performed using GraphPad Prism 6 (San Diego, CA, USA). Unless otherwise stated, all values quoted in the results are mean ± standard deviation (SD).

3.1 Characterisation of THP-1 In Vitro Macrophage Differentiation Model

3.1.1 PMA Inhibits Proliferation and Promotes Attachment of THP-1 cells

The effects of PMA activation on THP-1 cell line proliferation and metabolic activity were determined over a 7-day period (Supplementary Fig. 1). In the absence of PMA, the cell growth curve followed the standard pattern of lag, log, and stationary phase. However, in the presence of PMA, the growth curve plateaued showing only a slight increase in proliferation (Supplementary Fig. 1A). Total cell counts were significantly higher (p < 0.001) in GM^− PMA over 7 days compared to GM^+ PMA in both 21% O₂ and 10% O₂. PMA significantly (p < 0.05) downregulated MTT over 7 days in both 21% O₂ and 10% O₂ conditions (Supplementary Fig. 1B). Further, THP-1 exposure to PMA induced M0 differentiation resulting in a significant increase (p < 0.001) in cell attachment to tissue culture plastic surfaces (Supplementary Fig. 1C).

3.1.2 Activation of THP-1 Cells Results in IL-10/TNFa Secretion

IL-10 (pro-inflammatory and M1 marker) and TNFa (anti-inflammatory and M2 marker) markers were used to assess macrophage differentiation. PMA induced activation stimulated both IL-10 and TNFa secretion over a 7-day period. Elevated secretion of IL-10 was noted in 21% O₂ environment (p < 0.001) with higher secretion of TNFa in 10% O₂ cultured THP-1 cells (p < 0.001) (Supplementary Fig. 2A). Morphological assessment of THP-1 cells was scored according to their shape, either spindle-like or pancake-shaped, and the subsequent percentage of the total population determined. Polarisation of THP-1 cells in 21% O₂ lead to a significantly higher number of spindle-shaped (M2) cells whereas, culture in 10% promoted a significant increase in the number cells scored as pancake-shaped (M1) (p < 0.001) (Supplementary Fig. 2B). Surface marker expression of THP-1 cells in GM^+ PMA was performed via grouping of positive (CD45 and CD105), negative (CD19, CD25, CD34, CD86 and CD90), and target markers, representative of monocytes and M0 macrophages (CD14, CD73, and HLA-DR), M1 macrophages (CD197), and M2 macrophages (CD36, CD204 and CD206). PMA significantly upregulated CD14 and CD73 (p < 0.001) whilst significantly downregulating HLA-DR, CD45, and CD105 (p < 0.001) (Supplementary Fig. 2C).

3.1.3 THP-1 Gene Expression is Modulated by Oxygen Environment

Modulation of gene expression following THP-1 activation for chemotactic genes (CCL5, CCR5, IL-8, MCP1, and MIP1A), pro-inflammatory genes (TNFa, IL-1B, and IL-12B), and an anti-inflammatory gene (IL-10) was compared across both oxygen environments (Supplementary Fig. 3). PMA activation of THP-1 cells resulted in the weak upregulation of genes including, MCP1, MIP1A and IL-12B when compared to D0 in both 2% and 21% O₂. In 10% O₂, IL-8, CCL5, IL-1B, TNFa and IL-10 showed stronger band expression in comparison to 21% O₂. Conversely, L32 displayed stronger expression in 21% O₂ versus 10% O₂.

3.2 The Role of SFCM in Modulation of THP-1 Proliferation

3.2.1 SFCM Suppresses THP-1 Proliferation

In the absence of PMA, THP-1 exhibited normal proliferation patterns in both 21% O₂ and 10% O₂ and in SFM or SFCM cultured cells (Fig. 1A). The growth curve of THP-1 increased gradually in SFCM reaching a stationary phase at day 5 [21% O₂ (SFM^− PMA = 3.7×10⁵±1×10⁴ cells/mL, and SFCM^− PMA = 3.7×10⁵±2.1×10⁴ cells/mL) and 10% O₂ (SFM^− PMA = 3.9×10⁵±2.1×10⁴ cells/mL and SFCM^− PMA = 3.5×10⁵±1.5×10⁴ cells/mL)] and plateaued thereafter. Similarly, in the presence of PMA, the growth curve gradually increased reaching a maximum at day 5 [21% O₂ (SFM^+ PMA = 3.4×10⁵±7.6×10³ cells/mL, and SFCM^+ PMA = 2.5×10⁵±1.5×10⁴ cells/mL) and 10% O₂ (SFM^+ PMA = 3.4×10⁵±2.5×10⁴ cells/mL and SFCM^+ PMA = 2.3×10⁵±1.5×10⁴ cells/mL)]. The SFM growth curve showed significant upregulation compared to SFCM over 7 days (p < 0.001).

3.2.2 SFCM Suppresses THP-1 Metabolic Activity

In 21% O₂, non-activated THP-1 cell metabolic activity reached a maximum at day 3 (SFM = 1.2 ± 0.03 O.D. and SFCM = 1.2 ± 0.03 O.D.) in both SFM and SFCM with no significant differences noted (Fig. 1B). However, in 10% O₂, non-activated THP-1 cell metabolism in SFM was significantly upregulated (p < 0.001) reaching a maximum at day 3 (SFM = 1.2 ± 0.02 O.D. and SFCM = 1 ± 0.03 O.D.) and declined thereafter in comparison to SFCM, which remained stationary over 7 days. Moreover, in the presence of PMA, SFCM showed significant downregulation (p < 0.001) of metabolism in comparison to SFM in 21% O₂ and 10% O₂ environments. Maximum differences were observed at day 3 [21% O₂ (SFM = 1.1 ± 0.04 O.D. and SFCM = 0.9 ± 0.05 O.D.) and 10% O₂ (SFM = 1.2 ± 0.03 O.D. and SFCM = 0.9 ± 0.06 O.D.)].

3.2.3 SFCM Modulates the Adherence Potential of THP-1 Cells

We next sought to identify if SFCM had an impact on the differentiation potential of THP-1 cells. Adherent cells were counted in both SFM ± PMA and SFCM ± PMA in both 21% O₂ and 10% O₂ environments (Fig. 2). Adherent cell numbers increased at day 1 [21% O₂ (SFM^− PMA = 1.2×10⁴±2.5×10³ cells/mL, SFM^+ PMA = 5.7×10⁴±5.8×10³ cells/mL, SFCM^− PMA = 7.3×10³±5.8×10³ cells/mL, SFCM^+ PMA = 1.5×10⁵±1×10³ cells/mL)] and 10% O₂ (SFM^− PMA = 1×10⁴±2×10³ cells/mL, SFM^+ PMA = 2.2×10⁴±2.9×10³ cells/mL, SFCM^− PMA = 5.7×10⁴±5.8×10³ cells/mL, and SFCM^+ PMA = 1×10⁵±1×10³ cells/mL)] and plateaued thereafter in all culture conditions. SFCM induced significant upregulation of adherence (p < 0.001) over 7 days which was further increased by the addition of PMA.

3.3 SFCM Modulates THP-1 differentiation into M1 versus M2 in Oxygen-Dependent Manner

3.3.1 SFCM Induces Distinct THP-1 Cell Morphology in an Oxygen-Dependent Manner

To further determine the differentiation patterns of activated THP-1 cells, assessment of macrophage morphology was carried out (Supplementary Fig. 4 and Fig. 3A). SFM induced pancake or spindle-shaped THP-1 cells, reached a maximum at day 1 [21% O₂ (pancake shape = 10.5 ± 1.8% and spindle shape = 5 ± 1%) and 10% O₂ (pancake shape = 8.4 ± 1.5% and spindle shape = 6.8 ± 1.3%)] and plateaued thereafter (Fig. 3B). SFCM significantly upregulated (p < 0.001) the percentage of cells scored with pancake or spindle shape. THP-1 in SFCM displayed a significantly higher (p < 0.001) pancake-shaped percentage in 21% O₂ collected SFCM versus a significantly higher (p < 0.001) percentage of spindle-shaped cells in 10% O₂. Modulations induced by SFCM were noted at day 1 [21% O₂ (pancake shape = 28 ± 3.3% and spindle shape = 8.6 ± 1.6%) and 10% O₂ (pancake shape = 8.6 ± 1.6% and spindle shape = 36 ± 5.6%)] and plateaued thereafter.

3.3.2 SFCM Induced Distinct THP-1 Gene Transcription in an Oxygen-Dependent Manner

Application of SFCM downregulated the expression of the chemotactic gene CCR5 in THP-1 ± PMA, particularly in 10% O₂ versus SFM (Fig. 4). MCP-1 expression levels were broadly consistent in SFM ± PMA however, SFCM applied to physiological 10% O₂ cultures showed downregulation at later timepoints. Other chemotactic genes, IL-8 and CCL5, were seen to be consistent between SFCM and SFM in both oxygen concentrations.

SFCM generally downregulated the expression of pro-inflammatory genes including, IL-1B, IL-12B and TNFa compared to SFM in both oxygen concentrations but in particularly, physiological 10% O₂. The activation of THP-1 cells through the addition of PMA to SFM was associated with strong upregulation of pro-inflammatory TNFa gene expression, which was blocked through the application of SFCM ± PMA in 10% O₂ and to a lesser extent in 21% O₂. Moreover, SFCM upregulated the expression of the anti-inflammatory IL-10 gene in comparison to SFM in both 21% O₂ and 10% O₂, however, physiological oxygen collected SFCM induced stronger band expression in comparison to 21% O₂.

3.3.3 SFCM Induced Distinct THP-1 Surface Marker Expression in an Oxygen-Dependent Manner

We next sought to identify the role of SFCM in the induction of terminal differentiation. The effects of polarisation on THP-1 cell surface marker expression were assessed by flow cytometry (Fig. 5A). In 21% O₂, there was an overall trend of lower presence of differentiation markers; particularly the macrophage markers CD14, CD73 and HLA-DR and M2 markers. PMA significantly upregulated (p < 0.0001) the expression of CD73 (SFM^− PMA = 8.9 ± 1.4%, SFM^+ PMA = 37.8 ± 1.3%, SFCM^− PMA = 7.3 ± 0.4%, and SFCM^+ PMA = 33.4 ± 0.5%) and CD197 (SFM^− PMA = 9.6 ± 1%, SFM^+ PMA = 91.5 ± 1.2%, SFCM^− PMA = 40.6 ± 12.3%, and SFCM^+ PMA = 69.1 ± 6.4%) (Fig. 5). Further, THP-1 activation through the addition of PMA was seen to promote the highest levels of CD197 (M1 marker) expression in 21% O₂ (SFM^− PMA = 9.6 ± 1%, SFM^+ PMA = 91.5 ± 1.0%, SFCM^− PMA = 40.6 ± 12.3%, and SFCM^+ PMA = 69.1 ± 6.4%)].

In reduced 10% O₂, there was an overall trend towards increased markers of differentiation across all culture conditions; particularly CD36 and CD204 (M2 markers) (Fig. 5B). SFCM significantly upregulated the expression of CD14 (macrophage marker) (SFM^− PMA = 10.9 ± 0.7%, SFM^+ PMA = 13 ± 0.4%, SFCM^− PMA = 48.2 ± 1%, and SFCM^+ PMA = 43.1 ± 4.8%); CD73 (macrophage marker) (SFM^− PMA = 7.8 ± 0.5%, SFM^+ PMA = 14.8 ± 1.6%, SFCM^− PMA = 47 ± 0.4%, and SFCM^+ PMA = 45.3 ± 0.4%); and CD36 (M2 marker) (SFM^− PMA = 52.1 ± 0.9%, SFM^+ PMA = 25.3 ± 1.4%, SFCM^− PMA = 64.6 ± 0.7%, and SFCM^+ PMA = 61.3 ± 0.7%)(p < 0.0001).

3.3.4 SFCM Induces a Distinct THP-1 Cell Secretome in an Oxygen-Dependent Manner

The THP-1 secretome profile was assessed through quantification of the previously identified constituents TNFa and IL-10; markers for M1 and M2, respectively (Fig. 6). Secretion from polarised THP-1 indicated upregulation at day 1 for IL-10 [SFM (21% O₂ = 540 ± 248 pg/mL, and 10% O₂ = 2643 ± 413 pg/mL) and SFCM (21% O₂ = 3780 ± 560 pg/mL, and 10% O₂ = 9115 ± 889] (Fig. 6A). For TNFa, this was noted at day 2 [SFM (21% O₂ = 422 ± 26 pg/mL, and 10% O₂ = 304 ± 37 pg/mL) and SFCM (21% O₂ = 922 ± 49 pg/mL, and 10% O₂ = 373 ± 21 pg/mL)] (Fig. 6B). Downregulation was observed thereafter with a baseline at days 5–7 for IL-10 [SFM (21% O₂ = 171 ± 161 pg/mL, and 10% O₂ = 79 ± 24 pg/mL) and SFCM (21% O₂ = 295 ± 278 pg/mL, and 10% O₂ = 33 ± 19 pg/mL)] and TNFa [SFM (21% O₂ = 73 ± 26 pg/mL, and 10% O₂ = 61 ± 15 pg/mL) and SFCM (21% O₂ = 31 ± 19 pg/mL, and 10% O₂ = 50 ± 36 pg/mL)]. From day 1, IL-10 was significantly upregulated (p < 0.001) in SFCM collected under 2% O₂ when compared to 21% O₂ SFCM or SFM. Conversely, TNFa was significantly (p < 0.001) upregulated at day 2 in 21% O₂ SFCM when compared to 2% O₂ SFCM or SFM.

3.4 Cytokine Challenging During THP-1 Cell Differentiation

3.4.1 Anti-inflammatory cytokines modulate THP-1 surface marker expression

To help identify candidate biomolecule(s) responsible for driving M1/M2-macrophage lineage differentiation, four anti-inflammatory cytokines, IL-4, IL-10, IL-13, and TGFb, were either added to SFM or neutralised in SFCM with changes to surface marker expression assessed by flow cytometry (Supplementary Fig. 5 and Fig. 7). Target markers were selected to identify the differentiation potential of THP-1 cells toward either M1 (CD197 and HLA-DR) or M2 (CD36) macrophages following exposure to anti-inflammatory cytokines (Fig. 7). The combined exogenous addition of IL-4/IL-13 to SFM induced significant CD36-upregulation [CD36 (21% O₂ = 33 ± 3%), and (10% O₂ = 59 ± 5%), in comparison to control SFM [CD36 (21% O₂ = 12 ± 1%), and (10% O₂ = 25 ± 1%)] (p < 0.001). Neutralisation of IL-4/IL-13 in SFCM resulted in CD36-downregulation in both oxygen concentrations and CD197 upregulation in 10% O₂ [CD36 (21% O₂ = 7.8 ± 1%) and (10% O₂ = 31.9 ± 3%), CD197 (21% O₂ = 59 ± 2.4%) and (10% O₂ = 71 ± 14.3%), in comparison to neat SFCM [CD36 (21% O₂ = 29.5 ± 4.9%) and (10% O₂ = 61.3 ± 0.7%), CD197 (21% O₂ = 69.1 ± 6.4%) and (10% O₂ = 18.6 ± 1.5%).

Addition of IL-10 to SFM induced CD36-upregulation and CD197/HLA-DR-downregulation [CD36 (21% O₂ = 17.3 ± 3.6%) and (10% O₂ = 32.3 ± 1%), CD197 (21% O₂ = 21.4 ± 11%) and (10% O₂ = 40.8 ± 12.4%), and HLA-DR (21% O₂ = 20.2 ± 3.2%), and (10% O₂ = 10 ± 0.6%)]. IL-10 neutralisation in SFCM resulted in CD36/CD197/HLA-DR-downregulation [CD36 (21% O₂ = 9.8 ± 0.9%) and (10% O₂ = 19 ± 0.8%), CD197 (21% O₂ = 31.8 ± 6.2%) and (10% O₂ = 20.3 ± 3.5%), and HLA-DR (21% O₂ = 17.5 ± 2.5%) and (10% O₂ = 18 ± 1.5%)] compared to neat SFCM [CD36 (21% O₂ = 29.5 ± 4.9%) and (10% O₂ = 61.3 ± 0.7%), CD197 (21% O₂ = 69 ± 6.4%) and (10% O₂ = 18.6 ± 1.5%) and HLA-DR (21% O₂ = 49.7 ± 6.8%) and (10% O₂ = 45.8 ± 2.1%)]. Moreover, addition of TGFb to SFM induced significant (p < 0.001) CD197-downregulation [(CD197 = 15.8 ± 2.4%)] in 21% O₂ and slight CD36-upregulation [(CD36 = 34 ± 2.4%)] in 10% O₂.

3.4.2 Anti-inflammatory cytokines alter TNFa/IL-10 secretion in an oxygen-dependent manner

In 21% O₂, a combination of exogenous IL-4/IL-13 in SFM induced significant (p < 0.001) secretion of IL-10 and TNFa at day 2 [(IL-10 = 7846.8 ± 492 pg/mL) and (TNFa = 1452 ± 335 pg/mL)] in comparison to control SFM [(IL-10 = 3897.7 ± 354.5 pg/mL) and (TNFa = 422.1 ± 26.6 pg/mL)] (Fig. 8). In 10% O₂, the combination of supplemented IL-4/IL-13 in SFM induced significant (p < 0.001) secretion of TNFa at day 2 (TNFa = 677.3 ± 103 pg/mL)] with no effect on IL-10 (IL-10 = 2932.6 ± 65.6 pg/mL)] secretion in comparison to control SFM [(IL-10 = 2352.3 ± 108.1 pg/mL) and (TNFa = 304.7 ± 37 pg/mL)]. Neutralisation of IL-4/IL-13 in SFCM using specific polyclonal antibodies resulted in a downregulation of IL-10 at day 2 in both oxygen conditions [IL-10 (21% = 2505.4 ± 192.9 pg/mL) and (10% =320.2 ± 154.6 pg/mL)] and significant (p < 0.001) upregulation of TNFa [TNFa (21% = 2111.4 ± 86 pg/mL) and (10% =465 ± 118.3 pg/mL)] compared to neat SFCM [IL-10 (21% = 3204.4 ± 555 pg/mL) and (10% = 6500 ± 1129 pg/mL)] and [TNFa (21% = 922.3 ± 49.5 pg/mL) and (10% =373.2 ± 21.2 pg/mL].

Supplemented IL-10 in SFM induced significant (p < 0.001) secretion of both IL-10 and TNFa at day 2 in both oxygen conditions [IL-10 = (21% = 14895.6 ± 1481.3 pg/mL) and (10% = 5949.1 ± 1425.9 pg/mL)] and [TNFa 21% = 2053.6 ± 399 pg/mL) and 10% = 1779.7 ± 327.5 pg/mL)] in comparison to control SFM [IL-10 (21% = 3897.7 ± 354.5 pg/mL) and (10% = 2352.3 ± 108.1 pg/mL)] and [TNFa (21% = 422.1 ± 26.6 pg/mL) and (10% = 304.7 ± 37 pg/mL)]. IL-10 neutralisation from SFCM with a specific IL-10 polyclonal antibody resulted in significant (p < 0.001) downregulation of IL-10 secretion at day 2 in both oxygen conditions [IL-10 (21%= 1399 ± 122 pg/mL) and (10% = 798.4 ± 90.5 pg/mL)] compared to SFCM [IL-10 (21% = 3204.4 ± 555 pg/mL) and (10% = 6500 ± 1129.2 pg/mL)]. This was coupled with significant upregulation (p < 0.001) of TNFa [TNFa (21% = 1445 ± 42.60 pg/mL) and (10% = 735.6 ± 68.6 pg/mL)] compared to SFCM [TNFa (21% = 922.3 ± 49.5 pg/mL) and (10% = 373.2 ± 21.2 pg/mL)].

TGFb addition to SFM induced significant (p < 0.001) upregulation of TNFa secretion at day 2 in both oxygen conditions [TNFa (21% = 1645.5 ± 73 pg/mL) and (10% = 1040.9 ± 108.6 pg/mL)] compared to control SFM [TNFa (21% = 422.1 ± 27.8 pg/mL) and (10% = 304.7 ± 37 pg/mL)]. Addition of TGFb addition to SFM further induced the significant upregulation (p < 0.001) of IL-10 in 21% O₂ with no significant effect (p > 0.05) observed in 10% O₂ [IL-10 (21% = 6697 ± 411 pg/mL) and (10% = 3585.4 ± 1700 pg/mL)] compared to control SFM [IL-10 (21% = 3897.7 ± 377.4 pg/mL) and (10% = 2352.3 ± 108.1 pg/mL).

Currently, hMSCs are utilised in therapeutic approaches for a range of chronic diseases including graft versus host disease (GvHD)^22,23,52, ischemic heart disease^53,54, Crohn’s disease^55–57, diabetes mellitus⁵⁸; all sharing inflammation as an underlying pathology. Ongoing clinical trials explore the wider application of hMSCs to a number of therapeutic approaches⁵⁹. Early in vitro studies concerning the immunosuppressive action of MSCs on immune cells mainly focused on cells derived from lymphoid lineages (T cells, B cells and NK cells) with little or no attention paid to the cells from myeloid lineages, particularly monocytes and macrophages⁶⁰. Macrophages are the key player during the initiation and propagation of an immune response through antigen presentation (pro-inflammatory action) or regeneration (anti-inflammatory action)⁶. In vitro^61,62 and in vivo⁶³ studies have revealed that MSCs influence macrophage function and possess immunoregulatory activity.

The present study aimed to confirm whether a SFCM cell-free approach could reprogramme macrophage differentiation and ultimately, the direction of the immune response. Further, the identification of the factors responsible for the mode of action were dissected through either exogenous addition or neutralisation in SFCM. Throughout, we assessed whether the application of a physiological oxygen environment modulated the hMSC secretome and consequent reparative action of SFCM; reprogramming macrophage differentiation towards either a M1 or M2 fate. Consequently, we explored THP-1 cell sub-culture and activation in physiologically representative 10% O₂ versus 21% O₂. In parallel, SFCM was collected from hMSCs cultured in either physiological 2% O₂ or 21% O₂ and applied to the oxygen-matched THP-1 cell cultures.

Appropriate oxygen tension is an important parameter for hMSC isolation, recovery and routine culture as well as in vitro immune response models and a wide spectrum of other cell types³³. The oxygen concentration of inhaled air (~ 21% O₂) is reduced following entry to the lungs and circulation throughout the body; reaching much reduced concentrations on arrival to tissues, such as, bone marrow (1–6% O₂), adipose tissue (2–8% O₂), and neural tissues (1–8% O₂)⁶⁴. Some tissues and organs are under higher oxygen tension (4–14% O₂) than the bone marrow hMSC niche, however, it is still reduced compared to ambient oxygen tension⁶⁵. Moreover, inflammation stimulates the coagulation cascade initiating a localised hypoxic environment resulting in exposure of localised macrophages to reduced oxygen during their activation phase⁴³. Furthermore, phagocytosis, a characteristic function of macrophages and the overall processing, engulfment and digestion of pathogens, is oxygen dependent^66,67. The oxygen requirement for phagocytosis increases during respiratory burst, where reactive oxygen species are generated, serving as the basis of the defence mechanism^68,69. However, an anaerobic situation has no negative impact on phagocytosis processing suggesting that the mechanism of phagocytosis is multimodal. In the hypoxia environment microbial killing is performed based on the production of toxic acidic compounds to the microbe or their deprivation from important co-factor metals, such as iron⁷⁰.

The stimulation of the macrophage begins with the initiation of the immune response through antigen recognition, engulfment, digestion, and processing^71,72. Macrophage responses to stimuli include cell morphology changes, polarisation, modulation of gene expression, and changes to proliferation⁷³. Macrophages are tissue-specific showing differences in their morphology and nomenclature relevant to their resident tissues, such as Kupffer cells, microglia cells, alveolar macrophages, and Langerhans cells⁷⁴. Despite that, they share common properties regarding their functionalities, either initiating (M1 macrophage) or dampening (M2 macrophage) the immune response⁹. THP-1 cells, a human leukaemia monocytic cell line, has been extensively used as an in vitro model of macrophage functionality; proliferating normally as suspension cells, following lag, log, and stationary phases of growth^48,75. THP-1 proliferation is blocked through activation by PMA, resulting in adherence to culture surfaces⁷⁶. However, THP-1 activation with PMA has no impact on differentiation, indicating that that differentiation is mainly based on the localised cytokine environment⁸. THP-1 cells exposed to PMA showed no expression of M1 or M2 markers and minimal IL-10/TNFa secretion coupled with low CD14 expression⁵⁰ (Fig. 9A). PMA activated THP-1 cells differentiate into M0 macrophages, whilst their terminal differentiation requires the presence of cytokines; pro-inflammatory cytokines stimulate their differentiation into M1 macrophages whilst pro-healing cytokines stimulate their differentiation into M2 macrophages⁷.

In the present study, THP-1 cells were induced to achieve a terminally differentiated state through the application of SFCM, however, the differentiation phenotype was induced in an oxygen-dependent manner. SFCM, collected from 21% O₂ cultured hMSCs and applied to activated THP-1 cells, led to the production of an increased M1:M2 macrophage differentiation ratio; displaying distinct pancake-like morphology, increased TNFa secretion and highly positive surface marker expression of CD197 when compared to SFM controls. In contrast, SFCM collected from physiologically relevant 2% O₂ cultured hMSCs, led to the production of a greater M2:M1 macrophage differentiation ratio; displaying an elongated spindle shape, increased IL-10 secretion, and positive surface marker expression of CD3 and CD14, when compared to control SFM (Fig. 9B).

Cell morphology associated with macrophage differentiation has been related to cytoskeleton reorganisation and changes in geometrical shape following exposure to biochemical stimuli⁸. Macrophage exposure to proinflammatory factors, such as, lipopolysaccharide (LPS)/ IFNγ promoted a pancake-like shape phenotype, while pro-healing factors, such as, IL-4/IL-13 promoted an elongated spindle shaped morphology. In the present study, activated THP-1 cells cultured in SFM showed adherence potential without changing their morphological appearances. However, activated THP-1 cells cultured in SFCM were enlarged in size in both 21% O₂ and 10% O₂. Application of SFCM collected from hMSCs cultured under physiological 2% O₂ to THP-1 cells promoted an elongated spindle-shape morphology, indicating anti-inflammatory potential. In contrast, normoxic 21% O₂ SFCM induced pancake-shape morphology, indicative of a pro-inflammatory potential (Fig. 9). Taken together, these results demonstrate the anti-inflammatory and regenerative potential of SFCM collected under physiological oxygen conditions; highlighting the importance of in vitro culture conditions on cell behaviour including their secretome profile.

Differentiated macrophages exhibited modulation of their transcriptional profile including the expression of chemotactic, pro-inflammatory and anti-inflammatory genes^12,77. Activated THP-1 cells displayed a distinct chemotactic expression pattern in an oxygen-dependent manner, including; CCL5, IL-8, MCP1, MIP1A and in particular, CCR5 which was downregulated in SFCM compared to SFM in the present study and consistent with previously published literature concerning the immunosuppressive effects of MSC conditioned media^78,79. This downregulation was most evident in 2% O₂ SFCM regardless of activation state. Correspondingly, SFCM collected under 2% O₂ suppressed pro-inflammatory transcriptional profiles through downregulation of TNFa, IL-1B, and IL-12B compared to 21% O₂ collected SFCM. Conversely, 2% O₂ SFCM upregulated IL-10 transcription versus 21% O₂ SFCM. The transcriptional profile induced by SFCM was translated at the proteomic level where 21% O₂ SFCM induced elevated THP-1 secretion of pro-inflammatory TNFa in comparison to 2% O₂ SFCM, GM, and SFM. Further, 2% O₂ SFCM induced increased THP-1 secretion of anti-inflammatory IL-10 in comparison to 21% O₂ SFCM, GM and SFM. These results corroborate with Ke et al. (2019) who showed that a hypoxic culture environment promoted THP-1 macrophage polarization towards an M2 phenotype, and subsequently modified the inflammatory microenvironment through the decreased secretion of pro-inflammatory cytokines⁸⁰.

Macrophage differentiation state was tracked through distinct cell surface marker expression including positive (CD45+, CD73+, CD105+, HLA-DR+), negative (CD19−, CD25−, CD34−, CD86−, CD90−), and target (CD14, CD36, CD197, CD206, CD204) markers^6,9,50,62. THP-1 cells cultured across the different conditions showed no modulation in positive or negative monocyte markers while alterations were clearly observed in CD14 (macrophage marker), CD197 (M1 macrophage marker), and CD36 (M2 macrophage marker). Physiological 2% O₂ collected SFCM upregulated CD14 and CD36 while downregulating CD197; confirming terminal differentiation towards an M2 macrophage fate. Conversely, 21% air oxygen collected SFCM upregulated CD14 and CD197 coupled with the downregulation of CD36; confirming terminal differentiation towards the M1 macrophage. SFM control groups showed no alteration of these surface markers in either 21% O₂ or 10% O₂ cultured THP-1 cells. There is a lack of consensus on a standardised macrophage surface marker panel to identify the distinct differentiation state toward M1 or M2 macrophages^50,81. CD36 and CD163 are considered as an M2-specific markers and CD197 a M1-specific marker with CD14 classified as a monocyte to macrophage differentiation marker. CD204 and CD206 have been used in studies conducted on primary macrophages to identify terminal differentiation into M2 macrophages^10,82. However, THP-1 cells in this study displayed negative expression of these markers in both 21% O₂ and 10% O₂ culture. Consequently, the present study utilised multiple cellular aspects to confirm the direction of differentiation toward M1 or M2 macrophages based on proliferation, metabolic activity, morphology, secretome profile and surface marker expression^51,63,70.

Previous studies have reported that the macrophage localised milieu determines the functional differentiation with anti-inflammatory cytokines, such as IL-4 and IL-13 responsible for the direction toward M2 terminal differentiation, while pro-inflammatory factors, such as, LPS, IFNγ and TNFa being responsible for M1 terminal differentiation^6,9,50,51. To dissect the SFCM mode of action in the modulation of macrophage differentiation towards either an M1 or M2 fate, we examined the role of four anti-inflammatory cytokines: IL-4, IL-10, IL-13 and TGFb which we have previously confirmed as components of the MSC secretome^33,83. These cytokines were either added to SFM or specifically blocked from SFCM using specific polyclonal antibodies.

We noted that IL-4/13 and IL-10 added to SFM increased levels of anti-inflammatory IL-10 secretion compared to SFCM in in 21% O₂. This was accompanied with inducing the increased secretion of pro-inflammatory TNFa. Taken together, this suggests that IL-4/13 and IL-10 induce non-specific terminal differentiation of THP-1 cells, highlighted by elevated M1 and M2 macrophage markers. This trend was not reproduced in physiological oxygen culture where it was seen that SFCM more efficiently specified terminal differentiation towards an M2 fate indicated through the increased secretion of IL-10 compared to IL-4/13 supplemented SFM. Further, SFCM suppressed pro-inflammatory M1 differentiation evidenced through reduced TNFa secretion compared to IL-4/13 and IL-10 supplemented SFM. In 21% O2 the addition of IL-4/13 to SFM replicated SFCM-induced cell surface marker expression patterns with upregulation of CD14 and CD36. In 10% O₂, this was only partially replicated with increased levels of the M2 markers CD36 and CD204. However; SFCM cultured THP-1 cells showed additional increased levels of the monocytic cell markers CD14 and CD73 compared to IL-4/13 and IL-10 supplemented SFM.

SFCM collected from hMSCs exert their immunomodulation through several anti-inflammatory and pro-inflammatory cytokines. IL-10 is present in both 21% O₂ and 2% O₂ SFCM and appears to be the principle anti-inflammatory cytokine; exerting its immunosuppression activity on cellular aspects via chemokine synthesis, NO synthesis, HLA-DR expression and co-stimulatory molecules such as IL-12 and CD80/CD86^84–87. Combinations or individual application of IL-4/IL-13 have been used extensively to direct monocytes and/or THP-1 differentiation toward regulatory rather than pro-inflammatory subtypes^50,51. TGFb, also present in hMSC SFCM, has been previously implicated in having a role in modulation of immune response⁸⁸. The post-receptor translation pathways for these individual pathways include STAT3 for IL-10, STAT6 for IL-4 and IL-13 and TGFb induces Smad pathways^89,90. However, in the present study, these cytokines individually or in combination failed to induce THP-1 differentiation towards certain lineages in comparison to SFCM.

Collectively, it has been shown that MSC SFCM induced the terminal macrophage differentiation of activated THP-1 cells in an oxygen-dependent manner; physiological oxygen more strongly favours an M2 fate, whereas 21% air oxygen directs towards an M1 fate. SFCM more definitively specifies the terminal differentiation phenotype in comparison to the individually tested cytokines (IL-4, IL-10, IL-13 or TGFb). Taken together, this suggests an as yet undefined component(s) of MSC SFCM are responsible for the promotion of macrophage terminal differentiation; highlighting the complex effects of conditioned media products and the further work required to dissect and understand the mechanism of action to develop cell-free therapeutic tools to treat diseases with an inflammatory component.

hMSCs: Human mesenchymal stem/stromal cells

SFM: Serum-free media

SFCM: Serum-free conditioned media

IL: Interleukin

CD: Cluster of differentiation

TGF: Transforming growth factor

TNF: Tumour necrosis factor

LPS: Lipopolysaccharide

IFNγ: Interferon γ

GM-CS: Granulocyte macrophage colony-stimulating factor

NSAID: Non-steroidal anti-inflammatory drug

Tregs: Regulatory T cells

VEGF: Vascular endothelial growth factor

hiPSC: Human induced pluripotent stem cell

pO2: Physiological partial pressure of oxygen

PMA: Phorbol 12-myristate 13-acetate

MTT: (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)

O.D.: Optical density

GAPDH: Glyceraldehyde 3-phosphate dehydrogenase

L32: Ribosomal protein L32

HLA-DR: Human leukocyte antigen – DR isotype

CCL5: C-C motif chemokine ligand 5

CCR5: C-C motif chemokine receptor 5

MCP-1: Monocyte chemoattractant protein-1

MIP1A: C-C motif chemokine ligand 3

GvHD: Graft versus host disease

NO: Nitric oxide

STAT: Signal transducer and activator of transcription

Smad: Suppressor of mothers against decapentaplegic

BMA: Bone marrow aspirate

GM: growth media

DMEM: Dulbecco’s Modified Eagle Medium

FBS: Fetal bovine serum

NEAA: Non-essential amino acids

PSA: Penicillin-streptomycin-amphotericin B

DMSO: Dimethylsulfoxide

PBS: Phosphate-buffered saline

BSA: Bovine serum albumin

ABTS: (2,2′-Azino-bis (3-ethylbenzothiazoline-6- sulfonic acid)

PCR: Polymerase chain reaction

ANOVA: Analysis of variance

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Competing interests

The authors declare no competing interests.

Funding

Financial support provided by Ministry of Higher Education and Scientific Research, Iraq (S1443) and the Guy Hilton Asthma Trust. The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript

Authors' contributions

NRF and MMM designed the study; NRF and TPD supervised this study; MMM performed the experiments; MMM conducted formal analysis; MS wrote the original draft manuscript; MS, NRF, MMM and TPD edited the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

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The Human Mesenchymal Stem Cell Secretome Directs Macrophage Differentiation in an Oxygen-Dependent Manner

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Abstract

Background

Methods

Results

Conclusion

Figures

1 Background

2 Materials and methods

2.1 Cell Line Culture

2.1.1 hMSCs

2.1.2 THP-1

2.2 Cell Viability and Proliferation Assays

2.2.1. Cell Counting

2.2.2. MTT Assay

2.2.3. Cytospinning

2.3. Cytokine Challenge

2.4. Cytokine Blocking

2.5. ELISA Assay

2.6 Gene Expression

2.6.1 RNA Extraction and Processing

2.6.2 RT-PCR

2.6. Statistical Analysis

3 Results

3.1 Characterisation of THP-1 In Vitro Macrophage Differentiation Model

3.1.1 PMA Inhibits Proliferation and Promotes Attachment of THP-1 cells

3.1.2 Activation of THP-1 Cells Results in IL-10/TNFa Secretion

3.1.3 THP-1 Gene Expression is Modulated by Oxygen Environment

3.2 The Role of SFCM in Modulation of THP-1 Proliferation

3.2.1 SFCM Suppresses THP-1 Proliferation

3.2.2 SFCM Suppresses THP-1 Metabolic Activity

3.2.3 SFCM Modulates the Adherence Potential of THP-1 Cells

3.3 SFCM Modulates THP-1 differentiation into M1 versus M2 in Oxygen-Dependent Manner

3.3.1 SFCM Induces Distinct THP-1 Cell Morphology in an Oxygen-Dependent Manner

3.3.2 SFCM Induced Distinct THP-1 Gene Transcription in an Oxygen-Dependent Manner

3.3.3 SFCM Induced Distinct THP-1 Surface Marker Expression in an Oxygen-Dependent Manner

3.3.4 SFCM Induces a Distinct THP-1 Cell Secretome in an Oxygen-Dependent Manner

3.4 Cytokine Challenging During THP-1 Cell Differentiation

3.4.1 Anti-inflammatory cytokines modulate THP-1 surface marker expression

3.4.2 Anti-inflammatory cytokines alter TNFa/IL-10 secretion in an oxygen-dependent manner

4 Discussion

5 Conclusion

Abbreviations

Declarations

References

Supplementary Files

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