ADSC-conditioned media elicit an ex vivo anti-inflammatory macrophage response

in Journal of Molecular Endocrinology
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Obesity-associated inflammatory mechanisms play a key role in the pathogenesis of metabolic-related diseases. Failure of anti-inflammatory control mechanisms within adipose tissue and peripheral blood mononuclear cells (PBMCs) have been implicated in disease progression. This study investigated the efficacy of allogeneic adipose tissue-derived mesenchymal stem cells conditioned media (ADSC-CM) to counteract persistent inflammation by inducing an anti-inflammatory phenotype and cytokine response within PBMCs derived from patients with and without metabolic syndrome. Forty-six (n = 46) mixed ancestry females (18–45 years) were subdivided into (a) healthy lean (HL) (n = 10) (BMI <25 kg/m2), (b) overweight/obese (OW/OB) (BMI ≥25 kg/m2, <3 metabolic risk factors) (n = 22) and (c) metabolic syndrome (MetS) (visceral adiposity, ≥3 metabolic risk factors) (n = 14) groups. Body composition (DXA scan), metabolic (cholesterol, HDL, LDL, triglycerides, blood glucose) and inflammatory profiles (38-Plex cytokine panel) were determined. PBMCs were isolated from whole blood and treated ex vivo with either (i) autologous participant-derived serum, (ii) ADSCs-CM or (iii) a successive treatment regime. The activation status (CD11b+) and intracellular cytokine (IL6, IL10, TNFa) expression were determined in M1 (CD68+CD206−CD163−) and M2 (CD68+CD163+ CD206+) macrophage populations using flow cytometry. ADSC-CM treatment, promoted a M2 macrophage phenotype and induced IL10 expression, this was most pronounced in the OW/OB group. This response is likely mediated by multiple complementing factors within ADSC-CM, yet to be identified. This study is the first to demonstrate the therapeutic potential of ADSC-CM to restore the inflammatory balance in immune compromised obese individuals.

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  • Supplementary Table 1: Detectable and non-detectable cytokine levels within participant-derived serum.

 

      Society for Endocrinology

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    Body composition and metabolic profile of participants within the healthy lean (n = 10), overweight/obese (n = 22) and metabolic syndrome (n = 14) groups. (A) BMI (kg/m2). (B) Total body fat (%) (df 32). (C) Trunk-to-limb fat mass (TF/LF) ratio (df 32). (D) Waist-to-hip (WHR) ratio (df 32). (E) Android-to-gynoid fat mass (A/G) ratio (df 32). (F) FBG levels (mmol/L). (G) Serum HDL (mmol/L) (df 43). (H) Serum TGS (mmol/L) (df 43). (I) Serum cholesterol (mmol/L) (df 43) and (J) Serum LDL (mmol/L). Statistical analysis: One-way ANOVA with Tukey post hoc test (normal distribution), df – degrees of freedom. Non-parametric Kruskal–Wallis ANOVA with Dunns multiple comparison test (data not normally distributed). Level of significance accepted at P < 0.05. - - - indicates normal expected physiological range. A/G, android:gynoid ratio; BMI, body mass index; FBG, fasting blood glucose; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TF/LF, trunk fat:limb fat mass ratio; TGS, triglycerides. Groups: HL, healthy lean group; OW/OB, overweight/obese group; MetS, metabolic syndrome group.

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    Serum C-reactive protein levels. As indication of systemic inflammation in participants within the healthy lean (n = 10), overweight/obese (n = 22) and metabolic syndrome (n = 14) groups. Statistical analysis: One-Way ANOVA with Tukey post hoc test. Level of significance accepted at P < 0.05, df = 41. CRP, C-reactive protein. Groups: HL, healthy lean group; OW/OB, overweight/obese group; MetS, metabolic syndrome group.

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    Inflammatory cytokine profile of participants within the healthy lean (n = 10), overweight/obese (n = 22) and metabolic syndrome (n = 14) groups. Serum concentrations of cytokines that differed between groups are presented as box-and-whisker plots (data not normally distributed). (A) IFNa2 (pg/mL), (B) IL12 (p40) (pg/mL), (C) IL28A/IFN-λ2 (pg/mL), (D) MMP1 (pg/mL), (E) MMP3 (pg/mL) and (F) Osteopontin (ng/mL). Statistical analysis: Non-parametric Kruskal–Wallis ANOVA with Dunns multiple comparisons test or Mann–Whitney test (B). IFN, interferon; IL, interleukin; MMP, matrix metalloproteinase. Groups: HL, healthy lean group; OW/OB, overweight/obese group; MetS, metabolic syndrome group.

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    The responsiveness of the overall participant-derived PBMC population to ADSC-CM treatment. (A) Forward vs Side scatter (ungated) indicating the size vs granularity of the PBMC populations. (B, C and D) Representative flow cytometry histograms indicating regions of positive and negative staining for CD163-PerCP-Cy5.5 (B), CD206-BB515 (C) and IL10-PE (D). (E, F and G) The percentage of PBMCs positive for CD163, CD206 and IL10 following autologous serum, ADSC-CM or successive treatments within each group (HL n = 10; OW/OB n = 22; MetS n = 14). Statistical analysis: Non-parametric Kruskal Wallis ANOVA with Dunns multiple comparisons test (IL-10) or factorial ANOVA with Tukey post hoc test (CD163 and CD206) df = 129. CD, cluster of differentiation; FSC, forward scatter; IL, interleukin; PBMCs, peripheral blood mononuclear cells; SCC, side scatter. Groups: HL, healthy lean group; OW/OB, overweight/obese group; MetS, metabolic syndrome group. Treatments: ADSC-CM, adipose-derived stem cell conditioned media. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0078.

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    The responsiveness of CD68+ macrophages present within the participant-derived PBMCs to ADSC-CM treatment. (A) Representative flow cytometry histogram indicating regions of positive and negative staining for CD68-BV421 within the PBMC population. (B) Scatter plots indicating the ratio of M1 (CD206−CD163−) vs M2 (CD206+CD163+) macrophages within the CD68+ gated population following either autologous serum, ADSC-CM or successive treatment. (C) Representative flow cytometry histograms indicating intracellular cytokine (IL-6-APC, IL-10-PE and TNF-α-PE-Cy7) expression in naïve/unstimulated M1 (CD68+CD163CD206) or M2 (CD68+CD163+CD206+) macrophages following treatment. (D) Representative flow cytometry histograms indicating intracellular cytokine expression (IL-6-APC, IL-10-PE and TNF-α-PE-Cy7) upon stimulation with either unconditioned media, recombinant IL4 or Pam3CSK4. (E, F and G) Quantification of the percentage of M1 and M2 macrophages in the HL (n = 10), OW/OB (n = 22) and MetS (n = 14) groups following each treatment regime. Statistical analysis: Factorial ANOVA with Tukey post hoc test df = 129. CD, cluster of differentiation; IL, interleukin; PBMCs, peripheral blood mononuclear cells; TNF, tumour necrosis factor. Groups: HL, healthy lean group; OW/OB, overweight/obese group; MetS, metabolic syndrome group. Treatments: ADSC-CM, adipose-derived stem cell conditioned media. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0078.

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    The intracellular cytokine expression within the M2 population following exposure to ADSC-CM. M2 macrophages (CD68+CD206+CD163+) were regarded as activated when they also expressed the activation marker CD11b+. (A, B and C) Intracellular IL-10 expression, (D, E and F) intracellular IL-6 expression, (G, H and I) intracellular TNF-α expression. Statistical analysis: Non-parametric Kruskal Wallis ANOVA with Dunns multiple comparisons test. IL, interleukin; MFI, mean fluorescent intensity; TNF, tumour necrosis factor. Groups: HL, healthy lean group; OW/OB, overweight/obese group; MetS, metabolic syndrome group. Treatments: ADSC-CM, adipose-derived stem cell conditioned media.

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    The intracellular IL10 expression within the M2 population following exposure to (A) recombinant IL6 (400 pg/mL) and (B) ADSC-CM compared to treatment with unconditioned control media. (C and D) Intracellular M2 IL10 expression following ADSC-CM treatment with either anti-IL6 blocking antibody (C) or anti-PTX3 blocking antibody (D) compared to ADSC-CM treatment. Representative flow cytometry histograms are from a randomly selected healthy lean participant. A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0078.

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