Obesity is one of the major worldwide health challenges, considered a key risk factor for a number of age-related diseases, including diabetes. Recent evidence suggested that senescent cells (SCs) accumulate in obese adipose tissue. However, the phenotype and role of SCs in obesity-related inflammation have not been clearly described. Samples of abdominal visceral (VAT) and subcutaneous (SAT) adipose tissue obtained from 17 obese patients (BMI≥ 38 kg/m2) were provided from the Obesity Clinic, Ospedali Riuniti Ancona. The SCs number was evaluated counting SA-β-Gal positive cells in VAT and SAT samples and then the CD68 pan-macrophage marker was analysed. Besides, human monocytic THP-1 cells were differentiated in vitro into classically (M1) or alternatively (M2) activated macrophages, and hyperglycemia treatment was used to induce a senescent phenotype (HgSM). Co-cultures of macrophages with human multipotent adipose-derived stem cells (hMADS) was set up. A significantly higher number of SA-β-Galactosidase positive cells was observed in VAT compared to SAT. The majority of SCs were positive for CD68 and the number of SCs was positively correlated with BMI in VAT but not in SAT samples. HgSMs exhibited a polarization toward a mixed M1/M2-like phenotype. Moreover, HgSMs showed high SA-β-Gal activity, high p21 protein level and telomere shortening in comparison with M1 and M2 phenotypes. However, it seems not accompanied by a proper acquisition of the classical SASP. Surprisingly, when macrophages were co-cultured with hMADS, a significant increased expression of pro-inflammatory cytokines was observed in HgSMs, suggesting a shift into an M1-like phenotype. Furthermore, HgSMs fostered the inflammation in HMADs adipocytes at the mRNA level. Overall, our results support the hypothesis that in vivo SCs accumulate in VAT of obese patients and are mainly macrophages. In vitro results suggest that hyperglycemia induced a senescent phenotype in macrophages, not accompanied by the acquisition of the classical SASP. Interestingly, HgSMs polarized toward a mixed M1/M2 phenotype and they switch into M1-like phenotype when cultured with hMADS adipocytes, fueling inflammation.
L'obesità è una delle maggiori sfide sanitarie a livello mondiale, considerata fattore di rischio chiave per una serie di patologie legate all'età, incluso il diabete. Recenti studi hanno suggerito che le cellule senescenti (senescent cells, SCs) si accumulano nel tessuto adiposo dell’obeso. Tuttavia, il fenotipo e il ruolo delle SCs nell'infiammazione obesità-dipendente non sono stati chiaramente descritti. Campioni di tessuto adiposo addominale viscerale (visceral adipose tissue, VAT) e sottocutaneo (subcutaneous adipose tissue, SAT) ottenuti da 17 pazienti obesi (BMI≥ 38 kg / m2) sono stati forniti dalla Clinica per l'obesità, presso Ospedali Riuniti di Ancona. Il numero di SCs è stato valutato contando le cellule positive per l’attività della SA-β-Galattosidasi (SA-β-Gal) nei campioni di VAT e SAT ed è stato analizzato il marcatore pan-macrofagico CD68. Inoltre, le cellule monocitiche THP-1 umane sono state differenziate in vitro in macrofagi attivati classicamente (M1) o alternativamente (M2), e il trattamento con iperglicemia è stato utilizzato per indurre un fenotipo senescente (HgSM). Sono state messe a punto co-colture di macrofagi con cellule staminali umane multipotenti derivate da tessuto adiposo (hMADS). Un numero significativamente più elevato di cellule positive per l’attività della SA-β-Gal è stato osservato nel VAT rispetto al SAT. La maggior parte delle SCs erano positive per CD68 e il numero di SCs era positivamente correlato al BMI nel VAT ma non nei campioni di SAT. Le HgSM hanno esibito una polarizzazione verso un fenotipo misto M1 / M2. Inoltre, le HgSM hanno mostrato un'elevata attività della SA-β-Gal, un elevato livello di proteina p21 e un accorciamento dei telomeri rispetto ai fenotipi M1 e M2. Tuttavia, le HgSM non sembrano accompagnate da una classica acquisizione del fenotipo SASP. Sorprendentemente, quando i macrofagi sono stati messi in co-coltura con le hMADS, è stata osservata una espressione significativamente aumentata delle citochine pro-infiammatorie nelle HgSM, suggerendo l’acquisizione di un fenotipo simile agli M1. Inoltre, le HgSM hanno potenziato l'infiammazione degli adipociti HMADs a livello dell’ mRNA. Nel complesso, i nostri risultati supportano l'ipotesi che le SCs in vivo si accumulino nel VAT dei pazienti obesi e siano principalmente macrofagi. I risultati in vitro suggeriscono che l'iperglicemia ha indotto un fenotipo senescente nei macrofagi, non accompagnato dall'acquisizione della SASP. È interessante notare che le HgSM si sono polarizzate verso un fenotipo M1 / M2 misto e si trasformano in un fenotipo simile agli M1 quando vengono coltivate con adipociti hMADS, alimentando così l'infiammazione.
Senescent macrophages in adipose tissue: ex vivo and in vitro characterization / Matacchione, Giulia. - (2020 Mar 09).
Senescent macrophages in adipose tissue: ex vivo and in vitro characterization
MATACCHIONE, GIULIA
2020-03-09
Abstract
Obesity is one of the major worldwide health challenges, considered a key risk factor for a number of age-related diseases, including diabetes. Recent evidence suggested that senescent cells (SCs) accumulate in obese adipose tissue. However, the phenotype and role of SCs in obesity-related inflammation have not been clearly described. Samples of abdominal visceral (VAT) and subcutaneous (SAT) adipose tissue obtained from 17 obese patients (BMI≥ 38 kg/m2) were provided from the Obesity Clinic, Ospedali Riuniti Ancona. The SCs number was evaluated counting SA-β-Gal positive cells in VAT and SAT samples and then the CD68 pan-macrophage marker was analysed. Besides, human monocytic THP-1 cells were differentiated in vitro into classically (M1) or alternatively (M2) activated macrophages, and hyperglycemia treatment was used to induce a senescent phenotype (HgSM). Co-cultures of macrophages with human multipotent adipose-derived stem cells (hMADS) was set up. A significantly higher number of SA-β-Galactosidase positive cells was observed in VAT compared to SAT. The majority of SCs were positive for CD68 and the number of SCs was positively correlated with BMI in VAT but not in SAT samples. HgSMs exhibited a polarization toward a mixed M1/M2-like phenotype. Moreover, HgSMs showed high SA-β-Gal activity, high p21 protein level and telomere shortening in comparison with M1 and M2 phenotypes. However, it seems not accompanied by a proper acquisition of the classical SASP. Surprisingly, when macrophages were co-cultured with hMADS, a significant increased expression of pro-inflammatory cytokines was observed in HgSMs, suggesting a shift into an M1-like phenotype. Furthermore, HgSMs fostered the inflammation in HMADs adipocytes at the mRNA level. Overall, our results support the hypothesis that in vivo SCs accumulate in VAT of obese patients and are mainly macrophages. In vitro results suggest that hyperglycemia induced a senescent phenotype in macrophages, not accompanied by the acquisition of the classical SASP. Interestingly, HgSMs polarized toward a mixed M1/M2 phenotype and they switch into M1-like phenotype when cultured with hMADS adipocytes, fueling inflammation.File | Dimensione | Formato | |
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