Local and Systemic Cell Non-autonomous Mechanisms Uniquely Drive Intestinal Stem Cell Aging

Background: Ageing occurs as a complex interaction of cell autonomousand non-cell autonomous mechanisms. Functional decline is a hallmarkof aging in multiple tissues, including the intestine, and this process isthought to be driven in part by deterioration in resident stem cell function.The intestine has a rapid turnover of every 3-5 days, owing this to thepresence of Lgr5+ intestinal stem cells (ISCs), that resides at the bottomof the crypt along with their niche providing Paneth cells. Therefore, weinvestigated how this compartment is affected with aging.Methods: Four and 22 months old C57BL/6 male mice were used foraging phenotype studies. Young and old mice received rapamycin (4mg/kg) or salicylate (2 mg/mL) for 28 days. Neutralizing antibodies toIL-1β, TNF-α, IFN-γ or IgG1 were i.p. injected at a 300 μg dose for 3weeks. Isolated crypts from Lgr5-EGFP mice were re-suspended in TrypLEExpress with Rock inhibitor and DNAse I, filtered and centrifuged. Cellswere then re-suspended in FACS buffer containing PE-conjugated anti-CD24 antibody and APC-conjugated anti-Epcam antibody for 15 minat 4°C, and analyzed by MoFlo. ISC proliferation was assessed usingan ex vivo organoid assay. Briefly, isolated crypts were re-suspendedin matrigel, transferred to a 48-well plate to solidify at 37°C, and 250μL crypt culture medium (ADF, Pen/Strep, HEPES, Glutamax, N2, B27,N-acetyl-L-cysteine, Noggin, EGF and R-Spondin) was added to eachwell and maintained at 37°C. The organoid formation was quantified onday 9. Parabiosis surgery was carried out by the Einstein Chronobiosiscore.Results: As compared to young mice, old mice harbor declines inISC proliferation, impaired mucosal barrier integrity, and shifts in thegut immune cell composition, including increased CD8+ T cells anddendritic cells (P<0.05). We next generated isochronic (Y-Y, O-O) andheterochronic (Y-O) C57BL/6 male parabionts and observed impaired ISCfunction in young parabionts exposed to old blood (P<0.05). Rapamycinor salicylate treatment restored ISC function in old mice (P<0.05), withoutfurther suppressing mTOR signaling. However, rapamycin reducedplasma cytokines to more youthful levels (P<0.05), suggesting a potentialrole for inflammation in mediating the stem cell dysfunction with aging.Ex vivo screening assays confirmed that TNFα and IFNγ potently disruptISC proliferation (P<0.05), while in vivo treatment with a TNFα- or IFNγ-neutralizing-antibody restored ISC function in old mice (P<0.05). Uponsingle cell ablation in the crypts, the motion of neighboring cells wasdramatically impaired, and cell debris ‘lingered’ in the crypts of oldanimals (P<0.05).Conclusion: We propose that intestinal aging happens as a complexinterplay of cell autonomous and non-cell autonomous pathways.Moreover, IFN-γ and TNF-α can act as lead progeronic factors to drive adecline in intestinal stem cell function with aging.

کلیدواژه ها:

Ageing ، Intestinal stem cells ، Lgr5+ ، Inflammation ، Parabiosis