Systemic NF-κB-mediated inflammation promotes an aging phenotype in skeletal stem/progenitor cells

Abstract

Aging tissues undergo a progressive decline in regenerative potential. This decline in regenerative responsiveness has been attributed to changes in tissue-specific stem cells and their niches. In bone, aged skeletal stem/progenitor cell dysfunction is characterized by decreased frequency and impaired osteogenic differentiation potential. This aging phenotype ultimately results in compromised regenerative responsiveness to injury. The age-associated increase of inflammatory mediators, known as inflamm-aging, has been identified as the main culprit driving skeletal stem cell dysfunction. Here, we utilized a mouse model of parabiosis to decouple aging from inflammation. Using the Nfkb1^-/- mouse as a model of inflamm-aging, we demonstrate that a shared systemic circulation between a wild-type and Nfkb1^-/- mouse results in an aging phenotype of the wild-type skeletal stem and progenitor cells, shown by CFU-fs and osteogenic and adipogenic differentiation assays. Our findings demonstrate that exposure to an inflammatory secretome results in a phenotype similar to the one observed in aging.

Keywords: aging; inflammation; nuclear factor kappa B; regeneration; skeletal stem cell.

Figure 1

Exposure to Nfkb1^-/- circulation results in increased inflammation in bone marrow compartment. (A) Schematic representation of the parabiosis model in which one mouse shares circulation with another surgically-anatomized mouse. A wild-type mouse parabiosed to another wild-type mouse serves as the control (isochronic pair) and Nfkb1^-/- mouse parabiosed to a wild-type mouse serves as the experimental group (heterochronic pair). Parabionts are noted as such wild-type parabiosed to wild-type, WT^WT; Nfkb1^-/- mouse parabiosed to wild-type, Nfkb1^-/-WT; and wild-type parabiosed to Nfkb1^-/- mouse, WT^Nfkb1-/-. Green mice depict Beta-actin GFP mice (C57BL/6-Tg(CAG-EGFP)1Osb/J), brown mice represent wild-type mice. (B) Gating strategy demonstrating that parabiosis led to an insignificant transfer of SSPCs from one animal to the other. (C) After six weeks of shared circulation the bone marrow compartment of WT^Nfkb1-/- mice displayed higher expression of the inflammatory mediators Tnfa, Il1a and Il1b compared to WT^WT controls. Conversely, exposure of WT circulation to the Nfkb1^-/- bone marrow did not result in a reduced inflammatory status. (n=3, ns, non-significant, *P < 0.05, **P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001).

Figure 2

SSPC frequency declines in response to increased NF-κB-mediated inflammation. (A) SSPCs isolated from parabiosed animals six weeks post-surgery revealed that exposure to increased inflammation results in decreased SSPC number as revealed by colony forming unit (CFU) assays. (B) Nfkb1^-/-WT and WT^Nfkb1-/- SSPCs gave rise to significantly fewer colonies than those isolated from the WT^WT parabionts as revealed by CFU-f efficiency (n=3, *P < 0.05).

Figure 3

Increased NF-κB-mediated inflammation leads to impaired differentiation potential. (A, B) Osteogenic potential was assessed by Alizarin red staining (A), which demonstrated significantly reduced mineralization in Nfkb1^-/-WT and WT^Nfkb1-/- samples compared to control (B) (n=3, **** P < 0.0001). (C, D) Significantly increased lipid accumulation was observed in Nfkb1^-/-WT and WT^Nfkb1-/- samples as revealed by Oil Red O staining (C) and quantification (D) (n=3, *** P ≤ 0.001, **** P ≤ 0.0001).