Characterization of novel mouse models to study the role of necroptosis in aging and age-related diseases

Abstract

To study the impact of necroptosis-induced chronic inflammation on age-related diseases and aging, two knockin mouse models (Ripk3-KI and Mlkl-KI) were generated that overexpress two genes involved in necroptosis (Ripk3 or Mlkl) when crossed to Cre transgenic mice. Crossing Ripk3-KI or Mlkl-KI mice to albumin-Cre transgenic mice produced hepatocyte specific hRipk3-KI or hMlkl-KI mice, which express the two transgenes only in the liver. Ripk3 and Mlkl proteins were overexpressed 10- and fourfold, respectively, in the livers of the hRipk3-KI or hMlkl-KI mice. Treating young (2-month) hRipk3-KI or hMlkl-KI mice with carbon tetrachloride (CCl4), a chemical inducer of oxidative stress, resulted in increased necroptosis (Mlkl-oligomers) and inflammation in the liver compared to control mice receiving CCl4. Mlkl-oligomerization also was significantly increased in old (18-month) hRipk3-KI and hMlkl-KI mice compared to old control (Cre negative, Ripk3-KI and Mlkl-KI) mice. The increase in necroptosis was associated with an increase in inflammation, e.g., inflammatory cytokines (TNFα, IL-6) and macrophage markers (F4/80, CD68). Importantly, steatosis (triglycerides) and fibrosis (e.g., picrosirius red staining, hydroxyproline levels, and transcripts for TGFβ, Col1α1, and Col3α1) that increase with age were significantly higher in the livers of the old hRipk3-KI or hMlkl-KI mice compared to old control mice. In addition, markers of cellular senescence were significantly increased in the livers of the old hRipk3-KI and hMlkl-KI mice. Thus, the first mouse models have been developed that allow researchers to study the impact of inducing necroptosis in specific cells/tissues on chronic inflammation in aging and age-related diseases.

Lymphatic Vessels in Chronic Rhinosinusitis

“…Additional reduction in background staining was achieved by covering the sections with BLOXALL™ (Vector Laboratories, Inc.) for 10 minutes prior to a protein block with blocking solution (included in the kit). In order to localize MMP-14 and macrophages within the tissue sections, the antibodies mouse Anti-MMP-14 (R&D Systems, Bio-Techne, Wiesbaden, Germany) and mouse Anti-CD68 (macrophage marker protein; 26 Invitrogen, Karlsruhe, Germany) (see Additional Table 1 in additional data for detailed information) were incubated overnight at 4 °C. A nonspecific antibody (Cell Signaling Technology, Inc., Danvers, MA) served as negative control.…”

“…LYVE-1 and PDPN were co-stained using IF and the macrophage marker protein CD68 26 was stained using IHC in CRSwNP iTN (n=6), CRSwNP mTN (n=7), CRSwNP UP (n=7), CRSwNP ET (n=7), NP (n=8), CRSsNP iTN (n=7), CRSsNP mTN (n=7), CRSsNP UP (n=7), CRSsNP ET (n=7) and control iTN (n=13). The tissues for this analysis needed to be typical, as homogeneous as possible, similar in size and contain as few glands and bones as possible.…”