Non-Necroptotic Roles of MLKL in Diet-Induced Obesity, Liver Pathology, and Insulin Sensitivity: Insights from a High-Fat, High-Fructose, High-Cholesterol Diet Mouse Model.

Chronic inflammation is a key player in metabolic dysfunction-associated fatty liver disease (MAFLD) progression. Necroptosis, an inflammatory cell death pathway, is elevated in MAFLD patients and mouse models, yet its role is unclear due to the diverse mouse models and inhibition strategies. In our study, we inhibited necroptosis by targeting mixed lineage kinase domain-like pseudokinase (MLKL), the terminal effector of necroptosis, in a high-fat, high-fructose, high-cholesterol (HFHFrHC) mouse model of diet-induced MAFLD. Despite the HFHFrHC diet upregulating MLKL (2.5-fold), WT mice livers showed no increase in necroptosis markers or associated proinflammatory cytokines. Surprisingly, Mlkl-/- mice experienced exacerbated liver inflammation without protection from diet-induced liver damage, steatosis, or fibrosis. In contrast, Mlkl+/- mice showed a significant reduction in these parameters that was associated with elevated Pparα and Pparγ levels. Both Mlkl-/- and Mlkl+/- mice on the HFHFrHC diet resisted diet-induced obesity, attributed to the increased beiging, enhanced oxygen consumption, and energy expenditure due to adipose tissue, and exhibited improved insulin sensitivity. These findings highlight the tissue-specific effects of MLKL on the liver and adipose tissue, and they suggest a dose-dependent effect of MLKL on liver pathology.

Non-Necroptotic Roles of MLKL in Diet-Induced Obesity, Liver Pathology, and Insulin Sensitivity: Insights from a High Fat, High Fructose, High Cholesterol Diet Mouse Model.

Chronic inflammation is a key player in metabolic dysfunction-associated fatty liver disease (MAFLD) progression. Necroptosis, an inflammatory cell death pathway, is elevated in MAFLD patients and mouse models, yet its role is unclear due to diverse mouse models and inhibition strategies. In our study, we inhibited necroptosis by targeting mixed lineage kinase domain like pseudokinase (MLKL), the terminal effector of necroptosis, in a high-fat, high-fructose, high-cholesterol (HFHFrHC) mouse model of diet-induced MAFLD mouse model. Despite HFHFrHC diet upregulating MLKL (2.5-fold), WT mice livers showed no increase in necroptosis markers or associated proinflammatory cytokines. Surprisingly, Mlkl -/- mice experienced exacerbated liver inflammation without protection from diet-induced liver damage, steatosis, or fibrosis. In contrast, Mlkl +/- mice showed significant reduction in these parameters that was associated with elevated Pparα and Pparγ levels. Both Mlkl -/- and Mlkl +/- mice on HFHFrHC diet resisted diet-induced obesity, attributed to increased beiging, enhanced oxygen consumption and energy expenditure due to adipose tissue, and exhibited improved insulin sensitivity. These findings highlight the tissue specific effects of MLKL on the liver and adipose tissue, and suggest a dose-dependent effect of MLKL on liver pathology.

Intracellular iron accumulation facilitates mycobacterial infection in old mouse macrophages.

Aging has a significant impact on the immune system, leading to a gradual decline in immune function and changes in the body's ability to respond to bacterial infections. Non-tuberculous mycobacteria (NTM), also known as atypical mycobacteria or environmental mycobacteria, are commonly found in soil, water, and various environmental sources. While many NTM species are considered opportunistic pathogens, some can cause significant infections, particularly in individuals with compromised immune systems, such as older individuals. When mycobacteria enter the body, macrophages are among the first immune cells to encounter them and attempt to engulf mycobacteria through a process called phagocytosis. Some NTM species, including Mycobacterium avium (M. avium) can survive and replicate within macrophages. However, little is known about the interaction between NTM and macrophages in older individuals. In this study, we investigated the response of bone marrow-derived macrophage (BMMs) isolated from young (5 months) and old (25 months) mice to M. avium serotype 4, one of the main NTM species in patients with pulmonary NTM diseases. Our results demonstrated that BMMs from old mice have an increased level of intracellular iron and are more susceptible to M. avium serotype 4 infection compared to BMMs from young mice. The whole-cell proteomic analysis indicated a dysregulated expression of iron homeostasis-associated proteins in old BMMs regardless of mycobacterial infection. Deferoxamine, an iron chelator, significantly rescued mycobacterial killing and phagolysosome maturation in BMMs from old mice. Therefore, our data for the first time indicate that an intracellular iron accumulation improves NTM survival within macrophages from old mice and suggest a potential application of iron-chelating drugs as a host-directed therapy for pulmonary NTM infection in older individuals.

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

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 TGFß, 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.

A Genetically Heterogeneous Rat Model with Divergent Mitochondrial Genomes.

We generated a genetically heterogenous rat model by a 4-way cross strategy using 4 inbred strains (Brown Norway [BN], Fischer 344 [F344], Lewis [LEW], and Wistar Kyoto [KY]) to provide investigators with a highly genetically diverse rat model from commercially available inbred rats. We made reciprocal crosses between males and females from the 2 F1 hybrids to generate genetically heterogeneous rats with mitochondrial genomes from either the BN (OKC-HETB, a.k.a "B" genotype) or WKY (OKC-HETW a.k.a "W" genotype) parental strains. These two mitochondrial genomes differ at 94 nucleotides, more akin to human mitochondrial genome diversity than that available in classical laboratory mouse strains. Body weights of the B and W genotypes were similar. However, mitochondrial genotype antagonistically affected grip strength and treadmill endurance in females only. In addition, mitochondrial genotype significantly affected multiple responses to a high-fat diet (HFD) and treatment with 17α-estradiol. Contrary to findings in mice in which males only are affected by 17α-estradiol supplementation, female rats fed a HFD beneficially responded to 17α-estradiol treatment as evidenced by declines in body mass, adiposity, and liver mass. Male rats, by contrast, differed in a mitochondrial genotype-specific manner, with only B males responding to 17α-estradiol treatment. Mitochondrial genotype and sex differences were also observed in features of brain-specific antioxidant response to a HFD and 17α-estradiol as shown by hippocampal levels of Sod2 acetylation, JNK, and FoxO3a. These results emphasize the importance of mitochondrial genotype in assessing responses to putative interventions in aging processes.

Senolytic treatment reduces cell senescence and necroptosis in Sod1 knockout mice that is associated with reduced inflammation and hepatocellular carcinoma.

The goal of this study was to test the role cellular senescence plays in the increased inflammation, chronic liver disease, and hepatocellular carcinoma seen in mice null for Cu/Zn-Superoxide dismutase (Sod1KO). To inhibit senescence, wildtype (WT) and Sod1KO mice were given the senolytics, dasatinib, and quercetin (D + Q) at 6 months of age when the Sod1KO mice begin exhibiting signs of accelerated aging. Seven months of D + Q treatment reduced the expression of p16 in the livers of Sod1KO mice to WT levels and the expression of several senescence-associated secretory phenotype factors (IL-6, IL-1ß, CXCL-1, and GDF-15). D + Q treatment also reduced markers of inflammation in livers of the Sod1KO mice, for example, cytokines, chemokines, macrophage levels, and Kupffer cell clusters. D + Q treatment had no effect on various markers of liver fibrosis in the Sod1KO mice but reduced the expression of genes involved in liver cancer and dramatically reduced the incidence of hepatocellular carcinoma. Surprisingly, D + Q also reduced markers of necroptosis (phosphorylated and oligomerized MLKL) in the Sod1KO mice to WT levels. We also found that inhibiting necroptosis in the Sod1KO mice with necrostatin-1s reduced the markers of cellular senescence (p16, p21, and p53). Our study suggests that an interaction occurs between cellular senescence and necroptosis in the liver of Sod1KO mice. We propose that these two cell fates interact through a positive feedback loop resulting in a cycle amplifying both cellular senescence and necroptosis leading to inflammaging and age-associated pathology in the Sod1KO mice.

Necroptosis contributes to chronic inflammation and fibrosis in aging liver.

Inflammaging, characterized by an increase in low-grade chronic inflammation with age, is a hallmark of aging and is strongly associated with various age-related diseases, including chronic liver disease (CLD) and hepatocellular carcinoma (HCC). Because necroptosis is a cell death pathway that induces inflammation through the release of DAMPs, we tested the hypothesis that age-associated increase in necroptosis contributes to chronic inflammation in aging liver. Phosphorylation of MLKL and MLKL oligomers, markers of necroptosis, as well as phosphorylation of RIPK3 and RIPK1 were significantly upregulated in the livers of old mice relative to young mice and this increase occurred in the later half of life (i.e., after 18 months of age). Markers of M1 macrophages, expression of pro-inflammatory cytokines (TNFα, IL6 and IL1ß), and markers of fibrosis were all significantly upregulated in the liver with age and the change in necroptosis paralleled the changes in inflammation and fibrosis. Hepatocytes and liver macrophages isolated from old mice showed elevated levels of necroptosis markers as well as increased expression of pro-inflammatory cytokines relative to young mice. Short-term treatment with the necroptosis inhibitor, necrostatin-1s (Nec-1s), reduced necroptosis, markers of M1 macrophages, fibrosis, and cell senescence as well as reducing the expression of pro-inflammatory cytokines in the livers of old mice. Thus, our data show for the first time that liver aging is associated with increased necroptosis and necroptosis contributes to chronic inflammation in the liver, which in turn appears to contribute to liver fibrosis and possibly CLD.

Role of necroptosis in chronic hepatic inflammation and fibrosis in a mouse model of increased oxidative stress.

Mice deficient in the antioxidant enzyme Cu/Zn-superoxide dismutase (Sod1-/- or Sod1KO mice) have increased oxidative stress, show accelerated aging and develop spontaneous hepatocellular carcinoma (HCC) with age. Similar to humans, HCC development in Sod1KO mice progresses from non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH) with fibrosis, which eventually progresses to HCC. Oxidative stress plays a role in NAFLD to NASH progression, and liver inflammation is the main mechanism that drives the disease progression from NASH to fibrosis. Because necroptosis is a major source of inflammation, we tested the hypothesis that increased necroptosis in the liver plays a role in increased inflammation and fibrosis in Sod1KO mice. Phosphorylation of MLKL (P-MLKL), a well-accepted marker of necroptosis, and expression of MLKL protein were significantly increased in the livers of Sod1KO mice compared to wild type (WT) mice indicating increased necroptosis. Similarly, phosphorylation of RIPK3 and RIPK3 protein levels were also significantly increased. Markers of pro-inflammatory M1 macrophages, NLRP3 inflammasome, and transcript levels of pro-inflammatory cytokines and chemokines, e.g., TNFα, IL-6, IL-1ß, and Ccl2 that are associated with human NASH, were significantly increased. Expression of antioxidant enzymes and heat shock proteins, and markers of fibrosis and oncogenic transcription factor STAT3 were also upregulated and autophagy was downregulated in the livers of Sod1KO mice. Short term treatment of Sod1KO mice with necrostatin-1s (Nec-1s), a necroptosis inhibitor, reversed these conditions. Our data show for the first time that necroptosis-mediated inflammation contributes to fibrosis in a mouse model of increased oxidative stress and accelerated aging, that also exhibits progressive HCC development.

Effect of rapamycin on aging and age-related diseases-past and future.

In 2009, rapamycin was reported to increase the lifespan of mice when implemented later in life. This observation resulted in a sea-change in how researchers viewed aging. This was the first evidence that a pharmacological agent could have an impact on aging when administered later in life, i.e., an intervention that did not have to be implemented early in life before the negative impact of aging. Over the past decade, there has been an explosion in the number of reports studying the effect of rapamycin on various diseases, physiological functions, and biochemical processes in mice. In this review, we focus on those areas in which there is strong evidence for rapamycin's effect on aging and age-related diseases in mice, e.g., lifespan, cardiac disease/function, central nervous system, immune system, and cell senescence. We conclude that it is time that pre-clinical studies be focused on taking rapamycin to the clinic, e.g., as a potential treatment for Alzheimer's disease.