Necroptosis contributes to chronic inflammation and fibrosis in aging liver

May combine two claims.

Transcript levels of TNFα (fourfold), IL6 (fourfold), IL-1β (40-fold), and MCP1/CCL2 (fourfold) are significantly increased in hepatocytes isolated from old mice relative to young mice.

“Transcript levels were significantly increased for TNFα (fourfold), IL6 (fourfold) and IL‐1β (40‐fold) and MCP1/CCL2 (fourfold) in the hepatocytes isolated from old mice relative to hepatocytes from young mice (Figure 3h).”

May combine two claims.

The percent of liver cells undergoing early apoptosis (Annexin V positive) is significantly increased with age, starting from 12 months (2.5-fold), and remains elevated at 18 (2.7-fold) and 24 months (2.2-fold).

“The early apoptotic cell population was significantly higher in the liver starting from 12 months of age (2.5‐fold) and remained elevated at 18 (2.7‐fold) and 24 months of age (2.2‐fold)”

May combine two claims.

Transcript levels of F4/80 and MCP-1 are similar in 7- and 12-month-old mice, whereas they are significantly increased (twofold for F4/80 and fivefold and sixfold for MCP-1) in 18- and 24-month-old mice.

“Transcript levels of F4/80 and monocyte chemoattractant protein‐1 (MCP‐1) were similar in 7‐ and 12‐month‐old mice, whereas the levels are significantly increased (twofold for F4/80 and fivefold and sixfold for MCP‐1) in 18‐ and 24‐month‐old mice. (Figure S2a).”

May combine two claims.

P-MLKL and MLKL levels are both significantly elevated (twofold) in hepatocytes isolated from old mice relative to young mice.

“levels of P‐MLKL and MLKL were both significantly elevated (twofold) in the hepatocytes isolated from old mice relative to young mice (Figure 2b).”

May combine two claims.

Expression of M1 macrophage markers at 24 months is similar to 18 months, except for CD11c, which is increased between 18 and 24 months.

“Expression of M1 macrophage markers at 24 months was similar to those at 18‐months, except for CD11c, which was increased between 18 and 24 months of age (Figure S2b).”

May combine two claims.

P-RIPK3 and RIPK3 levels are significantly elevated in hepatocytes from old mice (twofold and 1.4-fold respectively).

“Levels of P‐RIPK3 and RIPK3 are significantly elevated in the hepatocytes from old mice (twofold and 1.4‐fold respectively)”

May combine two claims.

P-MLKL and P-RIPK3 levels show a slight reduction at 24 months relative to 18 months, but this is not statistically significant.

“The levels of P‐MLKL and P‐RIPK3 showed a slight reduction at 24 months relative to 18 months, however, this was not statistically significant.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The M2 macrophage population (CD45+CD11b+Ly6G-F4/80+CD206+ cells) is reduced in livers of old mice (5.06 ± 0.12) relative to young mice (6.48 ± 0.56), but this reduction did not reach statistical significance.

“The M2 macrophage population (CD45+CD11b+Ly6G‐ F4/80+ CD206+ cells) was reduced in the livers of old mice (5.06 ± 0.12) relative to young mice (6.48 ± 0.56), however, this reduction did not reach statistical significance (Figure 3d).”

May combine two claims.

Picrosirius red staining in old mice liver is significantly higher (2.7-fold) relative to young mice, suggesting increased collagen levels.

“Consistent with this, picrosirius red (PSR) staining in the old mice liver was significantly higher (2.7‐fold) relative to young mice, suggesting increased levels of collagen in old mice liver (Figure 4e).”

May combine two claims.

Nec-1s treatment had no effect on body weight or liver weight of old WT mice, indicating no obvious negative effect.

“Nec‐1s treatment had no effect on the body weight or liver weight of old WT mice (Figure S3a and Figure S3b), indicating no obvious negative effect of Nec‐1s on old mice.”

May combine two claims.

Serum ALT levels are significantly increased in old mice (2.2-fold) relative to young mice, and Nec-1s treatment significantly reduced serum ALT levels in old mice to levels comparable to young mice.

“We found a significant increase in the serum levels of ALT in the old mice (2.2‐fold) relative to young mice, and Nec‐1s treatment significantly reduced serum ALT levels in old mice to levels comparable to young mice (Figure 5f).”

May combine two claims.

Protein expression of F4/80 (fourfold) and CD68 (fivefold) is significantly increased in livers of old mice, and these are significantly reduced by Nec-1s treatment to levels similar to young liver.

“protein expression of expression of F4/80 (fourfold) and CD68 (fivefold) was significantly increased in the livers of old mice, which were significantly reduced by Nec‐1s treatment to levels similar to young liver as shown by immunofluorescence staining (Figure 5d).”

May combine two claims.

Expression of CD206 protein (M2 macrophage marker) is significantly reduced (80%) in livers of old mice relative to young mice, and Nec-1s had no effect on CD206 expression in old mice.

“In contrast, the expression of CD206 protein (M2 macrophage marker) was significantly reduced (80%) in the livers of old mice relative to young mice and Nec‐1s had no effect on CD206 expression in the livers of old mice (Figure 5d).”

May combine two claims.

Nec-1s treatment significantly reduced necroptosis, with P-MLKL and MLKL oligomer levels reduced to levels seen in young mice.

“Nec‐1s treatment significantly reduced necroptosis as predicted, that is, the levels of PMLKL and MLKL oligomers were reduced to the levels seen in young mice (Figure 5a, b).”

May combine two claims.

While P-MLKL expression was similar at 18 and 24 months, MLKL oligomer levels were significantly increased from 18 to 24 months.

“While the expression of P‐MLKL was similar at 18 and 24 months, MLKL oligomer levels were significantly increased from 18 to 24 months.”

May combine two claims.

Transcript levels of pro-inflammatory cytokines TNFα, IL6, and IL-1β, which were elevated in livers of old WT mice, are significantly reduced by Nec-1s treatment and are comparable to young mice.

“Similarly, levels of transcripts for pro‐inflammatory cytokines TNFα, IL6 and IL‐1β, which were elevated in the livers of old WT mice, were significantly reduced by Nec‐1s treatment and were comparable to the levels in young mice (Figure S3g).”

May combine two claims.

Nec-1s treatment of old mice resulted in reduced M1 macrophages and reduced expression of pro-inflammatory cytokines.

“we observed that Nec‐1s treatment of old mice resulted in reduced M1 macrophages and the expression of pro‐inflammatory cytokines.”

May combine two claims.

Total macrophage content and pro-inflammatory M1 macrophages increase with age, whereas anti-inflammatory M2 macrophages decline with age.

“the total macrophage content as well as pro‐inflammatory M1 macrophages increase with age, whereas anti‐inflammatory M2 macrophages decline with age.”

May combine two claims.

Pro-inflammatory cytokine/chemokine expression (TNFα, IL6, IL1β, and CCL2) is increased in isolated hepatocytes and isolated macrophages (IL1β and CCL2), but not in LSECs from old mice.

“We also found that pro‐inflammatory cytokine/chemokine expression (e.g., TNFα, IL6, IL1β, and CCL2) were increased in isolated hepatocytes, isolated macrophages (IL1β and CCL2), but not in LSECs from old mice.”

May combine two claims.

There is a strong age-related association between necroptosis and inflammation in the liver, as both increase in the latter half of life, around 18 months of age.

“We observed a strong age‐related association between necroptosis and inflammation in liver as both increase in the latter half of life, around 18 months of age when mice began to exhibit some age‐associated pathologies (Stahl et al., 2020).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Necroptosis increases with age in white adipose tissue of mice and is reduced by interventions that delay aging (dietary restriction or Ames dwarf mice).

“Previously, we reported that necroptosis increases with age in the white adipose tissue of mice and is reduced by interventions that delay aging (dietary restriction or in Ames dwarf mice) (Deepa et al., 2018; Royce et al., 2019).”

• cites Deepa et al., 2018Not checked yet

  Not checked in the free run.

• cites Royce et al., 2019Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Necroptosis has emerged as a novel mode of cell death in various chronic liver diseases such as NAFLD and NASH, conditions also associated with aging in liver.

“Necroptosis has emerged as a novel mode of cell death in various chronic liver diseases such as nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), conditions that are also associated with aging in liver (Saeed & Jun, 2014).”

• cites Saeed & Jun, 2014Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Necroptosis is initiated by necroptotic stimuli such as TNFα, oxidative stress or mTOR/Akt pathway, which sequentially phosphorylate and activate RIPK1 and RIPK3 that in turn phosphorylate MLKL.

“However, necroptosis is also initiated by necroptotic stimuli [e.g., TNFα, oxidative stress or mTOR/Akt pathway (Royce et al., 2019), which sequentially phosphorylate and activate receptor‐interacting protein kinase 1(RIPK1) and RIPK3 that in turn phosphorylate the pseudokinase mixed lineage kinase domain‐like (MLKL) protein.”

• cites Royce et al., 2019Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

DAMPs released from damaged or dying hepatocytes are one of the proposed mediators of chronic inflammation in the liver.

“Damage‐associated molecular patterns (DAMPs, example, HMGB1, mitochondrial DNA, nuclear DNA, ATP etc.) released from damaged or dying hepatocytes are one of the proposed mediators of chronic inflammation in the liver (Brenner et al., 2013).”

• cites Brenner et al., 2013Not checked yet

  Not checked in the free run.

May combine two claims.

Necroptosis and inflammation are increased in livers of Sod1-/- mice, and blocking necroptosis reduced inflammation in their livers.

“Recently, we found that necroptosis and inflammation are increased in the livers of a mouse model of accelerated aging (mice deficient in the antioxidant enzyme, Cu/Zn superoxide dismutase, Sod1‐/‐ mice) and blocking necroptosis reduced inflammation in the livers of Sod1‐/‐ mice (Mohammed et al., 2021).”

• cites Mohammed et al., 2021Not checked yet

  Not checked in the free run.

May combine two claims.

The changes in circulating HMGB1 levels parallel the changes in necroptosis markers in the liver, supporting that necroptosis increases with age in liver.

“The changes in circulating HMGB1 levels parallel the changes in the necroptosis markers in the liver, providing additional data supporting that necroptosis increases with age in liver.”

May combine two claims.

Chronic inflammation in the liver appears to contribute to liver fibrosis and possibly CLD.

“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.”

• AssumptionRests on an assumption: Chronic hepatic inflammation is a major driver of liver fibrosis.

Classified as probably not a substantial claim.

Absence of caspase-8 switches cell death from apoptosis to necroptosis.

“Absence of caspase‐8, an initiator of extrinsic apoptosis, switches cell death from apoptosis to necroptosis (Fritsch et al., 2019).”

• cites Fritsch et al., 2019Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

For mouse MLKL, tetramers fail to translocate to the plasma membrane and octamer formation is required for pore formation in the membrane.

“For mouse MLKL, tetramers fail to translocate to the plasma membrane and octamer formation is required for pore formation in the membrane (Huang et al., 2017).”

• cites Huang et al., 2017Not checked yet

  Not checked in the free run.

May combine two claims.

Nec-1s effectively blocks necroptosis and reduces inflammation in the liver as well as in other tissues.

“Mice were treated with Nec‐1s (RIPK1 inhibitor) for 30 days, which has been shown to effectively block necroptosis and reduce inflammation in the liver as well as in other tissues (Mohammed et al., 2021).”

• cites Mohammed et al., 2021Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Necroptosis most likely evolved as an alternative form of cell death to kill cells infected by viral pathogens and to promote inflammatory and immune responses to limit the spread of viruses.

“Necroptosis most likely evolved as an alternative form of cell death to kill cells infected by viral pathogens and to promote inflammatory and immune responses to limit the spread of the viruses (Dondelinger et al., 2016).”

• cites Dondelinger et al., 2016Not checked yet

  Not checked in the free run.

May combine two claims.

Short-term Nec-1s treatment can exert its effect on M1 macrophages and reduce age-associated hepatic inflammation and thereby liver damage and fibrosis.

“Thus, our data shows that short‐term Nec‐1s treatment can exert its effect on the M1 macrophages and reduce age‐associated hepatic inflammation and thereby liver damage and fibrosis.”

Classified as probably not a substantial claim. May combine two claims.

HMGB1 is a DAMP that has been shown to be released by hepatic necroptosis and is associated with acute liver injury and chronic liver disease.

“a DAMP that has been shown to be released by hepatic necroptosis and is associated with acute liver injury and chronic liver disease (Wen et al., 2020).”

• cites Wen et al., 2020Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Hepatocytes constitute 80% of the liver mass and are reported to undergo cell death in CLD.

“Hepatocytes constitute 80% of the liver mass and are reported to undergo cell death in CLD (Shojaie et al., 2020)”

• cites Shojaie et al., 2020Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Kupffer cells are reported to undergo necroptosis.

“Because Kupffer cells are reported to undergo necroptosis (Blériot et al., 2015), necroptosis in liver macrophages in aging was assessed.”

• cites Blériot et al., 2015Not checked yet

  Not checked in the free run.

May combine two claims.

Nec-1s is a specific and potent inhibitor of RIPK1 with no reported off-target effects.

“Nec‐1s is a specific and potent inhibitor of RIPK1 (Takahashi et al., 2012) with no reported off target effects.”

• cites Takahashi et al., 2012Not checked yet

  Not checked in the free run.

May combine two claims.

Chronic liver disease, especially fibrosis, increases with age in the livers of mice and humans and is associated with increased inflammation.

“CLD, especially fibrosis has been reported to increase with age in the livers of mice and humans (Kim et al., 2015) and is associated with increased inflammation.”

• cites Kim et al., 2015Not checked yet

  Not checked in the free run.

May combine two claims.

Blocking necroptosis genetically (Ripk3-/-) in mice fed a methionine-deficient diet or pharmacologically (RIPA56) in mice fed a high-fat diet reduced liver fibrosis.

“These data are consistent with previous reports showing that blocking necroptosis genetically (Ripk3‐/‐ ) in mice fed a methionine‐deficient diet or pharmacologically (RIPA56) in mice fed a high‐fat diet reduced liver fibrosis (Gautheron et al., 2014; Majdi et al., 2020).”

• cites Gautheron et al., 2014Not checked yet

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• cites Majdi et al., 2020Not checked yet

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Classified as probably not a substantial claim.

DAMPs are factors known to initiate macrophage activation and polarization.

“Because DAMPs are one of the factors that are known initiate macrophages activation and polarization (Lee et al., 2020)”

• cites Lee et al., 2020Not checked yet

  Not checked in the free run.

May combine two claims.

Exposure to chronic inflammation alters macrophage function, leading to a reduced ability to remove cells and increased production of inflammatory cytokines.

“Exposure to chronic inflammation has been shown to alter macrophage function leading to a reduction in their ability to remove cells, such as bacteria and senescent cells, and increase their production of inflammatory cytokines (Thevaranjan et al., 2017).”

• cites Thevaranjan et al., 2017Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

RIPK1 is involved in apoptosis as well as cell survival pathways such as NF-kB, Akt, and JNK.

“In addition to necroptosis, RIPK1 is also involved in apoptosis as well as cell survival pathways such as NF‐kB, Akt, and JNK (Mifflin et al., 2020).”

• cites Mifflin et al., 2020Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Several FDA-approved anti-cancer drugs such as Sorafenib and Dabrafenib have been identified as anti-necroptotic agents.

“several FDA‐approved anti‐cancer drugs such as Sorafenib and Dabrafenib have been identified as anti‐necroptotic agents (Fulda, 2018).”

• cites Fulda, 2018Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Nec-1s treatment reduces inflammation associated with necroptosis in multiple sclerosis and amyotrophic lateral sclerosis.

“Nec‐1s treatment has been reported to reduce inflammation associated with necroptosis in multiple sclerosis and amyotrophic lateral sclerosis (Mifflin et al., 2020).”

• cites Mifflin et al., 2020Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

TGFβ is a major player in the induction of fibrosis and is produced by several liver cells such as LSEC, Kupffer cells, platelets, hepatocytes and macrophages.

“Transforming growth factor beta (TGFβ) is a major player in the induction of fibrosis and is produced by several liver cells such as LSEC, Kupffer cells, platelets, hepatocytes and macrophages (Fabregat and Caballero‐Díaz, 2018).”

• cites Fabregat and Caballero‐Díaz, 2018Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Liver sinusoidal endothelial cells are known to produce inflammatory cytokines in chronic liver diseases.

“Liver sinusoidal endothelial cells (LSEC) are known to produce inflammatory cytokines in chronic liver diseases (Wang & Peng, 2021)”

• cites Wang & Peng, 2021Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Hepatocytes are also reported to produce pro-inflammatory cytokines.

“Because hepatocytes are also reported to produce pro‐inflammatory cytokines (Stahl et al., 2020), we measured the expression of pro‐inflammatory cytokines in isolated hepatocytes.”

• cites Stahl et al., 2020Not checked yet

  Not checked in the free run.

May combine two claims.

Short-term Nec-1s treatment effectively reduced necroptosis and inflammation in the livers of Sod1-/- mice.

“Short‐term Nec‐1s treatment effectively reduced necroptosis and inflammation in the livers of Sod1‐/‐ mice (Mohammed et al., 2021).”

• cites Mohammed et al., 2021Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Therapeutic applications of RIPK1 inhibitors for the treatment of a variety of human diseases are being tested in clinical trials.

“Currently, therapeutic applications of RIPK1 inhibitors for the treatment of a variety of human diseases are being tested in clinical trials (Mifflin et al., 2020).”

• cites Mifflin et al., 2020Not checked yet

  Not checked in the free run.

May combine two claims.

The study's data provide strong support for necroptosis playing a role in inflammaging in the liver and in the age-related increase in chronic liver disease.

“In summary, our data proved strong support for necroptosis playing a role in inflammaging in the liver and the age‐related increase in CLD.”

• AssumptionRests on an assumption: Chronic inflammation increases with age (inflammaging) and is associated with a large number of age-related diseases.
• AssumptionRests on an assumption: Necroptosis is a cell death pathway that can induce inflammation through the release of DAMPs from necroptotic cells.

Classified as probably not a substantial claim.

Macrophages play an important role in the removal of senescent cells.

“macrophages, which play an important role in removal of senescent cells (Kale et al., 2020).”

• cites Kale et al., 2020Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

p16 and p21 have been reported to increase with age.

“we assessed changes in cell senescence by measuring p16 and p21 expression, which have been reported to increase with age (Franceschi & Campisi, 2014).”

• cites Franceschi & Campisi, 2014Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

All procedures were approved by the Institutional Animal Care and Use Committee at the University of Oklahoma Health Sciences Center.

“All procedures were approved by the Institutional Animal Care and Use Committee at the University of Oklahoma Health Sciences Center (OUHSC).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The purity of isolated hepatocytes was checked by real-time PCR and western blotting for liver cell-specific markers.

“The purity of the isolated hepatocytes was checked by real‐time PCR and western blotting for the expression of liver cell specific markers: albumin (hepatocytes), F4/80 (macrophages), Clec4f (Kupffer cells), CD31 (endothelial cells) and desmin (hepatic stellate cells).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Necroptosis has dual effects in cancer, either promoting or reducing tumor growth depending on the type of cancer.

“Necroptosis has been reported to exhibit dual effects in cancer, that is, necroptosis can either promote or reduce tumor growth depending on the type of cancer (Gong et al., 2019).”

• cites Gong et al., 2019Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

The purity of isolated fractions was checked by real-time PCR and western blotting for cell-specific markers.

“The purity of the isolated fractions was checked by real‐time PCR and western blotting for cell specific markers.”

Probably not a substantial claim: may not pass S2 (checkable).

Necroptosis is a potential therapeutic target for treating chronic liver diseases associated with aging.

“Thus, necroptosis is a potential therapeutic target for treating chronic liver diseases associated with aging.”

• AssumptionRests on an assumption: Chronic inflammation increases with age (inflammaging) and is associated with a large number of age-related diseases.
• AssumptionRests on an assumption: Necroptosis is a cell death pathway that can induce inflammation through the release of DAMPs from necroptotic cells.