Gene expression markers of age-related inflammation in two human cohorts
Open accessAbstract
Introduction Chronically elevated circulating inflammatory markers are common in older persons but mechanisms are unclear. Many blood transcripts (>800 genes) are associated with interleukin-6 protein levels (IL6) independent of age. We aimed to identify gene transcripts statistically mediating, as drivers or responders, the increasing levels of IL6 protein in blood at older ages.Methods Blood derived in-vivo RNA from the Framingham Heart Study (FHS, n=2422, ages 40-92 yrs) and InCHIANTI study (n=694, ages 30-104 yrs), with Affymetrix and Illumina expression arrays respectively (>17,000 genes tested), were tested for statistical mediation of the age-IL6 association using resampling techniques, adjusted for confounders and multiple testing.Results In FHS, IL6 expression was not associated with IL6 protein levels in blood. 102 genes (0.6% of 17,324 expressed) statistically mediated the age-IL6 association of which 25 replicated in InCHIANTI (including 5 of the 10 largest effect genes). The largest effect gene (SLC4A10, coding for NCBE, a sodium bicarbonate transporter) mediated 19% (adjusted CI 8.9 to 34.1%) and replicated by PCR in InCHIANTI (n=194, 35.6% mediated, p=0.01). Other replicated mediators included PRF1 (perforin, a cytolytic protein in cytotoxic T lymphocytes and NK cells) and IL1B (Interleukin 1 beta): few other cytokines were significant mediators.Conclusions This transcriptome-wide study on human blood identified a small distinct set of genes that statistically mediate the age-IL6 association. Findings are robust across two cohorts and different expression technologies. Raised IL6 levels may not derive from circulating white cells in age related inflammation.
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- Findingown data
Three additional FHS mediator genes were validated by PCR in InCHIANTI.
“Of the 102 genes partial mediators of the age-IL6 association identified in FHS, 88 were available (Supplementary Table 1) in InCHIANTI and 22 of them significantly mediated the association of age with IL6 after adjustment for confounders (with 3 additionally validated by PCR, see below).”
Quote in the paperNot a substantial claim - Findingown data
In FHS, 29 genes mediated at least 5% of the age-IL6 association.
“Twenty-nine genes mediated ≥5% of the association, with SLC4A10 (solute carrier family 4, sodium bicarbonate transporter) mediating 19% of the association (CI 8.9 to 34.1%) in FHS (Table 2).”
Quote in the paperSubstantial claim - Findingown data
In InCHIANTI, the ratio of lymphocyte to neutrophil percentages significantly mediated the age-IL6 association.
“the ratio between lymphocyte and neutrophil percentages also significantly mediated the age~IL6 association (proportion of effect mediated= 0.06).”
Quote in the paperNot a substantial claim - Findingown data
IL6 transcript levels were mildly negatively associated with age in InCHIANTI.
“Expression of IL6 transcripts (from circulating blood leukocytes, see methods) was not associated with age in FHS and mildly negatively associated with age in InCHIANTI (Figure 1C and D) (FHS: n=2422, coefficient=0.013, p=0.12; InCHIANTI: n=186, coefficient=−4.7, p= 5.6 ×10−4).”
Quote in the paperNot a substantial claim - Findingown data
In InCHIANTI, lymphocyte and neutrophil percentages mediated the association between age and IL6.
“lymphocyte and neutrophil percentages mediated the association between age and IL6 (proportion of effect mediated= 0.09, 0.06 respectively);”
Quote in the paperSubstantial claim
- Findingown data
In FHS, SLC4A10 mediated 19% (CI 8.9 to 34.1%) of the age-IL6 association.
“Twenty-nine genes mediated ≥5% of the association, with SLC4A10 (solute carrier family 4, sodium bicarbonate transporter) mediating 19% of the association (CI 8.9 to 34.1%) in FHS (Table 2).”
Quote in the paperSubstantial claim - Findingown data
In InCHIANTI, 22 of the 88 available FHS mediator genes significantly mediated the age-IL6 association after adjustment for confounders.
“Of the 102 genes partial mediators of the age-IL6 association identified in FHS, 88 were available (Supplementary Table 1) in InCHIANTI and 22 of them significantly mediated the association of age with IL6 after adjustment for confounders (with 3 additionally validated by PCR, see below).”
Quote in the paperSubstantial claim - Findingown data
IL6 transcript levels were not associated with age in FHS.
“Expression of IL6 transcripts (from circulating blood leukocytes, see methods) was not associated with age in FHS and mildly negatively associated with age in InCHIANTI (Figure 1C and D) (FHS: n=2422, coefficient=0.013, p=0.12; InCHIANTI: n=186, coefficient=−4.7, p= 5.6 ×10−4).”
Quote in the paperSubstantial claim - Findingown data
IL6 transcript levels were not significantly correlated with serum IL6 protein levels in either FHS or InCHIANTI.
“Surprisingly, IL6 transcript levels were not significantly correlated with IL6 protein levels in serum in either study (FHS: coefficient=0.0064, p= 0.26; InCHIANTI: coefficient=0.021, p=0.76).”
Quote in the paperSubstantial claim - Findingown data
In FHS, 102 genes (0.6% of 17,324) significantly mediated a proportion of the age-IL6 association after adjustment for confounders and multiple testing.
“However, expression of only 102 (0.6% of the 17,324) genes significantly mediated a proportion of the association between age and IL6 (after adjustment for confounders – see methods - and multiple testing using 99.99972% confidence intervals, and after excluding 3 invalidly annotated transcripts, Supplementary Table 1).”
Quote in the paperSubstantial claim - Findingown data
In InCHIANTI, lymphocyte, neutrophil, and basophil percentages were associated with serum IL6 adjusting for age.
“lymphocyte, neutrophil and basophil percentages were associated with serum IL6 adjusting for age (Beta= −2.9, 2.8, −0.03; P= 4×10−14, 5×10−12, 1×10−3, respectively);”
Quote in the paperSubstantial claim - Findingown data
Serum IL6 protein levels were strongly positively associated with advancing age in both FHS and InCHIANTI.
“Interleukin-6 serum (protein) levels (IL6) were strongly positively associated with advancing age both in FHS (N=2422; unadjusted: beta=0.031, p=1×10−44) and InCHIANTI (N=694; unadjusted: beta=0.038, p=1 ×10−61, adjusted: beta=0.036, p= 1.1 ×10−36) (Figure 1 A and B).”
Quote in the paperSubstantial claim - Findingown data
In FHS, 4693 genes (27.1%) were associated with age at FDR<0.05.
“Transcripts for 17,324 unique genes could be tested in FHS, of which 4693 (27.1%) were associated with age (FDR<0.05) and 4140 (23.9%) were associated with IL6 (Lin and others 2014).”
Quote in the paperNot a substantial claim - Findingown data
No genes mediating at least 5% had significant associations in both cohorts but different directions of effect.
“No genes mediating ≥5% had significant associations in both cohorts but different directions of effect.”
Quote in the paperNot a substantial claim - Findingown data
In InCHIANTI, significant associations in the same direction were found for IL1b, VCAN, PRF1, and LTB.
“Significant associations in the same direction were found for IL1b, VCAN, PRF1 and LTB.”
Quote in the paperSubstantial claim - Findingown data
CDKN2A expression was not a significant mediator of the IL6-age association in either cohort.
“Expression of CDKN2A was not a significant mediator of the IL6-age association in either cohort.”
Quote in the paperNot a substantial claim - Findingown data
Many small effect genes were not significant in the InCHIANTI cohort.
“there were many small effect genes that were not significant in the InCHIANTI cohort.”
Quote in the paperNot a substantial claim - Findingown data
Eighteen clusters were identified by Scree analysis of the 102 significant mediators in FHS.
“Eighteen clusters were identified by the Scree analysis of the 102 significant mediators in FHS (Supplementary Figure 1).”
Quote in the paperNot a substantial claim - Findingown data
SLC4A10 expression is markedly lower in the older group than in the younger group.
“and also has markedly lower expression in the older group in general.”
Quote in the paperNot a substantial claim - Findingcitation
Serum IL6 protein concentration is strongly positively associated with age in both study cohorts.
“Very strong associations were present in both study cohorts between age and serum interleukin-6 (IL6) protein concentrations (Lin and others 2014).”
- Also stated in: § 4. Discussion · boosted
“although we confirmed a strong positive relationship between serum IL6 levels and age”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Lin and others 2014
Not checked in the free run.
- Also stated in: § 4. Discussion · boosted
- Findingown data
Those three genes did not meet background inclusion criteria in InCHIANTI microarray data.
“Three genes (SLC4A10, FLT3, DAAM2) with larger mediating effects (>7%) in the FHS analysis did not pass the background inclusion criteria in InCHIANTI microarray data.”
Quote in the paperSubstantial claim - Findingown data
Few interleukins, cytokines, or interferon gene transcripts were significant mediators of the age-IL6 association in FHS blood cells, with IL1B as the exception.
“Overall, very few interleukins or cytokines emerged as significant mediators (Figure 3) with all mediation effect sizes for these genes in FHS being <5%, with the sole exception of IL1B.”
- Also stated in: § 4. Discussion · plain
“we found strikingly few interleukins, cytokines (including mRNA for IL6) or interferon gene transcripts mediating the age-IL6 association in the studied blood white cells (Figure 3), with the exception of IL1B”
Quote in the paperSubstantial claim - Also stated in: § 4. Discussion · plain
- Findingown data
Only one KEGG pathway was significant in the functional annotation analysis of the 102 FHS probes: the NOD-like receptor signaling pathway (p=4.9 × 10−5, adjusted p=3.2 × 10−3).
“Only 1 KEGG pathway was significant in the functional annotation analysis; the NOD-like receptor signaling pathway (p=4.9 × 10−5, Benjamin-Hochberg multiple testing adjusted p=3.2 × 10−3).”
Quote in the paperNot a substantial claim - Findingown data
After WBC adjustment, the final list of genes mediating >5% that replicated in InCHIANTI remained the original list of 7.
“however the final list of genes mediating >5% found to replicate in InCHIANTI did not change from the original list of 7.”
Quote in the paperNot a substantial claim - Findingown data
In 194 InCHIANTI samples after exclusions and cleaning, SLC4A10 significantly partially mediated the age-IL6 association, with 35.6% mediated (p=0.01).
“In 194 samples (after exclusions/data cleaning) there was statistically significant partial mediation of the association between age and IL6 for all 3 genes (SLC4A10 % mediated=35.6%, p=0.01; DAAM2 %=−7.92%, p=0.0001; FLT3 %=−1.03%, p=0.0002)”
Quote in the paperSubstantial claim - Findingown data
In 194 InCHIANTI samples, DAAM2 significantly partially mediated the age-IL6 association, with −7.92% mediated (p=0.0001).
“In 194 samples (after exclusions/data cleaning) there was statistically significant partial mediation of the association between age and IL6 for all 3 genes (SLC4A10 % mediated=35.6%, p=0.01; DAAM2 %=−7.92%, p=0.0001; FLT3 %=−1.03%, p=0.0002)”
Quote in the paperSubstantial claim - Findingown data
The ZEB2 mediation effect was not significant in the InCHIANTI array data.
“The ZEB2 mediation effect was not significant in the InCHIANTI array data”
Quote in the paperNot a substantial claim - Findingown data
SLC4A10, the largest single mediator in FHS, clusters strongly only with KLRB1.
“Of particular note, SLC4A10, the largest single mediator in FHS, only clusters strongly with KLRB1”
Quote in the paperNot a substantial claim - Findingown data
Of the 29 genes mediating at least 5% in FHS, 22 were also present in the InCHIANTI microarray data.
“Of the 29 genes mediating ≥5% in FHS (Table 2), 22 were also in the InCHIANTI microarray data.”
Quote in the paperSubstantial claim - Findingown data
Both positive and negative mediators were identified.
“As noted previously, both positive and negative mediators were identified.”
Quote in the paperNot a substantial claim - Findingown data
Different mediators were positively or negatively associated with age and/or IL6.
“The plot illustrates that different mediators were positively or negatively associated with age and/or IL6.”
Quote in the paperSubstantial claim - Findingown data
SLC4A10 expression decreases as IL6 concentration increases.
“Figure 2 plots also show that SLC4A10 expression reduces with increasing IL6 concentration”
Quote in the paperSubstantial claim - Findingown data
Five of the ten most strongly significant FHS mediators replicated in InCHIANTI.
“Five of the ten most strongly significant mediators were replicated in InCHIANTI.”
Quote in the paperSubstantial claim - Findingown data
Few larger-effect CD marker mediators were found, other than exceptions such as KLRB1 (CD161).
“Similarly there were few larger effect CD marker mediators (Cluster of Differentiation, used to identify and characterize leukocyte subtypes) other than notable exceptions with updated gene symbols, such as KLRB1 (CD161).”
Quote in the paperNot a substantial claim - Findingown data
IL1B expression was the only replicated cytokine mediator in the analyses.
“IL1B expression emerged as the only replicated cytokine mediator in our analyses.”
Quote in the paperSubstantial claim - Findingown data
The correlation between proportions mediated by genes using a binary IL6 trait and linear IL6 concentrations was highly consistent, with R2 = 0.81.
“The correlation between the proportions mediated by the genes was highly consistent, the R2 = 0.81 (Supplementary Figure 2).”
Quote in the paperNot a substantial claim - Findingown data
SLC4A10 was the largest effect mediator, with 19.1% mediation in FHS and 35.6% in InCHIANTI.
“Our largest effect mediator SLC4A10 (19.1% in FHS, 35.6% in InCHIANTI) codes for NCBE”
Quote in the paperSubstantial claim - Findingown data
In 194 InCHIANTI samples, FLT3 significantly partially mediated the age-IL6 association, with −1.03% mediated (p=0.0002).
“In 194 samples (after exclusions/data cleaning) there was statistically significant partial mediation of the association between age and IL6 for all 3 genes (SLC4A10 % mediated=35.6%, p=0.01; DAAM2 %=−7.92%, p=0.0001; FLT3 %=−1.03%, p=0.0002)”
Quote in the paperSubstantial claim - Findingown data
The Illumina array appears less sensitive in the blood-derived RNA samples, as fewer gene expression measures passed quality control in InCHIANTI than FHS.
“The latter array appears less sensitive in our blood derived RNA samples as far fewer gene expression measures passed quality control in InCHIANTI than FHS”
Quote in the paperNot a substantial claim - Findingown data
Moderate (50%) replication of the most significant mediators was observed.
“Although moderate (50%) replication of the most significant mediators was observed”
Quote in the paperSubstantial claim - Findingown data
The study identified and independently replicated 7 partial mediators of the age-IL6 association with effect sizes ≥5%, including SLC4A10.
“Examining statistical mediators of the age-IL6 association, we identified and independently replicated 7 partial mediators with effect sizes ≥5%, including one larger effect transcript, namely SLC4A10.”
Quote in the paperSubstantial claim - Findingown data
In the FHS analysis, SLC4A10, FLT3, and DAAM2 had mediating effects greater than 7%.
“Three genes (SLC4A10, FLT3, DAAM2) with larger mediating effects (>7%) in the FHS analysis did not pass the background inclusion criteria in InCHIANTI microarray data.”
Quote in the paperSubstantial claim - Findingown data
The study did not find increased expression of IL6 with age in blood-derived data.
“We did not find increased expression of IL6 with age in our blood derived data”
Quote in the paperSubstantial claim - Study design and conductown data
There was no significant difference between results using measured and predicted CBCs in the 2,280 participants with both.
“We performed a test to compare results with measured and predicted CBCs on the 2,280 participants with both and found no significant difference.”
Quote in the paperSubstantial claim - Premisecitation
In FHS, 4140 genes (23.9%) were associated with IL6.
“Transcripts for 17,324 unique genes could be tested in FHS, of which 4693 (27.1%) were associated with age (FDR<0.05) and 4140 (23.9%) were associated with IL6 (Lin and others 2014).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Lin and others 2014
Not checked in the free run.
- Premisecitation
Chronically elevated circulating inflammatory markers are common in older persons.
“Chronically elevated circulating inflammatory markers are common in older persons but mechanisms are unclear.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites De Martinis and others 2006
Not checked in the free run.
- Premisecitation
Elevated IL6 and CRP are robust age-related risk factors for multiple adverse outcomes including several diseases, disability, cognitive impairment and death.
“Elevated Interleukin-6 (IL6) and C-reactive protein (CRP) have emerged as robust age-related risk factors for multiple adverse outcomes including several diseases, disability, cognitive impairment and death (Singh and Newman 2011).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Singh and Newman 2011
Not checked in the free run.
- Premisecitation
IL6 levels increase with advancing age even in the absence of detectable causes such as cardiovascular risk factors and disease.
“IL6 levels are often undetectable in young individuals but increase with advancing age, even in the absence of detectable causes, including cardiovascular risk factors and disease (Ferrucci and others 2005).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Ferrucci and others 2005
Not checked in the free run.
- Premisecitation
Many top genes associated with blood IL6 protein levels independent of age are in inflammation-related pathways or erythrocyte function, including the JAK/Stat and interleukin-10 signaling pathways.
“Many of the top genes generally associated with blood IL6 protein levels (independent of age) are in inflammation-related pathways or erythrocyte function, including the JAK/Stat signalling pathway and interleukin-10 signalling pathway.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Lin and others 2014
Not checked in the free run.
- Study design and conductreasoning
Multiple testing was controlled using Bonferroni-adjusted confidence intervals, set to 99.99972% in FHS.
“In FHS the number of array probes (and thus models) available was 17,873, so the CI were set to 99.99972% (1-(0.05/17873)).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Bonferroni 1936
Not checked in the free run.
- Premisecitation
The Sobel test provides higher power in small sample sizes than bootstrapping methods.
“we used the Sobel test, as it provides higher power in small samples sizes when compared to bootstrapping methods (Fritz and Mackinnon 2007).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Fritz and Mackinnon 2007 source
Not checked in the free run.
- Interpretationown data
The finding of IL1B and perforin among replicated mediators shows the study identified primary mechanisms involved in age-related chronic inflammation.
“Our finding of IL1B and perforin amongst the replicated mediators shows that our study was able to identify primary mechanisms involved in age-related chronic inflammation.”
Quote in the paperSubstantial claim - Interpretationown data
The alternate technologies provide evidence for the robustness of the positive findings across the two study cohorts.
“However the alternate technologies do provide evidence for the robustness of our positive findings across our two study cohorts.”
Quote in the paperSubstantial claim - Premisecitation
A high proportion of CD8+ T cells express Granzyme B but not PRF1 in response to an influenza challenge.
“A high proportion of CD8+ T cells express Granzyme B but not PRF1 in response to an influenza challenge”
Quote in the paperSubstantial claim0 of 1 sources checked- cites McElhaney and others 2012
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- Premisecitation
The absence of PRF1 results in Granzyme B degradation of the extracellular matrix and inflammation.
“and the absence of PRF1 results in Granzyme B degradation of the extracellular matrix and inflammation (McElhaney and others 2012).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites McElhaney and others 2012
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- Interpretationreasoning
The immune system responds to the age-associated increase in circulating IL6 in a specific way, distinct from the 807 genes seen in the analysis independent of age.
“However that we see a few specific gene expression mediators of age-related inflammation in whole blood suggests that the immune system responds to the age-associated increase in circulating IL6 in a specific way, distinct from the 807 genes seen in the analysis independent of age.”
Quote in the paperSubstantial claim - Interpretationown data
The primary origin of age-related inflammation, particularly the age-related increase in circulating IL6 protein, may not be circulating immune cells.
“These findings suggest that the primary origin of age-related inflammation, in particular the age-related increase in circulating IL6 protein, may not be circulating immune cells.”
Quote in the paperSubstantial claim - Study design and conductnone given
The analysis is cross-sectional.
“The analysis is cross-sectional, so more work will be needed to establish the direction of causation.”
Quote in the paperSubstantial claim - Interpretationown data
The study identified a novel larger effect partial mediator, SLC4A10, coding for NCBE, thought to be involved in pH homeostasis.
“We have identified a novel larger effect partial mediator (SLC4A10, coding for NCBE), thought to be involved in pH homeostasis.”
Quote in the paperSubstantial claim- Rests on an assumption: SLC4A10 codes for NCBE (NBCn2) and is a sodium bicarbonate transporter involved in intracellular acid-base homeostasis.
- Interpretationown data
Raised IL6 levels may not derive from circulating white cells in age-related inflammation.
“Raised IL6 levels may not derive from circulating white cells in age related inflammation.”
Quote in the paperSubstantial claim- Rests on an assumption: IL6 is a proxy for age-related inflammation.
- Premisecitation
Increased IL1B protein concentrations are a core feature of the pro-inflammatory state of aging.
“Increased Interleukin 1 beta protein concentrations (IL1B), a cytokine involved in initiation of most inflammatory responses and a product of inflammasome activation, is a core feature of the pro-inflammatory state of aging (Didier and others 2012).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Didier and others 2012
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- Premisecitation
Perforin expression per cell falls with advanced age.
“Perforin expression per cell falls with advanced age (Rukavina and others 1998).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Rukavina and others 1998
Not checked in the free run.
- Premisecitation
BioGPS shows similar SLC4A10 expression levels across studied human leukocyte subtypes.
“but the BioGPS database (Wu and others 2009) shows similar levels of expression in all studied leukocyte cell sub-types in humans”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Wu and others 2009
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- Study design and conductown data
In InCHIANTI, CDKN2A array expression was not above background.
“In InCHIANTI, CDKN2A array expression was not above background, so TaqMan Low Density Array real-time PCR measured expression in a subsample.”
Quote in the paperNot a substantial claim - Interpretationown data
The raised IL6 protein concentrations in blood may be predominantly generated from other body compartments.
“suggesting that the raised IL6 protein concentrations in blood may be predominantly generated from other body compartments.”
Quote in the paperSubstantial claim - Interpretationreasoning
The results suggest that age-related chronic inflammation has a discrete gene expression mediator signature.
“The results are very different from the widespread associations with general IL6 levels independent of age, suggesting that age-related chronic inflammation has a discrete gene expression mediator signature.”
Quote in the paperSubstantial claim
- May combine two claims. 23
- Classified as probably not a substantial claim. 24
May combine two claims.
Findingown dataIn InCHIANTI, directly measured lymphocyte, neutrophil, monocyte, and eosinophil percentages were each associated with higher mean age.
“In InCHIANTI, directly measured lymphocyte, neutrophil, monocyte and eosinophil percentages were each associated with higher mean age (Beta= −0.1, 0.09, 0.02, 0.02; P= 9×10−9, 1×10−4, 2×10−4, 3×10−3, respectively);”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThe FHS and InCHIANTI cohorts differ in size and age distributions but are otherwise broadly similar.
“The cohorts differ in size and age-distributions, but are otherwise broadly similar (Table 1).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataA small set of genes appear to have partially mediated the age-IL6 association, several of which have not previously been linked to the pro-inflammatory state of aging.
“A small set of genes appear to have partially mediated the age-IL6 association, several of which have not previously been linked to the pro-inflammatory state of aging.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataIL1B and ADM were closely related.
“IL1B and ADM were also closely related.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataExact effect estimates varied between models depending on the specific factor included.
“As expected, the exact effect estimates (the proportion of the IL6-age association mediated) varied between the models depending on the specific factor included (Supplementary Table 2).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataFour genes mediating less than 5% were significant with opposite directions of effect.
“Four of those mediating <5% were significant and with opposite directions of effect (Supplementary Table 1).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataIn InCHIANTI, after additional adjustment for potential confounders, all 7 replicated genes remained significant mediators (p<0.05) except SLC4A10, which was no longer significant after adjustment for waist (p=0.06) or physical activity (p=0.08).
“Additional adjustment for potential confounding factors in the analyses of the 7 replicated genes in InCHIANTI found that all genes were still statistically significant mediators (p<0.05), with the exception of SLC4A10 which was no longer significant after adjustment for waist or physical activity (p=0.06 and p=0.08 respectively)”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataFLT3 was the second largest effect gene in FHS and replicated in InCHIANTI.
“FTL3 (also called CD135) emerged as the second largest effect gene in FHS (replicated in InCHIANTI)”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataFew cytokines other than IL1B were significant mediators; other replicated mediators included PRF1 and IL1B.
“Other replicated mediators included PRF1 (perforin, a cytolytic protein in cytotoxic T lymphocytes and NK cells) and IL1B (Interleukin 1 beta): few other cytokines were significant mediators.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataIn an analysis restricted to the 7 replicated genes, DAVID returned no output.
“In an analysis restricted to the 7 replicated genes only, no output was returned by DAVID.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThe largest effect gene SLC4A10 replicated by PCR in InCHIANTI (n=194, 35.6% mediated, p=0.01).
“The largest effect gene (SLC4A10, coding for NCBE, a sodium bicarbonate transporter) mediated 19% (adjusted CI 8.9 to 34.1%) and replicated by PCR in InCHIANTI (n=194, 35.6% mediated, p=0.01).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataAdjusting for WBC count slightly altered the ordering by proportion mediated among significant FHS mediators.
“Adjusting for WBC count slightly altered the ordering (by proportion mediated) in the list of significant mediators in FHS”
Quote in the paperNot a substantial claimMay combine two claims.
Study design and conductreasoningThe study used two independent community-based cohorts with different microarray platforms, allowing replication of findings robust to cohort and microarray differences.
“The cohorts utilize two different microarray platforms, allowing replication of findings robust to cohort and microarray differences.”
Quote in the paperSubstantial claimMay combine two claims.
Study design and conductown dataThe PLS model for predicting blood cell fractions had testing-set prediction accuracy R2 values of 0.41, 0.61, 0.83, 0.83, 0.81, 0.87, 0.34, and 0.25 for red blood cell count, white blood cell count, percent neutrophil, percent lymphocyte, percent monocyte, percent eosinophil, percent basophil, and platelet count, respectively.
“The resulting model is tested on the testing set and gave the following testing-set prediction accuracy R2: 0.41, 0.61, 0.83, 0.83, 0.81, 0.87, 0.34, and 0.25 for red blood cell count, white blood cell count, percent neutrophil, percent lymphocyte, percent monocyte, percent eosinophil, percent basophil, and platelet count, respectively.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationAccumulation of cells expressing a senescence-associated secretory phenotype in different tissues may be a possible cause of age-related inflammation.
“The accumulation of cells expressing a senescence-associated secretory phenotype (Campisi 2013; Young and Narita 2009) in different tissues, possibly induced by age-related NF-kB dysregulation (Bektas and others 2013), may also be a possible cause.”
Quote in the paperNot a substantial claim0 of 3 sources checkedClassified as probably not a substantial claim.
Study design and conductreasoningThe discovery/replication procedure considered findings significant in discovery after multiple testing as independent tests for replication, accepting p<0.05 in InCHIANTI.
“We are therefore using a discovery/replication procedure, where any findings significant in the discovery analysis after adjustment for multiple testing are considered independent tests for replication, and thus we accepted p<0.05 (i.e. 95% confidence intervals) as significant.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationOverproduction of pro-inflammatory markers may occur in many sites beyond circulating leukocytes, including tissue resident macrophages, adipocytes, endothelial cells, cells within atherosclerotic plaques, and muscle cells.
“The overproduction of pro-inflammatory markers may occur in many sites beyond circulating leukocytes; candidates include tissue resident macrophages, adipocytes, endothelial cells, cells within atherosclerotic plaques and muscle cells (Howcroft and others 2013).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Howcroft and others 2013
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Classified as probably not a substantial claim. May combine two claims.
PremisecitationA prior study identified 4139 genes significantly associated with IL6 levels independent of age, sex, and blood cell components, of which 807 replicated in InCHIANTI.
“That analysis identified 4139 genes that were significantly associated with interleukin-6 levels (FDR<0.05), independent of age, sex and blood cell components, of which 807 genes replicated in the smaller InCHIANTI cohort.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Lin and others 2014
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductcitationAll microarray experiments and analyses complied with MIAME guidelines.
“All microarray experiments and analyses complied with MIAME guidelines (Brazma and others 2001).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Brazma and others 2001
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May combine two claims.
PremisecitationHigh IL6 levels predict all major age-associated diseases, physical and cognitive disability, and mortality.
“High levels of IL6 predict all major age-associated diseases, physical and cognitive disability and mortality (Economos and others 2013; Ferrucci and others 1999; Heikkila and others 2008; Ridker and others 2000).”
Quote in the paperSubstantial claim0 of 3 sources checked- cites Economos and others 2013
Not checked in the free run.
- cites Ferrucci and others 1999
Not checked in the free run.
- cites Heikkila and others 2008
Not checked in the free run.
Classified as probably not a substantial claim.
Study design and conductown dataEach mediation model was adjusted for sex, technical covariates, and leukocyte subtype proportions.
“Each model was adjusted for the following independent variables; sex, technical covariates (including microarray batches and study site – these are specific to each cohort) and, leukocyte sub-type proportions.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataThe findings are robust across two cohorts and different expression technologies.
“Findings are robust across two cohorts and different expression technologies.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThe study subjects were of European origin.
“The study subjects were of European origin, so further work will be needed to establish whether the results are applicable to other ethnic groups.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataThe results include known immune markers and several novel genes not previously linked to aging.
“Our results include known immune markers and several novel genes not previously linked to aging.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenChanges in cell subtype proportions were not adjusted for in the analyses.
“including changes in cell subtype proportions not already adjusted for in our analyses.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataThe mediator results differ from the many associations with general IL6 levels independent of age (807 replicated genes) and represent a mediator signature specific to age-related inflammation.
“These results are very different from the many associations with general IL6 levels independent of age (807 replicated genes) (Lin and others 2014) and represent a mediator signature specific to age-related inflammation.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Lin and others 2014
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationSLC4A10 expression is highest in the brain and has been linked to autism, epilepsy, and mental retardation.
“SLC4A10 expression is highest in the brain, and SLC4A10 has been linked to autism, epilepsy and mental retardation (Parker and Boron 2013).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Parker and Boron 2013
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
InterpretationcitationSLC4A10 mechanisms may be widespread.
“suggesting that SLC4A10 mechanisms may be widespread.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenThe RNA samples were from whole blood containing a mixture of white cells, with results reflecting overall expression patterns.
“The RNA samples were from whole blood containing a mixture of white cells with results reflecting overall expression patterns.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationZEB2 is mainly expressed in monocytes and is involved in epithelial-mesenchymal transition, which is characterized by downregulation of cellular adhesion complexes and is a key step in cancer development.
“The third larger effect gene, ZEB2, is mainly expressed in monocytes and is involved in epithelial-mesenchymal transition (ETM), which is characterized by the downregulation of cellular adhesion complexes and is a key step in cancer development (Gheldof and others 2012).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Gheldof and others 2012
Not checked in the free run.
Classified as probably not a substantial claim. May combine two claims.
Study design and conductown dataThe study includes two separate expression arrays plus three PCR validations, with different gene coverage and sensitivities.
“Our study includes two separate expression arrays (plus three PCR validations), with the arrays having different gene coverage and sensitivities, imposing some limitations.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataThe small replication sample size makes comparisons of effect sizes difficult.
“although this small sample size of the replication makes comparisons of effect sizes difficult.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim. May combine two claims.
PremisecitationThe Affymetrix Human Exon 1.0 ST array uses 25-mer probes grouped in sets of four per exon to estimate exon-specific expression, while the Illumina Human HT12v3 array uses 50-mer probes biased toward the 3′ end to estimate whole-gene expression.
“The Affymetrix Human Exon 1.0 ST array (used by FHS) uses 25-mer probes, typically grouped in sets of four per exon, to estimate exon-specific levels of expression, while the (InCHIANTI) Illumina Human HT12v3 array uses 50-mer probes biased towards the 3′ end of mRNA transcripts to estimate whole-gene levels of expression (Ramasamy and others 2013).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Ramasamy and others 2013
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationInflammation is associated with a local drop in pH from infiltration of inflammatory cells, and extracellular acidosis in inflammation may be a danger signal activating immune responses.
“Inflammation is associated with a local drop in pH resulting from infiltration of inflammatory cells, and extracellular acidosis in inflammation may be a ‘danger signal’ activating immune responses (Rajamaki and others 2013).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Rajamaki and others 2013
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationA higher anion gap and lower bicarbonate level were associated with higher leukocyte count and higher C-reactive protein level in the NHANES study.
“Farwell and Taylor (Farwell and Taylor 2010) showed that a higher anion gap and lower bicarbonate level were associated with a higher leukocyte count and higher C-reactive protein level, in the NHANES study (Farwell and Taylor 2010).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Farwell and Taylor 2010
Not checked in the free run.
May combine two claims.
PremisecitationAlterations to T-cell populations and other immune cell types with aging lead to reduced antibody response and increased incidence of infectious disease, contributing to age-related inflammation.
“Alterations to T-cell populations (and other immune cell types) with aging leads to reduced antibody response and increased incidence of infectious disease, contributing to age-related inflammation (Del Giudice and others 2014).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Del Giudice and others 2014
Not checked in the free run.
Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).
InterpretationreasoningSome genes that appeared not to replicate may be found in future studies.
“and thus some of the genes that appeared not to replicate may be found in future studies.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Interpretationown dataThe loss of significance for SLC4A10 after adjustment may be due to statistical power in the small sample (n=196).
“although this may be due to statistical power in the small sample size (n=196).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationCDKN2A expression increases in vivo with circulating lymphocyte cell senescence.
“CDKN2A (p16INK4a) expression is increased in-vivo with circulating lymphocyte cell senescence (Liu and others 2011).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Liu and others 2011
Not checked in the free run.
May combine two claims.
Interpretationown dataThe negative mediation value for IL1B reflects its expression increasing with IL6 but decreasing with age.
“The negative mediation value reflects IL1B expression increasing with IL6, but decreasing with age (as shown in Figure 2).”
Quote in the paperNot a substantial claim
The paper relies on this without support.
The mechanisms that cause and sustain high levels of inflammatory markers in aging are largely unknown.
“The mechanisms that cause and sustain high levels of inflammatory markers in aging are largely unknown.”
The paper relies on this without support.
Data from animal models on age-related inflammation are inconsistent and human data are scarce.
“Data collected in animal models have provided inconsistent results and there is a scarcity of data in humans.”
The paper relies on this without support.
IL6 is a proxy for age-related inflammation.
“IL6, a proxy for age-related inflammation”
The paper relies on this without support.
The study is the first larger-scale transcriptome-wide analysis of age-related inflammation in human blood.
“Our study presents the first larger-scale transcriptome-wide analysis of age-related inflammation in human blood.”
“To conclude, we have performed the first larger-scale transcriptome-wide analysis of age-related inflammation in two human population cohorts.”
The paper relies on this without support.
SLC4A10 codes for NCBE (NBCn2) and is a sodium bicarbonate transporter involved in intracellular acid-base homeostasis.
“Our largest effect mediator SLC4A10 (19.1% in FHS, 35.6% in InCHIANTI) codes for NCBE (also called NBCn2) and is a sodium bicarbonate transporter, part of a gene family involved in intracellular acid-base homeostasis.”
The paper relies on this without support.
FLT3 plays a key role in lymphocyte (B and T cell) development but not in development of other blood cells.
“FTL3 (also called CD135) emerged as the second largest effect gene in FHS (replicated in InCHIANTI), and plays a key role in lymphocyte (B and T cell) development but not for the development of other blood cells.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
A few gene transcripts mediate the age-IL-6 association, with SLC4A10 mRNA explaining as much as 19% of this association.
“A few gene transcripts mediate the age–IL-6 association, among which the largest affected transcript, SLC4A10 mRNA (encoding the sodium-driven chloride bicarbonate exchanger), explains as much as 19% of this association40.”
From Inflammageing: chronic inflammation in ageing, cardiovascular disease, and frailty (2018) · cites this paper as [40]
The same study did not detect an age-related increase in IL6 mRNA transcript levels.
“Interestingly, this study did not detect an age-related increase in IL6 mRNA transcript levels, suggesting that the overproduction of circulating proteins occurs in peripheral tissues rather than in blood cells.”
From Inflammageing: chronic inflammation in ageing, cardiovascular disease, and frailty (2018) · cites this paper as [40]
Levels of inflammatory cytokines such as IL-6, TNF-α, and IL-1β increase along with immune system dysregulation.
“Levels of inflammatory cytokines, such as IL‐6, TNF‐α, and IL‐1β, increase along with immune system dysregulation (ie, loss of vaccine efficiency, increased morbidity in infection/injury, increased incidence of cancer and autoimmune diseases). 28 , 94”
From Senescence, brain inflammation, and oligomeric tau drive cognitive decline in Alzheimer's disease: Evidence from clinical and preclinical studies (2024) · cites this paper as [94]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Two counts given for genes associated with IL6
MinorThe introduction reports 4,139 genes associated with IL6 in the prior analysis, while the results give 4,140 for that analysis.
“That analysis identified 4139 genes that were significantly associated with interleukin-6 levels (FDR<0.05), independent of age, sex and blood cell components”
“4140 (23.9%) were associated with IL6 (Lin and others 2014).”
PCR plate size changes within the same procedure
MinorThe procedure first describes 383-well PCR plates, then says expression was measured using 384-well plates.
“Firstly, PCR 383-well plates were used to assess the expression levels of SLC4A10, DAAM2 and FLT3”
“Expression levels for each target transcript were then measured in triplicate 5ul reactions using 384-well plates on the ABI Prism 7900HT platform”
The citation lists haven’t been fetched yet
References 42 · Cited by 24
Research works printed but not linked: 2
- Bonferroni, 1936, 8, 3
- Kannel, 1979, An investigation of coronary heart disease in families. The Framingham offspring study, Am. J. Epidemiol., 110, 281, 10.1093/oxfordjournals.aje.a112813 · DOI
- Authors
- Pilling, L. C., Joehanes, R., Melzer, D., Harries, L. W., Henley, W., Dupuis, J., Lin, H., Mitchell, M., Hernandez, D., Ying, S.-X., Lunetta, K. L., Benjamin, E. J., Singleton, A., Levy, D., Munson, P., Murabito, J. M., & Ferrucci, L.
- Affiliations
- University of Exeter (GB)
- Boston University (US)
- National Institutes of Health (US)
- National Institute on Aging (US)
- Corresponding author
- Ferrucci, Luigi
- ORCID
- Venue
- Experimental Gerontology
- Publisher
- Elsevier BV
- Published
- 2015-10
- Volume
- 70
- Pages
- 37-45
- ISSN
- 0531-5565 (ISSN-L)
- PMC
- PMC4600657
- OpenAlex
- W633498326
- Cited by
- 24
- References
- 42
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 1 · 2023: 2 · 2022: 4 · 2021: 1 · 2019: 4 · 2018: 3 · 2017: 5 · 2016: 2
- FWCI
- 1.34
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Experimental Gerontology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Wellcome Trust (WT097835MF)
- National Institutes of Health (n01hc25195-41-0-1)
- National Institute on Aging (N01AG-821336, R01AG029451, R01AG028321)
- National Heart, Lung, and Blood Institute (N01-HC-025195, R01HL076784)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
- Health Sciences › Medicine › Oncology › Cytokine Signaling Pathways and Interactions
- Health Sciences › Medicine › Epidemiology › Adipokines, Inflammation, and Metabolic Diseases
- Keywords
- interleukin-6, age-related inflammation, gene expression, mediation analysis, transcriptome-wide association study, SLC4A10, PRF1, perforin, IL1B, IL-1β, blood transcriptome, older adults, Framingham Heart Study
- MeSH terms
- Adult, Aged, Aged, 80 and over, Aging, Female, Genetic Markers, Humans, Inflammation, Male, Middle Aged, Cohort Studies, Biomarkers, Interleukin-6, Inflammation Mediators, Gene Expression Profiling
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