Journal: bioRxiv

Article Title: p16 High immune cell - controlled disease tolerance as a broad defense and healthspan extending strategy

doi: 10.1101/2024.07.15.603540

Figure Lengend Snippet: A. Analysis of the fraction of p16 High cells in different populations of the immune cells. Single cell suspensions were prepared from peritoneal cavity, spleen, liver, stromal-vascular fraction of abdominal fat (SVF), bone marrow, and peripheral blood from 2-, 12- and 18-month-old p16-Cre/R26-mTmG mice. Cell suspensions were stained with fluorescent-conjugated antibody against the corresponding markers of immune cells (CD3 for T lymphocytes, B220 for B lymphocyte, Ly6C for monocytes, Ly6G for neutrophils, CD11c for dendritic cells and F4/80 for tissue-resident macrophages) and analyzed by flow cytometry. Independent repeats n = 3 . A representative experiment is shown. Data are mean ± S.D. Statistical significance was analyzed using ANOVA plus Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001. B. Abundance and distribution of p16 High cells from the previously analyzed tissues in 18-month-old mice. Each pie chart represents the total number of p16 High cells and how they are distributed in the different cellular compartments. C. Percentage of p16 High cells in the populations expressing PD-1, PD-L1 and PD-L2. Peritoneal, liver, bone marrow and abdominal fat SVF single cell suspensions from 16-month-old mice were stained using fluorescent-conjugated antibody against PD-1 and PD-L1 and analyzed by flow cytometry. Bar graph represents the percentage of p16 High in populations expressing PD-1 and PD-L1. Pay charts show the distribution of the total p16 High cells expressing PD-1, PD-L1 or PD-L2. Independent repeats n = 3 . Data are mean. D. p16 High T lymphocytes express PD-1, PD-L1, PD-L2 and Foxp3. The percentage of p16 High cells was determined in T lymphocytes obtained from peritoneal cavity, liver, bone marrow and abdominal fat SVF defined as CD3 + CD4 + PD1 + , CD3 + CD4 + PD-L1 + , and CD3 + CD4 + Foxp3 + in 16-month-old mice. Data are mean ± S.D. E. Senescence-associated β-galactosidase activity was analyzed in 2-month-old and 12-month-old p16 High and p16 low F4/80-positive macrophages. Pictures were made with bright field – fluorescence microscope. The percentage of positive cells was calculated. Data are mean ± SD. Significant differences were determined by t-test. Independent repeats n = 6 . *p<0,05; **p<0,01 and ***p<0,001. F. p16 High peritoneal macrophages are not proliferative. Peritoneal macrophages from 12-month-old p16-Cre/R26-mTmG mice were incubated with 5-ethynyl-2’-deoxyuridine (EdU) for 24 h. The percentage of EdU positive cells was analyzed in p16 High and p16 Low populations. Data are mean ± SD. Significance was analyzed by t-test. **p<0,01. G. Immunostaining of different markers of senescence. Peritoneal macrophages from 12-16 month-old p16-Cre/R26-mTmG mice were stained for senescence markers ATM, 53bp1, and ϒ-H2AX. Pictures were obtained by high-definition or confocal microscopy. Cells were counted and the percentages of positive cells for p16 High and p16 low populations were calculated. Data are mean ± SD. Significant differences were determined by t-test. *p<0,05; **p<0,01 and ***p<0,001. H. Peritoneal macrophages from 12-16 month-old p16-Cre/R26-mTmG mice were stained for p21 and ϒ-H2AX. Pictures were obtained by high-definition or confocal microscopy. Cells were counted and the percentage of p16 High , p21 + or double positive cells were determined (left panel). The percentage of positive cells for ϒ-H2AX + , and ϒ-H2AX + in p21 + (right panel) were determined. Independent repeats n = 6. Data are mean ± SD. Significant differences were determined by t-test. ***p<0,001. I. Peritoneal F4/80 + macrophages from 12-month-old p16-Cre/R26-mTmG mice were isolated using magnetic cell sorting. Then, p16 High and p16 low populations were separated by fluorescence-activated cell sorting (FACS). RNA expression of both populations was analyzed by RNA sequencing. Kegg pathway analysis of downregulated genes from RNA-seq data set reveals a set of clusters of genes related with the negative control of cell cycle. Individually analyzed genes are shown. Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) were analyzed by t-test. n = 3 biologically independent samples. Data are mean ± SD. *p<0,05; **p<0,01 and ***p<0,001. Representative 8 genes are shown. j. Kegg pathway analysis of upregulated genes from RNA-seq data set reveals a set of clusters related with the negative control of immune cells and T-cells. Immunosuppressive and regulatory representative genes were individually analyzed and shown. Data are mean ± SD. Differences were established with t-test. **p<0,01 and ***p<0,001.

Article Snippet: Freshly isolated F4/80 + cells from 12 months p16-Cre/R26-mTmG mouse peritoneal cavity (Anti-F4/80 MicroBeads UltraPure, Miltenyi Order no. 130-110-443) were seeded in a 8 μm pore permeable insert (24 well plate; 3,2 mm diameter Transwells Corning) previously coated with matrigel in a migratory solution (DMEM 0,1% BSA).

Techniques: Staining, Flow Cytometry, Expressing, Activity Assay, Fluorescence, Microscopy, Incubation, Immunostaining, Confocal Microscopy, Isolation, FACS, RNA Expression, RNA Sequencing, Negative Control, Sequencing

Journal: bioRxiv

Article Title: p16 High immune cell - controlled disease tolerance as a broad defense and healthspan extending strategy

doi: 10.1101/2024.07.15.603540

Figure Lengend Snippet: A-B . Dextran sodium sulphate (DSS) induces p16 High cells in vivo in young mice. 2-3 month-old p16-Cre/R26-mTmG mice were treated with DSS 2.5% dissolved in drinking water for 7 days and the percentage of p16 High cells in different populations of immune system was determined. Single cell suspensions from peritoneal cavity and liver were stained with fluorescent conjugated antibody and analyzed by flow cytometry. Abundance and distribution of p16 High cells in the previously analyzed tissues (panel A) were determined and are shown as pie charts ( B ). n = 4 biologically independent samples. Data are mean ± SD. Statistical significance was determined using ANOVA plus Dunnett post hoc test. **p<0,01 and ***p<0,001. C-D . p16 High T lymphocytes express higher level of Foxp3, PD-1, and PD-L1 after DSS treatment. The percentage of p16 High cells was determined in T lymphocytes obtained from peritoneal cavity and liver defined as CD3 + CD4 + PD1 + , CD3 + CD4 + PD-L1 + , and CD3 + CD4 + Foxp3 + I in 12-month-old mice. Pie charts show abundance and distribution of Foxp3, PD-1, and PD-L1 in the total CD3 + p16 Hgh population ( D ). Data are mean. E-F . Different pattern of gene expression in the absence of p16 High cells. Control and p16-Cre/R26-DTA (DTA) were treated with 2.5% DSS for 7 days and RNA from peritoneal cells ( E ) and liver ( F ) were isolated. RNA was analyzed by quantitative SYBR-green based PCR (qPCR) to determine the level of expression of different genes. Data are mean +/- S.D. Independent repeats n = 3. Difference between groups was analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001. G-H . Specific TLRs induce p16 High state in vivo . 2-3 months p16-Cre/R26-mTmG mice were treated intraperitoneal (I.P) with a panel of agonist to different toll-like receptors (TLRs) (see Materials and Methods section for detailed treatments). 48 h later, peritoneal cells were stained with fluorescent-conjugated antibodies against F4/80 ( G ) and CD3 ( H ). The percentage of p16 High cells was determined by flow cytometry. Data are mean ± SD. n = 3 biologically independent samples. Statistical significance were determined using ANOVA plus Dunnett post hoc test. *p<0,05 and ***p<0,001.

Article Snippet: Freshly isolated F4/80 + cells from 12 months p16-Cre/R26-mTmG mouse peritoneal cavity (Anti-F4/80 MicroBeads UltraPure, Miltenyi Order no. 130-110-443) were seeded in a 8 μm pore permeable insert (24 well plate; 3,2 mm diameter Transwells Corning) previously coated with matrigel in a migratory solution (DMEM 0,1% BSA).

Techniques: In Vivo, Staining, Flow Cytometry, Gene Expression, Control, Isolation, SYBR Green Assay, Expressing

Journal: bioRxiv

Article Title: p16 High immune cell - controlled disease tolerance as a broad defense and healthspan extending strategy

doi: 10.1101/2024.07.15.603540

Figure Lengend Snippet: A-B . BNT162b2 mRNA COVID-19 vaccine induces p16 High cells in vivo . 2-3 month-old p16-Cre/R26-mTmG mice were treated intraperitoneal (I.P) with 5 µg of BNT162b2 mRNA COVID-19 vaccine. After 2 and 15 days the percentage of p16 High cells was determined in different immune subsets in peritoneal cavity ( A ). Abundance and distribution of different p16 High cells in analyzed tissues. Each pie chart represents the total number of p16 High cells and how they are distributed in different cellular compartments ( B ). Data are mean ± SD. n = 4 biologically independent samples. Statistical significance was determined using ANOVA plus Dunnett post hoc test. **p<0,01 and ***p<0,001 C. The percentage of p16 High positive cells in different populations of T cells was determined at the same time than experiment shown in A. Data are mean ± SD. Statistical significance were determined using ANOVA plus Dunnett’s post hoc test ***p<0,001. D. BNT162b2 vaccine induces p16 High cells that are sensitive to senolytic treatment. 2-3 month-old p16-Cre/R26-mTmG mice were treated I.P with 5 µg of BNT162b2 vaccine. 5 days later, mice were either treated orally with a senolytic combination of dasatinib (5 mg/kg) and quercetin (50 mg/kg) or vehicle (Mock). After 24h mice were euthanized and single cell suspensions from peritoneal cavity and liver were stained with conjugated antibodies against F4/80 and CD3. Cells suspensions were analyzed by flow cytometry and the percentage of p16 High cells in each population was determined. n = 3 biologically independent samples. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05 and ***p<0,001. E. Genetic ablation of p16 High cells modifies the expression of inflammatory genes after BNT162b2 treatment. Wild type and p16-Cre/R26-DTA (DTA) were treated with 5 µg of BNT162b2 vaccine. After 2 and 5 days, RNA from peritoneal cells was isolated. RNA was analyzed by qPCR to determine the level of expression of inflammatory genes. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001. F. p16 High cells protect against acute inflammation. Wild type and p16-Cre/R26-DTA (DTA) were either treated with 5 µg of BNT162b2 vaccine or saline (Mock). After 5 days, animals were injected with lipopolysaccharides (LPS) from Escherichia coli O55:B5 (LPS) 40 mg/kg. Animals were euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. *p<0,05 and **p<0,01. n used for each condition is showed in the plot. G. p16 High cells protect against Ionizing Irradiation. Wild type and p16-Cre/R26-DTA (DTA) were treated with either 5 µg of BNT162b2 vaccine or saline (Mock). Animals were exposed to 8 Gy of γ-irradiation. Animals were observed daily and euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. *p<0,05. n used for each condition is showed in the plot. H. BNT162b2 vaccine induces p16 High cells in the lung. 2-3 month-old p16-Cre/R26-mTmG mice were treated I.P with 5 µg of BNT162b2 vaccine. 2 and 15 days later, mice were euthanized and the percentage of p16 High immune cells was determined in the lung. Single cells suspensions from lung were stained with fluorescent conjugated-antibody against different markers of immune cells and analyzed by flow cytometry. Data are mean ± SD. Statistical significance were determined using ANOVA plus Dunnett post hoc test. **p<0,01 and ***p<0,001. n = 3 biologically independent samples. I. BNT162b2 vaccine increases p16 High cells and induces Foxp3 + , PD-1 + and PD-L1 + in CD3 + CD4 + populations. 2-3 month-old p16-Cre/R26-mTmG mice were treated I.P with 5 µg of BNT162b2 vaccine and after 5 days, single cell suspensions were stained with fluorescent-conjugated antibodies and the levels of CD3 + CD4 + PD1 + , CD3 + CD4 + PD-L1 + , and CD3 + CD4 + Foxp3 + populations and the percentage of p16 High cells were analyzed by flow cytometry. Data are mean ± SD. Statistical significance were determined using ANOVA plus Dunnett post hoc test. ***p<0,001. J. 2-3 months Balb/c mice were either treated with BNT162b2 vaccine or mock-treated and 3 days later were infected with a mouse-adopted MA10 virus. Analysis of probability of survival in experimental group as assessed, dead and mice that lost 70% and more body weight were considered as deceased. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. *p<0,05. n used for each condition is showed in the plot.

Article Snippet: Freshly isolated F4/80 + cells from 12 months p16-Cre/R26-mTmG mouse peritoneal cavity (Anti-F4/80 MicroBeads UltraPure, Miltenyi Order no. 130-110-443) were seeded in a 8 μm pore permeable insert (24 well plate; 3,2 mm diameter Transwells Corning) previously coated with matrigel in a migratory solution (DMEM 0,1% BSA).

Techniques: In Vivo, Staining, Flow Cytometry, Expressing, Isolation, Saline, Injection, Irradiation, Infection, Virus

Journal: bioRxiv

Article Title: p16 High immune cell - controlled disease tolerance as a broad defense and healthspan extending strategy

doi: 10.1101/2024.07.15.603540

Figure Lengend Snippet: A. p16 expression is dependent of TLR7 and STING. 2-3 months wilt type mice were treated with the BNT162b2 vaccine. One day before, in the same day, and day after treatment with BNT162b2, some animals were treated I.P with TLR7 inhibitor (M5049, 1 mg/kg) or STING inhibitor (H151, 10 mg/kg). p16 mRNA expression was determined after treatment in peritoneal cells and liver (left panel) by qPCR. 2-3 months MDA5 +/+ (wild type) and MDA5 -/- (MDA5-KO) mice were treated with the BNT162b2 vaccine. After 5 days of treatment p16 mRNA expression was determined after treatment in liver (right panel) by qPCR. Data are mean +/- S.D. Difference between groups were analyzed using ANOVA test and Tukey post hoc test. ***p<0,001. n = 3 biologically independent samples. B. 2-3 months p16-Cre/R26-mTmG mice were treated with the BNT162b2 vaccine. One day before, in the same day, and day after treatment with BNT162b2, some animals were treated I.P with TLR7 inhibitor (M5049, 1 mg/kg) or STING inhibitor (H151, 10 mg/kg). 5 days after BNT162b2 treatment, animals were euthanized and single cells suspensions were analyzed by flow cytometry to determine the percentage of p16 High cells on different immune subsets (left panel) and inside of Foxp3, PD-1 and PD-L1 (CD3 + CD4 + ) populations (right panel). n = 3 biologically independent samples. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001. C - D. 4-6 months p16-Cre/R26-mTmG mice were treated with the BNT162b2 vaccine. 5 days. 5 days after BNT162b2 treatment, animals were euthanized and single cells suspensions from peritoneal cavity ( C ) and liver ( D ) were analyzed by flow cytometry to determine the percentage of activated STING (p-STING) (phosphorylated at Ser366) in CD45 + p16 High , F4/80 + p16 High , and CD3 + p16 High populations (left panel). The percentage covered of F4/80 + p16 High , and CD3 + p16 High in the total CD45 + p16 High p-STING + is shown in the upper pie chart. The p-STING + p16 High F4/80 + and CD3 + abundance and distribution were determined in the total CD45+ p-STING + and is show in the lower pie chart. n = 3 biologically independent samples. Data are mean +/- S.D. E. STING activation induces p16 High subsets. 2-3 months p16-Cre/R26-mTmG mice were treated I.P with the STING agonist DMXAA at low or high dose and 5 days after treatment, animals were euthanized and cells suspensions from peritoneal cavity were analyzed by flow cytometry to determine the percentage of p16 High cells on different immune subsets (left panel). Differences between groups were analyzed using ANOVA test plus Tukey post hoc test. For the low dose condition, percentage of p16 High cells in Foxp3, PD-1 and PD-L1 (CD3 + CD4 + ) populations (right panel) was determined. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test plus Dunnett post hoc test. *p<0,05; **p<0,01 and ***p<0,001. n = 3 biologically independent samples. F. 2-3 months wild type mice were treated I.P with the STING agonist DMXAA (10 mg/kg) one time (low dose) or 2 consecutive days (high dose). 5 days after first treatment, animals were euthanized and RNA from peritoneal cells (P.C), and liver was isolated. RNA was analyzed by qPCR to determine the level of expression of inflammatory genes. Data are mean +/- S.D. Independent repeats n = 3. Differences between groups were analyzed using ANOVA test plus Dunnett post hoc test. *p<0,05; and ***p<0,001. G. Tonic STING activation promotes disease tolerance and tissue protection against severe inflammation. Wild type mice were either treated with saline (Mock); BNT162b2 vaccine (5 µg by mouse); BNT162b2 vaccine plus H151 (10 mg/kg, 3 consecutive days starting one day before BNT162b2 treatment); and DMXAA (10 mg/kg) for one (low dose) or two consecutive days subcutaneously (high dose). After 5 days, animals were injected with LPS O55:B5 30 mg/kg. Animals were euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. **p<0,01 and ***p<0,001. n used for each condition is showed in the plot.

Article Snippet: Freshly isolated F4/80 + cells from 12 months p16-Cre/R26-mTmG mouse peritoneal cavity (Anti-F4/80 MicroBeads UltraPure, Miltenyi Order no. 130-110-443) were seeded in a 8 μm pore permeable insert (24 well plate; 3,2 mm diameter Transwells Corning) previously coated with matrigel in a migratory solution (DMEM 0,1% BSA).

Techniques: Expressing, Flow Cytometry, Activation Assay, Isolation, Saline, Injection

Journal: bioRxiv

Article Title: p16 High immune cell - controlled disease tolerance as a broad defense and healthspan extending strategy

doi: 10.1101/2024.07.15.603540

Figure Lengend Snippet: A. The Nnmt gene is overexpressed in p16 High cells. RNA-seq data reveals overexpression of Nnmt gene in p16 High cells. Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) from p16 High and p16 low populations were analyzed by t-test. n = 3 biologically independent samples. Data are mean ± SD. ***p<0,001. B. The Nnmt gene is induced after DSS treatment. 2-3 month-old control and p16-Cre/R26-DTA (DTA) were treated with 2.5% DSS for 7 days and RNA from peritoneal cells and liver was isolated on day 8. RNA was analyzed by a quantitative SYBR-green based PCR (qPCR) to determine the level of expression Nnmt mRNA. Independent repeats n = 3. Data are mean +/- S.D. Difference between groups was analyzed using ANOVA test and Tukey post hoc test. ***p<0,001. C. BNT162b2 vaccine-induced Nnmt expression depends of p16 High cells. 2-3 month-old control and p16-Cre/R26-DTA (DTA) were treated with 5 µg of BNT162b2 vaccine. After 2 and 5 days, RNA from peritoneal cells, liver and lungs was isolated and analyzed by qPCR to determine the level of expression of Nnmt mRNA. Independent repeats n = 3. Data are mean +/- SD. Difference between groups was analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001. D. Nnmt expression is dependent of TLR7 and STING. 2-3 months wilt type mice were treated with the BNT162b2 vaccine. One day before, in the same day, and day after treatment with BNT162b2 some animals were treated I.P with TLR7 inhibitor (M5049, 1 mg/kg) or STING inhibitor (H151, 10 mg/kg). Nnmt mRNA expression was determined after treatment in peritoneal cells (P.C) and liver (left panel) by qPCR. Independent repeats n = 3. Data are mean +/- S.D. Difference between groups were analyzed using ANOVA test and Tukey post hoc test. ***p<0,001 E. Nnmt is required to maintain p16 High immune cells. 2-3 month-old p16-Cre/R26-mTmG and p16-Cre/Nnmt-cKO (Nnmt -/- conditional to the expression of p16) mice were treated intraperitoneal with 5 µg of BNT162b2 vaccine. After 5 days single cell suspensions from peritoneal cavity were stained with conjugated antibodies against different immune cell types. Cells suspensions were analyzed by flow cytometry and the percentage of p16 High cells in each population was determined. Independent repeats n = 3. Data are mean ± SD. Statistical significance were determined using ANOVA plus Tuckey post hoc test. ***p<0,001. F. Distribution and abundance of total p16 High cells from peritoneal cavity in p16-Cre/R26-mTmG and p16/Nnmt-cKO after BNT162b2 vaccine treatment are shown. G. Percentage of p16 High positive cells in different T cell populations was determined during experiment show in E . Data are mean ± SD. Statistical significance were determined using ANOVA plus Tukey post hoc test. ***p<0,001. H. Mice with selective inactivation of the Nnmt gene in p16 High cells cannot mount full immune response. 2-3 month-old control and p16/Nnmt-cKO mice were treated with 5 µg of BNT162b2 vaccine. 5 days after treatment, RNA from peritoneal cells was isolated and analyzed by quantitative PCR to determine the level of expression of Nnmt, p16 and inflammatory genes. Data are mean +/- S.D. Independent repeats n = 3. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001. I. Nnmt is necessary to protect against acute inflammation. 2-3 months wild type and p16/Nnmt-cKO mice were treated with 5 µg of BNT162b2 vaccine. After 5 days, animals were injected I.P with LPS O55:B5 40 mg/kg. Animals were euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. *p<0,05. n used for each condition is showed in the plot. J. Activation of p16 High program lower adenosine level. 2-3 months wild type mice were treated I.P with saline (Mock), BNT162b2 vaccine, one (low) or two doses (high) of DMXAA (10 mg/kg), and BNT162b2 (µg by mouse) plus orally supplemented L-methionine daily during 5 days (200 mg/kg). Additionally, 2-3 months p16/Nnmt-cKO mice (written as Nnmt-KO in graph label) were treated with saline or BNT162b2 vaccine. After treatment, peritoneal cells were collected and lysed, levels of adenosine were determined in the supernatant. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001. K. 3-4 months CDKN2A -/- (CDKN2A-KO) and CDKN2A +/+ (wild type) mice were either treated with saline (Mock) or BNT162b2 vaccine (5 µg by mouse). Single cell suspensions from peritoneal cavity were analyzed by flow cytometry to determine the percentage of cleaved-caspase-3 in CD45, F4/80 and CD3 populations. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001. L. High adenosine levels reduce BNT162b2 effect on survival. 2-3 months wild type mice were treated with 5 µg of BNT162b2 vaccine, or BNT162b2 plus daily supplementation with L-methionine (200 mg/kg). After 5 days, animals were injected I.P with LPS O55:B5 30 mg/kg. Animals were euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. *p<0,05. n used for each condition is showed in the plot. M. Adenosine level is increase during aging. 3-4 months and 20 months-old wild type mice were treated with saline or BNT162b2 vaccine (5 µg by mouse). 5 days after animals were euthanized and levels of adenosine were determined in CD45 + liver cells. Independent repeats n = 3. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05 and **p<0,01. N. Blocking adenosine receptor A 2A increase survival during aging. 2 year-old wild type mice were treated with 5 µg of BNT162b2 vaccine, or BNT162b2 plus adenosine receptor A 2A inhibitor (SCH58261, twice by day, 3 mg/kg) starting one day before LPS treatment. 5 days after BNT162b2 treatment, animals were injected I.P with LPS O55:B5 20 mg/kg. Animals were euthanized immediately once they reached the limit point of physical deterioration. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. **p<0,01. n used for each condition is showed in the plot.

Article Snippet: Freshly isolated F4/80 + cells from 12 months p16-Cre/R26-mTmG mouse peritoneal cavity (Anti-F4/80 MicroBeads UltraPure, Miltenyi Order no. 130-110-443) were seeded in a 8 μm pore permeable insert (24 well plate; 3,2 mm diameter Transwells Corning) previously coated with matrigel in a migratory solution (DMEM 0,1% BSA).

Techniques: RNA Sequencing, Over Expression, Sequencing, Control, Isolation, SYBR Green Assay, Expressing, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Injection, Activation Assay, Saline, Blocking Assay

Journal: bioRxiv

Article Title: p16 High immune cell - controlled disease tolerance as a broad defense and healthspan extending strategy

doi: 10.1101/2024.07.15.603540

Figure Lengend Snippet: A. Activated STING is increased in young MDA5-KO mice. 3-4 months Ifih1 +/+ (wild type) and Ifih1 -/- (MDA5-KO) littermates were treated with saline (Mock) or STING inhibitor (H151, 10 mg/kg). 48 h after, animals were euthanized and single cells suspensions from liver were analyzed by flow cytometry. Percentage of p-STING-Ser366 (and p-positive cells was determined in CD45, F4/80 and CD3 populations. n used for each condition is showed in the plot.Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001. B. p16 is overexpressed in MDA5-KO mice. 18-20 months MDA5 +/+ (wild type) and MDA5 -/- (MDA5-KO) littermates were treated with saline or H151 (10 mg/kg). 48 h after, animals were euthanized and mRNA from livers was isolated and analyzed by qPCR. Independent repeats n = 3. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. **p<0,01 and ***p<0,001. C. MDA5-KO mice exhibit reduced adenosine levels during aging. 3-4 months MDA5 +/+ (wild type); and 20 months Ifih1 +/+ (wild type) Ifih1 -/- (MDA5-KO) littermates were treated with saline or BNT162b2 vaccine (5 µg by mouse). 5 days after animals were euthanized and levels of adenosine were determined in peritoneal cells. Independent repeats n = 3. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001. D. MDA5-KO mice shown less basal inflammation during aging. Liver RNA from 3-4 months Ifih1 +/+ (wild type) and Ifih1 -/- (MDA5-KO) littermates was isolated and analyzed by qPCR. The level of expression of inflammatory genes was determined. Independent repeats n = 3. Data are mean +/- S.D. Differences between groups were analyzed using t-test. *p<0,05; **p<0,01 and ***p<0,001. E. Liver and muscle samples isolated from 24-month old wild type and MDA5-KO mice, stained for the endothelial marker CD31. Graph at the right represents mean ± S.D of CD31+ area. Area was determined among 3 animals per group. Differences between groups were calculated by unpaired, nonparametric Mann-Whitney test. ***p<0,001. Scale bar – 100 mm. F. Immunofluorescent analysis of fibrosis marker aSMA in liver and muscle samples (same as in E ). Graphs at the right represent mean ± S.D of aSMA area between groups, Area was determined among 3 animals per group. n = 3 biologically independent samples. Differences between groups were determined by unpaired, nonparametric Mann-Whitney test. *p<0,05; and ***p<0,001. Scale bars: 200 mm for liver, 100 mm for muscle. G. Muscle strength in 18 months wild type ( n = 4 ) and MDA5-KO ( n = 5 ); and 24 months old months wild type ( n = 10 ) and MDA5-KO ( n = 15 ) littermates mice was determined with a grip test. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Dunnett post hoc test. **p<0,01 and ***p<0,001. H. MDA5-KO mice show better physical fitness during natural aging. Frailty was assessed in 18 months wild type ( n = 4 ) and MDA5-KO ( n = 5 ); and 24 months old months wild type ( n = 10 ) and MDA5-KO ( n = 15 ) littermates using an clinically relevant index for frailty during aging (see material and methods). Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Dunnett post hoc test. **p<0,01 and ***p<0,001. I. A group of wild type ( n = 177 ) and MDA5-KO ( n = 222 ) animals were housed in the same conditions and followed during 25 months to determine survival during natural aging. Difference between groups was analyzed using Gehan-Breslow-Wilcoxon test. **p<0,01.

Article Snippet: Freshly isolated F4/80 + cells from 12 months p16-Cre/R26-mTmG mouse peritoneal cavity (Anti-F4/80 MicroBeads UltraPure, Miltenyi Order no. 130-110-443) were seeded in a 8 μm pore permeable insert (24 well plate; 3,2 mm diameter Transwells Corning) previously coated with matrigel in a migratory solution (DMEM 0,1% BSA).

Techniques: Saline, Flow Cytometry, Isolation, Expressing, Staining, Marker, MANN-WHITNEY

Journal: bioRxiv

Article Title: p16 High immune cell - controlled disease tolerance as a broad defense and healthspan extending strategy

doi: 10.1101/2024.07.15.603540

Figure Lengend Snippet: A-B. Expression of p16 and NNMT before (Day 0) and after BNT162b2 vaccine treatment (Day 7). RNA were isolated from PBMCs and analyzed by quantitative-PCR. (7 participants were involved in the study). Data were analyzed as a bulk (left panel) and as matched samples (right panel). t-test and paired t-test (right panel) were used to determine the differences between conditions. *p<0,05 and **p<0,01. C. Levels of p16 + cells in different immune populations before and after BNT162b2 treatment are shown. 7 volunteers (included in the CovImmune 2 cohort) with different range of age were involved in the study. Volunteers were treated intramuscularly with 30 µg of the BNT162b2 vaccine. Blood samples were collected before vaccination and after 7 and 30 days of treatment. Blood samples were analyzed by flow cytometry. Granulocytes, monocytes and lymphocytes populations were localized through size and granularity. CD45 + , Tregs (CD3 + CD4 + Foxp3 + ), PD1 + , and p16 + populations were localized with fluorescent conjugated antibodies. Data were analyzed as a bulk. Differences were determined using ANOVA test and Dunnetts test. *p<0,05 and ***p<0,001. D. Flow cytometry data were also treated as matched samples. The level of p16 + cells inside singles populations are shown in each plot. Paired t-test was used to determine the differences before (Day 0) and after (Day 7 and day 30) the BNT162b2 vaccine treatment for CD45 + , granulocytes, Tregs and PD1 + . For monocytes and lymphocytes, Shapiro-Wilk test was used to determine normality of the data. ANOVA plus Dunnett’s test or Friedman plus Dunńs test were used to determine difference before and after. *p<0,05 and **p<0,01. E. The total number of cells in CD45 + , lymphocytes, granulocytes and monocytes, was used to determine the distribution of p16 + cells among these populations. The percentage of contribution calculated for each population is showed in the pay graph at day 7. F. One cohort (CovImmune 1) of 41 unvaccinated patients and non-COVID volunteers older than 55 years were classified in negative for COVID-19 ( n = 17 ), moderate COVID-19 ( n = 6 ) and severe COVID-19 ( n = 18 ). IL6 concentration in serum was analyzed by ELISA. Data are mean +/- S.D. Differences between groups were analyzed using ANOVA test and Tukey post hoc test. ***p<0,001. G, H. One cohort of 41 unvaccinated patients in a older than 55 years were classified in negative for COVID-19 ( n = 17 ), moderate COVID-19 ( n = 6 ) and severe COVID-19 ( n = 18 ). RNA from PBMCs were isolated and analyzed by quantitative-PCR. Level of p16 ( G ) and NNMT ( H ) expression were determined. Differences among groups were determined using ANOVA test and Tukey post hoc test. *p<0,05; **p<0,01 and ***p<0,001.

Article Snippet: Freshly isolated F4/80 + cells from 12 months p16-Cre/R26-mTmG mouse peritoneal cavity (Anti-F4/80 MicroBeads UltraPure, Miltenyi Order no. 130-110-443) were seeded in a 8 μm pore permeable insert (24 well plate; 3,2 mm diameter Transwells Corning) previously coated with matrigel in a migratory solution (DMEM 0,1% BSA).

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: p16 High immune cell - controlled disease tolerance as a broad defense and healthspan extending strategy

doi: 10.1101/2024.07.15.603540

Figure Lengend Snippet: Both Tlr7 and Sting pathways as long as they induce low adenosine environment (via consumption of SAM and p16-dependent cell cycle arrest and protection from apoptosis (for more details see discussion)) induce beneficial senescence that provide heighten protection from different types of inflammation and tissue damage through a mechanism called disease tolerance. In contrast, excessive inflammation contributes to detrimental senescence and derails disease tolerance.

Article Snippet: Freshly isolated F4/80 + cells from 12 months p16-Cre/R26-mTmG mouse peritoneal cavity (Anti-F4/80 MicroBeads UltraPure, Miltenyi Order no. 130-110-443) were seeded in a 8 μm pore permeable insert (24 well plate; 3,2 mm diameter Transwells Corning) previously coated with matrigel in a migratory solution (DMEM 0,1% BSA).

Techniques: