Figure 4.
Microbially derived compounds are increased in blood of older mice, and older BM responds with increased and sustained IL-1 secretion upon LPS stimulation. (A) Relative levels of TLR and NOD2 ligands in heat-inactivated plasma samples from 2mo (n = 11) and 2y (n = 14) WT SPF mice measured by TLR/NOD HEK-Blue reporter cell lines. Selective ligands (n = 2) used as positive controls are indicated; unstimulated (PBS; n = 13) mice were used as negative controls. Measurements were taken from distinct animals, and distinct ligands were checked in the same plasma samples. (B) Experimental approach to test total BM and BM myeloid cell response to bacterial compounds in vitro. BM-resident iMono, CD11b+Gr1lowLy6Chigh; Gran, CD11b+Gr1high; and WBM nucleated cells were sorted from 2mo and 2y WT SPF and 2mo and 1y WT GF mice, seeded into 48-well plates (5 × 105 cells per well), and stimulated with PBS or LPS (100 ng/mL). After 10 and 24 hours, supernatants were harvested and IL-1a and IL-1b concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Measurements were taken from distinct samples. (C) IL-1a concentration in supernatants after PBS or LPS stimulation of indicated cell populations from WT SPF, IL-1R1KO SPF, and WT GF mice. For 2mo mice, iMono: n = 10 WT-PBS, 12 WT-LPS10h, 19 WT-LPS24h, 6 GF-PBS, 3 GF-LPS10h, 3 GF-LPS24h; Gran: n = 22 WT-PBS, 10 WT-LPS10h, 21 WT-LPS24h, 8 GF-PBS, 5 GF-LPS10h, 5 GF-LPS24h; WBM: n = 25 WT-PBS, 11 WT-LPS10h, 15 WT-LPS24h, 8 GF-PBS, 4 GF-LPS10h, 4 GF-LPS24h). For 1y mice, iMono: n = 15 WT-PBS, 9 WT-LPS10h, 9 WT-LPS24h, 9 GF-PBS, 7 GF-LPS10h, 6 GF-LPS24h; Gran: n = 24 WT-PBS, 12 WT-LPS10h, 9 WT-LPS24h, 18 GF-PBS, 9 GF-LPS10h, 9 GF-LPS24h; WBM: n = 14 WT-PBS, 8 WT-LPS10h, 6 WT-LPS24h, 11 GF-PBS, 7 GF-LPS10h, 5 GF-LPS24h. For 2y mice, iMono: n = 12 WT-PBS, 6 WT-LPS10h, 16 WT-LPS24h; Gran: n = 15 WT-PBS, 9 WT-LPS10h, 15 WT-LPS24h; WBM: n = 13 WT-PBS, 6 WT-LPS10h, 13 WT-LPS24h. (D). IL-1b concentration in supernatants after PBS or LPS stimulation of indicated cell populations from WT SPF, IL-1R1KO SPF, and WT GF mice. For 2mo, iMono: n = 14 WT-PBS, 10 WT-LPS10h, 19 WT-LPS24h, 3 GF-PBS, 4 GF-LPS10h, 3 GF-LPS24h; Gran: n = 17 WT-PBS, 11 WT-LPS10h, 16 WT-LPS24h, 8 GF-PBS, 4 GF-LPS10h, 5 GF-LPS24h; tBM: n = 19 WT-PBS, 8 WT-LPS10h, 12 WT-LPS24h, 7 GF-PBS, 4 GF-LPS10h, 3 GF-LPS24h. For 1y mice, iMono: n = 6 WT-PBS, 9 WT-LPS10h, 11 WT-LPS24h, 10 GF-PBS, 9 GF-LPS10h, 6 GF-LPS24h; Gran: n = 24 WT-PBS, 12 WT-LPS10h, 9 WT-LPS24h, 15 GF-PBS, 11 GF-LPS10h, 9 GF-LPS24h; tBM: n = 14 WT-PBS, 8 WT-LPS10h, 6 WT-LPS24h, 9 GF-PBS, 6 GF-LPS10h, 3 GF-LPS24h. For 2y mice, iMono: n = 13 WT-PBS, 6 WT-LPS10h, 17 WT-LPS24h; Gran: n = 14 WT-PBS, 6 WT-LPS10h, 10 WT-LPS24h; tBM: n = 21 WT-PBS, 9 WT-LPS10h, 18 WT-LPS24h. (E) Experimental approach to measure differences in LPS response between young and older mice in vivo. First, 2mo, 1y, and 2y mice were intraperitoneally injected with PBS or LPS (35 μg per mouse). PB and femur BM lysates were harvested 4, 14, or 20 hours later, and IL-1a and IL-1b concentrations were measured by ELISA. (F) IL-1a and IL-1b concentrations in BM lysates of 2mo, 1y, and 2y mice 4, 14, and 20 hours after injection with PBS or LPS. For 2mo mice, IL-1a: PBS0h, n = 11; LPS4h, n = 13; LPS14h, n = 4; LPS20h, n = 5. IL-1b: PBS0h, n = 9; LPS4h, n = 10; LPS14h, n = 5; LPS 20h, n = 5. For 1y mice, IL-1a: PBS0h, n = 7; LPS4h, n = 4; LPS14h, n = 5; LPS20h, n = 5; IL-1b: PBS0h, n = 5; LPS4h, n = 4; LPS14h, n = 5; LPS20h, n = 5. For 2y mice, IL-1a: PBS0h, n = 14; LPS4h, n = 10; LPS14h, n = 4; LPS20h, n = 5; IL-1b: PBS0h, n = 13; LPS4h, n = 11; LPS14h, n = 4; LPS20h, n = 5. Error bars represent SD in all panels. P values were calculated using Student t test. *P < .05; **P < .01; ***P < .001; ****P < .0001. OD, optical density; tBM, total BM.

Microbially derived compounds are increased in blood of older mice, and older BM responds with increased and sustained IL-1 secretion upon LPS stimulation. (A) Relative levels of TLR and NOD2 ligands in heat-inactivated plasma samples from 2mo (n = 11) and 2y (n = 14) WT SPF mice measured by TLR/NOD HEK-Blue reporter cell lines. Selective ligands (n = 2) used as positive controls are indicated; unstimulated (PBS; n = 13) mice were used as negative controls. Measurements were taken from distinct animals, and distinct ligands were checked in the same plasma samples. (B) Experimental approach to test total BM and BM myeloid cell response to bacterial compounds in vitro. BM-resident iMono, CD11b+Gr1lowLy6Chigh; Gran, CD11b+Gr1high; and WBM nucleated cells were sorted from 2mo and 2y WT SPF and 2mo and 1y WT GF mice, seeded into 48-well plates (5 × 105 cells per well), and stimulated with PBS or LPS (100 ng/mL). After 10 and 24 hours, supernatants were harvested and IL-1a and IL-1b concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Measurements were taken from distinct samples. (C) IL-1a concentration in supernatants after PBS or LPS stimulation of indicated cell populations from WT SPF, IL-1R1KO SPF, and WT GF mice. For 2mo mice, iMono: n = 10 WT-PBS, 12 WT-LPS10h, 19 WT-LPS24h, 6 GF-PBS, 3 GF-LPS10h, 3 GF-LPS24h; Gran: n = 22 WT-PBS, 10 WT-LPS10h, 21 WT-LPS24h, 8 GF-PBS, 5 GF-LPS10h, 5 GF-LPS24h; WBM: n = 25 WT-PBS, 11 WT-LPS10h, 15 WT-LPS24h, 8 GF-PBS, 4 GF-LPS10h, 4 GF-LPS24h). For 1y mice, iMono: n = 15 WT-PBS, 9 WT-LPS10h, 9 WT-LPS24h, 9 GF-PBS, 7 GF-LPS10h, 6 GF-LPS24h; Gran: n = 24 WT-PBS, 12 WT-LPS10h, 9 WT-LPS24h, 18 GF-PBS, 9 GF-LPS10h, 9 GF-LPS24h; WBM: n = 14 WT-PBS, 8 WT-LPS10h, 6 WT-LPS24h, 11 GF-PBS, 7 GF-LPS10h, 5 GF-LPS24h. For 2y mice, iMono: n = 12 WT-PBS, 6 WT-LPS10h, 16 WT-LPS24h; Gran: n = 15 WT-PBS, 9 WT-LPS10h, 15 WT-LPS24h; WBM: n = 13 WT-PBS, 6 WT-LPS10h, 13 WT-LPS24h. (D). IL-1b concentration in supernatants after PBS or LPS stimulation of indicated cell populations from WT SPF, IL-1R1KO SPF, and WT GF mice. For 2mo, iMono: n = 14 WT-PBS, 10 WT-LPS10h, 19 WT-LPS24h, 3 GF-PBS, 4 GF-LPS10h, 3 GF-LPS24h; Gran: n = 17 WT-PBS, 11 WT-LPS10h, 16 WT-LPS24h, 8 GF-PBS, 4 GF-LPS10h, 5 GF-LPS24h; tBM: n = 19 WT-PBS, 8 WT-LPS10h, 12 WT-LPS24h, 7 GF-PBS, 4 GF-LPS10h, 3 GF-LPS24h. For 1y mice, iMono: n = 6 WT-PBS, 9 WT-LPS10h, 11 WT-LPS24h, 10 GF-PBS, 9 GF-LPS10h, 6 GF-LPS24h; Gran: n = 24 WT-PBS, 12 WT-LPS10h, 9 WT-LPS24h, 15 GF-PBS, 11 GF-LPS10h, 9 GF-LPS24h; tBM: n = 14 WT-PBS, 8 WT-LPS10h, 6 WT-LPS24h, 9 GF-PBS, 6 GF-LPS10h, 3 GF-LPS24h. For 2y mice, iMono: n = 13 WT-PBS, 6 WT-LPS10h, 17 WT-LPS24h; Gran: n = 14 WT-PBS, 6 WT-LPS10h, 10 WT-LPS24h; tBM: n = 21 WT-PBS, 9 WT-LPS10h, 18 WT-LPS24h. (E) Experimental approach to measure differences in LPS response between young and older mice in vivo. First, 2mo, 1y, and 2y mice were intraperitoneally injected with PBS or LPS (35 μg per mouse). PB and femur BM lysates were harvested 4, 14, or 20 hours later, and IL-1a and IL-1b concentrations were measured by ELISA. (F) IL-1a and IL-1b concentrations in BM lysates of 2mo, 1y, and 2y mice 4, 14, and 20 hours after injection with PBS or LPS. For 2mo mice, IL-1a: PBS0h, n = 11; LPS4h, n = 13; LPS14h, n = 4; LPS20h, n = 5. IL-1b: PBS0h, n = 9; LPS4h, n = 10; LPS14h, n = 5; LPS 20h, n = 5. For 1y mice, IL-1a: PBS0h, n = 7; LPS4h, n = 4; LPS14h, n = 5; LPS20h, n = 5; IL-1b: PBS0h, n = 5; LPS4h, n = 4; LPS14h, n = 5; LPS20h, n = 5. For 2y mice, IL-1a: PBS0h, n = 14; LPS4h, n = 10; LPS14h, n = 4; LPS20h, n = 5; IL-1b: PBS0h, n = 13; LPS4h, n = 11; LPS14h, n = 4; LPS20h, n = 5. Error bars represent SD in all panels. P values were calculated using Student t test. *P < .05; **P < .01; ***P < .001; ****P < .0001. OD, optical density; tBM, total BM.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal