Aging-associated dysregulation of homeostatic immune response termination (and not initiation)

Abstract

Immunosenescence is a state of unbalanced immune responsiveness, characterized by a diverse repertoire of seemingly discreet and paradoxical alterations in all aspects of immunity arising in an aging-associated manner. We asked whether aging-associated alterations in the ability of apoptotic cells to elicit immunomodulatory responses (innate apoptotic immunity; IAI) or in IAI responses themselves might underlie the confounding aging-associated anomalies of immunosenescence. We explored this question by examining, as a function of animal age, responsiveness of murine macrophages on the single cell level. We monitored the expression of pro- and anti-inflammatory cytokines cytofluorimetrically in response to pro-inflammatory Toll-like receptor (TLR) stimulation and anti-inflammatory treatment with apoptotic cells. While we found no alterations with age in the potency of apoptotic cells or in the initiation and magnitude of IAI responses, we did identify a cell-intrinsic deficiency in anti-inflammatory IAI response termination linked with age and preceding manifestations of immunosenescence. Further, we found that an aging-associated deficiency in response termination also is evident following TLR stimulation. These surprising observations reveal that a loss of homeostatic immune control with animal age results from the dysregulation of response termination (as distinct from response initiation) and is exerted on the level of transcription. We suggest that, with advancing age, cells become locked into relatively longer-lived response states. Aging-associated immune dysfunctions may reflect a diminution in the cellular nimbleness of immune responsiveness.

Keywords: aging; apoptosis; homeostasis; immunosenescence; innate immunity; macrophages.

Figure 1

Aging does not alter the magnitude of intrinsic macrophage responsiveness to apoptotic cells – analysis of TNFα expression. (A) The magnitude of apoptotic suppression of TNFα expression in C57BL/6 macrophages isolated from mice of 2–3 months of age (‘young’, ) and mice of 24–25 months of age (‘old’, ) was determined. Elicited peritoneal macrophages from individual mice within each age cohort were cultured without or with apoptotic targets (splenocytes prepared from ‘young’ syngeneic mice, treated with staurosporine) at the indicated target: responder (T: R) ratio for 2 h before the addition of LPS (5 ng mL⁻¹) for another 5 h (Target cells were not removed.) Brefeldin A was included during the final 3 h of incubation. Results () are calculated from the mean fluorescence intensity (MFI) values of gated F4/80⁺ macrophages obtained cytofluorimetrically following intracellular immunostaining with a phycoerythrin (PE)‐conjugated TNFα‐specific antibody (MFI_PE) and are normalized with respect to the maximum stimulated level of intracellular TNFα (MFI_{PE ( LPS ‐Stimulated control)}). In addition, ‘background’ MFI values (MFI_{PE (Unstimulated control)}) are subtracted from experimental values. Calculations were made using the following formula: {MFI_{PE (Sample)} − MFI_{PE (Unstimulated control)}}/{MFI_{PE ( LPS ‐Stimulated control)} − MFI_{PE (Unstimulated control)}}. Alternatively, results () are expressed as the fraction of all F4/80⁺ cells that were TNFα‐positive by intracellular cytokine staining (see next). There were no statistically significant differences in the fractions of unstimulated cells that stained as TNFα positive among cell populations from mice of different ages. The data presented are compiled from the analysis of macrophages from 8 individual mice within each age cohort. Examples of the primary cytofluorimetric data are presented in Panels B–D. There were no statistically significant differences (NS: Ρ > 0.05) between age groups by either analytical method, as calculated by Student's t‐test. (B–D) Representative examples of the cytofluorimetric analysis of apoptotic suppression of LPS‐induced TNFα expression in viable F4/80⁺ elicited peritoneal macrophages from individual ♀ C57BL/6 mice of different ages are presented. (B) Macrophages from a 3‐month‐old (‘young’) mouse: (A) unstimulated, MFI_PE = 22.33; (B) LPS (5 ng mL⁻¹) and apoptotic targets (as above; T: R = 20: 1), MFI_PE = 26.23; (C) LPS and apoptotic targets (T: R = 2: 1), MFI_PE = 66.74; (D) LPS alone, MFI_PE = 980.38. (C) Macrophages from a 15‐month‐old (‘middle‐aged’) mouse: (A) unstimulated, MFI_PE = 19.78; (B) LPS and apoptotic targets (T: R = 20: 1), MFI_PE = 36.26; (C) LPS and apoptotic targets (T: R = 2: 1), MFI_PE = 109.75; (D) LPS alone, MFI_PE = 742.22. (D) Macrophages from a 24‐month‐old (‘old’) mouse: (A) unstimulated, MFI_PE = 28.58; (B) LPS and apoptotic targets (T: R = 20: 1), MFI_PE = 17.97; (C) LPS and apoptotic targets (T: R = 2: 1), MFI_PE = 29.42; (D) LPS alone, MFI_PE = 611.70. (E) TNFα secretion by cultured macrophages from ‘young’ (), ‘middle‐aged’ (), and ‘old’ () C57BL/6 mice after 20 h of incubation with target cells, as indicated, was determined by multiplex immunoassays. Compiled data are normalized within each age cohort. Data are normalized with respect to cultures stimulated with LPS alone. There were no statistically significant differences (NS: Ρ > 0.05) among age groups with respect to modulation of TNFα secretion, as calculated by one‐way ANOVA.

Figure 2

Aging does not alter the magnitude of intrinsic macrophage responsiveness to apoptotic cells – analysis of IL‐10 expression. (A) The magnitude of apoptotic induction of IL‐10 expression in C57BL/6 macrophages isolated from ‘young’ (•) and ‘old’ () mice was determined. Elicited peritoneal macrophages from individual mice within each age cohort were cultured as indicated with apoptotic targets (as in Fig. 1A) at the indicated T: R ratio and/or LPS; Brefeldin A was included during the last 3 h (Target cells were not removed.) Results are calculated from the MFI values obtained cytofluorimetrically following intracellular immunostaining with an IL‐10‐specific antibody coupled to a PE/cyanine 7 [PE/Cy7] conjugated fluor and are normalized with respect to the levels of intracellular IL‐10 in unstimulated cells. The data presented are compiled from the results of the analysis of macrophages from 6 individual mice within each age cohort. There were no statistically significant differences (NS: Ρ > 0.05) between age groups, as calculated by Student's t‐test. (B) IL‐10 secretion by cultured macrophages from ‘young’ (), ‘middle‐aged’ () and ‘old’ () C57BL/6 mice after 20 h of incubation with target cells, as indicated, was determined by multiplex immunoassays. Compiled data are normalized within each age cohort. Data are normalized with respect to unstimulated cultures. The data presented are compiled from the results of the analysis of macrophages from 6 individual mice within each age cohort. There were no statistically significant differences (NS: Ρ > 0.05) among age groups with respect to modulation of IL‐10 secretion, as calculated by one‐way ANOVA.

Figure 3

The potency of apoptotic cells to elicit IAI responses is not altered with age. The immunomodulatory activities of apoptotic targets derived from C57BL/6 mice of distinct ages were determined. Apoptotic suppression of TNFα expression was evaluated in syngeneic macrophages isolated from ‘young’ (; A) and ‘old’ (; B) mice, as in Fig. 1A. Apoptotic induction of IL‐10 expression in syngeneic macrophages isolated from ‘young’ (; C) and ‘old’ (; D) mice was determined as in Fig. 2A. Elicited peritoneal macrophages from individual mice of each age cohort were cultured with apoptotic targets (splenocytes prepared from ‘young’ [] or ‘old’ [] syngeneic mice, and treated with staurosporine) at the indicated T: R ratio. (Target cells were not removed.) The data presented are compiled from the results of the analysis of macrophages from 4 individual mice within each age cohort. The significance of differences between age groups, as calculated by Student's t‐test, is indicated (NS: Ρ > 0.05; *Ρ ≤ 0.05; **Ρ ≤ 0.01; ***Ρ ≤ 0.001).

Figure 4

Macrophage IAI response termination is impaired with age. The extent of apoptotic suppression of TNFα expression in elicited peritoneal macrophages isolated from ‘young’ (•) and ‘old’ () C57BL/6 mice was determined as in Fig. 1A. Macrophages were stimulated with LPS in the absence of apoptotic targets (‘no targets’) and following 20 h of interaction with apoptotic targets at T: R ratios of 20: 1 (A), 6: 1 (B), and 2: 1 (C). Targets remained throughout the subsequent incubation (‘not removed’) or were removed from macrophages by extensive washing before addition of LPS at the indicated times. In all cases, incubation with LPS (5 ng mL⁻¹) was for 5 h. Brefeldin A was included during the last 3 h. The data presented are compiled from the results of the analysis of macrophages from eight individual mice within each age cohort. The significance of differences between age groups, as calculated by Student's t‐test, is indicated (NS: Ρ > 0.05; *Ρ ≤ 0.05; **Ρ ≤ 0.01).

Figure 5

Macrophage TLR response termination is impaired with age. A representative example of the analysis of TNFα transcript levels in elicited peritoneal macrophages from individual ‘young’ (A; ) and ‘old’ (B; ) C57BL/6 mice, following incubation with LPS (5 ng mL⁻¹) alone, beginning at t₀ (), or with LPS, beginning at t₀, and TAK‐242 (2.5 μm), beginning at t _10 min (, TAK‐242 addition is indicated with arrows). TNFα transcript levels were assessed by RT–qPCR analysis and were normalized for the 18S rRNA content of each sample and normalized further to TNFα mRNA levels of uninduced macrophages of the same mouse. Values for LPS response persistence (Table 1) were derived from a compilation of these analyses, as described.