Adiposity induces lethal cytokine storm after systemic administration of stimulatory immunotherapy regimens in aged mice

Abstract

Aging is a contributing factor in cancer occurrence. We recently demonstrated that systemic immunotherapy (IT) administration in aged, but not young, mice resulted in induction of rapid and lethal cytokine storm. We found that aging was accompanied by increases in visceral fat similar to that seen in young obese (ob/ob or diet-induced obese [DIO]) mice. Yet, the effects of aging and obesity on inflammatory responses to immunotherapeutics are not well defined. We determine the effects of adiposity on systemic IT tolerance in aged compared with young obese mice. Both young ob/ob- and DIO-generated proinflammatory cytokine levels and organ pathologies are comparable to those in aged ad libitum mice after IT, culminating in lethality. Young obese mice exhibited greater ratios of M1/M2 macrophages within the peritoneal and visceral adipose tissues and higher percentages of TNF(+) macrophages in response to αCD40/IL-2 as compared with young lean mice. Macrophage depletion or TNF blockade in conjunction with αCD40/IL-2 prevented cytokine storms in young obese mice and protected from lethality. Calorie-restricted aged mice contain less visceral fat and displayed reduced cytokine levels, protection from organ pathology, and protection from lethality upon αCD40/IL-2 administration. Our data demonstrate that adiposity is a critical factor in the age-associated pathological responses to systemic anti-cancer IT.

Figure 1.

Aged AL mice show increased proinflammatory cytokines, rapid lethality to LD IT, and display increased fat volume ratios in comparison to aged CR mice. (A) Ad libitum–fed C57BL/6 mice were treated with LD αCD40/IL-2 or rIgG/PBS and survival was monitored, n = 5–8 mice/group. (B) Serum cytokines TNF, IL-6, and IFN-γ were measured day 2 by CBA after the start of αCD40/IL-2 or rIgG/PBS therapy in aged AL mice, n = 3–5 mice/group. (C) Young AL (3 mo) and aged AL (18 mo) mice were treated with CpG/IL-2 IT and survival was monitored, n = 5 mice/group. (D and E) Serum cytokine levels of TNF (D) and IL-6 (E) were measured by CBA on day 2 after the start of therapy, n = 5 mice/group. (F) Representative image of aged and young C57BL/6 mice showing size and waist circumference. (G) Total body weight (left graph) and visceral fat weight (right graph), was measured for young (2 mo) and aged AL mice (20 mo), n = 5 mice/group. (H) Total body weight (left graph) or visceral fat (right graph) was measured in naive 15-mo-old C57BL/6 mice on an AL or CR diet, n = 3–4 mice/group. (I) Visceral fat in young (4 mo), middle-aged (9 mo), and aged (15 mo) mice on either an AL (left) or CR (right) diet was evaluated by MRI. WS is indicative of water suppression, where fat is bright. FS is indicative of fat suppression, fat is dark/black. Data in all panels are representative of one of at least five experiments with similar results. MRI scans are representative of one of at least three mice imaged per group with similar results. Survival analysis was plotted according to the Kaplan-Meier method, and statistical differences were determined with the log-rank test. Bar graph (mean value ± SEM) statistics were performed using either one-way or two-way ANOVA with Bonferroni post-hoc tests. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Figure 2.

Calorie restriction in the aged confers protection by decreasing cytokine levels and IT-associated organ pathology, thereby allowing increased survival during IT. (A–C) Aged AL and age-matched aged CR mice (18 mo) were treated with HD αCD40/IL-2 or rIgG/PBS; at day 2 of IT, mice were sacrificed for histological analysis of livers (A) and intestines (C). Serum was collected on day 2 and assessed for ALT (B) levels, n = 3 mice/group. Representative H&E images of liver and intestines (gut) for aged AL and aged CR groups. Bars, 200 µm. Asterisks represent steatosis and arrows denote areas of severe immune infiltration and/or necrosis. (D–F) Serum was analyzed day 2 after treatment for TNF (D), IL-6 (E), and IFN-γ (F). (G) Aged (18 mo) C57BL/6 mice on AL or CR diet were treated with either LD or HD of αCD40/IL-2 and survival was monitored. Control groups received rIgG/PBS. All data panels are representative of one of four independent experiments with similar results for all panels. Survival analysis was plotted according to the Kaplan-Meier method and statistical differences were determined by using the log-rank test. Bar graph (mean value ± SEM) statistics were performed using one-way ANOVA with Bonferroni post-hoc test. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01.

Figure 3.

Increased adiposity independently of age results in systemic release of proinflammatory cytokines and organ pathology after IT in young ob/ob (obese) and aged AL mice. (A) Body weights of naive young (2 mo) WT and young ob/ob C57BL/6 mice, n = 6 mice/group. Representative pictures of young-AL WT mice and obese ob/ob naive mice are shown. (B) Total fat content in naive young WT (2 mo), young ob/ob (2 mo), and aged (18 mo) C57BL/6 mice was visualized by MRI. Fat is bright. (C and D) Young AL (2 mo), young ob/ob (2 mo), and aged AL (18 mo) mice were treated with HD αCD40/IL-2 and liver (C) and gut (D) were stained with H&E and scored for histopathology on day 2. Bars, 200 µm. Arrows indicate areas of steatosis and/or immune cell infiltration and asterisks indicate patchy necrosis and/or fibrosis. (E–G) Young AL (2 mo), young ob/ob (2 mo), aged AL (18 mo), and aged CR (18 mo) were treated with LD αCD40/IL-2 and serum was collected day 2 of treatment and analyzed by CBA for levels of: TNF (E), IL-6 (F), and IFN-γ (G), n = 3 mice/group. (H and I) Young AL (2 mo), young ob/ob (2 mo), aged AL (18 mo), and aged CR (18 mo) were treated with LD αCD40/IL-2 (H) or CpG/IL-2 (I) or rIgG/PBS control and survival was monitored. (J) Serum levels of TNF were measured on day 2 of CpG/IL-2 therapy by CBA. Data in all panels are representative of one of at least three experiments with similar results. MRI scans are representative of at least three mice imaged per group with similar results. Survival data were plotted using the Kaplan-Meier method and statistical differences were determined with the log-rank test. Bar graph (mean value ± SEM) statistics performed using Student’s t test or one-way ANOVA with Bonferroni post-hoc tests. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Figure 4.

IT toxicity is TNF-dependent through M1 macrophage accumulation. (A–D) Young (2 mo) WT and young (2 mo) ob/ob mice were treated with either control rIgG/PBS or LD αCD40/IL-2. Visceral adipose tissue and peritoneal lavages were collected on day 2 and assessed for the presence of macrophages identified as CD45⁺CD19⁻F4/80⁺CD11b⁺. Ratio of M1/M2 macrophages (A), and total numbers (B) and percentages (C) of TNF-producing macrophages. Macrophages identified as M1 macrophages were characterized as CD206⁻; M2 macrophages were gated as CD206⁺. (E–H) Young AL (2 mo) and young ob/ob (2 mo) were treated with either control or macrophage depleting clodronate liposomes before and during LD αCD40/IL-2 and survival was monitored; n = 5 mice/group. (F and G) On day 2, visceral adipose tissues and peritoneal lavages were collected and assessed for the percentages TNF⁺ macrophages (F and G) as characterized in experiments in A–D and serum TNF levels (H), n = 4 mice/group. (I–L) Young AL (2 mo) and young ob/ob (2 mo) mice were treated with either control hIgG/PBS or subcutaneous injection of 1.5 mg/0.1 ml etanercept before and during LD αCD40/IL-2 and survival was monitored (I), n = 5 mice/group. (J and K) On day 2, visceral adipose tissue and peritoneal lavages were collected and assessed by flow cytometry for the percentages of TNF⁺ macrophages (J and K) and serum TNF levels (L); n = 4 mice/group. Data in all panels are representative of one of at least three experiments with similar results. Survival data were plotted using the Kaplan-Meier method and statistical differences were determined with the log-rank test. Bar graph (mean value ± SEM) statistics performed using one-way ANOVA with Bonferroni post-hoc tests. **, P < 0.01; *, P < 0.05.

Figure 5.

DIO succumb to cytokine storm, organ pathology, and lethality similar to aged AL mice after IT. Young C57BL/6 mice were placed on a 60% fat diet starting at 3 wk of age to induce obesity (young DIO). Lean age-matched controls (young lean) were fed a 10% fat diet beginning at 3 wk of age. Young mice were analyzed at 5 mo of age, and aged AL mice were analyzed at 18 mo of age, n = 6 mice/group. Average body weight (A), representative pictures (B), and total fat content as measured by MRI (C) are shown for each group. (D–F) Young lean (5 mo), young DIO (5 mo), and aged AL (18 mo) mice were treated with HD αCD40/IL-2 or rIgG/PBS. Serum was collected day 2 after the start of treatment and analyzed by CBA for levels of TNF (C), IL-6 (D), and IFN-γ (E) and survival was analyzed (F). Data in A–C are representative of one of at least three experiments with similar results and data in D–G are representative of one of at least four experiments with similar results. Survival data were plotted using the Kaplan-Meier method and statistical differences were determined with the log-rank test. Bar graph (mean value ± SEM) statistics performed using one-way ANOVA with Bonferroni post-hoc tests. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.