Anti-PD-1 Antibody Administration following Hip Fracture Surgery Reverses Immune Dysfunction and Decreases Susceptibility to Infection

Abstract

The aim of this investigation was to assess expression of programmed death-1 (PD-1) and inflammatory status after hip fracture surgery in aged mice and to evaluate the effect of anti-PD-1 antibody intervention. Male C57BL/6 mice aged 22-28 months underwent hip fracture and femoral intramedullary pinning or a sham procedure. Expression of PD-1 was measured on CD4⁺ and CD8⁺ T cells. Additionally, the effects of anti-PD-1 antibody on lymphocyte apoptosis, cytokine production, bacterial clearance, and survival were determined. Expression of PD-1 on T cells was upregulated in mice after hip fracture and surgery compared to sham controls. Administration of anti-PD-1 antibody prevented T lymphocyte apoptosis, increased IFN-γ production in splenocytes, and decreased systemic inflammation. Antibody blockade of PD-1 significantly decreased susceptibility to bacteria and improved survival rates of aged mice after hip fracture and surgery followed by the induction of Pseudomonas aeruginosa pneumonia. This study showed that hip fracture and surgical trauma cause significant increases in PD-1 expression in aged mice. Antibody blockade of PD-1 partially reverses T cell apoptosis, decreases the systemic inflammatory response and susceptibility to bacteria, and reduces mortality.

Figure 1

(a, b) PD-1 expression on CD4⁺ and CD8⁺ T cells temporarily increases after hip fracture and surgery. Mice were euthanized 1, 3, and 7 days after hip fracture and surgery or anesthesia alone (sham). PD-1 expression was measured in isolated splenocytes using flow cytometry (n = 8 per group). Results are expressed as the mean ± SD. Analysis was performed using one-way ANOVA with the Tukey post hoc test. ^∗P < 0.001 when compared to the sham group.

Figure 2

(a, b) Antibody blockade of PD-1 inhibits splenic T cell apoptosis. Immediately following intramedullary nailing of hip fractures (Fx+Sur) or sham surgery (sham), mice were randomly assigned to receive anti-PD-1 antibody (anti-PD-1), isotype control antibody (Iso), or saline. Splenocytes were harvested 24 h after surgery. Apoptosis was quantified by active caspase 3 or TUNEL assays (n = 8 per group). Results are expressed as the mean ± SD. Analysis was performed using one-way ANOVA with the Tukey post hoc test. ^∗P < 0.001 when compared to the sham group and ^∗∗P < 0.001 when compared to the Fx+Sur+anti-PD-1 group.

Figure 3

Antibody blockade of PD-1 restores IFN-γ production in stimulated splenocytes after hip fracture and surgery (Fx+Sur). Splenocytes were harvested 24 h after surgery. IFN-γ levels were determined after stimulation with CD3 and CD28 (n = 8 per group). Results are expressed as the mean ± SD. Analysis was performed using one-way ANOVA with the Tukey post hoc test. ^∗P < 0.001 when compared to the Fx+Sur group and ^∗∗P < 0.001 when compared to the Fx+Sur+Iso group.

Figure 4

(a–d) Levels of IL-6, TNF-α, IL-1β, and IL-10 were significantly higher in mice that underwent fracture and surgery (Fx+Sur) than mice that underwent sham surgery. Animals treated with anti-PD-1 after surgery had significantly lower levels of IL-6, TNF-α, IL-1β, and IL-10 than animals treated with saline or isotype control (Iso) (n = 8 per group). Results are expressed as the mean ± SD. Analysis was performed using one-way ANOVA with the Tukey post hoc test. ^∗P < 0.001 when compared to the sham group and ^∗∗P < 0.001 when compared to the Fx+Sur+anti-PD-1 group.

Figure 5

Antibody blockade of PD-1 improves survival after infection. Mice were treated with anti-PD-1 antibody (anti-PD-1), saline, or isotype control antibody (Iso) immediately after hip fracture (Fx) and surgery (Sur). Twenty-four hours later, mice were treated with P. aeruginosa (ba). Survival was recorded for 14 days (n = 12 per group). Results are expressed as the mean ± SD. Analysis was performed using the log-rank test. ^∗P < 0.001 when compared to the Iso or saline group.

Figure 6

(a, b) Bacterial counts in BAL fluid and blood collected 24 h after P. aeruginosa infection (n = 8 per group). Results are expressed as the mean ± SD. Analysis was performed using one-way ANOVA with the Tukey post hoc test. ^∗P < 0.001 when compared to the sham group and ^∗∗P < 0.001 when compared to the Fx+Sur+anti-PD-1 group.