Frontline Science: Anti-PD-L1 protects against infection with common bacterial pathogens after burn injury

Abstract

Burn patients are susceptible to infections due, in part, to immune dysfunction. Upregulation of programmed death-1 (PD-1) receptor on T cells and programmed cell death ligand-1 (PD-L1) on myeloid cells contribute to immune dysfunction in nonburn-related sepsis. We hypothesized that PD-1/PDL1 interactions contribute to immune dysfunction after burn injury. To determine the impact of burn injury and infection on PD-L1, PD-1 and costimulatory receptor expression by leukocytes and its relationship to T cell functions. The efficacy of anti-PD-L1 antibody was evaluated in a clinically relevant mouse model of burn injury and bacterial infection. Mice underwent 35% scald burn followed by Pseudomonas aeruginosa or Staphylococcus aureus infection on day 4 postburn. Anti-PD-L1 was administered on day 3 postburn. Numbers and phenotype of leukocytes, plasma cytokine concentrations, bacterial clearance, organ injury, and survival were assessed. Burn injury and infection with P. aeruginosa caused a significant upregulation of PD-L1 on myeloid cells, along with a decrease in T cell numbers and function, significant multiorgan injury, and decreased survival. Treatment with anti-PD-L1 antibody improved bacterial clearance, reduced organ injury, and enhanced survival during Pseudomonas burn wound infection. Furthermore, anti-PD-L1 effectively protected against multiorgan injury, and improved bacterial clearance and survival following systemic S. aureus infection after burn injury. Blockade of PD-1/PD-L1 interactions might represent a viable treatment to improve outcomes among critically ill burn-injured subjects and increased leukocyte PD-L1 expression could serve as a valuable biomarker to select appropriate patients for such treatment.

Keywords: T cells; burn injury; checkpoint receptors; infection; myeloid cells; sepsis.

Figure 1

PD‐1 and PD‐L1 expression on leukocyte subsets in mice. PD‐1 and PD‐L1 expression by lymphocytes and antigen presenting cells in spleen and lymph nodes were measured using flow cytometry. [Sham = no burn and no infection; Burn = burn injury alone (no infection)]. Measurements were performed on day 4 after sham or burn procedures (before infection) or day 2 after wound infection. (A) The experimental timeline; (B, C, and D) the expression levels (%) of PD‐L1 on splenic CD11c⁺ dendritic cells, F4/80⁺ macrophages, and Ly6C⁺ inflammatory monocytes, respectively; (E and F) the expression levels (%) of PD‐1 on splenic CD4⁺ and CD8⁺ T cells, respectively. *Significantly (P < 0.05) different from sham group; and ^#significantly different from burn group (no infection)

Figure 2

Treatment with anti‐PD‐L1 antibody decreases systemic bacterial burden and attenuates organ injury in mice after P. aeruginosa infection. (A) Time line depicting the burn injury and wound infection model along with anti‐PD‐L1 antibody treatment (50 μg, i.p., 1 day before wound infection). P. aeruginosa CFU in the blood (B) and the lungs (C) were measured at 2 days after infection. Organ injury markers were measured on day 4 after burn injury and day 2 postburn wound infection. (D) Serum blood urea nitrogen, BUN; (E) serum alanine aminotransferase, ALT; and (F) serum aspartate aminotransferase, AST. n = 8–10 per group. *Significantly (P < 0.05) different from sham group; ^#significantly different from burn group (no infection); and ⁺significantly different from IgG group

Figure 3

Effect of burn injury and wound infection with P. aeruginosa on plasma cytokine levels. Measurements were performed on day 4 (before wound infection) or 2 days postwound infection. Mice were treated with IgG or anti‐PD‐L1 on day 3 (1 day prior to infection). (A), (B), (C), (D), and (E) show the levels of IL‐6, IL‐10, MIP‐2, KC, and IL‐17 respectively. n = 8–10 per group. *Significantly (P < 0.05) different from sham group; ^#significantly different from burn group (no infection); and ⁺significantly different from IgG group

Figure 4

Assessment of survival in burn and P. aeruginosa infected mice treated with anti‐PD‐L1 antibody. Mice were treated with IgG or anti‐PD‐L1 at 3 days after burn injury (1 day prior to infection) and survival was assessed for 7 days. Groups included burn alone (no infection, black line); burned and IgG treated followed by wound infection (blue line); and burned and anti‐PD‐L1 antibody treated followed by wound infection (red line). n = 15 in each group. ⁺Significantly (P < 0.05) different as compared with the IgG‐treated group

Figure 5

Anti‐PD‐L1 treatment protects against systemic S. aureus infection after burn injury. Anti‐PD‐L1 was administered 24 h before infection (on day 3 postburn). Mice were infected with S. aureus on day 4 after burn injury, and measurements performed on day 3 after infection. Organ injury markers were measured on day 4 after burn injury and day 3 postinfection. (A) Serum alanine aminotransferase, ALT; (B) serum aspartate aminotransferase, AST, and (C) serum blood urea nitrogen, BUN; S. aureus CFU in the lung (D) and the spleen (E) were measured at 3 days after infection. n = 8–10 per group. *Significantly (P < 0.05) different from sham group; ^#significantly different from burn group (no infection); and ⁺significantly different from IgG group

Figure 6

Measurement of CD4⁺, CD8⁺ T lymphocyte and CD19⁺ B lymphocyte counts in the spleen and burn wound draining lymph nodes. Upper panels represent cell counts in the spleen and lower panel represent cell counts in the burn wound draining lymph nodes. Flow cytometry was used to measure the individual CD4⁺ T (A, D), CD8⁺ T (B, E), and CD19⁺ B (C, F) cell counts. n = 10–15 per group. *Significantly (P < 0.05) different from sham group; ^#significantly different from burn group (no infection); and ⁺significantly different from IgG group

Figure 7

Effect of burn injury and P. aeruginosa wound infection on intracellular IFNγ production by CD4⁺ and CD8⁺ T cells. T cell function was assessed by measuring intracellular IFNγ production in ex vivo stimulated (PMA/ionomycin) T cells using flow cytometry. The percentage of intracellular IFNγ expressing CD4⁺ and CD8⁺ T cells in the spleen (A and B), and burn wound draining lymph nodes (C and D) was measured. n = 6–10 per group. *Significantly (P < 0.05) different from sham group; ^#significantly different from burn group (no infection); and ⁺significantly different from IgG group

Figure 8

Analysis of CD28 expression on CD4⁺ and CD8⁺ T lymphocytes. Upper and lower panels show the percentage of CD28 expression on CD4⁺ and CD8⁺ T cells in the spleen and burn wound draining lymph nodes, respectively. (A and C) CD28 expression on CD4⁺ T cells, and (B and D) CD28 expression on CD8⁺ T cells. n = 10–15 per group. *Significantly (P < 0.05) different from sham group; ^#significantly different from burn group (no infection); and ⁺significantly different from IgG group