Leukocyte infiltration and activation of the NLRP3 inflammasome in white adipose tissue following thermal injury

Abstract

Objectives: Severe thermal injury is associated with extreme and prolonged inflammatory and hypermetabolic responses, resulting in significant catabolism that delays recovery or even leads to multiple organ failure and death. Burned patients exhibit many symptoms of stress-induced diabetes, including hyperglycemia, hyperinsulinemia, and hyperlipidemia. Recently, the nucleotide-binding domain, leucine-rich family (NLR), pyrin-containing 3 (NLRP3) inflammasome has received much attention as the sensor of endogenous "danger signals" and mediator of "sterile inflammation" in type II diabetes. Therefore, we investigated whether the NLRP3 inflammasome is activated in the adipose tissue of burned patients, as we hypothesize that, similar to the scenario observed in chronic diabetes, the cytokines produced by the inflammasome mediate insulin resistance and metabolic dysfunction.

Design: Prospective cohort study.

Setting: Ross Tilley Burn Centre & Sunnybrook Research Institute.

Patients: We enrolled 76 patients with burn sizes ranging from 1% to 70% total body surface area. All severely burned patients exhibited burn-induced insulin resistance and hyperglycemia.

Interventions: None.

Measurements and main results: We examined the adipose tissue of control and burned patients and found, via flow cytometry and gene expression studies, increased infiltration of leukocytes-especially macrophages-and evidence of inflammasome priming and activation. Furthermore, we observed increased levels of interleukin-1β in the plasma of burned patients when compared to controls.

Conclusions: In summary, our study is the first to show activation of the inflammasome in burned humans, and our results provide impetus for further investigation of the role of the inflammasome in burn-induced hypermetabolism and, potentially, developing novel therapies targeting this protein complex for the treatment of stress-induced diabetes.

Figure 1. Hyperglycemia, hyperinsulenimia, and insulin resistance in burned patients

(Black line) Average daily glucose is shown for a portion of the patients included in this study. Burned patients exhibit sustained hyperglycemia following thermal injury. Dashed lines represent normal glucose range. (Grey line) Average exogenous insulin units administered during the course of standard burn care per day is shown for a portion of the patients included in this study.

Figure 2. Increased leukocyte infiltrates in white adipose tissue of patients with acute burn injuries

(A) A representative forward and side scatter profile of flow cytometric analysis of the stromal vascular fraction isolated from human white adipose tissue. (B) A histogram analysis to measure the percentage of CD45+ cells within the SVF. (C) A representative profile of CD14 and CD3 surface expression within the CD45+ population of the SVF. (D) Mean and SEM for the percentage of CD45+ cells in the SVF isolated from control (n=3) or burned patients (n=28). *p<0.05 vs. control.

Figure 3. Elevated expression of inflammasome priming and myeloid/macrophage markers in white adipose tissue, post-burn

(A) and (B) illustrate gene expression of inflammasome priming markers. Steady-state transcript levels for both NLRP3 (control, n=6 and burn, n=25) and IL-1β (control, n=10 and burn, n=34) are significantly increased in burned patients. (C) Expression of CD11b (control, n=3 and burn, n=9), a myeloid and activated lymphocyte marker was not significantly increased in burned patients, but (D) levels of EMR1 mRNA (control, n=3 and burn, n=9), a specific macrophage marker, were significantly higher in the WAT tissue of the burn group when compared with controls. All data is presented as mean values with error bars as SEM, *p<0.05 and **p<0.01.

Figure 4. Inflammasome activity is increased in the CD14+ portion of the stromal vascular fraction in white adipose tissue, post-burn

(A) Percentage of monocytes staining positive for inflammasome activity in the SVF isolated from WAT of control (n=2), acute minor burns (n=5), and acute major burns (n=4) patients. Results were first gated to exclude CD14- cells, then analyzed for FLICA+ staining in order to ascertain caspase I activity (refer to methods). Inflammasome activity was significantly greater in monocytes of the large burn group (***p<0.001) when compared to the small burn group. Control patients were unburned and donated subcutaneous fat tissue after undergoing liposuction or other surgical procedures for reconstructive purposes. All data is presented as mean values with error bars as SEM.

Figure 5. Elevated circulating protein levels of IL-1β, post-burn

Patients with acute minor burns (≤ 30% TBSA, n=42) and acute major burns (≥ 30% TBSA, n=34) exhibit significantly elevated levels of plasma IL-1β (***p<0.0001) when compared to controls (n=5), as measured by luminex technology. Comparing severity of burn injury, the large burn group demonstrated increased IL-1β (#p<0.05). All data is presented as mean values with error bars as SEM.