Intratracheal Administration of Acyl Coenzyme A Acyltransferase-1 Inhibitor K-604 Reduces Pulmonary Inflammation Following Bleomycin-Induced Lung Injury

Abstract

Acute lung injury (ALI) is characterized by epithelial damage, barrier dysfunction, and pulmonary edema. Macrophage activation and failure to resolve play a role in ALI; thus, macrophage phenotype modulation is a rational target for therapeutic intervention. Large, lipid-laden macrophages have been observed in various injury models, including intratracheal bleomycin (ITB), suggesting that lipid storage may play a role in ALI severity. The endoplasmic reticulum-associated enzyme acyl coenzyme A acyltransferase-1 (Acat-1/Soat1) is highly expressed in macrophages, where it catalyzes the esterification of cholesterol, leading to intracellular lipid accumulation. We hypothesize that inhibition of Acat-1 will reduce macrophage activation and improve outcomes of lung injury in ITB. K-604, a selective inhibitor of Acat-1, was used to reduce cholesterol esterification and hence lipid accumulation in response to ITB. Male and female C57BL6/J mice (n = 16-21/group) were administered control, control + K-604, ITB, or ITB + K-604 on d0, control or K-604 on d3, and were sacrificed on day 7. ITB caused significant body weight loss and an increase in cholesterol accumulation in bronchoalveolar lavage cells. These changes were mitigated by Acat-1 inhibition. K-604 also significantly reduced ITB-induced alveolar thickening. Surfactant composition was normalized as indicated by a significant decrease in phospholipid: SP-B ratio in ITB+K-604 compared with ITB. K-604 administration preserved mature alveolar macrophages, decreased activation in response to ITB, and decreased the percentage mature and pro-fibrotic interstitial macrophages. These results show that inhibition of Acat-1 in the lung is associated with reduced inflammatory response to ITB-mediated lung injury. SIGNIFICANCE STATEMENT: Acyl coenzyme A acyltransferase-1 (Acat-1) is critical to lipid droplet formation, and thus inhibition of Acat-1 presents as a pharmacological target. Intratracheal administration of K-604, an Acat-1 inhibitor, reduces intracellular cholesterol ester accumulation in lung macrophages, attenuates inflammation and macrophage activation, and normalizes mediators of surface-active function after intratracheal bleomycin administration in a rodent model. The data presented within suggest that inhibition of Acat-1 in the lung improves acute lung injury outcomes.

Graphical abstract

Fig. 1.

Intratracheal administration of K-604 reduces ITB-mediated loss of bodyweight. Difference between d0 and d7 bodyweight is shown as a percentage of d0 weight (n = 16–21 per group). Values are expressed as mean ± standard error of the mean. (*) represents significantly different from control (P < 0.05); (#) represents significantly different from ITB (P < 0.05).

Fig. 2.

Effect of K-604 on histologic markers of ITB-mediated injury. (A) Representative H&E-stained lung tissue sections derived from control (top left), K-604 (bottom left), ITB (top right) and ITB + K-604 (bottom right) treated animals (n = 16–21 per group). Images were obtained using a VS120 microscope (inset: 40x; magnified image: 400x). Randomly distributed images (n = 10) from each tissue preparation were analyzed for the number of nuclei present (B), percentage of white space (C), and average alveolar wall thickness (D). Values are expressed as mean ± standard error of the mean. (*) represents significantly different from control (P < 0.05); (#) represents significantly different from ITB (P < 0.05).

Fig. 3.

Effects of ITB on total protein, phospholipid and surfactant protein expression within the BAL. Protein concentration (A), phospholipid concentration (B), SP-D:SP-B ratio (C) and phospholipid:SP-B ratio were reported (D) accompanied by representative western blots (E) from analysis of BAL fluid. Values are expressed as mean ± standard error of the mean (n = 8-12 per group). (*) represents significantly different from control (P < 0.05); (#) represents significantly different from ITB (P < 0.05).

Fig. 4.

K-604 reduces ITB-mediated increases cholesterol content of BAL cells. Cells from the BAL were analyzed for total cholesterol, free cholesterol, and cholesterol esters (A) reported as cholesterol (μM) per 1 × 10⁴ cells. BAL cell expression of Abca1 (B) and Abcg1 (C) was determined by RT-qPCR and is shown as fold change over control. Values are expressed as mean ± standard error of the mean (n = 5–16 per group). (*) represents significantly different from control (P < 0.05); (#) represents significantly different from ITB (P < 0.05).

Fig. 5.

Flow cytometric analysis of macrophages in the BAL fluid. Cells were immunostained and analyzed by flow cytometry (Supplemental Fig. 1). Cells that were positively stained for both Siglec F and F4/80 were determined to be alveolar macrophages (AMs). Mature macrophages (CD11c+/CD11b-), migratory macrophages (CD11c+/CD11b+), and recruited macrophages (CD11c-/CD11b+) were identified from alveolar macrophages in the BAL (A). The percentage of mature (B), migratory (C), and recruited macrophages (D) was calculated from the total number of alveolar macrophages and were assessed for their Ly6c and CD206 expression. The percentage of CD11b+ alveolar macrophages that were identified as expressing the acutely activated phenotype (Ly6c+/CD206-) was determined (E). Values are expressed as mean ± standard error of the mean (n = 8-13 per group). (*) represents significantly different from control (P < 0.05); (#) represents significantly different from ITB (P < 0.05).

Fig. 6.

Flow cytometric analysis of macrophages isolated from lung tissue. Cells from digested lung tissue were immunomagnetically-separated based upon CD45 expression. CD45+ cells were isolated, immunostained, and analyzed (Supplemental Fig 2). Cells that expressed F4/80 and CD11b in the absence of Siglec F were categorized as interstitial macrophages and were analyzed for CD11c/CD206 expression (A). The percentage of mature, chronically activated cells (CD11c+/CD206+, green box) was calculated from the total number of interstitial macrophages (B). Values are expressed as mean ± standard error of the mean (n = 8-13 per group). (*) represents significantly different from control (P < 0.05); (#) represents significantly different from ITB (P < 0.05).

Fig. 7.

K-604 inhibits ITB-mediated induction of the inflammatory enzymes Nos2 and Arg1. BAL cells were analyzed for Nos2 and Arg1 expression by RT-qPCR. 2^-ΔΔct values were calculated using Gapdh as the control gene and PBS as the control condition to calculate fold change in expression. Values are expressed as mean ± standard error of the mean (n = 8 per group). (*) represents significantly different from control (P < 0.05); (#) represents significantly different from ITB (P < 0.05).