Haptoglobin-2 variant increases susceptibility to acute respiratory distress syndrome during sepsis

Abstract

Acute respiratory distress syndrome (ARDS) is an inflammatory lung disorder that frequently complicates critical illness and commonly occurs in sepsis. Although numerous clinical and environmental risk factors exist, not all patients with risk factors develop ARDS, raising the possibility of genetic underpinnings for ARDS susceptibility. We have previously reported that circulating cell-free hemoglobin (CFH) is elevated during sepsis, and higher levels predict worse outcomes. Excess CFH is rapidly scavenged by haptoglobin (Hp). A common HP genetic variant, HP2, is unique to humans and is common in many populations worldwide. HP2 haptoglobin has reduced ability to inhibit CFH-mediated inflammation and oxidative stress compared with the alternative HP1. We hypothesized that HP2 increases ARDS susceptibility during sepsis when plasma CFH levels are elevated. In a murine model of sepsis with elevated CFH, transgenic mice homozygous for Hp2 had increased lung inflammation, pulmonary vascular permeability, lung apoptosis, and mortality compared with wild-type mice. We then tested the clinical relevance of our findings in 496 septic critically ill adults, finding that HP2 increased ARDS susceptibility after controlling for clinical risk factors and plasma CFH. These observations identify HP2 as a potentially novel genetic ARDS risk factor during sepsis and may have important implications in the study and treatment of ARDS.

Keywords: Genetic variation; Genetics; Mouse models; Pulmonology.

Figure 1. Hp2-2 mice have decreased survival over 72 hours following experimental sepsis.

Hp1-1 (shown in red) and Hp2-2 (shown in blue) mice (n = 12 each group) were treated with intraperitoneal injection of 2.0 mg CS per gram body weight and intravenous injection of 100 μL of 0.15 mg/g CFH, then monitored for survival over 72 hours. (A) Survival curves showing survival was significantly worse in Hp2-2 mice. P = 0.03 by the Mantel-Cox log-rank test. (B) Plasma CFH levels were measured at 24 hours in Hp1-1 (n = 11) and Hp2-2 (n = 17) mice treated with CS and intravenous (IV) CFH. Dots represent individual values. In the box plots, the thick horizontal bars represent the median, boxes represent the IQR (25th and 75th percentiles), and whiskers represent the minimum and maximum values within 1.5 × IQR from the 25th and 75th percentiles. P = 0.09 by Mann-Whitney U test.

Figure 2. Hp2-2 mice have increased markers of lung inflammation.

Hp1-1 (red) and Hp2-2 (blue) mice were treated with CS and IV CFH. (A) Myeloperoxidase (MPO) activity was measured enzymatically in whole lungs. Reported values are normalized to the mean value for Hp1-1 mice. P = 0.014 by Mann-Whitney U test. (B) Whole-lung mRNA was extracted for CXCL1 and expression measured by real-time PCR. Values are reported as fold-change relative to GAPDH expression. P = 0.022 by Mann-Whitney U test. (C) CXCL1 protein levels were measured by ELISA in BAL samples. P = 0.011 by Mann-Whitney U test. Dots represent individual values. For the box plots, the horizontal bars represent the median, boxes represent the IQR (25th and 75th percentiles), and whiskers represent the minimum and maximum values within 1.5 × IQR from the 25th and 75th percentiles.

Figure 3. Hp2-2 increases lung microvascular permeability.

AngioSense, a fluorescent 70-kD macromolecule, was injected retroorbitally at the time of treatment. (A) After 24 hours, whole lungs were excised and imaged using a high-sensitivity charged coupled device camera. (B) Fluorescent signal per lung was normalized to body weight and quantified. Dots represent individual values. For the box plots, thick horizontal bars represent the median, boxes represent the IQR (25th and 75th percentiles), and whiskers represent the minimum and maximum values within 1.5 × IQR from the 25th and 75th percentiles. P = 0.037 by Mann-Whitney U test.

Figure 4. Hp2-2 mice have increased pulmonary apoptosis.

Hp1-1 and Hp2-2 mice were treated with CS and IV CFH. Lungs were harvested at 4 hours and examined for apoptotic cells by the TUNEL assay by a trained reviewer blinded to genotype. (A) Representative TUNEL stain images (left images) show increased number of TUNEL-positive cells (white arrows) in Hp2-2 mouse lungs compared with Hp1-1 mouse lungs. H&E stained sections from the same lung (right images). Scale bars on TUNEL images: 500 μm, scale bars on H&E images: 100 μm. (B) Hp2-2 mouse lungs (blue) demonstrated increased apoptosis of pulmonary cells following sepsis compared with Hp1-1 mice (red). Dots represent individual values. For the box plots, thick horizontal bars represent the median, boxes represent the interquartile range (IQR, 25th and 75th percentiles), and whiskers represent the minimum and maximum values within 1.5 × IQR from the 25th and 75th percentiles. *P = 0.004 by Mann-Whitney U test.

Figure 5. ARDS risk increases with higher plasma CFH during sepsis.

Risk of developing ARDS during sepsis increased with higher enrollment plasma CFH levels. Patients are grouped by enrollment plasma CFH quartile; height of bars and numbers over bars indicate percentage of patients who developed ARDS during the study period. Number of patients per quartile = 82, 130, 156, and 128, respectively (N = 496 in total). P = 0.032 by Cochran-Armitage test for trend of increasing ARDS risk ordered by CFH quartile. The lower limit of detection for the assay is 10 mg/dL.

Figure 6. HP genotype increases ARDS risk in septic adults.

In the entire study cohort (N = 496), HP2-2 patients and HP2-1 patients had increased risk of developing ARDS during the study period. Height of bars and numbers over bars indicate proportions of patients developing ARDS for each group. P = 0.029 by Cochran-Armitage test for increasing risk ordered by number of HP2 alleles.

Figure 7. HP genotype affects ARDS risk only in patients with detectable plasma cell-free hemoglobin.

(A) There was no association between HP genotype and ARDS risk in patients with undetectable plasma CFH. n = 82, and P = 0.46 by Cochran-Armitage test. (B) An association between HP genotype and ARDS risk was observed only in patients with detectable CFH. n = 414, and P = 0.026 by Cochran-Armitage test.

Figure 8. HP2 variant increases ARDS risk in septic adults when controlling for clinical factors.

Multivariable logistic regression model for ARDS in study population (N = 496). Circles represent point estimate odds ratios, and horizontal lines represent 95% CIs for each variable included in the model. Black dashed vertical line indicates odds ratio of 1.0 (no change in ARDS risk). P = 0.018 for HP2 variant.