Haptoglobin improves shock, lung injury, and survival in canine pneumonia

Abstract

During the last half-century, numerous antiinflammatory agents were tested in dozens of clinical trials and have proven ineffective for treating septic shock. The observation in multiple studies that cell-free hemoglobin (CFH) levels are elevated during clinical sepsis and that the degree of increase correlates with higher mortality suggests an alternative approach. Human haptoglobin binds CFH with high affinity and, therefore, can potentially reduce iron availability and oxidative activity. CFH levels are elevated over approximately 24-48 hours in our antibiotic-treated canine model of S. aureus pneumonia that simulates the cardiovascular abnormalities of human septic shock. In this 96-hour model, resuscitative treatments, mechanical ventilation, sedation, and continuous care are translatable to management in human intensive care units. We found, in this S. aureus pneumonia model inducing septic shock, that commercial human haptoglobin concentrate infusions over 48-hours bind canine CFH, increase CFH clearance, and lower circulating iron. Over the 96-hour study, this treatment was associated with an improved metabolic profile (pH, lactate), less lung injury, reversal of shock, and increased survival. Haptoglobin binding compartmentalized CFH to the intravascular space. This observation, in combination with increasing CFHs clearance, reduced available iron as a potential source of bacterial nutrition while decreasing the ability for CFH and iron to cause extravascular oxidative tissue injury. In contrast, haptoglobin therapy had no measurable antiinflammatory effect on elevations in proinflammatory C-reactive protein and cytokine levels. Haptoglobin therapy enhances normal host defense mechanisms in contrast to previously studied antiinflammatory sepsis therapies, making it a biologically plausible novel approach to treat septic shock.

Keywords: Bacterial infections; Clinical Trials; Drug therapy; Infectious disease; Innate immunity.

Figure 1. Kaplan-Meier survival curve for the 96-hour sepsis study.

The survival comparison in canines receiving haptoglobin or no haptoglobin with (A) or without (B) RBC exchange transfusion after S. aureus challenge. P values are denoted by asterisks indicating significance in comparison between each panel group using stratified log rank tests. (C and D) Mean shock scores (± SEM) at serial time points. The shock score accounts for the level of vasopressor support (norepinephrine) needed to maintain the mean arterial pressure at a preset normal level for canines (mean 80 mmHg). Shock score is compared over 96 hours in canines receiving haptoglobin or no haptoglobin with (C) or without (D) RBC exchange transfusion. Changes from baseline are shown for each study group plotted from a common origin the mean value for animals at baseline. P values indicate significance in each group comparison in each panel and are denoted by asterisks (for changes over time) or crosses (comparing haptoglobin vs. no haptoglobin at each time point). (E and F) Mean (± SEM) lung injury scores (LIS) at serial time points. The LIS detects pulmonary damage via measurements in mean pulmonary artery pressure, alveolar-arterial oxygen gradient, plateau pressure, oxygen saturation, and respiratory rate. The LIS is plotted over time (x axis) for animals receiving haptoglobin or no haptoglobin with (E) or without (F) RBC exchange transfusion. Changes from baseline are shown for each study group plotted from a common origin, with the mean value for animals at baseline. P values indicate significance in each group comparison in each panel and are denoted by asterisks (for changes over time) or crosses (comparing haptoglobin vs. no haptoglobin at each time point). Comparisons of all variables (except survival) were made based on contrasts in linear mixed models, which allow us to account for repeated measurements of each animal and the actual pairing of animals within each cycle.

Figure 2. Mean cell-free hemoglobin, nontransferrin bound iron, and haptoglobin cell–free hemoglobin binding at serial time points.

The format is similar to Figure 1, except that cell-free hemoglobin (A and B) and nontransferrin bound iron (C and D) are shown. In addition, in panel E, haptoglobin cell–free hemoglobin binding is shown. (A and B) The Drabkin’s assay was used to measures both unbound and bound (with haptoglobin) cell-free hemoglobin. (E) Absorbance profile for mixtures of haptoglobin and hemoglobin. The samples were spun at 163,000 g at 25.2°C, and absorbance was measured as a function of time and sedimentation distance. Absorbance was normalized to the maximum absorbance of all samples. The sample with a molar ratio of 68 to 1 of hemoglobin (in heme) to haptoglobin (in α-β dimer), essentially CFH, sedimented much more slowly than the others. The other 2 samples with much greater ratios of hemoglobin to haptoglobin sedimented more rapidly, consistent with a higher molecular weight and confirming that the canine hemoglobin binds to human haptoglobin. Analysis of the sedimentation data provided the percentage of fast (bound) and slow (unbound) sediment species. That percentage determination, together with the known total concentrations of haptoglobin and hemoglobin in each sample, was used to calculate the binding stoichiometry. For example, when 227 μM hemoglobin was mixed with 37 μM haptoglobin, 75% of the total hemoglobin (170.25 μM) was a slowly sedimenting species. Thus, 170.25/37 = 4.6 hemoglobin molecules (in heme) per haptoglobin (in α-β dimers). Comparisons of all variables (except survival) were made based on contrasts in linear mixed models, which allow us to account for repeated measurements of each animal and the actual pairing of animals within each cycle.

Figure 3. Human haptoglobin, central venous pressure, hematocrit, and pulmonary artery occlusion pressure measurements at serial time points.

The format is similar to Figure 1, except that human haptoglobin, central venous pressure, hematocrit, and pulmonary artery occlusion pressure are measured over 96 hours after S. aureus challenge in canines receiving haptoglobin or no haptoglobin with (A, C, E, G) or without RBC exchange transfusion (B, D, F, H) Comparisons of all variables (except survival) were made based on contrasts in linear mixed models, which allow us to account for repeated measurements of each animal and the actual pairing of animals within each cycle.

Figure 4. Arterial blood gas comparison over the 96-hour duration of the sepsis study.

The format is similar to Figure 1, except that quantitative arterial blood gas measurements (pH, pCO₂, base excess) and lactate levels are compared over 96 hours after S. aureus challenge in canines receiving haptoglobin or no haptoglobin with (A, C, E, G) or without RBC exchange-transfusion (B, D, F, H). Comparisons of all variables (except survival) were made based on contrasts in linear mixed models, which allow us to account for repeated measurements of each animal and the actual pairing of animals within each cycle.