Multi-Omic blood analysis reveals differences in innate inflammatory sensitivity between species.

Vertebrates differ greatly in responses to pro-inflammatory agonists such as bacterial lipopolysaccharide (LPS), complicating use of animal models to study human sepsis or inflammatory disorders. We compared transcriptomes of resting and LPS-exposed blood from six LPS-sensitive species (rabbit, pig, sheep, cow, chimpanzee, human) and four LPS-resilient species (mice, rats, baboon, rhesus), as well as plasma proteomes and lipidomes. Unexpectedly, at baseline, sensitive species already had enhanced expression of LPS-responsive genes relative to resilient species. After LPS stimulation, maximally different genes in resilient species included genes that detoxify LPS, diminish bacterial growth, discriminate sepsis from SIRS, and play roles in autophagy and apoptosis. The findings reveal the molecular landscape of species differences in inflammation, and may inform better selection of species for pre-clinical models.

Bifunctional Small Molecules Enhance Neutrophil Activities Against Aspergillus fumigatus in vivo and in vitro.

Aspergillosis is difficult to treat and carries a high mortality rate in immunocompromised patients. Neutrophils play a critical role in control of infection but may be diminished in number and function during immunosuppressive therapies. Here, we measure the effect of three bifunctional small molecules that target Aspergillus fumigatus and prime neutrophils to generate a more effective response against the pathogen. The molecules combine two moieties joined by a chemical linker: a targeting moiety (TM) that binds to the surface of the microbial target, and an effector moiety (EM) that interacts with chemoattractant receptors on human neutrophils. We report that the bifunctional compounds enhance the interactions between primary human neutrophils and A. fumigatus in vitro, using three microfluidic assay platforms. The bifunctional compounds significantly enhance the recruitment of neutrophils, increase hyphae killing by neutrophils in a uniform concentration of drug, and decrease hyphal tip growth velocity in the presence of neutrophils compared to the antifungal targeting moiety alone. We validated that the bifunctional compounds are also effective in vivo, using a zebrafish infection model with neutrophils expressing the appropriate EM receptor. We measured significantly increased phagocytosis of A. fumigatus conidia by neutrophils expressing the EM receptor in the presence of the compounds compared to receptor-negative cells. Finally, we demonstrate that treatment with our lead compound significantly improved the antifungal activity of neutrophils from immunosuppressed patients ex vivo. This type of bifunctional compounds strategy may be utilized to redirect the immune system to destroy fungal, bacterial, and viral pathogens.

Different Bactericidal and Inflammatory Activities of Human and Mouse Blood.

We performed side-by-side experiments to compare the behavior of four strains of Escherichia coli and one strain of Pseudomonas aeruginosa in fresh human and mouse blood. Bacteria were multiplied in mouse whole blood and plasma but were killed in human whole blood and plasma. The percentage of granulocytes associated with fluorescence-labeled heat-killed E coli relative to total leukocytes counted was higher in human compared to mouse blood as assessed by flow cytometry analysis. Concentrations of proinflammatory cytokines were high in human blood, but undetectable in mouse blood despite high concentrations of bacteria. We conclude that bacterial killing, phagocytosis, and cytokine induction in blood during human bacteremia with these organisms are probably not mimicked in mouse models of bacterial challenge. Understanding the mechanisms for low cytokine induction with high bacterial loads in mouse blood may be helpful to interpret murine models of bacteremia and develop new approaches for treating sepsis in humans.

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

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.

Endothelial dysfunction inhibits the ability of haptoglobin to prevent hemoglobin-induced hypertension.

Intravascular hemolysis produces injury in a variety of human diseases including hemoglobinopathies, malaria, and sepsis. The adverse effects of increased plasma hemoglobin are partly mediated by depletion of nitric oxide (NO) and result in vasoconstriction. Circulating plasma proteins haptoglobin and hemopexin scavenge extracellular hemoglobin and cell-free heme, respectively. The ability of human haptoglobin or hemopexin to inhibit the adverse effects of NO scavenging by circulating murine hemoglobin was tested in C57Bl/6 mice. In healthy awake mice, the systemic hemodynamic effects of intravenous coinfusion of cell-free hemoglobin and exogenous haptoglobin or of cell-free hemoglobin and hemopexin were compared with the hemodynamic effects of infusion of cell-free hemoglobin or control protein (albumin) alone. We also studied the hemodynamic effects of infusing hemoglobin and haptoglobin as well as injecting either hemoglobin or albumin alone in mice fed a high-fat diet (HFD) and in diabetic (db/db) mice. Coinfusion of a 1:1 weight ratio of haptoglobin but not hemopexin with cell-free hemoglobin prevented hemoglobin-induced systemic hypertension in healthy awake mice. In mice fed a HFD and in diabetic mice, coinfusion of haptoglobin mixed with an equal mass of cell-free hemoglobin did not reverse hemoglobin-induced hypertension. Haptoglobin retained cell-free hemoglobin in plasma, but neither haptoglobin nor hemopexin affected the ability of hemoglobin to scavenge NO ex vivo. In conclusion, in healthy C57Bl/6 mice with normal endothelium, coadministration of haptoglobin but not hemopexin with cell-free hemoglobin prevents acute hemoglobin-induced systemic hypertension by compartmentalizing cell-free hemoglobin in plasma. In murine diseases associated with endothelial dysfunction, haptoglobin therapy appears to be insufficient to prevent hemoglobin-induced vasoconstriction.NEW & NOTEWORTHY Coadministraton of haptoglobin but not hemopexin with cell-free hemoglobin prevents hemoglobin-induced systemic hypertension in mice with a normal endothelium. In contrast, treatment with the same amount of haptoglobin is unable to prevent hemoglobin-induced vasoconstriction in mice with hyperlipidemia or diabetes mellitus, disorders that are associated with endothelial dysfunction.

Alterations in Systemic Extracellular Heme and Hemopexin Are Associated With Adverse Clinical Outcomes in Ugandan Children With Severe Malaria.

BACKGROUND: Malaria remains a major cause of global mortality. Extracellular heme, released during malaria-induced hemolysis, mediates a number of pathogenic processes associated with vascular and organ injury. Hemopexin (hpx) facilitates the degradation of extracellular heme. In this study, we explore the hypothesis that dysregulation of the heme-hpx axis is associated with disease severity, acute kidney injury (AKI), and outcome. METHODS: Plasma levels of hemin and hpx (at admission, day 3, and day 14) were assessed in children with severe malaria in Jinja, Uganda. RESULTS: The ratio of heme to hpx was higher at admission and decreased with recovery (median, 0.043 [interquartile range {IQR}, 0.007-0.239] on day 1, 0.024 [IQR, 0.005-0.126] on day 3, and 0.008 [IQR, 0.002-0.022] on day 14; P < .001). Ratios of heme to hpx at admission were higher in children with as compared to those without severe anemia (median, 0.124 [IQR, 0.024-0.431] vs 0.016 [IQR, 0.003-0.073]; P < .0001), children with as compared to those without respiratory distress (median, 0.063 [IQR, 0.017-0.413] vs 0.020 [IQR, 0.004-0.124]; P < .01), and children with as opposed to those without stage 3 AKI (median, 0.354 [IQR, 0.123-2.481] vs 0.037 [IQR, 0.005-0.172], P < .01). The heme to hpx ratio at admission was associated with 6-month mortality (median, 0.148 [IQR, 0.042-0.500] vs 0.039 [IQR, 0.007-0.172]; P = .012). CONCLUSIONS: The ratio of heme to hpx is associated with disease severity and adverse clinical outcomes in Ugandan children, and dysregulation of the heme axis may contribute to malaria pathogenesis.

Haptoglobin or Hemopexin Therapy Prevents Acute Adverse Effects of Resuscitation After Prolonged Storage of Red Cells.

BACKGROUND: Extracellular hemoglobin and cell-free heme are toxic breakdown products of hemolyzed erythrocytes. Mammals synthesize the scavenger proteins haptoglobin and hemopexin, which bind extracellular hemoglobin and heme, respectively. Transfusion of packed red blood cells is a lifesaving therapy for patients with hemorrhagic shock. Because erythrocytes undergo progressive deleterious morphological and biochemical changes during storage, transfusion of packed red blood cells that have been stored for prolonged intervals (SRBCs; stored for 35-40 days in humans or 14 days in mice) increases plasma levels of cell-free hemoglobin and heme. Therefore, in patients with hemorrhagic shock, perfusion-sensitive organs such as the kidneys are challenged not only by hypoperfusion but also by the high concentrations of plasma hemoglobin and heme that are associated with the transfusion of SRBCs. METHODS: To test whether treatment with exogenous human haptoglobin or hemopexin can ameliorate adverse effects of resuscitation with SRBCs after 2 hours of hemorrhagic shock, mice that received SRBCs were given a coinfusion of haptoglobin, hemopexin, or albumin. RESULTS: Treatment with haptoglobin or hemopexin but not albumin improved the survival rate and attenuated SRBC-induced inflammation. Treatment with haptoglobin retained free hemoglobin in the plasma and prevented SRBC-induced hemoglobinuria and kidney injury. In mice resuscitated with fresh packed red blood cells, treatment with haptoglobin, hemopexin, or albumin did not cause harmful effects. CONCLUSIONS: In mice, the adverse effects of transfusion with SRBCs after hemorrhagic shock are ameliorated by treatment with either haptoglobin or hemopexin. Haptoglobin infusion prevents kidney injury associated with high plasma hemoglobin concentrations after resuscitation with SRBCs. Treatment with the naturally occurring human plasma proteins haptoglobin or hemopexin may have beneficial effects in conditions of severe hemolysis after prolonged hypotension.

Identification of CD163 as an antiinflammatory receptor for HMGB1-haptoglobin complexes.

Secreted by activated cells or passively released by damaged cells, extracellular HMGB1 is a prototypical damage-associated molecular pattern (DAMP) inflammatory mediator. During the course of developing extracorporeal approaches to treating injury and infection, we inadvertently discovered that haptoglobin, the acute phase protein that binds extracellular hemoglobin and targets cellular uptake through CD163, also binds HMGB1. Haptoglobin-HMGB1 complexes elicit the production of antiinflammatory enzymes (heme oxygenase-1) and cytokines (e.g., IL-10) in WT but not in CD163-deficient macrophages. Genetic disruption of haptoglobin or CD163 expression significantly enhances mortality rates in standardized models of intra-abdominal sepsis in mice. Administration of haptoglobin to WT and to haptoglobin gene-deficient animals confers significant protection. These findings reveal a mechanism for haptoglobin modulation of the inflammatory action of HMGB1, with significant implications for developing experimental strategies targeting HMGB1-dependent inflammatory diseases.

A Critical Reappraisal of Prolonged Neutropenia as a Risk Factor for Invasive Pulmonary Aspergillosis.

Prolonged neutropenia is generally thought to be the major factor for invasive pulmonary aspergillosis (IPA). In the present study, we characterize the frequency, severity, and duration of neutropenia that immediately precedes IPA. Prolonged neutropenia was identified in only one third of all IPA cases and occurred exclusively in hematologic patients.

Gut Microbiota-Induced Immunoglobulin G Controls Systemic Infection by Symbiotic Bacteria and Pathogens.

The gut microbiota is compartmentalized in the intestinal lumen and induces local immune responses, but it remains unknown whether the gut microbiota can induce systemic response and contribute to systemic immunity. We report that selective gut symbiotic gram-negative bacteria were able to disseminate systemically to induce immunoglobulin G (IgG) response, which primarily targeted gram-negative bacterial antigens and conferred protection against systemic infections by E. coli and Salmonella by directly coating bacteria to promote killing by phagocytes. T cells and Toll-like receptor 4 on B cells were important in the generation of microbiota-specific IgG. We identified murein lipoprotein (MLP), a highly conserved gram-negative outer membrane protein, as a major antigen that induced systemic IgG homeostatically in both mice and humans. Administration of anti-MLP IgG conferred crucial protection against systemic Salmonella infection. Thus, our findings reveal an important function for the gut microbiota in combating systemic infection through the induction of protective IgG.

Microfluidic assay for precise measurements of mouse, rat, and human neutrophil chemotaxis in whole-blood droplets.

Animal models of human disease differ in innate immune responses to stress, pathogens, or injury. Precise neutrophil phenotype measurements could facilitate interspecies comparisons. However, such phenotype comparisons could not be performed accurately with the use of current assays, as they require the separation of neutrophils from blood using species-specific protocols, and they introduce distinct artifacts. Here, we report a microfluidic technology that enables robust characterization of neutrophil migratory phenotypes in a manner independent of the donor species and performed directly in a droplet of whole blood. The assay relies on the particular ability of neutrophils to deform actively during chemotaxis through microscale channels that block the advance of other blood cells. Neutrophil migration is measured directly in blood, in the presence of other blood cells and serum factors. Our measurements reveal important differences among migration counts, velocity, and directionality among neutrophils from 2 common mouse strains, rats, and humans.

Purified and Recombinant Hemopexin: Protease Activity and Effect on Neutrophil Chemotaxis.

Infusion of the heme-binding protein hemopexin has been proposed as a novel approach to decrease heme-induced inflammation in settings of red blood cell breakdown, but questions have been raised as to possible side effects related to protease activity and inhibition of chemotaxis. We evaluated protease activity and effects on chemotaxis of purified plasma hemopexin obtained from multiple sources as well as a novel recombinant fusion protein Fc-hemopexin. Amidolytic assay was performed to measure the protease activity of several plasma-derived hemopexin and recombinant Fc-hemopexin. Hemopexin was added to the human monocyte culture in the presence of lipopolysaccharides (LPS), and also injected into mice intravenously (i.v.) 30 min before inducing neutrophil migration via intraperitoneal (i.p.) injection of thioglycolate. Control groups received the same amount of albumin. Protease activity varied widely between hemopexins. Recombinant Fc-hemopexin bound heme, inhibited the synergy of heme with LPS on tumor necrosis factor (TNF) production from monocytes, and had minor but detectable protease activity. There was no effect of any hemopexin preparation on chemotaxis, and purified hemopexin did not alter the migration of neutrophils into the peritoneal cavity of mice. Heme and LPS synergistically induced the release of LTB4 from human monocytes, and hemopexin blocked this release, as well as chemotaxis of neutrophils in response to activated monocyte supernatants. These results suggest that hemopexin does not directly affect chemotaxis through protease activity, but may decrease heme-driven chemotaxis and secondary inflammation by attenuating the induction of chemoattractants from monocytes. This property could be beneficial in some settings to control potentially damaging inflammation induced by heme.

Dysregulation of the haem-haemopexin axis is associated with severe malaria in a case-control study of Ugandan children.

BACKGROUND: Malaria is associated with haemolysis and the release of plasma haem. Plasma haem can cause endothelial injury and organ dysfunction, and is normally scavenged by haemopexin to limit toxicity. It was hypothesized that dysregulation of the haem-haemopexin pathway contributes to severe and fatal malaria infections. METHODS: Plasma levels of haemin (oxidized haem), haemopexin, haptoglobin, and haemoglobin were quantified in a case-control study of Ugandan children with Plasmodium falciparum malaria. Levels at presentation were compared in children with uncomplicated malaria (UM; n = 29), severe malarial anaemia (SMA; n = 27) or cerebral malaria (CM; n = 31), and evaluated for utility in predicting fatal (n = 19) vs non-fatal (n = 39) outcomes in severe disease. A causal role for haemopexin was assessed in a pre-clinical model of experimental cerebral malaria (ECM), following disruption of mouse haemopexin gene (hpx). Analysis was done using Kruskall Wallis tests, Mann-Whitney tests, log-rank tests for survival, and repeated measures ANOVA. RESULTS: In Ugandan children presenting with P. falciparum malaria, haemin levels were higher and haemopexin levels were lower in SMA and CM compared to children with UM (haemin, p < 0.01; haemopexin, p < 0.0001). Among all cases of severe malaria, elevated levels of haemin and cell-free haemoglobin at presentation were associated with subsequent mortality (p < 0.05). Compared to ECM-resistant BALB/c mice, susceptible C57BL/6 mice had lower circulating levels of haemopexin (p < 0.01), and targeted deletion of the haemopexin gene, hpx, resulted in increased mortality compared to their wild type littermates (p < 0.05). CONCLUSIONS: These data indicate that plasma levels of haemin and haemopexin measured at presentation correlate with malaria severity and levels of haemin and cell-free haemoglobin predict outcome in paediatric severe malaria. Mechanistic studies in the ECM model support a causal role for the haem-haemopexin axis in ECM pathobiology.

Hemopexin in severe inflammation and infection: mouse models and human diseases.

INTRODUCTION: Cell-free plasma hemoglobin is associated with poor outcome in patients with sepsis. Extracellular hemoglobin and secondarily released heme amplify inflammation in the presence of microbial TLR ligands and/or endogenous mediators. Hemopexin, a plasma protein that binds heme with extraordinary affinity, blocks these effects and has been proposed as a possible treatment approach to decrease inflammation in critically ill patients. METHODS: We studied mouse models of endotoxemia, burn wound infections and peritonitis in order to assess if a repletion strategy for hemopexin might be reasonable. We also measured hemopexin in small numbers of three patient populations that might be logical groups for hemopexin therapy: patients with sepsis and ARDS, patients with severe burns, and premature infants. RESULTS: Despite severe disease, mean plasma hemopexin levels were increased above baseline in each murine model. However, plasma hemopexin levels were decreased or markedly decreased in many patients in each of the three patient populations. CONCLUSIONS: Potentially different behavior of hemopexin in mice and humans may be important to consider when utilizing murine models to represent acute human inflammatory diseases in which heme plays a role. The findings raise the possibility that decreased hemopexin could result in insufficiently neutralized or cleared heme in some patients with ARDS, burns, or in premature infants who might be candidates to benefit from hemopexin administration.

Nutritional vitamin D supplementation in dialysis: a randomized trial.

BACKGROUND AND OBJECTIVES: Vitamin D (25-hydroxyvitamin D; 25[OH]D) deficiency is common in patients initiating long-term hemodialysis, but the safety and efficacy of nutritional vitamin D supplementation in this population remain uncertain. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This randomized, placebo-controlled, parallel-group multicenter trial compared two doses of ergocalciferol with placebo between October 2009 and March 2013. Hemodialysis patients (n=105) with 25(OH)D levels ≤32 ng/ml from 32 centers in the Northeast United States were randomly assigned to oral ergocalciferol, 50,000 IU weekly (n=36) or monthly (n=33), or placebo (n=36) for a 12-week treatment period. The primary endpoint was the achievement of vitamin D sufficiency (25[OH]D >32 ng/ml) at the end of the 12-week treatment period. Survival was assessed through 1 year. RESULTS: Baseline characteristics were similar across all arms, with overall mean±SD 25(OH)D levels of 21.9±6.9 ng/ml. At 12 weeks, vitamin D sufficiency (25[OH]D >32 ng/ml) was achieved in 91% (weekly), 66% (monthly), and 35% (placebo) (P<0.001). Mean 25(OH)D was significantly higher in both the weekly (49.8±2.3 ng/ml; P<0.001) and monthly (38.3±2.4 ng/ml; P=0.001) arms compared with placebo (27.4±2.3 ng/ml). Calcium, phosphate, parathyroid hormone levels, and active vitamin D treatment did not differ between groups. All-cause and cause-specific hospitalizations and adverse events were similar between groups during the intervention period. Lower all-cause mortality among ergocalciferol-treated participants was not statistically significant (hazard ratio, 0.28; 95% confidence interval, 0.07 to 1.19). CONCLUSIONS: Oral ergocalciferol can increase 25(OH)D levels in incident hemodialysis patients without significant alterations in blood calcium, phosphate, or parathyroid hormone during a 12-week period.

Spontaneous neutrophil migration patterns during sepsis after major burns.

Finely tuned to respond quickly to infections, neutrophils have amazing abilities to migrate fast and efficiently towards sites of infection and inflammation. Although neutrophils ability to migrate is perturbed in patients after major burns, no correlations have yet been demonstrated between altered migration and higher rate of infections and sepsis in these patients when compared to healthy individuals. To probe if such correlations exist, we designed microfluidic devices to quantify the neutrophil migration phenotype with high precision. Inside these devices, moving neutrophils are confined in channels smaller than the neutrophils and forced to make directional decisions at bifurcations and around posts. We employed these devices to quantify neutrophil migration across 18 independent parameters in 74 blood samples from 13 patients with major burns and 3 healthy subjects. Blinded, retrospective analysis of clinical data and neutrophil migration parameters revealed that neutrophils isolated from blood samples collected during sepsis migrate spontaneously inside the microfluidic channels. The spontaneous neutrophil migration is a unique phenotype, typical for patients with major burns during sepsis and often observed one or two days before the diagnosis of sepsis is confirmed. The spontaneous neutrophil migration phenotype is rare in patients with major burns in the absence of sepsis, and is not encountered in healthy individuals. Our findings warrant further studies of neutrophils and their utility for early diagnosing and monitoring sepsis in patients after major burns.

VaxCelerate II: rapid development of a self-assembling vaccine for Lassa fever.

Development of effective vaccines against emerging infectious diseases (EID) can take as much or more than a decade to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. Lassa is a prototypical emerging infectious disease endemic to West Africa for which no successful vaccine is available. We established the VaxCelerate Consortium to address the need for more rapid vaccine development by creating a platform capable of generating and pre-clinically testing a new vaccine against specific pathogen targets in less than 120 d A self-assembling vaccine is at the core of the approach. It consists of a fusion protein composed of the immunostimulatory Mycobacterium tuberculosis heat shock protein 70 (MtbHSP70) and the biotin binding protein, avidin. Mixing the resulting protein (MAV) with biotinylated pathogen-specific immunogenic peptides yields a self-assembled vaccine (SAV). To meet the time constraint imposed on this project, we used a distributed R&D model involving experts in the fields of protein engineering and production, bioinformatics, peptide synthesis/design and GMP/GLP manufacturing and testing standards. SAV immunogenicity was first tested using H1N1 influenza specific peptides and the entire VaxCelerate process was then tested in a mock live-fire exercise targeting Lassa fever virus. We demonstrated that the Lassa fever vaccine induced significantly increased class II peptide specific interferon-γ CD4(+) T cell responses in HLA-DR3 transgenic mice compared to peptide or MAV alone controls. We thereby demonstrated that our SAV in combination with a distributed development model may facilitate accelerated regulatory review by using an identical design for each vaccine and by applying safety and efficacy assessment tools that are more relevant to human vaccine responses than current animal models.