Stressed erythrophagocytosis induces immunosuppression during sepsis through heme-mediated STAT1 dysregulation.

Macrophages are main effectors of heme metabolism, increasing transiently in the liver during heightened disposal of damaged or senescent RBCs (sRBCs). Macrophages are also essential in defense against microbial threats, but pathological states of heme excess may be immunosuppressive. Herein, we uncovered a mechanism whereby an acute rise in sRBC disposal by macrophages led to an immunosuppressive phenotype after intrapulmonary Klebsiella pneumoniae infection characterized by increased extrapulmonary bacterial proliferation and reduced survival from sepsis in mice. The impaired immunity to K. pneumoniae during heightened sRBC disposal was independent of iron acquisition by bacterial siderophores, in that K. pneumoniae mutants lacking siderophore function recapitulated the findings observed with the WT strain. Rather, sRBC disposal induced a liver transcriptomic profile notable for suppression of Stat1 and IFN-related responses during K. pneumoniae sepsis. Excess heme handling by macrophages recapitulated STAT1 suppression during infection that required synergistic NRF1 and NRF2 activation but was independent of heme oxygenase-1 induction. Whereas iron was dispensable, the porphyrin moiety of heme was sufficient to mediate suppression of STAT1-dependent responses in human and mouse macrophages and promoted liver dissemination of K. pneumoniae in vivo. Thus, cellular heme metabolism dysfunction negatively regulated the STAT1 pathway, with implications in severe infection.

Effects of nitrite and far-red light on coagulation.

Nitric oxide, NO, has been explored as a therapeutic agent to treat thrombosis. In particular, NO has potential in treating mechanical device-associated thrombosis due to its ability to reduce platelet activation and due to the central role of platelet activation and adhesion in device thrombosis. Nitrite is a unique NO donor that reduces platelet activation in that it's activity requires the presence of red blood cells whereas NO activity of other NO donors is blunted by red blood cells. Interestingly, we have previously shown that red blood cell mediated inhibition of platelet activation by adenosine diphosophate (ADP) is dramatically enhanced by illumination with far-red light that is likely due to photolysis of red cell surface bound NO congeners. We now report the effects of nitrite, far-red light, and their combination on several measure of blood coagulation using a variety of agonists. We employed turbidity assays in platelet rich plasma, platelet activation using flow cytometry analysis of a fluorescently labeled antibody to the activated platelet fibrinogen binding site, multiplate impedance-based platelet aggregometry, and assessment of platelet adhesion to collagen coated flow-through microslides. In all cases, the combination of far-red light and nitrite treatment decreased measures of coagulation, but in some cases mono-treatment with nitrite or light alone had no effect. Perhaps most relevant to device thrombosis, we observed that platelet adhesions was inhibited by the combination of nitrite and light treatment while nitrite alone and far-red light alone trended to decrease adhesion, but the results were mixed. These results support the potential of combined far-red light and nitrite treatment for preventing thrombosis in extra-corporeal or shallow-tissue depth devices where the far-red light can penetrate. Such a combined treatment could be advantageous due to the localized treatment afforded by far-red light illumination with minimal systemic effects. Given the role of thrombosis in COVID 19, application to treatment of patients infected with SARS Cov-2 might also be considered.

Haptoglobin therapy has differential effects depending on severity of canine septic shock and cell-free hemoglobin level.

BACKGROUND: During sepsis, higher plasma cell-free hemoglobin (CFH) levels portend worse outcomes. In sepsis models, plasma proteins that bind CFH improve survival. In our canine antibiotic-treated Staphylococcus aureus pneumonia model, with and without red blood cell (RBC) exchange transfusion, commercial human haptoglobin (Hp) concentrates bound and compartmentalized CFH intravascularly, increased CFH clearance, and lowered iron levels, improving shock, lung injury, and survival. We now investigate in our model how very high CFH levels and treatment time affect Hp's beneficial effects. MATERIALS AND METHODS: Two separate canine pneumonia sepsis Hp studies were undertaken: one with exchange transfusion of RBCs after prolonged storage to raise CFH to very high levels and another with rapidly lethal sepsis alone to shorten time to treat. All animals received continuous standard intensive care unit supportive care for 96 hours. RESULTS: Older RBCs markedly elevated plasma CFH levels and, when combined with Hp therapy, created supraphysiologic CFH-Hp complexes that did not increase CFH or iron clearance or improve lung injury and survival. In a rapidly lethal bacterial challenge model without RBC transfusion, Hp binding did not increase clearance of complexes or iron or show benefits seen previously in the less lethal model. DISCUSSION: High-level CFH-Hp complexes may impair clearance mechanisms and eliminate Hp's beneficial effect during sepsis. Rapidly lethal sepsis narrows the therapeutic window for CFH and iron clearance, also decreasing Hp's beneficial effects. In designing clinical trials, dosing and kinetics may be critical factors if Hp infusion is used to treat sepsis.

Development of Zinc Chelating Resin Polymer Beads for the Removal of Cell-Free Hemoglobin.

Red blood cell (RBC) hemolysis is one of the most common storage lesions in packed RBCs (pRBC). Older units of pRBCs, especially those > 21 days old, have increasing levels of hemolysis leading to increased oxidative stress and premature platelet activation. This effect can mostly be attributed to the increase of cell-free hemoglobin (Hb). Therefore, removal of cell-free Hb from pRBCs prior to transfusion could mitigate these deleterious effects. We propose a new method for the removal of Hb from pRBCs using zinc beads. Prepared Hb solutions and pRBCs were treated with zinc beads using two different protocols. UV-Vis spectrophotometry was used to determine Hb concentrations, before and after treatment. Experiments were run in triplicate and paired t tests were used to determine significant differences between groups. Zinc beads removed on average 94% of cell-free Hb within 15 min and 78% Hb from pRBCs (p < 0.0001), demonstrating a maximum binding capacity ~ 66.2 ± 0.7 mg Hb/mL beads. No differences in RBC morphology or deformability were observed after treatment. This study demonstrates the feasibility of using zinc beads for the rapid and targeted removal of Hb from pRBC units. Further investigation is needed to scale this method for large volume removal.

Impact of different standard red blood cell storage temperatures on human and canine RBC hemolysis and chromium survival.

BACKGROUND: Storage temperature is a critical factor for maintaining red-blood cell (RBC) viability, especially during prolonged cold storage. The target range of 1 to 6°C was established decades ago and may no longer be optimal for current blood-banking practices. STUDY DESIGN AND METHODS: Human and canine RBCs were collected under standard conditions and stored in precision-controlled refrigerators at 2°C, 4°C, or 6°C. RESULTS: During 42-day storage, human and canine RBCs showed progressive increases in supernatant non-transferrin-bound iron, cell-free hemoglobin, base deficit, and lactate levels that were overall greater at 6°C and 4°C than at 2°C. Animals transfused with 7-day-old RBCs had similar plasma cell-free hemoglobin and non-transferrin-bound iron levels at 1 to 72 hours for all three temperature conditions by chromium-51 recovery analysis. However, animals transfused with 35-day-old RBCs stored at higher temperatures developed plasma elevations in non-transferrin-bound iron and cell-free hemoglobin at 24 and 72 hours. Despite apparent impaired 35-day storage at 4°C and 6°C compared to 2°C, posttransfusion chromium-51 recovery at 24 hours was superior at higher temperatures. This finding was confounded by a preparation artifact related to an interaction between temperature and storage duration that leads to removal of fragile cells with repeated washing of the radiolabeled RBC test sample and renders the test sample unrepresentative of the stored unit. CONCLUSIONS: RBCs stored at the lower bounds of the temperature range are less metabolically active and produce less anaerobic acidosis and hemolysis, leading to a more suitable transfusion product. The higher refrigeration temperatures are not optimal during extended RBC storage and may confound chromium viability studies.

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.

Parenteral irons versus transfused red blood cells for treatment of anemia during canine experimental bacterial pneumonia.

BACKGROUND: No studies have been performed comparing intravenous (IV) iron with transfused red blood cells (RBCs) for treating anemia during infection. In a previous report, transfused older RBCs increased free iron release and mortality in infected animals when compared to fresher cells. We hypothesized that treating anemia during infection with transfused fresh RBCs, with minimal free iron release, would prove superior to IV iron therapy. STUDY DESIGN AND METHODS: Purpose-bred beagles (n = 42) with experimental Staphylococcus aureus pneumonia rendered anemic were randomized to be transfused RBCs stored for 7 days or one of two IV iron preparations (7 mg/kg), iron sucrose, a widely used preparation, or ferumoxytol, a newer formulation that blunts circulating iron levels. RESULTS: Both irons increased the alveolar-arterial oxygen gradient at 24 to 48 hours (p = 0.02-0.001), worsened shock at 16 hours (p = 0.02-0.003, respectively), and reduced survival (transfusion 56%; iron sucrose 8%, p = 0.01; ferumoxytol 9%, p = 0.04). Compared to fresh RBC transfusion, plasma iron measured by non-transferrin-bound iron levels increased with iron sucrose at 7, 10, 13, 16, 24, and 48 hours (p = 0.04 to p < 0.0001) and ferumoxytol at 7, 24, and 48 hours (p = 0.04 to p = 0.004). No significant differences in cardiac filling pressures or performance, hemoglobin (Hb), or cell-free Hb were observed. CONCLUSIONS: During canine experimental bacterial pneumonia, treatment of mild anemia with IV iron significantly increased free iron levels, shock, lung injury, and mortality compared to transfusion of fresh RBCs. This was true for iron preparations that do or do not blunt circulating free iron level elevations. These findings suggest that treatment of anemia with IV iron during infection should be undertaken with caution.

Potential therapeutic action of nitrite in sickle cell disease.

Sickle cell disease is caused by a mutant form of hemoglobin that polymerizes under hypoxic conditions, increasing rigidity, fragility, calcium influx-mediated dehydration, and adhesivity of red blood cells. Increased red cell fragility results in hemolysis, which reduces nitric oxide (NO) bioavailability, and induces platelet activation and inflammation leading to adhesion of circulating blood cells. Nitric Oxide inhibits adhesion and platelet activation. Nitrite has emerged as an attractive therapeutic agent that targets delivery of NO activity to areas of hypoxia through bioactivation by deoxygenated red blood cell hemoglobin. In this study, we demonstrate anti-platelet activity of nitrite at doses achievable through dietary interventions with comparison to similar doses with other NO donating agents. Unlike other NO donating agents, nitrite activity is shown to be potentiated in the presence of red blood cells in hypoxic conditions. We also show that nitrite reduces calcium associated loss of phospholipid asymmetry that is associated with increased red cell adhesion, and that red cell deformability is also improved. We show that nitrite inhibits red cell adhesion in a microfluidic flow-channel assay after endothelial cell activation. In further investigations, we show that leukocyte and platelet adhesion is blunted in nitrite-fed wild type mice compared to control after either lipopolysaccharide- or hemolysis-induced inflammation. Moreover, we demonstrate that nitrite treatment results in a reduction in adhesion of circulating blood cells and reduced red blood cell hemolysis in humanized transgenic sickle cell mice subjected to local hypoxia. These data suggest that nitrite is an effective anti-platelet and anti-adhesion agent that is activated by red blood cells, with enhanced potency under physiological hypoxia and in venous blood that may be useful therapeutically.

Transfusion of older stored blood worsens outcomes in canines depending on the presence and severity of pneumonia.

BACKGROUND: In experimental pneumonia we found that transfused older blood increased mortality and lung injury that was associated with increased in vivo hemolysis and elevated plasma cell-free hemoglobin (CFH), non-transferrin-bound iron (NTBI), and plasma labile iron (PLI) levels. In this study, we additionally analyze identically treated animals that received lower or higher bacterial doses. STUDY DESIGN AND METHODS: Two-year-old purpose-bred beagles (n = 48) challenged intrabronchially with Staphylococcus aureus (0 [n = 8], 1.0 × 10(9) [n = 8], 1.25 × 10(9) [n = 24], and ≥1.5 × 10(9) [n = 8] colony-forming units/kg) were exchange transfused with either 7- or 42-day-old canine universal donor blood (80 mL/kg in four divided doses). RESULTS: The greater increases in CFH with older blood over days after exchange proved relatively independent of bacterial dose. The lesser increases in CFH observed with fresher blood were bacterial dose dependent potentially related to bacterial hemolysins. Without bacterial challenge, levels of CFH, NTBI, and PLI were significantly higher with older versus fresher blood transfusion but there was no significant measurable injury. With higher-dose bacterial challenge, the elevated NTBI and PLI levels declined more rapidly and to a greater extent after transfusion with older versus fresher blood, and older blood was associated with significantly worse shock, lung injury, and mortality. CONCLUSION: The augmented in vivo hemolysis of transfused older red blood cells (RBCs) appears to result in excess plasma CFH and iron release, which requires the presence of established infection to worsen outcome. These data suggest that transfused older RBCs increase the risks from infection in septic subjects.

Washing older blood units before transfusion reduces plasma iron and improves outcomes in experimental canine pneumonia.

In a randomized controlled blinded trial, 2-year-old purpose-bred beagles (n = 24), with Staphylococcus aureus pneumonia, were exchanged-transfused with either 7- or 42-day-old washed or unwashed canine universal donor blood (80 mL/kg in 4 divided doses). Washing red cells (RBC) before transfusion had a significantly different effect on canine survival, multiple organ injury, plasma iron, and cell-free hemoglobin (CFH) levels depending on the age of stored blood (all, P < .05 for interactions). Washing older units of blood improved survival rates, shock score, lung injury, cardiac performance and liver function, and reduced levels of non-transferrin bound iron and plasma labile iron. In contrast, washing fresh blood worsened all these same clinical parameters and increased CFH levels. Our data indicate that transfusion of fresh blood, which results in less hemolysis, CFH, and iron release, is less toxic than transfusion of older blood in critically ill infected subjects. However, washing older blood prevented elevations in plasma circulating iron and improved survival and multiple organ injury in animals with an established pulmonary infection. Our data suggest that fresh blood should not be washed routinely because, in a setting of established infection, washed RBC are prone to release CFH and result in worsened clinical outcomes.

Nitroxyl accelerates the oxidation of oxyhemoglobin by nitrite.

Angeli's salt (Na2N2O3) decomposes into nitroxyl (HNO) and nitrite (NO2(-)), compounds of physiological and therapeutic interest for their impact on biological signaling both through nitric oxide and nitric oxide independent pathways. Both nitrite and HNO oxidize oxygenated hemoglobin to methemoglobin. Earlier work has shown that HNO catalyzes the reduction of nitrite by deoxygenated hemoglobin. In this work, we have shown that HNO accelerates the oxidation of oxygenated hemoglobin by NO2(-). We have demonstrated this HNO mediated acceleration of the nitrite/oxygenated hemoglobin reaction with oxygenated hemoglobin being in excess to HNO and nitrite (as would be found under physiological conditions) by monitoring the formation of methemoglobin in the presence of Angeli's salt with and without added NO2(-). In addition, this acceleration has been demonstrated using the HNO donor 4-nitrosotetrahydro-2H-pyran-4-yl pivalate, a water-soluble acyloxy nitroso compound that does not release NO2(-) but generates HNO in the presence of esterase. This HNO donor was used both with and without NO2(-) and acceleration of the NO2(-) induced formation of methemoglobin was observed. We found that the acceleration was not substantially affected by catalase, superoxide dismutase, c-PTIO, or IHP, suggesting that it is not due to formation of extramolecular peroxide, NO2 or H2O2, or to modulation of allosteric properties. In addition, we found that the acceleration is not likely to be related to HNO binding to free reduced hemoglobin, as we found HNO binding to reduced hemoglobin to be much weaker than has previously been proposed. We suggest that the mechanism of the acceleration involves local propagation of autocatalysis in the nitrite-oxygenated Hb reaction. This acceleration of the nitrite oxyhemoglobin reaction could affect studies aimed at understanding physiological roles of HNO and perhaps nitrite and use of these agents in therapeutics such as hemolytic anemias, heart failure, and ischemia reperfusion injury.

Angeli's salt counteracts the vasoactive effects of elevated plasma hemoglobin.

Plasma hemoglobin (Hb) released during intravascular hemolysis has been associated with numerous deleterious effects that may stem from increased nitric oxide (NO) scavenging, but has also been associated with reactive oxygen species generation and platelet activation. Therapies that convert plasma oxyHb to metHb, or metHb to iron-nitrosyl Hb, could be beneficial because these species do not scavenge NO. In this study, we investigated the effects of Angeli's salt (AS; sodium α-oxyhyponitrite, Na2N2O3), a nitroxyl (HNO) and nitrite (NO2(-)) donor, on plasma Hb oxidation and formation of iron-nitrosyl Hb from metHb and on the vasoactivity of plasma Hb. We hypothesized that AS could ameliorate hemolysis-associated pathology via its preferential reactivity with plasma Hb, as opposed to red-cell-encapsulated Hb, and through its intrinsic vasodilatory activity. To test this hypothesis, we infused (n=3 per group) (1) cell-free Hb and AS, (2) cell-free Hb+0.9% NaCl, (3) AS+3% albumin, and (4) 3% albumin+0.9% NaCl (colloid controls for Hb and AS, respectively) in a canine model. Co-infusion of AS and cell-free Hb led to preferential conversion of plasma Hb to metHb, but the extent of conversion was lower than anticipated based on the in vivo concentration of AS relative to plasma Hb. This lower metHb yield was probably due to reactions of nitroxyl-derived AS with plasma components such as thiol-containing compounds. From a physiological and therapeutic standpoint, the infusion of Hb alone led to significant increases in mean arterial pressure (p=0.03) and systemic vascular resistance index (p=0.01) compared to controls. Infusion of AS alone led to significant decreases in these parameters and co-infusion of AS along with Hb had an additive effect in reversing the effects of Hb alone on the systemic circulation. Interestingly, in the pulmonary system, the decrease in pressure when AS was added to Hb was significantly less than would have been expected compared to the effects of Hb and AS alone, suggesting that inactivation of scavenging with AS reduced the direct vasodilatory effects of AS on the vasculature. We also found that AS reduced platelet activation when administered to whole blood in vitro. These data suggest that AS-like compounds could serve as therapeutic agents to counteract the negative vasoconstrictive consequences of hemolysis that occur in hemolytic anemias, transfusion of stored blood, and other diseases. Increases in metHb in the red blood cell, the potential of AS for neurotoxicity, and hypotension would need to be carefully monitored in a clinical trial.

Effects of a single sickling event on the mechanical fragility of sickle cell trait erythrocytes.

Hemolysis contributes to the pathology associated with sickle cell disease. However, the mechanism of hemolysis or relative contribution of sickling due to hemoglobin (Hb) polymerization vs. oxidative damage remains unknown. Earlier studies aimed at deciphering the relative importance of these two mechanisms have been complicated by the fact that sickle red cells (SS) have already been affected by multiple rounds of sickling and oxidative damage before they are collected. In our study, we examine the mechanical fragility of sickle cell trait cells, which do not sickle in vivo, but can be made to do so in vitro. Thus, our novel approach explores the effects of sickle Hb polymerization on cells that have never been sickled before. We find that the mechanical fragility of these cells increases dramatically after a single sickling event, suggesting that a substantial amount of hemolysis in vivo probably occurs in polymer-containing cells.

A novel role for cytochrome c: Efficient catalysis of S-nitrosothiol formation.

Although S-nitrosothiols are regarded as important elements of many NO-dependent signal transduction pathways, the physiological mechanism of their formation remains elusive. Here, we demonstrate a novel mechanism by which cytochrome c may represent an efficient catalyst of S-nitrosation in vivo. In this mechanism, initial binding of glutathione to ferric cytochrome c is followed by reaction of NO with this complex, yielding ferrous cytochrome c and S-nitrosoglutathione (GSNO). We show that when submitochondrial particles or cell lysates are exposed to NO in the presence of cytochrome c, there is a robust formation of protein S-nitrosothiols. In the case of submitochondrial particles protein S-nitrosation is paralleled by an inhibition of mitochondrial complex I. These observations raise the possibility that cytochrome c is a mediator of S-nitrosation in biological systems, particularly during hypoxia, and that release of cytochrome c into the cytosol during apoptosis potentially releases a GSNO synthase activity that could modulate apoptotic signaling.

Plasma nitrite response in older women to a physical function test.

BACKGROUND AND AIMS: Nitric oxide (NO) may play a critical role in facilitating the delivery of blood to active skeletal muscle, ultimately impacting functional health in older adults. Plasma nitrite is a useful marker of vascular NO bioavailability. The aim of the current investigation was to examine the effect of a widely used physical function test on plasma nitrite concentrations in older adults. METHODS: Venous blood was drawn before, immediately following, and 10 minutes following the completion of a 400-m walk test. Blood samples were added to heparin and frozen for subsequent analysis of nitrite levels using chemiluminescence. RESULTS: Twenty six (79±4 yrs) women participated in this study. Plasma nitrite levels decreased approximately 22% from baseline following a 400-m walk. Percent change in plasma nitrite was related to walking speed (r=-0.550, p=0.004). CONCLUSIONS: These data suggest an alteration in plasma nitrite concentration following a functional test which may impact functional health.

Distinct functional characteristics of the lateral/basolateral amygdala GABAergic system in C57BL/6J and DBA/2J mice.

It is generally understood that genetic mechanisms contribute to pathological anxiety and that C57BL/6 (B6) and DBA/2J (D2) mice, inbred strains differing markedly in their anxiety-like behaviors, may represent a model system to study these contributions. Because lateral/basolateral amygdala (BLA) GABA(A) receptors help regulate anxiety-like behaviors, we have tested the hypothesis that differences in receptor function/expression may be related to strain-specific differences in experimentally measured anxiety. First, we demonstrated that anxiety-like behaviors in two separate assays were more substantial in D2 mice. Then, using whole-cell electrophysiology of isolated neurons, we found that D2 BLA neurons expressed significantly greater GABA-gated responses than B6 BLA neurons. This was specific for GABA(A) receptors, because N-methyl-d-aspartate-gated responses were similar between strains. At the molecular level, this increased GABA(A) function was associated with higher levels of alpha 2 subunit mRNA expression in D2 BLA. Finally, to understand the ramifications of these functional and molecular biological differences, we examined both electrically evoked GABAergic responses and spontaneous synaptic currents using whole-cell recordings with in vitro slice preparations. Presynaptic GABAergic function was more robust in D2 compared with B6 slices. Together, our findings suggest that genetic mechanisms differentially represented in these two inbred mouse strains lead to robust differences in pre- and postsynaptic aspects of amygdala GABAergic function.