Alpha1-antitrypsin improves survival in murine abdominal sepsis model by decreasing inflammation and sequestration of free heme.

Background: Excessive inflammation, hemolysis, and accumulation of labile heme play an essential role in the pathophysiology of multi-organ dysfunction syndrome (MODS) in sepsis. Alpha1-antitrypsin (AAT), an acute phase protein with heme binding capacity, is one of the essential modulators of host responses to inflammation. In this study, we evaluate the putative protective effect of AAT against MODS and mortality in a mouse model of polymicrobial abdominal sepsis. Methods: Polymicrobial abdominal sepsis was induced in C57BL/6N mice by cecal ligation and puncture (CLP). Immediately after CLP surgery, mice were treated intraperitoneally with three different forms of human AAT-plasma-derived native (nAAT), oxidized nAAT (oxAAT), or recombinant AAT (recAAT)-or were injected with vehicle. Sham-operated mice served as controls. Mouse survival, bacterial load, kidney and liver function, immune cell profiles, cytokines/chemokines, and free (labile) heme levels were assessed. In parallel, in vitro experiments were carried out with resident peritoneal macrophages (MPMΦ) and mouse peritoneal mesothelial cells (MPMC). Results: All AAT preparations used reduced mortality in septic mice. Treatment with AAT significantly reduced plasma lactate dehydrogenase and s-creatinine levels, vascular leakage, and systemic inflammation. Specifically, AAT reduced intraperitoneal accumulation of free heme, production of cytokines/chemokines, and neutrophil infiltration into the peritoneal cavity compared to septic mice not treated with AAT. In vitro experiments performed using MPMC and primary MPMΦ confirmed that AAT not only significantly decreases lipopolysaccharide (LPS)-induced pro-inflammatory cell activation but also prevents the enhancement of cellular responses to LPS by free heme. In addition, AAT inhibits cell death caused by free heme in vitro. Conclusion: Data from the septic CLP mouse model suggest that intraperitoneal AAT treatment alone is sufficient to improve sepsis-associated organ dysfunctions, preserve endothelial barrier function, and reduce mortality, likely by preventing hyper-inflammatory responses and by neutralizing free heme.

Free heme and hemopexin in acute kidney injury after cardiopulmonary bypass and transient renal ischemia.

Free heme is released from hemoproteins during hemolysis or ischemia reperfusion injury and can be pro-inflammatory. Most studies on nephrotoxicity of hemolysis-derived proteins focus on free hemoglobin (fHb) with heme as a prosthetic group. Measurement of heme in its free, non-protein bound, form is challenging and not commonly used in clinical routine diagnostics. In contrast to fHb, the role of free heme in acute kidney injury (AKI) after cardiopulmonary bypass (CPB) surgery is unknown. Using an apo-horseradish peroxidase-based assay, we identified free heme during CPB surgery as predictor of AKI in patients undergoing cardiac valve replacement (n = 37). Free heme levels during CPB surgery correlated with depletion of hemopexin (Hx), a heme scavenger-protein. In mice, the impact of high levels of circulating free heme on the development of AKI following transient renal ischemia and the therapeutic potential of Hx were investigated. C57BL/6 mice were subjected to bilateral renal ischemia/reperfusion injury for 15 min which did not cause AKI. However, additional administration of free heme in this model promoted overt AKI with reduced renal function, increased renal inflammation, and reduced renal perfusion on functional magnetic resonance imaging. Hx treatment attenuated AKI. Free heme administration to sham operated control mice did not cause AKI. In conclusion, free heme is a predictor of AKI in CPB surgery patients and promotes AKI in transient renal ischemia. Depletion of Hx in CPB surgery patients and attenuation of AKI by Hx in the in vivo model encourage further research on Hx therapy in patients with increased free heme levels during CPB surgery.

Mitigation of therapeutic anti-CD38 antibody interference with fab fragments: How well does it perform?

BACKGROUND: Administration of anti-CD38 antibodies is a state-of-the-art therapy for patients diagnosed with multiple myeloma (MM). However, this treatment frequently leads to pan-agglutination of red blood cells (RBCs) in patients' serological testing making accurate blood typing and timely transfusion of compatible blood a challenging effort. The antigen masking indirect antiglobulin test (AMIAT) is an approach to address this diagnostic challenge. STUDY DESIGN AND METHODS: A new reagent, called DaraEx plus, uses anti-CD38 Fab fragments to mitigate the anti-CD38 antibody interference in serological assays by masking CD38 on the cell surface. Its performance is extensively examined with commercial sera as well as with patient samples, and compared to the current standard method using dithiothreitol (DTT), which denatures the CD38 antigens on test panel erythrocytes. RESULTS: In the Bio-Rad ID System, DaraEx plus effectively mitigated the interference caused by anti-CD38 antibodies in 86% of patient samples tested while DTT was successful in only 68%. Moreover, there was no negative influence on DTT-sensitive blood group systems such as KEL upon DaraEx plus treatment. The agglutination reactions of all tested anti-CD38 antibodies (Daratumumab, Felzartamab, and Isatuximab) were inhibited by DaraEx plus. The treatment was successful only if DaraEx plus was added to the test cells before the sample. Some of the other gel card systems tested showed background reactions with DaraEx plus-treated cells. CONCLUSION: DaraEx plus treatment is straightforward and quick to perform. In the Bio-Rad ID System, it is superior to DTT treatment in the prevention of anti-CD38 antibody interference.

Pleural CD14+ monocytes/macrophages of healthy adolescents show a high expression of metallothionein family genes.

Nowadays laparoscopic interventions enable the collection of resident macrophage populations out of the human cavities. We employed this technique to isolate pleural monocytes/macrophages from healthy young adults who underwent a correction of pectus excavatum. High quality CD14+ monocytes/macrophages (plMo/Mφ) were used for RNA-sequencing (RNA-seq) in comparison with human monocyte-derived macrophages (MDM) natural (MDM-0) or IL-4-polarized (MDM-IL4). Transcriptome analysis revealed 7166 and 7076 differentially expressed genes (DEGs) in plMo/Mφ relative to natural MDM-0 and polarized MDM-IL4, respectively. The gene set enrichment analysis, which was used to compare RNA-seq data from plMo/Mφ with single-cell (scRNA-seq) data online from human bronchial lavage macrophages, showed that plMo/Mφs are characterized by a high expression of genes belonging to the metallothionein (MT) family, and that the expression of these genes is significantly higher in plMo/Mφ than in MDM-0 or MDM-IL4. Our results provide additional insights on high MTs-expressing macrophage subsets, which seem to be present not only in bronchial lavage of healthy adults or in pleural exudates of lung cancer patients but also in pleural fluid of healthy young adults. Macrophage subsets expressing high MTs may have specific roles in lung defense, repair, and homeostasis, and require further investigations.

Cell-Free Hemoglobin in Acute Kidney Injury after Lung Transplantation and Experimental Renal Ischemia/Reperfusion.

Cell-free hemoglobin (CFH), a pro-oxidant and cytotoxic compound that is released in hemolysis, has been associated with nephrotoxicity. Lung transplantation (LuTx) is a clinical condition with a high incidence of acute kidney injury (AKI). In this study, we investigated the plasma levels of CFH and haptoglobin, a CFH-binding serum protein, in prospectively enrolled LuTx patients (n = 20) with and without AKI. LuTx patients with postoperative AKI had higher CFH plasma levels at the end of surgery compared with no-AKI patients, and CFH correlated with serum creatinine at 48 h. Moreover, CFH levels inversely correlated with haptoglobin levels, which were significantly reduced at the end of surgery in LuTx patients with AKI. Because multiple other factors can contribute to AKI development in the complex clinical setting of LuTx, we next investigated the role of exogenous CFH administration in a mouse model of mild bilateral renal ischemia reperfusion injury (IRI). Exogenous administration of CFH after reperfusion caused overt AKI with creatinine increase, tubular injury, and enhanced markers of renal inflammation compared with vehicle-treated animals. In conclusion, CFH is a possible factor contributing to postoperative AKI after LuTx and promotes AKI in an experimental model of mild transient renal ischemia. Targeting CFH might be a therapeutic option to prevent AKI after LuTx.

A Single Oral Dose of Diclofenac Causes Transition of Experimental Subclinical Acute Kidney Injury to Chronic Kidney Disease.

Nephrotoxic drugs can cause acute kidney injury (AKI) and analgesic nephropathy. Diclofenac is potentially nephrotoxic and frequently prescribed for pain control. In this study, we investigated the effects of single and repetitive oral doses of diclofenac in the setting of pre-existing subclinical AKI on the further course of AKI and on long-term renal consequences. Unilateral renal ischemia-reperfusion injury (IRI) for 15 min was performed in male CD1 mice to induce subclinical AKI. Immediately after surgery, single oral doses (100 mg or 200 mg) of diclofenac were administered. In a separate experimental series, repetitive treatment with 100 mg diclofenac over three days was performed after IRI and sham surgery. Renal morphology and pro-fibrotic markers were investigated 24 h and two weeks after the single dose and three days after the repetitive dose of diclofenac treatment using histology, immunofluorescence, and qPCR. Renal function was studied in a bilateral renal IRI model. A single oral dose of 200 mg, but not 100 mg, of diclofenac after IRI aggravated acute tubular injury after 24 h and caused interstitial fibrosis and tubular atrophy two weeks later. Repetitive treatment with 100 mg diclofenac over three days aggravated renal injury and caused upregulation of the pro-fibrotic marker fibronectin in the setting of subclinical AKI, but not in sham control kidneys. In conclusion, diclofenac aggravated renal injury in pre-existing subclinical AKI in a dose and time-dependent manner and already a single dose can cause progression to chronic kidney disease (CKD) in this model.

An Antibody-Aptamer-Hybrid Lateral Flow Assay for Detection of CXCL9 in Antibody-Mediated Rejection after Kidney Transplantation.

Chronic antibody-mediated rejection (AMR) is a key limiting factor for the clinical outcome of a kidney transplantation (Ktx), where early diagnosis and therapeutic intervention is needed. This study describes the identification of the biomarker CXC-motif chemokine ligand (CXCL) 9 as an indicator for AMR and presents a new aptamer-antibody-hybrid lateral flow assay (hybrid-LFA) for detection in urine. Biomarker evaluation included two independent cohorts of kidney transplant recipients (KTRs) from a protocol biopsy program and used subgroup comparisons according to BANFF-classifications. Plasma, urine and biopsy lysate samples were analyzed with a Luminex-based multiplex assay. The CXCL9-specific hybrid-LFA was developed based upon a specific rat antibody immobilized on a nitrocellulose-membrane and the coupling of a CXCL9-binding aptamer to gold nanoparticles. LFA performance was assessed according to receiver operating characteristic (ROC) analysis. Among 15 high-scored biomarkers according to a neural network analysis, significantly higher levels of CXCL9 were found in plasma and urine and biopsy lysates of KTRs with biopsy-proven AMR. The newly developed hybrid-LFA reached a sensitivity and specificity of 71% and an AUC of 0.79 for CXCL9. This point-of-care-test (POCT) improves early diagnosis-making in AMR after Ktx, especially in KTRs with undetermined status of donor-specific HLA-antibodies.

Genetic BACH1 deficiency alters mitochondrial function and increases NLRP3 inflammasome activation in mouse macrophages.

BTB-and-CNC homologue 1 (BACH1), a heme-regulated transcription factor, mediates innate immune responses via its functional role in macrophages. BACH1 has recently been shown to modulate mitochondrial metabolism in cancer cells. In the current study, we utilized a proteomics approach and demonstrate that genetic deletion of BACH1 in mouse macrophages is associated with decreased levels of various mitochondrial proteins, particularly mitochondrial complex I. Bioenergetic studies revealed alterations of mitochondrial energy metabolism in BACH1-/- macrophages with a shift towards increased glycolysis and decreased oxidative phosphorylation. Moreover, these cells exhibited enhanced mitochondrial membrane potential and generation of mitochondrial reactive oxygen species (mtROS) along with lower levels of mitophagy. Notably, a higher inducibility of NLRP3 inflammasome activation in response to ATP and nigericin following challenge with lipopolysaccharide (LPS) was observed in BACH1-deficient macrophages compared to wild-type cells. Mechanistically, pharmacological inhibition of mtROS markedly attenuated inflammasome activation. In addition, it is shown that inducible nitric oxide synthase and cyclooxygenase-2, both of which are markedly induced by LPS in macrophages, are directly implicated in BACH1-dependent regulation of NLRP3 inflammasome activation. Taken together, the current findings indicate that BACH1 is critical for immunomodulation of macrophages and may serve as a target for therapeutic approaches in inflammatory disorders.

A new lateral flow assay to detect sIL-2R during T-cell mediated rejection after kidney transplantation.

Kidney is the most frequently transplanted among all solid organs worldwide. Kidney transplant recipients (KTRs) undergo regular follow-up examinations for the early detection of acute rejections. The gold standard for proving a T-cell mediated rejection (TCMR) is a biopsy of the renal graft often occurring as indication biopsy, in parallel to an increased serum creatinine that may indicate deterioration of renal transplant function. The goal of the current work was to establish a lateral flow assay (LFA) for diagnosing acute TCMR to avoid harmful, invasive biopsies. Soluble interleukin-2 (IL-2) receptor (sIl-2R) is a potential biomarker representing the α-subunit of the IL-2 receptor produced by activated T-cells, e.g., after allogen contact. To explore the diagnostic potential of sIL-2R as a biomarker for TCMR and borderline TCMR, plasma and urine samples were collected from three independent KTR cohorts with various distinct histopathological diagnostic findings according to BANFF (containing 112 rsp. 71 rsp. 61 KTRs). Samples were analyzed by a Luminex-based multiplex technique and cut off-ranges were determined. An LFA was established with two specific sIL-2R-antibodies immobilized on a nitrocellulose membrane. A significant association between TCMR, borderline TCMR and sIL-2R in plasma and between TCMR and sIL-2R in urine of KTRs was confirmed using the Mann-Whitney U test. The LFA was tested with sIL-2R-spiked buffer samples establishing a detection limit of 25 pM. The performance of the new LFA was confirmed by analyzing urine samples of the 2nd and 3rd patient cohort with 35 KTRs with biopsy proven TCMRs, 3 KTRs diagnosed with borderline TCMR, 1 mixed AMR/TCMR rsp. AMR/borderline TCMR and 13 control patients with a rejection-free kidney graft proven by protocol biopsies. The new point-of-care assay showed a specificity of 84.6% and sensitivity of 87.5%, and a superior estimated glomerular filtration rate (eGFR) at the time point of biopsy (specificity 30.8%, sensitivity 85%).

Combined Targeted Proteomics and Oxylipin Metabolomics for Monitoring of the COX-2 Pathway.

The important role of inducible cyclooxygenase-2 (COX-2) in several diseases necessitates analytical tools enabling thorough understanding of its modulation. Analysis of a comprehensive oxylipin pattern provides detailed information about changes in enzyme activities. In order to simultaneously monitor gene expression levels, a targeted proteomics method for human COX-2 is developed. With limits of detection and quantification down to 0.25 and 0.5 fmol (on column) the method enables sensitive quantitative analysis via LC-MS/MS within a linear range up to 2.5 pmol. Three housekeeping proteins are included in the method for data normalization. A tiered approach for method development comprised of in silico and experimental steps is described for choosing unique peptides and selective and sensitive SRM transitions while avoiding isobaric interferences. This method combined with a well-established targeted oxylipin metabolomics method allows to investigate the role of COX-2 in the human colon carcinoma cell lines HCT-116, HT-29, and HCA-7. Moreover, the developed methodology is used to demonstrate the time-dependent prostanoid formation and COX-2 enzyme synthesis in lipopolysaccharide-stimulated human primary macrophages. The described approach is a helpful tool which will be further used as standard operation procedure, ultimately aiming at comprehensive targeted proteomics/oxylipin metabolomics strategies to examine the entire arachidonic acid cascade.

Early antihypertensive treatment and ischemia-induced acute kidney injury.

Acute kidney injury (AKI) frequently complicates major surgery and can be associated with hypertension and progress to chronic kidney disease, but reports on blood pressure normalization in AKI are conflicting. In the present study, we investigated the effects of an angiotensin-converting enzyme inhibitor, enalapril, and a soluble epoxide hydrolase inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea (TPPU), on renal inflammation, fibrosis, and glomerulosclerosis in a mouse model of ischemia-reperfusion injury (IRI)-induced AKI. Male CD1 mice underwent unilateral IRI for 35 min. Blood pressure was measured by tail cuff, and mesangial matrix expansion was quantified on methenamine silver-stained sections. Renal perfusion was assessed by functional MRI in vehicle- and TPPU-treated mice. Immunohistochemistry was performed to study the severity of AKI and inflammation. Leukocyte subsets were analyzed by flow cytometry, and proinflammatory cytokines were analyzed by quantitative PCR. Plasma and tissue levels of TPPU and lipid mediators were analyzed by liquid chromatography mass spectrometry. IRI resulted in a blood pressure increase of 20 mmHg in the vehicle-treated group. TPPU and enalapril normalized blood pressure and reduced mesangial matrix expansion. However, inflammation and progressive renal fibrosis were severe in all groups. TPPU further reduced renal perfusion on days 1 and 14. In conclusion, early antihypertensive treatment worsened renal outcome after AKI by further reducing renal perfusion despite reduced glomerulosclerosis.

Interplay of Heme with Macrophages in Homeostasis and Inflammation.

Macrophages are an integral part of the mononuclear phagocyte system that is critical for maintaining immune homeostasis. They play a key role for initiation and modulation of immunological responses in inflammation and infection. Moreover, macrophages exhibit a wide spectrum of tissue-specific phenotypes in steady-state and pathophysiological conditions. Recent clinical and experimental evidence indicates that the ubiquitous compound heme is a crucial regulator of these cells, e.g., in the differentiation of monocytes to tissue-resident macrophages and/ or in activation by inflammatory stimuli. Notably, heme, an iron containing tetrapyrrole, is essential as a prosthetic group of hemoproteins (e.g., hemoglobin and cytochromes), whereas non-protein bound free or labile heme can be harmful via pro-oxidant, pro-inflammatory, and cytotoxic effects. In this review, it will be discussed how the complex interplay of heme with macrophages regulates homeostasis and inflammation via modulating macrophage inflammatory characteristics and/ or hematopoiesis. A particular focus will be the distinct roles of intra- and extracellular labile heme and the regulation of its availability by heme-binding proteins. Finally, it will be addressed how heme modulates macrophage functions via specific transcriptional factors, in particular the nuclear repressor BTB and CNC homologue (BACH)1 and Spi-C.

TLR4 activation alters labile heme levels to regulate BACH1 and heme oxygenase-1 expression in macrophages.

Heme oxygenase (HO)-1, a stress-inducible enzyme that converts heme into carbon monoxide (CO), iron and biliverdin, exerts important anti-inflammatory effects in activated macrophages. HO-1 expression is mainly governed by a mutual interplay between the transcriptional factor NRF2 and the nuclear repressor BTB and CNC homology 1 (BACH1), a heme sensor protein. In the current study we hypothesized that alterations in the levels of intracellular labile heme in macrophages stimulated by lipopolysaccharide (LPS), a prototypical pro-inflammatory Toll-like receptor (TLR)4 agonist, are responsible for BACH1-dependent HO-1 expression. To this end, labile heme was determined in both mouse bone marrow-derived macrophages (mBMDMs) and human monocyte-derived macrophages (hMDMs) using an apo-horseradish peroxidase-based assay. We found that LPS raised the levels of labile heme, depressed BACH1 protein and up-regulated HO-1 in mBMDMs. In contrast, in hMDMs LPS decreased labile heme levels while increasing BACH1 expression and down-regulating HO-1. These effects were abolished by the TLR4 antagonist TAK-242, suggesting that TLR4 activation triggers the signaling cascade leading to changes in the labile heme pool. Studies using mBMDMs from BACH1-/- and NRF2-/- mice revealed that regulation of HO-1 and levels of labile heme after LPS stimulation are strictly dependent on BACH1, but not NRF2. A strong interplay between BACH1-mediated HO-1 expression and intracellular levels of labile heme was also confirmed in hMDMs with siRNA knockdown studies and following inhibition of de novo heme synthesis with succinylacetone. Finally, CORM-401, a compound that liberates CO, counteracted LPS-dependent down-regulation of HO-1 and restored levels of labile heme in hMDMs. In conclusion, alterations of labile heme levels in macrophages following TLR4 stimulation play a crucial role in BACH1-mediated regulation of HO-1 expression.

Human and murine macrophages exhibit differential metabolic responses to lipopolysaccharide - A divergent role for glycolysis.

Macrophages adopt different phenotypes in response to microenvironmental changes, which can be principally classified into inflammatory and anti-inflammatory states. Inflammatory activation of macrophages has been linked with metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis. In contrast to mouse macrophages, little information is available on the link between metabolism and inflammation in human macrophages. In the current report it is demonstrated that lipopolysaccharide (LPS)-activated human peripheral blood monocyte-derived macrophages (hMDMs) fail to undergo metabolic reprogramming towards glycolysis, but rely on oxidative phosphorylation for the generation of ATP. By contrast, activation by LPS led to an increased extracellular acidification rate (glycolysis) and decreased oxygen consumption rate (oxidative phosphorylation) in mouse bone marrow-derived macrophages (mBMDMs). Mitochondrial bioenergetics after LPS stimulation in human macrophages was unchanged, but was markedly impaired in mouse macrophages. Furthermore, treatment with 2-deoxyglucose, an inhibitor of glycolysis, led to cell death in mouse, but not in human macrophages. Finally, glycolysis appeared to be critical for LPS-mediated induction of the anti-inflammatory cytokine interleukin-10 in both human and mouse macrophages. In summary, these findings indicate that LPS-induced immunometabolism in human macrophages is different to that observed in mouse macrophages.

Inhibition of Heme Oxygenase-1 Activity Enhances Wilms Tumor-1-Specific T-Cell Responses in Cancer Immunotherapy.

Wilms tumor protein-1 (WT1) is an attractive target for adoptive T-cell therapy due to its expression in solid tumors and hematologic malignancies. However, T cells recognizing WT1 occur in low frequencies in the peripheral blood of healthy donors, limiting potential therapeutic possibilities. Tin mesoporphyrin (SnMP) is known to inhibit heme oxygenase-1 (HO-1), which has been shown to boost the activation and proliferation of human virus-specific T cells. We analyzed the influence of this effect on the generation of WT1-specific T cells and developed strategies for generating quantities of these cells from healthy donors, sufficient for adoptive T-cell therapies. HO-1 inhibition with SnMP increased WT1-specific T-cell frequencies in 13 (26%) of 50 healthy donors. To assess clinical applicability, we measured the enrichment efficiency of SnMP-treated WT1-specific T cells in response to a WT1-specific peptide pool and a HLA-A*02:01-restricted WT1 peptide by cytokine secretion assay. SnMP treatment resulted in a 28-fold higher enrichment efficacy with equal functionality. In conclusion, pharmacological inhibition of HO-1 activity with SnMP results in more efficient generation of functionally active WT1-specific T cells. This study demonstrates the therapeutic potentials of inhibiting HO-1 with SnMP to enhance antigen-specific T-cell responses in the treatment of cancer patients with WT1-positive disease.

Enhanced activation of interleukin-10, heme oxygenase-1, and AKT in C5aR2-deficient mice is associated with protection from ischemia reperfusion injury-induced inflammation and fibrosis.

Severe ischemia reperfusion injury (IRI) results in rapid complement activation, acute kidney injury and progressive renal fibrosis. Little is known about the roles of the C5aR1 and C5aR2 complement receptors in IRI. In this study C5aR1-/- and C5aR2-/- mice were compared to the wild type in a renal IRI model leading to renal fibrosis. C5a receptor expression, kidney morphology, inflammation, and fibrosis were measured in different mouse strains one, seven and 21 days after IRI. Renal perfusion was evaluated by functional magnetic resonance imaging. Protein abundance and phosphorylation were assessed with high content antibody microarrays and Western blotting. C5aR1 and C5aR2 were increased in damaged tubuli and even more in infiltrating leukocytes after IRI in kidneys of wild-type mice. C5aR1-/- and C5aR2-/- animals developed less IRI-induced inflammation and showed better renal perfusion than wild-type mice following IRI. C5aR2-/- mice, in particular, had enhanced tubular and capillary regeneration with less renal fibrosis. Anti-inflammatory IL-10 and the survival/growth kinase AKT levels were especially high in kidneys of C5aR2-/- mice following IRI. LPS caused bone marrow-derived macrophages from C5aR2-/- mice to release IL-10 and to express the stress response enzyme heme oxygenase-1. Thus, C5aR1 and C5aR2 have overlapping actions in which the kidneys of C5aR2-/- mice regenerate better than those in C5aR1-/- mice following IRI. This is mediated, at least in part, by differential production of IL-10, heme oxygenase-1 and AKT.

The macrophage heme-heme oxygenase-1 system and its role in inflammation.

Heme oxygenase (HO)-1, the inducible isoform of the heme-degrading enzyme HO, plays a critical role in inflammation and iron homeostasis. Regulatory functions of HO-1 are mediated via the catalytic breakdown of heme, which is an iron-containing tetrapyrrole complex with potential pro-oxidant and pro-inflammatory effects. In addition, the HO reaction produces the antioxidant and anti-inflammatory compounds carbon monoxide (CO) and biliverdin, subsequently converted into bilirubin, along with iron, which is reutilized for erythropoiesis. HO-1 is up-regulated by a plethora of stimuli and injuries in most cell types and tissues and provides salutary effects by restoring physiological homeostasis. Notably, HO-1 exhibits critical immuno-modulatory functions in macrophages, which are a major cell population of the mononuclear phagocyte system. Macrophages play key roles as sentinels and regulators of the immune system and HO-1 in these cells appears to be of critical importance for driving resolution of inflammatory responses. In this review, the complex functions and regulatory mechanisms of HO-1 in macrophages will be high-lighted. A particular focus will be the intricate interactions of HO-1 with its substrate heme, which play a contradictory role in distinct physiological and pathophysiological settings. The therapeutic potential of targeted modulation of the macrophage heme-HO-1 system will be discussed in the context of inflammatory disorders.

Alpha1-antitrypsin binds hemin and prevents oxidative activation of human neutrophils: putative pathophysiological significance.

Heme is a ubiquitous compound of human tissues, and it is involved in cellular physiology and metabolism. Once released from the cell, free heme oxidizes to the ferric state (hemin). High levels of hemin can cause oxidative stress and inflammation if not neutralized immediately by specialized scavenger proteins. Human alpha1-antitrypsin (A1AT), an acute-phase glycoprotein and important inhibitor of neutrophil proteases, is also a hemin-binding protein. A short-term exposure of freshly isolated human blood neutrophils to 4 µM hemin results in cell spreading, surface expression of filament protein, vimentin, free radical production, expression of heme oxygenase-1 (HO-1), release of IL-8, and enhanced neutrophil adhesion to human endothelial cells. Consequently, the phosphorylation of protein kinase C (PKC) occurs after 25 min. Under the same experimental conditions, addition of 1 mg/ml A1AT markedly reduces or abolishes neutrophil-activating effects of hemin and prevents PKC phosphorylation. In a mouse model of acute kidney injury (AKI) plus injection of hemin, monotherapy with 4 mg/mouse A1AT significantly lowered serum levels of free hemin at 2 h after surgery. Moreover, a tendency toward lower AKI scores, reduced infiltration of neutrophils, and lower levels of serum chemokine [CXCL1/keratinocyte-derived chemokine (KC)] was observed. Our findings highlight A1AT as a potential serum scavenger of hemin and suggest that the commercial preparations of human plasma A1AT might prove to be useful therapeutics in conditions associated with hemolysis.

A New Immunomodulatory Role for Peroxisomes in Macrophages Activated by the TLR4 Ligand Lipopolysaccharide.

Peroxisomes are proposed to play an important role in the regulation of systemic inflammation; however, the functional role of these organelles in inflammatory responses of myeloid immune cells is largely unknown. In this article, we demonstrate that the nonclassical peroxisome proliferator 4-phenyl butyric acid is an efficient inducer of peroxisomes in various models of murine macrophages, such as primary alveolar and peritoneal macrophages and the macrophage cell line RAW264.7, but not in primary bone marrow-derived macrophages. Further, proliferation of peroxisomes blocked the TLR4 ligand LPS-induced proinflammatory response, as detected by the reduced induction of the proinflammatory protein cyclooxygenase (COX)-2 and the proinflammatory cytokines TNF-α, IL-6, and IL-12. In contrast, disturbing peroxisome function by knockdown of peroxisomal gene Pex14 or Mfp2 markedly increased the LPS-dependent upregulation of the proinflammatory proteins COX-2 and TNF-α. Specifically, induction of peroxisomes did not affect the upregulation of COX-2 at the mRNA level, but it reduced the half-life of COX-2 protein, which was restored by COX-2 enzyme inhibitors but not by proteasomal and lysosomal inhibitors. Liquid chromatography-tandem mass spectrometry analysis revealed that various anti-inflammatory lipid mediators (e.g., docosahexaenoic acid) were increased in the conditioned medium from peroxisome-induced macrophages, which blocked LPS-induced COX-2 upregulation in naive RAW264.7 cells and human primary peripheral blood-derived macrophages. Importantly, LPS itself induced peroxisomes that correlated with the regulation of COX-2 during the late phase of LPS activation in macrophages. In conclusion, our findings identify a previously unidentified role for peroxisomes in macrophage inflammatory responses and suggest that peroxisomes are involved in the physiological cessation of macrophage activation.