Corticosteroid enhances heme oxygenase-1 production by circulating monocytes by up-regulating hemoglobin scavenger receptor and amplifying the receptor-mediated uptake of hemoglobin-haptoglobin complex

This study examined the relationship between steroid treatment and CD163-mediated downstream pathways linked to inflammatory resolution. Twelve patients referred for congenital heart disease surgery were divided into two groups based on the severity of intravascular hemolysis during cardiopulmonary bypass surgery. Patients with severe intravascular hemolysis were administered haptoglobin during the procedure. Flow cytometry indicated a peak in monocyte CD163 expression on post-operative day 1 in both groups. Enhanced and prolonged heme oxygenase-1 (HO-1) mRNA expression levels were observed in patients who received haptoglobin. Binding of hemoglobin-haptoglobin complex (Hb/Hp) to CD163 resulted in significant induction of HO-1 by peripheral blood mononuclear cells after exposure to dexamethasone prior to culture. This effect was significantly inhibited by anti-CD163 antibody. Our results demonstrated up-regulation of CD163 expression on the monocyte surface by steroid treatment. Steroid treatment was suggested to facilitate CD163-mediated endocytosis of hemoglobin to monocytes/macrophages and thereby induce acceleration of HO-1 synthesis.     

Expression of EAAT1 reflects a possible neuroprotective function of reactive astrocytes and activated microglia following human traumatic brain injury

Glutamate-mediated excitotoxicity is known to cause secondary brain damage following stroke and traumatic brain injury (TBI). However, clinical trials using NMDA antagonists failed. Thus, glial excitatory amino acid transporters (EAATs) might be a promising target for therapeutic intervention. METHODS AND RESULTS: We examined expression of EAAT1 (GLAST) and EAAT2 (Glt-1) in 36 TBI cases by immunohistochemistry. Cortical expression of both EAATs decreased rapidly and widespread throughout the brain (in lesional, adjacent and remote areas) following TBI. In the white matter numbers of EAAT1+ parenchymal cells increased 39-fold within 24h (p<0.001) and remained markedly elevated till later stages in the lesion (90-fold, p<0.01) and in peri-lesional regions (86-fold, p<0.01). In contrast, EAAT2+ parenchymal cells and EAAT1+ or EAAT2+ perivascular cells did not increase significantly. Within the first days following TBI mainly activated microglia and thereafter mainly reactive astrocytes expressed EAAT1. Perivascular monocytes and foamy macrophages lacked EAAT1 immunoreactivity. We conclude that following TBI i) loss of cortical EAATs contributes to secondary brain damage, ii) glial EAAT1 expression reflects a potential neuroprotective function of microglia and astrocytes, iii) microglial EAAT1 expression is restricted to an early stage of activation, iv) blood-derived monocytes do not express EAAT1 and v) pharmacological modification of glial EAAT expression might further limit neuronal damage.     

FTY720 attenuates accumulation of EMAP-II+ and MHC-II+ monocytes in early lesions of rat traumatic brain injury

FTY720 (Fingolimod) is a novel type of immunosuppressive agent inhibiting lymphocyte egress from secondary lymphoid tissues thereby causing peripheral lymphopenia. FTY720 can inhibit macrophage infiltration into inflammatory lesions under pathological conditions. FTY720 has been clinically evaluated for prophylaxis of allograft rejection and treatment of multiple sclerosis, showing promising immunosuppressive effects. A robust inflammatory response after traumatic brain injury (TBI) plays an important role in the secondary or delayed injuries of TBI. Here we have investigated by immunohistochemistry in a rat TBI model the effects of FTY720 on early cell accumulation into the inflammatory tissue response and on expression of major histo-compatibility complex class II (MHC-II) and endothelial-monocyte activating polypeptide II (EMAP-II). Accumulation of MHC-II(+) or EMAP-II(+) cells became significant 1 day after injury and continuously increased during the early time periods. Further, double-staining experiments confirmed that the major cellular sources of MHC-II were reactive macrophages, however MHC-II(+) cells only constituted a small subpopulation of reactive macrophages. Immediately after TBI, peripheral administration of FTY720 (1 mg/kg in 1 mL saline, every second day) significantly attenuated the accumulation of MHC-II(+) macrophages from Day 1 to 4 and significantly attenuated the accumulation of EMAP-II(+) macrophages/microglia at Day 4. Our findings show that FTY720 attenuates early accumulation of EMAP-II(+) and MHC-II(+) reactive monocytes following TBI, indicating that FTY720 might be a drug candidate to inhibit brain inflammatory reaction following TBI.     

Macrophages are comprised of resident brain microglia not infiltrating peripheral monocytes acutely after neonatal stroke

Macrophages can be both beneficial and detrimental after CNS injury. We previously showed rapid accumulation of macrophages in injured immature brain acutely after ischemia-reperfusion. To determine whether these macrophages are microglia or invading monocytes, we subjected post-natal day 7 (P7) rats to transient 3 h middle cerebral artery (MCA) occlusion and used flow cytometry at 24 and 48 h post-reperfusion to distinguish invading monocytes (CD45high/CD11b+) from microglia (CD45low/medium/CD11b+). Inflammatory cytokines and chemokines were determined in plasma, injured and contralateral tissue 1-24 h post-reperfusion using ELISA-based cytokine multiplex assays. At 24 h, the number of CD45+/CD11b+ cells increased 3-fold in injured compared to uninjured brain tissue and CD45 expression shifted from low to medium with less than 10% of the population expressing CD45high. MCA occlusion induced rapid and transient asynchronous increases in the pro-inflammatory cytokine IL-beta and chemokines cytokine-induced neutrophil chemoattractant protein 1 (CINC-1) and monocyte-chemoattractant protein 1 (MCP-1), first in systemic circulation and then in injured brain. Double immunofluorescence with cell-type specific markers showed that multiple cell types in the injured brain produce MCP-1. Our findings show that despite profound increases in MCP-1 in injured regions, monocyte infiltration is low and the majority of macrophages in acutely injured regions are microglia.     

The reaction of hydrogen peroxide with hemoglobin induces extensive alpha-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger pathways

Cell-free hemoglobin (Hb) enhances the oxidation-related toxicity associated with inflammation, ischemia, and hemolytic disorders. Hb is highly vulnerable to oxidative damage, and irreversible structural changes involving iron/heme oxidation, heme-adduct products, and amino acid oxidation have been reported. Specific structural features of Hb, such as unconstrained alpha-chains and molecular size, determine the efficiency of interactions between the endogenous Hb scavengers haptoglobin (Hp) and CD163. Using HPLC, mass spectrometry, and Western blotting, we show that H(2)O(2)-mediated Hb oxidation results in the formation of covalently stabilized globin multimers, with prominent intramolecular crosslinking between alpha-globin chains. These structural alterations are associated with reduced Hp binding, reduced CD163 interaction, and severely impaired endocytosis of oxidized Hb by the Hp-CD163 pathway. As a result, when exposed to oxidized Hb, CD163-positive HEK293 cells and human macrophages do not increase hemeoxygenase-1 (HO-1) expression, the physiological anti-oxidative macrophage response to Hb exposure. Failed Hb clearance, inadequate HO-1 expression, and the subsequent accumulation of oxidatively damaged Hb species might thus contribute to pathologies related to oxidative stress.     

CD163-L1 is an endocytic macrophage protein strongly regulated by mediators in the inflammatory response

CD163-L1 belongs to the group B scavenger receptor cysteine-rich family of proteins, where the CD163-L1 gene arose by duplication of the gene encoding the hemoglobin scavenger receptor CD163 in late evolution. The current data demonstrate that CD163-L1 is highly expressed and colocalizes with CD163 on large subsets of macrophages, but in contrast to CD163 the expression is low or absent in monocytes and in alveolar macrophages, glia, and Kupffer cells. The expression of CD163-L1 increases when cultured monocytes are M-CSF stimulated to macrophages, and the expression is further increased by the acute-phase mediator IL-6 and the anti-inflammatory mediator IL-10 but is suppressed by the proinflammatory mediators IL-4, IL-13, TNF-α, and LPS/IFN-γ. Furthermore, we show that CD163-L1 is an endocytic receptor, which internalizes independently of cross-linking through a clathrin-mediated pathway. Two cytoplasmic splice variants of CD163-L1 are differentially expressed and have different subcellular distribution patterns. Despite its many similarities to CD163, CD163-L1 does not possess measurable affinity for CD163 ligands such as the haptoglobin-hemoglobin complex or various bacteria. In conclusion, CD163-L1 exhibits similarity to CD163 in terms of structure and regulated expression in cultured monocytes but shows clear differences compared with the known CD163 ligand preferences and expression pattern in the pool of tissue macrophages. We postulate that CD163-L1 functions as a scavenger receptor for one or several ligands that might have a role in resolution of inflammation.     

Administration of DHA Reduces Endoplasmic Reticulum Stress-Associated Inflammation and Alters Microglial or Macrophage Activation in Traumatic Brain Injury

We investigated the effects of the administration of docosahexaenoic acid (DHA) post-traumatic brain injury (TBI) on reducing neuroinflammation. TBI was induced by cortical contusion injury in Sprague Dawley rats. Either DHA (16 mg/kg in dimethyl sulfoxide) or vehicle dimethyl sulfoxide (1 ml/kg) was administered intraperitonially at 5 min after TBI, followed by a daily dose for 3 to 21 days. TBI triggered activation of microglia or macrophages, detected by an increase of Iba1 positively stained microglia or macrophages in peri-lesion cortical tissues at 3, 7, and 21 days post-TBI. The inflammatory response was further characterized by expression of the proinflammatory marker CD16/32 and the anti-inflammatory marker CD206 in Iba1⁺ microglia or macrophages. DHA-treated brains showed significantly fewer CD16/32⁺ microglia or macrophages, but an increased CD206⁺ phagocytic microglial or macrophage population. Additionally, DHA treatment revealed a shift in microglial or macrophage morphology from the activated, amoeboid-like state into the more permissive, surveillant state. Furthermore, activated Iba1⁺ microglial or macrophages were associated with neurons expressing the endoplasmic reticulum (ER) stress marker CHOP at 3 days post-TBI, and the administration of DHA post-TBI concurrently reduced ER stress and the associated activation of Iba1⁺ microglial or macrophages. There was a decrease in nuclear translocation of activated nuclear factor kappa-light-chain-enhancer of activated B cells protein at 3 days in DHA-treated tissue and reduced neuronal degeneration in DHA-treated brains at 3, 7, and 21 days after TBI. In summary, our study demonstrated that TBI mediated inflammatory responses are associated with increased neuronal ER stress and subsequent activation of microglia or macrophages. DHA administration reduced neuronal ER stress and subsequent association with microglial or macrophage polarization after TBI, demonstrating its therapeutic potential to ameliorate TBI-induced cellular pathology.     

Lesional Accumulation of CD163-Expressing Cells in the Gut of Patients with Inflammatory Bowel Disease

Monocytes/macrophages displaying different markers of activation/differentiation infiltrate the inflamed gut of patients with inflammatory bowel diseases (IBD), but the role that each monocyte/macrophage subpopulation plays in the pathogenesis of IBD is not fully understood. The hemoglobin scavenger receptor CD163, a specific marker of monocytes/macrophages, has been associated with either anti-inflammatory or inflammatory functions of macrophages in several pathologies. In this study we examined the tissue distribution and function of CD163-expressing monocytes/macrophages in IBD. CD163 RNA and protein expression was more pronounced in IBD in comparison to normal controls, with no significant difference between Crohn''s disease and Ulcerative colitis. In IBD, over-expression of CD163 was restricted to areas with active inflammation and not influenced by current therapy. Immunohistochemical analysis confirmed the accumulation of CD163-expressing cells in IBD, mostly around and inside blood vessels, thus suggesting that these cells are partly recruited from the systemic circulation. Indeed, FACS analysis of circulating mononuclear cells showed that the fractions of CD163-positive monocytes were increased in IBD patients as compared to controls. Functionally, interleukin-6 up-regulated CD163 expression in lamina propria mononuclear cells and mucosal explants of normal subjects. In IBD blood and mucosal cell cultures, cross-linking of CD163 with a specific monoclonal anti-CD163 antibody enhanced tumor necrosis factor-α synthesis. These findings indicate that IBD mucosa is abundantly infiltrated with CD163-positive cells, which could contribute to amplify the inflammatory cytokine response.     

Regulation of surface CD163 expression and cellular effects of receptor mediated hemoglobin-haptoglobin uptake on human monocytes and macrophages.

Local dysregulation of iron metabolism is suggested to contribute to atherosclerotic lesion development through hemoglobin scavenging pathways. We evaluated the effects of CD163-mediated uptake of hemoglobin-haptoglobin (HbHp) complexes on surface CD163 and intracellular heme oxygenase-1 expression and the secretion of pro- and antiinflammatory cytokines by macrophages. We found that increased availability of HbHp complexes triggers the upregulation of surface CD163, and also results in a dose-dependent secretion of IL-6 and IL-10.     

The porcine 2A10 antigen is homologous to human CD163 and related to macrophage differentiation

The mAb 2A10 recognizes a 120-kDa protein with sequence homology to the human CD163 and whose expression is restricted to the cells of the porcine monocyte/macrophage lineage. While most of tissue macrophages express high levels of 2A10 Ag, bone marrow cells and a subset of blood monocytes are negative for this marker. The percentage of 2A10+ blood monocytes ranges between 5-50% depending on the donor. The phenotypic analysis indicates that these cells are more similar to mature macrophages than 2A10- monocytes. 2A10+ monocytes express higher levels of swine histocompatibility leukocyte Ag II, CD16, and the adhesion molecules very late Ag-4 (CD49d) and LFA-1 (CD11a) than 2A10- monocytes, while CD14 and SWC1 expression is lower. Both monocyte subsets also differ in their functional capabilities. 2A10+ monocytes induce a greater allogeneic response on T lymphocytes than 2A10- cells. LPS-stimulated 2A10+ and 2A10- monocytes both produce proinflammatory cytokines (TNF-alpha and IL-1alpha), but antiinflammatory IL-10 is only detected on the latter population. When 2A10- monocytes were cultured in medium containing pig serum, they acquired some phenotypic features of 2A10+ cells, expressing the 2A10 Ag. In contrast, when they were cultured in the presence of L929 supernatant as a source of GM-CSF, the 2A10 Ag expression remained low, scarcely increasing over basal levels. 2A10+ cells cultured with pig serum developed features that resemble monocyte-derived dendritic cells. These results indicate that 2A10+ monocytes could constitute a cell population in a more advanced maturation stage than 2A10- circulating monocytes.     

Translational event mediates differential production of tumor necrosis factor-alpha in hyaluronan-stimulated microglia and macrophages

Recent evidence has demonstrated that hyaluronan synthase 2 mRNA is up-regulated after brain ischemia. After a cerebral ischemic event, microglia and macrophages are the major inflammatory cells and are activated by hyaluronan (HA). However, it is unclear how these cells compare with regard to HA responsiveness. We show here that peritoneal macrophages and RAW 264.7 macrophages produced more than five- and 10-fold more tumor necrosis factor-alpha (TNF-alpha) than primary microglia and BV-2 microglia, respectively. Antibody blockade study showed that CD44, Toll-like receptor-4 receptor and the receptor for HA-mediated motility were responsible for HA-induced TNF-alpha release. Furthermore, HA induced higher levels of phosphorylated MAPK in RAW 264.7 cells when compared with BV-2 cells. HA-mediated TNF-alpha production required p38 MAPK, extracellular-regulated kinase and c-Jun N-terminal kinase phosphorylation in both cell types. The levels of HA-induced TNF-alpha mRNA expression in BV-2 cells were only twofold lower compared with RAW 264.7 cells, suggesting that a translational event is involved in the differential production of TNF-alpha. Western blot analysis revealed that HA treatment resulted in more rapid phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and more effective dissociation of 4E-BP1 from eukaryotic initiation factor 4E in RAW 264.7 cells than in BV-2 cells. Additionally, HA-induced phosphorylation of 4E-BP1 was dependent on MAPK signaling, indicating that RAW 264.7 cells exhibited higher levels of hyperphosphorylated 4E-BP1 possibly due to the overactivation of MAPK. The results suggest that resident microglia and blood-derived monocytes/macrophages exhibit differential sensitivities in response to extracellular mediators after brain ischemia.     

Elevated Cerebral Cortical CD24 Levels in Patients and Mice with Traumatic Brain Injury: A Potential Negative Role in Nuclear Factor Kappa B/Inflammatory Factor Pathway

Increasing evidence indicates that sterile inflammatory response contributes to secondary brain injury following traumatic brain injury (TBI). However, the specific mechanisms remain largely unknown, as is whether CD24, known as an important regulator in the non-infectious inflammatory response, plays a role in secondary brain injury after TBI. Here, the expression of CD24 was detected in samples from patients with TBI by quantitative real-time polymerase chain reaction (PCR), western blotting, immunohistochemistry and immunofluorescence. RNA interference was used to investigate the effects of CD24 on inflammatory response in a mouse model of TBI. Nuclear factor kappa B (NF-κB) DNA-binding activity was measured by electrophoretic mobility shift assay, and the levels of downstream pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and Interleukin 1β (IL-1β) were detected by real-time PCR. The results indicated that both the mRNA and protein levels of CD24 were markedly elevated after TBI in humans and mice, showing a time-dependent expression. The expression of CD24 could be observed in neurons, astrocytes and microglia in both humans and mice. Meanwhile, downregulation of CD24 significantly induced an increase of NF-κB DNA-binding activity and mRNA levels of TNF-α and IL-1β. These findings indicated that CD24 expression could negatively regulate the NF-κB/inflammatory factor pathway after experimental TBI in mice, thus providing a novel target for therapeutic intervention of TBI.     

L3T4(CD4)-, Lyt-2(CD8)- and Mac-1(CD11b)-phenotypic leukocytes in murine cryptococcal meningoencephalitis

An immunohistological study of L3T4(CD4)+ and LYT-2(CD8)+ lymphocytes, Mac-1(CD11b)+ monocytes and granulocytes in experimental murine cryptococcal meningoencephalitis was conducted. To assess the concomitant inflammatory reaction in an extracerebral site, livers were examined in parallel. Mice were infected i.v. withCryptococcus neoformans, group A/D, and organs were examined immunohistologically for CD4-, CD8- and monocyteand granulocyte-specific CD11b-phenotypic leukocytes over a period of 60 days. Intracerebrally, agglomerations of cryptococci formed pseudocysts that were surrounded by CD4+ and CD8+ lymphocytes at the end of the second week post-infection, followed by the invasion of monocytes and granulocytes into the lesions. After the fourth week post-infection, most of the invaded lesions were transformed into glious scars. Meningitis was usually marked and showed a homogenous distribution of CD4-, CD8- and CD11b-phenotypic cells, with a predominance of monocytes and CD4+ lymphocytes. Inflammatory infiltrates in the liver were found already 4 days post-infection. CD4+ lymphocytes and monocytes were distributed homogenously in the infiltrates, with a lower number of CD8+ lymphocytes being located rather in the periphery of the infiltrates. Comparing leukocyte kinetics in brain and liver, an important observation was the delayed immigration of immune cells at the intracerebral cryptococcal lesions as compared with the liver, and the different migration patterns of T-lymphocyte subgroups and macrophages. These results suggest that there are differential leukocyte migration patterns in the liver and brain following disseminated cryptococcosis. The immunological aspects of the observed leukocyte kinetics are discussed.     

Nitric oxide-mediated tumoricidal activity of murine microglial cells

Experimental metastases in the brain of mice are infiltrated by microglia, and parabiosis experiments of green fluorescent protein (GFP(+)) and GFP(-) mice revealed that these microglia are derived from circulating monocytes (GFP(+), F4/80(+), and CD68(+)). These findings raised the question as to whether microglia (specialized macrophages) possess tumoricidal activity. C8-B4 murine microglia cells were incubated in vitro in medium (control) or in medium containing both lipopolysaccharide and interferon-γ. Control microglia were not tumoricidal against a number of murine and human tumor cells, whereas lipopolysaccharide/interferon-γ-activated microglia lysed murine and human tumor cells by release of nitric oxide. Parallel experiments with murine peritoneal macrophages produced identical results. Neither activated microglia nor activated macrophages lysed nontumorigenic murine or human cells. Collectively, these data demonstrate that brain metastasis-associated microglia are derived from circulating mononuclear cells and exhibit selective and specific tumoricidal activity.     

Ghrelin reduces cerebral ischemic injury in rats by reducing M1 microglia/macrophages

The purpose of this study was to investigate the effect of Ghrelin on the polarization of microglia/ macrophages after cerebral ischemia (CI) in rats. 60 wild-type SD rats were randomly divided into sham group, CI group, CI+Ghrelin group, 20 rats in each group. The modified Longa suture method was used to establish the middle cerebral artery occlusion (MCAO) model in rats. Before surgery, Ghrelin was injected subcutaneously (100 μg/kg, twice a day) for 4 consecutive weeks. After modeling, neurological function scores were performed with three behavioral experiments: mNSS score, Corner test, and Rotarod test, to evaluate the recovery of neurological function after Ghrelin treatment. At the same time, the brain tissues were collected and stained with 2,3,5- triphenyltetrazolium chloride (TTC) to detect the cerebral infarct volume. RT-qPCR was used to detect the expression of TNF-α and IL-1β in the ischemic brain tissue, and the TUNEL staining was used to detect the apoptosis of brain tissue. Flow cytometry was used to detect the percentage of M1 type microglia/macrophages which were isolated by trypsin digestion of fresh cerebral cortex. Then, the Western blotting and immunofluorescence method were used to detect the phosphorylation level of AKT (P-AKT) and AKT. Compared with the CI group, the neurological function of the rats in the CI+Ghrelin group was dramatically improved, and the cerebral infarction area was dramatically reduced. At the same time, the expression of TNF-α and IL-1β in the ischemic brain tissue of rats in the CI+Ghrelin group decreased, and the apoptotic cells in the brain tissue also decreased. Compared with the CI treatment group, the activation of M1 microglia/macrophages in the cortex of the ischemic side of the infarct and the peri-infarct area in the CI+Ghrelin group was dramatically inhibited. At the same time, the ratio of P-AKT/AKT of the brain tissue in the CI+Ghrelin group was dramatically higher than that of the CI group. In the rat cerebral ischemia model, Ghrelin can promote the repair of brain damage and the recovery of neurological function after ischemia. Its mechanism may be related to activating AKT to selectively reduce M1 microglia/macrophages, reducing inflammation and cell apoptosis in brain tissue.Key words: Cerebral ischemia, ghrelin, microglia/macrophage     

即早基因c-fos在THP-1单核巨噬细胞亚型极化中的差异表达*

目的:探讨即早基因c-fos在THP-1巨噬细胞亚型极化过程中的表达变化。方法:运用PMA刺激诱导THP-1单核细胞极化为巨噬细胞,观察c-fos在单核细胞极化过程中的表达变化;在PMA刺激的基础上,分别运用LPS和IL-4诱导THP-1巨噬细胞向M1及M2亚型极化,实时定量PCR及Western blot技术分析刺激24 h时,细胞亚型标记物CD274、CD86和CD163的表达变化,并动态观察诱导极化过程中,c-fos的表达情况。结果:c-fos在PMA刺激THP-1单核细胞分化为巨噬细胞过程中蛋白和mRNA水平显示上调;LPS诱导THP-1巨噬细胞极化为M1型过程中,c-fos蛋白和mRNA水平表达降低,其特异性标记物在24 h呈现出M1型极化的特点(CD86蛋白表达升高,CD274、CD163蛋白表达降低);IL-4诱导THP-1巨噬细胞极化为M2型过程中,c-fos蛋白和mRNA水平表达升高,其特异性标记物在24 h表现出M2型极化的特点(CD86蛋白表达降低,CD274、CD163蛋白表达升高)。结论:c-fos参与了THP-1单核细胞向巨噬细胞极化的过程,并且可能通过抑制巨噬细胞M1亚型形成,促进巨噬细胞向M2亚型极化的作用参与巨噬细胞的亚型极化及其功能调节中。     

The intrathecal CD163-haptoglobin-hemoglobin scavenging system in subarachnoid hemorrhage

Delayed cerebral ischemia resulting from extracellular hemoglobin is an important determinant of outcome in subarachnoid hemorrhage. Hemoglobin is scavenged by the CD163-haptoglobin system in the circulation, but little is known about this scavenging pathway in the human CNS. The components of this system were analyzed in normal cerebrospinal fluid and after subarachnoid hemorrhage. The intrathecal presence of the CD163-haptoglobin-hemoglobin scavenging system was unequivocally demonstrated. The resting capacity of the CD163-haptoglobin-hemoglobin system in the normal CNS was 50 000-fold lower than that of the circulation. After subarachnoid hemorrhage, the intrathecal CD163-haptoglobin-hemoglobin system was saturated, as shown by the presence of extracellular hemoglobin despite detectable haptoglobin. Hemoglobin efflux from the CNS was evident, enabling rescue hemoglobin scavenging by the systemic circulation. Therefore, the CNS is not capable of dealing with significant intrathecal hemolysis. Potential therapeutic options to prevent delayed cerebral ischemia ought to concentrate on augmenting the capacity of the intrathecal CD163-haptoglobin-hemoglobin scavenging system and strategies to encourage hemoglobin efflux from the brain.     

Changes of the GPR17 receptor, a new target for neurorepair, in neurons and glial cells in patients with traumatic brain injury

Unveiling the mechanisms participating in the damage and repair of traumatic brain injury (TBI) is fundamental to develop new therapies. The P2Y-like GPR17 receptor has recently emerged as a sensor of damage and a key actor in lesion remodeling/repair in the rodent brain, but its role in humans is totally unknown. Here, we characterized GPR17 expression in brain specimens from seven intensive care unit TBI patients undergoing neurosurgery for contusion removal and from 28 autoptic TBI cases (and 10 control subjects of matched age and gender) of two university hospitals. In both neurosurgery and autoptic samples, GPR17 expression was strong inside the contused core and progressively declined distally according to a spatio-temporal gradient. Inside and around the core, GPR17 labeled dying neurons, reactive astrocytes, and activated microglia/macrophages. In peri-contused parenchyma, GPR17 decorated oligodendrocyte precursor cells (OPCs) some of which had proliferated, indicating re-myelination attempts. In autoptic cases, GPR17 expression positively correlated with death for intracranial complications and negatively correlated with patients’ post-traumatic survival. Data indicate lesion-specific sequential involvement of GPR17 in the (a) death of irreversibly damaged neurons, (b) activation of microglia/macrophages remodeling the lesion, and (c) activation/proliferation of multipotent parenchymal progenitors (both reactive astrocytes and OPCs) starting repair processes. Data validate GPR17 as a target for neurorepair and are particularly relevant to setting up new therapies for TBI patients.     

Differential expression of CD163 on monocyte subsets in healthy and HIV-1 infected individuals

CD163, a haptoglobin-hemoglobin (Hp-Hb) scavenger receptor, expressed by monocytes and macrophages, is important in resolution of inflammation. Age-related non-AIDS co-morbidities in HIV-infected individuals, particularly dementia and cardiovascular disease, result in part from effects of HIV-1 infection on monocyte and macrophage biology. CD163 co-expression on CD14+CD16++ monocytes has been proposed as a useful biomarker for HIV-1 disease progression and the presence of HIV associated dementia. Here we investigated CD163 expression on monocyte subsets ex vivo, on cultured macrophages, and soluble in plasma, in the setting of HIV-1 infection. Whole blood immunophenotyping revealed CD163 expression on CD14++CD16- monocytes but not on CD14+CD16++ monocytes (P = 0.004), supported by CD163 mRNA levels. Incubation with M-CSF induced CD163 protein expression on CD14+CD16++ monocytes to the same extent as CD14++CD16− monocytes. CD163 expression on CD14++CD16+ monocytes from HIV-infected subjects was significantly higher than from uninfected individuals, with a trend towards increased expression on CD14++CD16− monocytes (P = 0.019 and 0.069 respectively), which is accounted for by HIV-1 therapy including protease inhibitors. Shedding of CD163 was shown to predominantly occur from the CD14++CD16− subset after Ficoll isolation and LPS stimulation. Soluble CD163 concentration in plasma from HIV-1 infected donors was similar to HIV-1 uninfected donors. Monocyte CD163 expression in HIV-1 infected patients showed a complicated relationship with classical measures of disease progression. Our findings clarify technical issues regarding CD163 expression on monocyte subsets and further elucidates its role in HIV-associated inflammation by demonstrating that CD163 is readily lost from CD14++CD16− monocytes and induced in pro-inflammatory CD14+CD16++ monocytes by M-CSF. Our data show that all monocyte subsets are potentially capable of differentiating into CD163-expressing anti-inflammatory macrophages given appropriate stimuli. Levels of CD163 expression on monocytes may be a potential biomarker reflecting efforts by the immune system to resolve immune activation and inflammation in HIV-infected individuals.     

Human Cytomegalovirus Interleukin-10 Polarizes Monocytes toward a Deactivated M2c Phenotype To Repress Host Immune Responses

Several human cytomegalovirus (HCMV) genes encode products that modulate cellular functions in a manner likely to enhance viral pathogenesis. This includes UL111A, which encodes homologs of human interleukin-10 (hIL-10). Depending upon signals received, monocytes and macrophages become polarized to either classically activated (M1 proinflammatory) or alternatively activated (M2 anti-inflammatory) subsets. Skewing of polarization toward an M2 subset may benefit the virus by limiting the proinflammatory responses to infection, and so we determined whether HCMV-encoded viral IL-10 influenced monocyte polarization. Recombinant viral IL-10 protein polarized CD14⁺ monocytes toward an anti-inflammatory M2 subset with an M2c phenotype, as demonstrated by high expression of CD163 and CD14 and suppression of major histocompatibility complex (MHC) class II. Significantly, in the context of productive HCMV infection, viral IL-10 produced by infected cells polarized uninfected monocytes toward an M2c phenotype. We also assessed the impact of viral IL-10 on heme oxygenase 1 (HO-1), which is an enzyme linked with suppression of inflammatory responses. Polarization of monocytes by viral IL-10 resulted in upregulation of HO-1, and inhibition of HO-1 function resulted in a loss of capacity of viral IL-10 to suppress tumor necrosis factor alpha (TNF-α) and IL-1β, implicating HO-1 in viral IL-10-induced suppression of proinflammatory cytokines by M2c monocytes. In addition, a functional consequence of monocytes polarized with viral IL-10 was a decreased capacity to activate CD4⁺ T cells. This study identifies a novel role for viral IL-10 in driving M2c polarization, which may limit virus clearance by restricting proinflammatory and CD4⁺ T cell responses at sites of infection.