Youxi Ai

Neutrophils lacking phosphoinositide 3-kinase γ show loss of directionality during N-formyl-Met-Leu-Phe-induced chemotaxis

Confocal imaging and time-lapsed videomicroscopy were used to study the directionality, motility, rate of cell movement, and morphologies of phosphoinositide 3-kinase γ (PI3K)γ−/− neutrophils undergoing chemotaxis in Zigmond chambers containing N-formyl-Met-Leu-Phe gradients. Most of the PI3Kγ−/− neutrophils failed to translocate up the cheomotactic gradient. A partial reduction in cell motility and abnormal morphologies were also observed. In the wild-type neutrophils, the pleckstrin homology domain-containing protein kinase B (AKT) and F-actin colocalize to the leading edge of polarized neutrophils oriented toward the gradient, which was not observed in PI3Kγ−/− neutrophils. In PI3Kγ−/− neutrophils, AKT staining consistently failed to perfectly overlap with the F-actin. This failure was observed as an F-actin-filled region of 2.3 ± 0.5 μm between AKT and the cell membrane. These data suggest that PI3Kγ regulates neutrophil chemotaxis primarily by controlling the direction of cell migration and the intracellular colocalization of AKT and F-actin to the leading edge.

P47(phox)-deficient NADPH oxidase defect in neutrophils of diabetic mouse strains, C57BL/6J-m db/db and db/+.

Deficiencies in neutrophil NADPH oxidase proteins have been demonstrated in humans with chronic granulomatous disease. However, no spontaneous mutation in murine NADPH oxidase has been reported. In this study we report that neutrophils from the diabetic mouse strains, C57BL/6J‐m heterozygous lean (leprdb/+ ) and homozygous obese (leprdb/db) mice produced no superoxide on stimulation. An absence of intact p47phox but not other oxidase proteins was observed in both mouse strains through the use of immunoblotting. Molecular analysis by reverse transcriptase‐polymerase chain reaction identified three abnormal p47phox mRNA transcripts. Sequencing of genomic DNA of p47phox revealed a point mutation at the –2 position of exon 8, which is consistent with aberrant splicing of the p47phox transcript. These results indicate that the C57BL/6J‐m db/db and db/+ mice are the first spontaneously derived murine model of NADPH oxidase deficiency involving a p47phox mutation. J. Leukoc. Biol. 67: 210–215; 2000.

CCR2+Ly6Chi Inflammatory Monocyte Recruitment Exacerbates Acute Disability Following Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a devastating type of stroke that lacks a specific treatment. An intense immune response develops after ICH, which contributes to neuronal injury, disability, and death. However, the specific mediators of inflammation-induced injury remain unclear. The objective of the present study was to determine whether blood-derived CCR2+Ly6Chi inflammatory monocytes contribute to disability. ICH was induced in mice and the resulting inflammatory response was quantified using flow cytometry, confocal microscopy, and neurobehavioral testing. Importantly, blood-derived monocytes were distinguished from resident microglia by differential CD45 staining and by using bone marrow chimeras with fluorescent leukocytes. After ICH, blood-derived CCR2+Ly6Chi inflammatory monocytes trafficked into the brain, outnumbered other leukocytes, and produced tumor necrosis factor. Ccr2−/− mice, which have few circulating inflammatory monocytes, exhibited better motor function following ICH than control mice. Chimeric mice with wild-type CNS cells and Ccr2−/− hematopoietic cells also exhibited early improvement in motor function, as did wild-type mice after inflammatory monocyte depletion. These findings suggest that blood-derived inflammatory monocytes contribute to acute neurological disability. To determine the translational relevance of our experimental findings, we examined CCL2, the principle ligand for the CCR2 receptor, in ICH patients. Serum samples from 85 patients were collected prospectively at two hospitals. In patients, higher CCL2 levels at 24 h were independently associated with poor functional outcome at day 7 after adjusting for potential confounding variables. Together, these findings suggest that inflammatory monocytes worsen early disability after murine ICH and may represent a therapeutic target for patients.

Abnormal Migration Phenotype of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2−/− Neutrophils in Zigmond Chambers Containing Formyl-Methionyl-Leucyl-Phenylalanine Gradients

Time-lapsed video microscopy and confocal imaging were used to study the migration of wild-type (WT) and mitogen-activated protein kinase-activated protein kinase 2 (MK2)−/− mouse neutrophils in Zigmond chambers containing fMLP gradients. Confocal images of polarized WT neutrophils showed an intracellular gradient of phospho-MK2 from the anterior to the posterior region of the neutrophils. Compared with WT neutrophils, MK2−/− neutrophils showed a partial loss of directionality but higher migration speed. Immunoblotting experiments showed a lower protein level of p38 mitogen-activated protein kinase and a loss of fMLP-induced extracellular signal-related kinase phosphorylation in MK2−/− neutrophils. These results suggest that MK2 plays an important role in the regulation of neutrophil migration and may also affect other signaling molecules.

Leukocyte-specific gene 1 protein (LSP1) is involved in chemokine KC-activated cytoskeletal reorganization in murine neutrophils in vitro

Leukocyte‐specific gene 1 protein (LSP1) is a cytoskeletal‐associated protein of leukocytes that in vitro cross‐links F‐actin into extensively branched bundles of mixed polarity. In this study, we examined chemotaxis and superoxide production in neutrophils prepared from wild‐type (WT) and Lsp1 knockout mice. Compared to WT neutrophils, Lsp1‐/‐ neutrophils showed impairment in both migration speed and chemotaxis direction during chemokine KC‐directed chemotaxis. When examined by confocal microscopy, chemotaxing Lsp1‐/‐ neutrophils showed abnormal morphologies. They had discontinuous primary actin‐rich cortexes and large membrane protrusions. When stimulated by phorbol 12‐myristate 13‐acetate (PMA), Lsp1‐/‐ peritoneal neutrophils produce more superoxide than WT. The data presented suggest that LSP1 plays important roles in the regulation of neutrophil morphology, motility, and superoxide production.

TGF-β1 modulates microglial phenotype and promotes recovery after intracerebral hemorrhage

Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from the rupture of a blood vessel in the brain, leading to a mass of blood within the brain parenchyma. The injury causes a rapid inflammatory reaction that includes activation of the tissue-resident microglia and recruitment of blood-derived macrophages and other leukocytes. In this work, we investigated the specific responses of microglia following ICH with the aim of identifying pathways that may aid in recovery after brain injury. We used longitudinal transcriptional profiling of microglia in a murine model to determine the phenotype of microglia during the acute and resolution phases of ICH in vivo and found increases in TGF-&bgr;1 pathway activation during the resolution phase. We then confirmed that TGF-&bgr;1 treatment modulated inflammatory profiles of microglia in vitro. Moreover, TGF-&bgr;1 treatment following ICH decreased microglial Il6 gene expression in vivo and improved functional outcomes in the murine model. Finally, we observed that patients with early increases in plasma TGF-&bgr;1 concentrations had better outcomes 90 days after ICH, confirming the role of TGF-&bgr;1 in functional recovery from ICH. Taken together, our data show that TGF-&bgr;1 modulates microglia-mediated neuroinflammation after ICH and promotes functional recovery, suggesting that TGF-&bgr;1 may be a therapeutic target for acute brain injury.

α4 Integrin Is a Regulator of Leukocyte Recruitment After Experimental Intracerebral Hemorrhage

Background and Purpose— Intracerebral hemorrhage (ICH) is swiftly followed by an inflammatory response. A key component of this response is the recruitment of leukocytes into the brain, which promotes neurological injury in rodent models. However, the mechanisms by which leukocytes transmigrate across the endothelium into the injured brain are unclear. The present study examines leukocyte adhesion molecules (&agr;4 integrin, L-selectin, and &agr;L&bgr;2 integrin) on 4 leukocyte subtypes to determine which are important for leukocyte recruitment after ICH. Methods— We used the blood injection mouse model of ICH, whereby 25 &mgr;L of blood was injected into the striatum. Flow cytometry was used to quantify leukocyte populations and adhesion molecule expression in brain and blood. An &agr;4 integrin–blocking antibody was administered to evaluate the contribution of &agr;4 integrin in leukocyte migration and neurological injury. Results— &agr;4 integrin was elevated on all leukocyte populations in brain after ICH, whereas L-selectin was unchanged and &agr;L&bgr;2 was increased only on T cells. Antagonism of &agr;4 resulted in decreased leukocyte transmigration and lessened neurobehavioral disability. Conclusions— &agr;4 integrin is an important cell adhesion molecule involved in neuroinflammation after ICH.

Erythrocyte efferocytosis modulates macrophages towards recovery after intracerebral hemorrhage

Macrophages are a source of both proinflammatory and restorative functions in damaged tissue through complex dynamic phenotypic changes. Here, we sought to determine whether monocyte-derived macrophages (MDMs) contribute to recovery after acute sterile brain injury. By profiling the transcriptional dynamics of MDMs in the murine brain after experimental intracerebral hemorrhage (ICH), we found robust phenotypic changes in the infiltrating MDMs over time and demonstrated that MDMs are essential for optimal hematoma clearance and neurological recovery. Next, we identified the mechanism by which the engulfment of erythrocytes with exposed phosphatidylserine directly modulated the phenotype of both murine and human MDMs. In mice, loss of receptor tyrosine kinases AXL and MERTK reduced efferocytosis of eryptotic erythrocytes and hematoma clearance, worsened neurological recovery, exacerbated iron deposition, and decreased alternative activation of macrophages after ICH. Patients with higher circulating soluble AXL had poor 1-year outcomes after ICH onset, suggesting that therapeutically augmenting efferocytosis may improve functional outcomes by both reducing tissue injury and promoting the development of reparative macrophage responses. Thus, our results identify the efferocytosis of eryptotic erythrocytes through AXL/MERTK as a critical mechanism modulating macrophage phenotype and contributing to recovery from ICH.

CX3CR1 Signaling on Monocytes Is Dispensable after Intracerebral Hemorrhage

Intracerebral hemorrhage is a subset of stroke for which there is no specific treatment. The Ly6Chi CCR2+ monocytes have been shown to contribute to acute injury after intracerebral hemorrhage. The other murine monocyte subset expresses CX3CR1 and lower Ly6C levels, and contributes to repair in other disease models. We hypothesized that the Ly6Clo CX3CR1+ monocytes would contribute to recovery after intracerebral hemorrhage. Intracerebral hemorrhage was modeled by blood injection in WT and CX3CR1-null bone marrow chimeras. Neurological outcomes and leukocyte recruitment were quantified at various time points. Functional outcomes were equal at 1, 3, 7, and 14 days after intracerebral hemorrhage in both genotypes. No differences were observed in leukocyte recruitment between genotypes on either 3 or 7 days after intracerebral hemorrhage. A few hundred Ly6Clo monocytes were found in the ipsilateral hemisphere in each genotype and they did not change over time. Peripherally derived CX3CR1+ monocytes were observed in the perihematomal brain 7 and 14 days after intracerebral hemorrhage. Our data suggests CX3CR1 signaling on monocytes does not play an influential role in acute injury or functional recovery after intracerebral hemorrhage and therefore CX3CR1 is not a therapeutic target to improve outcome after intracerebral hemorrhage.