Proliferation of CD11b+ myeloid cells induced by TLR4 signaling promotes hepatitis B virus clearance.

BACKGROUNDS AND AIMS: Effective immune response plays a key role in the clearance of hepatitis B virus (HBV). However, the specific role of innate immune response in the clearance of virus is still unclear. Here we investigated the effect of TLR4 signaling on the proliferation and differentiation of CD11b+ myeloid cells, which contributes to virus clearance. METHODS: C57BL/6 mice were pretreated with TLR4 ligand lipopolysaccharide by intraperitoneal injection. Hydrodynamic injection (HI) was performed to establish HBV-replicated mice. The viremia was monitored. The immune cells were isolated from liver and spleen of the mice. The proliferation and differentiation of CD11b+ myeloid cells were analyzed by flow cytometry. The changes of CD11b+ myeloid cells and its role in virus clearance during HBV infection after LPS stimulation were analyzed. RESULTS: LPS stimulation induced the proliferation of CD11b+ myeloid cells which differentiated into neutrophils and inflammatory mononuclear macrophages. The expression of F4/80 protein on the surface of mononuclear macrophages in the liver of LPS-stimulated mice was significantly lower than that of control. It indicated that intrahepatic Kupffer cells were significantly decreased in the LPS-stimulated mice, which promoted the clearance of virus. CONCLUSION: LPS stimulation induces the proliferation of CD11b+ myeloid cells that differentiate into inflammatory neutrophils and monocytes, which inhibits HBV replication. And the decrease of intrahepatic Kupffer cells also contributes to the clearance of HBV during HBV infection.

Disruption of STAT6 Signal Promotes Cardiac Fibrosis Through the Mobilization and Transformation of CD11b+ Immature Myeloid Cells.

Cardiac fibrosis is an important pathological basis of various cardiovascular diseases. The roles of STAT6 signal in allergy, immune regulation, tumorigenesis, and renal fibrosis have been documented. However, the function and mechanism of STAT6 signal in sympathetic overactivation-induced cardiac fibrosis have not been fully elucidated. This study explores the novel role of STAT6 signal in isoproterenol (ISO)-induced cardiac fibrosis through the regulation of inflammatory response and the differentiation of macrophages from immature myeloid cells. The expression levels of STAT6, ß1-adrenergic receptor (ß1-AR), and inflammatory factors [interleukin α (IL-1α), IL-6, IL-18, and transforming growth factor ß (TGF-ß)] in CD11b+ myeloid cells were analyzed with a microarray study. The levels of IL-6 and TGF-ß1 in the CD11b+ myeloid cells-derived macrophages were detected with reverse transcriptase-polymerase chain reaction (RT-PCR). STAT6-knockout (KO) and WT mice were used to establish a murine cardiac fibrosis model by ISO injection. Cardiac fibroblasts were isolated from the hearts of newborn STAT6-KO and WT mice, and STAT6 expression was measured by Western blotting and RT-PCR after ISO stimulation, while α-smooth muscle actin (α-SMA) expression was detected by immunofluorescence and immunohistochemistry staining. Cardiac function and pathological characteristics were examined by echocardiography and immunohistochemistry staining, respectively. Immunohistochemistry staining with anti-CD11b was performed to detect the infiltration of CD11b+ myeloid cells in heart tissue. Flow cytometry analysis was used to measure the percentages of CD11b+ myeloid cells and CD11b+Ly6C+ macrophages in the peripheral blood. The results showed that STAT6 was highly expressed in CD11b+ myeloid cells located in injured hearts, and STAT6 expression in cardiac fibroblasts was down-regulated after ISO treatment. STAT6 deficiency further aggravated ISO-induced increased expression of α-SMA in cardiac fibroblasts, myocardial fibrosis, and cardiac dysfunction. The activation of ISO/ß1-AR signal aggravated cardiac inflammatory infiltration, promoted CD11b+ myeloid cell mobilization, and enhanced CD11b+Ly6C+/low macrophage differentiation, which was further exacerbated by STAT6 deficiency. Furthermore, ß1-AR mRNA expression significantly increased in splenic CD11b+ myeloid cells compared to their bone marrow-derived controls, and STAT6 deficiency promoted ß1-AR expression in an MI-induced sensitive cardiac fibrosis mouse model. The spleen-derived CD11b+ myeloid cells of STAT6-KO mice produced more IL-1α, IL-18, and TGF-ß than their WT counterparts. Taken together, these results suggest that STAT6 signal plays a critical role in ISO-induced ß1-AR overactivation and systemic inflammatory cascades, contributing to cardiac fibrogenesis. STAT6 should be a promising cardioprotective target against myocardial fibrosis and heart failure after ß1-AR overactivation-induced myocardial injury.

The role of physical trauma in social stress-induced immune activation.

It has been extensively studied in several mouse models how chronic, in particular chronic psychosocial, stressors facilitate the (re)activity of the innate immune system and, consequently, drive stress-associated pathologies. Here we first summarize the resulting concept and underlying mechanisms, proposing that social stress-induced bone marrow myelopoiesis, priming, emigration and activation of newly formed myeloid cells and accumulation of these cells in the spleen, gut, brain and fracture hematoma promote septic shock, colitis, anxiety and disturbed fracture healing, respectively. We further propose and discuss the hypothesis that it is not the social character of a particular stressor that promotes splenic invasion and subsequent full activation of stress-induced myeloid cells, but rather the occurrence of bite wounds as a result of direct physical interaction. Finally, we discuss the hypothesis that it is the combination of chronic stress, regardless of whether social or non-social in nature, and any kind of planned (i.e. surgery) or unplanned (i.e. bite wounds, injury) physical trauma that drives splenic invasion and subsequent full activation of stress-induced myeloid cells.

Epidermal loss of phospholipase Cδ1 attenuates irritant contact dermatitis.

Irritant contact dermatitis (ICD) is one of the most common inflammatory skin diseases caused by exposure to chemical irritants. Since chemical irritants primarily damage keratinocytes, these cells play a pivotal role in ICD. One of the phosphoinositide-metabolizing enzymes, phospholipase C (PLC) δ1, is abundantly expressed in keratinocytes. However, the role of PLCδ1 in ICD remains to be clarified. Here, we found that croton oil (CrO)-induced ear swelling, a feature of ICD, was attenuated in keratinocyte-specific PLCδ1 knockout mice (PLCδ1 cKO mice). Dendritic epidermal T cells (DETCs), which have a protective role against ICD, were activated in the epidermis of the PLCδ1 cKO mice. In addition, the skin of CrO-treated PLCδ1 cKO mice showed increased infiltration of Gr1+CD11b+ myeloid cells. Of note, elimination of Gr1+CD11b+ myeloid cells restored CrO-induced ear swelling in PLCδ1 cKO mice to a similar level as that in control mice. Taken together, our results strongly suggest that epidermal loss of PLCδ1 protects mice from ICD through induction of Gr1+CD11b+ myeloid cells and activation of DETCs.

Identification and establishment of sorting method for isolating CD11b+ myeloid cells in human hepatic metastases from colorectal cancer / 中国病理生理杂志

AIM:To establish a method for obtaining specific cells in solid tumor tissue by sorting of CD11b + myeloid cells in hepatic metastases from colorectal cancer.METHODS:Tumor tissues were prepared into single cell suspension by mechanical method combined with enzyme digestion,and then the CD11 b + myeloid cells were isolated by flow cytometry.The sorted cells were identified by immunocytochemistry.The viability and morphologiy of the sorted cells were evaluated by Giemsa and Typan blue staining.The cell purity was evaluated by flow cytometry.RESULTS:Sufficient numbers of CD11b+cells with high purity were isolated by sorting with flow cytometry from the single cell suspension prepared by mechanical and enzyme digestion.The purity of the cells was confirmed by statistical analysis (P ＜ 0.05).The positive rates of the cells before and after sorting were significantly different (P ＜0.01).The positive cells were verified by immunocytochemical method.Meanwhile,the sorted cells had complete morphology and good activity.CONCLUSION:The CD11b + myeloid cells in solid tumor tissue can be isolated by flow cytometry from the machine-enzyme digestion suspension with high purity,good activity and complete morphology.

Identification and establishment of sorting method for isolating CD11b+ myeloid cells in human hepatic metastases from colorectal cancer / 中国病理生理杂志

AIM:To establish a method for obtaining specific cells in solid tumor tissue by sorting of CD11b + myeloid cells in hepatic metastases from colorectal cancer.METHODS:Tumor tissues were prepared into single cell suspension by mechanical method combined with enzyme digestion,and then the CD11 b + myeloid cells were isolated by flow cytometry.The sorted cells were identified by immunocytochemistry.The viability and morphologiy of the sorted cells were evaluated by Giemsa and Typan blue staining.The cell purity was evaluated by flow cytometry.RESULTS:Sufficient numbers of CD11b+cells with high purity were isolated by sorting with flow cytometry from the single cell suspension prepared by mechanical and enzyme digestion.The purity of the cells was confirmed by statistical analysis (P ＜ 0.05).The positive rates of the cells before and after sorting were significantly different (P ＜0.01).The positive cells were verified by immunocytochemical method.Meanwhile,the sorted cells had complete morphology and good activity.CONCLUSION:The CD11b + myeloid cells in solid tumor tissue can be isolated by flow cytometry from the machine-enzyme digestion suspension with high purity,good activity and complete morphology.

Natural killer cells require monocytic Gr-1(+)/CD11b(+) myeloid cells to eradicate orthotopically engrafted glioma cells.

Malignant gliomas are resistant to natural killer (NK) cell immune surveillance. However, the mechanisms used by these cancers to suppress antitumor NK cell activity remain poorly understood. We have recently reported on a novel mechanism of innate immune evasion characterized by the overexpression of the carbohydrate-binding protein galectin-1 by both mouse and rat malignant glioma. Here, we investigate the cytokine profile of galectin-1-deficient GL26 cells and describe the process by which these tumors are targeted by the early innate immune system in RAG1(-/-) and C57BL/6J mice. Our data reveal that galectin-1 knockdown in GL26 cells heightens their inflammatory status leading to the rapid recruitment of Gr-1(+)/CD11b(+) myeloid cells and NK1.1(+) NK cells into the brain tumor microenvironment, culminating in tumor clearance. We show that immunodepletion of Gr-1(+) myeloid cells in RAG1(-/-) mice permits the growth of galectin-1-deficient glioma despite the presence of NK cells, thus demonstrating an essential role for myeloid cells in the clearance of galectin-1-deficient glioma. Further characterization of tumor-infiltrating Gr-1(+)/CD11b(+) cells reveals that these cells also express CCR2 and Ly-6C, markers consistent with inflammatory monocytes. Our results demonstrate that Gr-1(+)/CD11b(+) myeloid cells, often referred to as myeloid-derived suppressor cells (MDSCs), are required for antitumor NK cell activity against galectin-1-deficient GL26 glioma. We conclude that glioma-derived galectin-1 represents an important factor in dictating the phenotypic behavior of monocytic Gr-1(+)/CD11b(+) myeloid cells. Galectin-1 suppression may be a valuable treatment approach for clinical glioma by promoting their innate immune-mediated recognition and clearance through the concerted effort of innate myeloid and lymphoid cell lineages.

Dynamic Immune Cell Recruitment After Murine Pulmonary Aspergillus fumigatus Infection under Different Immunosuppressive Regimens.

Humans are continuously exposed to airborne spores of the saprophytic fungus Aspergillus fumigatus. However, in healthy individuals pulmonary host defense mechanisms efficiently eliminate the fungus. In contrast, A. fumigatus causes devastating infections in immunocompromised patients. Host immune responses against A. fumigatus lung infections in immunocompromised conditions have remained largely elusive. Given the dynamic changes in immune cell subsets within tissues upon immunosuppressive therapy, we dissected the spatiotemporal pulmonary immune response after A. fumigatus infection to reveal basic immunological events that fail to effectively control invasive fungal disease. In different immunocompromised murine models, myeloid, notably neutrophils, and macrophages, but not lymphoid cells were strongly recruited to the lungs upon infection. Other myeloid cells, particularly dendritic cells and monocytes, were only recruited to lungs of corticosteroid treated mice, which developed a strong pulmonary inflammation after infection. Lymphoid cells, particularly CD4(+) or CD8(+) T-cells and NK cells were highly reduced upon immunosuppression and not recruited after A. fumigatus infection. Moreover, adoptive CD11b(+) myeloid cell transfer rescued cyclophosphamide immunosuppressed mice from lethal A. fumigatus infection but not cortisone and cyclophosphamide immunosuppressed mice. Our findings illustrate that CD11b(+) myeloid cells are critical for anti-A. fumigatus defense under cyclophosphamide immunosuppressed conditions.

Gr-1+CD11b+ myeloid cells efficiently home to site of injury after intravenous administration and enhance diabetic wound healing by neoangiogenesis.

Vascularization is an important factor that affects diabetic wound healing. There is increasing evidence that myeloid cell lineages play a role in neovascularization. In this study, the efficiency of Gr-1+CD11b+ myeloid cells to home to the site of injury and enhance diabetic wound healing by neoangiogenesis after intravenous administration was investigated. Gr-1+CD11b+ myeloid cells were injected into tail vein after establishment of dorsal window chamber, hindlimb ischaemia and ear-punch injury in diabetic or non-diabetic mice. The Gr-1+CD11b+ myeloid cells efficiently homed to the site of injury after intravenous administration and increased neoangiogenesis. The chemokine receptor type 4 (CXCR4) is robustly expressed by Gr-1+CD11b+ myeloid cells. Inhibition of CXCR4 decreases the homing ability of Gr-1+CD11b+ myeloid cells to the site of injury, which indicates that the CXCR4/SDF-1 axis plays an important role in the homing of Gr-1+CD11b+ myeloid cells to the site of injury. In addition, Gr-1+CD11b+ myeloid cells were found to improve blood flow recovery of ischaemic limb and enhance wound healing in diabetic mice by neoangiogenesis after intravenous administration. Taken together, the results of this study suggest that Gr-1+CD11b+ myeloid cells may serve as a potential cell therapy for diabetic wound healing.

Tumor-conditioned Gr-1(+)CD11b(+) myeloid cells induce angiogenesis through the synergistic action of CCL2 and CXCL16 in vitro.

Gr-1(+)CD11b(+) cells can suppress innate and adaptive immunity, and the functional immunosuppressive characteristics of these cells can be modulated by the tumor microenvironment. Since Gr-1(+)CD11(+) cells are also involved in tumor-associated angiogenesis, we hypothesized that the angiogenic nature of Gr-1(+)CD11b(+) cells could be regulated by the tumor milieu. To address this hypothesis, we imitated a tumor microenvironment by exposing Gr-1(+)CD11b(+) cells isolated from spleen of 4T1 mammary carcinoma-bearing mice to tumor-conditioned medium. Supernatants from tumor-conditioned Gr-1(+)CD11b(+) cells significantly induced capillary-like tube formation and migration of human umbilical vein endothelial cells (HUVECs) compared to naive Gr-1(+)CD11b(+) cells. Incubation of Gr-1(+)CD11b(+) cells with tumor-conditioned medium induced production of pro-angiogenic chemokines CCL2 and CXCL16. Pretreatment with an anti-CCL2 antibody, but not an anti-CXCL16 antibody, suppressed the angiogenic effects of tumor-conditioned Gr-1(+)CD11b(+) cells on HUVECs. Simultaneous neutralization of CCL2 and CXCL16 significantly inhibited tube formation and migration of HUVECs compared to the sole neutralization against CCL2. Supernatants from tumor-conditioned Gr-1(+)CD11b(+) cells induced phosphorylation of ERK1/2 in HUVECs, and inhibition of the ERK pathway blocked angiogenic effects. ERK pathway activity was partially abrogated by neutralization of CCL2 and more suppressed by simultaneous neutralization of CCL2 and CXCL16. These results collectively indicate that CCL2 and CXCL16 chemokines produced by tumor-conditioned Gr-1(+)CD11b(+) myeloid cells synergistically induce angiogenesis in vitro by stimulating the ERK1/2 signaling pathway. Thus, regulation of Gr-1(+)CD11b(+) cells in the tumor microenvironment may contribute to angiogenesis through the secretion of pro-angiogenic chemokines.