Identification of triciribine as a novel myeloid cell differentiation inducer.

Differentiation therapy using all-trans retinoic acid (ATRA) for acute promyelocytic leukemia (APL) is well established. However, because the narrow application and tolerance development of ATRA need to be improved, we searched for another efficient myeloid differentiation inducer. Kinase activation is involved in leukemia biology and differentiation block. To identify novel myeloid differentiation inducers, we used a Kinase Inhibitor Screening Library. Using a nitroblue tetrazolium dye reduction assay and real-time quantitative PCR using NB4 APL cells, we revealed that, PD169316, SB203580, SB202190 (p38 MAPK inhibitor), and triciribine (TCN) (Akt inhibitor) potently increased the expression of CD11b. We focused on TCN because it was reported to be well tolerated by patients with advanced hematological malignancies. Nuclear/cytoplasmic (N/C) ratio was significantly decreased, and myelomonocytic markers (CD11b and CD11c) were potently induced by TCN in both NB4 and acute myeloid leukemia (AML) M2 derived HL-60 cells. Western blot analysis using NB4 cells demonstrated that TCN promoted ERK1/2 phosphorylation, whereas p38 MAPK phosphorylation was not affected, suggesting that activation of the ERK pathway is involved in TCN-induced differentiation. We further examined that whether ATRA may affect phosphorylation of ERK and p38, and found that there was no obvious effect, suggesting that ATRA induced differentiation is different from TCN effect. To reveal the molecular mechanisms involved in TCN-induced differentiation, we performed microarray analysis. Pathway analysis using DAVID software indicated that "hematopoietic cell lineage" and "cytokine-cytokine receptor interaction" pathways were enriched with high significance. Real-time PCR analysis demonstrated that components of these pathways including IL1ß, CD3D, IL5RA, ITGA6, CD44, ITGA2B, CD37, CD9, CSF2RA, and IL3RA, were upregulated by TCN-induced differentiation. Collectively, we identified TCN as a novel myeloid cell differentiation inducer, and trials of TCN for APL and non-APL leukemia are worthy of exploration in the future.

Alteration of functionality and differentiation directed by changing gene expression patterns in myeloid-derived suppressor cells (MDSCs) in tumor microenvironment and bone marrow through early to terminal phase of tumor progression.

Myeloid-derived suppressor cells are heterogenous immature myeloid lineage cells that can differentiate into neutrophils, monocytes, and dendritic cells as well. These cells have been characterized to have potent immunosuppressive capacity in neoplasia and a neoplastic chronic inflammatory microenvironment. Increased accumulation of myeloid-derived suppressor cells was reported with poor clinical outcomes in patients. They support neoplastic progression by abrogating antitumor immunity through inhibition of lymphocyte functions and directly by facilitating tumor development. Yet the shifting genetic signatures of this myeloid lineage cell toward immunosuppressive functionality in progressive tumor development remain elusive. We have attempted to identify the gene expression profile using lineage-specific markers of these unique myeloid lineage cells in a tumor microenvironment and bone marrow using a liquid transplantable mice tumor model to trace the changing influence of the tumor microenvironment on myeloid-derived suppressor cells. We analyzed the phenotype, functional shift, suppressive activity, differentiation status, and microarray-based gene expression profile of CD11b+Gr1+ lineage-specific cells isolated from the tumor microenvironment and bone marrow of 4 stages of tumor-bearing mice and compared them with control counterparts. Our analysis of differentially expressed genes of myeloid-derived suppressor cells isolated from bone marrow and the tumor microenvironment reveals unique gene expression patterns in the bone marrow and tumor microenvironment-derived myeloid-derived suppressor cells. It also suggests T-cell suppressive activity of myeloid-derived suppressor cells progressively increases toward the mid-to-late phase of the tumor and a significant differentiation bias of tumor site myeloid-derived suppressor cells toward macrophages, even in the presence of differentiating agents, indicating potential molecular characteristics of myeloid-derived suppressor cells in different stages of the tumor that can emerge as an intervention target.

CD11b Deficiency Favors Cartilage Calcification via Increased Matrix Vesicles, Apoptosis, and Lysyl Oxidase Activity.

Pathological cartilage calcification is a hallmark feature of osteoarthritis, a common degenerative joint disease, characterized by cartilage damage, progressively causing pain and loss of movement. The integrin subunit CD11b was shown to play a protective role against cartilage calcification in a mouse model of surgery-induced OA. Here, we investigated the possible mechanism by which CD11b deficiency could favor cartilage calcification by using naïve mice. First, we found by transmission electron microscopy (TEM) that CD11b KO cartilage from young mice presented early calcification spots compared with WT. CD11b KO cartilage from old mice showed progression of calcification areas. Mechanistically, we found more calcification-competent matrix vesicles and more apoptosis in both cartilage and chondrocytes isolated from CD11b-deficient mice. Additionally, the extracellular matrix from cartilage lacking the integrin was dysregulated with increased collagen fibrils with smaller diameters. Moreover, we revealed by TEM that CD11b KO cartilage had increased expression of lysyl oxidase (LOX), the enzyme that catalyzes matrix crosslinks. We confirmed this in murine primary CD11b KO chondrocytes, where Lox gene expression and crosslinking activity were increased. Overall, our results suggest that CD11b integrin regulates cartilage calcification through reduced MV release, apoptosis, LOX activity, and matrix crosslinking. As such, CD11b activation might be a key pathway for maintaining cartilage integrity.

ITGAM rs1143679 Variant in Systemic Lupus Erythematosus Is Associated with Increased Serum Calcification Propensity.

OBJECTIVES: CD11B/ITGAM (Integrin Subunit α M) mediates the adhesion of monocytes, macrophages, and granulocytes and promotes the phagocytosis of complement-coated particles. Variants of the ITGAM gene are candidates for genetic susceptibility to systemic lupus erythematosus (SLE). SNP rs1143679 (R77H) of CD11B particularly increases the risk of developing SLE. Deficiency of CD11B is linked to premature extra-osseous calcification, as seen in the cartilage of animals with osteoarthritis. Serum calcification propensity measured by the T50 test is a surrogate marker for systemic calcification and reflects increased cardiovascular (CV) risk. We aimed to assess whether the CD11B R77H gene variant is associated with a higher serum calcification propensity (i.e., a lower T50 value) in SLE patients compared to the wild-type allele (WT). METHODS: Cross-sectional study incorporating adults with SLE genotyped for the CD11B variant R77H and assessed for serum calcification propensity with the T50 method. Participants were included in a multicenter trans-disciplinary cohort and fulfilled the 1997 revised American College of Rheumatology (ACR) criteria for SLE. We used descriptive statistics for comparing baseline characteristics and sequential T50 measurements in subjects with the R77H variant vs. WT CD11B. RESULTS: Of the 167 patients, 108 (65%) were G/G (WT), 53 (32%) were G/A heterozygous, and 6 (3%) were A/A homozygous for the R77H variant. A/A patients cumulated more ACR criteria upon inclusion (7 ± 2 vs. 5 ± 1 in G/G and G/A; p = 0.02). There were no differences between the groups in terms of global disease activity, kidney involvement, and chronic renal failure. Complement C3 levels were lower in A/A individuals compared to others (0.6 ± 0.08 vs. 0.9 ± 0.25 g/L; p = 0.02). Baseline T50 did not differ between the groups (A/A 278 ± 42' vs. 297 ± 50' in G/G and G/A; p = 0.28). Considering all sequential T50 test results, serum calcification propensity was significantly increased in A/A individuals compared to others (253 ± 50 vs. 290 ± 54; p = 0.008). CONCLUSIONS: SLE patients with homozygosity for the R77H variant and repeated T50 assessment displayed an increased serum calcification propensity (i.e., a lower T50) and lower C3 levels compared to heterozygous and WT CD11B, without differing with respect to global disease activity and kidney involvement. This suggests an increased CV risk in SLE patients homozygous for the R77H variant of CD11B.

Daytime Light Deficiency Leads to Sex- and Brain Region-Specific Neuroinflammatory Responses in a Diurnal Rodent.

Seasonal changes in peripheral inflammation are well documented in both humans and animal models, but seasonal changes in neuroinflammation, especially the impact of seasonal lighting environment on neuroinflammation remain unclear. To address this question, the present study examined the effects of environmental lighting conditions on neuroinflammation in a diurnal rodent model, Nile grass rats (Arvicanthis niloticus). Male and female grass rats were housed in either bright (brLD) or dim (dimLD) light during the day to simulate a summer or winter light condition, respectively. After 4 weeks, microglia markers Iba-1 and CD11b, as well as pro-inflammatory cytokines TNF-α and IL-6, were examined in the anterior cingulate cortex (ACC), basolateral amygdala (BLA), and dorsal hippocampus (dHipp). The results revealed that winter-like dim light during the day leads to indicators of increased neuroinflammation in a brain site- and sex-specific manner. Specifically, relatively few changes in the neuroinflammatory markers were observed in the ACC, while numerous changes were found in the BLA and dHipp. In the BLA, winter-like dimLD resulted in hyper-ramified microglia morphology and increased expression of the pro-inflammatory cytokine IL-6, but only in males. In the dHipp, dimLD led to a higher number and hyper-ramified morphology of microglia as well as increased expression of CD11b and TNF-α, but only in females. Neuroinflammatory state is thus influenced by environmental light, differently in males and females, and could play a role in sex differences in the prevalence and symptoms of psychiatric or neurological disorders that are influenced by season or other environmental light conditions. Diurnal Nile grass rats were housed under bright or dim light during the day for 4 weeks, simulating seasonal fluctuations in daytime lighting environment. Dim light housing resulted in hyper-ramified morphology of microglia (scale bar, 15 µm) and altered expression of pro-inflammatory cytokines (TNF-α) in a sex- and brain region-specific manner.

A novel ZsGreen knock-in melanoma cell line reveals the function of CD11b in tumor phagocytosis.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is an efficient tool for establishing genetic models including cellular models, and has facilitated unprecedented advancements in biomedical research. In both patients and cancer animal models, immune cells infiltrate the tumor microenvironment and some of them migrate to draining lymph nodes to exert antitumor effects. Among these immune cells, phagocytes such as macrophages and dendritic cells engulf tumor antigens prior to their crosstalk with T cells and elicit adaptive immune response against tumors. Melanoma cells are frequently used as a tumor model because of their relatively high level of somatic mutations and antigenicity. However, few genetic models have been developed using melanoma cell lines to track tumor cell phagocytosis, which is essential for understanding protective immune response in vivo. In this study, we used CRISPR/Cas9-mediated DNA cleavage and homologous recombination to develop a novel knock-in tool which expresses the ultra-bright fluorescent probe ZsGreen in YUMM1.7 melanoma cells. Using this novel tool, we measured the macrophagic engulfment of melanoma cells inside the tumor microenvironment. We also found that in tumor-grafted mice, a subset of dendritic cells efficiently engulfed YUMM1.7 cells and was preferentially trafficking tumor antigens to draining lymph nodes. In addition, we used this knock-in tool to assess the impact of a point mutation of CD11b on phagocytosis in the tumor microenvironment. Our results demonstrate that the ZsGreen-expressing YUMM1.7 melanoma model provides a valuable tool for the study of phagocytosis in vivo.

Human Neutrophil Antigen Genotype and Allele Frequencies in Iranian Blood Donors.

OBJECTIVE: Human neutrophil antigens (HNAs) can be targeted by HNA-allo antibodies and cause a variety of clinical conditions such as transfusion-related acute lung injury (TRALI) and neonatal alloimmune neutropenia (NAIN). The current study is aimed at identifying the genotype and allele frequencies of HNAs in Iranian blood donors. METHODS: A total of 150 blood samples were obtained from healthy blood donors. HNA-1, HNA-3, HNA-4, and HNA-5 were genotyped, using the polymerase chain reaction sequence-specific primer (PCR-SSP) technique. The expression of the HNA-2 antigen on the neutrophil surface was evaluated by flow cytometry. RESULTS: The allele frequencies of FCGR3B∗1 (encoding HNA-1a), FCGR3B∗2 (encoding HNA-1b), and FCGR3B∗3 (encoding HNA-1c) were 0.34, 0.63, and 0.03, respectively. For HNA-3, the allele frequencies for SLC44A2∗1 (encoding HNA-3a) and SLC44A2∗2 (encoding HNA-3b) were 0.63 and 0.37, respectively. The frequencies of ITGAM∗1 (encoding HNA-4a) and ITGAM∗2 (encoding HNA-4b) alleles were 0.85 and 0.15, respectively. Furthermore, the frequencies of ITGAL∗1 (encoding HNA-5a) and ITGAL∗2 (encoding HNA-5b) alleles were 0.72 and 0.28, respectively. In the studied population, HNA-2 antigen was present on the neutrophil surface in 97.3% of the individuals, while no detectable HNA-2 expression was observed in 2.7% of the individuals. However, no significant difference in HNA-2 expression between different age groups was found. CONCLUSION: The present study provides the first report of the HNA allele and genotype frequencies among the Iranian population. All HNAs (HNA-1 to HNA-5) were typed using the PCR-SSP and flow cytometer. In the current cohort study, the determined HNA allele frequencies were similar to the previous reports from British, German, and Danish populations. Considering the presence of different Iranian ethnic groups, further studies with a larger sample size are needed to draw a total picture for HNA allele frequencies.

Specific Silencing of Microglial Gene Expression in the Rat Brain by Nanoparticle-Based Small Interfering RNA Delivery.

Microglia are the major innate immune cells in the brain and are essential for maintaining homeostasis in a neuronal microenvironment. Currently, a genetic tool to modify microglial gene expression in specific brain regions is not available. In this report, we introduce a tailor-designed method that uses lipid and polymer hybridized nanoparticles (LPNPs) for the local delivery of small interfering RNAs (siRNAs), allowing the silencing of specific microglial genes in the hypothalamus. Our physical characterization proved that this LPNP-siRNA was uniform and stable. We demonstrated that, due to their natural phagocytic behavior, microglial cells are the dominant cell type taking up these LPNPs in the hypothalamus of rats. We then tested the silencing efficiency of LPNPs carrying a cluster of differentiation molecule 11b (CD11b) or Toll-like receptor 4 (TLR4) siRNA using different in vivo and in vitro approaches. In cultured microglial cells treated with LPNP-CD11b siRNA or LPNP-TLR4 siRNA, we found a silencing efficiency at protein expression levels of 65 or 77%, respectively. In line with this finding, immunohistochemistry and western blotting results from in vivo experiments showed that LPNP-CD11b siRNA significantly inhibited microglial CD11b protein expression in the hypothalamus. Furthermore, following lipopolysaccharide (LPS) stimulation of cultured microglial cells, gene expression of the TLR4 downstream signaling component myeloid differentiation factor 88 and its associated cytokines was significantly inhibited in LPNP-TLR4 siRNA-treated microglial cells compared with cells treated with LPNP-scrambled siRNA. Finally, after LPNP-TLR4 siRNA injection into the rat hypothalamus, we observed a significant reduction in microglial activation in response to LPS compared with the control rats injected with LPNP-scrambled siRNA. Our results indicate that LPNP-siRNA is a promising tool to manipulate microglial activity locally in the brain and may serve as a prophylactic approach to prevent microglial dysfunction-associated diseases.

Association between complement gene polymorphisms and systemic lupus erythematosus: a systematic review and meta-analysis.

Complement dysfunction results in impaired ability in clearing apoptotic cell debris that may stimulate autoantibody production in systemic lupus erythematosus (SLE). Herein, we provided a comprehensive search to find and meta-analyze any complement gene polymorphisms associated with SLE. The ITGAM, C1q, and MBL gene polymorphisms were included in this meta-analysis to reveal the exact association with SLE risk. Electronic databases, including Scopus, PubMed, and Google Scholar, were searched to find studies investigating the ITGAM, C1q, and MBL gene polymorphisms and SLE risk in different populations. The pooled odds ratio (OR) and its corresponding 95% confidence interval (CI) were used to analyze the association between ITGAM, C1q, and MBL gene polymorphisms and susceptibility to SLE. According to inclusion criteria, a total of 24 studies, comprising 4 studies for C1QA rs292001, 5 studies for C1QA rs172378, 9 studies for ITGAM rs1143679, 8 studies for MBL rs1800450, 3 studies for MBL2 rs1800451, and 3 studies for MBL2 rs5030737, were included in the final meta-analysis. A significant positive association was found between rs1143679 and SLE risk, while rs1800451 significantly associated with decreased SLE susceptibility. In summary, ITGAM gene rs1143679 SNP and MBL gene rs1800451 SNP were positively and negatively associated with SLE risk, respectively.

Carbon Monoxide Regulates Macrophage Differentiation and Polarization toward the M2 Phenotype through Upregulation of Heme Oxygenase 1.

Carbon monoxide (CO) is generated by heme oxygenase (HO), and HO-1 is highly induced in monocytes and macrophages upon stimulation. Monocytes differentiate into macrophages, including pro-inflammatory (M1) and anti-inflammatory (M2) cells, in response to environmental signals. The present study investigated whether CO modulates macrophage differentiation and polarization, by applying the CO-releasing molecule-3 (CORM-3). Results showed that murine bone marrow cells are differentiated into macrophages by CORM-3 in the presence of macrophage colony-stimulating factor. CORM-3 increases expressions of macrophage markers, including F4/80 and CD11b, and alters the cell morphology into elongated spindle-shaped cells, which is a typical morphology of M2 cells. CORM-3 upregulates the expressions of genes and molecules involved in M2 polarization and M2 phenotype markers, such as STAT6, PPARγ, Ym1, Fizz1, arginase-1, and IL-10. However, exposure to CORM-3 inhibits the iNOS expression, suggesting that CO enhances macrophage differentiation and polarization toward M2. Increased HO-1 expression is observed in differentiated macrophages, and CORM-3 further increases this expression. Hemin, an HO-1 inducer, results in increased macrophage differentiation, whereas the HO-1 inhibitor zinc protoporphyrin IX inhibits differentiation. In addition, CORM-3 increases the proportion of macrophages in peritoneal exudate cells and enhances the expression of HO-1 and arginase-1 but inhibits iNOS. Taken together, these results suggest that the abundantly produced CO in activated macrophages enhances proliferation, differentiation, and polarization toward M2. It will probably help clear apoptotic cells, resolve inflammation, and promote wound healing and tissue remodeling.

Intestinal CD11b+ B Cells Ameliorate Colitis by Secreting Immunoglobulin A.

The intestinal mucosal immune environment requires multiple immune cells to maintain homeostasis. Although intestinal B cells are among the most important immune cells, little is known about the mechanism that they employ to regulate immune homeostasis. In this study, we found that CD11b+ B cells significantly accumulated in the gut lamina propria and Peyer's patches in dextran sulfate sodium-induced colitis mouse models and patients with ulcerative colitis. Adoptive transfer of CD11b+ B cells, but not CD11b-/- B cells, effectively ameliorated colitis and exhibited therapeutic effects. Furthermore, CD11b+ B cells were found to produce higher levels of IgA than CD11b- B cells. CD11b deficiency in B cells dampened IgA production, resulting in the loss of their ability to ameliorate colitis. Mechanistically, CD11b+ B cells expressed abundant TGF-ß and TGF-ß receptor II, as well as highly activate phosphorylated Smad2/3 signaling pathway, consequently promoting the class switch to IgA. Collectively, our findings demonstrate that CD11b+ B cells are essential intestinal suppressive immune cells and the primary source of intestinal IgA, which plays an indispensable role in maintaining intestinal homeostasis.

Homeostatic IL-13 in healthy skin directs dendritic cell differentiation to promote TH2 and inhibit TH17 cell polarization.

The signals driving the adaptation of type 2 dendritic cells (DC2s) to diverse peripheral environments remain mostly undefined. We show that differentiation of CD11blo migratory DC2s-a DC2 population unique to the dermis-required IL-13 signaling dependent on the transcription factors STAT6 and KLF4, whereas DC2s in lung and small intestine were STAT6-independent. Similarly, human DC2s in skin expressed an IL-4 and IL-13 gene signature that was not found in blood, spleen and lung DCs. In mice, IL-13 was secreted homeostatically by dermal innate lymphoid cells and was independent of microbiota, TSLP or IL-33. In the absence of IL-13 signaling, dermal DC2s were stable in number but remained CD11bhi and showed defective activation in response to allergens, with diminished ability to support the development of IL-4+GATA3+ helper T cells (TH), whereas antifungal IL-17+RORγt+ TH cells were increased. Therefore, homeostatic IL-13 fosters a noninflammatory skin environment that supports allergic sensitization.

Changes in Functional Glucocorticoid Sensitivity of Isolated Splenocytes Induced by Chronic Psychosocial Stress - A Time Course Study.

Chronic psychosocial stress is a risk factor for the development of numerous disorders, of which most are associated with chronic low-grade inflammation. Given the immunosuppressive effects of glucocorticoids (GC), one underlying mechanism might be the development of stress-induced GC resistance in certain immune cell subpopulations. In line with this hypothesis, male mice exposed to the chronic subordinate colony housing (CSC, 19 days) model develop GC resistance of in vitro lipopolysaccharide (LPS)-stimulated splenocytes, splenomegaly and an increased percentage of splenic CD11b+ cells. Here male C57BL/6N mice were euthanized at different days during CSC, and following 30 days of single housing after stressor termination to assess when CSC-induced splenic GC resistance starts to develop and whether this is a transient effect. Moreover, splenic CD11b, GC receptor (GR) and/or macrophage migration inhibiting factor (MIF) protein levels were quantified at respective days. While mild forms of CSC-induced GC resistance, increased splenic CD11b expression and/or splenomegaly were detectable on days 8 and 9 of CSC, more severe forms took until days 15 and 16 to develop, but normalized almost completely within 30 days following stressor termination (day 51). In contrast, splenic GR expression was decreased in CSC versus single-housed control (SHC) mice at all days assessed. While MIF expression was increased on days 15 and 16 of CSC, it was decreased in CSC versus SHC mice on day 20 despite persisting splenomegaly, increased CD11b expression and functional GC resistance. In summary, our data indicate that GC resistance and CD11b+ cell-mediated splenomegaly develop gradually and in parallel over time during CSC exposure and are transient in nature. Moreover, while we can exclude that CSC-induced reduction in splenic GR expression is sufficient to induce functional GC resistance, the role of MIF in CD11b+ cell-mediated splenomegaly and GC resistance requires further investigation.

Polymorphism of the ITGAM gene (rs7193943) and bioelectric impedance analysis as potential predictors of cachexia in chronic heart failure.

Cardiac cachexia (CC) is an unfavorable metabolic syndrome leading to exacerbation of chronic heart failure (CHF) and a higher risk of death. The main factor contributing to the development of cachexia is the ongoing inflammatory process mediated by genes (e.g. Integrin Subunit Alpha M-ITGAM). The study aimed to assess the relationship between a single nucleotide polymorphism (SNP) -323G > A of the ITGAM and the occurrence of nutritional disorders in patients with CHF. 157 CHF patients underwent clinical and nutritional screening. Body composition was evaluated by bioelectrical impedance analysis (BIA). Patients with cachexia were characterized by significantly lower weight, body mass index (BMI), lower fat mass (FM), albumin, and hemoglobin. Lower values of BIA parameters: capacitance of membrane (Cm), phase angle (PA), and impedance ratio (Z200/Z5) were noted in women. Those patients demonstrated significantly higher values of creatinine, c-reactive protein (CRP), N-terminal prohormone of brain natriuretic peptide (NT-proBNP), and pulmonary artery systolic pressure (PASP). A significantly higher risk of cachexia was reported in patients: aged ≥ 74 years (OR 3.55), with renal failure (OR 3.75), New York Heart Association classification (NYHA) III-IV (OR 2.83), with moderate or severe malnutrition according to the score of subjective global assessment (SGA) (OR 19.01) and AA genotype of ITGAM gene (OR 2.03). Determination of the -323G > A SNP in the ITGAM may prove to be a useful marker (after confirmation in further studies and appropriate validation) in the assessment of the risk of nutritional disorders in patients with CHF.

Macrophages exert homeostatic actions in pregnancy to protect against preterm birth and fetal inflammatory injury.

Macrophages are commonly thought to contribute to the pathophysiology of preterm labor by amplifying inflammation - but a protective role has not previously been considered to our knowledge. We hypothesized that given their antiinflammatory capability in early pregnancy, macrophages exert essential roles in maintenance of late gestation and that insufficient macrophages may predispose individuals to spontaneous preterm labor and adverse neonatal outcomes. Here, we showed that women with spontaneous preterm birth had reduced CD209+CD206+ expression in alternatively activated CD45+CD14+ICAM3- macrophages and increased TNF expression in proinflammatory CD45+CD14+CD80+HLA-DR+ macrophages in the uterine decidua at the materno-fetal interface. In Cd11bDTR/DTR mice, depletion of maternal CD11b+ myeloid cells caused preterm birth, neonatal death, and postnatal growth impairment, accompanied by uterine cytokine and leukocyte changes indicative of a proinflammatory response, while adoptive transfer of WT macrophages prevented preterm birth and partially rescued neonatal loss. In a model of intra-amniotic inflammation-induced preterm birth, macrophages polarized in vitro to an M2 phenotype showed superior capacity over nonpolarized macrophages to reduce uterine and fetal inflammation, prevent preterm birth, and improve neonatal survival. We conclude that macrophages exert a critical homeostatic regulatory role in late gestation and are implicated as a determinant of susceptibility to spontaneous preterm birth and fetal inflammatory injury.

Morphine promotes microglial activation by upregulating the EGFR/ERK signaling pathway.

Although they represent the cornerstone of analgesic therapy, opioids, such as morphine, are limited in efficacy by drug tolerance, hyperalgesia and other side effects. Activation of microglia and the consequent production of proinflammatory cytokines play a key pathogenic role in morphine tolerance, but the exact mechanisms are not well understood. This study aimed to investigate the regulatory mechanism of epidermal growth factor receptor (EGFR) on microglial activation induced by morphine in mouse microglial BV-2 cells. In this research, BV-2 cells were stimulated with morphine or pretreated with AG1478 (an inhibitor of EGFR). Expression levels of cluster of differentiation molecule 11b (CD11b), EGFR, and phospho-EGFR were detected by immunofluorescence staining. Cell signaling was assayed by Western blot. The migration ability of BV-2 cells was tested by Transwell assay. The production of interleukin-1beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) in the cell supernatant was determined by ELISA. We observed that the expression of CD11b induced by morphine was increased in a dose- and time- dependent manner in BV-2 cells. Phosphorylation levels of EGFR and ERK1/2, migration of BV-2 cells, and production of IL-1ß and TNFα were markedly enhanced by morphine treatment. The activation, migration, and production of proinflammatory cytokines in BV-2 cells were inhibited by blocking the EGFR signaling pathway with AG1478. The present study demonstrated that the EGFR/ERK signaling pathway may represent a novel pharmacological strategy to suppress morphine tolerance through attenuation of microglial activation.

Integrin CD11b Negatively Regulates B Cell Receptor Signaling to Shape Humoral Response during Immunization and Autoimmunity.

Our previous work has revealed the ability of CD11b to regulate BCR signaling and control autoimmune disease in mice. However, how CD11b regulates the immune response under normal conditions remains unknown. Through the use of a CD11b knockout model on a nonautoimmune background, we demonstrated that CD11b-deficient mice have an elevated Ag-specific humoral response on immunization. Deletion of CD11b resulted in elevated low-affinity and high-affinity IgG Ab and increases in Ag-specific germinal center B cells and plasma cells (PCs). Examination of BCR signaling in CD11b-deficient mice revealed defects in association of negative regulators pLyn and CD22 with the BCR, but increases in colocalizations between positive regulator pSyk and BCR after stimulation. Using a CD11b-reporter mouse model, we identified multiple novel CD11b-expressing B cell subsets that are dynamically altered during immunization. Subsequent experiments using a cell-specific CD11b deletion model revealed this effect to be B cell intrinsic and not altered by myeloid cell CD11b expression. Importantly, CD11b expression on PCs also impacts on BCR repertoire selection and diversity in autoimmunity. These studies describe a novel role for CD11b in regulation of the healthy humoral response and autoimmunity, and reveal previously unknown populations of CD11b-expressing B cell subsets, suggesting a complex function for CD11b in B cells during development and activation.

Sequential actions of EOMES and T-BET promote stepwise maturation of natural killer cells.

EOMES and T-BET are related T-box transcription factors that control natural killer (NK) cell development. Here we demonstrate that EOMES and T-BET regulate largely distinct gene sets during this process. EOMES is dominantly expressed in immature NK cells and drives early lineage specification by inducing hallmark receptors and functions. By contrast, T-BET is dominant in mature NK cells, where it induces responsiveness to IL-12 and represses the cell cycle, likely through transcriptional repressors. Regardless, many genes with distinct functions are co-regulated by the two transcription factors. By generating two gene-modified mice facilitating chromatin immunoprecipitation of endogenous EOMES and T-BET, we show a strong overlap in their DNA binding targets, as well as extensive epigenetic changes during NK cell differentiation. Our data thus suggest that EOMES and T-BET may distinctly govern, via differential expression and co-factors recruitment, NK cell maturation by inserting partially overlapping epigenetic regulations.

TYROBP, TLR4 and ITGAM regulated macrophages polarization and immune checkpoints expression in osteosarcoma.

We established a relationship among the immune-related genes, tumor-infiltrating immune cells (TIICs), and immune checkpoints in patients with osteosarcoma. The gene expression data for osteosarcoma were downloaded from UCSC Xena and GEO database. Immune-related differentially expressed genes (DEGs) were detected to calculate the risk score. "Estimate" was used for immune infiltrating estimation and "xCell" was used to obtain 64 immune cell subtypes. Furthermore, the relationship among the risk scores, immune cell subtypes, and immune checkpoints was evaluated. The three immune-related genes (TYROBP, TLR4, and ITGAM) were selected to establish a risk scoring system based on their integrated prognostic relevance. The GSEA results for the Hallmark and KEGG pathways revealed that the low-risk score group exhibited the most gene sets that were related to immune-related pathways. The risk score significantly correlated with the xCell score of macrophages, M1 macrophages, and M2 macrophages, which significantly affected the prognosis of osteosarcoma. Thus, patients with low-risk scores showed better results with the immune checkpoints inhibitor therapy. A three immune-related, gene-based risk model can regulate macrophage activation and predict the treatment outcomes the survival rate in osteosarcoma.