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ABSTRACT Murine macrophages are activated by interferon-γ (IFN-γ) and/or Toll-like receptor (TLR) agonists such as bacterial endotoxin (lipopolysaccharide [LPS]) to express an inflammatory (M1) phenotype characterized by the expression of nitric oxide synthase-2 (iNOS) and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-12. In contrast, Th2 cytokines IL-4 and IL-13 activate macrophages by inducing the expression of arginase-1 and the anti-inflammatory cytokine IL-10 in an IL-4 receptor-α (IL-4Rα)-dependent manner. Macrophages activated in this way are designated as “alternatively activated” (M2a) macrophages. We have shown previously that adenosine A 2A receptor (A 2A R) agonists act synergistically with TLR2, TLR4, TLR7, and TLR9 agonists to switch macrophages into an “M2-like” phenotype that we have termed “M2d.” Adenosine signaling suppresses the TLR-dependent expression of TNF-α, IL-12, IFN-γ, and several other inflammatory cytokines by macrophages and induces the expression of vascular endothelial growth factor (VEGF) and IL-10. We show here using mice lacking a functional IL-4Rα gene (IL-4Rα −/− mice) that this adenosine-mediated switch does not require IL-4Rα-dependent signaling. M2d macrophages express high levels of VEGF, IL-10, and iNOS, low levels of TNF-α and IL-12, and mildly elevated levels of arginase-1. In contrast, M2d macrophages do not express Ym1, Fizz1 (RELM-α), or CD206 at levels greater than those induced by LPS, and dectin-1 expression is suppressed. The use of these markers in vivo to identify “M2” macrophages thus provides an incomplete picture of macrophage functional status and should be viewed with caution.

Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. Here we show that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes are recruited to the intestine and secrete the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switch their response to DPP by inducing expression of Thickveins, a second type I receptor that has previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in ageing flies. We propose that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans. Jasper and colleagues report that following intestinal damage in Drosophila , haemocytes recruited to the intestine secrete Dpp, promoting intestinal stem cell proliferation and, at later stages of regeneration, the re-establishment of intestinal stem cell quiescence.

Cardiac tissue macrophages (cTMs) are a previously uncharacterised cell type that we have identified and characterise here as an abundant GFP(+) population within the adult Cx(3)cr1(GFP/+) knock-in mouse heart. They comprise the predominant myeloid cell population in the myocardium, and are found throughout myocardial interstitial spaces interacting directly with capillary endothelial cells and cardiomyocytes. Flow cytometry-based immunophenotyping shows that cTMs exhibit canonical macrophage markers. Gene expression analysis shows that cTMs (CD45(+)CD11b(+)GFP(+)) are distinct from mononuclear CD45(+)CD11b(+)GFP(+) cells sorted from the spleen and brain of adult Cx(3)cr1(GFP/+) mice. Gene expression profiling reveals that cTMs closely resemble alternatively-activated anti-inflammatory M2 macrophages, expressing a number of M2 markers, including Mrc1, CD163, and Lyve-1. While cTMs perform normal tissue macrophage homeostatic functions, they also exhibit a distinct phenotype, involving secretion of salutary factors (including IGF-1) and immune modulation. In summary, the characterisation of cTMs at the cellular and molecular level defines a potentially important role for these cells in cardiac homeostasis.

One of the most striking aspects of plant plasticity is the modulation of development in response to environmental changes. Plant growth and development largely depend on the phytohormone auxin that exerts its function through a partially redundant family of F-box receptors, the TIR1-AFBs. We have previously reported that the Arabidopsis double mutant tir1 afb2 is more tolerant to salt stress than wild-type plants and we hypothesized that down-regulation of auxin signaling might be part of Arabidopsis acclimation to salinity. In this work, we show that NaCl-mediated salt stress induces miR393 expression by enhancing the transcription of AtMIR393A and leads to a concomitant reduction in the levels of the TIR1 and AFB2 receptors. Consequently, NaCl triggers stabilization of Aux/IAA repressors leading to down-regulation of auxin signaling. Further, we report that miR393 is likely involved in repression of lateral root (LR) initiation, emergence and elongation during salinity, since the mir393ab mutant shows reduced inhibition of emergent and mature LR number and length upon NaCl-treatment. Additionally, mir393ab mutant plants have increased levels of reactive oxygen species (ROS) in LRs, and reduced ascorbate peroxidase (APX) enzymatic activity compared with wild-type plants during salinity. Thus, miR393 regulation of the TIR1 and AFB2 receptors could be a critical checkpoint between auxin signaling and specfic redox-associated components in order to coordinate tissue and time-specific growth responses and tolerance during acclimation to salinity in Arabidopsis.

Tumor-associated macrophages (TAMs) play an important role in the progression and prognostication of numerous cancers. However, the role and clinical significance of TAM markers in oral squamous cell carcinoma (OSCC) has not been elucidated. The present study was designed to investigate the correlation between the expression of TAM markers and pathological features in OSCC by tissue microarray. Tissue microarrays containing 16 normal oral mucosa, 6 oral epithelial dysplasia, and 43 OSCC specimens were studied by immunohistochemistry. We observed that the protein expression of the TAM markers CD68 and CD163 as well as the cancer stem cell (CSC) markers ALDH1, CD44, and SOX2 increased successively from the normal oral mucosa to OSCC. The expressions of CD68 and CD163 were significantly associated with lymph node status, and SOX2 was significantly correlated with pathological grade and lymph node status, whereas ALDH1 was correlated with tumor stage. Furthermore, CD68 was significantly correlated with CD163, SOX2, and ALDH1 (P<0.05). Kaplan-Meier analysis revealed that OSCC patients overexpressing CD163 had significantly worse overall survival (P<0.05). TAM markers are associated with cancer stem cell marker and OSCC overall survival, suggesting their potential prognostic value in OSCC.

Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.

Background Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of approximately 15 months. Semaphorin 3A (Sema3A), known for its axon guidance and antiangiogenic properties, has been implicated in GBM growth. We hypothesized that Sema3A directly inhibits brain tumor stem cell (BTSC) proliferation and drives invasion via Neuropilin 1 (Nrp1) and Plexin A1 (PlxnA1) receptors. Methods GBM BTSC cell lines were assayed by immunostaining and PCR for levels of Semaphorin 3A (Sema3A) and its receptors Nrp1 and PlxnA1. Quantitative BrdU, cell cycle and propidium iodide labeling assays were performed following exogenous Sema3A treatment. Quantitative functional 2-D and 3-D invasion assays along with shRNA lentiviral knockdown of Nrp1 and PlxnA1 are also shown. In vivo flank studies comparing tumor growth of knockdown versus control BTSCs were performed. Statistics were performed using GraphPad Prism v7. Results Immunostaining and PCR analysis revealed that BTSCs highly express Sema3A and its receptors Nrp1 and PlxnA1, with expression of Nrp1 in the CD133 positive BTSCs, and absence in differentiated tumor cells. Treatment with exogenous Sema3A in quantitative BrdU, cell cycle, and propidium iodide labeling assays demonstrated that Sema3A significantly inhibited BTSC proliferation without inducing cell death. Quantitative functional 2-D and 3-D invasion assays showed that treatment with Sema3A resulted in increased invasion. Using shRNA lentiviruses, knockdown of either NRP1 or PlxnA1 receptors abrogated Sema3A antiproliferative and pro-invasive effects. Interestingly, loss of the receptors mimicked Sema3A effects, inhibiting BTSC proliferation and driving invasion. Furthermore, in vivo studies comparing tumor growth of knockdown and control infected BTSCs implanted into the flanks of nude mice confirmed the decrease in proliferation with receptor KD. Conclusions These findings demonstrate the importance of Sema3A signaling in GBM BTSC proliferation and invasion, and its potential as a therapeutic target.

Key Points The strength and pervasive nature of Toll-like receptor (TLR) signalling necessitates a powerful and comprehensive negative regulatory mechanism to prevent autoimmune damage. This review discusses our current understanding of the negative regulators of TLR responses. There are at least five layers of negative regulation of TLR signalling. These range from extracellular decoy receptors to intracellular inhibitors, membrane-bound suppressors, degradation of TLRs, and TLR-induced apoptosis. Soluble decoy TLRs are potentially powerful competitors for TLR agonists, reminiscent of soluble chemokines and cytokine receptors. So far, only soluble TLR4 and TLR2 have been identified, and their role might be as an important first-line negative regulatory mechanism. Intracellular negative regulators so far identified include MyD88s, IRAKM, SOCS1, NOD2, phosphatidylinositol 3-kinase, TOLLIP and A20. This group is perhaps the best studied of all the regulators. They function at various stages of the TLR signalling cascade but concentrate principally on the MyD88-dependent pathway. Transmembrane protein regulators include ST2, SIGIRR, TRAILR and RP105. These proteins inhibit TLR functions either by sequestration of adaptor proteins (ST2) and transcription factors (TRAILR), or by interfering with the binding of TLR agonists to their respective TLRs (SIGIRR and RP105). Reduction of TLR expression could be by ubiquitylation (TRIAD3A), promoting proteolytic degradation of TLRs, or through inhibition of the transcription or stability of TLR-encoding mRNAs (interleukin-10, transforming-growth factor-β and lipopolysaccharide). The last line of negative regulation is self-destruction. Excessive TLR activation could lead to caspase-dependent (through the death domain of MyD88) and caspase-independent apoptosis. The existence of multiple and apparently non-redundant negative regulators of TLRs indicate that either the regulators function in a cascade manner or that each regulator is necessary but insufficient to control a particular TLR signalling pathway. The genetic polymorphism of the regulators and what regulates the negative regulator remains to be determined. The biological functions of some of the negative regulators in vivo also remain to be determined. Toll-like receptors (TLRs) are involved in host defence against invading pathogens, functioning as primary sensors of microbial products and activating signalling pathways that induce the expression of immune and pro-inflammatory genes. However, TLRs have also been implicated in several immune-mediated and inflammatory diseases. As the immune system needs to constantly strike a balance between activation and inhibition to avoid detrimental and inappropriate inflammatory responses, TLR signalling must be tightly regulated. Here, we discuss the various negative regulatory mechanisms that have evolved to attenuate TLR signalling to maintain this immunological balance.

Interleukin-33 (IL-33), a member of the IL-1 family of cytokines, binds to its plasma membrane receptor, heterodimeric complex consisted of membrane-bound ST2L and IL-1R accessory protein, inducing NFkB and MAPK activation. IL-33 exists as a nuclear precursor and may act as an alarmin, when it is released after cell damage or as negative regulator of NFκB gene transcription, when acts in an intracrine manner. ST2L is expressed on several immune cells: Th2 lymphocytes, NK, NKT and mast cells and on cells of myeloid lineage: monocytes, dendritic cells and granulocytes. IL-33/ST2 axis can promote both Th1 and Th2 immune responses depending on the type of activated cell and microenvironment and cytokine network in damaged tissue. We previously described and discuss here the important role of IL-33/ST2 axis in experimental models of type 1 diabetes, experimental autoimmune encephalomyelitis, fulminant hepatitis and breast cancer. We found that ST2 deletion enhance the development of T cell-mediated autoimmune disorders, EAE and diabetes mellitus type I. Disease development was accompanied by dominantly Th1/Th17 immune response but also higher IL-33 production, which suggest that IL-33 in receptor independent manner could promote the development of inflammatory autoreactive T cells. IL-33/ST2 axis has protective role in Con A hepatitis. ST2-deficient mice had more severe hepatitis with higher influx of inflammatory cells in liver and dominant Th1/Th17 systemic response. Pretreatment of mice with IL-33 prevented Con A-induced liver damage through prevention of apoptosis of hepatocytes and Th2 amplification. Deletion of IL-33/ST2 axis enhances cytotoxicity of NK cells, production of IFN-γ in these cells and systemic production of IFN-γ, IL-17 and TNF-α, which leads to attenuated tumor growth. IL-33 treatment of tumor-bearing mice suppresses activity of NK cells, dendritic cell maturation and enhances alternative activation of macrophages. In conclusion, we observed that IL-33 has attenuated anti-inflammatory effects in T cell-mediated responses and that both IL-33 and ST2 could be further explored as potential therapeutic targets in treatment of immune-mediated diseases.

Background Tumor-associated macrophages (TAMs) play an important role in growth, progression and metastasis of tumors. In non-small cell lung cancer (NSCLC), TAMs' anti-tumor or pro-tumor role is not determined. Macrophages are polarized into M1 (with anti-tumor function) and M2 (with pro-tumor function) forms. This study was conducted to determine whether the M1 and M2 macrophage densities in NSCLC are associated with patient's survival time. Methods Fifty patients with an average of 1-year survival (short survival group) and 50 patients with an average of 5-year survival (long survival group) were included in this retrospective study. Paraffin-embedded NSCLC specimens and their clinicopathological data including up to 8-year follow-up information were used. Immunohistochemical double-staining of CD68/HLA-DR (markers for M1 macrophages) and CD68/CD163 (markers for M2 macrophages) was performed and evaluated in a blinded fashion. The M1 and M2 macrophage densities in the tumor islets, stroma, or islets and stroma were determined using computer-aided microscopy. Correlation of the macrophage densities and patient's survival time was analyzed using the Statistical Package for the Social Sciences. Results Approximately 70% of TAMs were M2 macrophages and the remaining 30% were M1 macrophages in NSCLC. The M2 macrophage densities (approximately 78 to 113 per mm 2 ) in the tumor islets, stroma, or islets and stroma were not significantly different between the long survival and short survival groups. The M1 macrophage densities in the tumor islets (approximately 70/mm 2 ) and stroma (approximately 34/mm 2 ) of the long survival group were significantly higher than the M1 macrophage densities in the tumor islets (approximately 7/mm 2 ) and stroma (13/mm 2 ) of the short survival group (P < 0.001 and P < 0.05, respectively). The M2 macrophage densities were not associated with patient's survival time. The M1 macrophage densities in the tumor islets, stroma, or islets and stroma were positively associated with patient's survival time in a univariate analysis (P < 0.01 or 0.001). In a multivariate Cox proportional hazards analysis, the M1 macrophage density in the tumor islets was an independent predictor of patient's survival time. Conclusions The M1 macrophage density in the tumor islets is an independent predictor of survival time in NSCLC patients.