Properties of monocytes generated from haematopoietic CD34+ stem cells from bone marrow of colon cancer patients

Monocytes exhibit direct and indirect antitumour activities and may be potentially useful for various forms of adoptive cellular immunotherapy of cancer. However, blood is a limited source of them. This study explored whether monocytes can be obtained from bone marrow haematopoietic CD34(+) stem cells of colon cancer patients, using previously described protocol of expansion and differentiation to monocytes of cord blood-derived CD34(+) haematopoietic progenitors. Data show that in two-step cultures, the yield of cells was increased approximately 200-fold, and among these cells, up to 60 % of CD14(+) monocytes were found. They consisted of two subpopulations: CD14(++)CD16(+) and CD14(+)CD16(−), at approximately 1:1 ratio, that differed in HLA-DR expression, being higher on the former. No differences in expression of costimulatory molecules were observed, as CD80 was not detected, while CD86 expression was comparable. These CD14(+) monocytes showed the ability to present recall antigens (PPD, Candida albicans) and neoantigens expressed on tumour cells and tumour-derived microvesicles (TMV) to autologous CD3(+) T cells isolated from the peripheral blood. Monocytes also efficiently presented the immunodominant HER-2/neu(369–377) peptide (KIFGSLAFL), resulting in the generation of specific cytotoxic CD8(+) T lymphocytes (CTL). The CD14(++)CD16(+) subset exhibited enhanced cytotoxicity, though nonsignificant, towards tumour cells in vitro. These observations indicate that generation of monocytes from CD34(+) stem cells of cancer patients is feasible. To our knowledge, it is the first demonstration of such approach that may open a way to obtain autologous monocytes for alternative forms of adaptive and adoptive cellular immunotherapy of cancer

Phenotypic and functional analysis of monocyte populations in cattle peripheral blood identifies a subset with high endocytic and allogeneic T-cell stimulatory capacity

International audienceAbstractCirculating monocytes in several mammalian species can be subdivided into functionally distinct subpopulations based on differential expression of surface molecules. We confirm that bovine monocytes express CD172a and MHC class II with two distinct populations of CD14+CD16low/-CD163+ and CD14−CD16++CD163low- cells, and a more diffuse population of CD14+CD16+CD163+ cells. In contrast, ovine monocytes consisted of only a major CD14+CD16+ subset and a very low percentage of CD14−CD16++cells. The bovine subsets expressed similar levels of CD80, CD40 and CD11c molecules and mRNA encoding CD115. However, further mRNA analyses revealed that the CD14−CD16++ monocytes were CX3CR1highCCR2low whereas the major CD14+ subset was CX3CR1lowCCR2high. The former were positive for CD1b and had lower levels of CD11b and CD86 than the CD14+ monocytes. The more diffuse CD14+CD16+ population generally expressed intermediate levels of these molecules. All three populations responded to stimulation with phenol-extracted lipopolysaccharide (LPS) by producing interleukin (IL)-1β, with the CD16++ subset expressing higher levels of IL-12 and lower levels of IL-10. The CD14−CD16++ cells were more endocytic and induced greater allogeneic T cell responses compared to the other monocyte populations. Taken together the data show both similarities and differences between the classical, intermediate and non-classical definitions of monocytes as described for other mammalian species, with additional potential subpopulations. Further functional analyses of these monocyte populations may help explain inter-animal and inter-species variations to infection, inflammation and vaccination in ruminant livestock

The selective phosphodiesterase 4 inhibitor roflumilast and phosphodiesterase 3/4 inhibitor pumafentrine reduce clinical score and TNF expression in experimental colitis in mice.

The specific inhibition of phosphodiesterase (PDE)4 and dual inhibition of PDE3 and PDE4 has been shown to decrease inflammation by suppression of pro-inflammatory cytokine synthesis. We examined the effect of roflumilast, a selective PDE4 inhibitor marketed for severe COPD, and the investigational compound pumafentrine, a dual PDE3/PDE4 inhibitor, in the preventive dextran sodium sulfate (DSS)-induced colitis model. The clinical score, colon length, histologic score and colon cytokine production from mice with DSS-induced colitis (3.5% DSS in drinking water for 11 days) receiving either roflumilast (1 or 5 mg/kg body weight/d p.o.) or pumafentrine (1.5 or 5 mg/kg/d p.o.) were determined and compared to vehicle treated control mice. In the pumafentrine-treated animals, splenocytes were analyzed for interferon-γ (IFNγ) production and CD69 expression. Roflumilast treatment resulted in dose-dependent improvements of clinical score (weight loss, stool consistency and bleeding), colon length, and local tumor necrosis factor-α (TNFα) production in the colonic tissue. These findings, however, were not associated with an improvement of the histologic score. Administration of pumafentrine at 5 mg/kg/d alleviated the clinical score, the colon length shortening, and local TNFα production. In vitro stimulated splenocytes after in vivo treatment with pumafentrine showed a significantly lower state of activation and production of IFNγ compared to no treatment in vivo. These series of experiments document the ameliorating effect of roflumilast and pumafentrine on the clinical score and TNF expression of experimental colitis in mice

HDAC6 Regulates LPS-Tolerance in Astrocytes

Inflammatory tolerance is a crucial mechanism that limits inflammatory responses in order to avoid prolonged inflammation that may damage the host. Evidence that chronic inflammation contributes to the neuropathology of prevalent neurodegenerative and psychiatric diseases suggests that inflammatory tolerance mechanisms are often inadequate to control detrimental inflammation in the central nervous system. Thus, identifying mechanisms that regulate neuroinflammatory tolerance may reveal opportunities for bolstering tolerance to reduce chronic inflammation in these diseases. Examination of tolerance after repeated lipopolysaccharide (LPS) treatment of mouse primary astrocytes demonstrated that histone deacetylase (HDAC) activity promoted tolerance, opposite to the action of glycogen synthase kinase-3 (GSK3), which counteracts tolerance. HDAC6 in particular was found to be critical for tolerance induction, as its deacetylation of acetyl-tubulin was increased during LPS tolerance, this was enhanced by inhibition of GSK3, and the HDAC6 inhibitor tubacin completely blocked tolerance and the promotion of tolerance by inhibition of GSK3. These results reveal opposing interactions between HDAC6 and GSK3 in regulating tolerance, and indicate that shifting the balance between these two opposing forces on inflammatory tolerance can obliterate or enhance tolerance to LPS in astrocytes

Influenza Virus A Infection of Human Monocyte and Macrophage Subpopulations Reveals Increased Susceptibility Associated with Cell Differentiation

Influenza virus infection accounts for significant morbidity and mortality world-wide. Interactions of the virus with host cells, particularly those of the macrophage lineage, are thought to contribute to various pathological changes associated with poor patient outcome. Development of new strategies to treat disease therefore requires a detailed understanding of the impact of virus infection upon cellular responses. Here we report that human blood-derived monocytes could be readily infected with the H3N2 influenza virus A/Udorn/72 (Udorn), irrespective of their phenotype (CD14++/CD16−, CD14++/CD16+ or CD14dimCD16++), as determined by multi-colour flow cytometry for viral haemagglutinin (HA) expression and cell surface markers 8–16 hours post infection. Monocytes are relatively resistant to influenza-induced cell death early in infection, as approximately 20% of cells showed influenza-induced caspase-dependent apoptosis. Infection of monocytes with Udorn also induced the release of IL-6, IL-8, TNFα and IP-10, suggesting that NS1 protein of Udorn does not (effectively) inhibit this host defence response in human monocytes. Comparative analysis of human monocyte-derived macrophages (Mph) demonstrated greater susceptibility to human influenza virus than monocytes, with the majority of both pro-inflammatory Mph1 and anti-inflammatory/regulatory Mph2 cells expressing viral HA after infection with Udorn. Influenza infection of macrophages also induced cytokine and chemokine production. However, both Mph1 and Mph2 phenotypes released comparable amounts of TNFα, IL-12p40 and IP-10 after infection with H3N2, in marked contrast to differential responses to LPS-stimulation. In addition, we found that influenza virus infection augmented the capacity of poorly phagocytic Mph1 cells to phagocytose apoptotic cells by a mechanism that was independent of either IL-10 or the Mer receptor tyrosine kinase/Protein S pathway. In summary, our data reveal that influenza virus infection of human macrophages causes functional alterations that may impact on the process of resolution of inflammation, with implications for viral clearance and lung pathology

CD69 is a TGF-β/1α,25-dihydroxyvitamin D3 target gene in monocytes

CD69 is a transmembrane lectin that can be expressed on most hematopoietic cells. In monocytes, it has been functionally linked to the 5-lipoxygenase pathway in which the leukotrienes, a class of highly potent inflammatory mediators, are produced. However, regarding CD69 gene expression and its regulatory mechanisms in monocytes, only scarce data are available. Here, we report that CD69 mRNA expression, analogous to that of 5-lipoxygenase, is induced by the physiologic stimuli transforming growth factor-β (TGF-β) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in monocytic cells. Comparison with T- and B-cell lines showed that the effect was specific for monocytes. CD69 expression levels were increased in a concentration-dependent manner, and kinetic analysis revealed a rapid onset of mRNA expression, indicating that CD69 is a primary TGF-β/1α,25(OH)2D3 target gene. PCR analysis of different regions of the CD69 mRNA revealed that de novo transcription was initiated and proximal and distal parts were induced concomitantly. In common with 5-lipoxygenase, no activation of 0.7 kb or ~2.3 kb promoter fragments by TGF-β and 1α,25(OH)2D3 could be observed in transient reporter assays for CD69. Analysis of mRNA stability using a transcription inhibitor and a 3′UTR reporter construct showed that TGF-β and 1α,25(OH)2D3 do not influence CD69 mRNA stability. Functional knockdown of Smad3 clearly demonstrated that upregulation of CD69 mRNA, in contrast to 5-LO, depends on Smad3. Comparative studies with different inhibitors for mitogen activated protein kinases (MAPKs) revealed that MAPK signalling is involved in CD69 gene regulation, whereas 5-lipoxygenase gene expression was only partly affected. Mechanistically, we found evidence that CD69 gene upregulation depends on TAK1-mediated p38 activation. In summary, our data indicate that CD69 gene expression, conforming with 5-lipoxygenase, is regulated monocyte-specifically by the physiologic stimuli TGF-β and 1α,25(OH)2D3 on mRNA level, although different mechanisms account for the upregulation of each gene

Susceptibility and Response of Human Blood Monocyte Subsets to Primary Dengue Virus Infection

Human blood monocytes play a central role in dengue infections and form the majority of virus infected cells in the blood. Human blood monocytes are heterogeneous and divided into CD16− and CD16+ subsets. Monocyte subsets play distinct roles during disease, but it is not currently known if monocyte subsets differentially contribute to dengue protection and pathogenesis. Here, we compared the susceptibility and response of the human CD16− and CD16+ blood monocyte subsets to primary dengue virus in vitro. We found that both monocyte subsets were equally susceptible to dengue virus (DENV2 NGC), and capable of supporting the initial production of new infective virus particles. Both monocyte subsets produced anti-viral factors, including IFN-α, CXCL10 and TRAIL. However, CD16+ monocytes were the major producers of inflammatory cytokines and chemokines in response to dengue virus, including IL-1β, TNF-α, IL-6, CCL2, 3 and 4. The susceptibility of both monocyte subsets to infection was increased after IL-4 treatment, but this increase was more profound for the CD16+ monocyte subset, particularly at early time points after virus exposure. These findings reveal the differential role that monocyte subsets might play during dengue disease

Macrophage phenotype is associated with disease severity in preterm infants with chronic lung disease.

The etiology of persistent lung inflammation in preterm infants with chronic lung disease of prematurity (CLD) is poorly characterized, hampering efforts to stratify prognosis and treatment. Airway macrophages are important innate immune cells with roles in both the induction and resolution of tissue inflammation

Endotoxin receptor CD14 in PiZ α-1-antitrypsin deficiency individuals

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens