Differential role of basal keratinocytes in UV-induced immunosuppression and skin cancer.

Cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) comprise major UV-induced photolesions. If left unrepaired, these lesions can induce mutations and skin cancer, which is facilitated by UV-induced immunosuppression. Yet the contribution of lesion and cell type specificity to the harmful biological effects of UV exposure remains currently unclear. Using a series of photolyase-transgenic mice to ubiquitously remove either CPDs or 6-4PPs from all cells in the mouse skin or selectively from basal keratinocytes, we show that the majority of UV-induced acute effects to require the presence of CPDs in basal keratinocytes in the mouse skin. At the fundamental level of gene expression, CPDs induce the expression of genes associated with repair and recombinational processing of DNA damage, as well as apoptosis and a response to stress. At the organismal level, photolyase-mediated removal of CPDs, but not 6-4PPs, from the genome of only basal keratinocytes substantially diminishes the incidence of skin tumors; however, it does not affect the UVB-mediated immunosuppression. Taken together, these findings reveal a differential role of basal keratinocytes in these processes, providing novel insights into the skin's acute and chronic responses to UV in a lesion- and cell-type-specific manner.

Regulation of Intracellular Triiodothyronine Is Essential for Optimal Macrophage Function.

Innate immune cells, including macrophages, have recently been identified as target cells for thyroid hormone. We hypothesized that optimal intracellular concentrations of the active thyroid hormone triiodothyronine (T3) are essential for proinflammatory macrophage function. T3 is generated intracellularly by type 2 deiodinase (D2) and acts via the nuclear thyroid hormone receptor (TR). In zebrafish embryos, D2 knockdown increased mortality during pneumococcal meningitis. Primary murine D2 knockout macrophages exhibited impaired phagocytosis and partially reduced cytokine response to stimulation with bacterial endotoxin. These effects are presumably due to reduced intracellular T3 availability. Knockdown of the main TR in macrophages, TRα, impaired polarization into proinflammatory macrophages and amplified polarization into immunomodulatory macrophages. Intracellular T3 availability and action appear to play a crucial role in macrophage function. Our data suggest that low intracellular T3 action has an anti-inflammatory effect, possibly due to an effect on macrophage polarization mediated via the TRα. This study provides important insights into the link between the endocrine and innate immune system.

The dermal microenvironment induces the expression of the alternative activation marker CD301/mMGL in mononuclear phagocytes, independent of IL-4/IL-13 signaling.

Recently, we have shown that mononuclear phagocytes comprise the majority of interstitial cells in the mouse dermis, as indicated by their phenotypic and functional characteristics. In particular, these cells express the mouse macrophage galactose-/N-acetylgalactosamine-specific-lectin (mMGL)/CD301, identified by the monoclonal antibody ER-MP23, as well as other macrophage markers. As expression of mMGL is induced by IL-4 or IL-13 and is therefore a marker of alternatively activated macrophages, we asked whether dermal mononuclear phagocytes are genuinely alternatively activated. We observed that these cells expressed, next to mMGL, two other alternative activation markers, namely, the mannose receptor/CD206 and Dectin-1. Yet, as this expression profile was similar in IL-4 receptor alpha knockout mice, neither IL-4 nor IL-13 signaling appeared to be required for this phenotype. We also found that Langerhans cells (LC), which showed only a low level of mMGL in the epidermis, up-regulated mMGL expression upon migration through the dermis, allowing these cells to internalize limited amounts of mMGL ligands. LC isolated from epidermal preparations did not show this up-regulation when cultured in standard medium, but whole skin-conditioned medium did stimulate mMGL expression by LC. The vast majority of mMGL molecules was present in the cytoplasm, however. LC, which arrived in skin-draining lymph nodes, quickly down-regulated mMGL expression, and dermally derived cells retained significant mMGL levels. Taken together, these data suggest that the dermal microenvironment induces mononuclear phagocyte subpopulations to express mMGL and possibly other markers of alternatively activated macrophages, independent of IL-4/IL-13 signaling.

Study on inflammation-related genes and microRNAs, with special emphasis on the vascular repair factor HGF and miR-574-3p, in monocytes and serum of patients with T2D.

BACKGROUND: Recently, we reported signs of inflammation (raised IL-8, reduced miR-146a) and signs of vascular repair (raised HGF) in the serum of Ecuadorian patients with type 2 diabetes (T2D). In contrast, we found that the circulating monocytes lacked up-regulation of classical inflammatory genes (IL-1B, IL-6, and TNF) and there was even significant down-regulation of PTGS2. Notably, genes and a microRNA involved in adhesion, cell differentiation and morphology (CD9, DHRS3, PTPN7 and miR-34c-5p) were up-regulated in the T2D monocytes, suggesting a role of the anti-inflammatory cells in adhesion, vascular repair and invasion. AIM: To determine the gene expression of the vascular repair factor HGF in the circulating monocytes of patients with T2D and to investigate the relationship between HGF and the expression of the other previously tested monocyte genes and the contribution to the raised serum level of HGF. In addition, we tested the level of 6 microRNAs, which were previously found abnormal in the circulating monocytes, in the serum of the patients. METHODS: A gene and microRNA expression study in monocytes and serum of 64 Ecuadorian patients with T2D (37-85 years) and 44 non-diabetic controls (32-87 years). RESULTS: The gene expression of HGF was significantly raised in the monocytes of the patients with T2D and associated with the expression of genes involved in adhesion, cell differentiation and morphology. HGF gene expression did not correlate with the serum level of HGF. The monocyte expression of pro-inflammatory cytokine genes was also not associated with the serum levels of these cytokines. The level of miR-574-3p was significantly decreased in the serum of the patients with T2D, and correlated in expression with the decreased well-established inflammation-regulating miR-146a. The level of the microRNAs in serum did not correlate with their expression level in monocytes. CONCLUSION: In circulating monocytes of Ecuadorian T2D patients, the microRNA and gene expression of important inflammatory/chemotactic/motility/vascular repair factors differs from the expression in serum. While monocytes show a gene expression profile compatible with an anti-inflammatory state, serum shows a molecular profile compatible with an inflammatory state. Both compartments show molecular signs of vascular repair support, i.e. up-regulated HGF levels.

Type 2 Diabetes Monocyte MicroRNA and mRNA Expression: Dyslipidemia Associates with Increased Differentiation-Related Genes but Not Inflammatory Activation.

OBJECTIVE: To study the expression pattern of microRNAs and mRNAs related to inflammation in T2D monocytes. DESIGN: A microRNA finding study on monocytes of T2D patients and controls using array profiling was followed by a quantitative Real Time PCR (qPCR) study on monocytes of an Ecuadorian validation cohort testing the top over/under-expressed microRNAs. In addition, monocytes of the validation cohort were tested for 24 inflammation-related mRNAs and 2 microRNAs previously found deregulated in (auto)-inflammatory monocytes. RESULTS: In the finding study, 142 significantly differentially expressed microRNAs were identified, 15 having the strongest power to discriminate T2D patients from controls (sensitivity 66%, specificity 90%). However, differences in expression of these microRNAs between patients and controls were small. On the basis of >1.4 or <0.6-fold change expression 5 microRNAs were selected for further validation. One microRNA (miR-34c-5p) was validated as significantly over-expressed in T2D monocytes. In addition, we found over expression of 3 mRNAs (CD9, DHRS3 and PTPN7) in the validation cohort. These mRNAs are important for cell morphology, adhesion, shape change, and cell differentiation. Classical inflammatory genes (e.g. TNFAIP3) were only over-expressed in monocytes of patients with normal serum lipids. Remarkably, in dyslipidemia, there was a reduction in the expression of inflammatory genes (e.g. ATF3, DUSP2 and PTGS2). CONCLUSIONS: The expression profile of microRNAs/mRNAs in monocytes of T2D patients indicates an altered adhesion, differentiation, and shape change potential. Monocyte inflammatory activation was only found in patients with normal serum lipids. Abnormal lipid values coincided with a reduced monocyte inflammatory state.

Macrophages and dendritic cells constitute a major subpopulation of cells in the mouse dermis.

Macrophages and dendritic cells (DC) in tissues with close contact to the environment are of essential importance in host defense and are therefore present in sizeable numbers. Therefore, it is surprising that mononuclear phagocyte populations of the dermis have rarely been investigated in a quantitative manner. In this study, we examined mouse dermal skin immunophenotypically and related the observed numbers of observed cells to the total number of nucleated cells. These analyses show that about 70% of all dermal cells represent CD45+ leukocytes. The vast majority of these cells (approximately 60% of total) expresses the mononuclear phagocyte markers mMGL (ER-MP23), F4/80 and CD11b. In addition, these cells show avid phagocytic capacity and thus are identified as dermal macrophages. Different subpopulations can be defined using markers such as sialoadhesin, ER-HR3 and mSIGN-R1 (ER-TR9). Interestingly, MHC class II expression differs significantly between dermal cells from ear versus back skin. Moreover, we have identified small populations of dermal DC and migrating Langerhans cells (together approximately 10% of total). In summary, our findings show that mononuclear phagocyte populations form the majority of dermal cells and thus have been clearly underestimated so far.

Decreased serum level of miR-146a as sign of chronic inflammation in type 2 diabetic patients.

BACKGROUND: There is increasing evidence that chronic inflammation is an important determinant in insulin resistance and in the pathogenesis of type 2 diabetes (T2D). MicroRNAs constitute a newly discovered system of cell regulation and in particular two microRNAs (miR-146a and miR-155) have been described as regulators and biomarkers of inflammation. AIM: To determine a putative association between the levels of miR-146a and miR-155 in serum of T2D patients, clinical parameters and serological indicators of inflammation. METHODS: We performed quantitative Real Time PCR (qPCR) of microRNAs from serum (56 Ecuadorian T2D ambulatory patients and 40 non-diabetic controls). In addition, we evaluated T2D-related serum cytokines.chemokines and growth factors using a commercially available multi-analyte cytometric bead array system. We correlated outcomes to clinical parameters, including BMI, HbA1c and lipid state. RESULTS: The Ecuadorian non-diabetic controls appeared as overweight (BMI>25: patients 85%, controls 82.5%) and as dyslipidemic (hypercholesterolemia: patients 60.7%, controls 67.5%) as the patients. The serum levels of miR-146a were significantly reduced in T2D patients as compared to these non-diabetic, but obese/dyslipidemic control group (mean patients 0.61, mean controls set at 1; p = 0.042), those of miR-155 were normal.The serum levels of both microRNAs correlated to each other (r = 0.478; p<0.001) and to leptin levels. The microRNAs did not correlate to BMI, glycemia and dyslipidemia.From the tested cytokines, chemokines and growth factors, we found IL-8 and HGF significantly raised in T2D patients versus non-diabetic controls (p = 0.011 and 0.023 respectively). CONCLUSIONS: This study shows decreased serum anti-inflammatory miR-146a, increased pro-inflammatory IL-8 and increased HGF (a vascular/insular repair factor) as discriminating markers of failure of glucose control occurring on the background of obesity and dyslipidemia.

MicroRNA-Mediated Down-Regulation of M-CSF Receptor Contributes to Maturation of Mouse Monocyte-Derived Dendritic Cells.

Dendritic cell (DC) maturation is a tightly regulated process that requires coordinated and timed developmental cues. Here we investigate whether microRNAs are involved in this process. We identify microRNAs in mouse GM-CSF-generated, monocyte-related DC (GM-DC) that are differentially expressed during both spontaneous and LPS-induced maturation and characterize M-CSF receptor (M-CSFR), encoded by the Csf1r gene, as a key target for microRNA-mediated regulation in the final step toward mature DC. MicroRNA-22, -34a, and -155 are up-regulated in mature MHCII(hi) CD86(hi) DC and mediate Csf1r mRNA and protein down-regulation. Experimental inhibition of Csf1r-targeting microRNAs in vitro results not only in sustained high level M-CSFR protein expression but also in impaired DC maturation upon stimulation by LPS. Accordingly, over-expression of Csf1r in GM-DC inhibits terminal differentiation. Taken together, these results show that developmentally regulated microRNAs control Csf1r expression, supplementing previously identified mechanisms that regulate its transcription and protein surface expression. Furthermore, our data indicate a novel function for Csf1r in mouse monocyte-derived DC, showing that down-regulation of M-CSFR expression is essential for final DC maturation.

Kupffer cells express a unique combination of phenotypic and functional characteristics compared with splenic and peritoneal macrophages.

The immunostimulatory role of Kupffer cells in various inflammatory liver diseases is still not fully understood. In this study, phenotypic and functional aspects of Kupffer cells from healthy C57BL/6 mice were analyzed and compared with those of splenic and peritoneal macrophages to generate a blueprint of the cells under steady-state conditions. In the mouse liver, only one population of Kupffer cells was identified as F4/80(high)CD11b(low) cells. We observed that freshy isolated Kupffer cells are endocytic and show a relatively high basal ROS content. Interestingly, despite expression of TLR mRNA on Kupffer cells, ligation of TLR4, TLR7/8, and TLR9 resulted in a weak induction of IL-10, low or undetectable levels of IL-12p40 and TNF, and up-regulation of CD40 on the surface. Kupffer cells and splenic macrophages show functional similarities, in comparison with peritoneal macrophages, as reflected by comparable levels of TLR4, TLR7/8, and TLR9 mRNA and low or undetectable levels of TNF and IL-12p40 produced upon TLR ligation. The unique, functional characteristics of Kupffer cells, demonstrated in this study, suggest that Kupffer cells under steady-state conditions are specialized as phagocytes to clear and degrade particulates and only play a limited immunoregulatory role via the release of soluble mediators.