Joost B. Vos

During secondary immune responses, Ab-opsonized bacteria are efficiently taken up via FcRs by dendritic cells. We now demonstrate that this process induces cross-talk between FcRs and TLRs, which results in synergistic release of several inflammatory cytokines, as well as altered lipid metabolite profiles. This altered inflammatory profile redirects Th1 polarization toward Th17 cell responses. Interestingly, GM-CSF–producing Th cells were synergistically evoked as well, which suggests the onset of polyfunctional Th17 cells. Synergistic cytokine release was dependent on activation via MyD88 and ITAM signaling pathways through TLRs and FcRs, respectively. Cytokine regulation occurred via transcription-dependent mechanisms for TNF-α and IL-23 and posttranscriptional mechanisms for caspase-1–dependent release of IL-1β. Furthermore, cross-talk between TLRs and FcRs was not restricted to dendritic cells. In conclusion, our results support that bacteria alone initiate fundamentally different immune responses compared with Ab-opsonized bacteria through the combined action of two classes of receptors and, ultimately, may refine new therapies for inflammatory diseases.

M2 macrophages suppress inflammation in numerous disorders, including tumour formation, infection and obesity. However, the exact role of M2 macrophages in the context of several other diseases is still largely undefined. We here show that human M2 macrophages promote inflammation instead of suppressing inflammation on simultaneous exposure to complexed IgG (c-IgG) and TLR ligands, as occurs in the context of diseases such as rheumatoid arthritis (RA). c-IgG-TLR ligand co-stimulation of M2 macrophages selectively amplifies production of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 and promotes Th17 responses, which all play a critical role in RA pathology. Induction of pro-inflammatory cytokines on c-IgG co-stimulation mainly depends on Fc gamma receptor IIa (FcγRIIa), which selectively amplifies cytokine gene transcription and induces caspase-1 activation. These data indicate that FcγR-TLR cross-talk may be targeted for treatment to attenuate inflammation in RA, by restoring the anti-inflammatory function of M2 macrophages.

Blood–brain barrier (BBB) dysfunction is a major hallmark of many neurological diseases, including multiple sclerosis (MS). Using a genomics approach, we defined a microRNA signature that is diminished at the BBB of MS patients. In particular, miR-125a-5p is a key regulator of brain endothelial tightness and immune cell efflux. Our findings suggest that repair of a disturbed BBB through microRNAs may represent a novel avenue for effective treatment of MS.

MicroRNAs are negative regulators of gene expression that play a key role in cell‐type specific differentiation and modulation of cell function and have been proposed to be involved in neovascularization. Previously, using an extensive cloning and sequencing approach, we identified miR‐126 to be specifically and highly expressed in human endothelial cells (EC). Here, we demonstrate EC‐specific expression of miR‐126 in capillaries and the larger vessels in vivo. We therefore explored the potential role of miR‐126 in arteriogenesis and angiogenesis. Using miR‐reporter constructs, we show that miR‐126 is functionally active in EC in vitro and that it could be specifically repressed using antagomirs specifically targeting miR‐126. To study the consequences of miR‐126 silencing on vascular regeneration, mice were injected with a single dose of antagomir‐126 or a control ‘scramblemir’ and exposed to ischemia of the left hindlimb by ligation of the femoral artery. Although miR‐126 was effectively silenced in mice treated with a single, high dose (HD) of antagomir‐126, laser Doppler perfusion imaging did not show effects on blood flow recovery. In contrast, quantification of the capillary density in the gastrocnemius muscle revealed that mice treated with a HD of antagomir‐126 had a markedly reduced angiogenic response. Aortic explant cultures of the mice confirmed the role of miR‐126 in angiogenesis. Our data demonstrate a facilitary function for miR‐126 in ischemia‐induced angiogenesis and show the efficacy and specificity of antagomir‐induced silencing of EC‐specific microRNAs in vivo.

The epithelial surface of the airways is the largest barrier-forming interface between the human body and the outside world. It is now well recognized that, at this strategic position, airway epithelial cells play an eminent role in host defense by recognizing and responding to microbial exposure. Conversely, inhaled microorganisms also respond to contact with epithelial cells. Our understanding of this cross talk is limited, requiring sophisticated experimental approaches to analyze these complex interactions. High-throughput technologies, such as DNA microarray analysis and serial analysis of gene expression (SAGE), have been developed to screen for gene expression levels at large scale within single experiments. Since their introduction, these hypothesis-generating technologies have been widely used in diverse areas such as oncology and brain research. Successful application of these genomics-based technologies has also revealed novel insights in host-pathogen interactions in both the host and pathogen. This review aims to provide an overview of the SAGE and microarray technology illustrated by their application in the analysis of host-pathogen interactions. In particular, the interactions between epithelial cells in the human lungs and clinically relevant microorganisms are the central focus of this review.

Summary: The number of hematopoietic stem cells (HSC) and endothelial progenitor cells (EPC) is thought to be a marker for neovascularization and vascular repair. Because physical inactivity and aging are risk factors for cardiovascular diseases, these factors may influence the numbers of HSCs and EPCs. Therefore, we examined baseline and exercise‐induced levels of HSCs and EPCs in sedentary and trained young and older men. To study the role of aging in eight sedentary young (19–28 years) and eight sedentary older men (67–76 years), baseline and acute exercise‐induced numbers of HSCs (CD34⁺‐cells) and EPCs (CD34⁺/VEGFR‐2⁺‐cells) were quantified by fluorescence‐activated cell sorter (FACS) analysis. To examine the effect of chronic training, eight age‐matched trained young men (18–28 years) were compared with sedentary young men, whereas older men performed an 8‐week endurance training. Older men showed significantly lower baseline and exercise‐induced levels of HSCs/EPCs than the young men (P < 0.05). In young and older men, acute exercise significantly increased HSCs (P < 0.01), but not EPCs. The absolute increase in numbers of HSCs was attenuated in older men (P = 0.03). Apart from the lower baseline numbers of EPCs after chronic training in older men, training status did not alter baseline or exercise‐induced levels of HSCs/EPCs in young and older men. We concluded that advancing age results in lower circulating numbers of HSCs and EPCs and attenuates the acute exercise‐induced increase in HSCs. Interestingly, in young as well as in older men chronic endurance training does not affect baseline and exercise‐induced numbers of HSCs and EPCs.

IcroRNAs are 20- to 23-nucleotide RNA molecules that can regulate gene expression. Currently >400 microRNAs have been experimentally identified in mammalian genomes, whereas estimates go up to 1000 and beyond. Here we show that many more mammalian microRNAs exist. We discovered novel microRNA candidates using two approaches: testing of computationally predicted microRNAs by a modified microarray-based detection system, and cloning and sequencing of large numbers of small RNAs from different human and mouse tissues. Together these efforts experimentally identified 348 novel mouse and 81 novel human microRNA candidate genes. Most novel microRNAs candidates are not conserved beyond mammals, and ~10% are taxon-specific. Our analyses indicate that the entire microRNA repertoire is not remotely exhausted.

Several aspects of hippocampal cell function are influenced by adrenal‐secreted glucocorticoids in a delayed, genomic fashion. Previously, we used Serial Analysis of Gene Expression to identify glucocorticoid receptor (GR)‐induced transcriptional changes in the hippocampus at a fixed time point. However, because changes in mRNA levels are transient and most likely precede the effects on hippocampal cell function, the aim of the current study was to assess the transcriptional changes in a broader time window by generating a time curve of GR‐mediated gene expression changes. Therefore, we used rat hippocampal slices obtained from adrenalectomised rats, substituted in vivo with low corticosterone pellets, predominantly occupying the hippocampal mineralocorticoid receptors. To activate GR, slices were treated in vitro with a high (100 nM) dose of corticosterone and gene expression was profiled 1, 3 and 5 h after GR‐activation. Using Affymetrix GeneChips, a striking pattern with different waves of gene expression was observed, shifting from exclusively down‐regulated genes 1 h after GR‐activation to both up and down regulated genes 3 h after GR‐activation. After 5 h, the response was almost back to baseline. Additionally, real‐time quantitative polymerase chain reaction was used for validation of a selection of responsive genes including genes involved in neurotransmission and synaptic plasticity such as the corticotropin releasing hormone receptor 1, monoamine oxidase A, LIMK1 and calmodulin 2. This permitted confirmation of GR‐responsiveness of 15 out of 18 selected genes. In conclusion, direct activation of GR in hippocampal slices results in transient changes in gene expression. The pattern in which gene expression was modulated suggests that the fast genomic effects of glucocorticoids may be realised via transrepression, preceding a later wave of transactivation. Furthermore, we identified a number of interesting candidate genes which may underlie the glucocorticoid‐mediated effects on hippocampal cell function.

Background: Epithelia are barrier-forming tissues that protect the organism against external noxious stimuli. Despite the similarity in function of epithelia, only few common protective mechanisms that are employed by these tissues have been systematically studied. Comparative analysis of genome-wide expression profiles generated by means of Serial Analysis of Gene Expression (SAGE) is a powerful approach to yield further insight into epithelial host defense mechanisms. We performed an extensive comparative analysis of previously published SAGE data sets of two types of epithelial cells, namely bronchial epithelial cells and keratinocytes, in which the response to pro-inflammatory cytokines was assessed. These data sets were used to elucidate a common denominator in epithelial host defense. Results: Bronchial epithelial cells and keratinocytes were found to have a high degree of overlap in gene expression. Using an in silico approach, an epithelial-specific molecular signature of gene expression was identified in bronchial epithelial cells and keratinocytes comprising of family members of keratins, small proline-rich proteins and proteinase inhibitors. Whereas some of the identified genes were known to be involved in inflammation, the majority of the signature represented genes that were previously not associated with host defense. Using polymerase chain reaction, presence of expression of selected tissue-specific genes was validated. Conclusion: Our comparative analysis of gene transcription reveals that bronchial epithelial cells and keratinocytes both express a subset of genes that is likely to be essential in epithelial barrier formation in these cell types. The expression of these genes is specific for bronchial epithelial cells and keratinocytes and is not seen in non-epithelial cells. We show that bronchial epithelial cells, similar to keratinocytes, express components that are able to form a cross-linked protein envelope that may contribute to an effective barrier against noxious stimuli and pathogens.

Mucosal secretions contain a range of defense effector molecules including antimicrobial peptides and proteinase inhibitors. These molecules play a central role in host defense against infection, and in a variety of immune and inflammatory reactions. The aim of this study was to analyze the levels of neutrophil defensins, the cathelicidin hCAP-18/LL-37, and the proteinase inhibitors secretory leukocyte proteinase inhibitor, SKALP/elafin and cystatin M/E in various mucosal secretions and urine. We show here that especially seminal plasma is characterized by high concentrations of hCAP-18/LL-37, SLPI, SKALP/elafin and cystatin M/E. The results of this study demonstrate that each mucosal secretion is characterized by a unique profile of effector molecules, which may supply individual mucosal secretions with specific properties related to the control of local infection and inflammation.