The diverse roles of C-type lectin-like receptors in immunity

Our understanding of the C-type lectin-like receptors (CTLRs) and their functions in immunity have continued to expand from their initial roles in pathogen recognition. There are now clear examples of CTLRs acting as scavenger receptors, sensors of cell death and cell transformation, and regulators of immune responses and homeostasis. This range of function reflects an extensive diversity in the expression and signaling activity between individual CTLR members of otherwise highly conserved families. Adding to this diversity is the constant discovery of new receptor binding capabilities and receptor-ligand interactions, distinct cellular expression profiles, and receptor structures and signaling mechanisms which have expanded the defining roles of CTLRs in immunity. The natural killer cell receptors exemplify this functional diversity with growing evidence of their activity in other immune populations and tissues. Here, we broadly review select families of CTLRs encoded in the natural killer cell gene complex (NKC) highlighting key receptors that demonstrate the complex multifunctional capabilities of these proteins. We focus on recent evidence from research on the NKRP1 family of CTLRs and their interaction with the related C-type lectin (CLEC) ligands which together exhibit essential immune functions beyond their defined activity in natural killer (NK) cells. The ever-expanding evidence for the requirement of CTLR in numerous biological processes emphasizes the need to better understand the functional potential of these receptor families in immune defense and pathological conditions

Endothelial Gata5 transcription factor regulates blood pressure

Despite its high prevalence and economic burden, the aetiology of human hypertension remains incompletely understood. Here we identify the transcription factor GATA5, as a new regulator of blood pressure (BP). GATA5 is expressed in microvascular endothelial cells and its genetic inactivation in mice (Gata5-null) leads to vascular endothelial dysfunction and hypertension. Endothelial-specific inactivation of Gata5 mimics the hypertensive phenotype of the Gata5-null mice, suggestive of an important role for GATA5 in endothelial homeostasis. Transcriptomic analysis of human microvascular endothelial cells with GATA5 knockdown reveals that GATA5 affects several genes and pathways critical for proper endothelial function, such as PKA and nitric oxide pathways. Consistent with a role in human hypertension, we report genetic association of variants at the GATA5 locus with hypertension traits in two large independent cohorts. Our results unveil an unsuspected link between GATA5 and a prominent human condition, and provide a new animal model for hypertension

Positive regulation of plasmacytoid dendritic cell function via Ly49Q recognition of class I MHC

Plasmacytoid dendritic cells (pDCs) are an important source of type I interferon (IFN) during initial immune responses to viral infections. In mice, pDCs are uniquely characterized by high-level expression of Ly49Q, a C-type lectin-like receptor specific for class I major histocompatibility complex (MHC) molecules. Despite having a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, Ly49Q was found to enhance pDC function in vitro, as pDC cytokine production in response to the Toll-like receptor (TLR) 9 agonist CpG-oligonucleotide (ODN) could be blocked using soluble monoclonal antibody (mAb) to Ly49Q or H-2Kb. Conversely, CpG-ODN–dependent IFN-α production by pDCs was greatly augmented upon receptor cross-linking using immobilized anti-Ly49Q mAb or recombinant H-2Kb ligand. Accordingly, Ly49Q-deficient pDCs displayed a severely reduced capacity to produce cytokines in response to TLR7 and TLR9 stimulation both in vitro and in vivo. Finally, TLR9-dependent antiviral responses were compromised in Ly49Q-null mice infected with mouse cytomegalovirus. Thus, class I MHC recognition by Ly49Q on pDCs is necessary for optimal activation of innate immune responses in vivo

Proximity of transcription factor binding sites to predicted nucleosome centers within the Ly49 gene family.

<p>The C57BL/6 nucleosome map as generated in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004894#pcbi.1004894.g001" target="_blank">Fig 1</a> was compared individually to 17 transcription factors drawn from the TRANSFAC or JASPAR databases, plus TATA as a control. For factors with over 300 putative binding sites in the Ly49 gene family, a histogram is shown displaying each factor binding site’s distance to the nearest nucleosome. Dashed vertical lines indicate the nucleosome boundary. The kurtosis of each histogram is indicated. Histograms with a kurtosis less than -0.5 (blue) are considered to preferentially avoid nucleosome centers; those with kurtosis over 0.5 (red) are preferentially clustered in the center; and those between -0.5 and 0.5 (black) display no preference for nucleosome co-occupancy.</p

Nucleosome Presence at AML-1 Binding Sites Inversely Correlates with Ly49 Expression: Revelations from an Informatics Analysis of Nucleosomes and Immune Cell Transcription Factors

<div><p>Beyond its role in genomic organization and compaction, the nucleosome is believed to participate in the regulation of gene transcription. Here, we report a computational method to evaluate the nucleosome sensitivity for a transcription factor over a given stretch of the genome. Sensitive factors are predicted to be those with binding sites preferentially contained within nucleosome boundaries and lacking 10 bp periodicity. Based on these criteria, the Acute Myeloid Leukemia-1a (AML-1a) transcription factor, a regulator of immune gene expression, was identified as potentially sensitive to nucleosomal regulation within the mouse Ly49 gene family. This result was confirmed in RMA, a cell line with natural expression of Ly49, using MNase-Seq to generate a nucleosome map of chromosome 6, where the Ly49 gene family is located. Analysis of this map revealed a specific depletion of nucleosomes at AML-1a binding sites in the expressed Ly49A when compared to the other, silent Ly49 genes. Our data suggest that nucleosome-based regulation contributes to the expression of Ly49 genes, and we propose that this method of predicting nucleosome sensitivity could aid in dissecting the regulatory role of nucleosomes in general.</p></div

AML-1a shows specific nucleosome depletion <i>in vitro</i>.

<p>MNase-Seq analysis was performed on the Ly49A-expressing C57BL/6 mouse cell line, RMA, to determine the actual nucleosome positions within the Ly49 gene family. (A) Ly49 expression levels on RMA (green) compared to isotype (yellow), and compared to normal NK cells isolated from C57BL/6 splenocytes as a positive (blue) and negative (isotype, red) control. Note that RMA cells are only positive for Ly49A/D, but are negative for Ly49D, indicating the single expression of Ly49A. (B) Following MNase digest, mononucleosomes were isolated from the ~150 bp fragment and sequenced. (C) Nucleosomes from the MNase-Seq results were compared to the predicted nucleosome landscape to determine the degree of true positive nucleosomes at each Ly49 gene (‘Whole’, first column). This determination was repeated for each of the indicated transcription factors. For each column, a heatmap indicates the relative degree of accuracy of the predictions, with blue cells being more accurate and red being less. Regions with very low accuracy are believed to have had a nucleosome remodeling event. (D) A chi-square analysis was performed to assess the degree of specific nucleosome depletion at the indicated transcription factor binding site, taking the overall nucleosome accuracy (‘Whole’, first column of C) as the expected accuracy for that Ly49. Overall significance of the site-specific nucleosome depletion is indicated, and for each Ly49, the factors most contributing to that significance are indicated in red, with color intensity correlating with contribution to the chi-square statistic. A p value < 0.05 was considered significant.</p

Confounding variables unlikely to account for AML-1a-specific nucleosome depletion in Ly49A.

<p>(A) Schematic highlighting the possibility of confounding variables impacting transcription factor/nucleosome relationship studies. If an event occurs to deplete all nucleosomes covering a red factor, the coverage of the red factor drops from 100% to 0%. However, the coverage of all purple factors drops from 75% to 0%, and green from 75% to 50%. Taking nucleosome coverage as the readout, it is impossible to determine whether the red, purple, or green factors ‘caused’ the nucleosome depletion. However, if red depletions are always correlated with purple depletions, while purple depletions only occasionally predict red depletions, it is likely that red is causing the nucleosome remodeling. (B-C) Logistic regression analysis was performed for each of the four most depleted transcription factors from 4C (AML-1a, c-ETS-1, Lyf-1, and MZF-1), using (B) AML-1a as the predictor, or (C) AML-1a as the reporter. In each case, AML-1a was a better predictor of the other two nucleosome-preferring factors, Lyf-1 and MZF-1, than either were of AML-1a. AML-1a performed equally well as predictor or reporter against c-ETS-1, a factor that had no preference for nucleosome coverage, and so was unlikely to be involved as a confounding variable. (D) Chromatin immunoprecipitation (ChIP) using anti-AML-1 or an irrelevant isotype control was performed on RMA chromatin extract. ChIP using the anti-AML-1 antibody resulted in about a 10-fold enrichment of the Ly49A promoter region when compared to ChIP using the isotype control, indicating that AML-1 is likely bound to the promoter. As a control, a region of DNA approximately 6 kbp upstream of the Ly49A promoter displayed no such enrichment.</p

Convergence of actual and predicted nucleosomes at the indicated promoter.

<p>Convergence of actual and predicted nucleosomes at the indicated promoter.</p