CCR6, a CC Chemokine Receptor that Interacts with Macrophage Inflammatory Protein 3α and Is Highly Expressed in Human Dendritic Cells

Dendritic cells initiate immune responses by ferrying antigen from the tissues to the lymphoid organs for presentation to lymphocytes. Little is known about the molecular mechanisms underlying this migratory behavior. We have identified a chemokine receptor which appears to be selectively expressed in human dendritic cells derived from CD34+ cord blood precursors, but not in dendritic cells derived from peripheral blood monocytes. When stably expressed as a recombinant protein in a variety of host cell backgrounds, the receptor shows a strong interaction with only one chemokine among 25 tested: the recently reported CC chemokine macrophage inflammatory protein 3α. Thus, we have designated this receptor as the CC chemokine receptor 6. The cloning and characterization of a dendritic cell CC chemokine receptor suggests a role for chemokines in the control of the migration of dendritic cells and the regulation of dendritic cell function in immunity and infection

MAP3K19 Is Overexpressed in COPD and Is a Central Mediator of Cigarette Smoke-Induced Pulmonary Inflammation and Lower Airway Destruction.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung inflammation resulting in a progressive decline in lung function whose principle cause is cigarette smoke. MAP3K19 is a novel kinase expressed predominantly by alveolar and interstitial macrophages and bronchial epithelial cells in the lung. We found that MAP3K19 mRNA was overexpressed in a limited sampling of lung tissue from COPD patients, and a closer examination found it to be overexpressed in bronchoalveolar macrophages from COPD patients, as well as the bronchial epithelium and inflammatory cells in the lamina propria. We further found MAP3K19 to be induced in various cell lines upon environmental stress, such as cigarette smoke, oxidative and osmotic stress. Exogenous expression of MAP3K19 in cells caused an upregulation of transcriptionally active NF-κB, and secretion of the chemokines CXCL-8, CCL-20 and CCL-7. Inhibition of MAP3K19 activity by siRNA or small molecular weight inhibitors caused a decrease in cigarette smoke-induced inflammation in various murine models, which included a decrease in pulmonary neutrophilia and KC levels. In a chronic cigarette smoke model, inhibition of MAP3K19 significantly attenuated emphysematous changes in airway parenchyma. Finally, in a viral exacerbation model, mice exposed to cigarette smoke and influenza A virus showed a decrease in pulmonary neutrophilia, pro-inflammatory cytokines and viral load upon inhibition of MAP3K19. Collectively, these results suggest that inhibition of MAP3K19 may represent a novel strategy to target COPD that promises to have a potential therapeutic benefit for patients

MAP3K19 Is a Novel Regulator of TGF-β Signaling That Impacts Bleomycin-Induced Lung Injury and Pulmonary Fibrosis

<div><p>Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating disease for which two medications, pirfenidone and nintedanib, have only recently been approved for treatment. The cytokine TGF-β has been shown to be a central mediator in the disease process. We investigated the role of a novel kinase, MAP3K19, upregulated in IPF tissue, in TGF-β-induced signal transduction and in bleomycin-induced pulmonary fibrosis. MAP3K19 has a very limited tissue expression, restricted primarily to the lungs and trachea. In pulmonary tissue, expression was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. MAP3K19 was also found to be overexpressed in bronchoalveolar lavage macrophages from IPF patients compared to normal patients. Treatment of A549 or THP-1 cells with either MAP3K19 siRNA or a highly potent and specific inhibitor reduced phospho-Smad2 & 3 nuclear translocation following TGF-β stimulation. TGF-β-induced gene transcription was also strongly inhibited by both the MAP3K19 inhibitor and nintedanib, whereas pirfenidone had a much less pronounced effect. In combination, the MAP3K19 inhibitor appeared to act synergistically with either pirfenidone or nintedanib, at the level of target gene transcription or protein production. Finally, in an animal model of IPF, inhibition of MAP3K19 strongly attenuated bleomycin-induced pulmonary fibrosis when administered either prophylactically ortherapeutically. In summary, these results strongly suggest that inhibition of MAP3K19 may have a beneficial therapeutic effect in the treatment of IPF and represents a novel strategy to target this disease.</p></div

MAP3K19 is expressed in the atypical epithelium tissue that lies adjacent to the fibroblastic foci.

<p>MAP3K19 staining of a lung biopsy from a rapid IPF progressor patient showed clear, albeit lower intensity staining, of MAP3K19 (red staining, RabK19 Ab) in the atypical epithelium adjacent to the fibroblastic foci. This staining pattern of the atypical epithelium was found in multiple biopsy sections from IPF patients (n = 3). This section was counter-stained with SSEA-4 (brown stain).</p

The expression pattern and genomic structure of the MAP3K19 gene.

<p>(A) MAP3K19 expression in a panel of twenty human tissues was examined by RT-qPCR and normalized to GAPDH levels, showing the highest level of expression in the lung and testis. The MAP3K19 expression in the kidney was arbitrarily assigned a value of one and fold expression of MAP3K19 in all the other tissues examined were relative to kidney levels. This experiment was repeated twice and the average fold expression is shown ± SEM. The panel of human organ RNA was commercially obtained, and 17 of the 20 RNA samples are pooled from multiple donors (2–62 donors). (B) Deep sequencing of lung specific RNAs (n = 3) coupled with sequence analysis using GENCODE, the Human Genome Consortium HG19 and cufflinks revealed the genomic structure of MAP3K19, located on human chromosome 2. There were three upstream exons, that contain numerous start and stop codons and 10 coding exons. The kinase domain of MAP3K19 is encoded by exons 8 & 9. (C) RT-PCR analysis of MAP3K19 does not show evidence of differential splicing. MAP3K19 exons 1–7 (5’ Exons) and exons 7–10 (3’ Exons) were amplified by RT-PCR, and visualized on an ethidium bromide stained agarose gel, and all positive tissues showed amplified products of the expected size compared to the full length MAP3K19 gene. As a positive RT-PCR control, GAPDH was also amplified from each tissue sample.</p

The MAP3K19 inhibitor, Compound A, can block phosphorylated R-Smad nuclear accumulation, whereas pirfenidone has a negligible effect, following TGF-β1 stimulation.

<p>HeLa cells were cultured overnight on glass cover slips and pretreated with vehicle (DMSO), Compound A (1 μM) or pirfenidone (1 μM) for 30 minutes and treated with TGF-β1 (1 ng/ml) for one hour, prior to fixation and staining with anti-MAP3K19 (green staining, RabK19 polyclonal antibody) and anti-phospho-Smad2/3 (red staining). The cells were counterstained with DAPI nuclear stain. This experiment was repeated two independent times, and a representative experiment is shown. Similar results were observed with the A549 lung epithelial cell line.</p

MAP3K19 inhibition protected mice from bleomycin-induced pulmonary fibrosis when administered prophylactically (A-C).

<p>C57Bl/6J mice had saline (n = 5 mice) or bleomycin instilled intra-tracheally (i.t.) on Day 0 and for the prophylactic treatment, received either the vehicle control (n = 12) or Compound A (10 mg/kg; n = 12) delivered daily orally, dexamethasone administered intra-peritoneally (i.p.) every other day (3 mg/kg, n = 12), or no treatment (n = 5). On day 14, the mice were sacrificed, and histological criteria were used to assess (A) the pulmonary fibrosis determined using the Ashcroft scale [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154874#pone.0154874.ref031" target="_blank">31</a>], (B) the collagen deposition was scored using the method described by Faress et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154874#pone.0154874.ref032" target="_blank">32</a>], and (C) the soluble collagen content in lung homogenates was determined using a Sircol assay. Values shown are the mean ± SEM.</p

The majority of pulmonary macrophage co-express both MAP3K19 and CD68.

<p>Normal human lung was stained for MAP3K19 (Green, RabK19 polyclonal Ab) and CD68 (Red), and counterstained with DAPI.</p

MAP3K19 is over-expressed in bronchoalveolar lavage macrophages from IPF patients.

<p>Heatmap showing MAP3K19 expression in BAL macrophages isolated from mild-to-moderate IPF patients (n = 5), normal controls (n = 4) and healthy smokers (n = 6) as determined by RT-qPCR analysis. The data are normalized to GAPDH expression and the fold changes are depicted on the color scale. The RT-qPCR is representative of two independent experiments (biological replicates) performed in duplicate. Anova analysis of MAP3K19 expression levels among the different groups revealed that the difference in mRNA levels between the IPF group, with the lowest expressing patient omitted, and either the normal patients or smokers was significant, P<0.01.</p

Inhibition of MAP3K19 kinase activity by Compound A inhibits Smad2/3-mediated transcription.

<p>A549 cells were stably transfected with a construct containing the Smad binding element (SBE) linked to a luciferase reporter gene. The cells were plated out in 96-well plates, incubated with the designated concentrations of Compound A, and treated with 0.1 ng/ml of TGF-β1 for 18 hours. At that point, the cells were assayed for luciferase activity, indicative of Smad 2/3 transcriptional activation. Wells that received no Compound A, only TGF-β1, were normalized to 100% luciferase activity. The mean percentage of luciferase activity is shown ± SEM. A representative experiment is shown (n = 3).</p