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Rhinovirus stimulated IFN-? production: how important are plasmacytoid DCs, monocytes and endosomal pH?

ABSTRACT: Human rhinovirus (HRV) infection is a major cause of asthma exacerbations, which appears to be linked to a defective innate immune response to infection. Although the type I interferons (IFN-? and IFN-?) have a critical role in protecting against most viral infections, the cells responsible for IFN production in response to HRV and the relative importance of pattern recognition receptors located in endosomes has not been fully elucidated. In the current study we demonstrate that, using intracellular flow cytometry, >90% of the IFN-?-producing cells in human blood mononuclear cells following HRV16 exposure are plasmacytoid dendritic cells, whereas monocytes and myeloid dendritic cells contribute only 10% and <1%, respectively, of the IFN-? production. Bafilomycin and chloroquine, agents that inhibit the function of endosomal toll-like receptors (TLRs), significantly reduced the capacity of TLR3-, TLR7- and TLR-9-stimulated cells to produce IFN-? and the IFN-induced chemokine CXCL10 (IP-10). In contrast, only bafilomycin (but not chloroquine) effectively suppressed HRV16-stimulated IFN-? and IP-10 production, whereas neither bafilomycin or chloroquine inhibited HRV16-stimulated interleukin-6 release. Attempts to block IFN-? production with commercially available TLR-specific oligonucleotides were unsuccessful due to major 'off-target' effects. These findings suggest that among circulating haemopoietic cells, plasmacytoid dendritic cells and TLRs located within endosomes are critical for inducing efficient IFN-I production in response to HRVs.

SUBMITTER: Xi Y

PROVIDER: S-EPMC4673444 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Rhinovirus stimulated IFN-α production: how important are plasmacytoid DCs, monocytes and endosomal pH?

Xi Yang Y Finlayson Arvid A White Oliva J OJ Carroll Melanie L ML Upham John W JW

Clinical & translational immunology 20151030 10

Human rhinovirus (HRV) infection is a major cause of asthma exacerbations, which appears to be linked to a defective innate immune response to infection. Although the type I interferons (IFN-α and IFN-β) have a critical role in protecting against most viral infections, the cells responsible for IFN production in response to HRV and the relative importance of pattern recognition receptors located in endosomes has not been fully elucidated. In the current study we demonstrate that, using intracell ...[more]

PMID: 26682054

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Project description:Primary Sjögren's syndrome (pSS) is a systemic auto-immune disease typified by dryness of the mouth and eyes. A majority of patients with pSS have a type-I interferon (IFN)-signature, which is defined as the increased expression of IFN-induced genes in circulating immune cells and is associated with increased disease activity. As plasmacytoid dendritic cells (pDC) are the premier type-I IFN-producing cells and are present at the site of inflammation, they are thought to play a significant role in pSS pathogenesis. Considering the lack of data on pDC regulation and function in pSS patients, we here provided the first in-depth molecular characterization of pSS pDCs. In addition, a group of patients with non-Sjögren's sicca (nSS) was included; these poorly studied patients suffer from complaints similar to pSS patients, but are not diagnosed with Sjögren's syndrome. We isolated circulating pDCs from two independent cohorts of patients and controls (each n = 31) and performed RNA-sequencing, after which data-driven networks and modular analysis were used to identify robustly reproducible transcriptional "signatures" of differential and co-expressed genes. Four signatures were identified, including an IFN-induced gene signature and a ribosomal protein gene-signature, that indicated pDC activation. Comparison with a dataset of in vitro activated pDCs showed that pSS pDCs have higher expression of many genes also upregulated upon pDC activation. Corroborating this transcriptional profile, pSS pDCs produced higher levels of pro-inflammatory cytokines, including type-I IFN, upon in vitro stimulation with endosomal Toll-like receptor ligands. In this setting, cytokine production was associated with expression of hub-genes from the IFN-induced and ribosomal protein gene-signatures, indicating that the transcriptional profile of pSS pDCs underlies their enhanced cytokine production. In all transcriptional analyses, nSS patients formed an intermediate group in which some patients were molecularly similar to pSS patients. Furthermore, we used the identified transcriptional signatures to develop a discriminative classifier for molecular stratification of patients with sicca. Altogether, our data provide in-depth characterization of the aberrant regulation of pDCs from patients with nSS and pSS and substantiate their perceived role in the immunopathology of pSS, supporting studies that target pDCs, type-I IFNs, or IFN-signaling in pSS.

Project description:Recent studies have reported that the crosslinking of regulatory receptors (RRs), such as blood dendritic cell antigen 2 (BDCA-2) (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses the production of type I interferons (IFN-I, ?/?/?) and other cytokines in response to toll-like receptor 7 and 9 (TLR7/9) ligands. The exact mechanism of how this B cell receptor (BCR)-like signaling blocks TLR7/9-mediated IFN-I production is unknown. Here, we stimulated BCR-like signaling by ligation of RRs with BDCA-2 and ILT7 mAbs, hepatitis C virus particles, or BST2 expressing cells. We compared BCR-like signaling in proliferating pDC cell line GEN2.2 and in primary pDCs from healthy donors, and addressed the question of whether pharmacological targeting of BCR-like signaling can antagonize RR-induced pDC inhibition. To this end, we tested the TLR9-mediated production of IFN-I and proinflammatory cytokines in pDCs exposed to a panel of inhibitors of signaling molecules involved in BCR-like, MAPK, NF-?B, and calcium signaling pathways. We found that MEK1/2 inhibitors, PD0325901 and U0126 potentiated TLR9-mediated production of IFN-I in GEN2.2 cells. More importantly, MEK1/2 inhibitors significantly increased the TLR9-mediated IFN-I production blocked in both GEN2.2 cells and primary pDCs upon stimulation of BCR-like or phorbol 12-myristate 13-acetate-induced protein kinase C (PKC) signaling. Triggering of BCR-like and PKC signaling in pDCs resulted in an upregulation of the expression and phoshorylation of c-FOS, a downstream gene product of the MEK1/2-ERK pathway. We found that the total level of c-FOS was higher in proliferating GEN2.2 cells than in the resting primary pDCs. The PD0325901-facilitated restoration of the TLR9-mediated IFN-I production correlated with the abrogation of MEK1/2-ERK-c-FOS signaling. These results indicate that the MEK1/2-ERK pathway inhibits TLR9-mediated type I IFN production in pDCs and that pharmacological targeting of MEK1/2-ERK signaling could be a strategy to overcome immunotolerance of pDCs and re-establish their immunogenic activity.

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Rhinovirus stimulated IFN-? production: how important are plasmacytoid DCs, monocytes and endosomal pH?

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Rhinovirus stimulated IFN-α production: how important are plasmacytoid DCs, monocytes and endosomal pH?

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