Major and Minor Group Rhinoviruses Elicit Differential Signaling and Cytokine Responses as a Function of Receptor-Mediated Signal Transduction

Major- and minor-group human rhinoviruses (HRV) enter their host by binding to the cell surface molecules ICAM-1 and LDL-R, respectively, which are present on both macrophages and epithelial cells. Although epithelial cells are the primary site of productive HRV infection, previous studies have implicated macrophages in establishing the cytokine dysregulation that occurs during rhinovirus-induced asthma exacerbations. Analysis of the transcriptome of primary human macrophages exposed to major- and minor-group HRV demonstrated differential gene expression. Alterations in gene expression were traced to differential mitochondrial activity and signaling pathway activation between two rhinovirus serotypes, HRV16 (major-group) and HRV1A (minor-group), upon initial HRV binding. Variances in phosphorylation of kinases (p38, JNK, ERK5) and transcription factors (ATF-2, CREB, CEBP-alpha) were observed between the major- and minor-group HRV treatments. Differential activation of signaling pathways led to changes in the production of the asthma-relevant cytokines CCL20, CCL2, and IL-10. This is the first report of genetically similar viruses eliciting dissimilar cytokine release, transcription factor phosphorylation, and MAPK activation from macrophages, suggesting that receptor use is a mechanism for establishing the inflammatory microenvironment in the human airway upon exposure to rhinovirus

Phosphorylation kinetics of transcription factors after rhinovirus exposure.

<p>Primary MDMs (1×10⁶ cells/ml) were treated with HRV16 or HRV1A at an MOI of 10. Activation of A) CREB, B) CEBP-alpha and C) ATF-2 were assayed by SDS-PAGE and immunoblot using anti-phospho antibodies. Equal protein loading was ensured by using total GRB2 as an internal control probing with the appropriate antibody. Each blot is representative of five independent experiments.</p

Select gene expression from RNA sequencing data.

<p>Select gene expression from RNA sequencing data.</p

Infectious center assay in THP-1 cells.

<p>THP-1-derived macrophages were infected with the indicated HRV strains at an MOI of 10 and an infectious center assay was performed. The data are expressed as the mean percentage of infectious, virus-containing macrophages and error bars indicate the standard error of four replicates.</p

Cytokine mRNA expression by macrophages following 24 hours of exposure to HRV1A or HRV16.

<p>Primary MDMs (1×10⁶ cells/ml) were treated with HRV16 or HRV1A at an MOI of 10. Expression of A) CCL20, B) CCL2, C) IL-10 and D) CXCL10 were assayed in blood monocyte-derived macrophages by qPCR. The data are normalized to expression of the housekeeping gene β-actin and are expressed as gene expression fold change from untreated control. Error bars represent the standard error from five independent experiments.</p

Release of inflammatory cytokines by macrophages following 24 hours of exposure to HRV1A or HRV16.

<p>Primary MDMs (1×10⁶ cells/ml) from 34 healthy donors were treated with HRV16 or HRV1A at an MOI of 10. After 24 hours, cell supernatants were analyzed for A) CCL20, B) CCL2, C) IL-10, and D) CXCL10 protein by sandwich ELISA. Data from 50 donor cell populations were pooled and analyzed by Student’s t-tests for paired samples. Significant differences (p<0.05) are indicated by*.</p

Major- and minor-group HRV produce distinct gene expression profiles in primary macrophages.

<p>Human peripheral blood mononuclear cell-derived macrophages were exposed to HRV1A, HRV16, or mock at an MOI of 10 for 8 hours. Total RNA was extracted and ribosomal RNA was depleted and sequenced on the Illumina HiSeq platform. Raw read counts for were normalized to the transcript length and sequencing depth and quantile normalized. Gene expression profiles for macrophages exposed to A) HRV1A or B) HRV16 are presented as quantile normalized read counts per transcript per kb of transcript per million sequencing tags (RPKM), log₂ scale.</p