Human Rhinovirus Infection of the Respiratory Tract Affects Sphingolipid Synthesis

Abstract

The 17q21 asthma susceptibility locus includes asthma risk alleles associated with decreased sphingolipid synthesis, likely resulting from increased expression of ORMDL3. ORMDL3 inhibits serine-palmitoyl transferase (SPT), the rate-limiting enzyme of de novo sphingolipid synthesis. There is evidence that decreased sphingolipid synthesis is critical to asthma pathogenesis. Children with asthma and 17q21 asthma risk alleles display decreased sphingolipid synthesis in blood cells. Reduced SPT activity results in airway hyperreactivity, a hallmark feature of asthma. 17q21 asthma risk alleles are also linked to childhood infections with human rhinovirus (RV). This study evaluates the interaction of RV with the de novo sphingolipid synthesis pathway, and the alterative effects of concurrent SPT inhibition in SPT-deficient mice and human airway epithelial cells. In mice, RV infection shifted lung sphingolipid synthesis gene expression to a pattern that resembles genetic SPT deficiency, including decreased expression of Sptssa, a small SPT subunit. This pattern was pronounced in lung epithelial cellular adhesion molecule (EpCAM⁺) cells and reproduced in human bronchial epithelial cells. RV did not affect Sptssa expression in lung CD45⁺ immune cells. RV increased sphingolipids unique to the de novo synthesis pathway in mouse lung and human airway epithelial cells. Interestingly, these de novo sphingolipid species were reduced in the blood of RV-infected wild-type mice. RV exacerbated SPT deficiency-associated airway hyperreactivity. Airway inflammation was similar in RV-infected wild-type and SPT-deficient mice. This study reveals the effects of RV infection on the de novo sphingolipid synthesis pathway, elucidating a potential mechanistic link between 17q21 asthma risk alleles and rhinoviral infection.

Keywords: 17q21; asthma; early childhood wheeze; rhinovirus; sphingolipids.

Figure 1.

Clustering of lung and lung epithelial transcriptomes from sphingolipid-deficient and rhinovirus (RV)-infected mice. Serine-palmitoyl transferase (SPT) and control (Co) littermates were intranasally inoculated with RV-A1B (5 × 10⁶ Tissue Culture Infectious Dose 50 assay [TCID₅₀]/mouse) or equal volumes of mock-infected purified cell lysates. Lungs were harvested 24 hours after infection. Using magnetic bead separation, CD45⁺ cells and CD45⁻, epithelial cellular adhesion molecule (EpCAM⁺) cells were isolated from lung single-cell suspension. RNA sequencing was performed on RNA isolated from lung tissue, lung EpCAM⁺, and lung CD45⁺ cells. (A) Principal component analysis from whole lung, EpCAM⁺, and CD45⁺ cells irrespective of RV infection. (B) Sample-to-sample distances based on global gene expression of all groups. (C) Global differential gene expression (adjusted P value < 0.05) according to tissue type. Data represent four mice per group for all groups, except those removed after not meeting quality control standards including one Co sample in the lung and EpCAM⁺ cell analyses, and one SPT + RV sample in the CD45⁺ analyses. PC = principal component.

Figure 2.

RV infection and SPT deficiency induce similar changes in lung epithelial gene expression for sphingolipid synthesis. Gene ontology enrichment analysis of differentially expressed genes within the sphingolipid synthesis pathway (GO:0030148) for (A) lung, (B) lung EpCAM⁺ cells, and (C) lung CD45⁺ cells. Shown are results of hierarchical clustering of differentially expressed genes of Co + RV, SPT, and SPT + RV versus Co mice, with nominal P value less than 0.05. Data represent four mice per group for all groups, except those removed after not meeting quality control standards, including one Co sample in the lung and EpCAM⁺ cell analyses and one SPT + RV sample in the CD45⁺ analyses.

Figure 3.

Individual gene analysis following RV infection of wild-type and SPT-deficient lungs and lung epithelial and lung immune cells. In lung, lung EpCAM⁺ cells, and lung CD45⁺ cells, normalized expression values of de novo sphingolipid genes with differential expression were compared with false discovery threshold adjusted P value of 0.05. Normalized expression values calculated using variance-stabilizing transformation. Normalized expression values of Sptssa (small subunit of SPT A) in (A) lung, (B) lung EpCAM⁺ cells, and (C) lung CD45⁺ cells. Normalized expression values of Cers2 (ceramide synthase 2) in (D) lung, (E) lung EpCAM⁺ cells, and (F) lung CD45⁺ cells. Normalized expression values of Cers6 in (G) lung, (H) lung EpCAM⁺ cells, and (I) lung CD45⁺ cells. Shown are means ± SEM of four mice per group for all groups, except those removed after not meeting quality control standards, including one Co sample in the lung and EpCAM⁺ cell analyses and one SPT + RV sample in the CD45⁺ analyses. ns = not significant. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Figure 4.

RV infection induces a shift in de novo sphingolipid content in lung and blood. (A) Sphingolipid synthesis pathways contributing to the synthesis of ceramide metabolites. Blood and perfused, PBS-washed lungs were collected 24 hours after infection and analyzed by HPLC–mass spectrometry (MS)/MS. (B) Lung de novo synthesis species sphinganine and dihydroceramides C16:0, C18:0, and C18:1. (C) Blood de novo synthesis species sphinganine, sphinganine-1-phosphate, and dihydroceramides C16:0, C18:0, C18:1, C24:0, and C24:1. Data are representative of two independent experiments with 5–6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (two-way ANOVA with Tukey’s multiple comparison test). DH = dihydroceramide; S1P = sphinganine-1-phosphate; Sa = sphinganine.

Figure 5.

In human bronchial epithelial cells, RV induced similar shifts in gene expression and sphingolipid content in comparison to mouse lung epithelium. BCi NS1.1 cells grown in air–liquid interface were incubated with culture media or RV-A16 (1 × 10⁶ TCID₅₀) suspended in culture media for 1 hour at 33°C. After 1 hour, control and infectious media was removed, and incubation continued for 24 hours. Quantitative PCR analysis of mRNA concentrations for genes (A) Sptssa and (B) Cxcl-10. HPLC-MS/MS measurement of (C) de novo species sphinganine and DH-C16 and DH-C18. Data are representative of two independent experiments with five samples per group. **P < 0.01, ***P < 0.001, and ****P < 0.0001. Analyzed by (A and B) unpaired t test and (C) two-way ANOVA with Sidak’s post-test comparison.

Figure 6.

RV infection increases airway reactivity and inflammation in wild-type and SPT-deficient mice. SPT-deficient Sptlc2^+/− (SPT) and littermate Co mice were inoculated intranasally with RV-1B (5 × 10⁶ TCID₅₀) or mock-infected purified cell lysates. (A) Bronchial rings isolated 24 hours after infection were stimulated with increasing doses of methacholine. Shown are contractile responses expressed as the absolute force generated in millinewton (mN). (B) BAL cell composition analyzed 24 hours after infection by flow cytometry. (C) Relative lung weight 24 hours after infection. (D) From blood, total white blood cell count, and manual neutrophil and lymphocyte blood count measured before and after infection from the same mice. Data are means ± SEM or 5–8 animals per group. *P < 0.05, **P < 0.01, and ***P < 0.001 (two-way ANOVA with Tukey post-test comparison). WBC = white blood cells.