Crosstalk between CD4+ T Cells and Airway Smooth Muscle in Pediatric Obesity-related Asthma

Abstract

Rationale: Pediatric obesity-related asthma is a nonatopic asthma phenotype with high disease burden and few effective therapies. RhoGTPase upregulation in peripheral blood T helper (Th) cells is associated with the phenotype, but the mechanisms that underlie this association are not known. Objectives: To investigate the mechanisms by which upregulation of CDC42 (Cell Division Cycle 42), a RhoGTPase, in Th cells is associated with airway smooth muscle (ASM) biology. Methods: Chemotaxis of obese asthma and healthy-weight asthma Th cells, and their adhesion to obese and healthy-weight nonasthmatic ASM, was investigated. Transcriptomics and proteomics were used to determine the differential effect of obese and healthy-weight asthma Th cell adhesion to obese or healthy-weight ASM biology. Measurements and Main Results: Chemotaxis of obese asthma Th cells with CDC42 upregulation was resistant to CDC42 inhibition. Obese asthma Th cells were more adherent to obese ASM compared with healthy-weight asthma Th cells to healthy-weight ASM. Compared with coculture with healthy-weight ASM, obese asthma Th cell coculture with obese ASM was positively enriched for genes and proteins involved in actin cytoskeleton organization, transmembrane receptor protein kinase signaling, and cell mitosis, and negatively enriched for extracellular matrix organization. Targeted gene evaluation revealed upregulation of IFNG, TNF (tumor necrosis factor), and Cluster of Differentiation 247 (CD247) among Th cell genes, and of Ak strain transforming (AKT), Ras homolog family member A (RHOA), and CD38, with downregulation of PRKCA (Protein kinase C-alpha), among smooth muscle genes. Conclusions: Obese asthma Th cells have uninhibited chemotaxis and are more adherent to obese ASM, which is associated with upregulation of genes and proteins associated with smooth muscle proliferation and reciprocal nonatopic Th cell activation.

Keywords: Th cells; airway smooth muscle; asthma; children; obesity.

Figure 1.

Summary of experimental design. T-helper (Th) cells from obese children with asthma with CDC42 (Cell Division Cycle 42) upregulation (n = 10) and from healthy-weight children with asthma without CDC42 upregulation (n = 10) underwent quantification of (A) chemotaxis in response to SDF-1 (stromal differentiation factor 1) alone, and in the presence of pretreatment with ML141, a CDC42 inhibitor, because SDF-1 is a chemoattractant for LFA-1, which is upregulated with CDC42 upregulation; and (B) adhesion of Th cells to airway smooth muscle (ASM), comparing adhesion of obese asthma and healthy-weight asthma Th cells to obese and healthy-weight nonasthma ASM. (C) The obese asthma and healthy-weight asthma Th cell coculture with obese and healthy-weight nonasthma ASM also underwent transcriptomic and phosphoproteomic analysis to quantify differential gene and protein expression in each coculture condition. Comparisons were conducted between coculture of obese asthma Th cells with obese and healthy-weight nonasthma ASM and between coculture of healthy-weight asthma Th cells with obese and healthy-weight nonasthma ASM. LFA-1 = lymphocyte function–associated antigen 1.

Figure 2.

Comparison of chemotaxis of obese asthma T-helper (Th) cells and healthy-weight asthma Th cells. (A) There was no difference in the proportion of obese asthma and healthy-weight asthma Th cells that migrated in response to SDF-1 (stromal differentiation factor 1). Exposure to ML141, a chemical CDC42 inhibitor, inhibited migration of healthy-weight asthma Th cells but did not influence migration of obese asthma Th cells. (B) Representative image of actin polymerization and filopodia formation labeled with phalloidin CF555 (red) and the nuclei labeled with DAPI (blue) in cells after migration in response to SDF-1. (C) Representative image of actin polymerization in Th cells before migration. (D) Representative image of actin polymerization in Th cells after migration. (E) Comparison of phalloidin staining among the three experimental conditions of Th cell chemotaxis. (F) Comparison of LFA-1 (lymphocyte function–associated antigen 1) gene expression between obese and healthy-weight asthma Th cells. (G) Comparison of LFA-1 staining in migrated Th cells among the three experimental conditions of Th cell chemotaxis. CDC42 = Cell Division Cycle 42.

Figure 3.

Comparison of obese asthma T-helper (Th) cell and healthy-weight asthma Th cell adhesion to obese and healthy-weight airway smooth muscle (HASM). (A) Representative images from three biological replicates of healthy-weight asthma Th cell adhesion to healthy-weight ASM. (B) Representative images from three biological replicates of obese asthma Th cell adhesion to obese ASM. ASM was stained for actin (green) and Th cells were stained for Cluster of differentiation 4 (CD4) (red). The overlapping staining of Th cells with actin and CD4 led to the cells being visualized as yellow. (C) Comparison of adhesion of healthy-weight asthma Th cells to healthy-weight and obese ASM and of obese asthma Th cells to healthy-weight and obese ASM. (D) Representative images from two biological replicates of healthy-weight ASM staining for ICAM-1 (intracellular adhesion molecule 1) (green) and nuclei (labeled with DAPI [blue]). (E) Representative images from two biological replicates of obese ASM staining for ICAM-1 and nuclei. (F) The intensity of ICAM-1 staining was compared with DAPI staining for 10 representative cells in four biological replicates of healthy-weight and obese ASM. The ratio of ICAM staining to DAPI staining for each biological replicate was compared between obese and healthy-weight ASM. HASM = healthy-weight airway smooth muscle; HT = healthy-weight asthma T cell; OASM = obese airway smooth muscle; OT = obese asthma T cell.

Figure 4.

Comparison of transcriptomic differences between obese asthma T-helper (Th) cell coculture with obese airway smooth muscle (ASM) and healthy-weight ASM and between healthy-weight asthma Th cell coculture with obese ASM and healthy-weight ASM. (A) Enrichment analysis of differentially expressed genes and (B) top 20 Gene Ontology pathways identified when obese asthma Th cell coculture with obese ASM was compared with their coculture with healthy-weight ASM. (C) Enrichment analysis of differentially expressed genes and (D) top 20 Gene Ontology pathways identified when healthy-weight asthma Th cell coculture with obese ASM was compared with their coculture with healthy-weight ASM.

Figure 5.

Comparison of expression of biologically relevant T-helper (Th) cell genes and airway smooth muscle (ASM) genes between obese asthma Th cell coculture with obese ASM and healthy-weight ASM and between healthy-weight asthma Th cell coculture with obese ASM and healthy-weight ASM. Using quantitative RT-PCR, we compared biologically relevant (A) Th cell genes and (B) ASM genes between obese asthma Th cell coculture with obese ASM and healthy-weight ASM and between healthy-weight asthma Th cell coculture with obese ASM and healthy-weight ASM. AKT = ak strain transforming; CD247 = cluster of differentiation 247; HASM = healthy-weight airway smooth muscle; HT = healthy-weight asthma T cell; IFNG = interferon gamma; IP3R = inositol triphosphate receptor; OASM = obese airway smooth muscle; OT = obese asthma T cell; RHOA = ras homolog family member A.

Figure 6.

Comparison of proteomic differences between obese asthma T-helper (Th) cell coculture with obese airway smooth muscle (ASM) and healthy-weight ASM and between healthy-weight asthma Th cell coculture with obese ASM and healthy-weight ASM. (A) Enrichment analysis of differentially expressed proteins and (B) top 20 Gene Ontology pathways identified when obese asthma Th cell coculture with obese ASM was compared with their coculture with healthy-weight ASM. (C) Enrichment analysis of differentially expressed proteins and (D) top 20 Gene Ontology pathways identified when healthy-weight asthma Th cell coculture with obese ASM was compared with their coculture with healthy-weight ASM. MAPK = mitogen-activated protein kinase; UV = ultraviolet.

Figure 7.

Enrichment analysis of genes and proteins in obese asthma T-helper (Th) cell coculture with obese airway smooth muscle (ASM) and healthy-weight ASM. Enrichment analysis of transcriptome–proteome overlap that was positively enriched in obese asthma Th cell coculture with obese ASM was compared with their coculture with healthy-weight ASM using (A) cytoscape and (B) M-CODE. Enrichment analysis of transcriptome–proteome overlap that was negatively enriched in obese asthma Th cell coculture with obese ASM was compared with their coculture with healthy-weight ASM using (C) cytoscape and (D) M-CODE. CCT = chaperonin containing T-complex polypeptide; ECM = extracellular matrix; FcGR3A = fc gamma receptor 3A; PID = pathway interaction database; SLBP = stem-loop binding protein; TriC = t-complex protein ring complex; TC-NER = transcription-coupled nucleotide excision repair; VEGFA = vascular endothelial growth factor A; VEGFR2 = vascular endothelial growth factor receptor 2; WASP = Wiskott-Aldrich syndrome protein; WAVE = wasp-family verpolin-homologous protein.

Figure 7.

Enrichment analysis of genes and proteins in obese asthma T-helper (Th) cell coculture with obese airway smooth muscle (ASM) and healthy-weight ASM. Enrichment analysis of transcriptome–proteome overlap that was positively enriched in obese asthma Th cell coculture with obese ASM was compared with their coculture with healthy-weight ASM using (A) cytoscape and (B) M-CODE. Enrichment analysis of transcriptome–proteome overlap that was negatively enriched in obese asthma Th cell coculture with obese ASM was compared with their coculture with healthy-weight ASM using (C) cytoscape and (D) M-CODE. CCT = chaperonin containing T-complex polypeptide; ECM = extracellular matrix; FcGR3A = fc gamma receptor 3A; PID = pathway interaction database; SLBP = stem-loop binding protein; TriC = t-complex protein ring complex; TC-NER = transcription-coupled nucleotide excision repair; VEGFA = vascular endothelial growth factor A; VEGFR2 = vascular endothelial growth factor receptor 2; WASP = Wiskott-Aldrich syndrome protein; WAVE = wasp-family verpolin-homologous protein.