Role of TGF-β in anti-rhinovirus immune responses in asthmatic patients

Abstract

To the Editor: The majority of viral infections of the airways are associated with asthma exacerbations in children. Two thirds of these viral infections are caused by rhinovirus, and hospital admissions for asthma correlate with the seasonal peak of rhinovirus infections. 1 TGF-β is a cytokine known to induce forkhead box P3+ (FoxP3) regulatory T (Treg) cells and retinoic acid–related orphan receptor (ROR) γt+ TH17 cells in combination with IL-2 or IL-6, respectively, but also to inhibit the differentiation of TH1 and TH2 cells. 2 Because TGF-β and rhinovirus infection both influence asthma exacerbation and TGF-β also induces rhinovirus replication, 3 in this study we analyzed the effect of rhinovirus infection on TGF-β and the role of TGF-β on rhinovirus infection by analyzing asthmatic and nonasthmatic preschool children recruited in the European study Post-infectious Immune Reprogramming and Its Association with Persistence and Chronicity of Respiratory Allergic Diseases (PreDicta) and a murine model of asthma. The clinical data of the analyzed cohorts of children are reported in Table E1 and in the Methods section in this article's Online Repository at www.jacionline.org. In asthmatic children, in 66.6% of the cases, a viral infection was a triggering factor for development of the disease. Rhinovirus was the most common respiratory virus detected in the airways of these children (see Table E2 in this article's Online Repository at www.jacionline.org). To investigate the role of TGF-β in rhinovirus-induced asthma in children, we analyzed PBMCs from preschool children with and without asthma, which were cultured for 48 hours after 1 hour of in vitro exposure to rhinovirus 1B (RV1B) and subjected them to gene array (Fig 1, A, and see Table E3, Table E4, Table E5 in this article's Online Repository at www.jacionline.org). Because TGF-β induces Treg cells, 2 we first investigated which genes related to tolerance were significantly regulated by rhinovirus in PBMCs from these children. Here we found that in asthmatic children rhinovirus upregulated immunosuppressive genes, such as cytotoxic T lymphocyte–associated protein 4 (CTLA4) and indoleamine 2,3-dioxygenase (IDO), programmed death ligand 1 (PD-L1; CD274), and interferon-induced transmembrane protein 2 (IFITM2; Fig 1, B and C). Consistent with the array data, we found that IDO1 was upregulated in PBMCs of asthmatic children cultured with rhinovirus compared with those of control children (see Fig E1, A, in this article's Online Repository at www.jacionline.org). This regulation was found to be independent from steroids because dexamethasone significantly downregulated IDO in PBMCs (see Fig E1, B). Fig 1 PBMCs from asthmatic children exposed to RV1b in vitro upregulated IDO, PDL1, and LAP3. A, Experimental design for RNA arrays of PBMCs cultured in the presence or absence of rhinovirus (RV). B-E, Heat maps for asthmatic (Fig 1, B and C) and control (Fig 1, D and E) children and a differential expression analysis of the regulated genes are shown (asthma: n = 7, control: n = 5). Because TGF-β is secreted in a latent complex consisting of 3 proteins (TGF-β, the inhibitor latency-associated protein [LAP], and the ECM-binding protein LTBP), we also analyzed these and other TGF-β–inhibitory proteins. We noticed that TGF-β–inhibitory genes, such as TGIF2 and LAP3, were upregulated in rhinovirus-treated PBMCs from asthmatic children. Moreover, rhinovirus inhibited genes that cleave viruses, such as RNASE1, in PBMCs from children with asthma (Fig 1, B and C). By contrast, in control children rhinovirus did not significantly regulate these genes. In these children other factors were found to be significantly regulated by rhinovirus, such as lymphocyte antigen 6E (Fig 1, D and E), a protein involved in the TGF-β pathway. Moreover, we found that in PBMCs from control children, rhinovirus induced IL-32 (Fig 1, C and D). Expression of this protein is known to induce the production of IL-6 and TNF-α and might thereby modulate immune responses. 4 In subsequent experiments we analyzed in more detail the regulation of TGF-β in a larger group of children in the same cohort. Among PBMC supernatants, TGF-β protein was detected in high amounts in untreated cell-culture supernatants in both asthmatic and control children. However, after ex vivo challenge with rhinovirus, TGF-β protein expression was found to be significantly decreased (Fig 2, A), although TGFB mRNA expression remained constant (Fig 2, B). Because rhinovirus infection suppressed TGF-β release, we assumed that rhinovirus facilitates TGF-β binding to the cell membrane, and for this reason, we could not detect it in the supernatants of rhinovirus-infected PBMCs. Fig 2 Rhinovirus (RV) inhibits TGF-β release from PBMCs isolated from healthy and asthmatic children. A, TGF-β1 release from PBMCs of asthmatic and nonasthmatic children with or without in vitro rhinovirus infection analyzed by means of ELISA (n = 26-32 children per group, B0+F4). B-E, Relative mRNA expression of TGFB (Fig 2, B; n = 12-20), TGFBRII (Fig 2, C; n =3-6), FOXP3 (Fig 2, D; n = 19-31), and RORC (Fig 2, E; n = 19-31) in asthmatic and nonasthmatic children with or without in vitro rhinovirus infection (B0+F4) analyzed by means of real-time PCR. F-I, Correlation of RORC and FOXP3 mRNA expression in untreated and in vitro–infected PBMCs from asthmatic and nonasthmatic children. J and K, Relative TBX21 (Fig 2, J; n = 10-22) or IL6 (Fig 2, K; n = 10-27) mRNA expression from PBMCs in asthmatic and healthy children with or without in vitro rhinovirus treatment analyzed by using real-time PCR. The Student t test was used to calculate statistical significance. *P ≤ .05, **P ≤ .01, and ***P ≤ .001. Results are expressed as means ± SEMs. To prove this concept of a viral immune escape mechanism, we analyzed the expression of TGFBRII in PBMCs in the presence or absence of in vitro rhinovirus infection. We found that PBMCs isolated from control and asthmatic children and infected with rhinovirus expressed increased levels of TGFBRII compared with the respective controls (Fig 2, C). This finding suggests that rhinovirus induced TGF-β receptor II expression, thus increasing TGF-β binding to the cell membrane and in this way explaining why we could not detect it in the cell supernatants. To further analyze the influence of TGF-β signaling in molecules downstream of TGF-β, we then analyzed FOXP3 and RORC levels and found that PBMCs infected in vitro with rhinovirus express significantly more FOXP3 and RORC mRNA (Fig 2, D and E) in both control and asthmatic children. When we analyzed the correlation of FOXP3 and RORC mRNA expression, we found a positive correlation of these 2 transcription factors in rhinovirus-challenged PBMCs in both groups of children (Fig 2, F-I). Taken together, rhinovirus infection induced FOXP3 and RORC. We then asked whether T-box transcription factor (T-bet), a transcription factor known to regulate TH1/2, Treg, and TH17 cell development or activation, 5 could be regulated by rhinovirus in PBMCs of children with asthma. Although we previously detected decreased TBX21 mRNA expression in asthmatic patients, 6 here we found increased TBX21 mRNA levels in PBMCs isolated from asthmatic children after infection with rhinovirus compared with rhinovirus-infected PBMCs from control children (Fig 2, J). Thus TBX21 can be upregulated in asthmatic patients during active rhinovirus infection. IL-6 is an inflammatory cytokine that, together with TGF-β, can induce the differentiation of TH17 cells. 2 We found an upregulation of IL6 mRNA in control children after in vitro culture with rhinovirus. In contrast, asthmatic children showed a failure of such IL6 induction (Fig 2, K). By analyzing naive and asthmatic mice, we found that in vitro treatment of lung cells with rhinovirus increased the proportions of TC1 cells, whereas adding TGF-β to the culture inhibited T-bet expression in CD4+ T cells, as well as IDO expression in total lung cells. The experimental set up, as well as the results, are described in detail in Figs E2 and E3 in this article's Online Repository at www.jacionline.org. In summary, these data suggest that in patients with acute rhinovirus infections, endogenous TGF-β is retained intracellularly in rhinovirus-infected cells, resulting in a T-bet–mediated immune response. At the moment, we do not know which cells are infected by rhinovirus in the PBMC population we examined; however, we assume that plasmacytoid dendritic cells are infected because of the induction of IDO after rhinovirus challenge ex vivo. 6 However, rhinovirus infection also activates TGF-β present in the environment, as in patients with chronic asthma, to replicate and inhibit effective antiviral immune responses. Thus it is possible that children with acute asthma are able to induce an effective anti-rhinovirus immune response during acute exacerbation. By contrast, in patients with chronic asthma, TGF-β is increased in its active form and is released by structural cells. In this latter situation, when the rhinovirus infects plasmacytoid dendritic cells, this exogenous TGF-β inhibits TH1 and TC1 cells that carry the TGF-β receptor, resulting in TH1 cell depletion, and thus rhinovirus infection cannot be cleared. Although these data need further investigation, they open new avenues for our understanding of the role of rhinovirus-mediated asthma exacerbations in children.