IL-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo

Abstract

Rationale: Rhinoviruses are the major cause of asthma exacerbations; however, its underlying mechanisms are poorly understood. We hypothesized that the epithelial cell-derived cytokine IL-33 plays a central role in exacerbation pathogenesis through augmentation of type 2 inflammation.

Objectives: To assess whether rhinovirus induces a type 2 inflammatory response in asthma in vivo and to define a role for IL-33 in this pathway.

Methods: We used a human experimental model of rhinovirus infection and novel airway sampling techniques to measure IL-4, IL-5, IL-13, and IL-33 levels in the asthmatic and healthy airways during a rhinovirus infection. Additionally, we cultured human T cells and type 2 innate lymphoid cells (ILC2s) with the supernatants of rhinovirus-infected bronchial epithelial cells (BECs) to assess type 2 cytokine production in the presence or absence of IL-33 receptor blockade.

Measurements and main results: IL-4, IL-5, IL-13, and IL-33 are all induced by rhinovirus in the asthmatic airway in vivo and relate to exacerbation severity. Further, induction of IL-33 correlates with viral load and IL-5 and IL-13 levels. Rhinovirus infection of human primary BECs induced IL-33, and culture of human T cells and ILC2s with supernatants of rhinovirus-infected BECs strongly induced type 2 cytokines. This induction was entirely dependent on IL-33.

Conclusions: IL-33 and type 2 cytokines are induced during a rhinovirus-induced asthma exacerbation in vivo. Virus-induced IL-33 and IL-33-responsive T cells and ILC2s are key mechanistic links between viral infection and exacerbation of asthma. IL-33 inhibition is a novel therapeutic approach for asthma exacerbations.

Keywords: ILC2; Th2; infection; virus.

Figure 1.

Rhinovirus infection results in more severe upper and lower respiratory tract involvement, greater viral loads, and bronchial eosinophilia in asthma. Shown are the daily change from baseline in upper (A) and lower (B) respiratory symptoms of subjects with asthma (red) and healthy volunteers (black). The total lower respiratory symptom score (C) equates to the summation of daily scores over the 14-day postinoculation period and represents the severity of the exacerbation. As symptom scores were corrected for baseline and bronchoscopy-induced symptoms, a small number of subjects had a negative score (see online supplement for further details). Decreases in morning PEF are shown as percentage changes from baseline (D) following rhinovirus inoculation. The maximal decline in FEV₁ (E) represents the maximal change during the infection period for each subject. Viral load was measured at each study visit in nasal lavage (F). Bronchoalveolar lavage eosinophil counts were measured at baseline and on Day 4 postinoculation (G). Results shown are mean ± SEM (A–E); bars represent median values (F). Statistical comparisons between groups were performed at each time point, but have been left unmarked where nonsignificant to aid clarity. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2.

Rhinovirus infection in asthma leads to the induction of IL-33 and type 2 cytokines in vivo. Nasal levels of IL-33 and type 2 cytokines were measured by nasosorption (A) in subjects with asthma (red) and healthy subjects (black). The bronchosorption device (B) uses a strip of synthetic absorptive matrix similar to that used for nasosorption to sample bronchial mucosal lining fluid. In subjects with asthma, IL-33 levels were correlated with IL-5 and IL-13 levels (C), severity of the asthma exacerbation in vivo (D), and viral load (E). Bars represent median levels (A). The “infection” level (A) and “peak” levels (D and E) represent the greatest (maximal) level of induction during the infection for each subject. The correlations shown are for subjects with asthma only and are nonparametric (Spearman’s correlation). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.

Rhinovirus infection of BECs induces IL-33, which subsequently induces type 2 cytokine production by human T cells and ILC2s. (A) Levels of IL-33 in BEC supernatants 24 hours after rhinovirus 16 infection or culture with ultraviolet light–inactivated rhinovirus 16 or medium control. To determine whether IL-33 present in rhinovirus-infected BEC supernatants could induce Th2 responses in human T cells, naive (CD45RO⁻), activated (anti-CD2/CD3/CD28-stimulated), nonpolarized human CD4⁺ T cells (Th0 cells) were cultured in the presence of medium alone or in medium plus supernatant from either uninfected or rhinovirus 16–infected BECs, in the presence of blocking antibody to the IL-33 receptor (αST2) or isotype control antibody, before flow cytometric analysis. (B) Intracellular levels of IL-4, IL-5, IL-13, GATA-3, and IFN-γ in Th0 cells cultured in medium alone (white) or in medium plus supernatants from uninfected (gray) or in rhinovirus 16–infected (black) BECs, in the presence of isotype control or α-ST2 blocking antibody. (C) Levels of IL-4, IL-5, and IL-13 in supernatants from Th0 cells cultured in medium alone (white) or in medium plus supernatants from uninfected (gray) or rhinovirus 16–infected (black) BECs, in the presence of isotype control or α-ST2 blocking antibody. (D) Human peripheral blood mononuclear cells were enriched for ILC2s by magnetic depletion of CD3⁺, CD14⁺CD16⁺ and CD19⁺ cells and were then flow-sorted as lymphocyte-sized, lineage-negative (CD2⁻, CD3⁻, CD14⁻, CD16⁻, CD19⁻, CD56⁻, CD235a⁻, and CD123⁻) CRTH2⁺ cells. The top panel shows the forward and side scatter characteristics of the sorted population, and the lower panel shows the distinct lineage-negative, CRTH2⁺ ILC2 population. (E) Surface marker expression of ILC2s (red), lineage-negative CRTH2⁻ (gray) cells, and lineage-positive CRTH2⁻ (black) cells were compared using flow cytometry. Histograms show sorted ILC2s to have a distinct phenotype (lineage-negative CD34⁻, CRTH2⁺, cKit⁺, CD45⁺, CD127⁺, and CD25⁺). (F) Human ILC2s were cultured in medium alone (white) or in medium plus supernatants from uninfected (gray) and rhinovirus 16–infected (black) BECs, in the presence of isotype control or α-ST2–blocking antibody and cytokine levels were measured in the ILC2 culture supernatants. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n = 6.

Figure 3.

Rhinovirus infection of BECs induces IL-33, which subsequently induces type 2 cytokine production by human T cells and ILC2s. (A) Levels of IL-33 in BEC supernatants 24 hours after rhinovirus 16 infection or culture with ultraviolet light–inactivated rhinovirus 16 or medium control. To determine whether IL-33 present in rhinovirus-infected BEC supernatants could induce Th2 responses in human T cells, naive (CD45RO⁻), activated (anti-CD2/CD3/CD28-stimulated), nonpolarized human CD4⁺ T cells (Th0 cells) were cultured in the presence of medium alone or in medium plus supernatant from either uninfected or rhinovirus 16–infected BECs, in the presence of blocking antibody to the IL-33 receptor (αST2) or isotype control antibody, before flow cytometric analysis. (B) Intracellular levels of IL-4, IL-5, IL-13, GATA-3, and IFN-γ in Th0 cells cultured in medium alone (white) or in medium plus supernatants from uninfected (gray) or in rhinovirus 16–infected (black) BECs, in the presence of isotype control or α-ST2 blocking antibody. (C) Levels of IL-4, IL-5, and IL-13 in supernatants from Th0 cells cultured in medium alone (white) or in medium plus supernatants from uninfected (gray) or rhinovirus 16–infected (black) BECs, in the presence of isotype control or α-ST2 blocking antibody. (D) Human peripheral blood mononuclear cells were enriched for ILC2s by magnetic depletion of CD3⁺, CD14⁺CD16⁺ and CD19⁺ cells and were then flow-sorted as lymphocyte-sized, lineage-negative (CD2⁻, CD3⁻, CD14⁻, CD16⁻, CD19⁻, CD56⁻, CD235a⁻, and CD123⁻) CRTH2⁺ cells. The top panel shows the forward and side scatter characteristics of the sorted population, and the lower panel shows the distinct lineage-negative, CRTH2⁺ ILC2 population. (E) Surface marker expression of ILC2s (red), lineage-negative CRTH2⁻ (gray) cells, and lineage-positive CRTH2⁻ (black) cells were compared using flow cytometry. Histograms show sorted ILC2s to have a distinct phenotype (lineage-negative CD34⁻, CRTH2⁺, cKit⁺, CD45⁺, CD127⁺, and CD25⁺). (F) Human ILC2s were cultured in medium alone (white) or in medium plus supernatants from uninfected (gray) and rhinovirus 16–infected (black) BECs, in the presence of isotype control or α-ST2–blocking antibody and cytokine levels were measured in the ILC2 culture supernatants. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n = 6.