Critical role of caspase-8–mediated IL-1 signaling in promoting Th2 responses during asthma pathogenesis

Abstract

Allergic asthma is a chronic inflammatory disorder of the airways that affects more than 300 million people worldwide. The pro-inflammatory cytokines IL-1α and IL-1β play essential roles in the pathogenesis of asthma. However, the mechanisms underlying the production of IL-1 cytokines in allergic asthma remain unclear. In this study, we used a mouse model of ovalbumin (OVA)–induced asthma to identify a crucial role for caspase-8 in the development of allergic airway inflammation. We further demonstrated that hematopoietic cells play dominant roles in caspase-8–mediated allergic airway inflammation. Caspase-8 was required for the production of IL-1 cytokines to promote Th2 immune response, which promotes the development of pulmonary eosinophilia and inflammation. Thus, our study identifies caspase-8 as a master regulator of IL-1 cytokines that contribute to the pathogenesis of asthma and implicates caspase-8 inhibition as a potential therapeutic strategy for asthmatic patients.

INTRODUCTION

Allergic asthma is a heterogeneous, chronic inflammatory disease characterized by hyperresponsiveness, smooth muscle hypertrophy, obstruction and infiltration of inflammatory cells in the airway. T lymphocytes, particularly Th2 cells and Th2-type cytokines, such as IL-4, IL-5, and IL-13, are immunological signatures of allergic immune responses to inhaled antigens^{1, 2}. Accumulating evidence points to the involvement of pro-inflammatory cytokines within the IL-1 family, specifically IL-1α and IL-1β, in development of pulmonary Th2 immune responses and asthma^3–5. Patients with asthma often have excessive production of IL-1 cytokines⁶. In addition, administration of active human IL-1β to the mouse trachea induces airway inflammation and tissue remodeling⁷. The involvement of IL-1 in asthmatic diseases is further supported by the observation that genetic disruption of the IL-1 signaling pathway strongly attenuates allergic disease in murine models of asthma^{4, 8}. Moreover, recent studies revealed that IL-1 cytokines secreted in response to inhaled allergens can instruct pulmonary dendritic cells to induce Th2 responses and directly promote the activation of Th2 cells^{9, 10}. Collectively, results from these studies suggest that IL-1 is one of the apical signals of the cytokine cascade that drives dendritic cell activation and Th2 immunity in response to inhaled allergens. Despite important advances in understanding the crucial role of IL-1 cytokines in the genesis of allergic asthma, the mechanism of IL-1 production that contributes to inflammatory airway disease remains unknown.

Certain members of the IL-1 family, including IL-1β and IL-18, require proteolytic processing in order to exert their full biological potential^{11, 12}. These cytokines are generated as inactive pro-forms, cleaved by the cysteine protease caspase-1 within the inflammasome and are released as bioactive forms by the cell¹³. The nucleotide-binding domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome respond to a repertoire of activators and have been implicated in the development of asthma^14–17. Increased expression of components of the NLRP3 inflammasome has been found in the sputum of patients with asthma compared with healthy controls¹⁴. However, the precise role of the NLRP3 inflammasome in pathogenesis of allergic asthma is controversial¹⁵. Previous studies suggest that Nlrp3^−/−, Asc^−/−, and Caspase-1/11^−/− mice have dramatically reduced airway inflammation and expression of pro-inflammatory cytokines compared with wild-type (WT) mice in a model of ovalbumin (OVA)-induced allergic asthma^{16, 17}. In contrast, others found that the NLRP3 inflammasome or caspase-1/11 does not substantially contribute to either OVA–mediated or house dust mite–mediated allergic asthma^18–20.

Recently, several studies highlighted novel roles for caspase-8 in the processing of IL-1β and regulating inflammation beyond its role in cell death²¹. Caspase-8 is required for IL-1β production in response to fungal and bacterial infection and dysregulation of caspase-8 has been associated with cancer and autoinflammatory diseases^22–26. However, the role of caspase-8 in a chronic inflammatory lung disease is poorly understood.

In this study, we identified that caspase-8 mediates IL-1α and IL-1β production and plays a key role in the development of allergic lung inflammation in mice, via a mechanism independent of caspase-1 and -11. We found that caspase-8-mediated IL-1 signaling promotes Th2 immune responses, which contributes to the development of pulmonary eosinophilia and inflammation.

RESULTS

Caspase-8 and Fas-associated protein with death domain play pivotal roles in the OVA–induced pulmonary inflammatory response

Dysregulation of the pro-inflammatory cytokines IL-1α and IL-1β is associated with inflammatory and autoimmune diseases²⁷. Caspase-1/-11, cathepsin G, elastase, and proteinase-3 function at the intracellular or extracellular level to process IL-1¹². We used an OVA/alum–sensitized and OVA–challenged mouse model to analyze the role of these proteinases in promoting infiltration of inflammatory cells into the lungs of mice. The pulmonary infiltration of total inflammatory cells, eosinophils, and neutrophils was comparable between WT mice and mice doubly deficient in caspases-1 and 11 (Casp1/11^−/−), elastase (Elastase^−/−), elastase and proteinase 3 (Nepr3^−/−), and cathepsin G (Ctsg^−/−) (Supplementary Figure S1), suggesting that the caspase-1/-11 and the proteinases examined were dispensable for the OVA–induced allergic pulmonary inflammatory response.

A previous study suggested that inhibition of caspase activity with the use of a general caspase inhibitor can reduce airway inflammation²⁸. In addition to caspase-1/-11, caspase-8 is essential for priming and processing of pro-IL-1β^{22, 26, 29}. The embryonic lethality of caspase-8 deficient mice can be rescued by deleting receptor interacting protein kinase-3 (RIP3)^{30, 31}. To determine whether caspase-8 plays a role in inflammation during asthma, WT mice and mice deficient in RIP3 (Rip3^−/−) or RIP3 and caspase-8 (Rip3^−/−Casp8^−/−) were sensitized with OVA/alum and challenged with OVA. Genetic deletion of the gene encoding RIP3 did not substantially affect OVA–induced allergic pulmonary inflammation compared to WT mice, whereas Rip3^−/−Casp8^−/− mice had significantly reduced inflammatory cell infiltration in the lung. Markers of asthmatic lung inflammation, such as the numbers of total immune cells, eosinophils and neutrophils, in the lung of Rip3^−/−Casp8^−/− mice were reduced by 7.5-, 8-, and 11-fold, respectively, compared with those of WT mice (Figure 1a). Histopathological analysis of OVA-treated lung tissues showed remarkable peribronchial and perivascular infiltration of eosinophils, alveolar exudates, and vascular muscle hypertrophy in WT and Rip3^−/− mice but not in Rip3^−/−Casp8^−/− mice (Figure 1b,c). In line with these data, the level of allergy marker IgE was also significantly reduced in the absence of caspase-8 (Figure 1d). These results altogether suggested that caspase-8 promoted inflammatory responses in the lung during OVA–induced asthma.

Figure 1.

OVA–induced allergic pulmonary inflammation is significantly reduced in the absence of caspase-8 or FADD. (a) Wild-type (WT) mice, RIP3-deficient (Rip3^−/−) mice, and RIP3-caspase-8 double-knockout (R3/C8^−/−) mice were sensitized with OVA/alum and challenged with OVA. Fluorescence activated cell sorting (FACS) analysis was performed for pulmonary immune cell infiltration 24 h after the last OVA challenge. (b) Representative lung hematoxylin and eosin (H&E) sections from OVA–treated WT, Rip3^−/−, and Rip3^−/−Casp8^−/− mice. (c) Clinical scores of pulmonary disease on the basis of inflammation, eosinophils, alveolar exudates, and vascular muscle hypertrophy. (d) IgE level in the lung of OVA–treated WT, Rip3^−/−, and Rip3^−/−Casp8^−/− mice. (e) Rip3^−/− and RIP3-FADD double-knockout mice (R3/Fa^−/−) were sensitized with OVA/alum and challenged with OVA. FACS analysis was performed for pulmonary immune cell infiltration 24 h after the last OVA challenge. (f) Representative lung H&E sections from OVA–treated Rip3^−/− and Rip3^−/−Fadd^−/− mice. (g) Clinical scores of pulmonary disease on the basis of inflammation, eosinophils, alveolar exudates, and vascular muscle hypertrophy. (h) IgE level in the lung of OVA–treated Rip3^−/−, and Rip3^−/−Fadd^−/− mice. Arrow and arrowhead indicate immune cell infiltration and thick airway muscle, respectively. Each symbol indicates an individual mouse and mean ± SEM values are shown. Results are representative of 3 independent experiments for (a–d) and 2 independent experiments for (e–h). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Fas-associated protein with death domain (FADD) is a signaling adaptor protein for death receptor that plays an important role in apoptosis and necroptosis by recruiting and activating caspase-8. Genomic deletion of FADD is lethal, an effect which can be rescued by genetic deletion of RIP3³². Thus, we analyzed the involvement of FADD in OVA–induced allergic pulmonary inflammation response. Consistent with the data from Rip3^−/−Casp8^−/− mice, Rip3^−/−Fadd^−/− mice had substantially reduced pulmonary infiltration of total immune cells, eosinophils, and neutrophils compared with Rip3^−/− mice (Figure 1e). Furthermore, histopathological and ELISA analysis revealed that Rip3^−/−Fadd^−/− mice had decreased inflammation, eosinophilia, alveolar exudates, vascular muscle hypertrophy and IgE production compared with Rip3^−/− mice (Figure 1f–h). The reduced disease burden in Rip3^−/−Casp8^−/− mice and Rip3^−/−Fadd^−/− mice suggested that caspase-8 and FADD possibly function in the same complex to drive manifestation of allergic asthma.

Differentiation of Th2 cells is impaired in the absence of caspase-8

An exorbitant Th2 immune response and elaboration of Th2–type cytokines, such as IL-4, IL-5, and IL-13, contribute to the pathogenesis of allergic asthma¹. IFN-γ and IL-17 also act in conjunction with Th2–type cytokines to maintain allergic airway inflammation³. To further investigate the precise role of caspase-8 in OVA-induced Th2 immune responses, we analyzed populations of T cells producing either IL-4, IL-17 or IFN-γ in OVA-treated WT, Rip3^−/− and Rip3^−/− Casp8^−/− mice. Compared with WT mice, the percentage of CD4⁺ T cells producing IL-4, IFN-γ or IL-17 did not change substantially in Rip3^−/− mice (Figure 2a,b). In contrast, the percentage of CD4⁺ T cells producing IL-4 was significantly reduced in Rip3^−/−Casp8^−/− mice (2.94% ± 0.21% versus 1.18% ± 0.17%), whereas the percentage of CD4⁺ T cells producing IFN-γ was higher than that in WT and Rip3^−/− mice (Figure 2a,b). The percentage of CD4⁺ T cells producing IL-17 and the percentage of CD8⁺ T cells producing IFN-γ in Rip3^−/− Casp8^−/− mice were similar to that in WT and Rip3^−/− mice (Figure 2a,b). Notably, the total number of T cells producing IL-4, IL-17 and IFN-γ was substantially lower in Rip3^−/−Casp8^−/− mice than in WT and Rip3^−/− mice (Figure 2c), suggesting it is a consequence of less inflammatory cell infiltrates in the lung of protected Rip3^−/−Casp8^−/− mice. Furthermore, quantitative RT-PCR and ELISA analysis revealed that the gene expression and production of critical Th2-type cytokines IL-4, IL-5 and IL-13, but not of IFN-γ were dramatically lower in Rip3^−/− Casp8^−/− mice than in WT and Rip3^−/− mice (Figure 2d,e). Consistently, the production of IL-4, IL-5 and IL-13 was significantly reduced in the absence of FADD (Supplementary Figure S2a), but not in the mice deficient in caspase-1/-11, elastase, Nepr3, or cathepsin G (Supplementary Figure S2b). These results suggested that caspase-8 and FADD played critical roles in the differentiation and activation of Th2 cells, which contributed to OVA-induced allergic airway inflammation.

Figure 2.

Caspase-8 deficiency dramatically attenuates Th2 immune response in the allergic asthma model. (a) T cells from OVA–treated WT, Rip3^−/−, and Rip3^−/− Casp8^−/− mice were analyzed for IL-4, IFN-γ, and IL-17 expression by intracellular cytokine staining. (b) Percentage of T cells producing IL-4, IFN-γ, and IL-17 relative to total CD4⁺ or CD8⁺ T cells in (a). (c) The total number of T cells producing IL-4, IFN-γ, and IL-17 in (a). (d) RT-PCR analysis of Il4, Il5, Il13, and Ifng in OVA–treated WT, Rip3^−/−, and Rip3^−/−Casp8^−/− mice. (e) ELISA analysis of IL-4, IL-5, IL-13 and IFN-γ in OVA–treated WT, Rip3^−/−, and Rip3^−/−Casp8^−/− mice. Each symbol indicates an individual mouse and mean ± SEM values are shown. Results are representative of 3 independent experiments. **P < 0.01; ***P < 0.001; **** P < 0.0001; ns, not significant.

Hematopoietic cells play crucial roles in caspase-8–mediated allergic airway inflammation

To determine the cell type that contributed to preventing the progression of OVA–induced allergic airway inflammation in Rip3^−/−Casp8^−/− mice, we generated a series of bone marrow chimeric mice. Rip3^−/− and Rip3^−/−Casp8^−/− mice were lethally irradiated and received donor Rip3^−/− or Rip3^−/−Casp8^−/− bone marrow cells for hematopoietic reconstitution. Eight weeks later, the reconstitued mice were sensitized with OVA/alum and challenged with OVA. Notably, the level of inflammatory cell infiltration was significantly higher in Rip3^−/−Casp8^−/− mice that received Rip3^−/− bone marrow cells compared with Rip3^−/−Casp8^−/− mice that received Rip3^−/−Casp8^−/− bone marrow cells (Figure 3a). Moreover, the level of inflammatory cell infiltration was lower in Rip3^−/− mice that received Rip3^−/−Casp8^−/− bone marrow cells compared with Rip3^−/− mice that received Rip3^−/− bone marrow cells (Figure 3a). These analyses suggested that caspase-8 in the hematopoietic compartment contributed to the development of OVA–induced allergic airway disease. We also noticed that Rip3^−/−Casp8^−/− mice that received Rip3^−/−Casp8^−/− bone marrow cells had less inflammatory infiltrates in the lungs compared with Rip3^−/− mice that received Rip3^−/−Casp8^−/− bone marrow cells. This finding argued that caspase-8 in the radioresistant compartment may, in part, contribute to the development of the disease, however, caspase-8 plays a more dominant role in the hematopoietic cells in the development of allergic airway inflammation.

Figure 3.

Contribution of hematopoietic and radioresistant cells to the protected phenotype of Rip3^−/−Casp8^−/− mice in response to OVA administration. (a and b) Bone marrow chimeric mice (> indicates bone marrow donor cells transferred to recipient mice) were sensitized with OVA/alum and challenged with OVA. Pulmonary immune cell infiltration (a) and CD4⁺ T cells producing IL-4 (b) were analyzed by FACS 24 h after the last OVA challenge. (c and d) Dendritic cells sorted from Rip3^−/− and Rip3^−/−Casp8^−/− mice and co-cultured with OVA–primed CD4⁺ T cells in the presence or absence of the OVA peptide (1 µg/mL) in vitro for 5 days. The proliferated T cells were stimulated with PMA and ionomycin and analyzed for cytokine production. The percentage of total CD4⁺ T cells (c) and total CD4⁺ T cells producing IL-4, IFN-γ, and IL-17 (d) are shown. Results are pooled from 2 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P< 0.0001; ns, not significant.

In line with the inflammatory cell infiltration data, we found that transfer of Rip3^−/− bone marrow cells to Rip3^−/−Casp8^−/− mice restored the number of CD4⁺ T cells producing IL-4 in Rip3^−/−Casp8^−/− mice to normal level (Figure 3b). By contrast, transfer of Rip3^−/−Casp8^−/− bone marrow cells to Rip3^−/− mice dramatically reduced the capacity of CD4⁺ T cells producing IL-4 (Figure 3b). These results indicated that the hematopoietic compartment of Rip3^−/−Casp8^−/− mice plays a major role in preventing the development of the OVA–induced Th2 immune response.

To examine how caspase-8 controlled the Th2 immune response in OVA–induced allergic asthma, we assessed whether caspase-8 had a T-cell intrinsic role in controlling its polarization. To this end, Rip3^−/− and Rip3^−/−Casp8^−/− naive CD4⁺ T cells were subjected to an in vitro T helper cell polarization assay. The capacity for naive CD4⁺ T cells isolated from Rip3^−/− and Rip3^−/−Casp8^−/− mice to differentiate into Th1 and Th2 cells was comparable (Supplementary Figure S3). Next, we asked whether caspase-8 functioned within the dendritic cells to promote Th2 responses. To determine the role of caspase-8 in dendritic cells, we co-cultured dendritic cells from Rip3^−/− and Rip3^−/−Casp8^−/− mice with CD4⁺ T cells from OVA–primed WT mice for 5 days in the absence or presence of the OVA peptide and analyzed the differentiation of Th2 cells. The number of CD4⁺ T cells producing IL-4 induced by OVA treatment was significantly lower in WT CD4⁺ T cells co-cultured with dendritic cells from Rip3^−/−Casp8^−/− mice than in WT CD4⁺ T cells co-cultured with dendritic cells from Rip3^−/− mice (Figure 3c,d). The role of caspase-8 for Th2 cell differentiation was specific, because the differentiation of CD4⁺ T cells producing IFN-γ and IL-17 in the presence of dendritic cells from Rip3^−/−Casp8^−/− mice was similar to that in the presence of dendritic cells from Rip3^−/− mice (Figure 3c,d). Taken together, these results suggested that caspase-8 activity in dendritic cells was required for balancing Th1/Th2 responses and that the absence of caspase-8 in dendritic cells led to an attenuated Th2 immune response in OVA–treated Rip3^−/−Casp8^−/− mice.

Caspase-8–induced IL-1 production promotes a Th2 immune response during OVA treatment

To identify the molecule regulated by caspase-8 that accounts for the Th2 immune response, we investigated the expression of IL-1 cytokines in OVA–treated WT, Rip3^−/− and Rip3^−/−Casp8^−/− mice. The expression of the genes encoding IL-1α and IL-1β was much lower in OVA–treated Rip3^−/−Casp8^−/− mice compared with treated WT and Rip3^−/− mice (Figure 4a), suggesting that IL-1 cytokines regulated by caspase-8 could contribute to the development of a Th2 immune response during OVA–induced allergic airway inflammation. In agreement with a disease-associated role for IL-1 in asthma, the pulmonary infiltration of total inflammatory cells, eosinophils and neutrophils was significantly lower in OVA–treated Il1r^−/− mice than in OVA–treated WT mice (Figure 4b). The attenuated pulmonary inflammatory response in Il1r^−/− mice was confirmed by a lower score of pulmonary disease severity indicated by reduced lung inflammation, alveolar exudates and vascular muscle hypertrophy (Figure 4c). Consistently, the production of Th2-type cytokines and IgE were significantly reduced in Il1r^−/− mice compared with WT mice after OVA treatment (Figure 4d,e).

Figure 4.

Caspase-8–mediated IL-1 signaling is crucial for OVA–induced allergic pulmonary inflammation. (a) The expression of Il1a and Il1b in lung tissue from OVA–treated Rip3^−/− and Rip3^−/−Casp8^−/− mice were analyzed by qRT-PCR. (b) WT and IL-1R mutant mice (Il1r^−/−) were sensitized with OVA/alum and challenged with OVA. FACS analysis was performed for pulmonary immune cell infiltration 24 h after the last OVA challenge. (c) Representative lung H&E sections from OVA–treated WT and Il1r^−/− mice (left) and clinical scores of pulmonary disease on the basis of inflammation, alveolar exudates, and vascular muscle hypertrophy (right). (d and e) Cytokines (d) and IgE (e) in supernatants of lungs collected from OVA/alum sensitized and OVA-challenged (OVA), or PBS treated WT and Il1r^−/− mice. (f and g) IL-1α (f) and IL-1β (g) in supernatants of lungs collected from OVA/alum sensitized and OVA-challenged (OVA) WT, Rip3^−/−, Rip3^−/−Casp8^−/− (R3/C8^−/−), Rip3^−/−Fadd^−/− (F3/Fa^−/−), Caspase-1/11^−/− (C1/11^−/−), Elastase^−/−, Nepr3^−/− and Ctsg^−/− mice or PBS treated WT mice. Arrow and arrowhead indicate immune cell infiltration and thick airway muscle, respectively. Data represent mean ± SEM. Results are representative of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

To address whether the impaired IL-1 signaling is specific for Rip3^−/−Casp8^−/− or Rip3^−/−Fadd^−/− mice during asthma pathogenesis, we analyzed the production of IL-1α and IL-1β in WT, Rip3^−/−, Rip3^−/−Casp8^−/−, Rip3^−/−Fadd^−/−, caspase-1/11^−/−, Elastase^−/−, Nepr3^−/− and Ctsg^−/− mice after OVA treatment. Notably, reduced IL-1α and IL-1β was only observed in Rip3^−/−Casp8^−/− and Rip3^−/−Fadd^−/− mice, but not in WT, Rip3^−/−, caspase-1/11^−/−, Elastase^−/−, Nepr3^−/− or Ctsg^−/− mice (Figure 4f,g), suggesting reduced IL-1 production in Rip3^−/−Casp8^−/− or Rip3^−/−Fadd^−/− mice as the mechanism for attenuated pulmonary inflammation. In contrast, the production of another IL-1 family member, IL-18, was comparable between WT and Rip3^−/−Casp8^−/− mice after OVA treatment (Supplementary Figure S4a). Furthermore, Il18^−/− mice did not exhibit a significant change in airway inflammation compared with WT mice in response to OVA treatment (Supplementary Figure S4b), indicating that IL-1/IL-1R axis is the critical signaling axis in caspase-8–mediated allergic airway inflammation. IL-1 induced secretion of IL-33, TSLP and IL-25 from epithelial cells largely contributes to the development of allergic airway inflammation⁴. To determine whether caspase-8 deficiency results in reduced IL-33, TSLP and IL-25 production, we analyzed the gene expression of Il33, Tslp and Il25 in WT, Rip3^−/− and Rip3^−/−Casp8^−/− mice after OVA treatment and found the expression of Il33 but not Tslp or Il25 was significantly reduced in the absence of caspase-8 (Supplementary Figure S5a). Furthermore, the expression of chemokine (KC) that is essential for immune cell infiltration including neutrophils was also remarkably reduced in Rip3^−/−Casp8^−/− mice (Supplementary Figure S5b).

To confirm the contribution of IL-1 cytokines in driving a Th2 immune response, we co-cultured Rip3^−/−Casp8^−/− dendritic cells and CD4⁺ T cells in the presence or absence of IL-1 cytokines to investigate whether IL-1 cytokines can rescue dendritic cells from Rip3^−/−Casp8^−/− mice to promote the differentiation of Th2 cells in response to OVA treatment. Remarkably, the reduced differentiation of Th2 cells in dendritic cells from Rip3^−/−Casp8^−/− mice was rescued by the addition of exogenous recombinant IL-1α and/or IL-1β (Figure 5a,b). In line with previous reports³³, we found IL-1β promoted IFN-γ activation by T cells in response to OVA treatment (Figure 5b). In contrast, exogenous application of IL-1α and/or IL-1β had a modest effect on the differentiation of Th17 cells (Figure 5b). Taken together, our results indicated that caspase-8 controlled the Th2 immune response by driving the production of IL-1α and IL-1β (Supplementary Figure S6).

Figure 5.

The activity of Rip3^−/−Casp8^−/− dendritic cells for promoting Th2 cell differentiation is restored by exogenous application of IL-1α or IL-β. Dendritic cells sorted from naive Rip3^−/− and Rip3^−/−Casp8^−/− mice and co-cultured with OVA–primed CD4⁺ T cells with or without the exogenous application of IL-1α (40 ng/mL), IL-1β (40 ng/mL), or both in the presence of the OVA peptide (1 µg/mL) for 5 days. The proliferated T cells were stimulated with PMA and ionomycin and analyzed by FACS for cytokine production. (a) Representative plots of CD4⁺ T cells producing IL-4. (b) Total CD4⁺ T cells producing IL-4, IFN-γ, and IL-17. Results are representative of 3 independent experiments. *P < 0.05; **P < 0.01; ns, not significant.

DISCUSSION

Caspase-8 is a central component of the cell death and inflammatory pathways³⁴. Mounting evidence has demonstrated an involvement of caspase-8 in the proteolytic processing of IL-1β via caspase-1–dependent or –independent mechanisms²¹. A number of studies have shown that caspase-8–mediated production of IL-1 cytokines plays protective roles in the host defense against infection by pathogens^{22, 23}. Here, we showed that caspase-8 was required for IL-1 production in asthmatic mice, which had a detrimental role by promoting the development of an allergic lung disease. The severity of allergic airway inflammation and production of multiple IL-1 cytokines were markedly reduced in the absence of caspase-8 in OVA–induced mice, indicating that the inflammatory activity of caspase-8 largely contributed to disease development. Although the transcriptional level of the genes encoding IL-1α and IL-1β was reduced in OVA–treated Rip3^−/−Casp8^−/− mice, it is possible that caspase-8 also contributes to the proteolytic processing of IL-1β.

IL-1R signaling and its function within epithelial cells are crucial for the development of allergic disease via the release of the cytokines IL-33, GM-CSF, TSLP and IL-25^{4, 35}. Our study showed both IL-1α and IL-1β contributed to the OVA–induced Th2 immune response and allergic airway inflammation, and the production of IL-1 cytokines from hematopoietic cells had more important roles in caspase-8 mediated allergic airway inflammation. These data collectively suggested that IL-1α and IL-1β could have overlapping roles driving progression of asthmatic diseases and function in an autocrine loop acting on IL-1R to promote cytokine production. The role of NLRP3 inflammasome and caspase-1 meidated IL-1 signaling in allergic airway disease is controversial^{16, 19, 20}. In line with the report from Allen et al and Bruchard et al, we did not observe a positive role of caspase-1/-11 in OVA induced pulmonary inflammation. Thus, the dispensable role of NLRP3 inflammasome or caspase-1–mediated IL-1β in the development of allergic airway disease seen in some studies could be attributed to a redundant function between IL-1α and IL-1β¹⁹. While the role of caspase-8 seems to be dominant in the hematopoietic compartment, we also observed a partial role for caspase-8 in radioresistant cells. The modest effect of caspase-8 in the radioresistant compartment in the development of an asthmatic disease might be attributed to a caspase-8 dependent function on the production of IL-1 cytokines from lung epithelial cells⁴.

IL-1α and IL-1β are pleiotropic proinflammatory cytokines that have numerous roles in inflammatory and autoimmune diseases. In adaptive immunity, IL-1 cytokines have fundamental roles in Th1 and Th17 cell activation and differentiation in humans and mice³³. Surprisingly, we observed that IL-1 promoted the differentiation of Th2 cells in response to OVA treatment, but not in differentiation of Th1 or Th17 cells, indicating a context-specific role for IL-1 in priming Th2 immune responses. The capacity of IL-1 cytokines driving diverse T helper cell differentiation is directly related to the cytokine milieu and the genetic background of the host³⁶. For instance, the Th1-inducing proinflammatory cytokine IL-18 promotes Th2 cell differentiation in mice infected with Leishmania major³⁷. Indeed, our study uncovered a novel role for caspase-8–mediated IL-1 cytokines in promoting Th2 cell differentation during OVA-induced allergic airway inflammation.

Asthma is a complex disease; over 50 cytokines have been identified to be involved in its pathogenesis, including T cell derived cytokines, proinflammatory cytokines, growth factors, chemokines and anti-inflammatory cytokines³. The clinical outcomes of inhibiting specific cytokines with blocking antibodies in the treatment of asthma have been disappointing, probably because of the redundant effects of disease-associated cytokines³⁸. A useful future therapeutic approach might be to block multiple cytokines that are upstream in the cytokine cascade that drives the development of asthma. Importantly, human recombinant IL-1 receptor antagonist (anakinra) has been shown to be effective for asthma treatment³⁹. Our results showed that caspase-8–mediated IL-1 cytokines promoted Th2 immune response to induce asthma and demonstrated that caspase-8 was key in regulating multiple IL-1 cytokines in this process. Overall, our data raised the possibility that inhibition of caspase-8 might be beneficial in the treatment of asthma.

METHODS

Animals

Rip3^{−/− 40}, Rip3^−/−Casp8^{−/− 30}, Rip3^−/−Fadd^{−/− 32}, Il1r^{−/− 41}, Il18^{−/− 42}, and Casp1/11^{−/− 43} mice were generated as previously described. Mice were kept in specific pathogen-free conditions in the Animal Resource Center at St. Jude Children’s Research Hospital (St. Jude). Animal studies were conducted according to protocols approved by the St. Jude Institutional Animal Care and Use Committee.

OVA/Alum–Induced allergic pulmonary inflammation

OVA/alum sensitization and OVA challenge were performed as previously described⁴⁴. Briefly, wild-type (WT) and mutant mice were immunized with 100 µg OVA (Sigma-Aldrich, A5503) in Imject Alum (Thermo Scientific, 77161) at days 0 and 12, followed by intranasal challenge with 50 µg OVA in PBS at days 20, 21, 22 and 23. Twenty-four hours after the last challenge, mice were analyzed for inflammatory cell infiltration and antigen-specific T-cell response by flow cytometry.

Histopathologic studies

The inferior lobes of right lungs were fixed in formalin, and 5-µm sections were stained with haematoxylin and eosin and examined. The severity of lung disease was scored on the basis of the presence of inflammation, eosinophils, vascular muscle hypertrophy, and alveolar exudates by a pathologist blinded to the experimental groups.

Isolation of lung inflammatory cells and fluorescence activated cell sorting analysis

The left lungs of OVA–induced mice were processed by mincing and then passed through cell strainers. After Percoll density gradient centrifugation, the total number of cells per lung was determined. The single cell suspension was blocked with the blocking buffer (Biolegend, 101320) and stained with various antibodies for flow cytometry analysis (netrophils, CD11b⁺ Ly-6G⁺; eosinophils, CD11b⁺ SiglecF⁺). For intracelluar cytokine staining, cells were stimulated with phorbol myristate acetate (PMA) (100 ng/mL) and Ionomycin (1 µg/mL) in culture media with monensin (eBioscience, 00-4505-51). After 4 h, cells were washed, blocked with blocking buffer, and stained with the cell surface markers FITC-anti-mouse CD4 (RM4-5, BioLegend, 100510) and Pacific blue-anti-mouse CD8 (53-6.7, eBioscience, 48-0081-82) for 20 min, washed and fixed in 1% paraformaldehyde for 10 min, permeabilized in permeabilization buffer (eBioscience, 00-8333-56) for 5 min, and stained with specific cytokine antibodies APC-anti-IL-4 (11B11, eBioscience, 17-7041-82), PE-anti-IL-17 (ebio17B7, eBioscience, 12-7177-81) and Percp-anti-IFN-γ (G1.2, eBioscience, 45-7311-82) for 20 min. Dead cells were excluded by staining with the 7-AAD viability solution (BioLegend, 420402). Data were acquired on a BD FACS Calibur flow cytometer and analyzed by the FlowJo software.

Bone marrow chimeric mice

Rip3^−/− and Rip3^−/−Casp8^−/− mice were lethally irradiated with a dose of 1000 rads and transplanted with 5 × 10⁶ whole bone marrow cells from indicated donor mice by retro-orbital injection. Reconstitution was assessed after 6 weeks. After 8 weeks, reconstituted mice were sensitized with OVA/alum and challenged with OVA.

In vitro differentiation of T helper cells

CD4⁺ T cells were sorted from lymph node cells of WT mice 24 h after the last challenge with OVA. Dendritic cells (CD11c⁺MHCII⁺) were sorted from spleens of naive Rip3^−/− and Rip3^−/−Casp8^−/− mice. Sorted dendritic cells and CD4⁺ T cells were co-cultured in a 96-well plate in the absense or presense of 1 µg/mL OVA peptide 323–339. After 5 days, cells were stimulated with PMA and ionomycin for 4 h and populations of Th1, Th2, and Th17 cells were analyzed by flow cytometry as described above.

Cell polarization assay for Th1 and Th2 cells

Naïve CD4⁺ T cells (CD4⁺CD25⁻ CD44⁻CD62L⁺) were sorted from lymph nodes of Rip3^−/− and Rip3^−/−Casp8^−/− mice. WT splenocytes were irradiated for antigen presenting cells (APCs). Naive CD4⁺ T cells and irridated WT APCs were co-cultured in the presence of various cytokines and antibodies for T-helper-cell differentiation. For Th1 cells, anti-CD3 antibody (2 µg/mL), anti-CD28 antibody (2 µg/mL), anti-IL-4 antibody (10 µg/mL), IL-2 (100 µg /mL), and IL-12 (0.5 ng/mL) were used. For Th2 cells, anti-CD3 antibody (2 µg/mL), anti-CD28 antibody (2 µg/mL), anti-IFN-γ antibody (10 µg/mL), IL-2 (100 µg/mL), and IL-4 (10 ng/mL) were used. On day 5, cells were stimulated with PMA and ionomycin for 4 h and Th1 and Th2 cell populations were analyzed by flow cytometry as described above.

Real-Time quantitative PCR

Total RNA was isolated from lung tissues by using Trizol (Invitrogen) and followed by purification with RNeasy Kit (Qiagen). cDNA was reverse transcribed by using Superscript III (Invitrogen). Real-time quantitative PCR was performed on the ABI Prism 7500 sequence detection system (Applied Biosystems). Supplementary Table S1 lists the primer sequences.

ELISA

Supernatants from lung homogenates were analyzed for cytokine and chemokine release using ELISA kit (Biolegend ELISA MAX standard or Millipore multiplex assay) following the manufacturer’s instructions.

Statistical analyses

Data are given as mean ± standard error of the mean (SEM). Statistical analyses were performed by using the nonparametric Mann-Whitney test. P values ≤ 0.05 were considered significant.