CARD9 balances Aspergillus fumigatus‐induced anti‐fungal immunity and allergic inflammation

Aspergillus fumigatus (A. fumigatus), a ubiquitous pathogen and allergen, is involved in the pathogenesis of invasive aspergillosis, allergic bronchopulmonary aspergillosis (ABPA), etc. As it possesses pathogenicity and allergenicity, A. fumigatus exposure can induce either Th1/Th17 dominated anti-fungal immunity or Th2 dominated allergic inflammation, or both based on the situations.1, 2 However, some investigations show that type-2 immunity can suppress Th1 mediated immune-protection against fungal infection, which promote fungal persistence and disease progression.3 These data suggest that anti-fungal immunity and sensitization to fungal allergen are always in dynamic balance and overwhelming type-2 immune response may be detrimental for fungal elimination. Caspase recruitment domain-containing protein 9 (CARD9), a central adaptor protein mainly expressed in myeloid cells, relays downstream signalling of C-type lectin receptors (CLRs) via CARD9-Bcl10-Malt1 (CBM) complex, finally controlling canonical NF-κB activation and resultant pro-inflammatory cytokines production (TNF-α, IL-6, IL-12, etc.), which are critical for fungal clearance.1, 4 Accumulating clinical evidence have found that null or lose-of-function mutations in CARD9 gene increase the susceptibility to fungal infection.5 However, in these fungal infected patients carrying CARD9 mutations, higher serum IgE concentration and hyper-eosinophilia are always a common manifestation.6-8 In our previous work, we have identified CARD9 S12N mutation (c.35G > A, rs4077515) as a susceptible gene to ABPA.8 These investigations imply that CARD9 not only regulates host anti-fungal immunity but also involves in modulation of allergic inflammation. However, whether CARD9 tunes the balance between anti-fungal immunity and type-2 immune response remains unclear. To determine the role of CARD9 in modulation of anti-fungal immunity and allergic inflammation, wild type (WT) and CARD9 deficient mice were infected with A. fumigatus. The detailed data about fungal burden and anti-fungal immune parameters are available in the following repository (https://zenodo.org/record/7493754#.Y651P8hqilw). We observed that CARD9 deficient mice had lower survival and higher fungal burden in the lung after A. fumigatus infection. Consistently, PASM staining showed more fungal colonization in the lung from CARD9 deficient mice. Furthermore, CARD9 deficiency dramatically reduced the mRNA abundance of Tnf-α, Il-6 and Ifng, and decreased TNF-α and IL-6 production in the lung from mice after A. fumigatus infection. Moreover, we observed that neutrophil infiltration was significantly decreased in CARD9 deficient mice compared with those in WT mice. These data suggested that CARD9 deficiency impaired anti-fungal immune response after A. fumigatus exposure. However, we strikingly observed airway remodelling such as airway wall thickening and airway stenosis in the lung from CARD9 deficient mice compared with that in WT mice after A. fumigatus exposure (Figure 1A). Simultaneously, large amounts of eosinophils were scattered in the lung from CARD9 deficient mice (Figure 1A). These H&E staining data made us hypothesize that CARD9 deficiency may screw host anti-fungal immunity into allergic inflammation. To confirm this hypothesis, we first analysed the frequency of eosinophils in the lung both in WT and CARD9 deficient mice via flow cytometry. A higher frequency of eosinophils (Figure 1B) were accumulated in the lung from CARD9 deficient mice in comparison to those in WT mice after A. fumigatus challenge. Furthermore, we found that CARD9 deficiency dramatically elevated the expression of type-2 cytokines including Il-4, Il-5, Il-13 and Il-10 (Figure 1C) in the lung, and augmented serum IgE levels (Figure 1D) compared with those in WT mice. Collectively, these data suggested that CARD9 deficiency impaired anti-fungal immunity, but promoted A. fumigatus-induced airway allergic inflammation. Dectin-1 is a well-established receptor for A. fumigatus swollen conidia (SC). To determine whether Dectin-1 was involved in regulating pulmonary type-2 immunity in CARD9 deficient mice, we used α-Dectin-1 antibody to neutralize dectin-1 signalling. We found that α-Dectin-1 neutralization significantly blocked eosinophils accumulation (Figure 1E) in the lung from CARD9 deficient mice after A. fumigatus exposure. Consistently, α-Dectin-1 neutralization significantly decreased IL-4 and IL-5 concentration (Figure 1F) in the lung and serum IgE levels (Figure 1G) in comparison to those in IgG control mice. Together, CARD9 deficiency-mediated pulmonary type-2 immunity was dependent on Dectin-1 signalling after A. fumigatus exposure. Accumulating evidence have demonstrated that alveolar macrophages are involved in triggering allergic immune responses.8 To evaluate whether alveolar macrophages was essential for A. fumigatus-induced pulmonary type-2 immunity, we analysed the population of alveolar macrophages in the lung from WT and CARD9 deficient mice at day 2 after A. fumigatus exposure. We observed that alveolar macrophages were substantially depleted in WT mice, but not CARD9 deficient mice after A. fumigatus exposure (Figure 2A). To confirm the role of alveolar macrophages, we employed clodronate liposomes to deplete alveolar macrophages via intratracheal instillation. We found that depletion of alveolar macrophages in CARD9 deficient mice significantly abolished the pulmonary eosinophils infiltration in comparison to those in control mice after A. fumigatus exposure (Figure 2B). Further, depletion of alveolar macrophages significantly decreased IL-4 and IL-5 levels (Figure 2C) in the lung and serum IgE concentration (Figure 2D) in CARD9 deficient mice. Thus, these data indicated that alveolar macrophages functioned as primary initiators for A. fumigatus-induced pulmonary type-2 immunity in CARD9 deficient mice. Next, we sought to determine the underlying mechanism of how CARD9 modulated pulmonary type 2 immune responses after A. fumigatus infection. We firstly analysed phenotype and antigen presentation capability of alveolar macrophages in WT and CARD9 deficient mice with/without A. fumigatus exposure. The detailed data about the characteristics of alveolar macrophages are available in the following repository (https://zenodo.org/record/7493754#.Y651P8hqilw). We found that alveolar macrophages were marked by Siglec-H, Ly6C, CD64 and F4/80 both in WT and CARD9 deficient mice. Simultaneously, we found that the expression of CD80, CD86 and MHC-II were comparable between WT and CARD9 deficient mice with/without A. fumigatus exposure, hinting that CARD9 did not affect the antigen presentation capacity of alveolar macrophages. Next, we determined whether CARD9 deficiency could regulate canonical NF-κB and MAPK signalling pathways upon fungus stimulation. As expected, CARD9 deficiency dramatically blocked p65 and p50 nuclear translocation compared with those in WT BMDM after resting conidial (RC) and SC of A. fumigatus stimulation, but not candida albicans (C. albicans) yeast and LPS stimulation. Consistently, the protein levels of phosphorylated IκBα were decreased and total IκBα levels were increased after A. fumigatus stimulation (RC and SC), but not C. albicans. However, the protein levels of phosphorylated Syk and JNK were comparable between WT and CARD9 deficient BMDM after A. fumigatus and C. albicans stimulation. The detailed data about NF-κB and MAPK signalling are available in the following repository (https://zenodo.org/record/7493754#.Y651P8hqilw). Our previous work demonstrated that CARD9 S12N mutation induced non-canonical NF-κB activation, which regulated IL-5 production in alveolar macrophages after A. fumigatus challenge.8 Therefore, we sorted out alveolar macrophages from naïve WT and CARD9 deficient mice and then stimulated with A. fumigatus, and observed that A. fumigatus stimulation induced p65 (red) nuclear translocation in WT alveolar macrophages but not in CARD9 deficient alveolar macrophages. On the contrary, RelB (green) translocated into nuclear in CARD9 deficient, but not in WT alveolar macrophages after A. fumigatus stimulation. Moreover, we sorted out alveolar macrophages from WT and CARD9 deficient mice infected with/without A. fumigatus, and further confirmed that RelB activation only occurred in CARD9 deficient alveolar macrophages. Previous study have demonstrated Curdlan could regulate non-canonical NF-κB activation and Ccl17 expression, which can tune Th1 and Th2 balance.9 Thus, we challenged CARD9 deficient BMDMs with Curdlan, and found that CARD9 deficiency significantly increased the expression of Ccl17 and Ccl11. These data suggested that CARD9 promoted canonical NF-κB activation, but inhibited non-canonical NF-κB activation, which may modulate Ccl17 and Ccl11 expression in alveolar macrophages after A. fumigatus exposure. Generally, appropriate innate and adaptive immune responses are critical for fungal clearance, and impaired immune response will result in fungal infection. CARD9 functions as an adaptor protein, which integrates CLRs signalling after ligand engagement, finally orchestrating host innate and adaptive immunity against fungal infection.4 Consistently, we found that CARD9 deficient mice were susceptible to A. fumigatus infection. However, histological analysis and flow cytometry analysis showed large eosinophil accumulation in lungs from CARD9 deficient mice after A. fumigatus exposure. Elevated type 2 cytokines production in lung and serum IgE levels in CARD9 deficient mice further confirmed allergic airway inflammation after A. fumigatus challenge. These data suggested that CARD9 deficiency impaired host anti-fungal immunity, but boosted pulmonary type 2 immune responses after A. fumigatus exposure. In our previous study, we have provided direct evidence that revealed the correlation of CARD9 with pulmonary allergic inflammation, and identified CARD9 S12N as a susceptible gene to ABPA.8 Thus, combination with our previous work, we uncover an unidentified function of CARD9 that modulate the dynamic balance between A. fumigatus-induced anti-fungal immunity and allergic inflammation. Furthermore, our observation was also supported by some clinical studies or case reports of which these fungal infected-patients with CARD9 null mutations are also manifested by higher serum IgE concentration and hyper-eosinophilia in the peripheral blood.5-7 In summary, our data suggested that CARD9 deficiency impaired host anti-fungal immune response, but promoted A. fumigatus-induced type-2 dominant immunity via Dectin-1 signalling. Meanwhile, we also found that alveolar macrophages were essential for triggering allergic airway inflammation after A. fumigatus exposure. Our study uncovers the unrecognized function of CARD9 in modulating the balance between anti-fungal and allergic inflammation in response to A. fumigatus exposure. Jielin Duan, Fan Li, Ronghua Huang, Siyuan Wu and Xia Xu performed the experiments; Jielin Duan, Zhiwen Huang and Xia Xu performed statistics analysis; Xia Xu designed the study; Jielin Duan and Xia Xu wrote the paper. This work was supported by grants from the National Natural Science Foundation of China (81970036 to Xia Xu; 82201929 to Jielin Duan), and the Natural Science Foundation of Beijing (7202130 to Xia Xu), and China Postdoctoral Science Foundation (2022 M720906 to Jielin Duan). The authors declare that they have no conflicts of interest. The data that support the findings of this study are openly available in Additional information for methods and supplementary data-Cl at https://zenodo.org/record/7493754#.Y651P8hqilw, reference number https://doi.org/10.5281/zenodo.7493754.