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

To the Editor, 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 antifungal immunity or Th2 dominated allergic inflammation, or both based on the situations.1,2 However, some investigations show that type2 immunity can suppress Th1 mediated immuneprotection against fungal infection, which promote fungal persistence and disease progression.3 These data suggest that antifungal immunity and sensitization to fungal allergen are always in dynamic balance and overwhelming type2 immune response may be detrimental for fungal elimination. Caspase recruitment domaincontaining protein 9 (CARD9), a central adaptor protein mainly expressed in myeloid cells, relays downstream signalling of Ctype lectin receptors (CLRs) via CARD9Bcl10Malt1 (CBM) complex, finally controlling canonical NFκB activation and resultant proinflammatory cytokines production (TNFα, IL6, IL12, etc.), which are critical for fungal clearance.1,4 Accumulating clinical evidence have found that null or loseoffunction 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 hypereosinophilia 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 antifungal immunity but also involves in modulation of allergic inflammation. However, whether CARD9 tunes the balance between antifungal immunity and type2 immune response remains unclear. To determine the role of CARD9 in modulation of antifungal immunity and allergic inflammation, wild type (WT) and CARD9 deficient mice were infected with A. fumigatus. The detailed data about fungal burden and antifungal immune parameters are available in the following repository (https://zenodo.org/recor d/74937 54#. Y651P 8hqilw). 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 Tnfα, Il6 and Ifng, and decreased TNFα and IL6 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 antifungal 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 antifungal 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 type2 cytokines including Il4, Il5, Il13 and Il10 (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 antifungal immunity, but promoted A. fumigatusinduced airway allergic inflammation. Dectin1 is a wellestablished receptor for A. fumigatus swollen conidia (SC). To determine whether Dectin1 was involved in regulating pulmonary type2 immunity in CARD9 deficient mice, we used αDectin1 antibody to neutralize dectin1 signalling. We found that αDectin1 neutralization significantly blocked eosinophils accumulation (Figure 1E) in the lung from CARD9 deficient mice after A. fumigatus exposure. Consistently, αDectin1 neutralization significantly decreased IL4 and IL5 concentration (Figure 1F) in the lung and serum IgE levels (Figure 1G) in comparison to those in IgG control mice. Together, CARD9 deficiencymediated pulmonary type2 immunity was dependent on Dectin1 signalling after 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 (TNFα, 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][7][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/recor d/74937 54#. Y651P 8hqilw). 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 Tnfα, Il-6 and Ifng, and decreased TNFα 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.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 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 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  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

Key message
• CARD9 enhances pro-inflammatory immune response, but inhibits pulmonary type 2 immunity after A. fumigatus exposure.
• Alveolar macrophages trigger pulmonary type-2 immunity in CARD9 deficient mice after A. fumigatus exposure.
CARD9 null mutations are also manifested by higher serum IgE concentration and hyper-eosinophilia in the peripheral blood. [5][6][7] In summary, our data suggested that CARD9 deficiency impaired host anti-fungal immune response, but promoted A. fumigatusinduced 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. F I G U R E 2 Alveolar macrophages triggered pulmonary type-2 immunity in CARD9 deficient mice after A. fumigatus exposure. (A) Alveolar macrophage (CD11c + SiglecF + ) counts in lungs of WT and CARD9-deficient mice (n = 5) at day 2 after exposure to A. fumigatus conidia (1 × 10 7 ). (B-D) Eosinophil (SiglecF + ) counts (B), IL-4 and IL-5 levels in lung (C) and IgE levels in serum (D) of WT and CARD9-deficient mice (n = 6), which was intratracheally instilled with clodronate liposomes or control liposomes after exposure to A. fumigatus conidia (1 × 10 7 ). *p < .05; **p < .01 and ***p < .001. Data shown are representative of three independent experiments.