Extrapulmonary Aspergillus infection in patients with CARD9 deficiency

Abstract

Invasive pulmonary aspergillosis is a life-threatening mycosis that only affects patients with immunosuppression, chemotherapy-induced neutropenia, transplantation, or congenital immunodeficiency. We studied the clinical, genetic, histological, and immunological features of 2 unrelated patients without known immunodeficiency who developed extrapulmonary invasive aspergillosis at the ages of 8 and 18. One patient died at age 12 with progressive intra-abdominal aspergillosis. The other patient had presented with intra-abdominal candidiasis at age 9, and developed central nervous system aspergillosis at age 18 and intra-abdominal aspergillosis at age 25. Neither patient developed Aspergillus infection of the lungs. One patient had homozygous M1I CARD9 (caspase recruitment domain family member 9) mutation, while the other had homozygous Q295X CARD9 mutation; both patients lacked CARD9 protein expression. The patients had normal monocyte and Th17 cell numbers in peripheral blood, but their mononuclear cells exhibited impaired production of proinflammatory cytokines upon fungus-specific stimulation. Neutrophil phagocytosis, killing, and oxidative burst against Aspergillus fumigatus were intact, but neither patient accumulated neutrophils in infected tissue despite normal neutrophil numbers in peripheral blood. The neutrophil tissue accumulation defect was not caused by defective neutrophil-intrinsic chemotaxis, indicating that production of neutrophil chemoattractants in extrapulmonary tissue is impaired in CARD9 deficiency. Taken together, our results show that CARD9 deficiency is the first known inherited or acquired condition that predisposes to extrapulmonary Aspergillus infection with sparing of the lungs, associated with impaired neutrophil recruitment to the site of infection.

Figure 1. Intra-abdominal aspergillosis in CARD9 (caspase recruitment domain family member 9) deficiency.

Coronal image of computed tomography of the abdomen of patient 2 shows a large intra-abdominal mass (A, arrow), while chest computed tomography shows normal lungs (B). Biopsy of the intra-abdominal mass shows areas of palisading inflammation and necrosis on H&E stain (C; magnification, ×40) and acute-angle branching hyphae consistent with Aspergillus species on Grocott-Gomori’s methenamine silver stain (D; magnification, ×400). Coronal (E) and axial (F) images of computed tomography of the abdomen of patient 2 one year after the initial presentation shows a large right suprarenal mass (arrows).

Figure 2. The patients with extrapulmonary aspergillosis carry biallelic caspase recruitment domain family member 9 (CARD9) mutations that result in absent CARD9 protein.

Western blots of CARD9 protein expression in peripheral blood mononuclear cells from healthy donors and patient 1 (left panel) and 2 (right panel). GAPDH is shown as a loading control.

Figure 3. Caspase recruitment domain family member 9 (CARD9) deficiency results in impaired production of proinflammatory mediators upon fungal-specific stimulation.

IL-1β (A; ^*P = 0.0275, Mann-Whitney test) and IL-6 (B; ^*P = 0.0275, Mann-Whitney test) production by healthy donor (n = 6–12) and patient 1 (n = 2–4 independent experiments) peripheral blood mononuclear cells (PBMCs) after 24 hours of stimulation with live Candida albicans or Staphylococcus aureus or after 24 hours without stimulation. IL-1β (C; ^***P = 0.0002, unpaired t test), IL-6 (D; ^**P = 0.0019, unpaired t test), TNF-α (E; ^****P < 0.0001, unpaired t test) and GM-CSF (F; ^***P = 0.0008, unpaired t test) production by healthy donor (n = 7–10) and patient 2 (n = 3 independent experiments) PBMCs after 48 hours of stimulation with heat-killed C. albicans or LPS or after 48 hours without stimulation. Data represent the mean ± SEM.

Figure 4. The c.3G>C caspase recruitment domain family member 9 (CARD9) loss-of-function mutation impairs the production of proinflammatory mediators upon stimulation with Aspergillus fumigatus conidia.

TNF-α (^**P = 0.0091, Mann-Whitney test), GM-CSF (^*P = 0.0273, Mann-Whitney test), and IFN-γ (^**P = 0.0091, Mann-Whitney test) production by healthy donor (n = 9) and patient 2 (n = 3 independent experiments) peripheral blood mononuclear cells after 48 hours of stimulation with heat-killed A. fumigatus conidia. Data represent the mean ± SEM.

Figure 5. Caspase recruitment domain family member 9 (CARD9) deficiency does not impair neutrophil phagocytosis and killing of Aspergillus fumigatus.

(A) CARD9-deficient neutrophils from patient 1 exhibit normal conidial internalization as assessed by flow cytometry using FITC-labeled A. fumigatus conidia (n = 3 healthy donors; n = 2 independent experiments with patient 1 cells). (B and C) Control neutrophils and CARD9-deficient neutrophils from patient 1 have similar capacity to prevent the germination of opsonized (B) and nonopsonized (C) A. fumigatus conidia into hyphae. Fungal metabolic activity was measured using the MTT assay (n = 6 healthy donors; n = 2 independent experiments with patient 1 cells). (D) Killing of opsonized A. fumigatus hyphae by control neutrophils and CARD9-deficient neutrophils from patient 1. Fungal metabolic activity was measured using the MTT assay (n = 9 healthy donors; n = 2 independent experiments with patient 1 cells). (E and F) Opsonized (E) and nonopsonized (F) conidia or hyphae were incubated with neutrophils (ratio 1:1) from either healthy donors or Patient 2. Fungal metabolic activity was measured using the XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay (n = 4 healthy donors; n = 4 independent experiments with patient 2 cells). Data shown represent 1 of 3 independent replicates with similar patterns of results. Data represent the mean ± SEM (A–D) or mean ± SD (E and F).

Figure 6. Caspase recruitment domain family member 9 (CARD9) deficiency is associated with impaired neutrophil accumulation in the extrapulmonary infected tissue.

Biopsy of this suprarenal mass shows areas of palisading inflammation, in which giant cells/histiocytes (A) engulf Aspergillus hyphae (inset) and are CD68⁺ by immunohistochemistry (B). Surrounding the histiocytic infiltration are sheaths of plasma cells (C) and eosinophils (D). No neutrophils are seen. Magnification, ×200 (A); ×400 (A inset and B–D).