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. 2018 Jul 6;9(1):2636.
doi: 10.1038/s41467-018-04912-3.

Genetic deficiency of NOD2 confers resistance to invasive aspergillosis

Mark S Gresnigt  1   2   3 , Cristina Cunha  4   5 , Martin Jaeger  2 , Samuel M Gonçalves  4   5 , R K Subbarao Malireddi  6 , Anne Ammerdorffer  2 , Rosalie Lubbers  2 , Marije Oosting  2 , Orhan Rasid  1 , Grégory Jouvion  7 , Catherine Fitting  1 , Dirk J de Jong  8 , João F Lacerda  9   10 , António Campos Jr  11 , Willem J G Melchers  12 , Katrien Lagrou  13   14 , Johan Maertens  13   15 , Thirumala-Devi Kanneganti  6 , Agostinho Carvalho  4   5 , Oumaima Ibrahim-Granet  1 , Frank L van de Veerdonk  16
Affiliations

Genetic deficiency of NOD2 confers resistance to invasive aspergillosis

Mark S Gresnigt et al. Nat Commun. .

Abstract

Invasive aspergillosis (IA) is a severe infection that can occur in severely immunocompromised patients. Efficient immune recognition of Aspergillus is crucial to protect against infection, and previous studies suggested a role for NOD2 in this process. However, thorough investigation of the impact of NOD2 on susceptibility to aspergillosis is lacking. Common genetic variations in NOD2 has been associated with Crohn's disease and here we investigated the influence of these genetic variations on the anti-Aspergillus host response. A NOD2 polymorphism reduced the risk of IA after hematopoietic stem-cell transplantation. Mechanistically, absence of NOD2 in monocytes and macrophages increases phagocytosis leading to enhanced fungal killing, conversely, NOD2 activation reduces the antifungal potential of these cells. Crucially, Nod2 deficiency results in resistance to Aspergillus infection in an in vivo model of pulmonary aspergillosis. Collectively, our data demonstrate that genetic deficiency of NOD2 plays a protective role during Aspergillus infection.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Genetic variation in donor P268S (rs2066842) confers resistance to IA after HSCT. Shown are the results obtained in a cohort comprising 310 eligible patients and respective donors. Cumulative incidence of IA according to (a) donor or recipient genotypes at rs2066842 or b a dominant genetic model of donor genotypes at rs2066842. In a, the blue line represents the carriers of the reference (ref) CC genotype, the red line carriers of the heterozygous CT genotype, and the yellow line represents carriers of the homozygous TT genotype. In the dominant model (b) red line represents the carriers of both the CT and TT genotypes. Data were censored at 24 months, and relapse and death were considered competing events. p-values were calculated using Gray’s test. c IL-1β, TNF, IL-6, IL-8, IL-17A, and IL-10 levels measured in the BAL of patients with aspergillosis and stratified based on the NOD2 rs2066842 donor genotypes. Each dot represent an individual patient, with black filled dots representing HSCT donor carriers of the reference CC genotype, half filled black/gray dots representing carriers of the heterozygous CT genotype, and gray dots representing carriers of the homozygous TT genotype. Data are represented scatter dot plot with the median; with p-values were calculated using the Mann–Whitney U test, p-values of statistical tests are shown within the graphs
Fig. 2
Fig. 2
Human NOD2 polymorphisms influence Aspergillus-induced cytokine responses and fungal killing. a–d IL-1β, TNF, IL-6, IL-17A, IL-22, and IFNγ levels measured in culture supernatants of PBMCs stimulated with (a, c) live Aspergillus conidia for 24 h or (b, d) heat-inactivated (HI) Aspergillus conidia for 7 days. The PBMCs of individuals with various genotypes of the NOD2 gene were compared. These genotypes included (a, b) the P268S mutation (rs2066842; reference: CC n = 36, heterozygous: CT n = 28 and homozygous: TT n = 4) and (c, d) the 1007finsC mutation (rs2066847; reference n = 62 and heterozygous: insC n = 4). e, f Fungal killing capacity of human PBMCs assessed as CFU remaining of A. fumigatus (2 × 106) following exposure for 24 h to (5 × 105) PBMCs results are stratified based on the e P268S (rs2066842; ref: CC n = 49, heterozygous: n = 45 and homozygous: TT n = 7) and (f) 1007finsC (rs2066847; ref n = 98 insC n = 7) genotypes. g, h Area under the curve (AUC) of relative light units (RLU) induced by luminol oxidation by reactive oxygen species (ROS) released by PBMCs, results are stratified based on the (g) P268S (rs2066842; reference: CC n = 47, heterozygous: n = 50 and homozygous: TT n = 9) and (h) 1007finsC (rs2066847; ref n = 112 insC n = 5) genotypes. Data are represented scatter dot plot with the median. Each dot represent an individual patient, with (a, b, e, g) black filled dots representing carriers of the ancestral (reference) CC genotype, half-filled black/gray dots representing carriers of the heterozygous CT genotype, and gray dots representing carriers of the homozygous TT genotype, and (c, d, f, h) black filled dots representing carriers of the reference (ref) genotype without insertion and half-filled black/gray dots representing carriers of one Cysteine insertion (insC). The means were compared using the Mann–Whitney U test, p-values of statistical tests are shown within the graphs
Fig. 3
Fig. 3
NOD2-deficient individuals have impaired cytokine responses in response to Aspergillus. a IL-1β and TNF levels in culture supernatants of PBMCs from healthy controls (black dots) and NOD2-deficient patients (gray dots) that were stimulated for 24 h with A. fumigatus live conidia, HI conidia or HI hyphae (n = 11 controls and n = 9 patients for IL-1β and n = 9 controls and n = 8 patients for TNF). b IL-17A, IL-22, and IFNγ levels after 7 days of stimulation with HI conidia or HI hyphae (IL-17A n = 7 controls and n = 6 patients; IL-22 and IFNγ n = 10 controls, n = 8 patients). c IL-17A+, IL-22+, and IFNγ+ CD4 T-cell populations (n = 8 controls and n = 5 patients) after 7 days stimulation with HI conidia shown as the percentage of total CD4 cells. d The fungal killing capacity of PBMCs (5 × 105) from healthy controls and NOD2-deficient patients (n = 12 controls, n = 6 patients) counted as CFU remaining after 24-h stimulation with live A. fumigatus (2 × 106). e The area under the curve of the reactive oxygen species release of PBMCs from healthy controls and NOD2-deficient patients (controls n = 8, patients n = 4) in response to live Aspergillus conidia (1 × 107/mL) and zymosan (150 µg/mL) measured by luminescence signal from luminol conversion over 1 h. Data are represented as scatter dot plot with median and means were compared using the Mann–Whitney U test
Fig. 4
Fig. 4
Nod2−/− deficient mice have a reduced susceptibility to aspergillosis. Comparison of the susceptibility of wild-type mice (WT; black lines with circles) and Nod2-deficient mice (Nod2−/−; blue lines with squares) to invasive aspergillosis. a Kaplan–Meier survival curve of cyclophosphamide immunosuppressed WT (n = 12) and Nod2−/− (n = 9) mice infected intranasally with 5 × 104 conidia. p-values of the Kaplan–Meier curve were determined with the use of the log-rank test. Data represent the cumulative data of four separate experiments. b Percentage weight loss following cyclophosphamide immunosuppression and intranasal Aspergillus infection (5 × 104/mouse) in WT (n = 20) and Nod2−/− (n = 17) mice (p = 0.3515) (c, d) Luminescence signal at day 1 to 3 post infection from the luminescent Aspergillus originating from lung and sinus regions in WT (n = 20) and Nod2−/− (n = 17) mice. Curves were compared by repeated measurements two-way ANOVA. e Fungal burden as determined by amplification of Aspergillus ITS2 regions from lung homogenates. Data in graphs are represented as mean ± SEM or in scatterplots with a line indicating the median. The means were compared using the Mann–Whitney U test, p-values of statistical tests are shown within the graphs, luminescence and weight curves were compared for significance using a two-way repeated measurements ANOVA
Fig. 5
Fig. 5
Nod2−/− mice show reduced histological damage and fungal burden following Aspergillus infection. a Histology of lung sections of wild-type and Nod2−/− mice at day 3 pi, stained in HE (I, II), Grocott’s Methenamine Silver (III) or labelled using anti-F4/80 antibody (specific for macrophages), counterstained with Haematoxylin staining. Scale bars represent 1 mm (I) and 200 μm (II–IV). b Morphometric analysis of the lesions in the whole lung sections using Image J software to quantify the lesions in number and size. c Representative lung sections of two additional WT and Nod2−/− mice, stained in HE (I) and Grocott’s Methenamine Silver (II–IV). Scale bars represent 200 μm (I, II) and 50 μm (III, IV), means were compared for significance using the Mann-Whitney U test
Fig. 6
Fig. 6
histology of nasal sinuses Histology of Nasal Sinuses of (a–c) wild-type and (d–f) Nod2−/− mice at day 3 pi, stained by HE staining at (a, d) ×2 and (b, e) ×10 magnification and (c, f) Grocott’s Methenamine Silver staining at ×20 magnification. Scale bars represent (a, d) 500 μm, (b, e) 200 μm, and (c, f) 100 μm
Fig. 7
Fig. 7
NOD2 activation positively regulates cytokine production. a, b Aspergillus-induced IL-1β and TNF levels in the culture supernatants of human PBMCs (5 × 105) of (a) healthy volunteers (n = 13) represented as black dots or (b) NOD2-deficient patients (n = 6) represented as grey dots in the presence or absence of (10 μg/mL) MDP. Data is represented as scatter dot plot with median and means were compared using the Wilcoxon signed rank test was paired comparisons. c, d Aspergillus-induced IL-6, KC, and TNF levels in the culture supernatants of murine (c) BMDMs and (d) splenocytes of wild-type (WT, black dots) and Nod2-deficient (Nod2−/−, grey dots) mice. Data are represented scatterplots with a line indicating the median and means were compared using the Mann–Whitney U test
Fig. 8
Fig. 8
NOD2 negatively regulates, fungal killing, phagocytosis, and dectin-1 expression. a–c The fungal killing capacity of macrophages (1 × 105) assessed by CFU remaining of A. fumigatus (2 × 106) following exposure for 24 h, in (a) wild-type and Nod2−/− BMDMs (n = 16 WT, n = 12 Nod2−/−). b human GM-CSF differentiated MDMs treated (n = 9) for 48 h with siRNA targeting NOD2 or non-targeting siRNA, and c human GM-CSF differentiated MDMs (n = 9) treated for 48 h with the NOD2 ligand (10 μg/mL) MDP. Phagocytosis efficiency assessed as percentage of macrophages that engulfed FITC-labelled A. fumigatus conidia and mean fluorescence intensity of the total macrophage population in (d) wild-type and Nod2−/− BMDMs (n = 16 wt, n = 12 Nod2−/−), (e) human GM-CSF differentiated MDMs (n = 9) treated for 48 h with siRNA targeting NOD2 or non-targeting siRNA, and (f) human GM-CSF differentiated MDMs (n = 6) treated for 24 h with the NOD2 ligand (10 μg/mL) MDP. g, h The area under the curve of the reactive oxygen species release of (g) wild-type (n = 6) and Nod2−/− (n = 6) BMDMs or (h) human MDMs (n = 5) treated for 24 h with the NOD2 ligand (10 μg/mL) MDP in response to zymosan (150 µg/mL) measured by luminescence signal from luminol conversion over 1 h. i Dectin-1 (Clec7a) expression assessed by qPCR in wild-type and Nod2−/− BMDMs (n = 14 wt, n = 10 Nod2−/−), (j) NOD2 (n = 8) and CLEC7A (n = 6) mRNA expression in human GM-CSF differentiated MDMs treated for 48 h with siRNA targeting NOD2 or non-targeting siRNA, and (k) Surface dectin-1 expression measured by flow cytometry on human GM-CSF differentiated MDMs treated for 28 h with the NOD2 ligand (10 μg/mL) MDP. Data is represented as scatter dot plot with median with (a, d, g) black dots representing wild-type mice and grey dots representing Nod2 deficient mice, (b, e, j) black squares representing human macrophages treated with scrambled siRNA and grey squares human macrophages treated with NOD2 targeting siRNA, and black triangles representing MDMs without MDP pre-treatment and grey triangles MDMs with MDP pre-treatment. Means were compared using the Mann–Whitney U test for murine BMDMs (a, d, g, i) and the Wilcoxon signed-rank test was paired comparisons following siRNA treatment (b, e, j), or MDP stimulation (c, f, h, k)
Fig. 9
Fig. 9
MDP negatively affects antifungal host response only in the presence of functional NOD2. a, b The fungal killing capacity of (a) murine BMDMs (n = 8 wt and n = 6 Nod2−/−) and (b) human monocytes (1 × 105) assessed by CFU remaining of A. fumigatus (2 × 106) following exposure for 24 h. c Phagocytosis efficiency assessed as percentage of monocytes that engulfed FITC-labelled A. fumigatus conidia and mean fluorescence intensity of the total macrophage population, (d) surface dectin-1 expression measured by flow cytometry in cells of healthy controls (co) (n = 6) as well as NOD2-deficient patients (NOD2−/− PT) (n = 3). Data are represented as scatterplot with median, black dots represent wild-type and grey dots represent NOD2-deficient cells. Means were compared using the Wilcoxon signed-rank test for paired comparisons except for the comparison of WT with Nod2-/- cells which were compared using the Mann-Whitney U test
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