Neutrophil-Derived Tumor Necrosis Factor Drives Fungal Acute Lung Injury in Chronic Granulomatous Disease

Abstract

Chronic granulomatous disease (CGD) results from deficiency of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase and impaired reactive oxygen species (ROS) generation. This leads to impaired killing of Aspergillus and, independently, a pathologic hyperinflammatory response to the organism. We hypothesized that neutrophil-derived ROS inhibit the inflammatory response to Aspergillus and that acute lung injury in CGD is due to failure of this regulation. Mice with gp91phox deficiency, the most common CGD mutation, had more severe lung injury, increased neutrophilinfiltration, and increased lung tumor necrosis factor (TNF) after Aspergillus challenge compared with wild-types. Neutrophils were surprisingly the predominant source of TNF in gp91phox-deficient lungs. TNF neutralization inhibited neutrophil recruitment in gp91phox-deficient mice and protected from lung injury. We propose that, in normal hosts, Aspergillus stimulates TNF-dependent neutrophil recruitment to the lungs and neutrophil-derived ROS limit inflammation. In CGD, in contrast, recruited neutrophils are the dominant source of TNF, promoting further neutrophil recruitment in a pathologic positive-feedback cycle, resulting in progressive lung injury.

Keywords: Aspergillus; TNF; chronic granulomatous disease; neutrophil; reactive oxygen species.

Figure 1.

Lung injury in wild-type and gp91^phox-deficient (gp91^phox-knockout [KO]) mice in response to Aspergillus fumigatus. Wild-type and gp91^phox-KO mice were inoculated with nonviable A. fumigatus germlings intratracheally on day 0. A, Albumin content in bronchoalveolar lavage (BAL) fluid was measured using enzyme-linked immunosorbent assay. **P < .01 (mixed-effects analysis). Horizontal lines represent medians, and circles, individual animals; data are representative of 3 independent experiments. B, Lungs were fix inflated with 2% paraformaldehyde followed by paraffin embedding and hematoxylin-eosin staining. Representative sections shown are taken from lungs of wild-type and gp91^phox-KO mice untreated (left panels) and on day 3 after challenge (right panels) (original magnification ×40; scale bar represents 100 μm). Images shown are representative of 5 animals challenged with Aspergillus.

Figure 2.

Accumulation of leukocytes in the lungs of gp91^phox-deficient (gp91^phox-knockout [KO]) mice after Aspergillus challenge. A, Representative flow cytometry identification of neutrophils, alveolar macrophages, inflammatory dendritic cells (DCs), and recruited macrophages in single-cell suspension of whole lung after intratracheal challenge with nonviable Aspergillus germlings, gated on CD45⁺ cells (not shown). B, Time course of accumulation of leukocyte subsets in wild-type (WT) and gp91^phox-KO mouse lungs after Aspergillus challenge on day 0. Data represent means with standard errors of the mean for 5 per group per time point and are representative of 2 independent experiments; ‡P < .001 (2-way analysis of variance [ANOVA]). C, Lung leukocytes 3 days after challenge with nonviable Aspergillus germlings in bone marrow chimera, using WT and gp91^phox-KO mice. Horizontal lines represent medians, and circles, individual animals. P < .001 by 1-way ANOVA for both cell types. *P < .05; **P < .01 (Tukey multiple comparisons test). Data shown are pooled from 3 independent experiments.

Figure 3.

Lung tumor necrosis factor (TNF) production by neutrophils in gp91^phox-deficient (gp91^phox-knockout [KO]) mice after Aspergillus challenge. Wild-type (WT) and gp91^phox-KO mice were inoculated intratracheally with nonviable Aspergillus germlings on day 0. A, TNF protein concentration in bronchoalveolar lavage (BAL) fluid was measured with enzyme-linked immunosorbent assay (ELISA). Data represent means with standard errors of the mean for 4–6 mice per group per time point. ****P < .001 (mixed-effects analysis). B, Immunofluorescence images of lungs of WT and gp91^phox-KO mice, unchallenged or 2 days after Aspergillus challenge. Green represents neutrophils (7/4 antigen); red, TNF; blue, nuclei(DAPI) (original magnification ×40; [×100 for inset]; large scale bars represent 100 µm; small scale bar, 10 µm); data are representative of 2–3 animals per group. C, Representative intracellular staining for TNF in lungs from WT and gp91^phox-KO mice (gp91^phox) untreated and 2 days after Aspergillus challenge (gated on CD45⁺ cells) Numbers shown are percent of CD45+ cells except far right panel is percent of TNF+ cells. Abbreviation: SSC, side scatter. D, E, Summary data from flow cytometry shown in C. Data shown are pooled from 4 independent experiments; ****P < .001 (mixed-effects analysis). F, Representative DiffQuick stain of Cytospin preparation of Ly6G⁺ cells isolated from single-cell suspensions of gp91^phox-KO mouse lungs on day 2 after Aspergillus challenge by positive magnetic selection (original magnification ×20). G, TNF transcript in sorted lung leukocytes in gp91^phox-KO mice 2 days after Aspergillus challenge. Quantitative reverse-transcription polymerase chain reaction of >50 000 cells from each population was normalized to TNF messenger RNA expression in alveolar macrophages from untreated animals. P < .01 (2-way analysis of variance [ANOVA]); ***P < .001 (Sidak multiple comparisons test). Abbreviation: DCs, dendritic cells. H, Positively selected Ly6G⁺ cells from single-cell suspensions of WT or gp91phox-KO mouse lung on day 2 after Aspergillus challenge were cultured ex vivo. TNF was measured by ELISA in supernatant after overnight culture. *P = .03 (t test). I, The number of TNF⁺ neutrophils was determined with flow cytometry after overnight culture. P < .001 (1-way ANOVA); ***P < .001 and ****P < .0001 (Tukey multiple comparisons test). In D, E, and G–I, horizontal lines represent medians, and circles, individual animals.

Figure 4.

Effect of neutrophil depletion on lung tumor necrosis factor (TNF) production in gp91^phox-deficient (gp91^phox-knockout [KO]) mice after Aspergillus challenge. Gp91^phox-KO mice were treated with anti-Ly6G (PMN depletion) or isotype control antibody 1 day before intratracheal challenge with nonviable Aspergillus germlings. A–C, Number of lung neutrophils, inflammatory dendritic cells (DCs), and macrophages as determined with flow cytometry. Bar graphs represent means with standard errors of the mean for 3–4 animals per group; data shown are representative of 3 independent experiments. *P < .05; ****P < .001 (mixed-effects analysis). D, E, Bronchoalveolar lavage (BAL) fluid TNF and albumin concentrations by enzyme-linked immunosorbent assay. Data represent means with standard errors of the mean for 4 mice per group per time point. There were no significant differences between isotype control or PMN depletion groups for TNF or albumin. F, Intracellular staining for TNF in lung leukocytes 2 days after nonviable Aspergillus challenge (gated on CD45⁺ cells) in neutrophil-depleted gp91^phox-KO mouse. Percentages shown for TNF⁺ cells (left panel) represent percentage of total CD45⁺ cells; all other percentages, percentage of TNF⁺ cells. Abbreviation: side scatter,SSC. G, Summary data of total and TNF⁺ lung leukocyte populations in neutrophil-depleted gp91^phox-KO mice 2 days after Aspergillus challenge. Horizontal lines represent medians, and circles, individual animals. P < .01 by 1-way analysis of variance for both cell types; *P < .05 and **P < .01 by Tukey multiple comparisons test.

Figure 5.

Correlation between lung tumor necrosis factor (TNF)–producing neutrophils and lung injury in gp91^phox-deficient mice (gp91^phox-knockout [KO]) after Aspergillus challenge. A, Number of lung neutrophils, TNF-positive (TNF⁺) cells, and TNF⁺ neutrophils on day 2 after challenge with different inocula of nonviable Aspergillus germlings. ***P < .001 and ****P < .0001 for differences between groups and differences between inocula by mixed-effects analysis. B, Bronchoalveolar (BAL) fluid albumin, as measured with enzyme-linked immunosorbent assay. **P < .01 for association of BAL fluid albumin with inoculum (1-way analysis of variance). C, Association between TNF⁺ neutrophils and non-neutrophil leukocytes with BAL fluid albumin in gp91^phox-KO mice. Linear correlation (solid line) and 95% confidence intervals (dashed lines) are shown; r is the Pearson correlation coefficient. In A and B, horizontal lines represent medians, and circles, individual animals. Data are pooled from 2 independent experiments.

Figure 6.

Role of tumor necrosis factor (TNF) in mediating acute lung injury in gp91^phox-deficient mice after Aspergillus challenge. Wild-type (WT) and gp91^phox-deficient (KO) mice were treated with anti-TNF antibody (αTNF) or isotype control antibody and measurements were obtained on day 3. A, B, Total lung neutrophils, as determined with flow cytometry, and bronchoalveolar lavage (BAL) fluid albumin, as measured with enzyme-linked immunosorbent assay (ELISA). Antibodies were given 1 day before and 1 day after intratracheal challenge with nonviable Aspergillus germlings. C, BAL fluid neutrophils and albumin in gp91^phox-deficient mice with antibodies administered only 1 day after intratracheal challenge with nonviable Aspergillus germlings, with measurements obtained on day 3. D–G, BAL fluid cytokines as measured with ELISA (antibodies as in A and B). In all plots, horizontal lines represent medians, and circles, individual animals. For all panels, groups differed significantly by 1-way analysis of variance. *P < .05, **P < .01, ***P < .001, and ****P < .0001 (Tukey multiple comparisons test). Abbreviations: IL-1β , interleukin 1β ; IL-17A, interleukin 17A.