Neutrophils produce interleukin 17A (IL-17A) in a dectin-1- and IL-23-dependent manner during invasive fungal infection

Abstract

We have previously reported that compromised interleukin 17A (IL-17A) production in the lungs increased susceptibility to infection with the invasive fungal pathogen Aspergillus fumigatus. Here we have shown that culturing lung cells from A. fumigatus-challenged mice ex vivo demonstrated Dectin-1-dependent IL-17A production. In this system, neutralization of IL-23 but not IL-6, IL-1β, or IL-18 resulted in attenuated IL-17A production. Il23 mRNA expression was found to be lower in lung cells from A. fumigatus-challenged Dectin-1-deficient mice, whereas bone marrow-derived dendritic cells from Dectin-1-deficient mice failed to produce IL-23 in response to A. fumigatus in vitro. Addition of recombinant IL-23 augmented IL-17A production by wild-type (WT) and Dectin-1-deficient lung cells, although the addition of IL-6 or IL-1β did not augment the effect of IL-23. Intracellular cytokine staining of lung cells revealed lower levels of CD11b(+) IL-17A(+) and Ly-6G(+) IL-17A(+) cells in A. fumigatus-challenged Dectin-1-deficient mice. Ly-6G(+) neutrophils purified from the lungs of A. fumigatus-challenged Dectin-1-deficient mice displayed lower Il17a mRNA expression but surprisingly had intact Rorc and Rora mRNA expression. We further demonstrated that Ly-6G(+) neutrophils required the presence of myeloid cells for IL-17A production. Finally, upon in vitro stimulation with A. fumigatus, thioglycolate-elicited peritoneal neutrophils were positive for intracellular IL-17A expression and produced IL-17A in a Dectin-1- and IL-23-dependent manner. In summary, Dectin-1-dependent IL-17A production in the lungs during invasive fungal infection is mediated in part by CD11b(+) Ly-6G(+) neutrophils in an IL-23-dependent manner.

Fig. 1.

Whole-lung versus lung cell IL-17A production from A. fumigatus-challenged Dectin-1-deficient mice. (A) C57BL/6 wild-type (WT) and Dectin-1-deficient (KO) mice were challenged intratracheally with (5 to 7) × 10⁷ A. fumigatus conidia, and 48 h after exposure, IL-17A levels in lung homogenates were quantified by ELISA. Data are expressed as mean pg/ml + SEM. Cumulative data from three independent studies (n=5 mice/group for each study) are illustrated. “**” represents a P value of <0.01 (unpaired two-tailed Student's t test). (B) C57BL/6 wild-type (WT) and Dectin-1-deficient (KO) mice were challenged intratracheally with (5 to 7) × 10⁷ A. fumigatus conidia, and 18 h after exposure, lungs were collected and enzymatically digested. Immediately after single-cell suspensions were collected, total RNA was isolated from 1 × 10⁶ cells and transcribed to cDNA, and quantitative real-time PCR was performed for Il17a. Gene expression was normalized to Gapdh expression, and fold changes between WT (set at 1) and KO mice were determined using the 2^−ΔΔCT method. Cumulative data from five independent studies are shown. “***” represents a P value of <0.001 (paired two-tailed Student's t test). (C) Lung cells were isolated as for panel B, and 1 × 10⁶ cells were cultured for 24 h in a volume of 0.2 ml. IL-17A levels were quantified in clarified coculture supernatants by ELISA. Cumulative data from five independent studies are shown. Data are expressed as mean pg/ml + SEM. “**” represents a P value of <0.01 (unpaired two-tailed Student's t test). (D) Lung cells were isolated as described for panel B, Fc blocked, stained with a live/dead staining kit, and thereafter stained with fluorochrome-conjugated antibodies against various immune cell surface markers (see Materials and Methods). Cumulative data from three independent studies are shown. Data are expressed as absolute numbers of live cells in the lung digest cell preparation. “*” and “**” represent P values of <0.05 and 0.01, respectively (unpaired two-tailed Student's t test). PMNs, polymorphonuclear leukocytes; Eos, eosinophils; Monos, monocytes; Macs, macrophages.

Fig. 2.

IL-17A production by lung cells after A. fumigatus challenge partially requires IL-23. (A) Lung cells were isolated as described in the legend to Fig. 1B, and 1 × 10⁶ cells were cultured for 24 h in a volume of 0.2 ml. Neutralizing antibodies against IL-23 and IL-18 were added at a final concentration of 2 to 5 μg/ml at the beginning of the culture (α-18 and α-23). Rat (IL-18) or goat (IL-23) isotype antibodies (Iso 18 and Iso 23) were included as a control. IL-17A levels were quantified in clarified coculture supernatants by ELISA. Cumulative data from five independent studies are shown. Data are expressed as mean pg/ml + SEM. “**” and “***” represent P values of <0.01 and 0.001, respectively (unpaired two-tailed Student's t test). (B) Immediately after lung cells were collected, total RNA was isolated from 1 × 10⁶ cells and transcribed to cDNA, and quantitative real-time PCR was performed for Il23. Gene expression was normalized to that of Gapdh, and fold changes between WT (set at 1) and KO mice were determined using the 2^−ΔΔCT method. Cumulative data from five independent studies are shown. “***” represents a P value of <0.001 (paired two-tailed Student's t test). (C) Bone marrow-derived dendritic cells (BMDCs) were isolated and cultured at 1 × 10⁶ cells in a volume of 100 μl. A. fumigatus conidia (A.f) were added at a 1:1 ratio to BMDCs in a volume of 100 μl. Supernatants were collected after 24 h and clarified by centrifugation. IL-23 levels were quantified by ELISA. Cumulative data from three independent studies are shown. “*” represents a P value of <0.05 (unpaired two-tailed Student's t test). Unstim, unstimulated. (D) Lung cells were isolated from WT and KO mice as described for Fig. 1B, and 1 × 10⁶ cells were cultured for 24 h in a volume of 0.2 ml. Recombinant murine IL-23 was added at 1 and 10 ng/ml at the beginning of the culture. Controls included lung cells cultured in the absence of IL-23. IL-17A levels were quantified in clarified coculture supernatants by ELISA. Cumulative data from three independent studies are shown. Data are expressed as mean pg/ml + SEM. “*,” “**,” and “***” represent P values of 0.05, 0.01, and 0.001, respectively (unpaired two-tailed Student's t test).

Fig. 3.

IL-17A is not produced by T cells in a Dectin-1-dependent manner after A. fumigatus challenge. Lung cells were isolated as described for Fig. 1B and stimulated with PMA/I for 5 h in the presence of GolgiStop inhibitor. Cells were Fc blocked, stained with a live/dead staining kit, and thereafter stained with an anti-CD3 antibody, followed by fixation/permeabilization, and stained for intracellular IL-17A. Cumulative data from two independent studies are shown. Data are expressed as the percentages of CD3⁺ cells that are IL-17A⁺.

Fig. 4.

Lung CD11b⁺ Ly-6G⁺ cells are a source of Dectin-1-dependent IL-17A after A. fumigatus challenge. (A) Lung cells were isolated from WT and KO mice as described for Fig. 1B, and 1 × 10⁶ cells were cultured for 24 h in a volume of 0.1 ml (second half of the culture in the presence of GolgiStop inhibitor). Cells were Fc blocked, stained with a live/dead staining kit, and thereafter stained for CD11b, followed by fixation/permeabilization, and stained for intracellular IL-17A. Representative flow cytometric plots after gating on live cells followed by gating on CD11b⁺ IL-17A⁺ cells are shown. Double-positive populations (CD11b⁺ IL-17A⁺; square gates) were based on unstained controls. (B) Lung cells were isolated and cultured as described for Fig. 1B. Cells were Fc blocked, stained with a live/dead staining kit, and thereafter stained for Ly-6G, followed by fixation/permeabilization, and stained for intracellular IL-17A. Representative flow cytometric plots after gating on live cells followed by gating on Ly-6G⁺ IL-17A⁺ cells are shown. Double-positive populations (Ly-6G⁺ IL-17A⁺; square gates) were based on unstained controls. (C) Cumulative flow cytometric data from panel A from three independent studies with lung cells cultured and analyzed in triplicate. Data are expressed as the percentages of CD11b⁺ IL-17A⁺ cells. “***” represents a P value of <0.001 (unpaired two-tailed Student's t test). (D) Cumulative flow cytometric data from panel B from three independent studies with lung cells cultured and analyzed in triplicate. Data are expressed as the percentage of Ly-6G⁺ IL-17A⁺ cells. “**” represents a P value of <0.01 (unpaired two-tailed Student's t test). (E) Cumulative flow cytometric data from three independent studies with lung cells cultured and analyzed in triplicate. Data are expressed as the percentages of CD11b⁺ Ly-6G⁺ lung cells that are IL-17A⁺. “***” represents a P value of <0.001 (unpaired two-tailed Student's t test).

Fig. 5.

Lung Ly-6G⁺ cells require the presence of CD11b⁺ and CD11c⁺ cells for IL-17A production. (A) Lung cells were isolated from WT and KO mice as described for Fig. 1B, and Ly-6G⁺ cells were purified via magnetic bead isolation. Immediately thereafter, total RNA was isolated from 1 × 10⁶ cells and transcribed to cDNA, and quantitative real-time PCR was carried out for Il17a. Gene expression was normalized to that of Gapdh, and fold changes between WT (set at 1) and KO mice were determined using the 2^−ΔΔCT method. Cumulative data from three independent studies are shown. “*” represents a P value of <0.05 (paired two-tailed Student's t test). (B) Lung cells were isolated from WT mice as described for Fig. 1B, and Ly-6G⁺, CD11b⁺, and CD11c+ cells were purified by magnetic bead selection followed by culturing with A. fumigatus for 24 h individually or in various combinations. IL-17A levels were quantified in clarified coculture supernatants by ELISA. Cumulative data from three independent studies, with cultures performed in duplicate or triplicate in each, are represented. Data are expressed as mean pg/ml + SEM. “*” represents a P value of <0.05 (paired two-tailed Student's t test).

Fig. 6.

Dectin-1 is not required for Rorc, Rora, Ahr, or Irf4 mRNA expression in lung cells after A. fumigatus challenge. (A) Lung cells were isolated from WT and KO mice as described for Fig. 1B. Immediately after single-cell suspensions were collected, total RNA was isolated from 1 × 10⁶ cells and transcribed to cDNA, and quantitative real-time PCR was performed for Rorc, Rora, Ahr, and Irf4. Gene expression was normalized to that of Gapdh, and fold changes between WT (set at 1) and KO mice were determined using the 2^−ΔΔCT method. Cumulative data from 10 independent studies are shown. “**” represents a P value of <0.01 (paired two-tailed Student's t test). (B) Lung cells were isolated from WT and KO mice as described for Fig. 1B, and Ly-6G⁺ cells were purified via magnetic bead isolation. Immediately thereafter, total RNA was isolated from 1 × 10⁶ cells and transcribed to cDNA, and quantitative real-time PCR was performed for Rorc. Gene expression was normalized to that of Gapdh, and fold changes between WT (set at 1) and KO mice were determined using the 2^−ΔΔCT method. Cumulative data from six independent studies are represented. “*” represents a P value of <0.05 (paired two-tailed Student's t test).

Fig. 7.

Thioglycolate-elicited peritoneal neutrophils produce Dectin-1- and IL-23-dependent IL-17A in response to A. fumigatus in vitro. (A) Thioglycolate was injected intraperitoneally (3%; 1.5 ml) into naive WT and Dectin-1-deficient mice. After 4 h, mice were lavaged with 10 ml of prewarmed tissue culture medium, and cells were isolated via centrifugation. Neutrophil recruitment was assessed by Wright staining of cytospun peritoneal lavage cell preparations. Representative Wright stains of WT (left; 22/27 are neutrophils [81%]) and Dectin-1-deficient (right; 27/33 are neutrophils [77%]) mice are shown. (B) Cumulative cell differential data as determined by Wright staining of cytospun peritoneal lavage cell preparations. Cumulative data from three independent studies are shown. (C) Thioglycolate-elicited peritoneal neutrophils (1 × 10⁶) were cultured in a volume of 100 μl. A. fumigatus conidia were added at a 1:1 ratio to a volume of 100 μl. Supernatants were collected after 24 h and clarified by centrifugation. IL-17A levels were quantified by ELISA. Cumulative data from four independent studies are shown. “*” represents a P value of <0.05 (unpaired two-tailed Student's t test). (D) Thioglycolate-elicited peritoneal neutrophils (1 × 10⁶) from WT mice were cultured with A. fumigatus in the presence of neutralizing antibodies against IL-23. Goat isotype antibodies were included as a control. IL-17A levels were quantified in clarified coculture supernatants by ELISA after 24 h. Cumulative data from five independent studies with 1 to 2 mice per group analyzed in duplicate are shown. Data are expressed as mean pg/ml + SEM. “**” represents a P value of <0.01 (unpaired two-tailed Student's t test). (E) Representative flow cytometric data of intracellular IL-17A staining of thioglycolate-elicited Ly-6G⁺ cells from WT and Dectin-1-deficient mice after coculture with A. fumigatus in vitro.