IL-17A–Secreting Memory γδ T Cells Play a Pivotal Role in Sensitization and Development of Hypersensitivity Pneumonitis

Takashi Yamana, Tsukasa Okamoto, Masahiro Ishizuka, Satoshi Hanzawa, Masaru Ejima, Sho Shibata and Yasunari Miyazaki
J Immunol January 15, 2021, 206 (2) 355-365; DOI: https://doi.org/10.4049/jimmunol.2000198

This article has a correction. Please see:

Key Points

  • IL-17A is produced mainly by γδ T cells in hypersensitivity pneumonitis (HP).

  • The IL-17A/chemokines/CD11b+ alveolar macrophage axis underlies development of HP.

  • Memory responses of γδ T cells are involved in sensitization and development of HP.

Abstract

Hypersensitivity pneumonitis (HP) typically presents with interstitial inflammation and granulomas induced by an aberrant immune response to inhaled Ags in sensitized individuals. Although IL-17A is involved in the development of HP, the cellular sources of IL-17A and the mechanisms by which IL-17A contributes to granuloma formation remain unclear. Recent studies report that γδ T cells produce IL-17A and exhibit memory properties in various diseases. Therefore, we focused on IL-17A–secreting memory γδ T cells in the sensitization phase and aimed to elucidate the mechanisms by which IL-17A contributes to granuloma formation in HP. We induced a mouse model of HP using pigeon dropping extract (PDE) in wild-type and IL-17A knockout (IL-17A−/−) mice. IL-17A−/− mice exhibited reduced granulomatous areas, attenuated aggregation of CD11b+ alveolar macrophages, and reduced levels of CCL2, CCL4, and CCL5 in the bronchoalveolar lavage fluid. Among IL-17A+ cells, more γδ T cells than CD4+ cells were detected after intranasal PDE administration. Interestingly, the expansion of IL-17A–secreting Vγ4+ or Vγ1−Vγ4− cells of convalescent mice was enhanced in response to the sensitizing Ag. Additionally, coculture of macrophages with PDE and Vγ4+ cells purified from PDE-exposed convalescent mice produced significantly more IL-17A than coculture with Vγ4+ cells from naive mice. Our findings demonstrate that in the sensitization phase of HP, IL-17A–secreting memory γδ T cells play a pivotal role. Furthermore, we characterized the IL-17A/CCL2, CCL4, CCL5/CD11b+ alveolar macrophage axis, which underlies granuloma formation in HP. These findings may lead to new clinical examinations or therapeutic targets for HP.

Introduction

Hypersensitivity pneumonitis (HP) is a heterogeneous disorder caused by an aberrant immune response to repeated inhalation of Ags in sensitized individuals. A variety of organic/inorganic particles, including fungi, bacteria, animal proteins, and chemicals, are causative Ags (1, 2). HP is categorized by the type of Ag and clinical course. Bird-related HP, also called bird fancier’s lung, is one of the most frequent forms of HP, and the causative Ags are avian droppings, feathers, and serum (3–5). In acute HP, histological findings include interstitial cell inflammation and poorly formed granulomas. Although both Gell and Coomb type III (immunocomplex-mediated) and type IV (cell-mediated) hypersensitivity are thought to play central roles in the development of acute HP (6), detailed mechanisms of sensitization and development of HP have remained unclear.

Experimental models of HP show that IL-17A is involved in lung inflammation induced by Saccharopolyspora rectivirgula (7–11), pigeon dropping extract (PDE) (12), and Stachybotrys chartarum (13). IL-17A is involved in a variety of immunological responses, such as enhanced production of proinflammatory cytokines and chemokines and direct immune cell recruitment (14, 15). However, the detailed mechanism by which IL-17A contributes to the development of HP, especially in the process of granuloma formation, remains to be clarified because it has been thought to be induced mainly by the Th1 response (16, 17). Therefore, this study aimed to elucidate the mechanisms by which IL-17A contributes to granuloma formation.

Very few studies have identified the primary cellular source of IL-17A in the development of HP. In early studies, IL-17A was thought to be secreted mainly by Th17 lymphocytes; however, recent investigations revealed that IL-17A is also produced by several kinds of innate-like lymphocytes, including γδ T cells, type three innate lymphoid cells (ILC3s), and invariant NK cells (14). Among these innate-like lymphocytes, γδ T cells are a major source of IL-17A in various diseases, such as bacterial or fungal infection, autoimmunity, and stroke (18).

IL-17A production by γδ T cells has long been suspected to be an innate immune response. However, several recent studies focused on the memory responses of γδ T cells using murine models of enteric Listeria infection, Staphylococcus aureus–induced peritonitis, and Bordetella pertussis lung infection (19–22). In these studies, γδ T cells from mesenteric lymph nodes (19, 20), the peritoneum (21), and lungs (22) produced greater amounts of IL-17A from convalescent mice than from naive mice after reinfection with the same pathogen. Until recently, memory γδ T cells have not previously been explored in the development of HP. Therefore, we hypothesized that the main source of IL-17A in the lungs is γδ T cells and that IL-17A–secreting memory γδ T cells play a pivotal role in the sensitization and development of HP by producing more IL-17A in response to sensitized Ags.

To confirm our hypothesis and to achieve the aims mentioned above, we established an experimental model of HP induced by PDE using wild-type (WT) and IL-17A knockout (IL-17A−/−) mice. In this model, we found that IL-17A–secreting memory γδ T cells had enhanced properties of expansion and secretion and are major sources of IL-17A. Furthermore, we also demonstrated that IL-17A plays a pivotal role in granuloma formation via several chemokines and macrophages. These novel mechanisms of immunopathogenesis may lead to new clinical, experimental, and therapeutic targets in the future.

Materials and Methods

Mice

WT C57BL6/J mice were purchased from CLEA Japan (Tokyo, Japan). IL-17A−/− mice (C57BL6/J background) were donated by Prof. Y. Iwakura (Tokyo University of Science, Tokyo, Japan). All mice (female, 8–10 wk of age) were bred in the animal facility of Tokyo Medical and Dental University under specific pathogen-free conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (protocol number: A2019004).

Preparation of PDE as an intranasally administered Ag

PDE was acquired as previously described (23, 24). In brief, freshly collected pigeon droppings were stirred in a 20-fold volume of autoclaved PBS solution (pH 7.4) for 24 h and then dialyzed with distilled water. The obtained extract was sterilized via filtration (Millex-GV; Millipore, Bedford, MA) and lyophilized.

Protocols for the induction of HP (PDE mice)

Mice were immunized with 8 μg of PDE in 200 μl of saline via an i.p. injection without adjuvant. Subsequently, 8 μg of PDE in 40 μl of saline was administered intranasally 3 d a week for 3 wk under light anesthesia by isoflurane inhalation (Fig. 1A). Control mice were immunized with PDE and given 40 μl of saline in the same manner described for PDE-challenged mice. Mice were euthanized 24 h after the last PDE challenge after week 1 (WK1), week 2 (WK2), and week 3 (WK3) for serum, bronchoalveolar lavage, and histological analysis. To evaluate the effect of neutralization of IL-17A at each time point, 100 μg of anti–IL-17A Abs (50104; R&D Systems, Minneapolis, MN) was injected i.p. into WT PDE mice once on day −6, day 1, day 8, or day 15. Additionally, to evaluate the role of IL-17F in this model, 25 μg of rat anti–IL-17F mAb (clone RN17; Thermo Fisher Scientific, Waltham, MA) or normal rat IgG (clone eBRG1; Thermo Fisher Scientific) was injected i.p. into WT PDE mice on day 1, day 8, day 15, and day 22. For intracellular staining and flow cytometric analysis, the PDE mice were euthanized 2 h after the last PDE challenge on days 1, 8, 15, and 22. To examine the memory functions of γδ T cells, we prepared convalescent mice that were challenged with PDE as described above until day 8. Then, they were maintained without any procedures for 2 wk and then euthanized on day 23 (Fig. 1B).

Bronchoalveolar lavage

Mice were euthanized by i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Three 1-ml lavages with sterile PBS were obtained using a 23-gauge blunt-tipped needle inserted into the trachea. Samples were centrifuged at 200 × g for 15 min. The supernatants were stored at −20°C for multiplex cytokine and chemokine analysis. Cells were resuspended using sterile PBS. Cell counts were obtained by manual counting via light microscopy with a hemocytometer. Differential cell counts were determined by cytospin centrifugation and May-Grünwald-Giemsa staining. One hundred microliters of each sample was loaded onto a slide using a Cytospin 4 cytocentrifuge (Thermo Fisher Scientific). The slides were May-Grünwald-Giemsa stained, and at least 100 cells were counted to obtain differential cell counts. After that, the subsets of lymphocytes and monocyte-macrophage lineage cells were examined using flow cytometric analysis.

Cells in the bronchoalveolar lavage fluid (BALF) of PDE-challenged WT or IL-17A−/− mice and control mice at WK1 and WK3 were stained with PE-conjugated anti-CD4 (GK1.5; BD Biosciences, San Jose, CA), PE-Cy7–conjugated anti-CD3 (17A2; BioLegend, San Diego, CA), allophycocyanin-conjugated anti-CD8 (53-6.7; BD Biosciences), FITC-conjugated anti-CD68 (FA11; BioLegend), PE-conjugated anti-F4/80(BM8; BioLegend), PE-Cy7–conjugated anti-CD11b (M1/7; BioLegend), allophycocyanin-conjugated anti-CD45 (30-F11; BioLegend), and BV421-conjugated anti-CD11c (N418; BioLegend) Abs. After staining, the cells were analyzed using a FACSCanto 2 (BD Biosciences).

Histopathological analysis

Left lungs were harvested at WK1 and WK3. The lungs were filled with 10% neutral buffered formalin, placed in formalin for 24 h, and embedded in paraffin. The lungs were then sectioned at 4 μm and stained with H&E. The sections were evaluated by microscopy. The percentage granuloma area in the H&E-stained lung sections was analyzed using a BZ-4HC hybrid cell counting system (Keyence, Osaka, Japan).

Immunohistochemical analysis

Immunohistochemistry was conducted on the left lung of WT and IL-17A−/− mice euthanized at WK3 to examine the localization of lymphocytes, monocytes, and macrophages in granulomatous areas. Immunofluorescence staining for CD4 (D7D2Z; Cell Signaling Technology, Danvers, MA), CD8 (D4W2Z; Cell Signaling Technology), CD11c (DIV9Y; Cell Signaling Technology), CD11b (M1/70; BioLegend), and F4/80 (BM8; Thermo Fisher Scientific) was performed on paraffin-embedded 4-μm sections and for TCR γδ (GL3; BioLegend) and IL-17A (ab79056; Abcam, Cambridge, U.K.) on a 8-μm frozen section using the Opal Kit (PerkinElmer, Waltham, MA) according to the manufacturer’s protocol. The stained sections were analyzed using a BZ-X fluorescence microscope and the densities of CD4+, CD8+, and CD11c+ cells in a granulomatous area were quantified using BZ-4HC hybrid cell counting system (Keyence). Images of immunohistochemistry were processed using haze reduction in a BZ-X analyzer (Keyence). Additionally, we added immunohistochemistry of neutrophils in the lungs of WT and IL-17A−/− at WK3 to assess the distribution of neutrophils. Paraffin-embedded 4-μm sections were incubated with 3% H2O2 in PBS for 15 min to block endogenous peroxidases and then with normal goat serum for 20 min to block nonspecific binding of Abs. Consequently, the sections were incubated with anti-Ly6G/Ly6C (NIMP-R14; Thermo Fisher Scientific) overnight followed by the incubation with ImmPRESS HRP Goat Anti-Rat IgG (Vector Laboratories, Burlingame, CA). After that, ImmPACT DAB Substrate (Vector Laboratories) were added according to the manufacturer’s protocol. The stained sections were evaluated by microscopy.

Measurement of serum anti-PDE IgG

PDE-specific IgG in the serum was measured by ELISA. Each well of a polystyrene plate (Immulon 2HB; Thermo Fisher Scientific) was coated with 100 μl of 1 mg/ml PDE in carbonate buffer (pH 9.6) at 4°C for 12 h. The wells were washed three times with PBS containing 0.05% polysorbate-20 (Tween-20; PBS-T). Next, the wells were treated with 0.05% BSA–PBS-T to impede nonspecific binding. Then, 100 μl of the sample (1:1000 dilution) was added to each well and incubated at 37°C for 1 h. The wells were washed four times, and 100 μl of 1:1000–diluted rabbit anti-mouse IgG (Merck, Darmstadt, Germany) conjugated with HRP was added. Subsequently, the plate was incubated at 37°C for 1 h. Then, the wells were washed four times, and o-phenylenediamine was added as a substrate. The OD of each well was measured at 490 nm using a microplate reader (iMark; Bio-Rad Laboratories, Hercules, CA).

Multiplex cytokine and chemokine assays

BALF supernatants of PDE-challenged WT and IL-17A−/− mice and saline control mice harvested at each time point were examined using Milliplex Map Mouse Cytokine/Chemokine Panel 1 and the Mouse TH17 Panel (Merck) to measure the concentrations of mouse IFN-γ, IL-1β, IL-4, IL-5, IL-10, IL-17A, IL-17F, TNF-α, IL-21, IL-22, IL-23, CCL 2, CCL4, CCL5, CCL20, CXCL1, CXCL9, and CXCL10. The assays were performed on a Luminex 100 system with xPONENT 3.1 software (Luminex, Austin, TX).

Real-time quantitative PCR for an analysis of mRNA expression

The expression levels of mRNA coding for CCL2, CCL4, and CCL5 in the lung of mice were measured using quantitative real-time RT-PCR. First, right lungs were homogenized with TRIzol (Invitrogen, Carlsbad, CA) to extract total RNA of lungs. To purify the RNA, genomic DNA was removed by Qiagen RNeasy Kit (Qiagen, Mississauga, ON, Canada). Next, the purified RNA was reverse-transcribed using a SuperScript III First-Strand Synthesis SuperMix Kit (Invitrogen) (12, 24). We performed the PCR using a Mini Opticon (Bio-Rad Laboratories), operated by MJOpticon Monitor version 3.1 analysis software (Bio-Rad Laboratories), using ready-made fluorogenic probes (SYBR green; Bio-Rad Laboratories) according to the manufacturer’s protocol. For CCL2, the forward primer was 5′-CACTCACCTGCTGCTACTCA-3′ and the reverse primer was 5′-GCTTGGTGACAAAAACTACAGC-3′; for CCL4, the forward primer was 5′-GCCAGCTGTGGTATTCCTGA-3′ and the reverse primer was 5′-AGCTGCTCAGTTCAACTCCA-3′; for CCL5, the forward primer was 5′-CTCACCATATGGCTCGGACA-3′. Gene expression of each mice was quantified relative to the expression level of ribosomal protein S15 (RPS15; forward primer: 5′-TTCCGCAAGTTCACCTACC-3′, reverse primer: 5′-CGGGCCGGCCATGCTTTACG-3′) and to the expression level of each gene in control mice using the ΔΔCycle Threshold method.

Intracellular cytokine staining and flow cytometric analysis

For intracellular cytokine staining and flow cytometric analysis, WT mice were euthanized 2 h after PDE or saline administration on days 1, 8, 15, and 22. In addition, naive and convalescent mice were challenged with additional PDE and they were euthanized 2 h later. After mice were euthanized, their lungs were perfused through the right ventricle with 5 ml of PBS containing 0.5 mM EDTA. Then, the lungs were isolated and cut into small pieces. They were incubated for 1 h in RPMI 1640 (Sigma-Aldrich) containing collagenase type I (1 mg/ml; Sigma-Aldrich) and DNase I type II (60 U/ml; Sigma-Aldrich) at 37°C according to a previous report (25). Then, the lungs were homogenized and passed through a 40-μm nylon mesh to obtain a single-cell suspension. RBCs in the cell suspension were lysed with RBC lysis buffer (Thermo Fisher Scientific). The obtained cells were incubated for 2 h with Golgi Plug (BD Biosciences) containing brefeldin A for intracellular staining. Then, they were incubated for 30 min with normal rat serum containing an anti-CD16/32 Ab (2.4G2; Thermo Fisher Scientific) to impede the nonspecific binding of Abs. Subsequently, staining for surface markers was performed with PE-conjugated anti-CD3 (17A2; BD Biosciences) and FITC-conjugated anti-TCRδ (GL3; BD Biosciences) or anti-CD4 (GK1.5; BD Biosciences) Abs for γδ T cells and CD4+ lymphocytes, respectively, and with FITC-conjugated anti–Ly-6G (1A8; BD Biosciences) and anti–Siglec F (E50-2440; BD Biosciences) for neutrophils. Additionally, the staining of each γδ T cell surface phenotype was performed with PE-Cy7–conjugated anti-CD3 (17A2; BioLegend), BV510-conjugated anti-TCRδ (GL3; BD Biosciences), FITC-conjugated anti-Vγ4 (UC3-10A6; BioLegend), or CD27 (LG.3A10; BioLegend) Abs and PE-conjugated anti-Vγ1 (2.11; BioLegend) or anti-CD44 (IM7; BioLegend) Abs. Dead cells were excluded by staining with Fixable Viability Dye eFluor 660 (Thermo Fisher Scientific). The surface-stained cells were subjected to intercellular cytokine staining with BV421-conjugated anti–IL-17A following fixation and permeabilization using a Cytofix/Cytoperm Kit (BD Biosciences). The cells were analyzed using FACSCanto 2 (BD Biosciences).

In vitro coculture of purified lung γδ T cells with alveolar macrophages and PDE

Isolation of alveolar macrophages (AMs) was performed as previously described (21, 26). Cells in BALF from naive WT mice were obtained as described above and seeded into a 96-well flat-bottom plate (1.0 × 105 cells per well). AMs were allowed to adhere by incubation at 37°C with 5% CO2 in complete RPMI 1640 with FCS for 2 h. Next, the medium was removed and replaced with RPMI 1640 without FCS. The cells were then incubated with 0.2 mg/ml PDE. Lung Vγ1.1+ (5.0 × 103 per well), Vγ4+ (2.5 × 103 per well), or Vγ1−Vγ4− (5.0 × 103 per well) γδ T cells were purified as CD3+TCRγδ+Vγ1+, CD3+TCRγδ+Vγ4+, and CD3+TCRγδ+Vγ1−Vγ4− cells from naive or convalescent mice using an FACSAria2 (BD Bioscience). Surface markers were labeled with PE-Cy7 anti-mouse CD3 (17A2; BioLegend), FITC-conjugated anti-TCRδ, allophycocyanin-conjugated anti-TCR Vγ4 (UC3-10A6; BioLegend), and PE-conjugated anti-mouse Vγ1.1 (2.11; BioLegend) Abs. The purified cells were cocultured with AMs and PDE for 3 or 60 h. Subsequently, the concentration of IL-17A in the supernatant was measured using an ELISA Kit (R&D Systems).

Statistical analysis

The data are expressed as the means ± SEM. Differences between groups were evaluated by the Mann–Whitney U test. Statistical significance was defined as p < 0.05. The statistical analysis was performed using JMP (SAS, Tokyo, Japan).

Results

IL-17A−/− mice have fewer macrophages, monocytes, neutrophils, and CD8+ lymphocytes in the BALF after intranasal PDE administration

We examined the involvement of IL-17A in the aggregation of inflammatory cells in the BALF (Fig. 1). In WT mice, total inflammatory cell density was significantly increased in PDE mice compared with control mice at WK1, WK2, and WK 3 (Fig. 2A). In control mice, almost no lymphocytes, neutrophils, or eosinophils were observed. Compared with WT mice, in IL-17A−/− mice, the density of total cells and neutrophils was significantly lower at every time point, and the density of macrophages was significantly lower at WK2. The density of lymphocytes tended to be lower in IL-17A−/− mice than in WT mice, although the difference was not significant. There were no differences in the density of eosinophils between WT and IL-17A−/− mice. Flow cytometric analysis was used to identify subsets of lymphocytes (Fig. 2B, 2C). CD4+ lymphocytes were dominant at both WK1 and WK3 in both groups. The densities of CD8+ lymphocytes were lower, and the CD4/CD8 ratios at WK3 were significantly higher in IL-17A−/− mice than WT mice. Next, we evaluated the subsets of macrophages, dendritic cells, and monocytes in the BALF using flow cytometric analysis. After CD68– cells (polymorphonuclear cell and lymphocytes) were negatively gated, AMs were identified as CD11chighF4/80high cells, interstitial macrophages (IMs) as CD11cint F4/80low cells, and monocytes as CD11clowF4/80low cells (Fig. 2D), referring to a previous report (25). All of the densities of these subsets were significantly higher in WT mice at WK3 compared with IL-17A−/− and control mice (Fig. 2E). Interestingly, in WT mice, a substantial proportion of AM was positive for CD11b, and the percentage and absolute numbers of CD11b+ AM were significantly lower in IL-17A−/− mice and control mice. In addition, no dendritic cells (CD11c+F4/80−) were observed in the BALF of any group.

FIGURE 1.
FIGURE 1.

Protocols for the administration of PDE. (A) Protocol for the induction of HP by PDE (PDE mice). (B) Protocol for preparing convalescent mice.

FIGURE 2.
FIGURE 2.

(A) The density of total cells, macrophages, lymphocytes, neutrophils, and eosinophils in the BALF of PDE- or saline-challenged (control) mice. The data are presented as the mean ± SEM for each group (n = 4–5). Black bar, PDE-challenged WT mice; striped bar, PDE-challenged IL-17A−/− mice; gray bar, control WT mice. (B) The gating strategies of CD4+ or CD8+ lymphocytes gated on CD3+ cells. (C) The densities of CD4+ or CD8+ cells and the CD4/CD8 ratio in the BALF of WT and IL-17A−/− mice. The data are presented as the mean ± SEM for each group (n = 3). (D) The gating strategies of macrophages and monocytes gated on CD68+ cells. (E) The densities of AMs, IMs, and monocytes (Mono) in the BALF of WT, IL-17A−/−(17A−/−), or control mice (con). The data are presented as the mean ± SEM for each group (n = 3). These data are representative of two to three experiments. *p < 0.05.

IL-17A plays an important role in PDE-induced granuloma formation but not the production of Ag-specific IgG

Fig. 3A–C shows the lung histology of PDE mice. At WK1, mononuclear cell and neutrophil infiltration was observed in the peribronchial and perivascular areas of the lungs of WT mice. At WK2, although mononuclear cells and neutrophil infiltration were more prevalent than at WK1, clear granuloma formation was not seen yet. These features were milder in IL-17A−/− mice than in WT mice (Fig. 3A). At WK3, fewer and smaller granulomas developed in the lungs of IL-17A−/− mice compared with those in WT mice (Fig. 3A, 3B). The granulomatous area percentage at WK3 was significantly smaller in IL-17A−/− mice than in WT mice (Fig. 3B).

FIGURE 3.
FIGURE 3.

Lung histology of WT and IL-17A−/− mice given intranasal PDE or saline and the measurement of PDE-specific IgG. (A) H&E staining of lungs taken at WK1, WK2, and WK3 (original magnification, ×40). These are representative of four to five mice in each group. (B) The granuloma area percentage was analyzed using a BZ-4HC hybrid cell count system (Keyence). The data are presented as the mean ± SEM for each group (n = 4–5). *p < 0.05. (C) High-power field granuloma areas (H&E staining, original magnification ×200) and immunofluorescence staining of CD4 (green), CD8 (red), and CD11c (light blue) in the lungs at WK3. (D) Cell counts of CD4+, CD8+, and CD11c+ cells per 1.0 × 104 μm2 of granulomatous areas analyzed by the BZ-4HC hybrid cell count system (Keyence). The data are presented as the mean ± SEM for each group (n = 19–23 from three mice). (E) Immunofluorescence staining of CD11c (green), F4/80 (red), and CD11b (light blue) in the lungs at WK3 (original magnification, ×200). The data are representative of 8–10 pictures of three to four mice. (F) Immunohistochemical staining with anti–Ly-6G/Ly-6C Abs in the lungs of WT and IL-17A−/− mice at WK3 (left: original magnification, ×100; right: ×200). The pictures are representative of four mice. (G) OD data from ELISA for the measurement of PDE-specific IgG. The data represent the mean ± SEM for each group (n = 4). *p < 0.05. The data are representative of two experiments. Con, saline-challenged mice; IL-17A−/−, PDE IL-17A−/− mice; WT, PDE WT mice.

The anti–IL-17A Abs injected at day 1 or day 8 (but not at day 6 or day 15) significantly reduced the percentage of the granuloma area at WK3. In contrast, anti–IL-17F Abs injected at days 1, 8, 15, and 22 did not reduce the percentage of the granuloma area at WK3 (Supplemental Fig. 1).

Immunofluorescence staining revealed that CD4+, CD8+, and CD11c+ cells formed clusters in WT mouse granulomas at WK3, whereas fewer and scattered CD11c+ cells were identified in IL-17A−/− mouse granulomas (Fig. 3C). Quantification of the immunofluorescence staining revealed that the densities of CD8+ cells and CD11c+ cells in each granulomatous area in WT mice were significantly higher than those in IL-17A−/− mice, whereas there was no significant difference in the density of CD4+ cells between the two groups (Fig. 3D). Furthermore, Fig. 3E shows that most CD11c+ cells in the granulomatous areas were also stained with CD11b and F4/80, indicating that they were AMs or IMs. Furthermore, immunohistochemistry of neutrophils showed that, in the lungs of WT mice, neutrophils were not distributed within granulomas but mainly around blood vessels. Compared with WT, much fewer neutrophils were observed in the lungs of IL-17A−/− mice (Fig. 3F).

In contrast, although a significantly higher amount of PDE-specific IgG was detected in PDE mice than in control mice at WK2 and WK3, there were no significant differences between WT and IL-17A−/− PDE mice at any time point (Fig. 3G).

The concentrations of CCL2, CCL4, and CCL5 are reduced in the BALF of IL-17A−/− mice

To investigate the mediators involved in granuloma formation in PDE-treated mice, we measured the BALF concentrations of proinflammatory cytokines and chemokines, which act as chemoattractants for lymphocytes, monocytes, and macrophages. The concentrations of CCL2, CCL4, and CCL5 were significantly lower in IL-17A−/− mice than in WT mice at WK2 (Fig. 4). There were no significant differences in the concentrations of CXCL9, CXCL10, or IFN-γ between WT and IL-17A−/− mice at any time point. Compared with those in control mice, the concentrations of IL-17A in PDE mice were sequentially increased at WK1, WK2, and WK3. The concentrations of IL-17F were significantly higher in IL-17A−/− mice than in WT mice (Supplemental Fig. 2). However, the administration of anti–IL-17F Abs did not significantly suppress the expression of mRNA encoding CCL2, CCL4, or CCL5 (Supplemental Fig. 1).The concentrations of other cytokines and chemokines are shown in Supplemental Fig. 2.

FIGURE 4.
FIGURE 4.

The concentrations of CCL2, CCL4, CCL5, IL-17A, IFN-γ, CXCL9, and CXCL10 in the BALF of WT and IL-17A−/− mice given intranasal PDE or saline. The data are presented as the means ± SEM for each group (n = 4). Black bar, PDE WT mice; striped bar, PDE IL-17A−/− mice; gray bar, control WT mice. The data are representative of two experiments. *p < 0.05.

γδ T cells are a major source of IL-17A in PDE mice

To determine the major sources of IL-17A in PDE mice, lung cells isolated 2 h after the last PDE administration were evaluated by intracellular cytokine staining and flow cytometric analysis (Fig. 5A). The percentage of IL-17A+ γδ T cells was significantly higher than that of IL-17A+ CD4+ cells at any time point (Fig. 5B). Although there were fewer total γδ T cells than CD4+ cells, the absolute number of IL-17A+ γδ T cells was also significantly higher than that of IL-17A+ CD4+ T cells at day 1 and day 8, and the number of IL-17+ γδ T cells peaked on day 8 (Fig. 5C, 5D). Although there were only one or two samples, almost no IL-17A+ neutrophils were detected. In saline-administered mice (control mice), there were almost no IL-17A+ CD4+ cells or neutrophils (data not shown). Next, we evaluated cell numbers of IL-17A–secreting γδ T cells for each TCR phenotype over time in PDE and control mice. Fig. 5F shows that IL-17A+Vγ4+ and IL-17A+Vγ1−Vγ4− cells increased from day 1 to day 8 and then decreased thereafter. In contrast, there were almost no IL-17A+Vγ1+ cells over the time. In control mice, IL-17A+ γδ T cells with any TCR phenotype were not detected at any time point. Furthermore, the absolute numbers of CD44+ γδ T cells and the ratio of CD44+ to CD27+ γδ T cells peaked at day 8 and decreased thereafter in PDE mice (Fig. 5H). In control mice, an apparent increase of these cells was not observed over time. Additionally, immunofluorescence staining revealed that TCRγδ+IL-17A+ cells were well observed in areas where inflammatory cells infiltrated at WK1, but they were sparse in granulomatous areas at WK3 (Fig. 5I).

FIGURE 5.
FIGURE 5.

Intracellular IL-17A staining and flow cytometric analysis of lung cells collected from WT mice 2 h after intranasal PDE administration. (A) Representative gating strategy of IL-17A+ γδ T cells, CD4+ cells, and neutrophils. (B) The percentage of IL-17A+ cells in each cell population. (C) The total cell counts of γδ T cells and CD4+ cells. (D) IL-17A+ cell counts in each cell population. The data are presented as the means ± SEM and as dot plots for each group (n = 3–5 for γδ T cells and CD4+ cells, n = 1–2 for neutrophils). Except for neutrophils, the data are representative of two to three experiments. *p < 0.05. (E) Representative gating strategies of Vγ1+, Vγ4+, Vγ1−Vγ4− cells on CD3+TCRγδ+ cells at day 8. (F) The numbers of total cells and IL-17A+ cells in Vγ1+, Vγ4+, Vγ1−Vγ4− cells from day 1 to day 22 in the lungs of PDE or control mice. The data are presented as the mean ± SEM (n = 3) *p < 0.05. (G) Representative gating strategies of CD27+ or CD44+ cells on CD3+TCRγδ+ cells at day 8. (H) The numbers of CD27+ or CD44+ γδ T cells and the ratio of CD27+/CD44+ from day 1 to day 22 in PDE or control mice. The data are presented as the mean ± SEM (n = 3). *p < 0.05. (I) Immunofluorescence staining of TCRγδ (light blue), IL-17A (red), and DAPI (blue) in the lungs at WK1 and WK3. These are representative of four to five granulomatous or cell-infiltrated areas from three mice.

γδ T cells in mice sensitized with PDE exhibit enhanced proliferation and IL-17A production in response to PDE rechallenge

To evaluate the memory response of IL-17A–secreting γδ T cells, we prepared naive and convalescent mice. There were no differences between naive and convalescent mice in histological findings or BALF profiles (data not shown). Intracellular staining and flow cytometric analysis 2 h after intranasal PDE administration revealed that the percentages of IL-17A+ cells in both the γδ T and CD4+ cell populations of the convalescent mice were significantly greater than those of naive mice, and the absolute numbers of IL-17A+ γδ cells were also higher than those of naive mice (Fig. 6A–C).

FIGURE 6.
FIGURE 6.

Comparison of IL-17A production by γδ T cells in naive and convalescent mice. Naive mice had not been previously exposed to PDE, and convalescent mice were challenged with PDE until day 8 and harvested at day 22, as shown in Fig. 1. (A) Representative results of intracellular IL-17A staining and flow cytometric analysis of lung cells purified from naive or convalescent mice 2 h after intranasal PDE administration. (B and C) The percentages and absolute numbers of IL-17A+ cells in γδ T cells or CD4+ cells. (D) The percentages and absolute numbers of IL-17A+ cells of Vγ1+, Vγ4+, or Vγ1−Vγ4− cells in naive and convalescent mice (conv). (E) Absolute numbers of CD27+ or CD44+ cells in γδ T cells in naive and convalescent mice (conv). (F) Concentrations of IL-17A in supernatants after 60 h coculture of various combinations of γδ T cells (1.5 × 104 per well), macrophages (MΦ), and PDE. (G) Concentrations of IL-17A in supernatants after 3 or 60 h of coculture of AMs with Vγ1+, Vγ4+, or Vγ1−Vγ4− γδ T cells and PDE. The data are presented as the mean ± SEM for each group (n = 3). These data are representative of two experiments, except for those in (F). *p < 0.05.

To examine IL-17A production and Ag-elicited responses based on each γ-chain and surface phenotype, we also evaluated the percentages and absolute numbers of IL-17A–secreting Vγ1+, Vγ4+, or Vγ1−Vγ4− γδ T cells and CD27+ or CD44+ γδ T cells in the same experiment (Fig. 6D, 6E). The percentage and absolute numbers of IL-17A+ Vγ4+ and Vγ1−Vγ4− γδ T cells in convalescent mice were significantly higher than in naive mice. In addition, the absolute numbers of CD44+ γδ T cells and the ratio of CD44+ γδ T cells/CD27+ γδ T cells were significantly higher in convalescent mice compared with naive mice.

Additionally, we established in vitro cocultures of Vγ1+, Vγ4+, or Vγ1−Vγ4− γδ T cells and AMs with PDE. Fig. 6F shows that, if any element among γδ T cells, macrophages, or PDE was absent, the concentrations in the wells after 60 h of incubation were below the limit of detection of the ELISA Kit. Vγ1+ and Vγ4+ γδ T cells, but not Vγ1−Vγ4− γδ T cells, purified from convalescent mice produced significantly higher amounts of IL-17A compared with cells purified from naive mice after incubation with PDE for 3 and 60 h (Fig. 6G).

Discussion

The purpose of this study was to elucidate how IL-17A contributes to granuloma formation in HP and to verify our hypothesis that the memory response of IL-17A–secreting γδ T cells may play a key role in sensitization and development of HP via increased production of IL-17A in Ag-sensitized mice. Indeed, we found that the percentage of PDE-induced granulomas was reduced in IL-17A−/− mice and that the aggregation of CD11c+CD11b+F4/80+ cells, which were mainly considered CD11b+ AM, was suppressed compared with that in WT mice (Fig. 3E). Furthermore, the density of neutrophils, CD8+ lymphocytes, AMs, IMs, and monocytes in BALF was significantly lower in IL-17A−/− mice than in WT mice (Fig. 2). In particular, the difference in CD11b+ AM among these cells was remarkable. Together with the immunofluorescence results, the component of granulomas most strongly associated with IL-17A was CD11b+ AMs. Additionally, the concentrations of chemokines, including CCL2, CCL4, and CCL5, which mainly bind to CCR2 or CCR5 and act as chemoattractants for lymphocytes, monocytes, and macrophages (27), were significantly lower in the BALF of IL-17A−/− mice than WT mice (Fig. 4). Our results are consistent with those of previous papers. Joshi et al. (8) reported fewer lung CD11c+ cells in S. rectivirgula–challenged IL-17A−/− mice than in WT mice. CCL2 levels were also increased in the BALF of human summer-type HP (28) and S. rectivirgula–induced murine HP models (29). Similarly, Nance et al. (30) reported that Ccl4 and Ccl5 mRNA levels were increased in an S. lic>rectivirgula–induced HP model and were not independent of IFN-γ. Recently, IL-17A was shown to induce the production of these chemokines (31, 32). In addition, because CD11b expression was highlighted as a phenotype of AM, which expand in acute and chronic inflammation of the lung (33, 34), these cells are thought to be involved in PDE-induced inflammation and granuloma formation in this model. Taken together, these data indicate that the IL-17A/CCL2, CCL4, and CCL5/CD11b+ AM axis may play an important role in the development of HP, especially in the process of granuloma formation. Although the concentrations of IL-17F were elevated in the BALF, neutralization of IL-17F did not have a significant effect on granuloma formation or expression of mRNA encoding CCL2, CCL4, or CCL5 (Supplemental Fig. 1A–C). Therefore, IL-17F is not thought to be more dominant than IL-17A in the axis proposed in this study.

Next, we demonstrated that γδ T cells are a major source of IL-17A in this model (Fig. 5). Although neutrophils were reported to be the main source of IL-17A in the S. lic>rectivirgula–induced HP model (11, 12), almost no IL-17A+ neutrophils were detected in our flow cytometric analysis (Fig. 5A). Thus, different primary cellular sources of IL-17A contribute to HP development, at least the early phase. Although Th17 cells were the first identified source of IL-17A (35), IL-17A is also produced by innate immune cells, including γδ T cells, ILC3s, and NKT cells (36), in the early stage of the inflammatory immune response. In our study, intracellular cytokine staining showed that the absolute number of IL-17A+ γδ T cells was significantly higher than that of CD4+ cells at day 1 and day 8 (Fig. 5). The number of IL-17A+ Vγ4+ or Vγ1−Vγ4− cells peaked at day 8 and then decreased at day 15 and day 22, despite the gradual increase in IL-17A concentration in the BALF at WK2 and WK3. We hypothesize that, when the concentration of IL-17A reaches a certain level, some homeostasis systems downregulate the expansion of IL-17A–producing γδ T cells in the lungs. This hypothesis is supported by a previous report of an experimental murine model of obliterative bronchiolitis in which injection of an anti–IL-17A Ab increased the population of IL-17A–secreting γδ T cells (37). These results indicate that γδ T cells, particularly Vγ4+ or Vγ1−Vγ4− cells, are a major source of IL-17A, at least during the early phase of HP. The administration of anti–IL-17A Ab at only day 1 or day 8 significantly reduced the granuloma area (Supplemental Fig. 1), suggesting that the role of IL-17A–producing γδ T cells is particularly important at the early development phase and that IL-17A may be a therapeutic target of HP. These data are compatible with the result that IL-17A–secreting γδ T cells peaked at day 8 and subsequently decreased (Fig. 5).

A significantly higher percentage of IL-17A+ Vγ4− or Vγ1−Vγ4− γδ T cells was detected 2 h after PDE challenge in convalescent mice than in naive mice (Fig. 6), indicating that the proliferation of IL-17A–secreting γδ T cells is increased in response to the sensitizing Ag. Additionally, in vitro coculture of Vγ1+, Vγ4+, or Vγ1−Vγ4− γδ T cells with AMs and PDE revealed that Vγ1+ and Vγ4+ γδ T cells purified from convalescent mice produced markedly greater amounts of IL-17A compared with those purified from naive mice. Although the difference was not statistically significant, a similar trend was observed in Vγ1−Vγ4− cells. In addition, if any element among γδ T cells, macrophages, or PDE was absent, IL-17A production was not detected (Fig. 6F), suggesting that direct stimulation with PDE through non-TCR mechanisms or FcR-mediated crosslinking was minimal in the experiment.

Incidentally, the IL-17A production by Vγ1+ cells was incompatible with our intracellular staining. Although several previous reports have shown some production of IL-17A by Vγ1+ cells using intracellular cytokine staining (21) or in vitro incubation (22), the fact that Vγ1+ cells are minor producers of IL-17A is widely accepted (18). Considering our in vivo intracellular cytokine staining and previous findings, we concluded that Vγ4+ and Vγ1−Vγ4− cells are involved in the memory response in this model.

In the current study, CD44+CD27− γδ T cells increased from day 1 to day 8 and were found more abundantly in convalescent mice than in naive mice (Figs. 5H, 6E). These cells are known as an IL-17A–secreting phenotype (18) and have been described as an effector memory phenotype in several previous reports (19, 21, 22). Thus, in this model, CD44+CD27− γδ T cells are thought to act as effector memory cells and play important roles in the early development phase of HP.

Until recently, IL-17A production by γδ T cells was recognized as an innate response and thought to contribute mainly to immunity against bacterial or fungal infection at mucosal sites, such as the intestine, lung, skin, or tongue (38–40). Recent studies, however, have demonstrated that IL-17A–secreting γδ T cells also contribute to the memory response associated with adaptive immunity in different tissues (41). Several mouse infection studies, including enteric infection by Listeria monocytogenes (19, 20), peritonitis caused by S. aureus (21), and pulmonary infection by B. pertussis (22), have been reported. In the S. aureus–induced peritonitis model, prior infection with S. aureus augmented the recruitment of Vγ1+ or V4γ+ γδ T cells, an increased proportion of which produced IL-17A during subsequent infections (21). Additionally, in the model of B. pertussis pulmonary infection, Vγ4+ γδ T cells played an important role in the memory response by producing increased amounts of IL-17A during the second infection (22). Although memory γδ T cells are poorly documented in noninfectious diseases, an imiquimod-sensitized mouse model of psoriasis exhibited increased proliferation and enhanced IL-17A production by Vγ4+ γδ T cells upon imiquimod rechallenge, resulting in more severe skin inflammation (42). Therefore, our data support the hypothesis that IL-17A–secreting γδ T cells with PDE-elicited memory functions play crucial roles in the sensitization and development of HP by producing increased IL-17A in PDE-sensitized mice. These memory responses are similar to classically defined memory cells, such as T cells with TCR αβ and B cells, in that these cells expand rapidly and produce greater amounts of cytokines or Igs in response to sensitizing Ags. However, although Sheridan et al. (19) reported that the memory response of γδ T cells is dependent on mucosal APCs and TCR δ, the detailed mechanisms by which memory γδ T cells are generated from naive cells and recognized Ags are not yet elucidated. It is also possible that the memory response was induced by mechanisms other than the recognition of Ags by TCR of γδ T cells in the current study. Further investigations are needed to elucidate the mechanism of memory responses of γδ T cells in HP.

The memory function of IL-17A–secreting γδ T cells and the IL-17A/CCL2, CCL4, CCL5/CD11b+ AM axis may be a novel mechanism of immunopathogenesis in HP. The main immunopathogenesis of HP has long been recognized as type III or IV hypersensitivity (6). Positivity for circulating Ag-specific Abs and lymphocyte proliferation tests, which mainly reflect the Th1 response, is indicative of past exposure and partially supports the clinical diagnosis of HP (1, 43, 44). However, the sensitivity and specificity of these clinical tests are not sufficient (43). Interestingly, our data showed no significant differences in PDE-specific serum IgG levels (Fig. 3G) or Th1 cytokine and chemokine levels in BALF (Fig. 4) between IL-17A−/− mice and WT mice, indicating that IL-17A may not directly affect type III or type IV reactions. Therefore, our findings indicate that the detection of Ag-elicited γδ T cells may indicate the sensitized state and could contribute to the clinical diagnosis of HP. Further investigations of memory γδ T cells, especially in patients with HP, are required to develop new diagnostic methods for HP.

We examined the production of IL-17A by γδ T cells and CD4+ T cells in the lungs. Although other innate-like lymphocytes, such as NKT cells or ILC3s, have been reported to produce IL-17A (14), we focused on γδ T cells because γδ T cells have been investigated most frequently, and their memory function is better understood than that of the other innate-like lymphocytes. However, it is necessary to elucidate the involvement of other innate-like lymphocytes in the lung during the development of HP.

In conclusion, we demonstrated that IL-17A–secreting memory γδ T cells play crucial roles in the sensitization and development of HP. Furthermore, the IL-17A/CCL2, CCL4, and CCL5/CD11b+ AM axis underlies granuloma formation in HP. To the best of our knowledge, this is the first report on the memory response of IL-17A–secreting γδ T cells in an experimental model of HP. These findings may improve the understanding of HP immunopathogenesis and may lead to new clinical examinations or therapeutic targets for HP.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank all members of the Department of Respiratory Medicine of Tokyo Medical and Dental University and all staff of the Center for Experimental Animals. We also thank Prof. Y. Iwakura for providing the IL-17A−/− mice.

Footnotes

  • This work was supported by the Japan Society for the Promotion of Science (KAKENHI Grant Number 16K09576).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    AM
    alveolar macrophage
    BALF
    bronchoalveolar lavage fluid
    HP
    hypersensitivity pneumonitis
    IM
    interstitial macrophage
    PDE
    pigeon dropping extract
    WK1
    week 1
    WK2
    week 2
    WK3
    week 3
    WT
    wild-type.

  • Received February 26, 2020.
  • Accepted November 9, 2020.

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