Abstract
IL-17 plays a critical role in the immunological control of various infectious diseases; its function has been investigated in the removal of both extracellular and intracellular bacteria. Our group previously revealed the importance of IL-17 in neutrophil migration following Legionella infection by using IL-17AF knockout mice; however, aside from neutrophil infiltration, alternative causes for the reduced survival of these mice have not been characterized. In this study, we found that γδ T cells in IL-17AF knockout mice were markedly increased and produced the cytotoxic substances granzyme B and perforin. Moreover, the elimination of γδ T cells from these mice, via an anti-TCRδ Ab, caused a substantial reduction in the level of lactate dehydrogenase in bronchoalveolar lavage fluid, indicating that γδ T cells contribute to lung tissue damage. Moreover, although cells lysed by cytotoxic substances are typically eliminated by phagocytic cells, in IL-17AF knockout mice, lung homeostasis was not maintained because of a decrease in phagocytic cells that impaired the clearance of dead cells. Our results indicate that increased γδ T cells in IL-17AF knockout mice help eliminate Legionella by releasing cytotoxic substances and lysing infected cells; however, this results in tissue damage due to insufficient removal of dead cells by phagocytic cells. This study enhances our understanding of the protective response against Legionella and provides insights into γδ T cell–mediated protective immunity against various infectious diseases.
Introduction
γδ T cells, a unique and conserved population of lymphocytes, have attracted considerable attention because of their intrinsic contribution to several types of immune responses and immunopathology (1). γδ T cells express a TCR that is distinct from the TCR expressed by αβ T cells, and despite being T cells, γδ T cells serve as innate immune cells that target invading pathogens at the site of infection (2). Moreover, γδ T cells express various receptors and recognize nonself-antigens as well as stress-induced self-antigens. Activated γδ T cells also have the capacity to produce chemokines and cytokines (3, 4) and function as APCs (5–7).
Studies conducted using γδ TCR Ab or TCR δ-chain gene knockout (KO) mice revealed that resistance in mice was attenuated during Mycobacterium tuberculosis and Listeria monocytogenes infection; meanwhile, histologically, granuloma formation was not observed at the lesion site, but abscesses containing a large accumulation of neutrophils were detected (8, 9). In a Candida albicans systemic infection model, γδ T cells were activated in the lung on the first day of infection by IL-23 produced by TLR2-dependent macrophages and dendritic cells. Additionally, secreted IL-17 triggered neutrophil aggregation, thereby contributing to the first line of defense against infection (10). Furthermore, γδ T cells have been reported to express CD40L in mice infected with the malaria parasite and promote dendritic cell activation through CD40L/CD40 signaling (11).
In diverse mouse infection models, an increase in the number of bacteria in various organs has been found to be directly correlated with pathological deterioration, although excessive inflammation and immune responses are also recognized as inducers of tissue damage (12). Specifically, fragile tissues, including those of the lung, can be protected from microbial attack at the expense of chronic inflammatory damage. In mouse and human studies examining the protective effects against and virulence mechanisms of several fungi, including Aspergillus fumigatus, Cryptococcus neoformans, and Pneumocystis jiroveci, lung injury resulting from the inhalation of fungal spores was reported to be caused not by the fungal infection itself but rather by a pathology caused by an allergic pathway involving Th2, Th1, and Th17 cells (13).
Our group previously reported that IL-17A/IL-17F double-KO (IL-17AF KO) mice show increased susceptibility to Legionella pneumophila infection as compared with wild-type (WT) mice due to decreased production of inflammatory cytokines and chemokines and a subsequent reduction in neutrophil accumulation in the lungs (14). However, we did not previously characterize the involvement of innate immune cells, other than neutrophils, in the markedly reduced survival of IL-17AF KO mice during the early stages of infection, despite no differences in lung bacterial counts between the KO and WT mice.
In the current study, we therefore investigated whether the lower survival rate of IL-17AF KO mice during L. pneumophila infection is due to increased severity of lung tissue damage. We found that in IL-17AF KO mice the number of γδ T cells was congenitally and systemically increased and became further elevated after L. pneumophila infection. Moreover, these γδ T cells produced granzyme B and perforin rather than the normally produced IL-17A, thereby causing lysis of target cells. Our results indicate that γδ T cells contribute to bacterial elimination during L. pneumophila infection; however, they also cause tissue damage that adversely affects the host.
Materials and Methods
Animals
Specific pathogen–free female BALB/c mice were purchased from Charles River Laboratories (Kanagawa, Japan). IL-17A KO mice, IL-17F KO mice, and IL-17AF KO mice on BALB/c genetic background were previously established at the Institute of Medical Science, University of Tokyo (15, 16). All mice were housed under specific pathogen–free conditions in the animal care facility at Toho University School of Medicine (Tokyo, Japan) until the day of sacrifice. Animal and pathogen protocols were approved by the Institutional Care and Use Committee (approval numbers: 19-52-386, 19-52-101).
L. pneumophila inoculation and determination of bacterial counts
Clinical isolates of L. pneumophila Suzuki (serogroup 1) strain stocked at Toho University Hospital (17) were used for the pneumonia model, as previously reported (18). Mice were administered L. pneumophila as described previously (19). Briefly, mice were anesthetized through intramuscular injection of ketamine and xylazine at 50 and 10 mg/kg body weight, respectively; the trachea was exposed, and 30 μl of bacterial suspension was administered through a sterile 26-gauge needle. The skin incisions were then closed using surgical staples. To determine bacterial numbers, lungs were removed at various time points and homogenized (IKA Japan K.K., Osaka, Japan) in 1 ml of saline. Next, 10-μl aliquots of the homogenates were inoculated after 1:10 serial dilution in saline onto buffered charcoal yeast extract agar supplemented with α-ketoglutaric acid; the agar plates were incubated at 35°C for 3–4 d, and the bacterial colonies were counted visually.
Isolation of lung cells and flow cytometric analysis
Excised lung tissue separated from all associated lymph nodes was minced and incubated at 37°C in 5% CO2 for 50 min in RPMI 1640 containing 2% FBS, 0.5 mg/ml collagenase D (Roche Diagnostics GmbH, Mannheim, Germany), and 150 μg/ml DNase (Roche Diagnostics). The samples were then passed through a 70-μm cell strainer to prepare single-cell suspensions. Cell suspensions mixed with staining buffer (PBS containing 2% BSA and 2 mM EDTA) were incubated with an Fc-receptor–blocking Ab (anti-mouse CD16/32, clone 93) for 15 min on ice to reduce nonspecific Ab binding. Subsequently, the cells were washed with staining buffer and surface-stained for 30 min on ice with experimentally designed combinations of the following Abs: PerCP/Cy5.5 anti-mouse CD11b (clone M1/70), FITC anti-mouse Ly-6G (clone 1A8), PE/Cy7 anti-mouse F4/80 (clone BM8), allophycocyanin/Cy7 anti-mouse CD11c (clone N418), allophycocyanin anti–MHC class II (clone M5/114.15.2), PE/Cy7 anti-mouse B220 (clone RA3-6B2), FITC anti-mouse CD3e (clone 145-2C11), allophycocyanin/Cy7 anti-mouse CD3e (clone 145-2C11), PerCP anti-mouse CD4 (clone RM4-5), PE anti-mouse CD8a (clone 53-6.7), allophycocyanin/Cy7 anti-mouse CD49b (clone DX5), PE/Cy7 anti-mouse CD39 (clone Duha59), allophycocyanin anti-mouse CD25 (clone PC61.5), FITC anti-mouse TCRδ (clone GL3), PerCP/Cy5.5 anti-mouse TCRδ (clone GL3), allophycocyanin anti-mouse TCR Vγ1.1 (clone 2.11), and PE anti-mouse TCR Vγ2 (clone UC3-10A6). All Abs were purchased from BioLegend (San Diego, CA). Cells were washed, fixed with 4% paraformaldehyde, and detected and analyzed using a FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ) and FlowJo software (Tree Star, Ashland, OR).
Intracellular cytokine and granzyme B/perforin staining
Intracytoplasmic cytokine staining was performed using a Cytofix/Cytoperm Plus Kit according to the manufacturer’s protocol (BD Biosciences). For assessment of intracellular cytokine expression, PMA (25 ng/ml), ionomycin (1 μg/ml), and GolgiPlug (1 μg/ml) were added to cells at 37°C for 4 h. Cells were stained for cell surface molecules, fixed for 20 min on ice, washed, and then stained (for 30 min) for intracytoplasmic IFN-γ by using PE anti-mouse IFN-γ (clone XMG 1.2), allophycocyanin anti-mouse IL-17A (clone TC11-18H10.1), or IgG isotype control diluted in Perm/Wash solution. Intracytoplasmic staining for granzyme B and perforin was performed using the aforementioned kit; cells were stained with FITC anti-mouse granzyme B (clone GB11), PE anti-mouse perforin (clone S16009A), or isotype control IgG and detected using a FACSCanto II flow cytometer.
Purification of γδ T cells
γδ T cells from IL-17AF KO mouse spleen were defined as CD3+ TCRδ+ cells and were sorted using a FACSAria III (BD Biosciences). The sorted γδ T cells were determined to be >95% pure.
γδ T cell–killing assay
Mouse alveolar macrophage (AM) cell line MH-S cells (American Type Culture Collection, Manassas, VA) were seeded in 96-well round-bottom plates at 2.5 × 104 cells per well and rested overnight in complete RPMI 1640 (10% FBS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.5 mM 2-ME) at 37°C in 5% CO2. Cells were infected at a multiplicity of infection of 1 and incubated for 1 h after attachment through centrifugation. At the end of the infection period, nonphagocytosed and nonadherent bacteria were removed by washing three times with fresh medium, and after further incubation for 6 h, the infected cells were cocultured for 18 h with 1.3 × 105 cells per well γδ T cells from IL-17AF KO mice. Subsequently, culture supernatants were assayed for lactate dehydrogenase (LDH) levels by using a CyQUANT LDH Cytotoxicity Assay Kit (Invitrogen, Carlsbad, CA).
γδ T cell depletion in vivo
For γδ T cell depletion, GL3 mAb (a hamster anti-mouse IgG) was used with an isotype mAb included as a control. One day before bacterial infection, 100 μg of GL3 or control IgG was administered i.p.
Collection of bronchoalveolar lavage fluid
Bronchoalveolar lavage was performed as previously described (20). Briefly, the trachea was exposed, and a polyethylene catheter with a 1.7-mm outer diameter was inserted. Bronchoalveolar lavage fluid (BALF) was then collected three times (1.0 ml each). BALF was centrifuged to remove cells and debris, and the supernatant was used for LDH determination.
Histopathological analysis of lungs infected with L. pneumophila
Lungs were removed at 48 h postinfection and fixed in buffered 4% paraformaldehyde for histopathological examination. After embedding in paraffin wax, 6-μm-thick tissue sections were cut perpendicular to the anterior–posterior axis, and the sections were placed singly on polylysine-treated slides and stained with H&E, as previously reported (21). To detect apoptotic cells in the lung, staining was performed using a Colorimetric TUNEL System Kit according to the manufacturer’s protocol (Promega, Fitchburg, WI). The number of TUNEL-positive cells was counted under ×400 magnification with ImageJ Fiji software (National Institutes of Health, Bethesda, MD).
RNA isolation and gene expression analysis
Total RNA was isolated from mouse lungs using TRIzol reagent (Invitrogen) as per manufacturer instructions. For quantitative PCR analysis, 1 μg of total RNA was reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The SYBR Green real-time PCR technique was used, and analyses were performed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems). The following PCR primers were used: Gzmb (granzyme B), 5′-CATGTAGGGTCGAGAGTGGG-3′ (forward) and 5′-CCTCCTGCTACTGCTGACCT-3′ (reverse); Prf1 (perforin), 5′-TGGAGGTTTTTGTACCAGGC-3′ (forward) and 5′-TAGCCAATTTTGCAGCTGAG-3′ (reverse); Actb (β-actin), 5′-AGAGGGAAATCGTGCGTGAC-3′ (forward) and 5′-CAATAGTGATGACCTGGCCGT-3′ (reverse). Relative fold changes in transcript levels were calculated using the 2−ΔΔCT method (22), with the housekeeping gene β-actin serving as a reference standard for the loading amount and cDNA quality.
Statistical analysis
All results are expressed as means ± SE of mean. Statistical analyses were performed using GraphPad Prism 6 software (GraphPad, La Jolla, CA). A Student t test was used for comparisons between two groups, and one-way ANOVA followed by a Tukey multiple-comparison test was used for comparisons between more than two groups. Survival curves were constructed using the Kaplan–Meier method and analyzed using the log-rank (Mantel–Cox) test. Differences were considered significant at p < 0.05.
Results
Defects in both IL-17A and -17F attenuate protection against L. pneumophila infection
Our group previously reported that IL-17AF KO mice are more susceptible than WT mice to L. pneumophila infection (14). However, whether IL-17A or IL-17F is more important for defense against infection has remained unclear. To determine whether both IL-17A and IL-17F are required for L. pneumophila clearance in the lung, we inoculated IL-17A KO, IL-17F KO, IL-17AF KO, and WT mice with L. pneumophila. Following inoculation with a sublethal dose (1 × 106 CFU), the bacteria were gradually cleared from the lung in WT mice and also in all KO mice; however, at 7 d postinfection, bacterial numbers in the lung were higher in the IL-17AF KO mice than in the other groups (Fig. 1A). Upon lethal dose (1 × 107 CFU), bacterial numbers in the lungs of IL-17AF KO mice did not increase significantly compared with other groups, as in the sublethal dose (Fig. 1A). Next, mice were inoculated at a lethal dose and survival was assessed. The mice began to die on the second day of infection, and all IL-17AF KO mice had succumbed to infection by the fifth day; by contrast, 75% of WT mice survived, and 35% of IL-17A and IL-17F single-KO mice survived (Fig. 1B). These results indicate that both IL-17A and IL-17F are critical for protection against L. pneumophila infection and play complementary roles. Notably, in IL-17AF KO mice, mortality increased considerably before the time at which lung bacterial counts differed from those in WT mice, indicating that the elevation of bacterial counts in the lung was not the only cause of death in L. pneumophila–infected mice.
Pulmonary bacterial counts and survival after L. pneumophila infection in WT and various KO mice.
(A) Mice were infected intratracheally with 1.5 × 106 CFU (sublethal dose) or 2 × 107 CFU (lethal dose) of L. pneumophila. Bacterial numbers in the lungs were determined on days 2 and 7 postinfection. Bars represent means ± SE of mean (n = 4 mice per group). *p < 0.05 (compared with WT mice group) by Student t test, **p < 0.01 (compared with IL-17F KO mice group) by Student t test. (B) Kaplan–Meier survival curves (WT, n = 7 mice per group; KO, n = 6 mice per group). Mice were infected intratracheally with 2 × 107 CFU of L. pneumophila. Results were confirmed using two independent experiments.
In the acute phase of L. pneumophila infection, accumulation of innate immune cells is attenuated in IL-17AF KO mice, whereas T cells are increased
To examine the various lung cell populations at an early stage of infection, cells were isolated from the mouse lung on the second day of infection. The total number of lung cells at 2 d postinfection was the same in WT and KO mice (Fig. 2A). The gated leukocyte populations of isolated lung cells were analyzed using flow cytometry and defined as neutrophils, macrophages (divided into AMs and interstitial macrophages [IMs]), dendritic cells, B cells, and T cells (Fig. 2B). The number of cells in each population was calculated from the percentage of the total number of lung cells. Our quantified results (Fig. 2C) showed that, whereas the number of neutrophils was diminished in the lungs of IL-17AF KO mice, in agreement with previous reports (14), the total number of macrophages was similar in WT and IL-17AF KO mice, although separate AM/IM analysis revealed that the number of IM was decreased in IL-17AF KO mice. Moreover, among lymphocytes, the number of B cells did not differ between WT and KO mice, whereas the number of T cells was markedly increased in IL-17AF KO mice.
Flow cytometric analysis of lung cells from mice infected with L. pneumophila.
(A) Cells were collected from the left lung of mice on the second day of infection, and the absolute numbers of lung cells from each mouse group were determined. Bars represent means ± SEM (n = 4 mice per group). (B) Gating strategy for flow cytometric analysis. The leukocyte area was gated, and the cell population was defined as follows: neutrophils (CD11bhigh Ly-6Ghigh), macrophages (neutrophils ungated, F4/80+), AMs (macrophages gated, CD11b− CD11c+), IMs (macrophages gated, CD11b+ CD11clow), dendritic cells (neutrophils and macrophages ungated, CD11c+ MHC class II+), B cells (CD3− B220+), and T cells (CD3+ B220−). (C) Proportions of neutrophils, macrophages, dendritic cells, B cells, and T cells among leukocytes and of AMs and IMs among macrophages. Bars represent means ± SEM (n = 4 mice per group). Results were confirmed using three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
In the lungs of IL-17AF KO mice, large amounts of γδ T cells accumulate rather than αβ T cells postinfection
We next identified the T cell subsets that were increased in the lungs of IL-17AF KO mice by staining for T cell surface Ags (Fig. 3A). The number of helper T cells (CD3+ CD4+) and cytotoxic T cells (CD3+ CD8+) among lung cells from IL-17AF KO mice was increased as compared with WT mice. Furthermore, the number of γδ T cells (CD3+ TCRδ+) was increased ∼100-fold in IL-17AF KO mice (Fig. 3B).
γδ T cells are increased in L. pneumophila–infected IL-17AF KO mice.
(A) CD3+-gated lung cells on day 2 postinfection. Numbers in each panel represent proportions of CD4 T cells, CD8 T cells, and γδ T cells among CD3+ lymphocytes. (B) Proportions of CD4 T cells, CD8 T cells, and γδ T cells calculated from the data of the assay shown in (A). Bars represent means ± SEM (n = 4 mice per group). Results were confirmed using three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
The γδ T cells that accumulate in the lungs of IL-17AF KO mice are more abundant in the steady state than in WT mice and exhibit an IL-17 phenotype
To characterize the γδ T cells of IL-17AF KO mice, lungs were harvested and analyzed on days 2, 4, and 6 postinfection. The total number of lung cells did not differ between IL-17AF KO mice and WT mice up to day 4 postinfection, yet it was decreased in IL-17AF KO mice on day 6 (Fig. 4A). In accordance with the results of previous studies on BALF (14), the proportion of neutrophils was significantly decreased in IL-17AF KO mice beginning on day 2 postinfection (Fig. 4B). By contrast, the proportion of γδ T cells in the lungs of IL-17AF KO mice was higher than that in WT mice, even in uninfected mice, and the proportion was further increased postinfection (Fig. 4C). Similar to helper T cells that have been classified into different phenotypes with distinct functions, γδ T cells are classified into the CD27+ IFN-γ–producing type, CD25+ IL-17A–producing type (4, 23–25), and CD39+ immunosuppressive type (26). Therefore, we examined the phenotype and cytokine production of γδ T cells in the lungs of IL-17AF KO mice. First, analysis of the expression pattern of cell surface markers revealed that CD39 was not expressed in either uninfected or infected cells (Fig. 4D). However, CD25 was strongly expressed, with the percentage of CD25+ cells and the mean fluorescence intensity of staining for the marker found to be further increased on day 2 postinfection (Fig. 4D, 4E). Tissue-specific γδ T cell subsets show biased use of certain TCR V gene segments (2, 27), and the γδ T cells distributed in mouse lung are IL-17–producing Vγ4 or Vγ6 cells (28), according to the Heilig and Tonegawa nomenclature (29). Thus, we analyzed γδ T cells in the lungs of mice on day 2 postinfection by staining with Abs against Vγ4 and Vγ1 (control) (Fig. 4F). In accordance with the results of previous studies, we confirmed that the major population, CD3int TCRδhigh γδ T cells, was positive for Vγ4, whereas the minor population, CD3high TCRδint γδ T cells, was not (Fig. 4G). Next, analysis of cytokine production revealed that the γδ T cells of WT mice primarily produced IL-17A and not IFN-γ following L. pneumophila infection, whereas the γδ T cells of IL-17AF KO mice produced neither IFN-γ nor IL-17A (Fig. 4H, 4I). These results indicate that several CD25+ Vγ4+ γδ T cells (i.e., cells of the IL-17?–producing γδ T cell phenotype) accumulate in the lungs of IL-17AF KO mice after L. pneumophila infection, although as expected, IL-17A is not produced.
γδ T cells are activated during early stages of L. pneumophila infection in IL-17AF KO mice.
(A) Absolute numbers of total lung cells from WT and IL-17AF KO mice on days 0, 2, 4, and 6 postinfection. Bars represent means ± SEM (n = 4 mice per group). (B) Proportions of neutrophils in lungs from WT and IL-17AF KO mice on days 0, 2, 4, and 6 postinfection. Bars represent means ± SEM (n = 4 mice per group). (C) Proportions of γδ T cells in lungs from WT and IL-17AF KO mice on days 0, 2, 4, and 6 postinfection. Bars represent means ± SEM (n = 4 mice per group). (D) Proportion of CD25+ and CD39+ cells among γδ T cells from WT and IL-17AF KO mice on days 0, 2, and 4 postinfection. Numbers in each panel represent proportion of CD25+ cells among γδ T cells. (E) Proportion of CD25+ cells and mean fluorescence intensity (MFI) of allophycocyanin-conjugated mAbs against CD25 in γδ T cells from IL-17AF KO mice on days 0, 2, and 4 postinfection, calculated from the data of the assay shown in (D). Bars represent means ± SEM (n = 3 mice per group). (F) Dot plots showing CD3int TCRδhigh and CD3high TCRδint populations of γδ T cells in lungs from WT and IL-17AF KO mice on day 2 postinfection. (G) Expression profiles of TCR Vγ4 and TCR Vγ1 in γδ T cells in lungs from IL-17AF KO mice on day 2 postinfection. Dotted line represents data for unstained control. (H) Intracellular cytokine assay of γδ T cells in lungs from WT and IL-17AF KO mice on day 2 postinfection. (I) Proportions of γδ T cells producing IL-17A and IFN-γ, calculated from the data of the assay shown in (H). Bars represent means ± SEM (n = 3 mice per group). Results were confirmed using three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Following L. pneumophila infection, γδ T cells in the lung produce granzyme B and perforin
Detection of CD25+ Vγ4+ γδ T cells in the IL-17AF KO mouse lung raised the question of whether these γδ T cells perform effector functions other than cytokine production. Several mechanisms contribute to the stress surveillance of γδ T cells, one being the production of granzymes and perforin (30–33). Therefore, we hypothesized that γδ T cells release granzymes and perforin. We first confirmed this through quantitative RT-PCR analysis of gene expression, and we found that in the lung tissue of IL-17AF KO mice granzyme B and perforin mRNA levels were increased (Fig. 5A). Moreover, intracellular staining of collected lung cells revealed that, as expected, granzyme B and perforin were produced by γδ T cells (Fig. 5B, 5C). In contrast, there was no production from T cells, such as αβ T cells or NKT cells (data not shown). These results indicate that one function of the increased CD25+ Vγ4+ γδ T cells in the lungs of IL-17AF KO mice after L. pneumophila infection is the production of granzyme B and perforin.
γδ T cells enhance granzyme B and perforin production during early stages of L. pneumophila infection in IL-17AF KO mice.
(A) mRNA levels of granzyme B and perforin in lungs from WT and IL-17AF KO mice on days 0 and 2 postinfection. Bars represent means ± SEM (n = 3 mice per group). (B) Intracellular granzyme B and perforin staining of T cells in lungs from WT and IL-17AF KO mice on day 2 postinfection. (C) Proportions of γδ T cells producing granzyme B and perforin, calculated from the data of the assay shown in (B). Bars represent means ± SEM (n = 3 mice per group). Results were confirmed using three independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001.
γδ T cells recognize and lyse L. pneumophila–infected macrophages
γδ T cells can recognize (separately or synergistically) diverse stress-induced stimuli and maintain homeostasis by eliminating the cells that the host no longer requires at an early stage of infection. To examine whether γδ T cells recognize and directly lyse L. pneumophila–infected cells, γδ T cells sorted from the spleen of naive IL-17AF KO mice were cocultured with infected MH-S cells, after which the LDH level in culture supernatants was measured (Fig. 6A). In the case of infected MH-S cells, LDH levels in the supernatants were increased, as some of the cells were disrupted because of intracellular proliferation of L. pneumophila, and the LDH levels were further enhanced following the addition of γδ T cells. Conversely, bacterial counts were lower in the case of infected MH-S cells cocultured with γδ T cells compared with infected MH-S cells cultured alone (Fig. 6B). These results indicate that γδ T cells specifically recognize and lyse infected cells, thereby limiting the spread of L. pneumophila infection.
γδ T cells recognize and lyse L. pneumophila–infected macrophages.
(A) γδ T cells sorted from spleens of naive IL-17AF KO mice were cocultured for 18 h with L. pneumophila–infected or uninfected MH-S cells. LDH levels in culture supernatants were measured. Bars represent means ± SEM in triplicates. (B) Numbers of bacteria in infected MH-S cells. Bars represent means ± SEM in triplicates. Results were confirmed using two independent experiments. *p < 0.05, **p < 0.01.
Histopathological characteristics and accumulation of dead cells in IL-17AF KO mice after L. pneumophila infection
Dead cells generated through the action of granzyme B or perforin must be properly processed by phagocytes, such as macrophages; however, fewer phagocytic cells are present in the lungs of IL-17AF KO mice compared with WT mice after L. pneumophila infection. We hypothesized that this could cause an excessive inflammatory response. To assess the degree of tissue inflammation, we performed H&E staining for histopathological analysis of the mouse lung on day 2 of L. pneumophila infection (Fig. 7A). Postinfection, large numbers of infiltrating inflammatory cells were detected in the lungs of both WT and IL-17AF KO mice, and RBCs were found to have leaked into the alveolar cavity, indicating inflammation with hemorrhage. However, the lungs of IL-17AF KO mice clearly showed a stronger inflammatory response and marked alveolar wall thickening relative to the WT mouse lung. Notably, this enhanced inflammatory response was nearly eliminated after administration of an anti-TCRδ Ab (∼100% reduction; Fig. 7B). Next, we performed TUNEL assays to assess the frequency of dead cells (Fig. 7C, 7D) and found that larger numbers of dead cells had accumulated in the lungs of IL-17AF KO mice, which were removed following anti-TCRδ Ab administration. Furthermore, we collected BALF from mice on day 2 of L. pneumophila infection and measured LDH levels and found that LDH was significantly increased postinfection in both WT and IL-17AF KO mice; however, the levels were higher in IL-17AF KO mice (Fig. 7E). Moreover, when the anti-TCRδ Ab was administered, no change was detected in WT mice; however, the LDH level was significantly lowered in the IL-17AF KO mice. These results indicate that γδ T cells are involved in the severe tissue damage that occurs in IL-17AF KO mice following L. pneumophila infection.
Histopathological characteristics and accumulation of dead cells in IL-17AF KO mice after L. pneumophila infection.
(A) Histopathology of lung tissues assessed using H&E staining. Upper panel, Low-power field (original magnification ×100); lower panel, high-power field (original magnification ×400). Scale bars, 200 and 50 μm in low- and high-power fields, respectively. (B) One day before bacterial infection, 100 μg of GL3 or control IgG was administered i.p., and proportions of γδ T cells in lungs from IL-17AF KO mice were determined on day 2 postinfection. (C) TUNEL-based detection of dead cells in lung tissue. The arrow in each right upper inset indicates goblet cells. Scale bar, 50 μm. (D) Quantification of TUNEL-positive cells in lungs. The number of TUNEL-positive cells was counted under original magnification ×400 with ImageJ Fiji software. Cell counting was performed in at least six randomly selected separate areas for each mouse. Bars represent means ± SEM (n = 3 mice per group). (E) BALF from mice on day 2 of L. pneumophila infection and measured LDH levels. Bars represent means ± SEM (n = 3–4 mice per group). Results were confirmed using two independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001.
Depleting γδ T cells from IL-17AF KO mouse lungs increases bacterial counts in the lung and enhances survival
Last, we compared the number of bacteria in the lung and the survival rate postinfection in mice treated with the anti-TCRδ Ab. No change was observed in WT mice; however, in IL-17AF KO mice, bacterial counts in the lung were significantly higher in the anti-TCRδ Ab-treated group compared with the control group (Fig. 8A). Comparison of survival rates revealed that in the case of WT mice survival was almost unchanged in the anti-TCRδ Ab-treated group relative to the control group. In IL-17AF KO mice, administration of anti-TCRδ Ab had a modest although insignificant effect on survival (Fig. 8B). These results indicate that in IL-17AF KO mice γδ T cells contribute to the clearance of L. pneumophila and elicit a detrimental effect that could affect survival.
Depleting γδ T cells from IL-17AF KO mouse lungs increases bacterial numbers in lung and increases survival.
(A) Number of bacteria in lungs of WT and IL-17AF KO mice on day 2 postinfection. Bars represent means ± SEM (n = 3 mice per group). Results were confirmed using three independent experiments. (B) Kaplan–Meier survival curves (WT, n = 15 mice per group; KO, n = 12–13 mice per group). Mice were infected intratracheally with 2 × 107 CFU of L. pneumophila. Data from two independent experiments are summarized and shown. *p < 0.05.
Discussion
This study demonstrated that γδ T cells are involved in causing the death of IL-17AF KO mice infected with L. pneumophila. Our results show that, although γδ T cells in the lungs of infected IL-17AF KO mice contribute to bacterial elimination by killing infected cells, phagocytic cells do not effectively eliminate dead cells, leading to adverse side effects of tissue damage. The other key findings of this study is that γδ T cells are increased only in mice that lack both IL-17A and IL-17F but not in those lacking only one of these cytokines. Moreover, these cells produce granzyme B and perforin and contribute to protection against L. pneumophila infection.
IL-17A and IL-17F, which share the highest similarity (50%) at the amino acid level among IL-17-family members, were reported to induce the expression of various inflammatory cytokines and chemokines and participate in inflammation induction and infection defense (34). IL-17A was shown to contribute to host responses to diverse bacterial and fungal pathogens, including Klebsiella pneumoniae, Streptococcus pneumoniae, Bordetella pertussis, M. tuberculosis, and C. albicans (35), and both IL-17A and IL-17F were reported to be crucial for protection against mucoepithelial infections by Staphylococcus aureus and Citrobacter rodentium (16). However, in an L. pneumophila infection model developed using IL-17 KO mice (C57BL6/J background; resistant) and A/J mice (permissive) treated with anti–IL-17 Ab, IL-17F was found to be less critical than IL-17A in the defense against infection (36). In this study, we compared survival rates and lung bacterial counts in the context of the regulation of host defense against L. pneumophila infection, which clearly revealed that both IL-17F and IL-17A play an important role in defense. This disparity between our results and those from the previous work could be due to differences in the background of the KO mice or the bacterial strain used.
Experiments conducted using L. pneumophila pneumonia model mice have revealed the characteristics of innate immune cells and the mechanisms involved in host defense. Neutrophils and monocytes have been identified as sources of IL-12 production (37–39), whereas IFN-γ–producing lymphocytes, such as NK cells and Gr-1+ CD8+ T cells that appear early in severe infections, have been reported to be critical (40, 41). Although previously reported data have indicated that γδ T cells represent one of the sources of IFN-γ or IL-17 (36, 39), no study has demonstrated how γδ T cells contribute to host defense during L. pneumophila infection. Moreover, the physiological significance of these cells was not revealed by our experimental results obtained using γδ T cell–depleted WT mice, which is likely the result of γδ T cells not increasing substantially in number postinfection.
A limitation of this study is that we were unable to ascertain why γδ T cell numbers were increased in IL-17AF KO mice. Initially, we hypothesized that in IL-17AF KO mice infected with L. pneumophila γδ T cells were increased and played a compensatory role for the decreased neutrophils; however, the cells were also increased in the lung and spleen (data not shown) of uninfected IL-17AF KO mice, which indicated that this increase was not due to L. pneumophila infection. Conversely, the proportion of γδ T cells in the thymus was the same in the KO and WT mice (data not shown) and thus could not be readily regarded as a congenital phenotype. Nevertheless, these results were unexpected and could serve as the subject of future immunological studies. Another future challenge will be to identify the Ags recognized by γδ T cells. These findings may help to inform the design of new clinical treatments involving the adjustment of the proportions of distinct T cell populations or the overall populations of phagocytic cells.
We also observed a striking reduction in lung IMs of IL-17AF KO mice. IL-17 is reported to induce the production of GM-CSF, CCL2, and CCL7 chemokines (42–44). In a mouse model of malaria infection, IL-23 was found to induce IL-17 production in macrophages, which, in turn, stimulated macrophages and elicited the production of CCL2/7, likely in an autocrine fashion (45). Therefore, we examined the mRNA expression of GM-CSF and CCL2/7 in the lungs of IL-17AF KO mice after L. pneumophila infection; however, we did not detect a decrease in the mRNA levels (data not shown), suggesting that IL-17 maintains IM levels in a CCL2/7-independent manner in the case of L. pneumophila infection.
Studies of other infectious pathogens have demonstrated that the immune response triggered to eliminate pathogens does not unfailingly benefit the host. In patients with hepatitis B virus–associated acute-on-chronic liver failure, γδ T cells produce considerable levels of inflammatory cytokines and granzyme B and are cytotoxic (46). Similarly, neutrophils have been widely demonstrated to represent a factor in exacerbating the pathology in mouse models of intestinal infections (47, 48). Meanwhile, in mouse models of pneumonia caused by S. aureus infection, mortality is markedly increased in the case of IL-17A KO mice, although bacterial counts in the lung are similar between KO and WT mice (20). Moreover, inflammatory diseases are recognized to be caused when apoptotic cells are not properly phagocytosed by macrophages (49). Thus, for a host to overcome an infection, reducing tissue damage and maintaining homeostasis may prove to be more crucial than lowering the number of pathogenic micro-organisms.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank Sho Shimada for technical assistance. We thank Editage (http://www.editage.com) for English language editing.
Footnotes
This work was supported by Japan Society for the Promotion of Science KAKENHI grants (JP26860618 and JP19K17939 to C.K.), a Project Research Grant from Toho University School of Medicine (24-17 and 18-01 to C.K.), and a Grant-in-Aid for Private University Research Branding Project from the Ministry of Education, Culture, Sports, Science and Technology to K.T.
Abbreviations used in this article:
- AM
- alveolar macrophage
- BALF
- bronchoalveolar lavage fluid
- IL-17AF KO
- IL-17A/IL-17F double-KO
- IM
- interstitial macrophage
- KO
- knockout
- LDH
- lactate dehydrogenase
- WT
- wild-type.
- Received June 18, 2020.
- Accepted June 30, 2020.
- Copyright © 2020 The Authors
This article is distributed under the terms of the CC BY 4.0 Unported license.
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