Deletion of mTORC1 Activity in CD4+ T Cells Is Associated with Lung Fibrosis and Increased γδ T Cells
Deletion of mTORC1 Activity in CD4+ T Cells Is Associated with Lung Fibrosis and Increased γδ T Cells
Christine L. Vigeland 0 1
Samuel L. Collins 0 1
Yee Chan-Li 0 1
Andrew H. Hughes 0 1
Min- Hee Oh 1
Jonathan D. Powell 1
Maureen R. Horton 0 1
0 Department of Medicine, Johns Hopkins University School of Medicine , Baltimore , Maryland, United States of America, 2 Department of Oncology, Johns Hopkins University School of Medicine , Baltimore, Maryland , United States of America
1 Editor: Bernhard Ryffel, Centre National de la Recherche Scientifique , FRANCE
Pulmonary fibrosis is a devastating, incurable disease in which chronic inflammation and dysregulated, excessive wound healing lead to progressive fibrosis, lung dysfunction, and ultimately death. Prior studies have implicated the cytokine IL-17A and Th17 cells in promoting the development of fibrosis. We hypothesized that loss of Th17 cells via CD4-specific deletion of mTORC1 activity would abrogate the development of bleomycin-induced pulmonary fibrosis. However, in actuality loss of Th17 cells led to increased mortality and fibrosis in response to bleomycin. We found that in the absence of Th17 cells, there was continued production of IL-17A by γδ T cells. These IL-17A+ γδ T cells were associated with increased lung neutrophils and M2 macrophages, accelerated development of fibrosis, and increased mortality. These data elucidate the critical role of IL-17A+ γδ T cells in promoting chronic inflammation and fibrosis, and reveal a novel therapeutic target for treatment of pulmonary fibrosis.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: Support was provided by: National
Institutes of Health (https://www.nih.gov/) F32
HL124887 (CLV); National Institutes of Health (https://
www.nih.gov/) PO1 HL010342 (MRH); National
Institutes of Health (https://www.nih.gov/) R21
HL111783 (MRH); Flight Attendant Medical Research
Institute (http://www.famri.org/intro/) (MRH); and the
Osborne Family Research Fund (MRH). The funders
had no role in study design, data collection and
analysis, decision to publish, or preparation of the
Pulmonary fibrosis is a rapidly progressive, fatal lung disease. The pathogenesis of this disease
is not fully understood, but it is believed to develop as a result of dysregulated, excessive wound
]. Pro-inflammatory and pro-fibrotic cytokines such as TNF-α, IL-1β, and TGF-β,
as well as M2 macrophages are believed to play a role in promoting this response[
Currently, the only approved therapies slow the progression of disease, but do not arrest or reverse
]. Thus, there is an urgent need for improved understanding of the pathogenesis
of pulmonary fibrosis so that novel therapies can be developed. Prior work in both animal
models as well as humans has implicated IL-17A in driving the development of pulmonary
]. In animal models, IL-17A is produced by Th17 cells and γδ T cells within the
lungs after fibrotic stimuli[
7, 10, 11
]. However, the contributions of Th17 cells and γδ T cells to
the development of pulmonary fibrosis remains unclear. While Th17 cells have been implicated
in promoting the development of fibrosis through production of IL-17A, γδ T cells have been
found to ameliorate lung fibrosis[
7, 10, 12
]. This effect of γδ T cells has been hypothesized to
be mediated via production of IL-17A, CXCL10, IL-22, or IFN-γ[
It has been previously shown that mTOR is a critical regulator of CD4+ T cell
]. mTOR signals through two signaling pathways, mTORC1 and mTORC2. Loss of
mTORC1 signaling prevents the differentiation of naïve CD4+ T cells into Th1 and Th17 cells,
while loss of mTORC2 signaling prevents differentiation into Th2 cells[
]. The protein raptor
is necessary for mTORC1 signaling, but not mTORC2 signaling. Thus, selective deletion of
raptor linked to expression of CD4 creates CD4+ T cells that are unable to differentiate into a
Th1 or Th17 phenotype.
Previous work investigating the role of IL-17A-producing cells in pulmonary fibrosis has
utilized the intratracheal bleomycin model. This model causes acute inflammation that
progresses to chronic inflammation and fibrosis over 14 to 21 days[
]. These phases of injury
have been shown to have different mechanisms of regulation[
]. Thus, it is important to
differentiate between the acute and chronic inflammatory phases. Within human disease,
however, there is often not a recognized acute inflammatory phase[
]. Fibrosis gradually develops
over years. Thus, in order to mimic human pathology more closely, we employed a model
involving repeated intraperitoneal injections of bleomycin given over a period of four weeks to
induce pulmonary fibrosis with less acute inflammation[
]. This protocol produces
pulmonary fibrosis by day 42 that continues to progress through day 70[
]. By utilizing this model,
we can focus our investigation on the chronic inflammation and progressive fibrosis that is
more characteristic of human disease.
We hypothesized that, because they are unable to generate Th17 cells, our CD4 raptor
knockout mice would have attenuated injury from bleomycin. Surprisingly, in the CD4 raptor
knockout mice, in the absence of Th17 cells, we observed increased mortality and fibrosis.
Further investigation revealed robust IL-17A production by γδ T cells in the lungs. Our data
reveals the critical role of γδ T cells in promoting the development of fibrosis.
Loss of CD4 raptor expression causes increased mortality and fibrosis in response to bleomycin
To induce pulmonary fibrosis, wild type and CD4 raptor knockout mice were given
intraperitoneal (i.p.) injections of bleomycin over 28 days and harvested at day 42. Prior work by our
group has shown that this protocol induces pulmonary fibrosis by day 42 with little mortality
]. However, in response to bleomycin, the CD4 raptor knockout mice had significantly
increased mortality (Fig 1A). They developed fibrosis as shown by Masson-Trichrome staining,
and developed it earlier in the course of treatment (Fig 1B). By day 21, just halfway through the
protocol, the CD4 raptor mice had evidence of fibrosis with an increase in the number of lung
fibrocytes and expression of collagen 1A1 within the lungs (Fig 1C and 1D).
CD4 raptor knockout mice have increased γδ T cells within the lungs
In order to determine the cause of this difference in mortality and fibrosis, we assessed the
inflammatory response within the lungs at day 21. While there was no difference in the total
number of lung cells or the percent of CD4+ T cells, there was an increase in the percent of γδ
T cells (Fig 2A, 2B and 2C). Both wild type and CD4 raptor knockout mice had an increase in
γδ T cells following bleomycin exposure; however, this response was enhanced in the CD4
2 / 12
Fig 1. CD4 raptor knockout mice develop increased fibrosis and mortality in response to bleomycin. (A) Survival curves for
wild type and CD4 raptor knockout mice following i.p. bleomycin. Data from 3 pooled experiments, n = 15–19 mice per group. P = 0.03
by Log-rank test. (B) Masson’s trichrome staining of lungs on day 42 following i.p. bleomycin at x20 (upper panels) and x100 (lower
panels) magnification. (C) Lung fibrocytes on day 21 following i.p. bleomycin. Data shown from one representative experiment with a
total of three replicates and n = 3–6 per group. (D) Expression of collagen 1A1 within the lungs on day 21 following i.p. bleomycin.
Data shown from three pooled experiments with n = 11–17 per group. (C-D) Error bars represent one standard error. Significance
determined by one-way ANOVA followed by Sidak’s multiple comparison’s test.
3 / 12
Fig 2. CD4 raptor knockout mice have increased γδ T cells. Day 21 following i.p. bleomycin, lungs were
analyzed for (A) total lung cells, (B) percent of CD4+ cells by flow cytometry, (C) percent of γδ T cells by flow
cytometry. Data shown from one representative experiment with three replicates, n = 3–6 per group. Error
bars represent one standard error. Significance determined by one-way ANOVA followed by Sidak’s multiple
4 / 12
Loss of CD4 raptor expression causes a heightened IL-17A+ γδ T cell
Next, we sought to determine the phenotype of the CD4+ T cells and γδ T cells within the
lungs. Both wild type and CD4 raptor knockout mice had a paucity of Th1 and Th2 cells, and
this did not change after bleomycin treatment (S1A and S1B Fig). As expected, the CD4 raptor
knockout mice did not develop the Th17 response that is observed in wild type mice in
response to bleomycin (Fig 3A). Both groups did show similar increases in the population of
regulatory T cells (Fig 3B). In the absence of a Th17 response, the CD4 raptor knockout mice
had an enhanced IL-17A+ γδ T cell response (Fig 3C). These γδ T cells also exhibited
heightened production of IL-17A as measured by mean fluorescent intensity of IL-17A expression by
flow cytometry (Fig 3D).
Enhanced IL-17A+ γδ T cell response is associated with increased
inflammation and M2 macrophages
In addition to increasing mortality and fibrosis, this enhanced IL-17A+ γδ T cell response seen
in the CD4 raptor knockout mice was associated with an enhanced inflammatory response.
Fig 3. CD4 raptor knockout mice have increased IL-17A+ γδ T cells. Day 21 following i.p. bleomycin, flow cytometric analysis of lung T cells for (A)
Th17 cells, (B) regulatory T cells, (C) IL-17A+ γδ T cells. (D) Level of IL-17A expression quantified by mean fluorescence intensity by flow cytometry.
Data shown from one representative experiment with three replicates, n = 3–6 per group. Error bars represent one standard error. Significance
determined by one-way ANOVA followed by Sidak’s multiple comparison’s test.
5 / 12
These mice had more neutrophils within the lungs as well as increased expression of the
proinflammatory cytokines TNF-α and IL-1β (Fig 4A, 4F and 4G). While there was no difference
in the percentage of macrophages, there was a shift in macrophage phenotype, with an increase
in the expression of M2 markers Arginase 1 and YM1, as well as an increased percentage of
macrophages expressing the M2 marker CD206 (Fig 4B, 4C, 4D and 4E).
Our study reveals the key role of IL-17A+ γδ T cells in promoting the development of
pulmonary fibrosis. Prior studies have found that both Th17 cells and γδ T cells are upregulated in
animal models of pulmonary fibrosis and produce the cytokine IL-17A. In the absence of
IL17A, either via administration of a neutralizing antibody or use of IL-17A deficient animals,
mice develop less fibrosis after bleomycin exposure[
]. Therapies that reduce the population
of IL-17A producing cells are similarly able to abrogate and even reverse fibrosis[
8, 12, 20
order to clarify the roles of Th17 cells and IL-17A+ γδ T cells in this model of pulmonary
fibrosis, we utilized a cell-type specific deletion of the mTOR signaling protein raptor to selectively
block generation of Th17 cells.
For this study, we utilized a more chronic model of pulmonary fibrosis by administering
repeated intraperitoneal injections of bleomycin. We chose this approach because when
bleomcyin is administered via a single intratracheal dose, it induces acute inflammation, which
progresses to fibrosis[
]. Prior work has shown that the acute and chronic inflammatory
responses in bleomycin injury show differential mechanisms of regulation[
]. Thus, in this
study, we sought to focus on the chronic inflammation and fibrosis that characterizes human
Initially, we hypothesized that the CD4 raptor knockout mice would have decreased fibrosis.
Indeed our data revealed quite the opposite. The increase in pathology seen in the CD4 raptor
knockout mice was associated with a marked increase in IL-17A+ γδ T cells in the lungs, thus
implicating these cells in promoting inflammation and fibrosis. While Th17 cells also produce
the pro-fibrotic cytokine IL-17A, our data indicates that these cells are not necessary for
generation of pulmonary fibrosis in this model. Furthermore, our data point out the complexity of
responses even when using tissue specific Cre’s. If we had not examined the lungs for γδ T
cells, we might have overlooked their contribution with regard to IL-17A production and
promotion of disease in this model.
The precise role of γδ T cells in lung fibrosis has been somewhat confusing. One study
found that mice overexpressing the transcription factor Egr3 have a higher expression of γδ T
cells, and that these mice developed excessive bleomycin-induced lung injury in the setting of
an exaggerated response of both Th17 cells and IL-17A+ γδ T cells[
]. Other studies have
found that loss of γδ T cells enhances lung fibrosis, through loss of the anti-fibrotic mediators
CXCL10, IL-22, and IFN-γ[
]. Thus, previous models have purported that Th17 cells
promote fibrosis, while γδ T cells have anti-fibrotic effects. Furthermore, studying the role of
γδ T cells has been challenging because the antibodies that are used to deplete γδ T cells in fact
do not deplete these cells but rather lead to internalization of the γδ T cell receptor, masking
them from identification. Our data clarify the pathologic role of γδ T cell production of
IL17A. Additionally, our data indicates that IL-17A+ γδ T cells alone are sufficient to cause
pulmonary fibrosis in the absence of Th17 cells. Furthermore, we saw no protective role for these
cells despite the absence of Th17 cells. Of note, there was no change in the expression of the
cytokines CXCL10, IL-22, and IFN-γ, through which γδ T cells are believed to exert their
antifibrotic effects (S2A, S2B and S2C Fig).
6 / 12
Fig 4. CD4 raptor knockout mice have increased inflammation and M2 macrophages. Day 21 following i.p. bleomycin, lungs were analyzed
by flow cytometry for (A) neutrophils, (B) macrophages, (C) CD206+ macrophages. RT-PCR was performed on lungs on day 21 to quantify
expression of (D) Arginase 1, (E) YM1, (F) TNF-α, and (G) IL-1β, (A-C) Data shown from one representative experiment with three replicates,
n = 3–6 per group. (D-F) Data shown from three pooled experiments, n = 14–17 per group. Error bars represent one standard error. Significance
determined by t-test or one-way ANOVA followed by Sidak’s multiple comparison’s test.
7 / 12
Mechanistically, while the CD4 raptor knockout mice had an enhanced IL-17A+ γδ T cell
response, when balanced with the loss of Th17 cells, there was no change in the total expression
of IL-17A within the lungs by PCR and by ELISA (S2D and S2E Fig). However, there was an
increase in the expression of TNF-α and IL-1β, as well as an increased neutrophil infiltration of
the lungs, and increased M2 macrophage polarization. Taken together, we hypothesize that the
increased mortality and fibrosis in the CD4 raptor knockout mice was not due solely to
expression of IL-17A, but rather was due to a shift in the inflammatory response, via neutrophil
recruitment, M2 macrophage polarization, and upregulation of pro-inflammatory cytokines.
Other models of lung injury have similarly found that γδ T cells play a critical role in
promoting neutrophil recruitment and M2 macrophage polarization[
]. While these effects of γδ
T cells may be beneficial to respond to an acute insult such as a bacterial pneumonia, in our
chronic model of injury, we hypothesize that it is this persistent neutrophil recruitment,
enhanced production of cytokines with inflammatory and pro-fibrotic effects, and M2
macrophage polarization that perpetuates the chronic inflammation and fibrosis.
Prior work has highlighted the role of IL-1β, neutrophils, and M2 macrophages in promoting
]. IL-1β is an important pro-fibrotic cytokine, and direct instillation of IL-1β
intratracheally has been shown to induce pulmonary inflammation and fibrosis. This has
been hypothesized to be due to its effects on promoting IL-17A production both by γδ T cells
and Th17 cells[
]. Our study indicates, however, that in the absence of Th17 cells, IL-1β is still
able to promote inflammation and fibrosis. Further, the enhanced expression of IL-1β in the
absence of Th17 cells may indicate that Th17 cells act as a negative regulator on IL-1β expression.
Regarding neutrophils, in patients with IPF, an increase in BAL neutrophils has been found to
portend a worse prognosis[
]. Mechanistically, neutrophil elastase has been shown to promote
fibroblast and myofibroblast proliferation[
]. M2 macrophages have also been shown to
promote pulmonary fibrosis, and a reduction in M2 polarization results in less injury [
Our work reveals the important role that IL-17A+ γδ T cells play in promoting the
development of pulmonary fibrosis. Th17 cells have been identified as a potential target to treat
pulmonary fibrosis, and in animal models, therapies that diminish the Th17 response have been
shown to reduce or reverse fibrosis[
]. This data highlights the importance of targeting
both Th17 cells and IL-17A+ γδ T cells within the lungs to successful treat pulmonary fibrosis.
Additionally, it reveals a novel target for therapy in these IL-17A+ γδ T cells. Targeting these
γδ T cells specifically and altering their cytokine production away from IL-17A and towards a
phenotype that has been shown to be protective could represent a novel therapeutic avenue.
Materials and Methods
CD4 Cre mice and RAPTORfl/fl mice on the C57BL/6 background were purchased from The
Jackson Laboratory (Bar Harbor, ME), and bred to homozygosity. Mice were housed in a Johns
Hopkins University School of Medicine animal facility, kept at 72°F and 45% humidity, with
14 hour dark and 10 hour light exposure. Food was purchased from HARLAN (product
number 20185x). Mice had free access to food and water. Eight-to 10-week old female mice were
utilized. Mice were euthanized via injection of 100 μl of pentobarbital 20 mg/ml. All animal
protocols were approved by the Institutional Animal Care and Use Committee at Johns
Hopkins University (Baltimore, MD).
Bleomycin was purchased from App Pharmaceuticals (Schaumbrg, IL). Collagenase type I was
purchased from gibo (Waltham, MA). DNase I was purchased from Roche diagnostics
8 / 12
(Indianapolis, IN). PMA and Ionomycin were purchased from Sigma-Aldrich (St. Louis, MO).
Flow cytometry reagents were purchased from eBioscience (San Diego, CA). Antibodies
utilized were from eBioscience: Foxp3 FITC (clone FJK-16s), IL-17A PE (clone eBio17B7), Gr-1
PE (clone RB6-8C5), F4/80 PerCP-Cyanine5.5 (clone BM8), CD8a PerCP-Cyanine5.5 (clone
53–6.7), CD11b PerCP-Cyanine5.5 (clone M1/70), γδ TCR APC (clone eBIOGL3), CD11b
APC (clone M1/70); from BioLegend (San Diego, CA): CD4 FITC (clone GK1.5), CD206 FITC
(clone C068C2); and from BD Pharmingen (San Jose, CA): IFN-γ PE (clone XMG1.2), CD11c
PE (clone HL3), IL-4 PE (clone 11B11), CD4 PerCP (clone RM4-5), CD45 PerCP-Cy5.5 (clone
30-F11). Collagen 1 biotin antibody was purchased from Rockland (Limerick, PA). Strepavidin
FITC was purchased from BioLegend (catalog 405202). ELISA IL-17A kit was purchased from
eBioscience (San Diego, CA).
Pulmonary fibrosis model
As previously described, bleomycin was given via intraperitoneal injection (0.64 units per
injection in 400 μl PBS) on days 0, 3, 7, 10, 14, 21, and 28 to induce pulmonary fibrosis[
mice received an equivalent volume of PBS on each day. Mice were harvested on day 21 or day
42. The left lung was taken for lung ELISA, or lung PCR, or histology. The right lung was taken
for FACS analysis.
Lungs were inflated with formalin to atmospheric pressure 30 cm and sectioned and stained
for H&E staining and Masson’s trichrome staining. H&E staining was performed using the
Thermo Scientific Richard-Allen Scientific Histology Signature Series Stains and performed
according to manufacturer’s instructions. Masson’s trichrome staining was performed using
the American MasterTech Masson Trichrome Stain Kit per the manufacturer’s instructions.
Images were collected at x20 and x100 magnification using a Nikon Eclipse 80i microscope and
Nikon DS-fi1 camera.
Lungs were harvested, chopped with a straight razor, and digested in a solution of collagenase
type I (3 mg/ml) and DNase I (200 μl/10 ml) in RPMI media for 60 minutes at 37 C. The
homogenates were then filtered through a 70 μm cell strainer to produce a single-cell
suspension. Cells were stimulated with PMA (50 ng/ml) and Ionomycin (500 nM) in vitro for 3 hours
and then stained with the appropriate antibody for FACS analysis. eBioscience
fixation/permeabilization reagents were used. Cells were then analyzed with a BD FacsCaliber, BD
Biosciences (San Jose, CA), and analyzed using FlowJo software (Ashland, OR).
RNA extraction and quantitative real time PCR
Lungs were homozenized using an OMNI TH tissue homogenizer (OMNI International,
Kennesaw, GA) in Trizol reagent, Invitrogen (Carlsbad, CA), then RNA was extracted using
chloroform, precipitated with isopropanol, and finally washed with ethanol to isolate the RNA.
RNA was quantified using the NanoDrop 1000 Spectrophotometer (ThermoFisher Scientific,
Waltham, MA). cDNA was produced using the BIO-RAD (Hercules, CA) iScript cDNA
Synthesis kit per manufacturer’s instructions using a Labnet Multigene Gradient PCR Thermal
Cycler (Sigma-Aldrich, St. Louis, MO). Real time PCR was performed on a 7500 Applied
Biosystems (Carlsbad, CA) and normalized to 18s RNA. Reagents used for Real Time PCR were
purchased from Applied Biosystems. PCR primers were purchased from ThermoFisher
9 / 12
Scientific (Waltham, MA): Arg1 (catalogue Mm00475988_m1), Col1a1 (catalogue Mm008016
66_g1), CXCL10 (catalogue Mm00445235_m1), IFN-γ (catalogue Mm00801778_m1), IL-1β
(catalogue Mm00434228_m1), IL-17A (catalogue Mm00439619_m1), IL-22 (catalogue Mm01
226722_g1), TNF-α (catalogue Mm00443258_m1), YM1 (catalogue Mm00657889_mH).
Whole lung ELISA
Lungs were homogenized in RIPA buffer using an OMNI TH tissue homogenizer (OMNI
International, Kennesaw, GA). The total amount of protein was quantified via Bio Rad
(Hercules, CA) Quick Start Bradford Assay, and 500 μg of protein was diluted to a volume of 100 μl of
ELISA buffer and plated. ELISA was performed using the eBioscience (San Diego, CA) kit per
manufacturer’s instructions and analyzed on a Bio-Rad microplate reader model 680.
Statistical analysis was performed using Prism software (GraphPad Software), using a
Logrank test to analyze mortality differences, an unpaired t-test to compare means between two
groups, and a one-way ANOVA with a Sidak correction for multiple comparisons to compare
means between more than two groups. Statistical significant values were those where P<0.05.
S1 Fig. No change in Th1 and Th2 cells within the lungs following i.p. bleomycin. Day 21
following i.p. bleomycin, flow cytometric analysis of lung T cells for (A) Th1 cells, (B) Th2
cells. Data shown from one representative experiment with three replicates, n = 3–6 per group.
Error bars represent one standard error. Significance determined by one-way ANOVA
followed by Sidak’s multiple comparison’s test.
S2 Fig. No change in lung expression of CXCL10, IL-22, IFN-γ, or IL-17A in CD4 raptor
knockout mice. Day 21 following i.p. bleomycin, RT-PCR of lung tissue for expression of (A)
CXCL10, (B) IL-22, (C) IFN-γ, and (D) IL-17A. (E) Day 21 following i.p. bleomycin, lung
homogenates were analyzed by ELISA to quantify IL-17A. (A-D) Data shown from three
pooled experiments, n = 14–17 per group. (E) Data shown from one representative experiment
with three replicates, n = 3–6 per group. Error bars represent one standard error. Significance
determined by unpaired t-test.
Conceptualization: CLV JDP MRH.
Data curation: CLV JDP MRH.
Formal analysis: CLV MRH.
Funding acquisition: CLV MRH.
Investigation: CLV SLC AHH YC MO.
Methodology: CLV SLC JDP MRH.
Project administration: CLV SLC JDP MRH.
Resources: JDP MRH.
10 / 12
Supervision: JDP MRH.
Validation: CLV SLC AHH YC MO JDP MRH.
Writing – original draft: CLV JDP MRH.
Writing – review & editing: CLV JDP MRH.
11 / 12
1. Kuhn C. The pathogenesis of pulmonary fibrosis . Monogr Path . 1993 ; 78 : 1 .
2. Martinet Y , Menard O , Vaillant P , Vignaud JM , Martinet N. Cytokines in human lung fibrosis . Arch Toxicol Suppl . 1996 ; 18 : 127 - 39 . PMID: 8678788
3. Agostini C , Gurrieri C. Chemokine/cytokine cocktail in idiopathic pulmonary fibrosis . Proc Am Thorac Soc . 2006 ; 3 ( 4 ): 357 - 63 . Epub 2006/06/02. 3/4/357 [pii] doi: 10.1513/pats.200601-010TK PMID: 16738201 .
4. Wilson MS , Wynn TA . Pulmonary fibrosis: pathogenesis, etiology and regulation . Mucosal Immunol . 2009 ; 2 ( 2 ): 103 - 21 . Epub 2009/01/09. mi200885 [pii] doi: 10.1038/mi. 2008 .85 PMID: 19129758; PubMed Central PMCID : PMC2675823 .
5. King TE Jr, Bradford WZ , Castro-Bernardini S , Fagan EA , Glaspole I , Glassberg MK , et al. A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis . N Engl J Med . 2014 ; 370 ( 22 ): 2083 - 92 . Epub 2014/05/20. doi: 10 .1056/NEJMoa1402582 PMID: 24836312 .
6. Richeldi L , du Bois RM , Raghu G , Azuma A , Brown KK , Costabel U , et al. Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis . N Engl J Med . 2014 ; 370 ( 22 ): 2071 - 82 . Epub 2014/05/20. doi: 10 .1056/NEJMoa1402584 PMID: 24836310 .
7. Wilson MS , Madala SK , Ramalingam TR , Gochuico BR , Rosas IO , Cheever AW , et al. Bleomycin and IL-1beta-mediated pulmonary fibrosis is IL-17A dependent . J Exp Med . 2010 ; 207 ( 3 ): 535 - 52 . Epub 2010/02/24. jem. 20092121 [pii] doi: 10.1084/jem.20092121 PMID: 20176803; PubMed Central PMCID : PMC2839145 .
8. Collins SL , Chan-Li Y , Hallowell RW , Powell JD , Horton MR . Pulmonary vaccination as a novel treatment for lung fibrosis . PLoS One . 2012 ; 7 ( 2 ): e31299 . Epub 2012 /03/01. doi: 10 .1371/journal.pone. 0031299 PONE-D- 11-24654 [pii]. PMID: 22363610; PubMed Central PMCID : PMC3281950 .
9. Mi S , Li Z , Yang HZ , Liu H , Wang JP , Ma YG , et al. Blocking IL-17A promotes the resolution of pulmonary inflammation and fibrosis via TGF-beta1-dependent and -independent mechanisms . J Immunol . 2011 ; 187 ( 6 ): 3003 - 14 . Epub 2011/08/16. jimmunol. 1004081 [pii] doi: 10.4049/jimmunol.1004081 PMID: 21841134 .
10. Braun RK , Ferrick C , Neubauer P , Sjoding M , Sterner-Kock A , Kock M , et al. IL -17 producing gammadelta T cells are required for a controlled inflammatory response after bleomycin-induced lung injury . Inflammation . 2008 ; 31 ( 3 ): 167 - 79 . PMID: 18338242 . doi: 10 .1007/s10753-008-9062-6
11. Parkinson RM , Collins SL , Horton MR , Powell JD . Egr3 induces a Th17 response by promoting the development of gammadelta T cells . PLoS One . 2014 ; 9 ( 1 ): e87265 . Epub 2014 /01/30. doi: 10 .1371/ journal.pone.0087265 PONE-D- 12-24801 [pii]. PMID: 24475259; PubMed Central PMCID : PMC3901773 .
12. Collins SL , Chan-Li Y , Oh M , Vigeland CL , Limjunyawong N , Mitzner W , et al. Vaccinia vaccine-based immunotherapy arrests and reverses established pulmonary fibrosis . JCI Insight . 1 ( 4 ). doi: 10 .1172/jci. insight.83116
13. Pociask DA , Chen K , Choi SM , Oury TD , Steele C , Kolls JK. gammadelta T cells attenuate bleomycininduced fibrosis through the production of CXCL10 . Am J Pathol . 2011 ; 178 ( 3 ): 1167 - 76 . Epub 2011/ 03/02. S0002 - 9440 ( 10 ) 00180 - X [pii] doi: 10.1016/j.ajpath. 2010 . 11 .055 PMID: 21356368; PubMed Central PMCID : PMC3070585 .
14. Simonian PL , Wehrmann F , Roark CL , Born WK , O'Brien RL , Fontenot AP . gammadelta T cells protect against lung fibrosis via IL-22 . J Exp Med . 2010 ; 207 ( 10 ): 2239 - 53 . Epub 2010/09/22. jem. 20100061 [pii] doi: 10.1084/jem.20100061 PMID: 20855496; PubMed Central PMCID : PMC2947077 .
15. Segawa S , Goto D , Iizuka A , Kaneko S , Yokosawa M , Kondo Y , et al. The regulatory role of IFNgamma producing gammadelta T cells via the suppression of Th17 cell activity in bleomycin-induced pulmonary fibrosis . Clin Exp Immunol . 2016 . Epub 2016/04/17. doi: 10 .1111/cei.12802 PMID: 27083148 .
16. Delgoffe GM , Pollizzi KN , Waickman AT , Heikamp E , Meyers DJ , Horton MR , et al. The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1
17. Izbicki G , Segel MJ , Christensen TG , Conner MW , Breuer R . Time course of bleomycin-induced lung fibrosis . Int J Exp Pathol . 2002 ; 83 ( 3 ): 111 - 9 . Epub 2002/10/18. 220 [pii]. PMID: 12383190; PubMed Central PMCID : PMC2517673 .
18. Zhou Y , Schneider DJ , Morschl E , Song L , Pedroza M , Karmouty-Quintana H , et al. Distinct roles for the A2B adenosine receptor in acute and chronic stages of bleomycin-induced lung injury . J Immunol . 2011 ; 186 ( 2 ): 1097 - 106 . Epub 2010/12/15. jimmunol. 1002907 [pii] doi: 10.4049/jimmunol.1002907 PMID: 21149612; PubMed Central PMCID : PMC3607290 .
19. Daniil ZD , Gilchrist FC , Nicholson AG , Hansell DM , Harris J , Colby TV , et al. A histologic pattern of nonspecific interstitial pneumonia is associated with a better prognosis than usual interstitial pneumonia in patients with cryptogenic fibrosing alveolitis . Am J Respir Crit Care Med . 1999 ; 160 ( 3 ): 899 - 905 . PMID: 10471616 .
20. Conte E , Iemmolo M , Fagone E , Gili E , Fruciano M , Genovese T , et al. Thymosin beta4 reduces IL-17- producing cells and IL-17 expression, and protects lungs from damage in bleomycin-treated mice . Immunobiology . 2014 ; 219 ( 6 ): 425 - 31 . Epub 2014/03/13. S0171 - 2985 ( 14 ) 00025 - 4 [pii] doi: 10.1016/j. imbio. 2014 . 02 .001 PMID: 24613476 .
21. Koenecke C , Chennupati V , Schmitz S , Malissen B , Forster R , Prinz I. In vivo application of mAb directed against the gammadelta TCR does not deplete but generates "invisible" gammadelta T cells . Eur J Immunol . 2009 ; 39 ( 2 ): 372 - 9 . Epub 2009/01/09. doi: 10 .1002/eji.200838741 PMID: 19130484 .
22. Mathews JA , Kasahara DI , Ribeiro L , Wurmbrand AP , Ninin FM , Shore SA . gammadelta T Cells Are Required for M2 Macrophage Polarization and Resolution of Ozone-Induced Pulmonary Inflammation in Mice . PLoS One . 2015 ; 10 ( 7 ): e0131236 . Epub 2015 /07/03. doi: 10 .1371/journal.pone.0131236 PONE-D- 15-10718 [pii]. PMID: 26135595; PubMed Central PMCID : PMC4489797 .
23. Mathews JA , Williams AS , Brand JD , Wurmbrand AP , Chen L , Ninin FM , et al. gammadelta T cells are required for pulmonary IL-17A expression after ozone exposure in mice: role of TNFalpha . PLoS One . 2014 ; 9 ( 5 ): e97707 . Epub 2014 /05/16. doi: 10 .1371/journal.pone.0097707 PONE-D- 14-00636 [pii]. PMID: 24823369; PubMed Central PMCID : PMC4019643 .
24. Lo Re S , Dumoutier L , Couillin I , Van Vyve C , Yakoub Y , Uwambayinema F , et al. IL -17A-producing gammadelta T and Th17 lymphocytes mediate lung inflammation but not fibrosis in experimental silicosis . J Immunol . 2010 ; 184 ( 11 ): 6367 - 77 . Epub 2010/04/28. jimmunol. 0900459 [pii] doi: 10.4049/ jimmunol.0900459 PMID: 20421647 .
25. Cheng P , Liu T , Zhou WY , Zhuang Y , Peng LS , Zhang JY , et al. Role of gamma-delta T cells in host response against Staphylococcus aureus-induced pneumonia . BMC Immunol . 2012 ; 13 : 38 . Epub 2012/07/11. 1471 -2172-13-38 [pii] doi: 10.1186/ 1471 -2172-13-38 PMID: 22776294; PubMed Central PMCID : PMC3524664 .
26. Gasse P , Mary C , Guenon I , Noulin N , Charron S , Schnyder-Candrian S , et al. IL -1R1/MyD88 signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in mice . J Clin Invest . 2007 ; 117 ( 12 ): 3786 - 99 . Epub 2007/11/10. doi: 10 .1172/JCI32285 PMID: 17992263; PubMed Central PMCID : PMC2066195 .
27. Gharib SA , Johnston LK , Huizar I , Birkland TP , Hanson J , Wang Y , et al. MMP28 promotes macrophage polarization toward M2 cells and augments pulmonary fibrosis . J Leukoc Biol . 2014 ; 95 ( 1 ): 9 - 18 . Epub 2013/08/22. jlb. 1112587 [pii] doi: 10.1189/jlb.1112587 PMID: 23964118; PubMed Central PMCID : PMC3868192 .
28. Gregory AD , Kliment CR , Metz HE , Kim KH , Kargl J , Agostini BA , et al. Neutrophil elastase promotes myofibroblast differentiation in lung fibrosis . J Leukoc Biol . 2015 ; 98 ( 2 ): 143 - 52 . Epub 2015/03/07. jlb.3HI1014- 493R [pii] doi: 10.1189/jlb.3HI1014-493R PMID: 25743626; PubMed Central PMCID : PMC4763951 .
29. Gasse P , Riteau N , Vacher R , Michel ML , Fautrel A , di Padova F , et al. IL-1 and IL-23 mediate early IL17A production in pulmonary inflammation leading to late fibrosis . PLoS One . 2011 ; 6 ( 8 ): e23185 . Epub 2011 /08/23. doi: 10 .1371/journal.pone.0023185 PONE-D- 11-06354 [pii]. PMID: 21858022; PubMed Central PMCID : PMC3156735 .
30. Kinder BW , Brown KK , Schwarz MI , Ix JH , Kervitsky A , King TE Jr. Baseline BAL neutrophilia predicts early mortality in idiopathic pulmonary fibrosis . Chest . 2008 ; 133 ( 1 ): 226 - 32 . Epub 2007/12/12. S0012 - 3692 ( 15 ) 48982 - 2 [pii] doi: 10.1378/chest.07-1948 PMID: 18071016 .