Intestinal overgrowth of Candida albicans exacerbates bleomycin-induced
pulmonary fibrosis in mice with dysbiosis

Short running title:
Effect of changes in the intestinal fungal microbiota on pulmonary fibrosis

Authors:
Takahiro Yamada1, Taku Nakashima1*, Takeshi Masuda1, Shinjiro Sakamoto1,
Kakuhiro Yamaguchi1, Yasushi Horimasu1, Shintaro Miyamoto1, Hiroshi Iwamoto1,
Kazunori Fujitaka1, Hironobu Hamada2, Nobuhiko Kamada3,4, Noboru Hattori1

Affiliations:1

Department of Molecular and Internal Medicine, Graduate School of Biomedical and

Health Sciences, Hiroshima University, Hiroshima, Japan2

Department of Physical Analysis and Therapeutic Sciences, Graduate School of

Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
3

Division of Gastroenterology and Hepatology, Department of Internal Medicine,

University of Michigan, Ann Arbor, MI, USA.
4

Laboratory of Microbiology and Immunology, WPI Immunology Frontier ResearchCenter, Osaka University, Suita, Osaka, Japan1

*Corresponding author:
Taku Nakashima, M.D., Ph.D.
Department of Molecular and Internal Medicine, Graduate School of Biomedical and
Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 7348551, JAPAN;
Phone: +81-(82)-257-5196
Fax: +81-(82)-255-7360
E-mail: tnaka@hiroshima-u.ac.jp

Co-authors：
Takahiro Yamada (E-mail: y3htk@yahoo.co.jp)
Takeshi Masuda (E-mail: ta-masuda@hiroshima-u.ac.jp)
Shinjiro Sakamoto(E-mail: s-sakamoto@hiroshima-u.ac.jp)

Kakuhiro Yamaguchi (E-mail: yamaguchikakuhiro@gmail.com)
Yasushi Horimasu(E-mail: yasushi17@hiroshima-u.ac.jp)

Shintaro Miyamoto(E-mail: miyamos@hiroshima-u.ac.jp)

Hiroshi Iwamoto(E-mail: iwamotohiroshig@gmail.com)

Kazunori Fujitaka(E-mail: fujikazu@hiroshima-u.ac.jp)

Hironobu Hamada (E-mail: hirohamada@hiroshima-u.ac.jp)
2

Nobuhiko Kamada (E-mail: nkamada@umich.edu)
Noboru Hattori (E-mail: nhattori@hiroshima-u.ac.jp)

Data availability statement:
The sequences generated in this study are available in the DDBJ Sequence Read
Archive database (Accession Number: DRA015049 and DRA015055). The other data
sets generated and analyzed during this study are included in this published article.

Funding:
This work was partially supported by Satake Technology Promotion Foundation grant.
The funders had no role in study design, data collection and analysis, decision to
publish, or presentation of the manuscript.

Conflict of interest:
The authors declare that they have no competing interests.

Ethics approval:

3

All experimental procedures were approved by the Committee on Animal Research at
Hiroshima University (approval no. A21-98) and were conducted in accordance with
the Guide for the Care and Use of Laboratory Animals, 8th ed, 2010 (National Institutesof Health, Bethesda, MD, USA).

Word Count: 4106

4

ABSTRACT
Increasing evidence indicates an interaction between the intestinal microbiota anddiseases in distal organs. However, the relationship between pulmonary fibrosis and theintestinal microbiota, especially intestinal fungal microbiota, is poorly understood.
Thus, this study aimed to determine the effects of changes in the intestinal fungal
microbiota on the pathogenesis of pulmonary fibrosis.
Mice with intestinal overgrowth of Candida albicans (C. albicans), which wasestablished by oral administration of antibiotics plus C. albicans, showed accelerated
bleomycin-induced pulmonary fibrosis relative to the control mice (i.e., without C.
albicans treatment). In addition, the mice with intestinal overgrowth of C. albicans
showed enhanced Th17-type immunity, and treatment with IL-17A neutralizing
antibody alleviated pulmonary fibrosis in these mice but not in the control mice. This
result indicates that IL-17A is involved in the pathogenesis of C. albicans-exacerbatedpulmonary fibrosis. Even before bleomycin treatment, the expression of Rorc, themaster regulator of Th17, was already upregulated in the pulmonary lymphocytes of themice with intestinal overgrowth of C. albicans. Subsequent administration of bleomycin
triggered these Th17-skewed lymphocytes to produce IL-17A, which enhanced
endothelial–mesenchymal transition.
These results suggest that intestinal overgrowth of C. albicans exacerbates pulmonary
fibrosis via IL-17A-mediated endothelial–mesenchymal transition. Thus, it might be apotential therapeutic target in pulmonary fibrosis. This study may serve as a basis forusing intestinal fungal microbiota as novel therapeutic targets in pulmonary fibrosis.

5Keywords: Candida albicans, pulmonary fibrosis, Th17, IL-17A, endothelial–mesenchymal transition, fungal microbiota, intestinal microbiota

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INTRODUCTION
Idiopathic pulmonary fibrosis (IPF) is a refractory respiratory disease characterized byprogressive collagen deposition in the lungs. Antifibrotic agents reduce the decline inforced vital capacity in patients with IPF [1–4]. However, the prognosis of thesepatients remains poor, with a reported average survival of 3–5 years. Effective treatment
options for progressive pulmonary fibrosis are limited, and novel therapies need to be
developed urgently.
The microbiota is a promising therapeutic target for refractory diseases. The interaction
between gut microbiota and host immunity influences the progression and therapeutic
responsiveness of underlying diseases in distal organs, including the lungs, via
modification of immune cells and their cytokine production [5]. Furthermore, the lungs,
previously thought to be sterile, have their own microbiota associated with various
respiratory diseases [6,7]. The microbiota in bronchoalveolar lavage fluid (BALF) isaltered in murine bleomycin (BLM)-induced pulmonary fibrosis and human IPF.
Relative to conventionally housed mice, germ-free mice show alleviated BLM-induced
pulmonary fibrosis. Furthermore, IPF patients with low bacterial abundance in BALF
have better prognosis than those with high bacterial abundance in BALF [8–10]. A
recent study has shown that outer membrane vesicles produced by lung commensal
bacteria exacerbate murine pulmonary fibrosis, and eliminating these bacteria with
antibiotics alleviates the disease [11]. These studies suggest that targeting the
microbiota is a promising therapeutic strategy to improve pulmonary fibrosis.
In addition to commensal bacteria, fungi also play important roles in the microbiota[12]. Candida species are prototypic fungal pathogens indigenous in the intestine.
7

Antibiotics can alter the bacterial and fungal microbiota; for example, antibiotic
administration increases the proliferation of intestinal Candida [13]. Intestinal Candidaproliferation can exacerbate murine allergic airway inflammation via M2 macrophages
induction [14,15] and can induce Th17 immune reactions in human peripheral bloodmononuclear cells and exacerbate fungus-related airway inflammation via crossreactivity with other fungi [16]. These studies suggest that changes in the intestinal
fungal microbiota affect the distal organs, as well as the bacterial microbiota. M2
macrophages are increased in fibrotic areas in murine pulmonary fibrosis and human
IPF lungs and exacerbate fibrosis by producing IL-13 and TGF-β1 [17]. Meanwhile, IL17A exacerbates pulmonary fibrosis by mobilizing neutrophils and inducing
inflammatory cytokines [18–21]. It also induces epithelial–mesenchymal transition(EMT) and fibroblast activation in vitro [20,22,23]. Thus, antibiotic-induced changes in
the intestinal fungal microbiota, especially the genus Candida, may affect thepathogenesis of pulmonary fibrosis by increasing M2 macrophages or IL-17A.
However, little is known about the association between the intestinal fungal microbiota
and pulmonary fibrosis. We hypothesized that intestinal Candida proliferation
exacerbates pulmonary fibrosis. This study aimed to determine the effects of antibioticinduced changes in the intestinal fungal microbiota on the pathogenesis of pulmonary
fibrosis.

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MATERIALS AND METHODS
Mice and intestinal Candida proliferation
Specific pathogen-free male C57BL/6J mice (6–8 weeks old) were purchased fromCharles River Laboratories, Japan (Yokohama, Japan). The mice were housed in apathogen-free environment with free access to food and distilled water supplemented
with or without cefoperazone (CPZ, 0.4 mg/ml, Tokyo Chemical Industry, Tokyo,
Japan), clindamycin (CLDM, 0.5 mg/ml, Tokyo Chemical Industry), fluconazole(FLCZ, 0.5 mg/ml, Tokyo Chemical Industry), and Candida albicans (C. albicans). The
drinking water bottles were changed every 3 days. C. albicans was obtained from theInfection Diseases Laboratory of Hiroshima University Hospital. C. albicans cultured
on Sabouraud agar (2 × 108 colony forming units, Nissui Pharmaceutical, Tokyo, Japan)
was mixed in 200 ml of sterile water and administered to the mice ad libitum. To
confirm colonization, murine BALF and cecal contents were collected at the time ofsacrifice and then cultured on CHROMagar (Kanto Chemical, Tokyo, Japan) for 48 h.
Then, colony formation and color tone were evaluated.
All experimental procedures were approved by the Committee on Animal Research at
Hiroshima University (approval no. A21-98) and were conducted in accordance with
the Guide for the Care and Use of Laboratory Animals, 8th ed, 2010 (National Institutesof Health, Bethesda, MD, USA).

BLM-induced pulmonary fibrosis

9After being intraperitoneally anesthetized with medetomidine hydrochloride (0.3 mg/kg
body weight, Kyoritsu Seiyaku, Tokyo, Japan), midazolam (4.0 mg/kg body weight,
Sandoz K.K., Tokyo, Japan), and butorphanol tartrate (5.0 mg/kg body weight, Meiji
Seika Pharma, Tokyo, Japan), the mice were administered with BLM (2.0 mg/kg bodyweight, Nippon Kayaku, Tokyo, Japan) to induce pulmonary fibrosis. Except whereindicated, the mice were sacrificed on day 14 after BLM administration, and blood,
lung, BALF and intestinal samples were collected.

Histological examination
Murine lungs and ceca were fixed in a 2% formalin solution. After embedding inparaffin, the tissues were stained with hematoxylin–eosin, periodic acid Schiff (PAS),
and Masson’s Trichrome stain. Immunohistochemical staining was performed asfollows: paraffin-embedded ceca tissues were stained with anti-RORγ rabbit
monoclonal antibody (Clone: EPR20006, Abcam, Cambridge, UK) as primary
antibody, and peroxidase labeling anti-rabbit IgG polyclonal antibody (#424144,
Nichirei, Tokyo, Japan) as secondary antibody.

Hydroxyproline assay
Hydroxyproline content was measured to evaluate the severity of pulmonary fibrosis.
Murine left lungs were harvested and homogenized in 1 ml of PBS (Nacalai Tesque,
Kyoto, Japan) and 1 ml of 12 N HCl and then hydrolyzed at 120°C for 16 h. In a 96well plate, 5 μL of the hydrolyzed sample, 10 μL of citrate/acetate buffer (5% citric10acid, Sigma-Aldrich, St. Louis, MO, USA; 1.2% glacial acetic acid, Sigma-Aldrich;
7.25% sodium acetate, Sigma-Aldrich; and 3.4% sodium hydroxide, Nacalai Tesque),and 100 μL of chloramine T solution (0.141 g chloramine T [Sigma-Aldrich] added to 8
ml of citrate/acetate buffer, 1 ml of 1-propanol [Sigma-Aldrich], and 1 ml of deionizeddistilled water) were mixed. After incubation at room temperature for 30 min, 100 μL of
Ehrlich’s reagent (1.25 g p-dimethylaminobenzaldehyde [Sigma-Aldrich] in 4.65 ml of
1-propanol and 1.95 ml of 70% perchloric acid [Sigma-Aldrich]) was added, and the
mixture was incubated at 65°C for 30 min. The absorbance of each sample was obtained
at 540 nm on a plate reader (iMARK, Bio-Rad, Hercules, CA, USA).

Flow cytometry and fluorescence-activated cell sorting (FACS)
Single-cell suspensions were obtained as follows. The right lower lobe of the murine
lung was excised, cut into small pieces, and then incubated in RPMI (Thermo Fisher
Scientific, Waltham, MA, USA) supplemented with 1.0 mg/ml collagenase A (Roche,
Basel, Switzerland) at 37°C for 30 min. After passing through a 70 μm cell strainer, redblood cells were removed using RBC lysis buffer (Thermo Fisher Scientific). For
mouse blood samples, erythrocyte lysis was repeated twice to obtain samples for flow
cytometry. Cell suspensions were blocked with anti-CD16/32 antibody (FcγR, clone 93,
BioLegend, San Diego, CA, USA) and then reacted with fluorescent antibodies diluted
to the appropriate concentration. For intracellular staining, fixation and penetration were
performed using the Cytofix/Cytoperm Kit (BD Biosciences, San Jose, CA, USA). For
cytokine staining, the cells were added with GolgiStop (BD Biosciences) and thenincubated for 4 h before antibody attachment. The fluorescent antibodies used are listed11

in Table S1. Flow cytometry and FACS were performed using a FACS Aria II (BD
Biosciences), and data were analyzed using FlowJo (BD Biosciences).

Quantitative PCR (qPCR)
The excised lungs were stored in RNA later (Thermo Fisher Scientific) at −80°C before
use. Lungs or collected cells were homogenized with 1 ml of TRIzol Reagent (LifeTechnologies, Grand Island, NY, USA), and RNA was extracted using the RNeasy Mini
Kit (QIAGEN, Venlo, Netherlands). RNA was reverse-transcribed to complementary
DNA using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific) inaccordance with the manufacturer’s protocol. mRNA expression was evaluated using
the TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) and Applied
Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). The PCR
conditions were as follows: 120 s at 94°C, 10 s at 98°C, 30 s at 60°C, 120 s at 68°C,
repeated for 40 cycles. The primers used are listed in Table S2.

Enzyme-linked immunosorbent assay (ELISA)
Serum samples were centrifuged at 900 × g for 30 min, and the supernatant wascollected. The lower lobes of the right lung of each mouse were homogenized with 1 ml
of PBS and then stored at −80°C before measurement. IL-17A levels in the serum andlungs were measured using the LEGEND MAX Mouse IL-17A ELISA Kit (BioLegend)in accordance with the manufacturer’s protocol. TGF-β levels in the serum were12

measured using Mouse TGF-beta 1 DuoSet ELISA (R&D Systems, Minneapolis, MN,
USA).

Anti-IL-17A neutralizing antibody
To antagonize the effects of IL-17A, we intraperitoneally treated the mice with in vivo
MAb anti-mouse IL-17 (200 μg/mouse, clone 17F3, Bio X Cell, Lebanon, NH, USA)
three times a week from immediately before BLM administration until sacrifice [24].
The control group received the same dose of in vivo MAb mouse IgG1 isotype control(Bio X Cell).

Cell culture
The mouse microvascular endothelial cell line MS1 (MILE SVEN 1, ATCC CRL-2279)
was purchased from ATCC (Manassas, VA, USA) and grown in DMEM (Thermo
Fisher Scientific) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1%
penicillin–streptomycin (Thermo Fisher Scientific) at 37°C in a 5% CO2 incubator.
After being incubated with several concentrations of recombinant mouse IL-17A(BioLegend) for the indicated times, cells were harvested using trypsin-EDTA (Thermo
Fisher Scientific) and subjected to qPCR, fluorescent immunocytochemistry, and flow
cytometry.

Fluorescent immunocytochemistry

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Cells were cultured in 48-well plates as described above. After removing the culture
medium, the cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BDBiosciences), and then added with fluorescent antibodies. The antibodies used are listed
in Table S1. The cells were stained with DAPI (DOJINDO LABORATORIES,
Kumamoto, Japan) to identify the nuclei. All slides were examined under a fluorescencemicroscope BZ-9000 (KEYENCE, Osaka, Japan). Images were analyzed using ImageJsoftware.

DNA extraction and sequencing
For microbiological analysis, murine BALF, lungs, and cecal contents were collectedunder sterile conditions and promptly stored at −80°C. BALF pellets were centrifuged
at 10000 × g for 15 min, and the lungs were homogenized before use. Microbial DNA
was obtained from each specimen using the NucleoSpin DNA Stool (Takara Bio, Shiga,
Japan) in accordance with the manufacturer’s protocol. The extracted DNA wasmeasured using Nanodrop (Thermo Fisher Scientific) or Qubit (Thermo Fisher
Scientific). Libraries were created for 16S rRNA genes for the bacterial microbiota and
ITS for the fungal microbiota and sequenced using Miseq Reagent Kit v3 and MiSeq
Reagent Kit v2 (Illumina, San Diego, CA, USA), respectively. The primers used for thesequencing are listed in Table S3. Data analysis was performed using QIIME 2(https://qiime2.org/).

Statistical analyses
14All statistical analyses were performed using JMP Pro 16 software (SAS Institute Inc.,
Cary, NC, USA). Data are presented as box plots. To compare the groups, we
performed Mann–Whitney U tests. Statistical significance was set at P < 0.05.

15

RESULTS
Worsening of pulmonary fibrosis by intestinal C. albicans proliferation
To mimic intestinal C. albicans blooms in human, which requires intestinal dysbiosis,
we fed the intervention group mice with water containing antibiotics and exogenous C.
albicans from 1 week before BLM administration until sacrifice (Candida + group). In
contrast, the control group mice were fed water containing antibiotics and anti-fungal
drug (FLCZ) for the same period to eliminate the indigenous organisms (Candida −group) (Fig. 1A). The Candida + group mice showed no inflammatory cell infiltrationin the intestinal tract, but PAS-positive yeast fungi were found in the mucosal lamina
propria, suggesting colonization by C. albicans (Fig. 1B, S1A, S1B). In culture method,
we observed increased C. albicans in the feces obtained from Candida + but not
Candida − group (Fig. 1C). C. albicans was not detected in the BALF or lunghomogenates from either group (data not shown).
To clarify the effect of intestinal C. albicans proliferation on pulmonary fibrosis, we
compared hydroxyproline levels, the recommended indicator for lung collagen content,
and histological findings (Fig. 1D, E, F). We confirmed that fibrosis was not seen when
PBS was administered instead of BLM (Fig. S2). As indicated in the previous report[11], the antibiotic treatment suppressed pulmonary fibrosis (Fig. 1D, E, F: DW vs
Candida –). The fibrosis ameliorating effect of antibiotics was reversed by intestinal
overgrowth of C. albicans (Fig. 1D, E, F: Candida – vs Candida +). The severity of
pulmonary fibrosis was significantly higher in the mice from the Candida + group thanin the mice from the Candida – group. Comparison of BALF cell fractions showed anon-significant but increasing trend in neutrophils in the Candida + group (Fig. S3). To
16

exclude the possibility that FLCZ affected the severity of pulmonary fibrosis, we
confirmed that FLCZ alone did not affect hydroxyproline levels (Fig. S4).

Analysis of intestinal and pulmonary microbiota
To evaluate the changes caused by antimicrobials, antifungals, and BLM administration,
we analyzed the intestinal and pulmonary microbiota using samples obtained from thesegroups. Analysis of the fungal microbiota using DNA extracted from the specimens
revealed that all fungi detected in the stool samples of the mice from the Candida +group were Candida spp., including C. albicans. By contrast, Candida spp. were rarely
detected in the stool samples of the mice from the Candida − group, and various fungi,
including Saccharomyces spp., were identified (Fig. 2A, B, S5A). Although Candida
spp. were found in the BALF and lung samples of the mice from both groups, nosignificant difference in the percentage of Candida was detected (Fig. 2C, D, E, F, S5B,
S5C). Sequencing analysis of the bacterial microbiota revealed the presence ofFirmicutes spp. and Proteobacteria spp. in the stool samples of the mice from the
Candida − and Candida + groups, and no significant difference in the abundance of
these species was found between the two groups (Fig. S6A). The majority of bacteria
were difficult to identify in the BALF and lungs, and no particular difference wasobserved (Fig. S6B, S6C).

Pulmonary fibrosis aggravated by C. albicans is mediated by Th17 (IL-17A)17

We analyzed lung cell fractions through flow cytometry to determine the cell type that
contributed to the exacerbation of pulmonary fibrosis in the Candida + group (Fig. S7A,
S7B). No differences in M2 macrophage (CD45+/F4/80+/CD11c+/CD206+) or Treg
(CD3+/CD4+/CD25+) fractions were found between the groups, whereas Th17
(CD3+/CD4+/IL-17A+) was markedly elevated in the Candida + group (Fig. 3A).
Furthermore, there was a positive correlation between lung hydroxyproline content andTh17 (Fig. S8). Similarly, qPCR using lung tissues (Fig. 3B) and ELISA using serum
and lung tissues (Fig. 3C) revealed that the expression of the Th17 cytokine IL-17A(Il17a) was significantly higher in the mice from the Candida + group than in thosefrom the Candida – group. We found no difference in the expression of Tbx21 andGata3, the master regulators of Th1 and Th2 (Fig. S9A). TGF-β (Tgfb), one of the key
profibrotic mediators, also did not differ between groups (Fig. S9B, S9C). Based onthese results, we hypothesized that Th17/IL-17A mainly contributed to the difference inpulmonary fibrosis between the two groups. To test this hypothesis, we neutralized IL17A using anti-IL-17A neutralizing antibodies (Fig. S10). In fecal cultures, there was noapparent difference in Candida colonization regardless of the neutralizing antibody
administration (Fig. S11). However, results showed that the administration of IL-17A
neutralizing antibody significantly decreased the hydroxyproline levels and pathologicalfeatures in the mice from the Candida + group but not in those from the Candida −group (Fig. 3D, E, F), suggesting that the worsening of fibrosis in the mice from the
Candida + group was mediated by IL-17A.
To confirm whether the induction of Th17 was associated with intestinal C. albicans
proliferation, we evaluated the relationship between intestinal C. albicans detection andlung Th17 induction 14 days after BLM treatment under other settings. We compared18

pulmonary fibrosis established in mice fed water alone (DW group) versus mice fed
water containing C. albicans without antibiotics (C. albicans w/o ABx group) (Fig.
S12A). In the C. albicans w/o ABx group, the C. albicans colony formation was notnumerous but could be detected in fecal culture, and lung Th17 was significantly
increased compared to DW group (Fig. S12B, S12C). However, no difference inhydroxyproline levels between the two groups was detected (Fig. S12D). When themice were fed water containing antibiotics without C. albicans, fecal C. albicans was
irregularly detected in some mice as shown ABx (C. albicans +) in Figure S13A, andlung Th17 levels tended to increase in these mice (Fig. S13B).

Mechanism by which intestinal overgrowth of C. albicans induces lung Th17
We examined the mechanism by which the intestinal overgrowth of C. albicans
promotes Th17 in the lungs. Flow cytometric analysis of lung cells before BLM
administration did not show an increase in the number of Th17 (CD3+/CD4+/IL-17A+)
cells (Fig. 3G). C. albicans was not detected in the blood or BALF cultures (data notshown), which excluded the possibility that C. albicans migrated to the lungs. We also
explored the possibility that lymphocytes transformed to Th17 in the gut migrated to thelungs through the bloodstream, but flow cytometry results revealed no IL-17A+ cells inthe blood obtained before or after BLM administration (Fig. 3H). We then evaluated
RORγt (Rorc), a master regulator of Th17 [25]. Lung CD4+ lymphocytes (CD3+/CD4+)
were sorted before BLM administration, RNA was extracted, and qPCR was performed.
The mice in the Candida + group showed significantly higher Rorc mRNA expression
than those in the Candida − group, even though no difference in Il17a mRNA
19

expression was found between the mice from these two groups (Fig. 3I). To seek for theorigin of RORγt-expressing cells, immunohistochemical staining of ceca tissue obtained
from mice before BLM administration was performed. As a result, RORγt-positive cells
were exclusively found in the Candida + group compared to the Candida – group (Fig.
3J).

Involvement of endothelial–mesenchymal transition in the exacerbation of
pulmonary fibrosis mediated by IL-17A
We further investigated the mechanism by which IL-17A exacerbates pulmonary
fibrosis. IL-17A reportedly exacerbates pulmonary fibrosis by mobilizing neutrophils,
inducing inflammatory cytokines, promoting EMT, and activating fibroblasts in vitro[20,22,23]. To investigate whether these phenomena occurred in the present model, we
conducted flow cytometry using lung cells obtained from mice 7 days after BLM
treatment (Fig. S14A, S14B, S14C). In the Candida + group, the number of cells
expressing epithelial markers decreased, whereas that of epithelial cells co-expressing
mesenchymal markers tended to increase, suggesting that epithelial damage and EMT
occurred (Fig. 4A). Similarly, the number of αSMA+/Col1A1+ fibroblasts increased,
indicating that fibroblasts were converted to myofibroblasts, the activated form offibroblasts (Fig. 4B). Furthermore, the number of endothelial cells decreased, whereasthat of endothelial cells co-expressing mesenchymal markers increased, suggesting that
endothelial damage and endothelial–mesenchymal transition (EndMT) occurred (Fig.
4C). Moreover, EndMT was suppressed when mice were treated with anti-IL-17A
neutralizing antibody (Fig. 4D). To confirm whether this EndMT was caused by IL20

17A, we cultured murine endothelial cell line MS1 in a medium supplemented withvarious concentrations of IL-17A. Results of qPCR showed that the mRNA expressionof mesenchymal markers (Fn, Col1a1, and Acta2) and EndMT markers (Snail1 andSnail2) increased in the cells cultured with IL-17A (Fig. 4E). Flow cytometry revealed
that the supplementation of IL-17A decreased the expression of endothelial markers, but
increased the expression of EndMT markers (Fig. 4F). Furthermore, fluorescentimmunocytochemistry showed that the expression of αSMA and Col1a1 proteins was
upregulated in the MS1 cells cultured with IL-17A (Fig. 4G, H), suggesting that IL-17A
may exacerbate pulmonary fibrosis not only by promoting EMT and activating
fibroblasts, as previously reported, but also by inducing EndMT. Figure 5 summarizes
the findings of the current study.

21

DISCUSSION
Changes in the microbiota have been implicated not only in gastrointestinal diseases but
also in diseases in various organs, including the lungs. Regarding pulmonary fibrosis, ahigh bacterial load in BALF is associated with a poor prognosis in patients with IPF,
and removal of the pulmonary microbiota suppresses BLM-induced pulmonary fibrosis
in mice [11]. However, the role of the intestinal microbiota, especially fungal
microbiota, in the pathophysiology of pulmonary fibrosis remains unclear. In addition, aclinical trial involving antibiotics administration in patients with IPF failed to improvetheir prognosis [26]. In humans, administration of antibiotics causes yeast growth instools [13], and intestinal yeast growth, including Candida, due to antibiotic
administration may enhance pulmonary fibrosis, as we have shown in the present study.
To achieve the microbiota-targeted treatment of IPF, further studies need not only toeliminate specific bacteria but also to consider intestinal dysbiosis, especially antibioticinduced changes in the fungal microbiota, including C. albicans. The effects of theintestinal and fungal microbiota warrant further investigation.
Changes in the intestinal microbiota affect various pathophysiological conditions, suchas the efficacy of anticancer chemotherapy. Some studies have reported microbiotaassociated changes in T cell phenotype and cytokine production; however, the precise
mechanisms by which the intestinal microbiota affects distant organs have not been
fully elucidated. In a mouse model of allergic airway inflammation, intestinal
overgrowth of Candida and its produced factors, such as prostaglandins, results in theactivation of lung M2 macrophages [14], which was not observed in the current model.
Similarly, no changes in Th1, Th2 or TGF-β were observed (Fig. S9A, S9B, S9C).
22

Instead, RORγt (Rorc) expressing Th17-skewed lymphocytes in the ceca and lungs
increased even before induction of pulmonary fibrosis. Our results suggest that the
proliferation of C. albicans in the gut causes Rorc-expressing Th17-skewed
lymphocytes to patrol remote organs, including the lungs, and that BLM stimulates
these cells to produce IL-17A. It has been shown that C. albicans-reactive T cells are
involved in increased IL-17A expression via cross-reactivity with other fungi in the
peripheral blood of asthma and COPD patients and in the lungs of cystic fibrosis
patients [16]. Thus, the induction of IL-17A in the lung by intestinal C. albicans
proliferation is not fibrosis-specific and may be a common mechanism in a variety of
diseases. As we compared antibiotics +C. albicans (Candida + group) and antibiotics+FLCZ (Candida – group) in the current study, factors other than C. albicans,especially FLCZ administration or reduction of other specific fungi such as
Saccharomyces, may have influenced the induction of Th17-skewed lymphocytes.
However, in addition to the reported association between C. albicans and Th17 [27,28],
more IL-17A-producing cells were found in the mice fed water containing C. albicans
without antibiotics (Fig. S12C). Furthermore, mice in our facility have endogenous C.
albicans, and we occasionally detect C. albicans even when fed water containing
antibiotics only (Fig. S13A). We also showed that mice with C. albicans detected in the
fecal culture tend to induce more Th17 than mice without C. albicans (Fig. S13B).
These findings suggest that C. albicans contributes to the induction of Th17-skewed
lymphocytes.
IL-17A has been associated with worsening of pulmonary fibrosis [18,21,29]. Reported
mechanisms include promoting neutrophil mobilization through G-CSF induction,
producing chemokines, inducing EMT in alveolar epithelial cells [20,22], stimulating23

fibroblasts [23], and inhibiting autophagy [20]. The cell fractionation of BALF in thecurrent model showed a non-significant but increasing trend in neutrophils in the
Candida + group (Fig. S3). This neutrophilic tendency may be caused by an increase in
IL-17A, which may partially contribute to enhanced fibrosis. In addition to these known
mechanisms, we discovered that IL-17A can induce EndMT in vitro and in vivo.
Numerous studies have shown that EndMT aggravates pulmonary fibrosis [30–36],
indicating that this pathway could also be a promising therapeutic target. IL-17A is
involved in the pathogenesis of pulmonary fibrosis via several pathways, and IL-17A
inhibitors are already clinically available for the treatment of autoimmune diseases,
such as psoriasis. In patients with pulmonary fibrosis, inhibiting type 17 immunity has
no established efficacy, but this strategy may be effective in certain populations.
As shown in Figure S12D, the reason why pulmonary fibrosis is not worsened in themice fed water containing C. albicans but without antibiotics is unclear. The
percentages of CD3+/CD4+/IL-17A+ cells in the C. albicans w/o ABx group (Fig. S12C)
were increased at the same degree as that in the Candida + group (Fig. 3A), suggestingthat commensal bacteria can counteract the effects of Th17. Both C. albicans
proliferation and dysbiosis of the commensal microbiota seem necessary for C. albicans
to exert its virulence in pulmonary fibrosis. The specific commensal bacteria
counteracting C. albicans warrant further analyses.
In conclusion, this study shows that intestinal C. albicans proliferation exacerbates
pulmonary fibrosis under dysbiosis with antibiotics by mobilizing Th17 cells to thelungs and enhancing IL-17A production by these mobilized cells. This response isregulated by a stepwise mechanism, in which the proliferation of C. albicans in the gut
24

first leads to the circulation of Rorc-expressing cells to distant organs, followed by theactivation of Rorc-expressing cells triggered by a local stimulator such as BLM.
Furthermore, IL-17A exacerbates fibrosis by inducing EndMT, in addition to its
previously reported roles in promoting EMT and fibroblast activation. These findings
contribute to elucidating the importance of the intestinal and fungal microbiota as
therapeutic targets in pulmonary fibrosis.

25

ACKNOWLEDGEMENTS
We would like to thank Ms. Yukari Iyanaga (technical assistant from the Department of
Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences,
Hiroshima University) for providing technical assistance with the experiments and Ms.
Yumiko Koba (Section of Infection Diseases Laboratory, Department of ClinicalSupport, Hiroshima University Hospital) for providing microbiological support. We
would like to thank Mses. Yoko Hayashi, Nagisa Morihara and Shoko Hirano (technicalstaff from the Analysis Center of Life Science, Natural Science Center for Basic
Research and Development, Hiroshima University) for their technical assistance withflow cytometric analysis and microbial profiling. We also would like to thank Editage(www.editage.com) for English language editing. A part of this work was carried out at
Research Facilities for Laboratory Animal Science. This work was supported in part by
the Natural Science Center for Basic Research and Development (NBARD-00093), and
the Program of the Network-type Joint Usage/Research Center for Radiation Disaster
Medical Science.

AUTHOR CONTRIBUTIONS
TY: collection of data, data analysis and interpretation, manuscript writing. TN:conception and design, collection of data, financial support, data analysis andinterpretation, manuscript writing. TM, SS: data analysis and interpretation, reviewing
the manuscript for important intellectual content. KY, YH, SM, HI, KF, HH: reviewing
the manuscript for important intellectual content. NK: conception and design, data
analysis and interpretation, reviewing the manuscript for important intellectual content.
26

NH: conception and design, financial support, reviewing the manuscript for important
intellectual content. All authors read and approved the final manuscript.

CONSENT FOR PUBLICATION
Not applicable.

ABBREVIATIONSIPF = Idiopathic pulmonary fibrosis

BALF = bronchoalveolar lavage fluid

BLM = bleomycin

EMT = epithelial–mesenchymal transition

CPZ = cefoperazone

CLDM = clindamycin

FLCZ = fluconazole

C. albicans = Candida albicans

PAS = periodic acid Schiff
27

FACS = fluorescence-activated cell sorting
MS1 = MILE SVEN 1
EndMT = endothelial–mesenchymal transition

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34

FIGURE LEGENDS
Figure 1
Exacerbation of pulmonary fibrosis by Candida proliferation in the intestine(A) Experimental scheme and group naming for each antimicrobial treatment, fungal
administration and induction of pulmonary fibrosis. BLM, bleomycin; CPZ,
cefoperazone; CLDM, clindamycin; DW; distilled water; FLCZ, fluconazole.
(B) Periodic acid-Schiff staining of murine cecal tissue sections obtained from the
Candida − and Candida + groups. The red arrows indicate Candida in the lamina
propria.
(C) The evaluation of fecal culture on CHROMagar for 48 h. Green colonies indicate
growth of C. albicans. The colonies were observed in Candida + group (middle) but not
Candida − group. (left) Comparison of C. albicans CFUs in each group (n = 8–
11/group). (right)(D) Lung hydroxyproline content in each group (n = 4–5/group).
(E) Histological analysis using Masson's trichrome stain in lung tissue sections of eachgroup.
(F) Quantification of fibrosis by Ashcroft score (n = 5/group).

Figure 2
Analysis of fungal microbiota

35

(A)(C)(E) Taxonomic distribution of fungal genus using ITS sequencing in fecal (A),BALF (C), and lung (E) samples collected from mice in the Candida − and Candida +groups. Four representative examples per group are shown.
(B)(D)(F) Percentages of ITS transcripts for the Candida genus in fecal (B), BALF (D),and lung (F) samples of each group (n = 5–8/group).

Figure 3
IL-17A-mediated exacerbation of pulmonary fibrosis(A) Flow cytometric analysis (as shown in Fig. S7) for cell frequency of M2
macrophages, regulatory T cells (Treg), and T helper 17 cells (Th17). Lung cells are
obtained from each mouse at 14 days after bleomycin administration (n = 4–5/group).
(B) Real-time quantitative PCR analysis for the mRNA expression levels of Il17a inlung tissues obtained at 14 days after bleomycin administration. Values are expressed
relative to the expression of 18s mRNA level (n = 4–5/group).
(C) Serum and lung protein levels of IL-17A at 14 days after bleomycin administration(n = 4–5/group).
(D) Lung hydroxyproline content in each group administrated with or without IL-17A
neutralizing antibodies as shown in Fig. S10 (n = 6–12/group).
(E) Histological analysis using Masson's trichrome stain in lung tissue sections of eachgroup.
(F) Quantification of fibrosis by Ashcroft score (n = 5–6/group).
36

(G) Flow cytometric analysis of the percentages of Th17 in lung cells obtained fromeach mouse before bleomycin administration (n = 5/group).
(H) Flow cytometric analysis of the percentages of Th17 in blood cells obtained fromeach mouse before or 14 days after bleomycin administration (n = 4–5/group).
(I) Real-time quantitative PCR analysis of mRNA expression levels of Rorc and Il17a.
Nucleic acids are extracted from CD3+/CD4+ cells sorted using fluorescence-activatedcell sorting from each murine lung before bleomycin administration. Values are
expressed relative to the expression of 18s mRNA level (n = 6/group).
(J) Representative images of RORγt immunostaining in ceca obtained from each mouse
before bleomycin administration.

Figure 4
Endothelial–mesenchymal transition by IL-17A(A) Percentages of epithelial cells (EpCAM+ in CD45−/CD31−) and epithelial cells
undergoing epithelial–mesenchymal transition (αSMA+/Col1a1+ in
EpCAM+/CD45−/CD31−) defined as Fig. S14.
(B) Percentages of activated fibroblasts (myofibroblasts: αSMA+/Col1a1+ in
EpCAM−/CD45−/CD31−) defined as Fig. S14.
(C) Percentages of endothelial cells (CD45−/CD31+) and endothelial cells undergoingendothelial–mesenchymal transition (αSMA+/Col1a1+ in CD45−/CD31+) defined as Fig.
S14.
37

(D) Percentages of endothelial cells undergoing endothelial–mesenchymal transition inmice treated with isotype control antibody or anti-IL-17A antibody as shown in Fig.
S10 (n = 6–7/group).
(E) Real-time quantitative PCR analysis of the mRNA expression levels of
mesenchymal markers (Fn, Col1a1, and Acta2) and endothelial–mesenchymal transition
markers (Snail1 and Snail2) in MS1 cells cultured under defined conditions. Values are
expressed relative to the expression of 18s mRNA level (n = 4/group).
(F) Flow cytometric analysis for CD31 expression in MS1 cells (lower) andαSMA+/Col1a1+ in CD31+ MS1 cells (upper, MS1 undergoing endothelial–mesenchymal transition) (n = 3/group).
(G) Representative images of fluorescent immunocytochemistry of MS1. Cells are
incubated for 30 h in medium with or without IL-17A. Expression levels of αSMA
(red), Col1a1 (green), and nuclei (blue) are shown.
(H) Comparison of fluorescence intensity of each group (n = 6–11/group).

Figure 5
Graphical abstract of the current study

38

SUPPLEMENTAL FIGURE LEGENDS
Figure S1
Lack of inflammatory change in the cecum obtained from the Candida − andCandida + groups(A) Hematoxylin–eosin staining of murine cecal tissue sections obtained from the
Candida − and Candida + groups.
(B) Number of cells in the lamina propria of the cecum. Five high power fields for eachsample were analyzed, and the average were plotted (n = 5/group).

Figure S2
Candida − and Candida + groups without bleomycin administration
Experimental scheme for each antimicrobial treatment, fungal administration. CPZ,
cefoperazone; CLDM, clindamycin; DW; distilled water; FLCZ, fluconazole.

Figure S3
Analysis of bronchoalveolar lavage fluid in the Candida − and Candida + groups
Cellular fractionation of BALF was examined and shown for macrophages (A),lymphocytes (B), and neutrophils (C). BALF is obtained from each mouse at 14 days
after bleomycin administration (n = 5/group).

39

Figure S4
Effect of fluconazole administration on pulmonary fibrosis(A) Experimental scheme for each antimicrobial treatment and induction of pulmonary
fibrosis. BLM, bleomycin; DW, distilled water; FLCZ, fluconazole.
(B) Lung hydroxyproline content in each group (n = 5/group).

Figure S5
Analysis of fungal microbiota
Species-level phylogenetic classification in fecal (A), BALF (B), and lung (C) samples
collected from mice in the Candida − and Candida + groups (n = 4/group).

Figure S6
Analysis of bacterial microbiota
Phylum-level phylogenetic classification in fecal (A), BALF (B), and lung (C) samples
collected from mice in the Candida − and Candida + groups (n = 4/group). Microbialchanges are analyzed by 16S rRNA gene sequencing.

Figure S7
Flow cytometric gating strategy
Gating strategy used to identify M2 macrophages (A), Treg and Th17 (B).
40

Figure S8
Correlation between lung hydroxyproline content and cell frequency of Th17 cells
Correlation between lung hydroxyproline content and cell frequency of IL-17A+ in lungCD3+/CD4+ cells. Lung cells are obtained from the Candida − and Candida + groups at
14 days after bleomycin administration (n = 8).

Figure S9Factors other than Th17/IL-17A in Candida + and Candida – groups(A) Real-time quantitative PCR analysis of the mRNA expression levels of Tbx21 andGata3 in lung tissues obtained from the Candida − and Candida + groups at 14 days
after bleomycin administration. Values are expressed relative to the expression of 18s
mRNA level (n = 4–5/group).
(B) Real-time quantitative PCR analysis of the mRNA expression levels of Tgfb in lungtissues obtained from the Candida − and Candida + groups at 14 days after bleomycin
administration. Values are expressed relative to the expression of 18s mRNA level (n =4–5/group).
(C) Serum protein levels of TGF-β at 14 days after bleomycin administration (n =4/group).

Figure S10
41

IL-17A neutralization
Experimental scheme of IL-17A neutralization using the anti-IL-17A antibodies. BLM,
bleomycin; CPZ, cefoperazone; CLDM, clindamycin; DW, distilled water; FLCZ,
fluconazole.

Figure S11
Effect of anti-IL-17A neutralizing antibody administration on intestinal Candidacolonization
The evaluation of fecal culture on CHROMagar for 48 h. The box plots indicatecomparison of C. albicans CFUs in each group (n = 2/group).

Figure S12Intestinal Candida proliferation without antibiotic-induced dysbiosis(A) Experimental scheme for each fungal administration and induction of pulmonary
fibrosis. BLM, bleomycin; DW, distilled water.
(B) The evaluation of fecal culture on CHROMagar in C. albicans without ABx group.
(left) The box plots indicate comparison of C. albicans CFUs in DW and C. albicans
without ABx group (n = 2–3/group). (right)(C) Flow cytometric analysis for cell frequency of Th17. Lung cells are obtained fromeach mouse at 14 days after bleomycin administration (n = 4/group).

42

(D) Lung hydroxyproline content in each group (n = 4–6).

Figure S13
Changes in lung Th17 levels by whether or not Candida albicans is detected in fecal
culture(A) Experimental scheme for each antimicrobial treatment, fungal administration andinduction of pulmonary fibrosis. BLM, bleomycin; CPZ, cefoperazone: CLDM,
clindamycin; DW, distilled water.
(B) Flow cytometric analysis for cell frequency of Th17. Lung cells are obtained fromeach mouse at 14 days after bleomycin administration (n = 4/group).

Figure S14
Flow cytometric gating strategy(A) Lung cell subsets and their surface markers in flow cytometric analysis. Murine
lung cells were obtained at 7 days after bleomycin administration (n = 4–5/group).
(B)(C) Gating strategy used to identify fibroblasts, epithelial cells (B) and endothelial
cells (C).

SUPPLEMENTAL TABLES
Table S1
43

Fluorescent antibodies used in this study
Fluorescence and moleculeCompany

Clone

PE/Cyanine7 anti-CD3

BioLegend

17A2(San Diego, CA, USA)
FITC anti-CD4

BioLegend

RM4-5

PE anti-CD25

BioLegend

PC61

BV421 anti-RORγt

BD Biosciences

Q21-559(San Jose, CA, USA)
BV421 anti-mouse IgG2a, k

BD Biosciences

G155-178

APC anti-IL-17A

BioLegend

TC11-18H10.1

APC anti-rat IgG1, k

BioLegend

RTK2071

PerCP/Cyanine5.5 anti-CD45

BioLegend

30-F11

APC anti-CD11c

BioLegend

N418

FITC anti-F4/80

BioLegend

BM8

BV421 anti-CD80

BioLegend

BM8

PE anti-CD206/MMR

Thermo Fisher Scientific

MR6F3(Waltham, MA, USA)PerCP/Cyanine5.5 anti-CD31

BioLegend

MEC13.3

Pacific Blue anti-mouse CD31

BioLegend390

BV421 anti-CD326/EpCAM

BioLegend

G8.8

eFluor 660 anti-alpha smoothmuscle actin

Thermo Fisher Scientific

1A4

eFluor 660 anti-mouse IgG2a, k

Thermo Fisher Scientific

eBM2a

FITC anti-collagen type 1

Rockland Immunochemicals

N/A

44

(Pottstown, PA, USA)
FITC anti-rabbit IgG

Bioss

N/A

(Woburn, MA, USA)

Table S2
Primers used in this study
Gene Name

Company

Assay ID

Cdh1

Thermo Fisher Scientific

Mm01247357_m1(Waltham, MA, USA)
Fn1

Thermo Fisher Scientific

Mm01256744_m1

Acta2

Thermo Fisher Scientific

Mm00725412_m1

Vim

Thermo Fisher ScientificMM01333430_m1

Pecam1

Thermo Fisher Scientific

Mm01242576_m1

Cdh5

Thermo Fisher Scientific

Mm00486938_m1

Col1a1

Thermo Fisher Scientific

Mm00801666_g1

Il6

Thermo Fisher Scientific

Mm00446190_m1

Snail1

Thermo Fisher ScientificMM00441533_g1

Snail2

Thermo Fisher Scientific

Mm00441531_m1

Twist1

Thermo Fisher Scientific

Mm00442036_m1

Twist2

Thermo Fisher ScientificMM00492147_m1

Il17a

Thermo Fisher Scientific

Mm00439619_m1

Rorc

Thermo Fisher Scientific

Mm01261022_m1

Il17b

Thermo Fisher Scientific

Mm01258783_m1

45

Il6

Thermo Fisher Scientific

Mm00446190_m1

Il17f

Thermo Fisher Scientific

Mm00521423_m1

Tbx21

Thermo Fisher Scientific

Mm00450960_m1

Gata3

Thermo Fisher Scientific

Mm00484683_m1

Tgfb

Thermo Fisher Scientific

Mm01178820_m1

Candida a

Thermo Fisher Scientific

Fn04646233_s1

Actb

Thermo Fisher Scientific

Mm02619580_g1

Rn18s;R

Thermo Fisher ScientificMm03928990_g1

Gapdh

Thermo Fisher Scientific

Mm99999915_g1

Table S3
Primers used for microbiological analysis
Primers

Company

Sequences

16S fwd

Thermo Fisher Scientific

TCGTCGGCAGCGTCAGATGTGTATAA(Waltham, MA, USA)GAGACAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG

16S rev

Thermo Fisher Scientific

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTCTCGTGGGCTCGGAG

46

ATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC
ITS_frd_1

Thermo Fisher Scientific

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTTGGTCATTTAGAGGAAGTAA

ITS_frd_2

Thermo Fisher Scientific

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTCGGTCATTTAGAGGAAGTAA

ITS_frd_3

Thermo Fisher Scientific

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTTGGTCATTTAGAGGAAC
TAA

ITS_frd_4

Thermo Fisher Scientific

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCCGGTCATTTAGAGGAA
GTAA

47

ITS_frd_5

Thermo Fisher Scientific

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTAGGCTATTTAGAGGAAGTAAITS_frd_6

Thermo Fisher Scientific

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTTAGTTATTTAGAGGAAGTAA

ITS_frd_7

Thermo Fisher Scientific

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTACGTCATTTAGAGGAAGTAA

ITS_frd_8

Thermo Fisher Scientific

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTTGGTCATTTAGAGGTCGTAA

ITS_rev_1

Thermo Fisher Scientific

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTGCGTTCTTCATCGAT
GC

48

ITS_rev_2

Thermo Fisher Scientific

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTGCGTTCTTCATCGATGG

ITS_rev_3

Thermo Fisher Scientific

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTACGTTCTTCATCGATGC

ITS_rev_4

Thermo Fisher Scientific

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTGCGTTCTTCATCGATGT

ITS_rev_5

Thermo Fisher Scientific

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACTGTGTTCTTCATCGATGT

ITS_rev_6

Thermo Fisher Scientific

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTGCGTTCTTCATCGTTGC

49

ITS_rev_7

Thermo Fisher Scientific

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCGTTCTTCATCGATGC

50

A

−7

0

B

14

Days
BLM

Sacrifice

CPZ, CLDM and FLCZ in DW
Candida −100 μm

100 μm

CPZ, CLDM and C. albicans in DW
Candida +Candida +Candida −p = 0.0002

8.0

4.0
3.0

40
30
20
10

2.0

a+
BL :
M

C

an

di
d

F

1000 μm1000 μm

100 μm

100 μm

100 μm

100 μm

100 μmCandida −：PBS

Candida +：PBS

DW

Candida −Candida +

an
C

di
an
C

DW
:B

da
+
BL :
M

1000 μm

8
7
6
5
4
3
2
1
0

da
−
BL :
M

1000 μmp = 0.0157

LM1000 μm

Ashcroft score

E

di

an
C

di
d

a−
BL :
M

LM
:B

DW

an

Candida − Candida +

C

0

a+
PB :
S

1.0

di
d

a−
PB :
S

0
di
d

Candida +

5.0

50

an

Candida −6.0

p = 0.0209

60
Hydroxyproline(μg/Left lung)C. albicans(×10^5 CFU/ml)

7.0

D

C

C

C

Feces
g_Saccharomycesg_Candida

unidentified
g_Xeromyces

80
others

80

60

40

20

0

Candida −

60

60

40

g_Cryptococcus
k_Fungi
g_Metschnikowia

40

g_Schizophyllum
g_Malassezia

20

others0

Candida −100

p = 0.1778

80
g_Candida60

g_Metschnikowia
k_Fungi

40

g_Malassezia

20
0Candida +Candida −F

Candida +p = 0.7282
100

80

60

40

20

0

Candida +Candida +Candida -g_MalasseziaD

p = 0.0026
100

g_Schizophyllum
othersPercentage of Candida spp.
in BALF (%)

Candida +g_Fusarium

g_Cladosporium

80

Percentage of Candida spp.
in lung (%)

g_Wallemia

B
Percentage of Candida spp.
in feces (%)

g_Saccharomyces

g_Metschnikowia

Relative abundance (%)

g_Malassezia

g_Aspergillus

Candida −g_Metschnikowia
k_Fungi

1000g_Candida

k_Fungiothers20g_Candida

g_Cladosporium

g_Wallemia

g_Xeromyces

40

100

g_Candida
g_Cryptococcus

g_Fusarium

60

Lung

g_Aspergillus
unidentified

80

E

BALF

100

g_Candida

Relative abundance (%)

Relative abundance (%)

100

Relative abundance (%)

A

Candida −Candida +

80

60

40

20

0

Candida −Candida +20

0

Candida +Candida -Candida +

20
10
0

Candida −

1.0
0.8
0.6
0.4
0.2
0

Candida +Candida −

D

1.2
15

1.0
0.8
0.6
0.4

10
5

0.2
0

Candida +Candida −0

Candida +Candida −1000 μm1000 μm

40
30
20
10
C
an
+α did
IL a−
-1
7A
C
an
di
da
+
C
an
+α did
IL a+
-1
7A

da
−
di
an
C
1.2
1.0
0.8
0.6
0.4
0.2

Candida −Candida +

1.2
1.0
0.8
0.6
0.4
0.2
0

p = 0.0250

p = 0.6198

4
3
2

Candida −Candida +Candida +Candida +p = 0.2948

1.2
1.0
0.8
0.6
0.4
0.2
0

Candida ++αIL-17A

Candida −Candida +J

p = 0.3367

4

4

3

3

Il17a/18s

1.4

Candida −Lung day0I

Rorc/18s

p = 0.6714
IL-17A+ in CD4+ T cells (%)

IL17A+ in CD4+ T cells (%)

Blood day14

1.4

5

Candida ++ αIL-17A

Candida +100

Lung day0

p = 0.0059

100 μm

100 μm200

0Candida − Candida +

6

1
0

0

0

Ashcroft score

Hydroxyproline(μg/Left lung)

50

1.4

500

300

G
8
7

p = 0.0460

Blood day0

1000

F

60

H

1500

0

Candida +E
p = 0.3142

2000

IL-17A+ in CD4+ T cells (%)

30

1.2

p = 0.0088

2500

20

Il17a/18s

40

IL-17A+ in CD4+ T cells (%)

50

CD25+ in CD4+ T cells (%)

M2 in alveolar macrophages
(%)

1.4

1.4

p = 0.0143

p = 0.0500

p = 0.0127

IL-17A concentrationin lung (pg/ml)

p = 0.4624

p = 0.0864

C

B

Lung day14

IL-17A concentrationin serum (pg/ml)

A

2

1

0

0

Candida +100 μm

100 μm

100 μm2

1

Candida −100 μmCandida −Candida +Candida −Candida +p = 0.2087

20
10
0

Candida − Candida +

9

11
10
9
8
7
6

2
0

Candida+G

Candida++αIL-17A

0100

IL-17A (ng/ml)

αSMA-PE

7
6

4

Candida − Candida +20
10Snail2/18s

p = 0.0209

0

Candida − Candida +F

p = 0.0209

8

p = 0.0209

6
4
2
0
6

p = 0.0339

0Col1a1-FITC

20
100
IL-17A (ng/ml)
DAPI

Snail1/18s

4

Fn/18sCol1a1/18s

6

Acta2/18s

p = 0.0455

8

8
6
4
2
0
8
6
4
2
0
8
6
4
2
0

8

30

5

E

D
αSMA+/Col1a1+
in endothelial cells (%)

128

p = 0.0209

4
2
0

0

20
100
IL-17A (ng/ml)

MergedH

20

Candida −

10
5
0
98
96
94
92
90
88〜
86〜
84
0

30
20
10
0

2

15

40

0

4

p = 0.0495

p = 0.0495

020
IL-17A (ng/ml)

αSMA-PEp = 0.027050

p = 0.00283

60Candida − Candida +3.0
Relative fluorescenceintensity

30

10

p = 0.0900

40

αSMA+/Col1a1+
CD31+ in cells (%) in CD31+ cells (%)

40

C

p = 0.0275

13
αSMA+/Col1a1+
in fibroblasts (%)αSMA+/Col1a1+
in epithelial cells (%)

Epithelial cells
in CD45-/CD31- (%)

50

B

Endothelial cells

αSMA+/Col1a1+
in endothelial cells (%)

p = 0.0086

Relative fluorescenceintensity

A

Fibroblasts

Endothelial cells
in lung (%)

Epithelial cells

100

IL-17A (ng/ml)

Col1a1-FITC
p = 0.00208

2.5
2.0
1.5
1.0
0.5
0

0

100

IL-17A (ng/ml)

Candida +Lung

Intestinal tract

Bleomycin
EMT

Dysbiosis+
C. albicans proliferation

IL-17A

IL-17A producing
Th17 cells(IL-17A+/CD3+/CD4+)
Rorc expressing
Th17-skewed lymphocytes(Rorc+/IL-17A−/CD3+/CD4+)
Naive T cells

Fibroblast
activation

EndMT

FibrosisFig. S1A

100 μmCandida −Candida +Cells in the lamina propria /HPF

B
p = 0.9168
40

30

20

10

0

Candida −Candida +Fig. S2
A

−7

0

B

14

60

Days

p = 0.4005

PBS

Sacrifice

CPZ, CLDM and FLCZ in DW
Candida −CPZ, CLDM and C. albicans in DW

Hydroxyproline(μg/Left lung)

50
40
30
20
10

Candida +0

C：1000 μm1000 μm

100 μm

100 μmCandida −：PBS

Candida +：PBS

Candida −：PBS

Candida +：PBS

Fig. S3A

p = 0.2506

5

p = 0.0586

70

80
70

Lymphocytes (%)

Macrophages (%)

p = 0.2506

C

60
50
40
30
20

60
50
40
30
20
10

0

0

Candida +

3

2

1

10

Candida −4

Neutrophils (%)

90

B

Candida −Candida +0

Candida −Candida +Fig. S4
A
−7

0

14

Days
BLM

Sacrifice
DW

DW

FLCZ in DW
FLCZHydroxyproline (μg/Left lung)B60

p = 0.4647

50
40
30
20
10
0

DW

FLCZ

Fig. S5
A Feces100

Candida albicans
g_Candida
Saccaromyces cerevisiae

Relative abundance (%)

g_Saccharomyces

Candida albicans

Wallemia tropicalis

80

Aspergillus heterocaryoticus
unidentified
othersg_Candida
Saccaromyces cerevisiae60

g_Saccharomyces

40

Wallemia tropicalis
Aspergillus heterocaryoticus

20
0

B

unidentified
othersCandida −Candida +

BALF

100

Candida albicans
g_Candida
Cryptococcus uniguttulatus

Relative abundance (%)

k_Fungi

Candida albicans

g_Cladosporium

80

g_Metschnikowia

Schyzophyllum amplum
othersg_Candida
Cryptococcus uniguttulatus

60

k_Fungi40

g_Cladosporium
g_Metschnikowia

20
0

C

Schyzophyllum amplum
othersCandida −Candida +

Lung

100

Candida albicans
g_Candida

Relative abundance (%)

g_Metschnikowia
k_Fungi
Candida sake80

g_Malassezia

60

Candida albicans
g_Candida

40

g_Metschnikowia
k_Fungi

200Candida sakeg_Malassezia

Candida −Candida +Fig. S6
A Feces100

p_Firmicutes
p_Actinobacteriota
p_Proteobacteria

Relative abundance (%)

p_Cyanobacteria
unassigned

80

p_Firmicutes

p_Verrucomicrobiota
d_Eukaryota
othersp_Actinobacteriota
p_Proteobacteria

60

p_Cyanobacteria

40

unassigned
p_Verrucomicrobiota

20
0

B

d_Eukaryota
othersCandida −Candida +

BALF

100

unassigned
p_Proteobacteria
p_Actinobacteriota

Relative abundance (%)

d_Eukaryota

unassigned

p_Ascomycota

80

p_ Firmicutes

p_Cyanbacteria

p_Proteobacteriaothersp_Actinobacteriota

60

d_Eukaryota

40

p_Ascomycota
p_ Firmicutes

20
0

C

p_CyanbacteriaothersCandida −Candida +

Lung

100

unassigned
p_Proteobacteria

Relative abundance (%)

p_Firmicutesd_Eukaryota

unassigned

p_Actinobacteriota

80d_Bacteria

p_Cyanobacteriaothersp_Proteobacteria
p_Firmicutes

60

d_Eukaryota

40

p_Actinobacteriota
d_Bacteria

20

p_Cyanobacteria

Candida +0

Candida −Candida +othersFig. S7A
Whole lung cells

Lung CD45+ cells

IsotypeCD80 and CD206

SSC

F4/80

CD80

M1 Mφ

CD206

CD11c

CD45

M2 Mφ

Isotype

CD25

Isotype

IL-17A

B

Lung lymphocytes
SSC

Whole lung cells

CD4

SSC

CD25

CD3
SSC

FSC

IL-17A

Hydroxyproline (μg/Left lung)Fig. S8
A
60
50
40
30
20
10
0

p = 0.0041
0

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

IL-17A+ in CD4+ T cells (%)

Fig. S9A

p = 0.4624

p = 0.8065

B

p = 0.3272

C

p = 0.5637

1.2

1.0

1.0

1.0

0.8

0.8

0.8
0.6
0.4

0.6

0.6

0.4

0.4

0.2

0.2

0.2

0

0

0

Candida −Candida +Candida −Candida +35

TGF-β concentrationin serum (ng/ml)

1.2

Tgfb/18s

1.2

Gata3/18s

Tbx21/18s

40

30
25
20
15
10
5

Candida −Candida +0

Candida −Candida +Fig. S10
−7

0

14

Days
BLM

Candida −Sacrifice

CPZ, CLDM and FLCZ in DW
CPZ, CLDM and C. albicans in DW

Candida +αIL-17A or isotype control

Fig. S11
2.0

C. albicans(×10^5 CFU/ml)

1.5

1.0

0.5

0

Candida− Candida+ Candida++αIL-17A

Fig. S12
A

B

−7

0

2.0

14

BLM

C. albicans(×10^4 CFU/ml)

Days
Sacrifice
DW
DW
C. albicans
w/o ABx

C. albicans in DW

p = 0.0194

1.4

60

1.2

p = 0.8312

50

1.0
0.8
0.6
0.4

40
30
20

0.2

10

0

0

DW

C.albicans
w/o ABx

1.2
0.8
0.4
0

D

Hydroxyproline(μg/Left lung)

IL-17A+ in CD4+ T cells (%)

C

1.6

DW

C.albicans
w/o ABx

DW

C.albicans
w/o ABx

−7

0

B

14

Days
BLM

SacrificeABx (C. albicans −)CPZ and CLDM in DW
ABx

Fecal culture
ABx (C. albicans +)IL-17A+ in CD4+ T cells (%)

Fig. S13A0.8

p = 0.0814

0.6
0.4
0.2
0

ABx
ABx
(Candida −) (Candida +)Fig. S14A

B

Fibroblasts

Lung CD45−/CD31− cells
EpCAM−

Col1a1Whole lung cells

αSMA
SSC

SSC

Epithelial cells

EpCAM
Col1a1

CD45&CD31

EpCAM+αSMA

C
Whole lung cells

Lung CD45−/CD31+ cells

CD31

Col1a1

Endothelial cells

CD45

αSMA