ORIGINAL RESEARCH
published: 20 February 2020
doi: 10.3389/fvets.2020.00012

Upregulation of PD-L1 Expression by
Prostaglandin E2 and theEnhancement of IFN-γ by Anti-PD-L1
Antibody Combined With a COX-2
Inhibitor in Mycoplasma bovis
Infection
Edited by:Paul M. Coussens,Michigan State University,
United States
Reviewed by:
Jayne Hope,
University of Edinburgh,
United Kingdom
Robin James Flynn,
University of Liverpool,
United Kingdom*Correspondence:
Satoru Konnai
konnai@vetmed.hokudai.ac.jp
Specialty section:
This article was submitted to
Veterinary Infectious Diseases,
a section of the journal
Frontiers in Veterinary Science
Received: 09 October 2019
Accepted: 08 January 2020
Published: 20 February 2020
Citation:
Goto S, Konnai S, Hirano Y, Kohara J,
Okagawa T, Maekawa N, Sajiki Y,
Watari K, Minato E, Kobayashi A,
Gondaira S, Higuchi H, Koiwa M,
Tajima M, Taguchi E, Uemura R,
Yamada S, Kaneko MK, Kato Y,
Yamamoto K, Toda M, Suzuki Y,
Murata S and Ohashi K (2020)
Upregulation of PD-L1 Expression by
Prostaglandin E2 and theEnhancement of IFN-γ by Anti-PD-L1
Antibody Combined With a COX-2
Inhibitor in Mycoplasma bovis
Infection. Front. Vet. Sci. 7:12.
doi: 10.3389/fvets.2020.00012

Shinya Goto 1 , Satoru Konnai 1,2*, Yuki Hirano 3 , Junko Kohara 3 , Tomohiro Okagawa 2 ,Naoya Maekawa 2 , Yamato Sajiki 1 , Kei Watari 1 , Erina Minato 4 , Atsuhi Kobayashi 4 ,Satoshi Gondaira 5 , Hidetoshi Higuchi 5 , Masateru Koiwa 5 , Motoshi Tajima 5 , Eiji Taguchi 6 ,Ryoko Uemura 7 , Shinji Yamada 8 , Mika K. Kaneko 8 , Yukinari Kato 8,9 , Keiichi Yamamoto 2,10 ,Mikihiro Toda 2,11 , Yasuhiko Suzuki 2,12,13 , Shiro Murata 1,2 and Kazuhiko Ohashi 1,2
1

Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan, 2 Department ofAdvanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan, 3 Agriculture Research
Department, Animal Research Center, Hokkaido Research Organization, Shintoku, Japan, 4 Department of Veterinary Clinical
Medicine, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan, 5 School of Veterinary Medicine, Rakuno
Gakuen University, Ebetsu, Japan, 6 Shibetsu Animal Hospital, Shibetsu, Japan, 7 Department of Veterinary Medical Science,
Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan, 8 Department of Antibody Drug Development, Graduate Schoolof Medicine, Tohoku University, Sendai, Japan, 9 New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan,
10
Research and Development Center, Fuso Pharmaceutical Industries, Ltd., Osaka, Japan, 11 New Business and International
Business Development, Fuso Pharmaceutical Industries, Ltd., Osaka, Japan, 12 Division of Bioresources, Research Center for
Zoonosis Control, Hokkaido University, Sapporo, Japan, 13 Global Station for Zoonosis Control, Global Institution for
Collaborative Research and Education (GI-CoRE), Hokkaido University, Sapporo, Japan

Bovine mycoplasmosis caused by Mycoplasma bovis results in pneumonia and mastitis
in cattle. We previously demonstrated that the programmed death 1 (PD-1)/PD-ligand
1 (PD-L1) pathway is involved in immune dysfunction during M. bovis infection andthat prostaglandin E2 (PGE2 ) suppressed immune responses and upregulated PD-L1
expression in Johne’s disease, a bacterial infection in cattle. In this study, we investigated
the role of PGE2 in immune dysfunction and the relationship between PGE2 and thePD-1/PD-L1 pathway in M. bovis infection. In vitro stimulation with M. bovis upregulated
the expressions of PGE2 and PD-L1 presumably via Toll-like receptor 2 in bovine
peripheral blood mononuclear cells (PBMCs). PGE2 levels of peripheral blood in infected
cattle were significantly increased compared with those in uninfected cattle. Remarkably,plasma PGE2 levels were positively correlated with the proportions of PD-L1+ monocytesin M. bovis-infected cattle. Additionally, plasma PGE2 production in infected cattle wasnegatively correlated with M. bovis-specific interferon (IFN)-γ production from PBMCs.
These results suggest that PGE2 could be one of the inducers of PD-L1 expression andcould be involved in immunosuppression during M. bovis infection. In vitro blockade

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assays using anti-bovine PD-L1 antibody and a cyclooxygenase 2 inhibitor significantly
upregulated the M. bovis-specific IFN-γ response. Our study findings might contributeto the development of novel therapeutic strategies for bovine mycoplasmosis that target
PGE2 and the PD-1/PD-L1 pathway.
Keywords: immunoinhibitory molecules, PD-1, PD-L1, T-cell exhaustion, prostaglandin E2 , immune dysfunction,
Mycoplasma bovis, cattle

INTRODUCTION

of PGE2 in the immune dysfunction of bovine mycoplasmosis has
not yet been fully investigated.
In this study, we investigated the role of PGE2 in immune
dysfunction and the relationship between PGE2 and the PD1/PD-L1 pathway in M. bovis infection. We believe that ourfindings will help in the development of novel strategies forbovine mycoplasmosis.

Bovine mycoplasmosis caused by Mycoplasma bovis ischaracterized by chronic pneumonia, therapy-resistantmastitis, otitis, and arthritis (1–4). M. bovis has several
immunosuppressive characteristics in vitro, such as inhibition of
proliferative response of bovine peripheral blood mononuclear
cells (PBMCs), induction of apoptosis in bovine lymphocytes,
and delay of apoptosis in bovine monocytes, along with
suppressed production of interferon (IFN)-γ and tumor necrosis
factor (TNF)-α (5–7). These characteristics could cause chronic
progression of the disease and, especially during the lunginfection, and allow coinfection with other bacteria and viruses(2, 3). However, the mechanisms underlying the dysfunction in
M. bovis infection have remained unclear.
Programmed death-1 (PD-1) is an immunoinhibitory
receptor that is expressed on activated T cells and has been
involved in immune dysfunction during various chronic
infections (8–10). After binding of PD-ligand 1 (PD-L1), PD-1
induces T-cell dysfunction by inhibiting T-cell receptor signaling.
This immune dysfunction is called T cell exhaustion. On theother hand, treatment with monoclonal antibodies (mAbs)
specific to PD-1 or PD-L1 is capable of reactivating functions of
exhausted T cells. PD-1/PD-L1 could be a potential therapeutictarget in patients with chronic infections. We previously
demonstrated that the expression of PD-1 on T cells and PD-L1
on monocytes were significantly increased in M. bovis-infected
cattle (11). Furthermore, the blockade of PD-1/PD-L1 pathway
by antibodies in vitro activated immune responses in M. bovisinfected cattle (11). Therefore, the T cell exhaustion caused by
PD-1/PD-L1 might be involved in the immune dysfunction
during bovine mycoplasma.
Prostaglandin E2 (PGE2 ) is one of the lipid mediators thatare derived from arachidonic acid synthesized by cyclooxygenase
isoenzymes (COX-1 and COX-2) (12, 13). It is known that
PGE2 promotes the immune dysfunction associated with several
tumors and chronic inflammation (14, 15). Interestingly, recent
reports on cancer have shown a relationship between PGE2
and the PD-1/PD-L1 pathway (16, 17). Our previous study
demonstrated that PGE2 suppressed the Th1 response in cattle
and upregulated PD-L1 expression in bovine PBMCs in vitro(18). In addition, PGE2 showed immune dysfunction effects inother bovine chronic diseases, Johne’s disease (18), which is
known to be a chronic bovine disease by Mycobacterium avium
subsp. paratuberculosis (MAP) and bovine leukemia virus (BLV)
infection (19). Furthermore, the dual blocking of PGE2 andthe PD-1/PD-L1 pathway substantially enhanced the MAP and
BLV-specific T-cell reaction in cattle. However, the involvement

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MATERIALS AND METHODS
Bacterial Strain
M. bovis strain PG45 (ATCC25523) was used in the experimentsof this study. M. bovis was cultured in NK broth (Miyarisan
Pharmaceutical, Tokyo, Japan) at 37◦ C for 72 h and collectedby centrifugation. The bacteria were washed with phosphatebuffered saline (PBS), and colony-forming units were counted
using the NK agar plate (Miyarisan Pharmaceutical) by dilution
method. The bacteria were then resuspended in RPMI 1640
medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10%
heat-inactivated fetal bovine serum (FBS) (Thermo Fisher
Scientific, Waltham, MA, USA), 2 mM L-glutamine, 100
U/ml penicillin, and 100 µg/ml streptomycin (Thermo Fisher
Scientific) and stored at −80◦ C until use.

Ethical Approval
All experimental procedures were conducted following approval
from the local committee for animal studies according to the
Hokkaido University (17–24). Written informed consent was
obtained from all owners of cattle sampled in this study.

Bovine Samples
Peripheral blood samples of cattle were obtained from adult
Holstein-breed cattle in Hokkaido, Japan. Cattle infected with
M. bovis were diagnosed clinically and microbiologically at
Rakuno Gakuen University and Hokkaido University. M. bovis
infection was confirmed with PCR by using clinical samples
as described previously (21). The symptoms of infected cattle
included pneumonia, arthritis, and otitis media. Control blood
samples of M. bovis-uninfected cattle were obtained from adult
Holstein-breed cattle in Hokkaido, Japan. Negative control cattle
were serologically negative for M. bovis infection according to
enzyme-linked immunosorbent assay (ELISA). Briefly, ELISA
plates (Thermo Fisher Scientific) were coated with 100 µl ofsolubilized M. bovis (PG45, 50 µg/ml in carbonate buffer) as thetarget antigen at 37◦ C for 17 h. After washing the plates fourtimes with a wash solution (PBS with 0.1% Tween20), 100 µlof serum sample was added to each plate. After incubation at
37◦ C for 1 h, the plates were washed three times with TSB-T [PBS

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Pharmaceutical) for 72 h and the broth samples were tested with
PCR targeting M. bovis-specific gene (21). Then, fresh PBMCs
were isolated from the same individual and cultured in the 24-h
culture supernatants at 37◦ C under 5% CO2 for 24 h.

with 50 mM Tris, 0.1% bovine serum albumin (BSA), and 0.05%
Tween20] and incubated with skim milk (Wako, Osaka, Japan)
as a protein blocker at 37◦ C for 2 h. After washing the plates
thrice with TSB-T, protein G-conjugated horseradish peroxidase(Rockland Immunochemicals, Pottstown, PA, USA) was added
to the wells and the plates were incubated at 37◦ C for 1 h. Afterwashing the plates thrice with TSB-T, 3-ethylbenzothiazolin-6sulfonic acid (ABTS; Sera Care, Milford, MA, USA) was added to
the wells and the optical density was measured at 415 nm using aplate reader (iMarkTM Microplate Absorbance Reader, Bio-Rad,
Hercules, CA, USA).

Quantitation of PGE2 by ELISA
To investigate whether M. bovis antigen and TLR2 signaling
promote PGE2 production, PBMCs, CD14+ cells, or CD14−
cells (1 × 106 cells) from uninfected cattle were incubatedwith live M. bovis, heat-killed M. bovis, or FSL-1 (Adipogen
Life Sciences) as described above. Culture supernatants werecollected, and PGE2 levels were measured using Prostaglandin
E2 Express ELISA kit (Cayman Chemical), according to the
manufacturer’s instructions. The optical density was read at
450 nm in a microplate reader (Corona Electronics, Tokyo,
Japan). To compare PGE2 levels in peripheral blood between
infected cattle and uninfected cattle, PGE2 concentrations inserum or plasma were measured using ELISA.

Cell Preparation and Culture
Bovine PBMCs were purified from blood samples with density
gradient centrifugation using Percoll (GE Healthcare, Little
Chalfont, England, UK). As described previously (20), CD14+
cells were freshly isolated from bovine PBMCs using the
autoMACS Pro System (Miltenyi Biotec, Bergisch Gladbach,
Germany) with anti-bovine CD14 mAb (CAM36A, Washington
State University Monoclonal Antibody Center, Pullman, WA,USA), and anti-mouse IgG1 MicroBeads (Miltenyi Biotec).
CD14− cells were prepared from negative fractions of CD14+
cell sorting. The purity of cell populations was confirmed using
FACS Verse (BD Biosciences, San Jose, CA, USA). Only highly
pure populations (>90%) were used for experiments.
PBMCs, CD14+ cells, or CD14− cells (1 × 106 cells)
from uninfected cattle were seeded into each well of a 48well flat-bottom plate (Corning Inc., Corning, NY, USA) with
RPMI 1640 medium (Sigma-Aldrich) containing 10% heatinactivated FBS (Thermo Fisher Scientific), 2 mM L-glutamine,
100 U/ml penicillin, and 100 µg/ml streptomycin (Thermo Fisher
Scientific) and were cultured in the presence of live M. bovis ata multiplicity of infection (MOI) of 0.1:1, 1:1, or 10:1; 1.5 ng/mlof heat-killed M. bovis; 2.5 µM of PGE2 (Cayman Chemical,
Ann Arbor, MI, USA); or 100 ng/ml of fibroblast-stimulating
lipopeptide-1 (FSL-1; Adipogen Life Sciences, San Diego, CA,
USA) at 37◦ C under 5% CO2 for 24 h. Heat-killed M. bovis was
prepared by heating the bacteria to 70◦ C for 10 min.
To investigate the effects of blocking TLR2 signaling,
PBMCs (1 × 106 cells) from uninfected cattle were incubatedwith sparstolonin B (SsnB; Sigma-Aldrich) in the presence of
100 ng/ml of FSL-1 or 1.5 ng/ml of heat-killed M. bovis in a 48well flat-bottom plate (Corning Inc.) at 37◦ C under 5% CO2
for 24 h. DMSO (Nacalai Tesque, Kyoto, Japan) was used as
negative controls.
To assess the effect of soluble factors on PD-L1 expression, 24h culture supernatants of PBMCs incubated with live M. bovis
were collected and fresh PBMCs were cultured in these culture
supernatants. To prepare 24-h culture supernatants, PBMCs (1 ×106 cells) from uninfected cattle were seeded and were cultured
with or without live M. bovis at a MOI of 0.1:1, 1:1, or 10:1
as described above. After centrifugation (300× g for 3 min),the culture supernatants of PBMCs were collected. Bacteria
and cell-free supernatants were obtained by filtration througha 0.2-µm filter (Pall Life Sciences, Washington, NY, USA). To
confirm that the supernatants were not contaminated with M.
bovis, each supernatant was incubated in NK broth (Miyarisan

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Flow Cytometric Analysis of PD-L1
To investigate PD-L1 expression on immune cells, PBMCs were
blocked with PBS containing 10% goat serum (Thermo Fisher
Scientific) at 37◦ C for 15 min and were washed and stainedwith anti-bovine PD-L1 mAb (4G12, rat IgG2a ; 20) or rat IgG2aisotype control (R35-95, BD Biosciences) in the presence ofanti-CD11b mAb (CC126, mouse IgG2b , Bio-Rad, Hercules,
USA) at room temperature for 20 min. After washing with PBS
containing 1% BSA (Sigma-Aldrich), cells were stained with
PerCp/Cy5.5-conjugated anti-CD14 mAb (CAM36A, mouse
IgG1 , Washington State University Monoclonal Antibody
Center), APC-conjugated anti-rat immunoglobulin polyclonal
Ab (Southern Biotech, Birmingham, AL, USA), FITC-conjugatedanti-mouse IgG2b polyclonal Ab (Beckman Coulter, Fullerton,
CA, USA), and Fixable Viability Dye eFluor 780 (eBioscience,
San Diego, CA, USA) at room temperature for 20 min.
PerCp/Cy5.5-conjugated mouse IgG1 isotype control (15H6,Southern Biotech) and mouse IgG2b isotype control (A1, Southern Biotech) were used as isotype-matched control
antibodies for fluorescence minus one staining. mAbs CAM36A
and 15H6 were conjugated with PerCp/Cy5.5 using theLightning-Link PerCp/Cy5.5 Antibody Labeling Kit (Innova
Biosciences, Cambridge, England, UK). Cells were then washedand immediately analyzed using FACS Verse (BD Biosciences)and FACSuite Software (BD Biosciences).

IFN-γ Assay
To investigate IFN-γ levels in peripheral blood of infected cattle,
IFN-γ concentration in plasma were measured using Bovine
IFN-γ ELISA development kit (Mabtech, Nacka Strand, Sweden).
To investigate M. bovis-specific IFN-γ response in infected cattle,
PBMCs (4 × 105 cells) from infected cattle were incubated with
1.5 µg/ml of heat-killed M. bovis in 96-well plates (Corning,Inc.) at 37◦ C under 5% CO2 for 5 days. Subsequently, culture
supernatants were obtained from individual wells and were
tested for bovine IFN-γ using the ELISA kit (Mabtech) as
described above.

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Statistical Analysis

Immunohistochemical Assays of PD-L1
and PGE2

Differences were identified using Dunnett’s test, Mann–Whitney
U-test, and Steel–Dwass test. Correlation statistics wereanalyzed using Spearman correlation analysis. The statisticalanalysis program MEPHAS (http://www.gen-info.osaka-u.ac.jp/
MEPHAS/) was used to perform statistical analysis. A P < 0.05
was considered statistically significant.

Immunohistochemical assays were performed as previously
described (18), with slight modification. Briefly, lung tissue wascollected from M. bovis-infected cattle with pneumonia (JapaneseBlack, ale, 2 months old). M. bovis infection was confirmed withthe loop-mediated isothermal amplification assay at Miyazaki
University as described previously (22). Then, sections of the lunglesions were immunohistochemically stained for PGE2 and PDL1 using anti-PGE2 polyclonal Ab (ab2318, Abcam, Cambridge,
England, UK) and anti-PD-L1 mAb (6C11-3A11, Rat IgG2 a; 22).

RESULTS
M. bovis Infection Upregulated PD-L1
Expression via PGE2
Our previous study showed that expression of PD-L1 on
monocytes was increased in PBMCs of M. bovis-infected cattle(11). To confirm whether M. bovis directly upregulates PDL1 expression in bovine PBMCs during in vitro infection,
PBMCs from uninfected cattle were cultured with or without
live M. bovis and PD-L1 expression was analyzed on monocytesby flow cytometry. As shown in Figure S1, CD11b+ CD14+
monocytes were gated and analyzed for expression of PDL1. PD-L1 expression on CD11b+ CD14+ monocytes was
upregulated in the culture with live M. bovis when comparedwith that in the culture without live M. bovis (Figure 1A).
Our recent studies showed that PGE2 was one of the inducersof PD-L1 expression in Johne’s disease. Thus, to identify
whether PGE2 induced by live M. bovis upregulated PDL1 expression, PGE2 production in culture supernatants wasassessed using ELISA. PGE2 production was increased when
PBMCs were cultured with live M. bovis. Interestingly, PGE2
production was positively correlated with PD-L1 expression on
CD11b+ CD14+ monocytes (Figures 1B,C). To confirm whether
innate immune response against M. bovis contribute to PGE2
production and PD-L1 expression in the PBMC cultures,
PBMCs from uninfected cattle were cultured with or without
heat-killed M. bovis (Figures 1D–F). Heat-killed M. bovis also
upregulated PD-L1 expression on CD11b+ CD14+ monocytes
in line with increased PGE2 production in culture supernatants(Figures 1D–F). To test whether culture supernatants containing
PGE2 induced by M. bovis upregulated PD-L1 expression,
PBMCs from uninfected cattle were cultured in supernatantfrom PBMCs that were co-cultured with live M bovis for 24 h.
PD-L1 expression on CD11b+ CD14+ monocytes cultured in
24-h culture supernatants of PBMCs with live M. bovis was
upregulated when compared with that without live M. bovis(Figure 1G). For further confirmation, PBMCs from uninfected
cattle were incubated with PGE2 and PD-L1 expression on
monocytes was analyzed by flow cytometry. PD-L1 expression
on CD11b+ CD14+ monocytes incubated with PGE2 wasincreased when compared with that treated with negative
controls (Figure 1H). These results indicate that M. bovis could
upregulate PD-L1 expression on monocytes via PGE2 .
To identify the major cell type that produced PGE2 in the
PBMC culture, PGE2 productions from isolated CD14+ andCD14− cells cultured with live M. bovis were measured using
ELISA. PGE2 production from CD14+ cells was significantly
increased when compared with that from CD14− cells

Functional Analysis of Combined COX-2
Inhibition and PD-L1 Blockade
To evaluate T-cell response against the bacterial antigenic
stimulation, cell proliferation assay was performed. PBMCs
isolated from M. bovis-infected cattle were labeled withcarboxyfluorescein diacetate succinimidyl ester (CFSE) (Thermo
Fisher Scientific) and cultured with 1.5 µg/ml of heat-killed
M. bovis in 96-well plates (Corning, Inc.) with RPMI 1640
medium (Sigma-Aldrich) supplemented with 10% heatinactivated FBS (Thermo Fisher Scientific), 2 mM L-glutamine,
100 U/ml penicillin, and 100 µg/ml streptomycin (Thermo
Fisher Scientific) at 37◦ C with 5% CO2 for 5 days. Then,PBMCs were harvested and incubated in PBS containing
10% goat serum (Sigma-Aldrich) at room temperature for
15 min to prevent non-specific reactions. The cells were thenstained with anti-CD4-Alexa Fluor 647 (CC30; Bio-Rad),
anti-CD8-PerCp/Cy5.5 (CC63, Bio-Rad), anti-IgM-PE/Cy7
antibodies (IL-A30; Bio-Rad), and Fixable Viability Dye eFluor
780 (eBioscience, San Diego, CA, USA) at room temperaturefor 20 min. mAb CC30 was pre-labeled with Alexa Fluor 647
Zenon Mouse IgG1 Labeling Kit (Thermo Fisher Scientific).
mAbs CC63 and IL-A30 were conjugated with PerCp/Cy5.5
and PE/Cy7, respectively, with Lightning-Link Conjugation
Kits (Innova Biosciences). The cells were then washed with PBS
containing 1% BSA (Sigma-Aldrich) and analyzed immediately
by FACS Verse (BD Biosciences) and FACSuite Software(BD Biosciences).
To investigate the effects of dual blockade using a COX-2
inhibitor and anti-PD-L1 mAb, PBMCs (4 × 105 cells) from
infected cattle were incubated with 10 µg/ml of anti-bovine
PD-L1 mAb (4G12; 21) and/or 10 µM of meloxicam (SigmaAldrich) in the presence of 1.5 µg/ml of heat-killed M. bovis,
or 1.0 µg/ml of anti-CD3 mAb (MM1A, Washington State
University Monoclonal Antibody Center) and 1.0 µg/ml of antiCD28 mAb (CC220, Bio-Rad) in 96-well plates (Corning, Inc.)
with RPMI 1640 medium (Sigma-Aldrich) supplemented with
10% heat-inactivated FBS (Thermo Fisher Scientific), 2 mM Lglutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin(Thermo Fisher Scientific) at 37◦ C under 5% CO2 for 5 days.
DMSO (Nacalai Tesque) and rat IgG (Sigma-Aldrich) were usedas negative controls. Culture supernatants were obtained fromindividual wells and were tested for IFN-γ production using the
ELISA kit (Mabtech) as described above.

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FIGURE 1 | M. bovis upregulates PD-L1 and PGE2 expression. (A–F) PBMCs from M. bovis-uninfected cattle were incubated with (A–C) live M. bovis (MOI of 0.1, 1,
or 10) or (D–F) heat-killed M. bovis (1.5 ng/ml) for 24 h. (A) PD-L1 expression on CD11b+ CD14+ monocytes (untreated: n = 14, MOI 0.1: n = 14, MOI 1: n = 14, MOI
10: n = 14) was determined using flow cytometry. (B) PGE2 levels in culture supernatants (untreated: n = 9, MOI 0.1: n = 9, MOI 1: n = 9, MOI 10: n = 9) were
determined using ELISA. (C) Positive correlation is noted between PD-L1 expression on CD11b+ CD14+ monocytes and PGE2 levels under live M. bovis stimulation(MOI of 10, n = 11). (D) PD-L1 expression on CD11b+ CD14+ monocytes (untreated: n = 13, heat-killed M. bovis: n = 13) were determined using flow cytometry. (E)PGE2 levels in culture supernatants (untreated: n = 13, heat-killed M. bovis: n = 13) were determined using ELISA. (F) Positive correlation is noted between PD-L1
expression on CD11b+ CD14+ monocytes and PGE2 levels under heat-killed M. bovis stimulation (n = 13). (G) PD-L1 expression on CD11b+ CD14+ monocytes(Continued)

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FIGURE 1 | cultured in 24-h culture supernatant of PBMCs with or without live M. bovis (untreated: n = 9, MOI 0.1: n = 9, MOI 1: n = 9, MOI 10: n = 9) wereanalyzed by flow cytometry. (H) PBMCs from uninfected cattle were incubated with PGE2 , and PD-L1 expression on CD11b+ CD14+ monocytes (DMSO: n = 6,
PGE2 : n = 6) was determined using flow cytometry. The bars are the median of the values (A,B,D,E,G,H). Statistical significance was determined by the Steel–Dwass
test (A,B,G) or Mann–Whitney U-test (D,E,H). Correlation statistics were analyzed using Spearman’s correlation analysis (C,F). *P < 0.05.

FIGURE 2 | PGE2 production from CD14+ cells induced by M. bovis. CD14+ cells or CD14− cells from uninfected cattle were incubated with live M. bovis (MOI of 10)
for 24 h. (A) PGE2 production was determined by ELISA (CD14− cells untreated: n = 6, CD14− cells Live M. bovis: n = 6, CD14+ cells untreated: n = 6, CD14+ cells
M. bovis: n = 6). (B) PD-L1 expression on CD14+ cells (untreated: n = 6, live M. bovis: n = 6) was determined by flow cytometry. The bars are the median of thevalues (A,B). Statistical significance was determined by the Steel–Dwass test (A) or Mann–Whitney U-test (B). **P < 0.01.

(Figure 2A). In the CD14+ fraction, PD-L1 expression on
CD11b+ CD14+ monocytes cultured with live M. bovis wasincreased (Figure 2B). These results represent that CD14+ cells
could be a major cell type producing PGE2 against M. bovis.

stimulation (Figure 3D). CD14+ cells are considered as one of
the major cell types expressing TLR2 (29). These results suggestthat M. bovis could induce PGE2 production from monocytes
presumably via TLR2.

Induction of PGE2 via Toll-Like Receptor(TLR) 2

Upregulation of PGE2 Production in
M. bovis-Infected Cattle

Because PGE2 production was increased by heat-killed M. bovis,
we hypothesized that pattern-recognition receptors recognized
M. bovis and their signaling induced PGE2 production. Previous
research showed that other mycoplasma, M. fermentans, which
was first isolated from the human urogenital tract (25), induced
PGE2 production via TLR2 in human monocytes (26, 27).
Thus, to test whether PGE2 production and PD-L1 upregulation
were induced in bovine PBMCs under TLR2 stimulation,
PBMCs from uninfected cattle were incubated with a TLR2/6
agonist (FSL-1, 28), which is a synthetic lipoprotein of M.
salivarium, which been implicated in eye and ear disorders,oral infection, septic arthritis, and periodontal disease in human(28), and PGE2 production in culture supernatants and PDL1 expression on monocytes were analyzed. FSL-1 upregulated
PGE2 production from PBMCs and PD-L1 expression on
CD11b+ CD14+ monocytes (Figures 3A,B). Consistent withthe results of M. bovis stimulation, PGE2 production was
positively correlated with PD-L1 expression on CD11b+ CD14+
monocytes under FSL-1 stimulation (Figure 3C). Inhibition of
TLR2/4 signaling by a selective antagonist (sparstolonin B; SsnB)
decreased PGE2 production induced by FSL-1 and M. bovis

We found that M. bovis induced PGE2 production andupregulated PD-L1 expression in vitro. To confirm that PGE2 isassociated with immune dysfunction in bovine mycoplasmosis,
we analyzed serum PGE2 levels in cattle naturally infectedwith M. bovis, which were diagnosed in the previous studies(21, 30). Serum PGE2 levels were significantly increased in M.
bovis-infected cattle when compared with that in uninfected
cattle (Figure 4A). No differences in serum PGE2 levels wereobserved among the mycoplasmosis types with different clinicalsymptoms in the M. bovis-infected cattle (Figure 4B). Because
PBMC samples were not available for the previous clinicalsamples used in Figures 4A,B, we collected blood samples ofM. bovis-infected cattle and analyzed plasma levels of PGE2
and other immunological factors (Figures 4C–E). Interestingly,
plasma levels of PGE2 were positively correlated with theproportions of PD-L1+ CD11b+ CD14+ cells in infected cattle(Figure 4C). To investigate the relationship between PGE2 andimmune response, plasma IFN-γ levels and IFN-γ production
against heat-killed M. bovis from PBMCs of infected cattle wereanalyzed using ELISA and their correlation with plasma PGE2
levels. Plasma IFN-γ levels and IFN-γ production against M.

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FIGURE 3 | TLR2 signaling upregulates PD-L1 and PGE2 expression. PBMCs from uninfected cattle were incubated with FSL-1 (100 ng/ml). (A) PGE2 production
was determined by ELISA (PBS: n = 11, FSL-1: n = 11). (B) PD-L1 expression on CD14+ cells (PBS: n = 11, FSL-1: n = 11) was determined by flow cytometry. (C)Positive correlation is noted between PD-L1 expression on CD11b+ CD14+ monocytes and PGE2 levels under FSL-1 stimulation (n = 13). (D) PBMCs from uninfected
cattle were incubated with FSL-1 (100 ng/ml) or heat-killed M. bovis (1.5 ng/ml) under inhibition of TLR signaling by SsnB and PGE2 production was determined by
ELISA (DMSO: n = 9, SsnB: n = 9, DMSO and FSL-1: n = 9, SsnB and FSL-1: n = 9, DMSO and M. bovis: n = 9, SsnB and M. bovis: n = 9). The bars are themedian of the values (A,B,D). Statistical significance was determined by the Mann–Whitney U-test (A,B) or the Steel–Dwass test (D). Correlation statistics wereanalyzed using Spearman’s correlation analysis (C). *P < 0.05.

fibroblasts infiltrating the lesions (Figure 5A), and PGE2 wasproduced by epithelial cells and macrophages infiltrating thelesions (Figure 5C). On the other hand, the expressions of PD-L1
and PGE2 in the healthy lung of M. bovis-uninfected cattle werevery weak (Figures 5B,D).

bovis were negatively correlated with plasma PGE2 levels in
infected cattle (Figures 4D,E). These results suggest that PGE2
is associated with PD-L1 expression and immune dysfunction in
M. bovis-infected cattle.

Expressions of PD-L1 and PGE2
Associated With Pneumonic Lung Lesions
in M. bovis-Infected Cattle

Combined Blockade of the PD-1/PD-L1
Pathway and PGE2 Enhanced the M. bovis
IFN-γ Response

To determine the expressions of PD-L1 and PGE2 associated withlung lesions in infected cattle, immunohistochemical analysis
was conducted on pneumonic lung tissues from cattle with
M. bovis infection. PD-L1 was expressed on macrophages andFrontiers in Veterinary Science | www.frontiersin.org

We previously reported that PD-1/PD-L1 blockade by antibovine PD-1/PD-L1 mAbs enhanced the M. bovis-specific IFN-γ
response in vitro (11). Recent research showed that the combined

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FIGURE 4 | Analysis of PGE2 in cattle infected with M. bovis. (A,B) Serum PGE2 levels in M. bovis-infected cattle (n = 89; Mastitis: n = 62, Pneumonia: n = 21,
Pneumonia with arthritis: n = 6) and uninfected cattle (n = 18) was determined by ELISA. (C–E) Correlation between plasma PGE2 levels in M. bovis-infected cattlewith arthritis (n = 13), otitis (n = 10), pneumonia (n = 5), and other factors. (C) Correlation between plasma PGE2 levels and plasma IFN-γ levels (n = 29). (D)Correlation between the plasma levels of PGE2 and the proportions of PD-L1+ monocytes (n = 25). (E) Correlation between plasma PGE2 levels and IFN-γ production
from PBMCs against heat-killed M. bovis (n = 25). The bars are the median of the values (A,B). Statistical significance was determined by the Mann–Whitney U-test(A) or the Steel–Dwass test (B). Correlation statistics were analyzed using Spearman’s correlation analysis (C–E). *P < 0.05, **P < 0.01.

blockade of the PD-1/PD-L1 pathway and PGE2 by anti-PDL1 mAb and a COX-2 inhibitor enhanced immune responses
against several pathogens (18, 19, 31). Thus, we evaluatedthe immune enhancement of dual blockade of the PD-1/PDL1 pathway and PGE2 with regard to the M. bovis-specific

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IFN-γ response using anti-PD-L1 mAb and COX-2 inhibitor,
meloxicam. As shown in Figure S2, heat-killed M. bovis
stimulation induced the proliferative response of CD4+ andCD8+ T cells in the cultivated PBMCs isolated from M. bovisinfected cattle. IFN-γ production from PBMCs stimulated with

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PGE2 -Induced Immunodysfunction During Bovine Mycoplasmosis

FIGURE 5 | PD-L1 and PGE2 expression in the lung of M. bovis-infected cattle with pneumonia. Immunohistochemical staining of PD-L1 (A,B) and PGE2 (C,D) inlung tissues of cattle with or without M. bovis infection was performed using anti-bovine PD-L1 mAb (6C11-3A11) and human PGE2 polyclonal Ab (rabbit polyclonal).
Black arrowheads, PD-L1 positive macrophages; black arrows, PD-L1 positive fibroblasts; white arrowheads, PGE2 -positive macrophages; white arrows,
PGE2 -positive epithelial cells.

infection remains unknown. In this study, we revealed that
PGE2 produced by monocytes was one of the inducers of PD-L1
expression. In addition, we demonstrated that in vitro blockadeof the PD-1/PD-L1 pathway and PGE2 by anti-bovine PD-L1
Ab and a COX-2 inhibitor enhanced the IFN-γ response against
M. bovis. These results suggest that the PD-1/PD-L1 pathway
and PGE2 have therapeutic potentials for the control of M.
bovis infection.
PGE2 is typically known as a pro- and anti-inflammatory
mediator and its synthesis is induced by COX-2 and PGE
synthases (12, 13). Previous research in cancer showed
relationships between PGE2 and the PD-1/PD-L1 pathway (16,17). In human melanoma cells, COX-2 was positively correlatedwith PD-L1 expression (16). In a mouse model of bladdertumor, tumor-infiltrating PD-L1-expressing cells showed highlevels of the PGE2 -forming enzymes, namely, microsomal PGE2
synthase 1 (mPGES1) and COX-2 (17). Therefore, PGE2 wasrecently implicated in the induction of PD-L1 expression. Our
previous study showed that PGE2 upregulated PD-L1 expression
on bovine PBMCs and was associated with the progressionof Johne’s disease, which is a chronic bacterial infection of
cattle caused by MAP (18). Then, we hypothesized that thisimmune dysfunction via PGE2 -PD-L1 axis is commonly involvedin bovine chronic infections. Therefore, this study focusedon M. bovis infection, which is known to show immune

T-cell stimulators (combination of anti-CD3 mAb and anti-CD28
mAb; Figure 6A) or heat-killed M. bovis antigen (Figure 6B) was
significantly increased by anti-PD-L1 mAb or the combinationof meloxicam and anti-PD-L1 mAb when compared with thatin negative controls (combination of DMSO and rat IgG).
These results obtained by combining meloxicam and antiPD-L1 mAb support their potentials as novel treatments forM. bovis infection.

DISCUSSION
Bovine mycoplasmosis, especially associated with M. bovis
infection, is spreading globally, including in North America,
Europe, and Japan (32–36). M. bovis is a highly contagious
pathogen, and there are no effective vaccines owing to its
immunosuppressive effects. Although antibiotics are potentiallyeffective against M. bovis, strains resistant to antibiotics haverecently been emerging and spreading (37, 38). Therefore, a novel
strategy for the control of M. bovis infection is required.
Our previous study showed that PD-1-expressing T cells andPD-L1-expressing monocytes were increased in M. bovis-infected
cattle and that PD-1 and PD-L1 were closely associated with adecreased IFN-γ response against M. bovis (11). On the other
hand, the mechanism of PD-L1 upregulation during M. bovis

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results indicate that PGE2 induced by M. bovis could upregulate
PD-L1 expression on monocytes via STAT3 signaling. On
the other hand, many researchers have reported that PD-L1
expression can be induced by other factors, such as IFN-γ and
TNF-α production (41, 42). A previous report showed that M.
bovis induced IFN-γ and TNF-α production from PBMCs of
uninfected cattle (30). Further studies on other cytokines are
warranted to confirm the mechanisms of PD-L1 expression
during M. bovis infection.
TLRs are pattern-recognition receptors that play importantroles in early innate recognition and host immune responses
against several pathogens (43, 44). TLR2 plays the mostimportant role in Mycoplasma infection. TLR2 recognizes
lipoproteins from Mycoplasma spp. including M. bovis(25, 26, 45–48). TLR2/MyD88 signaling induces PGE2
production by COX-2 transcription via NF-κB (49), during not
only Mycoplasma infection (27, 50, 51) but also other bacterial
infection such as Mycobacterium bovis (52), Mycobacterium
leprae (53), and Staphylococcus aureus (54). TLR2 expression
has been demonstrated in several immune cells, such as
lymphocytes, monocytes (28), and some types of nonhematopoietic cells, such as epithelial cells (55). A previous
study in humans showed that CD14+ monocytes expressed
the highest level of TLR2 in blood (56). Indeed, the current
study revealed that CD14+ monocytes were a major cell typeproducing PGE2 in vitro response to M. bovis (Figure 2A). In
addition, immunohistochemical analysis demonstrated thatmacrophages infiltrating lung lesions and epithelial cells inlung lesions from infected cattle expressed PGE2 (Figure 5B).
These findings suggest that M. bovis could induce PGE2
from monocytes.
TLR4
recognizebacterial
lipopolysaccharide
andpeptidoglycan, which are the components of bacterial cellwall (30, 43). Although Mycoplasmas do not have cell wall(57), several researchers reported the relationships between
TLR4 and the inflammation during Mycoplasma infection(55, 58, 59). Shimizu et al. (60) have demonstrated thatM. pneumoniae induced TNF-α production from macrophages
through autophagy and TLR4. This inflammatory response could
be need to cytoadherence of live bacteria. Our current study
demonstrated that inactivated M. bovis and TLR2 stimulationinduced PGE2 production (Figures 1D, 3A). Inhibition of TLR
signaling by SsnB decreased PGE2 production induced by FSL-1
and inactivated M. bovis (Figure 3D). On the other hand, SsnB
selectively blocks not only TLR2- but also TLR4-mediatedinflammatory signaling (61). Taken together, although M. bovis
is recognized via TLR2 and/or TLR4, the results of this study
suggest that M. bovis could induce PGE2 production via mainly
TLR2 signaling. However, further studies focusing on TLR4 andM. bovis must be needed to elucidate the relationship between
M. bovis infection and PGE2 .
PGE2 suppresses Th1 immune responses, limiting thefunctions of natural killer (NK) cells, CD4 T cells, and cytotoxic
T cells via specific EP2 and EP4 receptors (14). After binding
EP2 or EP4, PGE2 induces the expression of anti-inflammatory
and immunosuppressive genes by activating the cyclic AMP(cAMP)/protein kinase A/cAMP response element binding

FIGURE 6 | Activation of IFN-γ responses by the combination of meloxicam
and anti-PD-L1 Ab. PBMCs from cattle infected with M. bovis (n = 7) wereincubated with meloxicam and anti-PD-L1 mAb in the presence of anti-bovine
CD3 and CD28 mAbs (A) or heat-killed M. bovis (B). IFN-γ production in
culture supernatants was determined by ELISA. The bars are the median ofthe values (A,B). Statistical significance was determined by the Steel–Dwass
test (A,B). *P < 0.05.

dysfunction induced by exhausted T cell during the diseaseprogression (11). In this study, we demonstrated that M. bovis
induced PGE2 production from monocytes in line with theupregulation of PD-L1 expression on monocytes (Figures 1A–F).
Additionally, we showed that the levels of plasma PGE2 andproportions of circulating PD-L1+ monocytes were positively
correlated (Figure 4C). These results indicate that PGE2 could
be associated with PD-L1 expression in M. bovis infection.
Several reports showed that PGE2 activates STAT3 signaling(18, 39). In tumor studies, STAT3 regulates PD-L1 expression
transcriptionally by binding to its promoter (40, 41). These

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proliferation of anti-BLV-specific CD4+ T cells, was restoredin cattle inoculated with chimeric Ab, while BLV provirus
loads were significantly reduced (19, 23). In addition, combined
treatment of chimeric Ab with COX-2 inhibitor synergisticallydecreased BLV provirus loads, clearly demonstrating that thistreatment induced antiviral activities (19). However, the invivo effect of combined treatment of chimeric Ab with COX2 inhibitor in M. bovis infection is still unclear and should
be determined in further experiments on infected cattle tosupport the efficacy of this novel treatment approach forclinical application.
In conclusion, the present study found that M. bovis can
induce PGE2 and upregulate PD-L1 expression on monocytes
and that M. bovis-specific IFN-γ response can be upregulatedby dual blockade of the PD-1/PD-L1 pathway and PGE2 .
Thus, PGE2 plays an important role in immune dysfunction
and is related with the PD-1/PD-L1 pathway. Our findings
might contribute to the development of novel strategies for
manipulating M. bovis-specific T-cell responses to prevent
disease progression.

protein pathway (14). In the current study, increased plasma
PGE2 levels were strongly correlated with lower M. bovis-specificIFN-γ production (Figure 4D). Additionally, plasma levels of
PGE2 and proportions of circulating PD-L1+ CD11b+ CD14+
monocytes were positively correlated (Figure 4C). These results
indicate that decreased IFN-γ levels in bovine mycoplasmosis
might be associated with the upregulation of PGE2 and PD-L1+
cells. On the other hand, PGE2 can inhibit not only adaptive
immunity but also innate immunity. A previous report inhumans demonstrated that PGE2 inhibited neutrophil functions,such as neutrophil extracellular trap (NET) formation, via EP2
and EP4 (58). Interestingly, in several reports, it is shownthat M. bovis could inhibit NET formation and escape fromneutrophil killing (59). Therefore, PGE2 in M. bovis infection
might play a role as not only an inducer of PD-L1 expression
but also a direct suppressor of other immune responses, suchas T cell and neutrophil responses. Further studies focusing on
inhibition induced by PGE2 might help elucidate the mechanismsof immune dysfunction in M. bovis infection.
Our previous studies demonstrated that blockade of the PD1/PD-L1 pathway and/or PGE2 effectively improved antigenspecific immune reactions in bovine chronic diseases, suchas bovine leukemia virus infection, Johne’s disease, andanaplasmosis (18–20, 23, 24, 60, 62, 63, 69). Effective reactivation
by dual blockade could be because of PGE2 function that
inhibition of T-cell activation and regulates PD-L1 expression.
Indeed, single blockade of the PD-1/PD-L1 pathway reactivatedthe M. bovis-specific IFN-γ response in vitro (Figure 6B).
Therefore, we evaluated the immune activation associated withthe combined treatment anti-PD-L1 mAb with a COX-2 inhibitor
in vitro. The combined treatment significantly increased IFNγ production in response to M. bovis. Although a significant
difference between single and dual blockade of the PD-1/PD-L1
pathway and PGE2 was not observed, IFN-γ production tended
to be increased by meloxicam and anti-PD-L1 mAb. To support
the efficacy of the combined treatment, further investigationis needed. The combination of PD-1/PD-L1 blockade with
COX-2 inhibition has therapeutic potential for controlling M.
bovis infection.
In other bovine mycoplasmosis, contagious bovine
pleuropneumonia caused by Mycoplasma mycoides subsp.
mycoides biotype Small Colony (MmmSC), the number of
MmmSC-specific IFN-γ-secreting CD4+ T cells was positively
correlated with disease recovery (64, 65). IFN-γ promotes
the activation and proliferation of T cells and macrophages,
which play vital roles in host defenses in the lungs. Previous
studies showed that macrophage dysfunction was induced by
Mycoplasma spp., including M. bovis, and promoted persistentinfection (2, 66–68). Immune reactivation by combination
therapy of anti-PD-L1 mAb with COX-2 inhibitor could help
in the development of potential methods for the control of M.
bovis infection.
Recently, our previous studies reported the establishmentof anti-bovine PD-L1 rat-bovine chimeric Ab, and a clinicalstudy on BLV infection was conducted (19, 23). Interestingly,
T cell function, which involved IFN-γ response and theFrontiers in Veterinary Science | www.frontiersin.org

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in thearticle/Supplementary Material.

AUTHOR CONTRIBUTIONSSK, SGot, SM, and KO were responsible for the conception
and design of the study. SGot, SK, YH, JK, TO, NM, YSa,
KW, EM, AK, RU, SY, MKK, YK, KY, and MTo performed
the experiments. SGot, SK, TO, YSu, SM, and KO analyzed
the data. SGon, HH, MK, MTa, ET, RU, SY, MKK, YK, KY,
and MTo provided intellectual input, field samples, laboratory
materials, reagents, and/or analytic tools. SGot and SK wrote themanuscript. SK, TO, SM, and KO contributed to the revisionof the manuscript. All authors reviewed and approved thefinal manuscript.

FUNDING
This work was supported by grants-in-aid for Scientific
Research from Japan Society for the Promotion of Science(JSPS) and by grants from the Project of the NARO,
Bio-oriented Technology Research Advancement Institution(Research program on development of innovative technology;
number 26058 BC to SK and the special scheme project
on regional developing strategy; grant 16817557 to SK).
This research was also supported by Platform Project for
Supporting Drug Discovery and Life Science Research [Basis for
Supporting Innovative Drug Discovery and Life Science Research(BINDS)] from AMED under Grant Number JP19am0101078
to YK. The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of
the manuscript.

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ACKNOWLEDGMENTS

SUPPLEMENTARY MATERIAL

We are grateful to Dr. Hideyuki Takahashi, Dr. Yasuyuki Mori,and Dr. Tomio Ibayashi for their valuable advice and discussions.
We would like to thank Enago for the English language review.

The Supplementary Material for this article can be foundonline at: https://www.frontiersin.org/articles/10.3389/fvets.
2020.00012/full#supplementary-material

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Goto et al.

PGE2 -Induced Immunodysfunction During Bovine Mycoplasmosis

Conflict of Interest: KY and MTo are employed by Fuso PharmaceuticalIndustries, Ltd. SGot, SK, TO, NM, YSa, YSu, SM, and KO are authors of apatent application covering materials and techniques described in this paper(PCT/JP2018/27041).

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The remaining authors declare that the research was conducted in the absence ofany commercial or financial relationships that could be construed as a potential
conflict of interest.
Copyright © 2020 Goto, Konnai, Hirano, Kohara, Okagawa, Maekawa, Sajiki,
Watari, Minato, Kobayashi, Gondaira, Higuchi, Koiwa, Tajima, Taguchi, Uemura,
Yamada, Kaneko, Kato, Yamamoto, Toda, Suzuki, Murata and Ohashi. This is an
open-access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) and the copyright owner(s) are credited and that theoriginal publication in this journal is cited, in accordance with accepted academic
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February 2020 | Volume 7 | Article 12