HOST RESPONSE AND INFLAMMATION

crossmProstaglandin E2 Induction Suppresses the Th1 Immune
Responses in Cattle with Johne’s Diseasea

Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japanb

Bacterial and Parasitic Disease Research Division, National Institute of Animal Health, Tsukuba, Japanc

North Lab, Sapporo, Japand

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japane

New Industry Creation Hatchery Center, Tohoku University, Sendai, Japanf

Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japang

Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education, Hokkaido
University, Sapporo, Japan

ABSTRACT Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis, is a bovine chronic infection that is endemic in Japan and many other countries.
The expression of immunoinhibitory molecules is upregulated in cattle with Johne’s
disease, but the mechanism of immunosuppression is poorly understood. Prostaglandin E2 (PGE2) is immunosuppressive in humans, but few veterinary data are
available. In this study, functional and kinetic analyses of PGE2 were performed toinvestigate the immunosuppressive effect of PGE2 during Johne’s disease. In vitro
PGE2 treatment decreased T-cell proliferation and Th1 cytokine production and upregulated the expression of immunoinhibitory molecules such as interleukin-10 andprogrammed death ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs)
from healthy cattle. PGE2 was upregulated in sera and intestinal lesions of cattlewith Johne’s disease. In vitro stimulation with Johnin puriﬁed protein derivative (JPPD) induced cyclooxygenase-2 (COX-2) transcription, PGE2 production, and upregulation of PD-L1 and immunoinhibitory receptors in PBMCs from cattle infected with
M. avium subsp. paratuberculosis. Therefore, Johnin-speciﬁc Th1 responses could be
limited by the PGE2 pathway in cattle. In contrast, downregulation of PGE2 with a
COX-2 inhibitor promoted J-PPD-stimulated CD8⫹ T-cell proliferation and Th1 cytokine production in PBMCs from the experimentally infected cattle. PD-L1 blockade
induced J-PPD-stimulated CD8⫹ T-cell proliferation and interferon gamma production in vitro. Combined treatment with a COX-2 inhibitor and anti-PD-L1 antibodies
enhanced J-PPD-stimulated CD8⫹ T-cell proliferation in vitro, suggesting that theblockade of both pathways is a potential therapeutic strategy to control Johne’s disease. The effects of COX-2 inhibition warrant further study as a novel treatment ofJohne’s disease.
KEYWORDS PGE2, immunoinhibitory molecules, PD-1, PD-L1, T-cell exhaustion,

immunotherapy, COX-2 inhibitor, Johne’s disease, cattleProstaglandin E2 (PGE2) is an inﬂammatory mediator (1) derived from arachidonic
acid by cyclooxygenase isoenzymes (COX-1 and -2) and PGE synthases (2). COX-1
is a widely expressed constitutive enzyme in many tissues, including the kidneys,
stomach, and vascular endothelium (3). COX-2 is an inducible enzyme, and its expression is regulated by inﬂammatory cytokines and growth factors via activation of nuclear
factor-kappa B (NF-␬B) (4). PGE2 has suppressive activity in T cells, natural killer (NK)May 2018 Volume 86 Issue 5 e00910-17

Infection and Immunity

Received 11 December 2017 Returned for
modiﬁcation 17 January 2018 Accepted 17
February 2018
Accepted manuscript posted online 26
February 2018
Citation Sajiki Y, Konnai S, Okagawa T,
Nishimori A, Maekawa N, Goto S, Ikebuchi R,
Nagata R, Kawaji S, Kagawa Y, Yamada S, Kato Y,
Nakajima C, Suzuki Y, Murata S, Mori Y, Ohashi
K. 2018. Prostaglandin E2 induction suppresses
the Th1 immune responses in cattle with
Johne's disease. Infect Immun 86:e00910-17.
https://doi.org/10.1128/IAI.00910-17.
Editor Sabine Ehrt, Weill Cornell Medical
College
Copyright © 2018 American Society for
Microbiology. All Rights Reserved.
Address correspondence to Satoru Konnai,
konnai@vetmed.hokudai.ac.jp.

iai.asm.org 1

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

Yamato Sajiki,a Satoru Konnai,a Tomohiro Okagawa,a Asami Nishimori,a Naoya Maekawa,a Shinya Goto,a Ryoyo Ikebuchi,aReiko Nagata,b Satoko Kawaji,b Yumiko Kagawa,c Shinji Yamada,d Yukinari Kato,d,e Chie Nakajima,f,g Yasuhiko Suzuki,f,g
Shiro Murata,a Yasuyuki Mori,b Kazuhiko Ohashia

Sajiki et al.

Infection and Immunity

May 2018 Volume 86 Issue 5 e00910-17

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

cells, dendritic cells (DCs), and macrophages via speciﬁc EP2 and EP4 receptors. Theinteraction of PGE2 and EP2 or EP4 activates the cyclic AMP (cAMP)/protein kinase A(PKA)/cAMP response element binding protein (CREB) pathway to induce the expression of anti-inﬂammatory and immunosuppressive genes (5, 6). Previous studies inhumans have demonstrated that PGE2 inhibits Th1 responses and the differentiation of
DCs and induces several subsets of immunosuppressive cells, including M2 macrophages, myeloid-derived suppressor cells (MDSCs), and regulatory T (Treg) cells (7–9).
Other studies have found an association of PGE2 with progression of chronic infections.
PGE2 upregulates the human immunodeﬁciency virus type 1 (HIV-1) long terminal
repeat-driven gene in T cells (10, 11). COX-2 expression is upregulated in hepatitis C
virus (HCV) infection, and PGE2 induces RNA replication of HCV (12). Because of theinvolvement of PGE2 in the immunopathogenesis of chronic infections, COX-2 inhibitors have been considered candidate immunotherapy drugs. COX-2 inhibitors activate
immune responses in vitro and in vivo (13, 14). COX-2 inhibition results in the activationof interleukin-12 (IL-12) production from antigen-presenting cells and the suppressionof IL-10 production in splenocytes of tumor-bearing mice (13). In patients with chronic
HIV-1 infection, COX-2 inhibitors were found to improve T-cell-dependent functionsand vaccine responses (14). Thus, the COX-2–PGE2 pathway limits Th1 responses during
chronic diseases.
Mycobacterium avium subsp. paratuberculosis is a facultative intracellular pathogen
causing Johne’s disease (paratuberculosis) in ruminants (15). The clinical signs ofJohne’s disease include chronic diarrhea, severe weight loss, reduction in milk production, and mortality (15). Johne’s disease is endemic worldwide; no country or region has
been found to be free of this disease (16). In early infections, M. avium subsp.
paratuberculosis induces strong Th1 responses characterized by interferon gamma(IFN-␥), and macrophages activated by IFN-␥ kill intracellular mycobacteria (17–19). The
Th1 response declines during the late subclinical stage, which allows bacterial growthand progression to clinical disease (20–22). The Th1 response is the key in the control
of progression of Johne’s disease.
Programmed death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) are immunoinhibitory receptors that act in a negative-feedback system to inhibit excessive
immune responses via interactions with their ligands, programmed death ligand 1(PD-L1) and major histocompatibility complex class II (MHC II) (23, 24). In chronic
infections, these immunoinhibitory molecules are involved in the exhaustion of
antigen-speciﬁc T cells (25, 26). PD-1 and LAG-3 are upregulated on CD4⫹ and/or CD8⫹
T cells during subclinical Johne’s disease in cattle, and an immunoinhibitory ligand,
PD-L1, is expressed on M. avium subsp. paratuberculosis-infected macrophages of theileum (27). In addition, antibody blockade of the PD-1 and LAG-3 pathways reactivates
M. avium subsp. paratuberculosis-speciﬁc Th1 cytokine production in vitro (27). The
dysfunction of the Th1 response during Johne’s disease is mediated by immunoinhibitory molecules on T cells, but it is not known how these immunoinhibitory molecules
are upregulated during the course of the disease.
The association of PGE2 and immunoinhibitory molecules has been investigatedin mouse models and in human patients (28–30). In a murine tumor model, PGE2
regulated PD-L1 expression in tumor-associated macrophages and MDSCs (28). Anotherstudy reported a positive correlation between COX-2 and PD-L1 expression in humanmelanoma cells (29). Additionally, in a mouse model of chronic infection, EP2 and EP4
were upregulated on CD8⫹ cytotoxic T cells (CTLs) and impaired CTL function andsurvival via PGE2 signaling (30). Concurrent blockade of the PGE2 and PD-1/PD-L1
pathways was shown to restore CTL function and improve viral control (30).
Few veterinary studies are available on the immunosuppressive effect of PGE2, theassociation of PGE2 and immunoinhibitory pathways, and the contribution to T-cell
dysfunction or chronic disease progression. This study investigated the immunosuppressive function and kinetics of PGE2 to investigate immunopathogenesis in Johne’s
disease in cattle.
iai.asm.org 2

T-Cell Exhaustion by PGE2 during Johne’s Disease

Infection and Immunity

RESULTS
Immunosuppressive effects of PGE2. To evaluate immunosuppression induced by
PGE2, T-cell proliferation, cytokine secretion, and gene expression (cytokine and STAT3
genes) were analyzed by cultivation assay of peripheral blood mononuclear cells(PBMCs) from uninfected cattle under PGE2 treatment. PGE2 inhibited proliferation of
CD4⫹ and CD8⫹ T cells (Fig. 1a and b) and IFN-␥ and TNF-␣ production from PBMCs(Fig. 1c and d). PGE2 downregulated the mRNA expression of IFN-␥, IL-2, and tumor
necrosis factor alpha (TNF-␣) (Fig. 1e to g) and upregulated IL-10 and STAT3 mRNA
expression (Fig. 1h and i). The results indicate that PGE2 promotes IL-10 signaling andinhibits Th1 responses in cattle. Since PGE2 is known to regulate PD-L1 expression inhumans (28), PGE2 regulation of PD-L1 expression was investigated in PBMCs of thehealthy cattle. As shown in Fig. 1j and k, PGE2 upregulated PD-L1 expression in PBMCs.
Overall, these results indicate that PGE2 has immunosuppressive activity against bovine
PBMCs.
Activation of immune responses by COX-2 inhibition. To demonstrate the effects
of COX-2 inhibition on T-cell function, production of IFN-␥ and TNF-␣ and T-cellproliferation were evaluated by the 3-day culture assay using PBMCs from uninfectedanimals in the presence of meloxicam. Meloxicam treatment signiﬁcantly increased
May 2018 Volume 86 Issue 5 e00910-17

iai.asm.org 3

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

FIG 1 Immunosuppressive effects of PGE2. (a to d) PBMCs from uninfected cattle (n ⫽ 6 [a to c] or 8 [d]) were cultured with PGE2 in the presence of anti-CD3
and anti-CD28 MAbs for 72 h. The proliferation of CD4⫹ cells (a) and CD8⫹ cells (b) was assayed by ﬂow cytometry. IFN-␥ (c) and TNF-␣ (d) production wasdetermined by ELISA. (e to k) PBMCs from uninfected cattle (n ⫽ 6 [e to i] or 7 [j and k]) were cultured with PGE2 for 24 h. Real-time PCR was performed in
duplicate to quantitate the mRNA expression of IFN-␥ (e), IL-2 (f), TNF-␣ (g), IL-10 (h), STAT3 (i), and PD-L1 (j). The expression of PD-L1 protein was measured
by ﬂow cytometry (k). Statistical signiﬁcance was determined by the Steel-Dwass test (a to c) or the Wilcoxon signed-rank test (d to k).

Sajiki et al.

Infection and Immunity

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

FIG 2 Functional analysis of the COX-2 inhibitor. PBMCs from uninfected cattle (n ⫽ 9 [a and b] or 8 [c])were cultured with meloxicam in the presence of anti-CD3 and anti-CD28 MAbs. CD8⫹ cell proliferation
was assayed by ﬂow cytometry (a). IFN-␥ (b) and TNF-␣ (c) production was determined by ELISA in
duplicate. Statistical signiﬁcance was determined by the Wilcoxon signed-rank test.

both IFN-␥ and TNF-␣ production in PBMCs and the proliferation of CD8⫹ T cells (Fig.
2a to c). This result indicates that meloxicam activates the T-cell response in cattle.
Kinetic analysis of PGE2 in cattle infected with M. avium subsp. paratuberculosis. In humans, PGE2 is associated with the progression of some chronic infections(10–12). Compared with that in uninfected cattle, the serum PGE2 concentration was
signiﬁcantly increased in cattle with natural infection of M. avium subsp. paratuberculosis (Fig. 3a). Johnin puriﬁed protein derivative (J-PPD) antigen stimulation increased
PGE2 production in PBMCs from the infected animals but not in PBMCs from theuninfected animals (Fig. 3c), and meloxicam inhibited PGE2 production under the J-PPD
stimulation (Fig. 3c). COX-2 expression in PBMCs from the infected animals was inducedby the stimulation of J-PPD (Fig. 3b). The results indicate that the COX-2–PGE2 pathway
May 2018 Volume 86 Issue 5 e00910-17

iai.asm.org 4

T-Cell Exhaustion by PGE2 during Johne’s Disease

Infection and Immunity

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

FIG 3 Kinetic analysis of PGE2 in cattle infected with M. avium subsp. paratuberculosis. (a) Serum PGE2
concentrations in cattle uninfected (n ⫽ 16) or naturally infected with M. avium subsp. paratuberculosis(n ⫽ 8) were determined by ELISA. (b) The expression of COX-2 mRNA in PBMCs from cattle experimentally infected with M. avium subsp. paratuberculosis (n ⫽ 7) cultured with J-PPD was assayed by real-time
PCR. (c) PBMCs of the uninfected (n ⫽ 6) and experimentally infected (n ⫽ 7) cattle were incubated with
J-PPD. The concentration of PGE2 in culture supernatants was determined by ELISA in duplicate.
Statistical signiﬁcance was determined by the Wilcoxon signed-rank test (a and b) or the Steel-Dwass test(c). N.S., no stimulation.

was activated by Johnin antigen stimulation in PBMCs of cattle infected with M. avium
subsp. paratuberculosis.
Upregulation of PD-L1, PD-1, and LAG-3 expression by J-PPD stimulation. The
expression of the immunoinhibitory molecules PD-L1, PD-1, and LAG-3 in PBMCs fromcattle experimentally infected with M. avium subsp. paratuberculosis was analyzed
following stimulation by J-PPD for 24 h. Compared with that in the uninfected cattle,
J-PPD signiﬁcantly increased PD-L1 expression at the protein and mRNA levels in PBMCs
of the experimentally infected animals (Fig. 4a and b). Flow cytometric analysis indicated that the expression of PD-L1 was signiﬁcantly upregulated in CD4⫹ T cells, CD8⫹
May 2018 Volume 86 Issue 5 e00910-17

iai.asm.org 5

Sajiki et al.

Infection and Immunity

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

FIG 4 Upregulation of PD-L1 expression by J-PPD. PBMCs from cattle experimentally infected with M. avium subsp.
paratuberculosis (n ⫽ 6) were incubated with J-PPD, and the expression of PD-L1 in PBMCs (a), CD4⫹ T cells (c),CD8⫹ T cells (d), IgM⫹ B cells (e), and CD14⫹ cells (f) was assayed by ﬂow cytometry. PBMCs of uninfected cattle(n ⫽ 5) were incubated with J-PPD, and PD-L1 expression in PBMCs was assayed by ﬂow cytometry (a). Expressionof PD-L1 mRNA in PBMCs of the experimentally infected cattle (n ⫽ 7) was assayed by real-time PCR in duplicate(b). Statistical signiﬁcance was determined by the Wilcoxon signed-rank test. N.S., no stimulation.

T cells, IgM⫹ B cells, and CD14⫹ cells of the infected animals by J-PPD stimulation (Fig.
4c to f). The expression of PD-1 and LAG-3 was also upregulated in CD4⫹ T cells andCD8⫹ T cells of the infected animals by J-PPD stimulation (Fig. 5a to d). In vitro
stimulation by Johnin antigen thus strongly induced the expression of immunoinhibitory molecules in PBMCs of cattle infected with M. avium subsp. paratuberculosis.
Expression of PGE2, EP2, and PD-L1 on M. avium subsp. paratuberculosisinfected cells of the ileum. A previous study conﬁrmed PD-L1 expression in macrophages infected with M. avium subsp. paratuberculosis in the ileum, with possible
association with the clinical disease (27). This study investigated the expression of PGE2
and EP2 in the intestinal macrophages of cattle with Johne’s disease. PGE2 expression
was detected in cattle naturally and experimentally infected with M. avium subsp.
May 2018 Volume 86 Issue 5 e00910-17

iai.asm.org 6

T-Cell Exhaustion by PGE2 during Johne’s Disease

Infection and Immunity

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

FIG 5 Upregulation of PD-1 and LAG-3 expression by J-PPD. PBMCs from cattle experimentally infected with M.
avium subsp. paratuberculosis (n ⫽ 7) were incubated with J-PPD, and the expression of PD-1 (a and b) and LAG-3(c and d) on CD4⫹ T cells (a and c) and CD8⫹ T cells (b and d) was assayed by ﬂow cytometry. Statistical signiﬁcancewas determined by the Wilcoxon signed-rank test. N.S., no stimulation.

paratuberculosis (1 and 65) but not in the uninfected cattle (C6) (Fig. 6a). Expressionof EP2, a PGE2 receptor, was detected in both the infected and uninfected animals (Fig.
6b), and both PD-L1⫹ and M. avium subsp. paratuberculosis-infected cells were observed in the same lesion (Fig. 6c and d). The results demonstrated coexpression of
PGE2 and PD-L1 in the ilea of cattle with Johne’s disease.
Activation of J-PPD-speciﬁc T-cell responses by a COX-2 inhibitor. J-PPD can
induce IFN-␥ production from T cells in cattle with M. avium subsp. paratuberculosis
infection (31). This study investigated whether the COX-2 inhibitor meloxicam activates
cytokine production and T-cell proliferation in response to J-PPD. As shown in Fig. 7,
J-PPD upregulated IFN-␥ and TNF-␣ production in meloxicam-treated cultures of
PBMCs from infected cattle (Fig. 7b and c). The proliferation of CD8⫹ cells was also
induced by meloxicam treatment (Fig. 7a). Meloxicam thus activated J-PPD-speciﬁc
immune responses.
Activation of J-PPD-speciﬁc T-cell responses by PD-L1 blockade. Blockade of thePD-1/PD-L1 pathway can activate IFN-␥ production from PBMCs in bovine chronic
infections, such as bovine leukemia virus (BLV) infection and bovine mycoplasmosis (32,33). To demonstrate the effects of PD-L1 blockade on cattle infected with M. avium
subsp. paratuberculosis, PBMCs from the experimentally infected cattle were incubatedwith an anti-PD-L1 monoclonal antibody (MAb) in the presence of J-PPD. As expected,
May 2018 Volume 86 Issue 5 e00910-17

iai.asm.org 7

Sajiki et al.

Infection and Immunity

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

FIG 6 PGE2, EP2, and PD-L1 expression and localization of M. avium subsp. paratuberculosis in the ileal mucosa of cattlewith or without Johne’s disease. (a to c) Immunohistochemical staining of PGE2 (a), EP2 (b), and PD-L1 (c) in ileal tissueof cattle naturally or experimentally infected with M. avium subsp. paratuberculosis (1 and 65) and uninfected cattle (C6)
was performed using anti-human PGE2 antibody (rabbit polyclonal), anti-human EP2 MAb [EPR8030(B)], and antibovine PD-L1 MAb (6C11-3A11). (d) Ziehl-Neelsen staining for acid-fast bacilli in ileal tissue of the infected anduninfected cattle was also performed. M, accumulations of M. avium subsp. paratuberculosis-infected macrophages.
Arrowheads, M. avium subsp. paratuberculosis-infected Langhans giant cells.

J-PPD-speciﬁc proliferation of CD8⫹ cells and IFN-␥ production were activated by theanti-PD-L1 MAb (Fig. 7d and e).
Combined effects of meloxicam and anti-PD-L1 antibody on activation of
J-PPD-speciﬁc T-cell response. Compared with that in negative controls, the proliferation of CD8⫹ cells was signiﬁcantly increased by the combination of meloxicam andanti-PD-L1 MAb (Fig. 8a). Stimulation by B-PPD, a negative-control antigen, had noeffect in either group (Fig. 8a). IFN-␥ production was increased by meloxicam andanti-PD-L1 MAb, but the difference from the results with negative controls did notreach signiﬁcance (Fig. 8b). A previous study found that an anti-PD-L1 chimericantibody (ChAb) activated immune responses in BLV infection in vitro and in vivo (34).
In this study, combined treatment with the COX-2 inhibitor and anti-PD-L1 ChAb
signiﬁcantly increased CD8⫹ cell proliferation compared with that for the negative
controls (Fig. 8c). The results obtained by combining a COX-2 inhibitor and an antiPD-L1 antibody support their potential as a novel treatment of Johne’s disease.
DISCUSSION
During the early phase of Johne’s disease in cattle, strong Th1 responses are elicitedby T cells speciﬁc to M. avium subsp. paratuberculosis that are activated by M. avium
subsp. paratuberculosis-infected macrophages following antigen presentation (17–19).
At the late subclinical stage, a decline in the Th1 response contributes to bacterial
May 2018 Volume 86 Issue 5 e00910-17

iai.asm.org 8

T-Cell Exhaustion by PGE2 during Johne’s Disease

Infection and Immunity

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

FIG 7 Activation of T-cell responses by the COX-2 inhibitor or anti-PD-L1 MAb. (a to c) PBMCs from cattle
experimentally infected with M. avium subsp. paratuberculosis (n ⫽ 7 [a and b] or 6 [c]) were cultured withmeloxicam in the presence of J-PPD. CD8⫹ cell proliferation (a) was assayed by ﬂow cytometry. IFN-␥ (b) and TNF-␣(c) production was determined by ELISA in duplicate. (d and e) PBMCs from the infected cattle (n ⫽ 7) were cultured
with anti-PD-L1 MAb in the presence of J-PPD. CD8⫹ cell proliferation (d) was assayed by ﬂow cytometry. IFN-␥
production (e) was determined by ELISA in duplicate. Statistical signiﬁcance was determined by the Wilcoxon
signed-rank test. Cont Ig, rat control IgG.

growth and progression to clinical disease (20–22). The loss of functional Th1 responses
during subclinical Johne’s disease is mediated by immunoinhibitory molecules (27), but
the mechanism of the upregulation of these molecules during the disease course is notclear. This study investigated the immunosuppressive effect of PGE2 because its
involvement in the regulation of immune responses in chronic human infections is well
documented (10–12). PGE2 inhibited the proliferation of CD4⫹ and CD8⫹ T cells and the
production of the Th1 cytokines IFN-␥ and TNF-␣ in cultures of PBMCs isolated fromcattle. PGE2 treatment downregulated the expression of IFN-␥, IL-2, and TNF-␣ mRNAs
and upregulated the expression of IL-10 and STAT3 mRNAs in cultured PBMCs. PGE2
May 2018 Volume 86 Issue 5 e00910-17

iai.asm.org 9

Sajiki et al.

Infection and Immunity

production was increased in the blood of animals infected with M. avium subsp.
paratuberculosis. The Th1 response plays a critical role in the control of M. avium subsp.
paratuberculosis infection and disease progression, but this infection induces IL-10,
which activates STAT3 via IL-10 receptor signaling, which in turn results in immunosuppression and bacterial persistence (35, 36). Our results show that PGE2 contributed
to the progression of Johne’s disease by the suppression of Th1 responses and theactivation of IL-10 production. Previous studies have associated PGE2 with mycobacterial infections (37, 38), but the role of PGE2 was not described. The results of this study
contribute to the understanding of PGE2 activity during infection with mycobacteria.
PD-L1 is upregulated in the macrophages in the peripheral blood and in the ilea ofcattle with Johne’s disease (27), but the mechanism underlying PD-L1 upregulation has
been unknown. In this study, PGE2 upregulated in vitro PD-L1 expression in the PBMCsof healthy cattle. Additionally, J-PPD stimulation induced PGE2 production by activating
COX-2 and promoting PD-L1 expression in the PBMCs of cattle infected with M. avium
subsp. paratuberculosis but not in uninfected cattle. In PBMC cultures of infectedanimals, J-PPD-speciﬁc CD4⫹ T cells secrete mainly IFN-␥ and TNF-␣ by J-PPD stimulation (17–19). COX-2 transcription is known to be induced by inﬂammatory cytokines(TNF-␣, IL-1, etc.), Toll-like receptor (TLR) signaling, and oxidative stress via the NF-␬B
pathway in human cells (39, 40). In addition, a previous paper has shown that IFN-␥induced TNF activates NF-␬B, which is required for full COX-2 expression in murine
macrophages (41). Therefore, TNF-␣ produced from J-PPD-speciﬁc T cells and/or IFN␥-stimulated macrophages might activate COX-2 transcription via the TNF–NF-␬B
May 2018 Volume 86 Issue 5 e00910-17

iai.asm.org 10

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

FIG 8 Activation of T-cell responses by the combination of meloxicam and anti-PD-L1 antibody. (a and b) PBMCs from cattle experimentally infected with M.
avium subsp. paratuberculosis (n ⫽ 6 [a] or 7 [b]) were incubated with meloxicam and anti-PD-L1 MAb in the presence of J-PPD or B-PPD. CD8⫹ cell proliferation
was assayed by ﬂow cytometry (a). IFN-␥ production was determined by ELISA in duplicate (b). (c and d) PBMCs from the infected cattle (n ⫽ 7) were incubatedwith meloxicam and anti-PD-L1 ChAb in the presence of J-PPD or B-PPD. CD8⫹ cell proliferation was assayed by ﬂow cytometry (c). IFN-␥ production wasdetermined by ELISA in duplicate (d). Statistical signiﬁcance was determined by the Steel-Dwass test.

T-Cell Exhaustion by PGE2 during Johne’s Disease

Infection and Immunity

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

pathway and result in increased PGE2 production in the PBMC cultivation assay with
J-PPD stimulation.
In this study, in vitro treatment of PGE2 upregulated transcription of the IL-10 and
STAT3 genes in the PBMCs of healthy cattle. The inhibition of STAT3 activity has beenshown to downregulate PD-L1 expression in non-small-cell lung cancer (42). The results
indicated that PGE2 may regulate PD-L1 expression in bovine PBMCs by activating
IL-10 –STAT3 signaling. PD-L1 expression in M. avium subsp. paratuberculosis-infected
macrophages has previously been associated with diseases involving the ileum (27). In
this study, PGE2 was expressed in M. avium subsp. paratuberculosis-infected macrophages and Langhans giant cells in the ilea of animals with clinical Johne’s disease. The
overall ﬁndings suggest that the PGE2–IL-10 –STAT3–PD-L1 axis is involved in thedownregulation of the Th1 response and the formation of pathological lesions in
the intestine during Johne’s disease. It is still unclear what types of cell subsets produce
PGE2 and what types of prostanoid receptors, EP1, EP2, EP3, or EP4, mediate immunosuppression by PGE2 on T cells during Johne’s disease. Further investigation is required
to determine the mechanism involved.
Macrophages and CD8⫹ cytotoxic T cells activated by Th1 cytokines are involved in
the killing of intracellular bacteria during Johne’s disease (17–19). COX-2 inhibition
induced the proliferation of CD8⫹ T cells and Th1 cytokine production in PBMCs from
M. avium subsp. paratuberculosis-infected cattle following J-PPD stimulation. Combined
blockade of PGE2 and PD-1/PD-L1 strongly stimulated the proliferation of CD8⫹ T cells
in the infected animals. IFN-␥ production tended to be activated by dual blockade of
PGE2 and PD-1/PD-L1, although signiﬁcance was not observed because of the limited
number of samples that were evaluated. The combination of COX-2 inhibition andPD-1/PD-L1 blockade has therapeutic potential for the control of Johne’s disease.
Selective COX-2 inhibitors, including meloxicam, are launched and widely used asanti-inﬂammatory compounds in the dairy and beef industries. Previous studies haveinvestigated the in vivo effect of meloxicam on T-cell subsets in healthy or vaccinatedcattle (43, 44). However, the in vivo effect of meloxicam on the Th1 response during
Johne’s disease is still unclear and should be determined in further experiments in
infected cattle. In addition, we are developing a method for large-scale production ofbovinized chimeric antibody using a mammalian expression system (34, 45), which
would enable antibody therapy even in cattle. Further study is warranted to investigate
the clinical effectiveness and cost-effectiveness of these inhibitors against Johne’s
disease. Previous studies have demonstrated that immunoinhibitory molecules mediate
T-cell exhaustion in cattle with other infectious diseases, including bovine leukosis,
mycoplasmosis, and anaplasmosis (33, 46–51), but the involvement of PGE2 in the
immunopathogenesis of those infections has not been investigated.
This is the ﬁrst study of the immunosuppressive effects of PGE2 in cattle and theimmunomodulatory effect of COX-2 inhibition during Johne’s disease in cattle. The
results suggest cotargeting PGE2 and PD-L1 as a novel immunotherapy for Johne’s
disease. Pilot clinical trials have shown that antibodies blocking the PD-1/PD-L1 pathway have immunomodulatory and antiviral effects (34, 45). Cost-effectiveness is an
obstacle to this approach because large amounts of the therapeutic antibodies are
required. If the antibody dose can be reduced by combination with a COX-2 inhibitor,
this approach could have a signiﬁcant impact. Further study will open up new avenues
for the treatment of bovine chronic infections, including Johne’s disease.
MATERIALS AND METHODS
Animals. Blood from Holstein cattle not infected with M. avium subsp. paratuberculosis was obtained
from dairy farms in Hokkaido, Japan, which have no history of Johne’s disease. Seven male Holsteincalves (animals 80 to 86) between 3 and 4 weeks of age were orally inoculated with intestinal tissue
homogenate from an infected cow containing M. avium subsp. paratuberculosis (6.8 ⫻ 106 CFU) once
daily for 20 days, as described previously with slight modiﬁcations (27, 52). All infected calves were kept
in a biosafety level 2 animal facility at the National Institute of Animal Health (Tsukuba, Ibaraki, Japan).
These cattle did not show clinical symptoms. All animal experiments were approved by the National
Institute of Animal Health Ethics Committee.
May 2018 Volume 86 Issue 5 e00910-17

iai.asm.org 11

Sajiki et al.

Infection and Immunity

May 2018 Volume 86 Issue 5 e00910-17

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

Cell preparation and culture. Peripheral blood samples were collected from cattle that were
uninfected or experimentally infected with M. avium subsp. paratuberculosis. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by density gradient centrifugation on Percoll (GEHealthcare, Buckinghamshire, England, UK). Cells were cultured in 200 ␮l RPMI 1640 medium (SigmaAldrich, St. Louis, MO, USA) including 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientiﬁc,Waltham, MA, USA), 200 IU/ml penicillin, 200 ␮g/ml streptomycin, and 0.01% L-glutamine (Thermo Fisher
Scientiﬁc) in 96-well culture plates (Corning Inc., Corning, NY, USA) at 37°C in a 5% CO2 atmosphere. The
culture assays of PBMCs from the experimentally infected animals were carried out using samples atvarious time points between 82 and 120 weeks after the ﬁrst inoculation.
Functional analysis of PGE2. To evaluate immunosuppressive functions of PGE2, PBMCs (4 ⫻ 105
cells/well) from uninfected cattle were labeled with carboxyﬂuorescein diacetate succinimidyl ester(CFSE) (Sigma-Aldrich) and incubated with 2.5 to 2,500 nM PGE2 (Cayman Chemical, Ann Arbor, MI, USA)for 72 h. Cultures were stimulated by adding 1 ␮g/ml of anti-CD3 monoclonal antibody (MAb) (MM1A;
Washington State University Monoclonal Antibody Center, Pullman, WA, USA) and 1 ␮g/ml of anti-CD28
MAb (CC220; Bio-Rad, Hercules, CA, USA) to each well.
To demonstrate the effects of PGE2 on IFN-␥ and TNF-␣ production, 72-h culture supernatants of
CFSE-labeled PBMCs were collected, and IFN-␥ and TNF-␣ were determined by enzyme-linked immunosorbent assay (ELISA) for bovine IFN-␥ (Mabtech, Nacka Strand, Sweden) and bovine TNF-␣ (Kingﬁsher
Biotech, St. Paul, MN, USA), following the manufacturer’s instructions.
To demonstrate the effects of PGE2 on cell proliferation, CFSE-labeled PBMCs were harvested after 72
h and incubated in phosphate-buffered saline (PBS) with 10% goat serum (Thermo Fisher Scientiﬁc) for
15 min at room temperature to prevent nonspeciﬁc reactions. Cells were washed with PBS containing 1%
bovine serum albumin (Sigma-Aldrich) (1% BSA-PBS) and stained with Alexa Fluor 647-conjugatedanti-CD4 MAb (CC30; Bio-Rad), peridinin-chlorophyll-protein complex/cyanin 5.5 (PerCP/Cy 5.5)conjugated anti-CD8 MAb (CC63; Bio-Rad), and phycoerythrin/cyanin 7 (PE/Cy7)-conjugated anti-IgM
MAb (IL-A30; Bio-Rad) for 25 min at room temperature. CC30 was prelabeled with a Zenon Alexa Fluor
647 mouse IgG1 labeling kit (Thermo Fisher Scientiﬁc). CC63 and IL-A30 were conjugated with PerCP/
Cy5.5 and PE/Cy7, respectively, by using Lightning-Link antibody labeling kits (Innova Biosciences,
Cambridge, England, UK). The stained cells were then washed with 1% BSA-PBS and analyzed immediately by FACS Verse (BD Biosciences, San Jose, CA, USA) and FCS Express 4 (De Novo Software, Glendale,
CA, USA). The primary antibodies used in this experiment are shown in Table S1 in the supplemental
material.
To investigate the effect of PGE2 on the transcriptional proﬁle of genes involved in the T-cell
response, PBMCs (4 ⫻ 105 cells/well) from uninfected cattle were incubated with 2,500 nM PGE2 (Cayman
Chemical) for 24 h. Total RNA was extracted from the cultivated cells in TRI Reagent (Molecular ResearchCenter, Cincinnati, OH, USA) following the manufacturer’s instructions. cDNA was synthesized from theobtained total RNA using PrimeScript reverse transcriptase (TaKaRa Bio, Otsu, Japan) following the
manufacturer’s instructions. The mRNA expression of COX-2, PD-L1, STAT3, and some cytokines in PBMCs
was assayed by real-time PCR performed with a LightCycler 480 system II (Roche Diagnostics, Mannheim,
Germany) using SYBR Premix DimerEraser (TaKaRa Bio) following the manufacturer’s instructions. The
GAPDH and ACTB genes were used as internal control genes. All assays were performed in duplicate. The
primer sequences used in this study are shown in Table S2 in the supplemental material.
Kinetic analysis of PGE2. The PGE2 concentrations in sera of uninfected animals and animals
naturally infected with M. avium subsp. paratuberculosis were measured using the Prostaglandin E2
Express enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemical) following the manufacturer’s
instructions with slight modiﬁcations. To indicate whether PGE2 production was promoted by Johnin
antigen stimulation, PBMCs (4 ⫻ 105 cells/well) from the infected cattle were incubated with 1 ␮g/ml ofJohnin puriﬁed protein derivative (J-PPD) for 5 days. J-PPD is the inactivated crude protein fraction
prepared from the culture supernatant of M. avium subsp. paratuberculosis, which is an immunogenic
antigen widely used to evaluate T-cell responses against the bacterial infection ex vivo. Total RNA thenwas extracted from the cultivated cells, and cDNA was synthesized from the obtained total RNA as
described above. The mRNA expression of COX-2 in PBMCs was then assayed by real-time PCR as
described above (Table S2). Culture supernatants were collected, and the PGE2 concentration wasdetermined by ELISA as described above.
Flow cytometry assay of PD-L1, PD-1, and LAG-3 expression. PBMCs (1 ⫻ 106 cells/well) fromcattle uninfected or experimentally infected with M. avium subsp. paratuberculosis were incubated with
1 ␮g/ml of J-PPD for 24 h. After 24 h, cells were harvested and incubated in PBS with 10% goat serum(Thermo Fisher Scientiﬁc) for 15 min at room temperature.
To analyze PD-L1 expression, the treated cells were washed with 1% BSA-PBS and stained with
anti-bovine PD-L1 MAb (4G12, rat IgG2a) (32) or a rat IgG2a isotype control (R35-95; BD Biosciences) for
25 min at room temperature. To detect PD-L1 on T cells and B cells, cells were washed with 1% BSA-PBS
after the reaction with primary antibody and then stained with phycoerythrin (PE)-conjugated anti-CD3
MAb (MM1A; Washington State University Monoclonal Antibody Center), ﬂuorescein isothiocyanate(FITC)-conjugated anti-CD4 MAb (CC8; Bio-Rad), PerCP/Cy5.5-conjugated anti-CD8 MAb (CC63; Bio-Rad),
PE/Cy7-conjugated anti-IgM MAb (IL-A30; Bio-Rad), and allophycocyanin (APC)-conjugated anti-rat immunoglobulin antibody (Southern Biotech, Birmingham, AL, USA) for 25 min at room temperature. MM1A
was prelabeled with the Zenon R-PE mouse IgG1 labeling kit (Thermo Fisher Scientiﬁc). CC63 and IL-A30
were conjugated with PerCP/Cy5.5 and PE/Cy7, respectively, by using Lightning-Link antibody labelingkits (Innova Biosciences). To detect PD-L1 on CD14⫹ cells, cells were washed with 1% BSA-PBS after thereaction with primary antibody and then stained with PerCP/Cy5.5-conjugated anti-CD14 MAb (CAM36A;
iai.asm.org 12

T-Cell Exhaustion by PGE2 during Johne’s Disease

Infection and Immunity

TABLE 1 Bacterial loads in fecal and tissue samples at necropsy
M. avium subsp.
paratuberculosis DNA (pg) ina:
AnimalC6
65b
1

StatusUninfected control
Experimentally infected
Naturally infected

Feces
ND
0.589
NA

Ileum tissue
ND
26.8
NA

aND, not detected; NA, not available.
bThe data for animal 65 have been published previously (27).

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

Washington State University Monoclonal Antibody Center) and APC-conjugated anti-rat immunoglobulin
antibody (Southern Biotech) for 25 min at room temperature. CAM36A was prelabeled using LightningLink PerCP/Cy5.5 antibody labeling kit (Innova Biosciences).
To analyze PD-1 expression, the treated cells were washed with 1% BSA-PBS and then stained with
anti-bovine PD-1 MAb (5D2, rat IgG2a) (46) or a rat IgG2a isotype control (R35-95; BD Biosciences) for 25
min at room temperature. To analyze LAG-3 expression, the treated cells were washed with 1% BSA-PBS
and then stained with anti-bovine LAG-3 MAb (71-2D8, rat IgG1) (27) or a rat IgG1 isotype control (R3-34;
BD Biosciences) for 25 min at room temperature. Cells were washed with 1% BSA-PBS after the reaction
with primary antibody and then stained with PerCP/Cy5.5-conjugated anti-CD3 MAb (MM1A; Washington
State University Monoclonal Antibody Center), FITC-conjugated anti-CD4 MAb (CC8; Bio-Rad), PEconjugated anti-CD8 MAb (CC63; Bio-Rad), PE/Cy7-conjugated anti-IgM MAb (IL-A30; Bio-Rad), andAPC-conjugated anti-rat immunoglobulin antibody (Southern Biotech) for 25 min at room temperature.
MM1A and IL-A30 were conjugated with PerCP/Cy5.5 and PE/Cy7, respectively, by using Lightning-Link
antibody labeling kits (Innova Biosciences). The stained cells were washed with 1% BSA-PBS and analyzed
immediately by FACS Verse (BD Biosciences) and FCS Express 4 (De Novo Software). The primary
antibodies used in this experiment are shown in Table S1 in the supplemental material.
Immunohistochemical assay of PD-L1, EP2, and PGE2 and Ziehl-Neelsen staining. Immunohistochemical assays were performed as previously described with slight modiﬁcations (27). Sections ofileum tissue from cattle experimentally or naturally infected with M. avium subsp. paratuberculosis were
evaluated. A naturally infected animal (naturally infected animal 1) had clinical symptoms of Johne’s
disease. Tissue samples from experimentally infected cattle were obtained from an animal (animal 65)
with bacterial shedding in feces and clinical symptoms such as diarrhea (Table 1) (27). Anti-prostaglandin
E2 antibody (Abcam, Cambridge, England, UK), anti-prostaglandin E receptor EP2 antibody [EPR8030(B);Abcam], or anti-PD-L1 MAb (6C11-3A11, rat IgG2a) (see Fig. S1 in the supplemental material) (32) wasused for the primary antibody for immunohistochemistry. Ziehl-Neelsen staining was performed aspreviously described (27).
Functional assay of COX-2 inhibition. To evaluate the immunostimulatory effects of COX-2
inhibition, PBMCs (4 ⫻ 105 cells/well) from cattle uninfected or experimentally infected with M. avium
subsp. paratuberculosis were labeled with CFSE (Sigma-Aldrich) and incubated with 1 ␮M meloxicam(Sigma-Aldrich) or dimethyl sulfoxide (DMSO) (Nacalai Tesque, Kyoto, Japan) for 3 days (uninfected cattle)
or 5 days (infected cattle). For stimulation of PBMCs from the uninfected cattle, 1 ␮g/ml of anti-CD3 MAb(MM1A; Washington State University Monoclonal Antibody Center) and 1 ␮g/ml of anti-CD28 MAb(CC220; Bio-Rad) were added to cultures; 1 ␮g/ml of J-PPD was added to cultures of PBMCs from theinfected cattle. After cultivation, IFN-␥ and TNF-␣ concentrations were measured by ELISA, and cellproliferation was measured by ﬂow cytometry as described above.
Functional analysis of combined COX-2 inhibition and PD-L1 blockade. To investigate theimmunostimulatory effects of combined treatment with COX-2 inhibitor and anti-PD-L1 blocking antibodies, PBMCs (4 ⫻ 105 cells/well) from cattle experimentally infected with M. avium subsp. paratuberculosis were labeled with CFSE (Sigma-Aldrich) and incubated with 1 ␮M meloxicam and 10 ␮g/ml ofanti-PD-L1 antibody in the presence of 1 ␮g/ml J-PPD for 5 days. DMSO (Nacalai Tesque) and rat IgG (10␮g/ml; Sigma-Aldrich) were used as negative controls. PPD puriﬁed from M. bovis BCG strain (B-PPD) (1␮g/ml) was used as a negative-control antigen. In this assay, two types of anti-PD-L1 blocking antibodies,anti-PD-L1 rat MAb (4G12) (32) and anti-PD-L1 rat-bovine chimeric antibody (ChAb) (Boch4G12) (34),were used to evaluate the combination effect. After 5 days of culture, the IFN-␥ concentration wasdetermined by ELISA, and cell proliferation was measured by ﬂow cytometry as described above.
Statistical analysis. Differences were evaluated by the Wilcoxon signed-rank test and the SteelDwass test. A P value of ⬍0.05 was considered statistically signiﬁcant.

SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00910-17.
SUPPLEMENTAL FILE 1, PDF ﬁle, 0.2 MB.
ACKNOWLEDGMENTS
We are grateful to Hideyuki Takahashi and Tomio Ibayashi for their valuable advice
and discussions. We thank Enago (http://www.enago.jp) for English language review.
May 2018 Volume 86 Issue 5 e00910-17

iai.asm.org 13

Sajiki et al.

Infection and Immunity

REFERENCES
1. Weller LC, Collington JS, Hartnell A, Conroy MD, Kaise T, Barker EJ,
Wilson SM, Taylor WG, Jose JP, Williams JT. 2007. Chemotactic actionof prostaglandin E2 on mouse mast cells acting via the PGE2 receptor
3. Proc Natl Acad Sci U S A 104:11712–11717. https://doi.org/10.1073/
pnas.0701700104.
2. Phipps RP, Stein SH, Roper RL. 1991. A new view of prostaglandin E
regulation of the immune response. Immunol Today 12:349 –352.
https://doi.org/10.1016/0167-5699(91)90064-Z.
3. Morita I. 2002. Distinct functions of COX-1 and COX-2. Prostaglandins
Other Lipid Mediat 68-69:165–175.
4. Subbaramaiah K, Telang N, Ramonetti TJ, Araki R, DeVito B, Weksler BB,
Dannenberg JA. 1996. Transcription of cyclooxygenase-2 is enhanced intransformed mammary epithelial cells. Cancer Res 56:4424 – 4429.
5. Honda A, Sugimoto Y, Namba T, Watabe A, Irie A, Negishi M, NarumiyaS, Ichikawa A. 1993. Cloning and expression of cDNA for mouse prostaglandin E receptor EP2 subtype. J Biol Chem 268:7759 –7762.
6. Yao C, Hirata T, Soontrapa K, Ma X, Takemori H, Narumiya S. 2013.
Prostaglandin E2 promotes Th1 differentiation via synergistic ampliﬁcation of IL-12 signaling by cAMP and PI3-kinase. Nat Commun 4:1685.
https://doi.org/10.1038/ncomms2684.
7. Betz M, Fox SB. 1991. Prostaglandin E2 inhibits production of Th1
lymphokines but not of Th2 lymphokines. J Immunol 146:108 –113.
8. Wang D, DuBois RN. 2013. An inﬂammatory mediator, prostaglandin E2,
in colorectal cancer. Cancer J 19:502–510. https://doi.org/10.1097/PPO.0000000000000003.
9. Wang D, DuBois RN. 2016. The role of prostaglandin E2 in tumor associated immunosuppression. Trends Mol Med 22:1–3. https://doi.org/10.1016/j.molmed.2015.11.003.
10. Dumais N, Barbeau B, Olivier M, Tremblay MJ. 1998. Prostaglandin E2
up-regulates HIV-1 long terminal repeat-driven gene activity in T cells
via NF-kappaB-dependent and -independent signaling pathways. J Biol
Chem 273:27306 –27314. https://doi.org/10.1074/jbc.273.42.27306.
11. Dumais N, Bounou S, Olivier M, Tremblay MJ. 2002. Prostaglandin E2mediated activation of HIV-1 long terminal repeat transcription in human T cells necessitates CCAAT/enhancer binding protein (C/EBP) bindMay 2018 Volume 86 Issue 5 e00910-17

ing sites in addition to cooperative interactions between C/EBP␤ andcyclic adenosine 5=-monophosphate response element binding protein.
J Immunol 168:274 –282. https://doi.org/10.4049/jimmunol.168.1.274.
12. Waris D, Siddiqui A. 2005. Hepatitis C virus stimulates the expression of
cyclooxygenase-2 via oxidative stress: role of prostaglandin E2 in RNA
replication. J Virol 79:9725–9734. https://doi.org/10.1128/JVI.79.15.9725-9734.2005.
13. Stolina M, Sharma S, Lin Y, Dohadwala M, Gardner B, Luo J, Zhu L,
Kronenberg M, Miller PW, Portanova J, Lee JC, Dubinett SM. 2000.
Speciﬁc inhibition of cyclooxygenase 2 restores antitumor reactivity byaltering the balance of IL-10 and IL-12 synthesis. J Immunol 164:361–370. https://doi.org/10.4049/jimmunol.164.1.361.
14. Pettersen FO, Torheim EA, Dahm AE, Aaberge IS, Lind A, Holm M,
Aandahl EM, Sandset PM, Taskén K, Kvale D. 2011. An exploratory trial of
cyclooxygenase type 2 inhibitor in HIV-1 infection: downregulated immune activation and improved T cell-dependent vaccine responses. J
Virol 85:6557– 6566. https://doi.org/10.1128/JVI.00073-11.
15. Rathnaiah G, Zinniel DK, Bannantine JP, Stabel JR, Gröhn YT, Collins MT,
Barletta RG. 2017. Pathogenesis, molecular genetics, and genomics of
Mycobacterium avium subsp. paratuberculosis, the etiologic agent ofJohne’s disease. Front Vet Sci 4:187. https://doi.org/10.3389/fvets.2017.00187.
16. Li L, Katani R, Schilling M, Kapur V. 2016. Molecular epidemiology of
Mycobacterium avium subsp. paratuberculosis on dairy farms. Annu Rev
Anim Biosci 4:155–176. https://doi.org/10.1146/annurev-animal-021815-111304.
17. Stabel JR. 2006. Host responses to Mycobacterium avium subsp.
paratuberculosis: a complex arsenal. Anim Heal Res Rev 7:61–70. https://doi.org/10.1017/S1466252307001168.
18. Coussens PM. 2004. Model for immune responses to Mycobacterium
avium subspecies paratuberculosis in cattle. Infect Immun 72:3089 –3096.
https://doi.org/10.1128/IAI.72.6.3089-3096.2004.
19. Sohal JS, Singh SV, Tyagi P, Subhodh S, Singh PK, Singh AV, Narayanasamy K, Sheoran N, Singh Sandhu K. 2008. Immunology of mycobacterial
infections: with special reference to Mycobacterium avium subspecies
iai.asm.org 14

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

Y. Sajiki, S. Konnai, S. Murata, Y. Mori, and K. Ohashi were responsible for theconception and design of the study. Y. Sajiki, S. Konnai, T. Okagawa, A. Nishimori, N.
Maekawa, S. Goto, R. Ikebuchi, R. Nagata, S. Kawaji, Y. Kagawa, S. Yamada, and Y. Kato
performed the experiments. Y. Sajiki, S. Konnai, T. Okagawa, Y. Suzuki, S. Murata, Y. Mori,
and K. Ohashi analyzed the data. R. Nagata, S. Kawaji, S. Yamada, Y. Kato, and Y. Mori
provided intellectual input, laboratory materials, reagents, and/or analytic tools. Y. Sajiki
wrote the manuscript. S. Konnai, T. Okagawa, Y. Suzuki, S. Murata, Y. Mori, and K. Ohashi
contributed to the revision of the manuscript. All authors reviewed and approved theﬁnal manuscript.
This work was supported by JSPS KAKENHI, Research Project for Improving Animal
Disease Prevention Technologies to Combat Antimicrobial Resistance 2017-2021 FY of
the Ministry of Agriculture, Forestry and Fisheries of Japan and by grants from the
Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry, Japan (number 26058B, to S. Konnai) and the NARO, Biooriented Technology Research Advancement Institution (the special scheme project onregional developing strategy; grant 16817557 to S. Konnai). This research was partially
supported by the Platform Project for Supporting Drug Discovery and Life Science
Research (Basis for Supporting Innovative Drug Discovery and Life Science Research[BINDS]) and a grant from the Ministry of Education, Culture, Sports, Science, andTechnology (MEXT), Japan, for the Joint Research Program of the Research Center for
Zoonosis Control, Hokkaido University, to Y. Suzuki.
The funders had no role in study design, data collection and interpretation, or thedecision to submit the work for publication.
S. Konnai, T. Okagawa, A. Nishimori, N. Maekawa, S. Goto, Y. Suzuki, S. Murata, and
K. Ohashi have a patent pending for materials and techniques described in this paper(Japanese patent application number 2017-140891).

T-Cell Exhaustion by PGE2 during Johne’s Disease

May 2018 Volume 86 Issue 5 e00910-17

in Mycobacterium avium subsp. paratuberculosis infection. Cell Commun
Signal 14:29. https://doi.org/10.1186/s12964-016-0152-z.
37. Edwards CK 3rd, Hedegaard HB, Zlotnik A, Gangadharam PR, Johnston
RB, Jr, Pabst MJ. 1986. Chronic infection due to Mycobacterium intracellulare in mice: association with macrophage release of prostaglandin E2
and reversal by injection of indomethacin, muramyl dipeptide, orinterferon-gamma. J Immunol 136:1820 –1827.
38. Chen M, Divangahi M, Gan H, Shin DS, Hong S, Lee DM, Serhan CN, Behar
SM, Remold HG. 2008. Lipid mediators in innate immunity against
tuberculosis: opposing roles of PGE2 and LXA4 in the induction ofmacrophage death. J Exp Med 205:2791–2801. https://doi.org/10.1084/
jem.20080767.
39. Schmedtje JF, Ji Y-S, Liu W-L, DuBois RN, Runge MS. 1997. Hypoxia
induces cyclooxygenase-2 via the NF-␬B p65 transcription factor inhuman vascular endothelial cells. J Biol Chem 272:601– 608.
40. Jobin C, Morteau O, Han DS, Balfour Sartor R. 1998. Speciﬁc NF-kappaB
blockade selectively inhibits tumour necrosis factor-alpha-induced
COX-2 but not constitutive COX-1 gene expression in HT-29 cells. Immunology 95:537–543.
41. Vila-del Sol V, Fresno M. 2005. Involvement of TNF and NF-␬B in thetranscriptional control of cyclooxygenase-2 expression by IFN-␥ in macrophages. J Immunol 174:2825–2833. https://doi.org/10.4049/jimmunol.174.5.2825.
42. Abdelhamed S, Ogura K, Yokoyama S, Saiki I, Hayakawa Y. 2016. AKTSTAT3 pathway as a downstream target of EGFR signaling to regulate
PD-L1 expression on NSCLC cells. J Cancer 7:1579 –1586. https://doi.org/
10.7150/jca.14713.
43. Maślanka T. 2014. Effect of dexamethasone and meloxicam on counts of
selected T lymphocyte subpopulations and NK cells in cattle—in vivo
investigations. Res Vet Sci 96:338 –346. https://doi.org/10.1016/j.rvsc.2014.02.006.
44. Van Engen NK, Platt R, Roth JA, Stock ML, Engelken T, Vann RC, Wulf LW,
Busby WD, Wang C, Kalkwarf EM, Coetzee JF. 2016. Impact of oral
meloxicam and long-distance transport on cell-mediated and humoral
immune responses in feedlot steers receiving modiﬁed live BVDV
booster vaccination on arrival. Vet Immunol Immunopathol 175:42–50.
https://doi.org/10.1016/j.vetimm.2016.05.006.
45. Okagawa T, Konnai S, Nishimori A, Maekawa N, Ikebuchi R, Goto S,
Nakajima C, Kohara J, Ogasawara S, Kato Y, Suzuki Y, Murata S, Ohashi K.
2017. Anti-bovine programmed death-1 rat-bovine chimeric antibody
for immunotherapy of bovine leukemia virus infection in cattle. Front
Immunol 8:650. https://doi.org/10.3389/ﬁmmu.2017.00650.
46. Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y,
Murata S, Ohashi K. 2013. Blockade of bovine PD-1 increases T cellfunction and inhibits bovine leukemia virus expression in B cells in vitro.
Vet Res 44:59. https://doi.org/10.1186/1297-9716-44-59.
47. Ikebuchi R, Konnai S, Shirai T, Sunden Y, Murata S, Onuma M, Ohashi K.
2011. Increase of cells expressing PD-L1 in bovine leukemia virus infection and enhancement of anti-viral immune responses in vitro via PD-L1
blockade. Vet Res 42:103. https://doi.org/10.1186/1297-9716-42-103.
48. Shirai T, Konnai S, Ikebuchi R, Okagawa T, Suzuki S, Sunden Y, Onuma M,
Murata S, Ohashi K. 2011. Molecular cloning of bovine lymphocyte
activation gene-3 and its expression characteristics in bovine leukemia
virus-infected cattle. Vet Immunol Immunopathol 144:462– 467. https://doi.org/10.1016/j.vetimm.2011.08.018.
49. Okagawa T, Konnai S, Ikebuchi R, Suzuki S, Shirai T, Sunden Y, Onuma M,
Murata S, Ohashi K. 2012. Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection. Vet Res 43:45. https://doi.org/10.1186/1297-9716-43-45.
50. Okagawa T, Konnai S, Deringer JR, Ueti MW, Scoles GA, Murata S, Ohashi
K, Brown WC. 2016. Cooperation of PD-1 and LAG-3 contributes to T-cell
exhaustion in Anaplasma marginale-infected cattle. Infect Immun 84:2779 –2790. https://doi.org/10.1128/IAI.00278-16.
51. Suzuki S, Konnai S, Okagawa T, Ikebuchi R, Nishimori A, Kohara J,
Mingala CN, Murata S, Ohashi K. 2015. Increased expression of theregulatory T cell-associated marker CTLA-4 in bovine leukemia virus
infection. Vet Immunol Immunopathol 163:115–124. https://doi.org/10.1016/j.vetimm.2014.10.006.
52. Nagata R, Kawaji S, Minakawa Y, Wang X, Yanaka T, Mori Y. 2010. A speciﬁc
induction of interleukin-10 by the Map41 recombinant PPE antigen of
Mycobacterium avium subsp. paratuberculosis. Vet Immunol Immunopathol
135:71–78. https://doi.org/10.1016/j.vetimm.2009.11.002.

iai.asm.org 15

Downloaded from http://iai.asm.org/ on April 23, 2018 by HOKKAIDO UNIV LIBRARY

paratuberculosis. Immunobiology 231:585–598. https://doi.org/10.1016/
j.imbio.2007.11.002.
20. Bassey EO, Collins MT. 1997. Study of T-lymphocyte subsets of healthy
and Mycobacterium avium subsp. paratuberculosis-infected cattle. InfectImmun 65:4869 – 4872.
21. Burrells C, Clarke CJ, Colston A, Kay JM, Porter J, Little D, Sharp JM. 1999.
Interferon-gamma and interleukin-2 release by lymphocytes derived fromthe blood, mesenteric lymph nodes and intestines of normal sheep andthose affected with paratuberculosis (Johne’s disease). Vet Immunol Immunopathol 68:139–148. https://doi.org/10.1016/S0165-2427(99)00022-7.
22. Weiss DJ, Evanson OA, Souza CD. 2006. Mucosal immune response incattle with subclinical Johne’s disease. Vet Pathol 43:127–135. https://doi.org/10.1354/vp.43-2-127.
23. Nishimura H, Nose M, Hiai H, Minato N, Honjo T. 1999. Development oflupus like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif carrying immunoreceptor. Immunity 11:141–151.
https://doi.org/10.1016/S1074-7613(00)80089-8.
24. Sierro S, Romero P, Speiser DE. 2011. The CD4-like molecule LAG-3,
biology and therapeutic applications. Expert Opin Ther Targets 15:
91–101. https://doi.org/10.1517/14712598.2011.540563.
25. Keir ME, Butte MJ, Freeman GJ, Sharpe AH. 2008. PD-1 and its ligands intolerance and immunity. Annu Rev Immunol 26:677–704. https://doi.org/10.1146/annurev.immunol.26.021607.090331.
26. Wherry EJ. 2011. T cell exhaustion. Nat Immunol 12:492– 499. https://doi.org/10.1038/ni.2035.
27. Okagawa T, Konnai S, Nishimori A, Ikebuchi R, Mizorogi S, Nagata R,
Kawaji S, Tanaka S, Kagawa Y, Murata S, Mori Y, Ohashi K. 2016. Bovine
immunoinhibitory receptors contribute to suppression of Mycobacterium avium subsp. paratuberculosis-speciﬁc T-cell responses. Infect Immun 84:77– 89. https://doi.org/10.1128/IAI.01014-15.
28. Prima V, Kaliberova LN, Kaliberov S, Curiel DT, Kusmartsev S. 2016.
COX2/mPGES1/PGE2 pathway regulates PD-L1 expression in tumorassociated macrophages and myeloid-derived suppressor cells. Proc Natl
Acad Sci U S A 144:1117–1122.
29. Botti G, Fratangelo F, Cerrone M, Liguori G, Cantile M, Anniciello AM,
Scala S, D’Alterio C, Trimarco C, Ianaro A, Cirino G, Caracò C, Colombino
M, Palmieri G, Pepe S, Ascierto PA, Sabbatino F, Scognamiglio G. 2017.
COX-2 expression positively correlates with PD-L1 expression in humanmelanoma cells. J Transl Med 15:46. https://doi.org/10.1186/s12967-017-1150-7.
30. Chen JH, Perry CJ, Tsui YC, Staron MM, Parish IA, Dominguez CX,
Rosenberg DW, Kaech SM. 2015. Prostaglandin E2 and programmed cell
death 1 signaling coordinately impair CTL function and survival during
chronic viral infection. Nat Med 21:327–334. https://doi.org/10.1038/nm.3831.
31. Plattner BL, Chiang YW, Roth JA, Platt R, Huffman E, Zylstra J, Hostetter
JM. 2011. Direct inoculation of Mycobacterium avium subspecies paratuberculosis into ileocecal Peyer’s patches results in colonization of theintestine in a calf model. Vet Pathol 48:584 –592. https://doi.org/10.1177/
0300985810383874.
32. Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y,
Murata S, Ohashi K. 2014. Inﬂuence of PD-L1 cross-linking on cell deathin PD-L1-expressing cell lines and bovine lymphocytes. Immunology
142:551–561. https://doi.org/10.1111/imm.12243.
33. Goto S, Konnai S, Okagawa T, Nishimori A, Maekawa N, Gondaira S,
Higuchi H, Koiwa M, Tajima M, Kohara J, Ogasawara S, Kato Y, Suzuki Y,
Murata S, Ohashi K. 2017. Increase of cells expressing PD-1 and PD-L1
and enhancement of IFN-␥ production via PD-1/PD-L1 blockade inbovine mycoplasmosis. Immun Inﬂamm Dis 5:355–363. https://doi.org/
10.1002/iid3.173.
34. Nishimori A, Konnai S, Okagawa T, Maekawa N, Ikebuchi R, Goto S, Sajiki
Y, Suzuki Y, Kohara J, Ogasawara S, Kato Y, Murata S, Ohashi K. 2017. In
vitro and in vivo antivirus activity of an anti-programmed death-ligand 1(PD-L1) rat-bovine chimeric antibody against bovine leukemia virus
infection. PLoS One 12:e0174916. https://doi.org/10.1371/journal.pone.0174916.
35. Weiss DJ, Evanson OA, de Souza C, Abrahamsen MS. 2005. A critical roleof interleukin-10 in the response of bovine macrophages to infection by
Mycobacterium avium subsp.paratuberculosis. Am J Vet Res 66:721–726.
https://doi.org/10.2460/ajvr.2005.66.721.
36. Hussain T, Shah SZ, Zhao D, Sreevatsan S, Zhou X. 2016. The role of IL-10

Infection and Immunity