TIGIT Negatively Regulates Inflammation by Altering

Immunobiology 221 (2016) 48–55

Contents lists available at ScienceDirect

Immunobiology

jo urnal homepage: www.elsevier.com/locate/imbio

TIGIT negatively regulates inﬂammation by altering macrophage phenotype

a,1 a,1 b c a a

Xi Chen , Pu-Han Lu , Lei Liu , Ze-Min Fang , Wu Duan , Zhe-Long Liu ,

d e a a,∗

Cong-Yi Wang , Ping Zhou , Xue-Feng Yu , Wen-Tao He

Department of Endocrinology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China

Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China

Department of Cardiothoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China

Center for Biomedical Research, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China

Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China

r t i c l e i n f o a b s t r a c t

Article history: Macrophages function as an essential component of innate immune system, contributing to both the

Received 18 March 2015

initiation and appropriate resolution of inﬂammation. The exposure of macrophages to the microbial

Received in revised form 22 June 2015

products, such as lipopolysaccharide (LPS), can strongly shift the balance between tissue homeostasis and

Accepted 11 August 2015

inﬂammation in favor of causing systemic damage, in which macrophage M1 polarization play important

Available online 17 August 2015

roles. Strategies aiming at restoring the balance of macrophage polarization remain to be further explored.

Herein, we have demonstrated that poliovirus receptor

Keywords:

(PVR), the receptor of TIGIT, was dramatically upregulated on the surface of mouse peritoneal

TIGIT

PVR macrophages when exposed to LPS. TIGIT-Fc fusion protein not only inhibited the macrophage acti-

Macrophage vation, but also skewed M1/M2 balance toward an anti-inﬂammatory proﬁle, especially enhanced the

Polarization secretion of IL-10. The activation of TIGIT/PVR pathway in macrophages correlated with increased nuclear

LPS translocation of c-Maf, which promotes IL-10 transcription. Treatment with ﬁbroblasts stably secreting

TIGIT-Fc fusion protein signiﬁcantly reversed the lethal and sublethal endotoxic shock, which facilitated

peritoneal macrophages to switch towards anti-inflammatory M2 cytokine profiles. These findings high-

light a novel role of the TIGIT/PVR pathway in macrophage M2 polarization and suggest that TIGIT may

have the potential to optimize the treatment of macrophage-involved inﬂammatory diseases.

1. Introduction

Abbreviations: TIGIT, T cell immunoglobulin and ITIM domain; PVR, poliovirus Macrophages play indispensable roles in host homeostasis

receptor; TLR, toll-like receptor; LPS, lipopolysaccharide; Fc, fragment crystalliz- and defense, which are widely distributed as sentinels for the

able region of immunoglobulin; TNF-␣, tumor necrosis factor-␣; MCP-1, monocyte

ﬁrst line alerts. They are functionally and phenotypically polar-

chemoattractant protein-1; IL, interleukin; CCL11, C-C motif chemokine 11; GM-

ized heterogeneous population under different stimulation, such

CSF, granulocyte-macrophage colony-stimulating factor; IFN-␥, interferon gamma;

as microbial products, cytokines or other immunomodulatory

CXCL1, C-X-C motif ligand 1; MIP-1␣, macrophage inﬂammatory protein-1␣; MIP-

1␤, macrophage inﬂammatory protein-1␤; RANTES, regulated on activation: normal molecules [1,2]. Polarized macrophages are classiﬁed into two

T cell expressed and secreted; GAPDH, glyceraldehyde-3-phosphate dehydroge-

major subtypes termed proinﬂammatory M1 (classically acti-

nase; TGF-␤, transforming growth factor ␤; Fgl2, ﬁbrinogen-like protein 2; Teff,

vated) and anti-inﬂammatory M2 (alternatively activated). M1

effector T cells; Treg, regulatory T cells; Akt, also known as PKB (protein kinase B);

macrophages are typically induced by exposure to IFN-␥ or TNF-

eGFP, enhanced green ﬂuorescent protein; DC, dendritic cells; G-CSF, granulocyte

␣

colony stimulating factor; GM-CSF, granulocyte-macrophage colony stimulating in the presence bacterial components such as lipopolysacchride

factor; ELISA, enzyme-linked immunosorbant assay; c-Maf, V-maf (Avian muscu- (LPS). M2 macrophages could be polarized by a number of stimuli,

loaponeurotic ﬁbrosarcoma oncogene) homolog; PVDF, polyvinylidene diﬂuoride;

including IL-4, IL-10 and glucocorticoid [3]. It has been demon-

Arg1, arginase 1.

∗ strated that elimination of M2 macrophages precipitates the

Corresponding author at: Department of Endocrinology, Tongji Hospital, Tongji

Medical College, Huazhong University of Science and Technology, 1095 Jiefang Road,

Wuhan 430030, China. Fax: +86 27 83662883.

E-mail address: [email protected] (W.-T. He).

These authors contributed equally to this study.

http://dx.doi.org/10.1016/j.imbio.2015.08.003

X. Chen et al. / Immunobiology 221 (2016) 48–55 49

LPS-induced lung inﬂammation [4]. In contrast, Akt2 ablation with 10% FBS. All non-adherent cells were removed before the fur-

resulting in an M2 phenotype promotes the resistance to LPS- ther experiments.

induced shock [5]. Delicate equilibrium of M1 and M2 macrophages

participates in orchestrating LPS-induced inﬂammation and its res-

2.3. Plasmid construction and lentiviral production

olution. However, the speciﬁc contributions of newly identiﬁed

molecules on macrophage polarization are not fully understood.

Total RNA was isolated from spleens by TRIzol Reagent (Invit-

It has been demonstrated that macrophages could upregulate

rogen, Carlsbad, CA) and cDNA was synthesized by reverse

poliovirus receptor (PVR, also known as CD155) expression in

transcription with a ReverTra Ace-␣-kit (TOYOBO, Osaka, Japan).

response to Toll-like receptors (TLRs) activation [6]. Given that PVR

The extracellular fragment of mouse TIGIT (amino acids 1–135)

serves as a receptor for T cell immunoglobulin and ITIM domain

excluding stop codon was PCR ampliﬁed (primers: 5 AAG CTT ATG

(TIGIT), a newly identiﬁed immunosuppressive molecule [7], we

CAT GGC TGG CTG CTC CTG GT3 and 5 GGA TCC GAA CTG AGC

suppose that TIGIT may possess the capability to negatively regu-

CAC TGA GCT TTC TT3 ) and cloned into an Ig␬-Fc-pSecTag2B vec-

late the function of macrophages.

tor which contains a human IgG3 Fc domain (generously provided

TIGIT has been identiﬁed as a new member of CD28 family. It is

by Prof. Jin Bo-Quan, the Fourth Military Medical University, China).

preferentially expressed on activated effector T cells (Teff), regula-

The Fc-fused TIGIT fragment (TIGIT-Fc) was then ampliﬁed by PCR

tory T cells (Treg), and NK cells [7,8]. The ligand for TIGIT is PVR,

using primers (5 GGA AGA TCT CCA CCA TGG AGA CAG ACA CAC

an immunoglobulin-like cell adhesion molecule and a member of

T3 and 5 GGA AGA TCT GAA TTC TCA TTT ACC CGG AGA CA3 )

the nectin-like family, which is expressed on dendritic cells (DCs),

containing the BglII restriction site and inserted into the lentivi-

endothelial cells and some tumor cells [7,9]. TIGIT/PVR interface

ral transfer vector pLOX (generously provided by Dr. Huang Min,

forms a conserved speciﬁc “lock-and-key” structure and transduces

Tongji Hospital, HUST, China). The lipofectamine 2000 (Invitrogen,

a bidirectional signaling. On one hand, TIGIT acts as a co-inhibitory

Carlsbad, CA) transfection method was used for lentivirus pro-

receptor on T cells and NK cells to inhibit their activation [10,11].

duction. HEK293T cells were seeded at a density of 8 × 10 cells

Agonistic anti-TIGIT has been shown to markedly inhibit the acti-

per 15 cm culture dish one day before transfection. Each indicated

vation of T cells through down-regulation of the TCR alpha chain

lentiviral transfer vector (24 ␮g) (TIGIT-Fc-pLOX or empty vec-

[10]. This novel ﬁnding supports the notion that TIGIT acts as a

tor pLOX) was mixed with pCMVR8.2 (18 ␮g) and pMD.G (9 ␮g)

co-inhibitory molecule which acts on the upstream of TCR signal-

for co-transfection. Supernatant containing recombinant lentivi-

ing. Moreover, the ligation of TIGIT on Treg induces a paracrine

ral particles was passed through a 0.45 ␮m ﬁlter (Corning, NY,

mechanism that secretion of ﬁbrinogen-like protein 2 (Fgl2) sup- ◦

USA) 48 h after transfection and then stored at −80 C. Multiplic-

press the Teff proliferation [12]. On the other hand, TIGIT acts as

ity of infection and viral titer of the supernatant were determined

a ligand for PVR expressed on DCs. Yu et al. have reported that

according to the method previously described [15]. Brieﬂy, 5 × 10

TIGIT could indirectly inhibit the T cell responses by modulating

293T cells were plated into a 96-well plate and lentivirus was

cytokine proﬁles of mature DCs via ligation on PVR [7]. In short,

−2 −1

added with dilutions ranging from 10 to 10 in the presence

TIGIT acts as an immunomodulatory molecule both on adaptive

of 6 ␮g/ml of polybrene (Sigma–Aldrich, St. Lous, MO). Four days

and innate immune cells. But the role(s) of TIGIT on another com-

after transduction, biological titer was calculated by the following

ponent of innate immune system, namely macrophages, remains

equation: transduction unit (TU/ml) = (% of enhanced Green Flu-

(remain) unknown.

orescent Protein (eGFP) positive cells × number of cells at time of

The response to endotoxin, also known as LPS, is an important

transduction) × dilution factor / (100 × volume of lentivirus added).

mechanism for macrophages to defense against Gram-negative

bacteria. However, excessive LPS challenge leads to life-threatening

endotoxic shock in which M1 macrophages play fundamental roles

2.4. Production of NIH/3T3 cell lines stably secreting TIGIT-Fc

[4,13]. Herein, we show in a rodent endotoxin shock model that

TIGIT leads to a shift from M1 to M2 polarization via ligation on PVR 5

NIH/3T3 cells (2 × 10 ) were suspended in 0.4 ml RPMI 1640

expressed on mature macrophages, resulting in protection against

medium containing 6 ␮g/ml polybrene in a 1.5 ml tube, and 0.1 ml

lethal shock. 7

of viral stock (2 × 10 TU/ml, MOI = 10) was added and incubated

◦ 2

at 37 C for 2 h. Infected cells were then transferred into a 25 cm

tissue culture ﬂask with 2 ml of RPMI 1640 medium and incu-

2. Materials and methods

◦

bated at 37 C with 5% CO2. The medium was replaced 24 h after

infection and transduction efﬁciencies were assessed on day 4.

2.1. Mice

The percentage of eGFP positive cells was estimated under a ﬂu-

orescence microscope (Nikon Eclipse TE2000-U, Tokyo, Japan) and

Speciﬁc pathogen-free (SPF) male BALB/c mice (6–8 weeks

determined by Flow cytometry (BD FACS Calibur, Bedford, MA). The

old) were purchased from Hunan SJA Laboratory Animal Company

cell line stably secreting TIGIT-Fc protein was designated as TIG-Fc-

(Changsha, China). Animal were maintained and used according to

3T3, with the cell line eGFP-3T3 which integrated the empty pLOX

the NIH Guide for the Care and Use of Laboratory Animals. All animal

vector as control. The concentration of TIGIT-Fc in the supernatant

studies conducted were approved by Animal Research Committee

was determined and calculated by automated immunoturbidimet-

of Tongji Medical College, HUST.

ric assay of human IgG3 (Beckman Coulter AU5800, CA, USA).

2.2. Cell lines and isolation of mouse peritoneal macrophages

2.5. Puriﬁcation of soluble TIGIT-Fc protein

Human embryonic kidney (HEK) 293T cells and NIH/3T3 mouse

fibroblasts were purchased from the Type Culture Collection of the Recombinant TIGIT-Fc was purified by Novoprotein Scientific

Chinese Academy of Sciences (Shanghai, China) and maintained in Inc. (Shanghai, China) for in vitro experiment. In brief, fusion TIGIT-

DMEM (HyClone, Logan, UT) supplemented with 10% FBS (GIBCO, Fc was constructed in a mammalian expression vector using the

Grand Island, NY). Mouse peritoneal macrophages derived from plasmids containing TIGIT-Fc we provided as a template. Then, the

BALB/c mice were prepared as described previously [14]. The cells mammalian expression was introduced into CHO cells and secreted

were cultured in RPMI 1640 (HyClone, Logan, UT) supplemented Fc-fusion proteins were puriﬁed by afﬁnity chromatography on

50 X. Chen et al. / Immunobiology 221 (2016) 48–55

protein A/G column. The human IgG3 (Sigma–Aldrich, St. Louis, MO) 2.10. Statistical analysis

was used as isotype control.

Data are summarized as mean ± standard deviation (SD). Dif-

ferences among groups were evaluated by ANOVA with the

2.6. Flow cytometry and intracellular staining Students–Newman–Keuls (SNK) post hoc test. The paired indi-

vidual means were compared with the Student t test. Statistical

Peritoneal macrophages were collected and stained with PE- analysis of survival rate was performed by log-rank test. P val-

anti-PVR, PE-anti-CD206, APC-anti-CD11c, APC-F4/80, or speciﬁc ues <0.05 were considered statistically signiﬁcant. SPSS software

isotype antibodies (eBioscience, San Diego, CA). For the intracellu- (version 20.0) was used for all statistical procedures.

lar staining of TIGIT, TIG-Fc-3T3 cells were ﬁxed and permeabilized

with Fixation/ Permeabilization buffer and then stained by an 3. Results

Alexa Fluor 647-conjugated anti-TIGIT (eBioscience). For indi-

rect surface staining of TIGIT-Fc, peritoneal macrophages were 3.1. The kinetics of PVR expression on macrophages stimulated by

challenged with LPS (100 ng/ml) for 12 h (to induce PVR expres- LPS

sion on macrophages) and then incubated with 1 ml supernatant

derived from TIG-Fc-3T3 for 30 min, followed by staining with PE- To assess the dynamic expression patterns of PVR on

␥

anti-human IgG (Fc -speciﬁc, eBioscience). BD FACS Calibur ﬂow macrophages, peritoneal macrophages were exposed to graded

cytometer was used and data were processed with FlowJo software doses of LPS or medium for 24 h. Firstly, we evaluated the purity of

(Tree Star Inc., Ashland, OR). macrophages with F4/80 as the surface marker. The adherent cells

cultured in vitro was qualiﬁed to further studies (Fig. 1A). As shown

in Fig. 1B, PVR was almost undetectable on resting macrophages

2.7. Cytokine assay (1.8 ± 0.7%). When exposed to a very low dose of LPS (10 ␮g/ml), the

percentage of PVR macrophages rose to 72.3 ± 4.3%. An increase of

Cultural supernatants concentrations for 23 analytes were LPS concentration from 10 to 100 ␮g/ml resulted in an increase to

determined using Bio-Plex Pro multiplex bead array (Bio-Rad, Her- 82.3 ± 2.2%. However, further increase of LPS (1000 ␮g/ml) showed

cules, CA). The 23-Plex kit was used to measure the concentration no further increase of PVR cells (83.0 ± 5.4%). Then, macrophages

of cytokines by a Luminex 200 Labmap system (Luminex, Austin, were stimulated with LPS (100 ␮g/ml) at indicated time points. PVR

␣

TX). The levels of TNF- and IL-10 were also measured with ELISA was obviously upregulated on macrophages as early as 6 h after

kits (MultiSciences, Hangzhou, China). All assays were performed exposure. The percentage of PVR cells appeared to be reaching

per the manufacturers’ instructions. toward a maximum level at 24 h post exposure (Fig. 1B). These data

support the notion that LPS promotes the upregulation of PVR on

primary macrophages in a dose- and time-dependent manner.

2.8. Quantitative real-time PCR (RT-PCR) analysis

3.2. TIGIT modulates LPS-induced cytokine production in

For ex vitro macrophage assays, cells from peritoneal cavi- macrophages

ties were cultured for 2 h to obtain the adherent cells as puriﬁed

macrophages. The cells were then subjected to RT-PCR analysis Further experiments were performed to determine whether

for LPS-challenged mice treated with TIG-Fc-3T3 or control. Total TIGIT, the ligand for PVR, mediates the functional swift of LPS-

RNA and cDNA were prepared as described above. Ampliﬁcations stimulated macrophages. Peritoneal macrophages were incubated

of cDNA were performed using SYBR Green real-time PCR Mas- with LPS (100 ␮g/ml) in the presence of TIGIT-Fc (100 ␮g/ml) or

ter Mix (Toyobo, Osaka, Japan). All the primers used are shown in isotype control for 24 h, followed by Luminex 23-Plex assays of

Supplementary Table 1. The quantitative PCR run consisted of ini- cytokine levels in the culture supernatants. As shown in Fig. 2A,

◦

tial denaturation at 95 C for 10 min followed by 40 cycles with TIGIT-Fc enhanced the level of IL-10 secretion and reduced the lev-

◦ ◦

denaturation at 95 C for 10 s, annealing at 60 C for 20 s, and poly- els of IL-1␤, IL-13, IL-17, IFN-␥, TNF-␣, MCP-1, MIP-1␣, MIP-1␤ and

◦ ◦

merization at 72 C for 20 s, and ﬁnally by extension at 72 C for G-CSF. To conﬁrm the results of the Luminex, ELISAs were further

5 min. All samples were normalized to GAPDH internal control. validated for IL-10 and TNF-␣. Peritoneal macrophages were stim-

ulated by LPS combined with TIGIT-Fc protein or isotype control for

increasing period of time, followed by ELISA analysis. Compared to

2.9. Western blotting

the isotype control or LPS control, the levels of TNF-␣ signiﬁcantly

decreased as early as 2 h post exposure, and persisted until 24 h,

Cytoplasmic and nuclear fractions were prepared from peri-

while the levels of IL-10 markedly rose at 12 h and continued to

toneal macrophages using a nuclear and cytoplasmic protein

be higher until 24 h (Fig. 2B and C). RT-PCR analyses showed the

extraction kit (Beyotime, Jiangsu, China). Nuclear and cytosolic pro-

early decrease of TNF-␣ mRNA and early increase of IL-10 mRNA

teins (∼10 ␮g) were separated by SDS-PAGE and then electrically

when macrophages were exposed to LPS and TIGIT-Fc (Fig. 2D).

transferred onto 0.45 ␮m PVDF (Polyvinylidene Diﬂuoride) mem-

However, the mRNA level of arginase 1 (Arg1), as a M2 marker,

branes. After blocking with 5% (w/v) bovine serum albumins in

began to increase at the later phase of incubation. These data

Tris-buffered saline (TBS) for 1 h, the membrane was washed and

have demonstrated that TIGIT-Fc promoted the functional remod-

then incubated with indicated primary antibodies (anti-␤-actin

eling of LPS-stimulated macrophages with preferential secretion of

and anti-histone H3 from Beyotime, Jiangsu, China; anti-c-Maf

anti-inﬂammatory cytokine IL-10 and signiﬁcantly reduced various

◦

from Santa Cruz, CA, USA) with appropriate dilutions at 4 C over

pro-inﬂammatory cytokines, such as TNF-␣.

night. After washes, the membranes were incubated at room tem-

perature with an HRP-conjugated goat anti-rabbit IgG (Beyotime,

3.3. TIGIT inhibits the LPS-induced macrophage activation and

Jiangsu, China) (dilution at 1:10000) for 1 h and then visualized by

reshapes macrophage polarization

using the ECL Plus western blotting detection reagents (Beyotime,

Jiangsu, China). ␤-actin and histone H3 were used as cytoplasmic

Resting macrophages (M0) could be activated by LPS/IFN-␥ and

and nuclear loading control, respectively.

polarized toward classically pro-inﬂammatory M1. On the other

X. Chen et al. / Immunobiology 221 (2016) 48–55 51

Fig. 1. The dynamic expression of PVR on LPS-stimulated macrophages appears to be in a time- and dose-dependent manner. (A) The purity of peritoneal macrophages is

shown for percentage of F4/80 positive cells. (B) The patterns of PVR cells are shown after stimulation with gradient concentrations of LPS for 24 h, or after exposure to LPS

(100 ng/ml) at indicated time points. Histograms represent surface staining of PVR (solid line) compared with isotype control (shaded gray).

Fig. 2. TIGIT modulates cytokine proﬁles of LPS-challenged macrophages ex vivo. Peritoneal macrophages were stimulated with LPS (100 ␮g/ml), and treated with TIGIT-Fc or

IgG isotype control. The resting macrophages (non-LPS) were served as negative control. (A) Levels of cytokines produced in the supernatant were measured by the multiplex

assay 24 h after the incubation. Data are expressed relatively to cytokine levels determined in LPS and control IgG-exposed macrophages, arbitrarily set the value of 100%.

Cytokine levels close to the lowest detectable limit were not shown, including IL-1␣, IL-2, IL-3, IL-4, IL-5 and IL-9. The levels of IL-6 and CXCL11 treated by LPS/TIGIT-Fc

and LPS/control IgG were not shown as they surpassed the highest limit. *p < 0.05, **p < 0.01, compared with the isotype control group. (B) TNF-␣ and (C) IL-10 levels were

determined by ELISA at the indicated time points. *p < 0.05, **p < 0.01, compared with the isotype control group and LPS group. (D) The relative mRNA levels of TNF-␣, IL-10

and Arg1 were normalized to control group (non-LPS treatment) at 2 h as baseline. *p < 0.05, compared with the isotype control group and LPS group.

52 X. Chen et al. / Immunobiology 221 (2016) 48–55

Fig. 3. TIGIT reduces the LPS-induced macrophage activation and alters macrophage polarization. Peritoneal macrophages were stimulated with LPS (100 ␮g/ml) in the

presence of TIGIT-Fc (100 ␮g/ml) or IgG3 isotype control for 24 h. Flow cytometry was used to detect the percentage of the inactivated and activated macrophages, the

+ − − +

percentage of M1 (CD11c CD206 ) and M2 (CD11c CD206 ) gated on the activated macrophages. The macrophages were gated based upon the light scatter properties on

forward scatter (FSC) and side scatter (SSC) dot plots. *p < 0.05, ** p < 0.01, compared with isotype control.

hand, IL-10/IL-4 was demonstrated to induce alternatively acti- 3.5. The functional study of recombinant TIGIT-Fc protein

vated anti-inﬂammatory M2 [16]. Based on our ﬁnding that TIGIT

could modulate LPS-induced cytokine production, especially could To fulﬁll the need for a large amount of TIGIT-Fc protein in

enhance IL-10 secretion in macrophages, we suppose that TIGIT animal studies, we constructed a stable cell line secreting soluble

might affect the macrophage polarization. To address this ques- TIGIT-Fc fusion protein. We infected NIH/3T3 ﬁbroblasts (BALB/c,

tion, peritoneal macrophages were isolated and stimulated with H-2 ) with a lentiviral vector encoding TIGIT-Fc (deﬁned as TIG-Fc-

LPS and TIGIT-Fc protein or isotype control for 24 h, followed by 3T3), or an empty lentiviral vector (deﬁned as eGFP-3T3) control

ﬂow cytometry analysis. As shown in Fig. 3, treatment with TIGIT- (Fig. 5A). As shown in Fig. 5B, intracellular expression of TIGIT-Fc

Fc significantly suppressed the activation of macrophage by LPS was confirmed by flow cytometry. Furthermore, we examined the

(62.3 ± 6.5% vs. 41.3 ± 4.34%, P < 0.05). Compared to the isotype secreted TIGIT-Fc in the supernatant of TIG-Fc-3T3 cell cultures.

control, incubation with TIGIT-Fc signiﬁcantly decreased the per- As shown in Fig. 5C, human IgG-Fc positive macrophages were

+ −

centage of CD11c CD206 M1 macrophages gated on the activated detected by indirect staining, indicating the binding of PVR by solu-

macrophages (19.1 ± 2.1% v.s. 3.0 ± 1.6%, P < 0.01), while obviously ble TIGIT-Fc. Using an immunoturbidimetric assay to determine the

− +

increased the ratio of CD11c CD206 M2 macrophages (3.1 ± 1.8% human IgG3 Fc, we calculated that the concentratioin of TIGIT-Fc

v.s 11.4 ± 1.9%, P < 0.01) (Fig. 3B). These results demonstrate that was around 4.35 ␮g/ml/24 h produced by every 5 × 10 TIG-Fc-3T3

TIGIT inhibits LPS-induced macrophage activation in favor of skew- cells in vitro.

ing macrophages to anti-inﬂammatory M2 phenotype.

3.6. TIGIT-Fc reversed the lethal and sublethal endotoxic shock

through modulating macrophage polarization

3.4. TIGIT promotes c-Maf nuclear translocation

Then, we evaluated the protective effect of TIGIT-Fc against

endotoxic shock in mice. As shown in Fig. 6A, in response to a

As c-Maf has been demonstrated to be an essential transcription

lethal dose of LPS (15 mg/kg), the survival rate of mice treated

factor promoting the IL-10 gene expression [17], we also evaluated

with TIG-Fc-3T3 was markedly improved compared with the con-

the nuclear translocation of c-Maf. With the addition of TIGIT-Fc,

trol group. A sublethal dose of LPS (10 mg/kg) challenge caused

nuclear translocation of c-Maf was substantially enhanced (Fig. 4)

about half of the mice died in the eGFP-3T3 control group, while

compared with the other two groups, while the LPS + Isotype group

all mice were rescued from death in the TIG-Fc-3T3 group. Based

induced the translocation to a lesser extent. These results demon-

on our previous ﬁndings, we further explored the cytokine pro-

strate that TIGIT-Fc suppress LPS-induced secretion of IL-10 maybe

ﬁles in macrophages from the peritoneal lavage ﬂuid. We sought to

through controlling the transcriptional activity of c-Maf.

examine the functional change of macrophages in the early phase

X. Chen et al. / Immunobiology 221 (2016) 48–55 53

Fig. 4. TIGIT regulates the nuclear translocation of c-Maf. Peritoneal macrophages were treated with LPS (100 ␮g/ml) in addition to TIGIT-Fc protein (100 ␮g/ml) or IgG3

isotype control for 12 h. (A) Western blot was performed to evaluate the levels of c-Maf in cytoplasm or nucleus. ␤-actin and histone H3 were respectively used as cytoplasmic

and nuclear fraction loading control. (B) Nuclear translocatioin of c-Maf was calculated by the intensity ratio of nuclear and cytoplastic c-Maf. Nuc, nuclear; Cyt, cytoplasm.

**P < 0.01, compared with the LPS + Isotype group.

Fig. 5. Establishment of a NIH/3T3 cell line which stably secretes soluable TIGIT-Fc. (A) TIG-Fc-3T3 persistently express eGFP tags. (B) TIGIT-Fc were detected by intracelluar

staining with anti-TIGIT targeting against the extracellular fragment of TIGIT in TIG-Fc-3T3 cells (anti-TIGIT: solid line; eGFP-3T3 control: shaded grey); (C) Peritoneal

◦

macrophages activated with LPS (100 ng/ml) for 2 h and then were incubated with 1 ml supernatant from eGFP-3T3 or TIG-Fc-3T3 for 30 min at 4 C. TIGIT-Fc was detected

with PE-labeled anti-human IgG-Fc (TIG-Fc-3T3: solid line; eGFP-3T3 control: shaded grey).

of endotoxemia. As expected, LPS induced a dramatic decrease of shown to be expressed on many other cells including ﬁbroblasts,

inﬂammatory cytokines TNF-␣, IL-1␤ and MCP-1. In sharp contrast, endothelial cells and some tumor cells [9,20]. The possible func-

treatment of mice with TIG-Fc-3T3 signiﬁcantly elevated mRNA tions of TIGIT on these cells remain unknown, which warrant

levels of anti-inﬂammatory IL-10 (Fig. 6C). These results indicated further investigations. TIGIT knockout mice were more susceptible

that TIGIT could protect mice against LPS-induced endotoxemia to experimental autoimmune encephalomyelitis [21]. In contrast,

through M2-biased polarization in favor of immune homeostasis. mice deﬁcient in PVR gene developed normally without obvious

autoimmune diseases [22]. In addition to TIGIT, PVR has another

receptor CD226 expressed on T cells and the activation of CD226

4. Discussion

by PVR transduces a co-stimulatory signaling to counteract the

PVR/TIGIT negative co-inhibitory signaling [23]. Thus, PVR serves

Here we demonstrate a regulatory role of TIGIT as a ligand in

as dual roles both in immune activation and inhibition, which

modulating the signaling pathway which facilitates increased M2

may explain the reason why PVR knockout mice have the nor-

polarization. This study speciﬁcally expands our knowledge on the

mal phenotype. Notably, the bidirectional TIGIT/PVR pathways

unsuspected ﬂexibility and plasticity of macrophages.

are widely involved in immunomodulatory responses in different

TIGIT has gained increasing attentions as feedback control

immune cells, including DCs, macrophages, NK cells and T lympho-

mechanisms of immune activation [7,8,12]. Previous studies have

cytes. The similar co-inhibitory molecular pairs are the well-known

largely focused on the function of TIGIT receptor/PVR ligand signal-

immune checkpoint CD80/86-CTLA-4 pathways [21], which have

ing [18,19], while the reversed TIGIT ligand/PVR receptor signaling

been demonstrated to be effective therapeutic targets in clinical

remains to be explored. In the present study, we found that PVR

settings [24,25]. It seems possible that TIGIT may be developed as a

was almost undetectable in naive macrophages, while it was highly

novel drug candidate like CTLA-4-Ig and antagonistic anti-CTLA-4.

induced upon LPS stimulation. Consistent with our ﬁnding, it has

Recently, it has been demonstrated that tumor-inﬁltrating T

been shown that PVR can be signiﬁcantly induced by various

cells preferentially expressed TIGIT and blocking antibody against

microbial products [6]. These data suggest that PVR may play

TIGIT showed promising tumor clearance in mice [26]. Tumor

physiological roles in inﬂammatory microenvironments. Moreover,

associated macrophages are often characterized as M2-like cells,

in our study, TIGIT-Fc strongly suppressed the pro-inﬂammatory

potentiating the tumor invasion by secreting the anti-inﬂammatory

cytokines both in vitro and in vivo and skewed the activated

cytokine IL-10 [27,28]. From the standpoint of TIGIT/PVR pathway

macrophages toward M2 phenotype through PVR. Therefore, the

in macrophages, the beneﬁts from TIGIT blockade may be partly

overexpression of PVR upon exposure to bacterial products, includ-

ascribed for the dampened M2 polarization.

ing LPS, may be an endogenous mechanism to avoid exaggerated

inﬂammation under the evolutionary pressure. PVR has also been

54 X. Chen et al. / Immunobiology 221 (2016) 48–55

Fig. 6. TIGIT protects mice from the LPS shock and attenuates the production of pro-inﬂammatory cytokines in peritoneal macrophages. BALB/c mice (H-2 ) were treated by

6 d d

intraperitoneally (i.p.) injection with 5 × 10 TIG-Fc-3T3 (H-2 ) or eGFP-3T3 (H-2 ) or IgG3 isotype control (100 ␮g/mice) 6 h prior to the LPS challenge. (A) A lethal dose of

LPS (15 mg/kg) or a sublethal dose of LPS (10 mg/kg) were administered by i.p. injection (n = 8 mice per group). (B) Peritoneal macrophages were assessed for mRNA levels

of IL-1␤, IL-6, IL-10, TNF-␣ and MCP-1 by real-time PCR 24 h after the LPS challenge. Relative mRNA levels are shown. N = 5 mice per group. *P < 0.05, **P < 0.01, compared to

the isotype, eGFP-3T3 and LPS control groups.

The anti-inﬂammatory cytokine IL-10 has also been found to be while DCs inclined to produce cytokines involved in T cell immune

elevated by TIGIT-Fc treatment in vitro, paralleled by an increase in responses [34]. It has been demonstrated that the functional phe-

c-Maf expression. It has been illustrated that c-Maf could directly notypes of polarized macrophages are reversible by changing

bind to the IL-10 promoter sequence and potentiate its tran- the cytokine microenvironments [35]. But the phenotypes of DCs

scription [17,29]. IL-10 seems to serve as an autocrine factor to treated by IL-10 seem to be more stable and can induce prolonged

maintain the M2-like phenotype [30]. Meanwhile, a number of pro- disease-free state [36]. We cannot exclude the possibility that TIGIT

inflammatory cytokines were found to be significantly reduced, may exert distinct epigenetic modifications of these two cell types,

␤

including IL-1 , IL-17, IFN-␥, TNF-␣, MCP-1, MIP-1␣ and MIP-1␤. which warrants further study.

Recently, IL-17 has also been found to be an important medi- In conclusion, our present study provides evidence for a pre-

ator secreted by macrophages which precipitates the endotoxin viously unrecognized role of TIGIT on macrophage polarization.

shock [31]. The TIG-Fc-3T3 treatment in vivo also potentiated The current study further conﬁrms that the interaction between

the IL-10 mRNA production in macrophages 24 h after LPS chal- TIGIT and PVR creates bidirectional signaling implicated in the

lenge, but in a different time-phase pattern compared with the negative regulation of immune responses. This negative regula-

in vitro assays. This phenomenon may be ascribed to the much tory mechanism may represent a promising therapy target in

more complicated interactions in vivo. Although various cytokines macrophage-involved inﬂammatory diseases.

are involved in the pathogenesis of endotoxin shock, it seems

that the balance between anti-inﬂammatory and pro-inﬂammatory

cytokines maintains the tissue homeostasis. Thus, TIGIT may exert

Conﬂicts of interest

immunoregluatory effects in macrophages mainly through reshap-

ing the cytokine proﬁles. None.

Our ﬁndings appear to be consistent with the report that

TIGIT modulates the regulatory function of mature DCs [7]. It

should be pointed out that macrophages and DCs share com- Acknowledgements

mon characteristics but they also have distinct functions in tissue

immunity. Macrophages are proﬁcient in ensuring tissue integrity,

This work was supported by grants from National Nature

while DCs focus on initiation of tissue immune responses [32].

Science Foundation of China (81102260,81102239) and the Funda-

In LPS shock model, macrophages seem to play more important

mental Research Funds for the Central Universities of China, HUST:

roles, because conditional delete of IL-10 receptor gene speciﬁ- 2012QN190.

cally on macrophages reversed the LPS tolerance mediated by IL-10

We express our gratitude to Professor Jin Bo-Quan, Dr. Ran

[33]. Under the stimulation by the same microbial component,

Zhuang and Dr. Min Huang for generously providing the experi-

macrophages preferentially secreted pro-inﬂammatory cytokines,

mental materials.

X. Chen et al. / Immunobiology 221 (2016) 48–55 55

Appendix A. Supplementary data Foks, A.C., Ran, I.A., Frodermann, V., et al., 2013. Agonistic anti-TIGIT treatment

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Levin, S.D., Taft, D.W., Brandt, C.S., et al., 2011. Vstm3 is a member of the CD28

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