M2 Macrophage Polarization and Gut Microbial Homeostasis

Article

YAP Aggravates Inflammatory Bowel Disease by Regulating M1/M2 Macrophage Polarization and Gut Microbial Homeostasis Graphical Abstract

Authors Xin Zhou, Weiyun Li, Shuang Wang, ..., Hongbin Ji, Bin Wei, Hongyan Wang

M1 LPS+IFNγ

Correspondence

M2

[email protected] (B.W.), [email protected] (H.W.)

IL-4+IL-13

YAP

YAP mRNA

JNK

YAP IL-6

p53 IL-6

YAP expression

IBD

?

ARG1 YM1 FIZZ

YAP Normal

Highlights d

Myeloid-specific knockout of YAP relieves inflammatory bowel disease (IBD)

d

YAP regulates the balance between M1 and M2 polarization

d

YAP expression is differentially regulated by LPS/IFN-g and IL-4/13 treatment

d

YAP in macrophages affects the abundance of gut microbiota in IBD mice

Zhou et al., 2019, Cell Reports 27, 1176–1189 April 23, 2019 ª 2019 The Author(s). https://doi.org/10.1016/j.celrep.2019.03.028

In Brief Zhou et al. show that deletion of YAP in macrophages relieves chemically induced inflammatory bowel disease (IBD) by enhancing M2 polarization, restraining IL-6 production in M1 macrophages, and changing gut microbiota homeostasis.

Cell Reports

Article YAP Aggravates Inflammatory Bowel Disease by Regulating M1/M2 Macrophage Polarization and Gut Microbial Homeostasis Xin Zhou,1 Weiyun Li,1 Shuang Wang,1 Panli Zhang,2 Qiong Wang,1,3 Jun Xiao,1 Chi Zhang,1 Xin Zheng,1 Xiaoyan Xu,1,4 Shengjie Xue,1 Lijian Hui,1 Hongbin Ji,1 Bin Wei,2,5,* and Hongyan Wang1,6,* 1State Key Laboratory of Cell Biology, Key Laboratory of Systems Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Innovation Center for Cell Signaling Network, Shanghai 200031, China 2College of Life Sciences, Shanghai University, Shanghai 200444, China 3Institute of Spine Disease, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China 4Experimental Immunology Branch, National Cancer Institute, NIH, Bethesda, MD, USA 5Cancer Center, Shanghai Tenth People’s Hospital, Tongji University, School of Medicine, Shanghai 200072, China 6Lead Contact *Correspondence: [email protected] (B.W.), [email protected] (H.W.) https://doi.org/10.1016/j.celrep.2019.03.028

SUMMARY

Inflammation, epithelial cell regeneration, macrophage polarization, and gut microbial homeostasis are critical for the pathological processes associated with inflammatory bowel disease (IBD). YAP (Yesassociated protein) is a key component of the Hippo pathway and was recently suggested to promote epithelial cell regeneration for IBD recovery. However, it is unclear how YAP regulates macrophage polarization, inflammation, and gut microbial homeostasis. Although YAP has been shown to promote epithelial regeneration and alleviate IBD, here we show that YAP in macrophages aggravates IBD, accompanied by the production of antimicrobial peptides and changes in gut microbiota. YAP impairs interleukin-4 (IL-4)/IL-13-induced M2 macrophage polarization while promoting lipopolysaccharide (LPS)/interferon g (IFN-g)-triggered M1 macrophage activation for IL-6 production. In addition, YAP expression is differently regulated during the induction of M2 versus M1 macrophages. This study suggests that fully understanding the multiple functions of YAP in different cell types is crucial for IBD therapy. INTRODUCTION Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease, is a chronic inflammatory disorder of the gastrointestinal tract (Lin et al., 2014). Large numbers of macrophages are present in colon samples from IBD patients and animal models, which play important roles in the initiation and resolution of inflammation (Krausgruber et al., 2011; Rauh et al., 2005). M1 macrophages are induced by interferon g (IFN-g) and bacterial lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-a) to produce a wide range of proinflammatory

cytokines, such as interleukin-6 (IL-6). M2 macrophages are induced by IL-4 and IL-13 to express ARG1, YM-1, and anti-inflammatory cytokines, including IL-10. M1 macrophages and proinflammatory cytokines aggravate IBD, whereas M2 macrophages promote tissue repair and inflammation resolution to reduce IBD symptoms (Lawrence and Natoli, 2011; Lin et al., 2014; Rauh et al., 2005). Because aberrant macrophage polarization occurs during the development of IBD, this process has been recently targeted as a potential therapeutic strategy for €hl et al., 2015; Lin et al., 2014; Shon et al., 2015; SteinIBD (Ku bach and Plevy, 2014; Weisser et al., 2011). Past efforts have elucidated the roles of the transcription factors involved in the regulation of M1/M2 polarization. IRF5 promotes M1 polarization (Krausgruber et al., 2011), whereas STAT6, C/EBPb, and IRF4 are crucial for enhancing the expression of M2-associated genes (Ruffell et al., 2009; Satoh et al., 2010). However, the mechanism underlying the regulation of the M1/M2 switch and the development of IBD is still incomplete and far from being systematic (Lawrence and Natoli, 2011). As a key transcription coactivator, YAP (Yes-associated protein) plays a vital role in the Hippo pathway and is well established to promote tumor formation (Murakami et al., 2017; Taniguchi et al., 2017; Wang et al., 2016a; Yimlamai et al., 2014). YAP is reported to be highly associated with inflammationrelated diseases, including atherosclerosis (Wang et al., 2016b) and pancreatitis (Murakami et al., 2017). In addition, YAP activation occurs during DSS-induced tissue damage or upon mucosal injury, which is beneficial for maintaining barrier function or widespread early-onset polyps (Taniguchi et al., 2015). Previous work has shown that YAP is crucial for epithelial progenitor cell proliferation and differentiation, and is involved in epithelial repair (Mahoney et al., 2014). The emerging data suggest that the Hippo-YAP pathway is also involved in the immune response. Hippo-Yorkie (Yorkie, a fly homolog of YAP) signaling is crucial for protecting Drosophila from gram-positive bacterial infections (Liu et al., 2016). We and others have demonstrated that STK4 (also termed Mst1 or Hippo), a serine and/or threonine kinase upstream of YAP, in macrophages

1176 Cell Reports 27, 1176–1189, April 23, 2019 ª 2019 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

A

C

Figure 1. YAP Deficiency in Macrophages Protects Mice from IBD

B

F

D

E

can clear bacterial infections (Geng et al., 2015) and inhibit Tolllike receptor 4 (TLR4)-induced inflammation, which protects the host from chronic inflammation-related hepatocellular carcinoma (Geng et al., 2015; Li et al., 2015b). However, how YAP in macrophages regulates M1/M2 polarization, inflammation, and IBD has remained elusive. In this study, we have demonstrated that YAP conditional knockout (cKO) mice have reduced DSS-induced colitis. YAP expression is differentially regulated during the induction of M1/M2 macrophages. Moreover, YAP inhibits M2 macrophage polarization while promoting IL-6 production in M1 macrophages. Despite the role of YAP in epithelial cells for tissue repair, we propose a function of YAP in macrophages in regulating the M1/M2 balance, inflammation, and gut microbiota during the pathological process of IBD.

(A) Body weight curves of YAP+/+(n = 8) and YAPDM/DM (n = 9) mice in an acute model of DSSinduced colitis for 7 days. (B) Stool consistency, fecal bleeding, and weight loss were observed on a daily basis, and the DAI (disease activity index) was scored for each YAP+/+ and YAPDM/DM mouse. The DAI score was graded on a scale of 0 to 12 as described in the STAR Methods. (C) Representative image of the DSS-induced colitis in YAP+/+ and YAPDM/DM mice. The colon lengths of the DSS-induced mice were measured on day 7 (n = 17). (D) Representative histopathological images and scores from colon tissue sections in DSS-induced WT and YAPDM/DM mice (n = 3). Scale bars: 2 mm (left), 100 mm (right). (E) RNA-seq analysis of differentially expressed genes in YAP+/+ and YAPDM/DM BMMs stimulated with IL-4/IL-13 for 24 h (n = 3). Differentially expressed genes (n = 451) were identified in all pairwise comparisons and exhibited a 2-fold change in expression with an adjusted p value of 0.05. (F) Pathway analysis of the RNA-seq data between YAP+/+ and YAPDM/DM macrophages using DAVID 6.8. See also Figure S1.

RESULTS YAP Deficiency in Macrophages Protects Mice from IBD Considering the significant role of macrophages in the colon for IBD (Steinbach and Plevy, 2014; Weisser et al., 2011), we were interested in exploring the function of YAP in macrophages with respect to IBD. Because a complete depletion of YAP in mice results in early embryonic lethality (Morin-Kensicki et al., 2006), YAP cKO mice were generated bearing two loxP sites flanking the first two exons of the YAP gene. We cross-bred YAP cKO mice with lysozyme-cre (Lysm-cre) mice to specifically eliminate YAP expression in the myeloid lineage, including macrophages (termed YAPDM/DM mice). The KO efficiency of YAP in macrophages was confirmed by western blotting assay (Figures S1A and S1B). Next, we used dextran sulfate sodium (DSS) to induce IBD in mice and monitored the body weight (Figure 1A), disease activity index (DAI) (Figure 1B), colon length (Figure 1C), and colon pathology (Figure 1D) of the DSS-treated wild-type (WT) or YAPDM/DM mice. Unexpectedly, YAP ablation in macrophages reduced the severity of IBD, which is the opposite of the protective function of YAP in epithelial cells from IBD (Taniguchi et al., 2015). Because YAP is also expressed in neutrophils, we next used an anti-Ly6G antibody to delete neutrophils in mice to exclude the potential effect from YAP-deficient neutrophils during the development of IBD (Figure S1C). The severity of IBD in

Cell Reports 27, 1176–1189, April 23, 2019 1177

Merge

CD206 /F4/80 (%)

B

+

+

YAP+/+

*

60 40 20 0

CD206 staining 40 30

10

43

2 ΔM /Δ M

0

YA

P

P

YA 2

P

ΔM /Δ M

4

YAPΔM /ΔM

*

1500 1000 500

13

0

/IL IL -4

-4 /IL 13

0

6

k

5

*

YAP+ /+

2000

M oc

10

Relavite Fizz 1 mRNA

P

+/ +

0.0

8

+/ +

0.5

YA

P

Relative Ym 1 mRNA

1.0

YA

Relative Il-10 mRNA

1.5

YA

IL

-4

/IL 13

0

*

2.0

**

15

k

20

2.5

YA

P YA

0

P

40

k

10

Relative Ym 1 mRNA

*

60

M oc

20

YA

P YA

YA

E

ΔM /Δ M

+/ +

0

P

ΔM /Δ M

+/ +

P 2

*

ΔM /Δ M

4

30

M oc

6

+/ +

0

YA

*

+/ +

Relavite Fizz 1 mRNA

8

P 4

β-actin

0

P

Relative Arg 1 mRNA

6

20

D

Relative Ar g 1 mRNA

43

ARG1

*

8

YAP+/+ YAPΔM/ΔM

ΔM /Δ M

YAPΔM/ΔM

10

P

YAP+/+

F

*

YA

CD206 cells(%)

50

IL

C

Relative ARG1 protein Y A

YA

P

+/ +

YAPΔM/ΔM

80

ΔM /Δ M

CD206

F4/80

A

Figure 2. YAP Deficiency Enhances M2 Macrophage Polarization In Vitro and in the Colon of IBD Mice (A and B) Immunostaining (A; scale bars: 100 mm) or FACS analysis (B) of CD206+F4/80+ macrophages in colonic tissues from DSS-induced colitis mice. (C) Immunohistochemical analysis of the M2 marker CD206 in paraffin-embedded colon sections from YAP+/+ and YAPDM/DM mice (scale bars: 200 mm). (D) The mRNA levels of Arg1, Ym-1, Fizz1, and Il-10 in immune cells from colonic lamina propria were detected by qRT-PCR.

(legend continued on next page)

1178 Cell Reports 27, 1176–1189, April 23, 2019

YAPDM/DM mice remained lower than that of WT mice (Figures S1D–S1F), demonstrating that a YAP deficiency in macrophages contributes to the protective phenotype. Despite being an oncogene involved in promoting tumor growth (Harvey et al., 2013), YAP did not significantly affect macrophage proliferation (Figure S1G) or macrophage development from bone marrow cells after being cultured in L929-conditioned complete DMEM (Figure S1H). We next explored the global differences in gene expression induced by YAP via RNA sequencing (RNA-seq). Because we observed that the expression of the IL-4, IL-13, and IL-4 receptors were enhanced in colonic tissue from IBD mice compared with those from the healthy mice (Figure S1I), WT and YAPDM/DM macrophages were stimulated with IL-4/IL-13 (Figure 1E). A total of 155 genes were upregulated in YAPDM/DM macrophages, including Arg1, Retnla, Mrc1, Fn1, Folr2, and Msx3, which are reported to be M2 markers or highly expressed in M2 macrophages (Figure 1E) (Jablonski et al., 2015; Puig-Kro¨ger et al., 2009). In contrast, some key genes related to the proinflammatory response, such as Il-6, Ila, Ccl5, and Nos2, were significantly reduced in YAPDM/DM macrophages (Figure 1E). When Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed, we observed that IBD-related genes, Salmonella infection, TNF-a signaling pathways, and TLR signaling pathways were highly enriched when comparing WT and YAPDM/DM macrophages (Figure 1F). In addition, some pathways were expressed at higher levels in the YAPDM/DM macrophages, including the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway (Figure S1J). These findings prompted us to investigate whether the YAP-specific deletion in macrophages may regulate M1/M2 macrophage polarization and inflammation, which could account for the observed protection of YAPDM/DM mice from IBD. YAP Deficiency Enhances M2 Macrophage Polarization In Vitro and in the Colon of IBD Mice We first characterized M2 macrophages in vivo in colonic tissue from DSS-induced IBD mice. An increase in the number of F4/ 80+CD206+ M2 macrophages was observed in the colons of YAPDM/DM mice by immunofluorescence staining, FACS analysis, and IHC staining (Figures 2A–2C). Next, the colonic lamina propria cells were isolated from WT and YAPDM/DM mice after the induction of IBD. Importantly, the mRNA levels of Arg1, Fizz, and Il-10 were significantly enhanced in YAPDM/DM mice (Figure 2D). To further confirm the function of YAP on the induction of M2 macrophages, we used IL-4 and IL-13 to treat macrophages in vitro. In agreement with the in vivo data, the expression of M2-associated genes, including Arg1, Fizz, and Ym1 at the mRNA level and ARG1 at the protein level, were significantly augmented in YAP-deficient BMMs (bone marrow-derived macrophages) (Figures 2E and 2F) or PEMs (peritoneal macrophages) (Figure S2A). Taken together, we showed that YAP can inhibit M2 macrophage polarization in response to DSS treatment in vivo or IL-4/IL-13 stimulation in vitro.

YAP Inhibits M2 Polarization via Promotion of p53 Transcription Because YAP acts as a transcription coactivator (Varelas, 2014; Zhao et al., 2008), we next explored whether the transcriptional regulatory activity of YAP is crucial to affect M2 polarization. A constitutively active YAP mutant (YAP5SA) and a YAP deletion mutant lacking the C-terminal transactivation domain (YAP5SA-DC) (Gao et al., 2014) were overexpressed in human THP1 cells (Figure 3A, upper panel shows protein expression levels). Overexpression of WT YAP dramatically repressed the expression of human M2 macrophage markers, including hCD163 and human CD209 (hDC-SIGN), which were further reduced by the overexpression of the constitutively active YAP-5SA mutant (Figure 3B). This inhibitory effect was significantly compromised when the inactive YAP5SA-DC mutant was overexpressed (Figure 3B). Our results indicate that transcriptional regulatory activity of YAP is indispensable for repressing M2 macrophage polarization. IL-4/IL-13 stimulation has been demonstrated to activate STAT6 through IL-4 receptor signaling for M2 polarization (Cosı´n-Roger et al., 2016; Li et al., 2015a). We therefore tested the levels of phosphorylated STAT6 (Figure 3C; Figure S3B) and observed no detectable differences between WT and YAP-deficient macrophages after IL-4/IL-13 treatment for the indicated time points. Because PEMs could adhere firmly to cover glasses, we used PEMs to perform immunofluorescence staining assay. We used PEM to examine the levels of nuclei-translocated STAT6 (Figure S3A) and observed no detectable differences between WT and YAP-deficient macrophages. Because antiYAP antibody is not suitable for immunoprecipitation in primary macrophages, we used human THP1 cells overexpressing hemagglutinin (HA)-tagged YAP to perform anti-HA immunoprecipitation. No interaction was identified between YAP and STAT6 (Figure S3C). Previous studies have reported that IRF4, CEBP-b, SOCS2, and SOCS3 are involved in the induction of alternatively activated macrophages (Odegaard et al., 2008; Satoh et al., 2010). Because YAP deficiency promoted M2 macrophage polarization, we unexpectedly observed a decrease in STAT6 and CEBP-b mRNA levels in YAP KO macrophages and comparable expression of IRF4, SOCS2, and SOCS3 when compared with WT controls (Figure 3D). To find out the underlying mechanism, we next examined whether YAP increased the transcription of negative regulators for M2 polarization. By analyzing the RNA-seq data, we observed downregulated expression of Trp53 and Tpt1 in YAP KO macrophages (Figure 1E). p53 was previously reported to act as a ‘‘brake’’ to inhibit M2 polarization (He et al., 2015; Li et al., 2015a). We therefore detected the mRNA and protein expression levels of p53 in WT and YAPDM/DM macrophages after IL-4 and IL-13 treatment, which were significantly reduced in YAPDM/DM macrophages compared with WT cells (Figure 3E). To further investigate whether YAP represses M2 macrophage polarization through p53, WT and YAP-deficient macrophages

(E and F) YAP+/+ and YAPDM/DM BMMs were stimulated with IL-4/IL-13 for 24 h, and the mRNA levels of Arg1, Fizz, and Ym-1 were detected by qRT-PCR (E), or ARG1 expression at the protein level was measured by immunoblotting analysis (F). The representative data from at least three independent experiments are shown. See also Figure S2.

Cell Reports 27, 1176–1189, April 23, 2019 1179

0

15

45

p -STAT6

95

STAT6

95

β-actin

43

E 1.5

* 1.0

YAP+/+ YAPΔM/ΔM 55

β-actin

43

*

4 3 2 1

si p

1.0 0.5 0.0

0.5 0.0 +/

40

*

YAPΔM/ΔM

20

NS

10 0 DMSO

NS

ΔM/ΔM

YAP

600

*

400 200 0 IL-4/13

* siNC -

siNC

sip53

+

+

were pretreated with the DMSO control or nutlin-3a, followed by IL-4 and IL-13 induction. Nutlin-3a was reported to accumulate p53 protein via inhibition of the E3 ligase Mdm2 (Li et al., 2015a). Nutllin-3a treatment indeed impaired Arg1 expression, and importantly, the effect of YAP depletion on Arg1 production was abolished after nutllin-3a treatment (Figure 3F). For the controls, Nutlin-3a treatment did not affect the expression of IL4R and MFG-E8, which are target genes of the p53 family members p63 and p73 (Figure S3D). In addition, knockdown of p53 in macrophages rescued the effect of YAP depletion on Arg1 production (Figure 3G). Because anti-YAP antibody is not suitable for a CHIP assay in primary macrophages, we next used anti-HA antibody to perform a CHIP assay in THP1 cells overexpressing HA-tagged YAP. We observed enhanced binding of YAP to the p53 promoter compared with the immunoglobulin G (IgG) control

1180 Cell Reports 27, 1176–1189, April 23, 2019

p53 promoter

**

3

(A) Diagram of WT or YAP mutations. The expression levels of YAP, YAP5SA, and YAP5SA-DC in THP1 cells were assessed by immunoblotting. (B) THP1 cells overexpressing YAP, YAP5SA, and YAP5SA-DC were stimulated with IL-4/IL-13 for 24 h to assess the levels of Cd163 and Dc-sign mRNA by qRT-PCR (n = 3). *p < 0.05 was calculated by one-way ANOVA with Holm-Sidak multiple comparisons test. (C) YAP+/+ and YAPDM/DM PEMs were stimulated with IL-4/IL-13, followed by immunoblotting with anti-pStat6 or anti-Stat6 antibodies. (D and E) YAP+/+ and YAPDM/DM BMMs were stimulated with IL-4/IL-13 for 24 h to assess the mRNA levels of Stat6, Irf4, Cebp-b, Socs3, and Socs2 (D) and p53 expression at both the mRNA and protein levels (E). (F) YAP+/+ and YAPDM/DM BMMs were incubated with the p53 activator Nutlin-3a, followed by IL-4/IL13 treatment for 24 h to detect Arg1 mRNA levels. (G) YAP+/+ and YAPDM/DM BMMs were transfected with p53 siRNA, followed by IL-4/IL-13 treatment to detect Arg1 mRNA levels. (H) ChIP assays were performed with anti-HA or the IgG control antibodies in THP1 cells overexpressing HA-YAP, followed by qPCR to detect the p53 promoter. The representative data from three independent experiments are shown. See also Figure S3.

Nutlin-3a

IL-4/IL13

YA P

YAP+/+

YAP+/+

30

H 800

53

0

R elative Arg 1 mRNA

5

si N C

Relative p5 3 mRNA

G

1.0

YA P

ΔM /Δ M

YA P

YA P

+/

+

0.0

***

*

*

1.5

Re la tive Enrichme nt

0.5

p53

YAPΔM/ΔM

2.0

F 1.5

+

45

0

YAP+/+

2.5

ΔM /Δ M

15

Rela tive mRN A levels

YAP

ΔM/ΔM

Relativ e p53 protein

IL-4 /13 (min) 0

5

D

YAP5SA-ΔC

YAP

10

So cs 2

YAP5SA

A A A tead binding

15

So cs 3

transcription

A

*

(Figure 3H). These results suggest that YAP inhibits M2 macrophage polarization by directly promoting p53 expression.

YAP Promotes M1 Polarization and Enhances IL-6 Production Except for the enhanced expression of the 1 M2 marker Arg1, we also observed from 0 the RNA-seq data that YAP KO macrophages reduced expression of inflammation-associated genes, such as Il6, Ccl5, Nos2, and Il1a (Figure 1E). In addition, KEGG analysis showed that YAP could regulate the expression of genes associated with TLR signaling pathways, Salmonella infection, chemokine, and TNF-a signaling pathways (Figure 1F), which are closely linked to the function of M1 macrophages (Jablonski et al., 2015; Krausgruber et al., 2011). Because we detected an enhanced production of IFN-g in the colons of DSSinduced IBD mice (Figure S4A), we next assessed whether YAP regulated M1 macrophage differentiation in response to gut bacterial infection and IFN-g stimulation. We observed that the numbers of F4/80+iNOS+ or CD80+CD86+ (Figures 4A and 4B) or CD68+CD80+ (Figure S4B) M1 macrophages were reduced in the colons of YAPDM/DM mice. In agreement with these results, serum IL-6 concentrations (Figure 4C) and IL-6 expression at the mRNA and protein levels (Figure 4D) in colon tissue were decreased in DSS-treated YAPDM/DM mice compared with WT mice. 2

H A

YAP

A A A tead binding

*

lg G

transcription

A

+/+

R elativ e hC d163 mRNA

0

tead binding

C

Relative p5 3 mRNA

1

20

Ve ct or YA Y YA A P P P5 5S SA A -Δ C

14-3-3 bd

2

Irf 4

43

β-actin

3

Figure 3. YAP Inhibits M2 Polarization via Promotion of p53 Transcription

NS

Ce bp -β

43

*

*

St at 6

55

YAP+MYC

4

Relative Arg 1 mRNA

Rela tiv e hDC-sign mRNA

Ve

72

NS

Ve ct or YA Y YA A P P P5 5S SA A -Δ C

-Δ C

B

ct YA or P YA P5 YA S P5 A SA

A

0.0

1.5

LP S

0

**

100

50

0

E.coli

*

1.0

0.5

0.0 6000

2.0

IF Nγ

5000 15000

+

***

ΔM /Δ M

10000

M oc k

20000

LP S

+

IF Nγ

M oc k

2

+

15000

TNF-α(pg/ml)

Relative Il-6 mRNA

0

10

YA P

IF Nγ

2

Relative Inos mRNA

+

4

Relative Il-6 mRNA

LP S

G

M

2.0 +/

M /Δ M

ΔM /Δ

+

M

+/

M

+/ + Δ M /Δ

YA P

P

YA P

YA

YA P

CD80+CD86+/F4/80+ (%)

Serum IL-6 (pg/ml)

0

Δ M /Δ

0.5

5

P

1.0

10

+/ +

*

15

P

Sl1344

20

YA

0

YA P

50

*

YA

100 150

M

*

Δ

*

Δ M /Δ

0

+

5

+/

150

M oc k

10

YA P YA P

15

IL-6(pg/ml)

20

Relative Ccl2 mRNA

IF N γ NS

Relative Il-6 mRNA

ΔM /Δ M

+/ +

ΔM /Δ M

P

YAPΔMΔM 25

P

0.0

YA P

YA

Relative Il-6 mRNA

YAP+/+

YA

0.5 25

B

+/ +

1.0

Merge

YA P

** 1.5

M

Aures

YA P

0

Δ M /Δ

500

P

** +

0

YA

LP S

5000

+

10000

+/

*

YA P

15000

M oc k

M /Δ M

0

Relative Tnf-α mRNA

IF Nγ

50

Relative Il-12 mRNA

+

+

100 6

+

+

ΔM /Δ M

+/

Δ

+/

**

Relative Il-6 mRNA

YA P

LP S

YA P

YA P

iNOS

+/

1.5

/Δ M

+

1500

Δ M

+/

YA P

M oc k

F4/80

YA P

YA P

H 150

YA P

1000

Relative Il-1β mRNA

F Colon IL-6 (pg/ml)

D

Relative Il-6 mRNA

E Relative Il-6 mRNA

A C 2000

*

1500

1000 500 0

*

8

Vector YAP

6

4

*

0

YAP+/+

YAPΔM/ΔM NS

10000 5000 0

*

4000

2000

0

Pao

1.5

*

1.0

0.5

0.0

(legend on next page)

Cell Reports 27, 1176–1189, April 23, 2019 1181

M1 macrophages are characterized by promoting the production of IL-6 and TNF-a (Krausgruber et al., 2011; Lawrence and Natoli, 2011). Compared with BMMs, PEMs are relatively easy to transfect with small interfering RNA (siRNA); we used siRNA to knock down YAP expression in PEMs to evaluate how YAP regulated LPS/IFN-g-induced IL-6 and TNF-a production. The YAP siRNA knockdown efficiency and specificity were measured at the mRNA level (Figure S4C), which demonstrated targeting of YAP and no effect on TAZ expression (the paralog of YAP). Knockdown of YAP decreased IL-6 mRNA levels in LPS/IFN-gstimulated macrophages (Figure S4D). This phenotype was confirmed in YAP-deficient macrophages, which exhibited profoundly reduced IL-6 production at the mRNA and protein levels (Figure 4E), but no effect on TNF-a production. In agreement with this result, markedly elevated IL-6 expression was observed in YAP-overexpressing THP1 cells after LPS/IFN-g treatment (Figure 4G). In addition, we observed reduced levels of Il1b, Il12, Ccl2, and iNOS mRNA in LPS/IFN-g-treated YAP-deficient macrophages (Figure 4F). Furthermore, YAP KO macrophages exhibited decreased IL-6 production when stimulated with polyinosinicpolycytidylic acid (poly(I:C)) (a TLR3 agonist) and CpG (a TLR9 agonist) (Figure S4E). The dysbiosis of the gut microbiota in IBD patients is related to higher levels of IL-6 in the colon (Saleh and Elson, 2011), and some gut-associated bacteria have been shown to trigger TLR-mediated pathways to induce IL-6 production. Therefore, we infected macrophages with the gram-positive bacterium Staphylococcus aureus (S. aureus) and the gram-negative bacteria Escherichia coli (E. coli), Pseudomonas aeruginosa (PAO), and Salmonella typhimurium (SL1344). Consistently, YAP-deficient macrophages decreased IL-6 production upon infection with these gut bacteria (Figure 4H). Taken together, our findings have demonstrated that YAP can promote M1 macrophage polarization with the enhancement of IL-6 production in response to microenvironmental challenges with gut bacteria and IFN-g. YAP Binds to the IL-6 Promoter We next determined whether YAP is dependent on its transcriptional regulatory activity to promote IL-6 production. Substantial enhancement of IL-6 production was observed in THP1 cells expressing either WT YAP or the constitutively active YAP5SA, whereas the inactive YAP 5SA-DC mutant failed to increase IL-6 levels (Figure 5A). To further confirm this result, we transfected HEK293T cells with an IL-6 luciferase reporter, YAP, YAP-5SA, or YAP 5SA-DC, together with MYD88, IRAK1,

TRAF6, TAK1, and p65. Either WT YAP or the constitutively active YAP5SA could cooperate with MYD88, IRAK1, TRAF6, and p65 to further enhance IL-6 luciferase readings, whereas the inactive YAP 5SA-DC failed to promote this effect (Figure 5B). Because TLR4/3/9 and bacterial infection could activate NFkB to induce IL-6 production (Bhattacharyya et al., 2010), we next assessed whether YAP regulates the transcription of the key molecules within the TLR-NF-kB pathway. We did not observe any differences in the mRNA levels of TLR4, MYD88, IRAK1, IRAK4, TRAF6, TAK1, and p65 between WT and YAPDM/DM macrophages after LPS/IFN-g stimulation (Figure S5A). In addition, we observed similar levels of inhibitor of nuclear factor kappa-a kinase (IKKa) and IKKb phosphorylation (Figure 5C; Figure S5B), as well as levels of p65 nuclear localization (Figure S5C) in the LPS/IFN-g-treated WT and YAPDM/DM macrophages. These results indicate that YAP may promote IL-6 production independently of NF-kB. Because MAPKs, including JNK, extracellular regulated protein kinase (ERK), and p38, can be activated after receptor engagement to promote cytokine production (Bhattacharyya et al., 2010), we examined whether YAP affected activation of MAPKs. LPS/IFN-g-treated YAP-deficient macrophages had decreased levels of phosphorylated JNK and p38, but not pERK (Figure 5C). The enhanced JNK phosphorylation was confirmed in YAP-overexpressing THP1 cells, which did not exhibit altered levels of ERK phosphorylation (Figure S5B). However, the JNK inhibitor (SP600125) only partially suppressed YAP-induced IL-6 expression (Figure S5D), indicating that other mechanisms may also participate in YAP-induced IL-6 expression. We next analyzed the IL-6 promoter region and identified two putative binding sites (50 -A/CATTC-30 ) for the transcription factor TEAD, i.e., nucleotides 424 to 420 (TEAD binding site 1, named T1) and 3 to 1 (TEAD binding site 2, named T2). In addition, YAP/TEAD has also been reported to interact with AP-1 to regulate gene expression (Zanconato et al., 2015), and we identified one AP-1 binding site (50 -TGAGTCA-30 ) at nucleotides 285 to 279 (named AP-1). The three sites were individually mutated and cloned into a luciferase reporter plasmid, which was subsequently cotransfected with YAP and IRAK1 into 293T cells. YAP was observed to promote IL-6 transcription, which was blocked by the T2 mutation, but not the T1 or AP1 mutations, indicating that the YAP/TEAD complex may bind to the T2 site to promote IL-6 transcription (Figure 5D). Importantly, we performed a CHIP assay using LPS/IFN-g-treated human THP1 cells and confirmed that YAP directly binds to the IL-6 promoter, with the classical YAP target gene CTGF used as a positive control (Figure 5E). Taken together, our results have

Figure 4. YAP Promotes M1 Polarization and Enhances IL-6 Production (A and B) Immunostaining or FACS analysis of iNOS+F4/80+ (A, scale bars: 100 mm) or CD80+CD86+ (B) macrophages in colonic tissues from DSS-induced IBD mice. (C and D) IL-6 levels in plasma (C) or in colon tissue (D) were evaluated from DSS-induced YAP+/+ (n = 8) and YAPDM/DM (n = 10) mice. (E) YAP+/+ and YAPDM/DM PEMs were stimulated with LPS/IFN-g to detect IL-6 and TNF-a expression by qRT-PCR or ELISA. (F) YAP+/+ and YAPDM/DM BMMs were stimulated with LPS/IFN-g to detect Il-1b, Il-12, Inos, and Ccl2 expression by qRT-PCR. (G) THP1 cells overexpressing the control or YAP were stimulated with LPS/IFN-g to measure Il-6 mRNA levels. (H) YAP+/+ and YAPDM/DM BMMs were infected with Escherichia coli (E. coli), Pseudomonas aeruginosa (PAO), Salmonella typhimurium (SL1344), or Staphylococcus aureus (S. aureus) for 4 h to detect Il-6 expression. The representative data from at least three independent experiments are shown. See also Figure S4.

1182 Cell Reports 27, 1176–1189, April 23, 2019

150

** *** 100

***

50

P P5 P5 SA SA -Δ C

YA

GAPDH

34

0

100 50 0 P YA P5 SA P5 SA -Δ C

43

**

10

YA

p-ERK

20

**

YA

43

p-p38

30

** ***

150

to r

55

p-JNK

** ***

40

p65 200

Ve c

95

p-IKKα/β

IRAK1 50

P YA P5 P5 SA SA -Δ C

45

Relative luciferase units(RLU)

YA

YA

YA

to r

0

YA

15

YA

0

to r

15 45

Ve c

LPS+IFN γ(min) 0

YAPΔMΔM

Relative luciferase units(RLU)

YAP+/+

(A) THP1 cells overexpressing vector, YAP, YAP5SA, or YAP5SA-DC were stimulated with LPS/ IFN-g for 4 h to assess Il-6 expression levels via qRT-PCR. (B) MYD88, IRAK1, TRAF6, and p65 were transfected into HEK293T cells with the vector, YAP, YAP5SA, YAP5SA-DC, and IL-6 luciferase reporter plasmids to measure luciferase readings. (C) The levels of phosphorylated IKKa/b, JNK, p38, and ERK in LPS/IFN-g-stimulated YAP+/+ and YAPDM/DM BMMs were detected by immunoblotting analysis, and the relative expression was analyzed. (D) The human IL-6 promoter region contains two putative TEAD-binding sites (T1, T2) and one AP-1 binding site. HEK293T cells were cotransfected with IRAK1 in combination with vector or YAP and the WT or mutant IL-6-luciferase reporter plasmid to measure luciferase readings. (E) ChIP assays were performed with anti-HA antibodies followed by qPCR to measure the IL-6 and CTGF promoters in LPS/IFN-g-treated THP1 cells overexpressing HA-YAP. The representative data from at least three independent experiments are shown. See also Figure S5.

TRAF6

Ve c

0

LP

C

Relative luciferase units(RLU)

γ IF N

20

S

+

***

40

P YA P5 P5 SA SA -Δ C

* *

60

to r

** ***

25 20 15 10 5 0

*** ***

80

YA

50

MYD88

Ve c

Vector YAP YAP5SA YAP5SA -ΔC

100

Figure 5. YAP Binds to the IL-6 Promoter Relative luciferase units(RLU)

B

** **

150

M oc k

Relative Il-6 mRNA

A

YAPΔM /ΔM

E

in in m m 15 45

0

in in m m 15 45

2 1

H A

0

in in m m 15 45

0

in in m m 15 45

CTGF promoter Relative Enrichment

*

lg G

Relative Enrichment

IL-6 promoter 3

0

***

4 3

3

2

53

Tead2

b-Catenin was previously reported to be able to bind to the YAP promoter to drive its transcription in colorectal carcinoma cells (Konsavage et al., 2012), and AKT is * NS important for b-catenin degradation (Yang et al., 2014). Thus, we next asked whether b-catenin is involved in IL-4/IL13-mediated YAP expression. LiCl, an activator of b-catenin (Wang et al., 2013), was used to treat cells during the induction of M2 macrophages by the IL-4/IL-13 treatment. LiCl stimulation enhanced the expression of YAP, as well as that of AXIN2, the b-catenin target gene (Figure S6B). In agreement with this result, the b-catenin inhibitor NCO43 decreased the levels of YAP and AXIN2 mRNA in IL-4/IL-13-induced M2 macrophages (Figure S6C). Moreover, knockdown of b-catenin reduced YAP mRNA expression in control macrophages, which failed to further affect the IL-4/IL-13-induced reduction of YAP expression (Figure S6D). To further confirm whether b-catenin is linked with the PI3KAKT pathway for YAP transcription, we pretreated macrophages with the b-catenin inhibitor NCO43 with or without the PI3K inhibitor LY294002, followed by induction into M2 macrophages via IL-4/IL-13 stimulation. Compared with the DMSO control, LY294002 administration enhanced YAP expression; however, the NCO43 treatment eliminated this effect (Figure S6E). In agreement with this result, IL-4/IL-13-stimulated macrophages exhibited enhanced phosphorylation of b-catenin, which was reduced to basal levels in response to the LY294002 treatment (Figure S6F). Vector YAP

**

2 1 0

T1

0

**

1 0 HA

0

4

AP-1

1

CATTC

ut at io n m ut at io n

1

Tead1

-3

-1

*

- 279

TGAGTCA

m

2

- 420 - 285

AATTC

AP

YAP

IL-6 promoter - 637 - 424

ut at io n

+ /+

T2

p-ERK

T

p-p38 *

W

p-JNK

m

p-IKKα/β

Relative luciferase units(RLU)

3

lg G

Relative protein levels

D

demonstrated that YAP promotes macrophages to produce IL-6 and polarize toward M1. YAP Expression Is Differentially Regulated during M1 and M2 Macrophage Polarization Because YAP regulates the balance of M1/M2 polarization, we wanted to investigate whether the expression of YAP is affected during the induction of M1/M2 macrophages. We first treated PEMs with IL-4/IL-13 in vitro. During the induction of M2 macrophages, YAP mRNA levels were downregulated (Figure 6A). Furthermore, IL-4/IL-13 treatment significantly elevated the levels of phosphorylated PI3K subunit p85 and AKT (Figure 6B). Interestingly, the treatment of cells with the p85 siRNA or PI3K inhibitor (LY294002) significantly reversed the reduction in YAP expression in response to the IL-4/IL-13 treatment (Figure S6A; Figure 6C). This result indicates that IL-4/IL-13 treatment may reduce YAP expression via activation of the PI3K-AKT pathway.

Cell Reports 27, 1176–1189, April 23, 2019 1183

B

*

*

IL-4/13 (min)

15

C

45 100

p-p85

1.0 0.5 0.0

0

72

p-AKT k oc M

2 M

h 12

2 M

43

β-actin

h 24

D

E

IL-4/13 (h)

0

12

F IL-4/13(min)

24

YAP

72

GAPDH

34

G

0

15

8

4 2 0

45

p-YAP S127

72

GAPDH

43

IL4/13 (h) 0

LPS+IFNγ (h)

24

YAP

72

p-YAP-Y357

GAPDH

34

β - actin

Hoechst

I

YAP

0

2

Merge

Mock

YAP

72

GAPDH

34

43

60 40 20 0

We observed reduced YAP expression not only at the mRNA level but also at the protein level in M2 macrophages (Figure 6D). Furthermore, IL-4/IL-13 treatment increased YAP phosphorylation at S127 in murine primary macrophages (Figure 6E) and human THP1 cells (Figure 6F). Next, we used large numbers of human THP1 cells to obtain enough cell lysates for nucleus-cytosol fractionation. YAP was dramatically enriched in the cytoplasm with the IL-4/IL-13 treatment (Figure S6G). Previous studies have reported that YAP is phosphorylated by upstream kinases in the Hippo pathway, including STK4/STK3 (the serine and threonine kinase 4/3, also termed MST1 and MST2) and LATS1/2 (the large tumor suppressor 1/2), which promotes YAP sequestration into the cytoplasm for future degradation (Taniguchi et al., 2015; Varelas, 2014). Interestingly, we observed enhanced phosphorylation of MST1/2 and LATS1/2 in IL-4/IL-13-stimulated macrophages (Figure S6H). Taken together, IL-4/IL-13 treatment reduces YAP expression at the mRNA and protein levels. Interestingly, we observed enhanced YAP expression at the protein level in macrophages after LPS/IFN-g treatment (Figure 6G). Furthermore, LPS/IFN-g stimulation enhanced YAP phosphorylation at the Y357 site (Figure 6H) and YAP translocation into the nuclei (Figure 6I). Thus, we suggest that during the development of IBD, different inflammatory microenvironments or gut bacterial infection can affect YAP expression in macrophages.

1184 Cell Reports 27, 1176–1189, April 23, 2019

(A) Yap expression at the mRNA level was assessed in PEMs that were stimulated with IL-4 and IL-13 for 12 or 24 h. (B) The levels of phosphorylated p85 and pAKT in IL-4/IL-13-stimulated PEMs were detected by immunoblotting analysis. (C) PEMs were pretreated with LY294002 for 1 h, followed by IL-4/IL-13 treatment for 24 h to measure Yap mRNA levels. (D–F) Expression of YAP and phosphorylated YAP S127 was assessed in PEMs (D and E) or THP1 (F) that were stimulated with IL-4 and IL-13 for the indicated times. (G and H) Expression in YAP (G) or phosphorylated YAP S357 (H) was assessed in LPS- and IFN-gtreated PEMs. (I) PEMs were stimulated with LPS/IFN-g, followed by immunostaining with Hoechst and anti-YAP (scale bars: 20 mm). A total of 10–15 viewing fields were analyzed in each experiment. The representative data from at least three independent experiments are shown. See also Figure S6.

**

80

LP S

LPS + IFNγ

72

1 72

4

YAP nuclei (%)

12

0.5

p-YAP S127

M oc k + IF Nγ

0

*

6

H

LPS+IFNγ(h)

Figure 6. YAP Expression Is Differentially Regulated during M1 and M2 Macrophage Polarization

NS

Relative Yap mRNA

1.5

M oc M 2+ k D M M 2+ LY SO 29 40 02

Relative Yap mRNA

A

YAP Regulates Colon Microbial Homeostasis To investigate the relevance of our findings with respect to human diseases, we analyzed samples from patients with UC, a type of IBD (GDS3268, GDS3119) (n = 71) (Hold et al., 2014; Lin et al., 2014; Steinbach and Plevy, 2014). YAP expression was positively correlated with IL-6 expression at the mRNA level in human IBD samples (Figure 7A). By analyzing IBD mouse array data (GDS3859), we also observed increasing amounts of IL-6 and YAP at day 6 after DSS induction (Figure 7B). Previous studies have suggested that the dynamic shifts in gut microbiota play a significant role during the development of IBD (Matsuoka and Kanai, 2015; Saleh and Elson, 2011). From the gene-pathway analysis of our RNA-seq data between WT and YAPDM/DM macrophages, we noticed a significant enrichment of genes involved in bacterial infection (Figure 1F). Therefore, we asked whether YAP cKO mice affected the diversity of the gut microbiota during the induction of IBD. The gut microbial composition in WT and YAP cKO mice showed significant differences, especially the decreased abundance of Bacteroidetes in the WT mice (Figure 7C, indicated in red). Interestingly, the lower level of Bacteroidetes in the gut microbiota is associated with IBD patients (Zhou and Zhi, 2016). In addition, the increased abundance of Prevotellaceae was observed in the WT mice (Figure 7C, top left), which could exacerbate IBD when transferred to WT mice (Ni et al., 2017). We then performed the 16S rDNA PCR assay to detect whether YAP affects other IBD-related bacteria abundances. We observed increased abundances of Lactobacillus, Bacteroides, and Bifidobacteria and decreased abundances of Prevotella, b-Proteobacteria, g-Proteobacteria, and

50

D

others:11.27%

Alistipes:2.21% Clostridiales_vadinBB60_ group:2.57% Bacteroidales_S24-7_ group:22.65%

Treponema_2:5.41% Rhodospirillaceae :5.38% Parasutterella:3.70% Ruminococcaceae _UCG-014 :9.03% Bacteroides:9.33%

YAPΔM/ΔM Alistipes:2.30% Clostridiales_vadinBB60_ group:2.45% Faecalibaculum:3.57% Bacteroidales_S24-7_ group:34.84%

Rhodospirillaceae :3.19% Parasutterella:7.46% Ruminococcaceae_ UCG-014:10.40% Bacteroides:13.41%

6

0

*

DSS

ΔM/ΔM

DSS YAP

*

** *

DSS YAP+/+

**

YAPΔM/ΔM REG3γ

8

*

6 4 2 0

An g4

M1

H2

H

R

eg III γ

0

M2 LPS +IFNγ

G

O

H2

O

5

R et nl β

Relative mRNA levels

**

H2O YAPΔM/ΔM

*

10

IL-4+IL-13 YAP mRNA

YAP

H2O YAP +/+

8

H2O YAP ΔM/ΔM

6

YAP

JNK

**

+/+

DSS YAP

p53

IL-6

DSS YAPΔM/ΔM

4

Il1 8

Il3 3

Il1 0

Il2 3

0

˛

ARG1 YM1 FIZZ

IL-6

2

Il2 2

Relative mRNA levels

*

YAP+/+

H2OYAP+/+

15

***

H2O

F

Akkermansia:8.23%

E 20

2

YAPΔM/ΔM

0

others:8.24%

Lachnospiraceae_NK4A136_ group:1.21%

**

3

1

Akkermansia:15.87%

unclassified_o__Bacteroidales:1.22%

YAP

M /Δ M

Faecalibaculum:2.09%

(day) +/+

YA YA P + / + DS P ΔM /Δ DS S M S YA YA P + / PΔ +

Gastranaerophilales :3.07%

1500

Relative REG3γ protein

unclassified_f__Prevotellaceae:2.19%

rDNA le ve ls

4

YAP+/+

(day)

αRelat ive 16S p P β- rot re pr eo vo ε- ote ba tel pr o ct la γ En -p ote bac eria te ro ob te ro te ac ria ba ob te r A cte act ia ct ri er Pe in ac ia pt P ob e os ep a ae tr to cte R ept co ria um o cc c in oc us oc cu oc s C cu lo s La tr E.c s ct idi oli o a B ba lI-V B act cil ifi e liu do ro s b id Fi ac es rm te ic ria ut es

C

2

0

0

2000

6

100

* 2500

4

**

**

3000

2

2000 1500 1000 500 150

Relative Yap mRNA

**

B

Relative Il-6 mRNA

A

YAP expression

IBD

YAP

Normal

(legend on next page)

Cell Reports 27, 1176–1189, April 23, 2019 1185

Enterobacteriaceae at day 7 in DSS-induced YAPDM/DM mice compared with WT control mice (Figure 7D). These bacteria with increased or reduced abundances have been reported in IBD patients and in animal models (Hold et al., 2014; Matsuoka and Kanai, 2015; Wang et al., 2014). In addition, without the DSS treatment, we observed no substantial changes in the abundance of these gut microbiota between YAPDM/DM and WT mice (Figure S7A). The antimicrobial peptides Ang4, Retn1b, and RegIIIg have been reported to promote homeostasis of the gut microbiota (Burger-van Paassen et al., 2012). We therefore tested whether YAP regulates the expression of antimicrobial peptides after the induction of IBD. Interestingly, the expression levels of Ang4, Retn1b, and RegIIIg were significantly higher in YAPDM/DM mice than in the WT controls (Figures 7E and 7F). Furthermore, DSS-treated colon epithelial cells exhibited significantly enhanced RegIIIg expression (Figure 7F; Figure S7B), which is consistent with previous studies (Burger-van Paassen et al., 2012; Xiao et al., 2017). IL-22, IL-23, IL-10, IL-33, and IL-18 have been reported to be associated with colon microbial homeostasis and play important roles in regulating RegIIIg expression (Nowarski et al., 2015; Parks et al., 2016; Pastorelli et al., 2013). Therefore, we measured the levels of these cytokines in colonic lamina propria, and the DSS treatment enhanced the expression of IL-22, IL-23, IL-10, and IL-33 in both WT and YAPDM/DM mice (Figure 7G). Taken together, our study has elucidated that YAP in macrophages plays a detrimental role in DSS-induced colitis by affecting M1/M2 macrophage polarization, as well as gut microbial homeostasis. Furthermore, YAP expression is differentially regulated by IL-4/IL-13 versus LPS/IFN-g during the induction of M2/M1 macrophages (Figure 7H). DISCUSSION Although it has been well studied about YAP acting as a transcriptional coactivator in tumor cells (Wang et al., 2016a; Yimlamai et al., 2014; Zhao et al., 2008), the investigation of YAP function in immune cells is limited. One of the possible reasons might be because of its low expression levels in immune cells. However, the emerging reports claim that YAP plays an important role in Treg-mediated suppression of antitumor immunity (Ni et al., 2018) and is necessary for B cell resistance to Salmonella-induced inflammasome (Perez-Lopez et al., 2013).

Macrophage polarization has been proposed to play important roles in many diseases, such as IBD (Cosı´n-Roger et al., 2016), tissue repair (Ruffell et al., 2009), and various cancers, suggesting that there is a precise and complex balance of macrophage polarization required for homeostasis. Some transcription factors have been reported to determine macrophage fate, such as STAT6, C/EBPb, and IRF4, which can upregulate M2-associated genes, whereas IRF5 is needed for M1 polarization. This study has provided evidence that YAP can play a dual role, driving macrophages toward M1 polarization while restricting M2 polarization. More interestingly, YAP expression levels were observed to be differentially regulated during the induction of M1 or M2 macrophages (i.e., YAP levels are reduced and enhanced in M2 and M1 macrophages, respectively, despite Figure S1B showing the low YAP expression in resting macrophages). Based on the model we summarize in Figure 7H, in response to local inflammation or gut bacterial infection, macrophages alter YAP expression levels, and YAP further regulates M1 versus M2 macrophage fate. Furthermore, M1 versus M2 polarization exacerbates the pre-existing imbalance of local inflammation or gut bacteria. This ‘‘feedback loop’’ effect of YAP in macrophage fate or function could be a crucial ‘‘controller’’ that determines whether the host returns to homeostasis or causes disease. We therefore suggest that YAP might be added to the list of key transcription factors and/or coactivators that manipulate macrophage fate or macrophage plasticity. In this study, we demonstrated that YAP can directly bind to the IL-6 promoter to promote IL-6 production in macrophages. YAP is also reported to participate in hemodynamics-mediated atherosclerosis via the promotion of IL-6 expression (Murakami et al., 2017; Wang et al., 2016b), and IL-6-gp130 signaling triggers YAP activation via Src family kinases (Taniguchi et al., 2015). Together, these findings suggest a positive feedback loop between YAP and IL-6, which explains why YAP is highly correlated with IL-6 in many diseases. Thus, YAP may be regarded as a diagnostic biomarker, as well as a drug target, for the treatment of inflammation-related diseases. Given the rapidly accumulating evidence supporting that promotion of M2 polarization and targeting of IL-6 may €hl et al., 2015; Lin et al., be a new strategy to treat IBD (Ku 2014), our data suggest that targeting of YAP in macrophages (i.e., the YAPDM/DM mice produced less IL-6 and promoted M2 macrophage polarization) should be a potential therapeutic manipulation to treat IBD.

Figure 7. YAP Regulates Colon Microbial Homeostasis (A) Microarray data from colitis patients (n = 71) were analyzed to assess the correlation between IL-6 and YAP expression using Pearson’s test. (B) The mRNA levels of IL-6 and YAP were analyzed at different time points during DSS-induced colitis in mice (n = 6). (C) Microbial composition of WT and YAP cKO mice on day 7 after DSS induction (n = 3). (D) Gut microbiota DNA was exacted from mouse stool, and the relative abundances of the interested bacteria were determined by qRT-PCR on day 7 after DSS induction (n = 3). (E) The mRNA levels of Retn1b, Ang4, and RegIIIg were assessed in colon tissues from DSS-induced YAP+/+ and YAPDM/DM mice (n = 5). (F) Representative images of REG3g immunostaining in paraffin-embedded colon sections from YAP+/+ and YAPDM/DM mice (scale bars: 50 mm). Quantitation was performed using Image-Pro Plus, and the average density was calculated from at least five areas of interest (AOIs) in each image. (G) The expression of Il-22, Il-23, Il-10, Il-33, and Il-18 in immune cells from colonic lamina propria was detected by qRT-PCR (n = 5). (H) Model: LPS/IFN-g treatment increases YAP expression, and YAP promotes IL-6 production via activating JNK or by directly binding to the IL-6 promoter. IL-4/ IL-13 treatment decreases YAP expression, and YAP inhibits M2 polarization. In addition, YAP cKO mice regulate antimicrobial peptides production and change gut microbial homeostasis. Together, YAP expression in macrophages promotes the development of IBD. See also Figure S7.

1186 Cell Reports 27, 1176–1189, April 23, 2019

In response to IL-4/IL-13 treatment, YAP acts as a ‘‘brake’’ to inhibit M2 polarization. Interestingly, a previous study suggests that in hepatocyte cells, YAP is inversely related with Arg1 expression (Yimlamai et al., 2014). Therefore, YAP negatively controls Arg1 expression in at least two types of cells. Arg1 is a key metabolic enzyme and is associated with diseases, including argininemia and intestinal schistosomiasis. Our and others’ findings may provide insights for further investigations into the function of YAP in Arg1-related diseases. Furthermore, in agreement with the suppressive role of p53 in M2 polarization (He et al., 2015; Li et al., 2015a), we observed that the p53 activator nutlin-3a or p53 siRNA could reverse the enhanced M2 polarization caused by YAP deficiency. YAP is recognized as a well-known oncogene, and p53 is a classical tumor suppressor. This study suggests that, in addition to their tumor suppressor or oncogenic roles in tumor cells, YAP and p53 may be differentially targeted in macrophages to treat other types of diseases, such as autoimmunity. Although YAP is recognized as an oncogene in many types of tumors, somatic or germline mutations in YAP from human patients are currently uncommon (Gao et al., 2014; Harvey et al., 2013; Murakami et al., 2017). By contrast, YAP is highly expressed in different human carcinomas and is correlated with tumor prognosis, indicating that abnormal YAP expression or activity is vital for these cancers. Previous studies reported that specific conditions, such as cell detachment, nutrition condition, and G protein-coupled receptor (GPCR) signaling, could activate the Hippo pathway to modulate YAP expression (Yu et al., 2012; Zhang et al., 2017). Our findings have demonstrated that IL-4/IL-13 treatment could inhibit YAP expression via the PI3K-AKT-b-catenin pathway. We also showed that LPS/IFN-g stimulation increases YAP protein expression in macrophages. Emerging data suggest that YAP phosphorylation is also affected by viral infection, and that YAP negatively controls IFN-b production (Kim et al., 2018; Zhang et al., 2017). Together, we and others have demonstrated how YAP expression is regulated in response to different stimuli, including cytokines or pathogens. Notably, recent work reported that the IL-6-STAT3 pathway and YAP increase intestinal epithelial regeneration and proliferation, promoting tissue healing and maintaining the barrier function of the epithelium to protect against IBD (Taniguchi et al., 2015). Our study suggests that in macrophages, YAP aggravates IBD via regulation of M1/M2 macrophage polarization. Therefore, YAP may play different roles in two types of key cells in the local gut microenvironment during the development of IBD. Patients with long-standing IBD have been reported to be at an increased risk of developing colorectal cancer, which is closely related to immune cell infiltration and chronic inflammation in the tumor microenvironment (He et al., 2015). Interestingly, the oncogene YAP is highly expressed in colorectal cancer (Taniguchi et al., 2017). In agreement with the above observations, our data suggest that pathogen infection might elevate YAP expression in macrophages, and YAP further promotes IL-6 production, which feeds back to promote the proliferation of colorectal cancer cells (Taniguchi et al., 2015, 2017). These observations indicate that YAP might promote the crosstalk between IL-6-producing macrophages and colorectal cancer cells to exacerbate inflammation-induced colon cancer. Despite this, we showed

that YAP also inhibits M2 macrophage polarization; this warns whether targeting YAP may facilitate tumor growth via promoting tumor-associated macrophages. In summary, YAP displays diverse functions in multiple cell types, including promoting intestinal epithelial regeneration and tumor proliferation or regulating M2/M1 macrophage polarization. Therefore, targeting YAP in the appropriate cell types should be considered in order to achieve a therapeutic effect to treat inflammation-related diseases. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

d d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Mice B DSS-Induced Colitis B Cell Culture METHOD DETAILS B Plasmids, Transfection and Luciferase Assays B Transfection and Lentiviral infection B Histology and Histopathological Score B Immunoprecipitation and Immunostaining B ChIP Assays B Nuclear and Cytoplasmic Protein Extraction B Isolation of Colonic Lamina Propria and Epithelial Cells B RNA-Seq Analysis B DNA Extraction Using a QIAamp Fast DNA Stool MiniKit B 16S rDNA Sequencing B RNA Interference and Real-Time PCR QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND SOFTWARE AVAILABILITY

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. celrep.2019.03.028. ACKNOWLEDGMENTS We thank Prof. Feng Shao at the National Institute of Biological Sciences, Beijing, for providing PAO and S. aureus. We also thank Prof. Lei Zhang of the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, for providing the p-Mst1/2 and p-Lats1/2 antibodies. This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000), the Ministry of Science and Technology of China (2016YFD0500207, 2016YFC0905902, and 2016YFD0500407), the National Natural Science Foundation of China (81825011, 81630043, 81571617, 81671572, 81571552, 81701569, 31700781, 81801575), and the State Key Laboratory of Cell Biology, SIBCB, CAS (SKL CBKF2013003). We thank the Genome Tagging Project (GTP) Center, Shanghai Institute of Biochemistry and Cell Biology, CAS for technical support. AUTHOR CONTRIBUTIONS Conceptualization, X. Zhou, W.L., and H.W.; Methodology, X. Zhou, W.L., and H.W.; Investigation, X. Zhou, W.L., S.W., P.Z., J.X., Q.W., C.Z., X. Zheng, X.Y.,

Cell Reports 27, 1176–1189, April 23, 2019 1187

and S.X.; Writing – Original Draft, X. Zhou, W.L., and P.Z.; Writing – Review & Editing, H.W.; Funding Acquisition, H.W.; Resources, L.H., B.W., and H.J.; Supervision, B.W. and H.W.

tory macrophage polarization and TH1-TH17 responses. Nat. Immunol. 12, 231–238. €hl, A.A., Erben, U., Kredel, L.I., and Siegmund, B. (2015). Diversity of IntesKu tinal Macrophages in Inflammatory Bowel Diseases. Front. Immunol. 6, 613.

DECLARATION OF INTERESTS

Lawrence, T., and Natoli, G. (2011). Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nat. Rev. Immunol. 11, 750–761.

The authors declare no competing interests.

Li, L., Ng, D.S., Mah, W.C., Almeida, F.F., Rahmat, S.A., Rao, V.K., Leow, S.C., Laudisi, F., Peh, M.T., Goh, A.M., et al. (2015a). A unique role for p53 in the regulation of M2 macrophage polarization. Cell Death Differ. 22, 1081–1093.

Received: December 4, 2017 Revised: December 17, 2018 Accepted: March 7, 2019 Published: April 23, 2019 REFERENCES Arranz, A., Doxaki, C., Vergadi, E., Martinez de la Torre, Y., Vaporidi, K., Lagoudaki, E.D., Ieronymaki, E., Androulidaki, A., Venihaki, M., Margioris, A.N., et al. (2012). Akt1 and Akt2 protein kinases differentially contribute to macrophage polarization. Proc. Natl. Acad. Sci. USA 109, 9517–9522. Bhattacharyya, S., Ratajczak, C.K., Vogt, S.K., Kelley, C., Colonna, M., Schreiber, R.D., and Muglia, L.J. (2010). TAK1 targeting by glucocorticoids determines JNK and IkappaB regulation in Toll-like receptor-stimulated macrophages. Blood 115, 1921–1931. Burger-van Paassen, N., Loonen, L.M.P., Witte-Bouma, J., Korteland-van Male, A.M., de Bruijn, A.C.J.M., van der Sluis, M., Lu, P., Van Goudoever, J.B., Wells, J.M., Dekker, J., et al. (2012). Mucin Muc2 deficiency and weaning influences the expression of the innate defense genes Reg3b, Reg3g and angiogenin-4. PLoS ONE 7, e38798. Cosı´n-Roger, J., Ortiz-Masia´, D., Calatayud, S., Herna´ndez, C., Esplugues, J.V., and Barrachina, M.D. (2016). The activation of Wnt signaling by a STAT6-dependent macrophage phenotype promotes mucosal repair in murine IBD. Mucosal Immunol. 9, 986–998. Gao, Y.J., Zhang, W.J., Han, X.K., Li, F.M., Wang, X.J., Wang, R., Fang, Z.Y., Tong, X.Y., Yao, S., Li, F., et al. (2014). YAP inhibits squamous transdifferentiation of Lkb1-deficient lung adenocarcinoma through ZEB2-dependent DNp63 repression. Nat. Commun. 5, 4629. Geng, J., Sun, X., Wang, P., Zhang, S., Wang, X., Wu, H., Hong, L., Xie, C., Li, X., Zhao, H., et al. (2015). Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity. Nat. Immunol. 16, 1142–1152. Harusato, A., Geem, D., and Denning, T.L. (2016). Macrophage Isolation from the Mouse Small and Large Intestine. Methods Mol. Biol. 1422, 171–180. Harvey, K.F., Zhang, X., and Thomas, D.M. (2013). The Hippo pathway and human cancer. Nat. Rev. Cancer 13, 246–257. He, X.Y., Xiang, C., Zhang, C.X., Xie, Y.Y., Chen, L., Zhang, G.X., Lu, Y., and Liu, G. (2015). p53 in the Myeloid Lineage Modulates an Inflammatory Microenvironment Limiting Initiation and Invasion of Intestinal Tumors. Cell Rep. 13, 888–897. Hold, G.L., Smith, M., Grange, C., Watt, E.R., El-Omar, E.M., and Mukhopadhya, I. (2014). Role of the gut microbiota in inflammatory bowel disease pathogenesis: what have we learnt in the past 10 years? World J. Gastroenterol. 20, 1192–1210. Jablonski, K.A., Amici, S.A., Webb, L.M., Ruiz-Rosado, Jde.D., Popovich, P.G., Partida-Sanchez, S., and Guerau-de-Arellano, M. (2015). Novel Markers to Delineate Murine M1 and M2 Macrophages. PLoS ONE 10, e0145342. Kim, N., Park, Y.Y., Joo, C.H., and Kim, H.S. (2018). Relief of YAP-mediated inhibition by IKKε promotes innate antiviral immunity. Cell. Mol. Immunol. 15, 642–644. Konsavage, W.M., Jr., Kyler, S.L., Rennoll, S.A., Jin, G., and Yochum, G.S. (2012). Wnt/b-catenin signaling regulates Yes-associated protein (YAP) gene expression in colorectal carcinoma cells. J. Biol. Chem. 287, 11730–11739. Krausgruber, T., Blazek, K., Smallie, T., Alzabin, S., Lockstone, H., Sahgal, N., Hussell, T., Feldmann, M., and Udalova, I.A. (2011). IRF5 promotes inflamma-

1188 Cell Reports 27, 1176–1189, April 23, 2019

Li, W., Xiao, J., Zhou, X., Xu, M., Hu, C., Xu, X., Lu, Y., Liu, C., Xue, S., Nie, L., et al. (2015b). STK4 regulates TLR pathways and protects against chronic inflammation-related hepatocellular carcinoma. J. Clin. Invest. 125, 4239– 4254. Lin, Y., Yang, X., Yue, W., Xu, X., Li, B., Zou, L., and He, R. (2014). Chemerin aggravates DSS-induced colitis by suppressing M2 macrophage polarization. Cell. Mol. Immunol. 11, 355–366. Liu, B., Zheng, Y., Yin, F., Yu, J., Silverman, N., and Pan, D. (2016). Toll Receptor-Mediated Hippo Signaling Controls Innate Immunity in Drosophila. Cell 164, 406–419. Mahoney, J.E., Mori, M., Szymaniak, A.D., Varelas, X., and Cardoso, W.V. (2014). The hippo pathway effector Yap controls patterning and differentiation of airway epithelial progenitors. Dev. Cell 30, 137–150. Matsuoka, K., and Kanai, T. (2015). The gut microbiota and inflammatory bowel disease. Semin. Immunopathol. 37, 47–55. Morin-Kensicki, E.M., Boone, B.N., Howell, M., Stonebraker, J.R., Teed, J., Alb, J.G., Magnuson, T.R., O’Neal, W., and Milgram, S.L. (2006). Defects in yolk sac vasculogenesis, chorioallantoic fusion, and embryonic axis elongation in mice with targeted disruption of Yap65. Mol. Cell. Biol. 26, 77–87. Murakami, S., Shahbazian, D., Surana, R., Zhang, W., Chen, H., Graham, G.T., White, S.M., Weiner, L.M., and Yi, C. (2017). Yes-associated protein mediates immune reprogramming in pancreatic ductal adenocarcinoma. Oncogene 36, 1232–1244. Ni, J., Wu, G.D., Albenberg, L., and Tomov, V.T. (2017). Gut microbiota and IBD: causation or correlation? Nat. Rev. Gastroenterol. Hepatol. 14, 573–584. Ni, X., Tao, J., Barbi, J., Chen, Q., Park, B.V., Li, Z., Zhang, N., Lebid, A., Ramaswamy, A., Wei, P., et al. (2018). YAP is essential for Treg-mediated suppression of antitumor immunity. Cancer Discov. 8, 1026–1043. Nowarski, R., Jackson, R., Gagliani, N., de Zoete, M.R., Palm, N.W., Bailis, W., Low, J.S., Harman, C.C.D., Graham, M., Elinav, E., and Flavell, R.A. (2015). Epithelial IL-18 Equilibrium Controls Barrier Function in Colitis. Cell 163, 1444–1456. Odegaard, J.I., Ricardo-Gonzalez, R.R., Red Eagle, A., Vats, D., Morel, C.R., Goforth, M.H., Subramanian, V., Mukundan, L., Ferrante, A.W., and Chawla, A. (2008). Alternative M2 activation of Kupffer cells by PPARdelta ameliorates obesity-induced insulin resistance. Cell Metab. 7, 496–507. Parks, O.B., Pociask, D.A., Hodzic, Z., Kolls, J.K., and Good, M. (2016). Interleukin-22 Signaling in the Regulation of Intestinal Health and Disease. Front. Cell Dev. Biol. 3, 85. Pastorelli, L., De Salvo, C., Vecchi, M., and Pizarro, T.T. (2013). The role of IL-33 in gut mucosal inflammation. Mediators Inflamm. 2013, 608187. Perez-Lopez, A., Rosales-Reyes, R., Alpuche-Aranda, C.M., and Ortiz-Navarrete, V. (2013). Salmonella downregulates Nod-like receptor family CARD domain containing protein 4 expression to promote its survival in B cells by preventing inflammasome activation and cell death. J. Immunol. 190, 1201– 1209. Puig-Kro¨ger, A., Sierra-Filardi, E., Domı´nguez-Soto, A., Samaniego, R., Corcuera, M.T., Go´mez-Aguado, F., Ratnam, M., Sa´nchez-Mateos, P., and Corbı´, A.L. (2009). Folate receptor beta is expressed by tumor-associated macrophages and constitutes a marker for M2 anti-inflammatory/regulatory macrophages. Cancer Res. 69, 9395–9403. Rauh, M.J., Ho, V., Pereira, C., Sham, A., Sly, L.M., Lam, V., Huxham, L., Minchinton, A.I., Mui, A., and Krystal, G. (2005). SHIP represses the generation of alternatively activated macrophages. Immunity 23, 361–374.

Ruffell, D., Mourkioti, F., Gambardella, A., Kirstetter, P., Lopez, R.G., Rosenthal, N., and Nerlov, C. (2009). A CREB-C/EBPbeta cascade induces M2 macrophage-specific gene expression and promotes muscle injury repair. Proc. Natl. Acad. Sci. USA 106, 17475–17480. Saleh, M., and Elson, C.O. (2011). Experimental inflammatory bowel disease: insights into the host-microbiota dialog. Immunity 34, 293–302. Satoh, T., Takeuchi, O., Vandenbon, A., Yasuda, K., Tanaka, Y., Kumagai, Y., Miyake, T., Matsushita, K., Okazaki, T., Saitoh, T., et al. (2010). The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection. Nat. Immunol. 11, 936–944. Shon, W.J., Lee, Y.K., Shin, J.H., Choi, E.Y., and Shin, D.M. (2015). Severity of DSS-induced colitis is reduced in Ido1-deficient mice with down-regulation of TLR-MyD88-NF-kB transcriptional networks. Sci. Rep. 5, 17305. Steinbach, E.C., and Plevy, S.E. (2014). The role of macrophages and dendritic cells in the initiation of inflammation in IBD. Inflamm. Bowel Dis. 20, 166–175. Taniguchi, K., Wu, L.W., Grivennikov, S.I., de Jong, P.R., Lian, I., Yu, F.X., Wang, K., Ho, S.B., Boland, B.S., Chang, J.T., et al. (2015). A gp130-SrcYAP module links inflammation to epithelial regeneration. Nature 519, 57–62. Taniguchi, K., Moroishi, T., de Jong, P.R., Krawczyk, M., Grebbin, B.M., Luo, H., Xu, R.H., Golob-Schwarzl, N., Schweiger, C., Wang, K., et al. (2017). YAPIL-6ST autoregulatory loop activated on APC loss controls colonic tumorigenesis. Proc. Natl. Acad. Sci. USA 114, 1643–1648. Varelas, X. (2014). The Hippo pathway effectors TAZ and YAP in development, homeostasis and disease. Development 141, 1614–1626. Wang, S., Yin, J., Chen, D., Nie, F., Song, X., Fei, C., Miao, H., Jing, C., Ma, W., Wang, L., et al. (2013). Small-molecule modulation of Wnt signaling via modulating the Axin-LRP5/6 interaction. Nat. Chem. Biol. 9, 579–585. Wang, W., Chen, L., Zhou, R., Wang, X., Song, L., Huang, S., Wang, G., and Xia, B. (2014). Increased proportions of Bifidobacterium and the Lactobacillus group and loss of butyrate-producing bacteria in inflammatory bowel disease. J. Clin. Microbiol. 52, 398–406.

Wang, G., Lu, X., Dey, P., Deng, P., Wu, C.C., Jiang, S., Fang, Z., Zhao, K., Konaparthi, R., Hua, S., et al. (2016a). Targeting YAP-Dependent MDSC Infiltration Impairs Tumor Progression. Cancer Discov. 6, 80–95. Wang, L., Luo, J.Y., Li, B., Tian, X.Y., Chen, L.J., Huang, Y., Liu, J., Deng, D., Lau, C.W., Wan, S., et al. (2016b). Integrin-YAP/TAZ-JNK cascade mediates atheroprotective effect of unidirectional shear flow. Nature 540, 579–582. Weisser, S.B., Brugger, H.K., Voglmaier, N.S., McLarren, K.W., van Rooijen, N., and Sly, L.M. (2011). SHIP-deficient, alternatively activated macrophages protect mice during DSS-induced colitis. J. Leukoc. Biol. 90, 483–492. Yang, W., Nam, K., Ju, J.H., Lee, K.M., Oh, S., and Shin, I. (2014). S100A4 negatively regulates b-catenin by inducing the Egr-1-PTEN-Akt-GSK3b degradation pathway. Cell. Signal. 26, 2096–2106. Yimlamai, D., Christodoulou, C., Galli, G.G., Yanger, K., Pepe-Mooney, B., Gurung, B., Shrestha, K., Cahan, P., Stanger, B.Z., and Camargo, F.D. (2014). Hippo pathway activity influences liver cell fate. Cell 157, 1324–1338. Yu, F.X., Zhao, B., Panupinthu, N., Jewell, J.L., Lian, I., Wang, L.H., Zhao, J., Yuan, H., Tumaneng, K., Li, H., et al. (2012). Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling. Cell 150, 780–791. Zanconato, F., Forcato, M., Battilana, G., Azzolin, L., Quaranta, E., Bodega, B., Rosato, A., Bicciato, S., Cordenonsi, M., and Piccolo, S. (2015). Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth. Nat. Cell Biol. 17, 1218–1227. Zhang, Q., Meng, F., Chen, S., Plouffe, S.W., Wu, S., Liu, S., Li, X., Zhou, R., Wang, J., Zhao, B., et al. (2017). Hippo signalling governs cytosolic nucleic acid sensing through YAP/TAZ-mediated TBK1 blockade. Nat. Cell Biol. 19, 362–374. Zhao, B., Ye, X., Yu, J., Li, L., Li, W., Li, S., Yu, J., Lin, J.D., Wang, C.Y., Chinnaiyan, A.M., et al. (2008). TEAD mediates YAP-dependent gene induction and growth control. Genes Dev. 22, 1962–1971. Zhou, Y., and Zhi, F. (2016). Lower Level of Bacteroides in the Gut Microbiota Is Associated with Inflammatory Bowel Disease: A Meta-Analysis. BioMed Res. Int. 2016, 5828959.

Cell Reports 27, 1176–1189, April 23, 2019 1189

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Rabbit anti-p-IKKa/b

Cell Signaling Technology

Cat# 2697, RRID:AB_2079382

Rabbit anti-p-JNK1/2

Cell Signaling Technology

Cat# 9251, RRID:AB_331659

Rabbit anti-p-p38

Cell Signaling Technology

Cat# 9211, RRID:AB_331641

Antibodies

Rabbit anti-p-ERK1/2

Cell Signaling Technology

Cat# 4377, RRID:AB_331775

Rabbit anti-p-AKT

Cell Signaling Technology

Cat# 4060, RRID:AB_2315049

Rabbit anti-b-catenine

Cell Signaling Technology

Cat# 9587, RRID:AB_10695312

Rabbit anti-p-b-catenine

Cell Signaling Technology

Cat# 9561, RRID:AB_331729

Rabbit anti-p-LATS1/2

Cell Signaling Technology

Cat# 8654, RRID:AB_10971635

Rabbit anti-YAP

ABclonal Biotech

Cat# A1002, RRID:AB_2757539

Rabbit anti-YAP1-S127

ABclonal Biotech

Cat# AP0489, RRID:AB_2771648

Rabbit anti-ARG1

Abways Technology

Cat# CY5101

Rabbit anti-STAT6

Abways Technology

Cat# CY5351

Rabbit anti-p-STAT6-Y641

Abways Technology

Cat# CY6509

HRP-b-actin

Abcam

Cat# ab49900, RRID:AB_867494

Rabbit anti-CD206

Abcam

Cat# ab64693, RRID:AB_1523910

Rabbit anti-MST1/2

Abcam

Cat# ab79199, RRID:AB_2271183

Rabbit anti-YAP1-S357

Abcam

Cat# ab62751, RRID:AB_956486

Mouse anti-CD80

Abcam

Cat# ab64116, RRID:AB_1640342

Rabbit anti-CD68

Abcam

Cat# ab955, RRID:AB_307338

Rabbit anti-INOS

Abcam

Cat# ab15323, RRID:AB_301857

Rat anti-F4/80

Abcam

Cat# ab6640, RRID:AB_1140040

Mouse anti-Myc

Abmart

Cat# M20002-19C2

Rabbit anti-p65

Santa Cruz Biotechnology

Cat# sc-372, RRID:AB_632037

Rabbit anti-REG3G

SAB

Cat# 38382

Rabbit anti-Lamin B1

ProteinTech

Cat# 12987-1-AP, RRID:AB_2136290

Rabbit anti-p53

ProteinTech

Cat# 10442-1-AP, RRID:AB_2206609

HRP-GAPDH

ProteinTech

Cat# 60004-1-Ig, RRID:AB_2107436

Mouse anti-Tubblin

Sigma

Cat# T5168, RRID:AB_477579

Mouse anti-HA

Sigma

Cat# H3663, RRID:AB_262051

PE-anti-rabbit-IgG

eBioscience

Cat# 12-4739-81, RRID:AB_1210761

CD4-APC

eBioscience

Cat# 17-0041-81, RRID:AB_469319

CD8a-PE

eBioscience

Cat# 12-0081-82, RRID:AB_465530

B220-FITC

eBioscience

Cat# 12-0452-81, RRID:AB_465670

F4/80-APC

eBioscience

Cat# 17-4801-80, RRID:AB_2688158

CD11b-FITC

eBioscience

Cat# 11-0112-82, RRID:AB_464935

Annexin V-APC

BioLegend

Cat# 640920, RRID:AB_2561515

Staphylococcus aureus

Laboratory of Feng Shao

ATCC 29213

Pseudomonas aeruginosa

Laboratory of Feng Shao

ATCC PAO BAA-47

Salmonella typhimurium

ATCC

ATCC 53647

Escherichia coli

ATCC

ATCC BAA-2471

LPS

Sigma

L2880

Ly294002

MCE

HY-10108

Bacterial and Virus Strains

Chemicals, Peptides, and Recombinant Proteins

(Continued on next page)

e1 Cell Reports 27, 1176–1189.e1–e5, April 23, 2019

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Nutlin 3a

MCE

HY-10029

Poly(I:C)

InvivoGen

Tlrl-picr

CpG

InvivoGen

Tlrl-1668

SP600125

Selleck

S1460

Mouse M-CSF

Peprotech

Cat# 315-02

Mouse IL-4

Peprotech

Cat# 214-14

Mouse IL-13

Peprotech

Cat# 210-13

Mouse IFN-g

Peprotech

Cat# 315-05

Human IL-4

Peprotech

Cat# 200-04

Human IL-13

Peprotech

Cat# 200-13

Protein G beads

GE healthcare

17-0618-01

Collagenase VIII

Sigma

C2139

DSS

MP Biomedicals

0216011080

IL-6 elisa kits

eBioscience

88-7064-88

TNF-a elisa kits

eBioscience

BMS607HS

Dual-Luciferase Reporter Assay

Promega

E1960

CellTiter-Glo

Promega

G7570

QIAamp Fast DNA stool MiniKit

QIAGEN

51604

Nuclear and cytoplasmic extracts kit

Beyotime

P0027

EZChIP kit

Millipore

17-371

16S rDNA sequencing

This paper

PRJNA516598

RNA-seq data

This paper

PRJNA523492

HEK293T cell

ATCC

ATCC ACS-4500

THP-1 cell

ATCC

ATCC TIB-202

Critical Commercial Assays

Deposited Data

Experimental Models: Cell Lines

Experimental Models: Organisms/Strains Mouse: C57BL/6J

AnimalCoreFacility, SIBCB

Jackson 000664

Mouse: YAP fl/fl

Laboratory of Hongbin Ji

Jackson 027929

Mouse: Lysmcre/wt

Laboratory of Bin Sun

Jackson 004781

This paper

N/A

Oligonucleotides See Table S1 for oligonucleotide information Recombinant DNA pCDH-CMV-EF1-Puro

Gao et al., 2014

System Biosciences KL-ZL-6999

pCDH-CMV-YAP1-EF1-Puro

Gao et al., 2014

pCDH-CMV-YAP1-EF1-Puro

pCDH-CMV-YAP15SA-EF1-Puro

Gao et al., 2014

pCDH-CMV-YAP1-EF1-Puro

pCDH-CMV-YAP15SADC-EF1-Puro

Gao et al., 2014

pCDH-CMV-YAP15SADC-EF1-Puro

Software and Algorithms Prism

Graphpad

https://www.graphpad.com/

David.6.8

Laboratory of Human Retrovirology and Immunoinformatics

https://david.ncifcrf.gov/home.jsp

Image-Pro 6.0

NIH

https://imagej.nih.gov/ij

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hongyan Wang ([email protected]).

Cell Reports 27, 1176–1189.e1–e5, April 23, 2019 e2

EXPERIMENTAL MODEL AND SUBJECT DETAILS Mice YAP fl/fl mice bearing two loxP sites flanking the first two exons of the YAP gene were cross-bred with Lysm-Cre mice to specifically KO YAP in the myeloid cell lineage, including macrophages (termed YAPDM/DM mice). We used YAPfl/fl Lysmcre/wt (heterozygous) as YAPDM/DM and YAPfl/fl Lysmwt/wt as the control mice. YAPWT/WT and YAPDM/DM mice were in a C57BL6 background. YAPfl/fl mice were generously provided by Dr. Ji at the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China. Mice were bred and maintained under specific pathogen free (SPF) conditions in the institutional animal facility of the Shanghai Institute of Biochemistry and Cell Biology. Age- and sex-matched littermates were used as control mice. All animal procedures were conducted in strict accordance with institutional guidelines and were approved by the Institutional Animal Care and Use Committee of Shanghai Institute of Biochemistry and Cell Biology (IBCB0057). DSS-Induced Colitis The age- and sex-matched YAPWT/WT and YAPDM/DM mice received 2.5% dextran sulfate sodium salt (DSS; MP Biomedicals, M.W. 36,000-50,000 kDa) for 78 days. The subsequent course of colitis development was evaluated by monitoring daily weight changes. Colitis severity was also scored by evaluating clinical disease activity through daily observations of the following parameters: weight loss (0 points = No weight loss or weight gain, 1 points = 5%–10% weight loss, 2 points = 11%–15% weight loss, 3 points = 16%–20% weight loss, and 4 points = > 21% weight loss); stool consistency (0 points = normal and well formed, 2 points = very soft and unformed, and 4 points = watery stool); and bleeding stool score (0 points = normal color stool, 2 points = reddish color stool, and 4 points = bloody stool). The disease activity index (DAI) was calculated based on the combined scores of weight loss, stool consistency, and bleeding and ranged from 0 to 12. All parameters were scored from day 0 to 7(Shon et al., 2015), with mice sacrificed on days 8. For each mouse, the entire colon was quickly removed, and the colon length was determined to be a marker of inflammation. Cell Culture Peritoneal elucidated macrophages (PEMs) were harvested from mice treated with an i.p. injection of 3% Brewer thioglycollate medium. Bone-marrow-derived macrophages (BMMs) were cultured with L929-conditioned completed DMEM medium for a week to generate BMMs as previously described(Li et al., 2015b). Primary murine PEMs, BMMs and HEK293T cell lines were cultured in complete DMEM supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin (100 U/ml) at 37 C with 5% CO2. THP1 cells were cultured using 1640 medium. METHOD DETAILS Plasmids, Transfection and Luciferase Assays HA-tagged human YAP, YAP5SA and YAP5SA-DC were cloned into the pCDH-CMV-EF1-Puro (Systems Biosciences) and were kindly provided by Dr.Ji (State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China). Plasmids expressing pRL-TK-Renilla were kindly provided by Drs. B. Sun, and R. Hu (Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academic of Sciences, Shanghai 200031, China). HEK293T cells were transfected with the IL-6 luciferase reporter plasmids (D2386 Beyotime), TK-Renilla, and the indicated plasmids. After 36 hr, luciferase readings were determined with a Dual-Luciferase Reporter Assay (Promega). Transfection and Lentiviral infection HA-YAP or its mutants and the lentiviral packaging plasmids psPAX2 and pMD2G were cotransfected into HEK293T cells, and supernatants were collected to infect THP1 cells to generate stable cells that were selected with medium containing puromycin (1 mg/ml) for 2 days. Histology and Histopathological Score For histological analyses, half of each distal colon was fixed in 4% paraformaldehyde for 24 h for subsequent paraffin embedding. Five-micrometer thick sections were stained with hematoxylin and eosin (H&E) using standard procedures. Disease severity was measured on the basis of a histopathological score. Colon sections were scored 0-4: 0, normal tissues; 1, mild inflammation in the mucosa with some infiltrating mononuclear cells; 2, increased level of inflammation in the mucosa with more infiltrating cells, damaged crypt glands and epithelium, mucin depletion from goblet cells; 3, extensive infiltrating cells in the mucosa and submucosa area, crypt abscesses present with increased mucin depletion and epithelial cell disruption; and 4, massive infiltrating cells in the tissue, complete loss of crypts(Arranz et al., 2012). Sections were analyzed by microscopy at 40 3 magnification. Three different sections from colon tissue sample in each mouse were examined.

e3 Cell Reports 27, 1176–1189.e1–e5, April 23, 2019

Immunoprecipitation and Immunostaining Immunoprecipitation was performed as described previously(Li et al., 2015b). In brief, cells were lysed in 1% NP-40 lysis buffer (50 mM Tris-HCl, 1% Nonidet-P40, 0.1% SDS, and 150 mM NaCl) together with freshly prepared protease and phosphatase inhibitors. Whole-cell lysates were incubated with the indicated antibodies, followed by incubation with protein G Sepharose beads at 4 C. The Sepharose beads were then washed three times with lysis buffer and resuspended in an appropriate amount of SDS-PAGE loading buffer. The samples were analyzed by immunoblotting. For immunostaining, LPS- and IFNg-stimulated PEMs were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.1% Triton X-100, then were stained with anti-p65 (1:200), anti-YAP (1:200) or anti-STAT6 (1:200) and Hoechst (1:10000). Images were taken using an Olympus BX-81 or BX-51 microscope and were processed with Image-Pro 6.0. ChIP Assays ChIP assays were performed with an EZChIP kit (Millipore), following the manufacturer’s instructions. In brief, cells were fixed with 0.9% formaldehyde and quenched with glycine, after which they were ultrasonicated with a Bioruptor (Diagenode). The supernatants were then collected and diluted, and the indicated antibodies were added for overnight incubation. The DNA-protein complexes were immunoprecipitated using protein G Sepharose, followed by washing, eluting, and reverse crosslinking steps. The DNA was then purified and quantified by qRT-PCR. Nuclear and Cytoplasmic Protein Extraction Nuclear and cytoplasmic extracts were prepared following manufacturer’s instructions (Beyotime P0027). Cells were harvested by adding 0.125% trypsin-EDTA, lysed in buffer A on ice for 15 min and then were centrifuged at 16,000 3 g for 5 min. The supernatants containing the cytoplasmic proteins were removed and stored for later use and the pellets were resuspended in buffer B. After incubation on ice for 30 min, lysates were centrifuged at 16,000 3 g for 15 min, and the supernatants containing the nuclear proteins were stored for later use. Isolation of Colonic Lamina Propria and Epithelial Cells Colonic lamina propria cells were isolated using a previously described protocol(Harusato et al., 2016). Briefly, dissected large intestines (colon) were cut into 2-mm pieces. After washing with iced Hank’s balanced salt solution (HBSS), the intestinal tissue samples were subjected to epithelial segregation by incubation in HBSS containing 30 mM EDTA at 37 C for 10 min. Next, the samples were digested in HBSS with 1.5 mg/ml collagenase VIII (Sigma) and 150 mg/ml DNase I (Sigma) for 50 min. The extracted cells were isolated with Percoll (40%–80%) and analyzed by flow cytometry or q-PCR. RNA-Seq Analysis Briefly, RNA preparation, library construction and sequencing on a BGISEQ-500 instrument were performed at Beijing Genomics Institute (BGI), and gene expression levels were quantified using RSEM. The NOISeq method was used to screen for differentially expressed genes between the samples, and hierarchical clustering was performed using Cluster. Heatmaps were generated using Java TreeView. GO and KEGG database were used to extrapolate differentially expressed pathways in a knowledge base-driven pathway analysis approach. DNA Extraction Using a QIAamp Fast DNA Stool MiniKit DNA extraction was performed according to the instructions of the manufacturer (QIAGEN 51604). In brief, 200 mg of fecal sample was mixed with 1 mL of InhibitEX buffer in a 2 mL tube and vortexed. After adding proteinase K, Buffer AL was mixed with the supernatants to form a homogeneous solution. Next, 600 ml of ethanol was added to the lysates, and the mixtures were subsequently loaded onto the spin columns provided in the kit. Finally, DNA was washed and diluted in 200 mL of water, and the samples were diluted to 1.0-1.5 ng/ml for q-PCR assays. 16S rDNA Sequencing The fecal sample DNA was sequencing by Shanghai Majorbio Bio-pharm Technology Co.,Ltd. The data were analyzed on the online platform of Majorbio I-Sanger Cloud Platform (https://www.i-sanger.com). RNA Interference and Real-Time PCR The synthesized siRNA was transfected into murine macrophages using Lipofectamine RNAiMAX (Invitrogen). Cellular RNA was extracted with RNAiso Plus (Takara), and cDNA was generated with M-MLV transcriptase (Promega) and Random Primer 50 -d(NNN NNN)-30 primers (Sangon). Quantitative RT-PCR was performed on a CFX-96 real-time PCR detection system (Bio-rad) with SYBR Green Master Mix (DBI Bioscience). Primer sequences were in Table S1.

Cell Reports 27, 1176–1189.e1–e5, April 23, 2019 e4

QUANTIFICATION AND STATISTICAL ANALYSIS Comparisons between two groups were performed by paired t test if the comparison was within animal. If not, comparison were performed by unpaired t test if both groups were normally distributed or by rank-sum test if at least one group was not normally distributed. Comparisons among three or more groups were performed with one-way ANOVA, followed by multiple comparison. Statistical analyses were performed with Graphpad Prism 6.0, and data are reported as the means ± SD *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. DATA AND SOFTWARE AVAILABILITY To obtain the 16S rDNA sequencing or RNA-seq data, check the website with the accession number PRJNA516598 or PRJNA523492 on https://www.ncbi.nlm.nih.gov/sra. The software used for data analysis are available upon request to Lead Contact, Hongyan Wang ([email protected]).

e5 Cell Reports 27, 1176–1189.e1–e5, April 23, 2019