Intestinal epithelial TLR4 signaling affects epithelial function, colonic ...

2 downloads 0 Views 4MB Size Report
Jan 11, 2016 - ... MTA has served as a consultant to AbbVie Laboratories, Hospira Inc.,. 24 ... Aventis, Janssen, GSK Holding Americas Inc., Eli Lilly, Mucosal ...
IAI Accepted Manuscript Posted Online 11 January 2016 Infect. Immun. doi:10.1128/IAI.01374-15 Copyright © 2016 Dheer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

1

Intestinal epithelial TLR4 signaling affects epithelial function, colonic microbiota and

2

promotes risk for transmissible colitis

3

Rishu Dheera, Rebeca Santaolallaa, Julie M Daviesa *, Jessica K Langa, Matthew C Phillipsb,

4

Cristhine Pastorinia ¶, Maria T Vazquez-Pertejoc, Maria T Abreua,b#

5 6 7 8 9 10

a

Division of Gastroenterology, Department of Medicine, University of Miami, Miller School of

Medicine, Miami, FL, USA b

Department of Microbiology and Immunology, University of Miami, Miller School of

Medicine, Miami, FL, USA c

Department of Pathology, Palms West Hospital, Loxahatchee, FL, USA

11 12 13

Running title: Role of epithelial TLR4 in regulating gut microbiota

14 15

# Address correspondence to: Maria T. Abreu, [email protected]

16 17 18 19 20

*

Current address: Mater Research Institute, The University of Queensland, Translational

Research Institute, Woolloongabba, QLD 4102. ¶

Current address: Division of Gastroenterology, Hepatology & Nutrition, University of

Pittsburgh, Pittsburgh, PA 15213

21 22

DISCLOSURE

1

23

We have read the journal's policy and the authors of this manuscript have the following

24

competing interests: MTA has served as a consultant to AbbVie Laboratories, Hospira Inc.,

25

Takeda, Prometheus Labs, Ferring Pharmaceuticals, Shire Pharmaceuticals, Pfizer, Sanofi

26

Aventis, Janssen, GSK Holding Americas Inc., Eli Lilly, Mucosal Health Board, Focus Medical

27

Communications and UCB. MTA serves on the scientific advisory board of Asana Medical Inc.

28

and Celgene Corp and on the board of directors for the GI Health Foundation. MTA serves as

29

trainer or Lecturer for Prova Education Inc., ECCO Congress, Pre-Med institute, LLC and

30

Imedex Inc. This does not alter the authors’ adherence to the journal’s policies on sharing data

31

and materials.

32

All other authors declare no conflict of interest.

33

2

34

ABSTRACT

35

Evidence obtained from gene knock-out studies support the role of TLR4 in intestinal

36

inflammation and microbiota recognition. Increased epithelial TLR4 expression is observed in

37

patients with inflammatory bowel diseases (IBD). However, little is known of the effect of

38

increased TLR4 signaling on intestinal homeostasis. Here, we examined the effect of increased

39

TLR4 signaling on epithelial function and microbiota by using transgenic villin-TLR4 mice that

40

over-express TLR4 in the intestinal epithelium. Our results revealed that villin-TLR4 mice are

41

characterized by increased density of mucosa-associated bacteria and bacterial translocation.

42

Furthermore, increased epithelial TLR4 signaling was associated with impaired epithelial barrier,

43

altered expression of anti-microbial peptide genes, and altered epithelial cell differentiation. The

44

composition of the colonic luminal and mucosa-associated microbiota differed between villin-

45

TLR4 and WT littermates. Interestingly, WT mice co-housed with villin-TLR4 mice displayed

46

increased susceptibility towards acute colitis relative to single-housed WT mice. The results of

47

this study suggest that epithelial TLR4 expression shapes the microbiota and affects the

48

functional properties of the epithelium. The changes in the microbiota induced by increased

49

epithelial TLR4 signaling are transmissible and exacerbate DSS-induced colitis. Together, our

50

findings imply that host innate immune signaling can modulate intestinal bacteria and ultimately

51

the host susceptibility towards colitis.

52

3

53

INTRODUCTION

54

Trillions of microbes co-exist with mammalian cells in the gastrointestinal tract of the host in a

55

relatively mutualistic environment (1, 2). The intestinal epithelium and its overlying mucus layer

56

provide the physical barrier that separates the commensal microbiota from the host (3). Pattern

57

recognition receptors including the toll like receptors (TLRs) expressed by epithelial cells

58

recognize microbe-associated molecular patterns (MAMPs) of the commensal bacteria and

59

regulate the cross talk between intestinal microbes and host (4, 5). Defects in TLR signaling and

60

an aberrant immune response to perturbed endogenous microbiota are a few of the major factors

61

that contribute towards the perpetuation of inflammation and tissue injury in patients with

62

inflammatory bowel diseases (IBD) (6, 7).

63

TLR4 recognizes lipopolysaccharide (LPS) in the cell wall of Gram-negative bacteria.

64

Studies have shown that TLR4 expression varies by region of the intestine and is determined

65

largely by the bacterial composition of that region (8). Conversely, TLR4 may play an important

66

role in maintaining the fine balance between tolerogenic and inflammatory properties of gut

67

microbiota by regulating innate immunity (9, 10). Although several studies have demonstrated

68

that the microbiota composition of the host is influenced by the status of TLRs and their adapter

69

proteins (11-13), others have reported no such effect (14, 15). In the context of TLR4 knock out

70

mice, two separate studies have reported that genetics, maternal transmission and housing

71

conditions rather than the absence of TLR4 have marked effect on the stool microbiota of these

72

mice (14, 15). However, these studies have focused on the microbiota composition of the stool

73

rather than mucosa-associated microbiota, which is in close proximity to the host and differs in

74

its composition from that of the luminal microbiota (16). More importantly, the relationship

75

between epithelial innate immune signaling and the microbiota, especially in the mucosa4

76

associated population, is not known. Thus the role of TLRs including TLR4 in determining

77

microbiota composition and associated baseline phenotypes remains ambiguous.

78

TLR4 is normally expressed at very low levels by various cell types of the intestine

79

including epithelial and lamina propria mononuclear (LPMN) cells (9, 17, 18). Increased

80

expression of TLR4 is observed in epithelial and lamina propria cells of IBD patients suggesting

81

an important role of TLR4 signaling in inflammation (19-21). In the context of infectious

82

enteritis, TLR4 signaling facilitates pathogen colonization and dissemination; and promotes

83

infection-induced colitis (22). Using TLR4 knock-out mice, we and others have demonstrated

84

that TLR4 signaling is required for protection against epithelial injury, inflammation and

85

bacterial invasion (23, 24). On the other hand, we have previously published that the mice

86

expressing a constitutively active form of TLR4 in the intestinal epithelium (villin-TLR4 mice)

87

exhibit increased susceptibility to chemically-induced colitis (25). We have also shown

88

activation of the NF-κB signaling pathway and increased expression of chemokines and

89

proinflammatory genes at baseline in the intestinal epithelial cells of villin-TLR4 mice (26).

90

These findings established the double-edged sword of TLR4 function in the intestine, wherein

91

both low and excessive TLR4 signaling can promote intestinal inflammation. We have also

92

previously shown that the mice expressing TLR4 in the colonic epithelial cells rather than

93

myeloid cells are more susceptible to inflammation associated colonic neoplasia (27).

94

Due to the close proximity of colonic microbiota to intestinal epithelium and the critical

95

role played by intestinal epithelial TLR4 in microbiota recognition and inflammation, the aim of

96

our study was to determine the impact of intestinal epithelial-specific TLR4 signaling on

97

microbial composition and the related host responses. Published studies by other research groups

98

have shown that mice lacking specific components of the innate immune system such as Nod2, 5

99

inflammasome genes, or Myd88 are more susceptible to chemically-induced colitis and possess

100

altered microbiota when compared to their WT littermates (28-30). Furthermore, using co-

101

housing strategies, it has been demonstrated that the microbiota of Nod2 knock-out mice is

102

capable of transmitting colitogenic properties to WT mice (28). However, studies detailing the

103

effect of increased innate immune signaling on microbiota and inflammation are not published.

104

Thus we hypothesized that intestinal epithelium-specific constitutive TLR4 signaling would

105

affect microbial composition, epithelial function and colitis susceptibility. By using villin-TLR4

106

mice as a model for excessive TLR4 signaling, we show that epithelial TLR4 mediated

107

interaction between the gut microbiota and the host is essential for protection against bacterial

108

dissemination, epithelial injury and maintenance of epithelial barrier function. We also show that

109

TLR4 regulates the expression of antimicrobial genes by distinct mechanisms in different parts

110

of the intestine. To our knowledge this is the first study to describe that enhanced TLR4

111

signaling promotes colonic inflammation through dysbiotic microbiota.

112

METHODS

113

Mice

114

Mice hemizygyous for the villin-TLR4 transgene that constitutively expresses TLR4 in the

115

intestinal epithelium are termed villin-TLR4 mice and were generated as described previously

116

(26). All experiments were conducted with 7-9 week old littermates generated by crossing villin-

117

TLR4 transgenic mice with C57BL/6 WT mice and obtained from consecutive litters of the same

118

parent pair. For cohousing and DSS exposure experiment, age and gender matched non-

119

littermate C57BL/6 and villin-TLR4 mice were housed in the same cages for the duration of the

120

experiment. Separately housed C57BL/6 mice were kept under similar conditions but without

6

121

any villin-TLR4 mice housed in their cages. All mice experiments were performed in accordance

122

to the institutional animal care and use committee at the University of Miami.

123

Bacterial culture

124

Spleen and MLNs were removed aseptically, weighed and homogenized using a handheld tissue

125

homogenizer in phosphate buffer saline. Homogenized samples were cultured on tryptic soy

126

sheep blood agar plates and incubated under aerobic and anaerobic conditions for 24-48 hours.

127

Colonization was expressed as the average number of colony-forming units on aerobic and

128

anaerobic plates per mg of tissue.

129

FITC-dextran assay

130

Intestinal epithelial integrity was determined using FITC-dextran assay. Mice were orally

131

gavaged with 4 kDa FITC-dextran (Sigma-aldrich, St Louis, MO) at a concentration of

132

60mg/100g body weight. The concentration of FITC-dextran in the serum was determined after 4

133

hours by Gemini EM fluorescence microplate reader (Molecular Devices, Sunnyvale, CA) at

134

490/525 nm.

135

Intestinal epithelial isolation and mRNA expression

136

Sections of small intestine and colon were excised following sacrifice. Small intestine was rinsed

137

with cold HBSS, cut longitudinally and transferred to 10mL of cold HBSS and gentle agitated to

138

removal additional debris. The tissue was then cut into 1cm pieces and transferred to cold HBSS

139

containing 3mM EDTA for 30 minutes on ice with 200rpm agitation on an orbital shaker. Pieces

140

were transferred to cold HBSS and shaken vigorously for 3-5 minutes. Released epithelial cells

141

in the supernatant were filtered through 70μm filter. Cells were washed twice with 1%FBS in

142

HBSS to remove any residual EDTA. Colons were processed similarly to small intestines, but

143

were incubated in 60mM EDTA in HBSS for one hour. Total RNA was extracted from IECs or 7

144

colon tissues using RNA bee according to manufacturer’s instructions (Tel-test, Friendswood,

145

TX). A total of 1 ug of RNA was reverse transcribed using transcriptor reverse transcriptase

146

enzyme and random hexamers (Roche life science, Indianapolis, IN). RT-qPCR was performed

147

using SYBR Premix Ex Taq (Clontech Laboratories, Mountain View, CA) and Roche

148

LightCycler 480 (Roche, Indianapolis, IN). Information for primer pairs used is provided in

149

supplementary methods. The relative expression levels were calculated by ΔΔCt method after

150

normalizing to the average of endogenous β-actin and Gapdh control genes or with Gapdh only

151

for DSS exposure experiments.

152

Western blot analysis

153

Total cell lysate from colon and small intestine epithelial cells was obtained by using M-PER

154

protein extraction reagent (Thermo Scientific, Waltham, MA) supplemented with protease

155

inhibitor cocktail set III (EMD Millipore Corp., Billerica, MA). Protein lysates were loaded into

156

precast, NuPAGE 10% Bis-Tris Gels (Novex by Life Technologies, NY), electrophoresed

157

(200V, 1 hr) and then transferred (30V, 1.25 hr) onto polyvinylidene fluoride microporous

158

membranes. Membranes were blocked for 1 hour in 5% bovine serum albumin (Sigma-Aldrich,

159

St Louis, MO) and incubated with their respective antibody (1:500 dilution) overnight at 4°C.

160

Rabbit anti-occludin and Rabbit anti-claudin-3 antibodies were obtained from Life Technologies

161

(Grand Island, NY). Membranes were washed and incubated with goat anti-rabbit antibody

162

conjugated to horseradish peroxidase (1:10,000 dilution; Invitrogen, MA) for 30 minutes. Anti-

163

β-Actin conjugated to horseradish peroxidase (1:10,000 dilution; Sigma, St Louis, MO) was used

164

as the loading control. Membranes were developed using SuperSignal West Dura Extended

165

Duration Substrate (Thermo Scientific, Waltham, MA) as per manufacturer instructions and

166

imaged on a myECL Imager (Thermo Scientific, Waltham, MA). 8

167

Tissue histology and immunofluorescence microscopy

168

Samples from ileum and colon were fixed in 10% neutral formalin. Paraffin sections were

169

stained with hematoxylin and eosin, periodic acid Schiff or immunofluorescence using standard

170

protocols. Antibodies used were: rabbit anti-muc2 and goat anti-lysozyme C (Santa cruz

171

Biotechnology, Dallas, TX). After antigen retrieval with citrate buffer, immunostaining for

172

MUC2 and lysozyme was performed by overnight staining with 1:100 dilution of primary

173

antibody at 4oC followed by staining with 1:200 dilution of secondary antibody conjugated with

174

Alexa fluor 488 (Life technologies, Grand Island, NY). The slides were mounted with slowfade

175

gold antifade reagent (Life technologies, Grand Island, NY) after counterstaining with DAPI.

176

Induction of colitis and colitis assessment

177

Acute colitis was induced in age matched mice with 3% DSS (molecular weight = 36-50 kDa;

178

MP biomedicals, Solon, OH) in drinking water for a period of 6 days. Body weight, stool

179

consistency and stool blood were recorded daily by a research technician (J.L.) blinded for the

180

study groups. Disease activity indices were calculated as described previously by averaging the

181

scores for weight loss, stool consistency and bleeding that were recorded on a scale of 0-4 (31).

182

The histological assessment for colitis was performed by a trained pathologist (M.V.) blinded for

183

the treatment groups and scored using the following criteria: architectural changes (0 = normal, 1

184

= mild focal abnormality, 2 = diffuse or multifocal mild to moderate abnormality, 3 = severe

185

diffuse or multifocal abnormality); basal plasmocytosis and neutrophil/eosinophil infiltration (0

186

= none, 1 = mild, 2 = moderate and 3 marked; Crypt abscesses and crypt destruction (0 =

187

absent,1 = < than 5% of crypts involved, 2 = 5 - 50% of crypts involved, 3 = > 50% of crypts

188

involved); and epithelial erosion or ulceration (0 = no erosion nor ulceration, 1 = superficial

9

189

erosion with epithelial repair, 2 = moderate ulceration with epithelial repair and 3 = ulcer with

190

granulation tissue and no epithelium).

191

Explant culture and IL-6 ELISA

192

A small segment of distal colon was washed in PBS supplemented with 50 mg/ml gentamicin.

193

The explant was cultured in 24 well plates containing RPMI 1640 medium supplemented with

194

penicillin and streptomycin. After 24 hours supernatant was collected, stored at -20oC and later

195

analyzed for IL-6 production by using Duoset mouse IL-6 ELISA kit (R&D systems,

196

Minneapolis, MN). All other cytokines were measured by customized luminex mouse magnetic

197

bead assay according to the manufacturer’s recommendations (R&D systems, Minneapolis,

198

MN).

199

Luminal and mucosal microbial analysis

200

Luminal samples comprised of fecal pellets that were collected from the distal portion of the

201

excised colon. Mucosal samples comprised of the tissues from the same distal region and were

202

collected after flushing the colon twice with PBS. Luminal fecal material and mucosal tissues

203

from distal portion of the ileum were collected in the same manner. Genomic DNA was extracted

204

from the lumen and mucosa of distal colon and ileum using QIAamp stool and genomic DNA

205

isolation kit (Qiagen, Valencia, CA). 16S copy number as an estimate for total bacterial number

206

and individual bacterial groups in the lumen and mucosal samples was determined using

207

universal and group-specific primers and qPCR as described above and previously (32). Analysis

208

for microbial richness, composition and diversity were conducted by Second genome Inc. (San

209

Francisco, CA) as described in supplementary methods. The sequences obtained in this paper

10

210

have been submitted to the European Bioinformatics Institute (EBI) database under accession

211

number PRJEB8110.

212

Terminal restriction fragment length polymorphism (T-RFLP)

213

Genomic DNA from luminal or stool samples was amplified using primers broad range forward

214

primer 8F (labelled with FAM dye at 5’ end) and reverse primer 1492R using GoTaq DNA

215

polymerase (Promega, Madison, WI). The amplified PCR product was purified and digested by

216

MspI restriction enzyme (Promega, Madison, WI). The restriction digest product was mixed with

217

GenScan 1200 LIZ size standard (Life technologies, Grand Island, NY) and deionized

218

formamide. The length and intensity of terminal restriction fragments (T-RF) were determined

219

on an ABI 3730 capillary sequencer (Life technologies, Grand Island, NY) and analyzed on peak

220

scanner software (Life technologies, Grand Island, NY). The T-RF profiles were subjected to

221

principal component analysis using Excel add-in Multibase package (Numerical Dynamics,

222

Japan).

223

Statistical analyses

224

Data were expressed as mean ± standard error of mean and analyzed in Graphpad prism 6.

225

Statistical significance was assessed using student's t-test, unless otherwise stated. For more than

226

two groups, analysis of variance with Tukey’s post-hoc test was used. Effect of time and

227

genotype in DSS exposure experiment was determined using 2-way ANOVA and Tukey’s post-

228

hoc test. Statistical significance for microbiota sequences comparison at various taxonomic

229

levels was determined by student’s t-test followed by Benjamini-Hochberg adjustment for false

230

discovery rate of 10%. Pearson correlation coefficient and significance was determined using

231

IBM SPSS statistics 22 software. Heat maps were constructed using CIMminer (Genomics and

11

232

Bioinformatics Group, Laboratory of Molecular Pharmacology (LMP), Center for Cancer

233

Research (CCR) National Cancer Institute (NCI)). Differences were considered significant when

234