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. 2017 Jun;76(6):1123-1132.
doi: 10.1136/annrheumdis-2016-210000. Epub 2017 Jan 9.

"V体育平台登录" Dysbiosis and zonulin upregulation alter gut epithelial and vascular barriers in patients with ankylosing spondylitis

Francesco Ciccia  1 , Giuliana Guggino  1 , Aroldo Rizzo  2 , Riccardo Alessandro  3 , Michele Maria Luchetti  4 , Simon Milling  5 , Laura Saieva  3 , Heleen Cypers  6   7 , Tommaso Stampone  2 , Paola Di Benedetto  8 , Armando Gabrielli  3 , Alessio Fasano  9 , Dirk Elewaut  6   7 , Giovanni Triolo  1
Affiliations

Dysbiosis and zonulin upregulation alter gut epithelial and vascular barriers in patients with ankylosing spondylitis

Francesco Ciccia et al. Ann Rheum Dis. 2017 Jun.

Abstract

Background: Dysbiosis has been recently demonstrated in patients with ankylosing spondylitis (AS) but its implications in the modulation of intestinal immune responses have never been studied VSports手机版. The aim of this study was to investigate the role of ileal bacteria in modulating local and systemic immune responses in AS. .

Methods: Ileal biopsies were obtained from 50 HLA-B27+ patients with AS and 20 normal subjects V体育安卓版. Silver stain was used to visualise bacteria. Ileal expression of tight and adherens junction proteins was investigated by TaqMan real-time (RT)-PCR and immunohistochemistry. Serum levels of lipopolysaccharide (LPS), LPS-binding protein (LPS-BP), intestinal fatty acid-BP (iFABP) and zonulin were assayed by ELISA. Monocyte immunological functions were studied in in vitro experiments. In addition the effects of antibiotics on tight junctions in human leukocyte antigen (HLA)-B27 transgenic (TG) rats were assessed. .

Results: Adherent and invasive bacteria were observed in the gut of patients with AS with the bacterial scores significantly correlated with gut inflammation. Impairment of the gut vascular barrier (GVB) was also present in AS, accompanied by significant upregulation of zonulin, and associated with high serum levels of LPS, LPS-BP, iFABP and zonulin V体育ios版. In in vitro studies zonulin altered endothelial tight junctions while its epithelial release was modulated by isolated AS ileal bacteria. AS circulating monocytes displayed an anergic phenotype partially restored by ex vivo stimulation with LPS+sCD14 and their stimulation with recombinant zonulin induced a clear M2 phenotype. Antibiotics restored tight junction function in HLA-B27 TG rats. .

Conclusions: Bacterial ileitis, increased zonulin expression and damaged intestinal mucosal barrier and GVB, characterises the gut of patients with AS and are associated with increased blood levels of zonulin, and bacterial products VSports最新版本. Bacterial products and zonulin influence monocyte behaviour. .

Keywords: Ankylosing Spondylitis; Infections; Inflammation. V体育平台登录.

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Conflict of interest statement

Competing interests: None declared.

Figures

Figure 1
Figure 1
Invasive and adherent bacteria are present in the ileum of patients with ankylosing spondylitis (AS) and are associated with alterations of tight junction proteins. (A-D) Representative microphotographs showing adherent (A) and invasive (B and C) bacteria in AS but not in controls (D). (E) Bacterial scores are directly correlated with the number of infiltrating mononuclear cells. (F-G) Representative images showing Gram staining in patients with AS demonstrating the presence of invading Gram-positive bacteria. (H-J) Representative images showing immunohistochemistry for lipopolysaccharide (LPS) in patients with AS (H and I) and controls (J). (K-M) Histological alterations are associated with the presence of bacteria such as haemorrhages (K and L) and detachment of epithelium from basal membrane (M). (N) Cultures of isolated bacteria displayed mainly Escherichia coli and Prevotella spp. (O–R) relative m-RNA levels of claudin1 (O), claudin 4 (P), occludin (Q) and zonula occludens 1 (R) were assessed by quantitative real-time (RT)-PCR in ileal samples obtained from all the patients and all the controls. Data are expressed as mean (SEM). (A-D, F-J): original magnification×250. Insert in (A-C) and (F-G) original magnification×630.
Figure 2
Figure 2
Occludin, claudin 4 and zonulin 1 tissue expression is altered on patients with ankylosing spondylitis (AS) and modulated by intestinal bacteria. (A and B) representative imaging showing occludin expression in the gut of patients with AS (A) and controls (B). (C) Higher numbers of occludin positive cells were observed in healthy controls compared with AS. (D and E) Representative imaging showing claudin 4 expression in the gut of patients with AS (D) and controls (E). (F) Higher numbers of claudin 4 positive cells were observed in healthy controls compared with AS. (G) relative m-RNA levels of zonulin 1 were assessed by real-time (RT)-PCR in the ileal samples obtained from all the patients with AS and HCs. (H and I) Representative imaging showing zonulin 1 expression in the gut of patients with AS (H) and controls (I). (J) Quantification of zonulin 1 positive cells was performed in the ileal biopsies of all the patients and the controls showing higher numbers of zonulin 1 positive cells in patients with AS. (K) The number of zonulin positive cells was significantly and directly correlated with the number of IL-8 positive cells. (L) Caco-2 cells were incubated with bacteria isolated from ileal biopsies obtained from five patients with AS and the modulation of zonulin expression assessed by RT-PCR. Data are expressed as mean (SEM) of five independent experiments. (A and B) Original magnification×630. (D and E) Original magnification×250. (H and I) Original magnification×400. Data are expressed as mean (SEM).
Figure 3
Figure 3
Gut vascular barrier (GVB) in patients with ankylosing spondylitis (AS). (A-C) relative m-RNA levels of VE-cadherin (A), junctional adhesion molecule (JAM)-1 (B) and PV1 (C) were assessed by RT-PCR in AS and HC ileal samples. (D-F) and (G-I) Representative confocal microscopy images showing CD31 and occludin co-localisation in HCs (D-F) and AS (G-I). (J-M and N-Q): Representative confocal microscopy images showing PV1, CD31 and GFAP co-localisation in HCs (J-M) and AS (N-Q). (D-Q): Original magnification×400. Data are expressed as mean (SEM).
Figure 4
Figure 4
Serum levels of zonulin in patients with ankylosing spondylitis (AS) and in vitro effects of zonulin on human umbilical vein endothelial cells (HUVECs) and peripheral monocytes. (A and B) MRNA expression of occludin (A) and VE-cadherin (B) was assessed in HUVEC cells treated or not with recombinant human zonulin by RT-PCR. Significant downregulation of Occludin and VE-cadherin was observed in HUVEC after incubation with zonulin. (C and D) Serum levels of zonulin were evaluated in 20 patients with AS and 20 controls (C) and correlated with LA/MA ratio (D). (E-H) Peripheral blood mononuclear cells (PBMCs) obtained from five patients with AS (E) and five controls (G) were incubated with recombinant zonulin and the percentage of CD163+c-MAF+ cells evaluated by flow cytometry; percentages of AS (F) and controls (H) CD163+c-MAF+ cells before and after zonulin stimulation. (A-B) Data are expressed as mean (SEM). (C, D, F and H): Data are expressed as individual data points.
Figure 5
Figure 5
Intestinal bacterial products translocate into ankylosing spondylitis (AS) bloodstream and modulate monocyte behaviour. (A-C) Serum levels of lipopolysaccharide (LPS) (A), LPS-binding protein (BP) (B) and intestinal fatty acid-BP (iFABP) (C) are increased in the sera obtained from patients with AS compared with controls. (D-F) Percentages of CD14+ cells is reduced in peripheral blood mononuclear cells (PBMCs) from patients with AS. (D) Representative dot plot showing the percentage of CD14+ cells gated on CD45 region among PBMCs in patients with AS and controls, (E) representative histogram showing CD14 MFI in patients with AS and HCs. (F) percentages of CD14+ cells in patients with AS and controls. (G and H) Percentage of HLA-antigen D Related (DR)+ cells is reduced in PBMCs from patients with AS. (G) Representative dot plot showing the percentage of HLA-DR+ cells gated on the monocytes region among PBMCs in patients with AS and controls, (H) percentages of CD14+ cells in patients with AS and controls. (I-K) Effects of monocyte stimulation with LPS alone, sCD14 alone or sCD14+LPS on CD14+ (H) and CD14− monocytes. Combination of LPS+sCD14 increased IL-23 production only in CD14− cells (I and J). (K) Representative dot plots showing the gating strategy and the percentage of IL-23 expressing cells. Results are showed as mean (SEM).
Figure 6
Figure 6
Ileal inflammation and dysbiosis in HLAB27 transgenic rats is modified by antibiotic treatment. (A-C) Representative images showing IL-23 staining in ileal samples obtaining from wild type (WT) rats (A), HLA-B27 transgenic (TG) rats (B) and HLA-B27 TG rats after antibiotic treatment (C). (D) Semiquantitative evaluation of IL-23+ cells. (E-F) Representative images showing IL-23 staining in ileal samples obtained from WT rats (E), HLA-B27 TG rats (F) and HLA-B27 TG rats after antibiotic treatment (G). (H) Semiquantitative evaluation of IL-23+ cells. (I-K) representative images showing Warthin starry staining for identifying bacteria in ileal samples obtained from WT rats (I), HLA-B27 TG rats (J) and HLA-B27 TG rats after antibiotic treatment (K). Higher numbers of adhering and sometimes invading bacteria were observed in HLA-B27 rats (J and insert in J). (L) Semiquantitative evaluation of bacteria in rats ileal samples. (A-C, E-G, I-K) original magnification ×250; J insert: original magnification ×630. Data are expressed as individual data points.
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