Re: 조눌린(글루텐, 스트레스, 장내유해균) 장내 투과성 증가 -- ＞ 자가면역염증 체크포인트 2020 네이처

[문형철]

1. nature  
2. nature communications  
3. articles  
4. article

Targeting zonulin and intestinal epithelial barrier function to prevent onset of arthritis

Download PDF

• Article
• Open access
• Published: 24 April 2020

Targeting zonulin and intestinal epithelial barrier function to prevent onset of arthritis

• Narges Tajik, 
• Michael Frech, 
• Oscar Schulz, 
• Fabian Schälter, 
• Sébastien Lucas, 
• Vugar Azizov, 
• Kerstin Dürholz, 
• Franziska Steffen, 
• Yasunori Omata, 
• Andreas Rings, 
• Marko Bertog, 
• Aroldo Rizzo, 
• Aida Iljazovic, 
• Marijana Basic, 
• Arnd Kleyer, 
• Stephan Culemann, 
• Gerhard Krönke, 
• Yubin Luo, 
• Klaus Überla, 
• Udo S. Gaipl, 
• Benjamin Frey, 
• Till Strowig, 
• Kerstin Sarter, 
• Stephan C. Bischoff, 
• …
• Mario M. Zaiss 

Show authors

Nature Communications volume 11, Article number: 1995 (2020) Cite this article

•  
•  
•  

Abstract

Gut microbial dysbiosis is associated with the development of autoimmune disease, but the mechanisms by which microbial dysbiosis affects the transition from asymptomatic autoimmunity to inflammatory disease are incompletely characterized. Here, we identify intestinal barrier integrity as an important checkpoint in translating autoimmunity to inflammation. Zonulin family peptide (zonulin), a potent regulator for intestinal tight junctions, is highly expressed in autoimmune mice and humans and can be used to predict transition from autoimmunity to inflammatory arthritis. Increased serum zonulin levels are accompanied by a leaky intestinal barrier, dysbiosis and inflammation. Restoration of the intestinal barrier in the pre-phase of arthritis using butyrate or a cannabinoid type 1 receptor agonist inhibits the development of arthritis. Moreover, treatment with the zonulin antagonist larazotide acetate, which specifically increases intestinal barrier integrity, effectively reduces arthritis onset. These data identify a preventive approach for the onset of autoimmune disease by specifically targeting impaired intestinal barrier function.

장 미생물 불균형은 

자가면역 질환의 발병과 연관되어 있지만, 

미생물 불균형이 무증상 자가면역 상태에서 

염증성 질환으로의 전환에 미치는 메커니즘은 완전히 규명되지 않았습니다. 

본 연구에서는 

장 장벽의 무결성이 

자가면역에서 염증으로의 전환을 조절하는

중요한 체크포인트임을 확인했습니다. 

intestinal barrier integrity

Zonulin family peptide (zonulin),

a potent regulator for intestinal tight junctions

--> 조눌린은 장투과성 증가시키는 단백질

     소장세포 특정 유전자에서 만들어지는 단백질. 

     글루텐이 조눌릴 분비를 자극

     장내세균 불균형. 조눌린 분비 자극 --> 장투과성 증가

--> 조눌린 분비를 촉진하는 약물은 NSAIDs

--> 스트레스는 코티솔 분비, 조눌린 분비 촉진

장 밀착 연결의 강력한 조절인자인 조눌린 가족 펩티드(조눌린)는

자가면역 마우스와 인간에서 고도로 발현되며,

자가면역에서 염증성 관절염으로의 전환을 예측하는 지표로 사용될 수 있습니다.

혈청 조눌린 수치 증가와 함께

장 장벽 기능 저하,

미생물 불균형 및 염증이 동반됩니다.

관절염 발병 전 단계에서

부티레이트 또는 cannabinoid type 1 수용체 작용제를 사용하여

장 장벽을 복원하면 관절염 발병이 억제됩니다.

또한,

장 장벽 무결성을 특이적으로 증가시키는

조눌린 억제제 라라조티드 아세테이트로 치료하면

관절염 발병이 효과적으로 감소합니다.

Restoration of the intestinal barrier in the pre-phase of arthritis using butyrate or a cannabinoid type 1 receptor agonist inhibits the development of arthritis. Moreover, treatment with the zonulin antagonist larazotide acetate, which specifically increases intestinal barrier integrity, effectively reduces arthritis onset.

이러한 결과는

장 장벽 기능 장애를 특이적으로 표적화하여

자가면역 질환 발병을 예방하는 접근법을 제시합니다.

Similar content being viewed by others

The double-edged sword of gut bacteria in celiac disease and implications for therapeutic potential

Article 14 January 2022

Early-life fingolimod treatment improves intestinal homeostasis and pancreatic immune tolerance in non-obese diabetic mice

Article 20 January 2021

Mannose ameliorates experimental colitis by protecting intestinal barrier integrity

Article Open access16 August 2022

Introduction

Altered intestinal microbiota composition, termed “dysbiosis”, is associated with autoimmune diseases, particularly type 1 diabetes1, multiple sclerosis2, systemic lupus erythematosus3 and rheumatoid arthritis (RA)4,5. Several findings support the hypothesis that the onset of RA is linked to the intestinal microbiota, for example that cell wall fragments from various intestinal bacteria have been found to be arthritogenic6,7, some drugs used to treat arthritis have antimicrobial effects (chloroquine, sulfasalazine and minocycline)8,9,10 and the restoration of eubiosis in arthritis patients showing clinical improvement7,11. Moreover, diet as the main microbiota influencing factor has been shown to influence arthritis12,13. However, direct mechanistic links between the gut microbiota and onset of autoimmune diseases are unclear.

소개

장내 미생물군집의 구성 변화,

즉 'dysbiosis'는 자가면역 질환과 연관되어 있으며,

특히 제1형 당뇨병1, 다발성 경화증2, 전신성 홍반성 루푸스3 및 류마티스 관절염(RA)4,5와 관련이 있습니다.

여러 연구 결과는

RA의 발병이 장내 미생물과 관련이 있다는 가설을 뒷받침합니다.

예를 들어,

다양한 장내 세균의 세포벽 조각이 관절염 유발 물질로 확인되었으며6,7,

관절염 치료에 사용되는 일부 약물(클로로퀸, 설파살라진, 미노사이클린)은

항균 효과를 나타냅니다8,9,10,

그리고

관절염 환자의 장내 미생물 균형 회복이 임상적 개선을 보였습니다7,11.

또한,

미생물군집에 영향을 미치는 주요 요인인

식단이 관절염에 영향을 미친다는 것이 밝혀졌습니다12,13.

그러나

장 미생물군집과 자가면역 질환 발병 사이의 직접적인 메커니즘적 연관성은

명확하지 않습니다.

The permeability against small substances of the intestinal epithelium depends on the regulation of intercellular tight junctions (TJ). Zonula occludens toxin (Zot, or zonulin), an enterotoxin secreted by intestinal epithelial cells following stimuli from diets or microbiota, was shown to be a potent regulator of TJ competency and intestinal barrier function. Importantly, breakdown of the intestinal barrier, e.g. by apoptosis of intestinal epithelial cells in response to microbial infection, enables a proinflammatory environment including differentiation of autoreactive Th17 cells and other T helper cells14. Our data show that in mice intestinal barrier function is impaired before the clinical onset of arthritis, while in humans serum markers associated with impaired intestinal barrier function are also increased before the onset of RA and associated with a higher risk to develop RA later on. We identify zonulin family peptide (zonulin) as molecular factor that triggers the onset of arthritis by regulating intestinal barrier function. Zonulin reduces the expression‎ of intestinal TJ proteins, induces T-cell-mediated mucosal inflammation and controls the transmigration of immune cells from the gut into the joints. Moreover, therapeutic restoration of intestinal barrier function, e.g. by specific targeting of zonulin, not only prevents the transmigration of immune cells from the gut to the joint but also partially protects from the onset of arthritis. These data unravel a mechanism that explains the transition from asymptomatic autoimmunity to inflammatory disease. Since early treatment of autoimmune diseases such as RA leads to better long-term outcomes and higher chances of drug-free remission15, the direct targeting of intestinal barrier function at onset of arthritis provides an opportunity to modulate the development of autoimmune diseases, i.e. the onset of inflammation in RA.

장 상피의 소분자 투과성은

세포 간 밀접 연결(TJ)의 조절에 의존합니다.

Zonula occludens toxin (Zot, 또는 조눌린)은

식이 또는 미생물총의 자극에 따라 장 상피 세포에서 분비되는 장 독소로,

TJ 기능 및 장 장벽 기능을 강력하게 조절하는 것으로 밝혀졌습니다.

중요한 것은,

미생물 감염에 대한 반응으로

장 상피 세포가 사멸하여 장 장벽이 파괴되면,

자가 반응성 Th17 세포 및 기타 T 헬퍼 세포의 분화를 포함한

전염증성 환경이 조성된다는 점입니다14.

Importantly, breakdown of the intestinal barrier, e.g. by apoptosis of intestinal epithelial cells in response to microbial infection, enables a proinflammatory environment including differentiation of autoreactive Th17 cells and other T helper cells14

미생물 감염은 자가면역 질환의 발병 전에 자주 발생합니다. 감염이 어떻게 자가면역 질환을 유발하는지는 아직 잘 이해되지 않고 있습니다. 우리는 감염이 자가 항원 펩타이드의 자극적 제시를 가능하게 하는 조건을 생성할 수 있으며, 이로 인해 자가반응성 T 세포 반응이 유발되어 자가면역 질환으로 이어질 수 있다는 가능성을 조사했습니다. 자가반응성 CD4+ T 세포는 자가면역 질환의 주요 유발 요인이지만, 일반적으로 자가 항원의 면역 자극적 제시를 제한하는 조절 메커니즘에 의해 활성화가 억제됩니다. 본 연구에서 우리는 감염된 호스트 세포의 아포토시스(세포 사멸)가 염증 환경에서 주요 조직 적합성 복합체(MHC) 클래스 II 분자를 통해 자가 항원의 제시를 가능하게 한다는 사실을 발견했습니다. 이는 자가반응성 TH17 보조 T 세포 하위 집단의 생성에 충분했으며, 이는 자가면역 질환과 밀접하게 연관되어 있습니다. 한 번 유도되면 자가반응성 TH17 세포는 자가염증과 자가항체 생성을 촉진했습니다. 본 연구 결과는 감염이 자가면역을 촉발하는 메커니즘에 대한 이해에 중요한 시사점을 제공합니다.

우리의 데이터에 따르면,

쥐에서는 관절염이 임상적으로 발병하기 전에 장 장벽 기능이 손상되는 반면,

인간에서는 RA 발병 전에 장 장벽 기능 장애와 관련된 혈청 마커가 증가하며,

이는 이후 RA 발병 위험이 높다는 것과 관련이 있습니다.

우리는

장 장벽 기능을 조절하여 관절염의 발병을 유발하는 분자 인자로

조눌린 패밀리 펩티드(조눌린)를 확인했습니다.

장내 미생물 불균형에 의해 촉진되는 장 투과성 증가와 염증은 류마티스 관절염(RA)의 발병에 기여하는 것으로 보입니다. 이 단일 센터 파일럿 연구는 시판 키트를 사용하여 RA 환자의 혈청 및 대변 샘플에서 장 투과성의 지표인 조눌린과 장 염증의 지표인 칼프로텍틴을 측정하는 것을 목표로 했습니다. 또한 장 투과성 및 염증의 지표인 혈장 지질 다당류(LPS) 수치를 분석했습니다. 또한, 조눌린과 칼프로텍틴이 LPS, BMI, 성별, 연령, RA 관련 매개변수, 섬유소 섭취량, 장내 단쇄 지방산과 관련이 있는지 여부를 확인하기 위해 단변량 및 다변량 회귀 분석을 실시했습니다. 혈청 조눌린 수치는 질병의 지속 기간이 길수록 비정상적인 경향이 더 높았고, 대변 조눌린 수치는 연령과 반비례하는 경향을 보였습니다. 남성에서 대변 칼프로텍틴과 혈청 칼프로텍틴 사이, 그리고 대변 칼프로텍틴과 LPS 사이의 강한 연관성이 관찰되었으나, 여성에서는 다른 생물학적 지표와 무관하게 이러한 연관성이 관찰되지 않았습니다. 이는 RA에서 장 염증의 지표로 혈청 칼프로텍틴보다 대변 칼프로텍틴이 더 특이적인 생물학적 지표일 수 있음을 시사합니다. 이 연구는 건강한 대조군이 없는 원리 증명 연구이므로, 다른 유망한 바이오마커와 비교하여 대변 및 혈청 조눌린이 RA의 유효한 바이오마커로 유효함을 검증하기 위해서는 추가 연구가 필요합니다.

조눌린은

장 TJ 단백질의 발현을 감소시키고,

T 세포 매개 점막 염증을 유도하며,

장에서 관절로 면역 세포의 이동을 제어합니다.

또한,

조눌린을 특이적으로 표적으로 하는 등

장 장벽 기능을 치료적으로 복원하면,

장에서 관절로 면역 세포의 이동을 방지할 뿐만 아니라

관절염 발병을 부분적으로 예방할 수 있습니다.

이 데이터는

무증상 자가면역에서 염증성 질환으로의 전환을 설명하는 메커니즘을 밝힙니다.

RA와 같은 자가면역 질환을 조기에 치료하면

장기적인 결과가 더 좋고 약물 없이 완치될 가능성이 높아지기 때문에15,

관절염 발병 시 장 장벽 기능을 직접 표적으로 하는 것은

자가면역 질환의 발병,

즉 RA의 염증 발병을 조절할 수 있는 기회를 제공합니다.

Results

Zonulin expression‎ correlates with onset of rheumatoid arthritis

The permeability and barrier function of the intestinal epithelium depends on the regulation of intercellular TJ. Among other factors, intestinal permeability is regulated by zonula occludens toxin (Zot, or zonulin)-mediated disengagement of the protein ZO-1 from the TJ protein complex16. Zonulin is secreted by intestinal epithelial cells following stimuli from diet or microbiota. We first assessed serum zonulin levels in two independent cohorts of established RA and found elevated zonulin levels compared to healthy controls (Fig. 1a, Supplementary Fig. 1a). Patients with chronic infections (hepatitis C infection; HCV) or cancer showed no elevation of zonulin (Fig. 1a). Furthermore, zonulin was already increased in two independent cohorts in a subset of patients with RA-specific autoimmunity that had not yet developed the disease (“pre-RA”) (Fig. 1b, Supplementary Fig. 1a). To add more evidence on intestinal barrier dysfunction, we also analyzed serum sCD14 levels, which has been described as marker of microbial translocation. Soluble CD14 levels were correlated with zonulin levels (Supplementary Fig. 1b), while no such correlation was found between the C-reactive protein (CRP) or the erythrocyte sedimentation rate (ESR) (Supplementary Fig. 1c). Longitudinal analysis revealed that pre-RA individuals with elevated zonulin levels (>10 ng/ml) had a high risk to develop RA within 1 year (Fig. 1c).

결과

조눌린 발현은

류마티스 관절염의 발병과 관련이 있습니다.

장 상피의 투과성 및 장벽 기능은

세포 간 TJ의 조절에 달려 있습니다.

다른 요인들 중에서도,

장 투과성은 조눌린 독소(Zot 또는 조눌린)에 의해 매개되는

단백질 ZO-1이 TJ 단백질 복합체에서 분리됨으로써 조절됩니다16.

조눌린은

식이 또는 미생물총의 자극에 따라

장 상피 세포에 의해 분비됩니다.

우리는

먼저 확립된 RA의 두 독립적인 코호트에서

혈청 조눌린 수치를 평가한 결과,

건강한 대조군에 비해 조눌린 수치가 높은 것을 발견했습니다 (그림 1a, 보충 그림 1a).

만성 감염 (C형 간염 감염, HCV)

또는 암이 있는 환자는

조눌린 수치의 상승이 나타나지 않았습니다 (그림 1a).

또한,

조눌린은 아직 질병이 발병하지 않은 RA 특이적 자가면역이 있는 환자들(“RA 전 단계”)의

두 독립적인 코호트에서 이미 증가되어 있었습니다(그림 1b, 보충 그림 1a).

장 장벽 기능 장애에 대한 추가적인 증거를 확보하기 위해,

미생물 이동의 지표로 알려진

혈청 sCD14 수치를 분석했습니다.

용해성 CD14 수치는

조눌린 수치와 상관 관계가 있는 반면(보충 그림 1b),

C-반응성 단백질(CRP)이나 적혈구 침강 속도(ESR)와는 상관 관계가 없었습니다(보충 그림 1c).

종단적 분석 결과,

조눌린 수치가 높은(10ng/ml 이상) RA 전 단계의 사람들은

1년 이내에 RA로 발전할 위험이 높은 것으로 나타났습니다(그림 1c).

Fig. 1: Zonulin family peptide levels and intestinal barrier dysfunction in human rheumatoid arthritis.

a Serum zonulin family peptide (zonulin) levels in healthy controls (controls) (n = 41), cancer (n = 19), hepatitis C virus (HCV) (n = 21), and rheumatoid arthritis (RA) (n = 40) patients.

 b Serum zonulin levels in healthy controls (n = 41) and pre-RA patients (n = 32). 

c Serum zonulin levels in pre-RA patients with (n = 12) or without (n = 53) later development of RA (left panel); Kaplan–Meier graph showing loss of healthy state and progression to RA patients with low (<10) or elevated (≥10) zonulin levels over month relapsed (right panel). 

d Fold reduction of occludin expression‎ determined in histological gut biopsy sections in healthy controls (n = 10), new-onset (n = 9) and established RA patients (n = 5) with representative microphotographs (occludin in brown) from three healthy control and three new-onset RA patients. Size bar: 20 µm. 

e Fold reduction of claudin-1 expression‎ determined in histological gut biopsy sections in healthy controls (n = 10), new-onset (n = 10) and established RA patients (n = 5) with representative microphotographs (claudin-1 in brown) from three healthy control and three new-onset RA patients. Size bar: 20 µm. 

f Immunohistological quantification of gut infiltrating T cells (CD3), B cells (CD19) and macrophages (CD68) in gut biopsies from healthy controls or new-onset RA patients from stained gut biopsies (n = 10). 

g Representative H&E-stained gut biopsy microphotographs from a new-onset RA patient; arrows: signs of inflammation. Size bar: 20 µm. 

h Intestinal permeability assessed by lactulose/mannitol recovery ratio in urine of healthy controls (n = 10), and new-onset (n = 10) and established (n = 5) RA patients after 24 h. Data are expressed as the mean ± s.d. Statistical difference was determined by one-way ANOVA. *p < 0.05; ***p < 0.001. Source data are provided as a Source Data file.

a 건강한 대조군(대조군) (n = 41), 암 (n = 19), C형 간염 바이러스 (HCV) (n = 21) 및 류마티스 관절염 (RA) (n = 40) 환자의 혈청 조눌린 가족 펩티드 (조눌린) 수준.

b 건강한 대조군 (n = 41) 및 RA 전 단계 환자 (n = 32)의 혈청 조눌린 수준.

c RA가 발병한 (n = 12) 또는 발병하지 않은 (n = 53) RA 전 단계 환자에서 혈청 조눌린 수치 (왼쪽 패널); 한 달 동안 조눌린 수치가 낮음 (<10) 또는 높음 (≥10)인 환자의 건강 상태 상실 및 RA로 진행을 나타내는 카플란-마이어 그래프 (오른쪽 패널).

d 건강한 대조군(n = 10), 신규 발병(n = 9) 및 확립된 RA 환자(n = 5)의 조직학적 장 생검 조직 절편에서 측정된 occludin 발현의 감소율. 대표적 미세사진(occludin은 갈색으로 표시)은 건강한 대조군 3명과 신규 발병 RA 환자 3명에서 촬영되었습니다. 크기 바: 20 µm.

e 건강한 대조군(n = 10), 신규 발병(n = 10) 및 확립된 RA 환자(n = 5)의 조직학적 장 생검 조직에서 클라우딘-1 발현의 감소율. 대표 미세사진(클라우딘-1은 갈색으로 표시)은 건강한 대조군 3명과 신규 발병 RA 환자 3명에서 추출되었습니다. 크기 바: 20µm.

f 건강한 대조군 또는 신규 발병 RA 환자의 염색된 장 생검 조직에서 장 침윤 T 세포(CD3), B 세포(CD19) 및 대식세포(CD68)의 면역조직화학적 정량화 (n = 10).

g 신규 발병 RA 환자의 H&E 염색 장 생검 미세사진; 화살표: 염증의 징후. 크기 바: 20µm.

h 건강한 대조군 (n = 10), 신규 발병 (n = 10) 및 확립된 (n = 5) RA 환자의 소변에서 24시간 후 락툴로오스/만니톨 회수 비율을 통해 장 투과성을 평가했습니다. 데이터는 평균 ± 표준편차로 표시되었습니다. 통계적 차이는 일원배치 분산분석 (ANOVA)으로 결정되었습니다. *p < 0.05; ***p < 0.001. 원본 데이터는 원본 데이터 파일로 제공됩니다.

Intestinal barrier changes at the onset of human RA

To further confirm‎ intestinal barrier dysfunction during RA development, ileal mucosal biopsies from ten new-onset treatment-naïve RA and ten established active RA patients were collected (see also Supplementary Table 1). New-onset and established RA patients revealed lower expression‎ of TJ proteins occludin and claudin-1 in intestinal epithelial cells than healthy controls on the RNA levels and in respective immune histology (Fig. 1d, e). No difference was observed between new-onset RA and established RA patients. Furthermore, the number of T cells (CD3), B cells (CD19) and macrophages (CD68) was increased in the lamina propria of new-onset treatment-naïve RA patients (Fig. 1f), which was associated with leukocyte infiltration in H&E-stained ileal biopsies (Fig. 1g). Functional assessment of intestinal permeability in these patients showed increased lactulose/mannitol recovery in the urine characteristic for impaired intestinal permeability (Fig. 1h). Taken together, these data point to a disruption of the intestinal epithelial barrier function early in the course of RA development.

인간 류마티스 관절염 발병 시 장 장벽 변화

RA 발병 과정에서 장 장벽 기능 장애를 추가로 확인하기 위해,

치료 경험이 없는 신규 발병 RA 환자 10명과 활동성 RA 환자 10명의 회장 점막 생검 조직을 수집했습니다(보충 표 1 참조).

신규 발병 및 활동성 RA 환자는

건강한 대조군에 비해 장 상피 세포에서 TJ 단백질인 occludin과 claudin-1의 발현 수준이

RNA 수준 및 면역 조직 화학 검사에서 모두 낮았습니다(그림 1d, e).

신규 발병 RA와 확립된 RA 환자 간 차이는

관찰되지 않았습니다.

또한,

신규 발병 치료 경험이 없는 RA 환자의 점막하층에서

T 세포(CD3), B 세포(CD19) 및 대식세포(CD68)의 수가 증가했으며(그림 1f),

이는 H&E 염색 소장 생검에서 백혈구 침윤과 연관되었습니다(그림 1g).

이 환자들에서 장 투과성 기능 평가 결과, 장 투과성 장애의 특징인 소변

중 락툴로오스/만니톨 회수율이 증가했습니다(그림 1h).

종합적으로,

이 데이터는 RA 발병 초기 단계에서 장 상피 장벽 기능의 손상을 시사합니다.

Intestinal inflammation precedes the onset of arthritis

We next performed time-course analysis of serum zonulin levels in collagen-induced arthritis (CIA) of mice. Zonulin levels increased well before the onset of CIA 25 days post immunization (dpi) (Fig. 2a). In line with these observations, CIA mice showed increased small intestine permeability for FITC-dextran MW 4000 (FD4) at day 15 (Fig. 2b) and for lactulose/mannitol at day 18 post immunization (Fig. 2c), which was later followed by increased colon permeability at day 32, as measured by sucralose recovery in the urine (Fig. 2d). Furthermore, expression‎ of TJ proteins ZO-1 (Fig. 2e–g) and occludin (Fig. 2h–j) was decreased before the onset of arthritis in the small intestine and shortly after onset of arthritis in the colon. Similar results were found at mRNA expression‎ level with downregulation of ZO-1 and occludin before the onset of arthritis (Supplementary Fig. 2a, b), while the expression‎ of ion and water channel-forming TJ proteins claudin-2 and -15 went up (Supplementary Fig. 2c, d). In addition, small intestine and colon length were transiently reduced at the onset of arthritis (Fig. 2k, l) along with crypt elongations in the small intestine (Fig. 2m–p) and reduced goblet cell numbers in the colon (Fig. 2q, r).

장 염증이 관절염 발병보다 먼저 발생

다음으로,

콜라겐으로 유발된 관절염(CIA)을 가진 마우스에서

혈청 조눌린 수치의 시간 경과에 따른 분석을 실시했습니다.

조눌린 수치는

면역 25일 후(dpi) CIA 발병보다 훨씬 전에 증가했습니다(그림 2a).

이 관찰 결과와 일치하게,

CIA 마우스는 면역화 후 15일째에 FITC-dextran MW 4000(FD4)에 대한 소장 투과성이 증가했으며(그림 2b),

면역화 후 18일째에 lactulose/mannitol에 대한 투과성이 증가했습니다(그림 2c).

이는 이후 면역화 후 32일째에

소변 내 sucralose 회복을 통해 측정된 대장 투과성 증가로 이어졌습니다(그림 2d).

또한, TJ 단백질 ZO-1(그림 2e–g)과 occludin(그림 2h–j)의 발현은 관

절염 발병 전 소장에서 감소했으며,

관절염 발병 후 대장에서 발병 직후 감소했습니다.

mRNA 발현 수준에서도 유사한 결과가 관찰되었으며,

관절염 발병 전 ZO-1과 occludin의 발현이 감소했습니다(보조 그림 2a, b),

반면 이온 및 수분 채널 형성 TJ 단백질인 claudin-2와 -15의 발현은 증가했습니다(보조 그림 2c, d).

또한, 관절염 발병 시 소장과 결장의 길이가 일시적으로 감소(그림 2k, l)하고,

소장에서 소장 융모가 길어지며(그림 2m–p),

결장에서 잔 세포 수가 감소했습니다(그림 2q, r).

Fig. 2: Zonulin upregulation, decrease in tight junction proteins and barrier dysfunction before onset of arthritis.

a Time course of serum zonulin levels in mice induced for collagen-induced arthritis (CIA) (n = 5). b–d Time course of intestinal permeability assessed by b the serum FITC-Dextran, c lactulose/mannitol urine recovery ratio and d urine sucralose recovery ratio in mice induced for CIA (n = 5). e, f Quantification of ZO-1 (red) expression‎ determined in histological sections of e the ileum (n = 6) and f the colon (n = 6) presented as floating bars (min to max) with line at median. g Representative microphotographs of ZO-1 (red) expression‎ in small intestine. Size bar: 50 µm. h, i Quantification of occludin expression‎ (red) in histological sections of h the ileum (n = 6) and i the colon (n = 6) presented as floating bars (min to max) with line at median with j representative microphotographs of occludin (red) expression‎ in small intestine. Size bar: 50 µm. k, l Time course of the length measurement of k the small intestine and l the colon in mice induced for CIA. m–o Time course of m duodenal, n jejunal and o ileal crypt elongation with p representative H&E-stained sections from the ileum. Size bar: 200 µm. q Time course of goblet cell numbers in the colon and r representative PAS-stained sections from the colon. Size bar: 200 µm. Data are derived from two (b–j), three (a), or four independent (k–r) experiments and expressed as the mean ± s.d. Statistical difference was determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001. Dashed red line: time point of clinical disease onset. Source data are provided as a Source Data file.

a 콜라겐으로 유발된 관절염 (CIA)을 유발한 마우스에서 혈청 조눌린 수치의 시간 경과 (n = 5). b–d CIA를 유발한 마우스에서 b 혈청 FITC-덱스트란, c 락툴로오스/만니톨 소변 회수율 및 d 소변 수크랄로스 회수율로 평가한 장 투과성의 시간 경과 (n = 5). e, f ZO-1 (적색) 발현량 측정 결과. e 소장 (n = 6) 및 f 결장 (n = 6)의 조직 절편에서 측정된 값을 중간값을 나타내는 선과 함께 부동 막대 그래프로 표시함. g 소장에서 ZO-1 (적색) 발현의 대표적 미세 사진. 크기 바: 50µm. h, i h 소장 (n = 6) 및 i 결장 (n = 6)의 조직 절편에서 오클루딘 발현 (빨간색)의 정량화, 부동 막대 (최소부터 최대)로 표시되며 중간에 선이 표시됨. j 소장에서 오클루딘 (빨간색) 발현의 대표적 미세 사진. 크기 바: 50µm. k, l CIA를 유도한 마우스의 소장(k)과 결장(l) 길이 측정 시간 경과. m–o 소장(m) 십이지장, n 공장, o 회장 크립트 연장 시간 경과와 회장 H&E 염색 단면도(p). 크기 바: 200µm. q 결장 내 고블릿 세포 수 시간 경과와 r 결장 PAS 염색 단면도. 크기 바: 200µm. 데이터는 두 번 (b–j), 세 번 (a), 또는 네 번의 독립적인 실험 (k–r)에서 유래되었으며 평균 ± 표준편차로 표시되었습니다. 통계적 차이는 일원배치 분산분석 (one-way ANOVA)으로 결정되었습니다. *p < 0.05; **p < 0.01; ***p < 0.001. 점선 빨간 선: 임상 질환 발병 시점. 원본 데이터는 원본 데이터 파일로 제공됩니다.

T cells accumulate in the intestine before arthritis onset

To investigate cellular intestinal alterations that precede the onset of arthritis in more detail, we analyzed T-cell subsets in the intestine at different time points during the CIA time course. The pathogenesis of T-cell-mediated inflammation in the intestine and the joints has been described to be Th1 and Th17 cell-dependent17,18. Interestingly, we observed increased frequencies of Th1 (CD4+IFNγ+) (Supplementary Fig. 3a) and Th17 (CD4+IL-17+) cells (Supplementary Fig. 3b) in the small intestine and colon of CIA mice during the initiation phase of arthritis. In contrast, Th2 (CD4+IL-4+) and Treg cells (CD4+Foxp3+), known to play critical roles counteracting inflammation in arthritis19,20, were increased only in the late stages of arthritis (Supplementary Fig. 3c, d)19,21. Detailed gating strategies are shown in Supplementary Fig. 3e, f. Collectively, histological and cellular signs of intestinal inflammation precede the onset of arthritis and may trigger the initiation of the disease. Analysis of the synovial tissue and popliteal lymph nodes (pLN) by flow cytometry analysis at this early time points (25 days after immunization) did not reveal any changes in the total synovial CD4+ T-cell or B220+ B-cell frequencies (Supplementary Fig. 4b), as well as no changes in pLN Th1 (CD4+IFNγ+) and Th2 (CD4+IL-4+) frequencies, while Th17 (CD4+IL-17+) cell frequencies were increased (Supplementary Fig. 4c).

관절염 발병 전 장에 T 세포가 축적됩니다

관절염 발병 전 장의 세포적 변화를 더 자세히 조사하기 위해,

우리는 CIA 진행 과정 중 다양한 시간점에

장 내 T 세포 하위 집단을 분석했습니다.

장과 관절에서의 T 세포 매개 염증의 병리 메커니즘은

Th1 및 Th17 세포에 의존적이라고 보고되었습니다17,18.

흥미롭게도,

관절염 발병 초기 단계에서 CIA 마우스의 소장과 대장에서

Th1 (CD4+IFNγ+) 세포 (보조 그림 3a)와 Th17 (CD4+IL-17+) 세포 (보조 그림 3b)의 빈도가 증가했습니다.

반면,

관절염에서 염증을 억제하는 데 중요한 역할을 하는 것으로 알려진

Th2 (CD4+IL-4+) 및 Treg 세포 (CD4+Foxp3+)는 관절염의 후기 단계에서만 증가했습니다 (보조 그림 3c, d)19,21.

세부적인 게이트 전략은 보충 그림 3e, f에 표시되어 있습니다.

종합적으로,

장 염증의 조직학적 및 세포학적 징후는

관절염 발병 전에 나타나며 질병의 발병을 유발할 수 있습니다.

면역화 후 25일 시점의 관절막 조직과 슬와 림프절(pLN)을 유세포 분석으로 분석한 결과,

총 관절막 CD4+ T 세포 또는 B220+ B 세포의 빈도에는 변화가 없었으며(보조 그림 4b),

또한 pLN의 Th1(CD4+IFNγ+) 및 Th2(CD4+IL-4+) 세포 빈도에도 변화가 없었으며,

Th17(CD4+IL-17+) 세포 빈도는 증가했습니다(보조 그림 4c).

Intestinal microbial changes in the pre-phase of arthritis

Among several potential stimuli that can trigger zonulin release, intestinal microbiota composition was identified as being one of the most potent16. Microbial dysbiosis is known to affect intestinal short chain fatty acid (SCFA) levels22,23. The SCFA butyrate has been shown to decrease bacterial translocation and strengthen intestinal barrier function24. CIA mice showed a change in the intestinal microbiota genera and families before the onset of arthritis at day 20 after immunization (Supplementary Fig. 5a–d). Moreover, such microbiota changes occurred already very early in the disease process finding differences in the small intestinal fecal content as early as day 3 after immunization, including an enrichment of Lactobacillaceae_OTU3 and decreased Lactobacillaceae_OTU1 or Bacteroidales S24_7_OTU9 (Supplementary Fig. 5e). PCoA plot analyses showed that these changes are mainly attributed to the day post immunization (Supplementary Fig. 5f). In accordance with these changes in the microbiota composition after CIA induction, butyrate levels changed, dropping just before the onset of arthritis indicative for a changed microbiota composition and decreased function of the intestinal barrier25 (Supplementary Fig. 5g).

관절염 전 단계의 장내 미생물 변화

조눌린 분비를 유발할 수 있는 여러 가지 잠재적 자극 중

장내 미생물 구성이 가장 강력한 자극 중 하나로 확인되었습니다16.

미생물 불균형은

장내 단쇄 지방산(SCFA) 수치에 영향을 미치는 것으로 알려져 있습니다22,23.

SCFA 부티레이트는

박테리아의 이동을 감소시키고

장의 방어 기능을 강화하는 것으로 나타났습니다24.

CIA 마우스는 관절염 발병 전인 면역화 후 20일째에 장내 미생물 군집의 속과 과에 변화가 관찰되었습니다(보조 그림 5a–d). 또한 이러한 미생물군 변화는 질병 과정의 매우 초기 단계에서 이미 발생했으며, 면역화 후 3일째에 소장 분변 내용물에서 Lactobacillaceae_OTU3의 풍부화와 Lactobacillaceae_OTU1 또는 Bacteroidales S24_7_OTU9의 감소가 관찰되었습니다(보조 그림 5e). PCoA 플롯 분석 결과, 이러한 변화는 주로 면역화 후 경과 일수에 기인하는 것으로 나타났습니다(보조 그림 5f). CIA 유도 후 미생물군집 구성 변화와 일치하여 부티레이트 수준이 관절염 발병 직전에 감소했으며, 이는 미생물군집 구성 변화와 장 장벽 기능 저하를 나타냅니다25(보조 그림 5g).

Breach in intestinal permeability is mediated by the microbiota

To further examine the link between the arthritis-related immune response, changed microbiota and intestinal epithelial barrier dysfunction, we applied different immunization protocols including (i) “arthritogenic” collagen type II + complete Freunds adjuvans (CII + CFA), (ii) denaturized CII + CFA (deCII + CFA) and (iii) CII without CFA (CII). We observed that barrier disruption only occurred in mice receiving the “arthritogenic” immunization and started as early as 7 days after immunization (Fig. 3a). Hence, arthritis only developed in mice, which developed increased intestinal permeability (based on lactulose/mannitol ratio measurements) and intestinal inflammation (based on small intestine length measurement) (Fig. 3b, c). Furthermore, only fecal ileal samples from mice immunized with CII + CFA, but not from the other regimen increased gut permeability (based on lactulose/mannitol ratio measurements) (Fig. 3d) when transferred into germ-free (GF) mice.

장 투과성 장애는 미생물군집에 의해 매개됩니다

관절염 관련 면역 반응,

미생물군집 변화 및 장 상피 장벽 기능 장애 간의 연관성을 추가로 조사하기 위해

다음과 같은 다양한 면역화 프로토콜을 적용했습니다:

(i) “관절염 유발성” 콜라겐 유형 II + 완전 Freund의 보조제 (CII + CFA),

(ii) 변성 콜라겐 유형 II + CFA (deCII + CFA), 및

(iii) CFA 없이 콜라겐 유형 II (CII).

장벽 손상은 “관절염 유발성” 면역화 프로토콜을 받은 마우스에서만 발생했으며,

면역화 후 7일 만에 시작되었습니다(그림 3a).

따라서

장 투과성 증가(락툴로오스/만니톨 비율 측정)와

장 염증(소장 길이 측정)이 관찰된 마우스에서만

관절염이 발생했습니다(그림 3b, c).

또한, CII + CFA로 면역화된 쥐의 분변 회장 샘플에서만 장 투과성(락툴로오스/만니톨 비율 측정 기반)이 증가했으며(그림 3d), 이는 무균(GF) 쥐로 이식된 경우에만 관찰되었습니다.

Fig. 3: Dysbiotic microbiota from arthritis mice transfers leaky barrier and mucosal inflammation to GF mice.

a Intestinal permeability assessed by the lactulose/mannitol urine recovery ratio in mice using different collagen-induced arthritis (CIA) immunization protocols namely collagen type II + CFA (CII + CFA), denaturated collagen type II + CFA (deCII + CFA) or collagen type II only (CII) (n = 5). b Arthritis scores in mice induced for CIA using different immunization protocols (n = 5). c Length measurement of the intestine at 20 days post immunization (dpi) in CIA mice (n = 4) presented as floating bars (min to max) with line at median. d Intestinal permeability assessed by the lactulose/mannitol urine recovery ratio in germ-free (GF) mice reconstituted with the donor microbiota from mice immunized with different CIA immunization protocols at 20 dpi (n = 4). e Time course of serum zonulin levels and western blot analysis of ZO-1 expression‎ in Caco-2 cells after 24 h stimulation with intestinal fecal supernatants (FSN) (n = 3). f Transepithelial electrical resistance (TEER) changes in mice induced for CIA treated with the zonulin agonist (AT-1002) (n = 6). g Intestinal organoids from wild-type mice treated with fecal supernatant (FSN) from mice with collagen-induced arthritis at 35 dpi, FSN and zonulin antagonist, or PBS (control). Upon addition of Lucifer yellow (457 Da), confocal fluorescent images were captured every 5 min for 120 min. At 105 min, EGTA was added as a positive control for the ability of the organoids to take up Lucifer yellow. Fluorescence was determined in the organoid lumen and outside of the organoid. Relative intensity values were calculated (fluorescence inside/outside) and are shown for each time point. Each point represents mean values, measured in ten organoids derived from two independent experiments (n = 10). Representative images at time point 55 min are shown. Upper panel: brightfield; lower panel: Lucifer yellow (green), size bar: 50 μm. Data are derived from two (d–g) or three (a–c) independent experiments and expressed as the mean ± s.d. Statistical difference was determined by one-way (d, e) or two-way ANOVA (a, b, g). *p < 0.05; **p < 0.01; ***p < 0.001. Dashed red line: time point of clinical disease onset. Source data are provided as a Source Data file.

Full size image

Moreover, in vitro stimulation of the intestinal epithelial Caco-2 cell line with fecal supernatants (FSN) of mice induced for CIA, showing elevated serum zonulin levels, effectively suppressed ZO-1 protein expression‎ (Fig. 3e). Overall, these data support the human observations and show increased zonulin levels, reduced TJ protein complex expression‎ and impaired intestinal barrier function preceding the onset of arthritis. As previously shown26, recombinant AT-1002, the active domain of Vibrio cholerae’s zonula occludens toxin (ZOT, zonulin) on average tended to decrease the transepithelial electrical resistance (TEER) in Caco-2 cell monolayers, indicating the permeability of epithelial cell monolayers (Fig. 3f). Hence, we tested whether blockade of zonulin overcomes the induction of epithelial permeability by FSN from mice induced for CIA. Lucifer yellow staining of intestinal organoids showed that FSN from mice induced for CIA enhanced permeability, which was reversed when zonulin was blocked by larazotide acetate (Fig. 3g), suggesting the microbial dysbiosis in mice induced for CIA impairs intestinal barrier via zonulin.

또한, CIA를 유발한 쥐의 대변 상청액(FSN)으로 장 상피 Caco-2 세포주를 체외에서 자극한 결과, 혈청 조눌린 수치가 상승하고 ZO-1 단백질 발현이 효과적으로 억제되었습니다(그림 3e).

전반적으로, 이러한 데이터는

인간에 대한 관찰 결과를 뒷받침하며,

관절염 발병에 앞서 조눌린 수치가 증가하고

TJ 단백질 복합체 발현이 감소하며

장 장벽 기능이 손상된다는 것을 보여줍니다.

이전에26에 제시된 바와 같이, Vibrio cholerae의 조눌린 독소(ZOT, 조눌린)의 활성 도메인인 재조합 AT-1002는 Caco-2 세포 단층에서 상피 세포의 투과성을 나타내는 세포 간 전기 저항(TEER)을 평균적으로 감소시키는 경향을 보였습니다(그림 3f). 따라서, 조눌린의 차단을 통해 CIA를 유발한 마우스에서 FSN에 의한 상피 투과성의 유도가 극복되는지 여부를 테스트했습니다. 장 오가노이드의 루시퍼 옐로우 염색 결과, CIA를 유발한 마우스에서 FSN이 투과성을 강화한 반면, 라라조티드 아세테이트로 조눌린을 차단한 경우 이러한 현상이 역전된 것으로 나타났습니다(그림 3g). 이는 CIA를 유발한 마우스에서 미생물 불균형이 조눌린을 통해 장 장벽을 손상시킨다는 것을 시사합니다.

Reducing intestinal barrier permeability attenuates arthritis

Since our results suggested that increased zonulin levels along with impaired intestinal permeability for lactulose and FITC-Dextran (FD4) are preceding the onset of murine and human arthritis, we sought an intervention that strengthens the intestinal barrier and may disrupt the transition from autoimmunity to inflammation and thereby inhibit the onset of arthritis. Therefore, we first strengthened intestinal barrier function by treatment with butyrate as it was described to have beneficial effects on gut permeability25. Butyrate treatment in the drinking water from day 0 post immunization prevented the early increase in intestinal permeability for FITC-Dextran (FD4) at day 15 after immunization (Fig. 4a). In addition, butyrate treatment attenuated CIA onset (Fig. 4b), decreased serum zonulin concentrations (Fig. 4c), and reduced inflammation-mediated small intestinal shortening (Fig. 4d). Moreover, butyrate treatment improved intestinal barrier function by modulating TJ protein mRNA expression‎ levels (Supplementary Fig. 6a). Given that the intestinal epithelial barrier function was found to be positively regulated by activation of the intestinal cannabinoid type 1 receptor (CB1R)27, we treated mice with ACEA (arachidonyl-2-chloroethylamide), a selective CB1R agonist28 during the autoimmune noninflammatory pre-phase of CIA from day 17 to day 27 after immunization. ACEA treatment led to reduced intestinal barrier permeability for FITC-Dextran (FD4) (Fig. 4e), attenuated clinical signs of arthritis (Fig. 4f) along with a reduction in serum zonulin levels (Fig. 4g) as well as no morphological signs of gut inflammation (Fig. 4h).

장 장벽 투과성을 감소시키면 관절염이 완화됩니다.

우리의 결과는 락툴로오스 및 FITC-덱스트란(FD4)에 대한 장 투과성 장애와 함께 조눌린 수치의 증가가 쥐와 인간의 관절염 발병에 선행한다는 것을 시사했기 때문에, 우리는 장 장벽을 강화하고 자가 면역에서 염증으로의 전환을 방해하여 관절염의 발병을 억제할 수 있는 중재 방법을 찾았습니다.

따라서 우리는 장 투과성에 유익한 영향을 미친다고 보고된 부티레이트로 장벽 기능을 강화하는 치료를 먼저 실시했습니다25. 면역화 후 0일부터 음용수에 부티레이트를 투여한 결과, 면역화 후 15일째에 FITC-데크스트란(FD4)에 대한 장 투과성의 초기 증가가 예방되었습니다(그림 4a). 또한, 부티레이트 치료는 CIA 발병을 약화시켰고 (그림 4b), 혈청 조눌린 농도를 감소시켰으며 (그림 4c), 염증에 의한 소장 단축을 감소시켰습니다 (그림 4d). 또한, 부티레이트 치료는 TJ 단백질 mRNA 발현 수준을 조절하여 장 장벽 기능을 개선했습니다 (보충 그림 6a). 장 상피 장벽 기능이 장 내 칸나비노이드 유형 1 수용체(CB1R) 활성화에 의해 긍정적으로 조절된다는 점27을 고려해, 우리는 CIA의 자가면역 비염증 전 단계인 면역화 후 17일부터 27일까지 마우스에 선택적 CB1R 작용제인 ACEA(arachidonyl-2-chloroethylamide)28를 투여했습니다. ACEA 치료는 FITC-덱스트란(FD4)에 대한 장 장벽 투과성을 감소시켰고(그림 4e), 관절염의 임상 증상을 약화시켰으며(그림 4f), 혈청 조눌린 수치를 감소시켰고(그림 4g), 장 염증의 형태학적 징후도 나타나지 않았습니다(그림 4h).

Fig. 4: Targeting intestinal barrier dysfunction before arthritis onset attenuates development of arthritis.

a, b Time course of a intestinal barrier permeability (n = 5) and b total arthritis scores in collagen-induced arthritis (CIA) or nontreated healthy (NT) mice with or without butyrate treatment (n = 5). c, d Effect of butyrate treatment of mice induced for CIA on their c serum zonulin levels (n = 5) and d intestinal length measurements (n = 5). e, f Time course of e intestinal barrier permeability (n = 5) and f total arthritis scores in CIA or nontreated healthy mice with or without CB1R treatment (n = 5). g, h Effect of CB1R agonist treatment of mice induced for CIA on g serum zonulin levels (n = 5) and h intestine length (n = 5). Data are derived from two (e–h) or four (a–d) independent experiments and expressed as the mean ± s.d. Statistical difference was determined by Students’ t test, two-tailed (c, d, g) or two-way ANOVA (a, b, e, f). *p < 0.05; **p < 0.01; ***p < 0.001. Dashed red line: time point of clinical disease onset. Source data are provided as a Source Data file.

Full size image

Targeting zonulin attenuates arthritis

Next, and most importantly, we directly blocked zonulin by larazotide acetate (AT-1001), a first-in-class TJ regulator, currently used in phase III clinical trials for celiac disease29. This octapeptide is related to the originally identified ZOT produced by V. cholera. It acts locally to decrease tight junction permeability by blocking zonulin receptors and promoting TJ assembly and actin filament rearrangement thus preventing actin rearrangement in response to gluten or microbial stimuli30,31. Ten days oral treatment of CIA mice with the zonulin antagonist larazotide shortly before the onset of arthritis prevented the observed increase in intestinal barrier permeability for FITC-Dextran (FD4) and attenuated arthritis symptoms (Fig. 5a, b). Moreover, zonulin antagonist treatment 17–27 dpi lead to a sustained decrease in serum zonulin levels (Fig. 5c) and positively modulated tight junction mRNA expression‎ in the ileum (Supplementary Fig. 6b). Conversely, further promoting zonulin action by short-term treatment with the agonist peptide AT-1002 led to an exacerbation of arthritis (Fig. 5d). Histologic eval‎uation of the joints revealed less severe synovitis (Fig. 5e), lower numbers of osteoclasts (Fig. 5f) and better preservation of bone (Fig. 5g), including a reduction of osteoclast-covered bone area (Fig. 5h) in mice receiving zonulin blockade by larazotide. These data indicate that specific restoration of intestinal epithelial integrity by zonulin blockade affects the development of arthritis.

Fig. 5: Short-term zonulin antagonist treatment improves bone homeostasis.

a–c Time course of a intestinal barrier permeability (n = 5), b total arthritis scores (n = 5) and c serum zonulin levels in collagen-induced arthritis (CIA) mice treated with Larazotide (zonulin antagonist) between 17 and 27 days post immunization (dpi) (n = 5, box and whiskers showing 1–99 percentile). d Time course of arthritis scores in mice induced for CIA treated with the zonulin agonist (AT-1002) (n = 5). e, f Histological analysis of tarsal joints after zonulin antagonist treatment showing e inflamed area and respective H&E-stained sections (size bar: 500 µm) (n = 5, showing 1–99 percentile) as well as f osteoclast numbers with respective TRAP-stained sections (size bar: 500 µm) (n = 5, showing 1–99 percentile). g Bone densities in mice treated with zonulin antagonist and respective micro-CT images (n = 5). h Osteoclast numbers per bone parameter in the tibia of CIA mice treated with zonulin antagonist between 17 and 27 days post immunization (n = 5, showing 1–99 percentile). i Schematic overview of cell trafficking experiments using photoconvertible Kaede mice. j Quantification of photoconverted cells from Kaede mice in lymphoid organs and the synovial tissue following short-term zonulin antagonist treatment (n = 8). k Cell populations identified in MLN, spleen and synovial tissue from Kaede CIA mice at day 26 post immunization (n = 6). Data are derived from three independent experiments and expressed as the mean ± s.d. Statistical difference was determined by Students’ t test, two-tailed (g) or two-way ANOVA (a–f, h, i). *p < 0.05; **p < 0.01; ***p < 0.001. Dashed red line: time point of clinical disease onset. Source data are provided as a Source Data file.

Full size image

Lamina propria lymphocytes migrate to synovial tissues

Next, we investigated the mechanism by which maintenance of intestinal barrier function inhibits arthritis onset. Indeed, short-term treatment with zonulin antagonist larazotide mitigated intestinal inflammation with abrogation of small intestine and colon shortening (Supplementary Fig. 7a). Also, T-cell homeostasis in the spleen and the intestine were shifted towards an anti-inflammatory proresolving phenotype by zonulin blockade during the initiation phase of CIA (Supplementary Fig. 7b, c). Hence, in the spleens CD4+FoxP3+ Tregs were increased, while proarthritic Th1 (CD4+Tbet+) and Th17 (CD4+RORγt+) were decreased (Supplementary Fig. 7b). In the lamina propria, Th1 cells were also decreased upon zonulin blockade, while CD4+Gata3+ Th2 cells were increased (Supplementary Fig. 7c). Of note, levels of CII-specific IgG, IgG2a and IgG2b were not changed by the zonulin antagonist (Supplementary Fig. 8a–c).

점막 고유 림프구가 활막 조직으로 이동

다음으로, 장 장벽 기능의 유지가 관절염 발병을 억제하는 메커니즘을 조사했습니다. 실제로, 조눌린 길항제 라라조티드를 단기간 투여한 결과, 소장 및 대장의 단축이 소멸되고 장 염증이 완화되었습니다 (보충 그림 7a). 또한, CIA의 초기 단계에서 조눌린을 차단함으로써 비장과 장의 T 세포의 항상성이 항염증성, 해결 성향으로 변화했습니다 (보충 그림 7b, c). 따라서, 비장에서 CD4+FoxP3+ Tregs는 증가한 반면, 관절염을 유발하는 Th1 (CD4+Tbet+) 및 Th17 (CD4+RORγt+)은 감소했습니다 (보충 그림 7b). 점막하층에서도 조눌린 차단에 따라 Th1 세포가 감소한 반면, CD4+Gata3+ Th2 세포는 증가했습니다 (보충 그림 7c). 주목할 점은, CII 특이적 IgG, IgG2a 및 IgG2b의 수준은 조눌린 길항제에 의해 변화되지 않았다는 것입니다 (보충 그림 8a–c).

To investigate the migration of T cells from the small intestine into the joints, we used mice engineered to ubiquitously express Kaede32. Kaede is a photoconvertible protein, which permanently changes its fluorescence emission from green (518 nm) to red (582 nm) upon photoactivation with near-UV light. After selective exposure of the small intestine, Kaede-photo-conversion was found to be specific to cells in the small intestine. As shown in the schematic overview of the experimental set-up (Fig. 5i), small intestinal cells in CIA Kaede mice were photoconverted before the onset of clinical arthritis at day 22 after immunization and tissues were analyzed 4 days later by flow cytometry. At day 26 after immunization, CIA Kaede mice showed migrated photoconverted Kaede red+ cells originating from the small intestine in the lymph nodes, the spleen and most prominent in the synovial tissue of the joints (Fig. 5j). Interestingly, the percentage of Kaede red+ CD4+ T cells was highest in the synovial tissues (Fig. 5k), demonstrating the preferential migration of T cells from the small intestine into the joints. Most importantly, this cellular trafficking of gut-primed immune cells to secondary lymphoid organs and the synovial tissues was prevented by short-term zonulin antagonist treatment (Fig. 5j, k). In summary, these data show the anzonulin-dependent migration of immune cells from the intestine to the joints during the onset of arthritis.

소장으로부터 관절로의 T 세포 이동을 조사하기 위해 Kaede32를 전신적으로 발현하도록 유전적으로 변형된 마우스를 사용했습니다. Kaede는 근자외선 광활성화 시 녹색(518 nm)에서 적색(582 nm)으로 형광 발광을 영구적으로 전환하는 광전환 단백질입니다. 소장을 선택적으로 노출시킨 후, Kaede 광변환은 소장 세포에 특이적으로 발생했습니다. 실험 설정의 개요도(그림 5i)에서 보듯이, CIA Kaede 마우스의 소장 세포는 면역화 후 22일째 임상 관절염 발병 전에 광변환되었으며, 4일 후 유세포 분석을 통해 조직을 분석했습니다. 면역화 후 26일째에 CIA Kaede 마우스는 소장에서 유래한 광변환된 Kaede 빨강+ 세포가 림프절, 비장, 특히 관절의 활막 조직에서 가장 두드러지게 이동한 것을 보여주었습니다(그림 5j). 흥미롭게도 Kaede red+ CD4+ T 세포의 비율은 관절막 조직에서 가장 높았으며(그림 5k), 이는 소장에서 관절로 T 세포의 선택적 이동을 보여줍니다. 가장 중요한 것은, 장에서 활성화된 면역 세포가 2차 림프 기관 및 활액 조직으로 이동하는 것이 단기 조눌린 길항제 치료에 의해 방지되었다는 점입니다 (그림 5j, k). 요약하면, 이러한 데이터는 관절염 발병 시 장에서 관절로 면역 세포가 조눌린에 의존하여 이동한다는 것을 보여줍니다.

Discussion

Here we show that zonula occludens toxin (Zot, or zonulin), a potent regulator for intestinal TJ competency and intestinal barrier function, is controlling the onset of murine and human arthritis. Changes in the intestinal barrier function have been reported in the clinical phase of inflammatory bowel disease but also in spondyloarthritis and type 1 diabetes mellitus33,34,35. Herein, we demonstrate that the disruption of the intestinal barrier function occurs before the onset of the inflammatory phase of autoimmune murine and human arthritis identifying it as checkpoint for the transition from autoimmunity to inflammation. We show that zonulin is a central factor in this process as it disrupts intestinal TJ proteins, enhances intestinal permeability and leads to Th1 and Th17 infiltration in the lamina propria before the onset of murine and human arthritis. While ZO-1, occludin and claudin-1 expression‎ was reduced, expression‎ of paracellular cation- and water- channel-forming claudins 2 and 15 was increased. Increased claudin-2 and claudin-15 mRNA levels (both forming paracellular cation and water channels) may explain the observed changes in in vitro TEER measurements following AT-1002 treatment of Caco-2 cell monolayers. In addition, in vivo, decreased occludin levels in combination with increased claudin-2 and claudin-1536 might in synergy result in the observed increased barrier leakiness in CIA mice. ZO-1 is an intracellular located adaptor protein that binds numerous transmembrane and cytoplasmic proteins and is required for assembly of both adherens and TJ. TJ paracellular barrier function is based on two independently regulated routes37, namely the pore pathway: a highly selective route for the flux of ions and water, that is regulated by claudins. This route could be experimentally measured by the assessment of TEER. While on the other hand there exist the leak pathway that regulates the passage of larger molecules and is measured by the passage of fluorescent tracers (FITC) across epithelial monolayers. These two routes can be regulated independently37. For example, depletion of ZO-1 destabilizes the barrier to large solutes, but does not disrupt the claudin-based pores38. Currently, the exact contribution of occludin in contrast to ZO-1 to the barrier function is debated and requires further research. Here, we could show that a zonulin-dependent transmigration of immune cells from the gut into the joints occurs during the onset of arthritis.

Our data also show that zonulin-dependent increase of intestinal permeability essentially results from microbial dysbiosis initiated by the arthritogenic combination of auto-antigen and adjuvant. This finding is in accordance with a previous study showing that adjuvant alone was not sufficient to initiate mucosal inflammation39. In support of the role of microbial dysbiosis, stool transfer was sufficient to initiate intestinal barrier changes in germ-free mice. Furthermore, functional experiments on intestinal epithelial cells showed that the permeability of intestinal organoids is increased by the feces of CIA mice in a zonulin-dependent manner. Recombinant zonulin was proven to increase the intestinal permeability ex vivo40 and in vitro16. In both settings this increase could be blocked by cleaving zonulin or treatment with a zonulin binding inhibitor. Moreover, as shown by the same group, recombinant zonulin-induced effects were completely reversible following its withdrawal in Rhesus monkey intestinal ileal tissues using Ussing chamber experiments and and the effects could specifically be blocked by preincubation of zonulin with anti-Zot antibodies41. Together, these data highlight the potential of zonulin, more specifically of its active domain which is shared with Zot42, to increase intestinal permeability. Here, we could demonstrate that blocking zonulin with the specific antagonist larazotide acetate was sufficient to restore the impaired barrier function in intestinal organoid cultures exposed to complete fecal supernatants from CIA mice containing high zonulin levels. This finding is of direct physiological relevance, as all other active metabolites in the used fecal supernatants were still present but mere inhibition of zonulin was sufficient to restore the impaired intestinal barrier function.

토론

여기에서는

장의 TJ 기능과 장 장벽 기능을 강력하게 조절하는

조눌린(Zot, 또는 조눌린)이

쥐와 인간의 관절염 발병을 제어하고 있음을 보여줍니다.

장 장벽 기능의 변화는

염증성 장 질환의 임상 단계뿐만 아니라

척추 관절염과 제 1형 당뇨병에서도 보고되었습니다33,34,35.

여기에서는,

자가 면역성 쥐와 인간 관절염의 염증 단계가 시작되기 전에

장 장벽 기능의 파괴가 발생하며,

이것이 자가 면역에서 염증으로의 전환의 체크포인트임을 확인했습니다.

조눌린이

이 과정에서 중심적인 역할을 한다는 것을 보여줍니다.

조눌린은

장 TJ 단백질을 파괴하고,

장 투과성을 강화하며,

쥐와 인간 관절염이 발병하기 전에 점막하층에

Th1 및 Th17이 침투하도록 유도합니다.

ZO-1, 오클루딘 및 클라우딘-1의 발현이 감소한 반면,

세포 간 이온 및 수분 채널을 형성하는 클라우딘 2 및 15의 발현은 증가했습니다.

클라우딘-2 및 클라우딘-15 mRNA 수준(모두 세포 간 이온 및 수분 채널을 형성함)의 증가는

AT-1002 처리 후 Caco-2 세포 단일층에서 관찰된

TEER 측정 변화의 원인을 설명할 수 있습니다.

또한,

체내에서 오클루딘 수준 감소와 클라우딘-2 및 클라우딘-1536 증가가 시너지 효과를 일으켜

CIA 마우스에서 관찰된 장벽 투과성 증가를 유발할 수 있습니다.

ZO-1은 세포 내부에 위치한 적응 단백질로,

수많은 막을 관통하는 단백질과 세포질 단백질에 결합하며,

접착 소체와 TJ의 조립에 필수적입니다.

TJ의 파라셀룰러 장벽 기능은

두 개의 독립적으로 조절되는 경로에 기반합니다37.

첫째, 이온과 물의 이동을 조절하는 고도로 선택적인 경로인 포어 경로로, 클라우딘에 의해 조절됩니다.

이 경로는 TEER 측정을 통해 실험적으로 측정될 수 있습니다.

반면, 더 큰 분자의 통과를 조절하는 누출 경로는 상피 단일층을 통해 형광 추적자(FITC)의 통과를 측정하여 평가됩니다.

이 두 경로는 독립적으로 조절될 수 있습니다37.

예를 들어, ZO-1이 고갈되면 큰 용질에 대한 장벽이 불안정해지지만, 클라우딘 기반의 기공은 파괴되지 않습니다38. 현재, ZO-1과 달리 오클루딘이 장벽 기능에 미치는 정확한 기여도는 논란의 여지가 있으며, 추가 연구가 필요합니다. 여기에서는, 관절염 발병 시 장에서 관절로 조눌린에 의존적인 면역 세포의 이동이 발생함을 보여줄 수 있었습니다.

또한, 우리의 데이터는 조눌린에 의존적인 장 투과성의 증가는 본질적으로 자가 항원과 보조제의 관절염 유발 조합에 의해 시작된 미생물 불균형에 기인한다는 것을 보여줍니다. 이 발견은 보조제만으로는 점막 염증을 유발하기에 충분하지 않다는 것을 보여주는 이전의 연구 결과와 일치합니다39. 미생물 불균형의 역할을 뒷받침하기 위해, 무균 마우스에서 대변을 이식하는 것만으로도 장 장벽의 변화를 유발할 수 있었습니다. 또한, 장 상피 세포에 대한 기능적 실험을 통해 CIA 마우스의 대변이 조눌린에 의존적인 방식으로 장 오가노이드의 투과성을 증가시키는 것으로 나타났습니다. 재조합 조눌린이 생체 외40 및 시험관 내16에서 장 투과성을 증가시키는 것으로 입증되었습니다. 두 환경 모두에서 이러한 증가는 조눌린을 절단하거나 조눌린 결합 억제제로 처리하여 차단할 수 있었습니다. 또한, 동일한 연구 그룹이 Ussing chamber 실험을 통해 Rhesus 원숭이의 장 회장에서 재조합 조눌린이 유발한 효과는 조눌린의 투여를 중단한 후 완전히 역전될 수 있으며, 조눌린을 항-Zot 항체로 사전 배양하면 그 효과를 특이적으로 차단할 수 있음을 입증했습니다41. 이러한 데이터를 종합하면, 조눌린, 보다 구체적으로는 Zot과 공유하는 활성 도메인이 장 투과성을 증가시키는 잠재력이 있음을 알 수 있습니다42. 여기에서, 우리는 특정 길항제인 라라조티드 아세테이트로 조눌린을 차단하는 것이, 조눌린 수치가 높은 CIA 마우스의 완전한 대변 상청액에 노출된 장 오르가노이드 배양에서 손상된 장벽 기능을 회복하기에 충분하다는 것을 입증할 수 있었습니다. 이 발견은 사용된 대변 상청액에 포함된 다른 모든 활성 대사 산물이 여전히 존재했지만, 조눌린을 억제하는 것만으로도 손상된 장 장벽 기능을 회복하기에 충분했기 때문에 생리학적으로 직접적인 관련이 있습니다.

Data from clinical observations and preclinical animal models suggest a step-wise process leading to autoimmune RA17. Genetic factors in conjunction with environmental triggers lead to breach of immune tolerance and a phase of clinically silent autoimmunity that can last for years. Later, in response to yet unidentified triggers, silent autoimmunity eventually turns into a clinically apparent inflammatory disease such as RA. Interfering with such triggers would allow retarding or even inhibiting the onset of the disease. Notably, currently available therapies for RA, such as cytokine (TNF and IL-6) blockers or inhibitors of the JAK-STAT pathway43, exclusively tackle the inflammatory phase of the disease but do not interfere with the underlying processes that initiate the onset of arthritis and therefore often need to be given life-long44.

We provide several lines of evidence that treatments that improve intestinal barrier function attenuate arthritic symptoms. Microbial metabolites such as butyrate, for instance, have shown to be potent regulators of zonulin and the intestinal barrier and appear to be essential mediators between microbial dysbiosis and barrier function. In accordance, treatment with butyrate not only restored epithelial barrier function and gastrointestinal permeability for FITC-Dextran (FD4) but also effectively inhibited arthritis. Even more specifically, specific zonulin inhibition during the preclinical phase of arthritis by larazotide reduced the later development of arthritis by nearly 50%. Larazotide treatment attenuated the enhanced intestinal permeability and blocked the migration of immune cells from the intestine to the joints (graphical summary in Supplementary Fig. 9). The implication of these findings is substantial, as it gives the chance to tackle the transition from autoimmunity to inflammation by restoring intestinal barrier function and thereby preventing the onset of RA. Such an approach also appears within short reach, as the zonulin antagonist larazotide is already used in phase III clinical trial for the treatment of celiac disease and thus represents an accessible target molecule to accomplish this task. Furthermore, elevated zonulin levels appear to be an interesting biomarker to identify those patients that are of highest risk to progress to RA, thus allowing a precision-medicine approach to target such individuals. In summary, these data support a gut-joint axis in RA, which is based on zonulin-mediated impairment of intestinal barrier function and which is drugable by the zonulin antagonist larazotide.

임상 관찰 및 전임상 동물 모델 데이터는 자가면역성 류마티스 관절염(RA)의 단계적 진행 과정을 제시합니다. 유전적 요인과 환경적 유발 요인이 결합되어 면역 관용이 깨지고 수년간 지속될 수 있는 임상적으로 무증상 자가면역 단계가 발생합니다. 이후 아직 밝혀지지 않은 유발 요인에 반응하여 무증상 자가면역이 결국 류마티스 관절염과 같은 임상적으로 명백한 염증성 질환으로 전환됩니다. 이러한 유발 요인을 차단하면 질병의 발병을 지연시키거나 심지어 억제할 수 있습니다. 특히 현재 RA 치료에 사용되는 사이토킨(TNF 및 IL-6) 차단제나 JAK-STAT 경로 억제제43 등은 질병의 염증 단계만을 표적으로 삼아 치료하며, 관절염 발병을 유발하는 근본적인 과정을 방해하지 않기 때문에 종종 평생 투여가 필요합니다44.

우리는 장벽 기능을 개선하는 치료가 관절염 증상을 완화한다는

여러 가지 증거를 제시합니다.

예를 들어,

부티레이트와 같은 미생물 대사 산물은

조눌린과 장벽을 강력하게 조절하는 것으로 밝혀졌으며,

미생물 불균형과 장벽 기능 사이의 필수적인 매개체 역할을 하는 것으로 보입니다.

이에 따라, 부티레이트로 치료한 결과,

상피 장벽 기능과 FITC-덱스트란(FD4)에 대한 위장 투과성이 회복될 뿐만 아니라

관절염도 효과적으로 억제되었습니다.

더 구체적으로 말하면,

라라조티드에 의한 관절염의 전임상 단계에서 조눌린을 특이적으로 억제하면,

이후 관절염의 발병이 거의 50%까지 감소했습니다.

라라조티드 치료는 장 투과성 증가를 약화시키고,

장에서 관절로 면역 세포의 이동을 차단했습니다 (보충 그림 9의 그래픽 요약).

이러한 연구 결과는

장의 장벽 기능을 회복하여 자가 면역에서 염증으로의 전환을 막고,

그로 인해 RA의 발병을 예방할 수 있는 가능성을 제시한다는 점에서 매우 중요합니다.

조눌린 길항제인 라라조티드는

이미 셀리악병 치료를 위한 임상 3상 시험에 사용되고 있으며,

이 과제를 달성하기 위한 접근 가능한 표적 분자로 자리매김하고 있어 이

러한 접근법이 가까운 미래에 실현될 가능성이 높습니다.

또한, 조눌린 수치의 상승은

RA로 진행될 위험이 가장 높은 환자를 식별하는 흥미로운 바이오마커로 보이며,

이를 통해 이러한 환자를 대상으로 정밀 의학 접근법을 적용할 수 있게 됩니다.

요약하면, 이러한 데이터는

조눌린에 의한 장 장벽 기능의 손상을 기반으로 하며,

조눌린 길항제인 라라조티드에 의해 치료 가능한 RA의 장-관절 축을 뒷받침합니다.

Methods

Study design

The objective of this study was to identify the effect of the TJ protein zonulin in CIA mice and RA patients as a mechanism contributing to the onset of clinical symptoms in arthritis. We hypothesized that preventing zonulin binding to intestinal epithelia cells decreased the here reported higher intestinal barrier permeability for FITC-Dextran (FD4) along with infiltration of proinflammatory cells in the lamina propria, leading to the prevention of arthritis. Using mice, human serum, and in vitro experiments, we analyzed the effect of short-term larazotide, a zonulin antagonist, treatment during preclinical phases of arthritis to show the existence of the gut−joint axis and open therapeutic opportunities to prevent arthritis. Except for 16S rRNA sequencing, all in vitro studies were based on at least three repeats and in vivo data on 5–10 mice per group with at least two independent repeats to achieve significant differences.

Human participants

Serum zonulin levels were analyzed by ELISA (Cusabio Technology LLC, USA) and compared in cross-sectional manner in two cohorts: (i) 41 healthy female controls, 65 female predisease patients and 40 female RA patients from the Department of Internal Medicine 3 of the University of Erlangen-Nürnberg/Germany as well as (ii) 11 healthy female controls, 21 female predisease patients and 28 female RA patients from the Department of Rheumatology & Immunology of the West China Hospital in Chengdu/China. Healthy controls had to have (1) no presence or history of chronic joint pain/swelling, (2) no presence of systemic diseases and (3) no positivity for ACPA or rheumatoid factor (RF). Predisease individuals were defined as having RA-specific autoimmunity (ACPA assessed by testing positive for anticyclic citrullinated peptide 2 IgG autoantibody reactivity) but no past or present evidence of joint swelling according to phases C and D of the RA-at risk definition45. In longitudinal, analyzed pre-RA patients of Erlangen were followed for the later development of RA. Diagnosis of RA was based on arthritis fulfilling the ACR/EULAR 2010 criteria (38). Disease activity of RA was assessed by disease activity score 28 (DAS28). In the Erlangen cohort, 17/40 (42.5%) RA patients received biological disease modifying antirheumatic drugs (bDMARDs), mostly TNF inhibitors. Interestingly, in patients treated with bDMARDs, zonulin levels were lower than in those receiving conventional treatments (p = 0.05). The three groups (healthy controls, pre-RA and RA patients) were balanced for age, sex and body mass index within their respective cohorts. None of these participants had present or past symptoms of a chronic inflammatory bowel disease or celiac disease. Participants were negative for HLA27 and none had clinical signs of spondyloarthritis or psoriasis. Baseline zonulin levels were compared between the groups as well as in pre-RA patients, who developed RA and those who did not develop RA (Erlangen cohort). In addition, serum samples from patients with chronic infections (hepatitis C virus, HCV; N = 21) and cancer (N = 19, NCT02600065 and NCT03453892) were analyzed for serum zonulin levels. Written informed consent was obtained from all participants. All analyses were approved by the institutional review board of the University Clinic of Erlangen (#4564) and the West China Hospital of the Sichuan University (ChiCTR1900022605).

In addition, serial ileal mucosal biopsies from ten healthy controls (undergoing ileo-colonoscopy for screening purpose), ten consecutive new-onset treatment-naïve RA patients (eight females, two males) and five patients with active (DAS28 > 3.2) established RA were analyzed. These procedures were approved by the institutional review board of the Azienda Ospedaliera Universitaria Paolo Giaccone of the University of Palermo and patients provided written informed consents. Patients were balanced for age, sex and body mass index. RA patients fulfilled the ACR-EULAR 2010 criteria (38) and had to be positive for anti-CCP2 antibodies. Also, none of these patients had present or past symptoms of a chronic inflammatory bowel disease or celiac disease. Patients had to be negative for HLA27 and clinical signs of spondyloarthritis or psoriasis. Paired paraffin-embedded tissue and RNA samples were prepared from all patients to allow cross-referencing between histological assessments and qPCR gene expression‎ analysis. Baseline characteristics of the patients are shown in Supplementary Table 1.

Histology and immunohistochemistry in humans

Paraffin-embedded ileal samples were cut into 4-μm sections and stained with hematoxylin and eosin (H&E). Immunohistochemical analysis for CD3, CD19, CD68, occludin and claudin-1 was performed on 5-µm-thick paraffin-embedded sections from these biopsies and from tonsils (used as positive controls) as previously described46. Briefly, following rehydration, antigen was unmasked for 45 min at 95 °C using Dako Target retrieval‎ solution (pH 6; Dako, Carpinteria, CA). Endogenous peroxidase was blocked for 10 min with Dako peroxidase blocking reagent, and nonspecific binding was blocked for 20 min with Dako protein block. The primary antibodies, mouse monoclonal anti-human CD3 (Leica Biosystems, dilution 1:100), CD19 (Dako, dilution 1:50), CD68 (Dako, dilution 1:100), occludin (Santa Cruz Biotechnology, dilution 1:100) and claudin-1 (Santa Cruz Biotechnology, dilution 1:100), were added and incubated for 1 h at room temperature. An isotype-matched irrelevant antibody was used as a negative control. Following three washes with Tris-buffered saline, slides were incubated for 30 min with peroxidase-conjugated Dako EnVision polymer. After three further washes, peroxidase activity was visualized using diaminobenzidine chromogen (Dako), and slides were lightly counterstained with hematoxylin before dehydration and mounting in DePex (VWR International, Oslo, Norway). The number of immune-reactive cells was determined by counting positively stained cells on photomicrographs obtained from three random high-power microscopic fields (×400 magnification) under a Leica DM2000 optical microscope, using a Leica DFC320 digital camera (Leica, Rijswijk, the Netherlands). AR and FC eval‎uated the ileal samples with no access to clinical data. The intra-rater agreement and the inter-rater agreement calculated by the Cohen’s K coefficient for the two observers were 0.88 and 0.78, respectively.

RT-PCR in humans

Soon after removal, ileal biopsies were stored in RNAlater® solution (Applied Biosystems, Foster City, CA, USA). RT-PCR was performed as previously described46. Master mix and Taqman® gene expression‎ assays for glyceraldehyde 3-phosphate dehydrogenase (GAPDH control) and target genes occludin (Hs05465837_g1) and claudin-1(Hs00221623_m1) were obtained from Applied Biosystems (Foster City, CA, USA). Data were quantified using sds 2.1 software and normalized using GAPDH as endogenous control. Relative changes in gene expression‎ between HCs and RA samples were determined using the ΔΔCt method. Levels of the target transcript were normalized to a GAPDH endogenous control, constantly expressed in both groups (ΔCt). For ΔΔCt values, additional subtractions were performed between RA (n = 10) and HCs (n = 10) ΔCt values. Final values were expressed as fold of induction.

RT-PCR in mice

Tissues were stored in RnaLater (Ambion) or directly transferred to TRIzol (Invitrogen). RNA was extracted according to the manufacturer’s instructions. Gene expression‎ results are expressed as arbitrary units relative to expression‎ of the house keeping gene GAPDH. Primer sequences are as follows: GAPDH: 5′-GGG TGT GAA CCA CGA GAA AT-3′ and 5′-CCT TCC ACA ATG CCA AAG TT-3′; ZO-1: 5′-CCA CCT CTG TCC AGC TCT TC-3′ and 5′-CAC CGG AGT GAT GGT TTT CT-3′; occludin: 5′-CCT CCA ATG GCA AAG TGA AT-3′ and 5′-CTC CCC ACC TGT CGT GTA GT-3′; claudin-1: 5′-TCC TTG CTG AAT CTG AAC A-3′ and 5′-AGC CAT CCA CAT CTT CTG-3′; claudin-2: 5′-TAT GTT GGT GCC AGC ATT GT-3′ and 5′-TCA TGC CCA CCA CAG AGA TA-3′; claudin-15: 5′-GCT TCT TCA TGT CAG CCC TG-3′ and 5′-TTC TTG GAG AGA TCC ATG TTG C-3′.

Lactulose to mannitol ratio in humans

The ratio of urinary excretion of lactulose to mannitol (LA/MA) was used to measure the intestinal mucosal permeability in ten RA patients and ten healthy controls who underwent colonoscopy, with higher ratios indicative of increased intestinal permeability as previously described33.

Animals

All mice were maintained under specific pathogen-free conditions at the Präklinisches Experimentelles Tierzentrum (PETZ), Erlangen, Germany and approved by the local ethics authorities of the Regierung of Unterfranken (#55.2-2532-2-424 and #55.2-2532-2-630). For all experiments, if not otherwise stated, DBA1/J female mice (8 weeks old) were used and purchased from Janvier Labs. Kaede mice were kindly provided by M. Tomura from the RIKEN Institute, Tokyo, Japan. Germ-free (GF) mice were kindly provided by M. Basic from the Hannover Medical School, Hannover, Germany. The animals were kept in the Franz-Penzoldt-Zentrum (FPZ) of the University Hospital Erlangen or the animal laboratory of Med3 under standardized husbandry conditions and hygiene management in accordance with Federation of European Laboratory Animal Science Associations (FELASA). The animals received water and feed ad libitum. The keeping rooms had a temperature of 22–23 °C and a humidity of 50–60%. There was also a 12-h light–dark rhythm in the holding rooms. Animals were kept in type II long cages, with maximum five animals.

Mice studies

For all experiments, mice were acclimated for 1 week, followed by a 2-week co-housing period before the experiments started. Supplementation of butyrate (all Sigma-Aldrich, Germany) was done in the drinking water at a final concentration of 150 mM and changed every 3 days and control mice received pH and sodium-matched water. Zonulin antagonist (known as Larazotide acetate, or AT-1001, or INN-202) was purchased from BOC Sciences, NY, USA or later synthesized by GENAXXON biosciences, Ulm, Germany and effectiveness was compared in one side-by-side experiment using both purchased zonulin antagonist sources (BOC Sciences, NY, USA vs. GENAXXON biosciences) to confirm‎ their biological function. Mice received 0.15 mg/ml zonulin antagonist in the drinking water and changed every day for 10 consecutive days between 17 and 27 dpi in the CIA model. For CAIA mouse model, C57BL/6 mice received 50 µg/mouse i.v. zonulin antagonist or vehicle (3% DMSO in phosphate‐buffered saline (PBS)) 1 day after CAIA induction. CB1 receptor agonist (ACEA; arachidonyl-2-chloroethylamide, Sigma-Aldrich, Germany) treatment was i.p. injections daily with 250 µg/mouse between 17 and 27 days post CII immunization. CIA was induced in 8-week-old female Kaede (C57BL/6J) or wild-type DBA/1J mice by subcutaneous injection at the base of the tail with 100 μl with 0.25 mg chicken type II collagen (Chondrex, Redmond, WA) in complete Freund adjuvant (Difco Laboratory, Detroit, MI), containing 5 mg/ml killed Mycobacterium tuberculosis (H37Ra). Mice were challenged after 21 days by intradermal immunization in the base of the tail with this emulsion. The paws were eval‎uated for joint swelling and grip strength three times per week. Each paw was individually scored using a 4-point scale: 0, normal paw; 1, minimal swelling or redness; 2, redness and swelling involving the entire forepaw; 3, redness and swelling involving the entire limp; 4, joint deformity or ankylosis or both. In addition, grip strength of each paw was analyzed on a wire 3 mm in diameter, using a score from 0 to −4 (0, normal grip strength; −1, mildly reduced grip strength; −2, moderately reduced grip strength; −3, severely reduced grip strength; −4, no grip strength at all). For photoconversion of Kaede-transgenic mice, the small intestine of anesthetized Kaede-transgenic mice was subjected to lighting using a BlueWave LED Prime UVA (Dymax).

In vivo intestinal permeability measurements

Once every week, mice were housed in metabolic cages after a 4 h fasting of food and water and immediately after a gavage of 0.2 ml of a 10 ml sugar probe containing 100 mg of sucrose, 12 mg of lactulose, 8 mg of mannitol and 6 mg of sucralose. After the collection of urine the animals were placed in their respective cages, and provided with food and water. For the FITC-Dextran measurements, after 4 h fasting of food and water, mice were immediately orally gavaged with 200 μl of FITC-dextran (FD4) (440 mg/kg body weight), and blood was collected 4 h later. The concentration of the FITC-dextran was determined using a fluorimeter with an excitation wavelength at 490 nm and an emission wavelength of 530 nm. Serially diluted FITC-dextran in serum was.used to establish a standard curve.

HPLC analysis

For HPLC analysis of lactulose and mannitol, urine samples were thawed, mixed thoroughly and centrifuged for 5 min at 20,000 × g and 4 °C. Fifty microliters of supernatant was mixed with 175 µl of HPLC-grade water and 25 µl of 20% sulfosalicylic acid fresh solution, followed by incubation for 15 min at room temperature. After storage at −20 °C overnight, diluted samples were thawed again, centrifuged for 10 min at 20,000 × g and 4 °C. An amount of 200 mg ion-exchange resins (TMD-8 hydrogen and hydroxide form, Sigma-Aldrich Chemie GmbH, Taufkirch, Germany) was added, mixed well and incubated for 30 min at room temperature. After centrifugation for 10 min at 20,000 × g and 4 °C, supernatants were subjected for HPLC analysis using a DIONEX UltiMate® 3000 system with Corona Veo SD Charged-Aerosol-Detector (Thermo Scientific GmbH, Dreieich, Germany). For chromatographic separation, a hydrophilic interaction liquid chromatography (HILIC) column (Acclaim™ Trinity™ P2, 3 µm, 3.0 × 100 mm, Thermo Scientific) was used. The eluent was a mixture of acetonitrile and 100 mM ammoniumformate (pH 3.65) with an isocratic ratio of 82:18 [vol/vol] at 0.6 ml/min and 60 °C column oven temperature. For determination of sucralose, urine samples were thawed, mixed thoroughly and centrifuged for 10 min at 20,000 × g and 4 °C. Fifty microliters of supernatant was mixed with 200 µl of HPLC-grade water, mixed thoroughly and centrifuged again for 10 min at 20,000 × g and 4 °C. Supernatants were analyzed by using a DIONEX UltiMate® 3000 system with Corona Veo SD Charged-Aerosol-Detector (Thermo Scientific GmbH, Dreieich, Germany). For chromatographic separation a Synergi 4µ Polar-RP 80A column was used (250 ×1.6 mm (Phenomenex, Aschaffenburg, Germany)). The eluent was a mixture of methanol and HPLC-grade water with an isocratic ratio of 30:70 [vol/vol] at 1.0 ml/min and 50 °C column oven temperature.

Histological grading of intestinal inflammation in mice

Upon sacrifice, proximal duodenal, jejunal, ileal and colon tissue were harvested and fixed in 10% phosphate-buffered formalin. These samples were embedded in paraffin, sectioned at 4 μm, and stained with H&E or PAS for light microscopy examination. The slides were reviewed in a blinded fashion by a pathologist and were assigned a histological score for intestinal inflammation.

Bone histology

Tibial bones and inflamed paws with tarsal joints were fixed in 4% formalin for 24 h and decalcified in Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich). Serial paraffin sections (2 μm) were stained for tartrate-resistant acid phosphatase (TRAP) using a Leukocyte Acid Phosphatase Kit (Sigma) according to the manufacturer’s instructions. Osteoclast numbers were quantified using a microscope (Carl Zeiss) equipped with a digital camera and an image analysis system for performing histomorphometry (Osteomeasure; OsteoMetrics).

Immunofluorescence microscopy

Paraffin-embedded tissue sections were deparaffinized with xylene and rehydrated through graded alcohol solutions. Heat-induced epitope retrieval‎ was performed by placing the sections in Retrieval‎ Solution pH 9, diluted 1:20 (cat. EB-DEPP-9; eubio) at 85 °C for 20 min, washed three times with PBS and blocked with PBS containing 0.2% bovine serum albumin (BSA) and 0.1% saponin for 1 h at room temperature. The sections were then incubated with primary antibodies against occludin (Abcam, dilution 1:100) and ZO-1 (ThermoFisher, dilution 1:100), diluted in PBS containing 0.2% BSA and 0.1% saponin at 4 °C overnight, washed, and incubated for 1 h with Alexa Fluor 647 donkey anti-rabbit IgG secondary antibody (Invitrogen, dilution 1:400). Some sections were incubated with PBS containing 0.2% BSA and 0.1% saponin alone in place of primary antibody as a negative control. After having been washed three times with PBS, the sections were mounted and visualized. Imaging was performed by using the Leica DMi8 TIRF Widefield Fluorescence of ×10 objective lens (Leica, Germany) and Zeiss Spinning Disc Axio Observer Z1 of ×63 objective lens (Carl Zeiss) microscopes.

To compare ZO-1 and occludin levels, we considered three samples for each of the consecutive time points (day 0, 25, 30, 35, 40, 45 and 50) in ileum and colon for each of the proteins with three images from different parts of each section. Microscopy was performed and fluorescence images were visualized and captured in DAPI and Cy5 channels. Quantification of the images was performed using ImageJ software based on the mean fluorescence intensity. We calculated for each of the samples and condition the mean intensity across the entire image and divided it by the mean intensity of the DAPI channel to account for varying tissue sizes.

Fecal supernatants preparation

Intestinal stool samples were obtained from healthy and CIA mice, at different time points of disease. Samples were suspended at 10% (wt/vol) in Dulbecco’s PBS, homogenized, and centrifuged at 4500 rpm for 10 min at 4 °C. Supernatants were passed through 1.2 µm membrane filters.

Cell culture

Caco-2 cells, a human colorectal carcinoma-derived epithelial cell line, were cultivated in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco) containing 10% fetal bovine serum (FBS) in a humidified incubator at 37 °C, 5% CO2. Cells were maintained for 14 days post confluency, serum-starved overnight and stimulated with FSN for 24 h or PBS as a control.

Transepithelial resistance measurements

Caco-2 cells were seeded onto 12 mm polycarbonate membrane inserts (PCF membrane inserts, Millipore, Germany) and bathed in DMEM/F12 medium which contained phenol red, 10% FBS, penicillin (100 units/ml) and streptomycin (10 µg/ml). Cells were continuously maintained in a humidified incubator at 37 °C, 5% CO2. To assess that these cells form monolayer on the membrane inserts, transepithelial resistance was routinely monitored using an epithelial volt-ohm-meter including STX-2 electrodes (EVOM, World Precision Instruments, Berlin, Germany). After 12–16 days post seeding, membrane inserts containing confluent monolayer of Caco-2 cells were transferred into Ussing chambers47,48. The basolateral bath contained 1.0 ml and the apical bath 0.5 ml of the supplemented DMEM/F12 medium. The transepithelial resistance was recorded every 10 s using a CVC6 clamp device (Fiebig, Berlin Germany)49 by measuring voltage deflections induced by injection of 20 µA symmetrical square pulses for 0.4 s. After an equilibration period of 10–40 min, AT-1002 (5 mg/ml) was applied to the apical bath by replacing 400 µl of the bath medium with 400 µl of medium containing the AT-1002 component. In time matched controls the dissolvent of AT-1002 was added to the apical bath. The transepithelial resistance was then continuously recorded for a further 3 h.

Flow cytometry

Small intestines (all data shown for the ileum) and colons were analyzed by flow cytometry. In short, tissues were incubated with PBS containing EDTA at 37 °C for 2 × 20 min, then digested twice for 35 min with a cocktail of collagenase D (1 mg/ml, Roche), dispase I (0.1 mg/ml, Roche), and DNase I (333 mg/ml, Sigma), smashed and filtered through a 40 mm gauze (BD Biosciences). Single-cell suspensions were then stimulated with phorbol 12-myristate 13 acetate and ionomycin (Sigma-Aldrich) for 6 h; monensin (BD-Bioscience) and brefeldin A (BioLegend) were added after 2 h or directly stained with the respective directly labeled FACS antibodies. All intracellular stainings were used in combination with a FoxP3staining kit (eBioscience). Flow cytometric analysis was performed on a Gallios Flow Cytometer (Beckman Coulter) or CytoFLEX (Beckmann Coulter) and eval‎uated using the Kaluza and cytExpert Flow Cytometry analysis software (Beckman Coulter). Antibodies used: Alexa Fluor® 647 anti-mouse CD3 antibody (BioLegend, 1:600); PE/Cy7 anti-mouse CD4 antibody (BioLegend, 1:400); Alexa Fluor® 700 anti-mouse CD8a antibody (BioLegend, 1:600); PE Mouse anti-Mouse RORγt (BD Pharmingen, 1:100); Pacific Blue™ anti-T-bet Antibody (BioLegend, 1:100); Alexa Fluor® 647 anti-mouse FOXP3 Antibody (BioLegend, 1:100); PE anti-GATA3 Antibody (BioLegend, 1:100); Brilliant Violet 421™ anti-mouse CD3ε Antibody (BioLegend, 1:200); APC/Cy7 anti-mouse/human CD45R/B220 Antibody (BioLegend, 1:200); Alexa Fluor 700 anti-mouse/human CD11b Antibody (BioLegend, 1:200); CD11c Monoclonal Antibody (N418), APC (eBioscience™, 1:400); APC anti-mouse IFN-Antibody (BioLegend, 1:50); BV421 Rat Anti-Mouse IL-4 (BD Biosciences, 1:200); PE anti-mouse IL17A Antibody (BioLegend, 1:200).

Intestinal organoid isolation and cultivation

The small intestine of wild-type C57Bl/6J was removed and washed with ice-cold PBS. It was opened longitudinally and villi were scraped out using a coverslip and discarded. The intestine was cut into 2–3 mm pieces and collected in ice-cold PBS. After sedimentation the supernatant was discarded and fresh ice-cold PBS was added to resuspend the intestinal pieces. This procedure was repeated 15–20 times until the supernatant was clear. The intestinal pieces were then incubated at 4 °C for 20–30 min in ice-cold PBS containing 2 mM EDTA (Merck) while agitating. Afterwards the tissue was sedimented by gravity and the supernatant was replaced by ice-cold PBS. The intestinal crypts were dissociated from the tissue by vigorous shaking. The supernatant was collected and screened for crypts by microscopy. This step was repeated until no crypts were left in the supernatant. The supernatant fractions enriched with crypts were passed through a 70 µm cell strainer and centrifuged at 300 × g for 5 min at 4 °C. The pelleted crypts were resuspended in 25 µl/well growth factor reduced Matrigel (Corning, New York, USA). The cell suspension was then distributed into a 48-well cell culture plate. The Matrigel was allowed to polymerize in the incubator at 37 °C and 5% CO2 for 20 min. Afterwards 300 µl of Intestinal Organoid Growth medium (mouse) (Stemcell, Köln, Germany) was added per well. Organoids were cultured in the incubator for at least 7 days before any experiments were performed. Medium was changed every 2–3 days and organoids were splitted once per week50.

Permeability assay

For imaging organoids were plated in eight-well chamber slides (Ibidi, Planegg, Germany). The organoids were imaged using a Zeiss Spinning Disc confocal microscope (Zeiss, Jena, Germany). At the beginning of the experiment 1 mM Lucifer yellow (VWR International GmbH, Darmstadt, Germany) was added to each of the chambers. Afterwards the organoids were imaged for 100 min with intervals of 5 min. To ensure the capability of the organoids to get permeable, 1 mM of EGTA (Merck) was added after the observation time. Following EGTA addition organoids were imaged for another 20 min. For each time point lucifer yellow fluorescence inside and outside the organoids was quantified using Fiji ImageJ. Only organoids that were able to take up Lucifer yellow after EGTA treatment were analyzed51.

Western blotting

Caco-2 cells were washed twice with PBS and subsequently lysed. Intestinal tissue samples were homogenized in protein lysis buffer and subsequently sonicated. Protein extracts were separated on 8% SDS-polyacrylamide or 4–12% bis−tris protein gel (Invitrogen), transferred on a nitrocellulose membrane, and stained with antibodies against ZO-1 (N1N2, GeneTex, dilution 1:3000). An antibody against β-actin (AC-15, abcam, dilution 1:25,000) was used as loading control. Supplementary Fig. 10 shows the uncropped gel scans of Western blots.

SCFA measurements

Five replicates of frozen cecal samples (100 mg) were weighed into a 2 ml polypropylene tube. The tubes were kept in a cool rack throughout the extraction. 33% HCl (50 µl for cecal contents or 5 µl for serum) was added and samples were vortexed for 1 min. One milliliter of diethyl ether was added, vortexed for 1 min and centrifuged for 3 min at 4 °C. The organic phase was transferred into a 2 ml gas chromatography (GC) vial. For the calibration curve, 100 μl of SCFA calibration standards (Sigma) were dissolved in water to concentrations of 0, 0.5, 1, 5 and 10 mM and then subjected to the same extraction procedure as the samples. For GCMS analysis 1 μl of the sample (4–5 replicates) was injected with a split ratio of 20:1 on a Famewax, 30 m x 0.25 mm iD, 0.25 μm df capillary column (Restek, Bad Homburg). The GC-MS system consisted of GCMS QP2010Plus gas chromatograph/mass spectrometer coupled with an AOC20S autosampler and an AOC20i auto injector (Shimadzu, Kyoto, Japan). Injection temperature was 240 °C with the interface set at 230 °C and the ion source at 200 °C. Helium was used as carrier gas with constant flow rate of 1 ml/min. The column temperature program started with 40 °C and was ramped to 150 °C at a rate of 7 °C/min and then to 230 °C at a rate of 9 °C/min, and finally held at 230 °C for 9 min. The total run time was 40 min. SCFA were identified based on the retention time of standard compounds and with the assistance of the NIST 08 mass spectral library. Full scan mass spectra were recorded in the 25–150 m/z range (0.5 s/scan). Quantification was done by integration of the extracted ion chromatogram peaks for the following ion species: m/z 45 for acetate eluted at 7.8 min, m/z 74 for propionate eluted at 9.6 min, and m/z 60 for butyrate eluted at 11.5 min. GCMS solution software was used for data processing.

Micro-computed tomography

µCT imaging was performed using the cone-beam Desktop Micro Computer Tomograph “µCT 40” by SCANCO Medical AG, Bruettisellen, Switzerland. The settings were optimized for calcified tissue visualization at 55 kVp with a current of 145 µA and 200 ms integration time for 500 projections/180°. For the segmentation of 3D-volumes, an isotropic voxel size of 8.4 µm and an eval‎uation script with adjusted grayscale thresholds of the operating system “Open VMS” by SCANCO Medical was used. Volume of interest tibia: The analysis of the bone structure was performed in the proximal metaphysis of the tibia, starting 0.43 mm from an anatomic landmark in the growth plate and extending 1.720 mm (200 tomograms) distally.

Specific IgG detection via ELISA

Collagen type II (CII)-specific antibodies were detected by ELISA using high-binding plates (Nunc) coated overnight with 10 μg/ml mouse or chicken CII. The detection of mouse CII-specific antibodies was carried out by Eu3+-labeled anti-mouse IgG antibody and the DELFIA system (PerkinElmer) according to the manufacturer’s recommendations.

16S rRNA microbiome analysis

For Supplementary Fig. 4a–d, genomic DNA was extracted according to Qiamp Fast DNA Stool extraction kit (Qiagen) and the V3–V4 region of the bacterial 16S rRNA gene was amplified with the NEBNext Q5 Hot Start Hifi PCR Master Mix (NEB) using a dual-index strategy. Amplified fragments were purified with AMPure XP Beads (Beckmann Coulter Genomics), combined and analyzed by “2 × 300 bp paired-end” sequencing using an Illumina MiSeq device. Quality control, OTU table generation and bioinformatics analysis was done using the Usearch10 (Edgar RC Flyvbjerg H, 2015) and the Microbiomeanalyst package52. The database of the “Ribosomal” database project (RDP release 16) was used for classification. For Supplementary Fig. 4e, microbial DNA extraction from fecal content was done using ZymoBIOMICS 96 Magbead DNA Kit automated on a Tecan Fluent 480 liquid handling system. Amplification of the V4 region (F515/R806) of the 16S rRNA gene was performed as in previously described protocols53. Samples were sequenced on an Illumina MiSeq platform (PE250). Barcode-based demultiplexing was performed using IDEMP software with default parameters (https://github.com/yhwu/idemp). Obtained reads were assembled, quality controlled, and clustered using Usearch8.1 software package (http://www.drive5.com/usearch/). Briefly, reads were merged using -fastq_mergepairs –with fastq_maxdiffs 30 and quality filtering was done with fastq_filter (-fastq_maxee 1), minimum read length 200 bp. The OTU clusters and representative sequences were determined using the UPARSE algorithm54, followed by taxonomy assignment using the Silva database v12855 and the RDP Classifier56 with a bootstrap confidence cutoff of 80% performed by using QIIME v1.8.057. OTU absolute abundance table and mapping file were used for statistical analyses and data visualization in the R statistical programming environment package phyloseq58 or web-based tool MicrobiomeAnalyst52.

Statistical analysis

Data are expressed as mean ± s.d. unless otherwise indicated in the figure legends. Analysis was performed using Student’s t test, single comparison, or analysis of variance (ANOVA) test for multiple comparisons (one-way or two-way ANOVA followed by Tukey’s or Bonferroni’s multiple comparisons test, respectively). All experiments were conducted at least two times. P values of 0.05 were considered significant and are shown as *p < 0.05, **p < 0.01, or ***p < 0.001. Graph generation and statistical analyses were performed using the Prism version 8 software (GraphPad, La Jolla, CA).

Data availability

All relevant data are available from the authors upon reasonable request. The source data underlying Figs. 1–6 and Supplementary Figs. 1–10 are provided as a Source data file. Sequence data are deposited in BioProject with the accession code PRJNA592061.

References

1.  
2.  
3.  
4.