Re: 장벽 tight junction의 역할, 장누수 2018 nature review 논문...

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Nat Rev Gastroenterol Hepatol

. 2023 Apr 25:1–16. Online ahead of print. doi: 10.1038/s41575-023-00766-3

Paracellular permeability and tight junction regulation in gut health and disease

Arie Horowitz 1, Sandra D Chanez-Paredes 2, Xenia Haest 2, Jerrold R Turner 2,✉

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PMCID: PMC10127193  NIHMSID: NIHMS1900769  PMID: 37186118

Abstract

Epithelial tight junctions define the paracellular permeability of the intestinal barrier. Molecules can cross the tight junctions via two distinct size-selective and charge-selective paracellular pathways: the pore pathway and the leak pathway. These can be distinguished by their selectivities and differential regulation by immune cells. However, permeability increases measured in most studies are secondary to epithelial damage, which allows non-selective flux via the unrestricted pathway. Restoration of increased unrestricted pathway permeability requires mucosal healing. By contrast, tight junction barrier loss can be reversed by targeted interventions. Specific approaches are needed to restore pore pathway or leak pathway permeability increases. Recent studies have used preclinical disease models to demonstrate the potential of pore pathway or leak pathway barrier restoration in disease. In this Review, we focus on the two paracellular flux pathways that are dependent on the tight junction. We discuss the latest evidence that highlights tight junction components, structures and regulatory mechanisms, their impact on gut health and disease, and opportunities for therapeutic intervention.

요약

상피의 촘촘한 접합부는 

장벽의 세포간 투과성을 결정합니다. 

분자들은 

크기와 전하 size-selective and charge-selective를 

선택적으로 통과하는 두 개의 세포간 경로인 

기공 경로와 누출 경로를 통해 촘촘한 접합부를 통과할 수 있습니다. 

the pore pathway and the leak pathway

이 두 경로는 선택성과 면역세포에 의한 차별적 조절에 따라 구분할 수 있습니다. 

그러나 

대부분의 연구에서 측정된 투과성 증가는 

상피 손상에 이차적으로 발생하며, 

이로 인해 제한되지 않은 경로를 통한 비선택적 흐름이 가능합니다. 

증가된 무제한 통로 투과성의 회복에는 점막의 치유가 필요합니다. 

반대로, 

단단한 접합 장벽의 손실은 

표적 개입을 통해 되돌릴 수 있습니다. 

기공 통로 또는 누출 통로 투과성 증가를 회복하기 위해서는 

구체적인 접근 방식이 필요합니다. 

최근의 연구에서는 전임상 질병 모델을 사용하여 질병에서 

기공 경로 또는 누출 경로 장벽 복원의 잠재력을 입증했습니다. 

이 리뷰에서는 조임 접합에 의존하는 두 개의 세포간 유출 경로에 초점을 맞춥니다. 

조임 접합 구성 요소, 구조 및 조절 메커니즘, 장 건강과 질병에 미치는 영향, 치료적 개입의 기회를 강조하는 최신 증거를 논의합니다.

Subject terms: Gastroenterology, Gastrointestinal system, Inflammatory bowel disease

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Increased intestinal permeability owing to tight junction barrier loss could be targeted in gastrointestinal diseases associated with increased permeability. In this Review, the authors discuss the molecular components and regulation of the tight junction, and consider the relevance to gut diseases and therapeutic opportunities.

장벽의 손실로 인한 장 투과성 증가는 투과성 증가와 관련된 위장 질환의 표적이 될 수 있습니다. 

이 리뷰에서 저자들은 장벽의 분자 구성 요소와 조절에 대해 논의하고, 

장 질환과의 관련성과 치료 기회에 대해 고려합니다.

Key points

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• 장 투과성 증가는 염증성 장 질환, 셀리악 병, 이식편 대 숙주병을 포함한 광범위한 질환에서 발생하지만, 장벽 기능 장애와 면역 반응의 상대적 기여도는 불분명합니다.
• 장벽 손실은 조인트 기능 장애 또는 상피 손상의 결과일 수 있습니다; 대부분의 연구에서 이러한 메커니즘은 구분되지 않습니다.
• 조인트의 세포간 수송은 기공 경로 또는 누출 경로를 통해 발생할 수 있으며, 이 두 경로는 크기 선택성과 전하 선택성이 뚜렷하고 면역 신호에 의해 다르게 조절됩니다.
• 클라우딘-2는 기공 경로를 통한 Na+ 및 물의 흐름을 증가시키지만, 더 큰 분자는 클라우딘-2 채널을 통과할 수 없습니다.
• 누출 경로는 거대 분자가 상피 장벽을 통과할 수 있도록 하며, 세포 골격과 상피 긴 미오신 경쇄 키나제 스플라이스 변이체 1(MLCK1)에 의해 조절됩니다.
• MLCK1의 타이트한 접합부 모집을 차단하면 상피 세포와 다른 유형의 세포에서 필수적인 MLCK 기능을 방해하지 않으면서 타이트한 접합부 장벽의 손실을 제한할 수 있습니다.

Introduction

Epithelia separate the organism from the external environment and define individual compartments within tissues. At some sites, the epithelia form a nearly complete barrier, disruption of which is catastrophic. For example, massive disruption of the epidermal (skin) barrier by burn injury or mutagenesis in animal models can be fatal. However, at other sites, such as renal tubules and intestines, the balance between permeability and barrier function is nuanced, as selective permeability is essential for physiological processes but must also be precisely regulated.

Flux across the epithelial barrier occurs via transepithelial transport, which involves transcellular and paracellular pathways. Transcellular transport involves movement of molecules through cells, and is mediated by apical and basolateral transmembrane transporters with exquisite substrate specificity. Paracellular transport is less selective and can involve movement of molecules across the epithelial barrier via the pore pathway or the leak pathway. A third permeability route, the unrestricted pathway, is created by epithelial damage (Box 1). Flux across the pore and leak pathways reflects permeability of tight junctions, which seal the space between adjacent epithelial cells and are the rate-limiting components of paracellular transport. Both pore and leak pathways are size-selective, and the pore pathway is charge-selective. However, both pathways lack the structural specificity of transcellular transport. For example, l-glucose can be absorbed paracellularly but is not recognized by transmembrane transport proteins and, therefore, cannot be absorbed via the transcellular route.

Although barrier loss is considered most often in the context of disease, insufficient selective permeability — that is, barrier enhancement — can also contribute to disease. For example, the first monogenic tight junction disease to be discovered — familial hypomagnesaemia with hypercalciuria and nephrocalcinosis — is caused by mutation of claudin-16, which forms paracellular cation channels within the renal tubule. In the absence of claudin-16, paracellular Mg2+ and Ca2+ absorption in the thick ascending limb of the nephron fails1,2. Conversely, loss of claudin-14, which enhances paracellular barriers, in the organ of Corti causes deafness in mice and autosomal recessive nonsyndromic hearing loss 29 (DFNB29) in humans3–6.

In the intestines, the epithelial monolayer separates subepithelial immune cells from the luminal microbiome7. The balance between paracellular permeability and barrier function is, therefore, especially delicate because the epithelial monolayer must prevent unregulated paracellular flux of potentially pathogenic luminal materials8 while also allowing the selective paracellular permeability that is required for nutrient and water absorption9. This situation contrasts with that in the nephron, where both the lumen of the renal tubule and the interstitial space are sterile under normal conditions. In the gut lumen, which contains a diverse microbiome, disruption of the balance between selective permeability and barrier function is associated with a wide range of intestinal and systemic disorders10,11.

In this Review, we consider several disorders that involve intestinal barrier dysfunction, focusing on changes to paracellular permeability and the function of tight junctions. We delineate molecular mechanisms that alter paracellular permeability and cause intestinal barrier loss. We also endeavour to differentiate between barrier loss as a contributor to disease pathogenesis and barrier loss as a consequence of disease processes.

소개

상피는

유기체를 외부 환경과 분리하고

조직 내의 개별 구획을 정의합니다.

일부 부위에서는

상피가 거의 완전한 장벽을 형성하며,

이 장벽이 파괴되면 치명적입니다.

예를 들어,

화상이나 동물 모델의 돌연변이로 인한

표피(피부) 장벽의 대규모 파괴는

치명적일 수 있습니다.

그러나

신장 세뇨관이나 장과 같은 다른 부위에서는

선택적 투과성이 생리학적 과정에 필수적이지만, 정

확하게 조절되어야 하기 때문에 투과성과 장벽 기능 사이의 균형이 미묘합니다.

상피 장벽을 통한 흐름은

세포간 및 세포내 경로를 포함하는 상피 투과를 통해 발생합니다.

세포간 수송은 세포를 통한 분자의 이동을 포함하며,

정교한 기질 특이성을 가진 정단 및 기저막 횡단 수송체에 의해 매개됩니다.

세포간 수송은 선택성이 낮고,

기공 경로 또는 누출 경로를 통해

상피 장벽을 가로지르는 분자의 이동을 포함할 수 있습니다.

세 번째 투과성 경로인 무제한 경로는

상피 손상에 의해 생성됩니다(박스 1).

기공 경로와 누출 경로를 통한 흐름은

인접한 상피 세포 사이의 공간을 밀봉하는 꽉 조이는 접합부의 투과성을 반영하며,

세포간 수송의 속도 제한 구성 요소입니다.

공극과 누출 통로는

모두 크기 선택적이며, 공극 통로는 전하 선택적입니다.

그러나

두 경로 모두 세포간 수송의 구조적 특이성이 부족합니다.

예를 들어,

l-글루코스는

세포간 수송을 통해 흡수될 수 있지만,

막 횡단 수송 단백질에 의해 인식되지 않으므로

세포간 경로를 통해 흡수될 수 없습니다.

장벽 손실은

질병의 맥락에서 가장 자주 고려되지만,

불충분한 선택적 투과성, 즉 장벽 강화도 질병의 원인이 될 수 있습니다.

예를 들어,

최초로 발견된 단일 유전자성 밀접 접합성 질환인

가족성 저마그네슘혈증과 고칼슘뇨증 및 신장석회증은

신장 세뇨관 내에 세포간 양이온 채널을 형성하는 클라우딘-16의 돌연변이에 의해 발생합니다.

클라우딘-16이 없으면,

네프론의 두꺼운 상승 사지에서

세포간 Mg2+와 Ca2+의 흡수가1,2 실패합니다.

반대로, 세포간 장벽을 강화하는 클라우딘-14가 코르티 기관에서 손실되면, 생쥐의 경우 청각 장애가 발생하고, 인간의 경우 상염색체 열성 비증후성 난청 29(DFNB29)가 발생합니다3-6.

장에서는

상피의 단층이 상피하 면역 세포와

내강 미생물 군집7을 분리합니다.

따라서,

상피의 단층은

잠재적으로 병원성 내강 물질8의 조절되지 않은 상피하 흐름을 방지해야 하는 동시에

영양분과 물의 흡수에 필요한 선택적 상피하 투과성을 허용해야 하기 때문에,

세포간 투과성과 장벽 기능 사이의 균형은

특히 섬세합니다9.

이 상황은

정상적인 조건에서 신장 세뇨관의 내강과 간질 공간이 모두 무균 상태인

네프론의 상황과 대조적입니다.

다양한 미생물 군집을 포함하고 있는 장 내강에서

선택적 투과성과 장벽 기능 사이의 균형이 깨지면

다양한 장 및 전신 질환과 관련이 있습니다10,11.

이 리뷰에서는

장벽 기능 장애와 관련된 여러 가지 장애를 고려하고,

세포간 투과성 변화와 꽉 조이는 접합부의 기능에 초점을 맞춥니다.

우리는

세포간 투과성을 변화시키고

장벽 손실을 유발하는 분자 메커니즘을 설명합니다.

또한

장벽 손실을

질병 발병 기전의 원인으로 간주할 것인지,

질병 과정의 결과로 간주할 것인지 구분하려고 노력합니다.

Box 1 The pore, leak and unrestricted permeability pathways.

Intestinal permeability can reflect contributions of three distinct pathways: the pore pathway, the leak pathway and the unrestricted pathway (see the figure). The pore and leak pathways reflect flux across tight junctions, whereas the unrestricted pathway is independent of tight junctions.

Pore pathway permeability is defined by claudin proteins, which form either channels or barriers at the tight junction291–294. Channels generated by pore-forming claudins are charge-selective and size-selective; the maximum diameter of solutes that can pass through them is 0.6 nm. Claudins form only cation-selective channels in the gastrointestinal tract, but anion-selective claudin channels are present at other sites, such as the nephron. Immune signals, including IL-13 and IL-22, lead to increased transcription and expression‎ of intestinal epithelial claudin-2, which increases pore pathway permeability (see the figure, top)45,50,86.

The tight junction leak pathway allows molecules with diameters up to ~12.5 nm to traverse the epithelial barrier. The molecular structure of the leak pathway is poorly understood, but its permeability can be regulated by long myosin light chain kinase splice variant 1 (MLCK1)125,140,141,157. MLCK1 phosphorylates myosin regulatory light chain to trigger endocytosis of the tight junction protein occludin, leading to an increase in tight junction permeability (see the figure, bottom left). MLCK1 expression‎ and enzymatic activity can be activated by cytokines that include IL-1β and tumour necrosis factor (TNF)124,125,135,140,158,295. Altered expression‎ of other tight junction proteins, including tricellulin or angulin 1, might also modify leak pathway permeability55,146,147,296.

The unrestricted pathway refers to the diffusion of material across regions that lack a continuous epithelial barrier owing to epithelial cell damage or death (see the figure, bottom right). This route is independent of tight junctions, as they are either absent or severely damaged at these sites. The unrestricted pathway allows flux of very large molecules and even intact bacteria.

In summary, the pore pathway is a high-capacity pathway that is exquisitely charge-selective and size-selective, whereas the leak pathway is a low-capacity pathway, is not charge-selective, and, although size-selective, allows flux of molecules 20-fold larger than those accommodated by the pore pathway. Thus, the two tight-junction-dependent pathways are complementary. The unrestricted pathway is tight-junction-independent, high-capacity and non-selective.

박스 1 기공, 누출 및 제한상실 투과성 경로.

장 투과성은 세 가지 뚜렷한 경로의 기여도를 반영할 수 있습니다:

기공 경로,

누출 경로,

제한상실 경로(그림 참조).

기공과 누출 경로는

단단한 접합부를 통과하는 흐름을 반영하는

반면, 무제한 경로는 단단한 접합부와 무관합니다.

기공 통로 투과성은

클라우딘 단백질에 의해 결정되는데,

이 단백질은 기공 형성 또는 장벽을 형성하는

단단한 접합부291-294를 형성합니다.

기공을 형성하는 클라우딘에 의해 생성된 기공은

전하 선택적이며 크기 선택적입니다.

이를 통과할 수 있는 용질의 최대 직경은 0.6nm입니다.

클라우딘은

위장관에서 양이온 선택성 채널만 형성하지만,

음이온 선택성 클라우딘 채널은 신장 같은 다른 부위에도 존재합니다.

IL-13과 IL-22를 포함한 면역 신호는

장 상피 세포의 클라우딘-2의 전사와 발현을 증가시켜,

기공 통로 투과성을 증가시킵니다(위 그림 참조)45,50,86.

밀접 접합 누출 통로는

직경이 최대 ~12.5nm인 분자가 상피 장벽을 통과할 수 있도록 합니다.

누출 통로의 분자 구조는 잘 알려져 있지 않지만,

긴 미오신 경쇄 키나제 스플라이스 변이체 1(MLCK1)125,140,141,157에 의해

그 투과성이 조절될 수 있습니다.

MLCK1은 미오신 조절 경쇄를 인산화하여, 꽉 조이는 접합 단백질인 오클루딘의 내포성 이입을 촉발함으로써, 꽉 조이는 접합 투과성을 증가시킵니다(그림의 왼쪽 아래 참조). MLCK1의 발현과 효소 활성은 IL-1β와 종양 괴사 인자(TNF)를 포함하는 사이토카인에 의해 활성화될 수 있습니다124,125,135,140,158,295. 트리셀룰린(tricellulin) 또는 앵굴린 1(angulin 1)을 포함한 다른 타이트 접합 단백질의 발현 변화도 누출 경로 투과성을 변화시킬 수 있습니다55,146,147,296.

제한상실 경로는

상피 세포의 손상이나 죽음으로 인해

연속적인 상피 장벽이 없는 영역을 가로지르는 물질의 확산(그림 오른쪽 아래 참조)을

의미합니다.

이 경로는 타이트 접합과 무관합니다.

왜냐하면 이러한 부위에는

타이트 접합이 없거나 심하게 손상되었기 때문입니다.

제한되지 않은 통로는

매우 큰 분자, 심지어는 온전한 박테리아의 흐름을 허용합니다.

요약하자면,

기공 통로는 정교하게 전하 선택적이며 크기 선택적인 고용량 통로인 반면,

누출 통로는 저용량 통로이고, 전하 선택적이지 않으며,

크기 선택적이기는 하지만 기공 통로에 수용되는 분자보다 20배 더 큰 분자의 흐름을 허용합니다.

따라서,

두 개의 긴밀 접합 의존적 경로는 상호 보완적입니다.

제한 없는 경로는 긴밀 접합 독립적이며,

용량이 크고 비선택적입니다.

Adapted from ref. 297, Springer Nature Limited.

Intestinal permeability in disease

Intestinal permeability is increased — that is, barrier function is reduced — in many intestinal and systemic diseases (Table 1). Of these diseases, the most well studied intestinal disorders are inflammatory bowel disease (IBD) and coeliac disease. Although tight junction permeability is increased in IBD, the extensive barrier loss seen in advanced, active disease is more likely to reflect epithelial damage12–16. Similarly, increases in intestinal permeability in advanced graft-versus host disease (GVHD) can reflect tight junction regulation or immune-mediated epithelial damage17. Although discrimination between these disparate mechanisms of barrier loss is possible, most studies have relied on the use of only a single probe, such as fluorescein isothiocyanate–4-kDa dextran in mouse models, to measure permeability. As a result, the data are insufficent to differentiate between leak pathway and unrestricted pathway flux. Thus, correlations observed between the extent of barrier loss and the severity of disease in IBD, coeliac disease and GVHD are most likely to be secondary to epithelial damage. By contrast, increased intestinal permeability in Crohn’s disease during remission is more likely to reflect increased tight junction permeability18,19, particularly as increased permeability can occur up to 1 year before disease reactivation. Notably, psychological stress, which can increase intestinal permeability in rodents, is a risk factor for reactivation of Crohn’s disease in patients20,21.

질병에 따른 장 투과성

장 투과성이 증가(장벽 기능이 감소)하는 것은

많은 장 및 전신 질환에서 관찰됩니다(표 1).

이러한 질병들 중에서 가장 잘 연구된 장 질환은

염증성 장 질환(IBD)과 셀리악 병입니다.

IBD에서 장 접합부 투과성이 증가하지만,

진행성 활성 질환에서 나타나는 광범위한 장벽 손실은

상피 손상을 반영할 가능성이 더 큽니다12-16.

마찬가지로, 진행성 이식편 대 숙주병(GVHD)에서 장 투과성의 증가는 밀접 접합 조절 또는 면역 매개 상피 손상을 반영할 수 있습니다17. 이러한 서로 다른 장벽 손실 메커니즘을 구분하는 것이 가능하지만, 대부분의 연구는 투과성을 측정하기 위해 마우스 모델에서 플루오레세인 이소티오시아네이트-4-kDa 덱스트란과 같은 단일 프로브만 사용했습니다.

그 결과,

누출 경로와 제한되지 않은 경로 흐름을 구분하기에는

데이터가 충분하지 않습니다.

따라서 장벽 손실 정도와 IBD, 셀리악 병, GVHD의 질병 중증도 사이의 상관관계는 상피 손상에 이차적인 것으로 보입니다.

대조적으로,

크론병의 장 투과성 증가는

특히 투과성 증가가 질병 재발 1년 전부터 발생할 수 있다는 점에서,

강화된 접합부 투과성18,19을 반영할 가능성이 더 큽니다.

특히, 설치류에서 장 투과성을 증가시킬 수 있는 심리적 스트레스는 크론병 환자의 재발 위험 요소입니다20,21.

Table 1.

Diseases associated with intestinal barrier defects

DiseaseFindings in patientsFindings in animal models

IBD (Crohn’s disease and ulcerative colitis)	Increased permeability is a risk factor for IBD in healthy relatives of patients with Crohn’s disease and for relapse in patients with Crohn’s disease18,19,22,24,174,182,183	Il10-knockout mice develop a permeability defect before disease onset; experimental IBD is more severe in genetically modified mice with increased intestinal permeability; genetic or pharmacological reduction of pore pathway or leak pathway function limits the severity of experimental IBD59,98,117,134,141,163,184,185
Graft-versus-host disease	Positive correlation of pre-conditioning gastrointestinal toxicity (presumed to indicate degree of transient barrier loss) with disease activity17,186	Gut damage is an essential driver of experimental disease; intestinal permeability is increased and tight junction protein organization is altered in experimental disease; disease progression and severity are attentuated in long MLCK-knockout mice137,187–191
Type 1 diabetes mellitus	Permeability is increased in patients with pre-diabetes and diabetes mellitus192–194	Permeability increases precede disease; barrier restoration can delay disease onset195,196
Metabolic syndrome (including type 2 diabetes mellitus, obesity and nonalcoholic fatty liver disease)	Increased intestinal permeability is a risk factor for type 2 diabetes mellitus and is associated with obesity and nonalcoholic fatty liver disease194,197–201	Hyperglycaemia, high-fat diet, nonalcoholic fatty liver disease and obesity are associated with increased intestinal permeability202–208
HIV/AIDS	Increased permeability in HIV enteropathy; positive correlation with disease stage; increased in patients with untreated HIV infection90,209,210	Increased intestinal permeability in simian immunodeficiency virus infection is associated with microbial translocation and systemic immune activation211,212
Multiple organ dysfunction syndrome	Correlates with increased disease severity213	Associated with shock; disease progression is limited in knockout mice that are protected from leak pathway permeability increases214–217
IBS	Increased in diarrhoea-predominant, post-infectious and non-post-infectious IBS; unaltered in constipation-predominant IBS28,218–229	Increased intestinal permeability is associated with and can cause changes in visceral sensitivity31,230,231
Coeliac disease	Positive correlation between increased permeability and disease activity; increased permeability in patients and healthy relatives; gluten-free diet can lead to barrier restoration232–235	Barrier loss is associated with disease in models of coeliac disease236–238
Environmental enteric dysfunction	Increased permeability; altered expression‎ of absorptive and barrier-enhancing proteins239–247	Barrier loss is associated with malnutrition240,248,249
Food allergy	Increased in people with food allergy250–252	Permeability increased in mice after food antigen challenge253–257
Sepsis	Increased permeability in sepsis; increased plasma zonulin258–261	Permeability increased in experimental sepsis; relationship to disease progression is not defined262–266
SARS-CoV-2 infection	Permeability is increased in severe systemic disease; some data suggest that barrier restoration using a zonulin antagonist is beneficial267–276	No direct measures of intestinal permeability
Parkinson disease	Permeability increased in a subset of patients277,278	No direct measures of intestinal permeability
Asthma	Permeability increased in people with asthma; IBD associated with increased risk of asthma279,280	Correlation between disease and intestinal permeability in some models281,282
Multiple sclerosis and amyotrophic lateral sclerosis	Increased intestinal permeability in a subset of patients283–285	Increased intestinal permeability in experimental allergic encephalitis model286
Rheumatic diseases (arthritis and ankylosing spondylitis)	Increased intestinal permeability in some patients287,288	Increased permeability in mouse models; barrier restoration can limit disease287,289,290

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IBD, inflammatory bowel disease; IBS, irritable bowel syndrome; MLCK, myosin light chain kinase.

Reports that some healthy first-degree relatives of patients with Crohn’s disease have modest intestinal barrier dysfunction led to the hypothesis that loss of intestinal barrier function is a primary event in Crohn’s disease pathogenesis. The idea that loss of barrier function is an early pathogenic event is supported by evidence that increased intestinal permeability in healthy first-degree relatives of patients with Crohn’s disease is associated with the risk-associated NOD2 3020insC polymorphism22. Perhaps the most convincing evidence comes from another study of healthy relatives of patients with Crohn’s disease, in which the risk of developing disease was twofold to threefold higher among those with increased intestinal permeability than among those with normal permeability23.

In healthy relatives of patients with Crohn’s disease, increased intestinal permeability was also associated with reduced microbial diversity and alterations in specific genera and microbial metabolic pathways24. By contrast, faecal calprotectin — a marker of mucosal inflammation but not of barrier loss — was increased in some healthy relatives but was not an independent risk factor for development of Crohn’s disease25. Reports of increased intestinal permeability up to 3 years before onset of Crohn’s disease suggest that barrier loss is the initial trigger that activates intermediate events, such as mucosal immune activation, that culminate in disease. Together, these observations suggest that barrier loss is a primary event in Crohn’s disease pathogenesis.

Some data suggest that increased intestinal permeability is associated with poorly understood conditions, including irritable bowel syndrome and autism spectrum disorders26–30. The mechanistic underpinnings of these associations have not been defined and, in both patients and animal models, the question of whether barrier loss is a cause or consequence of disease remains. The observation that mice with genetically induced increases in intestinal permeability develop anxiety-like behaviours, hyporesponsiveness to rectal distension and activation of neurons within the thalamus, hypothalamus and hippocampus31 demonstrates that increased intestinal permeability can effect changes in behaviour, visceral sensation and neurological activity. These mice also have an altered microbiome composition.

IBD, 염증성 장 질환; IBS, 과민성 대장 증후군; MLCK, 미오신 경쇄 키나제.

크론병 환자의 건강한 1차 친척 중 일부가 장벽 기능 장애가 있는 것으로 보고된 결과,

장벽 기능의 상실이

크론병 발병의 주요 원인이라는 가설이 제기되었습니다.

장벽 기능의 상실이 초기 발병 원인이라는 생각은

크론병 환자의 건강한 1차 친척의 장 투과성 증가가 위험 관련 NOD2 3020insC 다형성22와 관련이 있다는

증거에 의해 뒷받침됩니다.

아마도 가장 설득력 있는 증거는

크론병 환자의 건강한 친척을 대상으로 한

또 다른 연구에서 나온 것입니다.

이 연구에 따르면

장 투과성이 증가한 사람들은

투과성이 정상인 사람들보다 질병 발병 위험이 2~3배 더 높았습니다23.

크론병 환자의 건강한 친척에서 장 투과성 증가는

미생물 다양성의 감소 및 특정 속과 미생물 대사 경로의 변화와도 관련이 있었습니다24.

이에 반해, 장내 칼프로텍틴(faecal calprotectin)은

점막 염증의 지표이지만 장벽 손실의 지표는 아니며,

일부 건강한 친척의 경우 증가했지만

크론병 발병의 독립적인 위험 요인은 아니었습니다25.

크론병 발병 3년 전까지 장 투과성이 증가했다는 보고는

장벽 손실이 점막 면역 활성화와 같은 중간 사건을 활성화하여

질병을 유발하는 초기 유발 요인임을 시사합니다.

이러한 관찰 결과는

장벽 손실이 크론병 발병 기전에서

중요한 사건임을 시사합니다.

일부 데이터에 따르면 장 투과성 증가는 과민성 대장 증후군과 자폐 스펙트럼 장애를 포함한 잘 알려지지 않은 상태와 관련이 있는 것으로 나타났습니다26-30. 이러한 연관성의 메커니즘은 아직 밝혀지지 않았으며, 환자 및 동물 모델 모두에서 장벽 손실이 질병의 원인인지 결과인지에 대한 의문이 남아 있습니다. 유전적으로 장 투과성이 증가한 생쥐가 불안과 유사한 행동, 직장 팽창에 대한 반응성 저하, 시상, 시상하부, 해마 내 뉴런 활성화가 나타난다는 사실은 장 투과성 증가가 행동, 내장 감각, 신경 활동에 변화를 일으킬 수 있음을 보여줍니다31. 이 생쥐들은 또한 미생물 군집 구성이 변경되었습니다.

Tight junctions and intestinal barrier function

In the absence of epithelial damage, the tight junction is the rate-limiting determinant of passive paracellular transport. At the tight junction, the intercellular spaces between adjoining cells are eliminated and the outer leaflets of the plasma membrane lipid bilayer of adjacent epithelial cells are closely apposed and appear to fuse32 (Fig. 1). Subapical to the tight junction are the adherens junctions and desmosomes, which are linked to actin-based microfilaments and cytokeratin-based intermediate filaments, respectively. These cytoskeletal structures provide the tensile strength that supports tight junctions and maintains cell shape.

밀접 접합과 장벽 기능

상피 손상이 없는 경우,

밀접 접합은 수동적 세포간 수송의 속도 제한 결정 요인입니다.

밀접 접합에서,

인접한 세포 사이의 세포 간 공간이 제거되고

인접한 상피 세포의 원형질막 지질 이중층의 외엽이 밀접하게 부착되어 융합된 것처럼 보입니다32 (그림 1).

조밀한 접합부 위쪽에는 접착 접합부와 접합 소체가 있으며,

이들은 각각 액틴 기반 미세 섬유와 사이토케라틴 기반 중간 섬유에 연결되어 있습니다.

이러한 세포 골격 구조는 조밀한 접합부를 지지하고 세포의 형태를 유지하는 인장 강도를 제공합니다.

Fig. 1. Tight junction structure and morphology.

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a, Transmission electron micrograph showing the tight junction, adherens junction and desmosome, which, together, comprise the apical junctional complex. The tight junction is located just below the base of the microvilli. The magnification shows the transition from the luminal space, between the microvilli, into the tight junction, where morphologically detectable paracellular space is obliterated. b, Schematic of the apical junctional complex shown in part a, showing the location of the tight junction proteins zonula occludens 1 (ZO-1), occludin, claudin-2 and other claudin family members. Long myosin light chain kinase 1 (MLCK1) is associated with perijunctional F-actin and is a key regulator of tight junction permeability. c, Freeze–fracture electron micrograph showing tight junction strands at the base of apical microvilli.

a, 정점 접합 복합체를 구성하는 밀접 접합, 부착 접합, 데스모좀을 보여주는 투과 전자 현미경 사진. 밀접 접합은 미세 융모의 기저 바로 아래에 위치합니다. 확대된 사진은 미세 융모 사이의 내강 공간에서 형태학적으로 감지 가능한 세포간 공간이 사라지는 밀접 접합부로 이행하는 과정을 보여줍니다. b, 부분 a에 표시된 정단 접합 복합체의 도식도로, 밀접 접합 단백질인 조누라 오클루덴스 1(ZO-1), 오클루딘, 클라우딘-2 및 기타 클라우딘 계열 구성원의 위치를 보여줍니다. 긴 미오신 경쇄 키나아제 1(MLCK1)은 접합부 주변의 F-액틴과 관련이 있으며, 조임 접합부 투과성의 핵심 조절자입니다. c, 정지-파단 전자 현미경 사진으로 정점 미세 융모의 기저부에서 조임 접합부 가닥을 보여줍니다.

Tight junction pathways and proteins

The first tight junction protein to be discovered was zonula occludens 1 (ZO-1)33 (Fig. 1), followed by the two related proteins ZO-2 and ZO-3, and the unrelated protein cingulin34–37, though all of these proteins are intracellular peripheral membrane proteins. These discoveries were followed by the discovery of the tetraspan transmembrane tight junction proteins occludin38 and the claudins, which are encoded by 27 genes in mammals39,40.

When expressed in non-epithelial cells, claudins can self-assemble into large polymers to form structures that are reminiscent of tight junction strands seen by freeze–fracture electron microscopy41 (Fig. 1). Many claudins are critical for barrier function, but others form charge-selective and size-selective paracellular channels. The ensemble of expressed claudins dictates the size-selectivity and charge-selectivity of specific sites within tissues. Detailed characterization has demonstrated that the charge-selectivity of claudin channels is determined by specific residues within the first extracellular loop42,43. Regardless of whether they are cation-selective or anion-selective, all claudin channels studied to date are size-selective and allow paracellular flux of only molecules with a diameter of <0.6 nm (refs. 43–46). These channels define the pore pathway47 (Box 1) and are exemplified by claudin-2, which mediates paracellular flux of small cations, such as Na+, and water42,43,48,49. Claudin-2 cannot, however, accommodate the commonly used macromolecular probes lactulose, mannitol or 4 kDa dextran48,50,51, emphasizing the need to consider the physical characteristics of the solute being measured when assigning mechanisms of changes in paracellular permeability.

Although rejected by the pore pathway, molecules larger than 0.6 nm, including lactulose, mannitol and 4 kDa dextran, can cross tight junctions via a second paracellular flux route known as the leak pathway (Box 1), which can accommodate molecules with diameters of up to ~12.5 nm and is not charge-selective47,52. The specific molecular structure of the leak pathway has not been identified, but its permeability is regulated by occludin53,54, tricellulin (also known as MARVEL domain-containing protein 2, a member of the tight junction-associated MARVEL protein (TAMP) family)55, ZO-1 (refs. 52,56) and perijunctional actomyosin53,57–59.

단단한 접합 경로와 단백질

처음으로 발견된 단단한 접합 단백질은 zonula occludens 1(ZO-1)33(그림 1)이고, 그 다음으로 두 개의 관련 단백질인 ZO-2와 ZO-3, 그리고 관련이 없는 단백질인 cingulin34-37이 발견되었지만, 이 모든 단백질은 세포 내 주변막 단백질입니다. 그 후, 포유류에서 27개의 유전자에 의해 암호화되는 4중막 막관통 꽉 조이는 접합 단백질인 오클루딘38과 클라우딘이 발견되었습니다39,40.

상피세포가 아닌 세포에서 발현될 때, 클라우딘은 큰 폴리머로 자기 조립되어 전자 현미경으로 관찰했을 때, 단단한 접합 가닥을 연상시키는 구조를 형성합니다41 (그림 1). 많은 클라우딘은 장벽 기능에 중요하지만, 다른 클라우딘은 전하 선택적 및 크기 선택적 세포간 통로를 형성합니다. 발현된 클라우딘의 집합체는 조직 내 특정 부위의 크기 선택성과 전하 선택성을 결정합니다. 상세한 특성 분석에 따르면, 클라우딘 채널의 전하 선택성은 첫 번째 세포외 고리42,43 내의 특정 잔기에 의해 결정된다는 사실이 밝혀졌습니다. 양이온 선택성인지 음이온 선택성인지에 관계없이, 지금까지 연구된 모든 클라우딘 채널은 크기 선택적이며, 직경이 0.6nm 미만인 분자만 세포간 유출을 허용합니다(참고문헌 43-46). 이 채널들은 기공 통로를 정의합니다47 (박스 1) 그리고 클라우딘-2가 그 예입니다. 클라우딘-2는 Na+와 같은 작은 양이온과 물의 세포간 유출을 매개합니다42,43,48,49. 그러나 클라우딘-2는 일반적으로 사용되는 고분자 프로브인 락툴로오스, 만니톨 또는 4kDa 덱스트란48,50,51을 수용할 수 없으므로, 세포간 투과성의 변화 메커니즘을 할당할 때 측정되는 용질의 물리적 특성을 고려해야 한다는 점을 강조합니다.

모공 경로를 통해서는 통과할 수 없지만, 락툴로오스, 만니톨, 4kDa 덱스트런을 포함한 0.6nm보다 큰 분자들은 누출 경로(Box 1)로 알려진 두 번째 세포간 플럭스 경로를 통해 단단한 접합부를 통과할 수 있습니다. 이 경로는 직경이 최대 ~12.5nm인 분자를 수용할 수 있으며, 전하 선택적이지 않습니다47,52. 누출 경로의 구체적인 분자 구조는 밝혀지지 않았지만, 그 투과성은 occludin53,54, tricellulin(MARVEL domain-containing protein 2로도 알려져 있으며, Tight junction-associated MARVEL protein(TAMP) 계열의 일종)55, ZO-1(참고문헌 52,56) 및 perijunctional actomyosin53,57-59에 의해 조절됩니다.

Pore and leak pathway functions in health

The major pore-forming claudins expressed within the intestinal epithelium are claudin-2 and claudin-15 (refs. 9,60), both of which form cation-selective channels49,61,62. Mice that lack either claudin-2 or claudin-15 are viable, but mice that lack both claudins die before weaning9, consistent with the idea that these proteins are, at least partially, functionally redundant. Nevertheless, intestinal hypertrophy occurs in claudin-15-knockout, but not claudin-2-knockout, mice63,64.

Paracellular Na+ efflux via claudin-2 and claudin-15 channels is critical to transcellular nutrient transport. Brush border absorption is largely driven by Na+–nutrient cotransporters that rely on the Na+ gradient between the intestinal lumen and the cytoplasm of epithelial cells (Fig. 2). During this process, Na+ enters the cytoplasm and is exported across the basolateral membrane into the lamina propria by the Na+–K+ ATPase65. In the absence of paracellular Na+ transport, transcellular transport rapidly depletes luminal Na+ and nutrient cotransport across the apical brush border membrane stops. Flux across claudin-2 and claudin-15 channels allows Na+ efflux from the lamina propria to the lumen, where it can drive additional cycles of Na+–nutrient cotransport.

건강에 있어서 기공과 누출 통로 기능

장 상피 내에서 발현되는 주요 기공 형성 클라우딘은 클라우딘-2와 클라우딘-15(참고문헌 9,60)이며, 이 두 가지 모두 양이온 선택적 채널을 형성합니다49,61,62. 클라우딘-2 또는 클라우딘-15가 결핍된 생쥐는 생존할 수 있지만, 두 클라우딘이 모두 결핍된 생쥐는 이유기 전에 죽습니다9. 이는 이 단백질들이 적어도 부분적으로는 기능적으로 중복된다는 생각과 일치합니다. 그럼에도 불구하고, 클라우딘-15 결핍 생쥐에서는 장 비대가 발생하지만, 클라우딘-2 결핍 생쥐에서는 발생하지 않습니다63,64.

클라우딘-2와 클라우딘-15 채널을 통한 세포간 Na+ 유출은 세포간 영양소 수송에 매우 중요합니다. 융모막 흡수는 주로 장 내강과 상피 세포의 세포질 사이의 Na+ 구배에 의존하는 Na+ 영양 공동 수송체에 의해 주도됩니다(그림 2). 이 과정에서 Na+는 세포질로 들어가서 Na+-K+ ATPase65에 의해 기저막을 가로질러 실질층으로 수출됩니다. 세포간 Na+ 수송이 없을 경우, 세포내 수송은 관강 내 Na+를 빠르게 고갈시키고, 정점 브러시 경계막을 가로지르는 영양분 공동 수송은 중단됩니다. 클라우딘-2와 클라우딘-15 채널을 통한 플럭스는 층상피에서 내강으로 Na+의 유출을 허용하며, 이로 인해 Na+와 영양소의 공동 수송의 추가적인 사이클이 촉진될 수 있습니다.

Fig. 2. Coordination of transcellular and paracellular transport.

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The gradient of Na+ between the gut lumen and the cytoplasm of epithelial cells provides the driving force for nutrient absorption across the apical brush border membrane, such as glucose absorption via the intestinal epithelial Na+–glucose cotransporter SGLT1. Nutrients then exit the cell via facilitated exchange proteins, such as the glucose transporter GLUT2, and Na+ exits via the Na+–K+ ATPase. Na+–glucose cotransport also triggers signal transduction pathways that activate long myosin light chain kinase 1 (MLCK1) and increase tight junction permeability. The osmotic gradient generated by transcellular nutrient and Na+ transport draws water across the tight junction and, owing to the high concentration of nutrient monomers in the unstirred layer, nutrients are carried along with this fluid in a mechanism known as solvent drag71. This process would quickly exhaust luminal Na+ if not for claudin-2 and claudin-15, which form paracellular Na+ channels that enable efflux of absorbed Na+ in order to provide the driving force for continued transcellular nutrient absorption9,63.

The requirement for Na+ efflux in the intestine explains the critical roles of claudin-2 and claudin-15 in nutrient absorption but does not explain why expression‎ of these claudins is so precisely regulated. At birth, intestinal epithelial claudin-2 expression‎ is high throughout the crypt–villus axis of the intestinal epithelium60,63,66, but claudin-2 expression‎ is markedly diminished and limited to crypt epithelium after weaning. Concurrently, claudin-15 expression‎ increases throughout the crypt–villus axis. Although only subtle functional differences between these claudins have been detected in vitro48,62, this precise developmental regulation suggests that the in vivo properties of claudin-2 and claudin-15 channels must differ substantially. Hypothetically, claudin-2 might allow greater paracellular Na+ flux than claudin-15, which would be advantageous for the rapid growth that occurs during early postnatal development and depends on Na+–nutrient cotransport. Alternatively, channel open state probabilities or other subtle functional characteristics of claudin-2 and claudin-15 might differ. This question could be resolved by single-channel analysis but, unfortunately, such data have only been reported for claudin-2 (ref. 46).

Na+–nutrient cotransport triggers downstream signalling in the epithelial cell. These signalling events activate MLCK, which phosphorylates perijunctional myosin regulatory light chain (MLC) and increases leak pathway permeability67–69. This process amplifies paracellular nutrient transport via solvent drag (Fig. 2), whereby the osmotic gradient created by transcellular transport drives paracellular water absorption70. The absorbed water comes from the unstirred layer immediately adjacent to the epithelium, which is rich in small nutrient monomers owing to the activity of brush border digestive enzymes. This paracellular fluid absorption allows paracellular nutrient absorption to amplify transcellular transport71–74. Importantly, solvent drag only contributes substantially to nutrient absorption when luminal concentrations of nutrient monomers are high72. This subtlety probably explains why passive paracellular nutrient absorption via solvent drag has been detected in some studies and not others75–84. In contrast to transcellular transport, paracellular absorption via solvent drag is not stereospecific and can accommodate molecules for which there are no apical transporters, such as mannitol72,85.

Paracellular amplification of transcellular absorption, or solvent drag, probably explains why the rate of nutrient transport across the intestinal epithelium cannot be saturated72. Clinically, solvent drag contributes to the efficacy of simple Na+ and carbohydrate-containing oral rehydration solutions that have been used to treat countless individuals with potentially fatal, high-volume diarrhoeal diseases, such as cholera. By contrast, the glycosuria that occurs in diabetes mellitus suggests that no corresponding paracellular pathway for glucose resorption exists within the renal tubule.

장 내강과 상피 세포의 세포질 사이의 Na+의 농도 차이는 장 상피의 Na+–포도당 공동 수송체 SGLT1을 통한 포도당 흡수와 같은 정점 브러시 경계막을 통한 영양소 흡수를 위한 추진력을 제공합니다. 그런 다음 영양소는 포도당 수송체 GLUT2와 같은 촉진 교환 단백질을 통해 세포를 빠져나가고, Na+는 Na+-K+ ATPase를 통해 빠져나갑니다. Na+–포도당 공동 수송은 또한 긴 미오신 경쇄 키나아제 1(MLCK1)을 활성화하고, 꽉 조인 접합부의 투과성을 증가시키는 신호 전달 경로를 촉발합니다. 세포 간 영양분과 Na+ 수송에 의해 생성된 삼투압 구배는 꽉 조인 접합부를 통해 물을 끌어들이고, 교반되지 않은 층에 있는 영양분 단량체의 높은 농도 때문에, 용매 저항이라는 메커니즘에 따라 이 유체와 함께 영양분이 운반됩니다71. 이 과정은 흡수된 Na+의 유출을 가능하게 하는 세포간 Na+ 채널을 형성하는 claudin-2와 claudin-15가 없었다면 관상 Na+를 빠르게 고갈시켰을 것입니다. 이 세포간 Na+ 채널은 지속적인 세포간 영양소 흡수를 위한 추진력을 제공하기 위해 흡수된 Na+의 유출을 가능하게 합니다9,63.

장내 Na+ 유출에 대한 요구는 영양소 흡수에서 클라우딘-2와 클라우딘-15의 중요한 역할을 설명하지만, 왜 이 클라우딘들의 발현이 그렇게 정밀하게 조절되는지는 설명하지 못합니다. 출생 시, 장 상피 클라우딘-2의 발현은 장 상피의 크립토빌러스 축 전체에 걸쳐서60,63,66 높게 나타나지만, 이유 후에는 클라우딘-2의 발현이 현저하게 감소하고 크립토빌러스 상피로 제한됩니다. 동시에, 클라우딘-15 발현은 크립토빌러스 축 전체에서 증가합니다. 체외에서 이들 클라우딘 간의 미묘한 기능적 차이만이48,62 발견되었지만, 이러한 정확한 발달 조절은 클라우딘-2와 클라우딘-15 채널의 생체 내 특성이 상당히 달라야 함을 시사합니다. 가설적으로, 클라우딘-2는 클라우딘-15보다 더 큰 세포간 Na+ 유입을 허용할 수 있으며, 이는 출생 후 초기 발달 과정에서 발생하는 빠른 성장에 유리하며, Na+ 영양 이송에 의존합니다. 또는, 채널 개방 상태 확률 또는 클라우딘-2와 클라우딘-15의 다른 미묘한 기능적 특성이 다를 수 있습니다. 이 질문은 단일 채널 분석을 통해 해결할 수 있지만, 안타깝게도 이러한 데이터는 claudin-2(참고 46)에 대해서만 보고되었습니다.

Na+–영양 이송은 상피 세포에서 하류 신호 전달을 유발합니다. 이러한 신호 전달은 MLCK를 활성화시켜, 접합부 근위부 미오신 조절 경쇄(MLC)를 인산화시키고 누출 경로 투과성을 증가시킵니다67–69. 이 과정은 용매 끌기를 통한 세포간 영양소 수송을 증폭시킵니다(그림 2). 세포간 수송에 의해 생성된 삼투구배가 세포간 수분 흡수를 촉진합니다70. 흡수된 물은 상피 바로 옆에 있는 교반되지 않은 층에서 나오는데, 이 층은 소형 영양소 단량체가 풍부합니다. 이는 브러시 보더 소화 효소의 활동으로 인해 발생합니다. 이 세포간 수분 흡수는 세포간 영양소 흡수를 증폭시켜 세포간 수송을 촉진합니다71–74. 중요한 것은 용매 끌기가 영양소 단량체의 관내 농도가 높을 때만 영양소 흡수에 크게 기여한다는 사실입니다72. 이 미묘한 차이로 인해 일부 연구에서는 용매 끌기를 통한 수동적 세포간 영양소 흡수가 발견되었지만, 다른 연구에서는 발견되지 않았습니다75-84. 세포간 수송과는 달리, 용매 끌기를 통한 세포간 흡수는 입체 특이적이지 않으며, 만니톨과 같은 정점 수송체가 없는 분자를 수용할 수 있습니다72,85.

세포간 흡수 또는 용매 끌기의 세포간 증폭은 장 상피를 통한 영양소 수송 속도가 포화 상태에 이를 수 없는 이유를 설명해 줄 수 있을 것입니다72. 임상적으로, 용매의 끌림은 콜레라와 같이 치명적일 수 있는 다량의 설사 질환을 앓고 있는 수많은 사람들을 치료하는 데 사용되어 온 단순한 Na+ 및 탄수화물 함유 경구 재수화 용액의 효능에 기여합니다. 이와는 대조적으로, 당뇨병에서 발생하는 당뇨는 신장 세뇨관 내에 포도당 재흡수를 위한 상응하는 세포간 경로가 존재하지 않음을 시사합니다.

The pore pathway in disease

Intestinal epithelial claudin-2 has been the subject of intense scrutiny owing to its markedly increased expression‎ in a broad range of inflammatory disorders. Claudin-2 upregulation was first described in the contexts of ulcerative colitis and Crohn’s disease86,87. Subsequent work demonstrated that intestinal epithelial claudin-2 is also upregulated in coeliac disease88, irritable bowel syndrome89, HIV enteropathy90, enteric infection50, necrotizing enterocolitis91 and Whipple disease92. The factors that mediate claudin-2 upregulation are incompletely characterized, but in vitro and in vivo studies have implicated IL-1, IL-6, IL-13, IL-22 and tumour necrosis factor (TNF) as potential enhancers of claudin-2 expression‎50,86,93–96. By contrast, some in vitro studies suggest that butyrate can suppress claudin-2 expression‎ and increase barrier function via an IL-10 receptor-dependent mechanism97. Together, these data indicate that the mucosal immune system can fine-tune claudin-2 expression‎.

Administration of recombinant IL-13 to mice increases claudin-2 expression‎ and augments intestinal paracellular cation permeability98. Similarly, transgenic claudin-2 overexpression‎ within the intestinal epithelium increases paracellular permeability to cations to levels similar to those in IL-13-treated wild-type mice98. By contrast, IL-13 has no effect on intestinal permeability in mice in which the Cldn2 gene, which encodes claudin-2, is knocked out. Thus, claudin-2 upregulation is both necessary and sufficient to increase paracellular cation permeability in vivo. The impact of this effect on disease is discussed in the following sections.

질병의 기공 경로

장 상피 세포의 클라우딘-2는 광범위한 염증성 질환에서 현저하게 증가된 발현으로 인해 집중적인 조사의 대상이 되어 왔습니다. 클라우딘-2의 상향 조절은 궤양성 대장염과 크론병의 맥락에서 처음 설명되었습니다86,87. 이후의 연구에 따르면 장 상피 세포의 클라우딘-2도 셀리악 병88, 과민성 대장 증후군89, HIV 장병90, 장 감염50, 괴사성 장염91, 휘플병92에서 상향 조절된다는 사실이 밝혀졌습니다. 클라우딘-2의 상향 조절을 매개하는 요인은 아직 완전히 밝혀지지 않았지만, 체외 및 체내 연구에서 IL-1, IL-6, IL-13, IL-22 및 종양 괴사 인자(TNF)가 클라우딘-2 발현을 촉진하는 잠재적 인자로 밝혀졌습니다50,86,93–96. 반면에, 일부 시험관 내 연구에서는 부티레이트가 IL-10 수용체 의존성 메커니즘을 통해 클라우딘-2 발현을 억제하고 장벽 기능을 증가시킬 수 있다고 합니다97. 종합해 보면, 이러한 데이터는 점막 면역 체계가 클라우딘-2 발현을 미세 조정할 수 있음을 나타냅니다.

재조합 IL-13을 생쥐에게 투여하면 클라우딘-2 발현이 증가하고 장내 세포간 양이온 투과성이 증가합니다98. 마찬가지로, 장 상피 내에서 클라우딘-2의 유전자 변형이 과발현되면, IL-13을 투여한 야생형 생쥐와 유사한 수준으로 세포간 양이온 투과성이 증가합니다98. 반면, IL-13은 클라우딘-2를 암호화하는 Cldn2 유전자가 제거된 쥐의 장 투과성에 영향을 미치지 않습니다. 따라서, 클라우딘-2의 상향 조절은 생체 내 세포간 양이온 투과성을 증가시키는 데 필요하고도 충분합니다. 이 효과가 질병에 미치는 영향은 다음 섹션에서 논의됩니다.

Claudin-2 attenuates diseases induced by luminal insults

A pair of studies of Cldn2-transgenic and Cldn2-knockout mice provided initial evidence that increased claudin-2 expression‎ is beneficial in dextran sulfate sodium (DSS)-induced colitis98,99. However, claudin-2 overexpression‎ also increases faecal water content50, suggesting that increased claudin-2 expression‎ might reduce the severity of colitis simply by diluting DSS within the distal colon. An alternative possibility is that claudin-2 expression‎ promotes epithelial growth and mucosal repair100–102, as discussed below (see ‘Claudin-2 and epithelial proliferation’).

Claudin-2 expression‎ is increased during enteric infection in humans103. Similarly, the model pathogen Citrobacter rodentium triggers IL-22-dependent claudin-2 upregulation within 2 days of infection in mice50 (Fig. 3). Although IL-22 is pleiotropic, an increased number of mucosa-associated C. rodentium, delayed pathogen clearance and a greater severity of mucosal damage in Cldn2-knockout mice relative to wild-type mice demonstrate that IL-22-dependent claudin-2 upregulation contributes to host defence50. The observation that transgenic claudin-2 overexpression‎ limits C. rodentium-induced colitis provides further support for this conclusion.

클라우딘-2는 관 내 자극에 의해 유발되는 질병을 완화합니다.

Cldn2-transgenic과 Cldn2-knockout 마우스를 대상으로 한 두 가지 연구에서 클라우딘-2 발현이 증가하면 덱스트란 설페이트 나트륨(DSS)으로 유발된 대장염에 도움이 된다는 초기 증거가 제시되었습니다98,99. 그러나 클라우딘-2 과발현은 대변의 수분 함량을 증가시키기도 합니다50. 따라서 클라우딘-2 발현이 증가하면 단순히 원위 결장 내의 DSS를 희석함으로써 대장염의 심각성을 감소시킬 수 있다는 것을 시사합니다. 또 다른 가능성은 claudin-2 발현이 상피 성장과 점막 복구를 촉진한다는 것입니다100-102. 아래에서 이에 대해 논의합니다('Claudin-2와 상피 증식' 참조).

Claudin-2 발현은 인간의 장 감염 시 증가합니다103. 마찬가지로, 모델 병원균인 Citrobacter rodentium은 생쥐의 감염 후 2일 이내에 IL-22 의존적 claudin-2의 상향 조절을 유발합니다50(그림 3). IL-22가 다형성이기는 하지만, Cldn2-knockout 마우스의 경우, 야생형 마우스에 비해 점막 관련 C. rodentium의 수가 증가하고, 병원체 제거가 지연되며, 점막 손상의 심각도가 더 심하다는 사실은 IL-22 의존성 클라우딘-2의 상향 조절이 숙주 방어에 기여한다는 것을 보여줍니다50. 또한, 클라우딘-2의 형질전환 과발현이 C. rodentium에 의한 대장염을 제한한다는 사실은 이러한 결론을 뒷받침합니다.

Fig. 3. Promotion of pathogen clearance by paracellular fluid efflux.

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Citrobacter rodentium infection triggers an immune response that leads to IL-22 release within the lamina propria within 2 days of infection. IL-22 signalling activates claudin-2 transcription and increases claudin-2 channel-mediated Na+ and water efflux via the tight junction pore pathway, resulting in diarrhoea that promotes clearance of the infection. Adapted with permission from ref. 50, Elsevier.

The passage of either Na+ or water through claudin-2 channels104,105 could mediate the increased pathogen clearance and reduced disease severity associated with claudin-2 expression‎. In studies to dissect these mechanisms, wild-type, Cldn2-transgenic and Cldn2-knockout mice were infected with C. rodentium. Polyethylene glycol (PEG) was added to their drinking water 4 days later to create an osmotic gradient that increased fluid flow into the intestinal lumen. PEG treatment normalized the number of mucosa-associated C. rodentium, pathogen clearance and mucosal damage across genotypes, demonstrating that paracellular water efflux is the primary means by which claudin-2 promotes enteric pathogen clearance.

The effect of PEG was unlikely to have been a result of fluid efflux simply washing bacteria off the epithelial surface, as C. rodentium is an attaching and effacing pathogen that forms pedestals and is not easily displaced from intestinal epithelial cells. Moreover, intestinal epithelial cells turn over every few days during infection, so the cells present at the peak of disease (11 days after infection) are not the same cells that were initially colonized. Therefore, one possible explanation for the findings is that claudin-2-mediated paracellular water efflux reduces the efficiency with which new cells are infected. This hypothesis remains to be tested. Nevertheless, evidence indicates that the diarrhoea induced by claudin-2 upregulation is beneficial in the context of enteric infection. These results provide the first experimental data to show that diarrhoea promotes enteric pathogen clearance, an idea that has persisted for centuries despite a lack of supporting evidence106.

Na+ 또는 물이 claudin-2 채널104,105을 통과하면 claudin-2 발현과 관련된 병원체 제거율 증가와 질병 심각도 감소가 중재될 수 있습니다. 이러한 메커니즘을 분석하기 위한 연구에서, 야생형, Cldn2-transgenic 및 Cldn2-knockout 마우스를 C. rodentium에 감염시켰습니다. 4일 후, 식수에 폴리에틸렌글리콜(PEG)을 첨가하여 장 내강으로의 체액 흐름을 증가시키는 삼투압 구배를 만들었습니다. PEG 처리는 유전자형 전반에 걸쳐 점막 관련 C. rodentium의 수, 병원체 제거율, 점막 손상을 정상화하여, 세포간 수분 유출이 클라우딘-2가 장내 병원체 제거를 촉진하는 주요 수단임을 입증했습니다.

PEG의 효과는 단순히 세균을 상피 표면에서 씻어내는 유체 유출의 결과로 인한 것 같지 않습니다. C. rodentium은 부착 및 소멸 병원체로서 기둥을 형성하고 장 상피 세포에서 쉽게 제거되지 않기 때문입니다. 또한, 장 상피 세포는 감염 기간 동안 며칠마다 교체되기 때문에 질병의 최고점(감염 후 11일)에 존재하는 세포는 처음에 식민지화되었던 세포와 동일하지 않습니다. 따라서, 이 발견에 대한 한 가지 가능한 설명은 claudin-2에 의해 매개되는 세포간 수분 유출이 새로운 세포가 감염되는 효율성을 감소시킨다는 것입니다. 이 가설은 아직 검증되지 않았습니다. 그럼에도 불구하고, claudin-2의 상향 조절에 의해 유발되는 설사가 장 감염의 맥락에서 유익하다는 증거가 있습니다. 이 결과는 설사가 장내 병원균 제거를 촉진한다는 것을 보여주는 최초의 실험적 데이터를 제공하며, 이는 수세기 동안 근거가 부족함에도 불구하고 지속되어 온 생각입니다106.

Claudin-2 exacerbates immune-mediated colitis

Claudin-2 transcription is exquisitely responsive to cytokine stimulation and is increased in a wide range of human and experimental disorders associated with mucosal inflammation. Given its protective role during enteric infection and DSS-induced injury, claudin-2 upregulation might also be expected to be beneficial in inflammatory disease. This hypothesis was tested by inducing immune-mediated colitis by T cell transfer in immunodeficient, claudin-2 wild-type, transgenic and knockout mice98. In contrast to the effects of claudin-2 in infectious colitis, its overexpression‎ exacerbated immune-mediated colitis and was associated with severe weight loss, increased cytokine production, mucosal T cell infiltration and histopathological damage. Conversely, Cldn2 knockout attenuated all measures of colitis severity. Together with the effects of claudin-2 expression‎ on pathogen clearance, these data suggest that claudin-2 upregulation triggers defence mechanisms that include immune activation and, therefore, exacerbates immune-mediated disease. Although the mechanisms by which claudin-2-mediated paracellular Na+ and water flux enhances immune activation is unknown, this phenomenon could explain the observed exacerbation of immune-mediated disease by high-Na+ diets107–114.

The findings in Cldn2-knockout mice also provide further support for the idea that substantial functional differences exist between claudin-2 and claudin-15 in vivo. Although claudin-15 expression‎ is not altered in human IBD or experimental immune-mediated colitis, it was upregulated in colitic Cldn2-knockout mice98. However, Cldn2-knockout mice remained protected from immune-mediated colitis, indicating that claudin-15 cannot compensate for claudin-2 loss in this context.

Although disease severity was lower in Cldn2-knockout mice than in wild-type mice, survival was inferior98. The cause of death among Cldn2-knockout mice was intestinal obstruction. This observation could reflect an inability to increase luminal hydration, owing to lack of claudin-2-mediated water transport, that synergized with colitis-associated epithelial proliferation, mucosal expansion and luminal narrowing to allow formation of luminal faecaliths and intestinal obstruction. Consistent with this interpretation, induction of mild osmotic diarrhoea increased faecal water content, prevented intestinal obstruction and increased survival of the Cldn2-knockout mice50. Osmotic diarrhoea did not, however, affect overall disease severity in claudin-2 wild-type, transgenic or knockout mice98. Therefore, the increased survival due to osmotic diarrhoea does not reflect direct mitigation of immune activation or tissue damage.

The protection afforded by Cldn2 knockout suggests that pharmacological inhibition of claudin-2 function might be effective in immune-mediated disease. Of several reported approaches to claudin-2 channel inhibition115–118, only one has been assessed in vivo98. This approach relies on casein kinase 2 (CK2) inhibition, occludin dephosphorylation and assembly of a claudin-2–ZO-1–occludin complex that inactivates claudin-2 channels98,117. CK2 inhibition did not interfere with IL-13-induced increases in claudin-2 expression‎ but did prevent IL-13-induced changes in paracellular Na+ permeability. CK2 inhibition markedly attenuated the severity of immune-mediated colitis in claudin-2 wild-type mice98. Although CK2 is a ubiquitously expressed, promiscuous kinase, CK2 inhibition did not affect disease severity in Cldn2-knockout mice, indicating that its therapeutic benefit is largely due to claudin-2 channel inactivation. Notably, CK2 inhibition did not cause intestinal obstruction in Cldn2 wild-type mice, probably owing to incomplete claudin-2 channel inactivation. Together with the fact that the intestinal lumen diameter is much greater in humans than in mice, this observation suggests that pharmacological claudin-2 channel inhibition is unlikely to cause intestinal obstruction in humans.

Claudin-2 and epithelial proliferation

Some evidence suggests that claudin-2 promotes epithelial proliferation100,101,119,120. For example, the rate of intestinal epithelial proliferation was nearly doubled in one strain of mice with transgenic claudin-2 overexpression‎100. As mentioned above, this proliferation might contribute to the protection that claudin-2-transgenic mice have from DSS-induced colitis100. However, epithelial proliferation was not increased in a different Cldn2-transgenic mouse model50. The reasons for this discrepancy are unclear, as Cldn2 expression‎ was under the control of the 9 kB villin promoter121 and pore pathway permeability was increased in both models. However, the transgenic mice differed in that one expressed human claudin-2 at high levels100, whereas the other expressed EGFP-tagged mouse claudin-2 at lower levels50. Although further study is needed, this difference could underlie the discrepancy between the two models and could also explain increases in leak pathway permeability that occurred in the first, but not the second, model.

Other data that suggest a role for claudin-2 in regulating epithelial proliferation include studies of SW480 and HCT116 human colon cancer cells, which demonstrated that claudin-2 overexpression‎ increases proliferation in vitro, accelerates tumour growth in vivo and reduces apoptosis triggered by the chemotherapeutic agent 5-fluorouracil122. In a study in patients with colon cancer, high claudin-2 expression‎ correlated with lower overall and disease-free survival, further supporting the notion that claudin-2 can promote epithelial proliferation123. Thus, although the mechanisms are not defined, claudin-2 overexpression‎ might promote intestinal epithelial cell proliferation in some contexts.

The leak pathway in disease

Although the leak pathway is activated physiologically during Na+–nutrient cotransport, far greater increases in leak pathway permeability are induced by TNF124–126 (Box 1). The reasons for this difference between physiological and pathophysiological tight junction regulation are unclear because both depend on MLCK activation, but they might relate to the fact that occludin endocytosis occurs during TNF-induced barrier loss but occludin distribution is unaffected during Na+–nutrient cotransport-induced permeability increases53,68,125,127. Occludin is also internalized during MLCK-mediated barrier loss induced by the TNF-related cytokine LIGHT, IL-1β or lipopolysaccharide128–133.

In experimental, immune-mediated IBD, activation of intestinal epithelial MLCK accelerates disease progression, whereas genetic deletion of intestinal epithelial MLCK attenuates disease59,134. Interestingly, the claudin-2 upregulation that normally occurs in experimental, immune-mediated IBD is reduced in mice that lack intestinal epithelial MLCK134 and is restored by transgenic expression‎ of constitutively active MLCK within intestinal epithelia134. Thus, the leak pathway and pore pathway are linked in disease. Notably, transgenic expression‎ of constitutively active MLCK within intestinal epithelial cells, which modestly increases leak pathway permeability but does not induce disease, increases both mucosal IL-13 production and epithelial claudin-2 expression‎. Thus, increased leak pathway permeability can, via mucosal immune activation, trigger claudin-2 upregulation45. Conversely, as noted above, leak pathway permeability is increased in one of two claudin-2 overexpressing transgenic mice despite the absence of overt disease. Although the precise relationship between pore pathway and leak pathway regulation in the context of disease remains to be determined, the ability of distinct cytokines to specifically and independently regulate pore pathway or leak pathway permeability is striking.

TNF, LIGHT and IL-1β all trigger transcriptional and enzymatic activation of MLCK135–137, although reports differ as to whether the transcriptional activation is mediated by nuclear factor-κB, p38 mitogen-activated protein kinase (MAPK) or the transcription factor activator protein 1 (AP-1)135,138–140. Regardless of these discrepancies, MLCK activation clearly leads to perijunctional MLC phosphorylation and occludin endocytosis125,140,141. Despite ongoing debate regarding the functional significance of occludin, the consensus is that occludin, along with other proteins, is a critical regulator of leak pathway permeability. This role of occludin has been demonstrated in several in vitro studies52,142–144, but the strongest evidence comes from in vivo studies that have shown that blockade of occludin endocytosis or transgenic occludin overexpression‎ limits TNF-induced leak pathway barrier loss54. This observation suggests that reduced occludin expression‎ could explain the increased permeability of the leak pathway observed in human disease, including IBD86,145.

The tricellular tight junction proteins tricellulin and angulin 1 might also be important regulators of leak pathway permeability. In vitro studies have shown that deletion of either tricellulin or angulin-1 increases leak pathway permeability146–148. Similar to tricellulin, siRNA-mediated knockdown of the other TAMPs, MARVELD3 (refs. 146,149) or occludin142,143, also increased leak pathway permeability. Tricellulin redistribution from tricellular to bicellular tight junctions150 following occludin loss54,125 could, therefore, be an intermediate event that allows occludin to regulate leak pathway permeability. Thus, although a great deal has been learned about proteins that contribute to leak pathway barrier function and mechanisms of experimental and pathophysiological leak pathway regulation, the molecular structure of the leak pathway remains enigmatic.

Diverse occludin functions

Unexpectedly, intestinal epithelial specific occludin knockout protects mice from experimental colitis and epithelial injury driven by intrinsic and extrinsic TNF signalling pathways. This finding was ultimately explained by the observation that occludin enhances activity of the promoter for CASP3, which encodes caspase 3, through an undefined mechanism145. In cultured cell lines and mice, occludin downregulation led to a reduction of ~50% in caspase 3 expression‎ that conferred protection from a diverse range of pro-apoptotic stimuli145,151. Analyses of biopsy samples suggests that this process also occurs in human disease, as occludin downregulation correlates with reduced epithelial caspase 3 expression‎ in patients with ulcerative colitis or Crohn’s disease.

Thus, in addition to increasing leak pathway permeability, occludin downregulation can promote epithelial survival. However, this effect might not be entirely beneficial, as it could allow evolution of deleterious mutations that would have otherwise been eliminated by apoptosis. Consistent with this hypothesis, in vitro studies suggest that occludin functions as a tumour suppressor in some contexts151–155. Further exploration will, therefore, be required to fully understand extra-junctional functions of occludin.

Distinct functions of long MLCK splice variants

Epithelial MLCK is expressed from the same gene (MYLK) that encodes smooth muscle MLCK156. However, epithelial (long) MLCK is ~225 kDa (refs. 156,157), whereas smooth muscle (short) MLCK is only ~130 kDa (Fig. 4). Long MLCK transcription, which is activated by TNF, IL-1β and other stimuli135,158, generates mRNA transcripts that include additional 5′ exons that are not present in short MLCK transcripts156. This difference reflects the location of the short MLCK promoter within an intron of long MLCK. Nevertheless, the carboxy-terminal catalytic and calmodulin-binding regulatory domains are identical in long and short MLCK. The 5′ region that distinguishes long MLCK from short MLCK undergoes extensive alternative splicing156. Of the splice variants generated, only two — long MLCK1 and MLCK2 — are expressed in intestinal epithelial cells157 (Fig. 4). Although the underlying mechanisms have not been defined, splicing seems to be precisely regulated during differentiation, as MLCK2 is expressed throughout the crypt–villus axis but MLCK1 expression‎ is limited to the upper villus157. Moreover, the increased MLC phosphorylation in active Crohn’s disease is specifically associated with perijunctional MLCK1 recruitment159,160 (Fig. 4).

Fig. 4. Epithelial and smooth muscle myosin light chain kinase.

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a, The human MYLK gene encodes long (non-muscle) and short (smooth muscle) isoforms of myosin light chain kinase (MLCK) protein. Two long MLCK transcriptional start sites that result in expression‎ of the same protein have been identified. However, extensive alternative splicing within the 5′ half of the transcript occurs, which, in intestinal epithelial cells, results in expression‎ of two long MLCK splice variants, MLCK1 and MLCK2. These variants differ by a single exon (black), removal of which causes the third of the nine immunoglobulin-cell adhesion molecule (IgCAM) domains to be incomplete in long MLCK2. The short MLCK promoter is located within a long MLCK intron and drives transcription of smooth muscle MLCK, which lacks the six amino-terminal IgCAM domains that are present in long MLCK1. The kinase and calmodulin (CaM)-binding domains are encoded by sequences within the 3′ half of MYLK and are identical in long and short MLCK proteins. b, Inflammatory signals, such as tumour necrosis factor (TNF), trigger MLCK1 binding to FKBP8. This binding facilitates MLCK1 recruitment to the perijunctional actomyosin ring, where it phosphorylates MLC. This phosphorylation causes occludin internalization to increase leak pathway permeability. In contrast to MLCK1, MLCK2 distribution is not affected by TNF. c, MLCK1 expression‎ and recruitment to the perijunctional actomyosin ring (arrows) are increased in Crohn’s disease. The insets show the boxed areas. MLCK1 and total MLCK are shown, as the absence of unique MLCK2 sequences prevents generation of MLCK2-specific antibodies. Nuclei appear yellow. Part a adapted from ref. 141, Springer Nature Limited.

The two intestinal epithelial long MLCK splice variants differ by a single exon that is present in MLCK1 but not in MLCK2 (ref. 157). The 69 amino acids encoded by this exon complete the third immunoglobulin–cell adhesion molecule domain (IgCAM3)157. This domain must, therefore, contribute to preferential perijunctional localization of MLCK1 relative to MLCK2, which is distributed more diffusely through the cytoplasm141,157. TNF triggers even greater recruitment of MLCK1 to the perijunctional actomyosin ring141. Similarly, MLCK1 is concentrated within the perijunctional actomyosin ring in intestinal biopsy samples from patients with active IBD141,160,161. This inflammation-inducible perijunctional MLCK1 recruitment, together with increased barrier function after MLCK1-specific knockdown157, suggests that this splice variant is central to tight junction regulation.

These findings prompted solution of the IgCAM3 crystal structure, which was then used for in silico screening of small drug-like molecules that were predicted to bind to IgCAM3 (ref. 141). In vitro testing identified one compound that diverts MLCK1 from the perijunctional actomyosin ring and reverses cytokine-induced MLCK1 recruitment in vivo and in excised human intestine141. This molecule, known as divertin, blocks MLCK1-mediated phosphorylation of perijunctional MLC, thereby preventing subsequent occludin endocytosis and increases in leak pathway permeability141. Divertin does not, however, interfere with other functions of long or short MLCK, including its involvement in epithelial cell migration and smooth muscle contraction, because IgCAM3 is not present in short MLCK and is distant from the MLCK1 catalytic and regulatory domains141. This functional selectivity is critical, as in vivo inhibition of MLCK activity leads to hypotension and visceral paralysis, including aperistalsis, thereby precluding therapeutic use of enzymatic inhibitors162.

Therapeutic targeting of MLCK1 recruitment

Divertin was remarkably effective in a mouse model of immune-mediated IBD — its effects were equal or superior to those of anti-TNF therapy by all measures, including survival141. This result supports the hypothesis that divertin-mediated interference with IgCAM3-mediated protein–protein interactions prevents perijunctional MLCK1 recruitment and, ultimately, disease progression.

A screen for potential MLCK1 binding partners was used to identify protein–protein interactions targeted by divertin. This process led to the discovery of tacrolimus binding protein FKBP8 as an MLCK1-interacting protein160 (Fig. 4). MLCK1–FKBP8 interactions were specifically increased in TNF-treated intestinal epithelial monolayer cultures160. These interactions tended to occur near the perijunctional actomyosin ring160. Similarly, increases in perijunctional MLCK1–FKBP8 interactions were detected in biopsy samples from patients with Crohn’s disease160. Tacrolimus also prevented TNF-induced perijunctional MLCK1 recruitment, MLCK1–FKBP8 interactions and perijunctional MLC phosphorylation in human intestinal organoids160. Finally, tacrolimus prevented MLCK1 recruitment, occludin internalization and barrier loss after acute T cell activation in mice160. Surprisingly, divertin did not interfere with FKBP8 binding to recombinant MLCK1 IgCAM domains one to four in vitro. Thus, despite the efficacy of divertin in experimental colitis, agents that prevent MLCK1 interactions with FKBP8 or other MLCK1 binding partners should also be sought as potential therapeutics.

Conclusions

The first tight junction protein, ZO-1 (ref. 33), was discovered nearly 40 years ago, and numerous other tight junction proteins have been identified since37–39,149,163–167, leading to substantial data describing the molecular interactions responsible for selective permeability and barrier regulation144,146,147,168–172. This work has led to conceptual advances, including the pore and leak pathway model of paracellular permeability47,173, and foundational understanding of tight junction cell biology, physiology and pathobiology.

In the same year that ZO-1 was discovered33, increased intestinal permeability was identified in a subset of first-degree relatives of people with Crohn’s disease174. More recently, these modest leak pathway permeability increases were validated as an independent risk factor for IBD24. However, all human studies to date have relied on probes, such as lactulose and mannitol, that are too large to cross the pore pathway. Thus, despite increased claudin-2 expression‎ in human disease64,86,87,92,175,176 and experimental data showing that claudin-2-dependent pore pathway permeability increases exacerbate disease in mice50,98,100, the relevance of claudin-2 to human intestinal disease remains to be determined. Our understanding of how barrier function and disease are affected by polymorphisms in barrier-related genes associated with IBD, including INAVA177–179 and CDH1 (refs. 180,181), which encode innate immune activator and E-cadherin, respectively, is even more limited.

In conclusion, our understanding of how permeability of the pore and leak pathways contributes to health and disease remains relatively rudimentary. Thus, although much has been accomplished, much more remains to be discovered. Remaining challenges include identification of the sites and molecular structure of the leak pathway, elucidation of the differences that must exist between seemingly redundant claudins, and the definition of non-canonical tight junction protein functions. Nevertheless, the promise of tight junction-targeted therapeutics remains compelling, and implementation of such therapeutic approaches is growing progressively closer.

결론

첫 번째 밀접 접합 단백질인 ZO-1(참고 33)은 약 40년 전에 발견되었으며, 그 이후로37-39,149,163-167 등 수많은 밀접 접합 단백질이 확인되어 선택적 투과성과 장벽 조절을 담당하는 분자 상호작용을 설명하는 상당한 양의 데이터가 축적되었습니다144,146,147,168-172. 이 연구는 세포간 투과성의 기공 및 누출 경로 모델47,173을 포함한 개념적 발전을 가져왔고, 세포간 접합부 세포 생물학, 생리학 및 병리 생물에 대한 기초적인 이해를 가능하게 했습니다.

ZO-1이 발견된 33년 동안, 크론병 환자의 1차 친척 중 일부에서 장 투과성 증가가 확인되었습니다174. 최근에는 이러한 소폭의 누출 경로 투과성 증가가 IBD의 독립적 위험 요인으로 확인되었습니다24. 그러나 지금까지의 모든 인간 연구는 기공 경로를 통과하기에는 너무 큰 락툴로오스나 만니톨 같은 탐침에 의존해 왔습니다. 따라서 인간 질병에서 클라우딘-2 발현이 증가했음에도 불구하고64,86,87,92,175,176 그리고 클라우딘-2 의존성 기공 경로 투과성 증가가 생쥐의 질병을 악화시킨다는 실험 데이터가50,98,100 존재함에도 불구하고, 클라우딘-2와 인간 장 질환의 관련성은 아직 밝혀지지 않았습니다. 장벽 기능과 질병이 장벽 관련 유전자(INAVA177-179 및 CDH1(참조 180,181)의 다형성에 의해 어떻게 영향을 받는지에 대한 우리의 이해는 훨씬 더 제한적입니다.

결론적으로, 기공의 투과성과 누출 경로가 건강과 질병에 어떻게 기여하는지에 대한 우리의 이해는 상대적으로 기초적인 수준에 머물러 있습니다. 따라서 많은 성과가 있었지만, 아직 밝혀내야 할 것이 많이 남아 있습니다. 남아 있는 과제에는 누출 경로의 위치와 분자 구조의 확인, 중복된 것처럼 보이는 클라우딘 사이에 존재하는 차이점의 규명, 비정규적인 타이트 접합 단백질 기능의 정의 등이 포함됩니다. 그럼에도 불구하고, 타이트 접합을 표적으로 하는 치료법의 가능성은 여전히 매력적이며, 이러한 치료적 접근법의 구현은 점점 더 가까워지고 있습니다.

Acknowledgements

The authors are supported by National Institute of Diabetes and Digestive and Kidney Disease grants R01DK61931 and R01DK68271 (J.R.T.), U.S. Department of Defense award PR181271 (J.R.T.), and the Belgian American Educational Foundation (X.H.). The authors also greatly appreciate the contributions of S. Hagen, N. Shashikanth, L. Zuo, S. Abtahi, L.-S. Beier and G. Marsischky, as well as H. Marlatt (Nationwide Histology) and T. S. Davanzo (Slaybaugh Studios). The freeze–fracture electron micrograph in Fig. 1 was provided by the late E. E. Schneeberger (Harvard Medical School), an exceptional friend, pathologist and scientist.

Author contributions

A.H. and J.R.T. wrote the manuscript. All authors researched data for the article, made substantial contributions to discussion of content and reviewed and edited the manuscript before submission.

Peer reviewPeer review information

Nature Reviews Gastroenterology & Hepatology thanks A. Keshavarzian, R. Rao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Competing interests

J.R.T. is a consultant for Entrinsic and Kallyope. The other authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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