Re: 심한 염증상태 .. 중성구의 면역기능장애!!

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Crit Care

. 2016 Mar 23;20:73. doi: 10.1186/s13054-016-1250-4

The role of neutrophils in immune dysfunction during severe inflammation

Pieter H C Leliefeld 1,3,✉, Catharina M Wessels 1, Luke P H Leenen 1, Leo Koenderman 2,3, Janesh Pillay 3,4

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PMCID: PMC4804478  PMID: 27005275

Abstract

Critically ill post-surgical, post-trauma and/or septic patients are characterised by severe inflammation. This immune response consists of both a pro- and an anti-inflammatory component. The pro-inflammatory component contributes to (multiple) organ failure whereas occurrence of immune paralysis predisposes to infections. Strikingly, infectious complications arise in these patients despite the presence of a clear neutrophilia. We propose that dysfunction of neutrophils potentially increases the susceptibility to infections or can result in the inability to clear existing infections. Under homeostatic conditions these effector cells of the innate immune system circulate in a quiescent state and serve as the first line of defence against invading pathogens. In severe inflammation, however, neutrophils are rapidly activated, which affects their functional capacities, such as chemotaxis, phagocytosis, intra-cellular killing, NETosis, and their capacity to modulate adaptive immunity. This review provides an overview of the current understanding of neutrophil dysfunction in severe inflammation. We will discuss the possible mechanisms of downregulation of anti-microbial function, suppression of adaptive immunity by neutrophils and the contribution of neutrophil subsets to immune paralysis.

초록

수술 후, 외상 후 및/또는 패혈증 환자는

심각한 염증 반응을 특징으로 합니다.

이 면역 반응은

염증 촉진 및 억제 성분으로 구성됩니다.

염증 촉진 성분은 (다발성) 장기 부전에 기여하며,

면역 마비 발생은 감염 위험을 증가시킵니다.

흥미롭게도,

이러한 환자에서는 명확한 중성구 증가가 존재함에도 불구하고

감염 합병증이 발생합니다.

우리는 중성구의 기능 장애가

감염에 대한 감수성을 증가시키거나

기존 감염을 제거하는 능력을 상실시킬 수 있다고 제안합니다.

정상적인 항상성 상태에서

이 선천성 면역계의 효과 세포는

휴면 상태로 순환하며 침입하는 병원체에 대한 첫 번째 방어선 역할을 합니다.

그러나 심각한 염증 상태에서는

중성구가 급속히 활성화되어 화학유동, 식균작용, 세포 내 살상, NETosis, 적응 면역 조절 능력 등

기능적 역량이 손상됩니다.

이 리뷰는

심각한 염증에서의

중성구 기능 장애에 대한 현재의 이해를 개괄합니다.

우리는

중성구의 항미생물 기능 억제 메커니즘,

적응 면역 억제,

중성구 하위 집단의 면역 마비 기여도에 대해 논의할 것입니다.

Background

Severe inflammation induced by trauma, sepsis or ischemia/reperfusion injury is known to contribute to devastating complications such as acute respiratory distress syndrome (ARDS) and (multiple) organ failure [1]. This has been attributed to microvascular dysfunction, tissue damage and dysregulation of metabolism caused by severe inflammation [2]. In recent years, however, it has been recognised that severe systemic inflammation can also result in a profound ‘compensatory’ down-regulation of immune responses, rendering the host susceptible to infections or unable to clear existing infections [3]. Although conceivably an evolutionarily preserved response to protect the host from immune-mediated tissue damage, downregulation of anti-microbial immunity creates an unwanted consequence: susceptibility to bacterial infections such as caused by Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli as well as opportunistic fungal infections such as (disseminated) candidiasis [4–6]. In addition, reactivation of viruses such as cytomegalovirus are found in critically ill patients [7]. These findings clearly indicate that both the innate and the adaptive immune system are dysfunctional in these patients. Nosocomial infections in critically ill patients are associated with an increased length of hospital stay, increased health care costs and profound additional morbidity and mortality [8].

Neutrophils, effector cells of the innate immune system, are abundantly present in the circulation and comprise up to 50–70 % of total circulating leukocytes in humans. The enhanced frequency and severity of bacterial and fungal infections in patients with congenital neutrophil disorders demonstrate that neutrophils are indispensable for adequate protection against microbes [9]. Patients suffering from leucocyte adhesion deficiency (LAD)-I are at risk for development of necrotizing infections and sepsis because of inadequate neutrophil transendothelial migration to the site of infection [10]. The Chediak-Higashi syndrome and chronic granulomatous disease (CGD) underscore the eminent importance of intracellular bacterial killing by neutrophils. Chediak-Higashi syndrome is caused by a mutation in the LYST gene, which encodes a lysosomal trafficking regulator [11]. The mutation leads to the absence of a proper formation of phagolysosomes. Patients suffering from Chediak-Higashi are extremely susceptible to pyogenic infections and this syndrome is usually fatal before the age of 10 [11]. CGD is characterised by a defect in production of the bactericidal reactive oxygen species (ROS) due to defective NADPH oxidase and results in recurrent infections, reducing life-expectancy significantly [12]. In murine models of sepsis, knockout of essential neutrophil antimicrobial functions leads to rapid death. For instance, mice lacking the neutrophil granule proteins myeloperoxidase or elastase die more rapidly from sepsis [13, 14]. Apart from the severe phenotypes seen in patients with inborn errors and murine knockout models, more subtle effects were seen in a murine sepsis model where rapid death coincided with inadequate phagosomal acidification of neutrophils after phagocytosis [15]. These studies highlight the generally accepted importance of neutrophils in antimicrobial defence in acute inflammatory models. In addition, they demonstrate disturbances in the anti-microbial functionality of these cells during severe inflammation.

In this review we will discuss neutrophil functions required for adequate microbial defence and the mechanisms leading to neutrophil-mediated immune dysfunction.

배경

외상, 패혈증 또는 허혈/재관류 손상으로 인한 심각한 염증은

급성 호흡곤란 증후군(ARDS) 및 (다발성) 장기 부전과 같은

치명적인 합병증에 기여하는 것으로 알려져 있습니다 [1].

이는 심각한 염증에 의해 유발되는

미세혈관 기능 장애,

조직 손상 및 대사 조절 장애에 기인한다고 알려져 있습니다[2].

그러나 최근에는

심각한 전신 염증이 면역 반응의 심한 '보상성' 억제를 초래하여

호스트가 감염에 취약해지거나 기존 감염을 제거하지 못하게 한다는 것이 인정되었습니다[3].

진화적으로 보존된 반응으로 호스트를 면역 매개 조직 손상으로부터 보호하기 위한 메커니즘일 수 있지만,

항미생물 면역 반응의 억제는 원치 않는 결과를 초래합니다:

Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli와 같은 세균 감염 및

(확산성) 칸디다증과 같은 기회감염성 곰팡이 감염에 대한 취약성 [4–6].

또한 중증 환자에게서 사이토메갈로바이러스와 같은 바이러스의 재활성화가 관찰됩니다 [7].

이러한 결과는 중증 환자의 선천적 및 적응 면역 체계가 모두 기능 장애를 겪고 있음을 명확히 보여줍니다.

중증 환자의 병원 내 감염은 병원 체류 기간 연장, 의료 비용 증가, 심각한 추가적 morbidities 및 사망률과 연관되어 있습니다[8].

선천성 면역 체계의 효과 세포인 중성구는

혈액 순환계에 풍부하게 존재하며,

인간에서 순환하는 백혈구의 50–70%를 차지합니다.

선천성 중성구 장애를 가진 환자에서

세균 및 곰팡이 감염의 빈도와 중증도가 증가하는 것은

중성구가 미생물로부터 적절한 보호에 필수적임을 보여줍니다 [9].

백혈구 부착 결핍증(LAD)-I를 앓는 환자는

감염 부위로의 중성구 내피 세포 이동이 불충분하여

괴사성 감염 및 패혈증 발병 위험이 높습니다 [10].

체디아크-히가시 증후군과 만성 과립구 결핍증(CGD)은 중성구의 세포 내 세균 살상 기능의 중요성을 강조합니다. Chediak-Higashi 증후군은 리소좀 수송 조절 인자를 암호화하는 LYST 유전자의 돌연변이에 의해 발생합니다 [11]. 이 돌연변이는 식세포 소포의 적절한 형성을 방해합니다. Chediak-Higashi 증후군 환자는 화농성 감염에 매우 취약하며, 이 증후군은 보통 10세 이전에 사망에 이릅니다 [11]. CGD는 NADPH 산화효소 결함으로 인해 세균 살상 활성 산소 종(ROS)의 생산 결함이 특징이며, 이는 재발성 감염을 유발하여 기대 수명을 크게 감소시킵니다 [12]. 쥐의 패혈증 모델에서 중성구 항균 기능의 필수적 결손은 급속한 사망을 초래합니다. 예를 들어, 중성구 과립 단백질인 마이엘로퍼옥시다제나 엘라스테이스를 결여한 쥐는 패혈증으로 인해 더 빠르게 사망합니다 [13, 14]. 선천성 대사 장애 환자 및 마우스 유전자 결손 모델에서 관찰된 심각한 증상 외에도, 마우스 패혈증 모델에서 중성구 식작용 후 식작용 소체 산성화 부족과 급속한 사망이 동시에 발생하는 더 미묘한 효과가 관찰되었습니다 [15]. 이러한 연구는 급성 염증 모델에서 중성구의 항균 방어 기능의 중요성을 강조하며, 심각한 염증 시 이러한 세포의 항균 기능 장애를 보여줍니다.

이 리뷰에서는

적절한 미생물 방어를 위해 필요한

중성구 기능과 중성구 매개 면역 기능 장애로 이어지는 메커니즘을 논의할 것입니다.

Functions of neutrophils associated with anti-microbial defence

Chemotaxis

The controlled process of phagocytosis and killing of microbes by neutrophils firstly requires chemotaxis towards the site of infection. Chemotaxis is the propensity of cells to migrate in the direction of gradients of chemotactic stimuli [16]. The ability to adequately sense chemotactic gradients is one of the final capabilities acquired by neutrophils during maturation in the bone marrow and this functionality appears to be the most sensitive to perturbations in vivo and in vitro [17]. Impairment of chemotaxis has been described in a wide variety of diseases associated with increased susceptibility to infections: diabetes mellitus, viral infections (influenza), cytomegalovirus, HIV and tropical diseases (malaria) [18–22]. In sepsis, chemotaxis of neutrophils is impaired through various mechanisms [23–25]. Interleukin (IL)-33 limits this impairment by preventing downregulation of CXCR2 and improves outcome in a murine model [26]. In humans, extensive research has focused on the chemotactic capacity of neutrophils from burn patients. It has been shown that neutrophils from thermally injured subjects are characterised by impaired chemotaxis, both in vivo in the tissue and in vitro, towards the bacterial peptide fMLF, which is believed to contribute to the increased susceptibility to infections in this group of patients [27, 28].

항미생물 방어와 관련된 중성구 기능:

화학유동

중성구가 미생물을 식균작용으로 포식하고 죽이는 과정은

먼저 감염 부위로의 화학유동으로부터 시작됩니다.

화학유동은

세포가 화학유동 자극의 농도 차이에 따라 이동하는 경향입니다 [16].

화학유도 자극 농도 차이를 적절히 감지하는 능력은

골수에서 성숙 과정에서 중성구가 획득하는 최종 기능 중 하나이며,

이 기능은 체내 및 체외에서 교란에 가장 민감하게 반응하는 것으로 알려져 있습니다[17].

화학유동 장애는 감염에 대한 취약성이 증가하는 다양한 질환에서 보고되었습니다:

당뇨병, 바이러스 감염(인플루엔자), 사이토메갈로바이러스, HIV 및 열대 질환(말라리아)[18–22].

패혈증에서 중성구의 화학유동은

다양한 메커니즘을 통해 손상됩니다 [23–25].

인터루킨(IL)-33은 CXCR2의 하향 조절을 방지함으로써

이 손상을 제한하며, 쥐 모델에서 예후를 개선합니다 [26].

인간에서는 화상 환자의 중성구 화학유동 능력에 대한 광범위한 연구가 진행되었습니다.

열상 환자의 중성구는 조직 내 및 체외에서 세균 펩타이드 fMLF에 대한 화학유입이 손상되어 있으며,

이는 이 환자군의 감염 취약성 증가에 기여하는 것으로 추정됩니다 [27, 28].

Intracellular killing

Once neutrophils have found and recognised a pathogen, phagocytosis can take place and subsequent bacterial killing occurs in the phagolysosome. Neutrophils possess two separate but intercalating anti-microbial mechanisms, one dependent on oxygen and the other independent of it. Although categorisation of killing mechanisms in this manner creates a comprehensive understanding, it does not reflect the in vivo situation in which both systems operate simultaneously. Furthermore, it is likely that the individual significance of both killing mechanisms shifts during the course of inflammation. This is due to fluxes in oxygen demand and supply caused by dynamic tissue perfusion and oxygenation during the inflammatory response [29].

The oxygen-dependent mechanisms are mediated by ROS downstream of O2− formed by the NADPH oxidase complex [30]. In short, upon activation of a neutrophil, either via ingestion of bacteria or by extracellular stimuli, the NADPH oxidase complex is assembled from both cytosolic and membrane-bound components [31]. The active oxidase complex transports electrons from cytosolic NADPH across the membrane to the electron acceptor, molecular oxygen, generating superoxide anion [29]. This is in turn metabolises in the phagosome into highly bactericidal end products, such as hydroxyl radical, hydrogen peroxide and hypochlorous acid [31]. In sterile inflammation, such as trauma or acute liver failure, neutrophils are known to produce elevated levels of spontaneous ROS [32, 33]. Furthermore, ROS production in these patients in response to a stimulus is strongly elevated in comparison with that found in neutrophils from healthy controls; a process generally referred to as priming [27, 34–36]. Uncontrolled release of ROS by neutrophils accumulating in vascular beds can contribute to loss of endothelial barrier integrity and subsequent vascular leakage, predisposing patients to organ injury as a result of pro-inflammatory complications (acute lung injury, ARDS) [37, 38]. This hypothesis is in line with the findings of increased ROS production in trauma patients developing ARDS in comparison with control trauma patients [39]. In addition, the observation that neutrophils from patients with fatal sepsis are characterised by markedly increased production of ROS compared with survivors is noteworthy [40].

Granule products comprise the backbone of non-oxidative killing by neutrophils [41]. The azurophilic granule is a reservoir of serine proteases: neutrophil elastase, cathepsin G, proteinase 3, and azurocidin [42]. These digestive proteases are delivered into the phagolysosome upon fusion of granules with a phagosome containing bacteria. During maturation of the phagolysosome the intraphagosomal pH is rigorously altered. The early shift of intraphagosomal pH towards an alkaline level (pH 8.5–9.5) due to dismutation of O2− provides the initial milieu for the proper activation of proteases, leading to optimal microbicidal and digestive function of these enzymes [43]. Concomitant with the waning of production of ROS the phagosome progressively acidifies, coinciding with granule–phagosome fusion. These granules contain the Na+/H+-antiporter V-ATP-ase, which is responsible for pumping of protons into the phagosome [44–46]. Neutrophils of burn-injured patients are characterised by dysfunctional pH control of their phagolysosomes since these patients fail to demonstrate transient phagosomal alkalinisation in the first 5 minutes and acidify promptly after ingestion of bacteria [47]. This situation might lead to improper activation of the proteases and impaired killing of ingested microbes. On the other hand, deficient acidification of peritoneal neutrophils in a murine model of sepsis was associated with increased mortality [15]. These findings demonstrate the importance of adequate intraphagosomal pH regulation for microbial control.

The presence and proper function of granules intracellulary are crucial as these organelles supply neutrophils with an arsenal of antimicrobial mechanisms. However, uncontrolled activation of neutrophils in an inflammatory microenvironment can lead to collateral tissue damage by excessive extracellular degranulation and the release of neutrophil proteases. Neutrophil extravasation, homing and activation are mediated by activation of several surface receptors, including β2 integrins, complement receptors, Fcγ-receptors, and formyl peptide receptors. Uncontrolled activation of neutrophils is mediated through these same receptors by responding to aberrant production of chemokines, cytokines and release of extracellular peptides [48]. During this process granules fuse with the plasma membrane, releasing their content into the environment [49]. More tissue damage will lead to increased influx and activation of neutrophils, which then leads to a vicious cycle of tissue destruction [50].

세포 내 살균

중성구가 병원체를 발견하고 인식하면 식작용이 발생하며,

이후 식작용 소체에서

세균 살상이 이루어집니다.

중성구는

산소 의존적 및 산소 독립적 두 가지 서로 다른 항균 메커니즘을 갖추고 있습니다.

이러한 분류는 포괄적인 이해를 제공하지만,

두 시스템이 동시에 작동하는 체내 상황을 반영하지 않습니다.

또한, 두 살균 메커니즘의 개별적 중요성은 염증 과정 중에 변화할 가능성이 높습니다.

이는 염증 반응 중 동적 조직 혈류 및 산소화 변화로 인한 산소 수요와 공급의 변동 때문입니다 [29].

산소 의존적 메커니즘은

NADPH 산화효소 복합체에 의해 생성된 O2−로부터

하류에서 ROS에 의해 매개됩니다 [30].

요약하면,

중성구가 세균 섭취나 세포외 자극을 통해 활성화되면,

NADPH 산화효소 복합체는 세포질과 막 결합 성분에서 조립됩니다 [31].

활성 산화효소 복합체는

세포질 NADPH에서 전자를 막을 통해 전자 수용체인 분자 산소로 전달하여

슈퍼옥사이드 음이온을 생성합니다 [29].

이 과정은 파고소체 내에서

수산화 라디칼, 과산화수소, 차아염소산과 같은

고도로 세균 살상성 최종 산물로 대사됩니다 [31].

무균성 염증(예: 외상 또는 급성 간부전)에서 중성구는 자발적인 ROS 수준이 증가하는 것으로 알려져 있습니다 [32, 33]. 또한 이러한 환자의 자극에 대한 ROS 생성량은 건강한 대조군 중성구와 비교해 현저히 증가하며, 이 과정은 일반적으로 '프라이밍'이라고 불립니다 [27, 34–36].

혈관 내벽에 축적된 중성구가 ROS를 무절제하게 방출하면 내피 장벽의 무결성이 손상되고 혈관 누출이 발생해 염증성 합병증(급성 폐 손상, ARDS)으로 인한 장기 손상 위험이 증가합니다 [37, 38]. 이 가설은 외상 환자가 ARDS를 발병할 때 대조군 외상 환자보다 ROS 생산이 증가한다는 연구 결과와 일치합니다 [39]. 또한, 치명적 패혈증 환자의 중성구에서 생존자보다 ROS 생산이 현저히 증가했다는 관찰 결과도 주목할 만합니다 [40].

과립 제품은 중성구의 비산화성 살균 작용의 핵심 구성 요소입니다 [41]. 아즈로필릭 과립은 세린 프로테아제(중성구 엘라스테이스, 카테프신 G, 프로테아제 3, 아즈로시딘)의 저장고입니다 [42]. 이러한 소화 프로테아제는 과립이 세균을 포함하는 파고소좀과 융합될 때 파고리소좀으로 전달됩니다. 파고리소좀의 성숙 과정에서 파고소좀 내 pH는 엄격히 변화합니다. O2−의 분해로 인한 파고소좀 내 pH의 초기 알칼리화(pH 8.5–9.5)는 프로테아제의 적절한 활성화에 필요한 초기 환경을 제공하며, 이는 이러한 효소의 최적의 미생물 살상 및 소화 기능을 이끌어냅니다 [43]. ROS 생산이 감소함에 따라 파고소체는 점차 산성화되며, 이는 그란울-파고소체 융합과 동시에 발생합니다. 이 그란울에는 파고소체 내로 프로톤을 펌핑하는 Na+/H+-안티포터 V-ATP-ase가 포함되어 있습니다 [44–46]. 화상 환자의 중성구는 파고소좀의 pH 조절 기능 장애를 특징으로 하며, 이 환자들은 세균 섭취 후 첫 5분 내에 일시적인 파고소좀 알칼리화 현상을 보이지 않고 신속히 산성화됩니다 [47]. 이 상황은 프로테아제의 부적절한 활성화와 섭취된 미생물의 살균 기능 저하로 이어질 수 있습니다. 반면, 쥐의 패혈증 모델에서 복막 중성구의 산성화 결핍은 사망률 증가와 연관되었습니다 [15]. 이러한 결과는 미생물 통제를 위해 적절한 파고소 내 pH 조절의 중요성을 보여줍니다.

세포 내 소체(granules)의 존재와 정상적인 기능은 중성구에 항균 메커니즘의 무기를 공급하기 때문에 필수적입니다. 그러나 염증 미세환경에서 중성구의 무분별한 활성화는 과도한 세포외 과립 분비와 중성구 프로테아제의 방출로 인해 주변 조직 손상을 초래할 수 있습니다. 중성구의 혈관 외 유출, 귀소 및 활성화는 β2 인테그린, 보체 수용체, Fcγ 수용체, 포르밀 펩타이드 수용체 등 여러 표면 수용체의 활성화에 의해 매개됩니다. 중성구의 무분별한 활성화는 화학키닌, 사이토킨의 이상 생산 및 세포외 펩타이드 방출에 반응하여 동일한 수용체를 통해 매개됩니다 [48]. 이 과정에서 그레인들은 세포막과 융합되어 내용물을 환경으로 방출합니다 [49]. 조직 손상이 증가하면 중성구의 유입과 활성화가 증가하며, 이는 조직 파괴의 악순환으로 이어집니다 [50].

Neutrophil extracellular traps

In addition to conventional intracellular killing and degradation of individual bacteria, the concept of extracellular killing by neutrophils using neutrophil extracellular traps (NETs) has received much attention during the past decade [51, 52]. NETs consist of fibrils formed by active expulsion of DNA, chromatin and granule proteins from neutrophils [52, 53]. They are formed in response to a variety of pro-inflammatory stimuli of which IL-8, tumour necrosis factor-alpha and lipopolysaccharide are the most relevant [54]. During formation of NETs neutrophils die and this process is generally referred to as NETosis. This form of cell death is dependent on the NADPH-oxidase complex since neutrophils treated with the pharmacological NADPH-oxidase inhibitor DPI and CGD patients are unable to release NETs [53]. In vitro NETs were shown to be a cell-death-associated event occurring hours after stimulation [53]. However, intravital microscopy revealed viable neutrophils after formation of NETs and the resulting anuclear neutrophils were subsequently capable of phagocytosis and formation of mature phagosomes. These data indicate that NETosis does not necessarily result in cell death [55]. The direct bactericidal properties of NETs are a topic of discussion, and prevention of bacterial dissemination in vivo might be their main function [56]. Apart from this antimicrobial function, the cytotoxicity of NETs can be harmful to the host if their release is inappropriately controlled. NETs are released following sepsis, trauma and ischemia–reperfusion injury and a growing body of evidence shows they can contribute to tissue destruction, as reviewed by Liu et al. [57]. The potential of NETs to cause tissue destruction was elegantly demonstrated in a murine model of primary graft-dysfunction after lung transplantation [58]. In addition, several studies argue that NETs might be involved in triggering auto-immune diseases since auto-antibodies against NET constituents (e.g. DNA) are often found in these diseases [59, 60]. Although NETs have firmly established their tissue-damaging properties, scepticism still exists about the in vivo anti-microbial relevance of NETs [61].

중성구 세포외 트랩

전통적인 세포 내 살균 및 개별 세균의 분해 외에도,

중성구가 중성구 세포외 트랩(NETs)을 통해

세포외 살균을 수행한다는 개념은

지난 10년간 많은 관심을 받아왔습니다 [51, 52].

NETs는

중성구에서 DNA, 염색질 및 과립 단백질이

적극적으로 배출되어 형성된 섬유 구조물로 구성됩니다 [52, 53].

NETs는

IL-8, 종양 괴사 인자 알파 및 리포폴리사카라이드와 같은

다양한 염증 유발 자극에 반응하여 형성되며,

이 중 IL-8, 종양 괴사 인자 알파 및 리포폴리사카라이드가 가장 관련성이 높습니다 [54].

NETs 형성 과정에서 중성구는 사멸하며,

이 과정은 일반적으로 NETosis라고 불립니다.

이 형태의 세포 사멸은

NADPH-산화효소 복합체에 의존하며,

약리학적 NADPH-산화효소 억제제 DPI로 처리된 중성구나 CGD 환자는 NETs를 방출할 수 없습니다 [53].

중성구 세포외 트랩(NETs)은 감염, 특히 대형 병원체로부터 보호하는 역할을 하지만, 면역 매개 질환과 관련된 병리학적 과정에도 관여하는 것으로 알려져 있습니다. NET 형성은 선천성 면역 수용체를 통해 하류 세포 내 매개체(반응성 산소 종(ROS), NADPH 산화효소 또는 미토콘드리아에서 생성됨)에 의해 유발되며, 이는 골수 과산화효소(MPO), 중성구 엘라스테이스(NE) 및 단백질 아르기닌 디이미네이스 유형 4(PAD4)를 활성화하여 염색질 탈응축을 촉진합니다. NETosis는 미생물 신호와 내인성 위험 신호에 반응하여 유도되며, 급성 염증이나 만성 염증 및 자가면역 질환 동안 과도한 조직 손상을 방지하기 위해 엄격히 조절되어야 합니다. 미생물 크기, 미생물 독성 인자 및 사이토카인은 NETosis의 조절인자입니다. NET은 질병과 연관된 여러 면역 조절 기능을 가지고 있습니다. NET과 관련된 산화 DNA와 항균 펩타이드를 통해 인터페론 반응을 자극함으로써 무균성 염증을 유도하거나 자가면역 반응을 강화할 수 있습니다. NETs는 혈관 폐쇄를 촉진하여 혈전 형성을 유발하고 중요한 장기 부위를 차단하며, 전이성 종양을 포획하고 당뇨병에서 상처 치유를 지연시킬 수 있습니다. 초록 중성구는 선천성 면역 식세포로 면역 방어에 중심적인 역할을 합니다. 최근 몇 년간 중성구의 병원체 제거, 면역 조절 및 질환 병리학에서의 역할에 대한 이해가 급속히 발전했습니다. 중성구 세포외 트랩(NETs)으로 알려진 웹 모양의 염색질 구조는 중성구 생물학에 대한 새로운 관심의 중심에 있습니다. NETs의 방출을 조절하는 분자의 식별은 NETs의 면역 보호, 염증 및 자가면역 질환, 암에서의 역할을 이해하는 데 기여했습니다. 본 논문에서는 현재까지 NET 생물학 분야를 형성해 온 주요 발견과 개념을 논의합니다.

체외 실험에서 NETs는 자극 후 수 시간 후에 발생하는 세포 사멸 관련 사건으로 확인되었습니다 [53]. 그러나 생체 내 현미경 관찰 결과, NETs 형성 후 생존 가능한 중성구가 관찰되었으며, 결과적으로 핵이 없는 중성구는 이후 식작용과 성숙한 식소체 형성이 가능했습니다. 이 데이터는 NETosis가 반드시 세포 사멸로 이어지지 않을 수 있음을 시사합니다 [55].

NET의 직접적인 세균 살상 특성은 논의의 대상이며,

체내 세균 확산 방지 기능이 주요 역할일 수 있습니다 [56].

이 항균 기능 외에도,

NET의 세포 독성은 방출이 적절히 조절되지 않을 경우 호스트에게 해로울 수 있습니다.

NET은

패혈증, 외상 및 허혈-재관류 손상 후 방출되며,

Liu 등 [57]의 검토에 따르면 조직 파괴에 기여할 수 있다는 증거가 점점 늘고 있습니다.

NETs가 조직 파괴를 유발할 수 있다는 가능성은 폐 이식 후 일차 이식 기능 장애 마우스 모델에서 우아하게 입증되었습니다 [58]. 또한 여러 연구는 NETs가 자가면역 질환을 유발하는 데 관여할 수 있다고 주장합니다. 이는 이러한 질환에서 NET 구성 성분(예: DNA)에 대한 자가항체가 자주 발견되기 때문입니다 [59, 60]. NETs는 조직 손상 특성을 확고히 입증했지만, NETs의 체내 항미생물학적 관련성에 대한 회의론은 여전히 존재합니다 [61].

Neutrophil dysfunction in acute inflammation

The mechanisms involved in adequate anti-microbial defence can also disrupt subsequent immunity. This is caused by aberrant control of their own essential antimicrobial arsenal, such as: (1) auto- and paracrine cleavage of essential surface receptors; (2) downregulation of surface receptors and signalling pathways in non-resolving inflammation or during a second microbial hit following initial sterile inflammation (damage-associated molecular pattern (DAMP)–microbe-associated molecular pattern (MAMP) interference); and (3) the release of neutrophil populations with decreased microbicidal properties. In addition, neutrophils in inflammatory conditions can affect other immune cells and contribute to immune paralysis of the adaptive immune system.

Proteolytic cleavage by neutrophil-derived proteases and downregulation of immune receptors

Serine proteases released by neutrophils influence the expression‎ of receptors critical to neutrophil–microbial interactions (Fig. 1a). Apart from stimulatory effects through serine protease activated receptors (PARs), they can downregulate immune responses by cleaving essential receptors on the surface of both adaptive and innate immune cells [62]. For instance, neutrophil elastase cleaves CXCR1, a receptor for IL-8, on the surface of neutrophils [63, 64]. This mechanism is relevant during acute inflammation in which circulating neutrophils from trauma and sepsis patients selectively downregulate CXCR2, the only other neutrophil receptor for IL-8 [65, 66]. Tarlowe et al. [67] provided evidence that downregulation of this receptor preceded the occurrence of pneumonia in critically ill trauma patients. Downregulation of CXCR2 and cleavage of CXCR1 would result in severe hyporesponsiveness to IL-8, an important neutrophil chemoattractant.

급성 염증에서의 중성구 기능 장애

적절한 항미생물 방어 메커니즘은 후속 면역 반응을 방해할 수 있습니다. 이는 자체 필수 항미생물 무기의 이상적인 조절 장애로 인해 발생하며,

예를 들어:

(1) 필수 표면 수용체의 자가 및 파라크린 분해;

(2) 비해결성 염증이나 초기 무균 염증 후 두 번째 미생물 공격 시 표면 수용체 및 신호 전달 경로의 다운레귤레이션(손상 관련 분자 패턴(DAMP)과 미생물 관련 분자 패턴(MAMP) 간 간섭);

(3) 미생물 살상 능력이 감소된 중성구 집단의 방출. 또한 염증 상태에서 중성구는 다른 면역 세포에 영향을 미쳐 적응 면역계의 면역 마비를 유발할 수 있습니다.

중성구 유래 프로테아제에 의한 단백질 분해 및 면역 수용체 발현 감소

중성구가 분비하는 세린 프로테아제는 중성구-미생물 상호작용에 중요한 수용체의 발현에 영향을 미칩니다(그림 1a). 세린 프로테아제 활성화 수용체(PARs)를 통한 자극 효과 외에도, 이들은 적응 면역 세포와 선천 면역 세포 표면의 필수 수용체를 분해하여 면역 반응을 억제할 수 있습니다[62]. 예를 들어, 중성구 엘라스테이스는 중성구 표면의 IL-8 수용체인 CXCR1을 분해합니다[63, 64]. 이 메커니즘은 외상 및 패혈증 환자의 순환 중성구가 IL-8의 유일한 다른 중성구 수용체인 CXCR2를 선택적으로 억제하는 급성 염증 과정에서 관련이 있습니다[65, 66]. Tarlowe 등 [67]은 중증 외상 환자의 폐렴 발생 전에 이 수용체의 억제가 선행된다는 증거를 제시했습니다. CXCR2의 억제와 CXCR1의 분해는 중성구 화학유인물질인 IL-8에 대한 심각한 저반응성을 초래합니다.

Fig. 1.

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Downregulation of immune receptors by serine proteases from degranulated neutrophils and desensitisation by pattern recognition receptors. a Schematic representation of downregulation of receptors on neutrophils, monocytes and lymphocytes during inflammation due to cleavage by neutrophil serine proteases after degranulation. Binding of C5a to neutrophils results in internalisation of C5aR. Decreased expression‎ of these receptors impairs neutrophil effector functions during subsequent challenges. b Biological mimicry between DAMPs and MAMPs. Danger signals derived from necrotic tissue cells ("First hit") bind to pattern recognition receptors (PRRs) and limit subsequent responses to microbial signals ("Second hit") through homo- and heterologous desensitisation. DAMP damage-associated molecular pattern, IL interleukin, MAMP microbe-associated molecular pattern

Furthermore, neutrophil serine proteases can cleave complement receptors such as the CR1 receptor (CD35) and C5aR (CD88) on neutrophils [68, 69]. These receptors are important as they mediate chemotaxis, degranulation and proper recognition of opsonised microbial targets by CR1 and C5aR, respectively [70]. During inflammation, decreased expression‎ of C5aR is seen due to engagement and subsequent internalisation. This can result in a profound defect in neutrophil phagocytosis of subsequent pathogens as C5a-induced chemotaxis is important for neutrophils to find opsonised targets [71]. Proteases not only inhibit the function of neutrophils, they can also affect monocytes in the micro-environment. Neutrophil elastase cleaves CD14, a receptor necessary for the high affinity recognition of lipopolysaccharide by TLR4, thereby impairing proper bacterial recognition by monocytes [72]. Lastly, elastase and cathepsin G mediate shedding of cytokine receptors for IL-2 and IL-6 on T lymphocytes [73].

DAMP–MAMP interference

Trauma and ischemia/reperfusion injury can evoke the release of large amounts of cellular components from necrotic cells. These intracellular constituents are known as damage-associated molecular patterns (DAMPS). They are host-derived and serve as important pro-inflammatory non-microbial stimuli after injury [74]. Since the development of the ‘danger hypothesis’ by Matzinger [74], a large number of studies have focussed on molecules driving this response. The most extensively studied DAMPS are high-mobility group box 1, heat shock proteins, ATP, uric acid, formylated peptides from mitchondria and mitochondrial DNA [75–80]. Inflammation induced by pathogens on the other hand is mediated through microbial constituents referred to as microbe-associated molecular patterns (MAMPS), which resemble DAMPS and, importantly, share similar pattern recognition receptors (PRRs) on the neutrophil [81]. This biological mimicry and utilisation of similar receptors creates a problem for the immune system since injury (DAMPS) causes downregulation of many of these receptors by hetero- and homologous desensitisation. This can render neutrophils unable to mount an adequate response to a subsequent microbe (MAMP) (Fig. 1b). To illustrate the relevance of this phenomenon, Zhang et al. [80] showed the release of vast amounts of mitochondrial formylpeptides into the circulation of major trauma patients. These molecules stimulate neutrophils via formyl peptide receptor 1 (FPR1), an important receptor in recognizing microbes that produce danger signals by release of formyl-peptides [80] (Fig. 1b). It was shown that heterologous desensitisation of chemokine receptors and homologous desensitisation of FPR1 occurred simultaneously, predisposing trauma patients to infection [82].

Release of incompetent neutrophil populations

Much of the work detailed in the previous sections did not take into account the variations in functional phenotypes that appear in the circulating neutrophil compartment during severe inflammation. After maturation neutrophils are retained in the bone marrow via expression‎ of chemokine receptor CXCR4 (ligand CXCL12), whilst CXCR2 (ligands IL-8/CXCL1 and 2) controls release into the peripheral blood. Inflammatory stimuli can induce the release of neutrophils by disrupting the balance in CXCR4/CXCL12 signalling through various mechanisms [60]. In severe inflammation large numbers of neutrophils are released into the circulation from the bone marrow post-mitotic pool as well as from the marginated pool (i.e. neutrophils not freely circulating but attached to the microvasculature) [83]. Under these conditions we have previously shown that peripheral neutrophils consist of heterogeneous subsets with different priming states and function [84]. During severe inflammation a large number of immature or banded cells appear in the circulation and even neutrophil progenitor cells can be identified. As a result, persistent severe inflammation might lead to "bone marrow exhaustion" of neutrophils, which is thought to inevitably result in a state of compromised innate immunity [85]. At present, however, it is unclear how to interpret the presence of immature cells in the bloodstream in response to inflammation. It might be a compensatory response initiated by the depletion of mature neutrophils in the bone marrow or a dedicated inflammatory reaction to a bacterial stimulus. Our data support the first hypothesis since these immature neutrophils also show a pronounced decrease of various receptors in comparison with their mature circulating counterparts [84]. In addition to the IL-8 receptors (CXCR1 and CXCR2) and the C5a receptor, the Fc receptors (CD16 and CD32), which are important in pathogen recognition, phagocytosis and killing, are also downregulated on immature cells (Fig. 2) [84]. Relatively few studies have assessed the functionality of immature and progenitor neutrophils subsets in severe human inflammation. In septic patients, immature neutrophils were shown to have decreased phagocytic capacity [86]. Importantly, reduced phagocytosis and increased numbers of circulating neutrophil progenitors are both associated with poor outcome in septic patients as well as in patients with severe inflammation [87, 88].

Fig. 2.

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Circulating neutrophil subsets in severe inflammation. At least four types of neutrophils circulate in the bloodstream of patients during severe inflammation: immature, competent and suppressive neutrophils and myeloid-derived suppressor cells. Mechanisms contributing to immune dysfunction are displayed for neutrophils from different subsets. ROS reactive oxygen species

Suppression of adaptive immunity by neutrophils

Neutrophils have long been recognised as professional killers. Eradication of bacteria and fungi was thought to be their main task. Evidence is, however, accumulating that apart from their direct anti-microbial function, neutrophils participate in subsequent modulation of (adaptive) immune responses in severe inflammation [89–91]. Under these inflammatory conditions, neutrophils produce chemokines and secrete granule contents which can subsequently attract and modulate the function(s) of T cells both directly and indirectly [92, 93]. For instance, neutrophil elastase reduces expression‎ of co-stimulatory molecules by dendritic cells, limiting maturation and induction of a proper Th1 response [94]. In addition, T cells in the inflammatory microenvironment may be affected by neutrophil elastase by cleavage of their IL-2 and IL-6 receptors (Fig. 1a) [95]. Another mechanism of immune-modulation was observed in macrophages after phagocytosis of apoptotic neutrophils. Under these conditions immune responses of macrophages shift towards a more anti-inflammatory cytokine profile [96]. Furthermore, neutrophils themselves have been shown to produce anti-inflammatory cytokines such as IL-1ra and IL-10 [97]. However, the evidence regarding IL-10 production by neutrophils is controversial, as it has only been shown in mice with mycobacterial infections [98]. In humans neutrophils are unable to produce IL-10 [99]. Direct regulation of T-cell responses by neutrophils is slowly becoming an established concept. A large body of evidence demonstrates that a heterogeneous group of immature mononuclear cells and neutrophils termed myeloid-derived suppressor cells (MDSCs) can suppress T-cell responses in several murine tumour models. In addition, these cells have been shown to play a role in various models of infectious diseases, organ transplantation and autoimmune diseases [100]. Identification of human immature granulocytic MDSCs has proven to be challenging though. In particular, their differentiation from mature neutrophil phenotypes seen in the blood during acute inflammation remains to be established, as we have reviewed in detail elsewhere [101]. The mechanisms by which MDSCs can suppress T cells include the expression‎ and secretion of arginase-1, which depletes arginine from the microenvironment (Fig. 2) [102]. Depletion of L-arginine, which is an essential amino acid, results in cell cycle arrest of T cells in the G0–G1 phase [103]. Furthermore, in human inflammation we and others have observed a population of mature CD62Ldim neutrophils capable of suppressing T-cell responses through a mechanism which relies on ROS release in an immunological synapse [104]. Recently, similar neutrophils in septic shock patients have been found to express arginase-1 and suppress T-cell functions [105]. Another mechanism by which neutrophils might inhibit T-cell responses is through PD-L1 [106]. Neutrophils isolated from sepsis patients express the surface protein PD-L1, a potent inducer of apoptosis in T cells. The underlying mechanism of PD-L1 expression‎ is an interferon-gamma-dependent process [106]. The PD-1–PD-L1 axis is thought to be an important mechanism in immune suppression in septic patients by inducing lymphocyte apoptosis and monocyte dysfunction [107]. Blocking this axis after the induction of sepsis by administering a PD-1-blocking antibody improved survival in mice [108]. This suppressive mechanism might be protective in tissues with severe inflammatory infiltrates. On the other hand, this process might be unwanted when neutrophils migrate to lymph nodes and engage with adaptive immunity, as has been described under various conditions [109]. In these lymph nodes neutrophils are able to inhibit humoral immune responses through interaction with T and B lymphocytes [109, 110].

Conclusion

Severe inflammation can result in immune paralysis through various mechanisms. We propose that neutrophils play a central role in this process, either through decreased antimicrobial functions or through direct suppression of (adaptive) immunity. Many experimental studies have been performed addressing the damaging role of neutrophils, which contributes to organ failure in severe inflammation. However, their role in immune paralysis remains understudied. Studies to explore their causative role in susceptibility to infections in animal models of severe inflammation should be designed. Decreased neutrophil antimicrobial functions and their ability to suppress adaptive immunity in vitro should be considered as important patient outcomes. This approach is necessary to increase understanding of the role of neutrophils in immune paralysis leading to detrimental outcome in post-surgical, post-trauma and septic patients.

Acknowledgments

JP was supported by the Lung Foundation the Netherlands (grant number 5.2.14.058JO).

AbbreviationsARDS

acute respiratory distress syndrome

CGD

chronic granulomatous disease

DAMP

damage-associated molecular pattern

IL

interleukin

MAMP

microbe-associated molecular pattern

MDSC

myeloid-derived suppressor cell

NET

neutrophil extracellular trap

ROS

reactive oxygen species

Footnotes

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

PHCL designed search strategies, undertook search strategies, screened search results, organised retrieval‎ of papers, appraised quality of papers and extracted data from papers, drafted the manuscript and wrote the review. CMW designed search strategies, undertook search strategies, screened search results, organised retrieval‎ of papers, appraised quality of papers and extracted data from papers, drafted the manuscript and wrote the review. LPHL participated in designing this review, wrote the review and provided general advice on and coordinated the review. LK participated in designing this review, wrote the review and provided general advice on and coordinated the review. JP participated in designing this review, designed search strategies, undertook search strategies, screened search results, organised retrieval‎ of papers, appraised quality of papers and extracted data from papers, drafted the manuscript and wrote the review. All authors participated in editing of the manuscript and approved the final version.

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