Re: 중성구 세포외 트랩... 면역과 질병... 2017 nature

[문형철]

• Neutrophil extracellular traps (NETs) protect against infection, in particular by large pathogens, but they are also implicated in the pathology associated with a growing number of immune-mediated conditions.
• NET formation is triggered by innate immune receptors through downstream intracellular mediators that include reactive oxygen species (ROS), produced by NADPH oxidase or mitochondria, which activate myeloperoxidase (MPO), neutrophil elastase (NE) and protein-arginine deiminase type 4 (PAD4) to promote chromatin decondensation.
• NETosis is induced in response to microbial cues and endogenous danger signals and must be tightly regulated to prevent excessive tissue damage during acute inflammation or chronic inflammatory and autoimmune disease. Microorganism size, microbial virulence factors and cytokines are regulators of NETosis.
• NETs have several immune-modulatory functions that have been implicated in disease. They can prime other immune cells to induce sterile inflammation or potentiate autoimmunity by stimulating interferon responses owing to NET-associated oxidized DNA and antimicrobial peptides.
• NETs can also occlude the vasculature by promoting thrombosis and obstruct important organ areas, capture metastatic tumours and delay wound healing in diabetes.

Abstract

Neutrophils are innate immune phagocytes that have a central role in immune defence. Our understanding of the role of neutrophils in pathogen clearance, immune regulation and disease pathology has advanced dramatically in recent years. Web-like chromatin structures known as neutrophil extracellular traps (NETs) have been at the forefront of this renewed interest in neutrophil biology. The identification of molecules that modulate the release of NETs has helped to refine our view of the role of NETs in immune protection, inflammatory and autoimmune diseases and cancer. Here, I discuss the key findings and concepts that have thus far shaped the field of NET biology.

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.

Neutrophils are the most abundant innate immune effector cells of the human immune system. They are armed with broadly effective antimicrobials that are stored predominately in specialized granules. Given that this neutrophil arsenal can also damage host tissues, its deployment is tightly regulated through three major strategies: phagocytosis, degranulation and the release of neutrophil extracellular traps (NETs). NETs are large, extracellular, web-like structures composed of cytosolic and granule proteins that are assembled on a scaffold of decondensed chromatin1 . Although the majority of DNA in NETs originates from the nucleus, these structures also contain mitochondrial DNA2 . NETs trap, neutralize and kill bacteria1 , fungi3 , viruses4 and parasites5 and are thought to prevent bacterial and fungal dissemination6,7 . However, if dysregulated, NETs can contribute to the pathogenesis of immune-related diseases. Initially, 24 proteins were identified in NETs formed by stimulation of neutrophils with phorbol 12-myristate 13-acetate (PMA), a molecule that activates protein kinase C (PKC) and triggers the production of reactive oxygen species (ROS). Among these proteins were histones, the serine protease neutrophil elastase (NE; also known as ELANE), myeloperoxidase (MPO), calprotectin, cathelicidins, defensins and actin8 . Subsequent studies have extended this list, suggesting that the composition of NETs varies depending on the stimulus. For example, different Pseudomonas aeruginosa mucoid and non-mucoid strains induce the formation of NETs containing 33 common proteins and up to 50 variable proteins9 . Whether and how differences in NET composition impact NET function remains to be investigated. NET release occurs primarily through a cell death process termed NETosis10. To initiate this process, neutrophils arrest their actin dynamics and depolarize11. Next, the nuclear envelope disassembles, and nuclear chromatin decondenses into the cytoplasm of intact cells, mixing with cytoplasmic and granule components10 (FIG. 1). The plasma membrane then permeabilizes, and NETs expand into the extracellular space 3–8 hours after neutrophil activation. An alternative mechanism termed non-lytic NETosis leads to the rapid release of NETs within minutes of exposure to Staphylococcus aureus via the secretion of chromatin and granule contents12,13 and in the absence of cell death. This phenomenon has been observed by intravital microscopy in a small fraction of neutrophils during systemic S. aureus infection and generates NETs and anucleated cytoplasts that crawl and phagocytose bacteria13. This multitasking nonlytic rapid response is mounted by the first neutrophils to arrive at sites of infection.

중성구는 인간 면역 체계에서 가장 풍부한 선천성 면역 효과 세포입니다. 이들은 주로 특수한 과립에 저장된 광범위하게 효과적인 항균 물질을 갖추고 있습니다. 이 중성구의 무기가 호스트 조직을 손상시킬 수 있기 때문에, 그 활성화는 세 가지 주요 전략을 통해 엄격히 조절됩니다: 

식작용, 

과립 방출 및 중성구 세포외 트랩(NETs)의 방출입니다.

 NETs는 

세포질과 과립 단백질로 구성된 대형 세포외 웹 구조물로, 탈응축된 염색질 골격 위에 조립됩니다. 

NETs 내 DNA의 대부분은 핵에서 유래하지만, 

이 구조물에는 미토콘드리아 DNA도 포함됩니다. 

NETs는 세균1, 곰팡이3, 바이러스4, 기생충5를 

포획, 중화, 살상하며, 세균과 곰팡이의 확산을 방지하는 것으로 알려져 있습니다. 

그러나 

조절이 이상적으로 이루어지지 않을 경우, 

NETs는 면역 관련 질환의 병리 발생에 기여할 수 있습니다. 

초기 연구에서, 단핵구 자극제인 포르볼 12-미리스테이트 13-아세테이트(PMA)에 의해 형성된 NETs에서 24개의 단백질이 식별되었습니다. PMA는 단백질 키나제 C(PKC)를 활성화하고 활성산소종(ROS)의 생성을 유발하는 분자입니다. 이 단백질 중에는 히스톤, 세린 프로테아제 중 하나인 중성구 엘라스테이스(NE; ELANE로도 알려져 있음), 마이엘로퍼옥시다제(MPO), 칼프로텍틴, 카텔리시딘, 디펜신 및 액틴8이 포함되었습니다. 후속 연구는 이 목록을 확장했으며, NET의 구성은 자극에 따라 달라질 수 있음을 제안했습니다. 예를 들어, Pseudomonas aeruginosa의 점액성 및 비점액성 균주는 33개의 공통 단백질과 최대 50개의 변이 단백질을 포함하는 NET 형성을 유도합니다9. NET 구성의 차이가 NET 기능에 미치는 영향은 여전히 연구가 필요합니다. NET 방출은 주로 NETosis라고 불리는 세포 사멸 과정을 통해 발생합니다10. 이 과정을 시작하기 위해 중성구는 액틴 동역학을 중단하고 탈분극화됩니다11. 다음으로 핵막이 분해되고, 핵 염색질이 분해되어 완전한 세포의 세포질로 이동하며 세포질 및 과립 성분과 혼합됩니다10 (그림 1). 이후 세포막이 투과성이 되어, 중성구 활성화 후 3–8시간 내에 NET이 세포외 공간으로 확장됩니다. NETosis의 대체 메커니즘인 비용해성 NETosis는 Staphylococcus aureus에 노출된 후 몇 분 내에 염색질과 과립 내용물의 분비를 통해 NET이 급속히 방출됩니다12,13. 이 현상은 전신 S. aureus 감염 시 중성구의 소수 분획에서 생체 내 현미경 관찰을 통해 관찰되었으며, NET과 핵이 없는 세포질 조각이 박테리아를 기어다니며 식균하는 현상을 유발합니다13. 이 다기능성 비용해성 급속 반응은 감염 부위에 먼저 도착한 첫 번째 중성구들에 의해 유발됩니다.

The mechanisms that clear NETs are less well understood. During infection, NETs persist for several days7 and are thought to be dismantled by the secreted plasma nuclease DNase I (REF. 14). Injection of this enzyme during S. aureus infection leads to rapid degradation of NETassociated DNA15, but the dynamics of NET clearance by endogenous enzymes are unknown. Strikingly, NET proteins persist long after DNA degradation15, suggesting that they are cleared via additional mechanisms. These mechanisms might involve macrophage scavenging, as DNase I facilitates the ingestion of NETs by macrophages in vitro16. Here, I provide an overview of the mechanisms that regulate NET formation and clearance and describe recent advances in our understanding of how NETs protect against infection and cause pathology associated with several diseases. These topics are organized in a conceptual manner according to the immunological function of NETs. I pay attention to key findings, highlight open questions and discuss the controversies in the field.

NET를 제거하는 메커니즘은 아직 잘 이해되지 않고 있습니다. 감염 중 NET는 수일 동안 지속되며7, 분비된 플라즈마 핵산분해효소 DNase I에 의해 분해된다고 추정됩니다(REF. 14). S. aureus 감염 시 이 효소를 주입하면 NET와 관련된 DNA가 빠르게 분해되지만15, 내인성 효소에 의한 NET 제거의 역학은 알려지지 않았습니다. 주목할 점은 DNA 분해 후에도 NET 단백질이 장시간 지속된다는 점으로15, 이는 추가적인 메커니즘을 통해 제거된다는 것을 시사합니다. 이러한 메커니즘은 대식세포의 청소 작용을 포함할 수 있습니다. DNase I은 체외 실험에서 대식세포가 NET을 섭취하는 것을 촉진하기 때문입니다16. 본 논문에서는 NET 형성과 제거를 조절하는 메커니즘을 개괄적으로 설명하고, NET이 감염으로부터 보호하고 여러 질환과 관련된 병리를 유발하는 방식에 대한 최근 연구 성과를 소개합니다. 이 주제는 NET의 면역학적 기능에 따라 개념적으로 분류되었습니다. 주요 발견 사항을 강조하고, 미해결 문제를 지적하며, 해당 분야의 논쟁점을 논의합니다.

Mechanisms of NET formation

From ROS to chromatin decondensation.

Two enzymes in the ROS pathway have critical roles in NETosis. ROS generated by NADPH oxidase stimulate MPO to trigger the activation and translocation of NE from azurophilic granules to the nucleus, where NE proteolytically processes histones to disrupt chromatin packaging 17. Subsequently, MPO binds chromatin and synergizes with NE in decondensing chromatin independently of its enzymatic activity17 (FIG. 2). NADPH oxidase activity can be redundant in response to some stimuli, such as immune complexes in which mitochondrial ROS are sufficient to drive NETosis2 . NE release from azurophilic granules does not require membrane rupture or fusion. In resting neutrophils, a fraction of MPO is bound to NE as part of a complex called the azurosome, which spans granule membranes11. Hydrogen peroxide selectively releases NE into the cytosol in an MPO-dependent manner (FIG. 2). It is important to clarify that inhibition of the enzymatic activity of MPO does not block but only delays NETosis18, potentially owing to the role of MPO in activating the proteolytic activity of NE against large protein substrates. This oxidative activation is important because NE binds to F-actin filaments in the cytoplasm and must degrade them in order to enter the nucleus11. NE is sufficient to decondense nuclei in vitro17, but unknown mechanisms may help to disassemble the nuclear envelope in neutrophils. This MPO–NE pathway is induced by many NET stimuli, such as fungi and crystals19,20, and its role is supported by studies of neutrophils from patients with chronic granulomatous disease (CGD)10 and with complete MPO deficiency 18, as well as by studies using NE-deficient mice or NE inhibitors in mouse models of sepsis, cancer and pulmonary infection7,15,17,21. NET release is also abrogated in NADPH oxidase-deficient mice during pulmonary fungal infection, which stimulates robust NET release22. Similarly, NETosis is defective in neutrophils from patients with Papillon–Lefèvre syndrome caused by mutations in the cysteine protease cathepsin C (CTSC), which processes NE into its mature form23,24. Mice lacking CTSC fail to form NETs upon pulmonary Sendai virus infection25 and in aortic aneurism models26. Moreover, isolated CTSC-deficient neutrophils exhibit defects in NETosis, although the impairment is less striking than that observed after pharmacological NE inhibition. One study challenged the requirement of NE in NETosis27 on the basis of experiments with PMA-induced mouse neutrophils that yielded low levels of NETs. By contrast, NE deficiency and inhibitors attenuated NETosis upon stimulation with the Ca2+ ionophore ionomycin, which induced a robust response27. In the same study, NE deficiency did not reduce NET-mediated thrombosis in vivo27 , but this result contradicts prior literature28. Another nuclear chromatin-binding protein that has recently been implicated in NETosis is DEK. NETosis is defective in Dek-deficient neutrophils and can be rescued by addition of exogenous recombinant DEK protein, which suggests that DEK binding promotes chromatin decondensation in a similar manner to MPO29.

NET 형성의 메커니즘: 

ROS에서 염색질 해체까지. 

ROS 경로에 존재하는 두 가지 효소가 NETosis에 결정적인 역할을 합니다. NADPH 산화효소에 의해 생성된 ROS는 MPO를 자극하여 아즈로필릭 그란울에서 핵으로의 NE 활성화 및 이동을 유발합니다. 여기서 NE는 히스톤을 단백질 분해적으로 처리하여 염색질 포장 구조를 파괴합니다 17. 이후 MPO는 염색질에 결합하여 효소 활성과 무관하게 NE와 시너지 효과를 발휘해 염색질 분해를 촉진합니다 17 (그림 2). NADPH 산화효소의 활성은 일부 자극에 대해 중복적일 수 있습니다. 예를 들어 면역 복합체에서 미토콘드리아 ROS만으로도 NETosis를 유발할 수 있습니다2. 아즈로필릭 그레인에서 NE의 방출은 막 파열이나 융합을 필요로 하지 않습니다. 휴지 상태의 중성구에서 MPO의 일부는 아즈로소ーム이라는 복합체의 일부로 NE와 결합되어 그레인 막을 가로지릅니다11. 과산화수소는 MPO 의존적 방식으로 NE를 세포질로 선택적으로 방출합니다(그림 2). MPO의 효소 활성을 억제하는 것은 NETosis를 차단하지 않고 단지 지연시킬 뿐이라는 점을 명확히 하는 것이 중요합니다18. 이는 MPO가 NE의 단백질 분해 활성을 활성화하는 역할 때문일 수 있습니다. 이 산화 활성화는 NE가 세포질 내 F-액틴 필라멘트에 결합하고 이를 분해해야 핵으로 진입할 수 있기 때문에 중요합니다11. NE는 체외에서 핵을 분해하는 데 충분하지만, 중성구에서 핵막 분해에 기여하는 미지의 메커니즘이 존재할 수 있습니다. 이 MPO–NE 경로는 곰팡이와 결정체와 같은 다양한 NET 자극에 의해 유도되며, 만성 과립구증(CGD) 환자10 및 완전한 MPO 결핍 환자18의 중성구 연구, 그리고 NE 결핍 마우스나 NE 억제제를 사용한 패혈증, 암, 폐 감염 마우스 모델 연구7,15,17,21에서 그 역할이 지원됩니다. NADPH 산화효소 결핍 마우스에서 폐 곰팡이 감염 시 NET 방출이 억제되며, 이는 강력한 NET 방출을 자극합니다22. 마찬가지로, 시스테인 프로테아제 카테프신 C(CTSC)의 돌연변이로 인한 파피용-레프레브 증후군 환자의 중성구에서 NETosis가 결손되며, CTSC는 NE를 성숙형으로 처리합니다23,24. CTSC 결핍 마우스는 폐 센다이 바이러스 감염 시 NET을 형성하지 못하며25, 대동맥 동맥류 모델에서도 마찬가지입니다26. 또한, CTSC 결핍 중성구는 NETosis 결함을 보이지만, 약리학적 NE 억제 후 관찰된 결함보다 덜 두드러집니다. 한 연구는 PMA로 유도된 쥐 중성구에서 NET 수준이 낮은 실험을 바탕으로 NETosis에 NE의 필요성을 도전했습니다27. 반면, NE 결핍과 억제제는 Ca2+ 이온포어 이오노마이신으로 자극 시 강력한 반응을 유발한 NETosis를 억제했습니다27. 같은 연구에서 NE 결핍은 체내에서 NET 매개 혈전 형성을 감소시키지 않았습니다27, 이는 이전 문헌과 모순됩니다28. 최근 NETosis와 연관된 또 다른 핵 염색질 결합 단백질은 DEK입니다. DEK 결핍 중성구에서 NETosis가 결손되며, 외인성 재조합 DEK 단백질 추가로 회복됩니다. 이는 DEK 결합이 MPO와 유사한 방식으로 염색질 탈응축을 촉진함을 시사합니다29.

As mentioned above, some NET stimuli, such as immune complexes, ionomycin and nicotine, have been proposed to trigger NETosis independently of NADPH oxidase, relying instead on mitochondrial ROS2,30,31. Non-lytic NETosis is also thought to occur independently of ROS12. It is therefore important to consider the effects of ROS-blocking compounds on ROS generated by both the NADPH oxidase and the mitochondria. ROS do not only trigger chromatin decondensation. Chlorinated polyamines generated upon reaction with hypochlorous acid, produced by MPO, crosslink NET proteins, increasing NET stability and integrity and potentiating the capture of microorganisms32. This crosslinking reaction might explain why NET proteins persist longer than DNA following DNase I administration in vivo15. Interestingly, glycans in saliva induce NETs via an unknown mechanism that does not involve ROS or NE. These NETs are more resistant to nucleases and kill microorganisms more effectively than NETs generated with PMA33. Therefore, different pathways may generate NETs with different functional attributes.

위에서 언급된 바와 같이, 면역 복합체, 이오노마이신 및 니코틴과 같은 일부 NET 자극제는 NADPH 산화효소와 독립적으로 NETosis를 유발하며, 대신 미토콘드리아 ROS에 의존한다는 제안이 있습니다.2,30,31 비리틱 NETosis는 ROS12와 독립적으로 발생한다고 여겨집니다. 따라서 NADPH 산화효소와 미토콘드리아에서 생성되는 ROS에 대한 ROS 차단제의 영향을 고려하는 것이 중요합니다. ROS는 염색질 탈응축을 유발하는 것 외에도, MPO에 의해 생성된 차아염소산과 반응하여 생성되는 염소화 폴리아민이 NET 단백질을 교차결합시켜 NET의 안정성과 완전성을 증가시키고 미생물 포획을 강화합니다32. 이 교차결합 반응은 DNase I 투여 후 체내에서 NET 단백질이 DNA보다 더 오래 지속되는 이유를 설명할 수 있습니다15. 흥미롭게도, 타액 내 글리칸은 ROS나 NE와 무관한 미지의 메커니즘을 통해 NET을 유도합니다. 이러한 NET은 핵산 분해효소에 더 저항성이 있으며, PMA로 생성된 NET보다 미생물을 더 효과적으로 살균합니다33. 따라서 서로 다른 경로를 통해 기능적 특성이 다른 NET이 생성될 수 있습니다.

Another chromatin modification that is implicated in chromatin decondensation is histone deamination or citrullination, which is driven by protein-arginine deiminase type 4 (PAD4), a nuclear enzyme that citrullinates arginine residues, converting amine groups to ketones34,35 (FIG. 2). Despite evidence that PAD4 activity requires a reducing environment36, inhibition of NADPH oxidase decreases citrullination. Moreover, hydrogen peroxide is sufficient to activate PAD4 (REFS 37,38), which requires calcium39 and is activated by PKCζ40,41, a kinase that is implicated in the ROS burst. Together, these observations suggest that PAD4 lies downstream of ROS and calcium signalling during NETosis. The degree and specificity of citrullination seems to vary depending on the stimulus owing to the activation of different PKC isoforms that activate or suppress PAD4 (REFS 41–43). Physiological stimuli such as fungi and crystals induce histone citrullination during NETosis7,20. However, the contribution of citrullination to chromatin decondensation has been more difficult to eval‎uate38,44,45. Experiments with cell lines treated with PAD4 inhibitors or with mouse neutrophils derived from PAD4-deficient mice were initially difficult to interpret owing to low NET yields38,46. Mixed results have been reported with pharmacological PAD4 inhibition with Cl-amidine in human neutrophils known to be robust NET producers. For example, PAD4 inhibition blocks NETosis induced by nicotine but does not interfere in the formation of NETs induced by cholesterol crystals20,31. One complicating issue is that histone citrullination is often used as the sole marker to detect NETs in PAD4-deficient or PAD4-inhibited mice47,48. However, recent studies using multiple NET markers showed that PAD4 inhibition blocks NET release in mouse models of sepsis and cancer15,21. Moreover, PAD4-deficient mouse neutrophils fail to release NETs upon stimulation with lipopolysaccharide (LPS) and tumour necrosis factor (TNF)43. Whether histone citrullination is sufficient to promote chromatin decondensation in the absence of NE activity is unclear. NE inhibitors block chromatin de - condensation during pulmonary fungal infection without interfering with histone H3 citrullination7 , suggesting that histone citrullination occurs independently of NE activity, but histone citrullination might not be sufficient to drive chromatin decondensation. Interestingly, recent findings suggest that the repertoire of citrullinated proteins in NETosis induced by microorganisms or PMA is dominated by histones and is distinct from extensive protein hyper-citrullination associated with stress inducers such as ionomycin, pore-forming toxins and immune complexes42,49. Therefore, different NET-inducing stimuli might engage PAD enzymes in diverse ways, and the pattern of citrullinated substrates could help to determine the relevant immunopathogenic mechanisms in vivo. In summary, these pathways are implicated in NETosis, and their pharmacological inhibition blocks chromatin decondensation in a variety of scenarios. Nevertheless, examples of alternative mechanisms are also emerging. Furthermore, PMA and ionomycin are useful for mechanistic studies, but data obtained with these non-physiological stimuli should be viewed with caution until validated with physiological stimuli. PMA and fungi elicit common pathways downstream of ROS (TABLE 1). The ionomycin-induced pathway that involves calcium signalling and small conductance calcium-activated potassium channel protein 3 (SK3) may share features with pathways induced by immune complexes, platelets and other stimuli that elicit a faster mitochondrial ROS-dependent response with varying degrees of citrullination30,41.

크로마틴 해체에 관여하는 또 다른 크로마틴 변형은 히스톤 탈아미노화 또는 시트룰리네이션으로, 이는 핵 효소인 단백질-아르기닌 데이미네이스 유형 4(PAD4)에 의해 촉진됩니다. PAD4는 아르기닌 잔기를 시트룰린으로 변환하여 아민 그룹을 케톤으로 전환합니다(34,35)(그림 2). PAD4 활성이 환원 환경을 필요로 한다는 증거가 있음에도 불구하고(36), NADPH 산화효소 억제는 시트룰리네이션을 감소시킵니다. 또한 과산화수소는 PAD4를 활성화하는 데 충분하며(REFS 37,38), 이는 칼슘39를 필요로 하며 ROS 폭발과 관련된 키나아제인 PKCζ40,41에 의해 활성화됩니다. 이러한 관찰 결과는 NETosis 동안 PAD4가 ROS 및 칼슘 신호전달 경로의 하류에 위치함을 시사합니다. 시트룰리네이션의 정도와 특이성은 자극에 따라 달라지며, 이는 PAD4를 활성화하거나 억제하는 다양한 PKC 이소형의 활성화에 기인합니다(REFS 41–43). 곰팡이와 결정체와 같은 생리적 자극은 NETosis 동안 히스톤 시트룰리네이션을 유도합니다7,20. 그러나 시트룰리네이션이 염색질 해체에 기여하는 정도는 평가하기 어려웠습니다38,44,45. PAD4 억제제로 처리된 세포주나 PAD4 결핍 마우스에서 유래한 마우스 중성구로 수행된 초기 실험은 NET 생성량이 낮아 해석이 어려웠습니다38,46. 인간 중성구에서 강력한 NET 생성체로 알려진 Cl-amidine을 사용한 약리학적 PAD4 억제 실험에서는 혼합된 결과가 보고되었습니다. 예를 들어, PAD4 억제는 니코틴에 의해 유도된 NETosis를 차단하지만 콜레스테롤 결정에 의해 유도된 NET 형성에 간섭하지 않습니다20,31. 복잡한 문제 중 하나는 히스톤 시트룰리네이션이 PAD4 결핍 또는 PAD4 억제 마우스에서 NET을 탐지하는 유일한 지표로 자주 사용된다는 점입니다47,48. 그러나 다중 NET 지표를 사용한 최근 연구에서는 PAD4 억제가 패혈증 및 암 마우스 모델에서 NET 방출을 차단한다는 것이 밝혀졌습니다15,21. 또한, PAD4 결핍 마우스의 중성구는 리포폴리사카라이드(LPS)와 종양 괴사 인자(TNF) 자극 시 NET을 방출하지 않습니다43. 히스톤 시트룰리네이션이 NE 활성 없이 염색질 탈응축을 촉진하는 데 충분한지 여부는 명확하지 않습니다. NET 억제제는 폐 곰팡이 감염 시 히스톤 H3 시트룰리네이션에 영향을 주지 않으면서 염색질 탈응축을 차단합니다7, 이는 히스톤 시트룰리네이션이 NET 활성 없이 발생할 수 있음을 시사하지만, 히스톤 시트룰리네이션만으로는 염색질 탈응축을 유도하기에 충분하지 않을 수 있습니다. 흥미롭게도 최근 연구 결과는 미생물이나 PMA에 의해 유도된 NETosis에서 시트룰린화 단백질의 구성은 히스톤이 주를 이루며, 이온마이신, 구멍 형성 독소 및 면역 복합체와 같은 스트레스 유발제에 따른 광범위한 단백질 과시트룰린화와는 다르다는 것을 보여줍니다. 따라서 다양한 NET 유도 자극은 PAD 효소를 서로 다른 방식으로 활성화할 수 있으며, 시트룰린화 기질의 패턴은 체내에서 관련 면역 병리학적 메커니즘을 결정하는 데 도움이 될 수 있습니다. 요약하면, 이러한 경로는 NETosis와 연관되어 있으며, 약리학적 억제는 다양한 상황에서 염색질 탈응축을 차단합니다. 그럼에도 불구하고 대체 메커니즘의 예시가 등장하고 있습니다. 또한, PMA와 이오노마이신은 기전 연구에 유용하지만, 생리적 자극으로 검증되기 전까지 이러한 비생리적 자극으로 얻은 데이터는 신중하게 해석해야 합니다. PMA와 곰팡이는 ROS 하류에서 공통 경로를 유발합니다(표 1). 이오노마이신에 의해 유발되는 경로는 칼슘 신호전달과 소형 칼슘 활성화 칼륨 채널 단백질 3(SK3)을 포함하며, 면역 복합체, 혈소판 및 기타 자극에 의해 유발되는 경로와 유사한 특성을 공유할 수 있습니다. 이러한 경로는 다양한 정도의 시트룰리네이션을 동반한 더 빠른 미토콘드리아 ROS 의존적 반응을 유발합니다30,41.

Upstream signalling pathways.

The pathways that promote NETosis upstream of ROS are incompletely understood. A number of ROS-inducing receptors (BOX 1) and kinases, such as MEK (MAPK/ERK kinase), extracellular-signal-regulated kinase (ERK), IL-1 receptor-associated kinase (IRAK), PKC, phosphoinositide 3-kinase (PI3K) and AKT, have been linked to NETosis in response to PMA, microorganisms, parasites and immobilized immune complexes4,40,50–53 (FIG. 2; TABLE 1). The requirement for PI3K in NETosis has also implicated a role for autophagy, which also depends on this enzyme54. Consistent with this, promyelocytes that lack the autophagy-associated protein ATG7 exhibit a modest decrease in NET release55. By contrast, a requirement for mechanistic target of rapamycin (mTOR), which suppresses autophagy, has also been reported in NETosis56. Nevertheless, LC3B+ vacuoles that resemble autophagosomes have been observed in neutrophils undergoing NETosis54,55,57. Finally, ROS are known to induce autophagy 58, which in turn is required to sustain the ROS burst59 and might also help to tolerate ROS-induced stress. During NETosis, plasma membrane permeabilization occurs in a programmed manner and not as a consequence of physical disruption by the expanding chromatin11. This observation suggests that NETosis involves programmed cell death. Consistent with this observation, NET inducers, such as monosodium urate (MSU) crystals, promote necroptosis60, and neutrophils lacking receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3, two kinases involved in necroptosis, fail to form NETs without altering their ROS burst, which indicates that these enzymes act downstream or in parallel with the ROS pathway61. However, the role of these kinases in NETosis has been challenged by others62, and more evidence is needed to confirm‎ their role and mode of action.

상류 신호 전달 경로. 

ROS 상류에서 NETosis를 촉진하는 경로는 아직 완전히 이해되지 않았습니다. ROS를 유도하는 여러 수용체(BOX 1)와 키나제, 예를 들어 MEK (MAPK/ERK 키나제), 세포외 신호 조절 키나제 (ERK), IL-1 수용체 연관 키나제 (IRAK), PKC, 인산인오시타이드 3-키나제 (PI3K) 및 AKT, PMA, 미생물, 기생충 및 고정된 면역 복합체에 대한 반응으로 NETosis와 관련이 있는 것으로 밝혀졌습니다4,40,50–53 (그림 2, 표 1). NETosis에 PI3K가 필요한 것은 이 효소에 의존하는 자가포식도 관련이 있음을 의미합니다54. 이와 일치하여, 자가포식 관련 단백질 ATG7이 결핍된 전골수세포는 NET 방출이 소폭 감소하는 것을 보입니다55. 반면, 자가포식을 억제하는 mTOR (mechanistic target of rapamycin)의 필요성도 NETosis에서 보고되었습니다56. 그럼에도 불구하고, NETosis를 겪는 호중구에서 자가포식 소체와 유사한 LC3B+ 액포가 관찰되었습니다54,55,57. 마지막으로, ROS는 자가포식을 유도하는 것으로 알려져 있으며58, 이는 ROS의 폭발을 유지하는 데 필요하며59 ROS로 인한 스트레스를 견디는 데도 도움이 될 수 있습니다. NETosis 동안, 혈장 막 투과성은 확장된 염색질에 의한 물리적 파괴의 결과로 발생하지 않고, 프로그램된 방식으로 발생합니다11. 이 관찰은 NETosis에 프로그램된 세포 사멸이 관련되어 있음을 시사합니다. 이 관찰과 일치하게, NET 유도제인 모노소디움 우레이트(MSU) 결정은 네크로토시스를 촉진하며, 네크로토시스 관련 키나아제인 RIPK1과 RIPK3를 결여한 중성구는 ROS 급증을 변화시키지 않으면서도 NET을 형성하지 못합니다. 이는 이러한 효소가 ROS 경로 하류 또는 병행하여 작용함을 시사합니다. 그러나 이러한 키나제의 NETosis에서의 역할은 다른 연구자들에 의해 도전받았으며62, 그들의 역할과 작용 메커니즘을 확인하기 위해 추가적인 증거가 필요합니다.

Regulation of NETosis.

NETosis must be tightly regulated to prevent pathology. The size of microorganisms is one of several factors that influence NETosis. The sensing of pathogen size depends on the competition between NETosis and phagocytosis for access to NE. This mechanism enables neutrophils to preferentially deploy NETs against large microorganisms. Small microorganisms are taken up into phagosomes that fuse with azurophilic granules, sequestering NE away from the nucleus and blocking chromatin engage microorganisms that are too large to be ingested allows NE to translocate to the nucleus via the slower azurosome pathway and to drive NETosis. Furthermore, NE release into the cytosol promotes actin cytoskeleton degradation, blocking phagocytosis and committing cells to NETosis11 (FIG. 2). The influence of particle size on NETosis also applies to sterile stimuli. Larger, needleshaped urate crystals trigger NETosis more potently than urate microaggregates that are small enough to be ingested63. The selective induction of NETosis limits unnecessary tissue damage during infection by pathogens that are small enough to be killed intracellularly. Accordingly, mice that lack the antifungal phagocytic receptor dectin 1 are unable to selectively suppress NETosis and are susceptible to NET-mediated pathology in response to small microorganisms7 . However, NETosis induced by small bacteria has been widely reported. Other studies even report an increase in both phagocytosis and NET formation upon bacterial opsonization by IgA64 or disruption of the bacterial capsule65. Nevertheless, many of these microorganisms can survive and escape phagosomes66. It is therefore possible that NETosis is reserved for small virulent microorganisms that interfere with phagosomal killing. Consistent with this idea, virulent enteropathogenic bacteria induce NET formation, whereas non-virulent probiotic bacteria do not67. One strategy for small microorganisms to evade phagocytosis is aggregation. Large aggregates of Mycobacterium bovis Bacillus Calmette–Guérin drive NETosis in a microorganism size-dependent manner 7 . Similarly, S. aureus, which has been shown to stimulate NETosis in mouse models of sepsis12,13, forms large abscesses and aggregates upon exposure to plasma68,69. Aggregation might also explain early observations of pulmonary NET induction following infection with clumps of Klebsiella pneumoniae grown in solid phase17. Alternatively, microbial interference with phagosome maturation may also enable small microorganisms to induce NETosis. Neisseria gonorrhoeae delays the fusion of the phagosome with azurophilic granules and induces NETosis70. Virulence mechanisms are also involved in the ability of P. aeruginosa to induce NETosis, which depends on expression‎ of a motile flagellum71. Bacteria that lack flagella fail to elicit a potent ROS burst and NETosis, but flagella alone are not sufficient to induce NETosis. These findings appear to contradict the size-dependence principle. However, flagella are also known to alter host cell biology 72,73, and it will be interesting to investigate whether and how they might potentiate the translocation of NE to the nucleus. Several findings suggest that by altering neutrophil cell biology, microbial virulence factors affect NETosis74. Many virulent S. aureus serotypes kill neutrophils75 and might promote the association of NET components by physical lysis of cellular membranes. For example, the S. aureus pore-forming toxin leukotoxin GH is sufficient to drive NETosis, but it is unclear whether it is required for NET induction by bacteria76. Moreover, expression‎ of invasin, an adhesin that binds β- integrins, potentiates the ROS burst to induce NETosis in response to Yersinia pseudotuberculosis77. Finally, the observation that Porphyromonas gingivalis mutants that lack a phagocytosis-promoting protease drive NETosis78 is also consistent with the ability of phagocytosis to regulate NETosis.

decondensation7 (FIG. 2). The absence of phagosomes in neutrophils that In addition, microorganisms attenuate NETosis by engaging host receptors that suppress neutrophil activation. Both group A streptococci (GAS) and group B streptococci (GBS) deploy molecules that resemble sialic acids to dampen the ROS burst and reduce NETosis79,80 (FIG. 3; TABLE 1). Similarly, P. aeruginosa and GAS suppress NETosis through Siglec-9 by coating themselves with host sialylated glycoproteins80,81. Moreover, β-protein from GBS suppresses NETosis by binding to Siglec-5. However, engagement of Siglec-14 by β-protein antagonizes the repressive effects of Siglec-5 by activating mitogen-activated protein kinase (MAPK) signalling, which explains why polymorphisms that disrupt Siglec-14 increase host susceptibility to GBS82. Ligation of signal inhibitory receptor on leukocytes 1 (SIRL1) also attenuates NETosis by downregulating ROS production in response to S. aureus or MSU crystals83–85, but the physiological role of this pathway is unclear. In a similar manner, whereas bacterial biofilms induce NET formation86, fungal biofilms suppress NETosis by blocking ROS generation and increasing resistance to neutrophil killing87. The suppression of NETosis depends on mannosylation enzymes, but these enzymes are also important for fungal cell wall integrity, thereby making it difficult to attribute the virulence of these fungi solely to the suppression of NETosis87. Finally, the induction of immunosuppressive cytokines such as IL-10 can also inhibit NET release4 . In summary, microorganisms modulate NETosis through diverse mechanisms, depending on their size and the expression‎ of virulence factors.

In addition, microorganisms attenuate NETosis by engaging host receptors that suppress neutrophil activation. Both group A streptococci (GAS) and group B streptococci (GBS) deploy molecules that resemble sialic acids to dampen the ROS burst and reduce NETosis79,80 (FIG. 3; TABLE 1). Similarly, P. aeruginosa and GAS suppress NETosis through Siglec-9 by coating themselves with host sialylated glycoproteins80,81. Moreover, β-protein from GBS suppresses NETosis by binding to Siglec-5. However, engagement of Siglec-14 by β-protein antagonizes the repressive effects of Siglec-5 by activating mitogen-activated protein kinase (MAPK) signalling, which explains why polymorphisms that disrupt Siglec-14 increase host susceptibility to GBS82. Ligation of signal inhibitory receptor on leukocytes 1 (SIRL1) also attenuates NETosis by downregulating ROS production in response to S. aureus or MSU crystals83–85, but the physiological role of this pathway is unclear. In a similar manner, whereas bacterial biofilms induce NET formation86, fungal biofilms suppress NETosis by blocking ROS generation and increasing resistance to neutrophil killing87. The suppression of NETosis depends on mannosylation enzymes, but these enzymes are also important for fungal cell wall integrity, thereby making it difficult to attribute the virulence of these fungi solely to the suppression of NETosis87. Finally, the induction of immunosuppressive cytokines such as IL-10 can also inhibit NET release4 . In summary, microorganisms modulate NETosis through diverse mechanisms, depending on their size and the expression‎ of virulence factors.

NETs in host defence

Given that most of the proteins that are implicated in NETosis are also important for phagocytosis and cytokine regulation, it has been difficult to define the specific contribution of NETs to immune defence. The dependence of NETosis on microorganism size enabled us to study the role of NETs independently of phagocytosis. In humans, complete MPO deficiency leads predominately to recurrent fungal infections88. Experiments with MPO-deficient mice are also consistent with a crucial role for NETs against pathogens that are too large to be killed intracellularly, such as fungal hyphae7 . The importance of NETs in clearing systemic fungal infection is also supported by the restoration of NETosis in a patient with CGD following gene therapy89. Consistent with a selective antimicrobial role for NETs, only a small number of NET-deficient patients with Papillon–Lefèvre syndrome show susceptibility to pyogenic infections, and their neutrophils have no defects in bacterial killing23. Moreover, the reported lack of NETosis in PAD4-deficient mice does not affect bacteraemia and survival in polymicrobial sepsis90 or Burkholderia pseudomallei-induced sepsis91. Likewise, in the original study implicating NETs in protection against bacterial sepsis, NET degradation with DNase yielded only a twofold increase in bacteraemia following S. aureus skin infection13 and reduced bacterial load at the primary site of skin infection, which was interpreted as an increase in dissemination13. However, in most cases, dissemination is accompanied by uncontrolled growth at the primary site of infection. However, a reduction of skin bacteria upon DNase treatment may also be caused by biofilm breakdown or disruption of NET-mediated immune evasion mechanisms of S. aureus92. By contrast, impaired killing of Shigella flexneri and GAS has been reported in PAD4-deficient neutrophils alongside larger lesions in a model of GAS-induced necrotizing fasciitis38, which was attributed to defects in NETosis. Finally, many parasites trigger NETosis in vitro (reviewed in REF. 93), but it is unclear whether NETs offer protection against these pathogens. These studies suggest that NETs play a critical role against fungal infections and virulent bacteria that can subvert other neutrophil antimicrobial strategies.

NET release has also been observed in response to viruses such as HIV4 and respiratory syncytial virus or syncytial viral proteins94. NETs trap and reduce the infectivity of HIV virions4 , but evidence for an antiviral role for NETs in vivo is lacking95. Notably, NETs were absent in mild infection with influenza virus or co-infection with S. pneumoniae in wild-type mice96. Consistently, PAD4-deficient animals do not exhibit increased susceptibility to influenza virus95. On the contrary, NETs are thought to mediate pathology during severe influenza virus infection in mice deficient in viral sensing pathways97. Under these conditions, virus-induced tissue damage results in bacterial overgrowth associated with NET release and pathology. In this study, antibiotics, DNase treatment, neutrophil depletion and inhibition of neutrophil recruitment rescued mortality. However, given that the effect of antibiotics on NETosis was not examined, it is unclear whether NETs are  triggered directly by the microbiota or in response to host damage-associated molecular patterns (DAMPs), because alveolar epithelial cells isolated from influenza virusinfected lungs stimulate NETosis98. How NETs cause pathology in severe flu infection is unknown, but NETs have been associated with increased inflammation in pulmonary Sendai virus infections25. It is therefore evident that although NETs may be critical against specific infections, NET-driven pathology affects host survival. The molecular basis for the antimicrobial capacity of NETs is not well understood but is summarized along with several microbial NET countermeasures in BOX 2 and FIG. 3.