Neutrophils and neutrophil extracellular traps orchestrate initiation and resolution of inflammation J. Hahn1, J. Knopf1, C. Maueröder1, D. Kienhöfer1, M. Leppkes2, M. Herrmann1

1Department of Internal Medicine 3, ABSTRACT tion was thought to be limited: Phagocy- University of Erlangen-Nuremberg, Neutrophils, the most abundant leuko- tosis of pathogens with their subsequent 2 Erlangen; Department of Internal cytes in the human body, are considered killing and degradation, and release of Medicine 1, University of Erlangen- to be the first line of defense in the fight antimicrobial agents by degranulation. Nuremberg, Erlangen, Germany. against microorganisms. In this fight Both intra- and extracellular killing Jonas Hahn, MSc* neutrophils employ weaponry such as of microorganism is closely related to Jasmin Knopf, MSc* Christian Maueröder, MSc reactive oxygen species produced via production of reactive oxygen species Deborah Kienhöfer, MSc the NADPH oxidase complex 2 together (ROS) via the NADPH oxidase com- Moritz Leppkes, Dr. med.# with the release of intracellular gran- plex 2 (5, 6). However, recent research Martin Herrmann, Dr. rer. nat.# ules containing antimicrobial agents. revealed neutrophils as more sophisti- *JH and JK equally contributed to this The discovery that activated neutro- cated immune cells that are able to pre- work. phils release decondensed chromatin as cisely regulate their granular enzymes #ML and MH equally contributed to senior DNase-sensitive neutrophil extracellu- by ion fluxes, release immunomodula- authorship. lar traps (NETs) lead to a renewed inter- tory cytokines and chemokines (e.g., IL- Please address correspondence to: est in these leukocytes and the function 8, CXC chemokine receptor-2 ligands), Prof. Dr. Martin Herrmann, of NETs in vivo. In this review, we will interact with various components of the Department of Internal Medicine 3, focus on desirable as well as detrimen- immune system, and therefore can play University of Erlangen-Nuremberg, tal features of NETs by the example of a key role in (auto)immunity (7-11). Ulmenweg 18, 91054 Erlangen, Germany. gout and pancreatitis. In our models we Furthermore, neutrophils are involved E-mail: [email protected] observed that neutrophils drive the ini- in processes they were considered to be Received and accepted on July 3, 2016. tiation of inflammation and are required excluded from, like the killing of intra- for the resolution of inflammation. cellular pathogens and their participa- Clin Exp Rheumatol 2016; 34 (Suppl. 98): S6-S8. tion in the regulation of the adaptive Introduction immune system. They are also impor- © Copyright Clinical and Experimental Rheumatology 2016. The immune system is a complex net- tant players in several diseases includ- work of cells, molecules and organs ing allergy, atherosclerosis, thrombus Key words: tissues (A10), cells (A11), with the main function to protect the formation and metabolic disorders immune system (A15), animals (B01), body from invading and endogenous (12-14). Several of these new attributes immune system diseases (C20) pathogens. It further participates in the have been associated with the ability prevention of damage caused by attack- of activated neutrophils to release de- ing micro- and macroorganisms. Their condensed chromatin decorated with contribution in the resolution of inflam- granular content as DNase-sensitive mation has only recently been described neutrophil extracellular traps (NETs) in several models of health and disease. (9). NETs act as a scaffold for the aggre- However, so far few biological mecha- gation of viable, necrotic and apoptotic nisms are known that lead to the remov- cells as well as particulate matter such as Funding: this work was supported by the al of a specific target without causing crystals and microbes (15). The process German Research Council CRC-643-B5 & B8 (M.H.), CRC-1181-C03 (M.H.), collateral damage of surrounding cells of NETosis preferentially takes place in KFO257 (M.H., M.L.), SPP 1468 and tissues (1, 2). The neutrophil, the preformed cavities and to a lesser extent Osteoimmunology IMMUNOBONE most abundant leukocyte in the human inside the connective tissues. This addi- (M.H.), and the doctoral training body, is considered to be the first line of tional attribute of neutrophils has started programs GK1660 (M.H.) and SFB643 defense in the fight against microorgan- an intense discussion over the role and (M.H.). M.L. has received a research isms. Since neutrophils deploy a rather function of NETs in vivo. Specific con- scholarship by MSD Sharp & Dohme unspecific weaponry, controlling neu- ditions influence whether a NET will act GmbH. trophilic inflammation is strictly neces- inflammatory or anti-inflammatory and Competing interests: M. Leppkes has received research scholarship support from sary to avoid collateral damage (3, 4). the question for researchers and clini- Merck Sharp & Dohme GmbH, Germany. Neutrophils were long considered as cians remains, which conditions direct All the other authors declared no simple downstream executors of im- the mode of action of NETs in specific competing interests. mune responses and their mode of ac- models of disease.

S-6 Neutrophils initiate and resolve inflammation / J. Hahn et al.

‘Good’ NETs in gout lution of gouty inflammation and might The overexpression of IL-17A in- Gouty arthritis is an acute inflamma- be applied to other disease models like duced inflammatory pancreatitis which tory reaction which is initiated by systemic lupus erythematosus (SLE) was accompanied by neutrophilia, in- precipitation of oversaturated solu- (15). Mice with impaired ROS produc- creased mobilisation of neutrophils, tions of uric acid as monosodium urate tion via NOX2 show increased basal elevated tryptic activity of the pancreas crystals (MSU). Untreated, gout can levels of lupus auto-antibodies, and an homogenate and a striking myeloid in- cause significant structural damage, augmented deposition of complement flammatory infiltration (29). Closer ob- mostly of bone, cartilage and tendons C3 and IgG in glomeruli (23). Moreo- servation of the intraductal aggregates (16-18). Formation of MSU crystal ver, introduction of NOX2-deficiency revealed a strong co-localisation of cit- aggregates, denoted as tophi, are as- in the lupus-prone MRL.Faslpr mouse rullinated histone H3 (citH3) with ex- sociated with inflammation due to their strain precipitates the disease course tranuclear DNA, a hallmark character- ability to continuously remodel and (24). istic of NETs. Moreover, citH3, a sur- recruit monocytes (15, 19, 20). Re- rogate of PAD4 activity was predomi- cently, the involvement of NETosis in ‘Bad’ NETs in pancreatitis nantly observed inside pancreatic ducts gout has become of particular interest. Common risk factors of acute pancrea- and in the lumen of acini undergoing Schauer et al. observed that NETs re- titis, a leading cause for admissions to ductal metaplasia (29). PAD4 activ- lease IL-8, a key chemokine involved hospitals for gastrointestinal disorders, ity is crucial for neutrophil chromatin in the recruitment of neutrophils. At are formation of gallstones and alcohol decondensation during NETosis (30). high neutrophil concentration, solitary abuse (25). Obstruction of the pancre- Importantly, PADI4 deficiency strongly NETs form larger conglomerates, so atic duct leads to upstream blockage of protected mice from the development called aggregated NETs (aggNETs). pancreatic secretion, which is accom- of IL-17A-induced pancreatitis leading Moreover, Schauer et al. observed that panied by the premature activation of to the conclusion that PAD4-mediated pro-inflammatory mediators such as zymogens, mediating self-digestion of arginine citrullination vitally contrib- IL-1β and IL-6 are degraded by agg- the pancreas and thus causing severe utes to aggNET formation in the con- NETs in vitro via serine proteases at- inflammation (26). Neutrophils infil- text of ductal occlusion (29). The abil- tached to their meshwork (15). IL-1β trate the pancreatic parenchyma during ity of NETs to occlude ducts and poten- is a potent pro-inflammatory cytokine this inflammatory response (26). The tially cause pancreatitis allows for the and a strong stimulator of bone resorp- destroyed secretory parenchyma will possibility of therapeutic intervention tion via the upregulation of RANKL be replaced by fatty tissue, typical of to prevent excessive neutrophil activa- (21). Similarly, IL-6 promotes syno- chronic pancreatitis, if the underlying tion as a treatment option. vitis and joint destruction by stimulat- cause of the acute pancreatitis is not ing neutrophil migration and osteoclast resolved (27). Moreover, new forms of Conclusion maturation (22). Therefore, therapeutic pancreatitis, such as autoimmune pan- In recent years, the traditional view of targeting of either IL-1β or IL-6 might creatitis type 2, which displays granu- neutrophils as simple eliminators of be a promising way to ameliorate acute locytic epithelial lesions containing in- pathogens changed dramatically after inflammation and bone remodelling in tra- and periductal granulocyte aggre- they were found to be engaged in many this model. The authors further exam- gates, further support the involvement other physiological and pathological ined the in vivo relevance of cytokine of neutrophils in pancreatitis (28). processes. Most interestingly, many degradation via aggNETs using the Therefore, Leppkes and colleagues of these newly discovered functions of Ncf1** mouse strain that displays a hypothesised that the formation of in- neutrophils seem to be related to their drastically reduced capacity to execute traductal aggNETs and subsequent oc- ability to release nuclear content deco- ROS dependent NETosis upon stimu- clusion might be involved in the clini- rated with granular proteins via the for- lation with MSU. They found signifi- cal symptoms of chronic pancreatitis. mation of NETs. However, the release cantly higher concentrations of pro-in- Indeed, they observed that neutrophils of NETs is a double-edged sword. On flammatory mediators in MSU-injected enter the ducts under inflammatory the one hand, ‘bad’ NETs are involved air pouches of Ncf1** mice compared conditions and form aggNETs, which in the obstruction of vessels and ducts to wild type, suggesting an impaired in turn might hamper secretory flow as well as thrombus formation and degradation of cytokines/chemokines. and thereby drive focal pancreatitis and acute inflammation; on the other hand Strikingly, transfer of aggNETs from parenchymal remodelling (29). The au- ‘good’ NETs are able to contribute to WT mice into air pouches of Ncf1**mice thors further detected that these intra- the resolution of inflammation as dem- restored their ability to reduce inflam- ductal aggregates contained interleu- onstrated for gouty tophi. These recent matory mediators. Furthermore, Ncf1** kin-17A (IL-17A), a pro-inflammatory findings have led to the conclusion that mice showed a chronification of disease cytokine which triggers the recruitment NETs are neither the villains nor the in a murine model of MSU induced- of innate immune cells. These observa- hero in autoimmunity, but can act as a arthritis (15). This study highlights the tions lead to two different approaches, key component of initiation as well as importance of the oxidative burst and transgenic and vector-based, for the resolution of inflammation. It also be- the formation of aggNETs in the reso- systemic delivery of IL-17A in mice. came apparent that a certain shift in the

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