Epub ahead of print April 27, 2015 - doi:10.1189/jlb.4AB1114-543R
Brief Conclusive Report
The inhibitory effect of secretory leukocyte protease inhibitor (SLPI) on formation of neutrophil extracellular traps Katarzyna Zabieglo,* Pawel Majewski,* Monika Majchrzak-Gorecka,* Agnieszka Wlodarczyk,* Beata Grygier,* Aneta Zegar,* Monika Kapinska-Mrowiecka,† Antonina Naskalska,‡ Krzysztof Pyrc,‡,§ Adam Dubin,{ Sharon M. Wahl,‖ and Joanna Cichy*,1 *Department of Immunology, §Department of Microbiology, and {Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, and ‡Malopolska Centre of Biotechnology, Jagiellonian University, Krako´ w, Poland; †Department of Dermatology, Zeromski Hospital, Krako´ w, Poland; and ‖Cellular Immunology Section, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA RECEIVED NOVEMBER 13, 2014; REVISED MARCH 16, 2015; ACCEPTED MARCH 29, 2015. DOI: 10.1189/JLB.4AB1114-543R
ABSTRACT Neutrophil extracellular traps (NETs), web-like DNA structures, provide efficient means of eliminating invading microorganisms but can also present a potential threat to its host because it is a likely source of autoantigens or by promoting bystander tissue damage. Therefore, it is important to identify mechanisms that inhibit NET formation. Neutrophil elastase (NE)dependent chromatin decondensation is a key event in the release of NETs release. We hypothesized that inhibitors of NE, secretory leukocyte protease inhibitor (SLPI) and a1-proteinase inhibitor (a1-PI), has a role in restricting NET generation. Here, we demonstrate that exogenous human SLPI, but not a1-PI markedly inhibited NET formation in human neutrophils. The ability of exogenous SLPI to attenuate NET formation correlated with an inhibition of a core histone, histone 4 (H4), cleavage, and partial dependence on SLPI-inhibitory activity against NE. Moreover, neutrophils from SLPI2/2 mice were more efficient at generating NETs than were neutrophils from wild-type mice in vitro, and in experimental psoriasis in vivo. Finally, endogenous SLPI colocalized with NE in the nucleus of human neutrophils in vitro, as well as in vivo in inflamed skin of patients with psoriasis. Together, these findings support a controlling role for SLPI in NET generation, which is of potential relevance to infectious and autoinflammatory diseases. J. Leukoc. Biol. 98: 000–000; 2015.
Introduction Neutrophils, also referred to as polymorphonuclear leukocytes (PMNs), form a part of the front line of host defense against pathogens and are key components of tissue infiltrates in Abbreviations: a1-PI = a1-proteinase inhibitor, CatG = cathepsin G, H4 = histone 4, NE = neutrophil elastase, NETs = neutrophil extracellular traps, PAD4 = peptidylarginine deiminase 4, PMNs = polymorphonuclear leukocytes, SLPI = secretory leukocyte protease inhibitor, SLE = systemic lupus erythematosus, TSB = tryptic soy broth
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autoinflammatory diseases, such as systemic lupus erythematosus (SLE) or psoriasis [1–5]. Activated neutrophils contribute to immune responses through several mechanisms, including phagocytosis, production of reactive oxygen species, degranulation, and the formation of neutrophil extracellular traps (NETs) [6]. NETs are extracellular, decondensed, chromatin networks decorated with nuclear, granular, and cytoplasmic proteins. NETs were first reported to limit the growth and spread of microorganisms through mechanically trapping pathogens and facilitating the interaction of antimicrobial agents that are displayed on NETs with bacteria or fungi [6]. More recently, NETs were associated with tissue damage and autoinflammatory diseases, such as SLE, Wegener granulomatosis, and psoriasis [1–3, 5, 7]. The NET constituents, including self-DNA, histones, and myeloperoxidase, are potential autoantigens [1, 2]. In addition to their beneficial role in host defense, NETs may also be an underlying component of cell cytotoxicity and autoimmunity; therefore, specific counterregulatory mechanisms likely evolved to constrain NET formations. Before the nuclear content of neutrophils is expulsed to the extracellular milieu to form NETs, the cell nucleus disintegrates, and loose chromatin often fills the entire cell. The nuclear decondensation is attributed to peptidylarginine deiminase 4 (PAD4) and serine protease-neutrophil elastase (NE). PAD4 modifies the chromatin structure through conversion of histones’ arginine tail residues to citrulline, a nonconventional amino acid [8], whereas NE drives chromatin decondensation via proteolytic cleavage of specific histones, including the core histone 4 (H4) [9]. Under homeostatic conditions, NE is stored in primary (azurophilic) granules, but it translocates to the nucleus upon neutrophil activation. Once in the nucleus, NE can contribute to chromatin decondensation and subsequently to NET formation [9]. Activation of PMNs can also result in
1. Correspondence: Department of Immunology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7 St., 30-387 Krakow, Poland. E-mail: [email protected]
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deposition of NE into the extracellular environment through extracellular discharge of the granules’ contents [10]. Extracellular NE activity is controlled by several inhibitors; among which, a1-proteinase inhibitor (a1-PI) and secretory leukocyte proteinase inhibitor (SLPI) appear to have predominant roles in limiting excessive NE-mediated proteolysis. Although liverderived a1-PI is a major serine proteinase inhibitor in human plasma, SLPI is more likely to control NE activity at mucosal/ epithelial surfaces where it is abundantly present, likely as a secretory product of epithelial cells [11, 12]. Interestingly, neutrophils also produce a1-PI and SLPI [13, 14]. Thus, these inhibitors are well positioned to have affected the release of NETs. SLPI is the major inhibitor of NE in neutrophils [14]; however, its physiologic role in these cells is not well characterized. SLPI comprises 2 mutually homologous domains with its antiprotease activity, primarily localized to its C-terminal domain. Using SLPI mutants with various protease-inhibitory capacity against NE and other serine proteases, such as the closely related chymotrypsin and trypsin, Leu72, and to a lesser extent, Met73, were demonstrated to be critical sites for binding all 3 enzymes to SLPI [15, 16]. In this study, we demonstrated that SLPI is a potent regulator of NETs release by activated neutrophils in vitro and in vivo. Using antiprotease, active-site-mutated SLPI, we show that SLPI’s capacity to restrict NET formation is partially dependent on its anti-NE function. Our data suggest that SLPI-mediated restriction in NET release might be a mechanism to tailor neutrophil responses.
MATERIALS AND METHODS
Materials Recombinant human SLPI was purchased from R&D Systems (Minneapolis, MN, USA) or produced as described below. Human plasma a1-PI was isolated as previously described [17]. Human NE was isolated using fresh, whole human blood from healthy donors as a starting material, as previously described [18].
Production of recombinant wild-type SLPI and mutant forms of SLPI Wild-type SLPI encoding the mature secreted protein was generated by PCR, using RNA isolated from human alveolar epithelial cells as a template. The
PCR product was cloned into pPIC9 expression vector (Life Technologies, Carlsbad, CA, USA) with the addition of a hexahistidine tag and an enterokinase cleavage site to its N terminus, using the overlap-extension PCR method, as previously described [19]. Two mutants with substitutions of amino acids critical for SLPI’s antiprotease activity [16] (Table 1) were generated using the same PCR method. The identity of the created constructs was verified by sequencing (Genomed, Warsaw, Poland). Wild-type SLPI and its mutants were produced using Pichia pastoris strain GS115 (His2), and the purified form of the supernatants from His+ transformants were produced with Ni-Sepharose 6 Fast Flow (GE Healthcare, Uppsala, Sweden). The SLPI isoforms were eluted with 500 mM imidazole and dialyzed against PBS. The SLPI mutants obtained were routinely .90% pure as assessed by SDS-PAGE and Coomassie Blue staining.
Assessment of inhibitory activities of SLPI NE, trypsin, or chymotrypsin were preincubated for 10 min with various concentrations of wild-type SLPI and its mutants at 20°C in 0.1 M Tris-HCl (pH 8.0) with 0.5 M NaCl. The suitable substrate—MeO-Suc-Ala-Ala-Pro-ValpNA (Sigma-Aldrich, St. Louis) for NE, Bz-Ile-Glu-Gly-Arg-pNA (S-2222) (Chromogenex Technologies, Llanelli, Carmarthenshire, United Kingdom) for trypsin, and Suc-Ala-Ala-Pro-Phe- pNA (Sigma-Aldrich) for chymotrypsin— was then added to the final concentration of 1 mM and incubated for 10–30 min at 20°C in a total volume of 0.1 ml. Released p-nitroaniline was quantified at 405 nm using a Bio-Tek (Winooski, VT, USA) microplate reader.
Patients All human studies were performed in accordance with the guidelines established by the Jagiellonian University Institutional Bioethics Committee, under approved protocols. In total, six psoriasis patients (age 28.7 6 6.6 yr; F:M 2:4) and 20 healthy individuals (age 28.7 6 5.1 yr; F:M 7:13) were enrolled in these studies. The severity of the psoriatic skin lesions was assessed according to the Psoriasis Area Severity Index score (PASI) (minimum, 0 points; maximum, 72 points) and ranged from 25.3 to 39.6 (mean 6 SD 32.5 6 5.4).
Human neutrophils isolation and treatment Neutrophils were isolated from the blood of healthy individuals, as previously described [5]. Neutrophils were seeded on cover slips coated with poly-L-lysine or into 96-well plates and allowed to adhere for 30 min at 37°C under 5% CO2. The neutrophils were stimulated with the following factors: 40–100 nM PMA, 100 ng/ml TNF-a, 10% normal human serum, or Staphylococcus aureus (ATCC 8235; American Type Culture Collection, Manassas, VA, USA), added to neutrophil cultures at a 10:1 ratio. In the indicated experiments, SLPI, SLPI mutants, or a1-PI were added 10 min before the addition of stimulating factors at a final concentration 1.67 mM. This concentration was first optimized experimentally (0.08, 0.42, 0.84, and 1.67 mM) for SLPI. The neutrophils were
TABLE 1. Comparison of SLPI forms % Inhibition of proteolytic activity at 2:1 [SLPI:enzyme] molar ratioc Name SLPI (wild-type) Mut 1 Mut 2
Forms of SLPIa
% Survivalb
NE
Chymo-trypsin
Trypsin
% Neutrophils with SLPI in nucleusd
Leu72 Leu72 replaced with Lys Leu72Met73Leu74 replaced with LysGlyGly
49 6 20 55 6 15 40 6 11
71 6 14 0 0
79 6 9 15 6 6 0
43 6 6 44 6 3 22 6 10
78.4 6 12 77.8 6 1 52 6 18
a
Critical active-site amino acid residues with position in amino acid sequences are shown. b The CFU of SLPI-treated or mutant-treated S. aureus is presented as the CFU percentage of vehicle-treated S. aureus. Data are provided as means 6 SD of 3 independent measurements. c Data are provided as means 6 SD of 3 independent experiments. d The number of neutrophils demonstrating nuclear localization of the indicated SLPI forms is shown as a percentage of the total neutrophils treated with PMA for 30 min; 10 high-powered fields were analyzed per sample by fluorescence microscopy. Data are provided as means 6 SD of 2 independent experiments.
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Zabieglo et al. SLPI inhibits NETs then cultured at 37°C under 5% CO2 for 30 min or 3 h. The cell cultures were subjected to fluorescence microscopy or fluorimetry.
anti-human H4 antibodies (Millipore) and goat anti- rabbit IgG antibodies conjugated to HRP (Sigma-Aldrich).
Preparation of S. aureus and antimicrobial activity assay
Statistical analysis
S. aureus was grown in tryptic soy broth (TSB) (Sigma-Aldrich) to the midlogarithmic phase. For estimation of antimicrobial activity of wild-type SLPI and its mutants, bacteria were analyzed, as described previously [20]. In brief, bacterial suspensions in PBS supplemented with 1% TSB were mixed with diluent (PBS), wild-type SLPI, or its mutants, and were incubated at 37°C for 18–24 h. The diluted mixture was plated on TSB agar plates and incubated at 37°C overnight for enumeration of CFUs. CFUs of SLPI-treated bacteria are presented as a percentage of the PBS (diluent-treated) bacteria.
Evaluation of significance was performed using one-way ANOVA followed by a Bonferroni post hoc test, or 2-tailed Student’s t test.
Isolation and treatment of mouse neutrophils SLPI2/2 mice on a mixed C57BL/6-129/Sv background were obtained by interbreeding SLPI heterozygous mice originally generated by targeted disruption of the SLPI gene by homologous recombination [21, 22]. This recombinant strain is available at The Jackson Laboratory Repository (JAX Stock No. 010926; Bar Harbor, ME, USA). SLPI2/2 mice show no obvious pathologic phenotype under steady-state conditions. When challenged with proinflammatory stimuli, they show higher mortality and tissue destruction and exacerbated inflammatory responses compared with SLPI+/+ controls [21–23]. Mice (female or male, 8–12 wk old) were housed under pathogenfree conditions. Neutrophils were negatively isolated from bone marrow using an AutoMACS separator (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instruction. The purity of the isolated cells was examined by flow cytometry based on Ly6G immunoreactivity. Neutrophils were routinely .95% pure. Neutrophils were then treated with PMA and S. aureus, as described, for human neutrophils.
RESULTS AND DISCUSSION The regulatory role of SLPI and a1-PI on NET formation was first tested using human neutrophils isolated from healthy donors and stimulated with factors well known to induce NETs in vitro. As expected, the human pathogen S. aureus, phorbol ester (PMA), and TNF-a promoted NET generation at 3 h, a typical time frame for NET release [6] (Fig. 1). This was shown by fluorescence microscopy of the cultured neutrophils that were stained for NET components-DNA and CatG [7]. In addition, the levels of extracellular DNA in a conditioned medium of
Aldara model of psoriasis SLPI2/2 and SLPI+/+ control mice between 8–12 wk old were treated twice daily for up to 6 d with 15 mg Aldara (imiquimod cream; Meda, Solna, Sweden) on shaved and depilated back skin [24]. At indicated time points, skin sections comprising the center of treatment were snap-frozen and analyzed by fluorescence microscopy.
Fluorimetry Extracellular DNA in the conditioned medium of treated neutrophils was quantified using Quant PicoGreen dsDNA kit (Life Technologies), according to manufacturer’s recommendations.
Immunohistochemistry Frozen sections (6–10 mm) were prepared from skin biopsies. Human skin sections or purified human neutrophils were stained with the following antibodies: first, biotin-mouse anti-human SLPI (Abcam, Milton, Cambridge, United Kingdom) and rabbit anti-human NE or cathesin G (CatG) (both from Athens Research and Technology, Athens, GA, USA), followed by PEstreptavidin (BD Pharmingen, San Jose, CA, USA) and APC-goat anti-rabbit IgG F(ab9)2 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Mouse tissues or isolated neutrophils were stained with biotin-rat anti-mouse Ly6G (BD Pharmingen) mAb and rabbit anti-histone H3 (citrulline R2 + R8 + R17) Ab (Abcam) or rabbit anti-mouse histone 2a (Abcam), followed by PE-streptavidin and APC-goat anti-rabbit IgG F(ab9)2. The sections were counterstained with Hoechst dye 33258 (Life Technologies). Images were captured with a fully motorized fluorescence microscope (Eclipse; Nikon, Chiyoda, Tokyo, Japan) and were analyzed by NIS-Elements (Nikon) software.
Western blot analysis Neutrophils were resuspended in 13 SDS loading buffer. Samples were then resolved on SDS-16% PAGE under reducing conditions. Histone H4 cleavage was visualized by enhanced chemiluminescence after incubation with rabbit
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Figure 1. SLPI inhibits NET formation in human neutrophils. Purified PMNs were pretreated with the the indicated inhibitors or vehicle (2) for 10 min and were then left unstimulated (control) or were stimulated with the indicated factors for 3 h. The final concentrations of the factors were as follows: SLPI and a1-PI, 1.67 mM, PMA, 40 nM; TNF-a, 100 ng/ml. S. aureus was added in a 10:1 (bacteria:neutrophil) ratio. (A) Fluorescence microscopy of neutrophils stained to detect CatG (green) and DNA (blue). Data are from one experiment and are representative of at least 4 experiments. (B) Supernatants of stimulated neutrophils were analyzed for extracellular DNA by fluorimetry. OD data are presented relative to unstimulated cells. The means 6 SEM from 4–8 independent experiments are shown. Statistical significance comparing the inhibitor-treated cells vs. cells cultured in the absence of the inhibitors (**P , 0.01) was determined by ANOVA followed by a Bonferroni post hoc test.
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stimulated cells were analyzed by fluorimetry. As demonstrated in Fig. 1A, the number of netting cells, identified by extracellular, fibrous, DNA deposits and intracellular chromatin dispersion that was decorated with CatG, significantly increased following the treatment with PMA, TNF-a, or S. aureus. A similar tendency was observed when NETs were quantified by DNA fluorimetry, although the effect of TNF-a was less potent compared with PMA or S. aureus (Fig. 1B). Notably, in the presence of SLPI, NET formation in control-, PMA-, TNF-a, and S. aureus stimulated neutrophils, was markedly reduced (Fig. 1). On average, SLPI treatment resulted in significant reduction of NET formation from 100 to 38, 33, and 17% in human neutrophils stimulated with PMA, TNF-a, and S. aureus, respectively (Fig. 1B). In contrast to SLPI, a1-PI was not able to inhibit NET formation in either control or stimulated neutrophils (Fig.1). Taken together, these data suggest a selective effect of SLPI on constraining NET release. To determine whether endogenous SLPI restricts NET generation, we isolated neutrophils from bone marrow of SLPIdeficient (SLPI2/2) and wild-type (SLPI+/+) mice. Magnetically sorted neutrophils were then subjected to stimulation with PMA or S. aureus. As shown in Fig. 2A, SLPI2/2 neutrophils demonstrated greater ability to form NETs than SLPI+/+ neutrophils did. On average, 2- and 6-fold more SLPI2/2 neutrophils were found to release NETs following stimulation with PMA and S. aureus, respectively (Fig. 2B). To determine whether SLPI controls NET formation in vivo, we assessed the number of NET-forming neutrophils in the skin of SLPI+/+ and SLPI2/2 mice treated to induce psoriasis-like skin inflammation. Because infiltration of neutrophils into human skin is the hallmark of the autoinflammatory disease psoriasis [25] and because SLPI is a component of NETs in the lesional skin of psoriatic patients [5], these data suggest that SLPI has a role in this disease, possibly by regulating NET formation. A mouse model of psoriasiform dermatitis is based on the repeated topical application of Aldara, a cream containing 5% imiquimod, a ligand for TLR7 [24, 26]. NET-forming neutrophils were identified as Ly6G+ cells with dispersed or extruded DNA that costained with citrullinated histone H3 [27]. As shown in Fig. 2C, significantly more skin-infiltrating neutrophils formed NETs in SLPI2/2 mice, compared with SLPI+/+ mice. This was mainly seen in the first 2 d after application of Aldara (Fig. 2D). Together, these data suggest that a lack of endogenous SLPI results in a stronger ability of neutrophils to form NETs under autoinflammatory conditions, consistent with our in vitro data. Exogenous SLPI tends to localize to the nuclei of activated human neutrophils (Fig. 3A and B), indicating that its inhibitory effect on NET formation may depend on translocation to the nucleus. Therefore, we next asked whether endogenous human SLPI showed nuclear localization. Without stimulation, SLPI is dispersed throughout neutrophils, likely localizing to secondary granules or cytoplasm [14, 28] (Fig. 3C). Following treatment with PMA, SLPI was found to mainly migrate toward the cell surface and, occasionally, toward the cell nucleus, where most of the NE was found in approximately 10% of neutrophils (Fig. 3D; data not shown). Thus, in PMA-treated cells, NE and SLPI colocalize in either the cell nucleus or the cell membrane or were spatially separated, with NE present primarily in the cell 4
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nucleus and SLPI at the cell membrane (Fig. 3D). In a distinct scenario, SLPI and NE were typically found together in the nucleus or perinuclear area in approximately 5% of all neutrophils stimulated with normal serum (Fig. 3E; data not shown). These data suggest that SLPI follows NE to the nucleus, although localization of NE and SLPI is likely differently regulated, both spatially and kinetically, in activated neutrophils. To determine whether human SLPI colocalizes with NE in the nucleus in vivo, we examined lesional skin of patients with psoriasis. As shown in Fig. 3F, SLPI and NE could be found in the nucleus of neutrophils infiltrating psoriatic skin. Colocalization of SLPI with NE is found in most skin-infiltrating granulocytes at different stages of NET formation [5] (Fig. 3F; data not shown), but nuclear staining for both NE and SLPI was evident in approximately 0.5% of infiltrating PMNs at the time points evaluated. Together, these data suggest that SLPI controls NE activity in the nucleus of human inflammatory, skin-recruited neutrophils. To determine whether SLPI restricts NET release through regulating NE proteolytic activity in the nucleus, we analyzed cleavage of core H4, a well-described nuclear substrate of NE during NET formation [9, 29]. Using SLPI mutants that differ in NE-, chymotrypsin- and trypsin-inhibitory capacity (Table 1), we evaluated whether SLPI inhibitory activity against serine proteases was required for inhibition of NET formation in human neutrophils stimulated with PMA, TNF-a, and S. aureus. Because SLPI is equipped with antimicrobial activity that is likely to limit the growth of S. aureus and ultimately lower a neutrophil’s ability to generate NETs in response to S. aureus, we first determined, whether wild-type SLPI and SLPI mutants control S. aureus growth in a differential manner. As shown in Table 1, all tested SLPI forms displayed similar antibacterial activity against S. aureus, consistent with a previous report [30] that SLPI’s antimicrobial capacity is mainly located in the N-terminal, and not C-terminal, domain of SLPI that was mutated in the studies. Thus, SLPI’s antimicrobial potential is not likely to differentially influence the ability of the tested SLPI forms to control a neutrophil’s response to the bacteria. All SLPI mutants were similarly capable of migrating to the nucleus of PMA-activated neutrophils (Table 1). As expected, treatment of neutrophils with PMA, TNF-a, or S. aureus resulted in H4 cleavage as evidenced by the low-molecular-weight product (Fig. 4A), although the H4 degradation profile was the most pronounced in PMA-treated cells. In the presence of wild-type SLPI, H4 processing was partially or completely blocked in neutrophils activated with PMA, TNF-a, or S. aureus, respectively. SLPI-mediated inhibition of H4 cleavage coincided with nearly 63, 60, and 80% reduction in NET formation in PMA-, TNF-a-, and bacteria-stimulated neutrophils, respectively (Fig. 4B). Notably, SLPI mutants with a negligible anti-NE function (mutants 1 and 2) also significantly blocked H4 cleavage and NET release, although to a 1.2–1.9 times lesser extent, compared with the wild-type SLPI. Specifically, mutants 1 and 2 decreased NET release by 42 and 45% in PMA-activated neutrophils, by 42 and 50% in TNF-a-treated cells, and by 43 and 56% when S. aureus was used as the stimulus (Fig. 4B). Together, these data suggest that the suppressive effect of SLPI on NET formation is only partially dependent on its anti-NE activity.
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Zabieglo et al. SLPI inhibits NETs
Figure 2. SLPI-deficient mouse neutrophils showed higher propensity to form NETs in vitro and in vivo. (A) Bone marrow-derived neutrophils of SLPI+/+ and SLPI2/2 mice were left untreated (control) or were treated for 3 h with PMA or S. aureus. Neutrophils were then stained for NET components with Histone 2a (red) and DNA (blue) and were analyzed by fluorescence microscopy. Data are from one experiment and are representative of at least 4 experiments. Scale bar, 10 mm. (B) Frequency of bone marrow-derived neutrophils with extracellular NET deposits (% NETs) was examined by fluorescence microscopy. For each mouse (n = 4 per group), 3–4 high-power fields were analyzed. The means 6 SD are shown. *P , 0.05 comparing SLPI+/+ and SLPI2/2 neutrophils by a Student’s t test. (C) Mice were treated with Aldara twice daily for up to 6 d on back skin. The treated skin was collected at indicated time points, stained with anti-Ly6G (red), anti-citH3 (green) Abs, and Hoechst dye to detect DNA (blue), followed by fluorescence microscopy. Data are from one experiment and are representative of at least 5 experiments. Representative NET-forming neutrophils are indicated by arrowheads in enlargement of area outlined in top right panel. Scale bar = 10 mm. (D) NET-forming neutrophils are shown as a percentage of the total Ly6G+ cells (% NETs) for the indicated mice and time point. For each mouse (n = 3–6 per group), 10 high-power fields were analyzed. The means 6 SD are shown. *P , 0.05 comparing SLPI+/+ and SLPI2/2 mice (by a Student’s t test).
Here, we demonstrated that exogenous and endogenous SLPI is a potent, negative regulator of NET formation in neutrophils that respond to infection and inflammatory stimuli, such as S. aureus, TNF-a, or a surrogate inflammatory agent, PMA. Whereas
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S. aureus is a common human pathogen and often causes skin and soft-tissue infections, as well as sepsis [31, 32], TNF-a has a critical role in autoinflammatory diseases, including psoriasis [33]. Given a well-described role of NETs in S. aureus mediated
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as macrophages, and establish persistent infection [37]. Although these findings indicate a crucial role for NETs in various infection- or inflammation-associated pathologies, the mechanisms restricting NETs formation are poorly defined. To this end, interfering with key pathways underlying NET generation, such as the NADPH oxidase-mediated generation of reactive oxygen species or PAD-regulated histone deimination, were recently exploited to inhibit NETs [9, 29, 38]. Discovery of the critical role of NEs in promoting NET release also initiated a substantial interest in inhibitors of NE proteolytic function. Although small, b-lactam-based, cell-permeable NE inhibitors were already instrumental in demonstrating histone cleavage and chromatin decondensation by NEs [9, 29], regulatory mechanisms involving endogenous NE inhibitors, such as SLPI or SerpinB1, are likely to constrain NET formation. Notably, SerpinB1, a cytoplasmic serine protease inhibitor of neutrophils, was recently reported to be essential for restricting NET generation [39]. Likewise, exogenous SLPI was demonstrated to inhibit decondensation activity of NE-containing azurophilic granules in isolated nuclei of human neutrophils [9], suggesting its potential to regulate NET formation. Here, we demonstrated that SLPI colocalizes with NE in the nucleus of activated neutrophils in vitro and in vivo, blocks the cleavage of nuclear NE-substrate H4 in a manner
Figure 3. SLPI colocalizes with NE in cell nucleus in vitro and in vivo. Neutrophils were isolated from healthy donors and left untreated (2) or were treated for 30 min with exogenous SLPI (ex SLPI) (+) in the absence (A) or presence (B) of PMA. Cells were then stained for SLPI (yellow) and DNA (blue) and were analyzed by fluorescence microscopy under the acquisition settings (very short exposure), which did not detect endogenous SLPI. Neutrophils were left untreated (C) or were treated for 3 h with PMA (D) or normal serum (E). These cells, or frozen sections of psoriatic skin biopsy (F), were stained for human NE (HNE) (red), SLPI (green), and DNA (blue); single channels are shown as black and white images (C–F). Data in panels are from different donors and are representative of at least 3 (A–E) or 2 donors (F). Scale bar = 5 mm (A–E), 10 mm (F).
host invasion and autoinflammatory disorders, SLPI as a checkpoint in NET formation may have potential for controlling the pathologic outcome of these diseases. NETs emerge as crucial contributors to innate immune defense against microbes, but their excessive development during infection leads to pathology [34]. For example, hantaviruses, which cause severe kidney and lung damage in humans, were recently described to generate high levels of NETs and autoantibody production in response to virus-induced systemic NET overflow [35]. Likewise, excessive NET formation or impaired NET degradation was associated with autoinflammatory diseases [34], and a putative link between NETs and autoimmunity has been proposed [1, 2, 36]. On the other hand, S. aureus can hijack NETs to destroy host-defense effector cells, such 6
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Figure 4. Inhibition of H4 proteolytic processing and NET formation are partially dependent on anti-NE activity of SLPI. Neutrophils were isolated from blood of healthy donors and were left unstimulated (control) or were stimulated with PMA, TNF-a, or S. aureus for 3 h in the presence of wild-type SLPI, the indicated SLPI mutants, or a vehicle. (A) Cell lysates where subjected to Western blot analysis using Ab against H4. H4 degradation product is shown by arrowheads. (B) Neutrophil-conditioned medium was analyzed for extracellular DNA by fluorimetry. OD data are presented relative to unstimulated cells. The means 6 SEM from 5–14 independent experiments are shown. Statistically significant differences between SLPI- or mutant-treated vs. vehicle-treated cells are indicated with an asterisk (*P , 0.05; **P , 0.01; ANOVA followed by a Bonferroni post hoc test).
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partially dependent on its anti-NE inhibitory activity, and attenuates release of NETs in vitro and in vivo. Together, these findings implicate SLPI, a major NE inhibitor in human neutrophils [14], as a controlling agent in NET generation. It remains to be determined whether SLPI acts to control a threshold of NET formation or influences a neutrophil preference to a type of defense mechanism, such as degranulation or phagocytosis. SLPI acts to counteract excessive inflammatory responses and to initiate healing processes through several antiproteasedependent and -independent mechanisms. Our finding that SLPI-inactive mutants inhibit NET formation to some extent raises the question of whether the antiprotease-independent activity of SLPI might be involved in attenuating NET release. One such NE-separate pathway may involve inhibition of transcription factor NF-kB [21, 40], although these mechanisms in NET formation have yet to be explored. However, because SLPI mutants that were inactive against NE were not completely devoid of inhibitory potential against chymotrypsin or trypsin, it is also possible that SLPI controls NET formation through targeting other, yet-to be identified proteases. In conclusion, our findings suggest that SLPI is an important element for protection against aberrant NET formation and that its anti-NE activity is partially responsible for the protective effect.
AUTHORSHIP K.Z., P.M., M.M.-G., A.W., B.G., A.Z., and A.D. performed the experiments. M.K.-M., A.N., K.P., A.D., and S.M.W. contributed reagents/materials. J.C. and S.M.W. wrote the article.
ACKNOWLEDGMENTS This work was supported in part by the Polish National Science Center Grant 2011/02/A/NZ5/00337 and the Foundation for Polish Science Grant TEAM/2010-5 and was cofinanced by the European Union within a European Regional Development Fund Award (to J.C.). The Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University is a beneficiary of structural funds from the European Union (Grant POIG.02.01.0012-064/08) and a partner with the Leading National Research Center (KNOW) supported by the Polish Ministry of Science and Higher Education. This research was supported in part by the Intramural Research Program of the U.S. National Institutes of Health National Institute of Dental and Craniofacial Research (to S.M.W.).
DISCLOSURES
The authors declare no competing financial interests.
REFERENCES
1. Garcia-Romo, G. S., Caielli, S., Vega, B., Connolly, J., Allantaz, F., Xu, Z., Punaro, M., Baisch, J., Guiducci, C., Coffman, R. L., Barrat, F. J., Banchereau, J., Pascual, V. (2011) Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci. Transl. Med. 3, 73ra20.
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2. Lande, R., Ganguly, D., Facchinetti, V., Frasca, L., Conrad, C., Gregorio, J., Meller, S., Chamilos, G., Sebasigari, R., Riccieri, V., Bassett, R., Amuro, H., Fukuhara, S., Ito, T., Liu, Y. J., Gilliet, M. (2011) Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus. Sci. Transl. Med. 3, 73ra19. 3. Lin, A. M., Rubin, C. J., Khandpur, R., Wang, J. Y., Riblett, M., Yalavarthi, S., Villanueva, E. C., Shah, P., Kaplan, M. J., Bruce, A. T. (2011) Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis. J. Immunol. 187, 490–500. 4. Mantovani, A., Cassatella, M. A., Costantini, C., Jaillon, S. (2011) Neutrophils in the activation and regulation of innate and adaptive immunity. Nat. Rev. Immunol. 11, 519–531. 5. Skrzeczynska-Moncznik, J., Wlodarczyk, A., Zabieglo, K., KapinskaMrowiecka, M., Marewicz, E., Dubin, A., Potempa, J., Cichy, J. (2012) Secretory leukocyte proteinase inhibitor-competent DNA deposits are potent stimulators of plasmacytoid dendritic cells: implication for psoriasis. J. Immunol. 189, 1611–1617. 6. Brinkmann, V., Reichard, U., Goosmann, C., Fauler, B., Uhlemann, Y., Weiss, D. S., Weinrauch, Y., Zychlinsky, A. (2004) Neutrophil extracellular traps kill bacteria. Science 303, 1532–1535. 7. Skrzeczynska-Moncznik, J., Wlodarczyk, A., Banas, M., Kwitniewski, M., Zabieglo, K., Kapinska-Mrowiecka, M., Dubin, A., Cichy, J. (2013) DNA structures decorated with cathepsin G/secretory leukocyte proteinase inhibitor stimulate IFNI production by plasmacytoid dendritic cells. Am J Clin Exp Immunol 2, 186–194. 8. Wang, Y., Li, M., Stadler, S., Correll, S., Li, P., Wang, D., Hayama, R., Leonelli, L., Han, H., Grigoryev, S. A., Allis, C. D., Coonrod, S. A. (2009) Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J. Cell Biol. 184, 205–213. 9. Papayannopoulos, V., Metzler, K. D., Hakkim, A., Zychlinsky, A. (2010) Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J. Cell Biol. 191, 677–691. 10. Faurschou, M., Borregaard, N. (2003) Neutrophil granules and secretory vesicles in inflammation. Microbes Infect. 5, 1317–1327. 11. Reardon, C., Lechmann, M., Bru¨ stle, A., Gareau, M. G., Shuman, N., Philpott, D., Ziegler, S. F., Mak, T. W. (2011) Thymic stromal lymphopoetin-induced expression of the endogenous inhibitory enzyme SLPI mediates recovery from colonic inflammation. Immunity 35, 223–235. 12. Zhu, J., Nathan, C., Jin, W., Sim, D., Ashcroft, G. S., Wahl, S. M., Lacomis, L., Erdjument-Bromage, H., Tempst, P., Wright, C. D., Ding, A. (2002) Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair. Cell 111, 867–878. 13. Clemmensen, S. N., Jacobsen, L. C., Rørvig, S., Askaa, B., Christenson, K., Iversen, M., Jørgensen, M. H., Larsen, M. T., van Deurs, B., Ostergaard, O., Heegaard, N. H., Cowland, J. B., Borregaard, N. (2011) Alpha-1antitrypsin is produced by human neutrophil granulocytes and their precursors and liberated during granule exocytosis. Eur. J. Haematol. 86, 517–530. 14. Sallenave, J. M., Si Tahar, M., Cox, G., Chignard, M., Gauldie, J. (1997) Secretory leukocyte proteinase inhibitor is a major leukocyte elastase inhibitor in human neutrophils. J. Leukoc. Biol. 61, 695–702. 15. Eisenberg, S. P., Hale, K. K., Heimdal, P., Thompson, R. C. (1990) Location of the protease-inhibitory region of secretory leukocyte protease inhibitor. J. Biol. Chem. 265, 7976–7981. 16. Mulligan, M. S., Lentsch, A. B., Huber-Lang, M., Guo, R. F., Sarma, V., Wright, C. D., Ulich, T. R., Ward, P. A. (2000) Anti-inflammatory effects of mutant forms of secretory leukocyte protease inhibitor. Am. J. Pathol. 156, 1033–1039. 17. Travis, J., Salvesen, G. S. (1983) Human plasma proteinase inhibitors. Annu. Rev. Biochem. 52, 655–709. 18. Baugh, R. J., Travis, J. (1976) Human leukocyte granule elastase: rapid isolation and characterization. Biochemistry 15, 836–841. 19. Bryksin, A. V., Matsumura, I. (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques 48, 463–465. 20. Banas, M., Zabieglo, K., Kasetty, G., Kapinska-Mrowiecka, M., Borowczyk, J., Drukala, J., Murzyn, K., Zabel, B. A., Butcher, E. C., Schroeder, J. M., Schmidtchen, A., Cichy, J. (2013) Chemerin is an antimicrobial agent in human epidermis. PLoS ONE 8, e58709. 21. Ashcroft, G. S., Lei, K., Jin, W., Longenecker, G., Kulkarni, A. B., Greenwell-Wild, T., Hale-Donze, H., McGrady, G., Song, X. Y., Wahl, S. M. (2000) Secretory leukocyte protease inhibitor mediates nonredundant functions necessary for normal wound healing. Nat. Med. 6, 1147–1153. 22. McCartney-Francis, N., Jin, W., Belkaid, Y., McGrady, G., Wahl, S. M. (2014) Aberrant host defense against Leishmania major in the absence of SLPI. J. Leukoc. Biol. 96, 917–929. 23. Nakamura, A., Mori, Y., Hagiwara, K., Suzuki, T., Sakakibara, T., Kikuchi, T., Igarashi, T., Ebina, M., Abe, T., Miyazaki, J., Takai, T., Nukiwa, T. (2003) Increased susceptibility to LPS-induced endotoxin shock in secretory leukoprotease inhibitor (SLPI)-deficient mice. J. Exp. Med. 197, 669–674.
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24. van der Fits, L., Mourits, S., Voerman, J. S., Kant, M., Boon, L., Laman, J. D., Cornelissen, F., Mus, A. M., Florencia, E., Prens, E. P., Lubberts, E. (2009) Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis. J. Immunol. 182, 5836–5845. 25. Pinkus, H., Mehregan, A. H. (1966) The primary histologic lesion of seborrheic dermatitis and psoriasis. J. Invest. Dermatol. 46, 109–116. 26. Flutter, B., Nestle, F. O. (2013) TLRs to cytokines: mechanistic insights from the imiquimod mouse model of psoriasis. Eur. J. Immunol. 43, 3138–3146. 27. Branzk, N., Lubojemska, A., Hardison, S. E., Wang, Q., Gutierrez, M. G., Brown, G. D., Papayannopoulos, V. (2014) Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat. Immunol. 15, 1017–1025. 28. Sørensen, O., Arnljots, K., Cowland, J. B., Bainton, D. F., Borregaard, N. (1997) The human antibacterial cathelicidin, hCAP-18, is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils. Blood 90, 2796–2803. 29. Metzler, K. D., Goosmann, C., Lubojemska, A., Zychlinsky, A., Papayannopoulos, V. (2014) A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis. Cell Reports 8, 883–896. 30. Hiemstra, P. S., Maassen, R. J., Stolk, J., Heinzel-Wieland, R., Steffens, G. J., Dijkman, J. H. (1996) Antibacterial activity of antileukoprotease. Infect. Immun. 64, 4520–4524. 31. Kallen, A. J., Mu, Y., Bulens, S., Reingold, A., Petit, S., Gershman, K., Ray, S. M., Harrison, L. H., Lynfield, R., Dumyati, G., Townes, J. M., Schaffner, W., Patel, P. R., Fridkin, S. K.; Active Bacterial Core surveillance (ABCs) MRSA Investigators of the Emerging Infections Program. (2010) Health care-associated invasive MRSA infections, 2005-2008. JAMA 304, 641–648. 32. Wertheim, H. F., Melles, D. C., Vos, M. C., van Leeuwen, W., van Belkum, A., Verbrugh, H. A., Nouwen, J. L. (2005) The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect. Dis. 5, 751–762.
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33. Kircik, L. H., Del Rosso, J. Q. (2009) Anti-TNF agents for the treatment of psoriasis. J. Drugs Dermatol. 8, 546–559. 34. Bardoel, B. W., Kenny, E. F., Sollberger, G., Zychlinsky, A. (2014) The balancing act of neutrophils. Cell Host Microbe 15, 526–536. 35. Raftery, M. J., Lalwani, P., Krautkrmer, E., Peters, T., ScharffetterKochanek, K., Kru¨ ger, R., Hofmann, J., Seeger, K., Kru¨ ger, D. H., Schonrich, G. (2014) b2 integrin mediates hantavirus-induced release of neutrophil extracellular traps. J. Exp. Med. 211, 1485–1497. 36. Lande, R., Gregorio, J., Facchinetti, V., Chatterjee, B., Wang, Y. H., Homey, B., Cao, W., Wang, Y. H., Su, B., Nestle, F. O., Zal, T., Mellman, I., Schr¨oder, J. M., Liu, Y. J., Gilliet, M. (2007) Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 449, 564–569. 37. Thammavongsa, V., Missiakas, D. M., Schneewind, O. (2013) Staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death. Science 342, 863–866. 38. Knight, J. S., Subramanian, V., O’Dell, A. A., Yalavarthi, S., Zhao, W., Smith, C. K., Hodgin, J. B., Thompson, P. R., Kaplan, M. J. (2014) Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice. Ann. Rheum. Dis. [Epub ahead of print]. 39. Farley, K., Stolley, J. M., Zhao, P., Cooley, J., Remold-O’Donnell, E. (2012) A serpinB1 regulatory mechanism is essential for restricting neutrophil extracellular trap generation. J. Immunol. 189, 4574–4581. 40. Song, Xy., Zeng, L., Jin, W., Thompson, J., Mizel, D. E., Lei, K., Billinghurst, R. C., Poole, A. R., Wahl, S. M. (1999) Secretory leukocyte protease inhibitor suppresses the inflammation and joint damage of bacterial cell wall-induced arthritis. J. Exp. Med. 190, 535–542. KEY WORDS: serine inflammation autoimmune disease skin psoriasis •
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Brief Conclusive Report
The inhibitory effect of secretory leukocyte protease inhibitor (SLPI) on formation of neutrophil extracellular traps Katarzyna Zabieglo,* Pawel Majewski,* Monika Majchrzak-Gorecka,* Agnieszka Wlodarczyk,* Beata Grygier,* Aneta Zegar,* Monika Kapinska-Mrowiecka,† Antonina Naskalska,‡ Krzysztof Pyrc,‡,§ Adam Dubin,{ Sharon M. Wahl,‖ and Joanna Cichy*,1 *Department of Immunology, §Department of Microbiology, and {Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, and ‡Malopolska Centre of Biotechnology, Jagiellonian University, Krako´ w, Poland; †Department of Dermatology, Zeromski Hospital, Krako´ w, Poland; and ‖Cellular Immunology Section, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA RECEIVED NOVEMBER 13, 2014; REVISED MARCH 16, 2015; ACCEPTED MARCH 29, 2015. DOI: 10.1189/JLB.4AB1114-543R
ABSTRACT Neutrophil extracellular traps (NETs), web-like DNA structures, provide efficient means of eliminating invading microorganisms but can also present a potential threat to its host because it is a likely source of autoantigens or by promoting bystander tissue damage. Therefore, it is important to identify mechanisms that inhibit NET formation. Neutrophil elastase (NE)dependent chromatin decondensation is a key event in the release of NETs release. We hypothesized that inhibitors of NE, secretory leukocyte protease inhibitor (SLPI) and a1-proteinase inhibitor (a1-PI), has a role in restricting NET generation. Here, we demonstrate that exogenous human SLPI, but not a1-PI markedly inhibited NET formation in human neutrophils. The ability of exogenous SLPI to attenuate NET formation correlated with an inhibition of a core histone, histone 4 (H4), cleavage, and partial dependence on SLPI-inhibitory activity against NE. Moreover, neutrophils from SLPI2/2 mice were more efficient at generating NETs than were neutrophils from wild-type mice in vitro, and in experimental psoriasis in vivo. Finally, endogenous SLPI colocalized with NE in the nucleus of human neutrophils in vitro, as well as in vivo in inflamed skin of patients with psoriasis. Together, these findings support a controlling role for SLPI in NET generation, which is of potential relevance to infectious and autoinflammatory diseases. J. Leukoc. Biol. 98: 000–000; 2015.
Introduction Neutrophils, also referred to as polymorphonuclear leukocytes (PMNs), form a part of the front line of host defense against pathogens and are key components of tissue infiltrates in Abbreviations: a1-PI = a1-proteinase inhibitor, CatG = cathepsin G, H4 = histone 4, NE = neutrophil elastase, NETs = neutrophil extracellular traps, PAD4 = peptidylarginine deiminase 4, PMNs = polymorphonuclear leukocytes, SLPI = secretory leukocyte protease inhibitor, SLE = systemic lupus erythematosus, TSB = tryptic soy broth
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autoinflammatory diseases, such as systemic lupus erythematosus (SLE) or psoriasis [1–5]. Activated neutrophils contribute to immune responses through several mechanisms, including phagocytosis, production of reactive oxygen species, degranulation, and the formation of neutrophil extracellular traps (NETs) [6]. NETs are extracellular, decondensed, chromatin networks decorated with nuclear, granular, and cytoplasmic proteins. NETs were first reported to limit the growth and spread of microorganisms through mechanically trapping pathogens and facilitating the interaction of antimicrobial agents that are displayed on NETs with bacteria or fungi [6]. More recently, NETs were associated with tissue damage and autoinflammatory diseases, such as SLE, Wegener granulomatosis, and psoriasis [1–3, 5, 7]. The NET constituents, including self-DNA, histones, and myeloperoxidase, are potential autoantigens [1, 2]. In addition to their beneficial role in host defense, NETs may also be an underlying component of cell cytotoxicity and autoimmunity; therefore, specific counterregulatory mechanisms likely evolved to constrain NET formations. Before the nuclear content of neutrophils is expulsed to the extracellular milieu to form NETs, the cell nucleus disintegrates, and loose chromatin often fills the entire cell. The nuclear decondensation is attributed to peptidylarginine deiminase 4 (PAD4) and serine protease-neutrophil elastase (NE). PAD4 modifies the chromatin structure through conversion of histones’ arginine tail residues to citrulline, a nonconventional amino acid [8], whereas NE drives chromatin decondensation via proteolytic cleavage of specific histones, including the core histone 4 (H4) [9]. Under homeostatic conditions, NE is stored in primary (azurophilic) granules, but it translocates to the nucleus upon neutrophil activation. Once in the nucleus, NE can contribute to chromatin decondensation and subsequently to NET formation [9]. Activation of PMNs can also result in
1. Correspondence: Department of Immunology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7 St., 30-387 Krakow, Poland. E-mail: [email protected]
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Copyright 2015 by The Society for Leukocyte Biology.
deposition of NE into the extracellular environment through extracellular discharge of the granules’ contents [10]. Extracellular NE activity is controlled by several inhibitors; among which, a1-proteinase inhibitor (a1-PI) and secretory leukocyte proteinase inhibitor (SLPI) appear to have predominant roles in limiting excessive NE-mediated proteolysis. Although liverderived a1-PI is a major serine proteinase inhibitor in human plasma, SLPI is more likely to control NE activity at mucosal/ epithelial surfaces where it is abundantly present, likely as a secretory product of epithelial cells [11, 12]. Interestingly, neutrophils also produce a1-PI and SLPI [13, 14]. Thus, these inhibitors are well positioned to have affected the release of NETs. SLPI is the major inhibitor of NE in neutrophils [14]; however, its physiologic role in these cells is not well characterized. SLPI comprises 2 mutually homologous domains with its antiprotease activity, primarily localized to its C-terminal domain. Using SLPI mutants with various protease-inhibitory capacity against NE and other serine proteases, such as the closely related chymotrypsin and trypsin, Leu72, and to a lesser extent, Met73, were demonstrated to be critical sites for binding all 3 enzymes to SLPI [15, 16]. In this study, we demonstrated that SLPI is a potent regulator of NETs release by activated neutrophils in vitro and in vivo. Using antiprotease, active-site-mutated SLPI, we show that SLPI’s capacity to restrict NET formation is partially dependent on its anti-NE function. Our data suggest that SLPI-mediated restriction in NET release might be a mechanism to tailor neutrophil responses.
MATERIALS AND METHODS
Materials Recombinant human SLPI was purchased from R&D Systems (Minneapolis, MN, USA) or produced as described below. Human plasma a1-PI was isolated as previously described [17]. Human NE was isolated using fresh, whole human blood from healthy donors as a starting material, as previously described [18].
Production of recombinant wild-type SLPI and mutant forms of SLPI Wild-type SLPI encoding the mature secreted protein was generated by PCR, using RNA isolated from human alveolar epithelial cells as a template. The
PCR product was cloned into pPIC9 expression vector (Life Technologies, Carlsbad, CA, USA) with the addition of a hexahistidine tag and an enterokinase cleavage site to its N terminus, using the overlap-extension PCR method, as previously described [19]. Two mutants with substitutions of amino acids critical for SLPI’s antiprotease activity [16] (Table 1) were generated using the same PCR method. The identity of the created constructs was verified by sequencing (Genomed, Warsaw, Poland). Wild-type SLPI and its mutants were produced using Pichia pastoris strain GS115 (His2), and the purified form of the supernatants from His+ transformants were produced with Ni-Sepharose 6 Fast Flow (GE Healthcare, Uppsala, Sweden). The SLPI isoforms were eluted with 500 mM imidazole and dialyzed against PBS. The SLPI mutants obtained were routinely .90% pure as assessed by SDS-PAGE and Coomassie Blue staining.
Assessment of inhibitory activities of SLPI NE, trypsin, or chymotrypsin were preincubated for 10 min with various concentrations of wild-type SLPI and its mutants at 20°C in 0.1 M Tris-HCl (pH 8.0) with 0.5 M NaCl. The suitable substrate—MeO-Suc-Ala-Ala-Pro-ValpNA (Sigma-Aldrich, St. Louis) for NE, Bz-Ile-Glu-Gly-Arg-pNA (S-2222) (Chromogenex Technologies, Llanelli, Carmarthenshire, United Kingdom) for trypsin, and Suc-Ala-Ala-Pro-Phe- pNA (Sigma-Aldrich) for chymotrypsin— was then added to the final concentration of 1 mM and incubated for 10–30 min at 20°C in a total volume of 0.1 ml. Released p-nitroaniline was quantified at 405 nm using a Bio-Tek (Winooski, VT, USA) microplate reader.
Patients All human studies were performed in accordance with the guidelines established by the Jagiellonian University Institutional Bioethics Committee, under approved protocols. In total, six psoriasis patients (age 28.7 6 6.6 yr; F:M 2:4) and 20 healthy individuals (age 28.7 6 5.1 yr; F:M 7:13) were enrolled in these studies. The severity of the psoriatic skin lesions was assessed according to the Psoriasis Area Severity Index score (PASI) (minimum, 0 points; maximum, 72 points) and ranged from 25.3 to 39.6 (mean 6 SD 32.5 6 5.4).
Human neutrophils isolation and treatment Neutrophils were isolated from the blood of healthy individuals, as previously described [5]. Neutrophils were seeded on cover slips coated with poly-L-lysine or into 96-well plates and allowed to adhere for 30 min at 37°C under 5% CO2. The neutrophils were stimulated with the following factors: 40–100 nM PMA, 100 ng/ml TNF-a, 10% normal human serum, or Staphylococcus aureus (ATCC 8235; American Type Culture Collection, Manassas, VA, USA), added to neutrophil cultures at a 10:1 ratio. In the indicated experiments, SLPI, SLPI mutants, or a1-PI were added 10 min before the addition of stimulating factors at a final concentration 1.67 mM. This concentration was first optimized experimentally (0.08, 0.42, 0.84, and 1.67 mM) for SLPI. The neutrophils were
TABLE 1. Comparison of SLPI forms % Inhibition of proteolytic activity at 2:1 [SLPI:enzyme] molar ratioc Name SLPI (wild-type) Mut 1 Mut 2
Forms of SLPIa
% Survivalb
NE
Chymo-trypsin
Trypsin
% Neutrophils with SLPI in nucleusd
Leu72 Leu72 replaced with Lys Leu72Met73Leu74 replaced with LysGlyGly
49 6 20 55 6 15 40 6 11
71 6 14 0 0
79 6 9 15 6 6 0
43 6 6 44 6 3 22 6 10
78.4 6 12 77.8 6 1 52 6 18
a
Critical active-site amino acid residues with position in amino acid sequences are shown. b The CFU of SLPI-treated or mutant-treated S. aureus is presented as the CFU percentage of vehicle-treated S. aureus. Data are provided as means 6 SD of 3 independent measurements. c Data are provided as means 6 SD of 3 independent experiments. d The number of neutrophils demonstrating nuclear localization of the indicated SLPI forms is shown as a percentage of the total neutrophils treated with PMA for 30 min; 10 high-powered fields were analyzed per sample by fluorescence microscopy. Data are provided as means 6 SD of 2 independent experiments.
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Zabieglo et al. SLPI inhibits NETs then cultured at 37°C under 5% CO2 for 30 min or 3 h. The cell cultures were subjected to fluorescence microscopy or fluorimetry.
anti-human H4 antibodies (Millipore) and goat anti- rabbit IgG antibodies conjugated to HRP (Sigma-Aldrich).
Preparation of S. aureus and antimicrobial activity assay
Statistical analysis
S. aureus was grown in tryptic soy broth (TSB) (Sigma-Aldrich) to the midlogarithmic phase. For estimation of antimicrobial activity of wild-type SLPI and its mutants, bacteria were analyzed, as described previously [20]. In brief, bacterial suspensions in PBS supplemented with 1% TSB were mixed with diluent (PBS), wild-type SLPI, or its mutants, and were incubated at 37°C for 18–24 h. The diluted mixture was plated on TSB agar plates and incubated at 37°C overnight for enumeration of CFUs. CFUs of SLPI-treated bacteria are presented as a percentage of the PBS (diluent-treated) bacteria.
Evaluation of significance was performed using one-way ANOVA followed by a Bonferroni post hoc test, or 2-tailed Student’s t test.
Isolation and treatment of mouse neutrophils SLPI2/2 mice on a mixed C57BL/6-129/Sv background were obtained by interbreeding SLPI heterozygous mice originally generated by targeted disruption of the SLPI gene by homologous recombination [21, 22]. This recombinant strain is available at The Jackson Laboratory Repository (JAX Stock No. 010926; Bar Harbor, ME, USA). SLPI2/2 mice show no obvious pathologic phenotype under steady-state conditions. When challenged with proinflammatory stimuli, they show higher mortality and tissue destruction and exacerbated inflammatory responses compared with SLPI+/+ controls [21–23]. Mice (female or male, 8–12 wk old) were housed under pathogenfree conditions. Neutrophils were negatively isolated from bone marrow using an AutoMACS separator (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instruction. The purity of the isolated cells was examined by flow cytometry based on Ly6G immunoreactivity. Neutrophils were routinely .95% pure. Neutrophils were then treated with PMA and S. aureus, as described, for human neutrophils.
RESULTS AND DISCUSSION The regulatory role of SLPI and a1-PI on NET formation was first tested using human neutrophils isolated from healthy donors and stimulated with factors well known to induce NETs in vitro. As expected, the human pathogen S. aureus, phorbol ester (PMA), and TNF-a promoted NET generation at 3 h, a typical time frame for NET release [6] (Fig. 1). This was shown by fluorescence microscopy of the cultured neutrophils that were stained for NET components-DNA and CatG [7]. In addition, the levels of extracellular DNA in a conditioned medium of
Aldara model of psoriasis SLPI2/2 and SLPI+/+ control mice between 8–12 wk old were treated twice daily for up to 6 d with 15 mg Aldara (imiquimod cream; Meda, Solna, Sweden) on shaved and depilated back skin [24]. At indicated time points, skin sections comprising the center of treatment were snap-frozen and analyzed by fluorescence microscopy.
Fluorimetry Extracellular DNA in the conditioned medium of treated neutrophils was quantified using Quant PicoGreen dsDNA kit (Life Technologies), according to manufacturer’s recommendations.
Immunohistochemistry Frozen sections (6–10 mm) were prepared from skin biopsies. Human skin sections or purified human neutrophils were stained with the following antibodies: first, biotin-mouse anti-human SLPI (Abcam, Milton, Cambridge, United Kingdom) and rabbit anti-human NE or cathesin G (CatG) (both from Athens Research and Technology, Athens, GA, USA), followed by PEstreptavidin (BD Pharmingen, San Jose, CA, USA) and APC-goat anti-rabbit IgG F(ab9)2 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Mouse tissues or isolated neutrophils were stained with biotin-rat anti-mouse Ly6G (BD Pharmingen) mAb and rabbit anti-histone H3 (citrulline R2 + R8 + R17) Ab (Abcam) or rabbit anti-mouse histone 2a (Abcam), followed by PE-streptavidin and APC-goat anti-rabbit IgG F(ab9)2. The sections were counterstained with Hoechst dye 33258 (Life Technologies). Images were captured with a fully motorized fluorescence microscope (Eclipse; Nikon, Chiyoda, Tokyo, Japan) and were analyzed by NIS-Elements (Nikon) software.
Western blot analysis Neutrophils were resuspended in 13 SDS loading buffer. Samples were then resolved on SDS-16% PAGE under reducing conditions. Histone H4 cleavage was visualized by enhanced chemiluminescence after incubation with rabbit
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Figure 1. SLPI inhibits NET formation in human neutrophils. Purified PMNs were pretreated with the the indicated inhibitors or vehicle (2) for 10 min and were then left unstimulated (control) or were stimulated with the indicated factors for 3 h. The final concentrations of the factors were as follows: SLPI and a1-PI, 1.67 mM, PMA, 40 nM; TNF-a, 100 ng/ml. S. aureus was added in a 10:1 (bacteria:neutrophil) ratio. (A) Fluorescence microscopy of neutrophils stained to detect CatG (green) and DNA (blue). Data are from one experiment and are representative of at least 4 experiments. (B) Supernatants of stimulated neutrophils were analyzed for extracellular DNA by fluorimetry. OD data are presented relative to unstimulated cells. The means 6 SEM from 4–8 independent experiments are shown. Statistical significance comparing the inhibitor-treated cells vs. cells cultured in the absence of the inhibitors (**P , 0.01) was determined by ANOVA followed by a Bonferroni post hoc test.
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stimulated cells were analyzed by fluorimetry. As demonstrated in Fig. 1A, the number of netting cells, identified by extracellular, fibrous, DNA deposits and intracellular chromatin dispersion that was decorated with CatG, significantly increased following the treatment with PMA, TNF-a, or S. aureus. A similar tendency was observed when NETs were quantified by DNA fluorimetry, although the effect of TNF-a was less potent compared with PMA or S. aureus (Fig. 1B). Notably, in the presence of SLPI, NET formation in control-, PMA-, TNF-a, and S. aureus stimulated neutrophils, was markedly reduced (Fig. 1). On average, SLPI treatment resulted in significant reduction of NET formation from 100 to 38, 33, and 17% in human neutrophils stimulated with PMA, TNF-a, and S. aureus, respectively (Fig. 1B). In contrast to SLPI, a1-PI was not able to inhibit NET formation in either control or stimulated neutrophils (Fig.1). Taken together, these data suggest a selective effect of SLPI on constraining NET release. To determine whether endogenous SLPI restricts NET generation, we isolated neutrophils from bone marrow of SLPIdeficient (SLPI2/2) and wild-type (SLPI+/+) mice. Magnetically sorted neutrophils were then subjected to stimulation with PMA or S. aureus. As shown in Fig. 2A, SLPI2/2 neutrophils demonstrated greater ability to form NETs than SLPI+/+ neutrophils did. On average, 2- and 6-fold more SLPI2/2 neutrophils were found to release NETs following stimulation with PMA and S. aureus, respectively (Fig. 2B). To determine whether SLPI controls NET formation in vivo, we assessed the number of NET-forming neutrophils in the skin of SLPI+/+ and SLPI2/2 mice treated to induce psoriasis-like skin inflammation. Because infiltration of neutrophils into human skin is the hallmark of the autoinflammatory disease psoriasis [25] and because SLPI is a component of NETs in the lesional skin of psoriatic patients [5], these data suggest that SLPI has a role in this disease, possibly by regulating NET formation. A mouse model of psoriasiform dermatitis is based on the repeated topical application of Aldara, a cream containing 5% imiquimod, a ligand for TLR7 [24, 26]. NET-forming neutrophils were identified as Ly6G+ cells with dispersed or extruded DNA that costained with citrullinated histone H3 [27]. As shown in Fig. 2C, significantly more skin-infiltrating neutrophils formed NETs in SLPI2/2 mice, compared with SLPI+/+ mice. This was mainly seen in the first 2 d after application of Aldara (Fig. 2D). Together, these data suggest that a lack of endogenous SLPI results in a stronger ability of neutrophils to form NETs under autoinflammatory conditions, consistent with our in vitro data. Exogenous SLPI tends to localize to the nuclei of activated human neutrophils (Fig. 3A and B), indicating that its inhibitory effect on NET formation may depend on translocation to the nucleus. Therefore, we next asked whether endogenous human SLPI showed nuclear localization. Without stimulation, SLPI is dispersed throughout neutrophils, likely localizing to secondary granules or cytoplasm [14, 28] (Fig. 3C). Following treatment with PMA, SLPI was found to mainly migrate toward the cell surface and, occasionally, toward the cell nucleus, where most of the NE was found in approximately 10% of neutrophils (Fig. 3D; data not shown). Thus, in PMA-treated cells, NE and SLPI colocalize in either the cell nucleus or the cell membrane or were spatially separated, with NE present primarily in the cell 4
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nucleus and SLPI at the cell membrane (Fig. 3D). In a distinct scenario, SLPI and NE were typically found together in the nucleus or perinuclear area in approximately 5% of all neutrophils stimulated with normal serum (Fig. 3E; data not shown). These data suggest that SLPI follows NE to the nucleus, although localization of NE and SLPI is likely differently regulated, both spatially and kinetically, in activated neutrophils. To determine whether human SLPI colocalizes with NE in the nucleus in vivo, we examined lesional skin of patients with psoriasis. As shown in Fig. 3F, SLPI and NE could be found in the nucleus of neutrophils infiltrating psoriatic skin. Colocalization of SLPI with NE is found in most skin-infiltrating granulocytes at different stages of NET formation [5] (Fig. 3F; data not shown), but nuclear staining for both NE and SLPI was evident in approximately 0.5% of infiltrating PMNs at the time points evaluated. Together, these data suggest that SLPI controls NE activity in the nucleus of human inflammatory, skin-recruited neutrophils. To determine whether SLPI restricts NET release through regulating NE proteolytic activity in the nucleus, we analyzed cleavage of core H4, a well-described nuclear substrate of NE during NET formation [9, 29]. Using SLPI mutants that differ in NE-, chymotrypsin- and trypsin-inhibitory capacity (Table 1), we evaluated whether SLPI inhibitory activity against serine proteases was required for inhibition of NET formation in human neutrophils stimulated with PMA, TNF-a, and S. aureus. Because SLPI is equipped with antimicrobial activity that is likely to limit the growth of S. aureus and ultimately lower a neutrophil’s ability to generate NETs in response to S. aureus, we first determined, whether wild-type SLPI and SLPI mutants control S. aureus growth in a differential manner. As shown in Table 1, all tested SLPI forms displayed similar antibacterial activity against S. aureus, consistent with a previous report [30] that SLPI’s antimicrobial capacity is mainly located in the N-terminal, and not C-terminal, domain of SLPI that was mutated in the studies. Thus, SLPI’s antimicrobial potential is not likely to differentially influence the ability of the tested SLPI forms to control a neutrophil’s response to the bacteria. All SLPI mutants were similarly capable of migrating to the nucleus of PMA-activated neutrophils (Table 1). As expected, treatment of neutrophils with PMA, TNF-a, or S. aureus resulted in H4 cleavage as evidenced by the low-molecular-weight product (Fig. 4A), although the H4 degradation profile was the most pronounced in PMA-treated cells. In the presence of wild-type SLPI, H4 processing was partially or completely blocked in neutrophils activated with PMA, TNF-a, or S. aureus, respectively. SLPI-mediated inhibition of H4 cleavage coincided with nearly 63, 60, and 80% reduction in NET formation in PMA-, TNF-a-, and bacteria-stimulated neutrophils, respectively (Fig. 4B). Notably, SLPI mutants with a negligible anti-NE function (mutants 1 and 2) also significantly blocked H4 cleavage and NET release, although to a 1.2–1.9 times lesser extent, compared with the wild-type SLPI. Specifically, mutants 1 and 2 decreased NET release by 42 and 45% in PMA-activated neutrophils, by 42 and 50% in TNF-a-treated cells, and by 43 and 56% when S. aureus was used as the stimulus (Fig. 4B). Together, these data suggest that the suppressive effect of SLPI on NET formation is only partially dependent on its anti-NE activity.
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Figure 2. SLPI-deficient mouse neutrophils showed higher propensity to form NETs in vitro and in vivo. (A) Bone marrow-derived neutrophils of SLPI+/+ and SLPI2/2 mice were left untreated (control) or were treated for 3 h with PMA or S. aureus. Neutrophils were then stained for NET components with Histone 2a (red) and DNA (blue) and were analyzed by fluorescence microscopy. Data are from one experiment and are representative of at least 4 experiments. Scale bar, 10 mm. (B) Frequency of bone marrow-derived neutrophils with extracellular NET deposits (% NETs) was examined by fluorescence microscopy. For each mouse (n = 4 per group), 3–4 high-power fields were analyzed. The means 6 SD are shown. *P , 0.05 comparing SLPI+/+ and SLPI2/2 neutrophils by a Student’s t test. (C) Mice were treated with Aldara twice daily for up to 6 d on back skin. The treated skin was collected at indicated time points, stained with anti-Ly6G (red), anti-citH3 (green) Abs, and Hoechst dye to detect DNA (blue), followed by fluorescence microscopy. Data are from one experiment and are representative of at least 5 experiments. Representative NET-forming neutrophils are indicated by arrowheads in enlargement of area outlined in top right panel. Scale bar = 10 mm. (D) NET-forming neutrophils are shown as a percentage of the total Ly6G+ cells (% NETs) for the indicated mice and time point. For each mouse (n = 3–6 per group), 10 high-power fields were analyzed. The means 6 SD are shown. *P , 0.05 comparing SLPI+/+ and SLPI2/2 mice (by a Student’s t test).
Here, we demonstrated that exogenous and endogenous SLPI is a potent, negative regulator of NET formation in neutrophils that respond to infection and inflammatory stimuli, such as S. aureus, TNF-a, or a surrogate inflammatory agent, PMA. Whereas
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S. aureus is a common human pathogen and often causes skin and soft-tissue infections, as well as sepsis [31, 32], TNF-a has a critical role in autoinflammatory diseases, including psoriasis [33]. Given a well-described role of NETs in S. aureus mediated
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as macrophages, and establish persistent infection [37]. Although these findings indicate a crucial role for NETs in various infection- or inflammation-associated pathologies, the mechanisms restricting NETs formation are poorly defined. To this end, interfering with key pathways underlying NET generation, such as the NADPH oxidase-mediated generation of reactive oxygen species or PAD-regulated histone deimination, were recently exploited to inhibit NETs [9, 29, 38]. Discovery of the critical role of NEs in promoting NET release also initiated a substantial interest in inhibitors of NE proteolytic function. Although small, b-lactam-based, cell-permeable NE inhibitors were already instrumental in demonstrating histone cleavage and chromatin decondensation by NEs [9, 29], regulatory mechanisms involving endogenous NE inhibitors, such as SLPI or SerpinB1, are likely to constrain NET formation. Notably, SerpinB1, a cytoplasmic serine protease inhibitor of neutrophils, was recently reported to be essential for restricting NET generation [39]. Likewise, exogenous SLPI was demonstrated to inhibit decondensation activity of NE-containing azurophilic granules in isolated nuclei of human neutrophils [9], suggesting its potential to regulate NET formation. Here, we demonstrated that SLPI colocalizes with NE in the nucleus of activated neutrophils in vitro and in vivo, blocks the cleavage of nuclear NE-substrate H4 in a manner
Figure 3. SLPI colocalizes with NE in cell nucleus in vitro and in vivo. Neutrophils were isolated from healthy donors and left untreated (2) or were treated for 30 min with exogenous SLPI (ex SLPI) (+) in the absence (A) or presence (B) of PMA. Cells were then stained for SLPI (yellow) and DNA (blue) and were analyzed by fluorescence microscopy under the acquisition settings (very short exposure), which did not detect endogenous SLPI. Neutrophils were left untreated (C) or were treated for 3 h with PMA (D) or normal serum (E). These cells, or frozen sections of psoriatic skin biopsy (F), were stained for human NE (HNE) (red), SLPI (green), and DNA (blue); single channels are shown as black and white images (C–F). Data in panels are from different donors and are representative of at least 3 (A–E) or 2 donors (F). Scale bar = 5 mm (A–E), 10 mm (F).
host invasion and autoinflammatory disorders, SLPI as a checkpoint in NET formation may have potential for controlling the pathologic outcome of these diseases. NETs emerge as crucial contributors to innate immune defense against microbes, but their excessive development during infection leads to pathology [34]. For example, hantaviruses, which cause severe kidney and lung damage in humans, were recently described to generate high levels of NETs and autoantibody production in response to virus-induced systemic NET overflow [35]. Likewise, excessive NET formation or impaired NET degradation was associated with autoinflammatory diseases [34], and a putative link between NETs and autoimmunity has been proposed [1, 2, 36]. On the other hand, S. aureus can hijack NETs to destroy host-defense effector cells, such 6
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Figure 4. Inhibition of H4 proteolytic processing and NET formation are partially dependent on anti-NE activity of SLPI. Neutrophils were isolated from blood of healthy donors and were left unstimulated (control) or were stimulated with PMA, TNF-a, or S. aureus for 3 h in the presence of wild-type SLPI, the indicated SLPI mutants, or a vehicle. (A) Cell lysates where subjected to Western blot analysis using Ab against H4. H4 degradation product is shown by arrowheads. (B) Neutrophil-conditioned medium was analyzed for extracellular DNA by fluorimetry. OD data are presented relative to unstimulated cells. The means 6 SEM from 5–14 independent experiments are shown. Statistically significant differences between SLPI- or mutant-treated vs. vehicle-treated cells are indicated with an asterisk (*P , 0.05; **P , 0.01; ANOVA followed by a Bonferroni post hoc test).
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Zabieglo et al. SLPI inhibits NETs
partially dependent on its anti-NE inhibitory activity, and attenuates release of NETs in vitro and in vivo. Together, these findings implicate SLPI, a major NE inhibitor in human neutrophils [14], as a controlling agent in NET generation. It remains to be determined whether SLPI acts to control a threshold of NET formation or influences a neutrophil preference to a type of defense mechanism, such as degranulation or phagocytosis. SLPI acts to counteract excessive inflammatory responses and to initiate healing processes through several antiproteasedependent and -independent mechanisms. Our finding that SLPI-inactive mutants inhibit NET formation to some extent raises the question of whether the antiprotease-independent activity of SLPI might be involved in attenuating NET release. One such NE-separate pathway may involve inhibition of transcription factor NF-kB [21, 40], although these mechanisms in NET formation have yet to be explored. However, because SLPI mutants that were inactive against NE were not completely devoid of inhibitory potential against chymotrypsin or trypsin, it is also possible that SLPI controls NET formation through targeting other, yet-to be identified proteases. In conclusion, our findings suggest that SLPI is an important element for protection against aberrant NET formation and that its anti-NE activity is partially responsible for the protective effect.
AUTHORSHIP K.Z., P.M., M.M.-G., A.W., B.G., A.Z., and A.D. performed the experiments. M.K.-M., A.N., K.P., A.D., and S.M.W. contributed reagents/materials. J.C. and S.M.W. wrote the article.
ACKNOWLEDGMENTS This work was supported in part by the Polish National Science Center Grant 2011/02/A/NZ5/00337 and the Foundation for Polish Science Grant TEAM/2010-5 and was cofinanced by the European Union within a European Regional Development Fund Award (to J.C.). The Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University is a beneficiary of structural funds from the European Union (Grant POIG.02.01.0012-064/08) and a partner with the Leading National Research Center (KNOW) supported by the Polish Ministry of Science and Higher Education. This research was supported in part by the Intramural Research Program of the U.S. National Institutes of Health National Institute of Dental and Craniofacial Research (to S.M.W.).
DISCLOSURES
The authors declare no competing financial interests.
REFERENCES
1. Garcia-Romo, G. S., Caielli, S., Vega, B., Connolly, J., Allantaz, F., Xu, Z., Punaro, M., Baisch, J., Guiducci, C., Coffman, R. L., Barrat, F. J., Banchereau, J., Pascual, V. (2011) Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci. Transl. Med. 3, 73ra20.
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2. Lande, R., Ganguly, D., Facchinetti, V., Frasca, L., Conrad, C., Gregorio, J., Meller, S., Chamilos, G., Sebasigari, R., Riccieri, V., Bassett, R., Amuro, H., Fukuhara, S., Ito, T., Liu, Y. J., Gilliet, M. (2011) Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus. Sci. Transl. Med. 3, 73ra19. 3. Lin, A. M., Rubin, C. J., Khandpur, R., Wang, J. Y., Riblett, M., Yalavarthi, S., Villanueva, E. C., Shah, P., Kaplan, M. J., Bruce, A. T. (2011) Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis. J. Immunol. 187, 490–500. 4. Mantovani, A., Cassatella, M. A., Costantini, C., Jaillon, S. (2011) Neutrophils in the activation and regulation of innate and adaptive immunity. Nat. Rev. Immunol. 11, 519–531. 5. Skrzeczynska-Moncznik, J., Wlodarczyk, A., Zabieglo, K., KapinskaMrowiecka, M., Marewicz, E., Dubin, A., Potempa, J., Cichy, J. (2012) Secretory leukocyte proteinase inhibitor-competent DNA deposits are potent stimulators of plasmacytoid dendritic cells: implication for psoriasis. J. Immunol. 189, 1611–1617. 6. Brinkmann, V., Reichard, U., Goosmann, C., Fauler, B., Uhlemann, Y., Weiss, D. S., Weinrauch, Y., Zychlinsky, A. (2004) Neutrophil extracellular traps kill bacteria. Science 303, 1532–1535. 7. Skrzeczynska-Moncznik, J., Wlodarczyk, A., Banas, M., Kwitniewski, M., Zabieglo, K., Kapinska-Mrowiecka, M., Dubin, A., Cichy, J. (2013) DNA structures decorated with cathepsin G/secretory leukocyte proteinase inhibitor stimulate IFNI production by plasmacytoid dendritic cells. Am J Clin Exp Immunol 2, 186–194. 8. Wang, Y., Li, M., Stadler, S., Correll, S., Li, P., Wang, D., Hayama, R., Leonelli, L., Han, H., Grigoryev, S. A., Allis, C. D., Coonrod, S. A. (2009) Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J. Cell Biol. 184, 205–213. 9. Papayannopoulos, V., Metzler, K. D., Hakkim, A., Zychlinsky, A. (2010) Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J. Cell Biol. 191, 677–691. 10. Faurschou, M., Borregaard, N. (2003) Neutrophil granules and secretory vesicles in inflammation. Microbes Infect. 5, 1317–1327. 11. Reardon, C., Lechmann, M., Bru¨ stle, A., Gareau, M. G., Shuman, N., Philpott, D., Ziegler, S. F., Mak, T. W. (2011) Thymic stromal lymphopoetin-induced expression of the endogenous inhibitory enzyme SLPI mediates recovery from colonic inflammation. Immunity 35, 223–235. 12. Zhu, J., Nathan, C., Jin, W., Sim, D., Ashcroft, G. S., Wahl, S. M., Lacomis, L., Erdjument-Bromage, H., Tempst, P., Wright, C. D., Ding, A. (2002) Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair. Cell 111, 867–878. 13. Clemmensen, S. N., Jacobsen, L. C., Rørvig, S., Askaa, B., Christenson, K., Iversen, M., Jørgensen, M. H., Larsen, M. T., van Deurs, B., Ostergaard, O., Heegaard, N. H., Cowland, J. B., Borregaard, N. (2011) Alpha-1antitrypsin is produced by human neutrophil granulocytes and their precursors and liberated during granule exocytosis. Eur. J. Haematol. 86, 517–530. 14. Sallenave, J. M., Si Tahar, M., Cox, G., Chignard, M., Gauldie, J. (1997) Secretory leukocyte proteinase inhibitor is a major leukocyte elastase inhibitor in human neutrophils. J. Leukoc. Biol. 61, 695–702. 15. Eisenberg, S. P., Hale, K. K., Heimdal, P., Thompson, R. C. (1990) Location of the protease-inhibitory region of secretory leukocyte protease inhibitor. J. Biol. Chem. 265, 7976–7981. 16. Mulligan, M. S., Lentsch, A. B., Huber-Lang, M., Guo, R. F., Sarma, V., Wright, C. D., Ulich, T. R., Ward, P. A. (2000) Anti-inflammatory effects of mutant forms of secretory leukocyte protease inhibitor. Am. J. Pathol. 156, 1033–1039. 17. Travis, J., Salvesen, G. S. (1983) Human plasma proteinase inhibitors. Annu. Rev. Biochem. 52, 655–709. 18. Baugh, R. J., Travis, J. (1976) Human leukocyte granule elastase: rapid isolation and characterization. Biochemistry 15, 836–841. 19. Bryksin, A. V., Matsumura, I. (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques 48, 463–465. 20. Banas, M., Zabieglo, K., Kasetty, G., Kapinska-Mrowiecka, M., Borowczyk, J., Drukala, J., Murzyn, K., Zabel, B. A., Butcher, E. C., Schroeder, J. M., Schmidtchen, A., Cichy, J. (2013) Chemerin is an antimicrobial agent in human epidermis. PLoS ONE 8, e58709. 21. Ashcroft, G. S., Lei, K., Jin, W., Longenecker, G., Kulkarni, A. B., Greenwell-Wild, T., Hale-Donze, H., McGrady, G., Song, X. Y., Wahl, S. M. (2000) Secretory leukocyte protease inhibitor mediates nonredundant functions necessary for normal wound healing. Nat. Med. 6, 1147–1153. 22. McCartney-Francis, N., Jin, W., Belkaid, Y., McGrady, G., Wahl, S. M. (2014) Aberrant host defense against Leishmania major in the absence of SLPI. J. Leukoc. Biol. 96, 917–929. 23. Nakamura, A., Mori, Y., Hagiwara, K., Suzuki, T., Sakakibara, T., Kikuchi, T., Igarashi, T., Ebina, M., Abe, T., Miyazaki, J., Takai, T., Nukiwa, T. (2003) Increased susceptibility to LPS-induced endotoxin shock in secretory leukoprotease inhibitor (SLPI)-deficient mice. J. Exp. Med. 197, 669–674.
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24. van der Fits, L., Mourits, S., Voerman, J. S., Kant, M., Boon, L., Laman, J. D., Cornelissen, F., Mus, A. M., Florencia, E., Prens, E. P., Lubberts, E. (2009) Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis. J. Immunol. 182, 5836–5845. 25. Pinkus, H., Mehregan, A. H. (1966) The primary histologic lesion of seborrheic dermatitis and psoriasis. J. Invest. Dermatol. 46, 109–116. 26. Flutter, B., Nestle, F. O. (2013) TLRs to cytokines: mechanistic insights from the imiquimod mouse model of psoriasis. Eur. J. Immunol. 43, 3138–3146. 27. Branzk, N., Lubojemska, A., Hardison, S. E., Wang, Q., Gutierrez, M. G., Brown, G. D., Papayannopoulos, V. (2014) Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat. Immunol. 15, 1017–1025. 28. Sørensen, O., Arnljots, K., Cowland, J. B., Bainton, D. F., Borregaard, N. (1997) The human antibacterial cathelicidin, hCAP-18, is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils. Blood 90, 2796–2803. 29. Metzler, K. D., Goosmann, C., Lubojemska, A., Zychlinsky, A., Papayannopoulos, V. (2014) A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis. Cell Reports 8, 883–896. 30. Hiemstra, P. S., Maassen, R. J., Stolk, J., Heinzel-Wieland, R., Steffens, G. J., Dijkman, J. H. (1996) Antibacterial activity of antileukoprotease. Infect. Immun. 64, 4520–4524. 31. Kallen, A. J., Mu, Y., Bulens, S., Reingold, A., Petit, S., Gershman, K., Ray, S. M., Harrison, L. H., Lynfield, R., Dumyati, G., Townes, J. M., Schaffner, W., Patel, P. R., Fridkin, S. K.; Active Bacterial Core surveillance (ABCs) MRSA Investigators of the Emerging Infections Program. (2010) Health care-associated invasive MRSA infections, 2005-2008. JAMA 304, 641–648. 32. Wertheim, H. F., Melles, D. C., Vos, M. C., van Leeuwen, W., van Belkum, A., Verbrugh, H. A., Nouwen, J. L. (2005) The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect. Dis. 5, 751–762.
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33. Kircik, L. H., Del Rosso, J. Q. (2009) Anti-TNF agents for the treatment of psoriasis. J. Drugs Dermatol. 8, 546–559. 34. Bardoel, B. W., Kenny, E. F., Sollberger, G., Zychlinsky, A. (2014) The balancing act of neutrophils. Cell Host Microbe 15, 526–536. 35. Raftery, M. J., Lalwani, P., Krautkrmer, E., Peters, T., ScharffetterKochanek, K., Kru¨ ger, R., Hofmann, J., Seeger, K., Kru¨ ger, D. H., Schonrich, G. (2014) b2 integrin mediates hantavirus-induced release of neutrophil extracellular traps. J. Exp. Med. 211, 1485–1497. 36. Lande, R., Gregorio, J., Facchinetti, V., Chatterjee, B., Wang, Y. H., Homey, B., Cao, W., Wang, Y. H., Su, B., Nestle, F. O., Zal, T., Mellman, I., Schr¨oder, J. M., Liu, Y. J., Gilliet, M. (2007) Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 449, 564–569. 37. Thammavongsa, V., Missiakas, D. M., Schneewind, O. (2013) Staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death. Science 342, 863–866. 38. Knight, J. S., Subramanian, V., O’Dell, A. A., Yalavarthi, S., Zhao, W., Smith, C. K., Hodgin, J. B., Thompson, P. R., Kaplan, M. J. (2014) Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice. Ann. Rheum. Dis. [Epub ahead of print]. 39. Farley, K., Stolley, J. M., Zhao, P., Cooley, J., Remold-O’Donnell, E. (2012) A serpinB1 regulatory mechanism is essential for restricting neutrophil extracellular trap generation. J. Immunol. 189, 4574–4581. 40. Song, Xy., Zeng, L., Jin, W., Thompson, J., Mizel, D. E., Lei, K., Billinghurst, R. C., Poole, A. R., Wahl, S. M. (1999) Secretory leukocyte protease inhibitor suppresses the inflammation and joint damage of bacterial cell wall-induced arthritis. J. Exp. Med. 190, 535–542. KEY WORDS: serine inflammation autoimmune disease skin psoriasis •
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