Supplementary methods Flow cytometric analysis of DCs. DC were seeded into tissue culture dishes in IMDM 2% FCS, and added with PMN (1:1; PMN: DC) for 16h also in the presence of DNAse (100 U/ml); DC were evaluated by flow cytometry for the expression of CD40 and CD86 markers. FITC anti-cd40 (3/23), FITC anti-cd86 (gl1), and PE anti-cd11c (N418), were purchased from ebioscience. Surface staining reactions were performed in PBS supplemented with 2% FCS on ice for 30 min.
Supplementary Figures Supplementary. Fig. 1 Features of inflammation and PMN recruitment in s.c. implanted agar plugs. A. IHC of the skin surrounding implanted agar blocks showing an inflammatory condition characterized by accumulation of new blood vessels (CD31 staining) and leukocytes (CD11b staining), mainly PMN (GR-1 staining). PMN could be recovered and enriched up to the 95% by washing agar plugs and seeding harvested cells 30 min at 37 C on plastic. Immunocytochemistry (B) for GR-1 and (C) flow cytometry analysis of CD11b and GR-I co-expression in PMN after adhesion on plastic. D. Cumulative
data of one representative experiment out of five performed with 3 mice that have had the agar plug removed at the indicated time points.
Supplementary Fig. 2
Features of NETosis in agar-elicited PMN. A. Confocal microscopy analysis showing the time course of NET formation by agar PMN. PMN undergoing NETosis incorporate the dsdna dye SYTOX green. Red staining: Elastase Ab. Scale bars: 20 µm. B. Characterization of NET produced by agar PMN with Ab to MPO, Elastase and PR3 showing the localization of these proteins in the dsdna backbone (pan Histone Ab). Scale bars: 10 µm. C. Confocal microscopy analysis showing that NETosis by agar PMN initiates with a nuclear permeabilization allowing the colocalization of nuclear material, identified by a staining for histones, with cytoplasmic proteins like elastase (red staining). Scale bar: 5µm. b. The end point of NETosis is visualized as release of DNA-based fibers, decorated with cytoplasmic proteins, here shown with mab to His (green) and MPO (red). Scale bars: 10 µm.
Supplementary Fig.3 Quantification of NETosis by confocal microscopy. The method used to quantify NET and to distinguish NETosis from apoptosis is based on measuring the size of nuclear material that is stained with the DNA dye SYTOX green. SYTOX green is a cellimpermeable dye that in live cell imaging experiments, when added before PFA fixation, stains only dsdna released from NET. On the other hand, if added to cells after fixation, this dye stains also apoptotic cells as in the case showed in this picture. Particularly, to evaluate the relative amount of NETosis and apoptosis in our PMN preparation, cells were seeded onto poly-lysine coated glasses, fixed and stained with the SYTOX green, and then imaged through confocal microscopy or immunofluorescence. Using a software-assisted technique the size of nuclear material is measured. As shown in the picture, apoptotic cells (left) have smaller area, whereas NETotic cells (right) are characterized by a broad range of nuclear areas. By
plotting SYTOX + nuclear area of a significative number of cells (200-300) against the fraction of cells showing a given nuclear area interval, the result obtained is a distribution forming a narrow pick in case of prevalent apoptosis. Apoptotic cells have nuclear areas within the 10-300 µm 2 interval, whereas NETotic PMN spread their areas from the 10-100 up to the 700-800 µm 2 interval.
Supplementary Fig. 4 INFγ and TNF are needed for NETosis in agar-pmn. A. Quantification of NET formation by agar PMN obtained from INFγ-KO, TNF-KO and IL6-KO mice. Agar trap collected PMN from INFγ -KO and TNF-KO mice did not form NET. In such deficient mice many cells were still alive and excluded the SYTOX green dye. B. Representative IF analysis of the cells, quantified in A. Reduced NET formation in agar-elicited PMN from INFγ-KO and TNF-KO but not from IL6-KO mice. Number of nuclei analyzed/experiment= 200.
Supplementary Fig. 5 DC contacting netting neutrophils are activated and migrate to regional LN. A. Forward scatter (FSC)/side scatter (SSC) and annexin V/7-AAD representative plots for CD11b+GR-1+ spleen isolated PMN that have been treated with freeze-thaw or anti-fas mab methods in inducing necrosis or apoptosis, respectively. B. Flow cytometry analysis of of CD40 expression by mdc co-cultured with NETotic (in
presence or absence of DNAse), apoptotic or necrotic neutrophils. Cumulative data from 8 independent experiments are shown. C. Control mdc (unstimulated) or mdc stimulated with CpG or obtained from different mdc-pmn-co-cultures were labeled with fluorescent microbeads and injected i.p. into BALB/c naïve mice. After 24h mesenteric LN were analyzed for the presence of CD11c + /beads + cells. We found that DC treated with CpG or co-cultured with NETotic, apoptotic or necrotic PMN were all able to migrate to mesenteric LN. Representative plot obtained by analyzing a total of 2x10 6 /LN are shown. D. Activation of NADPH oxidase of agar PMN seeded onto poly-d-lysine coated glasses, in presence or absence of DNAse. Activation of NADPH oxidase was not affected by DNAse treatment as evaluated as the nitroblue tetrazolium (NBT) reduction test.
Supplementary Fig. 6
NET-loaded mdc immunization induces lung vasculities. A. Histopathological score of parenchymal damage (left) and extent of immune cells infiltration (right) in mice immunized with mdc co-cultured with NETotic, apoptotic or necrotic PMN, and in control mice immunized with DC or PMN alone (n=9/group; p<0.05 and **p<0.01, one-way Anova with post test Dunn s correction. Data are shown as mean ± SD). Autoimmune lpr/lpr mice have been included in the analysis for comparison. B. Representative histological H&E pictures showing the perivascular neutrophilic infiltration characterizing the lungs of mice immunized with DC co-cultured with NEtotic PMN, but not the other groups. This phenotype was prevented in mice immunized with mdc co-cultured with NET in the presence of DNAse. Autoimmune lpr/lpr mice have been included in the analysis for comparison. Original Magnification: x20.
Supplementary. Fig.7 Histopathological features of the renal small vessel vasculitis induced by immunization with NET-loaded mdc. A) prominent Gr1+ granulocyte infiltrates are detected throughout the renal parenchyma where they show vascular aggression (arrows). B) Foci of granulocyte infiltration are associated with small vessels (arrows) in the renal parenchyma of mice immunized with NET-loaded mdcs. C) The vascular and peri-vascular inflammatory infiltrates show a pleomorphous composition and is populated by polymorphonuclear leukocytes (black arrows). D-E) Small necrotic foci are detected in the glomerular tufts and extraglomerular vessels. Inset: Some glomeruli show signs of extracapillary proliferation and crescent formation (red arrow). The pictures are representative of the 9 mice analyzed for SVV features among those immunized with DC+NETotic PMN.
Supplementary Fig. 8 Immunoglobulin deposition in kidney from immunized mice. Renal frozen sections from mice immunized with DC+ NETotic, apoptotic or necrotic PMN as well as from lpr/lpr mice and mice have been stained with Alexa 488 coniugated mab to mouse IgG (H+L). IF analysis shows glomerural IgG deposition in mice immunized with mdc co-cultured with NETotic, apoptotic or necrotic PMN but not in mice immunized with PMN or DC alone or with DC+ NETotic PMN in the presence of
DNAse. Overall glomerular IgG deposition in immunized mice is lower than in autoimmune lpr/lpr mice.
Supplementary Fig. 9 Correlation between perivascular neutrophils infiltration and development of ANCA in mice immunized with mdc co-cultured with NETotic PMN. A. Immunohistochemical analysis for Gr-1 expression in frozen renal sections from 6 individual mice. B. Overall histological damage and extent of immune cells infiltration in kidneys from the same mice. C. The six mice have been also analyzed for serum MPO-ANCA. The level of circulating ANCA correlated with that of granulocyte/immune cells infiltration in kidneys.
Supplementary Fig. 10 The serum of mice immunized with DC+NETotic PMN recognizes PMA-induce NET. Spleen purified PMN were seeded onto poly-d-lysine coated glasses and treated with PMA, in the presence or absence of DNAse (100U/ml), such to induce or prevent NET formation. PMN were than stained with sera from mice immunized with DC+NETotic PMN or from lpr-lpr mice. Sera from DC+NETotic PMN recognize PMA-treated PMN only in the absence of DNAse, whereas sera from lpr/lpr mice still recognize them even in the presence of DNAse, a result consistent with their high ds- DNA and ss-dna ANA titers. A. Representative IF (Scale bars: 25 µm) and B. Software assisted micrograph quantification of the above staining performed on a total of 200 cells that were imaged by confocal microscopy. One representative experiment out of three performed is shown. D. The serum from mice immunized with DC+NEtotic PMN, similarly to that of lpr/lpr mice is poly-reactive and capable to recognize human neutrophils in peripheral blood smears. Peripheral blood smears from healthy donors have been fixed with PFA 4%, treated with 0.1% TRITON and stained with serum from mice immunized with DC+NETotic PMN or lpr/lpr mice
(1/20 dilution). The staining with serum from the lpr/lpr mouse (mouse number 54 of the previous experiments) is characterized by a nuclear and peri-nuclear pattern, whereas that from immune mice shows a more diffuse cytoplasmic staining. The serum from lpr/lpr, but not that from immunized mice, stains also nuclear material in circulating monocytes.
Supplementary Table 1. Histophatological and serological parameters of immune mice. PMN alone DC alone DC+Netotic PMN DC+Netotic PMN+DNAse DC+Apoptotic PMN DC+ Necrotic PMN lpr/lpr b) MPO-ANCA 5,57±4,54* 2,89±3,89*** 23,83±14 4,49±4,2** 8,89±6,9* 2,91±1,9** 9,5±10,75* PR3-ANCA 3,2± 3,4* ND 23,40 ±23,44 4,3 ± 3,0* 9,39 ± 15,6 3,7 ± 4,9* 42,5 ±26,12 anti-ss DNA Ab a) 265,8±169 852,3±272 995,9±105,3 631,8±105 1360±265 633,5±73 1972±142 anti-ds DNA Ab a) 103,1±74 152,2±68 503,46±182 142,9±125 548,9±202 375,05±69 1054±293 anti-ana DNA Ab a) 1111±166 1108±368 1383±365 999,0±401 1541±275 1367±65 3817±1282 Nephritic lesions score Interstitial lesions score 1,00±0 0,33±0,511* 4±1,48 1±0 0,33±0,52* 0,833±1,33* 2,125±1,64 1,33±0,577* ND 8±2,045 2,25±0,957 1,333±1,36* 2,33±1,86 9,25±3,24 Albuminuria 22,27±16,73 24,27±7,65 30,27±20 27,87±10,3 37,7±27 29,15±4,3 32,77±22 Skin lesions(%) ND ND Pulmonary lesions score 22,2±4 ND 8,33±0,0 1,5±2,12 ND 4,71±1,03 2,66±0,577 1,33±1,15* 0,33±0* 5±1,19 For statistical analysis DC-NETotic PMN group is compared to the other type of immunization. Dunn's Multiple Comparison Test. Mean ± SD are shown. * p< 0,05; ** p< 0,01; *** p< 0,001 a) U/ml 1/400 serum dilution; b) Months of age : 9,14±1,41. Mean ± SD ND ND
Supplementary table2. Characteristics of subjects with microscopic poliangiitis, SLE, and psoriasis, whose skin lesion biopsies were analyzed for NET/DC spatial interaction. Patient ID Gender Age Clinical diagnosis ANCA pattern (IIF) ANCA type (ELISA) Skin lesions NET/DC interaction #1 F 31 MPA p-anca MPO+ P,E + #2 M 68 MPA n.a. MPO+ E - #3 M 53 MPA p-anca MPO+ P,E + #4 F 76 MPA p-anca MPO+ P,E,U + #5 M 34 MPA p-anca MPO+ P,E - #6 F 42 MPA n.a. MPO+ P + #7 M 47 SLE n.a. n.a. V/B - #8 M 40 SLE n.a. n.a. E,P,M + #9 F 59 SLE n.a. n.a. E,M - #10 F 39 SLE p-anca MPO+ E,P,M + #11 F 42 SLE n.a. n.a. E,PL + #12 F 53 Psoriasis n.a. n.a. E,M - #13 M 26 Psoriasis n.a. n.a. E,M - #14 M 42 Psoriasis n.a. n.a. E - #15 F 55 Psoriasis n.a. n.a. E,PL - Abbreviations: ANCA, anti-neutrophil cytoplasmatic antibodies; MPA, microscopic poliangiitis; SLE, systemic lupus erithematosus; p-anca, peri-nuclear ANCA staining on immunofluorescence; MPO, myeloperoxidase; P, purpura; E, erythema; U, ulcers; V/B, vesciculae/bullae; M, maculae; PL, plaques; NET, neutrophil extracellular trap; DC, dendritic cell.