Periodontitis Associates with a Type 1 IFN Signature in Peripheral Blood Neutrophils Helen J. Wright, John B. Matthews, Iain L. C. Chapple, Nic Ling-Mountford and Paul R. Cooper This information is current as of September 23, 2021. J Immunol 2008; 181:5775-5784; ; doi: 10.4049/jimmunol.181.8.5775 http://www.jimmunol.org/content/181/8/5775 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Periodontitis Associates with a Type 1 IFN Signature in Peripheral Blood Neutrophils1

Helen J. Wright, John B. Matthews, Iain L. C. Chapple, Nic Ling-Mountford, and Paul R. Cooper2

Peripheral blood neutrophils from periodontitis patients exhibit a hyperreactive and hyperactive phenotype (collectively termed hyperresponsivity) in terms of production of reactive oxygen species (ROS). The molecular basis for this phenomenon, however, has yet to be determined. Our objectives were to identify differentially expressed in hyperresponsive peripheral blood neutrophils from chronic periodontitis patients relative to periodontally healthy controls and use these data to identify potential contributory pathways to the hyperresponsive neutrophil phenotype. Using microarray technology we demonstrated differential expression of 163 genes (149 increased, 14 decreased) representing a range of ontological classes. There was increased expression of a significant number of IFN-stimulated genes (ISG). RT-PCR analysis of ISG transcripts in individual and pooled samples Downloaded from further corroborated these data, and indicated that levels decreased to near those of controls following successful therapy. Significantly enhanced Fc␥R-stimulated ROS production was subsequently achieved by priming control neutrophils with IFN- ␣/-␤/-␥, but not LPS, and expression analysis indicated that exposure to the type I IFN (in particular IFN-␣) better replicated the mRNA profile observed in vivo. Further studies demonstrated that plasma levels of IFN-␣ were significantly higher in samples from patients relative to unaffected controls. Following successful periodontitis treatment, plasma IFN-␣ levels, neutrophil ISG expression, and Fc␥R-stimulated neutrophil ROS output of patients, all decreased to levels comparable with those of controls. In conclusion, http://www.jimmunol.org/ although chronic periodontitis is a complex disease, raised IFN-␣ may be one determinant of the distinct molecular phenotype and hyperresponsivity exhibited by patients’ peripheral blood neutrophils. The Journal of Immunology, 2008, 181: 5775–5784.

eripheral blood neutrophils (PBN)3 from patients with a lated ROS production (7–10). Although a host molecular defect in range of inflammatory disorders have been shown to have intracellular lipid signaling may explain peripheral neutrophil ROS P enhanced chemotactic and extracellular proteolytic activ- hyperreactivity in the relatively rare form of the disease, localized ity as well as the ability to produce increased amounts of reactive aggressive periodontitis (11), this mechanism does not explain the oxygen species (ROS; Refs. 1–3). It has been proposed that the

patient predisposition observed in chronic periodontitis. The un- by guest on September 23, 2021 accumulation of these activated neutrophils at sites of tissue irritation, derlying mechanism(s) responsible for the hyperinflammatory neu- Ag localization, or bacterial infection subsequently leads to the tissue trophil phenotype seen in chronic periodontitis (the commonest damage that underpins the clinical course of these diseases. form of the disease) is currently unknown, although analyses in- Periodontitis is one of the most prevalent human inflammatory dicate that it is not a result of altered adhesion molecule (7) or diseases and is characterized by an aberrant and exaggerated neu- Phox gene (10) expression, polymorphisms in Fc␥R (8, 12), or in trophilic response to microbial plaque present at the gingival mar- vitro priming by cytokines or LPS (13, 14). Furthermore, the as- gin indicating dysregulation of the hosts’ innate immune response sociation of periodontitis with increased relative risk for cardio- (4, 5). The resultant collateral host tissue damage to the supporting vascular disease, fatal coronary events (15), and ischemic stroke periodontal tissues leads to progressive periodontitis and ulti- (16), and the demonstrable medium-term reductions in vascular mately culminates in tooth loss (6). Several studies have demon- endothelial dysfunction following aggressive periodontal therapies strated that PBN from chronic periodontitis patients are not only (17), emphasize the potential impact of inflammatory periodontitis ␥ hyperreactive, in response to Fc R stimulation by periodontal on peripheral macrovascular disease. pathogens, but also hyperactive, with respect to baseline unstimu- To identify factors predisposing to, or biomarkers of disease activity, peripheral blood levels of several acute phase response (APR) in periodontitis patients have been analyzed. Dur- Periodontal Research Group, School of Dentistry, University of Birmingham, Bir- mingham, United Kingdom ing the APR, following local release of factors such as TNF-␣, ␤ Received for publication November 27, 2007. Accepted for publication August IL-1 , and IL-6, systemic changes occur that include significant 6, 2008. hepatic release of plasma proteins (e.g., C-reactive and The costs of publication of this article were defrayed in part by the payment of page serum amyloid A), activation of complement proteins, and several charges. This article must therefore be hereby marked advertisement in accordance other metabolic events (18, 19). Thus far, whereas plasma C-re- with 18 U.S.C. Section 1734 solely to indicate this fact. active protein levels have generally been found to be higher in 1 This work was supported by the Medical Research Council (MRC UK-G0000797). periodontitis patients compared with healthy controls, findings for 2 Address correspondence and reprint requests to Dr. Paul R. Cooper, Oral Biology, ϳ School of Dentistry, University of Birmingham, St. Chad’s Queensway, Birmingham, any of the other 40 recognized APR proteins are inconsistent, B4 6NN, U.K. E-mail address: [email protected] mainly due to the biological heterogeneity of patients (20–22). 3 Abbreviations used in this paper: PBN, peripheral blood neutrophil; ROS, reactive Notably, analysis of circulating APR-associated cytokine levels, oxygen species; APR, acute phase response; ISG, IFN-stimulated gene; RLU, relative including TNF-␣, IL-1␤, IL-2, IL-4, and IL-10, has therefore yet light unit; CL, chemiluminescence; bDNA, bacterial DNA. to identify any useful biomarkers, nor has it been able to provide Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 a mechanistic explanation for disease pathogenesis (23–26). www.jimmunol.org 5776 ELEVATED IFN-␣ LEVELS IN PERIODONTITIS

Table I. Details of primer sequences and semi-quantitative RT-PCR conditionsa

Gene Symbol Sequence Product Size Cycle No.

Myxovirus (influenza virus) resistance 1, MX1 F-AGCCACTGGACTGACGACTT 203 bp 30 IFN-inducible protein p78 R-GAGGGCTGAAAATCCCTTTC IFN induced with tetratricopeptide repeats 4 IFIT4 F-CCCTTCAGGCATAGGCAGTA 185 bp 30 R-CTCCTACCCGTCACAACCAC IFN-stimulated protein, 15 kDa (ISG15) G1P2 F-CGCAGATCACCCAGAAGATT 185 bp 33 R-GCCCTTGTTATTCCTCACCA IFN induced with tetratricopeptide repeats 1 IFIT1 F-GGTCGGTTTCAGGAATTTCA 236 bp 33 R-GCCCGCTCATAGTACTCCAG CMV-inducible gene 5/viperin cig5 F-TAGCTGGTTGGCCCTACATC 237 bp 33 R-TGCTTGCTTTCTCTGAGCTG IFN-induced protein 44-like IFI-44 like F-AGCGGTGGGCTAAGATAGGT 245 bp 33 R-TCCACGCTGTTTCTCAAAGA GAPDH GAPDH F-TCTAGACGGCAGGTCAGGTCC 391 bp 26 R-CCACCCATGGCAAATTCCATG

a All DNA sequences are shown in the 5Ј to 3Ј orientation. F ϭ Forward primer; R ϭ Reverse primer. Downloaded from Nevertheless, it is hypothesized that the APR, which may arise determine the priming effects of IFN on Fc␥R-stimulated ROS production chronically in patients, is responsible for the association between and expression of IFN-stimulated genes (ISG). Ethical approval was periodontitis and other systemic disorders such as cardiovascular granted by South Birmingham Local Research Ethics Committee (LREC ␥ 5643). Informed consent to participate was initially obtained, followed by disease (20, 27). Although IFN- may be important in eliciting the the completion of a medical questionnaire. APR, and its levels have been shown to be higher in tissues, serum, and gingival crevicular fluid from periodontitis patients (23), the Collection of venous blood and preparation of plasma and http://www.jimmunol.org/ type I IFNs have received little attention. Although IFN-␣ and -␤ neutrophils are generally associated with autoimmune diseases and host re- sponses to viral infection (28), both can also modulate neutrophil Venous blood was collected from patients and corresponding age/gender matched controls (simultaneously) from the ante-cubital fossa into Vacu- behavior. Notably, type I IFN can prolong neutrophil life span by tainer lithium heparin (17 IU/ml) tubes and following an overnight fast inhibiting apoptosis via PI3K, protein kinase C-␦, NF-␬B, and where subjects were also asked to refrain from drinking (except water) or STAT3 signaling pathways (29, 30). IFN-␣ is also able to enhance chewing gum. Platelet-depleted plasma was prepared (1000 ϫ g, 30 min, the respiratory burst in the presence of other stimuli, such as 4°C) and subsequently stored in liquid nitrogen. Neutrophils were isolated from venous blood (lithium heparin) as pre- fMLP, leukotriene B4, or influenza A virus (31, 32). viously described (9, 10) using a discontinuous Percoll gradient (␦ ϭ

Recently, high-throughput approaches compar- 1.079:1.098) followed by erythrocyte lysis (0.83% NH4Cl containing 1% by guest on September 23, 2021 ing peripheral blood cells from control subjects and patients have KHCO3, 0.04% Na2 EDTA.2H2O, and 0.25% BSA). Isolated cells were contributed to improved diagnostic procedures and molecular un- resuspended in PBS supplemented with glucose (1 mM) and cations (1 mM MgCl , 1.5 mM CaCl )at1ϫ 106 cells/ml. Cell viability, typically Ͼ98%, derstanding for a range of chronic inflammatory diseases, such as 2 2 was determined using dye exclusion (trypan blue), and the purity of the systemic erythematosus and ulcerative colitis (33, 34). The neutrophils was in excess of 95% using this methodology (37). aim of this study, therefore, was to use microarray technology to analyze the gene expression signature of hyperresponsive PBN ECL for assay of total ROS production from periodontitis patients to identify factors potentially important All assays were performed as previously described (9, 10). In brief, neu- for disease pathogenesis. trophils (1 ϫ 105) were placed in preblocked (PBS with 1% BSA, over- night at 4°C) white microwells (Microlite 2; Dynex) with supplemented Materials and Methods PBS or supplemented PBS containing 25 IU of IFN-␣,-␤,-␥ (R&D Sys- ␮ ␮ Study populations tems), or 0.1 g of LPS (Sigma-Aldrich; serotype 026:B6; 35 l), luminol (3 mmol/L; 30 ␮l) for detection of total ROS production, or isoluminol (3 Subjects with chronic periodontitis (n ϭ 19, 5 males and 14 females; mean mmol/L; 60 ␮l)with6UofHRPfordetection of extracellular ROS re- age ϭ 47.2 Ϯ 6.1 years, range ϭ 36–61 years) were recruited from pa- lease. Following equilibration in the microplate reader (37°C, Bertold mi- tients referred to the periodontal department of Birmingham’s Dental Hos- croplate-luminometer; LB96v) for 30 min, cells were stimulated with op- pital (Birmingham, U.K.). The diagnostic criteria for chronic periodontitis sonized Staphylococcus aureus (NCTC 6571; 300 bacteria/neutrophil; 25 were as previously reported (35). Age- and gender-matched periodontally ␮l) or PBS as control. All analyses were performed in triplicate. Light healthy control subjects (n ϭ 19, 5 males and 14 females; mean age ϭ emission in relative light units (RLU) was recorded during the priming 46.4 Ϯ 5.4 years, range ϭ 37–62 years) were recruited from staff of the phase (30 min) to study unprimed radical release and after stimulation (150 Dental Hospital. All subjects were systemically healthy and exclusion cri- min) with the peak signal determined. Experiments using IFN-␣ as a prim- teria included a course of nonsteroidal anti-inflammatory drugs or antimi- ing agent were repeated using lower amounts (0.025, 0.25, and 2.5 IU) to crobial drugs within a 3-mo period before enrollment, pregnancy, use of give more physiologically relevant priming concentrations of 0.14, 1.4, 14 mouthwashes or vitamin supplements within the previous 3 mo. Venous IU/ml and 0.12, 1.2, 12 IU/ml for luminol and isoluminol reactions, blood (see below) was collected from patients and controls immediately respectively. following initial patient presentation and diagnosis. Samples were also col- lected 3 mo following completion of successful periodontal treatment Nitrite assay where active disease was no longer apparent, and before relapse of indi- vidual sites during periodontal maintenance (postreview; Ref. 36). All vol- Neutrophils (2 ϫ 107 in 1150 ␮l of PBS supplemented with glucose) were unteers were never smokers with no history of recreational drug use and no primed with 50 ␮l IFN␣ (5000 IU, equivalent to 25 IU/1 ϫ 105 cells) or special dietary requirements. This population has been investigated in both PBS and incubated at 37°C for 30 min. Subsequently, neutrophils were cross-sectional and longitudinal studies of the effects of nonsurgical peri- stimulated with 800 ␮l of fMLP (1 mM, equivalent to 4 nM/1 ϫ 105 cells) odontal therapy upon PBN ROS hyperresponsiveness (9, 10). orPBSfor2hat37°C. Following stimulation, cells were centrifuged (2 A further 10 periodontally and systemically healthy subjects (five males min, 100 ϫ g), the supernatants removed and assayed in triplicate for and five females; mean age ϭ 37.8 Ϯ 11.4 years, range ϭ 22–60 years) nitrite, the stable end product of NO metabolism, by the Greiss reaction as were recruited from staff of the Dental Hospital as a source of PBN to described previously (38). The Journal of Immunology 5777

Table II. Genes more abundantly (2-fold or greater; n ϭ 149) expressed in periodontitis as compared with healthy neutrophils and determined using GeneSpring softwarea

Affymetrix Identifier Fold Change p Value Gene Gene Symbol IFN

204439_at 29.56 0.0104 IFN-induced protein 44-like IFI44-like ␣, ␤, ␥ 222068_s_at 13.48 0.0099 Similar to RIKEN cDNA 4930457P18 LOC123872 200844_s_at 10.36 0.0382 Peroxiredoxin 6 PRDX6 213797_at 8.745 0.0187 Viperin cig5 ␣, ␤ 201682_at 7.618 0.0073 Peptidase (mitochondrial processing) ␤ PMPCB 208622_s_at 7.247 0.0117 Villin 2 (ezrin) VIL2 205483_s_at 6.783 0.0454 IFN, ␣-inducible protein (clone IFI-15K) G1P2 ␣ 203153_at 6.614 0.0125 IFN-induced protein with tetratricopeptide repeats 1 IFIT1 ␣, ␤ 202145_at 5.236 0.0352 Lymphocyte Ag 6 complex, E LY6E 209104_s_at 4.781 0.0158 Nucleolar protein family A, member 2 (H/ACA small NOLA2 nucleolar ribonucleoproteins) 214453_s_at 4.63 0.0415 IFN-induced protein 44 IFI44 ␣, ␤ 202086_at 4.275 0.0385 Myxovirus (influenza virus) resistance 1, MX1 ␣, ␤ IFN-inducible protein p78 (mouse) 217502_at 4.131 0.0258 IFN-induced protein with tetratricopeptide repeats 2 IFIT2 ␣, ␤ 214329_x_at 4.118 0.00942 xs95h07.x1 NCI_CGAP_Ut4 Homo sapiens cDNA TNFSF10 ␣␤␥ clone IMAGE:2777437 3Ј similar to contains Alu repetitive element; contains element HGR Downloaded from repetitive ele 204502_at 4.006 0.0139 SAM domain and HD domain 1 SAMHD1 ␣, ␤, ␥ 203596_s_at 3.992 0.0339 IFN-induced protein with tetratricopeptide repeats 5 IFIT5 ␣ 217933_s_at 3.966 0.0151 Leucine aminopeptidase 3 LAP3 ␥ 200036_s_at 3.949 0.0299 Ribosomal protein L10a RPL10A 202340_x_at 3.882 0.0127 Nuclear receptor subfamily 4, group A, member 1 NR4A1

219691_at 3.709 0.0283 Sterile ␣ motif domain containing 9 SAMD9 http://www.jimmunol.org/ 219863_at 3.679 0.0189 Cyclin-E binding protein 1 CEB1 ␣, ␤ 206133_at 3.634 0.0134 XIAP associated factor-1 BIRC4BP ␣␤ 206881_s_at 3.574 0.0199 Leukocyte Ig-like receptor, subfamily A (without TM LILRA3 domain), member 3 204747_at 3.556 0.0241 IFN-induced protein with tetratricopeptide repeats 4 IFIT4 ␣, ␤ 208856_x_at 3.393 0.0205 Ribosomal protein, large, P0 RPLP0 207375_s_at 3.323 0.0331 IL-5 receptor, ␣ IL15RA ␣, ␤ 211972_x_at 3.299 0.00504 Ribosomal protein, large, P0 RPLP0 218978_s_at 3.262 0.0423 Solute carrier family 25, member 37 SLC25A37 213377_x_at 3.252 0.0392 Ribosomal protein S12 RPS12 200823_x_at 3.206 0.0358 Ribosomal protein L29 RPL29 by guest on September 23, 2021 201049_s_at 3.2 0.00548 Ribosomal protein S18 RPS18 200697_at 3.149 0.0404 Hexokinase 1 HK1 208623_s_at 3.147 0.0251 Villin 2 (ezrin) VIL2 218136_s_at 3.138 0.0226 Solute carrier family 25, member 37 SLC25A37 202446_s_at 3.12 0.0293 Phospholipid scramblase 1 PLSCR1 ␣␤ 203063_at 3.093 0.0222 Protein 1F (PP2C domain containing) PPM1F 213137_s_at 2.984 0.05 Protein tyrosine phosphatase, nonreceptor type 2 PTPN2 201566_x_at 2.972 0.0407 Inhibitor of DNA binding 2, dominant negative ID2 helix-loop-helix protein 213293_s_at 2.971 0.0246 Tripartite motif-containing 22 TRIM22 ␣, ␤ 212952_at 2.962 0.0282 Calreticulin CALR 202687_s_at 2.931 0.000782 TNF (ligand) superfamily, member 10 TNFSF10 ␣, ␤ 211720_x_at 2.927 0.0119 Ribosomal protein, large, P0 RPLP0 200002_at 2.899 0.0348 Ribosomal protein L35 RPL35 208116_s_at 2.894 0.0429 Mannosidase, ␣, class 1A, member 1 MAN1A1 209369_at 2.858 0.049 Annexin A3 ANXA3 204221_x_at 2.853 0.0276 GLI pathogenesis-related 1 (glioma) GLIPR1 212734_x_at 2.847 0.0242 Ribosomal protein L13 RPL13 211623_s_at 2.841 0.0393 FBL 205040_at 2.822 0.0102 1 ORM1 200937_s_at 2.743 0.000951 Ribosomal protein L5 RPL5 205965_at 2.742 0.0286 Basic leucine zipper transcription factor, activating BATF ␣, ␤, ␥ transcription factor-like 215783_s_at 2.721 0.0131 , liver/bone/kidney ALPL 212469_at 2.708 0.0108 Nipped-B homolog (Drosophila) NIPBL 222326_at 2.706 0.0154 4B, cAMP-specific PDE4B ␥ ( phosphodiesterase E4 dunce homolog Drosophila) 212039_x_at 2.683 0.0447 Ribosomal protein L3 RPL3 200093_s_at 2.67 0.0489 Histidine triad nucleotide binding protein 1 HINT1 34689_at 2.661 0.0138 Three prime repair 1 TREX1 212191_x_at 2.655 0.0219 Ribosomal protein L13 RPL13 201033_x_at 2.637 0.0114 Ribosomal protein, large, P0 RPLP0 201560_at 2.595 0.0171 Chloride intracellular channel 4 CLIC4 44790_s_at 2.581 0.0433 13 open reading frame 18 C13orf18 (Table continues) 5778 ELEVATED IFN-␣ LEVELS IN PERIODONTITIS

Table II. (Continued)

Affymetrix Identifier Fold Change p Value Gene Gene Symbol IFN

209723_at 2.574 0.0197 Serine (or cysteine) proteinase inhibitor, clade B SERPINB9 (ovalbumin), member 9 213036_x_at 2.564 0.0469 Homo sapiens SERCA3 gene, exons 1–7 (and joined ATP2A3; CDS) SERCA3 201217_x_at 2.537 0.0296 Ribosomal protein L3 RPL3 200949_x_at 2.532 0.0334 Ribosomal protein S20 RPS20 201030_x_at 2.496 0.0191 Lactate dehydrogenase B LDHB 205668_at 2.486 0.0117 Lymphocyte antigen 75 LY75 209670_at 2.485 0.0174 Human mRNA for T-cell receptor ␣-chain (TCR-␣) TRAC 200624_s_at 2.471 0.0442 Matrin 3 MATR3 221775_x_at 2.464 0.037 nah24c06.y1 NCI_CGAP_HN21 Homo sapiens RPL22 cDNA clone IMAGE:4232050 5Ј similar to SW: RL22_HUMAN P35268 60S RIBOSOMAL PROTEIN L22 203501_at 2.45 0.0161 Aminopeptidase; go_component: extracellular space PGCP ͓goid 0005615͔͓evidence E͔͓pmid 10206990͔; go_component: cytoplasm ͓goid 0005737͔ ͓evidence͔ 212065_s_at 2.442 0.0405 Ubiquitin specific protease 34 USP34 Downloaded from 204236_at 2.423 0.00912 Friend leukemia virus integration 1 FLI1 ␥ 200834_s_at 2.404 0.0134 Ribosomal protein S21 RPS21 213294_at 2.4 0.00421 Hypothetical protein FLJ38348 FLJ38348 213347_x_at 2.389 0.0499 Ribosomal protein S4, X-linked RPS4X 221475_s_at 2.372 0.0224 Ribosomal protein L15 RPL15 200088_x_at 2.354 0.0406 Homo sapiens cDNA: FLJ22838 fis, clone RPL12

KAIA4494, highly similar to HUML12A Human http://www.jimmunol.org/ ribosomal protein L12 mRNA 200081_s_at 2.351 0.0462 Ribosomal protein S6 RPS6 218157_x_at 2.329 0.0497 Small protein effector 1 of Cdc42 SPEC1 210873_x_at 2.325 0.00135 Apolipoprotein B mRNA editing , catalytic APOBEC3A polypeptide-like 3A 201435_s_at 2.317 0.0259 Eukaryotic translation initiation factor 4E EIF4E 200889_s_at 2.304 0.0493 Signal sequence receptor, ␣ (translocon-associated SSR1 protein ␣) 213080_x_at 2.3 0.00321 Ribosomal protein L5 RPL5 213193_x_at 2.287 0.0474 TCR ␤-chain BV20S1 BJ1–5 BC1 mRNA, TCRB ␥ complete cds by guest on September 23, 2021 201559_s_at 2.281 0.0211 Chloride intracellular channel 4 CLIC4 200089_s_at 2.278 0.0181 Ribosomal protein L4 RPL4 200869_at 2.271 0.0232 Ribosomal protein L18a RPL18A 201565_s_at 2.271 0.0246 Inhibitor of DNA binding 2, dominant negative ID2 helix-loop-helix protein 217719_at 2.247 0.046 Eukaryotic translation initiation factor 3, subunit 6 EIF3S6IP interacting protein 200029_at 2.243 0.0282 Ribosomal protein L19 RPL19 220960_x_at 2.235 0.00318 Ribosomal protein L22 RPL22 209476_at 2.233 0.02 Thioredoxin domain containing TXNDC 201385_at 2.218 0.0448 DEAH (Asp-Glu-Ala-His) box polypeptide 15 DHX15 204415_at 2.208 0.0456 IFN, ␣-inducible protein (clone IFI-6–16) G1P3 ␣ 213980_s_at 2.204 0.0451 C-terminal binding protein 1 CTBP1 200028_s_at 2.199 0.0414 START domain containing 7 STARD7 206099_at 2.196 0.00477 Protein kinase C, eta PRKCH 209417_s_at 2.193 0.0129 IFN-induced protein 35 IFI35 ␣, ␥ 38157_at 2.189 0.00538 Dom-3 homolog Z (Caenorhabditis elegans) DOM3Z 200626_s_at 2.186 0.0431 Matrin 3 MATR3 203739_at 2.182 0.000624 Zinc finger protein 217 ZNF217 221816_s_at 2.179 0.0259 PHD finger protein 11 PHF11 213687_s_at 2.161 0.0476 Ribosomal protein L35a RPL35A 201094_at 2.16 0.00556 Ribosomal protein S29 RPS29 212461_at 2.158 0.0382 Ornithine decarboxylase antizyme inhibitor OAZIN 202864_s_at 2.151 0.00952 Nuclear antigen Sp100 SP100 ␣, ␤ 212716_s_at 2.151 0.0171 Eukaryotic translation initiation factor 3 subunit k eIF3k 212240_s_at 2.145 0.0144 Phosphoinositide-3-kinase, regulatory subunit, PIK3R1 polypeptide 1 (p85 ␣) 202731_at 2.139 0.0397 Programmed cell death 4 (neoplastic transformation PDCD4 inhibitor) 201665_x_at 2.13 0.0144 Ribosomal protein S17 RPS17 213084_x_at 2.121 0.0465 Ribosomal protein L23a RPL23A 203595_s_at 2.119 0.00977 IFN-induced protein with tetratricopeptide repeats 5 IFIT5 ␣ 217887_s_at 2.119 0.0463 Epidermal growth factor receptor pathway EPS15 substrate 15 222150_s_at 2.119 0.0379 Hypothetical protein LOC54103 LOC54103 (Table continues) The Journal of Immunology 5779

Table II. (Continued)

Affymetrix Identifier Fold Change p Value Gene Gene Symbol IFN

201155_s_at 2.109 0.0139 Mitofusin 2 MFN2 200038_s_at 2.105 0.0273 Ribosomal protein L17 RPL17 208965_s_at 2.102 0.0416 IFN, ␥-inducible protein 16 IFI16 ␥ 208834_x_at 2.102 0.0295 Ribosomal protein L23A RPL23A 214787_at 2.102 0.0177 c-myc promoter-binding protein IRLB 200016_x_at 2.099 0.00723 Heterogeneous nuclear ribonucleoprotein A1 HNRPA1 200017_at 2.095 0.0243 Ribosomal protein S27a RPS27A 202863_at 2.091 0.0425 Nuclear Ag Sp100 SP100 ␣, ␤ 210972_x_at 2.09 0.00338 TCR ␣ locus TCRA ␥ 211339_s_at 2.084 0.014 IL2-inducible T-cell kinase ITK 221920_s_at 2.082 0.0111 Solute carrier family 25, member 37 SLC25A37 200716_x_at 2.081 0.0333 Ribosomal protein L13a RPL13A 217650_x_at 2.077 0.0287 oz96b09.x1 Soares_parathyroid_tumor_NbHPA Homo SIAT4B sapiens cDNA clone IMAGE:1683161 3Ј similar to contains Alu repetitive element;, mRNA sequen 213969_x_at 2.072 0.0445 Transcribed sequence with strong similarity to RPL29 protein sp:P47914 (Homo sapiens) RL29_HUMAN 60S ribosomal protein L29 208549_x_at 2.07 0.0344 Prothymosin a14 LOC51685 Downloaded from 217414_x_at 2.064 0.0293 Human ␣-globin gene with flanks. HBA2 206036_s_at 2.056 0.0285 v-Rel reticuloendotheliosis viral oncogene REL homolog (avian) 208047_s_at 2.053 0.034 NGFI-A binding protein 1 (EGR1 binding protein 1) NAB1 221478_at 2.048 0.0385 BCL2/adenovirus E1B 19kDa interacting protein BNIP3L 3-like

212185_x_at 2.048 0.0183 Metallothionein 2A MT2A ␣ http://www.jimmunol.org/ 201184_s_at 2.048 0.0184 Chromodomain helicase DNA binding protein 4 CHD4 212773_s_at 2.044 0.00607 Translocase of outer mitochondrial membrane 20 TOMM20 homolog (yeast) 212297_at 2.043 0.0115 ATPase family homolog up-regulated in senescence AFURS1 cells 208934_s_at 2.026 0.0146 Lectin, galactoside-binding, soluble, 8 (galectin 8) LGALS8 217826_s_at 2.026 0.000397 Ubiquitin-conjugating enzyme E2, J1 (UBC6 UBE2J1 homolog, yeast) 209112_at 2.025 0.00572 Cyclin-dependent kinase inhibitor 1B (p27, Kip1) CDKN1B 215719_x_at 2.019 0.0465 Fas (TNFR superfamily, member 6) FAS ␣, ␥ 201258_at 2.018 0.0451 Ribosomal protein S16 RPS16 by guest on September 23, 2021 200639_s_at 2.012 0.0347 Tyrosine 3-monooxygenase/tryptophan YWHAZ 5-monooxygenase activation protein, ␨ polypeptide 203435_s_at 2.012 0.0323 Membrane metallo-endopeptidase (neutral MME endopeptidase, enkephalinase, CALLA, CD10) 201924_at 2.01 0.0127 AF4/FMR2 family, member 1 AFF1 200796_s_at 2.007 0.0311 Myeloid cell leukemia sequence 1 (BCL2-related) MCL1 ␣ 201254_x_at 2.004 0.0143 Ribosomal protein S6 RPS6 218383_at 2.002 0.00751 Chromosome 14 open reading frame 94 C14orf94

a Genes set in boldface indicate transcripts representing ribosomal proteins, and genes set in italics indicate ISG. IFN regulation for ISG as determined from de Veer et al. (Ref. 42) and www.lerner.ccf.org/labs/williams/oligo.cgi is shown in final column.

IFN treatment of neutrophils for RNA extraction 27,061 RLU), and these data have been published elsewhere (9, 10). RNA samples were analyzed using human Affymetrix HG_U133A oli- ϫ 6 Neutrophils (1 10 cells; 1 ml) were added to supplemented PBS (500 gonucleotide arrays, as described at www.affymetrix.com/products/ar- ␮ ␣ ␤ ␥ ␮ l) containing either IFN- ,- ,- (250 IU), or LPS (1 g) in Eppendorf rays/specific/hgu133.affx. Total RNA from each sample was used to pre- tubes, which were then incubated uncapped at 37°C for 3 h. Following ϫ pare biotinylated target RNA, according to the manufacturer’s instructions stimulation, cells were centrifuged (2 min, 100 g), the supernatant re- (www.affymetrix.com/support/technical/manual/expression_manual.affx). moved, and the cell pellet resuspended in 1 ml of TRIzol (Sigma-Aldrich). In brief, DNase digested total RNA (5 ␮g) was used to generate double- After phenol/chloroform extraction (Sigma-Aldrich), the aqueous phase stranded cDNA using SuperScript reagents (Life Technologies) and a T7- was combined with 70% ethanol and added to an RNeasy minicolumn linked oligo(dT) primer. cRNAs were synthesized using the ENZO Bio- (Qiagen). Subsequent purification and DNase treatment were performed as ␮ Array High Yield RNA Transcript Labeling Kit (Affymetrix), and resulting recommended by the manufacturer (Qiagen). RNA was eluted in 30 lof biotinylated labeled cRNA was subsequently fragmented into 35- to sterile water, and concentrations were determined from absorbance values 200-bp lengths using fragmentation buffer (Affymetrix). As recommended at 260 nm using a BioPhotometer (Eppendorf). RNA integrity was verified by the manufacturers, RNA, cDNA, and cRNA quality and size distribution by visual inspection of samples on 1% nondenaturing agarose gels stained were visually confirmed by agarose gel electrophoresis. Spike controls B2, with SYBR Gold (Molecular Probes). bio-B, bio-C, bio-D, and Cre-x were added to the hybridization mixture Microarray target preparation, hybridization, and analysis before overnight hybridization at 45°C for 16 h. Arrays were stained and washed on the Fluidics Station 400 (Affymetrix) using the EukGE-WS2 Total patient and control RNA were obtained by pooling RNA from four protocol (dual staining) before being scanned twice on the GeneChip Scan- pairs of PBN samples, obtained from patients and controls in the study ner 3000 at an excitation wavelength of 488 nm. The integrity and quality population. Patient and control pairs were previously demonstrated to ex- of prepared samples was confirmed using Affymetrix Test3 GeneChips. hibit a high hyperresponsive differential (both hyperreactivity to Fc␥R- HG_U133A microarrays (Affymetrix) were subsequently hybridized with stimulation and baseline unstimulated hyperactivity) as determined by ECL the cRNA samples. Analysis of control parameter data (as described for (mean differential periodontitis vs health ϭ 13,897 RLU, range ϭ 8,300– Test3 GeneChip analysis) confirmed hybridization success (data not 5780 ELEVATED IFN-␣ LEVELS IN PERIODONTITIS Downloaded from

FIGURE 1. Relative levels of MX1, IFIT4, G1P2, IFIT1, CIG5, and IFI44-like gene expression (shown as ratio to GAPDH levels). A, Periodontitis patients pre-/postreview and healthy controls (n ϭ 4, individual pooled samples). B, Individual patient-control pair IFIT4 expression analysis (n ϭ 9). C, Mean (ϮSD) gene expression for periodontitis patients pre-/post-review and controls (n ϭ 9). http://www.jimmunol.org/ shown) and scaling factors were well within Affymetrix recommended nonsurgical therapy (n ϭ 12 patient-control pairs). Plasma samples were guidelines. diluted 1/2 in sample diluent and levels measured in duplicate using a high sensitivity Biotrak ELISA (Amersham Biosciences) according to the man- Microarray data analysis ufacturer’s instructions. This assay has a detection limit of 0.01 pg/well and For analysis of hybridization results, which all met minimum requirements for a range of 0.63–20.0 pg/ml. data quality and distribution, raw data files were exported from Affymetrix Data handling and statistical analysis microarray suite 5.0 software (MAS5.0; Affymetrix) into GeneSpring 5.1 soft- ware (Silicon Genetics). Values were normalized to the median signal values Chemiluminescent data were recorded automatically and transferred to Mi- for each array. Based on previous experiences, Affymetrix and GeneSpring crosoft Excel spreadsheets. Manipulation of data was performed in Excel by guest on September 23, 2021 recommendations, and published literature (39, 40), genes with at least a 2-fold and statistical evaluation performed using Instat 3.2 (GraphPad). Between change in expression level and with a signal intensity value of Ͼ100 on either group plasma IFN-␣ differences were assessed using Mann-Whitney U the control or test arrays were classified as being differentially expressed. The tests. Within group (periodontitis pre- vs posttherapy) differences in plasma remaining genes were considered informative and were subjected to t test IFN-␣ and primed neutrophil chemiluminescence, data were analyzed by using a global error model with the variance statistic derived from replicates. Wilcoxon test. A level of p Ͻ 0.05 was used to assign statistical Finally, to reduce false-differential gene expression a Bonferroni multiple test significance. correction filter was applied. Microarray analyses were Minimum Information About a Microarray Experiment compliant, and raw data files are available in Results the Gene Expression Omnibus database under the series number GSE12484 Gene expression analysis in patient and control neutrophils (www.ncbi.nlm.nih.gov/geo/). Initial assessment of microarray data revealed that whereas gran- cDNA synthesis and semi-quantitative RT-PCR analysis ulocyte CSFR, CD45, and liver/bone/kidney alkaline phosphatase RT-PCR was performed using pooled RNA from individual pre-/post-re- transcripts were detected at high signal levels, macrophage CSFR ϭ view patient and control RNA samples (n 9) as well as pooled healthy transcript was absent (data not shown), confirming that there was control samples primed with IFN and Escherichia coli LPS. For cDNA synthesis, 1–5 ␮g of total RNA was used for oligo(dT) reverse transcrip- no detectable monocytic contamination of the original neutrophil tion to generate single stranded cDNA (Omniscript kit; Qiagen). cDNA RNA samples. Pairwise analysis of hybridization data indicated concentrations were determined using a BioPhotometer (Eppendorf). that of the 5680 genes detected as being present in both targets, Primer sequences and cycling conditions for the genes analyzed are pro- 163 genes (2.87% of detected genes) were 2-fold or greater dif- vided in Table I. The housekeeping gene GAPDH was used as a normal- ferentially expressed between healthy and periodontitis patient ization control. Primers were designed from the Affymetrix probe target identifier sequences using the Primer3 program (frodo.wi.mit.edu/cgi-bin/ samples. Of this dataset, 14 were more highly expressed in neu- primer3/primer3_www.cgi). Typically cDNA (50 ng) was used to seed 50 trophils from healthy patients than those with periodontitis (age- ␮l of REDTaq PCR mixes (Sigma-Aldrich) and subjected to between 25 and gender-matched), and 149 were up-regulated in periodontitis and 40 cycles. Amplification cycles of 95°C for 20 s, 61° for 20 s, and 72° patient neutrophils relative to healthy patients. The majority of the for 20 s were performed using a Mastercycler thermal cycler (Eppendorf). Following the designated number of cycles 7 ␮l of the reaction product was differentially expressed genes (96.8%) were 2-to 10-fold differen- removed and PCR products separated and visualized on a 1.5% agarose gel tially expressed with 3.2% exhibiting Ͼ10-fold differential expres- containing ethidium bromide (0.5 ␮g/ml). Scanned gel images were im- sion. Whereas genes more abundant in patient neutrophils (n ϭ ported into AIDA image analysis software (Fuji) and the volume density of 149; Table II) mapped to a range of biological processes and mo- amplified products calculated and normalized against GAPDH control lecular functions, genes representing ribosomal function and trans- values. lational mechanisms were highly represented (n ϭ 35; 23%, data IFN-␣ ELISA not shown), as were those representing ISG (n ϭ 33; 22.2%). IFN- ␣ levels were determined in all the available liquid nitrogen-stored Notably, of the differentially expressed ISG, 11 of the 33 corre- plasma samples from the study population before and 3 mo after successful sponded to genes reported to have increased expression due to The Journal of Immunology 5781 Downloaded from

FIGURE 2. Mean (ϮSD) luminol-dependent, total (A), and isoluminol-dependent extracellular (B) CL generated by peripheral neutrophils stimulated .(p Ͻ 0.05 compared with no primer control ,ء ;with opsonized S. aureus with and without priming with 25 IU of IFN or 0.1 ␮gofE. coli LPS (n ϭ 5 C, MX1, IFIT4, G1P2, IFIT1, CIG5, and IFI44-like gene expression in neutrophils treated with IFN-␣,-␤,-␥, and E. coli LPS. http://www.jimmunol.org/

IFN-␥ exposure, whereas 25 corresponded to genes reported to IFN-␣ plasma levels in periodontitis patients and orally healthy increase due to type I IFN exposure (41, 42). controls To further investigate the gene expression data obtained by mi- Due to the observed ability of type I IFN to prime neutrophils for croarray analysis in a larger panel of patient and controls pairs the respiratory burst and to consistently increase expression of all ϭ (n 9), six differentially expressed genes, representing ISG from the investigated ISG identified as up-regulated in the periodontitis a range of ontological groups and fold changes, were selected for neutrophil dataset, IFN-␣ levels in pre- and post-therapy patient

RT-PCR analysis (Table I). The gene expression data confirmed (n ϭ 12) and control (n ϭ 12) plasma samples (platelet-depleted by guest on September 23, 2021 that transcript levels for all six genes were increased in a pooled plasma) were investigated. Plasma IFN-␣ levels in periodontitis ϭ ϭ RNA sample (from n 4; Fig. 1A) and also in individual (n 9; subjects were significantly higher pre-nonsurgical therapy (0.97 Ϯ Fig. 1C) periodontitis samples compared with controls, supporting 0.31 pg/ml) than those of periodontally healthy controls (0.47 Ϯ the data obtained by microarray analysis. These latter results also 0.22 pg/ml, p ϭ 0.0045). After therapy, plasma IFN-␣ concentra- demonstrated the heterogeneity of gene expression levels in peri- tions (0.53-Ϯ0.31pg/ml) reduced to levels not significantly differ- odontitis and healthy individuals (Fig. 1B). After therapy, gene ent from matched healthy controls ( p ϭ 0.603; Fig. 3). Although expression levels within the periodontitis patient neutrophils our initial studies (Fig. 2) were performed using 25 IU (equivalent decreased, approaching control values, in both the pooled (n ϭ 4; to IFN-␣ priming concentrations of 143 and 122 IU/ml for the Fig. 1A) and individual (n ϭ 9; Fig. 1C) samples. luminol and isoluminol assays, respectively), we subsequently wanted to determine whether levels found within the peripheral circulation in patients of ϳ1 pg/ml (approximately equivalent to Analysis of neutrophil responses to IFN 0.37 IU/ml) were able to prime PBN with regard to the oxidative Due to the relatively high proportion of ISG represented in the burst. Data indicated (Fig. 4) that IFN-␣ concentrations as low as PBN from periodontitis patients (33 of 149 genes; Table II), the 0.14 (luminol) and 0.12 IU/ml (isoluminol) were able to prime effect of IFN-␣,-␤,-␥, and LPS on ROS and gene expression was neutrophils for both intra- and extracellular ROS production at analyzed in neutrophils from periodontally and systemically levels statistically significantly higher than controls ( p ϭ 0.03). healthy individuals (n ϭ 5). LPS stimulation was included in these analyses as a positive control as it has previously been reported to elicit an ISG expression response (42). Enhancement of total and extracellular Fc␥R-stimulated chemiluminescence (CL) by neutro- phils was induced by priming with IFN-␣ ( p ϭ 0.031 total CL; p ϭ 0.031 extracellular CL), IFN-␤ ( p ϭ 0.031; p ϭ 0.063), and IFN-␥ ( p ϭ 0.031; p ϭ 0.031). By comparison, E. coli LPS caused a small, insignificant increase in mean total and extracellular CL ( p ϭ 0.063, p ϭ 0.094; Fig. 2, A and B). Gene expression analysis showed that IFN-␣ and IFN-␤ priming increased expression of all six ISGs by between 49 and 209%. Priming with IFN-␥ or LPS increased expression of five ISGs by between 9 and 101%. IFI44- FIGURE 3. Mean (ϮSD) plasma IFN-␣ levels for periodontitis patients like and Mx1 gene expression appeared unaffected by IFN-␥ and pre-/post-therapy and periodontally healthy matched controls (n ϭ 12). p .indicates statistical significance ء ,LPS priming, respectively (Fig. 2C). values are shown 5782 ELEVATED IFN-␣ LEVELS IN PERIODONTITIS

neutrophil dataset. Analysis of the literature indicated that the most likely candidates for stimulating this profile were type-I IFN, data supported by our PCR analyses (Figs. 1 and 2C). Indeed, the IFI44-like transcript (whose function remains unknown), which ex- hibited the highest change in expression level, was up-regulated only by type I IFN and not by IFN-␥ under the conditions tested (Fig. 2C). Consistent with these data were our results demonstrating the ability of IFN to enhance the oxidative burst. Although IFN-␥ has previously been shown to “prime” for neutrophil ROS generation in a stimulus- dependant manner (46), we now demonstrate that type I IFN can perform a similar function. Taken together, these data indicate that increased peripheral blood type I IFN levels in periodontitis patients have the potential to serve as a priming factor and could contribute to the reported neutrophil hyperresponsivity with respect to Fc␥R-me- diated ROS generation (7–10). Notably, it has been shown that neutrophil maturation is accompa- nied by heightened expression of genes that increase their response to type I and type II IFN, which act as priming agents on mature neu-

trophils enabling the formation of extracellular traps upon further ap- Downloaded from FIGURE 4. Mean (ϮSD) luminol-dependent, total (A), and isoluminol- propriate stimulation (47). Thus, the heightened expression of ISG by dependent extracellular (B) CL generated by peripheral neutrophils stimulated neutrophils in periodontitis patients may indicate that they are primed with opsonized S. aureus with and without priming with increasing concen- and hyperresponsive in terms of neutrophil extracellular trap produc- .(p Ͻ 0.05 compared with no primer control ,ء ;trations of IFN-␣ (n ϭ 5 tion, a possibility currently under investigation. It is known that following local infection, bacteria and/or their ␣ Experiments were also performed to determine whether IFN- components can enter the systemic circulation (48, 49). It is there- http://www.jimmunol.org/ could prime neutrophil NO production induced by fMLP, a known fore conceivable that this process may also directly or indirectly stimulator of NO (38). These results demonstrated that IFN-␣,at contribute to the peripheral activation of neutrophils and poten- concentrations that primed Fc␥R-stimulated ROS generation, was tially the patient gene expression signature observed in this study. unable to prime fMLP NO release (data not shown). Indeed, infectious bacteria (e.g., Chlamydia, S. aureus, E. coli) have previously been reported to increase IFN-␣ levels (50, 51). Discussion Comparison of our results with published data for neutrophils Global gene expression profiling of peripheral blood cells has al- stimulated with LPS indicates similarities in gene expression data- ready proven to be a valuable tool not only for identifying novel sets with respect to ISG (52). Notably, ISG induction by LPS was clinically useful biomarkers, but also for determining pathogenic shown to be independent of type I IFN or other unidentified sol- by guest on September 23, 2021 mechanisms in chronic inflammatory human diseases that provide uble autocrine factors released by the neutrophils (53). Our anal- new therapeutic targets (33, 34). To our knowledge the current yses, however, demonstrate that LPS does not best recapitulate the study is the first to apply this technology to study transcriptional ISG profile observed in vivo (Fig. 2C), and therefore does not patterns in unstimulated, unprimed PBN from chronic periodontitis represent the most likely source of neutrophil priming. It is also patients. By using microarrays not tailored for the disease we were unlikely that neutrophils are the source of elevated IFN levels po- able to elucidate transcriptional changes that were previously not part tentially causing autocrine activation. Indeed, it is known that plas- of current understanding. These studies have identified several func- macytoid dendritic cells are the more probable source of this mol- tional groups of genes up-regulated in periodontitis patients’ neutro- ecule as they express 1000-fold more IFN-␣/-␤ than other cell phils compared with healthy controls, providing a better understand- types. Notably, other bacterial products (e.g., DNA) can also en- ing of the molecular processes underpinning this highly prevalent hance plasmacytoid dendritic cells’ IFN expression (54). disease in which peripheral neutrophils generate increased levels of Another potential mechanism for priming of peripheral neutro- ROS in the presence and absence of stimulation (7–10). phils in periodontitis is that of bacterial DNA (bDNA). Recently, Previous studies comparing the transcriptional response of neu- GeneChip studies of peripheral blood monocytes identified that trophils to fMLP stimulation in health and periodontitis (aggres- bDNA CpG motifs induced ISG expression. Subsequent blockade sive and chronic disease) have demonstrated that patients’ cells of the IFN-␣/-␤ receptor on these cells strongly inhibited ISG in- showed a preferential up-regulation of transcripts representing ri- duction, indicating that this receptor was capable of recognizing bosomal protein-encoding genes. These transcriptional changes bDNA (55). In addition, bDNA directly affects neutrophil behavior may indicate an enhanced capacity for protein synthesis and/or by activating changes in cell shape and migration and inhibiting other functions these molecules mediate in periodontitis (43). In- apoptosis (56, 57). Animal experiments have also shown that mice terestingly, members of this same group of genes were also up- pretreated with bDNA exhibit enhanced neutrophil influx at sites regulated in neutrophils primed with GM-CSF (12), a growth fac- of infection. The recruited neutrophils were phenotypically hyper- tor known to be associated with neutrophil-mediated pathology active, exhibited up-regulation of phagocytic receptors and activ- (44, 45) and recently implicated in production of heightened base- ity, and elevated ROS production (58). Although our data strongly line levels of extracellular ROS by neutrophils in chronic peri- implicates type I IFN in priming PBN, the systemic involvement of odontitis (9). It is therefore interesting to speculate that the rela- periodontal bacteria or their components in the chronic disease tively high proportion of ribosomal protein-encoding genes cycle cannot be excluded. represented in our periodontitis dataset indicate that patients’ PBN Although peripheral blood ISG transcript levels vary in appar- are already in a “primed” state. ently healthy individuals within the general population (59), our Our most significant finding, however, was the relatively high combined findings support the hypothesis that elevated plasma proportion of up-regulated ISGs within our periodontitis patient IFN-␣ levels and subsequent ISG expression are associated with The Journal of Immunology 5783 periodontal inflammation. The data clearly demonstrate that fol- this approach would enable a more comprehensive understanding lowing conventional periodontal treatment, plasma IFN-␣ levels of the local and peripheral molecular and cellular mechanisms un- and neutrophil ISG expression decrease in periodontitis patients to derpinning the pathogenesis of periodontitis. levels comparable with those of healthy controls, reflecting the reduc- In conclusion, the role of a dysregulated host immune response, tion in neutrophil ROS hyperreactivity after therapy previously re- in particular neutrophil hyperresponsiveness in the generation of ported in the same patients (10). Our functional studies (Figs. 2 and 3) tissue damage in periodontitis, has been recognized for some time, indicate that peripheral IFN-␣ could contribute to the previously re- however, the molecular mechanisms underlying this phenomenon ported hyperreactive neutrophil phenotype (9, 10). have yet to be elucidated. Our studies now indicate for the first Although it is possible that IFN-␣ levels are elevated in a similar time that PBN from patients exhibit a distinct molecular pheno- manner to that of other acute phase proteins following periodontal type, potentially indicating a “primed” state. The ability of type I infection, it is also conceivable that IFN-␣ levels were initially IFN to “prime” the oxidative burst of neutrophils combined with elevated due to other as yet unidentified factors, initiating peri- the elevated levels of IFN-␣ found in patients blood during disease odontal inflammation. The mechanism by which IFN-␣ levels be- indicate this molecule is potentially involved in pathogenesis in come raised within the peripheral circulation may therefore be key certain patient subsets. The implications this may have for peri- to understanding host susceptibility and pathogenesis of the dis- odontitis diagnosis and therapy remain to be elucidated, as the ease. Notably, of the genes analyzed in Fig. 1, and in general for cause of the IFN increase is yet to be determined. ISG identified by microarray analysis, the majority, whose func- tion is known, encode proteins involved in antiviral responses Acknowledgments and/or have been implicated in autoimmune disease. Indeed, MX1 We thank Drs. Iwona Bujalska and Tanja Stankovic for help with microar- Downloaded from has been shown to provide a key protective function against vi- ray experiments, Dr. Rachael Sammons for the provision of S. aureus, and ruses, in particular influenza, by interfering with its replication Dr. Melissa Grant and Mike Milward for manuscript review. cycle (60). Significantly, Cig5 has been shown to have antiviral Disclosures activity against several viruses including influenza, hepatitis C, The authors have no financial conflict of interest. and human CMV (61–63). G1P2 has also been demonstrated to enhance the innate antiviral response via regulation of IFN-stim- References http://www.jimmunol.org/ ulated intracellular signaling pathways (64). Notably, for other 1. Quint, J. K., and J. A. Wedzicha. 2007. The neutrophil in chronic obstructive genes including IFIT1, IFIT4, and IFI44, there is limited knowl- pulmonary disease. J. Allergy Clin. Immunol. 119: 1065–1071. edge regarding their biochemical function or biological effect for 2. D’Odorico, A., R. D’Inca, C. Mestriner, V. Di Leo, A. Ferronato, and G. C. Sturniolo. 2000. Influence of disease site and activity on peripheral neu- the host, although it has been noted that their expression is in- trophil function in inflammatory bowel disease. Dig. Dis. Sci. 45: 1594–1600. creased in the autoimmune disease systemic lupus erythematosus 3. Jarvis, J. N., H. R. Petty, Y. Tang, M. B. Frank, P. A. Tessier, I. Dozmorov, K. Jiang, A. Kindzelski, Y. Chen, C. Cadwell, et al. 2006. 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