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The Journal of Immunology, 2008, 181, 5775 -5784
Copyright © 2008 by The American Association of Immunologists, Inc.
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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

Periodontal Research Group, School of Dentistry, University of Birmingham, Birmingham, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 genes 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 further corroborated these data, and indicated that levels decreased to near those of controls following successful therapy. Significantly enhanced Fc{gamma}R-stimulated ROS production was subsequently achieved by priming control neutrophils with IFN-{alpha}/-β/-{gamma}, but not LPS, and gene expression analysis indicated that exposure to the type I IFN (in particular IFN-{alpha}) better replicated the mRNA profile observed in vivo. Further studies demonstrated that plasma levels of IFN-{alpha} were significantly higher in samples from patients relative to unaffected controls. Following successful periodontitis treatment, plasma IFN-{alpha} levels, neutrophil ISG expression, and Fc{gamma}R-stimulated neutrophil ROS output of patients, all decreased to levels comparable with those of controls. In conclusion, although chronic periodontitis is a complex disease, raised IFN-{alpha} may be one determinant of the distinct molecular phenotype and hyperresponsivity exhibited by patients’ peripheral blood neutrophils.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Peripheral blood neutrophils (PBN)3 from patients with a range of inflammatory disorders have been shown to have enhanced chemotactic and extracellular proteolytic activity as well as the ability to produce increased amounts of reactive oxygen species (ROS; Refs. 1, 2, 3). It has been proposed that the accumulation of these activated neutrophils at sites of tissue irritation, Ag localization, or bacterial infection subsequently leads to the tissue damage that underpins the clinical course of these diseases.

Periodontitis is one of the most prevalent human inflammatory diseases and is characterized by an aberrant and exaggerated neutrophilic response to microbial plaque present at the gingival margin indicating dysregulation of the hosts’ innate immune response (4, 5). The resultant collateral host tissue damage to the supporting periodontal tissues leads to progressive periodontitis and ultimately culminates in tooth loss (6). Several studies have demonstrated that PBN from chronic periodontitis patients are not only hyperreactive, in response to Fc{gamma}R stimulation by periodontal pathogens, but also hyperactive, with respect to baseline unstimulated ROS production (7, 8, 9, 10). Although a host molecular defect in intracellular lipid signaling may explain peripheral neutrophil ROS hyperreactivity in the relatively rare form of the disease, localized aggressive periodontitis (11), this mechanism does not explain the patient predisposition observed in chronic periodontitis. The underlying mechanism(s) responsible for the hyperinflammatory neutrophil phenotype seen in chronic periodontitis (the commonest form of the disease) is currently unknown, although analyses indicate that it is not a result of altered adhesion molecule (7) or Phox gene (10) expression, polymorphisms in Fc{gamma}R (8, 12), or in vitro priming by cytokines or LPS (13, 14). Furthermore, the association of periodontitis with increased relative risk for cardiovascular disease, fatal coronary events (15), and ischemic stroke (16), and the demonstrable medium-term reductions in vascular endothelial dysfunction following aggressive periodontal therapies (17), emphasize the potential impact of inflammatory periodontitis on peripheral macrovascular disease.

To identify factors predisposing to, or biomarkers of disease activity, peripheral blood levels of several acute phase response (APR) proteins in periodontitis patients have been analyzed. During the APR, following local release of factors such as TNF-{alpha}, IL-1β, and IL-6, systemic changes occur that include significant hepatic release of plasma proteins (e.g., C-reactive protein and serum amyloid A), activation of complement proteins, and several other metabolic events (18, 19). Thus far, whereas plasma C-reactive protein levels have generally been found to be higher in periodontitis patients compared with healthy controls, findings for any of the other ~40 recognized APR proteins are inconsistent, mainly due to the biological heterogeneity of patients (20, 21, 22). Notably, analysis of circulating APR-associated cytokine levels, including TNF-{alpha}, IL-1β, IL-2, IL-4, and IL-10, has therefore yet to identify any useful biomarkers, nor has it been able to provide a mechanistic explanation for disease pathogenesis (23, 24, 25, 26). Nevertheless, it is hypothesized that the APR, which may arise chronically in patients, is responsible for the association between periodontitis and other systemic disorders such as cardiovascular disease (20, 27). Although IFN-{gamma} may be important in eliciting the APR, and its levels have been shown to be higher in tissues, serum, and gingival crevicular fluid from periodontitis patients (23), the type I IFNs have received little attention. Although IFN-{alpha} and -β are generally associated with autoimmune diseases and host responses to viral infection (28), both can also modulate neutrophil behavior. Notably, type I IFN can prolong neutrophil life span by inhibiting apoptosis via PI3K, protein kinase C-{delta}, NF-{kappa}B, and STAT3 signaling pathways (29, 30). IFN-{alpha} is also able to enhance the respiratory burst in the presence of other stimuli, such as fMLP, leukotriene B4, or influenza A virus (31, 32).

Recently, high-throughput gene expression approaches comparing peripheral blood cells from control subjects and patients have contributed to improved diagnostic procedures and molecular understanding for a range of chronic inflammatory diseases, such as systemic lupus erythematosus and ulcerative colitis (33, 34). The aim of this study, therefore, was to use microarray technology to analyze the gene expression signature of hyperresponsive PBN from periodontitis patients to identify factors potentially important for disease pathogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Study populations

Subjects with chronic periodontitis (n = 19, 5 males and 14 females; mean age = 47.2 ± 6.1 years, range = 36–61 years) were recruited from patients referred to the periodontal department of Birmingham’s Dental Hospital (Birmingham, U.K.). The diagnostic criteria for chronic periodontitis were as previously reported (35). Age- and gender-matched periodontally healthy control subjects (n = 19, 5 males and 14 females; mean age = 46.4 ± 5.4 years, range = 37–62 years) were recruited from staff of the Dental Hospital. All subjects were systemically healthy and exclusion criteria included a course of nonsteroidal anti-inflammatory drugs or antimicrobial drugs within a 3-mo period before enrollment, pregnancy, use of mouthwashes or vitamin supplements within the previous 3 mo. Venous blood (see below) was collected from patients and controls immediately following initial patient presentation and diagnosis. Samples were also collected 3 mo following completion of successful periodontal treatment where active disease was no longer apparent, and before relapse of individual sites during periodontal maintenance (postreview; Ref. 36). All volunteers were never smokers with no history of recreational drug use and no special dietary requirements. This population has been investigated in both cross-sectional and longitudinal studies of the effects of nonsurgical periodontal therapy upon PBN ROS hyperresponsiveness (9, 10).

A further 10 periodontally and systemically healthy subjects (five males and five females; mean age = 37.8 ± 11.4 years, range = 22–60 years) were recruited from staff of the Dental Hospital as a source of PBN to determine the priming effects of IFN on Fc{gamma}R-stimulated ROS production and expression of IFN-stimulated genes (ISG). Ethical approval was granted by South Birmingham Local Research Ethics Committee (LREC 5643). Informed consent to participate was initially obtained, followed by the completion of a medical questionnaire.

Collection of venous blood and preparation of plasma and neutrophils

Venous blood was collected from patients and corresponding age/gender matched controls (simultaneously) from the ante-cubital fossa into Vacutainer lithium heparin (17 IU/ml) tubes and following an overnight fast where subjects were also asked to refrain from drinking (except water) or chewing gum. Platelet-depleted plasma was prepared (1000 x g, 30 min, 4°C) and subsequently stored in liquid nitrogen.

Neutrophils were isolated from venous blood (lithium heparin) as previously described (9, 10) using a discontinuous Percoll gradient ({delta} = 1.079:1.098) followed by erythrocyte lysis (0.83% NH4Cl containing 1% KHCO3, 0.04% Na2 EDTA.2H2O, and 0.25% BSA). Isolated cells were resuspended in PBS supplemented with glucose (1 mM) and cations (1 mM MgCl2, 1.5 mM CaCl2) at 1 x 106 cells/ml. Cell viability, typically >98%, was determined using dye exclusion (trypan blue), and the purity of the neutrophils was in excess of 95% using this methodology (37).

ECL for assay of total ROS production

All assays were performed as previously described (9, 10). In brief, neutrophils (1 x 105) were placed in preblocked (PBS with 1% BSA, overnight at 4°C) white microwells (Microlite 2; Dynex) with supplemented PBS or supplemented PBS containing 25 IU of IFN-{alpha}, -β, -{gamma} (R&D Systems), or 0.1 µg of LPS (Sigma-Aldrich; serotype 026:B6; 35 µl), luminol (3 mmol/L; 30 µl) for detection of total ROS production, or isoluminol (3 mmol/L; 60 µl) with 6 U of HRP for detection of extracellular ROS release. Following equilibration in the microplate reader (37°C, Bertold microplate-luminometer; LB96v) for 30 min, cells were stimulated with opsonized Staphylococcus aureus (NCTC 6571; 300 bacteria/neutrophil; 25 µl) or PBS as control. All analyses were performed in triplicate. Light emission in relative light units (RLU) was recorded during the priming phase (30 min) to study unprimed radical release and after stimulation (150 min) with the peak signal determined. Experiments using IFN-{alpha} as a priming agent were repeated using lower amounts (0.025, 0.25, and 2.5 IU) to give more physiologically relevant priming concentrations of 0.14, 1.4, 14 IU/ml and 0.12, 1.2, 12 IU/ml for luminol and isoluminol reactions, respectively.

Nitrite assay

Neutrophils (2 x 107 in 1150 µl of PBS supplemented with glucose) were primed with 50 µl IFN{alpha} (5000 IU, equivalent to 25 IU/1 x 105 cells) or PBS and incubated at 37°C for 30 min. Subsequently, neutrophils were stimulated with 800 µl of fMLP (1 mM, equivalent to 4 nM/1 x 105 cells) or PBS for 2 h at 37°C. Following stimulation, cells were centrifuged (2 min, 100 x g), the supernatants removed and assayed in triplicate for nitrite, the stable end product of NO metabolism, by the Greiss reaction as described previously (38).

IFN treatment of neutrophils for RNA extraction

Neutrophils (1 x 106 cells; 1 ml) were added to supplemented PBS (500 µl) containing either IFN-{alpha}, -β, -{gamma} (250 IU), or LPS (1 µg) in Eppendorf tubes, which were then incubated uncapped at 37°C for 3 h. Following stimulation, cells were centrifuged (2 min, 100 x g), the supernatant removed, and the cell pellet resuspended in 1 ml of TRIzol (Sigma-Aldrich). After phenol/chloroform extraction (Sigma-Aldrich), the aqueous phase was combined with 70% ethanol and added to an RNeasy minicolumn (Qiagen). Subsequent purification and DNase treatment were performed as recommended by the manufacturer (Qiagen). RNA was eluted in 30 µl of sterile water, and concentrations were determined from absorbance values at 260 nm using a BioPhotometer (Eppendorf). RNA integrity was verified by visual inspection of samples on 1% nondenaturing agarose gels stained with SYBR Gold (Molecular Probes).

Microarray target preparation, hybridization, and analysis

Total patient and control RNA were obtained by pooling RNA from four pairs of PBN samples, obtained from patients and controls in the study population. Patient and control pairs were previously demonstrated to exhibit a high hyperresponsive differential (both hyperreactivity to Fc{gamma}R-stimulation and baseline unstimulated hyperactivity) as determined by ECL (mean differential periodontitis vs health = 13,897 RLU, range = 8,300–27,061 RLU), and these data have been published elsewhere (9, 10). RNA samples were analyzed using human Affymetrix HG_U133A oligonucleotide arrays, as described at www.affymetrix.com/products/arrays/specific/hgu133.affx. Total RNA from each sample was used to prepare biotinylated target RNA, according to the manufacturer’s instructions (www.affymetrix.com/support/technical/manual/expression_manual.affx).

In brief, DNase digested total RNA (5 µg) was used to generate double-stranded cDNA using SuperScript reagents (Life Technologies) and a T7-linked oligo(dT) primer. cRNAs were synthesized using the ENZO BioArray High Yield RNA Transcript Labeling Kit (Affymetrix), and resulting biotinylated labeled cRNA was subsequently fragmented into 35- to 200-bp lengths using fragmentation buffer (Affymetrix). As recommended by the manufacturers, RNA, cDNA, and cRNA quality and size distribution were visually confirmed by agarose gel electrophoresis. Spike controls B2, bio-B, bio-C, bio-D, and Cre-x were added to the hybridization mixture before overnight hybridization at 45°C for 16 h. Arrays were stained and washed on the Fluidics Station 400 (Affymetrix) using the EukGE-WS2 protocol (dual staining) before being scanned twice on the GeneChip Scanner 3000 at an excitation wavelength of 488 nm. The integrity and quality of prepared samples was confirmed using Affymetrix Test3 GeneChips. HG_U133A microarrays (Affymetrix) were subsequently hybridized with the cRNA samples. Analysis of control parameter data (as described for Test3 GeneChip analysis) confirmed hybridization success (data not shown) and scaling factors were well within Affymetrix recommended guidelines.

Microarray data analysis

For analysis of hybridization results, which all met minimum requirements for data quality and distribution, raw data files were exported from Affymetrix microarray suite 5.0 software (MAS5.0; Affymetrix) into GeneSpring 5.1 software (Silicon Genetics). Values were normalized to the median signal values for each array. Based on previous experiences, Affymetrix and GeneSpring recommendations, and published literature (39, 40), genes with at least a 2-fold change in expression level and with a signal intensity value of >100 on either the control or test arrays were classified as being differentially expressed. The remaining genes were considered informative and were subjected to t test using a global error model with the variance statistic derived from replicates. Finally, to reduce false-differential gene expression a Bonferroni multiple test correction filter was applied. Microarray analyses were Minimum Information About a Microarray Experiment compliant, and raw data files are available in the Gene Expression Omnibus database under the series number GSE12484 (www.ncbi.nlm.nih.gov/geo/).

cDNA synthesis and semi-quantitative RT-PCR analysis

RT-PCR was performed using pooled RNA from individual pre-/post-review patient and control RNA samples (n = 9) as well as pooled healthy control samples primed with IFN and Escherichia coli LPS. For cDNA synthesis, 1–5 µg of total RNA was used for oligo(dT) reverse transcription to generate single stranded cDNA (Omniscript kit; Qiagen). cDNA concentrations were determined using a BioPhotometer (Eppendorf). Primer sequences and cycling conditions for the genes analyzed are provided in Table I. The housekeeping gene GAPDH was used as a normalization control. Primers were designed from the Affymetrix probe target identifier sequences using the Primer3 program (frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Typically cDNA (50 ng) was used to seed 50 µl of REDTaq PCR mixes (Sigma-Aldrich) and subjected to between 25 and 40 cycles. Amplification cycles of 95°C for 20 s, 61° for 20 s, and 72° for 20 s were performed using a Mastercycler thermal cycler (Eppendorf). Following the designated number of cycles 7 µl of the reaction product was removed and PCR products separated and visualized on a 1.5% agarose gel containing ethidium bromide (0.5 µg/ml). Scanned gel images were imported into AIDA image analysis software (Fuji) and the volume density of amplified products calculated and normalized against GAPDH control values.


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

 
IFN-{alpha} ELISA

IFN- {alpha} levels were determined in all the available liquid nitrogen-stored plasma samples from the study population before and 3 mo after successful nonsurgical therapy (n = 12 patient-control pairs). Plasma samples were diluted 1/2 in sample diluent and levels measured in duplicate using a high sensitivity Biotrak ELISA (Amersham Biosciences) according to the manufacturer’s instructions. This assay has a detection limit of 0.01 pg/well and a range of 0.63–20.0 pg/ml.

Data handling and statistical analysis

Chemiluminescent data were recorded automatically and transferred to Microsoft Excel spreadsheets. Manipulation of data was performed in Excel and statistical evaluation performed using Instat 3.2 (GraphPad). Between group plasma IFN-{alpha} differences were assessed using Mann-Whitney U tests. Within group (periodontitis pre- vs posttherapy) differences in plasma IFN-{alpha} and primed neutrophil chemiluminescence, data were analyzed by Wilcoxon test. A level of p < 0.05 was used to assign statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Gene expression analysis in patient and control neutrophils

Initial assessment of microarray data revealed that whereas granulocyte CSFR, CD45, and liver/bone/kidney alkaline phosphatase transcripts were detected at high signal levels, macrophage CSFR transcript was absent (data not shown), confirming that there was no detectable monocytic contamination of the original neutrophil RNA samples. Pairwise analysis of hybridization data indicated that of the 5680 genes detected as being present in both targets, 163 genes (2.87% of detected genes) were 2-fold or greater differentially expressed between healthy and periodontitis patient samples. Of this dataset, 14 were more highly expressed in neutrophils from healthy patients than those with periodontitis (age- and gender-matched), and 149 were up-regulated in periodontitis patient neutrophils relative to healthy patients. The majority of the differentially expressed genes (96.8%) were 2-to 10-fold differentially expressed with 3.2% exhibiting >10-fold differential expression. Whereas genes more abundant in patient neutrophils (n = 149; Table II) mapped to a range of biological processes and molecular functions, genes representing ribosomal function and translational mechanisms were highly represented (n = 35; 23%, data not shown), as were those representing ISG (n = 33; 22.2%). Notably, of the differentially expressed ISG, 11 of the 33 corresponded to genes reported to have increased expression due to IFN-{gamma} exposure, whereas 25 corresponded to genes reported to increase due to type I IFN exposure (41, 42).


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

 

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  		Table 2A. (Continued)

 

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  		Table 2B. (Continued)

 
To further investigate the gene expression data obtained by microarray analysis in a larger panel of patient and controls pairs (n = 9), six differentially expressed genes, representing ISG from a range of ontological groups and fold changes, were selected for RT-PCR analysis (Table I). The gene expression data confirmed that transcript levels for all six genes were increased in a pooled RNA sample (from n = 4; Fig. 1A) and also in individual (n = 9; Fig. 1C) periodontitis samples compared with controls, supporting the data obtained by microarray analysis. These latter results also demonstrated the heterogeneity of gene expression levels in periodontitis and healthy individuals (Fig. 1B). After therapy, gene expression levels within the periodontitis patient neutrophils decreased, approaching control values, in both the pooled (n = 4; Fig. 1A) and individual (n = 9; Fig. 1C) samples.


Figure 1
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  		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 = 4, individual pooled samples). B, Individual patient-control pair IFIT4 expression analysis (n = 9). C, Mean (±SD) gene expression for periodontitis patients pre-/post-review and controls (n = 9).

 
Analysis of neutrophil responses to IFN

Due to the relatively high proportion of ISG represented in the PBN from periodontitis patients (33 of 149 genes; Table II), the effect of IFN-{alpha}, -β, -{gamma}, and LPS on ROS and gene expression was analyzed in neutrophils from periodontally and systemically healthy individuals (n = 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{gamma}R-stimulated chemiluminescence (CL) by neutrophils was induced by priming with IFN-{alpha} (p = 0.031 total CL; p = 0.031 extracellular CL), IFN-β (p = 0.031; p = 0.063), and IFN-{gamma} (p = 0.031; p = 0.031). By comparison, E. coli LPS caused a small, insignificant increase in mean total and extracellular CL (p = 0.063, p = 0.094; Fig. 2, A and B). Gene expression analysis showed that IFN-{alpha} and IFN-β priming increased expression of all six ISGs by between 49 and 209%. Priming with IFN-{gamma} or LPS increased expression of five ISGs by between 9 and 101%. IFI44-like and Mx1 gene expression appeared unaffected by IFN-{gamma} and LPS priming, respectively (Fig. 2C).


Figure 2
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  		FIGURE 2. Mean (±SD) luminol-dependent, total (A), and isoluminol-dependent extracellular (B) CL generated by peripheral neutrophils stimulated with opsonized S. aureus with and without priming with 25 IU of IFN or 0.1 µg of E. coli LPS (n = 5; *, p < 0.05 compared with no primer control). C, MX1, IFIT4, G1P2, IFIT1, CIG5, and IFI44-like gene expression in neutrophils treated with IFN-{alpha}, -β, -{gamma}, and E. coli LPS.

 
IFN-{alpha} plasma levels in periodontitis patients and orally healthy controls

Due to the observed ability of type I IFN to prime neutrophils for the respiratory burst and to consistently increase expression of all the investigated ISG identified as up-regulated in the periodontitis neutrophil dataset, IFN-{alpha} levels in pre- and post-therapy patient (n = 12) and control (n = 12) plasma samples (platelet-depleted plasma) were investigated. Plasma IFN-{alpha} levels in periodontitis subjects were significantly higher pre-nonsurgical therapy (0.97 ± 0.31 pg/ml) than those of periodontally healthy controls (0.47 ± 0.22 pg/ml, p = 0.0045). After therapy, plasma IFN-{alpha} concentrations (0.53-±0.31pg/ml) reduced to levels not significantly different from matched healthy controls (p = 0.603; Fig. 3). Although our initial studies (Fig. 2) were performed using 25 IU (equivalent to IFN-{alpha} priming concentrations of 143 and 122 IU/ml for the luminol and isoluminol assays, respectively), we subsequently wanted to determine whether levels found within the peripheral circulation in patients of ~1 pg/ml (approximately equivalent to 0.37 IU/ml) were able to prime PBN with regard to the oxidative burst. Data indicated (Fig. 4) that IFN-{alpha} concentrations as low as 0.14 (luminol) and 0.12 IU/ml (isoluminol) were able to prime neutrophils for both intra- and extracellular ROS production at levels statistically significantly higher than controls (p = 0.03).


Figure 3
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  		FIGURE 3. Mean (±SD) plasma IFN-{alpha} levels for periodontitis patients pre-/post-therapy and periodontally healthy matched controls (n = 12). p values are shown, * indicates statistical significance.

 

Figure 4
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  		FIGURE 4. Mean (±SD) luminol-dependent, total (A), and isoluminol-dependent extracellular (B) CL generated by peripheral neutrophils stimulated with opsonized S. aureus with and without priming with increasing concentrations of IFN-{alpha} (n = 5; *, p < 0.05 compared with no primer control).

 
Experiments were also performed to determine whether IFN-{alpha} could prime neutrophil NO production induced by fMLP, a known stimulator of NO (38). These results demonstrated that IFN-{alpha}, at concentrations that primed Fc{gamma}R-stimulated ROS generation, was unable to prime fMLP NO release (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Global gene expression profiling of peripheral blood cells has already proven to be a valuable tool not only for identifying novel clinically useful biomarkers, but also for determining pathogenic mechanisms in chronic inflammatory human diseases that provide new therapeutic targets (33, 34). To our knowledge the current study is the first to apply this technology to study transcriptional patterns in unstimulated, unprimed PBN from chronic periodontitis patients. By using microarrays not tailored for the disease we were able to elucidate transcriptional changes that were previously not part of current understanding. These studies have identified several functional groups of genes up-regulated in periodontitis patients’ neutrophils compared with healthy controls, providing a better understanding of the molecular processes underpinning this highly prevalent disease in which peripheral neutrophils generate increased levels of ROS in the presence and absence of stimulation (7, 8, 9, 10).

Previous studies comparing the transcriptional response of neutrophils to fMLP stimulation in health and periodontitis (aggressive and chronic disease) have demonstrated that patients’ cells showed a preferential up-regulation of transcripts representing ribosomal protein-encoding genes. These transcriptional changes may indicate an enhanced capacity for protein synthesis and/or other functions these molecules mediate in periodontitis (43). Interestingly, members of this same group of genes were also up-regulated in neutrophils primed with GM-CSF (12), a growth factor known to be associated with neutrophil-mediated pathology (44, 45) and recently implicated in production of heightened baseline levels of extracellular ROS by neutrophils in chronic periodontitis (9). It is therefore interesting to speculate that the relatively high proportion of ribosomal protein-encoding genes represented in our periodontitis dataset indicate that patients’ PBN are already in a "primed" state.

Our most significant finding, however, was the relatively high proportion of up-regulated ISGs within our periodontitis patient 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 exhibited the highest change in expression level, was up-regulated only by type I IFN and not by IFN-{gamma} 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-{gamma} 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{gamma}R-mediated ROS generation (7, 8, 9, 10).

Notably, it has been shown that neutrophil maturation is accompanied by heightened expression of genes that increase their response to type I and type II IFN, which act as priming agents on mature neutrophils enabling the formation of extracellular traps upon further appropriate stimulation (47). Thus, the heightened expression of ISG by neutrophils in periodontitis patients may indicate that they are primed and hyperresponsive in terms of neutrophil extracellular trap production, a possibility currently under investigation.

It is known that following local infection, bacteria and/or their components can enter the systemic circulation (48, 49). It is therefore conceivable that this process may also directly or indirectly contribute to the peripheral activation of neutrophils and potentially the patient gene expression signature observed in this study. Indeed, infectious bacteria (e.g., Chlamydia, S. aureus, E. coli) have previously been reported to increase IFN-{alpha} levels (50, 51). Comparison of our results with published data for neutrophils stimulated with LPS indicates similarities in gene expression datasets with respect to ISG (52). Notably, ISG induction by LPS was shown to be independent of type I IFN or other unidentified soluble autocrine factors released by the neutrophils (53). Our analyses, however, demonstrate that LPS does not best recapitulate the ISG profile observed in vivo (Fig. 2C), and therefore does not represent the most likely source of neutrophil priming. It is also unlikely that neutrophils are the source of elevated IFN levels potentially causing autocrine activation. Indeed, it is known that plasmacytoid dendritic cells are the more probable source of this molecule as they express 1000-fold more IFN-{alpha}/-β than other cell types. Notably, other bacterial products (e.g., DNA) can also enhance plasmacytoid dendritic cells’ IFN expression (54).

Another potential mechanism for priming of peripheral neutrophils in periodontitis is that of bacterial DNA (bDNA). Recently, GeneChip studies of peripheral blood monocytes identified that bDNA CpG motifs induced ISG expression. Subsequent blockade of the IFN-{alpha}/-β receptor on these cells strongly inhibited ISG induction, indicating that this receptor was capable of recognizing bDNA (55). In addition, bDNA directly affects neutrophil behavior by activating changes in cell shape and migration and inhibiting apoptosis (56, 57). Animal experiments have also shown that mice pretreated with bDNA exhibit enhanced neutrophil influx at sites of infection. The recruited neutrophils were phenotypically hyperactive, exhibited up-regulation of phagocytic receptors and activity, and elevated ROS production (58). Although our data strongly implicates type I IFN in priming PBN, the systemic involvement of periodontal bacteria or their components in the chronic disease cycle cannot be excluded.

Although peripheral blood ISG transcript levels vary in apparently healthy individuals within the general population (59), our combined findings support the hypothesis that elevated plasma IFN-{alpha} levels and subsequent ISG expression are associated with periodontal inflammation. The data clearly demonstrate that following conventional periodontal treatment, plasma IFN-{alpha} levels and neutrophil ISG expression decrease in periodontitis patients to levels comparable with those of healthy controls, reflecting the reduction in neutrophil ROS hyperreactivity after therapy previously reported in the same patients (10). Our functional studies (Figs. 2 and 3) indicate that peripheral IFN-{alpha} could contribute to the previously reported hyperreactive neutrophil phenotype (9, 10).

Although it is possible that IFN-{alpha} levels are elevated in a similar manner to that of other acute phase proteins following periodontal infection, it is also conceivable that IFN-{alpha} levels were initially elevated due to other as yet unidentified factors, initiating periodontal inflammation. The mechanism by which IFN-{alpha} levels become raised within the peripheral circulation may therefore be key to understanding host susceptibility and pathogenesis of the disease. Notably, of the genes analyzed in Fig. 1, and in general for ISG identified by microarray analysis, the majority, whose function is known, encode proteins involved in antiviral responses and/or have been implicated in autoimmune disease. Indeed, MX1 has been shown to provide a key protective function against viruses, in particular influenza, by interfering with its replication cycle (60). Significantly, Cig5 has been shown to have antiviral activity against several viruses including influenza, hepatitis C, and human CMV (61, 62, 63). G1P2 has also been demonstrated to enhance the innate antiviral response via regulation of IFN-stimulated intracellular signaling pathways (64). Notably, for other genes including IFIT1, IFIT4, and IFI44, there is limited knowledge regarding their biochemical function or biological effect for the host, although it has been noted that their expression is increased in the autoimmune disease systemic lupus erythematosus (65, 66). It is therefore interesting to speculate that periodontitis could exhibit an autoimmune component that may result from inefficient tissue autophagy (67). Combined with data from the literature, it is also reasonable to suggest that another potential etiological factor is viral infection that could transiently raise type I IFN levels (68). Several studies have implicated herpesviruses in the pathogenesis of periodontitis, with infected tissues harboring increased amounts of periodontopathogenic bacteria (e.g., Porphyromonas gingivalis, Tannerella forsythus, Prevotella intermedia; Ref. 69). Notably, as the majority of adults are herpesvirus carriers, it is conceivable that following periods of viral latency subsequent activation and replication of the virus, triggered by periods of immunosuppression, stress, trauma, or indeed other common viral infections known to raise IFN-{alpha} levels such as influenza (70), disease activity may be initiated in periodontitis-susceptible individuals.

This current study used a relatively small number of patients, and therefore despite this group being clinically representative, the results should be regarded as providing pilot data with more comprehensive studies now being planned. In addition, it would now be of interest to correlate neutrophil gene expression signatures with those within diseased tissues, along with determining the composition of the patients’ plaque biofilm, their peripheral viral loads, and a more thorough monitoring of blood IFN-{alpha} levels throughout the treatment duration. However, a recent study (71) has demonstrated that IFN-{alpha}, as well as TLR-9, gene expression is elevated in biopsy tissues from periodontitis compared with gingivitis lesions, further supporting our findings. Notably, peripheral blood levels of IFN-{alpha} were not measured and seropositivity for herpesvirus was not found to be different between periodontitis patients and gingivitis controls (71). Analysis of IFN-β blood levels may also be of interest as type I IFN are generally raised in concert (72), and their combined levels may be important to periodontitis pathogenesis. Although the proposed simultaneous analyses on large numbers of subjects may be technically demanding, this approach would enable a more comprehensive understanding of the local and peripheral molecular and cellular mechanisms underpinning the pathogenesis of periodontitis.

In conclusion, the role of a dysregulated host immune response, in particular neutrophil hyperresponsiveness in the generation of tissue damage in periodontitis, has been recognized for some time, however, the molecular mechanisms underlying this phenomenon have yet to be elucidated. Our studies now indicate for the first time that PBN from patients exhibit a distinct molecular phenotype, potentially indicating a "primed" state. The ability of type I IFN to "prime" the oxidative burst of neutrophils combined with the elevated levels of IFN-{alpha} found in patients blood during disease indicate this molecule is potentially involved in pathogenesis in certain patient subsets. The implications this may have for periodontitis diagnosis and therapy remain to be elucidated, as the cause of the IFN increase is yet to be determined.


   Acknowledgments
 
We thank Drs. Iwona Bujalska and Tanja Stankovic for help with microarray experiments, Dr. Rachael Sammons for the provision of S. aureus, and Dr. Melissa Grant and Mike Milward for manuscript review.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Medical Research Council (MRC UK-G0000797). Back

2 Address correspondence and reprint requests to Dr. Paul R. Cooper, Oral Biology, School of Dentistry, University of Birmingham, St. Chad’s Queensway, Birmingham, B4 6NN, U.K. E-mail address: p.r.cooper{at}bham.ac.uk Back

3 Abbreviations used in this paper: PBN, peripheral blood neutrophil; ROS, reactive oxygen species; APR, acute phase response; ISG, IFN-stimulated gene; RLU, relative light unit; CL, chemiluminescence; bDNA, bacterial DNA. Back

Received for publication November 27, 2007. Accepted for publication August 6, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Quint, J. K., J. A. Wedzicha. 2007. The neutrophil in chronic obstructive pulmonary disease. J. Allergy Clin. Immunol. 119: 1065-1071. [Medline]
  2. D'Odorico, A., R. D'Inca, C. Mestriner, V. Di Leo, A. Ferronato, G. C. Sturniolo. 2000. Influence of disease site and activity on peripheral neutrophil function in inflammatory bowel disease. Dig. Dis. Sci. 45: 1594-1600. [Medline]
  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. Evidence for chronic, peripheral activation of neutrophils in polyarticular juvenile rheumatoid arthritis. Arthritis Res. Ther. 8: R154[Medline]
  4. Page, R. C., K. Kornman. 1997. The pathogenesis of human periodontitis: an introduction. Periodontol. 2000 14: 9-11.
  5. Socransky, S. S., A. D. Haffajee, M. A. Cugini, C. Smith, R. L. Kent, Jr. 1998. Microbial complexes in subgingival plaque. J. Clin. Periodontol. 25: 134-144. [Medline]
  6. Chapple, I. L., J. B. Matthews. 2007. The role of reactive oxygen and antioxidant species in periodontal tissue destruction. Periodontol. 2000 43: 160-232.
  7. Gustafsson, A. K., B. E. Asman. 1996. Increased release of oxygen free radicals from peripheral neutrophils in adult periodontitis after Fc{gamma}-receptor stimulation. J. Clin. Periodontol. 23: 38-44. [Medline]
  8. Fredriksson, M. I., A. K. Gustafsson, K. G. Bergstrom, B. E. Asman. 2003. Constitutionally hyperreactive neutrophils in periodontitis. J. Periodontol. 74: 219-224. [Medline]
  9. Matthews, J. B., H. J. Wright, A. Roberts, P. R. Cooper, I. L. Chapple. 2007. Hyperactivity and reactivity of peripheral blood neutrophils in chronic periodontitis. Clin. Exp. Immunol. 147: 255-264. [Medline]
  10. Matthews, J. B., H. J. Wright, A. Roberts, N. Ling-Mountford, P. R. Cooper, I. L. Chapple. 2007. Neutrophil hyper-responsiveness in periodontitis. J. Dent. Res. 86: 718-722. [Abstract/Free Full Text]
  11. Gronert, K., A. Kantarci, B. D. Levy, C. B. Clish, S. Odparlik, H. Hasturk, J. A. Badwey, S. P. Colgan, T. E. Van Dyke, C. N. Serhan. 2004. A molecular defect in intracellular lipid signaling in human neutrophils in localized aggressive periodontal tissue damage. J. Immunol. 172: 1856-1861. [Abstract/Free Full Text]
  12. Kobayashi, T., N. A. Westerdaal, A. Miyazaki, W. L. van der Pol, T. Suzuki, H. Yoshie, J. G. van de Winkel, K. Hara. 1997. Relevance of immunoglobulin G Fc receptor polymorphism to recurrence of adult periodontitis in Japanese patients. Infect. Immun. 65: 3556-3560. [Abstract]
  13. Fredriksson, M., A. Gustafsson, B. Åsman, K. Bergstrom. 1998. Hyperreactive peripheral neutrophils in adult periodontitis: generation of chemiluminescence and intracellular hydrogen peroxide after in vitro priming and Fc{gamma}R-stimulation. J. Clin. Periodontol. 25: 394-398. [Medline]
  14. Gustafsson, A., B. Åsman, K. Bergstrom. 1997. Priming response to inflammatory mediators in hyperreactive peripheral neutrophils from adult periodontitis. Oral Dis. 3: 167-171. [Medline]
  15. Beck, J., R. Garcia, G. Heiss, P. S. Vokonas, S. Offenbacher. 1996. Periodonal disease and cardiovascular disease. J. Periodontol. 67: 1123-1137. [Medline]
  16. Scannapieco, F. A., R. B. Bush, S. Paju. 2003. Associations between periodontal disease and risk for atherosclerosis, cardiovascular disease, and stroke: a systematic review. Ann. Periodontol. 8: 38-53. [Medline]
  17. Tonetti, M. S., F. D'Aiuto, L. Nibali, A. Donald, C. Storry, M. Parkar, J. Suvan, A. D. Hingorani, P. Vallance, J. Deanfield. 2007. Treatment of periodontitis and endothelial dysfunction. N. Eng. J. Med. 356: 911-920. [Abstract/Free Full Text]
  18. Ceciliani, F., A. Giordano, V. Spagnolo. 2002. The systemic reaction during inflammation: the acute-phase proteins. Protein Pept. Lett. 9: 211-223. [Medline]
  19. Gabay, C., I. Kushner. 1999. Acute-phase proteins and other systemic responses to inflammation. N. Engl. J. Med. 340: 448-454. [Free Full Text]
  20. Loos, B. G.. 2005. Systemic markers of inflammation in periodontitis. J. Periodontol. 76: 2106-2115. [Medline]
  21. Slade, G. D., S. Offenbacher, J. D. Beck, G. Heiss, J. S. Pankow. 2000. Acute-phase inflammatory response to periodontal disease in the US population. J. Dent. Res. 79: 49-57. [Abstract/Free Full Text]
  22. Noack, B., R. J. Genco, M. Trevisan, S. Grossi, J. J. Zambon, E. De Nardin. 2001. Periodontal infections contribute to elevated systemic C-reactive protein level. J. Periodontol. 72: 1221-1227. [Medline]
  23. Gorska, R., H. Gregorek, J. Kowalski, A. Laskus-Perendyk, M. Syczewska, K. Madalinski. 2003. Relationship between clinical parameters and cytokine profiles in inflamed gingival tissue and serum samples from patients with chronic periodontitis. J. Clin. Periodontol. 30: 1046-1052. [Medline]
  24. Ide, M., D. McPartlin, P. Y. Coward, M. Crook, P. Lumb, R. F. Wilson. 2003. Effect of treatment of chronic periodontitis on levels of serum markers of acute-phase inflammatory and vascular responses. J. Clin. Periodontol. 30: 334-340. [Medline]
  25. Orozco, A., E. Gemmell, M. Bickel, G. J. Seymour. 2006. Interleukin-1β, interleukin-12 and interleukin-18 levels in gingival fluid and serum of patients with gingivitis and periodontitis. Oral Microbiol. Immunol. 21: 256-260. [Medline]
  26. Albandar, J. M., A. M. DeNardin, M. R. Adesanya, D. M. Winn, S. R. Diehl. 2002. Associations of serum concentrations of IgG, IgA, IgM and interleukin-1β with early-onset periodontitis classification and race. J. Clin. Periodontol. 29: 421-426. [Medline]
  27. D'Aiuto, F., M. Parkar, G. Andreou, J. Suvan, P. M. Brett, D. Ready, M. S. Tonetti. 2004. Periodontitis and systemic inflammation: control of the local infection is associated with a reduction in serum inflammatory markers. J. Dent. Res. 83: 156-160. [Abstract/Free Full Text]
  28. Bach, J. F.. 2005. Infections and autoimmune diseases. J. Autoimmun. 25: (Suppl):74-80. [Medline]
  29. Wang, K., D. Scheel-Toellner, S. H. Wong, R. Craddock, J. Caamano, A. N. Akbar, M. Salmon, J. M. Lord. 2003. Inhibition of neutrophil apoptosis by type 1 IFN depends on cross-talk between phosphoinositol 3-kinase, protein kinase C-{delta}, and NF-{kappa}B signaling pathways. J. Immunol. 171: 1035-1041. [Abstract/Free Full Text]
  30. Sakamoto, E., F. Hato, T. Kato, T. C. Sakamoto, M. Akahori, M. Hino, S. Kitagawa. 2005. Type I and type II interferons delay human neutrophil apoptosis via activation of STAT3 and up-regulation of cellular inhibitor of apoptosis 2. J. Leukocyte Biol. 78: 301-309. [Abstract/Free Full Text]
  31. Gyllenhammar, H., I. Hafstrom, B. Ringertz, A. M. Uden, J. Palmblad. 1988. Recombinant human leukocyte interferon modulates neutrophil function in vitro. J. Interferon Res. 8: 441-449. [Medline]
  32. Little, R., M. R. White, K. L. Hartshorn. 1994. Interferon-{alpha} enhances neutrophil respiratory burst responses to stimulation with influenza A virus and FMLP. J. Infect. Dis. 170: 802-810. [Medline]
  33. Burczynski, M. E., R. L. Peterson, N. C. Twine, K. A. Zuberek, B. J. Brodeur, L. Casciotti, V. Maganti, P. S. Reddy, A. Strahs, F. Immermann, et al 2006. Molecular classification of Crohn’s disease and ulcerative colitis patients using transcriptional profiles in peripheral blood mononuclear cells. J. Mol. Diagn. 8: 51-61. [Abstract/Free Full Text]
  34. Centola, M., M. B. Frank, A. I. Bolstad, P. Alex, A. Szanto, M. Zeher, T. O. Hjelmervik, R. Jonsson, B. Nakken, G. Szegedi, P. Szodoray. 2006. Genome-scale assessment of molecular pathology in systemic autoimmune diseases using microarray technology: a potential breakthrough diagnostic and individualized therapy-design tool. Scand. J. Immunol. 64: 236-242. [Medline]
  35. Brock, G. R., C. J. Butterworth, J. B. Matthews, I. L. Chapple. 2004. Local and systemic total antioxidant capacity in periodontitis and health. J. Clin. Periodontol. 31: 515-521. [Medline]
  36. Papapanou, P. N., M. H. Sedaghatfar, R. T. Demmer, D. L. Wolf, J. Yang, G. A. Roth, R. Celenti, P. B. Belusko, E. Lalla, P. Pavlidis. 2007. Periodontal therapy alters gene expression of peripheral blood monocytes. J. Clin. Periodontol. 34: 736-747. [Medline]
  37. Lichtenberger, C., S. Zakeri, K. Baier, M. Willheim, M. Holub, W. Reinisch. 1999. A novel high-purity isolation method for human peripheral blood neutrophils permitting polymerase chain reaction-based mRNA studies. J. Immunol. Methods 227: 75-84. [Medline]
  38. Masini, E., S. Nistri, A. Vannacci, T. Bani Sacchi, A. Novelli, D. Bani. 2004. Relaxin inhibits the activation of human neutrophils: involvement of the nitric oxide pathway. Endocrinology 145: 1106-1112. [Abstract/Free Full Text]
  39. McLachlan, J. L., A. J. Smith, I. J. Bujalska, P. R. Cooper. 2005. Gene expression profiling of pulpal tissue reveals the molecular complexity of dental caries. Biochim. Biophys. Acta 1741: 271-281. [Medline]
  40. Wodicka, L., H. Dong, M. Mittmann, M. H. Ho, D. J. Lockhart. 1997. Genome-wide expression monitoring in Saccharomyces cerevisiae. Nat. Biotechnol. 15: 1359-1367. [Medline]
  41. Der, S. D., A. Zhou, B. Williams, R. Silverman. 1998. Identification of genes differentially regulated by interferon {alpha}, β or {gamma} using oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 95: 15623-15628. [Abstract/Free Full Text]
  42. de Veer, M. J., M. Holko, M. Frevel, E. Walker, S. Der, J. M. Paranjape, R. H. Silverman, B. R. Williams. 2001. Functional classification of interferon-stimulated genes identified using microarrays. J. Leukocyte Biol. 69: 912-920. [Abstract/Free Full Text]
  43. Kubota, T., T. Morozumi, K. Shimizu, N. Sugita, T. Kobayashi, H. Yoshie. 2001. Differential gene expression in neutrophils from patients with generalized aggressive periodontitis. J. Periodontal Res. 36: 390-397. [Medline]
  44. Bussolino, F., J. M. Wang, P. Defilippi, F. Turrini, F. Sanavio, C. J. Edgell, M. Aglietta, P. Arese, A. Mantovani. 1989. Granulocyte- and granulocyte-macrophage-colony stimulating factors induce human endothelial cells to migrate and proliferate. Nature 337: 471-473. [Medline]
  45. Takematsu, H., H. Tagami. 1990. Granulocyte-macrophage colony-stimulating factor in psoriasis. Dermatologia 181: 16-20.
  46. Ellis, T. N., B. L. Beaman. 2004. Interferon-{gamma} activation of polymorphonuclear neutrophil function. Immunology 112: 2-12. [Medline]
  47. Martinelli, S., M. Urosevic, A. Daryadel, P. A. Oberholzer, C. Baumann, M. F. Fey, R. Dummer, H. U. Simon, S. Yousefi. 2004. Induction of genes mediating interferon-dependent extracellular trap formation during neutrophil differentiation. J. Biol. Chem. 279: 44123-44132. [Abstract/Free Full Text]
  48. Kinane, D. F., M. P. Riggio, K. F. Walker, D. MacKenzie, B. Shearer. 2005. Bacteraemia following periodontal procedures. J. Clin. Periodontol. 32: 708-713. [Medline]
  49. Ide, M., D. Jagdev, P. Y. Coward, M. Crook, G. R. Barclay, R. F. Wilson. 2004. The short-term effects of treatment of chronic periodontitis on circulating levels of endotoxin, C-reactive protein, tumor necrosis factor-{alpha}, and interleukin-6. J. Periodontol. 75: 420-428. [Medline]
  50. Devitt, A., P. A. Lund, A. G. Morris, J. H. Pearce. 1996. Induction of {alpha}/β interferon and dependent nitric oxide synthesis during Chlamydia trachomatis infection of McCoy cells in the absence of exogenous cytokine. Infect. Immun. 64: 3951-3956. [Abstract]
  51. Eloranta, M. L., K. Sandberg, P. Ricciardi-Castagnoli, M. Lindahl, G. V. Alm. 1997. Production of interferon-{alpha}/β by murine dendritic cell lines stimulated by virus and bacteria. Scand. J. Immunol. 46: 235-241. [Medline]
  52. Fessler, M. B., K. C. Malcolm, M. Duncan, G. S. Worthen. 2002. A genomic and proteomic analysis of activation of the human neutrophil by lipopolysaccharide and its mediation by p38 mitogen-activated protein kinase. J. Biol. Chem. 277: 31291-31302. [Abstract/Free Full Text]
  53. Chow, J. C., D. W. Young, D. T. Golenbock, W. J. Christ, F. Gusovsky. 1999. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J. Biol. Chem. 274: 10689-10692. [Abstract/Free Full Text]
  54. Lee, P. Y., W. H. Reeves. 2006. Type I interferon as a target of treatment in SLE. Endocr. Metab. Immune Disord. Drug Targets 6: 323-330. [Medline]
  55. Kato, A., T. Homma, J. Batchelor, N. Hashimoto, S. Imai, H. Wakiguchi, H. Saito, K. Matsumoto. 2003. Interferon-{alpha}/β receptor-mediated selective induction of a gene cluster by CpG oligodeoxynucleotide 2006. BMC Immunol. 4: 8[Medline]
  56. József, L., T. Khreiss, J. G. Filep. 2004. CpG motifs in bacterial DNA delay apoptosis of neutrophil granulocytes. FASEB J. 18: 1776-1778. [Abstract/Free Full Text]
  57. Trevani, A. S., A. Chorny, G. Salamone, M. Vermeulen, R. Gamberale, J. Schettini, S. Raiden, J. Geffner. 2003. Bacterial DNA activates human neutrophils by a CpG-independent pathway. Eur. J. Immunol. 33: 3164-7314. [Medline]
  58. Weighardt, H., C. Feterowski, M. Veit, M. Rump, H. Wagner, B. Holzmann. 2000. Increased resistance against acute polymicrobial sepsis in mice challenged with immunostimulatory CpG oligodeoxynucleotides is related to an enhanced innate effector cell response. J. Immunol. 165: 4537-4453. [Abstract/Free Full Text]
  59. Radich, J. P., M. Mao, S. Stepaniants, M. Biery, J. Castle, T. Ward, G. Schimmack, S. Kobayashi, M. Carleton, J. Lampe, P. S. Linsley. 2004. Individual-specific variation of gene expression in peripheral blood leukocytes. Genomics 83: 980-988. [Medline]
  60. Haller, O., P. Staeheli, G. Kochs. 2007. Interferon-induced Mx proteins in antiviral host defense. Biochimie 89: 812-818. [Medline]
  61. Wang, X., E. R. Hinson, P. Cresswell. 2007. The interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts. Cell Host Microbe 2: 96-105. [Medline]
  62. Jiang, D., H. Guo, C. Xu, J. Chang, B. Gu, L. Wang, T. M. Block, J. T. Guo. 2008. Identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis C virus. J. Virol. 82: 1665-1678. [Abstract/Free Full Text]
  63. Chin, K. C., P. Cresswell. 2001. Viperin (cig5), an IFN-inducible antiviral protein directly induced by human cytomegalovirus. Proc. Natl. Acad. Sci. USA 98: 15125-15130. [Abstract/Free Full Text]
  64. Lu, G., J. T. Reinert, I. Pitha-Rowe, A. Okumura, M. Kellum, K. P. Knobeloch, B. Hassel, P. M. Pitha. 2006. ISG15 enhances the innate antiviral response by inhibition of IRF-3 degradation. Cell. Mol. Biol. 52: 29-41. [Medline]
  65. Ye, S., H. Pang, Y. Y. Gu, J. Hua, X. G. Chen, C. D. Bao, Y. Wang, W. Zhang, J. Qian, B. P. Tsao, et al 2003. Protein interaction for an interferon-inducible systemic lupus associated gene, IFIT1. Rheumatology 42: 1155-1163. [Abstract/Free Full Text]
  66. Huang, X., N. Yang, X. Ou, D. Li, Z. Wang, Q. Xie, Y. Chen, H. Lin, G. Yin, F. Wen. 2008. Sequential activation of protein kinase C {delta} and JNK is required for interferon-{alpha}-induced expression of IFIT4. Cell. Signal. 20: 112-119. [Medline]
  67. Rioux, J. D., R. J. Xavier, K. D. Taylor, M. S. Silverberg, P. Goyette, A. Huett, T. Green, P. Kuballa, M. M. Barmada, L. W. Datta, et al 2007. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nat. Genet. 39: 596-604. [Medline]
  68. Haller, O., G. Kochs, F. Weber. 2006. The interferon response circuit: induction and suppression by pathogenic viruses. Virology 344: 119-130. [Medline]
  69. Slots, J.. 2007. Herpesviral-bacterial synergy in the pathogenesis of human periodontitis. Curr. Opin. Infect. Dis. 20: 278-283. [Medline]
  70. Kawada, J., H. Kimura, Y. Kamachi, K. Nishikawa, M. Taniguchi, K. Nagaoka, H. Kurahashi, S. Kojima, T. Morishima. 2006. Analysis of gene-expression profiles by oligonucleotide microarray in children with influenza. J. Gen. Virol. 87: 1677-1683. [Abstract/Free Full Text]
  71. Kajita, K., T. Honda, R. Amanuma, H. Domon, T. Okui, H. Ito, H. Yoshie, K. Tabeta, T. Nakajima, K. Yamazaki. 2007. Quantitative messenger RNA expression of Toll-like receptors and interferon-alpha1 in gingivitis and periodontitis. Oral Microbiol. Immunol. 22: 398-402. [Medline]
  72. Biron, C. A.. 2001. Interferons {alpha} and β as immune regulators: a new look. Immunity 14: 661-664. [Medline]




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