Rheumatoid arthritis (RA) is one of the commonest autoimmune diseases, with a prevalence of 0.5–1% [1, 2]. RA is a chronic and severely disabling disease of unknown etiology, although both environmental [3] and genetic factors [4] are believed to play roles in its cause. There is presently no cure for RA, although a variety of different drugs is used to treat the symptoms. The most common treatments include disease-modifying antirheumatic drugs such as,methotrexate [5, 6]. Other efficient antirheumatic drugs are the recently developed biological-response modifiers, such as tumor necrosis factor (TNF)-α blockers, which reduce both the established inflammation and joint destruction [7]. The drawback of these treatments is an increased risk of infections because the body's defense system is impaired. Other biological-response modifiers targeting other cytokines or costimulatory molecules are currently being clinically tested and evaluated [8]. Despite this range of antirheumatic drugs, there is still a large number of RA patients for whom none of these treatments is effective [9, 10], thus making the development of new therapies essential.

Identification of new targets for development of antirheumatic drugs is hampered by the heterogeneity of RA and the complexity of its molecular pathology. However, recent efforts with linkage-association studies, both in human populations and in animal models, are now giving results in the form of successful identification of autoimmunity-regulating genes [11–15]. The combined results from such studies will eventually lead to a greater understanding of the molecular regulation of this complex disease and they present potential new targets for drug development.

Neutrophil cytosolic factor 1 (Ncf1), also known as p47phox, was one of the first single genes identified to regulate arthritis severity. This finding was a result of linkage analysis and positional cloning in an arthritis model in rats using a cross between the arthritis-susceptible dark agouti (DA) rat and the arthritis-resistant E3 strains DA.Ncf1 ^E3([14, 16]. NCF-1 is the activating component of the NADPH oxidase complex, which, upon activation, produces reactive oxygen species (ROS) [17]. We found that the dramatic increase in arthritis severity was caused by a decreased capacity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex to produce ROS. Interestingly, the lower oxidative burst led to a reduction in cell membrane proteins and activation of autoreactive and arthritogenic T cells [18]. Thus, the identified Ncf1 polymorphism and its effect on the immune system by decreased ROS production was concluded to be associated with the regulation of arthritogenic CD4 T cells in the immune priming phase [14, 19].

NADPH oxidase-activating substances such as phytol (3,7,11,15-tetramethyl-2-hexadecene-1-ol), were identified from studies performed on a human neutrophil cell line, and have subsequently been shown to be very efficacious in the treatment of arthritis in rats [20]. However, the mechanism through which these compounds act is still not completely understood.

The intention of this study was to obtain a molecular insight into the anti-inflammatory mechanism of NADPH oxidase activation by phytol in an experimental arthritis model in rats, the pristane-induced arthritis (PIA) model [21]. To analyze the in vivo effects of the NADPH oxidase activator phytol versus the effects of the arthritis-inducing compound pristane, global gene-expression profiling was performed. A biodistribution analysis of phytol was performed to determine which tissue and time point would be most relevant to investigate. Phytol was observed to accumulate slowly in inguinal lymph nodes, with peak levels being reached approximately 11 days after subcutaneous administration, which coincides with the onset period of arthritis. In the global gene-expression profiling, inguinal lymph nodes obtained 10 days after administration were analyzed. This analysis revealed several genes that were differentially expressed in rats with PIA compared with rats injected with phytol. A group of interesting genes was selected and verified by quantitative real-time PCR analysis in three further separate biological experiments, including a comparative study between the DA and DA.Ncf1 ^E3 congenic rats. The comparison between the two strains was aimed at determining whether the selected genes were differentially expressed in the arthritis-susceptible DA rat versus the arthritis-resistant congenic DA.Ncf1 ^E3 rat. Such data would indicate whether the NADPH oxidase-activating compound phytol is activating a pathway that is normally inactive in the absence of a fully functional Ncf1.

By studying gene-expression profiles and performing pathway analysis, we have identified the importance of an IFN-β-dependent pathway as one molecular mechanism for the arthritis-ameliorating efficacy of NADPH oxidase-activating compounds.

The discovery that NADPH oxidase-derived ROS have disease-protecting effects in arthritis opens up new possibilities for drug development against autoimmune and inflammatory diseases. To obtain a more detailed understanding of the molecular mechanism of this regulation, gene-expression profiling experiments were performed on inguinal lymph nodes from rats with pharmacologically (phytol) and genetically (Ncf1) modified NADPH oxidase activity.

Biodistribution and NADPH oxidase activation

Phytol has previously been identified as a potent NADPH oxidase activator [14, 20]. The in vivo distribution of phytol was analyzed and examined by subcutaneous injection of tritium-labeled phytol into the base of the tail in DA rats. The rats were killed and dissected 2, 4, 8, 11 and 17 days after administration. Various organs (inguinal lymph nodes, spleen, heart, thymus, kidney, liver, lung, fat, muscle and peripheral blood) were collected, homogenized and analyzed in a beta counter to determine tissue-incorporated tritiated phytol. Most of the administered phytol remained as a depot at the injection site (> 90%, data not shown), whereas phytol distributed to tissues was recovered mainly from the inguinal lymph nodes. Furthermore, the accumulation of phytol in the lymph nodes reached its maximum level 11 days after administration (Figure 1). No phytol was detected in peripheral blood at the time points studied or in blood collected at earlier time points (2, 6, 13 or 25 hours after administration, data not shown). Further analysis of the biodistribution of phytol at the microscopic level in the inguinal lymph nodes was performed by microautoradiography. It was shown that phytol was distributed primarily to the cortical regions and also in the sinusoidal space of the inguinal lymph nodes (data not shown). Moreover, the staining seemed to appear between cells or in the cell membrane, but not intracellularly.

Global gene-expression profiling

Previous results have shown that the NADPH oxidase activator phytol has ameliorating properties on arthritis when administrated prior to arthritis induction and when given as therapeutic treatment [20]. To obtain a molecular understanding of the protective effects of phytol on PIA, four groups of animals were subjected to different conditions; (i) induced arthritis by injection with pristane, (ii) injection with phytol, (iii) injection with both pristane and phytol, and (iv) no treatment. All animals were killed close to disease onset, i.e.10 days after administration. Global gene-expression profiling using chip arrays (Genechip^®;Affymetrix, Santa Clara, CA, USA) was performed on the collected inguinal lymph nodes from the described animals. The gene-expression pattern was quite different between treated rats and naïve controls. Comparing any of the group of treated animals and untreated animals, > 350 differentially expressed genes were detected (at significance level p < 0.05 and absolute fold change > 1.4; data not shown). However, as the most interesting differences were to be found between the different treatments, the analysis focused on these groups. When comparing phytol-treated versus pristane-treated animals, the expression of 29 genes was significantly changed (p < 0.05, with absolute fold change > 1.4). Of these 29 genes, 13 showed expression in phytol-treated rats, whereas 16 genes showed higher expression in pristane-treated rats (Figure 2). Because more complex gene induction was observed in animals given phytol plus pristane, further studies concentrated on analyses of single-compound administrations.

Time study

To analyze how the expression of the selected genes (Table 1) varies over time, a third biological experiment was performed. In this experiment, only pristane-treated or phyto- treated animals were studied, and inguinal lymph nodes were isolated at 0, 3, 6, 8, 10, 13, 15 and 19 days after injection. This time study enabled us to follow the expression of these genes during the development of arthritis induced by pristane (Figure 3).

The three genes associated with increased expression after pristane administration (Ass, Cxcl9 and Mmp12) showed significantly increased expression (p < 0.01) after disease onset (i.e. days 13–19 after pristane administration) compared with the phytol-treated group. Phytol induced a small increase in expression of these genes around the day of disease onset (day 8–13) and then decreased to the same low expression as seen at day 0 (Figure 4). The fact that the highest expression of these genes occurs after arthritis onset makes it plausible that the increased expression of these genes is associated with the inflammatory response. However, as seen for the expression of Cxcl9 (Figure 4B), which also increased significantly (p < 0.05) at days 3–6 after pristane injection, the expression of such genes also plays a role in the early response after injection.

All genes associated with increased expression after phytol administration (Best5, Irf7, Ifit3, Oas1, Mx2 and S100a9) showed significantly increased expression (p < 0.05) compared with pristane (Figure 5) in the early disease-onset period (day 8–10). The expression levels then decreased to the same low level as for the pristane-treated rats.

Besides the genes described above, S100a8 and the Mx2-related gene Mx1 were also analyzed in this time study and shown to be expressed in a similar pattern to that of S100a9 and Mx2, respectively (data not shown). As these genes are closely related, only one of each was followed for further expression characterization. The expression of Ncf1 was also included in this analysis as a control. However, no marked difference in Ncf1 expression was detected between the groups of animals (data not shown).

The expressions of the Ifn genes were also analyzed over time (Figure 6). The expression of Ifnγ was significantly increased in the rats injected with pristane (p < 0.01) and expression of Ifnα and Ifnβ was increased after injection with phytol. To further validate the significance of the increased expression of Ifnα and Ifnβ in DA rats at day 10 after treatment with phytol, RNA samples produced from all four separate experiments was analyzed for Ifnα and Ifnβ expression levels in DA rats treated with either pristane (n = 19) or phytol (n = 18). This combined dataset showed an upregulation of IFN-α (p < 0.05) and an upregulation of IFN-β (p < 0.01) in phytol-treated compared with pristane-treated animals. The increased expression of Ifnα/β coincided with the most pronounced increase in expression of Best5, Irf7, Ifit3, Oas1, Mx2 and S100a9, i.e. day 8–10 after treatment (Figure 5 and Figure 6A, B). Hence, Ifnα and Ifnβ could be postulated to be common regulatory genes for this molecular pathway of genes that were all induced by phytol treatment. However, it should be noted that the expression levels of the studied type I interferons were very low and that the difference in expression levels could only be detected using quantitative real-time PCR (Figures 6 and 9).

TNF-α and IL-1 are other cytokines identified as important immunological regulators in the same molecular pathways as the interferons. Transcription of Tnfα and IL-1 is increased in macrophages activated by IFN-α to act synergistically with IFN-γ in initiating a chronic inflammatory response. The expression of Tnfα and IL-1 was also analyzed, but only a small decrease in the expression of Tnfα could be observed after phytol administration, and the expression of IL-1 was below the detection limit (data not shown).

Verification of Ifn type I pathway in Ncf1congenic rats

The selected genes that were identified as being associated with either the disease state (pristane) or treatment state (phytol) of DA rats, were further analyzed in another biological experiment including a comparison between DA and congenic DA.Ncf1 ^E3 rats. These rats carry different alleles of the Ncf1 gene, and differ dramatically in arthritis severity; the DA rat is highly susceptible and the congenic DA.Ncf1 ^E3 is almost completely resistant to arthritis [14]. This analysis was included to study the importance of the selected genes for the difference in arthritis susceptibility caused by different Ncf1 alleles, and thus also NADPH oxidase functionality in PIA compared with phytol treatment. Administration with either pristane or phytol leads to increased expression of Ass, Cxcl9 and Mmp-12 in both strains, compared with the untreated controls (p < 0.01) (Figure 7). Further, a clear and significant difference (p < 0.001) between the pristane-treated and phytol-treated animals was observed, although no significant difference was found between the two rat strains.

Expression of Best5, Irf7, Ifit3, Mx2, Oas1 and S100a9 were all induced by phytol, compared with untreated controls (p < 0.01). Interestingly, after pristane injection, there was significantly higher expression (p < 0.05) of Best5, Irf7, Ifit3 and Mx2 in DA.Ncf1 ^E3 rats than in DA rats. No difference in expression levels between the strains was observed for S100a9. Overall, the gene expression of Best5, Irf7, Ifit3, Mx2 and Oas1 showed the highest expression levels in rats that had been injected with a combination of pristane and phytol (Figure 8).

Ifnα was expressed at a lower level in both the pristane-treated and phytol-treated animals, and in the rats with combined treatment, compared with the untreated control rats (p < 0.01) (Figure 9A). The expression of Ifnβ was significantly higher (p < 0.01) in the pristane-injected or phytol-injected groups than in the untreated control group, while the combined treatment induced even higher expression (Figure 9B). The expression of Ifnγ was significantly higher (p < 0.01) for the rats injected with pristane than for both control and phytol-treated rats (Figure 9C). Interestingly, only Ifnβ showed different expression in the DA.Ncf1 ^E3 rats compared with DA rats after pristane injection (p < 0.05). The expression level of Ifnβ in DA rats after pristane injection was comparable with that of untreated control rats; however, after phytol administration, the level of Ifnβ was increased to a level equal to that of DA.Ncf1 ^E3 rats (Figure 9B).

Cell distribution in Ncf1congenic rats

The levels of different cell populations in the studied tissue may affect the outcome of gene-expression profiling. Therefore, fluorescence-activated cell-sorting (flow cytometry, BD Biosciences) analyses were performed on isolated inguinal lymph nodes to determine whether the observed differentially expressed genes could be explained by a skewed cell population. In both the pristane-treated and phytol-treated rats, the number of B and NK cells were raised compared with untreated controls, whereas the relative number of T cells were lower. Significantly more B and NK cells were observed after phytol treatment than after pristane treatment (p < 0.01, DA and DA.Ncf1^E3 combined). No significant alterations in the subset of cells containing macrophages and dendritic cell populations (i.e. CD4+TCR- cells) could be detected (data not shown). No difference between the strains was observed for all of the studied groups of animals (Figure 10). These effects on cell populations could have a major impact on the gene-expression profiles, but as no difference between the strains was observed, the differentially expressed genes assigned to the Ifnβ pathway are unlikely to be the result of differences in these cell populations.

Ncf1 is a gene encoding the activating component of the NADPH oxidase complex. By positional cloning in rat models of arthritis, a polymorphism of Ncf1 was found to regulate arthritis severity [14]. In fact, it was shown that a less functional Ncf1, and the resulting decrease in NADPH oxidase capacity to produce ROS, is a major cause of increased arthritis severity in both rat and mouse models of arthritis, an observation that challenges the general dogma of the inflammatory role of ROS. Furthermore, NADPH-activating substances have both preventive and therapeutic effects on arthritis, which opens up new approaches for disease treatment [20]. However, from the results of the positional cloning of Ncf1 and the data showing strong ameliorating effects of ROS-inducing compounds in animal models, we are still a long way from fully understanding the underlying mechanism of action.

One approach to obtaining a molecular insight into a complex biological system is to use global gene-expression profiling [51]. An advantage of this method is the large set of genes that can be analyzed in the same experimental setup, making it possible to use clustering and pathway analysis of large sets of genes [52, 53]. The assembled biological information can then be stratified into molecular pathways that can be studied and thoroughly validated.

However, the intricate choice of time point and tissue/cell type to be selected for analysis presents a significant hurdle [54]. A further difficulty is the need for biological replicates and the genetic heterogeneity that is involved in human studies. This has naturally led to some skepticism regarding the utility of gene-expression profiling as a method to achieve an understanding of rheumatic diseases [55]. However, recent analysis of gene-expression fingerprints of individual patients with RA might give hope for this still technically evolving method for understanding complex disorders [56–59].

In this study, we used global gene-expression analysis and quantitative real-time PCR techniques in four separate arthritis experiments in rats to investigate the downstream effects of preventive arthritis treatment with the NADPH oxidase activator phytol. Based on biodistribution analyses of phytol after SC administration, global gene-expression analysis was performed on inguinal lymph nodes at a time point just before the estimated day of disease onset. We observed that the expression of our set of differentially expressed genes in our material varies dramatically during the disease progress (Figures 4, 5, 6). Therefore, it is necessary to have information about which time point and tissue to analyze before initiating gene-expression profiling. Furthermore, we also analyzed, in other tissues (i.e. thymus, blood and spleen) the expression of the genes identified as differentially expressed in inguinal lymph nodes, and observed that the expression levels could be quite different or even undetectable in these tissues (data not shown). As a result, it is not possible to extrapolate the information between tissues and time points. In addition, the number of biological replicates used in the compared groups should be high enough to enable statistical analysis. Altogether, these issues make these studies extremely cumbersome to perform, especially when analyzing human samples. By using animal models, a more optimized study design may be provided [60], as a number of identical (inbred) individuals under the same treatment and environmental conditions are compared and tissue collected at the same time point by the same researcher.

In this study, we identified a molecular mechanism that links the protective effects observed from treatment with NADPH oxidase activators with a relevant inflammatory mechanism. However, it is not the differentially expressed genes per se that are most interesting, but the molecular pathways that can be extracted (Figure 11). From our comparison between pristane (arthritis inducer [21]) and phytol (NADPH oxidase-activating and arthritis-ameliorating compound [20]), two main pathways were identified. The pristane-induced pathway was marked by increased expression of Ass, Cxcl9 and Mmp12 (Table 1). These genes all share IFN-γ as a common regulator [30, 61, 62], which was also shown to be upregulated in the inguinal lymph nodes, in a similar pattern, after induction of arthritis with pristane (Figures 4, 6C, 7 and 9C). IFN-γ is known to effect the balance between Th1 and Th2 cells, and increased expression of this pro-inflammatory cytokine plays an important role in the progression of RA [63]. Although pristane stimulates the expression of these cytokine-regulated genes, no direct conclusions about whether these genes are involved in the cause of arthritis can be drawn, as no difference in gene expression could be seen between the arthritis-susceptible strain (DA) and the arthritis-protected strain (DA.Ncf1 ^E3) (Figures 7 and 9C). As the DA.Ncf1 ^E3 congenic rats carry the arthritis-protecting variant of Ncf1, one might speculate that the effect caused by the functional Ncf1 is to generate a resistance to the effect from induction of Ass, Cxcl9 and Mmp12 genes, which might otherwise contribute to the development of arthritis. Further investigation of the effect on disease regulation is needed to be able to propose a more exact role for these genes as potential markers of arthritis [64].

Six genes (Best5, Irf7, Ifit3, Mx2, Oas1 and S100a9) were identified as genes induced by phytol (Table 1). The common inducers of these are IFN-α and IFN-β. These interferons are anti-inflammatory cytokines expressed after pathogenic infections [35, 65, 66]. From the gene-expression data it is not possible to determine whether IRF-7 is upregulated by an increased level of IFN-α/β as a downstream feedback regulator of the IFN-α/β receptor or if increased IFN-β level is the result of an upregulation of IRF-7 [35, 41, 36]. Oas1 and Mx2 are both induced upon viral invasion and IFN type I activation, and are thus regulated by the same pathway [67]. The expression of Best5 can be induced by both IFN-α/β and IFN-γ, but studies have shown IFN-α/β to have the strongest enhancing effect on the expression [32].

Interestingly, a clear tendency in the expression of Ifnβ between DA and DA.Ncf1 ^E3 strains was observed after injection with pristane, with the DA.Ncf1 ^E3 rats having higher expression of Ifnβ than the DA rats (Figure 9B). This difference in expression level clearly resembles the differences observed for the phytol-induced genes Best5, Irf7, Ifit3, Mx2 and Oas1 (Figure 8). It is therefore likely that the downstream effect of the polymorphism in Ncf1, as well as the therapeutic effect of phytol, involves regulation of an IFN-β pathway. This IFN-β-regulated gene profile is interesting with respect to similarities to the patterns that have been identified in patients with SLE and in patients with juvenile arthritis treated with anti-TNF-α [68–71], and in mouse models of SLE [72]. Subpopulations with active RA have also been shown to express increased levels of Ifn type I signature genes in peripheral blood [59]. These reports show an Ifnα/β expression signature, which is very similar to that identified in the rats treated with phytol. However, in our study, the Ifn type I expression profile was linked to disease protection while the SLE Ifn type I profile was linked to disease progression. This may indicate that the interferon balance could be an important threshold denominator of autoimmune diseases, functioning as an essential balance for immune regulation, with an imbalance resulting in either SLE or arthritis. This is also exemplified by a recent study in which pristane was used to induce SLE in mice. In that study, chronic peritoneal administration of pristane elicited increased expression of the type I interferon-inducible genes Mx1, Irf7, IP-10 and Isg-15 as a consequence of SLE [72]. However, despite the similarities in expression pattern, it must be noted that the SLE studies were performed on tissues other than inguinal lymph nodes and on established disease, so that differences in gene profiles could be caused by tissue differences or time-dependent regulation. Also interesting is the fact that the present study points towards a disease-ameliorating pathway that is regulated by IFN-β. Therefore, as the present observation of increased Ifn type I signature genes was observed to be a signal for prevention of disease onset, in contrast to the studies in ongoing SLE [68]and arthritis [59], one might speculate that increased IFN type I regulation is a way to downregulate an ongoing immune response. Such an attempt to limit the inflammatory response has been suggested previously [73], and has also been indicated by experiments in our laboratory using Ifnβ-deficient mice, where a prolonged arthritis severity was observed due to Ifnβ deficiency [74]. In fact, treatment with recombinant IFN-β significantly reduces cartilage destruction and bone destruction in collagen-induced arthritis in mice, which suggested a beneficial effect in patients also [70]. However, to date no positive outcome of clinical trials using recombinant IFN-β in RA has been presented [75, 76]. As we have shown that the effect of phytol on Ifnβ-related genes is time-dependent and also correlates with the biodistribution of phytol to the inguinal lymph nodes, it is possible that tissue distribution as well as the dose and frequency of administration are crucial for the efficacy of arthritis treatment via this pathway.

The data presented here, together with gene-expression profiles of SLE, [68–71] and arthritis [59] strongly suggests the importance of the IFN-α/β pathway as a key mechanism in autoimmune conditions [77].

The fact that no difference in cell populations was observed to explain the differential levels of mRNA for IFN-β-regulated genes does not prove that alterations in cell populations in the inguinal lymph nodes are not crucial. As IFN-β is mainly produced by plasmacytoid dendritic cells [77], which were not specifically addressed in this study, more careful analysis of this cell population might provide further insight into the disease-protecting effects of phytol in arthritis.

Increased expression of S100a9 was observed in rats treated with phytol (Figure 5F). However, the expression of S100a9 did not show any difference between DA and DA.Ncf1 ^E3 rats (Figure 8F), and the expression profile was not similar to that of Ifnβ (Figure 9B). As a result, we consider S100a9 not to be regulated by IFN-β. It is more likely that the increased levels of S100a9 expression is a direct effect of increased concentrations of intracellular Ca²⁺ induced by the NADPH oxidase-produced ROS [78, 79]. Increased expression of S100a9 is mostly reported to be proinflammatory. However, this knowledge is based on analyses from tissues and blood during ongoing inflammation, while these studies analyzed inguinal lymph nodes prior to disease onset. Hence, the expression levels for different timepoints of the disease process could differ. Even so, S100a9 may be a good biomarker for the in vivo efficacy of administered phytol as an activator of NADPH oxidase specifically in lymph node tissues.

By targeting the NADPH oxidase complex with activating compounds such as phytol, we highlight a new mechanism to treat autoimmune conditions such as arthritis. By extracting and verifying a relevant biological molecular mechanism from gene-expression profiling data, we also indicate a plausible relation between increased levels of ROS and an anti-inflammatory response regulated by an IFN-β pathway. The use of gene-expression profiling to compare treatments in animal models of complex diseases also points to a useful pharmacogenomic approach to extract relevant information about the mechanism of action and to identify potential molecular biomarkers to be used in future animal experiments and clinical trials.

The global gene-expression profiling data is deposited at ArrayExpress (accession number E-MEXP-78).