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. 2007 May 9:5:19.
doi: 10.1186/1741-7007-5-19.

Arthritis suppression by NADPH activation operates through an interferon-beta pathway

Peter Olofsson  1 , Annika Nerstedt, Malin Hultqvist, Elisabeth C Nilsson, Sofia Andersson, Anna Bergelin, Rikard Holmdahl
Affiliations

Arthritis suppression by NADPH activation operates through an interferon-beta pathway

Peter Olofsson et al. BMC Biol. .

Abstract

Background: A polymorphism in the activating component of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, neutrophil cytosolic factor 1 (NCF1), has previously been identified as a regulator of arthritis severity in mice and rats. This discovery resulted in a search for NADPH oxidase-activating substances as a potential new approach to treat autoimmune disorders such as rheumatoid arthritis (RA). We have recently shown that compounds inducing NCF1-dependent oxidative burst, e.g. phytol, have a strong ameliorating effect on arthritis in rats. However, the underlying molecular mechanism is still not clearly understood. The aim of this study was to use gene-expression profiling to understand the protective effect against arthritis of activation of NADPH oxidase in the immune system.

Results: Subcutaneous administration of phytol leads to an accumulation of the compound in the inguinal lymph nodes, with peak levels being reached approximately 10 days after administration. Hence, global gene-expression profiling on inguinal lymph nodes was performed 10 days after the induction of pristane-induced arthritis (PIA) and phytol administration. The differentially expressed genes could be divided into two pathways, consisting of genes regulated by different interferons. IFN-gamma regulated the pathway associated with arthritis development, whereas IFN-beta regulated the pathway associated with disease protection through phytol. Importantly, these two molecular pathways were also confirmed to differentiate between the arthritis-susceptible dark agouti (DA) rat, (with an Ncf-1DA allele that allows only low oxidative burst), and the arthritis-protected DA.Ncf-1E3 rat (with an Ncf1E3 allele that allows a stronger oxidative burst).

Conclusion: Naturally occurring genetic polymorphisms in the Ncf-1 gene modulate the activity of the NADPH oxidase complex, which strongly regulates the severity of arthritis. We now show that the Ncf-1 allele that enhances oxidative burst and protects against arthritis is operating through an IFN-beta-associated pathway, whereas the arthritis-driving allele operates through an IFN-gamma-associated pathway. Treatment of arthritis-susceptible rats with an NADPH oxidase-activating substance, phytol, protects against arthritis. Interestingly, the treatment led to a restoration of the oxidative-burst effect and induction of a strikingly similar IFN-beta-dependent pathway, as seen with the disease-protective Ncf1 polymorphism.

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Figures

Figure 1
Figure 1
Tissue distribution of tritium-labeled phytol in rats. Biodistribution was estimated as relative counts per minute per gram of tissue. A large fraction of phytol remained as a depot in the injection site (> 90%, not shown). Besides that, the inguinal lymph nodes were the primary tissue for accumulation of phytol. The distribution of phytol to the inguinal lymph nodes showed the highest accumulation > 1 week after injection and showed a reduction after 2 weeks. Values are means from groups of four animals.
Figure 2
Figure 2
Hierarchical clustering of differentially expressed genes in phytol-treated compared with pristane-treated rats. Euclidean distance was used as a similarity measure. Each column represents one individual rat, and each horizontal stripe represents a gene transcript. The Affymetrix probe set identification, the gene symbol, the average fold change (FC) for phytol versus pristane together with the p value, and the average fold change for phytol/pristane versus pristane and its p value are given to the right. The colors in the clustering represent the gene-expression level in each individual rat compared with the average expression in all arrays, where green indicates low expression and red represents high expression.
Figure 3
Figure 3
Arthritis development in rats after subcutaneous administration of pristane (circles) or phytol (squares) in the time study experiment. Only animals injected with pristane developed arthritis. Values are means ± SEM from groups of four animals. Levels of significance were calculated using Student's t-test (***p < 0.001).
Figure 4
Figure 4
The relative mRNA expression of (A) Ass, (B) Cxcl9 and (C) Mmp12 in lymph nodes at 0, 3, 6, 8, 10, 13, 15 and 19 days after injection with pristane (circles) or phytol (squares). Values are means ± SEM from groups of four animals. Levels of significance were calculated using Student's t-test (*p < 0.05; **p < 0.01; ***p < 0.001).
Figure 5
Figure 5
The relative mRNA expression of (A) Best5, (B) Irf7, (C) Ifit3, (D) Oas1, (E) Mx2 and (F) S100a9 in lymph nodes at 0, 3, 6, 8, 10, 13, 15 and 19 days after injection with pristane (circles) or phytol (squares). Values are means ± SEM from groups of four animals. Levels of significance were calculated using Student's t-test (*p < 0.05; **p < 0.01).
Figure 6
Figure 6
The relative mRNA expression of (A) Ifnα, (B) Ifnβ and (C) Ifnγ in lymph nodes at 0, 3, 6, 8, 10, 13, 15 and 19 days after injection with pristane (circles) or phytol (squares). Values are means ± SEM from groups of four animals. Levels of significance were calculated using Student's t-test (**p < 0.01).
Figure 7
Figure 7
Gene-expression comparison between DA (open bars) and DA. Ncf1E3 (filled bars) of (A) Ass, (B) Cxcl9, and (C) Mmp12 in inguinal lymph nodes 10 days after injection. Values are means ± SEM of 4–5 animals per group. No significant difference between the strains was detected.
Figure 8
Figure 8
Gene-expression comparison between DA (open bars) and DA. Ncf1E3 (filled bars) of (A) Best5, (B) Irf7, (C) Ifit3, (D) Oas1, (E) Mx2 and (F) S100a9 in inguinal lymph nodes 10 days after injection. Values are means ± SEM of 4–5 animals per group. Levels of significance between the strains were calculated using Student's t-test (*p < 0.05).
Figure 9
Figure 9
Gene-expression comparison between DA (open bars) and DA. Ncf1E3 (filled bars) of (A) Ifnα, (B) Ifnβ and (C) Ifnγ in inguinal lymph nodes 10 days after injection. Values are means ± SEM of 4–5 animals per group. Levels of significance between the strains were calculated using Student's t-test (*p < 0.05).
Figure 10
Figure 10
Relative (%) number of lymphocytes. Comparison between DA (open bars) and DA.Ncf1E3 (filled bars) of (A) B cells, (B) T cells and (C) NK cells in inguinal lymph nodes 10 days after treatment injection. Values are means ± SEM of 4–5 animals per group. No significant difference between the strains was detected.
Figure 11
Figure 11
Schematic representation of the differentially expressed genes and how they are induced upon injection by (A) pristane or (B) phytol. The molecular pathway that is shared between the arthritis resistant DA.Ncf1E3 rat strain and the arthritis treatment achieved with phytol is the Ifn-β-dependent pathway, which results in the upregulation of Best5, Irf7, Ifit3, Oas1 and Mx2. The inflammatory arthritis that is induced by an injection with pristane is characterized by increased expression of the Ifnγ-connected signature genes Ass, Cxcl9 and Mmp12, which potentially could be used as molecular markers for an initiating inflammatory response. As this pathway is also induced by pristane in the DA.Ncf1E3 strain this upregulation per se does not cause arthritis. Most important in this mechanism is how administration of phytol induces arthritis protection via the Ifn-β-regulated pathway in the arthritis-susceptible DA rats.
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