Enhanced autoimmunity, arthritis, and encephalomyelitis in mice with a reduced oxidative burst due to a mutation in the Ncf1 gene

Abstract

The Ncf1 gene was recently identified as a strong regulator of severe arthritis in rat. This finding was surprising, because the disease-promoting allele mediated a lower level of reactive oxygen species in NADPH oxidase-expressing cells. We have now investigated a splice mutation of the Ncf1 gene in B10.Q mice, causing a truncated and nonfunctional Ncf1 protein. We found that the mutated Ncf1 led to a more severe and chronic relapsing collagen-induced arthritis. Enhanced IgG and delayed-type hypersensitivity responses against type II collagen were seen, indicating increased activity of autoreactive T cells. Interestingly, female Ncf1-mutated mice spontaneously developed severe arthritis during the postpartum period. The arthritis was accompanied by an increased antibody response to type II collagen, with the same fine specificity as in collagen-induced arthritis. The enhancing effect of the mutated Ncf1 could also be shown to be more general in that it enhanced myelin oligodendrocyte glycoprotein protein-induced experimental autoimmune encephalomyelitis, a model for multiple sclerosis. These results show that Ncf1, a gene important for oxidative burst, regulates the susceptibility and severity of both arthritis and encephalomyelitis and modulates, directly or indirectly, the level of T cell-dependent autoimmune responses.

Fig. 1.

Mice with mutated Ncf1 develop enhanced CII autoimmunity and chronic CIA. Development of CIA in B10.Q.Ncf1*/* (n = 19) mice compared with B10.Q.Ncf1+/* (n = 28) and B10.Q.Ncf1+/+ (n = 11) littermates. Mice were immunized with 100 μg of rat CII in CFA injected intradermally at the base of the tail. At day 35, a boosting with an intradermal injection containing 50 μg of rat CII in incomplete Freund's adjuvant was performed. After day 58, fewer animals are scored due to scarification for histology; B10.Q.Ncf1*/* (n = 10), B10.Q.Ncf1+/* (n = 19), B10.Q.Ncf1+/+ (n = 4). (A) Arthritis scores plotted against the different days after CII immunization. (B) Serum concentration of COMP used as a measurement of ongoing cartilage destruction days 40 and 102. Filled circles represent mice with arthritis, and lines represent the mean values [B10.Q.Ncf1*/* (n = 19), B10.Q.Ncf1+/* (n = 28), B10.Q.Ncf1+/+ (n = 11)]. #, fewer animals analyzed on day 102 [B10.Q.Ncf1*/* (n = 10), B10.Q.Ncf1+/* (n = 19), B10.Q.Ncf1+/+ (n = 4)]. P > 0.001 for both wild-type and heterozygous mice in comparison with mutated mice on day 40. Day 102 P < 0.01 for wild-type and P < 0.5 for heterozygous mice. (C) Representative morphological sections of ankle joints taken at day 102 after CIA induction. Sections were stained with hematoxylin and erythrosine (×100). Severe arthritis with cartilage and bone destruction is seen in the B10.Q.Ncf1*/* mice, whereas there was milder cartilage destruction in the B10Q.Ncf1+/* and B10.Q.Ncf1+/+ control mice.

Fig. 2.

T cell-dependent autoimmune response to CII, but not to OVA, is enhanced in Ncf1-mutated mice. Antibody response directed against (A) CII and (B) epitopes of CII was measured in serum at day 40 after CIA induction in B10.Q.Ncf1*/* (n = 19), B10.Q.Ncf1+/* (n = 28) and B10.Q.Ncf1+/+ (n = 11) mice. A significant difference (P < 0.05) in levels of antibodies against C1, J1, and U1 can be seen between wild-type and homozygously mutated mice. (C) DTH response against CII in B10.Q.Ncf1*/* (n = 17), B10.Q.Ncf1+/* (n = 20) and B10.Q.Ncf1+/+ (n = 7) was evaluated for reactivity 48 h after stimulation with CII in the ear. DTH result is presented as percent volume increase of the stimulated ear in comparison to control injected left ear. A significantly lower response can be seen in B10.Q.Ncf1+/* and B10.Q.Ncf1+/+ compared with B10.Q.Ncf1*/* (P < 0.05). (D) DTH response presented as percent volume increase of the ear stimulated with OVA in comparison with control-injected left ear after 48 h and (E) antibody response measured in sera on day 18 after immunization with OVA in B10.Q.Ncf1+/* (n = 7) and B10.Q.Ncf1*/* (n = 11).

Fig. 3.

Enhancement of EAE induced with native MOG but reduction of EAE induced with a MOG peptide in the Ncf1-mutated mice. Influence of the Ncf1 mutation on experimental EAE. (A) Development of EAE in mice immunized with the MOG peptide 79–96 in CFA B10.Q.Ncf1*/* (n = 13), B10.Q.Ncf1+/* (n = 9) and B10.Q.Ncf1+/+ (n = 12). (B) Development of EAE in mice immunized with native MOG recombinant protein in CFA B10.Q.Ncf1*/* (n = 13), B10.Q.Ncf1+/* (n = 7) and B10.Q.Ncf1+/+ (n = 4).

Fig. 4.

Spontaneous arthritis and CII autoimmunity develops in B10.Q.Ncf1*/* mice. (A) Development of arthritis in three individual mice followed by clinical scoring. (B) A naïve Ncf1-mutated mouse with spontaneous arthritis. (C) Representative morphological sections of ankle joints taken from the mouse shown in B and a control mouse. The sections were stained with hematoxylin and erythrosine (×100). (D) The antibody response directed against CII and epitopes of CII in B10.Q.Ncf1*/* mice with spontaneous arthritis compared to healthy B10.Q.Ncf1+/* and B10.Q.Ncf1*/* mice (n = 3). The mutated mice with spontaneous arthritis have significantly higher levels of C1, J1, and U1 epitopes (P < 0.05).