Effect of H2 treatment in a mouse model of rheumatoid arthritis‐associated interstitial lung disease

Abstract Rheumatoid arthritis (RA)‐associated interstitial lung disease (ILD), a primary cause of mortality in patients with RA, has limited treatment options. A previously established RA model in D1CC transgenic mice aberrantly expressed major histocompatibility complex class II genes in joints, developing collagen II‐induced polyarthritis and anti‐cyclic citrullinated peptide antibodies and interstitial pneumonitis, similar to those in humans. Molecular hydrogen (H2) is an efficient antioxidant that permeates cell membranes and alleviates the reactive oxygen species‐induced injury implicated in RA pathogenesis. We used D1CC mice to analyse chronic lung fibrosis development and evaluate H2 treatment effects. We injected D1CC mice with type II collagen and supplied them with H2‐rich or control water until analysis. Increased serum surfactant protein D values and lung densities images were observed 10 months after injection. Inflammation was patchy within the perilymphatic stromal area, with increased 8‐hydroxy‐2ʹ‐deoxyguanosine‐positive cell numbers and tumour necrosis factor‐α, BAX, transforming growth factor‐β, interleukin‐6 and soluble collagen levels in the lungs. Inflammatory and fibrotic changes developed diffusely within the perilymphatic stromal area, as observed in humans. H2 treatment decreased these effects in the lungs. Thus, this model is valuable for studying the effects of H2 treatment and chronic interstitial pneumonia pathophysiology in humans. H2 appears to protect against RA‐ILD by alleviating oxidative stress.

damage. 1,4 The main manifestation of RA is joint disease; however, recent estimates suggest that more than half of all patients with RA develop some form of extra-articular lung disease. 5 In addition, approximately 30% of patients with RA with lung involvement develop RA-associated interstitial lung disease (RA-ILD), a serious diffuse parenchymal lung disease associated with impaired gas exchange and fibrotic injury of alveolar septa. The most common histological patterns of RA-ILD are usual and non-specific interstitial pneumonia, 6 which are the primary causes of morbidity and mortality in such patients 7 ; however, the pathogenesis of RA-ILD is unclear, and treatment options are limited.
We previously established a model of arthritis-prone D1CC mice to provide a basis for the development of therapeutic interventions for RA-ILD. This novel mouse RA model is transgenic for the DBA/1 background. The model uses a class II transactivator and collagen type II (CII) promoter and features aberrant major histocompatibility complex class II gene expression in the joints. 8 The administration of a low CII concentration to D1CC mice, which are highly susceptible to arthritogenic stimuli, induces the gradual development of chronic inflammatory arthritis. This contrasts with the conventional mouse model of collagen-induced arthritis, which is characterized by acute inflammation. D1CC mice exhibit interstitial pneumonitis, in addition to RA-like synovitis with pannus formation and joint destruction, and develop anti-cyclic citrullinated peptide antibodies. 8 Thus, we expected D1CC mice to serve as a model that is considerably similar to RA-ILD in humans and function as an important investigative tool to model human RA-ILD and chronic interstitial pneumonia. However, to date, we have only obtained data on lung histology 6 months after CII injection in D1CC mice using elastica and Kernechtrot staining. 8 Autoreactive T cells that infiltrate the synovial tissue in RA promote an immune response and result in the overproduction of proinflammatory cytokines such as tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Thus, therapy in early RA targets aggressive biological disease alteration by regulating synovial T cells and decreasing the levels of cytokines associated with the disease. 9 Current treatment options for RA-ILD include disease-modifying anti-rheumatic drugs and biological anti-TNF-α therapies. However, evidence suggests that although these treatments benefit joint disease, they can exacerbate pulmonary dysfunction as a side effect. 10 In addition to these immunogenic factors, reactive oxygen species (ROS) are important therapeutic targets because they occur upstream of cytokine-mediated inflammatory cascades. 11 The synovial fluid and peripheral blood of patients with RA contain high levels of ROS and ROS-induced molecules such as superoxide, hydroxyl radicals (•OH) and peroxynitrite (ONOO − ). 1,[12][13][14][15][16][17] Molecular hydrogen (H 2 ) only quenches harmful ROS, including •OH and ONOO − , and permeates cell membranes; thus, it can easily target organelles, including mitochondria and nuclei.
Inhaled H 2 suppresses oxidative stress-induced injury in several organs, such as ischaemia/reperfusion injury in the brain, 18 liver 19 and heart, 20 and irradiation-induced injury in the lungs. 21 Furthermore, continuous consumption of H 2 -rich water protects against oxidative damage, including manifestations of oxidative stress associated with diabetes in humans, 22 cisplatin-induced renal injury in mice, 23 naphthalene-evoked acute lung injury in mice 24 and non-alcoholic steatohepatitis 25,26 in animal models.
Thus, H 2 may be used clinically as a safe and effective antioxidant with few side effects.
In this study, we re-evaluated fibrotic lung lesions in D1CC mice as a model of RA-ILD. Furthermore, we investigated the effect of H 2 treatment in this model to evaluate its therapeutic use against chronic lung fibrosis.

| Production of H 2 -rich water
H 2 -rich water was prepared using a previously described method. 21 Briefly, H 2 gas (purity > 99.999%; Iwatani) was dissolved in reverse osmosis water under high pressure (0.4 MPa) to a super-saturated level in a stainless steel tank (Unicontrols). During the preparation of H 2 -rich water, the H 2 concentration in the air was carefully monitored using a H 2 sensor with an alarm for safety. The saturated water was poured into 70-mL glass vessels equipped with an outlet line containing two ball bearings, which prevented the water from being degassed. The H 2 concentration in the water was measured using a

| Evaluation of joint arthritis
D1CC mice with or without H 2 treatment were monitored twice weekly. The clinical severity of arthritis was quantified according to a previously reported simple scoring system 8 (Table 1) as follows: 0, no clinical symptom; 1, swelling and redness of one or two joints; 2, moderate swelling and redness of three or more joints; and 3, severe swelling and redness of the entire paw. All scores were summed to generate the total clinical score, which had a maximum value of 12 (four severely affected joints, 4 × 3).

| Sample collection
We anesthetized the mice and exsanguinated them via the abdominal aorta at specific times after bCII injection. After blood sampling and micro-computed tomography (micro-CT), we cannulated the trachea and removed the lungs. For each animal, the left lung (two lobes) was immediately frozen at − 80°C and later used for Western blot analyses and real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The right lung (three lobes), which was used for microscopic analyses, was fixed for 8 hours at 4°C in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) at a H 2 O inflation pressure of 20 cm and then embedded in paraffin. Lipid peroxide (LPO) levels were determined using the thiobarbituric acid-reactive substance method according to the protocol provided by Naito and Yamanaka. 28

| Histology and fibrosis score
We utilized haematoxylin and eosin to stain paraffin-embedded sections for routine histological examinations and elastica Masson-Goldner staining to investigate collagen deposition. For immunohistochemical studies, we utilized the remaining sections of the right lungs. Ashcroft scores were used to evaluate the severity of lung fibrosis (a lung lesion with inflammatory and fibrotic changes), as described previously. 29 Briefly, we scanned eight fields of sections of the right lung (three lobes) at ×100, and we visually graded each field using scores of 0 (normal) to 8 (complete obliteration of the field by fibrosis). We defined the visual fibrosis score as the mean value of grades obtained from all inspected fields. Certain histological parameters of all samples were independently analysed by two blinded observers (N. K. and M. T.).

| Collagen assay
The soluble collagen content of the left lung was determined using a Sircol assay (Biocolor) according to the manufacturer's instructions.
Bound Sircol dye was assessed using a microplate reader (iMark) at 555 nm. Collagen standards supplied with the kit were used as assay controls.
F I G U R E 1 Study experimental design. D1CC mice were immunized using four 10-µg injections of bovine type II collagen (bCII) and evaluated 10 months later. Mice consumed H 2 -rich or control water from day 0 after the first injection until analysis. The mice were randomly assigned to three groups (n = 20 in each group): (a) control, normal water and not injected with bCII; (b) H 2 treatment (−), normal water and injected with bCII; and (c) H 2 treatment (+), H 2 -rich water and injected with bCII

| Immunohistochemistry
We microwaved lung sections in DakoCytomation Target Retrieval Solution (Dako) for 10 minutes at 100°C and restricted endogenous peroxidase activity using the method of Brown et al. 30 We immunostained sections using a Histofine Simple Stain kit

| Micro-CT
We performed micro-CT using an LCT-200 micro-CT system

| Western blot analysis
Western blotting was performed in each experiment according to the standard procedure. We quantified proteins using a BCA Protein

| RT-qPCR
We performed RT-qPCR to analyse IL-6 mRNA expression. We ex-

| Statistical analysis
We calculated arithmetic means and standard errors of the means for each data set and applied Student's t test to compare paired or independent variables. We determined the statistical differences among groups using one-way ANOVA and considered P < .05 to be statistically significant.

| Serum SP-D and pathological analysis of the RA-ILD model
Serum SP-D levels were significantly increased approximately 10 months (40 weeks) after the first injection of bCII ( Figure S1).

| Analyses of the RA-ILD model with and without H 2 treatment
Up to 8 weeks after immunization ( Figure S2), we found that D1CC mice developed inflammatory arthritis with redness, swelling and fever in the joints after immunization with bCII as previously reported, 8 whereas H 2 treatment suppressed inflammatory arthritis development in D1CC mice induced by bCII. Ten months after the first bCII injection, we also found an increase in lung density on lung CT images ( Figure 3A,D,J) and increased serum SP-D levels ( Figure   S1 and Figure 3K), which were consistent with the histological findings of active alveolitis with pneumocyte hyperplasia (Figures 2 and   3B,C,E,F). H 2 treatment protected against lung damage as evidenced by a reversal of the increases in serum SP-D levels ( Figure 3K) and lung density on CT images ( Figure 3D,G,J and Figure S3) induced by bCII injection; furthermore, the Ashcroft score ( Figure 3L) and soluble collagen levels ( Figure 4F) were significantly decreased in DICC mice that consumed H 2 -treated water compared with the findings in control mice.

| D ISCUSS I ON
We previously reported that D1CC transgenic mice aberrantly express major histocompatibility complex class II genes in their joints, developing RA signs such as erosive inflammatory polyarthritis, with anti-cyclic citrullinated peptide antibody production and Accordingly, in this study, we confirmed that bCII injections induce lung damage in D1CC mice (Figure 2), resulting in increased serum SP-D levels ( Figure S1 and Figure 3K) and lung density on CT images ( Figure 3D,G). Furthermore, we demonstrated that  Consistent with these results, we observed that H 2 protects against lung damage by decreasing soluble collagen levels and TNF-α, IL-6, BAX and TGF-β expression (Figures 4 and 5). These inflammatory,   25,26 In this study, the RA-ILD model is a chronic lung fibrosis model induced by the immunization of D1CC mice via several type II collagen injections; however, the precise molecular mechanisms underlying the protective effects of H 2 remain to be elucidated. There is no satisfactory in vitro cell culture model of RA, although H 2 has been reported to inhibit the lipopolysaccharide (LPS)-and IFN-γ-induced production of nitric oxide in macrophages in vitro by controlling signal transduction. In addition, consuming H 2 -rich water for 2 weeks ameliorates anti-type II collagen antibody production and LPS-induced arthritis in mice, 39 similar to our observations illustrating that H 2 treatment suppressed inflammatory arthritis development in D1CC mice induced by bCII up to 8 weeks after immunization ( Figure   S2). Because reactive nitrogen species such as ONOO − and reactive nitrogen dioxide mediate tissue damage in patients with RA through nitric oxide/nitric oxide synthase, 40 the effect of H 2 treatment in our model may be associated with the modulation of signal transduction by reactive nitrogen species in addition to the scavenging of ONOO − , which we previously reported. 18 In conclusion, we reported that the D1CC mouse model of RA-ILD is considerably similar to RA-ILD in humans, and it could be valuable as a model of lung fibrosis, using specific markers such as serum SP-D and CT to detect treatment effects. Furthermore, this model can be used in studies of the treatment and general pathophysiology of chronic interstitial pneumonias in humans. Our study revealed that H 2 might protect against RA-ILD by decreasing oxidative stress.