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. 2011 Jun 7;108(23):9548-53.
doi: 10.1073/pnas.1012645108. Epub 2011 May 18.

Spontaneous and aging-dependent development of arthritis in NADPH oxidase 2 deficiency through altered differentiation of CD11b+ and Th/Treg cells

Kihyun Lee  1 , Hee Yeon Won, Myung Ae Bae, Jeong-Ho Hong, Eun Sook Hwang
Affiliations

Spontaneous and aging-dependent development of arthritis in NADPH oxidase 2 deficiency through altered differentiation of CD11b+ and Th/Treg cells

Kihyun Lee et al. Proc Natl Acad Sci U S A. .

Abstract

Emerging evidence indicates that NADPH oxidase (NOX) and its reactive oxygen species (ROS) products modulate a variety of cellular events, including proliferation, differentiation, and apoptosis. In this study, we investigated the functions of NOX2 and ROS in immune modulation using NOX2 knockout (KO) mice. Interestingly, NOX2 KO mice spontaneously developed arthritis with onset at 6-7 wk of age and high incidence (60%) at 15-18 wk of age. Arthritis severity in NOX2 KO mice was proportionally increased with age and higher in females than in males. Bone destruction was confirmed by microcomputed tomography scanning and histological analyses of joints. Inflammatory factors, including TNF-α, IL-1β, and RANKL, and serum level of anti-type II collagen IgG were significantly increased in NOX2 KO mice. In addition, NOX2 deficiency perturbed the immune system upon aging VSports手机版. NOX2 KO mice demonstrated preferred development of CD11b+Gr-1+ myeloid cells with profound production of proinflammatory cytokines and augmented expression of IL-17 through the activation of STAT3 and RORγt in vivo. NOX2 deficiency increased differentiation of effector Th cells in vitro and decreased CD25+FoxP3+ Treg cells both in vitro and in vivo. Furthermore, adoptive transfer of NOX2-deficient CD4(+) T cells into RAG KO mice increased arthritic inflammation compared with WT cells. These results demonstrated that NOX2 deficiency affected the development of CD11b+ myeloid cells and Th17/Treg cells, and thus promoted inflammatory cytokine production and inflammatory arthritis development, strongly supporting a crucial role for ROS generation in the modulation of Th17/Treg cell development and its related inflammatory immune response upon aging. .

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Conflict of interest statement

The authors declare no conflict of interest.

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Fig. 1.
Fig. 1.
Aging-dependent arthritis development in NOX2 KO mice. All mice were examined in a blind manner. (A) Arthritis incidence was calculated in male (n = 73) and female (n = 63) mice. (B) Arthritis severity was scored in arthritic KO mice (n = 25 males, n = 34 females). *P < 0.05. (C) Pictures of swollen paws in KO mice. (D) Arthritis severity was determined in two female groups (n = 10): those with birth experience (birth) and those with no experience (no birth). ns, not significant.
Fig. 2.
Fig. 2.
Characterization of inflammatory arthritis in KO mice. All mice were analyzed at 15–18 wk of age. (A) WT and KO mice (n = 6) were anesthetized and scanned with the eXplore Locus in vivo micro-CT scanner. Representative images of paw and knee joint are presented. (B) Knee joint was sectioned and stained with H&E (n = 5). BM, bone marrow; C, chondrocyte. (Scale bars: Upper, 100 μm; Lower, 50 μm.) (C) BMD was calculated using the micro-CT scanner, and mean ± SEM for six mice was determined. (D) Bone marrow cells were stained with anti-RANKL Ab, followed by flow cytometry (n = 4). (E and F) Reverse transcription and quantitative real time-PCR determined the expression level of TRAP and MMP-9 (E) and proinflammatory cytokines IL-1β and TNF-α (F) in total joint cells. Data are expressed as mean ± SEM for four mice. (G) Whole blood was collected by an eye bleeding, and the resulting serum was subjected to ELISA to measure IgE and IgA. (H) ELISA plate precoated with CII collagen was incubated with serum and subsequently incubated with anti-mouse Ig subtypes. Color changes with substrate were determined, and data were obtained from at least three independent experiments (n = 6). *P < 0.05, **P < 0.005, ***P < 0.0005.
Fig. 3.
Fig. 3.
Aberrant immune cell development in NOX2 deficiency. All mice were analyzed at 15–18 wk of age. (A) Hind limbs were collected from WT and KO mice (n = 5). Bone marrow cells were isolated from femur and tibia and stained with anti-CD11b or anti-Ly6C. (B) Comparison of lymph node and spleen between WT and KO mice. Total cell number was determined as mean ± SEM for five mice. *P < 0.05, ***P < 0.0005. (C–E) Single-cell suspensions of spleen were stained with surface marker Abs as indicated, and then subjected to flow cytometry analysis. (F) Spleen cells (n = 4) were stimulated in vitro with LPS (0.1 μg/mL) for 6 h and incubated with anti–TNF-α or anti–IL-6 for intracellular cytokine staining.
Fig. 4.
Fig. 4.
Increased IFN-γ and IL-17 production in NOX2-deficient cells. WT and KO mice were analyzed at 12–18 wk of age. (A) Total lymph node cells were stimulated with PMA and ionomycin for 6 h, and subjected to measurement of IFN-γ and IL-17 by ELISA (n = 5) and intracellular cytokine staining by gating CD4+ T cells. nd, not detected. **P < 0.005, ***P < 0.0005. (B) The expression of NOX2, pSTAT3, STAT3, RORγt, and β-actin in spleen was determined by immunoblotting. (C) CD11b+ cells were isolated, stimulated with LPS (0.1 μg/mL) for 6 h (n = 6), and harvested for total RNA preparation. Relative transcript levels of IL-6, IL-1β, and TNF-α were determined. (D) WT and KO CD4+ T cells were activated with anti-CD3 and anti-CD28 for 72 h along with CD11b+ cells that were stimulated with LPS for 6 h. Culture supernatants were analyzed by ELISA to determine IFN-γ and IL-17 levels. (E) CD4+ T cells were labeled with CFSE and cultured in the presence of LPS-stimulated CD11b+ cells. After 72 h, cells were analyzed using a FACSCalibur flow cytometer. CD4+ and CD11b+ cells in C, D, and E were isolated from spleen. **P < 0.005.
Fig. 5.
Fig. 5.
Promoter Th17 cell development in the absence of NOX2. (A) Naïve CD4+ T cells were isolated and stimulated with plate-bound anti-CD3 and anti-CD28. Cells were additionally cultured under Th1-, Th17-, or Th2-skewing conditions for 5 d. After restimulation with anti-CD3 for 24 h, signature cytokines IFN-γ and IL-17, and Th2 cytokines IL-4, IL-5, and IL-13, were determined in Th1, Th17, and Th2 cells, respectively. (B) CD4+ T cells were harvested by stimulation with anti-CD3 and anti-CD28 for 30 h. Relative expression levels of T-bet, GATA-3, RORγt, and FoxP3 were determined by quantitative real-time PCR. (C–E) Th17 cells were generated and treated with vehicle (–) or 10 μM DPI (+) for 4 h. Cells were stained with CM-dichlorodihydrofluorescein diacetate (DCFDA) (C) or harvested for measuring the relative expression of IL-17 mRNA (D). (E) CD4+ T cells were activated for 3 d and analyzed with anti-RANKL Ab. Three independent experiments were conducted for all data in A–D. One representative result is presented in E. All mice were killed at 12–18 wk of age. *P < 0.05, ***P < 0.0005.
Fig. 6.
Fig. 6.
Attenuation of FoxP3+ Treg cells by NOX2 deficiency. (A–C) Total spleen cells obtained from WT and KO mice were stained with anti-CD4, anti-CD25, and anti-Foxp3 according to the manufacturer's protocol. CD4- and CD25-expressing cells are presented in dot plots (A) and FoxP3-expressing CD4+ cells are expressed in histogram plots (B). (C) The level of FoxP3 in spleen cells was determined by immunoblotting assay. (D) Inducible Treg cells were generated from naïve CD4+ T cells of WT and KO mice in the presence of anti-IFN-γ, anti-IL-4, IL-2, and TGF-β and stained with anti-CD25 and anti-Foxp3. (E and F) Inducible Treg cells were treated with vehicle (–) or DPI (+) for 4 h and incubated with CM-DCFDA dye (E) or anti-CD25 and anti-Foxp3 (F). (G and H) CD4+ T cells were isolated from WT and KO mice at 16 wk of age and transferred into RAG KO mice (n = 6), followed by injection with CII. Mice were analyzed 4 wk after adoptive transfer. (G) Paws were evaluated by histological staining, and infiltrating immune cells were quantitatively analyzed using the Tissue FAXS program. ***P < 0.0005. (H) Paw thickness was measured, and the difference was calculated in a time-dependent manner. *P < 0.05. (I) Cells were harvested from lymph node of reconstituted RAG KO mice, stimulated with PMA and ionomycin for 4 h, and then subjected to staining for IFN-γ and IL-17 production.
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