Loss of WDFY3 ameliorates severity of serum transfer-induced arthritis independently of autophagy

Abstract

WDFY3 is a master regulator of selective autophagy that we recently showed to interact with TRAF6 and augment RANKL-induced osteoclastogenesis in vitro and in vivo via the NF-κB pathway. Since the NF-κB pathway plays a major role in inflammation herein, we investigate the role of WDFY3 in an arthritis animal model. Our data show that WDFY3 conditional knockout mice (Wdfy3^loxP/loxP-LysM-Cre+) were protected in the K/BxN serum transfer-induced arthritis animal model. These effects were independent of alterations in starvation-induced autophagy as evidenced by Western blot analysis of the autophagy marker LC3, autophagosome formation in osteoclast precursors and lysosome formation in osteoclasts derived from WDFY3-cKO mice compared to controls. Moreover, we demonstrate by immunofluorescence and co-immunoprecipitation that WDFY3 interacts with SQSTM1 in macrophages and osteoclasts. Collectively, our data suggest that loss of WDFY3 in myeloid cells leads to reduced severity of inflammatory arthritis independently of WDFY3 function in starvation-induced autophagy.

Keywords: ALFY; Autophagy; Autophagy-linked FYVE containing protein; Musculoskeletal diseases; Osteoclast; WDFY3.

Figure 1

Reduced disease severity in K/BxN serum-transfer arthritis in Wdfy3-cKO mice. Eight weeks old Wdfy3-cKO mice and wild type littermates were injected with 200 µl pooled K/BxN serum. (A) Representative pictures of swollen paws derived from wild type or Wdfy3-cKO mice at day 8 post serum transfer. (B) Diseases severity score and (C) increased ankle thickness of the K/BxN serum transferred animals (n=6 for each group). The results were pooled from two independent experiments. Error bars represent mean ± SEM. 2-way ANOVA is used for statistical analysis in B, C. *p ≤ 0.05, **p ≤ 0.01.

Figure 2

WDFY3 deficient macrophages demonstrate no difference in starvation-induced autophagy, lysosome and autophagosome formation. (A, B) Western blot analysis of total cell lysates from wild type and WDFY3 deficient bone marrow derived macrophages (pre-osteoclasts) starved for 0, 2 and 4 hours in the absence or (C, D) in the presence of 10µM chloroquine. (E) Immunofluorescent photomicrographs of wild type or WDFY3 deficient macrophages in serum free HBSS for 2 to 4 hours with 10 µM chloroquine treatment and stained with Cyto-ID kit to visualized autophagosomes (green) and nuclei (blue). (White arrowheads indicate autophagosomes). (F) Multinucleated giant cells cultured from wild type or Wdfy3-cKO mice were stained with LysoTracker DND-99 (red) and Hoechst (blue) (White arrowheads indicate lysosomes. Scale bars represent 20 µm in E and 30 µm in F.

Figure 3

WDFY3 interacts with SQSTM1 in macrophages and multinucleated giant cells. (A) Immunofluorescent photomicrographs of macrophages (pre-osteoclasts) or (B) multinucleated giant cells (osteoclasts) were stained with anti-SQSTM1 (green), anti-WDFY3 (red), and DAPI (blue). (White arrowheads indicate WDFY3 and SQSTM1 co-localization). (C) Total cell lysates from osteoclast cultures co-immunoprecipitated with anti-WDFY3 antibodies and probed with SQSTM1 antibodies. Scale bars represent 10 µm in A, 20 µm in B.

Figure 4

Loss of WDFY3 leads to decrease level of SQSTM1 in macrophages. (A) Western blot analysis of total cell lysates from macrophages and multinucleated giant cells culture from wild type or Wdfy3-cKO mice. (B) Quantification of LC3-II/I ratio and SQSTM1 to β-actin ratio. Student t-test is used for statistical analysis in B. **p ≤ 0.01.