The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice

Abstract

The growth factor progranulin (PGRN) has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation, but its receptors remain unidentified. We report that PGRN bound directly to tumor necrosis factor receptors (TNFRs) and disturbed the TNFα-TNFR interaction. PGRN-deficient mice were susceptible to collagen-induced arthritis, and administration of PGRN reversed inflammatory arthritis. Atsttrin, an engineered protein composed of three PGRN fragments, exhibited selective TNFR binding. PGRN and Atsttrin prevented inflammation in multiple arthritis mouse models and inhibited TNFα-activated intracellular signaling. Collectively, these findings demonstrate that PGRN is a ligand of TNFR, an antagonist of TNFα signaling, and plays a critical role in the pathogenesis of inflammatory arthritis in mice. They also suggest new potential therapeutic interventions for various TNFα-mediated pathologies and conditions, including rheumatoid arthritis.

Figures

Fig. 1

PGRN directly binds to TNFR and antagonizes TNFα actions. (A) PGRN interacts with TNFR2 in chondrocytes (Co-IP assay). The cell lysates of human chondrocytes were incubated with anti-PGRN, anti-TNFR2 or control IgG antibodies, and bound protein was examined by Western blotting with the corresponding antibodies, as indicated. (B) FastStep Kinetic Assay for binding of PGRN and TNFα to TNFR1 and TNFR2. Samples were injected using FastStep injection, and dissociation of analyte-ligand complexes was monitored. KD for each interaction is indicated. (C) PGRN inhibits the binding of TNFα to TNFR1 and TNFR2 (solid phase binding). Microtiter plate coated with TNFα was incubated with TNFR1 or TNFR2 in the presence of various amounts of rhPGRN, and the bound TNFR to TNFα was detected by corresponding antibodies. Values are mean ± s.d. (D) Flow cytometry analysis of Raw264.7 cells after staining with 50 ng biotinylated human TNF-α (Bt-TNFα) and different doses of rhPGRN pretreatment. (E) PGRN deletion potentiates TNFα-induced H2O2 production (neutrophil activation). Wild type (WT) or PGRN-deficient (KO) neutrophils were treated with TNFα, and H2O2 production was measured. Values are mean ± s.d. **P <0.01; n=4. (F) PGRN deletion potentiates TNFα-induced nitrite production in bone marrow derived macrophages (BMDMs). M-CSF pretreated wild type (WT) or PGRN-deficient (KO) BMDMs were incubated with TNFα, and the supernatants were tested for NO production. Values are mean ± s.d. ***P <0.001; n=4.

Fig. 2

PGRN-deficient mice are highly susceptible to collagen-induced arthritis, and administration of PGRN reverses the severe inflammatory arthritis seen in collagen-challenged PGRN-deficient mice. (A) Paws of wild type (WT) and Grn−/− (KO) mice derived from C57BL/6 (n=10/group) immunized with collagen II for 15 weeks. (B) Radiography of ankle joints of WT and KO collagen II-immunized mice. Arrow indicates areas of severe joint destruction in PGRN-deficient CIA mice. (C) Clinical arthritis scores in WT and KO mice with CIA. The data are presented as the mean clinical score ± s.e.m. *P<0.05, **P<0.01 versus the control WT group. (D) Incidence of arthritis in the indicated groups. (E) H&E stained sections and evaluation of synovitis, pannus and erosion of ankle joints in WT and KO mice with CIA 15 weeks following primary immunization. Scale bar, 200μm. Values are mean ± s.d. *P<0.05, **P<0.01 versus the control WT group. (F) Paws of KO mice treated with PBS or rhPGRN from 4 to 15 weeks following collagen II immunization. (G) Clinical arthritis scores in KO mice with CIA treated with PBS or rhPGRN (n=10/group). Data are presented as the mean clinical score ± s.e.m. **P<0.01, ***P<0.001 versus the control PBS group. (H) Incidence of arthritis in each experimental group.

Fig. 3

Deletion of PGRN exacerbates, whereas recombinant PGRN prevents, the spontaneous development of inflammatory arthritis in TNF transgenic mice. (A) Incidence of arthritis in TNF-Tg, TNF-Tg/Grn+/−, and TNF-Tg/Grn−/−, mice (n=8/group). (B) Clinical arthritis scores. Data are presented as the mean clinical score ± s.e.m. *P<0.05, **P<0.01 and ***P<0.001 versus the control TNF-Tg group. (C) Photographs of paws of TNF-Tg mice with mild arthritis treated with either PBS or rhPGRN for 4 weeks. (D) Effect of PGRN in TNF-Tg mice. TNF-Tg mice with established mild arthritis (Clinical score is around 5) were treated with PBS or rhPGRN (n=8/group). The treatment type was then switched between the two groups, and the switch time point is indicated with arrows. Development of arthritis was then scored. The data are presented as the mean clinical score ± s.e.m. The statistics were compared between untreated (PBS) and rhPGRN-treated group before the switch time point (black star). After that statistics were compared to the switch time point in each group (green star). *P<0.05, **P<0.01, ***P<0.001.

Fig. 4

Atsttrin exhibits selective TNFR binding and inhibits TNFα/TNFR interactions. (A) FastStep Kinetic Assay for binding of Atsttrin and TNFα to TNFR1 and TNFR2. Samples were injected using FastStep injection, and dissociation of analyte-ligand complexes was monitored. KD for each interaction was indicated. (B) Atsttrin inhibits the binding of TNFα to TNFR1 and TNFR2 (solid phase binding). Microtiter plate coated with TNFα was incubated with TNFR1 or TNFR2 in the presence of various amounts of Atsttrin, and the bound TNFR to TNFα was detected by corresponding antibodies. Values are mean ± s.d. (C) Flow cytometric analysis of Raw264.7 cells after staining with 50 ng biotinylated human TNFα (Bt-TNFα) in the presence of different doses of Atsttrin.

Fig. 5

Effects of PGRN and Atsttrin in CIA. (A) Photographs of paws of CIA mice treated with PBS, rhPGRN, or Atsttrin. (B) Clinical arthritis scores in PBS (n=9), rhPGRN (n=8), Atsttrin (n=12) or Etanercept (n=8) treated CIA mice. Data are presented as the mean clinical score ± s.e.m. ***P<0.001 versus the control PBS group. (C) Incidence of arthritis in each treatment group. (D) H&E stained sections and evaluation of synovitis, pannus formation, and erosion of tarsal joints in CIA mice sacrificed at day 41 following primary immunization and treatment (starting day 19) with PBS, rhPGRN or Atsttrin. Scale bar, 200μm. Values are mean ± s.d. ***P<0.001 versus the control PBS group. (E) Therapeutic effects of Atsttrin in established CIA mice receiving intraperitoneal injections of indicated amounts of Atsttrin (mg per kg bodyweight once a week; n=8/group). Values are mean ± s.e.m. ***P<0.001 versus the group of Atsttrin at a dose of 0. (F) Therapeutic effects of Atsttrin in established CIA of wild type, Tnfrsf1a−/−, Tnfrsf1b−/− mice. Atsttrin was administered at 0.02, 0.1or 0.5 mg per kg body weight once a week; n=8/group. Values are mean ± s.e.m. *P<0.05, **P<0.01, *** P<0.001 versus the 0mg/kg treatment group.

Fig. 6

PGRN and Atsttrin inhibit TNFα-mediated activation of NF-κB and MAPK signaling. (A) BMDMs were incubated with TNFα in the presence or absence of rhPGRN or Atsttrin, and phosphorylation and expression of the indicated signaling molecules at various time points were determined by immunoblotting. Tubulin is shown as a loading control. (B) Immunohistochemistry for phosphorylated IκBα in the articular cartilage of CIA mice on day 41 following primary immunization and treatment with PBS, rhPGRN or Atsttrin. Arrows indicate phosphorylated IκBα. Scale bar, 25μm. (C) NF-κB amounts were analyzed by Western blotting with p65 antibody and assessed using cytoplasmic (CE) and nuclear (NE) extracts of TNFα-treated BMDMs in the presence and absence of rhPGRN or Atsttrin. Tubulin and lamin A serve as cytoplasmic and nuclear controls, respectively. (D) BMDMs were incubated with TNFα in the presence or absence of rhPGRN or Atsttrin for 6 hours, and analyzed by chromatin immunoprecipitation (ChIP) assay. (E) BMDMs transfected with the NF-κB-dependent reporter construct were incubated with TNFα (10 ng/ml) in the presence of increasing concentrations of rhPGRN or Atsttrin (0.1, 0.5, 2.5nM), and the luciferase activity was measured. Values are mean ± s.d. **P<0.01, ***P<0.001 versus TNFα-stimulated cells. (F) The order change of mRNA expression relative to unstimulated cells, as assessed by real time PCR. (G) PGRN and Atsttrin inhibit TNFα-induced ERK1/2, p38 and JNK phosphorylation. BMDMs were stimulated with TNFα in the presence or absence of rhPGRN or Atsttrin. At the indicated time points, cell lysates were probed using specific antibodies against total and phosphorylated Erk1/2, p38, and JNK.