Nlrp3 mice

Il-1 receptor deficient (Il-1r^−/−) mice and Nlrp3 null (Nlrp3^−/−) mice were purchased from Jackson Laboratory. All mice were on C57BL6 background, and mouse genotyping was performed by PCR. To induce estrogen deficiency, bilateral ovariectomy (OVX) or sham surgery (in which ovaries were exteriorized, but replaced intact) were performed under general anesthesia in 4-month old WT or Nlrp3^−/− mice, and bones were analyzed 8 weeks after surgery. Estrogen deficiency was confirmed by uterine atrophy. For PTH experiments, 80 μg/kg/day human PTH1-34 or vehicle were delivered to 4-month old WT or Nlrp3^−/− male mice for 2 weeks via subcutaneously implanted ALZET osmotic pumps (model-1002, DURECT Corporation) with a delivery rate of 0.25 ml/h as previously described^{46 (link)}. GST-RANKL or vehicle was administrated intra-peritonally to 3-month old WT or Nlrp3^−/− male mice at a dose of 1 µg/kg, daily for 2 days. For pharmacologic inhibition of bone resorption, 40 mg/kg zoledronate (Novartis #484188) were injected subcutaneously to 3-month old WT mice, once a week, for 4 weeks prior to GST-RANKL injection. All procedures were approved by the Institutional Animal Care and Use Committee of Washington University School of Medicine in St. Louis. All experiments were performed in accordance with the relevant guidelines and regulations described in the IACUC-approved protocol #20160245.

Wild-type (WT) female 6-8-week-old C57BL/6 mice were purchased from Liaoning Changsheng Experimental Animal Centre. Nlrp3^-/- mice with C57BL/6 genetic background were obtained from the Jackson Laboratory (4 (link)). WT or Nlrp3^-/- mice were intraperitoneally injected with 2 ml of 5% thioglycolate medium (BD Biosciences, CA, USA) per mouse, and 3 days later peritoneal macrophages (PMs) from the peritoneal cavity were collected by flushing twice with 6 ml ice sterile PBS. PMs were counted and plated into 6-well-plate at 2.5×10⁶ cells per well with RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). The supernatant was discarded the next day, and adherent cells were further cultures as PMs (27 (link)).

Wild-type C57BL/6, caspase-1^−/− and NLRP3^−/− mice were obtained from The Jackson Laboratory, USA [8 (link),13 (link)]. The knockout mice were crossed onto the C57BL/6 background for at least nine generations. Pregnant mice were injected with EVs at days 10.5 and 11.5 p.c., as described earlier [8 (link),14 (link)]. Anakinra or aspirin was injected intraperitoneally 30 min prior to each EV injection [8 (link),15 (link)]. All animal experiments were conducted following standards and procedures approved by the local Animal Care and Use Committee (Landesverwaltungsamt, Halle, Germany).

C57BL/6 wild-type (WT), Thy-1-YFP transgenic mice (003782; The Jackson Laboratory, Bar Harbor, ME), and NLRP3^−/− mice (021302; The Jackson Laboratory) of either sex were used in this study. Thy-1-YFP H line, NLRP3^−/− mice were genotyped by polymerase chain reaction using forward primers to detect mutant: 5′-TGCCTGCTCTTTACTGAAGG-3′, and wide type: 5′-TCAGTTTCCTTGGCTACCAGA-3′; and the common reverse primer: 5′-TTCCATTACAGTCACTCCAGATGT-3′29 (link). NLRP3^−/− mice (2–6-months old) were crossed with Thy-1-YFP mice, which had a small number of RGC labeled30 (link)31 (link), permit visualization of RGCs in vivo. All animal procedures conformed to the guidelines on the Use of Animals from the NIH and the Society for Neuroscience and were approved by the Northwestern University Institutional Animal Care and Use Committee.