Sterile particle-induced inflammation is mediated by macrophages releasing IL-33 through a Bruton's tyrosine kinase-dependent pathway

Abstract

Initiation of the innate sterile inflammatory response that can develop in response to microparticle exposure is little understood. Here, we report that a potent type 2 immune response associated with the accumulation of neutrophils, eosinophils and alternatively activated (M2) macrophages was observed in response to sterile microparticles similar in size to wear debris associated with prosthetic implants. Although elevations in interleukin-33 (IL-33) and type 2 cytokines occurred independently of caspase-1 inflammasome signalling, the response was dependent on Bruton's tyrosine kinase (BTK). IL-33 was produced by macrophages and BTK-dependent expression of IL-33 by macrophages was sufficient to initiate the type 2 response. Analysis of inflammation in patient periprosthetic tissue also revealed type 2 responses under aseptic conditions in patients undergoing revision surgery. These findings indicate that microparticle-induced sterile inflammation is initiated by macrophages activated to produce IL-33. They further suggest that both BTK and IL-33 may provide therapeutic targets for wear debris-induced periprosthetic inflammation.

Figure 1:. The microparticle (MP) induced type 2 innate response is dependent on BTK signaling

Wild type (CBA/CaJ) (W) or BTK mutant (CBA/N^XID) (m) mice were inoculated i.p. with either PBS or microparticles (MPs). Forty-eight hours later peritoneal cells (PECs) were collected and stained for neutrophils (CD11b PerCP/Cy5.5⁺, Ly6G FITC⁺), eosinophils (c-Kit APC⁻, Siglec-F PE⁺), and alternatively activated (M2) macrophages (F4/80 APC⁺, CD206 Alexa Fluor 488⁺). FACS analyses are presented as representative plots (A) or graphically as the percentage of total peritoneal cells in each sample (B). RNA extracted from PECs was analyzed by qPCR for mRNA species characteristic of M2 macrophages (Arg-1, Chi3l3, Retnla) and cytokines representative of type 1, 2, and 17 responses (C). The mean and se is shown for each treatment group along with the exact p values and number (N) of individual mice, 4 or 5/treatment group, which varied with availability of mice. No outliers were removed from analyses and measurements were taken from distinct samples. Statistical analyses were performed by One way ANOVA followed by Tukey multiple comparisons using GrapPad-Prism software. All experiments were performed two times with similar results.

Fig 2:. Macrophages are the primary source of IL-33 and IL-33 blockade inhibits the type 2 response

Five C57BL/6 mice were inoculated i.p. with vehicle or microparticles (MPs) and peritoneal cells (PECs) were collected after 48 hours. PECs were pooled from individual mice and neutrophils were electronically sorted as CD11b PerCP/Cy5.5⁺, Ly6G FITC⁺ cells. Non-neutrophil cells (CD11b PerCP/Cy5.5⁺,Ly6G FITC⁻) were sort purified for eosinophils (Siglec-F PE⁺, F4/80 APC⁻), macrophages (F4/80 APC⁺, Siglec-F PE⁻), and cells negative for above markers. RNA extracted from sorted cell populations was analyzed by qPCR for Il-4, Il-5, Il-13, Il-33, and Il-1b mRNA (A). Total PECs and sorted macrophages from PBS or MP treated mice (5/treatment group) were incubated overnight and supernatants assessed for IL-33 by ELISA (B). Experiments A and B were performed twice with similar results. In a separate experiment, C57BL/6 mice were inoculated i.p. with vehicle or MP and administered recombinant ST2/IL-33R Fc or control IgG1Fc. Forty-eight hours later PECs were collected and stained for flow cytometry with antibodies specific for neutrophils (CD11b PerCP/Cy5.5⁺, Ly6G FITC⁺), eosinophils (c-Kit APC⁻, Siglec-F PE⁺), and alternatively activated (M2) macrophages (F4/80 APC⁺, CD206 Alexa Fluor 488⁺) and plotted either as a representative plot (C) or as a graph showing the percentage of individual cells/total PECs (D). PEC RNA was analyzed by qPCR for markers characteristic of M2 macrophages and cytokines representative of type 1,type 2, and type 17 responses (E). For D-E, the mean and se are shown for each treatment group along with the exact P values and the number (N) of individual mice, 4 or 5/treatment group, which varied with availability of mice. No outliers were removed from analyses and measurements were taken from distinct samples. Statistical tests are as described in Fig. 1. All experiments were performed two times with similar results.

Fig 3:. IL-33 restores the microparticle (MP) induced response following Bruton’s tyrosine kinase (BTK) inhibition

C57BL/6 mice were inoculated i.p. with vehicle or MPs and treated with either vehicle or with the BTK inhibitor, ibrutinib. Recombinant IL-33 or vehicle (1μg/day) was administered (i.p.) to ibrutinib treated mice. Forty-eight hours after MP inoculation, peritoneal cells (PECs) were collected and then stained for flow cytometry with specific antibodies for detection of neutrophils (CD11b PerCP/Cy5.5⁺, Ly6G FITC⁺), eosinophils (c-Kit APC⁻, Siglec-F PE⁺), and alternatively activated (M2) macrophages (F4/80 APC⁺, CD206 Alexa Fluor 488⁺) and plotted as a representative plot (A) and as a graph showing the percentage of individual innate immune cells/total PEC population (B). RNA extracted from the PECs was analyzed by qPCR for mRNA species characteristic of M2 macrophages and other cytokines representative of the type 1, 2, and 17 responses (C). For B-C, the mean and se is shown for each treatment group along with the exact p values and the number (N) of individual mice, 4 or 5/treatment group, which varied with availability of mice. No outliers were removed from analyses and measurements were taken from distinct samples. Statistical tests are as described in Fig. 1. All experiments were performed two times with similar results.

Fig 4:. Macrophages rescue type 2 inflammation in BTK deficient (XID) mice

Control wild type (CBA/CaJ) (W) or BTK mutant (CBA/N^XID) (m) mice were inoculated i.p. with vehicle 4 hours prior to macrophage isolation. 24 hours before MP inoculation, 1×10⁶ peritoneal macrophages either from XID (m) or wild type (w) mice were transferred to XID (m) recipient mice. Forty-eight hours after MP inoculation, peritoneal cells were collected and then stained for flow cytometry with specific antibodies for neutrophils (CD11b PerCP/CY5.5⁺, Ly6G FITC⁺), eosinophils (c-Kit APC⁻, Siglec-F PE⁺), and alternatvely activated (M2) macrophages (F4/80 APC⁺, CD206 Alexa Fluor 488⁺) and plotted either as a representative plot (A) or as a graph showing the percentage of individual innate immune cells/total peritoneal cell population (B). RNA extracted from the peritoneal cells was analyzed by qPCR for mRNA species characteristic of M2 macrophages and other cytokines representative of the Type 1, Type 2, and Type 17 responses (C). For B-C, the mean and se is shown for each treatment group along with the exact p values and the number (N) of individual mice, (5)/treatment group. No outliers were removed from analyses and measurements were taken from distinct samples. Statistical tests are as described in Fig. 1. All experiments were performed two times with similar results.

Fig 5:. Microparticle (MP) induced articular tissue inflammation is SYK, BTK and IL33 dependent.

C57BL/6 mice were administered bilateral intra-articular knee injections with PBS vehicle or MPs. For SYK inhibition studies (A), either vehicle or Bay 61–3606 was administered 2 days prior to MP inoculation. For BTK inhibition studies (B), either vehicle or Ibrutinib was administered 1 day prior to MP inoculation. For blockade of IL33 signaling (C), the mice were administered either Isotype control Ab or recombinant sST2 by Intra-peritoneal and intra-articular knee injection. For all studies, whole hind limbs were collected 2 days after MP inoculation. For pathologic analysis, formalin fixed tissue samples were decalcified with EDTA, sections were H&E stained and digitally imaged. Scale bars (50μm) are shown for each image. The extent of inflammation was digitally quantitated as area of synovial inflammation/total area of synovium and graphed. For FACs analysis, samples were digested at 37°C for 1 hour with collagenase and single cell suspensions were stained for flow cytometry with specific antibodies for neutrophils (CD11b PerCP/CY5.5⁺, Ly6G FITC⁺), eosinophils (c-Kit APC⁻, Siglec-F PE⁺), and alternatively activated (M2) macrophages (F4/80 APC⁺, CD206 Alexa Fluor 488⁺). The mean and se is shown for each treatment group along with the exact p values and the number (N) of individual mice, 4 or 5/treatment group, which varied with availability of mice.. No outliers were removed from analyses and measurements were taken from distinct samples. Statistical tests are as described in Fig. 1. All experiments were performed two times with similar results.

Fig 6:. Increased M2 macrophage markers detected in periprosthetic tissues

Histological analysis of infiltrate surrounding wear debris in revision joint replacement or control primary implant. (A-B) Slides were stained with H&E and infiltrates surrounding polyethylene particles identified by polarized light microscopy (indicated by green arrows). Frozen sections were dual stained to identify M2 macrophages using anti-CD68 in combination with either anti-CD206 (C, D) or anti-CD163 (E, F). M1 macrophages were identified using anti-CD68 and anti-iNOS (G, H). All photos were digitally imaged at 400× and scale bar of 50μm size is shown in each image. Quantitative real-time PCR showed significant increases in gene expression of alternatively activated (M2) macrophage markers, type 2 cytokines, and associated wound healing related genes in joint tissue of revision patients compared to primary patients (I). The mean and se is shown for each marker along with the exact p value and number (N) of individual clinical samples (N=6). GraphPad Prism software was used to perform unpaired two sided T test. Pri = Primary arthroplasty; Rev = Revision arthroplasty.