Minimal asbestos exposure in germline BAP1 heterozygous mice is associated with deregulated inflammatory response and increased risk of mesothelioma

Abstract

Germline BAP1 mutations predispose to several cancers, in particular malignant mesothelioma. Mesothelioma is an aggressive malignancy generally associated with professional exposure to asbestos. However, to date, we found that none of the mesothelioma patients carrying germline BAP1 mutations were professionally exposed to asbestos. We hypothesized that germline BAP1 mutations might influence the asbestos-induced inflammatory response that is linked to asbestos carcinogenesis, thereby increasing the risk of developing mesothelioma after minimal exposure. Using a BAP1(+/-) mouse model, we found that, compared with their wild-type littermates, BAP1(+/-) mice exposed to low-dose asbestos fibers showed significant alterations of the peritoneal inflammatory response, including significantly higher levels of pro-tumorigenic alternatively polarized M2 macrophages, and lower levels of several chemokines and cytokines. Consistent with these data, BAP1(+/-) mice had a significantly higher incidence of mesothelioma after exposure to very low doses of asbestos, doses that rarely induced mesothelioma in wild-type mice. Our findings suggest that minimal exposure to carcinogenic fibers may significantly increase the risk of malignant mesothelioma in genetically predisposed individuals carrying germline BAP1 mutations, possibly via alterations of the inflammatory response.

Figure 1. MΦ polarization is altered in BAP1^+/− mice exposed to low doses of asbestos fibers

Macrophages and macrophage subtypes were identified using a separate tube of peritoneal cells stained for general MΦ markers CD11b (anti-CD11b-Bv711, 563168, BD Biosciences) and F4/80, CD206 (M2 marker; anti-CD206-APC, 141707, BioLegend), and CD86 (M1 marker; anti-CD86-PE, 561963, BD Biosciences). (a) Representative flow cytometry dot plot of peritoneal MΦ in BAP1^+/− mice and wild type littermates after short-term treatment with glass beads or crocidolite asbestos. (b) Mean fluorescence intensities of CD86 and CD206. (c) Percentage of MΦ subpopulations: M1 (CD86+ CD206-), M2 (CD86-CD206+), Double positive (DP) (CD86+ CD206+). (d) M2/M1 ratio (overall percentage of CD206+ cells divided by overall percentage of CD86+ cells). Comparisons between heterozygous and wild-type groups were calculated using Mann-Whitney U test for rank comparisons. * (P < 0.05). The experiment was replicated two times.

Figure 2. Several cytokines and chemokines are differentially expressed in lavage from BAP1^+/− mice

The supernatants recovered from the peritoneal lavages were concentrated 45–60 times using Amicon Ultra Centrifuge Filters with a 3,000 Dalton cutoff. Levels of 32 cytokines and chemokines were detected in concentrated lavages using human cytokine multiplex kits (EMD Millipore Corporation, Billerica, MA). Levels of MCP-1 (a), LIF (b), KC (c), eotaxin (d) and IL-6 (e) in lavages from BAP1 wild type and heterozygous mice after short-term exposure to glass beads or crocidolite fibers. Comparisons between heterozygous and wild type groups were calculated using Mann-Whitney U test for rank comparisons. * (P < 0.05), ** (P < 0.01) (f) Correlation of IL-6 and LIF levels (both belonging to the IL-6 family of cytokines) calculated using linear regression. The experiment was replicated two times.

Figure 3. BAP1^+/− mice develop more MMs and have shorter survival compared to wild type littermates

Briefly, BAP1^+/+ mice (n = 50 per group) and BAP1^+/− mice (n = 25 per group) were injected intraperitoneally every week for ten weeks with 0.05 mg (low dose) or 0.5 mg (standard dose) of UICC crocidolite. 0.5 mg of glass beads were injected at the same schedule as control. Sample size was estimated to detect a difference in MM incidence between the low-exposed groups ≥ 25%. Mice were monitored daily for clinical evidence of abdominal swelling, and euthanized in presence of respiratory distress, gait instability, unresponsiveness to pain stimuli, or when tumor burden was obvious. Upon detection of illness, mice were sacrificed by CO2 asphyxiation, and all the major organs were evaluated histologically. (a) MM incidence in BAP1^+/− mice and wild type littermates after long-term exposure to glass beads or asbestos fibers (standard and low dose) was compared using Fisher’s exact test. * (P < 0.05) (b) Formalin-fixed/paraffin-embedded samples were cut into 5 μm sections and stained with Hematoxylin and Eosin (H&E) according to standard procedure. The pathological diagnosis of mesothelioma was based on H&E staining and supported by WT1 nuclear staining in tumor cells. H&E and immunostainings were blindly interpreted by M.C and A.P., both US board specialized pathologists with expertise in human and animal mesotheliomas^{, ,} (c) Tumors were also stained with a rabbit polyclonal anti-BAP1 antibody to evaluate presence and localization of BAP1. (d) Survival curves of BAP1^+/− mice and wild type littermates after long-term exposure to asbestos fibers (standard and low dose) were compared using log-rank (Mantel-Cox) test. . ** (P < 0.01), *** (P < 0.001). The experiment was performed one time.