NF-kappaB dysregulation in microRNA-146a-deficient mice drives the development of myeloid malignancies

Abstract

MicroRNA miR-146a has been implicated as a negative feedback regulator of NF-κB activation. Knockout of the miR-146a gene in C57BL/6 mice leads to histologically and immunophenotypically defined myeloid sarcomas and some lymphomas. The sarcomas are transplantable to immunologically compromised hosts, showing that they are true malignancies. The animals also exhibit chronic myeloproliferation in their bone marrow. Spleen and marrow cells show increased transcription of NF-κB-regulated genes and tumors have higher nuclear p65. Genetic ablation of NF-κB p50 suppresses the myeloproliferation, showing that dysregulation of NF-κB is responsible for the myeloproliferative disease.

Fig. 1.

miR-146a–deficient mice develop myeloid and lymphoid malignancies. Mice were 18- to 22-mo-old miR-146a^−/− mice (KO) and sex- and age-matched C57BL/6 control mice (WT). (A) Incidences of myeloid and lymphoid malignancies observed in wild-type (n = 39) and KO (n = 43) mice. (B) Photograph, FACS plot, and histological analysis of a representative myeloid tumor from a KO spleen. Panels 3 and 4 show an H&E-stained spleen section. [Scale bars, (Left) 100 μm; (Right) 40 μm.] Arrows, mitotic figures. (C) Photograph, FACS plot, and histological analysis of a representative B-cell lymphoma from a KO gastrointestinal tract. Panels 3 and 5 show an H&E-stained tumor section; panel 4 shows positive immunohistochemical staining for B220 [Scale bars, (Left to Right) 100 μm in panels 3 and 4 and 40 μm in panel 5.] (D) Photograph, FACS plot, and histological analysis of a representative mixed T- and B-cell lymphoma from a KO liver. Panels 3–5 show H&E-stained liver sections. Lym, Lymphoma; Liv, relatively uninvolved liver. [Scale bars (Left to Right) are 400 μm, 100 μm, and 40 μm.]

Fig. 2.

Myeloid sarcoma is transplantable into immunocompromised Rag2^−/−γC^−/− recipient mice, causing lethal myeloid pathology. WT designates Rag2^−/−γC^−/− mice transplanted with wild-type splenocytes; KO designates Rag2^−/−γC^−/− mice transplanted with miR-146a KO splenocytes (n = 4 for wild-type and n = 4 for KO; data are representative of three independent experiments). (A) Representative bioluminiscence images of Rag2^−/−γC^−/− recipient mice splenic side view. (B) Quantification of whole-body bioluminiscence intensity from splenic side view of one representative experiment. Vertical axis is in logarithmic scale. Transduction efficiency is determined by flow cytometric analysis of GFP⁺ cells before injection. The bioluminescence intensity is normalized to the percentage of initially transduced cells. (C) Spleen weight of Rag2^−/−γC^−/− recipient mice (Student t test, *P < 0.05). (D) Photographs of spleens, kidneys, and livers from representative Rag2^−/−γC^−/− recipient mice. (E) Flow cytometric analysis of myeloid cells (defined as CD11b⁺) in representative recipient kidneys and livers. SSC, side scatter.

Fig. 3.

Chronic myeloproliferation and myelofibrosis occur in miR-146a–deficient bone marrow. Mice were 18- to 22-mo-old miR-146a^−/− mice (KO) and sex- and age-matched wild-type control mice (WT). Data are shown as Mean ± SEM. Each individual dot represents one individual mouse. (A) Flow cytometric analysis of nucleated bone marrow cells from one representative wild-type mouse and one representative KO mouse for B cells (defined as CD19⁺) and myeloid cells (defined as CD11b⁺). (B) Percentage of B cells (defined as CD19⁺) and myeloid cells (defined as CD11b⁺) in nucleated bone marrow cells by flow cytometric analysis (n = 12 for WT and n = 17 for KO from at least three independent experiments). (C) Absolute numbers of total white blood cells, lymphocytes, red blood cells, and platelets by complete blood count analysis (n = 8 for WT and n = 8 for KO). (D) Representative H&E-stained tibia sections from KO mice showing myelofibrosis and wild-type control. WT, wild-type bone marrow; adi, adipose tissue. KO1, markedly hypercellular KO bone marrow that contains virtually no megakaryocytres (arrowhead, Lower Left) or erythroid islands (outlined by dashed line, Lower Left). KO2: fibrotic KO bone marrow; arrows in the Upper Right, a broad band of fibrosis, of which there are many in this field; within the dotted line, an area of new bone formation that may represent end-stage fibrosis; (Lower Right) the interface between an area of fibrosis (fib) with entrapped myeloid cells and new bone formation (bone). [Scale bars, (Upper) 200 microns; (Lower) 40 μm.]

Fig. 4.

Spleen and bone marrow cells from miR-146a–deficient mice (KO) show increased activation of the NF-κB–mediated transcription. Mice were 18- to 22-mo-old miR-146a^−/− mice (KO) and sex- and age-matched wild-type control mice (WT). Data are shown as mean ± SEM. n represents the number of mice analyzed from at least two independent experiments. Student t test, *P < 0.05, ***P < 0.005. (A) Gene-expression analysis of NF-κB–responsive genes in wild-type (n = 7) nucleated splenocytes, KO (n = 13) nucleated splenocytes, and myeloid tumor cells (Tumor, n = 7) isolated from KO spleen by gross dissection. (B) Gene-expression analysis of NF-κB–responsive genes in wild-type (n = 9) and KO (n = 11) bone marrow cells. (C) Gene-expression analysis of NF-κB–responsive genes in CD11b⁺ population purified with MACS beads (WT n = 9 and KO n = 6). (D) Western blot analysis of the nuclear protein extracts from wild-type or KO spleen. Data are representative of three independent experiments.

Fig. 5.

Reduction in the NF-κB level by deleting the p50 subunit of NF-κB effectively rescues the myeloproliferative phenotype in miR-146a–deficient mice. All mice were 6- to 7-mo-old miR-146a^+/+, p50^+/+ (WT), miR-146a^−/−, p50^+/+ (miRKO), miR-146a^−/−, p50^+/− (miRKO p50HET), or miR-146a^−/−, p50^−/− (DKO) mice (n = 17 for WT, n = 24 miRKO, n = 10 for miRKO p50HET, and n = 9 for DKO). Data are shown as mean ± SEM from at least three independent experiments. Student t test, *P < 0.05, **P < 0.01, and ***P < 0.005. (A) Spleen weight of wild-type, miRKO, miRKO p50HET, and DKO mice. (B) Percentage of T cells (defined as CD3ε⁺), B cells (defined as CD19⁺), myeloid cells (defined as CD11b⁺), and erythroid cells (defined as Ter119⁺) in nucleated spleen and bone marrow cells from wild-type, miRKO, miRKO p50HET, and DKO mice by flow cytometric analysis.