MicroRNA-146a acts as a guardian of the quality and longevity of hematopoietic stem cells in mice
- California Institute of Technology, United States
- University of California, Los Angeles, United States
- University of Utah, United States
Figures
Figure 1 with 1 supplement
Accelerated HSC decline and myeloproliferation in miR-146a–deficient mice during chronic inflammation.
(A) MiR-146a and miR-146b expression in FACS-sorted HSPC populations by Taqman RT-qPCR. Lin-BM, lineage negative bone marrow cells; L−S+K+ (LSK), Lin−Sca1+cKit+; HSC, LSK CD150+CD48−; L−S−K+, Lin−Sca…
Figure 1—figure supplement 1
HSPC FACS analysis and colony-forming ability in 6-week-old and 4-month-old WT and miR-146a KO mice.
(A) Quantification of number of white blood cells (CD45), T cells (CD3ε), myeloid cells (CD11b), B cells (CD19), nucleated erythrocytes (Ter119), LSK cells, LSK CD150+CD48− HSCs and LSK CD150+CD48−EP…
Figure 2 with 1 supplement
Progressive functional decline of miR-146a-deficient HSCs.
(A) Ratio of CD45.2+ over CD45.1+ cells of various lineages in pretransplanted bone marrow (BM) mixtures consisted of equal numbers of CD45.2+ WT and CD45.1+ WT total BM cells (blue bar, CD45.2 …
Figure 2—figure supplement 1
Functional decline of miR-146a-deficient HSCs.
(A) Related to Figure 2C–E. Equal numbers of BM HSCs (LSK CD150+CD48−) were sorted from 8-week-old CD45.2+ WT and CD45.2+ miR-146a KO mice. Fifty WT or KO HSCs were mixed with 500,000 CD45.1+ WT …
Figure 3 with 1 supplement
Hematopoietic-intrinsic and hematopoietic-extrinsic contribution to hematopoietic defects.
(A)–(J) Reciprocal bone marrow (BM) transplant performed by transferring WT donor BM cells to WT recipient mice (WT to WT), WT donor BM cells to miR-146a KO recipient mice (WT to KO), miR-146a KO …
Figure 3—figure supplement 1
Hematopoietic-intrinsic and hematopoietic-extrinsic contribution to hematopoietic defects.
(A)–(U) The same reciprocal BM transplant experiment as Figure 3A–J. (A) Kaplan–Meier survival curve of the four transplant groups (WT to WT, WT to KO, KO to WT, and KO to KO). (B) FACS plot of a …
Figure 4
MiR-146a-deficient lymphocytes contribute to the HSC defect and myeloproliferation.
(A)–(M) Age-and-sex-matched WT, miR-146a KO (miR KO), Rag1 KO and miR-146a/Rag1 double KO (miR/Rag1 DKO) mice were allowed to age to 10-month-old before harvested for analysis. (A) Representative …
Figure 5 with 2 supplements
NF-κB regulates HSC homeostasis during chronic inflammation.
(A)–(C) 8-Week-old WT (WT-GFP), Mir146a+/− (miRHET-GFP), and Mir146a−/− (miRKO-GFP) NF-κB-GFP reporter mice were subjected to repeated intraperitoneal LPS stimulation (3 mg LPS/kg of body weight …
Figure 5—figure supplement 1
NF-κB activity in HSPCs and peripheral blood of NF-κB-GFP reporter mice.
(A) Regular WT mice and WT NF-κB-GFP reporter mice were challenged with one dose of LPS intraperitoneally (2 mg LPS/kg body weight). GFP+ cells in various lineages were quantified by FACS 6 hr after …
Figure 5—figure supplement 2
NF-κB regulates HSPC homeostasis during chronic inflammation.
(A)–(C) Related to Figure 5A–C. 8-Week-old WT-GFP, miRHET-GFP, and miRKO-GFP reporter mice were subjected to repeated intraperitoneal LPS stimulation (3 mg LPS/kg of body weight every other day) for …
Figure 6 with 1 supplement
NF-κB-regulated pro-inflammatory cytokine IL-6 is an important driver of HSC depletion and myeloproliferation.
Gene expression of IL-6 in bone marrow cells (BM) (A) and spleen cells (B) of aging WT, miR KO, and miR/p50 DKO mice measured by RT-qPCR. All mice are age- and sex-matched 6- to 10-month-old female …
Figure 6—figure supplement 1
NF-κB–regulated proinflammatory cytokine IL-6 is an important driver of HSC depletion and myeloproliferation.
(A) Related to Figure 6A,B. Gene expression of TNFα in BM cells and spleen cells of aging WT, miRKO, and miR/p50 DKO mice measured by RT-qPCR. All mice are age- and sex-matched 6- to 10-month-old …
Figure 7
Analysis of cellular source and direct cellular target of IL-6.
Serum level of IL-6 measured by ELISA in 2-month-old WT, miR KO, Rag1 KO, and miR/Rag1 DKO mice stimulated with LPS (1 mg LPS /kg body weight) intraperitoneally for 6 hr (A) IL-6 concentration …
Figure 8 with 1 supplement
Derepression of TRAF6, a miR-146a target, is responsible for bone marrow failure.
Transcript levels of TRAF6 (A) and IRAK1 (B) in WT and miR-146a KO (miR KO) bone marrow–derived macrophages (BMMs) stimulated with LPS, which was added to the culture medium at 0 and 48 hr (black …
Figure 8—figure supplement 1
Gene expression analysis of miR-146a targets in BM HSPCs.
(A) Related to Figure 8D. Expression of TRAF6 and IRAK1 in BM HSPCs tranduced with pMIG-Luc, pMIG-TRAF6, or pMIG-IRAK viruses (∼50% GFP+). (B) Related to Figure 8C. Gene expression of STAT1 in …
