Promotion of Expansion and Differentiation of Hematopoietic Stem Cells by Interleukin-27 into Myeloid Progenitors to Control Infection in Emergency Myelopoiesis.

Emergency myelopoiesis is inflammation-induced hematopoiesis to replenish myeloid cells in the periphery, which is critical to control the infection with pathogens. Previously, pro-inflammatory cytokines such as interferon (IFN)-α and IFN-γ were demonstrated to play a critical role in the expansion of hematopoietic stem cells (HSCs) and myeloid progenitors, leading to production of mature myeloid cells, although their inhibitory effects on hematopoiesis were also reported. Therefore, the molecular mechanism of emergency myelopoiesis during infection remains incompletely understood. Here, we clarify that one of the interleukin (IL)-6/IL-12 family cytokines, IL-27, plays an important role in the emergency myelopoiesis. Among various types of hematopoietic cells in bone marrow, IL-27 predominantly and continuously promoted the expansion of only Lineage-Sca-1+c-Kit+ (LSK) cells, especially long-term repopulating HSCs and myeloid-restricted progenitor cells with long-term repopulating activity, and the differentiation into myeloid progenitors in synergy with stem cell factor. These progenitors expressed myeloid transcription factors such as Spi1, Gfi1, and Cebpa/b through activation of signal transducer and activator of transcription 1 and 3, and had enhanced potential to differentiate into migratory dendritic cells (DCs), neutrophils, and mast cells, and less so into macrophages, and basophils, but not into plasmacytoid DCs, conventional DCs, T cells, and B cells. Among various cytokines, IL-27 in synergy with the stem cell factor had the strongest ability to augment the expansion of LSK cells and their differentiation into myeloid progenitors retaining the LSK phenotype over a long period of time. The experiments using mice deficient for one of IL-27 receptor subunits, WSX-1, and IFN-γ revealed that the blood stage of malaria infection enhanced IL-27 expression through IFN-γ production, and the IL-27 then promoted the expansion of LSK cells, differentiating and mobilizing them into spleen, resulting in enhanced production of neutrophils to control the infection. Thus, IL-27 is one of the limited unique cytokines directly acting on HSCs to promote differentiation into myeloid progenitors during emergency myelopoiesis

STAT1 and STAT3 are important for expansion and differentiation of LSK cells by IL-27 and SCF.

<p>(A) mRNA expression of <i>STAT1</i> and <i>STAT3</i> in the LSK cells expanded by IL-27 and SCF for 2 weeks and in primary LSK cells. (B) Flow cytometry histogram analysis of primary LSK cells after stimulation with IL-27 and SCF for 60 min using anti-pY-STAT1 or anti-pY-STAT3 (solid line) and control antibody (plain line with shading). (C-D) Dispensable role for STAT1 in the expansion of LSK cells in response to IL-27 and SCF. LSK cells (1 × 10³) purified from BM cells of WT (129) mice and <i>STAT1</i>-deficient mice were expanded by IL-27 and SCF for 2 weeks and analyzed for expression of LSK phenotype (C). The cell numbers of LSK and LS⁻K cell populations were counted. Purified cells of the <i>STAT1</i>-deficient LSK and LS⁻K cells (1 × 10³) were further stimulated by IL-27 and SCF for more 1 week and the cell number of expanded cells was counted (D). (E-G) Contribution of STAT1 to the differentiation into mDC (E) and myeloid cells (F) of LSK cells expanded by IL-27 and SCF for 2 weeks together with their mRNA expression of transcription factors (G). (H) Indispensable role for STAT3 in the expansion of LSK cells in response to IL-27 and SCF. Purified GFP⁻ <i>STAT3</i>^{<i>flox/flox</i>} LSK cells and GFP⁺ <i>STAT3</i> cKO LSK cells (1 × 10²) were expanded by IL-27 and SCF for 10 days and analyzed for expression of LSK phenotype and their cell numbers. (I-K) Critical role for STAT3 in the differentiation into mDC (I) and myeloid cells (J) of LSK cells expanded by IL-27 and SCF for 10 days together with their mRNA expression of transcription factors (K). Data are shown as mean ± SEM (n = 3–4) and representative of two to three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.005.</p

IL-27 and SCF most strongly expand only LSK cells among various kinds of BM progenitors and differentiate them into myeloid progenitors retaining the LSK phenotype.

<p>(A) Expansion of only the c-Kit⁺CD11b⁻Lin⁻ population by IL-27 and SCF in BM cells. Total BM cells were divided into two populations positive or negative for Lin markers, and the Lin⁻ population was further divided into four populations positive for either c-Kit or CD11b, both positive, or both negative. Each population (5 × 10³) purified by sorting was stimulated by either IL-27 or SCF alone, or both, in a round 96-well plate and photographed 2 weeks later. (B-E) Expansion of only the LSK cell population by IL-27 and SCF among various BM progenitors. BM progenitors (4–5 × 10³) purified by sorting were stimulated with IL-27 and SCF. Cell expansion was photographed at indicated periods (B) and cell number of the expanded LSK cell population retaining the LSK phenotype was counted at 1 week (C). Time kinetic analysis of cell numbers in the LSK population retaining the LSK phenotype (D). Representative flow cytometry dot plot analysis of c-Kit and Lin (upper) and of c-Kit and Sca-1 in the Lin⁻c-Kit⁺ population (lower) of expanded LSK cells and progenitor cells at 1 week (E). (F) Expansion of LSK populations <i>in vivo</i> by IL-27 in <i>IL-27</i> Tg mice. LSK cells were purified by sorting from BM cells of <i>GFP</i> Tg mice and transferred into non-lethally irradiated WT and <i>IL-27</i> Tg mice. Twenty days later, BM and spleen in these recipient mice were analyzed for GFP⁺ LSK populations. Data are shown as mean ± SEM (n = 2–3) and are representative of at least two independent experiments. *<i>P</i> < 0.05. (G-H) Augmented and prolonged expansion of the LSK cell population by only IL-27 and SCF <i>in vitro</i>. LSK cells (1 × 10³) from WT mice were stimulated by various cytokines together with SCF for 1 to 4 weeks, the stimulated cells were analyzed by flow cytometry (G), and cell number was counted with time course (H). Data are representative of at least two independent experiments.</p

IL-27 plays an important role in expansion, differentiation, and mobilization of LSK cells to control malaria infection.

<p>(A-D) Reduced induction of LSK cell population in <i>WSX-1</i>-deficient mice after malaria infection, accompanied by increased parasitemia and comparable production of IFN-γ. WT or <i>WSX-1</i>-deficient mice were infected with the blood stage of <i>P</i>. <i>berghei</i> XAT. Seven days later, parasitemia (A) and serum IFN-γ level (B) were determined, and LSK populations in the BM and spleen were analyzed by flow cytometry, and representative dot plots of c-Kit and Sca-1 in the Lin⁻ population are shown (C). Cell number of the LSK cell population was counted (D). (E) Augmented potential of LSK cells to differentiate into myeloid cells by malaria infection. LSK cells (1 × 10³) in the BM and spleen of the malaria-infected or non-infected WT mice were purified and differentiated into myeloid cells <i>in vitro</i> by IL-3 and SCF, and cell number of differentiated cells was counted. (F-G) Reduced cell number of neutrophils in <i>WSX-1</i>-deficient mice after malaria infection. The BM and spleen cells were analyzed for expression of Gr-1 and CD11b at 7 days after the infection (F), and cell number of neutrophils (Gr-1⁺CD11b⁺) was counted (G). (H-I) Decreased parasitemia in the <i>WSX-1</i>-deficient mice transferred with LSK cells purified from BM cells of the malaria-infected WT mice. LSK cells purified from BM cells of the infected WT CD45.1 mice were transferred into non-lethally irradiated <i>WSX-1</i>-deficient CD45.2 mice 7 days before infection. Neutrophil population in the BM and spleen was analyzed by flow cytometry, and representative dot plots of CD11b and Gr-1 in the CD45.1⁺ population are shown (H). Parasitemia was measured 4 and 7 days after the infection (I). Data are shown as mean ± SEM (n = 3–9) and are representative of at least two independent experiments. *<i>P</i> < 0.05, ***<i>P</i> < 0.005.</p