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

IL-27 and SCF expand CD34⁻CD150⁺ LSK cells into multipotent myeloid progenitor cells.

<p>(A-B) Enhanced expansion of CD34⁻ LSK cells by IL-27 and SCF. LSK cells from WT mice were divided into two populations according to the expression of CD34, CD34⁻ LSK, and CD34⁺ LSK cells, and each population (1 × 10²) was stimulated with IL-27 and SCF. One to 4 weeks later, these stimulated cells were analyzed by flow cytometry; representative dot plots of c-Kit and Sca-1 in the Lin⁻ population at 2 weeks are shown (A). Cell numbers of these stimulated cells were counted with time course (B). (C-E) Augmented expansion of CD34⁻CD150⁺ LSK cells by IL-27 and SCF. LSK cells were further divided into eight populations (F1-F8) according to the expression of CD34, CD150, and CD41 (C), and each population (50 cells) purified by sorting was stimulated with IL-27 and SCF. One to 4 weeks later, these stimulated cells were analyzed by flow cytometry. Representative dot plots of c-Kit and Sca-1 in the Lin⁻ population at 4 weeks are shown, and the cell number of the LSK cell population in these stimulated cells was counted with time course (D). LSK populations (1 × 10³) purified from primary or IL-27/SCF-expanded F1, F4, and F5 LSK cells were differentiated into myeloid cells by IL-3 and SCF, and cell number was counted (E). Data are shown as mean ± SEM (n = 3–4) and are representative of two to three independent experiments. *<i>P</i> < 0.05, ***<i>P</i> < 0.005.</p

Malaria infection enhances IL-27 expression through IFN-γ production to promote the expansion, differentiation, and mobilization of LSK cells.

<p>(A-B) Indispensable role for IFN-γ in the expansion of LSK cells after malaria infection. WT and <i>IFN-γ</i>-deficient mice were infected with the blood stage of <i>P</i>. <i>berghei</i> XAT. Seven days later, parasitemia was determined (A) and LSK populations were analyzed by flow cytometry (B). (C-D) IFN-γ-dependent induction of IL-27 p28 subunit expression by malaria infection. RNA was prepared 7 days after the infection and analyzed for expression of <i>p28</i> by real-time RT-PCR (C), and serum p28 levels were determined by ELISA (D). (E-G) Decreased parasitemia and augmented expansion of LSK cell population in <i>IFN-γ</i>-deficient mice by IL-27. <i>IFN-γ</i>-deficient mice were hydrodynamically injected with IL-27-expression vector or control vector at days 0 and 4 after infection; at day 7, parasitemia was measured (E), LSK population was analyzed by flow cytometry (F), and cell numbers of LSK cells and neutrophils were counted (G). Data are shown as mean ± SEM (n = 3–5) and are representative of at least two independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.005.</p

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

LSK cells expanded by IL-27 and SCF are multipotent myeloid Progenitors.

<p>(A) Flow cytometry histogram analysis of cell surface markers in the LSK cells expanded by IL-27 and SCF for 4 weeks and primary BM cells using antibodies as indicated (solid line) and their control antibodies (plane line with shading). (B-C) Augmented potential of the LSK cells expanded by IL-27 and SCF to differentiate into mDCs. LSK populations (3 × 10³) purified from the LSK cells expanded by IL-27 and SCF for 2 and 4 weeks and primary BM cells were stimulated with GM-CSF. After the indicated time, these stimulated cells were analyzed for the expression of MHC class II and CD11c (B), and the cell numbers of mDC (MHC class II⁺CD11c⁺) were counted (C). (D-E) Decreased potential of the LSK cells expanded by IL-27 and SCF to differentiate into pDCs (Siglec H⁺PDCA1⁺CD11c⁺) and cDCs (Siglec H⁻PDCA1⁻CD11c⁺). (F-G) Enhanced potential of the LSK cells expanded by IL-27 and SCF for 2 weeks to differentiate into neutrophils. Neutrophil; Ly6G⁺CD11b⁺, macrophage; F4/80⁺CD11b⁺, mast cell; c-Kit⁺FcεR1α⁺CD11b⁻, basophil; CD49b⁺FcεR1α⁺CD11b⁻. (H) Abrogated potential of the LSK cells expanded by IL-27 and SCF for 2 weeks to differentiate into T (day 18) and B (day 15) cells. (I) Enhanced potential of the LSK cells expanded by IL-27 and SCF to differentiate into neutrophils <i>in vivo</i>. LSK populations purified from the LSK cells from CD45.1 congenic mice expanded by IL-27 and SCF for 2 weeks were transferred into sublethally irradiated CD45.2 recipient mice with the same congenic BM cells. Development of Gr-1⁺CD11b⁺ neutrophils in the BM and spleen were analyzed by flow cytometry after 9 days, and the percentage of neutrophils in each CD45.1⁺ and CD45.2⁺ cells was compared. (J) Increased expression of transcription factors critical for the differentiation into myeloid cells in the LSK cells expanded by IL-27 and SCF. RNA was prepared from the LSK population purified from LSK cells expanded for 2 weeks together with other progenitors and subjected to real-time RT-PCR. Data are shown as mean ± SEM (n = 2–4) and are 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

Contribution of IL-12/IL-35 Common Subunit p35 to Maintaining the Testicular Immune Privilege

<div><p>The testis is an organ with immune privilege. The comprehensive blood–testis barrier formed by Sertoli cells protects autoimmunogenic spermatozoa and spermatids from attack by the body’s immune system. The interleukin (IL)-6/IL-12 family cytokines IL-12 (p35/p40), IL-23 (p19/p40), IL-27 (p28/Epstein-Barr virus−induced gene 3 [EBI3]), and IL-35 (p35/EBI3) play critical roles in the regulation of various immune responses, but their roles in testicular immune privilege are not well understood. In the present study, we investigated whether these cytokines are expressed in the testes and whether they function in the testicular immune privilege by using mice deficient in their subunits. Expression of EBI3 was markedly increased at both mRNA and protein levels in the testes of 10- or 12-week-old wild-type mice as compared with levels in 2-week-old mice, whereas the mRNA expression of p40 was markedly decreased and that of p35 was conserved between these two groups. Lack of EBI3, p35, and IL-12 receptor β2 caused enhanced infiltration of lymphocytes into the testicular interstitium, with increased interferon-γ expression in the testes and autoantibody production against mainly acrosomal regions of spermatids. Spermatogenic disturbance was more frequently observed in the seminiferous tubules, especially when surrounded by infiltrating lymphocytes, of these deficient mice than in those of wild-type mice. In particular, p35-deficient mice showed the most severe spermatogenic disturbance. Immunohistochemical analyses revealed that endothelial cells and peritubular cells in the interstitium were highly positive for p35 at both ages, and CD163⁺ resident macrophages positive for p35 and EBI3, possibly producing IL-35, were also detected in the interstitium of 12-week-old mice but not those of 2-week-old mice. These results suggest that p35 helps in maintaining the testicular immune privilege, in part in an IL-35-dependent manner.</p></div

Spermatogenic disturbance in mice deficient in EBI3, p35, and IL-12Rβ2.

<p>(A) Sections of testes of WT mice and deficient mice (12 weeks old; <i>n</i> = 10 per group) were stained with hematoxylin and eosin. Representative histology images are shown. Infiltrating lymphocytes (arrow heads) were observed in the interstitium around the seminiferous tubules, especially with exhibition of spermatogenic disturbance (*), in deficient mice. (B) More than 100 round or oval seminiferous tubules were counted in each mouse, and the percentage of seminiferous tubules showing the disappearance of mature germ cells and desquamation of germinal epithelium was calculated. Data are shown as mean ± SD. *<i>P</i><0.05 and **<i>P</i><0.001 compared with WT mice, respectively. (C) Sections of testes of WT mice and IL-10-deficient mice were also similarly stained with hematoxylin and eosin. Representative histology images are shown. Similar results were obtained in two independent experiments.</p

Increased mRNA expression of EBI3 and decreased mRNA expression of p40 and WSX-1 in testes of adult WT mice.

<p>Total RNA was prepared from various tissues of 2- and 10-week-old WT mice (<i>n</i> = 3 per group) and subjected to real-time RT-PCR using various sets of primers. Ratio of relative mRNA expression to the mRNA expression level in testes of 2-week-old mice was calculated. Data are shown as mean ± SD. *<i>P</i><0.05 compared with 2-week-old mice. Similar results were obtained in two independent experiments.</p

Enhanced infiltration of lymphocytes in testes of mice deficient in EBI3, p35, and IL-12Rβ2.

<p>(A) Cryostat sections of testes of WT mice and deficient mice (12 weeks old; <i>n</i> = 5 per group) were immunohistochemically stained with anti-CD4, anti-CD8, or anti-B220. The sections were also counterstained with methyl green. Representative histology images are shown. Positive cells are shown as gray spots. Dashed line indicates basal lamina of the seminiferous tubules. (B) The histopathological changes were evaluated by counting the number of infiltrating lymphocytes per square millimeter of testicular interstitium. Data are shown as mean ± SD. *<i>P</i><0.05. Similar results were obtained in more than two independent experiments. KO, knockout.</p