IL-27 limits HSPC differentiation during infection and protects from stem cell exhaustion
- University of Pennsylvania School of Veterinary Medicine, United States
- Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, United States
- University of Colorado, Anschutz Medical Campus, United States
Figures
Figure 1 with 2 supplements
IL-27 regulates monopoiesis during infection.
(A) Schematic of infection and TAM dosing strategy for Procr-Ai6 mice. (B) Procr-Ai6 mice were infected and representative flow plots of long-term hematopoietic stem cells (LTHSCs) (Lin (CD3, NK1.1, B220, Ly6G)−, Sca-1+, CD117 (cKit)+, CD135−, CD48−, CD150+) from naive (left) and infected (right) mice are shown. Proportions of zsGreen+ LTHSCs were then quantified in both groups. (C) Proportions of zsGreen+ monocytes (CD3−, B220−, CD11b+, Ly6C+, Ly6G−) in the spleen and peritoneal exudate cells (PECs) of naive and infected mice. (D) Schematic of infection of WT and Il27–/– mice for E and F. (E) Numbers of LTHSCs and granulocyte progenitors (GPs) (CD3−, NK1.1−, B220−, CD117+, CD34+, CD16/32hi, Ly6C+, CD135−, CD115−) in the bone marrow of WT and Il27–/– infected mice throughout infection. (F) Monocyte progenitors (MPs) (CD3−, NK1.1−, B220−, CD117+, CD34+, CD16/32hi, Ly6C+, CD135−, CD115+; orange box) in the bone marrow of infected WT and Il27−/− mice throughout infection. Representative flow plots are shown (left) and quantified (right). Statistical significance was tested by one-way ANOVA with Sidak’s correction. *, **, and *** correspond to p-values ≤0.05, 0.01, and 0.001, respectively. N = 3–5 mice/group and data shown are representative of two to three repeated experiments. Error bars indcate standard error of the mean (SEM).
Figure 1—figure supplement 1
IL-27 regulates monopoiesis during infection.
(A) The proportion of zsGreen+ monocyte dendritic cell progenitors (MDPs) (CD3−, NK1.1−, B220−, CD117+, CD34+, CD16/32lo, CD115+), monocytes (CD3−, B220−, Ly6G−, CD115+, CD135−, CD117−, CD11b+, Ly6C+), and neutrophils (CD3−, B220−, CD11b+, CD115−, Ly6G+) in the BM of naive and infected Procr-Ai6 mice. (B) The proportion of zsGreen+ CCR2hi/lo CX3CR1hi/lo monocytes in the spleen and peritoneal exudate cells (PECs) in naive and infected Procr-Ai6 mice. (C) Number of MDPs in WT and Il27–/– mice in the bone marrow during infection. (D) Numbers of monocytes in the liver in WT and Il27−/− throughout infection. (E) WT and Il27–/– mice were injected I.V. with fluorescent anti-CD45 to label immune cells in the vasculature. Numbers of I.V. label- and CCR2hiCX3CR1lo monocytes in the liver were then quantified throughout infection. (F) IFNγ-Thy1.1 reporter mice were infected and treated with an anti-p28 blocking antibody. Numbers of IFNγ+ cells in the bone marrow were then assessed by flow cytometry. Representative flow plots are shown (left) and quantified (right). Statistical significance was tested by Welch’s t-test (A, F) or one-way ANOVA with Sidak’s correction (B–E). *, **, ***, and **** correspond to p-values ≤0.05, 0.01, 0.001, and 0.0001, respectively. N = 3–5 mice/group and data shown are representative of two to three repeated experiments. Error bars indcate SEM.
Figure 1—figure supplement 2
Gating strategy for flow cytometric analysis of cells in the bone marrow and periphery.
Representative flow plots from the bone marrow (top) and spleen (bottom) are shown indicating the gating strategy used to identify various cell populations. The combination of surface markers used is listed in the tables (bottom) and is color-coded to correspond to both the developmental schematic pictured as well as the corresponding flow gate on the representative plots.
Figure 2 with 1 supplement
IL-27 regulates monopoiesis during infection in a cell-intrinsic manner.
(A) CD4-IL-27R mice were infected and MPs (orange box) measured at 5 dpi in the BM. Representative flow plots are shown (left) and quantified (right). (B) Schematic of BM chimeras generated from WT (CD45.1) and Il27ra–/– (CD45.1.2) bulk bone marrow that were then infected and used in C and D. Schematic created with BioRender.com. (C) The proportion of long-term hematopoietic stem cells (LTHSCs) (left) and MPs (right) from each respective donor lineage in the bone marrow of both the single (left two bars) and mixed (right two bars) chimeras at 5 dpi. (D) Representative flow plots pre-gated on splenic monocytes (left plot) and then down-gated on CCR2hiCX3CR1lo (right plot) monocytes at 5 dpi. WT-derived monocytes are shown in the solid-dark gray, while Il27ra–/–-derived cells are in the dashed-light gray. The proportion of each donor lineage that contributes to mature CCR2hiCX3CR1lo splenic monocytes at 5 dpi was then quantified (far right). Statistical significance was tested by either Welch’s t-test (A) or one-way ANOVA with Sidak’s correction (C, D). * and **** correspond to p-values ≤0.05 and 0.0001, respectively. N = 3–5 mice/group and data shown are representative of two to three repeated experiments. Error bars indcate SEM.
Figure 2—figure supplement 1
IL-27 regulates monopoiesis during infection and post-irradiation in a cell-intrinsic manner.
(A) IL-27R⍺ expression on CD4+ (CD19-CD3+CD8⍺-) T cells, CD8+ (CD19-CD3+CD4-) T cells, and B cells (CD19+CD3-) in CD4-IL27R mice at 5 dpi. (B) Reconstitution and chimerism in the peripheral blood of 1:1, WT:Il27r–/– chimeras was measured over time. T (top) and B (bottom) cells from each lineage are shown. (C) The proportion of long-term hematopoietic stem cells (LTHSCs) (left) and MPs (right) from each respective donor lineage in the bone marrow post-reconstitution and pre-infection. (D) The proportion of CCR2hiCX3CR1lo monocytes from each respective donor lineage in the spleen and liver post-reconstitution and pre-infection. Statistical significance was tested by one-way ANOVA with Sidak’s correction. ** , ***, and **** correspond to p-values ≤0.01, 0.001, and 0.0001, respectively. N = 3–5 mice/group, and data shown are representative of two repeated experiments. Error bars indcate SEM.
Figure 3 with 1 supplement
IL-27p28 is produced in the BM during infection.
(A) WT mice were infected, and the bone marrow harvested throughout infection. BM was either washed with media or cultured for 24 hr, and supernatant collected. Both the wash and supernatant were analyzed by ELISA for IL-27p28. (B) Il27GFP reporter mice were infected and their bone marrow analyzed by flow cytometry throughout infection. A representative flow plot at 5 dpi of bulk marrow is shown (left) and proportion of immune cell contribution to the GFP+ population measured (right). N=3 biological replicates, with two technical replicates performed. (C) Numbers of GFP+ cells in the spleen, blood, and BM of infected mice were quantified throughout infection (left). Proportions of GFP+ CD45+ cells were analyzed by cell type in the blood, spleen, and peritoneal exudate cell (PEC) during infection (right). (D) Femurs from infected ProcrtdTomato+Il27GFP+ mice were harvested, sectioned, and imaged at ×40 magnification. The total imaged femur is shown (top), with zoomed-in sections shown below for increased detail of cellular localization (bottom). Arrows indicate areas of localization with ProcrtdTomato+ and Il27GFP+ cells. Numbers of ProcrtdTomato+ and Il27GFP+ cells were then counted in eight randomly selected regions throughout the marrow (see Figure 3—figure supplement 1). GFP+ cells were normalized to tdTomato+ cells and the eight counted regions classified as areas of hyper- or hypo-vascularization according to proximity of the region to the labeled vasculature. This allowed quantification of the localization of cells (bottom right). N = 3–5 mice/group, and data shown are representative of two repeated experiments. Error bars indcate SEM.
Figure 3—figure supplement 1
IL-27p28 is produced in the bone marrow during infection.
(A) Il27GFP mice were infected, and the numbers of each CD45+, immune cell type that expressed GFP in the BM or periphery were quantified throughout infection. (B) Levels of GFP expression were measured in bulk monocytes (red) and dendritic cells (blue) in the bone marrow (left) and spleen (right). (C) Femurs from naive Il27GFP mice were stained for Sca-1 and imaged at ×40 magnification. Zoomed-in images are shown (right) of the indicated areas, with the yellow arrows indicating positively stained, Sca-1+ cells. (D) The merged image shown in Figure 3D is separated by channel (GFP, tdTomato, and AF647, from top to bottom). Eight randomly selected regions that were used for quantification of either GFP+ or tdTomato+ cells are shown (top), and the quantification of cells in each region is shown next to the corresponding fluorescence channel (right). N = 3–5 mice/group, and data shown are representative of two repeated experiments. Error bars indcate SEM.
Figure 4 with 1 supplement
Expression of the IL-27R subunits gp130 and IL-27Rα during hematopoietic development.
(A) Schematic ball-and-stick model of hierarchical hematopoietic development. Cell type colors are maintained for reference in the following panels. (B) Progenitors and progeny shown in (A) were analyzed in WT and infected mice (at 5 dpi) by flow cytometry for the gp130 receptor. 10,000 live cells were concatenated from n = 3 mice, and representative flow plots are shown. The fold change of the MFI of the receptor in each cell type over the FMO control for that cell type was then quantified. Error bars indicate SEM. (C) Cells in (B) were analyzed for expression of the IL-27Rα and analyzed as above. (D) Bulk bone marrow was isolated from Irgm1dsRed reporter mice. 22 × 106 cells were plated per condition and stimulated with 20 ng/ml of IL-27 for 0, 6, or 48 hr and RFP expression measured by flow cytometry. Data are representative of two repeated experiments.
Figure 4—figure supplement 1
IL-27Rα transcript expression during hematopoiesis and differential sensitivity of long-term hematopoietic stem cell (LTHSC) and common myeloid progenitor (CMP) to IL-27.
(A) Expression levels of Il27ra are shown from the Haemosphere RNA-seq dataset and online tool as well as the Blood Spot dataset and visualizer (B). (C) Representative flow plots of RFP expression in LTHSCs, CMPs, and lin+ T, NK, and B cells 48 hr post-stimulation with IL-27 are shown (top). In vivo treatment of Irgm1RFP reporter mice with 1 μg of recombinant IFN-γ was performed and all populations analyzed as in (C). Representative flow plots are shown.
Figure 5 with 1 supplement
Developmental expression of hematopoietic cytokine receptors.
(A) Expression of CD132 (IL-2Rγ) and (B) CD131 (GM-CSF/IL-3/IL-5Rβ) was analyzed by flow cytometry on progenitors and progeny in the BM of naive and 5 dpi mice as in Figure 4. (C) Expression of IL-6R⍺ and (D) GM-CSFR⍺ was measured as in (A).
Figure 5—figure supplement 1
Developmental expression of additional cytokine receptors.
Figure 6 with 2 supplements
IL-27 limits infection-induced hematopoietic stem and progenitor cell (HSPC) polarization.
WT and Il27ra–/– mice were infected for 5 days, long-term hematopoietic stem cells (LTHSCs) sorted, and cultured in MethoCult for 10–12 days before being analyzed by flow cytometry. (A) 10,000 live cells from n = 3–4 mice/group (WT (naive +5 dpi) and Il27ra–/– (5 dpi)) were concatenated and dimensionally reduced via UMAP. (B) Numbers of cells in each cluster from (A) were measured. (C) X-shift analysis was used to identify the expression level of each marker used in the clustering performed in (A). Five of the dominant clusters from (B) are shown. (D) Clusters 1, 10, and 11, all involved in monocyte development, are highlighted. (E) Contribution of clusters 1, 10, and 11 to each genotype is shown (left) and quantified (right). (F) Single bone marrow chimeras were generated with bone marrow from infected WT and Il27ra–/– mice or naive controls (left). The numbers of single-donor-derived, mature monocytes were then measured at 14 weeks post-transplant and compared based on WT vs Il27ra–/– donor (right). Statistical significance was tested by one-way ANOVA with Sidak’s correction. *, ***, and **** correspond to p-values ≤0.05, 0.001, and 0.0001, respectively. N = 3–5 mice/group, and data shown are representative of two repeated experiments. Error bars indicate SEM. Schematic in (F) created with BioRender.com.
Figure 6—figure supplement 1
Gating strategy for sorting of long-term hematopoietic stem cells (LTHSCs) used in monocyte differentiation.
LTHSCs were sorted from WT naive, WT 5 dpi, and Il27ra–/– 5 dpi mice. The gating strategy is shown as well as the purity of sorted samples (last panel).
Figure 6—figure supplement 2
Development and validation of an unbiased flow cytometry approach for the analysis of MethoCult colonies.
WT and Il27ra-/- mice were infected, long-term hematopoietic stem cells (LTHSCs) sorted, and cultured in MethoCult for either 5 or 10 days. (A) Colony phenotypes were analyzed by microscopy and counted by eye. (B) Colonies were collected at either 5 or 10 days post-culture (dpc) and analyzed by flow cytometry. 500 live cells from n = 3–4 mice/group were concatenated and dimensionally reduced via UMAP. UMAP plots are shown, and Ki67 expression is overlayed. (C) X-shift analysis was used to identify the expression level of each marker used in the clustering performed in (B). (D) Numbers of cells in each cluster from (B) were measured. (E) Contribution of each genotype to each cluster was quantified. Statistical significance was tested by one-way ANOVA with Sidak’s correction. *, ***, and **** correspond to p-values ≤0.05, 0.001, and 0.0001, respectively. N = 3–4 mice/group, and data shown are representative of two repeated experiments. Error bars indicate SEM.
Figure 7 with 1 supplement
Testing the impact of IL-27 on functionality and fitness of hematopoietic stem and progenitor cells (HSPCs) during infection.
(A) WT and Il27ra–/– were infected, treated with 5 mg BRDU, and kept on 1 mg/ml BRDU in the drinking water throughout infection. Incorporation of BRDU was then measured in Lin-Sca-1+cKit+ (LSK) progenitor cells in comparison to Ki67 at 5 dpi. Representative flow plots are shown (left) and the proportion of BRDU+Ki67+ cells quantified (right). (B) Schematic for the generation of BM chimeras from infected mice used in (C), schematic created with BioRender.com. (C) As in Figure 6F, numbers of long-term hematopoietic stem cells (LTHSCs) from recipients receiving single-transfer donor marrow from either uninfected or infected mice were measured based on genotype. (D) Secondary transplant chimeras from mice that originally received naive or infected marrow were generated as shown (left) image created with BioRender.com. Survival of recipients was then assessed (right). Statistical significance was tested by Welche’s t-test (A) or one-way ANOVA with Sidak’s correction (C). * corresponds to p-values ≤0.05. N = 3–5 mice/group, and data shown are representative of two experiments. Error bars indicate SEM.
Figure 7—figure supplement 1
Testing hematopoietic stem and progenitor cell (HSPC) survival and fitness.
(A) FMO gating controls for Ki67 and BRDU in the LSK population corresponding to Figure 7A. (B) Long-term hematopoietic stem cells (LTHSCs) from WT and Il27ra–/– BM at 5 dpi were stained for Annexin V (AV) expression and free-amine staining viability dye (LD). This allowed the measurement of apoptotic (AV+LD-), late apoptotic (AV+LD+), and necrotic (AV-LD+) cells. The proportion of LTHSCs in each of these stages was quantified. (C) Monocyte genotype was analyzed at 9 weeks post-transplant (wpt) from either naive WT (CD45.1) or Il27ra–/– (CD45.1.2) donors (top) or from infected donors (bottom), including a 1:1 mix of infected WT and Il27ra–/– donors. Statistical significance was tested by Welch’s t-test. NS indicates results are “not significant.” N = 5 mice/group, and data shown are representative of two repeated experiments. Error bars indicate SEM.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/105876/elife-105876-mdarchecklist1-v1.docx
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Source data 1
These are the raw data points are all graphed figures.
- https://cdn.elifesciences.org/articles/105876/elife-105876-data1-v1.xlsx
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IL-27 limits HSPC differentiation during infection and protects from stem cell exhaustion
eLife 14:RP105876.
https://doi.org/10.7554/eLife.105876.3
