Figure 5. Deletion of H1.2 or H1.4 biases murine hematopoietic cells towards an eosinophil lineage.
(a–c) Analysis of circulating neutrophils and eosinophils in wild type (wt) and H1.2/H1.4-double deficient mice (H1.2/H1.4 -/-). p Values, derived from unpaired two-tailed T tests, are indicated. (a) Flow cytometry analysis of neutrophils (Gr1 positive, CD115 negative) in whole blood of adult age-matched animals (wt n = 15, H1.2/H1.4 - / - n = 14), other immune cell types are shown in Figure 5—figure supplement 1. (b) Flow cytometry analysis of absolute amounts of neutrophils (Ly-6G positive cells) in whole blood (n = 4). (c) Flow cytometry analysis of amounts of eosinophils (SiglecF positive) in whole blood (n = 4). H1.2/H1.4 - / - animals show enhanced eosinophil numbers in circulation. d-f Lineage-negative hematopoietic stem cells were sorted from murine bone marrow and cultured for 6 days in the presence of various cytokines allowing differentiation into several immune cell lineages. Depicted are CD11b positive cells (d) and the fraction of neutrophils (e) or eosinophils (f) within CD11b-positive cells. Each dot represents an independent experiment consisting of two mice per genotype, the mean of the two wild type animals was set to one and the mean of the two H1.2/H1.4 - / - animals is depicted relative to wt. (a–f) Error bars are SEM.
Figure 5—figure supplement 1. Gating strategies and circulating leukocytes.
(a, b) Gating strategy for different subsets of immune cells in whole blood and bone marrow. (a) Single cells are gated for expression of CD45, then for CD3. CD3 positive cells are divided into CD4 and CD8 positive T cells, CD3 negative fractions are divided into neutrophils (GR1 positive, CD115 negative), monocytes (GR1 positive CD115 positive) and ‘rest’ (B cells, NK cells and others). (b) Gating strategy for neutrophils, eosinophils and ageing markers of neutrophils. Single cells are gated for viable (DAPI negative) cells and subsequently into neutrophils (Ly-6G positive) and eosinophils (SiglecF positive). Neutrophils are divided into young and aged populations by expression of surface markers CD62L and CXCR4.(c–f) Abundance of the indicated leukocytes in whole blood of wild type (wt, n = 15) and H1.2/H1.4 double-deficient (H1.2/H1.4 -/-, n = 14) animals, each dot represents a mouse, error bars are mean -/+ SEM. p values are indicated and derived from an unpaired two-tailed t test. The profile of circulating immune cells in H1.2/H1.4 - / - animals looks normal.
Figure 5—figure supplement 2. Leukocytes in circulation and bone marrow in homeostasis.
(a–n) Analysis of leukocytes in blood (a–g) and bone marrow (h–n) of wild type and H1.2/H1.4-deficient female mice (n = 4). Animals were sacrificed, bone marrow and blood was isolated and directly stained with the indicated surface markers (for gating strategies see Figure 5—figure supplement 1a). Each dot represents a mouse, values are calculated as cells/μl blood (a–g) or as cells per femur and tibia (h–n). Error bars are mean +/- SD. Note that g) essentially displays the same population of cells also shown in Figure 5a. Figure 5a shows the percentage of neutrophils in blood (with corresponding other leukocyte percentages shown in Figure 5—figure supplement 1c–f), whereas in Figure 5—figure supplement 2 the counts of neutrophils are shown.
Figure 5—figure supplement 3. Leukocytes in circulation and bone marrow upon casein injection.
(a–n) Same cell populations and staining procedure as in Figure 5—figure supplement 2, but upon injection of 2 × 1 ml 7% casein into the peritoneal cavity of female mice (n = 4). Animals were sacrificed at 24 hr after the first injection and blood and bone marrow were analyzed for the indicated immune cell populations. Error bars are mean +/- SD.
Figure 5—figure supplement 4. Eosinophil counts in bone marrow upon casein injection and in circulation.
(a) Counts of eosinophils in the blood of casein-injected animals, showing that the enhanced number of eosinophils seen in homeostatic H1.2/H1.4-deficient mice is not seen in inflammatory conditions. (b, c) Counts of eosinophils in the bone marrow of wild type and H1.2/H1.4-deficient mice during homeostasis (b) and upon casein injection (c), the numbers of eosinophils do not differ between genotypes. Depicted are mean +/- SEM, each data point represents one mouse. p values are indicated and derived from an unpaired, two-tailed t test.
Figure 5—figure supplement 5. Differentially regulated ageing markers in H1.2/H1.4-deficient mice.
a Counts of neutrophils in the bone marrow of wild type and H1.2/H1.4-deficient animals, showing similar numbers of neutrophils in both genotypes. (b) Mean fluorescence intensity (MFI) of CD62L surface expression in bone marrow neutrophils of the indicated genotype. (c) MFI of CXCR4 surface expression in bone marrow neutrophils of the indicated genotype. d-f Same analysis as in a–c), but in casein-injected animals. There is a difference in CD62L (e) and CXCR4 (f) expression in H1.2/H1.4-deficient animals. (g, h) Percentage of CD62L+ CXCR4- (young) neutrophils in the bone marrow of homeostatic (g) and casein-injected (h) animals of the indicated genotype. a,d,g,h Each data point represents one mouse. p values are indicated and derived from an unpaired, two-tailed t test. b,c,e,f each histogram represents one mouse.
Figure 5—figure supplement 6. Differentially regulated cytokines in H1.2/H1.4-deficient mice.
a, b mRNA expression of the chemokine CXCL1 (a) and the inflammatory cytokine IL6 (b) in homeostatic (left panel) and casein-injected (right panel) animals. H1.2/H1.4-deficient bone marrow cells transcribe more CXCL1 and less IL6. (c–e) protein levels of the indicated cytokines and chemokines in the serum of homeostatic and casein-injected mice. The enhanced expression of CXCL1 is also seen in serum (c). Casein injection leads to an upregulation of IL-17 (d) and G-CSF (e) and the upregulation of IL-17 is stronger in H1.2/H1.4-deficient animals. Depicted is the mean -/+ SD, each data point represents one mouse. p values are indicated and derived from an unpaired, two-tailed t test.
Figure 5—figure supplement 7. Enhanced survival and granularity of H1.2/H1.4-deficient bone marrow cells and neutrophils.
(a) Bone marrow of casein-injected wild type and H1.2/H1.4-deficient mice was incubated overnight in the presence or absence of G-CSF (100 ng/ml). G-CSF enhanced the survival of cells, measured by adding SYTOX Green and analyzing SYTOX Green positive cells by flow cytometry. The effect of G-CSF on cell survival was stronger on H1.2/H1.4-deficient cells. (b) Granularity of bone marrow cells after overnight incubation as depicted by side scatter (SSC) in flow cytometry. H1.2/H1.4-deficient cells show enhanced granularity. (c, d) Flow cytometry analysis of granularity of neutrophils (Ly-6G+ cells) in the bone marrow (c) and blood (d) of wild type and H1.2/H1.4-deficient mice during homeostasis (left panel) or upon casein injection (right panel). Bone marrow neutrophils of H1.2/H1.4-deficient mice are more granular than wild type cells. (a–d) Each data point represents a mouse, error bars are mean +/- SD.