Figure 3. Hypocretin controls pre-neutrophil CSF1 production in the bone marrow.

Hypocretin receptor-1 (Hcrtr1) mRNA in cells sorted from bone marrow (n=4 except neutrophils n=7). b, HcrtR1 mRNA expression in bone marrow and blood neutrophil populations (n=4). c, Flow cytometry plot of HCRTR1⁺ pre-neutrophils in the bone marrow of WT mice transplanted with HcrtR1^Gfp/Gfp BM. d, Colony stimulating factor-1 (Csf1) expression in sorted bone marrow cells (for Ly-6C^hi monocytes, B cells, and other leukocytes n=3; for neutrophils and CD45⁻ cells n=5). e, Csf1 expression in sorted bone marrow neutrophil populations (n=4). f, CSF1 production by pre-neutrophils sorted from WT mice exposed to LPS and/or HCRT-1 (for untreated and HCRT-1 n=4 per group; for LPS and LPS+HCRT-1 n=6 per group) *p<0.05, ***p<0.001, One-way ANOVA. g, CSF1 production by pre-neutrophils sorted from WT and Hcrt^−/− mice (n=4 per group). h, BM CSF1 concentration in Apoe^−/− SF and Hcrt^−/− mice (n=4 Apoe^−/−; n=6 Apoe^−/−SF; n=4 WT; n=7 Hcrt^−/−). i, Enumeration of blood Ly-6C^hi monocytes over 24 hours in WT^{bmHcrtR1Gfp/Gfp} mice (n=3 per group) ***p<0.001, Two-way ANOVA. j, BM LSKs and LSK proliferation in WT mice transplanted with WT or HcrtR1^Gfp/Gfp BM cells (for LSKs/leg n=5 per group; for proliferation n=4 WT^bmWT and n=5 WT^{bmHcrtR1Gfp/Gfp}). k, BM CSF1 concentration in WT mice transplanted with WT or HcrtR1^Gfp/Gfp BM cells (n=4 WT^bmWT; n=5 WT^{bmHcrtR1Gfp/Gfp} ). l, Cross section images of aortic roots stained with oil-red-o and quantification of atherosclerosis in Ldlr^−/− mice transplanted with WT or HcrtR1^Gfp/Gfp BM cells and fed a high cholesterol diet for 12 weeks (n=8 Ldlr^−/−bmWT; n=9 Ldlr^{−/−bmHcrtR1Gfp/Gfp} ). Data presented as mean ± s.e.m., *p<0.05, **p<0.01,***p<0.001, Mann-Whitney two-tailed tests unless otherwise indicated.