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G. Hernandez et al.
translation, was decreased (Figure 4H). Collectively, these data identify a global downregulation of HSC activation pathways despite chronic inflammation.
To identify the functional impact, we analyzed the cell cycle distribution of HSC in control and CIA mice (Figure 4I). Strikingly, HSC from CIA mice retained a quiescent cell cycle phenotype (Figure 4J-K). Likewise, the cell cycle distribution of phenotypic MPP or Lin-cKit+Sca-1-CD34+ myeloid progenitors (MyPro) was unaltered (Online Supplementary Figure S5E). This suggests that myeloid expansion in CIA mice likely arises from preferential dif- ferentiation into myeloid-biased progenitors from HSC and/or MPP, rather than from increased proliferative activ- ity. Thus, despite ongoing inflammatory arthritis, HSC retain a quiescent phenotype associated with a prolifera- tion arrest gene program that could serve to prevent cell cycle entry during chronic inflammatory stress.
(Figure 5B). Using qRT-PCR, we found IFN target genes unchanged in HSC from CIA mice, consistent with unchanged Sca-1 surface expression (Figure 5C, Online Supplementary Figure S4A, B).16 In contrast, IL-1 receptor (Il1r1), which is a target of multiple cytokines including IL- 1 itself, was increased in HSC from CIA mice (Figure 5D). These data suggest that systemic production of pro- inflammatory cytokines is a feature of CIA mice, similar to human RA patients.
Impact of cytokine blockade on myeloid expansion in mice with collagen-induced arthritis
Cytokine blockade therapy, particularly against IL-1 and TNF, is efficacious in reducing inflammatory arthritis. It can also normalize blood parameters in RA patients,29 although its impact on hematopoiesis is not well described. We used anakinra, a recombinant form of human IL-1 receptor antagonist (IL-1Ra) that is approved for treatment of RA in human patients and is considered a paradigm for cytokine blockade therapy.30 We induced CIA in C57BL/10.RIII (B10.RIII) mice (Figure 6A), a C57BL/6-related strain that develops a severe and highly penetrant autoimmune arthritis following CIA induction.31 These mice are thus ideal for testing therapeutic interven- tions. Consistent with prior mouse studies, anakinra treat- ment significantly reduced arthritis severity based on clin- ical scoring of paw swelling (Figure 6B).32 Strikingly, anakinra normalized peripheral blood neutrophil and, to a lesser extent, red blood cell counts (Figure 6C, Online Supplementary Figure S7A). Anakinra treatment also nor- malized granulocyte and B-cell numbers in the BM, with a
Systemic pro-inflammatory cytokine production in mice with collagen-induced arthritis
RA-associated cytokines, such as IL-1, TNF, IFN-γ, and myeloid growth factors, such as granulocyte-colony stim- ulating factor (G-CSF), can activate myeloid gene pro- grams in hematopoietic stem and progenitor cells, leading to altered blood lineage output.8,10 We therefore used a Luminex-based 36-plex array to analyze cytokine levels in the serum of control and CIA mice (Figure 5A, Online Supplementary Figure S6). Several cytokines were increased in the serum of CIA mice, including TNF, G-CSF and IFN- γ. On the other hand, IL-1β, which is often produced local- ly at the joint synovia in RA patients,28 was not detected
AB
CD
Figure 5. Systemic inflamma- tion in mice with collagen- induced arthritis. (A) Experimental design. (B) Cytokine levels in serum from control mice (Ctrl) and mice with collagen-induced arthritis (CIA) (n=7 Ctrl and 4 CIA). Serum sample data were com- piled from two independent experiments. (C,D) Gene expression analysis of hematopoietic stem cells (HSC) from Ctrl and CIA mice showing (C) IFN/STAT signal- ing genes and (D) IL-1/TNF signaling genes. The data are presented as log10 fold expres- sion in CIA HSC versus Ctrl HSC. (n=14-16 per group) *P<0.05; **P<0.01, as deter- mined by the Mann-Whitney U- test.
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