Page 72 - Haematologica March 2020

G. Hernandez et al.
Maintenance of the blood system requires a carefully orchestrated interaction between blood-forming hematopoietic stem cells (HSC) and cell-extrinsic signals provided by their microenvironment in the bone marrow (BM).6 Such signals regulate HSC quiescence and/or direct HSC differentiation into lineage-biased multipotent pro- genitors (MPP) and mature blood cells.7 Pro-inflammatory cytokines can activate 'emergency' gene programs in HSC associated with overproduction of myeloid- and platelet- biased MPP subsets that, in turn, overproduce myeloid cells and platelets at the expense of other lineages.8-10 Along these lines, hematopoietic defects in RA patients have been ascribed to pro-inflammatory cytokines.11 However, the underlying molecular mechanism(s) and impact of therapeutic intervention on HSC function have not been well characterized.
Here, we used the type II collagen-induced arthritis (CIA) mouse model of human RA to identify the impact of chronic autoimmune arthritis on HSC function. We found that CIA leads to expansion of myeloid progenitors in the BM and overproduction of myeloid cells. HSC from CIA mice activated a myeloid gene program, although they retained their quiescence and long-term repopulating function. Interestingly, HSC quiescence was associated with a proliferation arrest program, characterized by downregulation of cell cycle and mRNA translation genes, which may serve to limit spurious HSC activation. Notably, we showed that pharmacological cytokine blockade using the recombinant IL-1 receptor antagonist anakinra alleviated inflammatory arthritis and myeloid expansion in the blood and BM of mice with CIA. In addi- tion, anakinra treatment reduced activation of inflamma- tion-induced myeloid and proliferation arrest gene pro- grams in HSC. Taken together, our findings suggest that anti-inflammatory therapies such as cytokine blockade can restore hematopoietic function in the context of chronic inflammatory diseases such as RA.
Methods
The methods are described in detail in the Online Supplement.
Mice and in vivo treatments
Male, 6- to 12-week old C57BL/6J (strain #000664) and
B10.RIII (strain #000457) mice from The Jackson Laboratory (Bar Harbor, ME, USA) were maintained in a temperature- and light- controlled environment with irradiated chow and water ad libi- tum for all experiments. Arthritis was induced by intradermal injection of type II chicken (for C57BL/6J mice) or bovine (for B10.RIII mice) collagen (Sigma-Aldrich) emulsified in Complete Freund Adjuvant (CFA; Sigma-Aldrich).12 Anakinra (Swedish Orphan Biovitrum) was administered daily at a dose of 50 mg/kg via subcutaneous injection. All animal procedures were approved by the University of Colorado Denver Anschutz Medical Campus Institutional Animal Care and Use Committee (IACUC).
Flow cytometry and hematopoietic stem cell isolation
BM was flushed from femora and tibiae with Hank’s balanced salt solution (HBSS) without calcium or magnesium salts but containing 2% heat-inactivated fetal bovine serum (FBS). BM cells were depleted of red blood cells using ACK lysis except for the erythroblast analysis. Next, 1×107 cells were stained for hematopoietic stem and progenitor cells or 1×106 for mature BM
cells and were analyzed on a BD FACSCelesta or LSRII instru- ment. For HSC isolation, posterior limb, anterior limb and pelvic bones were crushed in HBSS + 2% FBS, treated with ACK, placed on a Histopaque 1119 gradient and enriched in c-Kit cells using anti-c-Kit microbeads (Miltenyi) and separation on an AutoMACS Pro (Miltenyi). Cells were double-sorted to purity on a FACSAria IIu or FACSAria Fusion (Becton Dickenson) at 20 psi using a 100 mm nozzle.
Hematopoietic stem cell and bone marrow transplantation assays
Lethally irradiated (11 Gy, split dose 3 h apart) CD45.1+ Boy/J congenic recipient mice were transplanted with either 250 CD45.2+ donor HSC plus 5×105 CD45.1+ Sca-1 depleted cells, or 5×105 unfractionated BM cells, via the retro-orbital vein in a 100 mL volume of HBSS + 2% FBS. Recipient mice were maintained on autoclaved water containing Bactrim for 4 weeks following transplantation, and donor blood chimerism was assessed every 4 weeks up to 16 weeks via bleeding from the tail vein or retro- orbital sinus.
Gene expression analyses
RNA-sequencing analysis was performed on three bio- logical replicate pools of 4×103 to 1×104 HSC isolated from groups of two mice per condition (control and CIA). DESeq2-1.12.3 within R-3.3.0 was used for data normal- ization and differential expression analysis with an adjusted P-value threshold of 0.05. Fluidigm analyses were performed using commercially designed DeltaGene primer sets on a Biomark instrument (Fluidigm). Relative gene expression was calculated using the ΔCt method. Data were normalized to Actb.
Cytokine analysis
Serum cytokine levels were determined using a Luminex 36- analyte ProCartaPlex cytokine array (Thermo Fisher) according to the manufacturer’s instructions.
Statistical analysis
Statistical analyses were performed using Prism 7 software (GraphPad). P-values were determined using a Mann-Whitney U-test or one-way analysis of variance. P-values ≤0.05 were con- sidered statistically significant.
Results
Aberrant blood system in mice with collagen-induced arthritis
The CIA system is the most commonly studied model of RA, and faithfully recapitulates several disease features, including B- and T-cell-driven autoimmunity and chronic production of pathogenic cytokines.12 To assess the impact of RA on the blood system, C57BL/6 mice were injected intradermally at the base of the tail twice 21 days apart with an emulsion of CFA and type II collagen (Figure 1A). Non-arthritic control mice were injected with CFA alone on the same treatment schedule. After the second (boost) injection, mice rapidly developed polyarthritis character- ized by swelling and erythema of the front and rear paws, metatarsals and/or ankles within a week of the boost injection (Figure 1A, Online Supplementary Figure S1A). Peripheral blood parameters in CIA mice 21 days after the boost injection showed a significant increase in neu- trophils, while lymphocyte and platelet numbers were not
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