Page 75 - Haematologica March 2020

Cytokine blockade reduces myeloid expansion in RA
Collagen-induced arthritis leads to myeloid expansion and activation of a myeloid gene program in hematopoietic stem cells
We next assessed BM lineage potential using methylcel- lulose-based colony-forming unit (CFU) assays (Figure 2A). Strikingly, the numbers of granulocyte-macrophage colonies (CFU-GM) (Figure 2A) were significantly increased, whereas erythroid colony formation was impaired (Figure 2A). CFU-GM were also significantly increased in the spleens of CIA mice alongside a signifi- cant increase in splenocyte numbers, indicative of extramedullary hematopoiesis16 (Online Supplementary Figure S2A-C). While granulocyte-macrophage progenitors (GMP; Lin-c-Kit+CD41-CD150-FcγR+) were unchanged in the BM, common lymphoid progenitors (CLP; Lin- Flk2+IL7R+c-KitintSca1int) and phenotypic CFU-E cells (Lin-c- Kit+CD41-FcγR-CD150-CD105+) were significantly reduced (Figure 2B-D and Online Supplementary Figure S1C). Recently, distinctive lineage-biased MPP populations downstream of HSC were identified and termed MPP2, MPP3 and MPP4, (LSK Flk2-CD48+CD150+, LSK Flk2- CD48+CD150-, and LSK Flk2+, respectively); these popula- tions exhibit megakaryocyte/erythroid, myeloid and lym- phoid lineage priming, respectively.7 CIA mice exhibited expansion of myeloid-biased MPP3 in the BM (Figure 2E and Online Supplementary Figure S1C), consistent with myeloid overproduction. Within the phenotypic LSK Flk2- CD48-CD150+ compartment, hereafter referred to in the text as HSC, the distribution of CD34- long-term HSC (HSCLT) and metabolically-activated CD34+ MPP1 subsets was unchanged (Figure 2G and Online Supplementary Figure S1C). Despite some heterogeneity between individual mice, we also did not observe a significant increase in the abundance of CD41-expressing (CD41+) HSC, which rap- idly differentiate into megakaryocytes in the context of inflammation (Figure 2H, I).17,18 Likewise, the numbers of short-term HSC (HSCST; LSK Flk2-CD48-CD150-) were unchanged (Figure 2F and Online Supplementary Figure S1C). Since remodeling of BM stroma occurs in RA patients and animal models,19 we analyzed stromal cell populations comprising the endosteal HSC niche6 and observed a significant decrease in bone-forming mes- enchymal stromal cells (MSC), consistent with reduced bone-forming activity in a genetic mouse model of RA (Online Supplementary Figure S2D, E).19,20 Lastly, we con- firmed activation of a myeloid lineage gene program in HSC from CIA mice using custom Fluidigm real-time quantitative polymerase chain reaction (qRT-PCR) assays (Figure 2J). Consistent with prior reports,21 expression of myeloid genes was increased in HSC from CIA mice, including the myeloid master regulator Spi1/PU.1 and its target genes Itgam1 and Csf2rb, as well as Mpo (Figure 2K). On the other hand, other lineage determinant genes were minimally altered (Figure 2L). Altogether, these data iden- tify aberrant activation of an 'emergency' myeloid differ- entiation pathway in CIA mice, characterized by BM remodeling, myeloid expansion and activation of a myeloid gene program in HSC.
Impact of collagen-induced arthritis on long-term reconstitution of hematopoietic stem cells
We and others previously showed that chronic inflam- mation impairs HSC long-term reconstitution capacity.8 To interrogate long-term HSC potential, we transplanted purified CD45.2+ HSC from control and CIA donor mice
into lethally-irradiated (11 Gy) CD45.1+ recipient mice (Figure 3A and Online Supplementary Figure S3A). Strikingly, overall reconstitution capacity of HSC from CIA mice 16 weeks after transplantation was not signifi- cantly different from that of controls (Figure 3B), consis- tent with short-term CFU assays on purified HSC (Online Supplementary Figure S3B). Interestingly myeloid lineage output at week 16 was significantly increased (Figure 3C), with a significantly increased proportion of CIA donor- derived phenotypic MPP3, consistent with a myeloid biased phenotype (Online Supplementary Figure S3C, D). In parallel, we assessed HSC function independently of sur- face markers via transplantation of unfractionated BM cells from control and CIA mice (Figure 3D). We observed no defect in reconstitution, save for a slight myeloid bias (Figure 3E-F) and a decreased frequency of CIA donor- derived phenotypic HSC, likely related to a decreased fre- quency of HSC in the BM of CIA mice (Online Supplementary Figures S1D and S3E, F). These results sug- gest that long-term HSC potential is not compromised, although a degree of myeloid bias is present, consistent with prior published results in the KRN×G7 arthritis model.21 Likewise, expression of genes associated with HSC identity and self-renewal was largely unchanged (Figure 3G-I) except for an increase in Cd48 expression, suggesting that HSC from CIA mice may be primed toward differentiation into MPP (Figure 3I). However, at the protein level CD48 and other key surface markers associated with lineage bias or differentiation, such CD150, were unchanged,22 suggesting that such MPP priming could be restricted primarily to the mRNA level (Online Supplementary Figure S4A, B). In addition, reactive oxygen species, which accompany differentiation and can impair HSC function if chronically elevated,23,24 were unchanged in HSC from CIA mice (Online Supplementary Figure S4C). Taken together, these data indicate that, apart from myeloid priming, the functional and molecular prop- erties of HSC are largely unperturbed in CIA mice.
A proliferation arrest gene program is associated with quiescence in hematopoietic stem cells from mice with collagen-induced arthritis
To gain additional insight into the impact of CIA on HSC molecular regulation, we performed RNA-sequenc- ing analysis on HSC isolated from control and CIA mice (Figure 4A). Differential expression analysis identified 292 significantly downregulated genes and 237 upregulated genes based on an adjusted P-value (Padj) of >0.05 (Figure 4B and Online Supplementary Table S1). To uncover poten- tial mechanisms regulating HSC function in CIA mice, we used the Upstream Regulator analysis function in Ingenuity Pathway Analysis (IPA) software. Strikingly, few regulatory pathways were significantly activated in HSC from CIA mice, most notably APC and PTEN, which both suppress HSC activity and enforce quiescence (Figure 4C and Online Supplementary Table S2).25 On the other hand, pathways activated in response to inflammatory and mitogenic cues were downregulated, including ERK, MYC and IL-1/NF-κB (Figure 4C and Online Supplementary Table S2).26 In parallel, gene ontology (GO) analysis27 iden- tified enrichment of downregulated genes involved in pro- tein translation initiation, G1/S cell cycle transition, posi- tive regulation of gene expression and transcription (Figure 4D and Online Supplementary Table S3). Conversely, significantly upregulated genes in HSC from CIA mice
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