Transcriptome reprogramming and myeloid skewing in haematopoietic stem and progenitor cells in systemic lupus erythematosus

Objectives Haematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to both myeloid and lymphoid cell lineages. We reasoned that the aberrancies of immune cells in systemic lupus erythematosus (SLE) could be traced back to HSPCs. Methods A global gene expression map of bone marrow (BM)-derived HSPCs was completed by RNA sequencing followed by pathway and enrichment analysis. The cell cycle status and apoptosis status of HSPCs were assessed by flow cytometry, while DNA damage was assessed via immunofluorescence. Results Transcriptomic analysis of Lin−Sca-1+c-Kit+ haematopoietic progenitors from diseased lupus mice demonstrated a strong myeloid signature with expanded frequencies of common myeloid progenitors (CMPs)—but not of common lymphoid progenitors—reminiscent of a ‘trained immunity’ signature. CMP profiling revealed an intense transcriptome reprogramming with suppression of granulocytic regulators indicative of a differentiation arrest with downregulation trend of major regulators such as Cebpe, Cebpd and Csf3r, and disturbed myelopoiesis. Despite the differentiation arrest, frequencies of BM neutrophils were markedly increased in diseased mice, suggesting an alternative granulopoiesis pathway. In patients with SLE with severe disease, haematopoietic progenitor cells (CD34+) demonstrated enhanced proliferation, cell differentiation and transcriptional activation of cytokines and chemokines that drive differentiation towards myelopoiesis, thus mirroring the murine data. Conclusions Aberrancies of immune cells in SLE can be traced back to the BM HSPCs. Priming of HSPCs and aberrant regulation of myelopoiesis may contribute to inflammation and risk of flare. Trial registration number 4948/19-07-2016.


AbSTrACT
Objectives Haematopoietic stem and progenitor cells (HsPcs) are multipotent cells giving rise to both myeloid and lymphoid cell lineages. We reasoned that the aberrancies of immune cells in systemic lupus erythematosus (sle) could be traced back to HsPcs. Methods a global gene expression map of bone marrow (BM)-derived HsPcs was completed by rna sequencing followed by pathway and enrichment analysis. The cell cycle status and apoptosis status of HsPcs were assessed by flow cytometry, while Dna damage was assessed via immunofluorescence. results Transcriptomic analysis of lin − sca-1 + c-Kit + haematopoietic progenitors from diseased lupus mice demonstrated a strong myeloid signature with expanded frequencies of common myeloid progenitors (cMPs)but not of common lymphoid progenitors-reminiscent of a 'trained immunity' signature. cMP profiling revealed an intense transcriptome reprogramming with suppression of granulocytic regulators indicative of a differentiation arrest with downregulation trend of major regulators such as Cebpe, Cebpd and Csf3r, and disturbed myelopoiesis. Despite the differentiation arrest, frequencies of BM neutrophils were markedly increased in diseased mice, suggesting an alternative granulopoiesis pathway. in patients with sle with severe disease, haematopoietic progenitor cells (cD34 + ) demonstrated enhanced proliferation, cell differentiation and transcriptional activation of cytokines and chemokines that drive differentiation towards myelopoiesis, thus mirroring the murine data. Conclusions aberrancies of immune cells in sle can be traced back to the BM HsPcs. Priming of HsPcs and aberrant regulation of myelopoiesis may contribute to inflammation and risk of flare. Trial registration number 4948/19-07-2016.

InTrOduCTIOn
In systemic lupus erythematosus (SLE), an interplay between environmental, genetic and epigenetic factors leads to perturbation of complex biological networks culminating into diverse clinical phenotypes. In this disease, interferon-alpha (IFN-α)driven immunological alterations result in persistent immune responses against autologous nucleic acids, mimicking a sustained antiviral response. Intractable tissue damage caused by autoantibodies or immune-complex depositions affects several organs, leading to significant morbidity. 1 2 Haematopoietic stem and progenitor cells (HSPCs) represent the most primitive multipotent population giving rise to all blood cell types. 3 HSPCs reside in the bone marrow (BM) niche and remain in a quiescent state. Under stress or inflammatory conditions, they respond by proliferating and differentiating to replenish any progeny needed. [4][5][6] A key observation in SLE is that most cells participating in its pathogenesis, such as lymphocytes, monocytes and neutrophils, originate from HSPCs. In a congenic strain of lupus mice, the function of hematopoietic stem cells is altered by both genetic and inflammatory factors with evidence of increased self-renewal and resistance to stress. 7 In patients with SLE with cytopenias, BM exhibits necrosis, stromal alterations, hypocellularity, dyspoiesis and distortion of normal architecture with abnormal localisation of immature precursors aggregates. 8 Gene expression studies from our group have demonstrated upregulation of genes involved in cell death and granulopoiesis, providing further evidence of the role of apoptosis and granulocytes in its pathogenesis. 9 Within the BM niche, inflammatory cytokines and myeloidspecific growth factors, including interleukin (IL)-1 and granulocyte macrophage growth factor, 10 drive the reprogramming of HSPCs towards myeloid lineage by epigenetic modifications and induction of lineage-specific transcription factors. These alterations increase their adaptation to inflammatory and haematopoietic stress, promoting the generation of myeloid cells that confer protection to secondary infection, in a phenomenon termed 'trained innate immunity' or 'innate immune memory'. 11 12 Perturbed immunological imprinting (mediated by hyperactive and myeloid priming) could be detrimental, exaggerating immune responses in autoimmune and inflammatory diseases such as arthritis, 13 14 SLE 15 or atherothrombosis. [16][17][18] We reasoned that the fundamental molecular aberrations in SLE (genetic or epigenetic) may be traced back in the HSPCs within the BM. To this end, we used the NZBW/F1 mouse model of SLE to investigate the lupus transcriptome of HSPCs and compared it with transcriptomic data from human SLE CD34 + cells. Herein, we report reprogramming of HSPCs towards myeloid lineage-with evidence of a 'trained immunity' signature-and propose that this may contribute to exaggerated immune responses and flares in SLE.

Human subjects
BM aspirates and PBMCswere obtained from SLE and gender matched healthy controls (HCs). Patients met the 1999 American College of Rheumatology revised criteria for the classification of SLE. 20 Patients' clinical and serological characteristics are summarised in online supplementary tables 1 and 5.

rnA sequencing
LSKs and CMPs were sorted from BM of NZBW/F1 and C57BL/6 mice. CD34 + cells were isolated from BMMCs. Genes with a false discovery rate of ≤0.05 and a fold change of >1.5 were considered statistically significantly upregulated/downregulated, respectively. For the human-mouse comparison, human genes were converted to mouse orthologos and pathways were compared based on their ID.

Statistics
Statistical analyses were performed using unpaired two-tailed Student's t-test, while Mann-Whitney U test was used for the comparison of two groups. Data are presented as mean±SD. Differences were considered statistically significant at p<0.05. All data were analysed using GraphPad Prism V.5 software.

Study approval
Informed consent was obtained from all patients and HC prior to sample collection (Athens, Greece, protocol 10/22-6-2017). All procedures in mice were in accordance with institutional guidelines and were reviewed and approved by the Greek Federal Veterinary Office (Athens, Greece).

The transcriptional profile of murine lupus HSPCs demonstrates myeloid skewing
To study whether HSPCs in SLE exhibit transcriptional alterations, we used the spontaneous mouse model NZBW/F1 21-23 at two time points: preclinical stage (F1-P) and clinical stage, defined as the point with proteinuria of >100 ng/dL (F1-L). Age-matched female C57BL/6 mice served as controls (B6-Y and B6-O, respectively). Gene profiling was performed in murine LSK compartment-representing HSPCs in mice-sorted by flow cytometry from BM of NZBW/F1 lupus and C57BL/6 control mice (figure 1A). A total of 758 differentially expressed genes (DEGs) between F1-P and F1-L mice were identified (figure 1B and online supplementary table 2), including enriched GO terms per cluster: haematopoietic cell lineage, neutrophil degranulation and cell adhesion (red); and lymphocyte activation, extracellular region and immunoglobulin heavy chain variable region genes (IGVH) repertoire (green/blue). Gene set enrichment analysis (GSEA) showed a positive correlation with signatures related to inflammatory response, activation of innate immune response and platelet degranulation (figure 1C and online supplementary table 3) in F1-L mice. These categories are crucial for the adaptation of stem cell phenotype to inflammation with studies showing expansion of stem cell-like megakaryocyte committed cells within HSPCs under inflammatory conditions. 24 Gene ontology and pathway analysis demonstrated DEGs implicated in myeloid leukocyte-mediated immunity, cytokine secretion, granulocyte/neutrophil activation and migration in F1-L mice (online supplementary figure 1A). Notably, F1-L LSK demonstrated increased proliferation and strong myeloid signature ( figure 1D). IFN-associated genes (Gbp6 and Ciita) were upregulated in F1-L LSK, showing a strong type I IFN signature, a hallmark of active SLE 25 ( figure 1D). Comparison with publicly available gene sets-encompassing CMP 26 and granulocyte-macrophage progenitor (GMP) 27 signatures from wild-type mice as reference-demonstrated positive enrichment in F1-L LSK for CMP and GMP signatures (online supplementary figure 1B) indicative of priming towards this direction. Together, these transcriptomic data indicate increased proliferation and strong differentiation of F1-L LSK towards myeloid/ granulocytic lineage. 28

Active proliferation and replicative stress of murine lupus HSPCs cells
To validate transcriptomic data, we analysed the BM LSK compartment and its subpopulations. LSK were increased by almost twofold in F1-L mice compared with F1-P ( figure 2A,B). Within the LSK compartment, short-term HSCs and multipotent progenitor cells (MPPs) had higher frequency in F1-L mice (figure 2A,C). Compared with their control counterparts, we found enhanced frequency of circulating LSK in the peripheral blood-but not in the spleen-of F1-L mice (online supplementary figure 1C,D), suggesting that BM-derived LSK may be activated, may exit the niche and then migrate to the periphery.
During steady state, HSPCs are relatively quiescent, maintaining a low number of cycling cells that will differentiate into mature blood cells. 29 Cell cycle analysis of lupus LSK revealed increased proliferation with fewer F1-L LSK in G0 phase compared with B6-O control (figure 2D). The frequency of apoptotic LSK from NZBW/F1 mice was also increased compared with B6 with no significant difference in F1-P versus F1-L stage (online supplementary figure 1E). In view of the enhanced proliferation, γ-H2AX was measured as a marker of proliferative stress. Lupus LSK exhibited increased double-strand DNA breaks compared with B6 (figure 2E), suggesting that LSK in lupus mice is under replicative stress (see discussion).

differentiation arrest of CMPs with lupus disease progression
To delineate the haematopoietic differentiation after the LSK stage, we characterised the progenitors of each lineage. CMPs were increased by 2.5-fold (**p≤0.01), while GMPs were 2-fold (***p≤0.001) reduced in F1-L versus F1-P mice (figure 3A). There was no significant difference in common lymphoid progenitors (online supplementary figure 2A). Both LSK and CMP frequencies were increased in F1-L mice, a profile reminiscent of emergency granulopoiesis. 30 To further investigate the regulation of myeloid differentiation, we performed transcriptional analysis. Most of 721 DEGs were downregulated at F1-L CMPs (figure 3B and online supplementary table 2), including enriched GO terms per cluster: response to IFN-beta and nucleotide signalling (green); immune response/ immunoglobulins (blue); and cytokine signalling, neutrophil degranulation and haematopoietic cell lineage (red). Myeloid markers were downregulated and proliferation markers were not differentially expressed ( figure 3C). DEGs were involved in pathways related to myeloid-mediated immunity, granulocyte activation, neutrophil migration and complement activation (online supplementary figure 2B). Thus, we checked the expression of specific granulocytic markers (figure 3D). Chemokines and regulators of IL-1 family 11 31 32 were downregulated in F1-L CMPs. Indicatively, the expression of main regulators such as Cebpe, Cebpd, Csf3r and Csf2rα was dampened in CMPs of F1-L stage ( figure 3D). Collectively, these results suggest differentiation arrest at the level of myeloid progenitors.

Increased neutrophils in the lupus bM: evidence of 'granulocytic priming'
In view of the differentiation arrest, we assumed that terminally differentiated cells may be decreased. However, neutrophils exhibited a 1.6-fold increase in the F1-L mice compared with F1-P, while there were comparable monocyte levels in BM ( figure 4A,B). Ageing accounted for only an increase of 1.16-fold in neutrophils of control mice. In contrast, there was marked decrease of neutrophils in blood and spleen of F1-L mice ( figure 4C,D, respectively), while monocytes did not differ significantly in the periphery (online supplementary figure 3A,B, respectively). Together these data suggest priming in the lupus BM towards neutrophils.

deregulation of differentiation of primed HSPCs indicates an alternative granulopoiesis pathway in lupus mice
To investigate how 'granulocytic priming' evolves during differentiation of haematopoiesis, we performed a comparative analysis between LSK and CMP transcriptomes. We used Regulatory Network Enrichment Analysis (RNEA) algorithm 33 to report enrichment of transcription factors and regulators by combining previous studies with our data. We identified 13 common differentially expressed transcription factors and regulators (online supplementary figure 4A), predominantly downregulated in the F1-L CMP stage (online supplementary figure 4B), mainly of myeloid and granulocytic differentiation. Therefore, we looked into expression of major regulators of granulocytic and neutrophilic differentiation, such as Cebpα, Cebpe, Irf8, Mpo and Elane, and found them upregulated in F1-L LSK while downregulated in F1-L CMPs (online supplementary figure 4C). Collectively, these data indicate a deregulation of differentiation at the CMP level and 'priming' of LSK towards granulocytes, indicative of an alternative granulopoiesis pathway.

Human Sle Cd34 + transcriptome demonstrates active proliferation and myeloid skewing
We next asked whether we could trace the 'lupus LSK signature' in human disease. To this end, we purified CD34 + cells from BM of female patients with SLE 34 35 (online supplementary tables 1 and 5) and HC. In humans, the CD34 + compartment comprises a cluster of 0.5%-2.0% of BM, which encompasses both stem and progenitor cells of different lineages. 36 We identified 2364 DEGs between patients with SLE and HC, which contained 832 upregulated and 1532 downregulated genes (online supplementary table 2). Enriched GO terms per cluster include extracellular vesicle-mediated signalling in recipient (blue); myeloid leucocyte migration and chemokine signalling pathway (turquoise); integrinmediated signalling and regulation of cell-cell adhesion (red); antigen processing and presentation, autoimmunity and abnormal inflammatory response (green); and cell surface receptor signalling pathway (yellow) (figure 5A). Lymphoid markers were downregulated in SLE, while expression of myeloid markers exhibited considerable variation within the patients. Combining these two panels, we found that early haematopoiesis in humans is characterised by skewing towards myeloid lineage (figure 5B). SLE CD34 + cells exhibited enhanced proliferation (figure 5B), while GSEA indicates a positive correlation with 'activation of ATR in response to replication stress', 'cell cycle' and 'DNA-dependent DNA replication' sets ( figure 5C and online supplementary table 4). In view of enhanced proliferation, 37 γ-H2AX was assessed to check if CD34 + cells are in proliferative stress; indeed, SLE HSPCs exhibited increased double-strand DNA breaks ( figure 5D). Collectively, CD34 + cells of patients with SLE exhibited enhanced proliferation  with increased DNA damage and unbalanced differentiation towards myeloid axis.
Subgroup analysis showed that patients with SLE with both severe and moderate disease (online supplementary figure 6) showed variable expression of myeloid markers ( figure 5E). Notably, expression of specific markers for granulopoiesis, such as CEBPZ, CEBPD, GATA2, was increased in a subgroup of patients with severe SLE compared with those with moderate disease (figure 5E), a result consistent with our findings in murine lupus LSK. GSEA revealed a positive correlation with 'cytokine-cytokine receptor interaction' and 'positive regulation of locomotion' sets in patients with severe SLE (figure 5F and online supplementary table 4). Enrichment analysis using upregulated genes in severe SLE revealed an over-representation of chemotaxis and migration of granulocytes and neutrophilic GO terms ( figure 5G). Together, HSPCs are activated and more proliferative in patients with SLE compared with HC with a distinct transcriptional differentiation profile in patients with severe disease.

Comparison of human lupus Cd34 + with murine lupus CMP transcriptome reveals common attributes with evidence of arrest at the progenitor stage
Next, we compared human CD34 + transcriptome to LSK and CMP data. 38 We found a significant overlap of LSK DEGs Systemic lupus erythematosus (6.7%) and CMPs DEGs (8.5%) with the human CD34 + DEGs (representation factor (RF)=3.2, p<2.7×10 −29 and RF=4, p<4.7×10 −18 , respectively) ( figure 6A,B). The majority of shared DEGs (26) between mouse LSK and human CD34 + cells were downregulated in patients with human SLE, but not in murine lupus ( figure 6C). However, the expression of common DEGs (54) between mouse CMPs and human CD34 + cells exhibited commonalities ( figure 6D). Thus, the SLE CD34 + transcriptomic profile is more reminiscent of murine lupus CMPs than murine lupus LSK. This is likely due to the fact that the haematopoietic stem cell signature might be diluted within the heterogeneous CD34 + population in humans. 39 At pathway level, the overlap between mouse and human data was more prominent. Compared with gene-level results, there was a fivefold higher representation of the enriched pathways in LSK and a twofold higher representation of the enriched pathways in CMPs (RF=81, p<6.8×10 −172 , and RF=43.3, p<4.8×10 −178 , respectively), which were also found enriched in human HSPCs ( figure 6A,B). Key pathways such as cell activation, regulation of cell differentiation, immune system development and leucocyte migration were shared. Using RNEA, we found 15 suppressed regulators in both lupus CMPs and CD34 + SLE cells. Among them, there were significant disease-specific effectors such as IFNγ and IL-6, as well as regulators for the homeostasis-differentiation balance of stem cells like Lif, Ets1 and Pax5 ( figure 6E).
Collectively, human CD34 + cells from patients with SLE display more commonalities in their transcripthmic profile with murine lupus CMPs rather than LSK. This comparison indicates an arrest at the progenitor level both in murine and human lupus haematopoiesis as important regulators for terminal differentiation are downregulated.

dISCuSSIOn
Blood and immune cells derive from HSPCs, which reside in the BM in quiescent state, being ready to respond to stress, such as severe infection, systemic inflammation or iatrogenic myeloablation. 40 Recent data suggest significant heterogeneity within the HSPCs with evidence of early lineage segregation, lineage-biassed existence and containment of lineage-restricted progenitors. 41 These lineage-biassed and lineage-restricted cells within the phenotypical HSPC compartment might serve as an emergency backup for stress conditions, capable of efficiently and specifically counterbalance the sudden loss of a particular lineage. Herein, we provide evidence of dysregulated differentiation during haematopoiesis in SLE. Transcriptomic data demonstrate enhanced activation and differentiation preference towards myeloid/granulocytic lineage after disease onset. NZBW/F1 exhibit detectable levels of type I IFN compared with other SLE mouse models. 42 We show that, indeed, IFN signature is present in the NZBW/F1 model and that lupus HSPCs can sense and respond to IFN. Chronic activation of the IFN-α pathway in HSPCs impairs their function, whereas acute IFN-α treatment promotes the proliferation of dormant HSPCs. 43 Our flow cytometric analysis revealed enhanced proliferation of LSK in lupus mice and increase in their subpopulations. This finding is consistent with findings by Niu et al 7 in a congenic lupus model where they found a genetic polymorphism on the Cdkn2c gene related to cell cycle. In the context of stem cell proliferation and activation, Walter et al 37 showed direct response of HSPCs by exiting quiescence with concomitant DNA damage. In agreement to this, lupus LSK had more DNA damage compared with their controls, which could be detrimental for their maintenance and self-renewal. Pronounced cell cycle entry and consequent proliferative stress may result in impaired HSC self-renewal potential. 44 45 To confirm the transcriptomic results on LSK differentiation, we profiled CMPs. Physiological myelopoiesis evolves through MPPs to lineage-restricted CMPs and then converges to GMPs. 46 Phenotypical analysis of progenitors showed increased frequency of CMPs but decreased frequency of GMPs, as evidenced in RNA sequencing by 'silencing' of differentiation after the CMP stage. Myeloid skewing is, in part, expected due to inflammation and ageing, 47 both operant in our model. Our results suggest 'priming' of LSK with a pronounced 'myeloid/ granulocytic signature' but downregulation as the differentiation evolves towards canonical myelopoiesis ( figure 6F).
Increased neutrophils in lupus BM suggest deregulation of homeostatic mechanisms in the level of CMPs with priming of LSK towards the granulocytic differentiation at the expense of lymphopoiesis. These results are consistent with our earlier findings of strong granulopoiesis signature in the BM by using DNA arrays. 9 Priming of LSK highly correlates with the signature of HSPCs after 'training' with β-glucan 11 (online supplementary figure 3), strongly indicating that 'SLE inflammatory milieu' promotes the immune training memory of BM progenitor cells. Accordingly, we found differentially methylated regions from lupus HSPCs overlapping with transcription factor binding sites relevant to haematopoietic development, including Cebpα (data not shown). Innate immune memory, while beneficial to host defence against pathogens, could also lead to maladaptation of the immune system in chronic inflammation, leading to perpetuation of chronic inflammatory disorders and predisposing to flares in response to environmental stimuli such as infections or stress. 48 Myeloid cells are crucial for disease progression. In the periphery of lupus mice, we found increased circulating LSK but decreased neutrophils. This could be due to either extensive destruction of neutrophils in the periphery or migration to target tissues. This might act as a positive feedback loop where an inflammatory environment triggers priming and exit of HSPCs to periphery, driving them to increased myeloid output, which in turn circulates and perpetuates the inflammation as proposed by Oduro et al 14 in an arthritis mouse model. It is conceivable that neutrophils may migrate to the inflamed tissues, hence their relative paucity in the periphery. The release of neutrophil extracellular traps represents a novel neutrophil effector function contributing to thromboinflammation and fibrosis in SLE. 49 It has been assumed that various blood cell lineages arise via a hierarchical scheme-starting with HSPCs-and that their differentiation potential becomes increasingly restricted through oligopotent and then unipotent progenitors. However, recent work suggests a developmental shift to an adult 'two-tier' hierarchy whereby HSPCs can generate restricted subsets of terminally differentiated progeny, bypassing the stepwise progression through common progenitor stage. 50 Yammamoto et al 51 proposed a revised model of haematopoietic differentiation with the existence of progenitors within the HSPC compartment, mostly myeloid-committed ones. Our results are in agreement with this model. It is reasonable to assume that SLE LSK are already predefined to differentiate towards granulocytic lineage, creating an alternative granulopoiesis pathway in the haematopoietic tree (figure 6F). In parallel, differentiation arrest at the intermediate stage of CMPs blocks flow of haematopoiesis towards GMPs.
In summary, we have presented evidence for deregulation of granulopoiesis and priming of HSPCs, which may contribute

Systemic lupus erythematosus
to persistent inflammation in SLE and risk of flare once the disease is in remission. Myeloid skewing of HSPCs, associated with epigenetic tinkering, is also typical of HSPCs during ageing, 51 52 contributing to decreased adaptive immunity and enhanced cardiovascular mortality of the elderly population. [53][54][55] Re-establishment of the appropriate lymphoid versus myeloid balance in systemic autoimmune diseases may improve immune function, decreasing risk of infection or atherosclerosis and resolution of inflammation.