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could be further addressed by determining whether HSC from CIA mice fail to proliferate in response to a concurrent inflammatory stimulus. Future studies could also address the functional significance of disease-induced changes in key BM niche populations such as MSC in hematopoiesis.
Interestingly, these results are distinct from the increased HSC cell cycle activity and HSC depletion observed in a model of chronic M. avium infection.40 The difference could be due to ongoing consumption of mature immune cells (particularly granulocytes) during the active infection that drives continuous HSC proliferation. Notably, serum levels of IFN-γ in mice infected with M. avium are also over 20- fold higher than those in our mice with CIA, which may be sufficient to activate HSC and overwhelm quiescence- enforcing mechanisms. While we found elevated serum IFN-γ levels in CIA mice, these did not translate into activa- tion of IFN target genes in HSC, suggesting that either the IFN-γ level was not sufficiently high to activate HSC, or or HSC had become refractory to such signals. Altogether, our data suggest that chronic inflammation could actually induce protection of the HSC pool (at least up to a point) by activating stress response mechanisms that maintain quies- cence. Such a model is in line with an emerging body of work showing that pro-inflammatory signaling can play a positive role in hematopoiesis.8 This model could explain the rarity of BM failure in RA patients despite some increased risk of myelodysplastic syndrome,41 as well as the rarity of graft failure in RA patients undergoing autologous BM transplantation.42
While concurrent activation of myeloid differentiation and proliferation arrest gene programs in HSC seems para- doxical, cell cycle arrest is crucial for myeloid differentiation by promoting accumulation of factors needed for differen- tiation.43 Myeloid transcription factors, including PU.1, pro- mote cell cycle arrest via induction of cell cycle inhibitors such as Cdkn1a while repressing cell cycle activators like Ccnd1.43,44 Thus, induction of myeloid gene programs could serve a dual purpose during chronic inflammation: protect- ing the HSC pool from excess proliferation while promot- ing myeloid differentiation of actively cycling hematopoiet- ic progenitor cells. In this way, HSC may participate mini- mally in day-to-day blood system maintenance45 during chronic inflammation, with occasional HSC divisions nonetheless leading to preferential myeloid progenitor pro- duction. On the other hand, telomere attrition has been identified in CD34+ hematopoietic stem and progenitor cells from human RA patients.46 RA is typified by years of ongoing disease, including ‘flares’ and therapies which could induce premature replicative ‘aging' in the HSC com- partment.11 Further analyses should clarify the extent to which HSC are affected by telomere attrition and whether such effects are due to increased proliferation versus impaired telomere maintenance. Divisional tracing approaches, such as H2B-GFP labeling47 in mouse models including the CIA model, could therefore provide valuable insights into the long-term impact of inflammatory disease on HSC proliferation.
Cytokine blockade therapies, including anakinra, have been used for over a decade to treat chronic inflammatory disorders, such as RA.5 Anakinra can correct aberrant blood parameters in RA patients.29 However, the impact of cytokine blockade on hematopoiesis in the BM has not been closely studied. Here, we show that anakinra treat- ment reduces myeloid progenitor expansion in mice with CIA, characterized by partial reduction of MPP3 and GMP.
It should be noted that MPP3 is not a transient population; clonal analyses and transplantation assays have shown that MPP3 can persist in mice and continue to produce GMP for weeks if not months. Hence, it is possible that the impact of cytokine blockade on hematopoiesis is gradual, with fur- ther reduction in MPP3 and GMP numbers as these popula- tions turn over and are not replaced due to reduced activity of ‘emergency’ differentiation pathways in HSC. Indeed, anakinra reduced activation of myeloid and proliferation arrest gene programs activated in HSC from CIA mice. This suggests that HSC molecular responses to inflammation are reversible and may be regulated in proportion to the level of inflammatory signaling and/or BM remodeling in the indi- vidual.
Our data are agnostic as to whether anakinra acts directly on HSC, or orthogonally by reducing inflammation at the joints, which in turn translates into less systemic inflamma- tion. Indeed, we did not detect increased IL-1 levels in the serum of CIA mice, consistent with studies in human patients indicating that IL-1 production is often localized to the joint synovia.28 On the other hand, we found increased Il1r1 expression in HSC from CIA mice, suggesting that HSC could be sensitized to tonic IL-1 production in the BM, even if IL-1 levels themselves do not increase. Along these lines, anakinra treatment reduces expression of IL1R1 and other IL-1 target genes in blood cells from breast cancer patients.48 Hence, anakinra could also contribute to restored HSC function by breaking a feedback loop between IL-1 and Il1r1 expression in HSC from CIA mice. Future studies could determine the extent to which anakinra reduces the effects of inflammation on HSC via direct versus indirect mechanisms such as reduction in downstream cytokine production. Notably, other cytokine blocking drugs, such as the TNF inhibitor etanercept, also alleviate the symptoms of inflammatory arthritis in RA patients.49 It is therefore possible that blockade of other cytokines elicits a similar restoration of hematopoiesis. In addition, blockade of mul- tiple inflammatory cytokines may further normalize hematopoiesis in RA and other diseases, although such interventions could also increase the risk of infection or cytopenia in patients.50 Taken together, our findings show that pro-inflammatory cytokine blocking treatment can reverse inflammatory-induced changes in hematopoiesis and HSC gene regulation, with the degree of impact likely related to the duration of treatment and/or the extent to which inflammatory arthritis is alleviated in the patient. Our data thus provide further rationale for the use of anti- cytokine therapies to redirect HSC fate and restore normal hematopoiesis in patients with RA and, potentially, other chronic inflammatory diseases.
Acknowledgments
The authors would like to thank Phillip Bendele, Shannon Spiegel, Kyle Rothermel and Connor Ohlsen for expert technical assistance. This work was supported by K01 DK098315, R01 DK119394 (to EMP), the Cleo Meador and George Ryland Scott Chair of Medicine in Hematology (to EMP), the Boettcher Foundation Webb-Waring Early Career Investigator Award (to EMP and KAK), F31 HL138754 (to JLR), an NIH Institutional National Service Award 2T32 AI07449 (to CJF), a National Science Foundation Predoctoral Research Fellowship (to TSM), R01 AI098417 (to JRH), and R01 AR51749 (to VMH). This work was also supported in part by the University of Colorado Cancer Center Flow Cytometry Shared Resource, funded by NCI grant P30 CA046934.
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