Pro-inflammatory cytokine blockade attenuates myeloid expansion in a murine model of rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation and progressive destruction of joint tissue. It is also characterized by aberrant blood phenotypes including anemia and suppressed lymphopoiesis that contribute to morbidity in RA patients. However, the impact of RA on hematopoietic stem cells (HSC) has not been fully elucidated. Using a collagen-induced mouse model of human RA, we identified systemic inflammation and myeloid overproduction associated with activation of a myeloid differentiation gene program in HSC. Surprisingly, despite ongoing inflammation, HSC from arthritic mice remain in a quiescent state associated with activation of a proliferation arrest gene program. Strikingly, we found that inflammatory cytokine blockade using the interleukin-1 receptor antagonist anakinra led to an attenuation of inflammatory arthritis and myeloid expansion in the bone marrow of arthritic mice. In addition, anakinra reduced expression of inflammation-driven myeloid lineage and proliferation arrest gene programs in HSC of arthritic mice. Altogether, our findings show that inflammatory cytokine blockade can contribute to normalization of hematopoiesis in the context of chronic autoimmune arthritis.

Figure 1

Altered blood system in mice with collagen-induced arthritis. (A) The strategy for producing collagen-induced arthritis (CIA) and representative images showing swelling and anklyosis in the hind paws of a CIA mouse and a control (Ctrl) mouse 21 days after disease induction. (B) Peripheral blood (PB) parameters, determined by a complete blood count, of Ctrl and CIA mice (n=5 per group). (C) Total bone marrow (BM) cellularity of hind legs, and (D-F) numbers of the indicated populations (see Online Supplementary Figure S1 for FACS gating and surface marker definitions) expressed as number per million BM cells (n=6 Ctrl and 11 CIA). (G) Red blood cell parameters in PB (n=5 per group). (H) Representative image of flushed BM. (I) Erythroblast number per million BM cells (n=6 Ctrl and 11 CIA). *P<0.05; **P<0.01 ***P<0.001, as determined by the Mann-Whitney U-test. The data were compiled from three independent experiments. CFA: complete Freund adjuvant; Gr: mature granulocytes; Pre-Gr: immature granulocytes; Mon: monocytes; RBC: red blood cells.

Figure 2

Myeloid expansion in the bone marrow of mice with collagen-induced arthritis. (A) Experimental design and numbers of colony-forming units (CFU)-granulocyte-macrophage (GM), CFU-burst (CFU-B) (n=4 per group) and CFU-erythroid (CFU-E) (n=8/group) in unfractionated bone marrow cells from control mice (Ctrl) and mice with collagen-induced arthritis (CIA). (B-I) Numbers of the indicated populations (see Online Supplementary Figure S1 for FACS gating and surface marker definitions) expressed as number per million cells. (H) Representative FACS plots showing the gating strategy for identifying the CD41⁺ fraction of hematopoietic stem cells (HSC) (n=6 Ctrl and 11 CIA). The data were compiled from three independent experiments. (J) Experimental design and (K, L) Fluidigm gene expression analysis of HSC from Ctrl and CIA mice showing (K) myeloid and (L) megakaryocyte (Mk), erythroid (E) and lymphoid (Ly) lineage genes. The data are presented as log10 fold expression in CIA HSC versus Ctrl HSC. Ct values were normalized to Actb (n=8-16 per group). Data are compiled from two independent experiments. *P<0.05; **P<0.01 ***P<0.001, as determined by the Mann-Whitney U-test. BM: bone marrow; Pre GM: precursor granulocyte-macrophage; GMP: granulocyte-macrophage progenitors, CLP; common lymphoid progenitors; Pre MkE: precursor megakaryocyte/erythroid; MkP: megakaryocyte progenitors; MPP: multipotent progenitors; qRT-PCR: real-time quantitative polymerase chain reaction.

Figure 3

Hematopoietic stem cells from mice with collagen-induced arthritis retain reconstituting capacity. (A-C) Long-term engraftment of purified hematopoietic stem cells (HSC) isolated from control mice (Ctrl) and mice with collagen-induced arthritis (CIA). (A) Experimental design. (B) Donor chimerism and (C) lineage distribution in peripheral blood of recipient mice over time (n=8 Ctrl and 7 CIA recipient mice). (D-F) Long-term engraftment of unfractionated bone marrow isolated from Ctrl and CIA mice. (D) Experimental design. (E) Donor chimerism and (F) lineage distribution in peripheral blood of recipient mice over time (n=10 Ctrl and 9 CIA recipient mice). The data are representative of one of two independent experiments. (G) Experimental design and (H-I) Fluidigm gene expression analysis of HSC from Ctrl and CIA mice showing (H) HSC genes and (I) HSC surface marker genes. The data are presented as log₁₀ fold expression in CIA HSC versus Ctrl HSC. Ct values were normalized to Actb (n=8-16 per group). *P<0.05, as determined by the Mann-Whitney U-test. The data were compiled from two independent experiments.

Figure 4

Activation of a proliferation arrest gene program in hematopoietic stem cells from mice with collagen-induced arthritis. (A) Experimental design. (B) Volcano plot showing significantly differentially expressed genes (shaded areas) based on P_adj <0.05 (n=3 per group); hematopoietic stem cell (HSC) pools were sorted from three independent sets of mice. (C) Significantly differentially activated upstream regulators as determined by Ingenuity Pathway Analysis. (D, E) Gene ontology (GO) analysis of significantly differentially (D) downregulated and (E) upregulated gene sets. (F-H) Fluidigm gene expression analysis of HSC from control mice (Ctrl) and mice with collagen-induced arthritis showing (F) cell cycle activator genes; (G) cell cycle repression genes; (H) mRNA translation genes. The data are presented as log₁₀ fold expression in CIA HSC versus Ctrl HSC. Ct values were normalized to Actb (n=8-16 per group). The data were compiled from two independent experiments. (I) Experimental design. (J) Representative FACS plots and (K) cell cycle distribution of HSC in Ctrl and CIA mice (n=8 Ctrl and 7 CIA mice). The data were compiled from two independent experiments. See also Online Supplementary Tables S1-S6. *P<0.05; **P<0.01 ***P<0.001, as determined by the Mann-Whitney U-test.

Figure 5

Systemic inflammation 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 compiled from two independent experiments. (C,D) Gene expression analysis of hematopoietic stem cells (HSC) from Ctrl and CIA mice showing (C) IFN/STAT signaling genes and (D) IL-1/TNF signaling genes. The data are presented as log₁₀ fold expression in CIA HSC versus Ctrl HSC. (n=14-16 per group) *P<0.05; **P<0.01, as determined by the Mann-Whitney U-test.

Figure 6

Cytokine blockade reduces myeloid expansion in mice with collagen-induced arthritis. (A) Induction of collagen-induced arthritis (CIA) and anakinra (Ana) treatment strategy in B10.RIII mice. (B) Representative images showing swelling and anklyosis in the hind paws of mice 17 days after disease induction (top left, CIA mice not treated with Ana; bottom left, CIA mice treated with Ana) and impact of Ana treatment on arthritis score (right) (n=9 per group). (C) Peripheral blood neutrophil count and (D-I) number of the indicated bone marrow populations expressed as number per million BM cells of control, CIA and CIA+Ana mice (n=9 per group). The data were compiled from two independent experiments. *P<0.05; **P<0.01 ***P<0.001, as determined by one-way analysis of variance or the Mann-Whitney U-test. CFA: complete Freund adjuvant; Col: collagen; Ctrl: control; PB: peripheral blood; Gr: mature granulocytes; Pre Gr: immature granulocytes; Mon: monocytes, GMP: granulocyte-macrophage progenitors, CLP; common lymphoid progenitors; MPP: multipotent progenitors.

Figure 7

Cytokine blockade attenuates inflammation-induced hematopoietic stem cell gene programs. (A) Experimental design. (B) Representative FACS plots and (C) cell cycle distribution of hematopoietic stem cells (HSC) from control mice (Ctrl), mice with collagen-induced arthritis (CIA) and CIA mice treated with anakinra (+Ana) (n=5 per group). (D) Fluidigm analysis of proliferation arrest gene programs in HSC from Ctrl, CIA and CIA +Ana mice. The data are presented as log10 fold expression versus Ctrl HSC (n=12-16 per group). (E) Fluidigm analysis of myeloid gene expression in HSC from Ctrl, CIA and CIA +Ana mice. The data are presented as log₁₀ fold expression versus Ctrl HSC. Ct values were normalized to Actb (n=12-16 per group). The data are presented as log10 fold expression versus Ctrl HSC (n=12-16 per group). The data were compiled from two independent experiments. *P<0.05; **P<0.01 ***P<0.001, ****P<0.0001 as determined by one-way analysis of variance or the Mann-Whitney U-test. BM: bone marrow.

Figure 8

Model of inflammation-driven hematopoietic alterations in mice with collagen-induced arthritis. Under homeostatic conditions, hematopoietic stem cells (HSC) are largely quiescent but occasionally enter the cell cycle and give rise to lineage-biased multipotent progenitor (MPP) subsets, leading to a balanced lineage output and nominal HSC self-renewal capacity. In mice with collagen-induced arthritis (CIA), chronic inflammation leads to expansion of myeloid-biased MPP3 and granulocyte-macrophage progenitors (GMP), resulting in increased myeloid cell production. Concurrently, inflammation induces a myeloid lineage gene program in HSC that may bias HSC toward further overproduction of MPP3. Despite ongoing inflammation, HSC are maintained in a quiescent state characterized by repression of cell cycle and mRNA translation genes alongside induction of cell cycle inhibitor genes. Notably, pro-inflammatory cytokine blockade, here using anakinra, attenuates myeloid expansion and altered gene expression in HSC. These data indicate that chronic inflammation drives aberrant hematopoiesis in rheumatoid arthritis, and this phenotype can be attenuated by cytokine-blocking therapy.