Overproduced bone marrow neutrophils in collagen-induced arthritis are primed for NETosis: An ignored pathological cell involving inflammatory arthritis

Abstract

Objectives: Bone marrow edema is a universal manifestation of rheumatoid arthritis (RA), and its pathological essence is a bone marrow lesion (BML) formed by various bone marrow (BM) immune cells. Neutrophils play an important role in inflammatory arthritis, but the role and mechanism of neutrophils in BML are not clear.

Materials and methods: Granulocyte colony-stimulating factor (G-CSF) -/- mice and wild type (WT) C57BL/6 mice were immunized for collagen-induced arthritis (CIA). Histological scores of arthritis were evaluated. Immunohistochemistry staining with anti-Ly6G was conducted. Neutrophil extracellular traps (NETs) in joint sections were determined by immunofluorescence staining. BM neutrophils were isolated for flow cytometry and NETosis induction in vitro.

Results: Histological study showed significant neutrophil infiltrations in BML of CIA mice. Inhibition of BM neutrophil production by G-CSF knock out can obstruct the induction of BML and CIA. In addition to abundant infiltrated NETs intra-articular, remarkable NETosis primed BM neutrophils were infiltrated in BML of CIA mice, which was positively related to bone erosion. Neutrophils derived from G-CSF-/- mice have diminished ability of NETs formation in vitro, while G-CSF induction can enhance its capacity of NETs formation.

Conclusions: We propose for the first time that the overproduced BM neutrophils in CIA mice are primed for NETosis in a G-CSF dependent manner, and these pathogenic cells may have an important role in inflammatory arthritis. Blocking this pathological process could be a potential strategy for the treatment of RA.

Keywords: G-CSF; bone marrow lesion; neutrophil extracellular traps; rheumatoid arthritis.

FIGURE 1

Collagen‐induced arthritis (CIA) mice showed significant neutrophil infiltration in bone marrow lesion (BML). A, Representative images of hematoxylin and eosin‐stained sections of arthritis and subchondral BML obtained from the ankle joints of CIA and normal control mice. B, Representative pictures of neutrophils infiltrating the ankle joints and subchondral bone marrow (BM) of CIA and normal control mice, by Ly6G staining (in brown). C, The number and percentage of subchondral BM Ly6G‐ positive cells per field in CIA vs normal control mice were analyzed by Image‐pro plus 6.0, as determined by two‐tailed, unpaired Student's t test (mean ± SEM). Results shown are representative for five fields in different regions of each section and two independent experiments with 8 individual mice per group

FIGURE 2

Suppression of bone marrow (BM) neutrophil production by granulocyte colony‐stimulating factor (G‐CSF) knockout can significantly obstruct the induction of collagen‐induced arthritis (CIA). A, The cumulative incidence of arthritis and (B) clinical scores (mean ± SEM) in immunized WT and G‐CSF−/− mice during days 21‐51 of CIA induction. Results shown are from 8 individual mice per group and two independent experiments. C, Representative histopathological sections of ankle joints from immunized WT and G‐CSF−/− mice by hematoxylin and eosin staining. D, The semi‐quantitative histological score was evaluated and compared, as determined by two‐tailed, unequal variance Student's t test (mean ± SEM). Results shown are from 8 individual mice per group and two independent experiments. E, Representative immunohistochemical staining of Ly6G in subchondral BM. Brown staining indicates Ly6G positive cells. F, The percentage and number of Ly6G positive cells per field in subchondral BM from normal control or immunized WT and G‐CSF−/− mice were enumerated for at least five different fields of each section by Image‐pro plus 6.0, as determined by two‐tailed, unpaired Student's t test (mean ± SEM). Results shown are from 8 individual mice per group and two independent experiments

FIGURE 3

Significant NETosis primed phenomenon of bone marrow (BM) neutrophils, in addition to neutrophil extracellular traps (NETs) intra‐articular. A, Representative confocal images of NETs formation in joints of collagen‐induced arthritis (CIA) or non‐CIA by immunofluorescent staining. B, Representative confocal images of NETs formations in cartilage, articular cavity, synovium. C, Representative confocal images of neutrophils primed for NETosis in subchondral BM of WT CIA mice and magnified NETosis primed neutrophils

FIGURE 4

Bone marrow (BM) neutrophils may be involved in inflammatory arthritis through NETosis. A, Representative confocal images of neutrophils primed for NETosis in subchondral BM and BM cavity of immunized or normal control WT and granulocyte colony‐stimulating factor (G‐CSF)−/− mice. B, The number of BM neutrophils primed for NETosis per field in immunized or normal control WT compare to G‐CSF−/− mice, as determined by Mann‐Whitney U test (mean ± SEM). C, Correlation between the number of NETosis primed BM neutrophils and the pathological score of bone erosion in WT mice. Results shown are representative of three different fields of subchondral BM and three different fields of BM cavity in each section from 8 individual mice per group and 2 independent experiments were repeated. BM Neutrophils primed for NETosis were defined as colocalized cit‐H3, Ly6G, and DNA. All pictures were taken by confocal microscopy (Nikon Eclipse Ti)

FIGURE 5

Bone marrow (BM) neutrophils are primed for NETosis. A, Representative confocal images of BM neutrophils isolated from collagen‐induced arthritis (CIA) mice and normal control mice before and after induction of serum. B, Percentage of BM neutrophils primed for NETosis per field in neutrophils from CIA mice as compared to neutrophils from normal control mice before induction, (C) Percentage of BM neutrophils forming neutrophil extracellular traps (NETs) per field from normal control and CIA mice before induction as compared to that of after induction with their corresponding serum, as determined by two‐tailed, unequal variance Student's t test (mean ± SEM). Results shown are representative of at least eight fields in different regions for each condition, and representative of two independent experiments. D, Magnified representative confocal images of BM neutrophils from CIA mice forming NETs in vitro after induction. E, Magnified representative confocal images of NETosis primed neutrophils in BM and NETs formation in cartilage and joint cavity

FIGURE 6

Impaired maturity of bone marrow (BM) neutrophils from granulocyte colony‐stimulating factor (G‐CSF)−/− mice. A, Representative dot plots of the relative proportion of mature neutrophils. B, The percentage of mature neutrophils relative to the total number of WT BM cells as compared to G‐CSF−/− BM cells, as determined by two‐tailed, unequal variance Student's t test (mean ± SEM). Results shown are from 8 individual mice per group and two independent experiments

FIGURE 7

Granulocyte colony‐stimulating factor (G‐CSF) affects the ability of bone marrow (BM) neutrophils to form neutrophil extracellular traps (NETs) in vitro. A, Representative confocal images of immunofluorescent staining of NETs in BM neutrophils isolated from WT and G‐CSF−/− mice with the induction of PBS, phorbol 12‐myristate 13‐acetate (PMA), PMA + G‐CSF. B, Percentage of neutrophils forming NETs per field in WT neutrophils as compared to G‐CSF−/− neutrophils with the induction of PBS, PMA, PMA + G‐CSF, as determined by two‐tailed, unequal variance Student's t test (mean ± SEM). Results shown are representative of at least eight fields in different regions for each condition and represent three independent experiments

FIGURE 8

Under inflammation, bone marrow (BM) neutrophils are produced in large quantities under the action of granulocyte colony‐stimulating factor (G‐CSF), meanwhile primed for NETosis. Once released into the periphery and subjected to chemotaxis of inflammation, these pathological cells can infiltrate into inflammatory joints and form neutrophil extracellular traps (NETs), promoting joint inflammation and autoantibody production, eventually leading to bone erosion