Fibrocyte enrichment and myofibroblastic adaptation causes nucleus pulposus fibrosis and associates with disc degeneration severity

Abstract

Fibrotic remodeling of nucleus pulposus (NP) leads to structural and mechanical anomalies of intervertebral discs that prone to degeneration, leading to low back pain incidence and disability. Emergence of fibroblastic cells in disc degeneration has been reported, yet their nature and origin remain elusive. In this study, we performed an integrative analysis of multiple single-cell RNA sequencing datasets to interrogate the cellular heterogeneity and fibroblast-like entities in degenerative human NP specimens. We found that disc degeneration severity is associated with an enrichment of fibrocyte phenotype, characterized by CD45 and collagen I dual positivity, and expression of myofibroblast marker α-smooth muscle actin. Refined clustering and classification distinguished the fibrocyte-like populations as subtypes in the NP cells - and immunocytes-clusters, expressing disc degeneration markers HTRA1 and ANGPTL4 and genes related to response to TGF-β. In injury-induced mouse disc degeneration model, fibrocytes were found recruited into the NP undergoing fibrosis and adopted a myofibroblast phenotype. Depleting the fibrocytes in CD11b-DTR mice in which myeloid-derived lineages were ablated by diphtheria toxin could markedly attenuate fibrous modeling and myofibroblast formation in the NP of the degenerative discs, and prevent disc height loss and histomorphological abnormalities. Marker analysis supports that disc degeneration progression is dependent on a function of CD45⁺COL1A1⁺ and αSMA⁺ cells. Our findings reveal that myeloid-derived fibrocytes play a pivotal role in NP fibrosis and may therefore be a target for modifying disc degeneration and promoting its repair.

References

[1.] Sun Y, Lyu M, Lu Q, Cheung K, Leung V. Current perspectives on nucleus pulposus fibrosis in disc degeneration and repair Int. J. Mol. Sci., 2022, 23: 6612.

[2.] Zhang S, et al.. The role of structure and function changes of sensory nervous system in intervertebral disc-related low back pain Osteoarthritis Cartilage, 2021, 29: 17-27.

[3.] Leung VY, et al.. Mesenchymal stem cells reduce intervertebral disc fibrosis and facilitate repair Stem Cells, 2014, 32: 2164-2177.

[4.] Au TYK, et al.. Transformation of resident notochord-descendent nucleus pulposus cells in mouse injury-induced fibrotic intervertebral discs Aging Cell, 2020, 19. e13254

[5.] Zhang Y, et al.. Directed differentiation of notochord-like and nucleus pulposus-like cells using human pluripotent stem cells Cell Rep., 2020, 30: 2791-2806.e2795.

[6.] Zhou T, et al.. Spatiotemporal characterization of human early intervertebral disc formation at single-cell resolution Adv. Sci., 2023, 10. e2206296

[7.] Han S, et al.. Single-cell RNA sequencing of the nucleus pulposus reveals chondrocyte differentiation and regulation in intervertebral disc degeneration Front. Cell Dev. Biol., 2022, 10: 824771.

[8.] Kuppe C, et al.. Decoding myofibroblast origins in human kidney fibrosis Nature, 2021, 589: 281-286.

[9.] Lv FJ, et al.. Matrix metalloproteinase 12 is an indicator of intervertebral disc degeneration co-expressed with fibrotic markers Osteoarthritis Cartilage, 2016, 24: 1826-1836.

[10.] Chen C, et al.. Autologous fibroblasts induce fibrosis of the nucleus pulposus to maintain the stability of degenerative intervertebral discs Bone Res., 2020, 8: 7.

[11.] Wang D, et al.. Single-cell transcriptomics reveals heterogeneity and intercellular crosstalk in human intervertebral disc degeneration iScience, 2023, 26: 106692.

[12.] Tu J, et al.. Single-cell transcriptome profiling reveals multicellular ecosystem of nucleus pulposus during degeneration progression Adv. Sci., 2022, 9. e2103631

[13.] Ling Z, et al.. Single-cell RNA-Seq analysis reveals macrophage involved in the progression of human intervertebral disc degeneration Front. Cell Dev. Biol., 2021, 9: 833420.

[14.] Gan Y, et al.. Spatially defined single-cell transcriptional profiling characterizes diverse chondrocyte subtypes and nucleus pulposus progenitors in human intervertebral discs Bone Res., 2021, 9: 37.

[15.] Sakai D, et al.. Migration of bone marrow-derived cells for endogenous repair in a new tail-looping disc degeneration model in the mouse: a pilot study Spine J., 2015, 15: 1356-1365.

[16.] Melrose J, et al.. Spatial and temporal localization of transforming growth factor-beta, fibroblast growth factor-2, and osteonectin, and identification of cells expressing alpha-smooth muscle actin in the injured anulus fibrosus: implications for extracellular matrix repair Spine, 2002, 27: 1756-1764.

[17.] Esteve D, et al.. Lobular architecture of human adipose tissue defines the niche and fate of progenitor cells Nat. Commun., 2019, 10. 2549

[18.] Hinz B, Lagares D. Evasion of apoptosis by myofibroblasts: a hallmark of fibrotic diseases Nat. Rev. Rheumatol., 2020, 16: 11-31.

[19.] Li XC, et al.. Investigation of macrophage polarization in herniated nucleus pulposus of patients with lumbar intervertebral disc herniation J. Orthop. Res., 2023, 41: 1335-1347.

[20.] Kim J, et al.. Circulating and renal fibrocytes are associated with interstitial fibrosis in lupus nephritis Rheumatology, 2023, 62: 914-923.

[21.] Abe S, et al.. Functional analysis of human fibrocytes derived from monocytes reveals their profibrotic phenotype through paracrine effects J. Med. Investig., 2020, 67: 102-112.

[22.] Reinhardt JW, Breuer CK. Fibrocytes: a critical review and practical guide Front. Immunol., 2021, 12: 784401.

[23.] Ozono Y, et al.. Neoplastic fibrocytes play an essential role in bone marrow fibrosis in Jak2V617F-induced primary myelofibrosis mice Leukemia, 2021, 35: 454-467.

[24.] White MJ, Roife D, Gomer RH. Galectin-3 binding protein secreted by breast cancer cells inhibits monocyte-derived fibrocyte differentiation J. Immunol., 2015, 195: 1858-1867.

[25.] Abu El-Asrar AM, et al.. Myofibroblasts in proliferative diabetic retinopathy can originate from infiltrating fibrocytes and through endothelial-to-mesenchymal transition (EndoMT) Exp. Eye Res., 2015, 132: 179-189.

[26.] Li D, et al.. HtrA1 upregulates the expression of ADAMTS-5 in HNPCs via the ERK/NF-kappaB/JNK signaling pathway Am. J. Transl. Res., 2019, 11: 5114-5121

[27.] Liu FJ, et al.. Expression of ANGPTL4 in nucleus pulposus tissues is associated with intervertebral disc degeneration Dis. Markers, 2021, 2021: 3532716.

[28.] Reich B, et al.. Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model Kidney Int., 2013, 84: 78-89.

[29.] Andersson-Sjoland A, et al.. Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis Int. J. Biochem. Cell Biol., 2008, 40: 2129-2140.

[30.] Basatvat S, et al.. Harmonization and standardization of nucleus pulposus cell extraction and culture methods JOR Spine, 2023, 6. e1238

[31.] Srakocic S, et al.. Proposed practical protocol for flow cytometry analysis of microglia from the healthy adult mouse brain: systematic review and isolation methods’ evaluation Front. Cell Neurosci., 2022, 16: 1017976.

[32.] Qin C, et al.. Causal relationship between the blood immune cells and intervertebral disc degeneration: univariable, bidirectional and multivariable Mendelian randomization Front. Immunol., 2023, 14: 1321295.

[33.] Niedermeier M, et al.. CD4⁺ T cells control the differentiation of Gr1⁺ monocytes into fibrocytes Proc. Natl. Acad. Sci. USA, 2009, 106: 17892-17897.

[34.] Bermudez-Lekerika P, et al.. Immuno-modulatory effects of intervertebral disc cells Front. Cell Dev. Biol., 2022, 10: 924692.

[35.] Sinha M, et al.. Direct conversion of injury-site myeloid cells to fibroblast-like cells of granulation tissue Nat. Commun., 2018, 9. 936

[36.] Meng XM, et al.. Inflammatory macrophages can transdifferentiate into myofibroblasts during renal fibrosis Cell Death Dis., 2016, 7. e2495

[37.] Burt KG, Kim MKM, Viola DC, Abraham AC, Chahine NO. Nuclear factor kappaB overactivation in the intervertebral disc leads to macrophage recruitment and severe disc degeneration Sci Adv., 2024, 10: eadj3194.

[38.] Hong HS, Ahn W, Son Y. CD45⁺/CD11b⁺ monocytes are required for mesenchymal stem cell proliferation Tissue Eng. Regen. Med., 2014, 11: 224-229.

[39.] Jin, L. et al. CCR2 monocytes as therapeutic targets for acute disc herniation and radiculopathy in mouse models. Osteoarthritis Cartilage 32, 52–65 (2023).

[40.] Mouton AJ, et al.. Mapping macrophage polarization over the myocardial infarction time continuum Basic Res. Cardiol., 2018, 113: 26.

[41.] Zhang S, et al.. Inhibiting heat shock protein 90 attenuates nucleus pulposus fibrosis and pathologic angiogenesis induced by macrophages via down-regulating cell migration-inducing protein Am. J. Pathol., 2023, 193: 960-976.

[42.] Yasuma T, Arai K, Yamauchi Y. The histology of lumbar intervertebral disc herniation. The significance of small blood vessels in the extruded tissue Spine, 1993, 18: 1761-1765.

[43.] Ishida Y, et al.. Essential roles of the CC chemokine ligand 3-CC chemokine receptor 5 axis in bleomycin-induced pulmonary fibrosis through regulation of macrophage and fibrocyte infiltration Am. J. Pathol., 2007, 170: 843-854.

[44.] Phillips RJ, et al.. Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis J. Clin. Investig., 2004, 114: 438-446.

[45.] Liu W, et al.. Annulus fibrosus cells express and utilize C-C chemokine receptor 5 (CCR5) for migration Spine J., 2017, 17: 720-726.

[46.] Phillips KL, et al.. The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc Arthritis Res. Ther., 2013, 15: R213.

[47.] Wangler S, et al.. CD146/MCAM distinguishes stem cell subpopulations with distinct migration and regenerative potential in degenerative intervertebral discs Osteoarthritis Cartilage, 2019, 27: 1094-1105.

[48.] Leite Pereira, C. et al. Stromal cell derived factor-1-mediated migration of mesenchymal stem cells enhances collagen Type II expression in intervertebral disc. Tissue Eng. Part A 24, 1818–1830 (2018).

[49.] Gruber HE, et al.. Production and expression of RANTES (CCL5) by human disc cells and modulation by IL-1-beta and TNF-alpha in 3D culture Exp. Mol. Pathol., 2014, 96: 133-138.

[50.] Zhang H, et al.. SDF1/CXCR4 axis facilitates the angiogenesis via activating the PI3K/AKT pathway in degenerated discs Mol. Med. Rep., 2020, 22: 4163-4172

[51.] Liu Z, et al.. SDF‑1/CXCR4 axis induces apoptosis of human degenerative nucleus pulposus cells via the NF‑kappaB pathway Mol. Med. Rep., 2016, 14: 783-789.

[52.] Supakul S, et al.. Pericytes as a source of osteogenic cells in bone fracture healing Int. J. Mol. Sci., 2019, 20: 1079.

[53.] Chen Y, et al.. Type-I collagen produced by distinct fibroblast lineages reveals specific function during embryogenesis and Osteogenesis Imperfecta Nat. Commun., 2021, 12. 7199

[54.] Saraswati S, Marrow SMW, Watch LA, Young PP. Identification of a pro-angiogenic functional role for FSP1-positive fibroblast subtype in wound healing Nat. Commun., 2019, 10. 3027

[55.] Mitsuhashi A, et al.. Identification of fibrocyte cluster in tumors reveals the role in antitumor immunity by PD-L1 blockade Cell Rep., 2023, 42: 112162.

[56.] Niu XH, et al.. IL-18/IL-18R1 promotes circulating fibrocyte differentiation in the aging population Inflamm. Res., 2020, 69: 497-507.

[57.] Henrot P, et al.. Muscarinic receptor M3 activation promotes fibrocytes contraction Front. Pharmacol., 2022, 13: 939780.

[58.] Ashley SL, Wilke CA, Kim KK, Moore BB. Periostin regulates fibrocyte function to promote myofibroblast differentiation and lung fibrosis Mucosal Immunol., 2017, 10: 341-351.

[59.] Kuroda N, et al.. Infiltrating CCR2⁺ monocytes and their progenies, fibrocytes, contribute to colon fibrosis by inhibiting collagen degradation through the production of TIMP-1 Sci. Rep., 2019, 9. 8568

[60.] Gan Y, et al.. Role of semaphorin 7a signaling in transforming growth factor beta1-induced lung fibrosis and scleroderma-related interstitial lung disease Arthritis Rheum., 2011, 63: 2484-2494.

[61.] Binch AL, et al.. Class 3 semaphorins expression and association with innervation and angiogenesis within the degenerate human intervertebral disc Oncotarget, 2015, 6: 18338-18354.

[62.] Vo NV, et al.. Expression and regulation of metalloproteinases and their inhibitors in intervertebral disc aging and degeneration Spine J., 2013, 13: 331-341.

[63.] Zhu D, et al.. Periostin: an emerging molecule with a potential role in spinal degenerative diseases Front. Med., 2021, 8: 694800.

[64.] Furtwangler T, Chan SC, Bahrenberg G, Richards PJ, Gantenbein-Ritter B. Assessment of the matrix degenerative effects of MMP-3, ADAMTS-4, and HTRA1, injected into a bovine intervertebral disc organ culture model Spine, 2013, 38: E1377-E1387.

[65.] Tam V, et al.. Histological and reference system for the analysis of mouse intervertebral disc J. Orthop. Res., 2018, 36: 233-243.

[66.] Lattouf R, et al.. Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues J. Histochem. Cytochem., 2014, 62: 751-758.

Funding

The General Research Fund of the Research Grant Council of Hong Kong, #17121619; The Scientific Research Foundation of Peking University Shenzhen Hospital, #KYQD202100X; Guangdong Basic and Applied Basic Research Foundation, #2024A1515010104