Chronic MRSA periprosthetic joint infection induces formation of mature tertiary lymphoid structures and osteolysis in mice

doi:10.12122/j.issn.1673-4254.2026.07.09

Abstract

Abstract:

Objective To investigate the histomorphological and functional characteristics of tertiary lymphoid structures (TLS) in a mouse model of chronic knee periprosthetic joint infection (PJI) with methicillin-resistant Staphylococcus aureus (MRSA) and explore their association with local osteolysis. Methods C57BL/6J mice receiving prosthesis implantation in the knee joint were randomized into control group and PJI group for injection of sterile PBS and of MRSA USA300 (1×10⁴ CFU) into the knee joint cavity, respectively. Peripheral blood leukocyte counts, erythrocyte sedimentation rates, and C-reactive protein levels were monitored regularly. Sixty days after the injections, periprosthetic tissues were harvested for HE staining to measure the cross-sectional area of mature TLS. Multiplex immunofluorescence staining was used to analyze spatial segregation of T and B cells (CD3/B220) and the network of follicular dendritic cells (FDCs) and detect Ki67 and PNAd expressions for assessing TLS functionality. The number of osteoclast per unit of bone surface (N.Oc/BS) in the periprosthetic region was determined using histomorphometric analysis with TRAP staining. Results At 21 days post-surgery, the mice in PJI group showed significant elevation of all the peripheral blood inflammatory markers. Numerous mature TLS were observed in the periprosthetic tissues in PJI group with an average cross-sectional area significantly greater than that in the control group. The TLS in PJI group exhibited a typical spatial organization, characterized by a B-cell-centered core surrounded by T cells with an FDC network; abundant Ki67-positive proliferating cells were observed within the TLS, and PNAd-positive high endothelial venules were visible at the periphery and within the parenchyma. The N.Oc/BS was significantly greater in PJI group than in the control group (28.75±4.13 vs 1.63±0.74 mm^-1, P<0.0001). Conclusion Chronic MRSA infection induces formation of mature TLS with well-defined structural organization and functional activity in the periprosthetic region of mice, accompanied by significant bone loss.

Key words: methicillin-resistant Staphylococcus aureus, periprosthetic infection, tertiary lymphoid structures, mouse model, immune microenvironment

Maihemuti Munila, Lisi HUANG, Jiakai LI, Rulong CAI, Qi FENG, Yudi LIN, Zehong HUANG, Sijia WU, Muhammad Umar, Hang FANG. Chronic MRSA periprosthetic joint infection induces formation of mature tertiary lymphoid structures and osteolysis in mice[J]. Journal of Southern Medical University, 2026, 46(7): 1553-1562.

Figures/Tables 9

Fig.1 Design and manufacturing of the murine knee prosthesis. A: The micro-prosthesis model designed via CAD software, tailored to the distal femoral anatomy of C57BL/6J mice. B: Macroscopic view of the final Ti6Al4V implant produced by selective laser melting.

Tab.1 Chemical composition of Ti6Al4V powder (wt%)

Powder Ti Al V O N C H Fe Zn P Cu Ag
Ti6Al4V powder	- 5.5-6.5 3.5-4.5 <0.13 <0.03 <0.08 <0.012 <0.25 - - - -

Fig.2 Schematic diagram of construction workflow for establishing mouse models of chronic knee periprosthetic joint infection (PJI) with methicillin-resistant Staphylococcus aureus (MRSA). Left: Experimental grouping and modeling procedure. Right: Schematic illustration of the periprosthetic microenvironment in the presence of infection (indicated by the red box).

Fig.3 Establishment and postoperative evaluation of the mouse knee PJI model. A: Surgical procedures of the PJI model. B: X-ray images of mouse knee joints at 28 days post-surgery. C: Quantitative analysis of peripheral blood WBC, ESR, and CRP levels. Data are presented as Mean±SD, n=6. *P<0.05, ****P<0.0001.

Fig.4 HE staining and quantitative analysis of tertiary lymphoid structures (TLS) in periprosthetic soft tissues of mouse knee joints (n=6). A-C: HE staining showing massive dense inflammatory cell infiltration and formation of typical lymphoid follicle-like aggregate structures in PJI group. D-F: HE staining showing only sparse inflammatory cells in the prosthesis (control) group. Scale bar=100 μm. Data are presented as Mean±SD. **P<0.01.

Fig.5 HE staining and multiplex immunofluorescence (mIF) staining of periprosthetic tissues. A-D: HE staining and merged mIF images in PJI group (CD21, green; CD45, red; CD3, yellow). E, F: HE staining and merged mIF image in prosthesis group (Scale bar=50 μm).

Fig.6 Histological features and spatial organization of T cells, B cells, and follicular dendritic cells (FDCs) in periprosthetic tissues of the mice, using spleen tissue as the positive control. A, C, E: Representative HE staining images of the PJI group (A), prosthesis group (C), and the spleen (E). B, D, F: Corresponding merged mIF images showing immune cell distribution in PJI group (B), prosthesis group (D), and the spleen (F). The markers are colored as follows: CD3 (red), B220 (yellow), CD21 (green), and nuclei are counterstained with DAPI (blue) (Scale bar=50 μm).

Fig.7 Expression of Ki67 and PNAd in periprosthetic tissues of the mice (Scale bar=50 μm). A: Representative mIF images of Ki67 and B220 in PJI group (top two rows) and prosthesis group (bottom row). B: Representative mIF images of PNAd and B220 in PJI group (top two rows) and prosthesis group (bottom row). The markers are colored as follows: Ki67 or PNAd (green), B220 (red), and nuclei are counterstained with DAPI (blue).

Fig.8 TRAP staining and quantitative analysis of osteoclasts in periprosthetic bone tissues of the mice in PJI and prosthesis groups (Scale bar=100 μm). Data are presented as Mean±SD (n=6). ****P<0.0001.

References 38

[1]	Borens O, Yusuf E, Trampuz A. Surgical site infections (SSIs): risk factors and prevention strategies[M]//European Instructional Lectures. Berlin, Heidelberg: Springer, 2013: 15-24. doi：10.1007/978-3-642-36149-4_2
[2]	Kurtz SM, Lau E, Watson H, et al. Economic burden of periprosthetic joint infection in the United States[J]. J Arthroplasty, 2012, 27(8 ): 61-5.e1. doi：10.1016/j.arth.2012.02.022
[3]	Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections[J]. Science, 1999, 284(5418): 1318-22. doi：10.1126/science.284.5418.1318
[4]	Foster TJ. Immune evasion by staphylococci[J]. Nat Rev Microbiol, 2005, 3(12): 948-58. doi：10.1038/nrmicro1289
[5]	de Mesy Bentley KL, Trombetta R, Nishitani K, et al. Evidence of Staphylococcus aureus deformation, proliferation, and migration in canaliculi of live cortical bone in murine models of osteomyelitis[J]. J Bone Miner Res, 2017, 32(5): 985-90. doi：10.1002/jbmr.3055
[6]	Visperas A, Santana D, Klika AK, et al. Current treatments for biofilm-associated periprosthetic joint infection and new potential strategies[J]. J Orthop Res, 2022, 40(7): 1477-91. doi：10.1002/jor.25345
[7]	Muthukrishnan G, Masters EA, Daiss JL, et al. Mechanisms of immune evasion and bone tissue colonization that make Staphylococcus aureus the primary pathogen in osteomyelitis[J]. Curr Osteoporos Rep, 2019, 17(6): 395-404. doi：10.1007/s11914-019-00548-4
[8]	Tang HD, Zhu MZ, Qiao J, et al. Lymphotoxin signalling in tertiary lymphoid structures and immunotherapy[J]. Cell Mol Immunol, 2017, 14(10): 809-18. doi：10.1038/cmi.2017.13
[9]	Mueller CG, Nayar S, Gardner D, et al. Cellular and vascular components of tertiary lymphoid structures[M]//Tertiary Lymphoid Structures. New York, NY: Springer New York, 2018: 17-30. doi：10.1007/978-1-4939-8709-2_2
[10]	Dieu-Nosjean MC, Goc J, Giraldo NA, et al. Tertiary lymphoid structures in cancer and beyond[J]. Trends Immunol, 2014, 35(11): 571-80. doi：10.1016/j.it.2014.09.006
[11]	Song IH, Heo SH, Bang WS, et al. Predictive value of tertiary lymphoid structures assessed by high endothelial venule counts in the neoadjuvant setting of triple-negative breast cancer[J]. Cancer Res Treat, 2017, 49(2): 399-407. doi：10.4143/crt.2016.215
[12]	Wang XQ, Sadovskaya I, Leterme D, et al. A comparative study of antibodies against proteins extracted from staphylococcal biofilm for the diagnosis of orthopedic prosthesis-related infections in an animal model and in humans[J]. Diagn Microbiol Infect Dis, 2013, 75(2): 124-9. doi：10.1016/j.diagmicrobio.2012.10.013
[13]	Carli AV, Ross FP, Bhimani SJ, et al. Developing a clinically representative model of periprosthetic joint infection[J]. J Bone Joint Surg Am, 2016, 98(19): 1666-76. doi：10.2106/jbjs.15.01432
[14]	Inzana JA, Schwarz EM, Kates SL, et al. A novel murine model of established Staphylococcal bone infection in the presence of a fracture fixation plate to study therapies utilizing antibiotic-laden spacers after revision surgery[J]. Bone, 2015, 72: 128-36. doi：10.1016/j.bone.2014.11.019
[15]	Taniguchi N, Fujibayashi S, Takemoto M, et al. Effect of pore size on bone ingrowth into porous titanium implants fabricated by additive manufacturing: an in vivo experiment[J]. Mater Sci Eng C Mater Biol Appl, 2016, 59: 690-701. doi：10.1016/j.msec.2015.10.069
[16]	Gbejuade HO, Lovering AM, Webb JC. The role of microbial biofilms in prosthetic joint infections[J]. Acta Orthop, 2015, 86(2): 147-58. doi：10.3109/17453674.2014.966290
[17]	Chen ZT, Klein T, Murray RZ, et al. Osteoimmunomodulation for the development of advanced bone biomaterials[J]. Mater Today, 2016, 19(6): 304-21. doi：10.1016/j.mattod.2015.11.004
[18]	Heim CE, Vidlak D, Kielian T. Interleukin-10 production by myeloid-derived suppressor cells contributes to bacterial persistence during Staphylococcus aureus orthopedic biofilm infection[J]. J Leukoc Biol, 2015, 98(6): 1003-13. doi：10.1189/jlb.4vma0315-125rr
[19]	Buckley CD, Barone F, Nayar S, et al. Stromal cells in chronic inflammation and tertiary lymphoid organ formation[J]. Annu Rev Immunol, 2015, 33: 715-45. doi：10.1146/annurev-immunol-032713-120252
[20]	Pitzalis C, Jones GW, Bombardieri M, et al. Ectopic lymphoid-like structures in infection, cancer and autoimmunity[J]. Nat Rev Immunol, 2014, 14(7): 447-62. doi：10.1038/nri3700
[21]	Cao J, Xu F, Lin SH, et al. IL-27 controls sepsis-induced impairment of lung antibacterial host defence[J]. Thorax, 2014, 69(10): 926-37. doi：10.1136/thoraxjnl-2014-205777
[22]	Ruddle NH. Lymphoid neo-organogenesis: lymphotoxin's role in inflammation and development[J]. Immunol Res, 1999, 19(2/3): 119-25. doi：10.1007/bf02786481
[23]	Denton AE, Linterman MA. Stromal networking: cellular connections in the germinal centre[J]. Curr Opin Immunol, 2017, 45: 103-11. doi：10.1016/j.coi.2017.03.001
[24]	Aloisi F, Pujol-Borrell R. Lymphoid Neogenesis in chronic inflammatory diseases[J]. Nat Rev Immunol, 2006, 6(3): 205-17. doi：10.1038/nri1786
[25]	Rangel-Moreno J, Carragher DM, de la Luz Garcia-Hernandez M, et al. The development of inducible Bronchus-associated lymphoid tissue depends on IL-17[J]. Nat Immunol, 2011, 12(7): 639-46. doi：10.1038/ni.2053
[26]	Moyron-Quiroz JE, Rangel-Moreno J, Kusser K, et al. Role of inducible Bronchus associated lymphoid tissue (iBALT) in respiratory immunity[J]. Nat Med, 2004, 10(9): 927-34. doi：10.1038/nm1091
[27]	Heesters BA, Myers RC, Carroll MC. Follicular dendritic cells: dynamic antigen libraries[J]. Nat Rev Immunol, 2014, 14(7): 495-504. doi：10.1038/nri3689
[28]	Krimpenfort LT, Degn SE, Heesters BA. The follicular dendritic cell: At the germinal center of autoimmunity?[J]. Cell Rep, 2024, 43(3): 113869. doi：10.1016/j.celrep.2024.113869
[29]	Kranich J, Krautler NJ. How follicular dendritic cells shape the B-cell antigenome[J]. Front Immunol, 2016, 7: 225. doi：10.3389/fimmu.2016.00225
[30]	Dapunt U, Maurer S, Giese T, et al. The macrophage inflammatory proteins MIP1α (CCL3) and MIP2α (CXCL2) in implant-associated osteomyelitis: linking inflammation to bone degradation[J]. Mediators Inflamm, 2014, 2014: 728619. doi：10.1155/2014/728619
[31]	Teillaud JL, Dieu-Nosjean MC. Tertiary lymphoid structures: an anti-tumor school for adaptive immune cells and an antibody factory to fight cancer?[J]. Front Immunol, 2017, 8: 830. doi：10.3389/fimmu.2017.00830
[32]	Scherr TD, Heim CE, Morrison JM, et al. Hiding in plain sight: interplay between staphylococcal biofilms and host immunity[J]. Front Immunol, 2014, 5: 37. doi：10.3389/fimmu.2014.00037
[33]	Titorencu I, Jinga VV, Constantinescu E, et al. Proliferation, differentiation and characterization of osteoblasts from human BM mesenchymal cells[J]. Cytotherapy, 2007, 9(7): 682-96. doi：10.1080/14653240701561329
[34]	Meednu N, Zhang HW, Owen T, et al. Production of RANKL by memory B cells: a link between B cells and bone erosion in rheumatoid arthritis[J]. Arthritis Rheumatol, 2016, 68(4): 805-16. doi：10.1002/art.39489
[35]	Takayanagi H. Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems[J]. Nat Rev Immunol, 2007, 7(4): 292-304. doi：10.1038/nri2062
[36]	Okamoto K, Nakashima T, Shinohara M, et al. Osteoimmunology: the conceptual framework unifying the immune and skeletal systems[J]. Physiol Rev, 2017, 97(4): 1295-349. doi：10.1152/physrev.00036.2016
[37]	Sato K, Suematsu A, Okamoto K, et al. Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction[J]. J Exp Med, 2006, 203(12): 2673-82. doi：10.1084/jem.20061775
[38]	Marriott I. Osteoblast responses to bacterial pathogens: a previously unappreciated role for bone-forming cells in host defense and disease progression[J]. Immunol Res, 2004, 30(3): 291-308. doi：10.1385/ir:30:3:291