{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T23:47:53Z","timestamp":1791244073421,"version":"4.1.0"},"reference-count":45,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T00:00:00Z","timestamp":1769644800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002428","name":"Austrian Science Fund","doi-asserted-by":"publisher","award":["10.55776\/P25338"],"award-info":[{"award-number":["10.55776\/P25338"]}],"id":[{"id":"10.13039\/501100002428","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Validated biomarkers for clinical decision-making in spondyloarthritis (SpA) remain limited, and exploratory experimental studies may help prioritize candidate immune and bone-related readouts for future validation. In this pilot study, cytokine and bone-related biomarker profiles were analyzed in a proteoglycan-induced SpA model using Th1-prone C57BL\/6J wild-type (WT) mice (non-immunized n = 8; immunized n = 16) and Th2-prone BALB\/c WT mice (non-immunized n = 7; immunized n = 9), as well as immunized TLR2-knockout (KO) (n = 7), TLR3-KO (n = 8), and TLR4-KO (n = 3) strains on the C57BL\/6J background. Serum cytokines were quantified longitudinally with a 26-plex immunoassay, and ELISA measured bone metabolism markers (DKK1, Wnt3a, Noggin). Cytokine analysis revealed distinct Th1\/Th2 polarization: immunized Th1-prone C57BL\/6J WT mice exhibited high Th1- and Th17-type cytokines (TNF-\u03b1, IFN\u03b3, IL-12p70, IL-17A, and IL-22), whereas immunized Th2-prone BALB\/c WT mice showed elevated Th2- and eosinophil-related cytokines (IL-4, IL-9, IL-13, IL-5, and RANTES). In TLR2-KO and TLR3-KO, Th1- and Th17-associated cytokines were markedly reduced, while Th2 cytokines were increased, confirming that TLR2 is essential for maintaining pro-inflammatory signaling. DKK-1 and Noggin levels were significantly higher in TLR2-KO mice, indicating altered terminal serum bone-marker profiles under immunized conditions. These findings indicate that Th1\/Th2 immune backgrounds and TLR-associated contexts are associated with distinct cytokine patterns and differences in terminal bone markers in this experimental SpA model. Given the pilot design, small and imbalanced groups, missing non-immunized TLR-KO controls, and exploratory statistics without multiplicity adjustment, the results should be interpreted as hypothesis-generating and require confirmation in appropriately controlled, statistically powered studies incorporating longitudinal and structural endpoints, as the present findings are exploratory and not directly translatable to clinical biomarker use or therapeutic decision-making.<\/jats:p>","DOI":"10.3390\/ijms27031337","type":"journal-article","created":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T09:12:01Z","timestamp":1769677921000},"page":"1337","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Exploratory Cytokine and Bone-Marker Patterns in a Proteoglycan-Induced Spondyloarthritis Mouse Model: Th1\/Th2 Strain Comparison and TLR2\/3\/4 Knockout Readouts"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0203-213X","authenticated-orcid":false,"given":"Johannes Dominikus","family":"Pallua","sequence":"first","affiliation":[{"name":"Department of Orthopaedics and Traumatology, Medical University of Innsbruck, 6020 Innsbruck, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9208-7809","authenticated-orcid":false,"given":"Michael","family":"Schirmer","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine, Medical University of Innsbruck, 6020 Innsbruck, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1320","DOI":"10.1002\/acr.22831","article-title":"Global Prevalence of Spondyloarthritis: A Systematic Review and Meta-Regression Analysis","volume":"68","author":"Stolwijk","year":"2016","journal-title":"Arthritis Care Res."},{"key":"ref_2","first-page":"3","article-title":"One year in review 2016: Spondyloarthritis","volume":"35","author":"Parma","year":"2017","journal-title":"Clin. Exp. Rheumatol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.molimm.2013.07.013","article-title":"HLA-B27 misfolding and ankylosing spondylitis","volume":"57","author":"Colbert","year":"2014","journal-title":"Mol. Immunol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"631","DOI":"10.1038\/s41584-018-0091-8","article-title":"Effects of the IL-23-IL-17 pathway on bone in spondyloarthritis","volume":"14","author":"Gravallese","year":"2018","journal-title":"Nat. Rev. Rheumatol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"R292","DOI":"10.1186\/ar793","article-title":"Prevalence, clinical relevance and characterization of circulating cytotoxic CD4+CD28- T cells in ankylosing spondylitis","volume":"5","author":"Duftner","year":"2003","journal-title":"Arthritis Res. Ther."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Costantino, F., Breban, M., and Garchon, H.J. (2018). Genetics and Functional Genomics of Spondyloarthritis. Front. Immunol., 9.","DOI":"10.3389\/fimmu.2018.02933"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1080\/14397595.2017.1366006","article-title":"Update upon efficacy and safety of etanercept for the treatment of spondyloarthritis and juvenile idiopathic arthritis","volume":"28","author":"Murdaca","year":"2018","journal-title":"Mod. Rheumatol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"adv00698","DOI":"10.2340\/actadv.v102.563","article-title":"Long-term Safety of Secukinumab Over Five Years in Patients with Moderate-to-severe Plaque Psoriasis, Psoriatic Arthritis and Ankylosing Spondylitis: Update on Integrated Pooled Clinical Trial and Post-marketing Surveillance Data","volume":"102","author":"Gottlieb","year":"2022","journal-title":"Acta Derm. Venereol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"990","DOI":"10.1136\/annrheumdis-2013-204655","article-title":"Efficacy and safety of the anti-IL-12\/23 p40 monoclonal antibody, ustekinumab, in patients with active psoriatic arthritis despite conventional non-biological and biological anti-tumour necrosis factor therapy: 6-month and 1-year results of the phase 3, multicentre, double-blind, placebo-controlled, randomised PSUMMIT 2 trial","volume":"73","author":"Ritchlin","year":"2014","journal-title":"Ann. Rheum. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1069","DOI":"10.1038\/nm.2817","article-title":"IL-23 induces spondyloarthropathy by acting on ROR-\u03b3t+ CD3+CD4-CD8- entheseal resident T cells","volume":"18","author":"Sherlock","year":"2012","journal-title":"Nat. Med."},{"key":"ref_11","first-page":"488","article-title":"Interleukin-22 drives the proliferation, migration and osteogenic differentiation of mesenchymal stem cells: A novel cytokine that could contribute to new bone formation in spondyloarthropathies","volume":"56","author":"Jones","year":"2017","journal-title":"Rheumatology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"15","DOI":"10.3899\/jrheum.120234","article-title":"Th17 and interleukin 23 in the pathogenesis of psoriatic arthritis and spondyloarthritis","volume":"89","author":"Cauli","year":"2012","journal-title":"J. Rheumatol. Suppl."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"647","DOI":"10.1136\/ard.2005.042085","article-title":"Preferential type 1 chemokine receptors and cytokine production of CD28- T cells in ankylosing spondylitis","volume":"65","author":"Duftner","year":"2006","journal-title":"Ann. Rheum. Dis."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.gene.2004.06.044","article-title":"Wnt signaling in osteoblasts and bone diseases","volume":"341","author":"Westendorf","year":"2004","journal-title":"Gene"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"572","DOI":"10.1136\/annrheumdis-2011-200216","article-title":"High level of functional dickkopf-1 predicts protection from syndesmophyte formation in patients with ankylosing spondylitis","volume":"71","author":"Heiland","year":"2012","journal-title":"Ann. Rheum. Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1186\/ar2642","article-title":"Progress in spondylarthritis. Mechanisms of new bone formation in spondyloarthritis","volume":"11","author":"Lories","year":"2009","journal-title":"Arthritis Res. Ther."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1016\/j.intimp.2018.03.002","article-title":"Toll-like receptors in immunity and inflammatory diseases: Past, present, and future","volume":"59","author":"Vijay","year":"2018","journal-title":"Int. Immunopharmacol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"R1412","DOI":"10.1186\/ar1840","article-title":"Between adaptive and innate immunity: TLR4-mediated perforin production by CD28null T-helper cells in ankylosing spondylitis","volume":"7","author":"Raffeiner","year":"2005","journal-title":"Arthritis Res. Ther."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Antoniou, M., Jorgensen, A.L., and Kolamunnage-Dona, R. (2016). Biomarker-Guided Adaptive Trial Designs in Phase II and Phase III: A Methodological Review. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0149803"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"8630871","DOI":"10.1155\/2019\/8630871","article-title":"Biomarkers in Inflammatory Bowel Disease-Associated Spondyloarthritis: State of the Art and Unmet Needs","volume":"2019","author":"Benfaremo","year":"2019","journal-title":"J. Immunol. Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1002\/art.38291","article-title":"Review: The interleukin-23\/interleukin-17 axis in spondyloarthritis pathogenesis: Th17 and beyond","volume":"66","author":"Smith","year":"2014","journal-title":"Arthritis Rheumatol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1186\/s12967-019-1774-x","article-title":"Protein inhibitor of activated STAT3 reduces peripheral arthritis and gut inflammation and regulates the Th17\/Treg cell imbalance via STAT3 signaling in a mouse model of spondyloarthritis","volume":"17","author":"Min","year":"2019","journal-title":"J. Transl. Med."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1689","DOI":"10.1002\/art.38636","article-title":"Animal models of spondyloarthritis: Do they faithfully mirror human disease?","volume":"66","author":"Breban","year":"2014","journal-title":"Arthritis Rheumatol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1002\/art.1780300211","article-title":"Proteoglycan-induced arthritis in BALB\/c mice. Clinical features and histopathology","volume":"30","author":"Glant","year":"1987","journal-title":"Arthritis Rheum."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1136\/ard.46.5.403","article-title":"Raised circulating levels of the eosinophil cationic protein in ankylosing spondylitis: Relation with the inflammatory activity and the influence of sulphasalazine treatment","volume":"46","author":"Feltelius","year":"1987","journal-title":"Ann. Rheum. Dis."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"943","DOI":"10.1016\/j.jsps.2018.05.001","article-title":"The relationship between blood eosinophil count and disease activity in ankylosing spondylitis patients treated with TNF-alpha inhibitors","volume":"26","author":"Sargin","year":"2018","journal-title":"Saudi Pharm. J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"464","DOI":"10.1038\/mi.2015.2","article-title":"Eosinophils in mucosal immune responses","volume":"8","author":"Travers","year":"2015","journal-title":"Mucosal Immunol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2694","DOI":"10.1016\/j.mayocp.2021.04.025","article-title":"Eosinophils in Health and Disease: A State-of-the-Art Review","volume":"96","author":"Wechsler","year":"2021","journal-title":"Mayo Clin. Proc."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1038\/nri3341","article-title":"Eosinophils: Changing perspectives in health and disease","volume":"13","author":"Rosenberg","year":"2013","journal-title":"Nat. Rev. Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"981","DOI":"10.1136\/ard.2004.029710","article-title":"A longitudinal study on an autoimmune murine model of ankylosing spondylitis","volume":"64","author":"Czipri","year":"2005","journal-title":"Ann. Rheum. Dis."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1038\/nri3831","article-title":"Type 2 cytokines: Mechanisms and therapeutic strategies","volume":"15","author":"Wynn","year":"2015","journal-title":"Nat. Rev. Immunol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1303","DOI":"10.1093\/intimm\/dxp102","article-title":"IL-5- and eosinophil-mediated inflammation: From discovery to therapy","volume":"21","author":"Kouro","year":"2009","journal-title":"Int. Immunol."},{"key":"ref_33","first-page":"121","article-title":"CXCL10\/IP-10 in infectious diseases pathogenesis and potential therapeutic implications","volume":"22","author":"Liu","year":"2011","journal-title":"Cytokine Growth Factor Rev."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1378\/chest.10-3297","article-title":"IL-27 Is Elevated in Patients With COPD and Patients With Pulmonary TB and Induces Human Bronchial Epithelial Cells to Produce CXCL10","volume":"141","author":"Cao","year":"2012","journal-title":"Chest"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1016\/S1359-6101(97)00015-4","article-title":"The immunobiology of interferon-gamma inducible protein 10 kD (IP-10): A novel, pleiotropic member of the C-X-C chemokine superfamily","volume":"8","author":"Neville","year":"1997","journal-title":"Cytokine Growth Factor Rev."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"968","DOI":"10.1136\/ard.61.11.968","article-title":"Atopic disorders in ankylosing spondylitis and rheumatoid arthritis","volume":"61","author":"Rudwaleit","year":"2002","journal-title":"Ann. Rheum. Dis."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"819","DOI":"10.1016\/j.berh.2014.10.019","article-title":"Treatment of spondyloarthritis beyond TNF-alpha blockade","volume":"28","author":"Deodhar","year":"2014","journal-title":"Best Pract. Res. Clin. Rheumatol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1007\/s11926-019-0856-3","article-title":"The Pathogenesis of Ankylosing Spondylitis: An Update","volume":"21","author":"Pedersen","year":"2019","journal-title":"Curr. Rheumatol. Rep."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1007\/s10067-010-1647-4","article-title":"Increased serum IL-17 and IL-23 in the patient with ankylosing spondylitis","volume":"30","author":"Mei","year":"2011","journal-title":"Clin. Rheumatol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1073","DOI":"10.3899\/jrheum.091469","article-title":"Anti-tumor necrosis factor therapies reduce serum macrophage inflammatory protein-1alpha in ankylosing spondylitis","volume":"37","author":"Akbulut","year":"2010","journal-title":"J. Rheumatol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"R53","DOI":"10.1186\/ar3766","article-title":"Is IL-6 an appropriate target to treat spondyloarthritis patients refractory to anti-TNF therapy? A multicentre retrospective observational study","volume":"14","author":"Lekpa","year":"2012","journal-title":"Arthritis Res. Ther."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Nocturne, G., Pavy, S., Boudaoud, S., Seror, R., Goupille, P., Chanson, P., van der Heijde, D., van Gaalen, F., Berenbaum, F., and Mariette, X. (2015). Increase in Dickkopf-1 Serum Level in Recent Spondyloarthritis. Data from the DESIR Cohort. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0134974"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"S66","DOI":"10.2144\/Oct0208","article-title":"Toll-like receptor signaling and regulation of cytokine gene expression in the immune system","volume":"33","author":"Ozato","year":"2002","journal-title":"Biotechniques"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"5067","DOI":"10.4049\/jimmunol.167.9.5067","article-title":"Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo","volume":"167","author":"Pulendran","year":"2001","journal-title":"J. Immunol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Roth, K., Pallua, J.D., Degenhart, G., De Zordo, T., Kremser, C., Reif, C., Streif, W., and Schirmer, M. (2025). Reduced Bone Quality of Sacrum and Lumbal Vertebrae Spongiosa in Toll-like Receptor 2- and Toll-like Receptor 4-Knockout Mice: A Blinded Micro-Computerized Analysis. Biomolecules, 15.","DOI":"10.3390\/biom15020239"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/3\/1337\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T09:12:40Z","timestamp":1769677960000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/3\/1337"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,29]]},"references-count":45,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["ijms27031337"],"URL":"https:\/\/doi.org\/10.3390\/ijms27031337","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,29]]}}}