# Exploratory Cytokine and Bone-Marker Patterns in a Proteoglycan-Induced Spondyloarthritis Mouse Model: Th1/Th2 Strain Comparison and TLR2/3/4 Knockout Readouts

## TL;DR

This study explores immune and bone marker patterns in a mouse model of spondyloarthritis, comparing different mouse strains and TLR gene knockouts.

## Contribution

The study identifies distinct cytokine and bone marker profiles associated with Th1/Th2 immune polarization and TLR gene knockout effects in an experimental spondyloarthritis model.

## Key findings

- Immunized Th1-prone C57BL/6J mice showed elevated Th1/Th17 cytokines like TNF-α and IFNγ.
- TLR2 and TLR3 knockouts exhibited reduced Th1/Th17 cytokines and increased Th2 cytokines.
- TLR2-KO mice had higher DKK-1 and Noggin levels, indicating altered bone metabolism markers.

## Abstract

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-α, IFNγ, 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.