Role of TLR4 activation and signaling in bone remodeling, and afferent sprouting in serum transfer arthritis

In the murine K/BxN serum transfer rheumatoid arthritis (RA) model, tactile allodynia persists after resolution of inflammation in male and partially in female wild type (WT) mice, which is absent in Toll-like receptor (TLR)4 deficient animals. We assessed the role of TLR4 on allodynia, bone remodeling and afferent sprouting in this model of arthritis.

K/BxN sera were injected into male and female mice with conditional or stable TLR4 deletion and controls. Paw swelling was scored and allodynia assessed by von Frey filaments. At day 28, synovial neural fibers were visualized with confocal microscopy and bone density assayed with microCT. Microglial activity and TLR4 dimerization in spinal cords were examined by immunofluorescence and flow cytometry.

In the synovium, K/BxN injected WT male and female mice showed robust increases in calcitonin gene related-peptide (CGRP⁺), tyrosine hydroxylase (TH)⁺ and GAP43⁺ nerve fibers. Trabecular bone density by microCT was significantly decreased in K/BxN WT female but not in WT male mice. The number of osteoclasts increased in both sexes of WT mice, but not in Tlr4^-/- K/BxN mice. We used conditional strains with Cre drivers for monocytes/osteoclasts (lysozyme M), microglia (Tmem119 and Cx3CR1), astrocytes (GFAP) and sensory neurons (advillin) for Tlr4^f/f disruption. All strains developed similar arthritis scores after K/BxN serum injection with the exception being the Tlr4^Tmem119 mice which showed a reduction. Both sexes of Tlr4^Lyz2, Tlr4^Tmem119 and Tlr4^Cx3cr1 mice displayed a partial reversal of the chronic pain phenotype but not in Tlr4^Avil, and Tlr4^Gfap mice. WT K/BxN male mice showed increases in spinal Iba1, but not GFAP, compared to Tlr4^-/- male mice. To determine whether spinal TLR4 was indeed activated in the K/BxN mice, flow cytometry of lumbar spinal cords of WT K/BxN male mice was performed and revealed that TLR4 in microglia cells (CD11b⁺ /TMEM119⁺) demonstrated dimerization (e.g. activation) and a characteristic increase in lipid rafts.

These results demonstrated a complex chronic allodynia phenotype associated with TLR4 in microglia and monocytic cell lineages, and a parallel spinal TLR4 activation. However, TLR4 is dispensable for the development of peripheral nerve sprouting in this model.