Spinal cord Toll-like receptor 4 mediates inflammatory and neuropathic hypersensitivity in male but not female mice

Abstract

The innate immune system is increasingly appreciated to play an important role in the mediation of chronic pain, and one molecule implicated in this process is the Toll-like receptor 4 (TLR4). Here, using pharmacological and genetic manipulations, we found that activating TLR4 in the spinal cord, with the agonist lipopolysaccharide (LPS), causes robust mechanical allodynia but only in male mice. Spinal LPS had no pain-producing effect in female mice. TLR4 also has a sex-specific role in inflammatory (complete Freund's adjuvant) and neuropathic (spared nerve injury) pain: pain behaviors were TLR4 dependent in males but TLR4 independent in females. The sex differences appear to be specific to the spinal cord, as LPS administered to the brain or the hindpaw produces equivalent allodynia in both sexes, and specific to pain, as intrathecal LPS produces equivalent hypothermia in both sexes. The involvement of TLR4 in pain behaviors in male mice is dependent on testosterone, as shown by gonadectomy and hormone replacement. We found no sex differences in spinal Tlr4 gene expression at baseline or after LPS, suggesting the existence of parallel spinal pain-processing circuitry in female mice not involving TLR4.

Figure 1.

a–c, Female CD-1 mice do not exhibit mechanical allodynia after intrathecal LPS administration (a, b), despite equivalent levels of Tlr4 expression in the spinal cord (c). d–h, The sex difference is testosterone dependent (d) and TLR4 dependent (e), and is not seen after intracerebroventricular (f) or intraplantar (g) LPS injection, nor in hypothermia from intrathecal LPS (h). Symbols in a represent the mean ± SEM threshold (in grams) to withdraw from von Frey filaments. Symbols or bars in graphs b and d–g represent mean ± SEM percentages of the maximum possible allodynia (see main text) from time course data collected exactly as shown in graph a. Bars in graph c represent mean spinal cord Tlr4 mRNA expression in arbitrary units (AU) compared with Gapdh. Symbols in graph h represent the mean ± SEM maximum hypothermia (°C) exhibited by each mouse over the 24 h testing period. *p < 0.05, ***p < 0.001 compared with relevant opposite-sex group, or baseline, by t test; ^•p < 0.05 compared with all other same-sex groups by Tukey's post hoc test. GDX, Gonadectomy; TP, testosterone propionate.

Figure 2.

a–d, Effects of pharmacological (LPS-RS) blockade of spinal TLR4 on mechanical allodynia induced by LPS (a), zymosan (b), CFA (c), and SNI (d) in male and female CD-1 mice, and male wild-type (B10ScSnJ; +/+) and null mutant (B10ScNJ; −/−) mice. Symbols represent mean ± SEM threshold (g) to withdraw from von Frey filaments before injection or surgery [baseline (BL)], at the time point of maximal allodynia (see text), and 10–120 min after injection of 2 μg (i.t.) LPS-RS. Bars represent mean ± SEM percentages of maximum anti-allodynia (i.e., reversal of allodynia already developed) by LPS-RS at any time point. *p < 0.05, **p < 0.01 compared with opposite sex or opposite Tlr4 genotype group by t test.