Effects of gut-derived endotoxin on anxiety-like and repetitive behaviors in male and female mice

Christopher T. Fields, Benoit Chassaing, Alexandra Castillo-Ruiz, Remus Osan, Andrew T. Gewirtz, Geert J. de Vries

Gut dysbiosis is observed in several neuropsychiatric disorders exhibiting increases in anxiety behavior, and recent work suggests links between gut inflammation and such disorders. One source of this inflammation may be lipopolysaccharide (LPS), a toxic component of gram-negative bacteria. Here, we (1) determine whether oral gavage of LPS, as a model of gut-derived endotoxemia, affects anxiety-like and/or repetitive behaviors; (2) test whether these changes depend on TLR4 signaling; and (3) test the extent to which gut-derived endotoxin and TLR4 antagonism affects males and females differently. In experiment 1, male wild-type (WT) and Tlr4-/- mice were tested for locomotor, anxiety-like, and repetitive behaviors in an automated open field test apparatus, 2 h after oral gavage of LPS or saline. In experiment 2, male and female WT mice received an oral gavage of LPS and an injection of one or two TLR4 antagonists that target different TLR4 signaling pathways ((+)-naloxone and LPS derived from R. sphaeroides (LPS-RS)). Univariate and multivariate analyses were used to identify effects of treatment, sex, and genotype and their interaction. In experiment 1, oral gavage of LPS increased anxiety-like behavior in male WT mice but not in Tlr4-/- mice. In experiment 2, oral gavage of LPS increased anxiety-like and decreased repetitive behaviors in WT mice of both sexes. Neither antagonist directly blocked the effects of orally administered LPS. However, treatment with (+)-naloxone, which blocks the TRIF pathway of TLR4, had opposing behavioral effects in males and females (independent of LPS treatment). We also identified sex differences in the expression of interleukin-6, a pro-inflammatory cytokine, in the gut both in basal conditions and in response to LPS. In spite of the ubiquitous nature of LPS in the gut lumen, this is the first study to demonstrate that intestinally derived LPS can initiate behavioral aspects of the sickness response. While an increased enteric load of LPS increases anxiety-like behavior in both sexes, it likely does so via sex-specific mechanisms. Similarly, TLR4 signaling may promote baseline expression of repetitive behavior differently in males and females. This study lays the groundwork for future interrogations into connections between gut-derived endotoxin and behavioral pathology in males and females.