Abstract
BackgroundGut 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.MethodsIn 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.ResultsIn 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.ConclusionIn 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.
Highlights
Gut dysbiosis is observed in several neuropsychiatric disorders exhibiting increases in anxiety behavior, and recent work suggests links between gut inflammation and such disorders
Intravenous injections of LPS facilitate its fast and robust interaction with splenic immune cells and circulating leukocytes. It is unknown whether elevations of serum LPS levels originating from gut barrier dysfunction, observed in rodent models of gut dysbiosis (such as emulsifier-fed mice [13], mice with dextran sodium sulfate (DSS)-induced colitis [14, 15], high-fat diet-fed mice [16], and toll-like receptor 2 knockout (Tlr2−/−) mice [17]), are responsible for increases in anxiety-like behavior observed in these models
2 × 2 univariate ANOVAs across all behavioral measures showed that oral gavage of LPS significantly increased anxiety-like behavior in WT mice but not Tlr4−/− mice
Summary
Gut dysbiosis is observed in several neuropsychiatric disorders exhibiting increases in anxiety behavior, and recent work suggests links between gut inflammation and such disorders. Intravenous injections of LPS facilitate its fast and robust interaction with splenic immune cells and circulating leukocytes It is unknown whether elevations of serum LPS levels originating from gut barrier dysfunction, observed in rodent models of gut dysbiosis (such as emulsifier-fed mice [13], mice with dextran sodium sulfate (DSS)-induced colitis [14, 15], high-fat diet-fed mice [16], and toll-like receptor 2 knockout (Tlr2−/−) mice [17]), are responsible for increases in anxiety-like behavior observed in these models. As even a 10-μg/kg dose of LPS (10 times lower than in most published studies) is sufficient to increase serum levels of LPS to 25× above baseline [19], it is questionable whether intraperitoneal injections recapitulate the dynamics of LPS-induced inflammation observed in “metabolic endotoxemia.” the site of action may make a difference, as an inflammatory stimulus injected intraperitoneally may differ in its neurobehavioral effects from an inflammatory stimulus administered orally
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