Lipopolysaccharide‐induced sickness behavior is not altered in male Fmr1‐deficient mice

Abstract Objectives Fragile X syndrome is the main monogenetic cause of intellectual disability and autism. Alterations in the immune system are commonly found in these developmental disorders. We and others have demonstrated that Fmr1 mutant mice present an altered response to immune stimuli. However, whether this altered immune response can influence the Fmr1 mutant behavioral outcomes in response to inflammation has not been fully investigated. Materials and methods In the current study, we examine the behavioral sickness response of male wildtype and knockout mice to the innate immune stimulus lipopolysaccharide (LPS) (0.1 mg/kg) to determine if Fmr1 mutants have altered sickness behavior. We used an enzyme‐linked immunosorbent assay (ELISA) to measure changes in the cytokine interleukin‐6 (IL‐6) to determine that inflammation was induced in the mice. Sickness behavior was assessed in a wheel‐running paradigm, and a tail suspension test was used to assess the depressive‐like phenotype that follows sickness behavior in response to LPS. Results The ELISA using blood serum confirmed a significant increase in IL‐6 in mice that were treated with LPS. Treated Fmr1 mutants exhibited decreased distance traveled in the wheel running after LPS administration, similar to treated controls. Another cohort of animals treated with LPS were tested in the tail suspension test and exhibited no alterations in immobility time in response to LPS. Conclusion Together, our data suggest that Fmr1 mutant mice do not have altered sickness behavior in response to a low dose of LPS.


INTRODUCTION
Fragile X syndrome (FXS) is an X-linked neurodevelopmental disorder that is caused by the functional loss of the fragile X messenger ribonucleoprotein 1 (Fmr1) gene, resulting in a reduction or absence of RNA-binding protein fragile X messenger ribonucleoprotein (FMRP) (Hagerman, 2006;Hagerman & Hagerman, 2002). It is the most common monogenetic cause of autism spectrum disorder (ASD), with the disorders sharing several cognitive and behavioral phenotypes, including social communication deficits and stereotypical behavior (Harris et al., 2008). Evidence suggests that immune dysfunction could play a role in the etiology of both disorders (Ashwood et al., 2010;Eissa et al., 2020;Masi et al., 2017;Petrelli et al., 2016). The molecular and physiological basis of neuroimmune alterations in FXS and ASD has been studied extensively. However, whether dysregulated immune function could impact the behavioral phenotype observed in neurodevelopmental disorders is largely unknown.
A significant component of an immune response is the coordinated set of behavioral changes initiated by the insult, collectively known as sickness behavior (Dantzer et al., 2008). In response to peripheral infection, cells of the innate immune system release proinflammatory cytokines that act on neural pathways in the brain and trigger a highly organized signaling cascade to fight infection (Layé et al., 1994). The key inflammatory cytokines known to be involved in the sickness response are interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNFα), and interleukin-6 (IL-6) (Dantzer, 2009). Behavioral changes associated with the sickness response include lethargy, loss of appetite, sleepiness, social withdrawal, and depressed mood (Dantzer, 2001). While these adaptive behavioral changes are often viewed as unpleasant, they play a critical role in facilitating recovery from the infection.
Systemic administration of the endotoxin lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is a classical way of mimicking a peripheral bacterial infection in rodent models (Bassi et al., 2012). It induces a peripheral immune response that is accompanied by similar neuroimmune and endocrine changes that mimic the human response to infection. The behavioral sickness response is similar in rodents, which includes fatigue, anhedonia, little to no interest in their physical and social environment, and rodents exhibiting a hunched posture often in the corner of their home cage (Hart, 1988). This response can also lead to a reduction in food and water intake by rodents. To investigate sickness behavior, several behavioral paradigms are used, including burrowing of food and sucrose preference tasks. Wheel running can also be utilized to examine changes in motor activity, another measure of the sickness response (Biesmans et al., 2013). LPS administration can also induce depressive-like behavior at a later timepoint, characterized by increased immobility time in the tail suspension test and forced swimming test (Lasselin et al., 2020).
We and others have previously shown that Fmr1 knockout (KO) mice in the Fvb background strain exhibit altered immune functions and responses to LPS (Hodges et al., 2020;Parrott et al., 2021). Immune regulation of behavior has been shown by several studies that demonstrate that inflammation can alter neurotransmission (Vezzani & Viviani, 2015). Neurotransmitter signaling can be regulated by inflammatory cytokines such as IL-6. Exposure to IL-6 downregulates serotonergic transporters (SERT) in cell culture, while IL-6 KO mice have elevated levels of SERT and decreased depressive-like behavior (Kong et al., 2015). IL-6 can also regulate ion channel activity, such as Nav1.7 by increasing the number of spikes and decreasing the latency to the first action potential (Yan et al., 2012). Additionally, a large body of literature suggests that IL-6 plays an important role in the pathophysiology of depression, where both in clinical and pre-clinical studies, increased IL-6 levels are associated with a depressive phenotype (Roohi et al., 2021).
Based on previous studies that have shown the role of IL-6 in behavior (Kong et al., 2015;Sukoff Rizzo et al., 2012) and that have identified increased levels of IL-6 in Fmr1 KOs in response to LPS (Hodges et al., 2020;Parrott et al., 2021), we hypothesized that Fmr1 KOs will present with more severe sickness behavior and depressive-like phenotypes than wild-type (WT) mice. Hence, in the present study, we examined the behavioral sickness response in a mouse model of FXS, utilizing Fmr1 KO and WT mice on a C57BL/6J background strain. Following a period of baseline running, mice were administered either a single dose of the bacterial mimetic LPS (0.1 mg/kg) or of saline, followed by having access to running wheels for ∼2 days. Total voluntary wheel running activity, in addition to differences in the pattern of fatigue across the testing period, was examined between genotypes. To also examine potential depressive-like behavior in Fmr1 KO and WT mice, one cohort of mice was tested 24 h following LPS administration in the tail suspension task. This study will provide insight into whether a mouse model of FXS, a common neurodevelopmental disorder, displays an altered sickness behavioral response following an immune insult.

Animals
Experimental subjects included male Fmr1 KO and WT mice on a C57BL/6J background strain (Jackson Labs). We used only male mice based on our previous study that showed altered immune response in male Fmr1 KO mice with the FvB/NJ background (Hodges et al., 2020).
Mice used in the study were between 43 and 106 days old. The sam-

Lipopolysaccharide stimulation
The bacterial endotoxin LPS was administered to examine the effects of innate immune stimulation on sickness and depressive-like behavior in Fmr1 mutant and WT mice. LPS was prepared at a stock concentration of 1 mg/mL in 0.9% physiological saline and further diluted in saline prior to administration. All mice received either a single intraperitoneal injection of 0.1 mg/kg LPS from Escherichia coli serotype O127:B8 purified by gel-filtration chromatography (Sigma) or an equivalent volume of filtered 0.9% physiological saline as a control. All injections were approximately 200 μL. The 0.1 mg/kg LPS dose was chosen based on preliminary wheel running experiments in C57BL/6J mice that demonstrated this as an ideal dose to examine the recovery from an LPS insult.
Additionally, our previous study indicated that a higher dose of LPS (0.33 mg/kg) could induce a flooring effect in the sickness behavior test.
The 0.1 mg/kg dose was used in the present study to potentially reveal any subtle behavioral differences between genotypes, while avoiding a potential floor effect with all mice exhibiting significant signs of sickness behavior.
In the first cohort of mice, injections were administered at ∼12 p.m., and blood was collected 24 h later. In the second cohort of mice, injections were given at 12 p.m. following 4 days of baseline wheel running. Following injections, mice were placed back in their individual cages with a running wheel for approximately 50 h of testing. In the third cohort of mice, LPS injections were given at 12 p.m., and the tail suspension test was performed 24 h later ( Figure 1).

Enzyme-linked immunosorbent assay
The first cohort of animals was treated with LPS (0.1 mg/kg), and blood was collected 24 h after treatment to measure IL-6 levels and confirm an immune response to the dose used in the study. Trunk blood was collected and an hour later centrifugated for 10 min at 1000 g at room temperature. Serum was isolated, frozen, and stored at −20C • until further analysis. IL-6 was measured in serum samples using a commercially available mouse IL-6 enzyme-linked immunosorbent assay (ELISA) kit (ELISA MAX™ Deluxe Set, Biolegend) following the manufacturer's protocol. Data are expressed in pg/mL.

Voluntary wheel running
In the second cohort of animals, mice went through 4 days of baseline running prior to administration of LPS or saline, followed by a testing period of ∼2 days (50 h) in which mice were able to voluntarily run (Figure 1b)

Tail suspension
Tail suspension was conducted to examine depressive-like behavior following administration of LPS in Fmr1 mutant and WT mice (Figure 1c).
Each mouse was suspended in a visually isolated area by fastening their tail to a padded close pin attached to an elevated shelf in a room controlled for background noise, temperature, and light levels. To prevent tail climbing behaviors, mouse tails were passed through a piece of cut straw (∼4 cm) prior to suspension. Total time spent moving during a 6min period was recorded by an experimenter blinded to the genotype of each subject. Immobility was calculated by subtracting the seconds the mouse was mobile from the total duration of the testing session (360 s). Measurements of immobility were defined as the absence of hind limb movement (Can et al., 2012;Cryan et al., 2005). Small movements confined to the front legs that did not involve the hind limbs, or swinging due to momentum gained during earlier mobility, were not counted as mobility.

Statistics
All statistical analyses were performed using GraphPad Prism 7 software (La Jolla) or SPSS 25.0 (IBM). The levels of IL-6 in response to LPS were analyzed with a two-way analysis of variance (ANOVA) to compare controls and Fmr1 mutant IL-6 levels. Changes in weight from the beginning of baseline wheel running, injection date, and tail suspension were analyzed with a two-way repeated measures ANOVA, with a within-subject factor of time and between-subject factors of genotype and treatment. Tail suspension was analyzed with a twoway ANOVA to examine differences in immobility across genotype and treatments. Average baseline wheel running was analyzed with a t-test.
Total wheel running following immune stimulation during the testing period was analyzed with a two-way ANOVA. A two-way repeated measures ANOVA with a within subject factor of time and between subject factors of genotype and treatment was also conducted to examine changes in running patterns over time after immune stimulation, followed by t-tests to determine differences at individual time points. This study was not preregistered.

Immune response
To confirm the immune response to LPS and assess if there were any genotype-specific effects in the response to LPS, blood was collected 24 h after treatment and the levels of the cytokine IL-6 were measured in the serum. A main effect of LPS treatment was identified (F [1,29] = 20.77, p < 0.0001, Figure 2a). No main effect of genotype or interaction between genotype and treatment was identified.

Body weight
Changes in weight of wheel running mice were examined by performing a repeated-measures ANOVA, with a within-subjects factor of "time"  (Figure 2b).
In the cohort tested 24 h following immune stimulation, a significant interaction between time and treatment for weight was detected  Figure 2c).

Wheel running
Prior to immune stimulation, all mice went through a period of baseline  Figure 3b).

F I G U R E 3
Immune stimulus with lipopolysaccharide (LPS) decreases total distance travelled in the wheel running test. (a) Baseline total distance travelled in Fmr1 knockouts (KOs) is lower than wild-type (WT) mice (N = 23-24). (b) LPS treatment decreased the total distance ran independent of genotype (N = 11-12). (c) Representation of wheel running across time after LPS administration. Data are expressed as mean ± SEM. Total distance ran at baseline is analyzed by t-test. Total distance ran after LPS administration is analyzed by two-way analysis of variance (ANOVA). Main effect of treatment is represented by *p < 0.05.

Tail suspension
Twenty-four hours after LPS administration, tail suspension test was performed. There was no effect of treatment (F [1,46] = 0.00, p = 0.98).
However, there was a significant effect of genotype KO mice also present with an altered behavioral response to inflammation using the bacterial endotoxin LPS as an immune stimulus. To assess alterations in sickness and depressive-like behavior in response to LPS, mice were tested in the wheel running and tail suspension test.
LPS is a widely used inflammatory stimulus that can induce sickness behavior and depressive-like behavior that varies in severity according to the dose administered (Lasselin et al., 2020;Xu et al., 2007). We Weight was measured to assess if weight loss could account for the behavioral deficits we identified, as it is possible that a reduction in weight resulted in fatigue which could impact wheel running. Treated mice did not present a significant weight loss in response to LPS when measured at 1 and 2 days after the immune stimulus occurred. writing-original draft, and writing-review and editing: Joaquin N. Lugo.

ACKOWLEDGMENTS
The research was funded by NIH NINDS grant R15NS088776 (Joaquin N. Lugo). The graphical abstract was created using Biorender.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.