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Published in final edited form as: Physiol Behav. 2023 Mar 16;265:114159. doi: 10.1016/j.physbeh.2023.114159

The effects of lipopolysaccharide exposure on social interaction, cytokine expression, and alcohol consumption in male and female mice

E B Decker Ramirez 1, ME Arnold 1, KT McConnell 1, MG Solomon 1, KN Amico 1, JR Schank 1
PMCID: PMC10121933  NIHMSID: NIHMS1885676  PMID: 36931488

Abstract

Much recent research has demonstrated a role of inflammatory pathways in depressive-like behavior and excess alcohol consumption. Lipopolysaccharide (LPS) is a cell wall component of gram-negative bacteria that can be used to trigger a strong inflammatory response in rodents in a preclinical research setting to study the mechanisms behind this relationship. In our study, we exposed male and female mice to LPS and assessed depressive-like behavior using the social interaction (SI) test, alcohol consumption in the two-bottle choice procedure, and expression of inflammatory mediators using quantitative PCR. We found that LPS administration decreased SI in female mice but had no significant impact on male mice when assessed 24 hours after injection. LPS resulted in increased proinflammatory cytokine expression in both male and female mice; however, some aspects of the cytokine upregulation observed was greater in female mice as compared to males. A separate cohort of male and female mice underwent drinking for 12 days before receiving a saline or LPS injection, which we found to increase alcohol intake in both males and females. We have previously observed a role of the neurokinin-1 receptor (NK1R) in escalated alcohol intake, and in the inflammatory and behavioral response to LPS. The NK1R is the endogenous target of the neuropeptide SP, and this system has wide ranging roles in depression, anxiety, drug/alcohol seeking, pain, and inflammation. Thus, we administered a NK1R antagonist prior to alcohol access. This treatment reduced escalated alcohol consumption in female mice treated with LPS but did not affect drinking in males. Taken together, these results indicate that females are more sensitive to some physiological and behavioral effects of LPS administration, but that LPS escalates alcohol consumption in both sexes. Furthermore, NK1R antagonism can reduce alcohol consumption that is escalated by LPS treatment, in line with our previous findings.

Keywords: Inflammation, Alcohol, Sex Differences, Neurokinin-1 receptor, Cytokines

Introduction

Much recent work has shown a critical role of inflammatory processes in the development of psychiatric disorders such as depression (Kim et al., 2016; Miller et al., 2009; Troubat et al., 2021). For example, higher levels of peripheral proinflammatory cytokines and elevated central nervous system inflammatory markers are observed in patients with depression and associate with depression severity (Dowlati et al., 2010; Felger, 2018; Holmes et al., 2018; Richards et al., 2018). Accordingly, drugs that reduce inflammatory signaling have shown efficacy in reducing depressive symptoms (Kappelmann et al., 2018). Preclinically, inflammatory mediators and their downstream signaling mechanisms influence the behavioral, cellular, and molecular phenotypes induced by models of depression-like behavior in rodents (Christoffel et al., 2011; Christoffel et al., 2012; Hodes et al., 2014; Koo & Duman, 2008; Koo et al., 2010).

Alcohol use disorder (AUD) is often expressed comorbidly with major depression, and approximately one third of people in treatment for AUD meet criteria for major depressive disorder (MDD; McHugh & Weiss, 2019). One longitudinal study found that AUD in adolescent participants predicted MDD in early adulthood, and MDD in early adulthood predicted later AUD (Briere et al., 2014). Interestingly, women are more likely than men to have the co-occurrence of AUD and MDD (McHugh & Weiss 2019). As with depression, inflammation plays a role in the development of AUD (Crews et al., 2017). Preclinically, the effects of chronic alcohol heighten the response of inflammatory pathways, and neuroimmune signaling systems mediate alcohol consumption (Harris & Blednov, 2013; Qin & Crews, 2012; Qin et al., 2008; Robinson et al., 2014). For example, the inflammation-associated toll like receptor 4 (TLR4) mediates alcohol responses, and manipulating downstream signaling processes of this receptor, such as MyD88 and IKKbeta can influence alcohol consumption (Blednov et al., 2017; Truitt et al., 2016). IKKbeta is upstream regulator of a transcription factor known as nuclear factor kappa-light-chain-enhancer of activated B (NF-kB), which regulates many genes related to inflammation and addiction, and plays a role in alcohol reward (Nennig et al., 2017; Nennig & Schank, 2017).

Inflammatory processes and their role in complex behaviors are often studied in the lab by exposing animals to lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria that induces a major inflammatory response and upregulation of cytokines and other inflammatory mediators. Endogenously, LPS can enter the bloodstream from the gut due to stress and binds to TLR4, resulting in an inflammatory response (de Punder & Pruimboom, 2015). Exogenous LPS administration has been shown to induce depressive-like behavior in human and rodent models (Lasselin et al., 2020). For example, rodents injected with LPS show behavioral responses such as social withdrawal and anhedonia (Bluthe et al., 1999; Bluthe et al., 1992; Bluthe et al., 2000; Frenois et al., 2007; Fulenwider et al., 2018; Haba et al., 2012; Henry et al., 2008; Jangra et al., 2014; Orlandi et al., 2015; Reis et al., 2022). Similar effects on depressive-like behaviors have been observed in human studies as well, with low dose endotoxin administration resulting in decreased ventral striatum activation to reward cues, and increased observer-rated depressed mood (Eisenberger et al., 2010). Important for our particular study is the fact that LPS exposure also induces a long lasting increase in alcohol consumption in mice (Blednov et al., 2011b). Although much research suggests that TLR4 signaling is a driver in escalated alcohol consumption following the stimulation of inflammatory signaling by LPS, Harris et al. found that TLR4 signaling was more involved in the acute effects rather than a cause of excess alcohol consumption (2017). However, the experiment in this study that examined LPS-induced escalation in alcohol intake utilized TLR4 knockout rats that self-administered alcohol in an operant task. Thus, this study differs from much earlier work in the species and drinking model used, which have generally studied mice in models of voluntary homecage consumption. Also, it is possible that additional signaling mechanisms are recruited either directly or indirectly by LPS to trigger escalation in voluntary alcohol intake.

Many of the studies referenced above included only male subjects. However, some studies suggest that females show greater inflammatory responses than their male counterparts. For example, females may show prolonged LPS-induced cytokine expression compared to males (Sharma et al., 2018). Increased sensitivity to LPS in females has also been observed in aged mice, with LPS resulting in greater proinflammatory cytokine levels in plasma and more significant depressive-like phenotypes compared to male mice (Dockman et al., 2022). In humans, administration of endotoxin decreased activity in the ventral striatum during reward anticipation in female but not male subjects, and cytokine levels negatively correlated with striatal activity (Moieni et al., 2019). For preclinical models of alcohol consumption, some studies have demonstrated that certain inflammatory mechanisms preferentially effect drinking in female mice. For example, manipulations of inflammatory mediators including the cytokine IL6, chemokine receptors Ccr2 and Ccl2, and the NLRP3 inflammasome influenced drinking in female mice but not male mice (Blednov et al., 2005; Harris & Blednov, 2013; Lowe et al., 2020). Taken together, these findings suggest that females may be more sensitive to the behavioral effects induced by immune stimulation.

In our previous work, we have found that both excessive alcohol intake and the neuroinflammatory response to LPS are mediated by the neurokinin-1 receptor (NK1R; Fulenwider et al., 2018; Nelson et al., 2019; Nelson et al., 2017; Schank et al., 2013; Sequeira et al., 2018). The NK1R is the primary endogenous target of the neuropeptide substance P, and this system mediates complex behaviors such as anxiety, stress responses, pain processing, and drug/alcohol seeking (Ebner & Singewald, 2006; Schank, 2014, 2020; Schank et al., 2012). For alcohol specifically, our group and others have shown that the NK1R mediates escalated alcohol consumption induced by multiple interventions, as well as stress-induced reinstatement of seeking to several classes of drugs (Fulenwider et al., 2019; Nelson et al., 2019; Nelson et al., 2017; Schank et al., 2014; Schank et al., 2015; Schank et al., 2011; Schank et al., 2013; Sequeira et al., 2018). In regards to LPS effects specifically, we have shown that inhibiting the NK1R prevented the effects of LPS on sucrose preference and proinflammatory cytokine levels in the hippocampus (Fulenwider et al., 2018). In that study, LPS increased NF-kb DNA binding activity, but an NK1R antagonist pretreatment impeded this effect. This suggests that NF-kb activation is at least partially dependent on NK1R activation. Taken together, this provides strong evidence to suggest that NK1R inhibition may attenuate LPS-induced escalation in alcohol intake.

In the current study we used LPS exposure as a model to examine sex differences in inflammatory signaling, and its effect on social behavior and alcohol consumption. Additionally, we assessed the ability of a NK1R antagonist to reduce LPS-induced escalation in alcohol intake. We hypothesized that female mice would have a stronger inflammatory and behavioral response to LPS and that NK1R antagonism could attenuate LPS-induced escalation in alcohol consumption.

Methods

Animals

Male and female C57BL6/J mice arrived from Jackson Laboratories at 8-10 weeks of age, and were allowed to habituate for 7 days. Mice were housed 4-5 per cage in standard rodent cages with food and water provided ad libitum. The housing room was on a 12:12 light/dark cycle (lights off at 11:00 am). The social interaction test and bottle measurements were conducted during the dark cycle. All experiments were approved by the University of Georgia Institutional Animal Care and Use Committee. The experiments proceeded as depicted by Figure 1. The experiments were performed in separate cohorts of mice with one cohort used for behavioral and cytokine expression analysis (Fig. 1a) and another cohort for alcohol consumption (Fig. 1b). Mice in drinking experiments were individually housed, which is necessary for two bottle choice consumption models, so that intake of individual mice can be tracked.

Figure 1.

Figure 1.

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Timeline of experiments.

LPS Treatment

Lipopolysaccharides from Escherichia coli O111:B4 from Sigma-Aldrich (St. Louis, MO; product #2630), was diluted in 0.9% sterile saline, then was administered via i.p. injection at a dose of 1 mg/kg and volume of 10 mL/kg. This dose was selected based on the work of Blednov and colleagues, who used this treatment dose to induce escalated alcohol consumption (2011a). Mice were randomly assigned to saline or LPS treatment groups.

Social Interaction Test

The Social Interaction (SI) test was conducted during the dark cycle approximately 24 hours after LPS injection (n=16/group). The test took place in a plexiglass testing box with a perforated metal cage which allowed for sensory contact in the presence of a target mouse. The dimensions of the testing box and the marked corner and target zones followed the dimensions previously reported by our group and others (Golden et al., 2011; Nelson et al., 2017).

Each SI test consisted of a pre-test habituation period and test period with a social target mouse present. For the pre-test, the mouse was placed into the middle of the testing container to habituate for 150 seconds with the empty cage in the target zone. The test mouse was placed in its home cage for 30 seconds to place the target mouse into the cage. Then, the test mouse is placed into the arena with the target mouse present in the enclosure for another 150 seconds. An adult C57BL6/J target mouse which corresponded to the same sex and approximate age as the test mouse was used as the social target during the test. The testing box was cleaned and disinfected in between each test. Behavioral tests were recorded and scored by an experimenter that was blind to treatment group. The dependent variable used for analysis was the amount of time spent in the interaction zone that included the social target mouse enclosure.

Quantitative Polymerase-chain reaction

To confirm neuroimmune activation by LPS, and to examine any potential sex differences in the magnitude of response to LPS administration at this dose and time point, we assessed the expression of IL6 and TNFα following LPS injection and SI testing. We selected these specific transcripts because TNFα shows strong activation in rodent brain following LPS injection (Fulenwider et al., 2018) and IL6 has been shown to play a critical role in stress-induced effects on social interaction (Hodes et al., 2014). Here, one cohort of the mice exposed to injections and SI testing was immediately sacrificed following the SI test (n=8/group) to analyze gene expression. Brain tissue punches measuring 1.5mm were taken from the ventral striatum (VS), hippocampus (HPC), and prefrontal cortex (PFC), flash frozen with isopentane, then transferred to dry ice. Samples were homogenized with a mechanical homogenizer and pestle and passed through an 18-gauge needle, then RNA extracted using the Purelink™ RNA Mini Kit (Invitrogen™) following the manufacturer’s instructions. RNA concentration was measured with the Nanodrop 1000 and calculations used to ensure cDNA samples of each brain region consisted of the same concentration. RNA was synthesized into cDNA using the Maxima First Strand cDNA synthesis kit for RT-PCR (Thermo Scientific™) according to the manufacturer’s instructions. IL-6 (Mm00416190_ml) and TNFα (Mm00443258_ml) FAM-labeled TaqMan™ primers from Applied Biosystems™ were used to analyze gene expression with Gapdh (Mm99999915_g1) used as the housekeeping gene. Samples were run in triplicate and measured in a Biosystems Quantstudio 6 Flex machine. Samples for a particular brain region were excluded if they had too low of an RNA concentration as measured by Nanodrop. Every group in every brain region had at least 6 samples. Additionally, out of 163 total data points from brain regions and transcripts, 4 were removed as outliers as detected by Grubb’s test (one in the male saline group for TNFα in the PFC, one from the female saline group for IL6 in the VS, one from the female saline group for TNF in the VS, and one from the female LPS group for TNF in the VS). Data were expressed as fold change from saline treated controls and calculated using the 2−ΔΔCT method.

Two-Bottle Choice

Mice (n=8/group) were single housed with two water bottles for three days before switching one bottle out with a 20% alcohol bottle. Bottles were weighed and sides were switched daily to prevent effects of side preference. Alcohol consumption (g/kg) was calculated based on amount consumed and individual animal weight. Drinking continued for 12 days until the alcohol consumption stabilized with less than 15% variability over a three-day period. Mice were then randomly assigned to receive saline vehicle or LPS injections. Alcohol bottles were taken off 24 hours prior to the LPS injection and returned 72 hours after the injection. This 72 hour delay was to ensure that alcohol access was not given in the first few days after LPS injection, when sickness behavior may suppress food/fluid consumption. First, we aimed to determine the effect of NK1R antagonism on LPS-induced escalation in alcohol consumption. After 5 days of post-LPS drinking, mice were injected with vehicle or L-733060 hydrochloride prior to alcohol availability. L-733060 was diluted in Milli-Q ultrapure water and administered via i.p. injection at a dose of 15 mg/kg and volume of 10 mL/kg. Each mouse received both treatments in a repeated measures design with 1 day of drinking without pretreatment in between test days. Next, we aimed to determine the effect on NK1R antagonism under conditions where drinking had not been escalated by LPS injected. To accomplish this objective, we gave saline pretreated mice 14 days of two bottle choice, after which mice were treated with L-733060 (15 mg/kg) or vehicle, as above.

Statistical Analysis

Data were analyzed using GraphPad prism 9 (San Diego, California). Two way ANOVAs were performed to analyze treatment and sex differences, followed by Bonferroni multiple comparison test for posthoc analysis. Data was considered significant when the p value was less than 0.05. Graphs are shown as mean ± SEM.

Results

Social Interaction (SI)

The SI test indicated that LPS had a greater impact on female mice (Figure 2). Mice (n=16/group) were tested in the SI task approximately 24 hours after LPS or vehicle injection. Two way ANOVA analysis revealed a main effect of treatment (F1,60=10.6, p=0.002) and a sex by treatment interaction (F1.60=4.2, p=0.04). The main effect of sex did not reach statistical significance (F1, 60= 0.018, p=0.89). Bonferroni’s posthoc test indicated that female mice treated with LPS spent significantly less time in the interaction zone when compared to saline treated controls (p=0.002). Time spent in the interaction zone was not affected by LPS treatment in male mice (p>0.99).

Figure 2. LPS reduces social interaction in female mice.

Figure 2.

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Shown is the time (in seconds) that male and female mice spent in the interaction zone during the SI test. Main effects of treatment (p=0.002), and interaction effect (p=0.04) were observed. **p<0.01 compared to saline treated females.

Cytokine expression

Brain tissue was extracted immediately following SI testing and mRNA transcripts for the cytokines TNFα and IL-6 were assessed in the VS, HPC, and PFC (Figure 3). For IL6 expression, two way ANOVA revealed a main effect of LPS treatment in the HPC (F1,22=22.6, p<0.0001; Figure 3A), PFC (F1,23=10.8, p=0.003; Figure 3B), and VS (F1,24=7.3, p=0.013; Figure 3C), with expression levels in the LPS treated animals being significantly higher. No main effect of sex was observed in any of these regions (HPC: F1,22=0.09, p=0.77; PFC: F1,23=3.0, p=0.097; VS: F1,24=3.8, p=0.064). No significant sex by treatment interaction was detected for the HPC (F1,22=0.005, p=0.94) or PFC (F1,23=3.2, p=0.086). However, a nearly significant interaction effect was observed for the VS (F1,24=4.1, p=0.054). Posthoc tests comparing males and females treated with LPS indicated significantly higher expression of IL6 in the VS of female mice (p=0.046).

Figure 3. TNFα and IL6 expression following LPS injection.

Figure 3.

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Expression of IL-6 and TNFα proinflammatory cytokines in the hippocampus (HPC), prefrontal cortex (PFC), and ventral striatum (VS), a-c Expression of IL-6 in the HPC, PFC, and VS. d-f Expression of TNF in the HPC, PFC, and VS. Data expressed as fold change in expression compared to saline treated mice of same sex. *p<0.05, **p<0.01, ***p<0.001 compared to saline treatment. + p<0.05 compared to male mice treated with LPS.

For TNFα expression two way ANOVA revealed a main effect of LPS treatment in the HPC (F1,21=12.8, p=0.0018; Figure 3D), PFC (F1,23=29.9, p<0.0001; Figure 3E), and VS (F1,22=31.1, p<0.0001; Figure 3F), with expression levels in the LPS treated animals being significantly higher. No main effect of sex was observed in the PFC (F1,23=2.1, p=0.16) or VS (F1,22=0.42, p=0.52), but this effect nearly reached significance for the HPC (F1,21=4.2, p=0.052). No significant sex by treatment interaction was detected for the PFC (F1,23=2.1, p=0.16) or VS (F1,22=0.46, p=0.50). However, a nearly significant interaction effect was observed for the HPC (F1,21=4.2, p=0.052). Posthoc tests comparing males and females treated with LPS indicated higher expression of TNFα in the HPC of female mice that nearly reached signficance (p=0.057). Taken together, these data show that LPS increases the proinflammatory cytokines strongly, with the effect being greater in female mice for IL6 expression in the VS and perhaps also for TNFα in the HPC.

Bodyweight Change

After 12 days of baseline drinking, mice (n=8/group) were injected with LPS. After treatment with LPS, three-way repeated measures ANOVA revealed a main effect of treatment (F1,28=227.2, p<0.0001), day post-LPS administration (F7,196=75.9, p<0.0001), and sex (F1,28=8.65, p=0.0065) on percent change of bodyweight (Figure 4). Three way ANOVA revealed significant interactions between day and sex (F7,196=5.42, p<0.0001), day and treatment (F7,196=71.2, p<0.0001), sex and treatment (F1,28=6.16, p=0.019), and a three-way interaction between day, sex, and treatment (F7,196=2.24, p=0.032). Posthoc test showed that on days 1-4 post injection both the male and female LPS groups had a significantly decreased weight compared to their respective saline group (P<0.001 for all). The male and female mice lost a similar amount of weight due to LPS treatment, however the female mice were able to regain body weight slightly faster. The female LPS group’s percent weight change was not significantly different from the female saline group by day five. Male mice which received LPS had lower weights than their counterparts for days 5-7 (p<0.001 for days 5-6, p<0.01 for day 7).

Figure 4. Body Weights after LPS treatment.

Figure 4.

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Body weights were tracked for 7 days following injection of LPS. **p<0.01, ***p<0.001 male saline versus male LPS; ###p<0.001 female saline versus female LPS.

Alcohol consumption

To assess LPS-induced escalation in consumption we compared the average of the last 3 days of alcohol consumption prior to LPS to the average of the first 3 days after LPS treatment. After treatment with LPS, drinking was significantly increased in both sexes with two way ANOVA revealing an effect of treatment (F1,28=27.7, p<0.0001) and sex (F1,28=21.5, p<0.0001), but no significant treatment by sex interaction (F1,28=0.0037, p=0.95; Figure 5). Overall, female mice consumed more alcohol than male mice (saline treated males versus saline treated females p=0.02, LPS treated males versus LPS treated females p=0.02) and LPS greatly increased alcohol intake (saline treated males versus LPS treated males p=0.006, saline treated females versus LPS treated females p=0.005), as has been reported previously. Daily averages in alcohol consumption over the 12 days prior to LPS injection and the 5 days following injection are shown in supplemental figure 1. We do not think that LPS-induced escalation in g/kg alcohol intake is the result of body weight change, as the absolute grams of alcohol consumed (not corrected for body weight) is higher in LPS treated mice as compared to their pretreatment baseline, but this is not observed in saline treated mice. Specifically, two way ANOVA revealed a main effects of drinking phase (F1,28=11.8, p=0.0019) and LPS treatment (F1,28=5.9, p=0.022), as well as a phase by treatment interaction (F1,28=19.8, p=0.0001; supplemental figure 2). Posthoc tests indicated that mice treated with LPS show increases in grams consumed compared to their pre-injection baseline (male LPS: p=0.0056, female LPS: p=0.033).

Figure 5. Alcohol consumption following LPS injection.

Figure 5.

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Ethanol consumption, averaged over the three-day periods immediately before and after LPS injection, were compared in male and female mice with a treatment of saline or LPS (main effects of treatment p<0.0001 and sex: p<0.0001). *p<0.05, **p<0.01.

Next, mice were pretreated with L-703060 or vehicle prior to alcohol drinking and intake over the next 24-hour period was analyzed. In mice treated with LPS, we observed a main effect of antagonist treatment (F1,13=5.7, p=0.03) and sex (F1,14=12.4, p=0.003), as well as an interaction effect (F1,13=5.5, p=0.04; Figure 6A). Post hoc analysis showed a significant difference between the vehicle and antagonist treated females (p=0.009), and between the male and female vehicle group (p=0.0004). Antagonist treated male mice did not differ from vehicle treated controls. For saline treated mice, two-way repeated measures ANOVA revealed main effects of treatment (F1,13=21.0, p=0.0005) and sex (F1,14=10.9, p=0.005), but no interaction effect (F1,13=1.1, p=0.31; Figure 6B). Posthoc tests indicated a significant difference between the female vehicle and antagonist groups (p=0.002), and between males and females following treatment with vehicle (p=0.004) or antagonist (p=0.02). Antagonist treated male mice did not differ from vehicle treated controls.

Figure 6. Effects of NK1R antagonist on alcohol consumption.

Figure 6.

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Alcohol consumption is reported in g/kg consumed over the 24 hours following treatment. a LPS treated mice were given pretreatment injections with vehicle or NK1R antagonist (main effects of antagonist treatment: p=0.03, sex: p=0.003, interaction: p=0.04). b Saline treated mice were given pretreatment injections with vehicle or NK1R antagonist (main effects of antagonist treatment p=0.0005, sex: p=0.005). *p<0.05, **p<0.01, ***p<0.001.

Discussion

LPS administration triggers a rapid and intense immune response that induces cytokine expression, weight loss, and depression-like behaviors. In line with this literature, we found that LPS induced social avoidance and increased expression of the inflammatory mediators IL6 and TNFα. Interestingly, these effects were more pronounced in female mice. We also found that LPS injection induced increased alcohol consumption, as has been shown previously (Blednov et al., 2011b). When pretreating mice with an NK1R antagonist, we observed an interesting pattern of effects in that this intervention reduced drinking in females treated with LPS as well as those treated with saline, and did not seem to have any effect on alcohol intake in male mice under either condition. Taken together, these findings suggest that LPS can induce inflammation and increased alcohol consumption, with some of these effects being more pronounced and more strongly influenced by the NK1R in female animals.

It is well known that LPS can induce sickness behavior that includes depression-like symptoms such as anhedonia and social avoidance. We found that LPS treatment had this effect in female mice. We were a bit surprised that this effect was not observed in males. However, it is important to note that our behavioral measure was taken 24 hours after LPS injection. Most groups that have examined post-LPS social behavior in adult male rodents observed these effects at earlier timepoints after injection (typically 2 to 6 hours), and that this behavior was mostly normalized after 24 hours (Bluthe et al., 1999; Bluthe et al., 1992; Fishkin & Winslow, 1997; Henry et al., 2008; Jangra et al., 2014; Konsman et al., 2008; Orlandi et al., 2015; Reis et al., 2022). However, some groups have reported longer effects of LPS on SI lasting up to 24 hours in some mouse strains (Haba et al., 2012). Notably, all of the studies referenced above assessed this response in male rodents. Some studies have assessed the effect of LPS at early timepoints on SI in female rodents, but it is unclear how long this response persists (Painsipp et al., 2008). Based on our data, we would suggest that this pattern would persist for up to 24 hours. A primary motivation for testing at this later timepoint was to ensure that SI behavior was not confounded by general locomotor suppression that occurs in the immediate response to LPS exposure. Taken together, these data suggest that female mice have a longer lasting effect of LPS exposure on SI behavior as compared to males.

We also observed a strong activation of cytokine mRNA expression in both male and female mice. Some aspects of this response seemed to be stronger in females as compared to males. Specifically, IL6 expression was more strongly stimulated in female mice in the VS. This is interesting in light of the fact that IL6 has been shown to play a strong role in social stress and subsequent interaction behavior (Hodes et al., 2014), and the nucleus accumbens, a core region of the VS, it a critical mediator of depression-like behavior following stress (Chaudhury et al., 2013; Christoffel et al., 2011; Christoffel et al., 2012; Fox et al., 2020; Francis et al., 2015). TNFα expression was also elevated in the HPC of LPS-treated female mice, but this measure did not exceed the threshold for statistical significance. One limitation of our cytokine measures is that they were performed in animals that were alcohol-naïve, and do not directly examine possible interactions between alcohol exposure history and LPS-induced effects. This interesting question will be addressed in future experiments.

In line with our findings, other groups have found a similar increase in sensitivity to LPS in females in regards to expression of inflammatory mediators and behavioral effects. For example, Dockman and colleagues (Dockman et al., 2022), showed elevated sickness behavior and cytokine activation in aged female mice relative to males. In support of this increased sensitivity to LPS in female rodents, Tonelli and colleagues (Tonelli et al., 2008) show increased sensitivity to intranasally administered LPS in adult female rats as measured by cytokine expression, glucocorticoid release, and depressive-like behavior. Taken together, these findings demonstrate that female rodents may respond more strongly to multiple effects of LPS.

In agreement with the work of Blednov and colleagues (Blednov et al., 2011b), we observed significantly increased alcohol consumption following injection of LPS. This was observed in both sexes and to similar degrees. However, when we treated mice with an NK1R antagonist, we found that this significantly reduced LPS-induced escalation in alcohol intake in females only. A similar pattern was observed in saline treated mice. Together this shows that NK1R antagonism can reduce alcohol consumption under conditions of high intake. We were a bit surprised to see no effect of the NK1R antagonist in male mice, given prior reports (Thorsell et al., 2010). However, the current study differed from those prior studies in terms of alcohol concentration (20% versus a range of concentrations with a maximum of 15%), antagonist used (L733060 versus L703606), and duration of access (17 days versus several weeks).

In summary, we show an ability of LPS administration to increase cytokine expression, social avoidance, and alcohol intake, with increased sensitivity to some aspects of this response in female mice. Escalated alcohol consumption could be reversed in females treated with NK1R antagonist, but this effect was not observed in males. Taken together, this suggests an increased sensitivity to inflammatory stress in females that is partially mediated by the NK1R.

Supplementary Material

1

Supplemental Figure 1. Daily alcohol consumption in g/kg over 12 days prior to saline or LPS injection, and 5 days following this treatment. Red arrow indicates time of experimental treatment.

2

Supplemental Figure 2. Grams of alcohol consumed before and after LPS injection, not corrected for body weight. *p<0.05, **p<0.01.

Highlights.

  • LPS injection induces social avoidance in female mice

  • LPS induces cytokine expression in both males and females

  • LPS increases alcohol intake in both males and females

  • Neurokinin-1 receptor antagonism reduces LPS-induced escalation in alcohol intake

Acknowledgements

This work funded by NIH grant R01 AA026362 (JRS).

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Supplemental Figure 1. Daily alcohol consumption in g/kg over 12 days prior to saline or LPS injection, and 5 days following this treatment. Red arrow indicates time of experimental treatment.

NIHMS1885676-supplement-1.jpg (111.6KB, jpg)
2

Supplemental Figure 2. Grams of alcohol consumed before and after LPS injection, not corrected for body weight. *p<0.05, **p<0.01.

NIHMS1885676-supplement-2.jpg (122.4KB, jpg)

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