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. Author manuscript; available in PMC: 2022 Feb 1.
Published in final edited form as: Brain Behav Immun. 2020 Dec 9;92:184–192. doi: 10.1016/j.bbi.2020.12.008

Lipoteichoic acid, a cell wall component of Gram-positive bacteria, induces sleep and fever and suppresses feeding

Éva Szentirmai 1,2,*, Ashley R Massie 1, Levente Kapás 1,2
PMCID: PMC7897295  NIHMSID: NIHMS1653519  PMID: 33307170

Abstract

Fragments of the bacterial cell wall are bioactive microbial molecules that have profound effects on the function of the brain. Some of the cell wall constituents are common to both Gram-positive and Gram-negative bacteria, e.g. peptidoglycans, while other cell wall components are specific to either Gram-positive or Gram-negative microbes. Lipopolysaccharide (LPS), also called endotoxin, is found exclusively in Gram-negative bacteria, while lipoteichoic acid (LTA) is specific to Gram-positive bacteria. The effects of peptidoglycans, their fragments, and LPS are well characterized, they induce sleep, fever and anorexia. In the present study, we investigated the sleep, body temperature and food intake modulating effects of LTA. We found that intraperitoneal injection of 100 and 250 μg LTA from B. subtilis and S. aureus increases non-rapid-eye movement sleep (NREMS) in mice. The effects were dose-dependent, and the changes were accompanied by decreased motor activity and feeding as well as febrile responses. Intraperitoneal injection of 10 μg LTA induced monophasic increases in body temperature, while 100 and 250 μg LTA from B. subtilis induced initial hypothermia followed by fever. Treatment with 250 μg LTA from S. aureus elicited monophasic hypothermia. Administration of 300 μg/kg LTA from S. aureus directly into the portal vein elicited similar sleep responses in rats but did not affect body temperature. The sleep-modulating effects of LTA were similar to that of LPS in mice, although LTA appears to be less potent. These findings suggest that the role of LTA in signaling by Gram-positive bacteria in the host body is analogous to the role of LPS/endotoxin in signaling by Gram-negative microbes. LTA may play a role in the development of sickness response in clinically manifest Gram-positive bacterial infections and may contribute to sleep signaling by the commensal intestinal microbiota.

Introduction

Microbial infections caused by bacteria, viruses, fungi or parasites activate the immune system and cause characteristic central nervous system-mediated responses collectively called sickness response (Dantzer, 2009). The classic symptoms and signs of this complex adaptive behavioral and autonomic response include fever, sleepiness, reduced feeding and social withdrawal. Sickness response during bacterial infections can be caused by a variety of bacterial molecules such as toxins, virulence factors and particularly by componenets of the cell wall.

A primary component and structural scaffold of the cell wall of Gram-positive and -negative bacteria is peptidoglycan (PGN) (Beveridge, 1999; Shockman and Barrett, 1983). In Gram negative bacteria, the cell wall is composed of a thin layer of peptidoglycan surrounded by an outer membrane. Lipopolysaccharide (LPS), also called endotoxin, is the major component of the outer membrane of Gram-negative bacteria and is responsible for the initiation of Gram-negative sepsis. The Gram-positive cell wall, however, lacks LPS, and consists of several layers of peptidoglycan and large amounts of wall-associated polymers such as teichuronic acids and teichoic acids. Teichoic acids, which are amphipathic molecules similarly to LPS, compose 50% of the cell wall mass (Garimella et al., 2009). They are present in two forms: cell wall-anchored teichoic acids are attached to PGNs, while membrane teichoic acids, are hydrophobically anchored to a glycolipid moiety of the plasma membrane and named lipoteichoic acids (LTA).

Bacterial cell wall molecules are recognized by the innate immune system by detecting evolutionarily conserved microbial molecular signatures, so-called microbial associated molecular patterns through pattern recognition receptors (PRRs). LPS, PGNs and LTAs activate the host defense mechanisms by engaging with PRRs of the innate immune system. Toll-like receptors (TLRs) represent the best characterized PPRs. Of the known members of TLR family, LTAs signals via TLR2, while TLR4 recognizes LPS (Akira and Takeda, 2004). TLR2 is predominantly expressed by monocytes, macrophages and neutrophils and it also recognizes PGNs and lipoproteins (Seki and Schnabl, 2012). Further, LTA binds to the complement receptor of immunoglobulin superfamily (CRIg) expressed by Kupffer cells. This binding is key to the capture of circulating Gram-positive bacteria by the liver and thus to prevent systemic bacterial dissemination (Zeng et al., 2016).

The biological effects of PGNs, their fragments, and LPS are well characterized in many species. LPS and PGNs have been long recognized as somnogenic microbial products (reviewed in Krueger and Opp, 2016), they induce fever (reviewed in Roth and Blatteis, 2014) and suppress feeding (reviewed in Langhans, 1996). During clinically manifest bacterial infections LPS and PGNs stimulate cells of the innate immune system to release cytokines and other proinflammatory mediators which are likely responsible for the initiation of the acute phase response of the sickness response (Dantzer, 2009).

The clinical symptoms of Gram-positive and Gram-negative sepsis are strikingly similar (Draing et al., 2008). Sickness response to Gram-negative bacteria is commonly attributed to their LPS component, but the pathogenesis of Gram-positive sepsis has not been linked unequivocally to a such a single, key microbial molecule. We hypothesized that LTA may have an analogous role to that of LPS in triggering sickness response in Gram-positive bacterial infections. Surprisingly, despite the great abundance of teichoic acids in the cell wall of Gram-positive bacteria and their importance in the removal of these microbes, their impact on the physiology of the host has been scarcely investigated at the organismal level. To our knowledge, their actions on sleep and feeding, key components of sickness response, have never been studied. In the present study, we investigated the effects of LTA on sleep, body temperature and food intake in mice and rats. We report that systemic administration of LTA from B. subtilis and S. aureus induces typical sickness response, including robust, dose-dependent sleep and fever responses and suppression of feeding. These findings are consistent with the proposed role of LTA in sickness response to Gram-positive bacteria.

Methods

Animals

All procedures involving the use of animals followed the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal husbandry and experimental procedures were carried out in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care and approved by the Institutional Animal Care and Use Committee of the Washington State University (protocol number 6031). Breeding pairs of C57BL/6J mice were purchased from The Jackson Laboratories, Inc. and were bred in-house at Washington State University. Male Sprague-Dawley rats were purchased from Envigo. During the experiments, the animals were housed individually in temperature-controlled (29 ± 1°C for mice, 23 ± 1°C for rats), sound-attenuated environmental chambers on a 12:12-hour light-dark cycle (lights on at 3 AM), with the humidity maintained in the 35–50% range. Food (Harlan Teklad, Product no. 2016) and water were available unrestricted throughout all experiments.

Surgery

All surgical procedures were performed using ketamine-xylazine anesthesia [87 and 13 mg/kg, respectively, intraperitoneally (ip)]. For pain management, mice received 0.5 mg/kg buprenorphine subcutaneously (sc) postoperatively, and rats received 0.05 mg/kg buprenorphine sc preoperatively and 1 mg/kg buprenorphine and 5 mg/kg carprofen, both sc, after the surgeries. For sleep-wake activity recordings, 3-month old mice (25.5 ± 0.9 g) and rats (325–350 g) were implanted with three cortical electroencephalographic (EEG) electrodes, placed over the frontal and parietal cortices, and two electromyographic (EMG) electrodes in the neck muscles. Leads from the EEG and EMG electrodes were anchored to the skull with dental cement. Telemetry transmitters were implanted ip for body temperature and motor activity measurements. In addition, the rats were implanted with an intraportal cannula three weeks prior to the sleep surgery. Briefly, a biocompatible polyurethane was inserted into the superior mesenteric vein and the tip of the cannula routed to the main stream of the portal vein. The free end of the cannula was routed subcutaneously to the dorsal surface of the neck and exteriorized. The cannula was sutured to the portal vein, the abdominal muscles and the neck skin. The patency was maintained by daily flushing with 0.2 ml isotonic saline followed by 0.08 ml of lock solution containing 500 IU/ml heparin in 50% glycerol solution. The animals were allowed to recover from surgery for at least 10 days before any experimental manipulation started and handled daily to adapt them to the experimental procedures.

Sleep recordings and analyses

The animals were connected to the recording system through a lightweight, flexible tether plugged in into a commutator, which was further routed to Grass Model 15 Neurodata amplifier system (Grass Instrument Division of Astro-Med, Inc., West Warwick, RI). The amplified EEG and EMG signals were digitized at 256 Hz and recorded by computer. The high-pass and low-pass filters for EEG signals were 0.5 and 30.0 Hz, respectively. The EMG signals were filtered with low and high cut-off frequencies at 100 and 10,000 Hz, respectively. The outputs from the 12A5 amplifiers were fed into an analog-to-digital converter and collected by computer using SleepWave software (Biosoft Studio, Hersey, PA). Sleep-wake states were scored visually off-line in 10-s segments. The vigilance states were defined as non-rapid-eye movement sleep (NREMS), rapid-eye movement sleep (REMS) and wakefulness according to standard criteria as described previously (Szentirmai and Krueger, 2014). Artifact-free EEG epochs were subjected to off-line spectral analysis by fast Fourier transformation. EEG power data in the range of 0.5 to 4.0 Hz during NREMS were used to compute EEG slow-wave activity (SWA). EEG SWA data were normalized for each animal by using the average EEG SWA across 24 h on the baseline day as 100.

Telemetry recordings of body temperature and motor activity

Core body temperature and locomotor activity were recorded by MiniMitter telemetry system recordings (Starr Life Sciences Corp., model G2 Emitter for mice, PDT 4000 E-Mitter for rats and ER-4000 receiver) using VitalView software. Temperature and activity values were collected every 1 and 10 min, respectively, throughout the experiment and were averaged over 1-h time blocks.

Experimental procedures

Experiment 1: The effects of systemic administration of LTA from B. subtilis on sleep-wake activity, body temperature and metabolic parameters in mice

A group of mice (n = 8) was habituated to the injection procedure by administering 0.3 ml isotonic saline daily for 7 days 5–10 min before dark onset. On the baseline days, the animals were injected ip with 0.3 ml isotonic NaCl. On the test day, the animals received 10, 100 or 250 μg/mouse (~0.4, 4 and 10 mg/kg) LTA from B. subtilis (Millipore Sigma) dissolved in 0.3 ml isotonic NaCl. The LTA doses were chosen based on their potency to induce proinflammatory cytokine production in mice (Zhao et al., 2007; Mayerhofer et al., 2017). The three doses of LTA were tested in the same group of mice in increasing order with at least one week recovery between the treatments. The injections were performed 5–10 min before dark onset. Sleep and telemetry recordings started at the onset of the dark phase and continued for 23.5 h. Due to the malfunction of a telemetry plate, motor activity data were obtained from only 7 animals after each treatment. Mice are nocturnal animals, and they sleep about twice as much during the day as compared to the dark period. We performed LTA treatments at dark onset, because a potentially sleep-promoting treatment should be administered at the beginning/during the dark period, since the light period administration might be confounded with a possible ceiling effect.

Oxygen consumption (VO2, ml/kg/h), CO2 output (ml/kg/h) and respiratory exchange ratio (RER) were measured via indirect calorimetry; food intake was measured simultaneously by an automated system (Oxymax System, Columbus Instruments, Columbus, OH). Heat production (HEAT, kcal/h) was calculated from oxygen uptake and the caloric value of oxygen (CV) as follows: Heat = CV × VO2, where CV = 3.815 +1.232 × RER. Mice (n = 8) were housed in individual calorimetry cages and habituated to the experimental conditions for at least three days before the recordings. On the baseline day, 0.3 ml isotonic NaCl was administered, on the test day 250 μg (n = 8) LTA from B. subtilis was injected ip. All treatments were performed 5–10 min before the onset of the dark period. Food intake values were obtained from only 7 animals due to the malfunction of one of the balances.

Experiment 2: The effects of systemic administration of 250 μg LTA from S. aureus on sleep-wake activity, body temperature in mice

A group of mice (n = 8) was habituated to the injection procedure as described above. On the baseline day, the animals were injected ip with 0.3 ml isotonic NaCl. On the test day, 250 μg/mouse LTA from S. aureus (Millipore Sigma) was administered ip dissolved in 0.3 ml isotonic NaCl (n = 8) 5–10 minutes before dark onset. Sleep and telemetry recordings started at onset of the dark phase and continued for 23.5 h. Due to the malfunction of a telemetry plate, motor activity data were obtained from only 7 animals.

Experiment 3: The effects of portal vein administration of 300 μg/kg LTA from S. aureus on sleep-wake activity, body temperature in rats

To mimic the translocation of LTA from the intestines to the portal circulation, intraportal injections were performed in rats. Eight rats were habituated to the injection procedure by daily flushing of the cannula with isotonic saline 5–20 min before dark onset. On the baseline day, 1 ml/kg isotonic NaCl (vehicle) was administered through the cannula. On the test day, the animals received 300 μg/kg LTA from S. aureus, in a volume of 1 ml/kg. The treatments were performed 5–20 min before dark onset. Sleep and telemetry recordings started at dark onset and continued for 23.5 h. Due to artefacts in the EEG, SWA was obtained and analyzed from only 7 animals.

Statistics

Time spent in wakefulness, NREMS and REMS, as well as body temperature and motor activity, VO2, HEAT and RER were calculated in 1-h blocks and EEG SWA was calculated in 2-h blocks. Two-way repeated measures ANOVA was performed across 24 h between test days and the corresponding baselines (factors: treatment and time, both repeated). When appropriate, Tukey’s Honestly Significant Difference test was applied post hoc. Body temperature responses to LTS during the first 12 h after treatments were also calculated in 10-min averages. Two-way repeated measures ANOVA was performed followed by paired t-tests with Bonferroni correction. An α-level of P < 0.05 was considered to be significant.

Results

Experiment 1: The effects of LTA from B. subtilis on sleep-wake activity, body temperature and metabolic parameters in mice

Intraperitoneal injection of LTA at the beginning of the dark phase brought about marked, dose-dependent increases in NREMS, and changes in body temperature and motor activity in mice (Figure 1, Table S1). Administration of 10 μg LTA did not affect sleep-wake activity, EEG SWA or motor activity. The effects of 100 μg LTA on sleep were manifested after a latency of one hour; starting from h 2, wakefulness decreased, NREMS and REMS significantly increased above baseline for 2 hours. Sleeping animals were easily arousable, they showed all the characteristic behavioral signs of natural sleep, they slept curled-up or slightly stretched out. Time spent in NREMS in the first 3 hours after injection increased by 42.5% above baseline at the expense of wakefulness (NREMS baseline: 60.19 ± 7.8 min/3 h, LTA: 85.77 ± 4.1 min/3 h, p < 0.05). The amount of REMS increased by 133.9% above baseline (REMS baseline: 2.5 ± 0.5 min/3 h, LTA: 5.9 ± 0.6 min/3 h, p < 0.01). ANOVA analysis of EEG SWA indicated significant changes after 100 μg LTA treatment; post hoc analysis revealed significantly elevated EEG SWA for the last 2-hour period of the dark phase. The effects of 250 μg LTA on sleep were similar to those of the 100 μg. NREMS increased by 57.6% above baseline in the first 3 hours after a 1-h latency. Time spent in REMS did not change after the highest dose of LTA. EEG SWA was significantly suppressed during the first 6 hours after LTA administration.

Figure 1.

Figure 1.

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Effects of intraperitoneal (ip) administration of 10, 100 and 250 μg/mouse lipoteichoic acid (LTA) from B. subtilis on wakefulness, non-rapid-eye movement sleep (NREMS), rapid-eye movement sleep (REMS), electroencephalographic slow wave activity (EEG SWA), body temperature and motor activity in mice. Data are expressed in 1-h time blocks, except EEG SWA values which are shown in 2-h blocks. LTA and saline were injected at time “0”. Grey shaded areas represent the dark period. Asterisks: significant difference between control and LTA treatments, Tukey’s HSD test; error bar: SE.

After the administration of 10 μg LTA, body temperature was slightly but significantly elevated starting from h 4 for 3 hours (Figures 1 and S1, Tables S1 and S4). Changes in body temperature after 100 μg LTA showed biphasic pattern, manifested as an initial hypothermia lasting for 2 hours followed by significantly elevated temperature for 3 hours, then returned to baseline for the remainder of the recording period. Body temperature responses were also biphasic in response to 250 μg/kg LTA. The initial drop in temperature in hours 1–2 was followed by significant increases lasting for 5 hours.

Motor activity was significantly suppressed in first 3 h after the middle and in hours 2 and 3 after the treatment high dose of LTA (Figure 1, Table S1).

To gain further insight into the action of LTA on metabolism, we investigated the effects of ip administration of LTA on O2 consumption (VO2), heat production, RER and food intake. Intraperitoneal injection of 250 μg LTA induced marked suppression of VO2, HEAT, RER and feeding lasting for 3 hours after treatment (Figure 2, Table S1).

Figure 2.

Figure 2.

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Effects of ip administration of 250 μg/mouse LTA from B. subtilis on oxygen consumption (VO2), heat production (HEAT), respiratory exchange ratio (RER) and food intake in mice. Data are expressed in 1-h time blocks. See legend to Figure 1 for details.

Experiment 2: The effects of 250 μg LTA from S. aureus on sleep-wake activity, body temperature in mice

The sleep effects of 250 μg LTA from S. aureus were similar to those of the 250 μg LTA from B. subtilis (Figures 3 and S1, Tables S2 and S4). NREMS increased by 57.6% in the first 3 hours after LTA injection. There was no significant change in time spent in REMS. EEG SWA slightly, but significantly decreased after LTA injection. Body temperature response was slightly different from what we observed after LTA from B. subtilis; LTA from S. aureus elicited only the hypothermic response immediately after injection. Simultaneously, motor activity was significantly suppressed in response to LTA treatment.

Figure 3.

Figure 3.

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Effects of ip administration of 250 μg LTA from S. aureus on wakefulness, NREMS, REMS, EEG SWA, body temperature and motor activity in mice. See legend to Figure 1 for details.

Experiment 3: The effects of portal vein administration of 300 μg/kg LTA from S. aureus on sleep-wake activity and body temperature in rats

Intraportal injection of 300 μg/kg LTA induced prompt and robust NREMS increases in rats (Figure 4, Table S3). Overall, NREMS increased by 53.3% in the first 3 hours after the LTA injection at the expense of wakefulness (NREMS baseline: 50.3 ± 3.7 min/3 h, LTA: 77.2 ± 5.5 min/3 h; p < 0.01). The time spent in REMS was increased only in the third hour and EEG SWA was significantly suppressed after a 4-hour latency following LTA administration. Motor activity was suppressed for 3 hours immediately after LTA injection. Intraportal administration of LTA failed to induce any significant changes in body temperature (Figures 4 and S1, Tables S3 and S4).

Figure 4.

Figure 4.

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Effects of portal vein administration of 300 μg/kg LTA from S. aureus on wakefulness, NREMS, REMS, EEG SWA, body temperature and motor activity in rats. See legend to Figure 1 for details.

Discussion

Our main finding is that systemic injection of LTA, a major component of the Gram-positive bacterial cell wall, increases NREMS in mice. The effects were dose-dependent, and the changes in NREMS were accompanied by decreased EEG SWA, motor activity, food intake and biphasic body temperature responses. Collectively, these data suggest that LTA induces acute phase responses, and its activities may underlie sickness response during Gram-positive bacterial infections.

Bioactive microbial molecules, such as fragments of the bacterial cell wall, have long been implicated in the pathophysiological consequences of bacterial infections. The effects of LPS, which is found exclusively in the cell wall of Gram-negative bacteria, are extensively investigated and it is implicated in the pathogenesis of Gram-negative sepsis. It is much less understood which microbial molecules are involved in sickness responses to Gram-positive bacteria. PGNs are common structural components in both Gram-negative and -positive bacteria. PGNs and their fragments also induce sleep, fever and anorexia (Langhans et al., 1990; Krueger et al., 1982; Kadlecova et al., 1972) and thus they likely contribute to the symptoms of both Gram-positive and -negative bacterial infections.

Teichoic acid glycopolymers are specific to the cell wall of Gram-positive bacteria such as S. aureus or B. subtilis. They make up about 2% of the dry cell weight (Gutberlet et al., 1997) and 50–60% of the total cell wall mass (van Dalen et al., 2020). LTA shares many of its pathogenetic properties with LPS, and it also triggers inflammatory responses. It is released during natural death of bacteria and by bacteriolysis induced by bactericidal molecules and antibiotics. It stimulates the release of reactive oxygen and nitrogen species, acid hydrolases, highly cationic proteinases, bactericidal cationic peptides, growth factors, and proinflammatory cytokines from neutrophils and macrophages (reviewed in Van Dalen et al., 2020; Ginsburg, 2002).

While the effects of LPS, PGNs and their fragments on sleep have been thoroughly characterized, LTA, a highly abundant bacterial wall component, have, to our knowledge, never been investigated for its somnogenic properties, effects on feeding and its potential contribution to sickness response in Gram-positive infections. Systemic injection of LPS induces sleep in rats (Kadlecova et al., 1972x; Opp and Toth, 1998; Kapas et al., 1998), mice (Toth and Opp, 2001; Morrow and Opp, 2005; Szentirmai and Krueger, 2014x; Szentirmai and Kapas, 2018), rabbits (Krueger et al., 1986) and humans (Pollmacher et al., 1993; Trachsel et al., 1994; Mullington et al., 2000). Peripheral administration of PGNs and their peptide fragments induce sleep in rats (Kadlecova et al., 1972; Masek et al., 1973), mice (Massie et al., 2018), rabbits (Krueger et al., 1982; Shoham and Krueger, 1988) and squirrel monkeys (Wexler and Moore-Ede, 1984). In our experiments, the sleep-modulating effects of LTA were similar to that of LPS in mice, although LTA appears to be less potent. While ip injection of 0.4–3 μg/mouse LPS induces significant NREMS increases in mice (Szentirmai and Krueger, 2014; Szentirmai and Kapas, 2018), in the present study 10 μg LTA was subthreshold dose in terms of somnogenic actions. EEG SWA activity is often regarded as a measure of NREMS intensity, but it can also change independently of sleep, e.g., related to changes in cerebral blood flow (Davis et al., 2011). It has been shown that LPS and pneumococcal cell wall components containing teichoic acid produce changes in regional cerebral blood flow (Weiner, 1970, Pfister et al., 1992). Products of the bacterial cell wall elicit diverse effects on EEG SWA, for example the effects of LPS differ among species and depend on the route of administration and the type of pathogen. In humans, both slightly elevated (Mullington et al., 2000) and unaltered EEG SWA (Pollmächer et al., 1993) were observed after LPS administration. In rabbits, EEG SWA increases were observed (Krueger et al., 1986), while in mice dark onset administration of LPS induced decreases in EEG delta power during NREMS (Toth and Opp, 2001, Morrow and Opp, 2005; Szentirmai and Kapás, 2018). In rats, both biphasic responses and decreased SWA were reported (Kapás et al., 1998; Lancel et al., 1995). In our experiments, EEG SWA was suppressed after LTA treatment, a response similar to that observed after LPS in mice. The extent to which this change reflects altered sleep intensity or changes in cerebral blood flow or other parameters is unknown.

Although LTA is often referred to as a pyrogen (e.g., Daneshian et al., 2008; Stang et al., 2014), its effects on core temperature, heat production and metabolism are not well characterized. Body temperature effects of LTA have been reported only for rabbits in two separate studies. In one study, two of the four animals did not show any response to intravenously administered 500 μg LTA, and the other two developed monophasic fever (Atkins and Morse, 1967). In a second report, intravenously administered LTA induced fever in the 50–500 μg/kg range (Gimenes et al., 2015). In our study, 10–250 μg (~ 0.4–10 mg/kg) LTA from B. subtilis induced 0.5–0.8°C increase in body temperature in mice indicating that the sensitivity of mice to the febrile actions of LTA is much lower than that of rabbits. This is not unexpected as the sensitivity of rabbits to other pyrogens, such as LPS, is very high compared to other laboratory or domestic animals (van Miert and Frens, 1968). TLR2 is one of the receptor types LTA activates (Schwander et al., 1999). Similarly to LTA, synthetic TLR2 agonists induce fever and suppresses activity in rats (Hubschle et al., 2006; Jin et al., 2016; Murayama et al., 2019).

Fever was preceded by a drop in body temperature in the first two hours after the middle and high doses of LTA. These effects are similar to those of LPS, which also elicits biphasic response on body temperature in mice (Kozak et al., 1994; Wang et al., 1997, Morrow and Opp, 2005; Szentirmai and Kapás, 2018). Oxygen consumption (VO2) was decreased during the first 2–3 h after the administration of LTA which suggests that decreased heat production may underly the initial hypothermic phase. During the same period, food intake and RER were also significantly suppressed. Bacterial infections and LPS or PGN treatment are also associated with anorexia (reviewed in Langhans, 1996). Since heat production/metabolic rate is very sensitive to the acute caloric intake in mice, it is possible that anorexia is the primary effect of LTA, leading to suppressed heat production, which, in turn, is manifested in decreased body temperature. Reduced RER is likely also the consequence of suppressed feeding and indicates enhanced lipolysis to meet energy demand. Increased lipolysis has already been reported for LTA (Nonogaki et al., 1995) and LPS in vivo (Zu et al., 2009). We cannot discount the possibility that in addition to suppressed heat production, increased heat loss via vasodilation can also contribute to the hypothermic response to LTA. During the febrile phase, VO2 remained at baseline levels indicating that increased heat production is not the primary cause of elevated body temperature. It is likely that the febrile phase is due to decreased heat loss.

In experiment 1, the same group of mice was injected with 3 doses of LTA in increasing order, with at least 1 week apart between treatments. Repeated injections of high doses of LPS lead to tolerance to its effects, including its pyrogenic action (Beeson, 1947). Tolerance, however, does not develop to all bacterial pyrogens. For example, repeated injections of MDP elicit similar peak fever responses in guinea pigs, although the time course of the response is affected by repeated treatments (Roth et al., 1997). Further, no tolerance developed to the febrile or serum iron response following repeated daily injections of killed S. aureus in rabbits (Goelst and Laburn, 1991). In our study, we observed dose-dependently enhanced febrile and somnogenic effects after increasing doses of LTA in the same animals. This suggests that there is no over tolerance to the actions of LTA in the dose range used in the present experiments, but the possibility cannot be ruled out that some attenuation of the effects occurred with repeated injections.

There are several possible receptor mechanisms that may mediate the actions of LTA. LTA activates TLR2 receptors (Schwander et al., 1999) as well as macrophage scavenger receptors (Greenberg et al., 1996), the platelet-activating factor receptor (Lemjabbar and Basbaum, 2002) and CRIg (Zeng et al., 2016). LTA-induced activation of these receptors on macrophages leads to inflammatory responses and secretion of interleukin-1β, tumor necrosis factor α (TNFα) and nitric oxide (Draing et al., 2008). All these proinflammatory signal molecules have potent somnogenic, pyrogenic and anorectic activities (Shoham et al., 1987; Opp et al., 1991; Kapas and Krueger, 1996; Kapas et al., 1992; reviewed in Kelley et al., 2003), thus they are likely candidates for mediating the actions of LTA on sleep, body temperature and feeding. Proinflammatory cytokines are also thought to play a role in the somnogenic and febrile actions of other bacterial molecules, such as LPS (Krueger and Majde, 2017). The stimulatory activity of LTA for these cytokines is at least three orders of magnitude less potent than that of LPS (Ginsburg 2002) which is likely the explanation for its less robust sleep- and fever-promoting actions.

Among components of the bacterial cell wall, the mechanisms of LPS-induced fever have been thoroughly investigated. The first phase of the biphasic febrile response to LPS is linked to peripherally produced PGE2 (Steiner et al., 2006; Blatteis, 2007), while later phases of fever are mediated by PGE2 produced by perivascular and endothelial cells in the central nervous system (Saper et al., 2012; Garami et al., 2018). It requires further investigation to determine if similar PGE2-mediated mechanisms also contribute to LTA-induced fever.

The significance of bacterial cell wall molecules has been generally viewed as the mediators of the symptoms of clinically manifest bacterial infections. In rabbits, both Gram-positive infections, induced by inoculating S. aureus or S. pyogenes, and Gram-negative infections, induced by whole E. coli or P. multocida, elicit robust sleep increases and fever (Toth and Krueger, 1988; Toth and Krueger, 1989; Toth and Krueger, 1990). Similarly to our findings with LTA, inoculation with S. aureus increased time spent in NREMS, induced fever and biphasic changes in EEG SWA. The sleep effects of Gram-negative bacteria are thought to be induced by the LPS and PGN components of the bacterial cell wall (Krueger and Opp, 2016). Our findings strongly suggest that in Gram-positive infections, the LTA component of the cell wall may also serve as an important contributor to the observed sleep effects as well as to fever and reduced feeding.

In addition to their pathogenic role in infections, bacterial molecules released from the intestinal microbiota likely play a role in modulating brain functions under healthy conditions (Cryan et al., 2019) and their continuous translocation from the intestines to the host’s internal environment may provide an important signal to the brain for the daily regulation of sleep, body temperature, appetite and other functions. Depletion of the intestinal microbiota reduces spontaneous sleep in mice and rabbits suggesting that the microbiota is a source of sleep-promoting signals (Brown et al., 1990; Millican et al., 2018). The somnogenic effects of the intestinal microbiota could be mediated by cell wall fragments of bacteria and/or bacterial metabolites, such as short-chain fatty acids (SCFAs), translocated into the portal circulation. We previously demonstrated that butyrate, a SCFA product of anaerobic bacterial fermentation of non-digestible carbohydrates, enhances sleep in rats and mice (Szentirmai et al., 2019). To mimic translocation from the intestines to the portal circulation, we injected butyrate, bacterial cell wall fragments, such as LPS, iEDAP and muramyl dipeptide, and in the present experiment LTA, directly into the portal circulation. Intraportal injection of butyrate, and bacterial cell wall components, including LTA, all increased NREMS in rats (Millican et al., 2019; Szentirmai et al., 2019). These findings indicate the existence of a hepatoportal sleep-promoting viscerosensory mechanism. Hepatoportal sensors have been described for several signal molecules, such as glucose, amino acids and various gastrointestinal hormones (Niijima, 1996; Mithieux et al., 2005; Niijima and Meguid, 1995; Niijima, 1988). Our results suggest that similar hepatoportal sensors exist for SCFAs and bacterial cell wall fragments, including LTA, and these sensors provide a peripheral input to sleep circuits in the brain. Since liver macrophages express receptors for LTA (Wu et al., 2010; Canton et al., 2013; Helmy et al., 2006), and LTA-stimulated macrophages secrete somnogenic signal molecules, such as TNFα and prostaglandins (Keller et al., 1991; Wakabayashi et al., 1991; Card et al., 1994), it is possible that the primary target for LTA in the host to induce sleep is the liver. Not only LTA translocated from the intestines or injected directly to the portal circulation reaches the liver, but LTA from the systemic circulation is also quickly taken up by the liver, mainly by hepatic macrophages (van Oosten et al., 2001).

TLR2 receptors are expressed by neurons of the hypothalamic arcuate nucleus and by microglia in other circumventricular organs in mice under baseline conditions (Shechter et al., 2013; Murayama et al., 2019), and their expression in microglia can be further induced by the administration of synthetic agonists of the receptor (Jin et al., 2016) and components of the bacterial cell wall (Laflamme et al., 2001). Since relatively large molecules, such as components of the bacterial cell wall, can diffuse across the capillary walls of circumventricular organs (Banks, 1999), a direct action of LTA on central targets to induce the observed responses cannot be ruled out.

In summary, current findings indicate that LTA, similarly to LPS and PGNs, is a bacterial cell wall component with significant sleep- and fever-inducing as well as anorectic potencies and thus likely contributes to the development of the acute phase response to Gram-positive bacterial infections as well as signaling from the intestinal microbiota.

Supplementary Material

1

Highlights.

  • Systemic injection of LTA induces sickness responses in mice.

  • LTA induces increased non-rapid eye movement sleep in mice.

  • LTA elicits biphasic body temperature responses.

  • LTA suppresses feeding and decreases energy expenditure.

  • Injection of LTA into the portal vein induces sleep in rats.

Acknowledgements

This work was supported by the National Institute of Health, National Heart, Lung, and Blood Institute, grant numbers R01HL122390 and R0151853.

Footnotes

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Competing Interests

The authors declare no competing interests.

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