Abstract
While diarrhea, the hallmark symptom of rotavirus infection, has been considered to occur only due to intrinsic intestinal effects, we show evidence for central control underlying the symptomology. With large-scale 3D volumetric tissue imaging a mouse model, we show that rotavirus infection disrupts the autonomic balance by downregulating the noradrenergic sympathetic nervous system in ileum, concomitant with increased intestinal transit. A most interesting observation was that nervous response from CNS occurs pre-symptomatically, an observation that bring new understanding to how virus give raise to clinical symptoms. In the CNS of infected animals, we found increased pS6 immunoreactivity in the area postrema and decreased phosphorylated STAT5-immunoreactive neurons in the bed nucleus of the stria terminalis, which are associated with autonomic control including stress response. Our observations bring new and important knowledge of how rotavirus virus infection induce gut-nerve-brain crosstalk giving raise to sickness symptoms.
Introduction
Rotavirus is the major cause of paediatric gastroenteritis, resulting in acute diarrhoea and vomiting. In 2019, rotavirus was estimated to have caused more than 150,000 dehydration-associated child deaths and the hospitalization of millions of children younger than 5 years old (Debellut et al., 2021). The disease mechanisms behind rotavirus-induced diarrhoea and vomiting are still not fully understood and no symptomatic treatment are available. While it is well established that diarrhoea and vomiting are the hallmarks of rotavirus infections, the extent of infection and the involvement of the central nervous system (CNS) in the illness have remained elusive.
Rotavirus non-structural protein 4 (NSP4) stimulates the enterochromaffin (EC) cells of the small intestine to release serotonin (Bialowas et al., 2016; Hagbom et al., 2011), which is sensed by neurons and leads to direct and indirect activation of both the enteric and central nervous systems (ENS and CNS, respectively). Consequently, it has been suggested that vomiting is elicited by gut–brain cross-talks involving the ascending and descending vagal pathways relayed through the vomiting centre in the brain (Crawford et al., 2017; Hagbom et al., 2011). Moreover, illness is not only associated with diarrhoea and vomiting, but also triggers other symptoms such as nausea, fever, anorexia and sickness symptoms, revealing a complex mechanism of disease and further indicating the participation of the CNS.
While intrinsic factors of rotavirus-induced diarrhoea have been investigated (Istrate, Hagbom, Vikström, Magnusson, & Svensson, 2014; S. Kordasti, Sjövall, Lundgren, & Svensson, 2004; Ove Lundgren et al., 2000) the role of CNS in rotavirus illness symptoms remain uncharted. Although the ENS drives intestinal motility independently (Wood, Alpers, & Andrews, 1999), it is de facto modulated centrally by the autonomic and endocrine nervous systems (Browning & Travagli, 2014). The inhibitory and excitatory effects of the autonomic nervous system on the small intestine through the sympathetic and the parasympathetic systems are well established (O. Lundgren, 2000; Sharkey & Pittman, 1996; Wood et al., 1999). Normal conditions are defined by the proper balance between these two opposing systems, and balance disruption by either up- or downregulation in either system can disrupt proper motility control and lead to either diarrhoea or constipation.
Recent developments in tissue clearing techniques, such as iDISCO (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) (Renier et al., 2014), together with volumetric imaging of large samples with light-sheet microscopy (Fadero et al., 2018) and computer-aided analysis of big data, have enabled 3D organ-wide investigation. Here, we used these techniques to study the extent of organ-wide rotavirus infection. Furthermore, we used the same techniques to investigate the effect of rotavirus infection on the sympathetic innervation and activity of the infected small intestine in ways previously not possible. We demonstrate that rotavirus infection of the small intestine pre-symptomatically disrupts the autonomic balance by downregulating the noradrenergic sympathetic nervous system in ileum, concomitant with increased intestinal transit.
Methods
Animals
Five to seven–day-old neonatal mice of both sexes and 8–10-week-old female adult BALB/c mice were used. All animal experiments had been approved by the local ethical committee in Linköping, Sweden (approval no.: N141/15 and 55-15).
Rotavirus infection
The mice were orally infected with 100 diarrhoea doses (100DD) of EDIM rotavirus in 10 µL 0.9% saline as described previously (Hagbom et al., 2011; Istrate et al., 2014). Non-infected control mice were mock-infected with 10 µL 0.9% saline. The groups were kept in separate litters, and whole litters were infected simultaneously and housed with their mother during the entire experimental period.
Tissue preparation
For iDISCO, segments of the small intestine were placed in 4% formaldehyde at room temperature for 24 h, and then transferred to phosphate-buffered saline (PBS) and stored at 4°C until tissue clearing was started.
For immunofluorescence, infant mice were sacrificed, and the brains were resected and fixed for 48 h in 4% formaldehyde solution (Histolab, Sweden). Adult animals were perfused, and their brains resected and fixed for 2 h in 4% formaldehyde. Subsequently, the brains were transferred to 15% sucrose in PBS for 7 days at 4°C, then rapidly frozen and stored at -80°C until sectioning was performed. The brains were cut into 14-µm thick sections on a cryostat (Microm; Walldorf, Germany), mounted on chrome alum gelatin-coated slides and stored at -20°C for subsequent immunofluorescence processing.
Immunofluorescence
The slides were thawed to room temperature, incubated in PBS, and processed for conventional indirect immunofluorescence or tyramide signal amplification (TSA; Perkin Elmer, Waltham, MA, USA) protocols as described previously (Foo, Hellysaz, & Broberger, 2014). All reactions were performed at room temperature unless otherwise stated. Primary antisera cocktails were prepared in staining buffer containing 0.03% Triton X-100 in 0.01 M PBS with 1% bovine serum albumin at least 24 h before use.
For conventional immunofluorescence, the sections were incubated in primary antisera at 4°C for 16 h, rinsed in PBS for 30 min, incubated for 1 h in secondary antisera cocktail, diluted in staining buffer, incubated in 4’,6-diamidino-2-phenylindole (DAPI, 1:10,000 in PBS), rinsed in PBS for 30 min, and mounted with 2.5% 1,4-diazabicyclo[2.2.2]octane (DABCO, Sigma, St. Louis, MO, USA) anti-fade agent in glycerol.
For TSA, antigen retrieval was initially performed with Tris-HCl (pH 8.0) at 95°C for 5 min. The sections were subsequently washed in Tris-sodium chloride-Tween buffer (TNT; 0.1 M Tris, 0.15 M NaCl, 0.05% Tween 20), incubated in primary antisera at 4°C for 42 h, washed in TNT, pre-incubated with blocking buffer (TNB) supplied in the TSA Plus kit (Perkin Elmer) for 30 min, incubated for 2 h in secondary antisera cocktail diluted in TNB, rinsed with TNT buffer, and incubated for 10 min with tyramide-conjugated fluorescein (1:500 in amplification diluent as supplied with the TSA Plus kit). The sections were then stained for DAPI and mounted as described above.
iDISCO
Approximately 5-mm long intestinal tissue samples from the duodenum and ileum were processed for iDISCO (Renier et al., 2014) according to the May 2016 updated protocol (available at http://www.idisco.info/), with some modifications. Briefly, the samples were dehydrated with gradient methanol, bleached in chilled fresh 5% H2O2 in methanol overnight at 4°C, and rehydrated and washed in PBS with 10 mg/L heparin and 0.2% Tween 20. Subsequently, the samples were permeabilized for 1 day, blocked for 1 day, incubated in primary antibody at 42°C for 7 days, washed, incubated in secondary antisera at 42°C for 7 days, and washed.
Following methanol gradient dehydration, the samples were incubated for 3 h in 66% dichloromethane in methanol, 2 × 15 min in 100% dichloromethane, and transferred to dibenzyl ether. All samples used for final analysis were processed in parallel and treated with the same buffers and solutions.
FITC-dextran transit
At 16 h p.i., the animals were orally administered 10 µL freshly prepared 4-kDa FITC-dextran (FD-4s, Sigma) dissolved in Milli Q water, at a dose of 0.25 mg/animal. After 15 min, the animals were sacrificed and the entire intestine, from the stomach to the rectum, was removed and visualized with ultraviolet light in a ChemiDoc XRS system (Bio-Rad, Sweden). The front part of the main accumulating FITC-dextran was defined from the photo, and the software program Adobe Illustrator was used to exactly measure the intestinal length and migration of the FITC-dextran probe. Intestinal transit was calculated on how far the FITC-dextran probe has passed as a percentage of the entire length of the intestine, from the pylorus to the rectum (Istrate et al., 2014).
Antisera
All antisera used in the different protocols are presented in Table 1. For detection, Alexa Fluor-conjugated secondary antisera (Life Technologies, Carlsbad, California, United States) for conventional detection, and horseradish peroxidase-conjugated secondary antisera (Dako, Glostrup, Denmark) for TSA were used. All secondary antisera were diluted to 1:500 for IHC and TSA and 1:250 for iDISCO.
Microscopy
Wide-field image montages were automatically generated in Neurolucida computer software (MBF Bioscience, Williston, VT, USA) by taking consecutive pictures with an automated stage controller mounted on a Zeiss Axio Imager M1 (Carl Zeiss, Oberkochen, Germany). Confocal micrographs were captured using a Zeiss LSM 800 Airyscan microscope with Zen Blue computer software. Light-sheet micrographs were acquired with a UltraMicroscope II (LaVision Biotec, Bielefeld, Germany) setup using ImSpector computer software. All intestinal tissues were randomized and sampled consecutively with the same acquisition settings. Post-acquisition brightness/contrast adjustments were performed uniformly on all light-sheet micrographs.
Micrograph analysis
The fluorescence micrographs were post-processed for rotation and brightness/contrast in Photoshop (Adobe, San Jose, California, United States) and analyzed in QuPath computer software (Bankhead et al., 2017).
We performed 3D confocal and light sheet analyses in Imaris. To maintain uniform tissues and measurements between animals, intestinal tissue integrity was visually confirmed in 3D, and damaged segments lacking an intact myenteric plexus (Figure S4) were excluded from analysis. Furthermore, the reconstructed 3D models were trimmed in silica, and only fragments with fully intact submucosa and myenteric plexuses were used. Therefore, mucosal immunofluorescence from, for example, enteric dopaminergic cells (Figure 2g) and intense fluorescence from incoming axon bundles (compare Figure 1j, k) were not included in the analysis and did not falsely skew the results.
Two different approaches were used to assess the level of infection in the small intestine. First, the number of infected cells per volume was estimated from the total number of infected surfaces and the total volume of the analyzed tissue. For a more accurate estimation, we set the infected surface creation pipeline to consider cell diameter and split touching objects (see parameters and threshold settings in Figure S5). In the second approach, the tissue infection ratio was estimated based on the total volume occupied by rotavirus relative to the total tissue volume. This approach for estimating the level of infection is independent of cell size and is therefore prone to methodological errors introduced by the splitting algorithm, from which the first approach might suffer from. Both non-infected and infected sample were analyzed with the same analysis pipeline.
Statistical analysis
Statistical analysis was performed with Prism (GraphPad, San Diego, California, United States) computer software. Statistical significance was set at p < 0.05 and was determined using the statistical tests described in the figures (*p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant). The statistics are reported as the mean ± standard error of the mean (SEM); n corresponds to the number of animals unless indicated otherwise.
Data availability
Data is available from the corresponding author upon request.
Results
Rotavirus infection is widespread throughout the entire length of the small intestine at 16 h post infection
Light-sheet micrograph stacks of the duodenum (Figure 1a, b, Supplementary Video 1) with 3D reconstruction (Figure 1c), and the ileum (Figure 1d, Supplementary Video 2-3) with 3D reconstruction (Figure 1e, Supplementary Video 4), immunostained for rotavirus structural viral protein 6 (VP6), indicated uniform and widespread infection throughout the entire length of the small intestine. VP6 immunoreactivity was not observed in non-infected animals (Figure 1f). Notably, the presence of VP6 was restricted to the mucosa, and no immunoreactivity was observed in the intestinal wall (Figure 1g).
Next, the extent of infection was investigated. To quantify the level of rotavirus infection, light-sheet micrographs were processed in Imaris (Bitplane, Zürich, Switzerland), and 3D surface models based on voxel fluorescence intensity were automatically created (Figure 1c, e, h–k). The tissue was modelled using autofluorescence. The level of infection was assessed with two different approaches, where number of infected cells (Figure 1l) or tissue infection ratio (Figure 1m) for non-infected (n = 5) and infected (n = 5) duodenum and ileum was estimated. Notably, both approaches generated similar results and yielded the same conclusions (compare Figure S1).
The estimated number of infected cells was 8504 ± 1615 in the duodenum and 17,458 ± 6058 in the ileum (Figure 1l). Likewise, the estimated tissue infection ratio was 5.8 ± 1.0‰ in the duodenum and 8.6 ± 3.5‰ in ileum (Figure 1m). Therefore, our data show no statistically significant differences in the level of rotavirus infection between the duodenum and the ileum.
Rotavirus infection induces downregulation of the noradrenergic sympathetic neurons in ileum
The main clinical outcome of gastrointestinal rotavirus infection is diarrhoea, which is caused by altered intestinal secretion and motility (Crawford et al., 2017). As both secretion and motility can be modulated by the autonomic nervous system (Browning & Travagli, 2014), we determined whether rotavirus infection would affect the sympathetic nervous afferents innervating the small intestine.
Within the intestinal wall, all tyrosine hydroxylase (TH), i.e., the rate-limiting enzyme of noradrenalin biosynthesis (Levitt, Spector, Sjoerdsma, & Udenfriend, 1965; Nagatsu, Levitt, & Udenfriend, 1964), reside within the axons of the sympathetic neurons, and extrinsic sympathetic denervation of the ileum abolishes all traces of TH (Mann & Bell, 1993). We measured the total TH immunoreactivity in 3–4 mm long pieces of the intestinal wall with volumetric 3D imaging (see Supplementary Video 1-7) to assess the extent of sympathetic modulation of the rotavirus-infected small intestine.
Surprisingly, in non-infected animals (Figure 2a–d), we observed a clear difference in TH immunoreactivity between the duodenum and ileum. This difference was not obvious in the infected animals (Figure 1e–h). The measured fluorescence intensity (Figure 1i) was 2155 ± 89 au/µm3 in the duodenum (n = 5) and was significantly higher (4601 ± 483 au/µm3) in the ileum (n = 5) of the non-infected animals. In the duodenum of the infected animals (n = 5), the fluorescence intensity was 2319 ± 128 au/µm3. Accordingly, no significant differences in TH immunoreactivity could be observed in the duodenal wall of the infected vs. non-infected animals.
The immunoreactivity in the ileum of the infected animals (n = 5), however, was 2850 ± 309 au/µm3. Hence, rotavirus infection led to a significant decrease of TH immunoreactivity in the ileum, but not in the duodenum (see Figure 2a–i). Relative to the average immunoreactivity levels of the uninfected animals, we observed this decrease, which ranged 15–50%, in all infected animals (Figure S2). These data show that rotavirus infection causes robust downregulation of the sympathetic nervous system innervating the ileum.
Downregulation of the sympathetic nervous system is concomitant with increased intestinal motility
As intestinal motility can be both increased and decreased by the autonomic nervous system (O. Lundgren, 2000; Sharkey & Pittman, 1996; Wood et al., 1999), we next investigated if the rotavirus-induced alteration of the sympathetic nervous system was concomitant with altered intestinal motility in vivo by utilizing the well-established fluorescein isothiocyanate (FITC)-dextran intestinal transit model (Hagbom et al., 2020; Istrate et al., 2014). Spectro photographs of resected intestines from animals 16 h post-infection (h p.i.), which had received oral FITC-dextran 15 min prior to termination, clearly showed increased FITC-dextran transit in infected vs. non-infected animals (Figure 2j).
The estimated mean relative transit distance (Figure 2k) was 19.1% in the non-infected animals (n = 3) and 89.2% in the infected animals (n = 3). Hence, the infected animals exhibited statistically significantly increased intestinal motility (p = 0.0001) concomitant with reduced sympathetic activity. Notably, the infected animals also showed signs of delayed gastric emptying, visualized by high amounts of remnant FITC-dextran in the stomach (Figure 2j).
Oral rotavirus infection modulates discrete regions of the brain
The cell bodies of postganglionic sympathetic neurons that innervate the small intestine wall are located in the prevertebral ganglia (Jänig, 1988; Mann & Bell, 1993; Trudrung, Furness, Pompolo, & Messenger, 1994) and receive innervation from the CNS (Berthoud & Powley, 1996; Trudrung et al., 1994). Therefore, we hypothesized that the increased intestinal motility associated with the downregulation of sympathetic nerves during rotavirus infection might be partly controlled by the CNS. To address this question, we investigated the brains of infected and non-infected adult mice using immunohistochemistry for markers of nerve activity.
First, ribosomal protein S6, whose phosphorylated state (pS6) is emblematic of active neurons and parallels expression of the immediate early gene cFos (Knight et al., 2012), was investigated throughout the entire brain. Although a full rostro-caudal survey of the brains of the infected and non-infected animals revealed few differences in the immunoreactivity pattern of pS6 (see e.g. Figure 3a, b), we observed a significant (p = 0.0233) increase in pS6 immunoreactivity in the area postrema of the infected animals at 48 h p.i. (Figure 3c–f). Within the area postrema, number of pS6-immunoreactive cells per section significantly increased (p = 0.0233) from 21.3 ± 9.2 in non-infected animals (n = 3) to 154.7 ± 47.7 in infected animals (n = 3).
As immediate early genes such as cFos, and likewise phosphorylation of S6, mark activation in short time frames (hours), while rotavirus infection lasts for days, we next investigated evidence for transcriptional modulations in select brain areas known to control endocrine and autonomic nervous systems. Members of the signal transducer and activator of transcription (STAT) protein family are primarily phosphorylated by the activation of Janus kinase-associated membrane receptors, and the activation of several hypothalamic pathways, particularly regarding feeding behaviour (Furigo, Ramos-Lobo, Frazão, & Donato, 2016), is associated with phosphorylated STAT5 (pSTAT5). We therefore investigated the number of cells expressing pSTAT5 in various brain areas of infected and non-infected animals.
We found pSTAT5 immunoreactive cell somata (Figure 4) in the bed nucleus of stria terminalis (BNST) of all non-infected animals (n = 4) with an average of 6.8 ± 1.7 cells per 14-µm section. Conversely, in the BNST of infected animals (n = 4), we observed a complete and robust absence (p = 0.0286) of pSTAT5 immunoreactive cells (Figure 4). No significant difference was observed in pSTAT5-expressing hypothalamic nuclei, including the arcuate, paraventricular, and periventricular nuclei, as well as the medial preoptic and the anteroventral periventricular areas (Figure S3). Notably, some of these regions showed a high degree of variability among the animals.
Rotavirus-induced modulation of the CNS is not caused by brain infection
While our data suggest that rotavirus-induced increase of intestinal motility is associated with nervous gut–brain communication, we cannot completely rule out the idea that the virus can reach the brain via the blood and thereby trigger the CNS. Despite little previous evidence for extramucosal spread of EDIM rotavirus (Uhnoo et al., 1990), and the lack of reports of viremia at 16 h p.i., we investigated this possibility with immunohistochemistry. Full rostro-caudal immunohistochemical investigation of fixed neonatal brains at 16 h p.i. (n = 5) and 48 h p.i. (n = 4) did not revealed any evidence of rotavirus VP6 antigen (Figure 5), nor perfused adult brains (n = 5) at 48 h p.i. (data not shown).
Discussion
Previous studies have investigated the mechanisms of rotavirus diarrhoea mainly by focusing on the intrinsic intestinal effects (Ball, Tian, Zeng, Morris, & Estes, 1996; Chang-Graham et al., 2019, 2020; Hagbom et al., 2020, 2011; Istrate et al., 2014; Shirin Kordasti et al., 2006; Ove Lundgren et al., 2000). Although these observations are compelling and have provided important mechanistic information of rotavirus diarrhoea, no information is available on how the gut communicate with CNS before the onset of diarrhoea nor how this communication initiates the illness. By using novel, large-scale volumetric 3D tissue clearing and imaging techniques, we studied the pathophysiology of rotavirus gastroenteritis. We show that rotavirus infection pre-symptomatically disrupts the autonomic balance by downregulating the noradrenergic sympathetic nervous system in ileum, concomitant with increased intestinal transit. In the CNS of infected animals, we found increased pS6 immunoreactivity in the area postrema, and decreased phosphorylated STAT5-immunoreactive neurons in the BNST, which has been associated with autonomic control including stress response. Altogether, these observations reveal that rotavirus signal to CNS before onset of diarrhoea a surprising observation that bring new understanding to how virus give raise to clinical symptoms.
Our 3D illustrations (compare Supplementary Video 1-7) identify a previously unappreciated early widespread infection. Furthermore, our data show that all segments of the small intestine are infected synchronously and demonstrate that the infection triggers neuronal circuitries through the CNS many hours before the development of diarrhoea. These observations are supported clinically, as the well-established early symptoms of rotavirus illness preceding diarrhoea are fever, and nausea/vomiting (stanfordchildrens.org), which are likely to be caused by early gut–brain cross-talks.
The endpoint neurotransmitter of the sympathetic nervous system is noradrenalin (Gershon, 1967; Mann & Bell, 1993). However, as measuring released noradrenalin in the small intestine of infected neonatal mice is challenging due to technical limitations, and released noradrenalin cannot be visualized easily, we chose to investigate the sympathetic system by targeting TH. Since TH is the rate-limiting enzyme of catecholamine biosynthesis (Daubner, Lauriano, Haycock, & Fitzpatrick, 1992; Levitt et al., 1965; Nagatsu et al., 1964), its expression level defines the maximum amount of available neurotransmitter in the cell. Moreover, within the small intestinal wall, TH can only be found in the sympathetic axons (Mann & Bell, 1993), and extrinsic sympathetic denervation of the ileum abolishes all TH immunoreactivity in the intestinal wall. Therefore, our measurements do not appear to be attributed to intrinsic intestinal nerves or any other systems than the sympathetic system. Furthermore, the cell somata of intestinal sympathetic axons receive input from the CNS and are located in the prevertebral ganglia in close proximity to the spinal cord (Jänig, 1988), far from the site of action and shielded from direct viral influence.
By targeting TH, our 3D reconstructions are directly and exclusively correlated to the noradrenergic sympathetic outputs to the small intestine, which we found were downregulated specifically in the ileum, but not in the duodenum, of the rotavirus-infected animals. Occurring within 16 h p.i., this downregulation ranged 15–50% compared to non-infected animals. How this downregulation translates to actual noradrenalin concentration in the cell, and how much noradrenalin is released at the axon terminals, cannot be elucidated from our data. Nonetheless, both clinical data and animal experiments (Istrate et al., 2014) show that the post-infection onset of diarrhoea can vary and occurs between 24 and 48 h.
Notably, we could not find any significant differences in TH immunoreactivity in the duodenum between the infected and non-infected animals, suggesting a tissue-specific rather than general downregulation. Indeed, intestinal segment-specific regulation was reported in 1857 by Eduard Pflüger, who noted that the activation of sympathetic innervation inhibited motility but constricted sphincters (Browning & Travagli, 2014; Jänig, 1988).
The inhibitory effect of the noradrenalin from the sympathetic nervous system on the small intestine is well established (Gershon, 1967; Kadowaki, Yoneda, & Takaki, 2003). Early histochemical investigations have determined that axons of the sympathetic postganglionic neurons are present in the submucosal and myenteric plexuses, and also extend to the villi in the mucosa (Schultzberg et al., 1980). Furthermore, functional and pharmacological studies show that noradrenalin mainly acts on α1-adrenergic receptors to excite myenteric neurons and thereby increase intestinal motility (Furuichi et al., 2001; Schemann, 1991). Further, enteric glia both regulate gastrointestinal motility (Gulbransen & Sharkey, 2012) and express adrenergic receptors (Nasser, Ho, & Sharkey, 2006). Indeed, rotavirus activates enteric glia cells via serotonin (Hagbom et al., 2020). Altogether, this suggests that noradrenalin simultaneously acts on enteric neurons and glia cells, parasympathetic axons and smooth muscle cells to coordinately inhibit intestinal motility. In accordance with our data, reducing the available noradrenalin will remove these inhibitions, i.e. remove the brake, and shift the balance towards increased intestinal motility, as illustrated in Figure 6.
We observed increased pS6 immunoreactivity in area postrema and decreased number of pSTAT5 immunoreactive cell somata in the BNST and no rotavirus antigen in CNS. Based on these observations we conclude that the sympathetic downregulation in the intestine resulted from gut–brain nervous signalling rather than direct infection and/or cytokine stimulation. This conclusion is supported by the fact that rotavirus is associated with limited inflammatory response in both human and mice (Greenberg & Estes, 2009; Hagbom et al., 2021; O. Lundgren, 2000; Ove Lundgren & Svensson, 2001; Morris & Estes, 2001), and that the EDIM murine rotavirus strain used in the present study has not been associated with extramucosal spread earlier than 72 h p.i. (Kraft, 1958) nor hepatic infiltration (Uhnoo et al., 1990).
Abnormal gastric motor function, as manifested by delayed emptying, has been reported in rotavirus-infected children (Bardhan, Salam, & Molla, 1992), and has been proposed to be associated with gastrointestinal hormones, neuronal pathways (including non-cholinergic and non-adrenergic), vagal neurons, and CNS control. The precise mechanisms, remain unresolved (Crawford et al., 2017). The FITC-dextran remnants in the stomach of the infected animals (Figure 2j, k) suggest the occurrence of delayed gastric emptying during the early stages of infection. Together with our other data showing downregulation of the sympathetic nervous system in the ileum, it strengthens the view of nerves participating in rotavirus illnesses. Altogether, our data suggest altered autonomic control as the underlying cause of other symptoms as well, and further investigation of the stomach, for example, is warranted.
Interestingly, we found a strong reduction of pSTAT5 immunoreactive cell somata in the BNST of infected animals (Figure 4). Spinal neuron projections directly to the BNST have been reported (Menétrey & de Pommery, 1991). Further, BNST sends projections to the dorsal motor nucleus of the vagus (Hopkins & Holstege, 1978), the nucleus ambiguous (Holstege, Meiners, & Tan, 1985), and the nucleus of the solitary tract (Hopkins & Holstege, 1978), i.e., the brain centra involved in controlling gastrointestinal motility (Browning & Travagli, 2014; Gillis, Quest, Pagani, & Norman, 2011). Furthermore, the BNST is involved in several autonomic regulations responding to non-fear-associated stress, and alters both blood pressure (Koikegami, Kimoto, & Kido, 1953) and heart rate. Our data showing modulation of the BNST in response to rotavirus infection strengthens the view of BNST involvement in intestinal motility and possibly symptoms of illness.
EC cells of the small intestine modulate neuronal signalling, including intestinal motility and secretion. Rotavirus, as well as NSP4, stimulates serotonin release from EC cells (Chang-Graham et al., 2019, 2020; Hagbom et al., 2011) and directly modulates ascending vagal pathways (Crawford et al., 2017; Hagbom et al., 2011). EC cells also receive direct sympathetic input, and noradrenalin excites EC cells to release serotonin (Bellono et al., 2017). Based on these reports and our collective observations, we propose EC cells as an intestinal sensor using vagal outputs and sympathetic intestinal sensory feedback to modulate gastrointestinal motility. This proposal provides both molecular and systemic explanations for how rotavirus infection can disrupt the autonomic balance. Furthermore, we suggest the nucleus of the solitary tract, the area postrema, and the BNST as central relay points of this feedback loop (Figure 7).
Conclusions
We showed and quantified the extent of rotavirus infection of the small intestine in 3D and identified centrally relayed downregulation of the sympathetic innervation of ileum, concomitant with increased intestinal transit and altered brain activity before onset of diarrhoea. We found increased pS6 immunoreactivity in area postrema and decreased phosphorylated STAT5-immunoreactive neurons in the BNST, which has been associated with autonomic control including stress response. Collectively, our data provide novel information how rotavirus causes illness and communicate with nerves and the brain.
Author contributions
A.H., L.S., and M.H. designed the studies; A.H., L.S., and M.H. conducted the experiments; A.H. and M.H. analysed the data; A.H. wrote the manuscript with input from all authors. All authors read and approved the final manuscript.
Competing interests
The authors declare no conflicts of interest.
Supplementary Videos
Acknowledgements
Financial support for this study was provided by the Swedish Research Council (Grants 2014-02827, 2017-01479, 2018-02862).