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. 2021 Aug 12;16(8):e0247738. doi: 10.1371/journal.pone.0247738

Rotavirus susceptibility of antibiotic-treated mice ascribed to diminished expression of interleukin-22

Daniel Schnepf 1,*, Pedro Hernandez 2, Tanel Mahlakõiv 1, Stefania Crotta 3, Meagan E Sullender 4, Stefan T Peterson 4, Annette Ohnemus 1, Camille Michiels 5, Ian Gentle 6,7, Laure Dumoutier 5, Celso A Reis 8,9, Andreas Diefenbach 10,11, Andreas Wack 3, Megan T Baldridge 4, Peter Staeheli 1,7,*
Editor: Michael Nevels12
PMCID: PMC8360596  PMID: 34383769

Abstract

The commensal microbiota regulates susceptibility to enteric pathogens by fine-tuning mucosal innate immune responses, but how susceptibility to enteric viruses is shaped by the microbiota remains incompletely understood. Past reports have indicated that commensal bacteria may either promote or repress rotavirus replication in the small intestine of mice. We now report that rotavirus replicated more efficiently in the intestines of germ-free and antibiotic-treated mice compared to animals with an unmodified microbiota. Antibiotic treatment also facilitated rotavirus replication in type I and type III interferon (IFN) receptor-deficient mice, revealing IFN-independent proviral effects. Expression of interleukin-22 (IL-22) was strongly diminished in the intestine of antibiotic-treated mice. Treatment with exogenous IL-22 blocked rotavirus replication in microbiota-depleted wild-type and Stat1-/- mice, demonstrating that the antiviral effect of IL-22 in animals with altered microbiome is not dependent on IFN signaling. In antibiotic-treated animals, IL-22-induced a specific set of genes including Fut2, encoding fucosyl-transferase 2 that participates in the biosynthesis of fucosylated glycans which can mediate rotavirus binding. Interestingly, IL-22 also blocked rotavirus replication in antibiotic-treated Fut2-/- mice. Furthermore, IL-22 inhibited rotavirus replication in antibiotic-treated mice lacking key molecules of the necroptosis or pyroptosis pathways of programmed cell death. Taken together, our results demonstrate that IL-22 determines rotavirus susceptibility of antibiotic-treated mice, yet the IL-22-induced effector molecules conferring rotavirus resistance remain elusive.

Introduction

The microbiota dramatically alters host susceptibility to multiple viral infections via diverse mechanisms [1–4]. Physical interaction with bacteria enhances viral infectivity for some enteric viruses. Binding to bacterial surface lipopolysaccharides (LPS) enhances the stability of poliovirus virions, resulting in decreased susceptibility of mice treated with an antibiotic cocktail compared to mice with undisturbed microbiota [5, 6]. Binding to bacteria further promotes poliovirus attachment to target cells, thereby facilitating simultaneous infection with multiple virions per cell and promoting viral fitness by enhancing the frequency of genetic recombination between different virus strains [7]. Similar mechanisms may account for the observed reduction of reovirus infectivity in antibiotic-treated mice [5]. Reduced vertical transmission of mouse mammary tumor virus through maternal milk in antibiotic-treated mice has also been observed, attributed to a lack of virus-bound bacterial LPS which stimulate the production of the immunosuppressive cytokine IL-10 [8, 9].

For other enteric viruses, both direct and indirect interactions with the microbiota may be at work. Human norovirus infection of B cells was enhanced in the presence of histo-blood group antigen-expressing enteric bacteria [10], and direct binding of human and murine norovirus to commensal bacteria appear to contribute to the proviral effects of the microbiota [11]. Other mechanisms of regulating virus susceptibility rely on modulation of host signaling by the microbiota. Antibiotic treatment decreased replication of persistent murine norovirus in the intestines of wild-type mice but not of mice with defective interferon-λ (IFN-λ) signaling [12], indicating that the bacterial microbiota limits the efficacy of IFN-λ-dependent innate immunity by an unknown mechanism. Interestingly, commensal bacteria were found to facilitate acute murine norovirus infection of distal gut regions while simultaneously inhibiting virus infection of the proximal small intestine [10, 13]. Virus inhibition in the proximal gut is secondary to priming of IFN-λ expression by bacteria-biotransformed bile acids [13].

The microbiota was further shown to restrict rather than to promote viral growth in several other experimental systems [4, 14, 15]. In these cases, commensal-derived signals provided tonic type I IFN-mediated immune stimulation that lowered the activation threshold of the innate immune system required for optimal antiviral immunity. Accordingly, mice treated with an antibiotic cocktail exhibited impaired epithelial, innate and adaptive antiviral responses resulting in delayed viral clearance after exposure to systemic LCMV or mucosal influenza virus infections [4, 15]. Mononuclear phagocytes of microbiome-depleted mice no longer expressed inflammatory response genes so that priming of natural killer cells and antiviral immunity to murine cytometagalovirus was severely compromised [14].

How the microbiota modulates rotavirus replication is incompletely understood, despite this being a pathogen of great clinical importance [16]. Antibiotic-mediated microbiome modulation enhanced fecal shedding of a rotavirus live vaccine in adult volunteers [17], and gram-negative probiotics protected against human rotavirus infection of gnotobiotic pigs [18]. However, antibiotic treatment has been reported to suppress rotavirus infection of gut epithelial cells, with an overall reduction in the incidence and duration of diarrhea in suckling mice [19]. Conversely, some mouse colonies have been found to be highly resistant to rotavirus infection, a resistance that can be transferred to susceptible mice via fecal microbial transplantation [20], indicating that a specific component of the microbiome can mediate antiviral effects. Various probiotics including Bifidobacterium sp. have been identified as protective against rotavirus infection [21, 22], and particular segmented filamentous bacteria (SFB) were shown to be sufficient to protect mice against rotavirus infection and associated diarrhea [20]. Interestingly, SFB-mediated protection was independent of known rotavirus-impeding factors such as IFN-λ [23–25], interleukin-18 (IL-18) [26] and IL-22 [26, 27], and instead rotavirus resistance correlated with accelerated epithelial cell turnover in this experimental system [20]. Bacterial flagellin alone is also sufficient to mediate potent antiviral effects against rotavirus, acting through TLR5 to promote IL-22- and IL-18-dependent antiviral activity [26]. Thus, the relative pro- and antiviral effects of the commensal microbiota on rotavirus infection remain unclear.

IFN-λ and IL-22 are both members of the IL-10 cytokine family. Their specific receptor chains (IFN-λR1 and IL-22Rα, respectively) both associate with IL-10Rβ to form functional heterodimeric receptor complexes, and the Ifnlr1 and Il22ra1 genes are close relatives located in adjacent positions in the mouse and human genomes [28, 29]. The IFN-λ and the IL-22 receptors are highly expressed in epithelial cells, and both cytokines cooperate for the induction of IFN-stimulated genes and the control of rotavirus infection in mice [27]. IL-22 dramatically alters epithelial cell expression of a variety of genes, ultimately stimulating proliferation and migration of intestinal epithelial cells toward villus tips, driving increased extrusion of the highly differentiated enterocytes in which rotavirus replicates [26, 30]. However, the antiviral activity of IL-22 is less well-understood compared with its well-described function as a key mediator of anti-bacterial responses [31, 32]. IL-22 expression is controlled by the commensal microbiota [33] and, in turn, IL-22 can modulate the composition of the microbiota [34].

To better understand the role of commensal bacteria in host defense against rotavirus, we performed infections of microbiota-depleted mice and we evaluated the antiviral activity of IL-22 under gnotobiotic conditions. Using antibiotic-treated and germ-free mice we found that the microbiota strongly repressed rotavirus replication in the intestinal tract. Our data indicate that rotavirus susceptibility of microbiota-depleted mice results from poor expression of IL-22 in the intestine. We further show that IL-22 confers rotavirus resistance in microbiota-depleted mice by a mechanism that is independent of IFN-λ or STAT1 signaling, fucosyl-transferase 2, or mediators of inducible cell death.

Material & methods

Mice

Conventional specific pathogen free (SPF) C57BL/6J mice of both sexes were purchased from Janvier Labs. Germ-free (GF) C57BL/6 mice were obtained from the Clean Mouse Facility of the University of Bern, Bern, Switzerland. GF mice were kept in autoclaved individually ventilated cages, and sterile water and food was provided. GF mice were handled exclusively in a biosafety cabinet. C57BL/6J mice lacking functional Fut2, Ripk3 or Casp1/11 genes [35–37] as well as mutant B6.A2G-Mx1-Ifnlr1-/- [38], B6.A2G-Mx1-Ifnlr1-/-Il22-/- [27], B6.A2G-Mx1-Stat1-/- [39] and corresponding B6.A2G-Mx1 wild-type mice [38] were bred and housed in the animal facilities of the University Medical Center Freiburg. Other C57BL/6J mice originally purchased from Jackson Laboratories (stock 000664; Jackson Laboratories, Bar Harbor, ME) were bred and housed in animal facilities of Washington University in Saint Louis under specific-pathogen-free (including murine norovirus-free) conditions. Generation of Ifnlr1−/− mice was previously described [40]; briefly, these mice were established by interbreeding Ifnlr1tm1a(EUCOMM)Wtsi mice and deleter-Cre mice, followed by backcrossing by speed congenics onto a C57BL/6J background. Ifnar1-/- (B6.129.Ifnar1tm1) were crossed with these Ifnlr1−/− mice to generate Ifnar1-/-Ifnlr1−/− double knockout mice. Sentinel animals of our housing facilities were routinely checked for unwanted pathogens by serological and molecular technologies. These observations yielded no evidence for spontaneous infections of our mice with rotavirus.

Ethics statement

All experiments with mice were carried out in accordance with the guidelines of the Federation for Laboratory Animal Science Associations and the national animal welfare body. Experiments were in compliance with the German animal protection law and were approved by the animal welfare committee of the Regierungspräsidium Freiburg (permit G-16/98). All experiments at Washington University were conducted according to regulations stipulated by the Washington University Institutional Animal Care and Use Committee and to animal protocol 20190126, approved by the Washington University Animal Studies Committee.

Antibiotic treatment of mice

To deplete the commensal microbiota, four- to six-week old mice received drinking water ad libitum containing 1 mg/ml Cefoxitin (Santa Cruz Biotechnology, Inc), 1 mg/ml gentamycin sulfate (Sigma-Aldrich), 1 mg/ml metronidazole (Sigma-Aldrich) and 1 mg/ml vancomycin (HEXAL®) for 4 weeks. For experiments at Washington University, six-week old mice received drinking water with 1 mg/ml ampicillin, 1 mg/ml metronidazole, 1 mg/ml neomycin, 0.5 mg/ml vancomycin (Sigma, St. Louis, MO) in 20 mg/ml grape Kool-Aid (Kraft Foods, Northfield, IL), or with Kool-Aid alone, ad libitum for 9–14 days. In most experiments we confirmed the absence of live bacteria in fecal samples by plating the material on suitable agar plates.

Virus stocks and infections

To produce the rotavirus used in Freiburg, six day old suckling BALB/c mice were infected orally with 680 infectious dose 50 (ID50) (5 μl of a 1:250 dilution) from a stock containing 3.4x107 ID50/ml of murine rotavirus strain EDIM [23]. Four days post-infection, mice were sacrificed and colon samples with content were homogenized twice for 18 sec at 6 m/s using a FastPrep®-12 homogeniser (MP Biomedicals). The homogenate was cleared by centrifugation at 5,000 rpm for 15 min. Supernatants were pooled and filtered using a 10 ml syringe and 4.5 μm sterile filters. The filtrate was aliquoted and stored at -80°C. Viral protein VP6 was quantified by RIDASCREEN® ELISA in comparison to the parental stock. The infectious dose 50 (ID50) was determined by orally infecting groups of six C57BL/6 suckling mice with 5 μl samples of 10-fold serial dilutions, ranging from 10−3 to 10−7. Mice were sacrificed 24 h post-infection and intestines were analyzed for the presence of RNA encoding for VP6 by RT-qPCR. Adult mice (8–10 week old) were orally infected with 2.4x104 ID50 in 100 μl by oral gavage using a gavage needle (19G).

To produce the rotavirus used in Saint Louis, four day old suckling CD-1 mice (Charles River) were infected orally with 400 diarrhea dose 50 (5 μl of a 1:10 dilution in PBS + Evan’s blue) from a stock of murine rotavirus strain EC-RV received from Estes Lab at Baylor College of Medicine. Three days post-infection, mice were sacrificed and the entire small intestine and colon with contents were harvested and pooled for weighing. A 20% homogenate was prepared in homogenization media of DMEM (Gibco 11995–040) supplemented with 1% penicillin/streptomycin (Gibco 15140–122) by three rounds of homogenization on ice (homogenize for 1 min, then allow to settle for 30 sec). Homogenate was pooled on ice, aliquoted, and then stored at -80˚C until use. The shedding dose 50 (SD50) of the stock was determined by orally infecting groups of five adult mice (6–8 weeks old), of both BALB/c and C57BL/6 genotypes, with 100 μl of 10-fold serial dilutions, ranging from 10−5 to 10−9 for BALB/c and 100 to 10−5 for C57BL/6, and analyzing fecal pellets for RV genome copies by RT-qPCR. Adult mice (8–10 weeks old) were orally infected with 104 SD50 in 100 μl by oral gavage, preceded by 100 μl of sterile 1.33% sodium bicarbonate solution.

Viral protein quantification in feces by RIDASCREEN® Rotavirus ELISA

Fecal samples were homogenized according to weight with RIDASCREEN® sample dilution buffer 1 and homogenized twice for 11 sec with 6 m/s using a FastPrep®-12 homogenizer. Homogenates were cleared by centrifugation [5,000 rpm, 5 min, 4°C] and 100 μl of fecal homogenate supernatants were used for RIDASCREEN® Rotavirus ELISA. ELISA was performed according to the manufacturer’s instructions. Optical density was measured at 450 nm wave length by using a Tecan Infinite 200 plate reader.

RNA isolation and RT-qPCR

RNA was isolated using the Direct-zol™ RNA MiniPrep Kit (Zymo Research). Two-three cm pieces of ilea were homogenized in 1 ml TriFast™ twice for 18 sec with 6 m/s using a FastPrep®-12 homogenizer (MP Biomedicals). Homogenates were centrifuged at 12,000 g for 5 min and supernatant were diluted 1:10 in TriFast™. RNA was then isolated following the manufacturer’s instructions. LunaScript™ RT SuperMix Kit (New England Biolabs) was used to generate cDNA from 850 ng of total RNA following the manufacturer’s instructions. Resulting cDNA served as template for the amplification of transcripts from Ubc (QT00245189, QuantiTect Primer Assay, Qiagen), Hprt (mm00446968_m1, Applied Biosystems), Il22 (forward: 5’- CATGCAGGAGGTGGTACCTT -3’; reverse: 5’- CAGACGCAAGCATTT CTCAG -3’), Reg3b (forward: 5’- GCTGGAAGTTGGACACCTCAA -3’; reverse: 5’- GACATAGGGCAACTTCACCTCACA -3’), Reg3g (forward: 5’- TTCCTGTCCTCCATGATCAAAA -3’; reverse: 5’- CATCCACCTCTGTTGGGTTCA -3’), Saa1 (forward: 5’- AAATCAGTGATGGAAGAGAGGC -3’; reverse: 5’- CAGCACAACCTACTGAGCTA -3’), Fut2 (forward: 5’- ACCTCCAGCAACGAATAGTGA -3’; reverse: 5’- GCCGATGGAATTGATCGTGAA -3’), Rps29 (forward 5′-GCAAATACGGGCTGAACATG-3′; reverse 5′-GTCCAACTTAATGAAGCCTATGTC-3′) by real-time PCR using TaqMan Gene Expression Assays (Applied Biosystems), Universal PCR Master Mix (Applied Biosystems) and the QuantStudio 5 Real-Time PCR system (Applied Biosystems by Thermo Fisher Scientific). Increase in transcript levels were determined by the 2-ΔCt method relative to expression of the indicated housekeeping genes.

Intestinal epithelial cell enrichment

Small intestines were collected and cut into 3–6 cm long pieces, mechanically cleaned, rinsed with PBS using a gavage needle (19G) and inverted on customized inoculating loops. Samples were incubated in 30 mM EDTA for 10 min at 37°C. Intestinal epithelia cells were detached from organ pieces by centripetal force using a customary electric drill [approximately 1,000 rpm]. Samples were centrifuged with 2–3 short impulses in 50 ml PBS with 5% FCS. After 1 h of sedimentation, the lower half of the suspension was isolated, cells were pelleted [1,300 rpm, 10 min, 4°C], lyzed in TriFast™ and total RNA isolated using the Direct-zol™ RNA MiniPrep Kit (Zymo Research).

Transcriptome analysis

RNA for transcriptome analysis was isolated using the RiboPure™ Kit (Invitrogen) according to the manufacturer’s instructions. In brief, 2–3 cm pieces of ilea were homogenized in 1 ml TriFast™ twice for 18 sec with 6 m/s using a FastPrep®-12 homogenizer (MP Biomedicals). Homogenates were centrifuged with 12,000 g for 10 min at 4°C, 600 μl of supernatant mixed with 500 μl TriFast™, centrifuged again with 12,000 g for 10 min at 4°C and 1 ml of supernatant was mixed with 100 μl of 1-bromo-3-chloropropane and incubated for 5 min at room temperature. Phase separation was achieved by centrifugation with 12,000 g for 10 min at 4°C. 400 μl of the aqueous phase was then mixed with 200 μl of ethanol and passed through the filter cartridge by centrifugation (12,000 g for 1 min), the filter cartridge was then washed twice with 500 μl wash solution and RNA was finally eluted with 100 μl elution buffer.

MicroArray

Total RNA harvested from intestinal epithelia cells was hybridized using Illumina Mouse WG-6_V2_0_R0_11278593_A arrays. The raw intensity values for each entity were preprocessed by RMA normalization against the median intensity in mock-treated samples. Using GeneSpring 11.5, all transcripts were then filtered based on flags (present or marginal, in at least 2 out of 10 samples). Moderate t-test (IL22-treated versus mock-treated) was performed to identify genes differentially expressed relative to controls (≥5-fold change; p<0.01, Benjamini-Hochberg multiple test correction). Raw microarray data can be accessed at GEO under accession number GSE166400.

Statistical analysis

For statistical analyses the GraphPad Prism 8 software (GaphPad Software, USA) was used. Testing for statistical significance was performed on log-transformed values by using ordinary one-way ANOVA with Tukey’s multiple comparisons or the unpaired Student’s t-test. Asterisks indicate p-values: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001.

Results

Germ-free and antibiotic-treated mice are highly susceptible to oral infection with rotavirus

To investigate the effect of the microbiota on rotavirus susceptibility, we monitored the levels of rotavirus antigen in the feces of three experimentally infected groups of adult C57BL/6 mice, namely conventionally-housed specific pathogen-free (SPF) animals, SPF animals treated with an antibiotic cocktail (Abx), and germ-free (GF) animals. During the 6-day observation period following oral infection with rotavirus, viral antigen was nearly undetectable in fecal samples of SPF mice by ELISA (Fig 1). In contrast, fecal samples from the majority of infected Abx and GF mice contained high levels of rotavirus antigen (Fig 1), indicating that the microbiota has an inhibitory effect on rotavirus replication.

Fig 1. Germ-free and antibiotic-treated mice are highly susceptible to oral infection with rotavirus.

Fig 1

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Conventional specific pathogen free (SPF, n = 10), antibiotic-treated (Abx, n = 9) and germ-free (GF, n = 8) C57BL/6 mice were infected orally with 2.4x104 ID50 of murine rotavirus strain EDIM. Fecal pellets of individual mice were collected daily and levels of viral antigen were determined by ELISA. Four animals of the Abx and the GF groups were sacrificed on day 3 post-infection for analysis of tissue in the context of a different project.

Since an earlier study indicated that the microbiota promotes rather than inhibits rotavirus replication [19], we sought to confirm this finding in an unrelated colony of C57BL/6 wild-type mice at a second independent institution, using an independently prepared rotavirus stock. By measuring viral RNA in fecal samples on day 5 post-infection by RT-qPCR analysis we again found that wild-type (WT) mice with undisturbed microbiota shed only low amounts of virus whereas virus shedding of antibiotic-treated WT mice was strongly enhanced (Fig 2). Prior work indicated that the type I and the type III IFN systems inhibit the replication of rotavirus in the mouse intestine [24, 25]. Therefore, we assessed the possibility that microbiota-mediated rotavirus resistance might depend on IFN signaling. In agreement with earlier studies [24, 25], we observed that animals lacking functional receptors for type I IFN (Ifnar1-/-), for type III IFN (Ifnlr1-/-) or for both IFN types (DKO) were more susceptible to rotavirus infection compared with WT mice (Fig 2). Importantly, however, treating these mutant mice with antibiotics also enhanced rotavirus levels on average at least 50-fold (Fig 2), demonstrating that enhanced intestinal replication of rotavirus in mice with reduced microbiota is not only due to impaired activation of type I IFN and/or type III IFN pathways by commensal bacteria. Rotavirus shedding by untreated mutant mice was surprisingly heterogeneous (Fig 2), raising the possibility that the microbiota composition of individual animals could matter. Since our animal facilities are not free of segmented filamentous bacteria (SFB) which are known to confer rotavirus resistance [20], we speculate that the degree of SFB colonization in individual animals could influence intrinsic rotavirus susceptibility.

Fig 2. Rotavirus susceptibility of wild-type and IFN receptor-deficient C57BL/6 mice is greatly enhanced if the microbiome is depleted with antibiotics.

Fig 2

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WT (n = 7), Ifnar1-/- (n = 6), Ifnlr1-/- (n = 8) and Ifnar1-/- Ifnlr1-/- (DKO, n = 10) mice were left untreated or treated with antibiotics (Abx) before oral infection with 104 SD50 of the EC strain of murine rotavirus. On day 5 post-infection, fecal pellets were collected and viral RNA levels were determined by RT-qPCR. Symbols represent individual mice, and bars represent means ± SEM. Line represents limit of detection of the RT-qPCR assay. Statistical analysis: Ordinary one-way ANOVA with Tukey’s multiple comparisons; ****p<0.0001.

IL-22 confers rotavirus resistance in antibiotic-treated mice via a mechanism that is not dependent on IFN-λ or STAT1 signaling

To identify IFN-independent antiviral factors which are present in the intestine of SPF mice but absent or strongly diminished in GF mice, we performed RT-qPCR analysis of cytokines previously linked to rotavirus resistance [24–26]. We found that expression of Il22 was on average about 200 fold lower in ileum samples from uninfected GF (Fig 3A) or Abx mice (Fig 3B) than in samples from uninfected SPF mice. Furthermore, compared with SPF mice, expression of the IL-22-induced Reg3b and Reg3g genes was on average 10–20 fold reduced in the ileum of GF (Fig 3A) and Abx mice (Fig 3B), demonstrating that the high rotavirus susceptibility of animals with diminished microbiota correlates with diminished baseline expression of IL-22.

Fig 3. IL-22 confers short-lived rotavirus resistance in antibiotic-treated C57BL76 mice via a mechanism that is not dependent on IFN signaling.

Fig 3

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(A, B) RT-qPCR analysis of tonic expression of Il22 and IL-22-regulated genes Reg3b and Reg3g in enriched intestinal epithelial cell fractions of (A) germ-free (GF) or in ileum samples of (B) antibiotic-treated (Abx) mice compared to animals with undisturbed microbiota (SPF). (C, D) Antibiotic-treated WT mice were subjected to brief (n = 5) or extended (n = 10) IL-22 treatment regimens (1 μg per injection, time points indicated by arrows) before infection with 2.4x104 ID50 of murine rotavirus strain EDIM by oral gavage. Control animals (C, n = 5; D, n = 9) were treated with saline. Fecal pellets were collected at the indicated time points and viral antigen in the samples was quantified by ELISA. (E, F) Same experimental setup as in panel D, except that (E) Ifnlr1-/- (saline n = 9; IL-22 n = 10) or (F) Stat1-/- (saline n = 7; IL-22 n = 9) mice were used. Symbols represent individual mice, and bars in (A) and (B) represent means ± SEM. Statistical analysis: Unpaired t-test; ****p<0.0001.

We next tested whether IL-22 can confer rotavirus resistance by treating Abx mice with recombinant IL-22 one day before and on days 0, 1, 2 and 4 after oral infection with rotavirus, then monitoring fecal virus shedding by ELISA for the next 12 days. All saline-treated control mice acutely shed high amounts of viral antigen, whereas all IL-22-treated animals exhibited low levels of fecal viral antigen during the first 5 days post-infection (Fig 3C). However, four of the five IL-22-treated mice shed virus with delayed kinetics, indicating that the protective effect of exogenous IL-22 was short-lived. Therefore, in a second experiment, the IL-22 treatment period was extended and daily injections of IL-22 were continued until day 8 post-infection. Under these more stringent conditions, IL-22-mediated suppression of virus shedding was very effective (Fig 3D).

As synergistic activity between IL-22 and IFN-λ to control rotavirus has previously been reported [27], we assessed whether the rotavirus-inhibitory effect of IL-22 was direct or mediated by IFNs. We repeated the IL-22 treatment study using mice in which either IFN-λ (Ifnlr1-/-) or all IFN subtypes (Stat1-/-) are no longer functional, and found that daily injections of IL-22 were still highly effective at suppressing rotavirus shedding in antibiotic-treated Ifnlr1-/- (Fig 3E) and Stat1-/- (Fig 3F) mice. Thus, IL-22 confers virus resistance in microbiota-depleted mice by a mechanism that is not dependent on IFN-λ or STAT1 signaling.

Microbes modulate the IL-22 response of the intestinal epithelium

To identify IL-22-induced effector molecules which confer rotavirus resistance in microbiota-depleted mice, we compared gene expression profiles in the ileum of antibiotic-treated Ifnlr1-/- mice which received either saline or IL-22. We identified 22 genes that showed at least 5-fold increased expression and 3 genes with at least 5-fold decreased expression after IL-22 treatment compared with mock-treated mice (Fig 4A). Enhanced expression of several genes, including Saa1, Fut2 and Reg3g, in tissue of IL-22-treated animals was confirmed by RT-qPCR (Fig 4B).

Fig 4. IL-22 regulates a distinct set of genes in antibiotic-treated Ifnlr1-/- mice, but the Fut2 gene plays no decisive role in rotavirus resistance.

Fig 4

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(A) Four h prior to preparation of ileum samples for transcriptome analysis, antibiotic-treated Ifnlr1-/- mice (n = 5 per group) were treated with either buffer or 1 μg of IL-22. Genes up- or downregulated at least 5-fold on average in IL-22-treated mice are listed. For all listed genes, variation between samples from the different mice was small (p value <0.01 for every data point) and was part of the original filter used to acquire the data. (B) Verification of IL-22-mediated induction of Saa1, Fut2 and Reg3g by RT-qPCR. Symbols represent individual mice, and bars represent means ± SEM. (C) Fut2-/- mice with undisturbed microbiota (n = 6) were orally infected with 2.4x104 ID50 of murine rotavirus strain EDIM and viral antigen levels in fecal samples were analyzed by ELISA. WT (n = 6) and Ifnlr1-/- Il22-/- (n = 6) mice served as negative and positive controls, respectively. (D) Fut2-/- mice with depleted microbiota (n = 6 per group) were treated daily with saline or IL-22 (extended treatment as described in Fig 3) prior to infection with 2.4x104 ID50 of murine rotavirus strain EDIM. Antibiotic-treated WT mice (n = 4–5 per group) served as controls. Symbols in (C) and (D) represent means ± SEM.

The Fut2 gene was of particular interest because it encodes the enzyme fucosyl-transferase 2 (FUT2) that participates in the biosynthesis of histo-blood group antigens which can be recognized by rotavirus [41]. To investigate whether IL-22 inhibits rotavirus growth in the intestine of antibiotic-treated mice by regulating FUT2 expression, we first determined whether Fut2-/- mice with normal gut flora were more susceptible to oral infection with rotavirus than standard wild-type mice, using highly susceptible Ifnlr1-/-Il22-/- mice as a positive control (Fig 4C). Fut2-/- mice exhibited no defects in rotavirus control. Next, we tested whether IL-22 might no longer be able to inhibit rotavirus replication in antibiotic-treated Fut2-/- mice, but found IL-22 treatment to be highly effective in these animals (Fig 4D). We thus concluded that IL-22-mediated upregulation of Fut2 in microbiota-depleted mice does not play a decisive role in rotavirus resistance.

Since SFB increase the turnover of intestinal epithelial cells that results in rotavirus resistance [20], microbiota-mediated acceleration of infected epithelial cell death might be responsible for the intrinsically high rotavirus resistance of mice with intact microbiota. Therefore, we first evaluated the possibility that the antiviral activity of IL-22 in antibiotic-treated mice results from accelerated death of intestinal epithelial cells by necroptosis [42]. However, we found that IL-22 was effective in inhibiting rotavirus growth in antibiotic-treated Ripk3-/- mice (Fig 5A), in which cell death by necroptosis cannot occur [43]. This finding excluded the possibility that IL-22 induces premature death of rotavirus-infected cells by necroptosis in antibiotic-treated mice. An alternative form of induced cell death is pyroptosis, in which caspase-1 plays a key role [44]. To evaluate the possibility that IL-22 acts by inducing pyroptosis in infected cells, possibly via regulation of IL-18 expression [26, 45], we tested IL-22-induced antiviral effects in antibiotic-treated Casp1/11-/- mice. We found that IL-22 remained active in these mice (Fig 5B), precluding the possibility that IL-22 inhibits rotavirus by triggering pyroptotic cell death in antibiotic-treated mice.

Fig 5. No role for RIPK3-induced necroptosis or caspase 1-mediated death in IL-22-mediated clearance of rotavirus from antibiotic-treated mice.

Fig 5

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Antibiotic-treated Ripk3-/- (A) and Casp1/11-/- (B) mice were treated with saline or IL-22 and infected with rotavirus strain EDIM as in the experiment described in Fig 3D. Viral antigen levels in fecal samples were analyzed by ELISA. Numbers of animals in each group are indicated. Symbols represent means ± SEM.

Discussion

Our work revealed that the microbiota protects against rotavirus infection and inhibits virus replication in the small intestine. These results are distinct from conclusions of an earlier study [19], which indicated that antibiotic treatment renders mice resistant to rotavirus infection. Of interest, our observation of a protective effect of the microbiota against rotavirus recapitulates observations in neonatal gnotobiotic piglets, wherein the microbiota has been shown to protect against diarrhea and viral shedding [46]. Our use of mice from two separate animal facilities located on different continents, as well as two independently-generated rotavirus stocks of EDIM and EC, makes it unlikely that housing conditions or unrecognized mutations in the virus genome are responsible for this discrepancy. We speculate that delayed passage of content through the intestines of antibiotic-treated mice [5, 12, 47] may contribute to differences in virus excretion between microbiota-replete and -depleted mice, which ultimately resulted in differential interpretations regarding the influence of the microbiota on virus resistance. Of note, our results are highly consistent with recent work that attributed the presence of particular SFB with rotavirus resistance of mice [20].

Our study revealed that the rotavirus susceptibility phenotype of antibiotic-treated mice cannot be explained by the involvement of known mediators of rotavirus resistance such as IFN-λ or type I IFN. We confirmed earlier findings [24, 25] that mice with defective IFN receptors are intrinsically more susceptible to rotavirus infection than wild-type mice. Importantly, however, when such mutant mice were treated with antibiotics, their susceptibility to infection with rotavirus still increased, indicating that poorly defined additional factors also contribute to rotavirus resistance. Our analysis suggests that IL-22 plays an important role in this phenomenon. First, we observed that poor expression of IL-22 and IL-22-regulated genes in the ileum correlates with rotavirus susceptibility of antibiotic-treated mice. This observation is consistent with previous work demonstrating decreased intestinal IL-22 expression in the intestine in germ-free or antibiotics-treated mice that can be rescued with short-chain fatty acid administration [48], which drives IL-22 production by CD4+ T cells and innate lymphoid cells [49, 50].

Second, we found that application of IL-22 readily restored rotavirus resistance of antibiotic-treated mice. Interestingly, unlike in mice with undisturbed microbiota in which the antiviral activity of IL-22 was strongly dependent on IFN-λ [27], the anti-rotavirus effect of IL-22 in antibiotic-treated mice was not dependent on IFN-λ or transcription factor STAT1. Thus, IL-22 can negatively affect rotavirus replication by at least two mechanisms, of which only one is dependent on IFN. The IFN-independent mechanism plays a dominant role in antibiotic-treated mice, whereas the IFN-λ-dependent mechanism dominates in mice with undisturbed microbiome.

We currently do not understand how IL-22 inhibits the replication of rotavirus in antibiotic-treated mice. Our transcriptome analysis showed that Fut2 is strongly induced by IL-22 under such conditions. This finding is in good agreement with earlier work indicating that antibiotic-mediated down-regulation of Fut2 alters the glycosylation pattern of intestinal epithelial cells [51, 52]. Such changes in protein glycosylation resulted in enhanced susceptibility of mice to infection with several pathogenic bacteria, including Citrobacter rodentium [53], Helicobacter pylori [36, 52] and Salmonella typhimurium [51]. However, our experiments with Fut2-deficient mice indicate that antiviral defense is not reduced in these animals and that fucosyltransferase-2 is not the elusive IL-22-induced mediator of rotavirus resistance.

IL-22 is known to act through the STAT3 transcription factor in intestinal epithelial cells to drive expression of numerous antimicrobial genes, though it remains unclear which are particularly critical for antiviral effects against rotavirus [26, 54]. Since the intestinal epithelium consists of short-lived cells, we envisaged the possibility that IL-22 might inhibit rotavirus replication by inducing premature death of infected cells. Yet, we found that IL-22 effectively blocked rotavirus replication in antibiotic-treated mice lacking key factors for necroptosis or pyroptosis, rendering such a mechanism unlikely. However, since these programmed cell death pathways are highly interconnected, we cannot exclude the possibility of compensatory effects [55]. Available data suggest that SFB prevents and cures rotavirus infection by enhancing the proliferation of infected epithelial cells in the terminal ileum, promoting their shedding from the tissue [20]. We speculate that IL-22 may provide similar growth-promoting stimuli to epithelial cells in antibiotic-treated mice and that enhanced cell proliferation might explain the antiviral effect of IL-22 that we observed. It has also been suggested that IL-22 may induce extrusion of intestinal epithelial cells [30], which remains a possibility in the context of microbiota-mediated regulation of rotavirus infection. Recently, reversal of viral appropriation of autophagic flux has been suggested as an antiviral mechanism for IL-22 in control of respiratory syncytial virus, a possibility that remains open for its regulation of rotavirus [56]. Our study reveals a clear role for the microbiota in maintenance of a homeostatic antiviral state, driven by IL-22, against rotavirus infection, and future studies delineating the specific pathways required for this activity may help reveal additional therapeutic approaches against this pathogen.

Acknowledgments

We thank the Clean Mouse Facility (CMF), University of Bern, Bern, Switzerland, for providing GF mice, Ana Magalhães, University of Porto, Portugal for providing Fut2-deficient mice, and Otto Haller for helpful comments on the manuscript.

Data Availability

All relevant data are within the manuscript. Raw microarray data can be accessed at GEO under accession number GSE166400.

Funding Statement

This work was supported by a grant from the Deutsche Forschungsgemeinschaft to P.S., as well as grants from the NIH (R01 AI141716 and R01 OD024917), the Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital Interdisciplinary Research Initiative (MI-II-2019-790), and The Mathers Foundation to M.T.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Rotavirus susceptibility of antibiotic-treated mice ascribed to diminished expression of interleukin-22

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Reviewer #1: This work presents data that indicates that C57BL/6 mice are highly, if not completely resistant to infection by WT murine rotavirus and that this resistance can be alleviated by ablation of microbiota by either antibiotics or germfree approaches. They then go on to show that such treatments reduce IL-22 and that exogenous IL-22 protects against rotavirus, and that all of these findings are independent of type 1 IFN. There is not really all that much new here but, nonetheless, the work cold revised to make a reasonable contribution to the literature but major revisions in how they present and interpret their findings are needed.

Infection of C57BL/6 mice with murine rotavirus EC strain is a very widely used model of rotavirus infection, used for many years. Accordingly, when such mice are directly purchased from most commercial vendors and bred at many institutions, they will display clear shedding of RV antigens and genomes. Here, the authors report that such mice bred at 2 different institutions were not infectable by RV unless first exposed to antibiotics. I don’t doubt the observation they made but how they present it and interpret it is highly problematic. Firstly, they don’t actually acknowledge that the untreated mice were not, in fact, infected with RV at all and thus that they their positive control did not work as it has in the literature for over 20 years. Consequently, they have not really interpreted and properly contextualized the antibiotic data. The proper interpretation is that, for some reason, their mice are highly RV resistant, relative to mice long used in the literature which can be readily infected by this RV strain. They mention “timing of virus excretion” as a possible explanation but I have no idea what they mean by this. Countless studies have shown adult C57BL/6 shed mice within a few days of inoculation and their assay period certainly covers this time. It is certainly appropriate to cite reference 19 to speculate that the mice studied here might also be RV-resistant to their specific microbiota composition but again they need to start by acknowledging that their mice contradict the well-established phenotype. Moreover, this makes it very difficult to compare their findings with published papers that started with the typical readily-infectable mice and then examined impacts of antibiotics and germfree conditions.

Reviewer #2: Dear Authors,

It is a very important scientific work and would definitely contribute in the advancement of the current knowledge about the influence of microbiome and its corelation with antibiotics on viral infections.

I would strongly suggest you the improvement of all figures. I am unable to clearly read what is written along x axis and y axis. Kindly improve it. It is in bold letters, it will be more convenient for the readers if it appears in normal letters.

Moreover at line number 142 (Antibiotic treatment of Mice), kindly mention the mode of adminstration of antibiotics, as I could not get it completely. you are requested to make it clear weather the antibiotics were adminsitered orally or intramuscular or intravenous.

The rest is fine to my understanding.

Reviewer #3: The manuscript titled “Rotavirus susceptibility of antibiotic-treated mice ascribed to diminished expression of IL-22” by Schnepf et al describes the finding that rotavirus, a common intestinal viral infection, replicates more efficiently in the intestines of germ free and antibiotic treated mice compared to wild type mice. The authors ascribe this finding to the fact that the microbiome is altered in the germ free and antibiotic treated mice. The effect was associated with the diminished expression of IL-22 and when IL-22 was administered to germ free mice, the replication of the virus was blocked presenting with a more wild type phenotype. They present several experiments that exclude specific pathways that could explain this finding including interferon, glycosylation patterns, necroptosis, and pyroptosis. Although the findings are interesting and provocative, the authors do not provide an explanation for their findings beyond the association with IL22. There is a vast volume of literature in the murine model of rotavirus over the last 35 years and a recent publication from Shi et al (Cell 2019) that directly contradict what is presented here. This descriptive observation, which lacks full development and explanation, adds little to the scientific field. In addition, discrepancies in the experimental approach raises some concerns.

Major Concerns:

This paper needs several crucial modifications before it can be published. Overall, the introduction needs to be more informative about rotavirus-specific interactions with bacteria and IL-22. The presented results are strong and the discovery that IL-22 is dependent on gut microbiota is excellent but more information is needed about the strain of rotavirus used for each experiment and more discussion is needed about variation in susceptibility of replicate mice. The discussion largely repeats the results and should instead be put into context with the other viral interactions currently in the discussion. Although the mechanism remains elusive, the authors do not postulate a possible pathway based on literature detailing what is known about the interplay between bacteria and immune effectors.

1. This paper directly contradicts over 35 years of work in the murine model by at least 5 major research groups and many more smaller groups. Recently, it was published that antibiotic treatment reduced rather than promoted rotavirus infection (Shi et al Cell 2019). Although the authors do acknowledge this publication, a more depth discussion is necessary to provide explanation beyond IL22 as to why these data differ from the vast volume of previous studies.

2. The gnotobiotic piglet models (Yuan et all JV 1998) has been used for many years and does not seem to model the authors findings here. Discussing the findings in the context of these published results is warranted and should be included in the discussion.

3. Lines 238-286: Although the authors are looking at the immune effectors that contribute to this resistance rather than specific microbiome composition, and they reference Shi et all Cell 2019, the authors do not test for the presence of SFB. Thus it is not clear whether this work is extending the findings of Shi et al or the authors have found a completely independent protective mechanism from RV. The mice should be tested for the presence of SFB either through SFB-specific qPCR or another 16S sequencing approach. More evidence that this extends the work of Shi et al is needed before publication. Rewording the introduction and the discussion is recommended as well as adding qPCR specific for SFB.

4. There are major differences in virulence between the EDIM strain and the EC strain. An explanation should be included in the discussion as to the differences in virulence of these strains and how it might affect the interpretation of the results. Line 239 and 248: Since authors are using 2 different strains of rotavirus, they should specify which strain they are referring to (EDIM or EC). Also reference strain in the figure legends

5. Microarray is an outdated method in which to assess transcriptional changes. State of the art approaches should utilize RNAseq based transcriptomics

6. Since viral stocks were made from intestinal and fecal isolates that were rich in microbial organisms, some description is necessary as to how the authors ensured that the inoculum was bacteria free.

7. Immunofluorescence for RV antigen in intestinal sections should be included to demonstrate there is no infection in the wild type animals.

Minor Concerns:

1. With antibiotics in the drinking water, how was the amount of antibiotic that each animal received controlled for and how would this affect the microbiome differently in each animal. This could perhaps explain the large differences in the shedding curves of each animal.

2. 16S sequencing should be done before and after antibiotic treatment in order to verify that large changes in the microbiome were occurring with treatment. The different mouse strains used may not have comparable microbiomes

3. Rotavirus is a small intestinal infection and so it is not clear why colonic samples were used for the viral stock of EDIM rather than small intestine. An explanation should be included in the materials and methods.

4. An upaired student’s t test does not seem the appropriate test to assess statistical significance. Studies using this statistical test should have more than six samples in each group and with this data, the ability to generalize to a larger population is difficult. Appropriate statistical tests should be performed.

5. It is not clear whether all the animals were on the same food and water. This would be important as it could explain some of the differences the authors observed. The description of food and water should be included in the materials and methods.

6. Were the animals prechecked for the presence of serum RV antibodies that might suggest the animals had previously seen RV? This should be done and stated in the materials and methods.

7. Line 291 Why were IFNLR1-/- mice used instead of WT mice for these studies. More rationale should be included.

8. In Figure 3 A and B why is there such a large difference in expression levels in the SPF controls. A discussion of this should be included in the results.

9. Figures 3 C and D need complete shedding curves and area under the curve calculated as performed in O’Neal et all Virology 1997.

10. In Figure 2, day 5 seems too late to assess viral shedding based on the work of Offit, Greenberg, Ward, Estes, Connor. Earlier timepoints should be presented.

11. What does the statement in lines 325-326 mean about the timing? The timing of shedding of rotavirus in the murine model of rotavirus infection has been well documented over the last 30 years by multiple groups (Offit, Greenberg, Ward, Estes, Connor)

12. Line 94-98: The authors should mention that IL-22 is known to be protective from rotavirus infection (reference 18)

13. Line 242 and 249: Loss of infectivity in C57BL6 adult mice is highly surprising. The SFB protection from RV reported by Shi et al was the first identification of a resistant mouse model. A short statement such as “this was surprising given reports of consistent infection in previous studies” should be added to put the discovery in context for readers.

14. Line 249 and Figure 2: The authors should make it clear whether the baseline of 3e10 genome copies per fecal pellet is the limit of detection or the expected amount of RV shedding. It is unclear whether the mice at the second independent institution were also resistant to RV infection or simply had an increase from baseline infectivity.

15. Line 255-256 and Figure 2: No reference is made to the variability in the -Abx condition, despite the fact that this hints at variation either in the microbiota composition or the IL-22 signaling. Some discussion on this point should be included. Comparison between mice that are highly susceptible and those that are less susceptible may even be helpful in determining the mechanism.

16. Line 265-266: It should be clarified that these are basal levels in the absence of infection

17. Line 295-304: As Fut2 enables RV entry, it is not clear why an increase in Fut2 would confer resistance to RV infection. More rationale should be included to understand the logic behind this statement.

18. Line 315 and Figure 5B: The authors do not discuss why there is reduced shedding in saline treated Casp1/11-/- Abx mice as compared to other mouse models treated with Abx. A discussion should be included to address this.

19. Figure 5 legend: The question mark following pyroptosis is unnecessary.

20. Lines 46 and 296: The comments about O glycosylation are incorrect. Fut 2 adds fucose moieties to glycoproteins and glycolipid so they are fucosylated glycans on the cell surface. This enzyme has nothing to do with O glycans. There are no O molecules linked to rotavirus entry. The wording should be modified

**********

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Reviewer #2: No

Reviewer #3: No

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PLoS One. 2021 Aug 12;16(8):e0247738. doi: 10.1371/journal.pone.0247738.r002

Author response to Decision Letter 0

28 Apr 2021

We wished to thanks the reviewers for their constructive criticism. We addressed all concerns as detailed in the rebuttal letter. The various text changes clearly improved the quality of our manuscript and will facilitate assessing the main scientific conclusions resulting from our work.

Rebuttal letter:

Reviewer #1:

This work presents data that indicates that C57BL/6 mice are highly, if not completely resistant to infection by WT murine rotavirus and that this resistance can be alleviated by ablation of microbiota by either antibiotics or germfree approaches. They then go on to show that such treatments reduce IL-22 and that exogenous IL-22 protects against rotavirus, and that all of these findings are independent of type 1 IFN. There is not really all that much new here but, nonetheless, the work cold revised to make a reasonable contribution to the literature but major revisions in how they present and interpret their findings are needed.

Infection of C57BL/6 mice with murine rotavirus EC strain is a very widely used model of rotavirus infection, used for many years. Accordingly, when such mice are directly purchased from most commercial vendors and bred at many institutions, they will display clear shedding of RV antigens and genomes. Here, the authors report that such mice bred at 2 different institutions were not infectable by RV unless first exposed to antibiotics. I don’t doubt the observation they made but how they present it and interpret it is highly problematic. Firstly, they don’t actually acknowledge that the untreated mice were not, in fact, infected with RV at all and thus that they their positive control did not work as it has in the literature for over 20 years. Consequently, they have not really interpreted and properly contextualized the antibiotic data. The proper interpretation is that, for some reason, their mice are highly RV resistant, relative to mice long used in the literature which can be readily infected by this RV strain.

Response: We would like to clarify that we did not intend to claim that our WT mice are “not infectable” by RV unless treated with antibiotics. It has previously been reported by multiple other groups that adult C57BL/6 mice infected with EDIM or EC exhibit “low and inconsistent levels of” viral shedding (PMID 15003858), and that “the ID50 for C57BL/6 mice was approximately 1000× the dose of ECwt required for the BALB/c mice” (PMID 16191453), consistent with our observations of low viral shedding in adult C57BL/6 mice at two independent sites. The key aspect of these experiments we intended to emphasize that our data demonstrate that RV replicates much better in germ-free or antibiotic-treated mice than in mice with an undisturbed microbiota. We apologize for not having emphasized this important point well enough. We rephrased the corresponding sections of the revised manuscript (current lines 270-274).

They mention “timing of virus excretion” as a possible explanation but I have no idea what they mean by this.

Response: We suggest that differences in “timing of virus excretion” between microbiota-replete and -depleted mice might explain the discrepancy between our data and published work by other authors. It has been reported that antibiotic treatment can delay the passage of content through the intestinal tract of mice (PMID 25431490, 21998395, 24237703). In the revised manuscript (lines 342-345), we rephrased the critical sentence to clarify this point, and cite the work which demonstrated reduced speed of content passage in mice with depleted microbiota.

Countless studies have shown adult C57BL/6 shed mice within a few days of inoculation and their assay period certainly covers this time. It is certainly appropriate to cite reference 19 to speculate that the mice studied here might also be RV-resistant to their specific microbiota composition but again they need to start by acknowledging that their mice contradict the well-established phenotype. Moreover, this makes it very difficult to compare their findings with published papers that started with the typical readily-infectable mice and then examined impacts of antibiotics and germfree conditions.

Response: As discussed above, multiple other groups have also reported low levels of viral shedding in adult C57BL/6 mice, and we have now clarified that our WT mice “shed only low amounts of virus” unless treated with antibiotics. We have also added in the statement, “Since our animal facilities are not free of segmented filamentous bacteria (SFB) which are known to confer rotavirus resistance [20], we speculate that the degree of SFB colonization in individual animals could regulate intrinsic rotavirus susceptibility” (lines 271-274) to emphasize the possibility of already-present microbial factors that could contribute to low levels of viral infection in mice with replete microbiota.

Reviewer #2:

It is a very important scientific work and would definitely contribute in the advancement of the current knowledge about the influence of microbiome and its corelation with antibiotics on viral infections.

I would strongly suggest you the improvement of all figures. I am unable to clearly read what is written along x axis and y axis. Kindly improve it. It is in bold letters, it will be more convenient for the readers if it appears in normal letters.

Response: High-resolution versions of the figures were provided, but required downloading from the PLoS One server. These versions contain axis labels that are easily readable.

Moreover at line number 142 (Antibiotic treatment of Mice), kindly mention the mode of adminstration of antibiotics, as I could not get it completely. You are requested to make it clear weather the antibiotics were adminsitered orally or intramuscular or intravenous.

Response: We apologize for any confusion. Antibiotics were added to the drinking water at concentrations described in the methods section. We rephrased this section to now make the administration method clearer (lines 151-158).

Reviewer #3:

The manuscript titled “Rotavirus susceptibility of antibiotic-treated mice ascribed to diminished expression of IL-22” by Schnepf et al describes the finding that rotavirus, a common intestinal viral infection, replicates more efficiently in the intestines of germ free and antibiotic treated mice compared to wild type mice. The authors ascribe this finding to the fact that the microbiome is altered in the germ free and antibiotic treated mice. The effect was associated with the diminished expression of IL-22 and when IL-22 was administered to germ free mice, the replication of the virus was blocked presenting with a more wild type phenotype. They present several experiments that exclude specific pathways that could explain this finding including interferon, glycosylation patterns, necroptosis, and pyroptosis. Although the findings are interesting and provocative, the authors do not provide an explanation for their findings beyond the association with IL22.

Response: The last notion is certainly correct: we did not manage to elucidate the mechanism by which IL-22 limits rotavirus replication in the intestinal tract of antibiotic-treated mice. However, we addressed several hypotheses which, unfortunately, proved invalid. A better understanding of this interesting new activity of IL-22 at the molecular level can hopefully be achieved in future studies.

There is a vast volume of literature in the murine model of rotavirus over the last 35 years and a recent publication from Shi et al (Cell 2019) that directly contradict what is presented here.

Response: We respectfully disagree with the view that our work would directly contradict the findings of Shi and coworkers. These researchers reported that specific segmented filamentous bacteria (SFB) that heavily colonizes the intestine of certain mouse strains are able to confer a very high degree of RV resistance to immunocompromised (Rag1-/-) mice, a resistance which is not mediated by IL-22. However, these authors did not dispute earlier findings of several other labs that IL-22 can impede RV replication in the intestinal tract of mice, nor did this work address the cumulative effects of the endogenous microbiota in regulating RV infection.

This descriptive observation, which lacks full development and explanation, adds little to the scientific field. In addition, discrepancies in the experimental approach raises some concerns.

Response: We are not sure which discrepancies are meant.

Major Concerns:

This paper needs several crucial modifications before it can be published. Overall, the introduction needs to be more informative about rotavirus-specific interactions with bacteria and IL-22.

Response: The introduction has been substantially expanded to now include additional details and references related to bacteria-rotavirus interactions, as well as to further discuss what is known to date about IL-22-mediated antiviral effects upon rotavirus.

The presented results are strong and the discovery that IL-22 is dependent on gut microbiota is excellent but more information is needed about the strain of rotavirus used for each experiment and more discussion is needed about variation in susceptibility of replicate mice.

Response: Detailed information on the rotavirus strains used in each particular figure is now provided in the figure legends. We have also now added statements addressing the variation among mice with the following: “Rotavirus shedding by untreated mutant mice was surprisingly heterogeneous (Fig. 2), raising the possibility that the microbiota composition of individual animals could matter. Since our animal facilities are not free of segmented filamentous bacteria (SFB) which are known to confer rotavirus resistance [20], we speculate that the degree of SFB colonization in individual animals could regulate intrinsic rotavirus susceptibility.”

The discussion largely repeats the results and should instead be put into context with the other viral interactions currently in the discussion. Although the mechanism remains elusive, the authors do not postulate a possible pathway based on literature detailing what is known about the interplay between bacteria and immune effectors.

Response: The discussion has been expanded to include additional discussion of the mechanisms by which the microbiota maintains IL-22 expression.

1. This paper directly contradicts over 35 years of work in the murine model by at least 5 major research groups and many more smaller groups. Recently, it was published that antibiotic treatment reduced rather than promoted rotavirus infection (Shi et al Cell 2019). Although the authors do acknowledge this publication, a more depth discussion is necessary to provide explanation beyond IL22 as to why these data differ from the vast volume of previous studies.

Response: These authors showed that GSU-RAG mice (which carry SFB that block infection with rotavirus) could not be rendered rotavirus susceptible by prolonged treatment with antibiotics. However, as mentioned above, our wild-type mice are not fully resistant to rotavirus infection. SFB is present in most mice of our colony, and recent PCR data indicate that the extent of SFB colonization is most prominent in immune-deficient mice. We now discuss this finding in the revised manuscript (lines 270-274). Nevertheless, we continue to believe that the limited RV susceptibility of our mice cannot be explained entirely by the presence of SFB in our colonies mainly because, unlike Shi and coworkers, we do not observe complete resistance to RV infection. However, the unexplained heterogeneity of RV susceptibility of some individual mice in our experiments could well be due to a higher abundance of SFB in these individuals. To account for these observations, we rephrased the text to indicate that the role of SFB remains unclear in our experimental setting.

2. The gnotobiotic piglet models (Yuan et all JV 1998) has been used for many years and does not seem to model the authors findings here. Discussing the findings in the context of these published results is warranted and should be included in the discussion.

Response: We would suggest that findings from gnotobiotic piglets in fact nicely recapitulate what we have observed in mice. Specifically, it has been reported that colonized piglets exhibit reduced HRV-induced diarrhea and viral shedding compared to their noncolonized germ-free counterparts, which indicates a protective role for the microbiota against RV (PMID 29929472). This is highly compatible with our observations, and we agree that this helpful point should be added to the discussion (lines 336-339).

3. Lines 238-286: Although the authors are looking at the immune effectors that contribute to this resistance rather than specific microbiome composition, and they reference Shi et all Cell 2019, the authors do not test for the presence of SFB. Thus it is not clear whether this work is extending the findings of Shi et al or the authors have found a completely independent protective mechanism from RV. The mice should be tested for the presence of SFB either through SFB-specific qPCR or another 16S sequencing approach. More evidence that this extends the work of Shi et al is needed before publication. Rewording the introduction and the discussion is recommended as well as adding qPCR specific for SFB.

Response: As proposed by this reviewer, we tested for the presence of SFB in our mice and found that the majority of animals in our two facilities are colonized by these bacteria. These results are mentioned in the revised manuscript and possible implications of these findings are discussed (lines 270-274).

4. There are major differences in virulence between the EDIM strain and the EC strain. An explanation should be included in the discussion as to the differences in virulence of these strains and how it might affect the interpretation of the results. Line 239 and 248: Since authors are using 2 different strains of rotavirus, they should specify which strain they are referring to (EDIM or EC). Also reference strain in the figure legends

Response: We have now more clearly specified which virus strains were used for the experiment in the various graphs, and have included the point that we observed similar findings for both EDIM and EC in our discussion.

5. Microarray is an outdated method in which to assess transcriptional changes. State of the art approaches should utilize RNAseq based transcriptomics

Response: We agree that microarrays represent an outdated method. However, at the time when this particular experiment was performed, this technique was still used in many laboratories. Since this approach yielded clear hits which could be validated by standard RT-qPCR technology, the resulting information was useful and triggered the evaluation of new hypotheses. Although state of the art approaches might be more sensitive and might yield additional hits, we do not believe that such alternative technology could provide entirely novel insights with regard to IL-22-mediated gene regulation in our system.

6. Since viral stocks were made from intestinal and fecal isolates that were rich in microbial organisms, some description is necessary as to how the authors ensured that the inoculum was bacteria free.

Response: The methods section contains this information: the virus stocks were filtered before use.

7. Immunofluorescence for RV antigen in intestinal sections should be included to demonstrate there is no infection in the wild type animals.

Response: As discussed above, we have now fully clarified in the text that our WT mice with intact microbiota are not fully resistant to infection with RV, and are instead focused in this study on the increased viral levels observed when the microbiota is depleted.

Minor Concerns:

1. With antibiotics in the drinking water, how was the amount of antibiotic that each animal received controlled for and how would this affect the microbiome differently in each animal. This could perhaps explain the large differences in the shedding curves of each animal.

Response: We did not monitor the drinking behavior of individual animals. However, since the antibiotic cocktail was the only source of water and since the antibiotic treatment lasted several weeks, we assume that sufficient amounts of antibiotics were present in all mice. Prior experiments using the same methods have demonstrated consistent depletion of the microbiota below the limit of detection of assays (PMID 25431490). In most experiments we confirmed the virtual absence of live bacteria in the feces by plating the material onto suitable agar petri dishes. This latter fact is mentioned in the revised manuscript (lines 157-158).

2. 16S sequencing should be done before and after antibiotic treatment in order to verify that large changes in the microbiome were occurring with treatment. The different mouse strains used may not have comparable microbiomes

Response: As mentioned above, in most experiments we confirmed the efficacy of treatment by plating fecal samples on BHI agar-containing petri dishes, and the capacity of antibiotics treatment to dramatically deplete WT and Ifnlr1-/- mice of bacteria has been previously demonstrated (PMID 25431490). While microbiota compositions are broadly similar between WT and Ifnlr1-/- mice (PMID 25431490), we have not carefully compared the initial microbiota composition of some mouse strains used here in our facilities. Although we cannot exclude variations between strains, it is unlikely that such differences in the microbiota could affect our principal conclusion that microbiota-driven production of IL-22 can limit RV replication in the intestinal tract.

3. Rotavirus is a small intestinal infection and so it is not clear why colonic samples were used for the viral stock of EDIM rather than small intestine. An explanation should be included in the materials and methods.

Response: Although RV predominately replicates in the small intestine, large amounts of virus are present in the stool of infected pups. Therefore, to produce virus stocks, we used the complete intestine including both small intestine and colon with content as virus source, a method described clearly in the materials and methods section. This approach is consistent with a general preference by our institutions for avoiding use of excessive mice.

4. An upaired student’s t test does not seem the appropriate test to assess statistical significance. Studies using this statistical test should have more than six samples in each group and with this data, the ability to generalize to a larger population is difficult. Appropriate statistical tests should be performed.

Response: Here, statistical analyses with unpaired student’s t test were performed exclusively with data shown in figures 3A and 3B. In these experiments, the group sizes were larger than 6, consistent with the reviewer’s point.

5. It is not clear whether all the animals were on the same food and water. This would be important as it could explain some of the differences the authors observed. The description of food and water should be included in the materials and methods.

Response: The same diet was used for all animals, but some animals received antibiotics with the drinking water. This fact has now been stated more clearly in the revised manuscript (lines 151-158).

6. Were the animals prechecked for the presence of serum RV antibodies that might suggest the animals had previously seen RV? This should be done and stated in the materials and methods.

Response: No testing for the presence of RV-specific antibodies was performed. Animals were housed in a clean facility that is routinely screened for the presence of RV using sentinel mice with no evidence for spontaneous RV infections. This fact is mentioned in the revised manuscript (lines 138-141).

7. Line 291 Why were IFNLR1-/- mice used instead of WT mice for these studies. More rationale should be included.

Response: We used Ifnlr1-ko mice for this experiment to exclude possible complications from the fact that IL-22 can act by influencing the activity of IFN-λ (ref. 27). We have detailed this rationale in our manuscript text.

8. In Figure 3 A and B why is there such a large difference in expression levels in the SPF controls. A discussion of this should be included in the results.

Response: Data shown in Figure 3A was derived from analyzing enriched intestinal epithelial cell fractions and results were normalized to the housekeeping gene Hprt. Data shown in Figure 3B was derived from ileum samples and results were normalized to a different housekeeping gene (Rps29). Therefore, absolute Il22 expression levels cannot be assessed by simply comparing Figures 3A and B.

9. Figures 3 C and D need complete shedding curves and area under the curve calculated as performed in O’Neal et all Virology 1997.

Response: The shedding curves cover the complete observation period of 12 days. We believe that the graph nicely illustrates our findings and that no additional calculations are required.

10. In Figure 2, day 5 seems too late to assess viral shedding based on the work of Offit, Greenberg, Ward, Estes, Connor. Earlier timepoints should be presented.

Response: In our hands, day 5 post-infection proved to be an effective time point for assessing differences in fecal RV shedding.

11. What does the statement in lines 325-326 mean about the timing? The timing of shedding of rotavirus in the murine model of rotavirus infection has been well documented over the last 30 years by multiple groups (Offit, Greenberg, Ward, Estes, Connor)

Response: We suggest that differences in “timing of virus excretion” between microbiota-replete and -depleted mice might explain the discrepancy between our data and published work by other authors. It has been reported that antibiotic treatment can delay the passage of content through the intestinal tract of mice (PMID 25431490, 21998395, 24237703). In the revised manuscript (lines 342-345), we rephrased the critical sentence to clarify this point, and cite the work which demonstrated reduced speed of content passage in mice with depleted microbiota.

12. Line 94-98: The authors should mention that IL-22 is known to be protective from rotavirus infection (reference 18)

Response: In the introduction we have further expanded our discussion of the relevant literature which indicates that IL-22 can protect from RV infection.

13. Line 242 and 249: Loss of infectivity in C57BL6 adult mice is highly surprising. The SFB protection from RV reported by Shi et al was the first identification of a resistant mouse model. A short statement such as “this was surprising given reports of consistent infection in previous studies” should be added to put the discovery in context for readers.

Response: We did not report a “loss of infectivity” in C57BL/6 mice. We demonstrate that if WT mice with standard microbiota were orally infected with a given dose of RV, we could detect how levels of virus in fecal samples by ELISA. However, virus shedding was detected in the majority of WT mice if they were treated with antibiotics or if germ-free animals were used for the experiments. Based upon previous reports demonstrating poor infection of C57BL/6 mice by other groups, discussed above, we were not particularly surprised to find low levels of viral infection in our WT mice with replete microbiota.

14. Line 249 and Figure 2: The authors should make it clear whether the baseline of 3e10 genome copies per fecal pellet is the limit of detection or the expected amount of RV shedding. It is unclear whether the mice at the second independent institution were also resistant to RV infection or simply had an increase from baseline infectivity.

Response: As mentioned above, we did not claim that our mice are completely resistant to infection with RV if they had a normal microflora. We used experimental conditions under which mice with standard microbiota showed a very low level of viral infection. Under these same conditions, mice with diminished microbiota were highly susceptible to RV infection. We have now further clarified that the line represents the limit of detection of the RT-qPCR assay in the figure legend.

15. Line 255-256 and Figure 2: No reference is made to the variability in the -Abx condition, despite the fact that this hints at variation either in the microbiota composition or the IL-22 signaling. Some discussion on this point should be included. Comparison between mice that are highly susceptible and those that are less susceptible may even be helpful in determining the mechanism.

Response: The Freiburg and St. Louis laboratories used slightly different antibiotics cocktails to effectively suppress the gut microbiota. In both laboratories we noted some heterogeneity with few individual animals in almost every group showing different rotavirus susceptibility. We do not understand the molecular basis of this phenomenon and we did not try to link it to possible changes in the composition of the microbiota of individual mice. We agree further studies examining the source of heterogeneity among similar mice would be of interest, but is beyond the scope of our current study.

16. Line 265-266: It should be clarified that these are basal levels in the absence of infection

Response: As suggested by this reviewer, we modified the text to better indicate that non-infected mice were analyzed (lines 281-282).

17. Line 295-304: As Fut2 enables RV entry, it is not clear why an increase in Fut2 would confer resistance to RV infection. More rationale should be included to understand the logic behind this statement.

Response: The working hypothesis for this particular experiment was rephrased to better explain the rationale (lines 311-312).

18. Line 315 and Figure 5B: The authors do not discuss why there is reduced shedding in saline treated Casp1/11-/- Abx mice as compared to other mouse models treated with Abx. A discussion should be included to address this.

Response: Since no side-by-side experiments were performed, we do not know whether virus shedding of antibiotic-treated Casp1/11-ko mice was reduced compared with antibiotic-treated WT mice. This particular experiment did not aim at answering this particular question. Rather, this experiment aimed at answering the question whether IL-22 remained active in antibiotic-treated Casp1/11-ko mice. Our results clearly demonstrate that this was the case.

19. Figure 5 legend: The question mark following pyroptosis is unnecessary.

Response: Thanks for noting this mistake, which has been corrected in the revised manuscript.

20. Lines 46 and 296: The comments about O glycosylation are incorrect. Fut 2 adds fucose moieties to glycoproteins and glycolipid so they are fucosylated glycans on the cell surface. This enzyme has nothing to do with O glycans. There are no O molecules linked to rotavirus entry. The wording should be modified

Response: We rephrased the critical sentences to better explain the role of FUT-2 during rotavirus cell entry (lines 46-47 and 311-312).

Attachment

Submitted filename: PONE-D-21-04729_Response to Reviewers.docx

Click here for additional data file. (43.8KB, docx)

Decision Letter 1

Michael Nevels

27 Jul 2021

Rotavirus susceptibility of antibiotic-treated mice ascribed to diminished expression of interleukin-22

PONE-D-21-04729R1

Dear Dr. Staeheli,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Michael Nevels

Academic Editor

PLOS ONE

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Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: I Don't Know

Reviewer #2: N/A

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: While the provide an explanation that they don't mean to declare C57BL/6 with a microbiota un-infectable by RV their data seems to do exactly that. Indeed, this could reflect a low inoculum but, given this work generally lacks novelty and is trying to reconcile some previously published work, they need to at least start with the widely used previous conditions in which C57BL6 mice purchased from standard vendors shed copious amounts of RV. Fine to then use a lower inoculum, but starting with a POSITIVE CONTROL IS ESSENTIAL.

Reviewer #2: In the research article entitled Rotavirus susceptibility of antibiotic-treated mice ascribed to diminished expression of interleukin-22, the authors have incorporated all the suggested changes except the fonts of the figures that are still bold and one can not read them. It is requested to make those fonts readable.

Regards

Reviewer #3: The authors have substantially expanded both the introduction and discussion to put their findings in the context of the field. Additionally, they have clarified experimental details.

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Reviewer #1: No

Reviewer #2: Yes: Nazish Bostan.

Reviewer #3: No

Acceptance letter

Michael Nevels

4 Aug 2021

PONE-D-21-04729R1

Rotavirus susceptibility of antibiotic-treated mice ascribed to diminished expression of interleukin-22

Dear Dr. Staeheli:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Michael Nevels

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Associated Data

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    Supplementary Materials

    Attachment

    Submitted filename: PONE-D-21-04729_Response to Reviewers.docx

    Click here for additional data file. (43.8KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript. Raw microarray data can be accessed at GEO under accession number GSE166400.

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