Homeostatic interferon-lambda response to bacterial microbiota stimulates preemptive antiviral defense within discrete pockets of intestinal epithelium
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Evaluation Summary:
The paper provides information about the relative importance of the type I and type III interferon-driven gene expression and anti-viral responses, particularly focused on the role of the Intestinal microbiota to maintain background levels of type III (interferon lambda) signaling. Type III-driven gene expression is highly discontinuous in the epithelial layer and mainly at the villous tips with consequent effects on the kinetics of rotavirus model infections.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript.The reviewers remained anonymous to the authors.)
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Abstract
Interferon-lambda (IFN-λ) protects intestinal epithelial cells (IECs) from enteric viruses by inducing expression of antiviral IFN-stimulated genes (ISGs). Here, we find that bacterial microbiota stimulate a homeostatic ISG signature in the intestine of specific pathogen-free mice. This homeostatic ISG expression is restricted to IECs, depends on IEC-intrinsic expression of IFN-λ receptor ( Ifnlr1 ), and is associated with IFN-λ production by leukocytes. Strikingly, imaging of these homeostatic ISGs reveals localization to pockets of the epithelium and concentration in mature IECs. Correspondingly, a minority of mature IECs express these ISGs in public single-cell RNA sequencing datasets from mice and humans. Furthermore, we assessed the ability of orally administered bacterial components to restore localized ISGs in mice lacking bacterial microbiota. Lastly, we find that IECs lacking Ifnlr1 are hyper-susceptible to initiation of murine rotavirus infection. These observations indicate that bacterial microbiota stimulate ISGs in localized regions of the intestinal epithelium at homeostasis, thereby preemptively activating antiviral defenses in vulnerable IECs to improve host defense against enteric viruses.
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Evaluation Summary:
The paper provides information about the relative importance of the type I and type III interferon-driven gene expression and anti-viral responses, particularly focused on the role of the Intestinal microbiota to maintain background levels of type III (interferon lambda) signaling. Type III-driven gene expression is highly discontinuous in the epithelial layer and mainly at the villous tips with consequent effects on the kinetics of rotavirus model infections.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript.The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this paper the authors provide new information about the relative importance of the type I and type III interferon-driven gene expression and anti-viral responses, particularly focused on the role of the Intestinal microbiota to maintain background levels of type III (interferon lambda) signaling. They show with interferon lambda administration as a positive control, and antibiotic-mediated microbiota biomass depletion that low background levels of type III interferon-driven gene expression are mediated by the microbiota. Heterozygous mouse strain combinations for epithelial specific either type I or type III interferon receptor deficiency shows that the effect is type III mediated. In-situ hypbridisation shows that type III-driven gene expression is highly discontinuous in the epithelial layer and mainly …
Reviewer #1 (Public Review):
In this paper the authors provide new information about the relative importance of the type I and type III interferon-driven gene expression and anti-viral responses, particularly focused on the role of the Intestinal microbiota to maintain background levels of type III (interferon lambda) signaling. They show with interferon lambda administration as a positive control, and antibiotic-mediated microbiota biomass depletion that low background levels of type III interferon-driven gene expression are mediated by the microbiota. Heterozygous mouse strain combinations for epithelial specific either type I or type III interferon receptor deficiency shows that the effect is type III mediated. In-situ hypbridisation shows that type III-driven gene expression is highly discontinuous in the epithelial layer and mainly at the villous tips. Rotavirus infection shows slightly accelerated kinetics in the absence of the type III receptor-signaling. Since intestinal type I and type III interferon responses are well described to occur, this provides a distinction between the two signaling pathways the consequent antiviral responses and the role of the microbiota in maintaining a basal level of type III signaling.
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Reviewer #2 (Public Review):
In this work, Van Winkle et al. examine contributions of interferons and microbiota to innate responses in the intestine, and which cell types are involved. Previous work by this team and others demonstrated that microbiota influence interferon responses in the intestine, which can affect infection with several enteric viruses, either increasing infection or decreasing infection depending on the viral system. Here, the team profiles expression of ISGs, examines which cells produce IFNs and express ISGs, whether these responses are microbiota dependent, and examines the effect on infection with murine rotavirus.
Strengths:
The most interesting aspect of this study is the observation that ISG expression in the intestine is extremely patchy and limited to a few mature enterocytes. Fig. 6 is stunning, and these …
Reviewer #2 (Public Review):
In this work, Van Winkle et al. examine contributions of interferons and microbiota to innate responses in the intestine, and which cell types are involved. Previous work by this team and others demonstrated that microbiota influence interferon responses in the intestine, which can affect infection with several enteric viruses, either increasing infection or decreasing infection depending on the viral system. Here, the team profiles expression of ISGs, examines which cells produce IFNs and express ISGs, whether these responses are microbiota dependent, and examines the effect on infection with murine rotavirus.
Strengths:
The most interesting aspect of this study is the observation that ISG expression in the intestine is extremely patchy and limited to a few mature enterocytes. Fig. 6 is stunning, and these data are supported by beautiful controls (robust/broad ISG expression everywhere in the intestine of mice treated IP with IFNlambda in Fig. S4, but loss of expression in mice treated with ABX, and confirmation in public single cell RNA-seq data sets). This is the most unique and significant contribution of the study.
Other aspects of the study are also well done with appropriate controls (multiple panels with ISG levels throughout, etc.). The team uses a variety of mouse strains, treatments, etc. to support their claims.
Weaknesses:
The primary weakness lies in significance, partially based on past work. The observation about patchy ISG expression in mature enterocytes is very cool, but it remains unknown why/how this happens and whether there are any functional consequences. This reviewer understands that this is a very tough problem and may take time to figure out. Additionally, much work has already been done with microbiota and IFNlambda effects on enteric virus infection, making many of the findings here overlapping or redundant with prior work: IFNlambda effects on IECs and viral infection (Sommereyns Plos Path 2008, Baldridge JVI 2017), leukocytes as the source of IFNlambda (Mahlakoiv Plos Path 2015), and microbiota-mediated innate immune modulation and effects of IFNlambda (e.g., Baldridge Science 2015, Nice Science 2015).
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Reviewer #3 (Public Review):
The authors convincingly show by RNAseq, that microbiota ablation by antibiotics treatment deprives WT mice of a tonic and interferon lambda induced interferon stimulated gene (ISG) response. Alongside they test interferon lambda receptor ko mice, which do not show a tonic induction of ISGs or a response to antibiotics dependent removal of microbiota. Induction of ISGs in intestinal tissue depends on TLR signaling, which would support a microbiota dependent induction. Intriguingly this ISG response is highly localised in very focussed areas of the intestine. Fecal transfer or LPS treatment restore the antibiotics induced phenotype of basal ISG expression loss and partially restore antiviral protection in intestinal epithelial cells.
These data complement recent publications [PMID: 32380006][PMID: 31269444] …
Reviewer #3 (Public Review):
The authors convincingly show by RNAseq, that microbiota ablation by antibiotics treatment deprives WT mice of a tonic and interferon lambda induced interferon stimulated gene (ISG) response. Alongside they test interferon lambda receptor ko mice, which do not show a tonic induction of ISGs or a response to antibiotics dependent removal of microbiota. Induction of ISGs in intestinal tissue depends on TLR signaling, which would support a microbiota dependent induction. Intriguingly this ISG response is highly localised in very focussed areas of the intestine. Fecal transfer or LPS treatment restore the antibiotics induced phenotype of basal ISG expression loss and partially restore antiviral protection in intestinal epithelial cells.
These data complement recent publications [PMID: 32380006][PMID: 31269444] of tonic type I interferon signaling induced by microbiota could explain a series of publications showing the importance of microbiota for antiviral defence in the gut.
The authors build a line of arguments based on the correlation of data from IFNLR -/- mice and ABX treated mice. They omit however an important alternative explanation, which might be qualitative differences in the microbiota composition between IFNLR -/- and WT mice.
An explanation for the focussed ISG response in the intestine and how this explains the reduced resistance to enteric viruses is not provided.
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Reviewer #4 (Public Review):
In this paper from Van Winkle et al, the authors determined that murine intestinal epithelial cells produce several homeostatic ISGs. These homeostatic ISGs are located in the mature enterocytes and are stimulated by the presence of bacterial microbiota. This is an exciting concept as it becomes clearer that all cells in a tissue are not the same. The authors nicely demonstrate that these homeostatic ISGs are only present when the commensal microbiota is also present and that they depend on IFNl signaling.
A main weakness of the paper is that the authors use IFNl treatment to stimulate ISGs and then use this as a basis to determine homeostatic gene levels. However, it has been shown that stem cells have basal ISGs that are IFN independent (Wu...Rice, Cell 2018), which means that the only cells that could …
Reviewer #4 (Public Review):
In this paper from Van Winkle et al, the authors determined that murine intestinal epithelial cells produce several homeostatic ISGs. These homeostatic ISGs are located in the mature enterocytes and are stimulated by the presence of bacterial microbiota. This is an exciting concept as it becomes clearer that all cells in a tissue are not the same. The authors nicely demonstrate that these homeostatic ISGs are only present when the commensal microbiota is also present and that they depend on IFNl signaling.
A main weakness of the paper is that the authors use IFNl treatment to stimulate ISGs and then use this as a basis to determine homeostatic gene levels. However, it has been shown that stem cells have basal ISGs that are IFN independent (Wu...Rice, Cell 2018), which means that the only cells that could respond to IFN would be the mature cells in the villi. Additionally, as the bacteria would normally only be in contact with the enterocytes and not the crypts then one would not expect to find bacterial induced homeostatic ISGs lower in the crypt-villi axis. Given these, the authors findings while interesting are expected due to the experimental set-up. While several experiments contain complete controls to make all interpretations, several experiments lack controls and many conclusions suffer from over statements, lack of convincing imaging and correlations between infections and ISGs.
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