Leukocyte-Derived IFN-α/β and Epithelial IFN-λ Constitute a Compartmentalized Mucosal Defense System that Restricts Enteric Virus Infections

<div><p>Epithelial cells are a major port of entry for many viruses, but the molecular networks which protect barrier surfaces against viral infections are incompletely understood. Viral infections induce simultaneous production of type I (IFN-α/β) and type III (IFN-λ) interferons. All nucleated cells are believed to respond to IFN-α/β, whereas IFN-λ responses are largely confined to epithelial cells. We observed that intestinal epithelial cells, unlike hematopoietic cells of this organ, express only very low levels of functional IFN-α/β receptors. Accordingly, after oral infection of IFN-α/β receptor-deficient mice, human reovirus type 3 specifically infected cells in the lamina propria but, strikingly, did not productively replicate in gut epithelial cells. By contrast, reovirus replicated almost exclusively in gut epithelial cells of IFN-λ receptor-deficient mice, suggesting that the gut mucosa is equipped with a compartmentalized IFN system in which epithelial cells mainly respond to IFN-λ that they produce after viral infection, whereas other cells of the gut mostly rely on IFN-α/β for antiviral defense. In suckling mice with IFN-λ receptor deficiency, reovirus replicated in the gut epithelium and additionally infected epithelial cells lining the bile ducts, indicating that infants may use IFN-λ for the control of virus infections in various epithelia-rich tissues. Thus, IFN-λ should be regarded as an autonomous virus defense system of the gut mucosa and other epithelial barriers that may have evolved to avoid unnecessarily frequent triggering of the IFN-α/β system which would induce exacerbated inflammation.</p></div

IFN-λ restricts reovirus replication in the epithelium and determines virus shedding in feces, whereas type I IFN blocks replication in the lamina propria and inhibits systemic dissemination.

<p>(A-D) Adult wild-type (n = 25), <i>Ifnar1</i>^<i>-/-</i> (n = 18) and <i>Ifnlr1</i>^<i>-/-</i> (n = 27) mice were inoculated intragastrically with 10⁸ pfu of reovirus T3D. Data pooled from several independent experiments. (A) At day 4 post-infection, reovirus replication in small intestinal tissue was analyzed by virus titration. (B) Reovirus titers in feces samples collected from wild-type and mutant mice at day 4 post-infection. (C, D) Immunostaining for reovirus antigen (green), E-cadherin (red) and DAPI (blue) in (C) small intestinal tissue or (D) Peyer’s patches. Images are representative of three independent experiments. Bar = 100 μm. ns = non-significant, ** p<0.01, *** p<0.001.</p

Intestinal epithelial cells minimally express IFN-α/β receptor and do not respond to type I IFN.

<p><b>(A) RT-qPCR analysis of IFN-α/β receptor chains (<i>Ifnar1</i> and <i>Ifnar2</i>) and IFN-λ receptor chains (<i>Ifnlr1</i> and <i>Il10r2</i>) in intestinal epithelial cells (IEC) and lamina propria lymphocytes (LPL) isolated from whole intestinal tissue of adult wild-type mice (n = 4–8).</b> (B) IFNAR1 expression analyzed by flow cytometry on IEC or LPL fractions harvested from wild-type or <i>Ifnar1</i>^<i>-/-</i> mice. (C) RT-qPCR analysis of two representative ISGs at steady state in IEC and LPL isolated from wild-type, <i>Ifnar1</i>^<i>-/-</i> and <i>Ifnlr1</i>^<i>-/-</i> mice (n = 3). (D) Adult <i>Ifnar1</i>^<i>-/-</i> and <i>Ifnlr1</i>^<i>-/-</i> mice were treated twice subcutaneously with 1 μg of mouse IFN-λ2 or human IFN-αB/D, respectively, at 24 h and 12 h prior to sacrifice as indicated. IFN-induced Mx1 in tissue sections from the gastrointestinal tract was visualized by immunofluorescence. IFN-responsive cells contain nuclear Mx1 (dotty structures in green). Epithelial cells express E-cadherin (red). DAPI (blue) stains nuclei. Data is representative for two to three independent experiments. Mean ± SEM. Bar = 100 μm. ns = non-significant, * p<0.05, ** p<0.01, *** p<0.001.</p

Reovirus replicates extensively in epithelial cells of intestine and biliary tract and induces fatal liver disease in suckling mice lacking functional IFN-λ receptors.

<p>Suckling wild-type (n = 7), <i>Ifnar1</i>^<i>-/-</i> (n = 8) and <i>Ifnlr1</i>^<i>-/-</i> (n = 11) mice were infected orally with 5 x 10⁶ pfu of reovirus T3D. Data from two independent experiments were pooled. (A) Reovirus titers in the small intestine on day 4 post-infection. (B) Immunostaining of small intestinal tissue at day 4 post-infection for reovirus antigen (green), E-cadherin (red) and DAPI (blue). (C) Survival kinetics of wild-type (n = 11), <i>Ifnar1</i>^<i>-/-</i> (n = 13) and <i>Ifnlr1</i>^<i>-/-</i> (n = 15) mice. (D) Immunostaining of liver tissue harvested on day 4 post-infection. Reovirus antigen (green), cytokeratin (red) and DAPI (blue). Arrows point to intrahepatic bile ducts. (E) Immunostaining of extrahepatic bile ducts for reovirus antigen (green), cytokeratin (red) and DAPI (blue) at day 4 post-infection. (F) Immunostaining for reovirus antigen of an extrahepatic bile duct from a diseased <i>Ifnlr1</i>^<i>-/-</i> mouse on day 8 post-infection. Note that the duct is filled with material seemingly originating from virus-infected cells. Bar = 100 μm. ** p<0.01, *** p<0.001.</p

IECs are potent producers of IFN-λ but not type I IFN.

<p>(A) Base line expression of <i>Ifna5</i>, <i>Ifnb</i> and <i>Ifnl2/3</i> genes in IEC and LPL isolated from adult wild-type mice (n = 3) assessed by RT-qPCR. (B) Adult wild-type (n = 3) mice were injected intraperitoneally with 100 μg of polyI:C and intestinal tissue was harvested at 2 and 6 h post-treatment. Expression of <i>Ifna5</i>, <i>Ifnb</i> and <i>Ifnl2/3</i> was analyzed by RT-qPCR in IEC and LPL fractions. (C) Steady state IFN-λ gene expression analysis by RT-qPCR in FACS-sorted EpCAM⁺CD45^- and EpCAM^-CD45⁺ cells from the epithelial fraction. Data is representative for two to three independent experiments. Mean ± SEM. * p<0.05, ** p<0.01. Different letters above bars mark significant differences (p<0.05).</p

Timely IFN-λ production by epithelial cells drives rapid clearance of intestinal reovirus infection.

<p>Suckling wild-type, <i>Ifnar1</i>^<i>-/-</i> and <i>Ifnlr1</i>^<i>-/-</i> mice (n = 3–4) were orally infected with 5 x 10⁶ pfu of reovirus T3D, and tissue was harvested at either day 1 or day 4 post-infection. (A) Kinetics of reovirus replication by titration. (B) Expression of IFN-responsive genes <i>Isg15</i> and <i>Oasl2</i> in whole intestinal tissue analyzed by RT-qPCR. (C) Expression of <i>Ifna5</i>, <i>Ifnb</i> and <i>Ifnl2/3</i> genes in the IEC fraction of wild-type and <i>Ifnlr1</i>^<i>-/-</i> mice assessed by RT-qPCR at day 1 post-infection. (D) Immunostaining of small intestinal tissue for reovirus antigen (red), Mx1 (green) and DAPI (blue). Dotted line marks the border of villi. Bar = 50 μm. d.p.i. = days post infection. Data representative for two individual experiments are shown. Mean ± SEM. Different letters above bars mark significant differences (p<0.05). * p<0.05.</p