A murine intestinal intraepithelial NKp46-negative innate lymphoid cell population characterized by group 1 properties

The Ly49E receptor is preferentially expressed on murine innate-like lymphocytes, such as epidermal V gamma 3 T cells, intestinal intraepithelial CD8 alpha alpha(+) T lymphocytes, and CD49a(+) liver natural killer (NK) cells. As the latter have recently been shown to be distinct from conventional NK cells and have innate lymphoid cell type 1 (ILC1) properties, we investigated Ly49E expression on intestinal ILC populations. Here, we show that Ly49E expression is very low on known ILC populations, but it can be used to define a previously unrecognized intraepithelial innate lymphoid population. This Ly49E-positive population is negative for NKp46 and CD8 alpha alpha, expresses CD49a and CD103, and requires T-bet expression and IL-15 signaling for differentiation and/or survival. Transcriptome analysis reveals a group 1 ILC gene profile, different from NK cells, iCD8 alpha cells, and intraepithelial ILC1. Importantly, NKp46(-)CD8 alpha alpha(-)Ly49E(+) cells produce interferon (IFN)-gamma, suggesting that this previously unrecognized population may contribute to Th1-mediated immunity

Interleukin-12 and -23 Control Plasticity of CD127+ Group 1 and Group 3 Innate Lymphoid Cells in the Intestinal Lamina Propria

SummaryHuman group 1 ILCs consist of at least three phenotypically distinct subsets, including NK cells, CD127+ ILC1, and intraepithelial CD103+ ILC1. In inflamed intestinal tissues from Crohn’s disease patients, numbers of CD127+ ILC1 increased at the cost of ILC3. Here we found that differentiation of ILC3 to CD127+ ILC1 is reversible in vitro and in vivo. CD127+ ILC1 differentiated to ILC3 in the presence of interleukin-2 (IL-2), IL-23, and IL-1β dependent on the transcription factor RORγt, and this process was enhanced in the presence of retinoic acid. Furthermore, we observed in resection specimen from Crohn’s disease patients a higher proportion of CD14+ dendritic cells (DC), which in vitro promoted polarization from ILC3 to CD127+ ILC1. In contrast, CD14− DCs promoted differentiation from CD127+ ILC1 toward ILC3. These observations suggest that environmental cues determine the composition, function, and phenotype of CD127+ ILC1 and ILC3 in the gut

Group 3 Innate Lymphoid Cells Program a Distinct Subset of IL-22BP-Producing Dendritic Cells Demarcating Solitary Intestinal Lymphoid Tissues

Solitary intestinal lymphoid tissues such as cryptopatches (CPs) and isolated lymphoid follicles (ILFs) constitute steady-state activation hubs containing group 3 innate lymphoid cells (ILC3) that continuously produce interleukin (IL)-22. The outer surface of CPs and ILFs is demarcated by a poorly characterized population of CD11c+ cells. Using genome-wide single-cell transcriptional profiling of intestinal mononuclear phagocytes and multidimensional flow cytometry, we found that CP- and ILF-associated CD11c+ cells were a transcriptionally distinct subset of intestinal cDCs, which we term CIA-DCs. CIA-DCs required programming by CP- and ILF-resident CCR6+ ILC3 via lymphotoxin-β receptor signaling in cDCs. CIA-DCs differentially expressed genes associated with immunoregulation and were the major cellular source of IL-22 binding protein (IL-22BP) at steady state. Mice lacking CIA-DC-derived IL-22BP exhibited diminished expression of epithelial lipid transporters, reduced lipid resorption, and changes in body fat homeostasis. Our findings provide insight into the design principles of an immunoregulatory checkpoint controlling nutrient absorption. Keywords: cryptopatches; dendritic cells; innate lymphoid cells; interleukin 22 binding protein; isolated lymphoid follicles; solitary intestinal lymphoid tissues

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

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

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

Interleukin-22 protects intestinal stem cells against genotoxic stress

Environmental genotoxic factors pose a challenge to the genomic integrity of epithelial cells at barrier surfaces that separate host organisms from the environment. They can induce mutations that, if they occur in epithelial stem cells, contribute to malignant transformation and cancer development1–3. Genome integrity in epithelial stem cells is maintained by an evolutionarily conserved cellular response pathway, the DNA damage response (DDR). The DDR culminates in either transient cell-cycle arrest and DNA repair or elimination of damaged cells by apoptosis4,5. Here we show that the cytokine interleukin-22 (IL-22), produced by group 3 innate lymphoid cells (ILC3) and γδ T cells, is an important regulator of the DDR machinery in intestinal epithelial stem cells. Using a new mouse model that enables sporadic inactivation of the IL-22 receptor in colon epithelial stem cells, we demonstrate that IL-22 is required for effective initiation of the DDR following DNA damage. Stem cells deprived of IL-22 signals and exposed to carcinogens escaped DDR-controlled apoptosis, contained more mutations and were more likely to give rise to colon cancer. We identified metabolites of glucosinolates, a group of phytochemicals contained in cruciferous vegetables, to be a widespread source of genotoxic stress in intestinal epithelial cells. These metabolites are ligands of the aryl hydrocarbon receptor (AhR)6, and AhR-mediated signalling in ILC3 and γδ T cells controlled their production of IL-22. Mice fed with diets depleted of glucosinolates produced only very low levels of IL-22 and, consequently, the DDR in epithelial cells of mice on a glucosinolate-free diet was impaired. This work identifies a homeostatic network protecting stem cells against challenge to their genome integrity by AhR-mediated ‘sensing’ of genotoxic compounds from the diet. AhR signalling, in turn, ensures on-demand production of IL-22 by innate lymphocytes directly regulating components of the DDR in epithelial stem cells