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Operating and Biocontainment Procedures of a Facility for Laboratory Mice with a Natural Microbiome: Immunophenotyping Procedure

The use of laboratory mice with a natural microbiome, such as &#34;Wildling mice&#34;, offers a promising research tool for both basic and applied science ...

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin

The intestinal mucosa constitutes the biggest surface area of the body. It is constantly challenged by bacteria, commensal and pathogenic, protozoa, and food-derived irritants. In order to maintain homeostasis, a complex network of signaling circuits has evolved that includes contributions of immune cells. In recent years a subset of lymphocytes, which belong to the innate immune system, has caught particular attention. These so-called innate lymphoid cells (ILC) reside within the lamina propria of the small and large intestines and rapidly respond to environmental challenges. They provide immunity to various types of infections but may also contribute to organ homeostasis as they produce factors acting on epithelial cells thereby enhancing barrier integrity. Here, we describe how these cells can be isolated from their environment and provide an in-depth protocol how to visualize the various ILC subsets by flow cytometry.

Isolation and Flow Cytometry Analysis of Innate Lymphoid Cells from the Intestinal Lamina Propria.

Immunology has recently witnessed several new developments in understanding the biology of innate lymphocytes. In particular, the discovery of innate lymphoid cells (ILCs) has opened entirely new avenues for research. The exciting new developments in this rapidly expanding field were the focus of the 2EMBO Conference on Innate Lymphoid Cells, which took place from November 30 to December 2, 2016 in Berlin, Germany. Here, we summarize the key new developments reported at the conference.

The ILC World Revisited.

Innate and adaptive immunity are two complementary systems that work together to protect the host organism. A new study unravels how innate lymphoid cells and adaptive T lymphocytes act sequentially to establish microbial commensalism and ensure tissue and metabolic homeostasis.

Gut Immunity: Passing on the Baton from Innate to Adaptive Immunity.

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 development. 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 apoptosis. 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), 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.

Interleukin-22 protects intestinal stem cells against genotoxic stress.

Innate lymphoid cells (ILC) are a recently identified group of innate lymphocytes. ILC are subdivided into cytotoxic ILC (i.e., conventional natural killer (NK) cells) and helper ILC. Helper ILC are tissue-resident cells that have been involved in various physiological and pathological processes of organs and tissues. While their roles during inflammation are well studied, their contribution to tumor immunity is not well known. Here, we will provide an overview of the various helper ILC subsets, their development and function and discuss their potential roles during tumorigenesis and for anti-tumor immunity at border surface with the environment.

Innate lymphoid cells and cancer at border surfaces with the environment.

The intestines have the essential but challenging mission of absorbing nutrients, restricting damage from food-derived toxins, promoting colonization by symbionts, and expelling pathogens. These processes are often incompatible with each other and must therefore be prioritized in view of the most crucial contemporary needs of the host. Recent work has shown that tissue-resident innate lymphoid cells (ILCs) constitute a central sensory module allowing adaptation of intestinal organ function to changing environmental input. Here, we propose a conceptual framework positing that the various types of ILC act in distinct modules with intestinal epithelial cells, collectively safeguarding organ function. Such homeostasis-promoting circuitry has high potential to be plumbed for new therapeutic approaches to the treatment of immune-mediated inflammatory diseases.

Innate Lymphoid Cell-Epithelial Cell Modules Sustain Intestinal Homeostasis.

Enabling anti-tumor immunity by unleashing ILC2.

Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment.

Microbiota-Induced Type I Interferons Instruct a Poised Basal State of Dendritic Cells.

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.

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

SARS-CoV-2 is a single-stranded RNA virus that causes COVID-19. Given its acute and often self-limiting course, it is likely that components of the innate immune system play a central part in controlling virus replication and determining clinical outcome. Natural killer (NK) cells are innate lymphocytes with notable activity against a broad range of viruses, including RNA viruses. NK cell function may be altered during COVID-19 despite increased representation of NK cells with an activated and adaptive phenotype. Here we show that a decline in viral load in COVID-19 correlates with NK cell status and that NK cells can control SARS-CoV-2 replication by recognizing infected target cells. In severe COVID-19, NK cells show defects in virus control, cytokine production and cell-mediated cytotoxicity despite high expression of cytotoxic effector molecules. Single-cell RNA sequencing of NK cells over the time course of the COVID-19 disease spectrum reveals a distinct gene expression signature. Transcriptional networks of interferon-driven NK cell activation are superimposed by a dominant transforming growth factor-β (TGFβ) response signature, with reduced expression of genes related to cell-cell adhesion, granule exocytosis and cell-mediated cytotoxicity. In severe COVID-19, serum levels of TGFβ peak during the first two weeks of infection, and serum obtained from these patients severely inhibits NK cell function in a TGFβ-dependent manner. Our data reveal that an untimely production of TGFβ is a hallmark of severe COVID-19 and may inhibit NK cell function and early control of the virus.

Untimely TGFβ responses in COVID-19 limit antiviral functions of NK cells.

Innate effector cells are immune cells endowed with host protective features and cytotoxic functions. By sensing the tissue environment, innate cells have an important role in regulating the transition from homeostasis to inflammation and the establishment of pathological states, including the onset and development of cancer. The tumor microenvironment induces molecular and functional modifications in innate cells, dampening their capability to initiate and sustain anti-tumor immune responses. Emerging studies clearly showed a contribution of the microbiota in modulating the functions of innate cells in cancer. Commensal microorganisms can not only directly interact with innate cells in the tumor microenvironment but can also exert immunomodulatory features from non-tumor sites through the release of microbial products. The microbiota can mediate the priming of innate cells at mucosal tissues and determine the strength of immune responses mediated by such cells when they migrate to non-mucosal tissues, having an impact on cancer. Finally, several evidences reported a strong contribution of the microbiota in promoting innate immune responses during anti-cancer therapies leading to enhanced therapeutic efficacy. In this review, we considered the current knowledge on the role of the microbiota in shaping host innate immune responses in cancer.

Regulation of innate immune system function by the microbiome: Consequences for tumor immunity and cancer immunotherapy.

In systemic lupus erythematosus, loss of immune tolerance, autoantibody production and immune complex deposition are required but not sufficient for organ damage. How inflammatory signals are initiated and amplified in the setting of autoimmunity remains elusive. Here we set out to dissect layers and hierarchies of autoimmune kidney inflammation to identify tissue-specific cellular hubs that amplify autoinflammatory responses. Using high-resolution single-cell profiling of kidney immune and parenchymal cells, in combination with antibody blockade and genetic deficiency, we show that tissue-resident NKp46 innate lymphoid cells (ILCs) are crucial signal amplifiers of disease-associated macrophage expansion and epithelial cell injury in lupus nephritis, downstream of autoantibody production. NKp46 signalling in a distinct subset of group 1 ILCs (ILC1s) instructed an unconventional immune-regulatory transcriptional program, which included the expression of the myeloid cell growth factor CSF2. CSF2 production by NKp46 ILCs promoted the population expansion of monocyte-derived macrophages. Blockade of the NKp46 receptor (using the antibody clone mNCR1.15; ref. ) or genetic deficiency of NKp46 abrogated epithelial cell injury. The same cellular and molecular patterns were operative in human lupus nephritis. Our data provide support for the idea that NKp46 ILC1s promote parenchymal cell injury by granting monocyte-derived macrophages access to epithelial cell niches. NKp46 activation in ILC1s therefore constitutes a previously unrecognized, crucial tissue rheostat that amplifies organ damage in autoimmune hosts, with broad implications for inflammatory pathologies and therapies.

Article	Year
Isolation and Flow Cytometry Analysis of Innate Lymphoid Cells from the Intestinal Lamina Propria. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28063049	2017
The ILC World Revisited. Immunity\| PubMed ID: 28329694	2017
Gut Immunity: Passing on the Baton from Innate to Adaptive Immunity. Current biology : CB\| PubMed ID: 29738732	2018
Interleukin-22 protects intestinal stem cells against genotoxic stress. Nature\| PubMed ID: 30700914	2019
Innate lymphoid cells and cancer at border surfaces with the environment. Seminars in immunology\| PubMed ID: 31473072	2019
Innate Lymphoid Cell-Epithelial Cell Modules Sustain Intestinal Homeostasis. Immunity\| PubMed ID: 32187516	2020
Enabling anti-tumor immunity by unleashing ILC2. Cell research\| PubMed ID: 32367044	2020
Microbiota-Induced Type I Interferons Instruct a Poised Basal State of Dendritic Cells. Cell\| PubMed ID: 32380006	2020
Group 3 Innate Lymphoid Cells Program a Distinct Subset of IL-22BP-Producing Dendritic Cells Demarcating Solitary Intestinal Lymphoid Tissues. Immunity\| PubMed ID: 33207209	2020
Untimely TGFβ responses in COVID-19 limit antiviral functions of NK cells. Nature\| PubMed ID: 34695836	2021
Regulation of innate immune system function by the microbiome: Consequences for tumor immunity and cancer immunotherapy. Seminars in immunology\| PubMed ID: 36758379	2023
Amplification of autoimmune organ damage by NKp46-activated ILC1s. Nature\| PubMed ID: 39137897	2024

Andreas Diefenbach

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