IL-10 Suppression of NK/DC Crosstalk Leads to Poor Priming of MCMV-Specific CD4 T Cells and Prolonged MCMV Persistence

IL-10 is an anti-inflammatory cytokine that regulates the extent of host immunity to infection by exerting suppressive effects on different cell types. Herpes viruses induce IL-10 to modulate the virus-host balance towards their own benefit, resulting in prolonged virus persistence. To define the cellular and molecular players involved in IL-10 modulation of herpes virus-specific immunity, we studied mouse cytomegalovirus (MCMV) infection. Here we demonstrate that IL-10 specifically curtails the MCMV-specific CD4 T cell response by suppressing the bidirectional crosstalk between NK cells and myeloid dendritic cells (DCs). In absence of IL-10, NK cells licensed DCs to effectively prime MCMV-specific CD4 T cells and we defined the pro-inflammatory cytokines IL-12, IFN-γ and TNF-α as well as NK cell activating receptors NKG2D and NCR-1 to regulate this bidirectional NK/DC interplay. Consequently, markedly enhanced priming of MCMV-specific CD4 T cells in Il10-/-mice led to faster control of lytic viral replication, bu

TGF-beta Is Required To Maintain the Pool of Immature Langerhans Cells in the Epidermis

The pivotal role of TGF-beta in Langerhans cell (LC) development has been previously established in TGF-beta-deficient mice, which lack epidermal LCs. As to whether TGF-beta also governs LC homeostasis and function remains elusive. To assess the role of TGF-beta-mediated control of cutaneous dendritic cells (DCs) in vivo, we generated mice with a conditional knockout of the TGF-beta receptor 1 (T beta R1) under a DC-specific promoter (DC-T beta R1(del) mice). While initial LC seeding occurred in DC-T beta R1(del) mice, the cells disappeared from the epidermis during the first week of life. T beta R1-deficient LCs demonstrated spontaneous maturation and gained migratory potential based on increased surface expression of MHC class II, costimulatory molecules, and CCR7 and downregulation of E-cadherin. In parallel to their early loss from the epidermis, migrating LCs were reduced in the dermis and skin-draining lymph nodes of adult DC-T beta R1(del) mice, whereas the number of Langerin(+) dermal DCs was similar to wild-type. In the absence of LCs, low-dose contact hypersensitivity in DC-T beta R1(del) mice was significantly diminished. In contrast, ear swelling was restored to wild-type levels when a higher hapten dose was applied to efficiently target T beta R1-deficient dermal DCs. In conclusion, TGF-beta inhibits in vivo LC maturation and migratory phenotype, identifying TGF-beta as a critical factor controlling LC homeostasis in the steady state. The Journal of Immunology, 2010, 185: 3248-325

Optimized Protocol for Characterization of Mouse Gut Innate Lymphoid Cells

© 2020 Valle-Noguera, Gómez-Sánchez, Girard-Madoux and Cruz-Adalia.Since their discovery, innate lymphoid cells (ILCs) have gradually been gaining greater relevance in the field of immunology due to their multiple functions in the innate immune response. They can mainly be found in mucosal and barrier organs like skin, gut, and lungs, and have been classified into five main types (NKs, ILC1s, ILC2s, ILC3s, and Lti cells) according to their function and development. They all play major roles in functions such as tissue homeostasis, early pathogen defense, regulation of inflammation, or tissue remodeling. ILCs are mostly tissue-resident cells tightly bound to the tissue structure, a fact that requires long and complex protocols that do not always provide sufficient yield for analysis. This suggests the need for optimized approaches aimed at ensuring that enriched and viable ILC samples are obtained, in order to furnish quality results. Herein a detailed protocol is established for obtaining a single-cell suspension highly enriched in lymphoid cells from mouse gut in order to identify the different subsets of ILCs by means of flow cytometry. The cell marker panel and flow cytometry gating strategies for identification and quantification of all the different ILC populations are provided for simultaneous analysis. Moreover, the protocol described includes a procedure for studying the different cytokines produced by ILC3s involved in maintaining the integrity of the gut barrier and defending against extracellular pathogens. As a result, herein an efficient method is presented for studying mouse ILCs within the lamina propria of the small intestine and colon; this can constitute a useful tool for future investigations in the field.The present research was supported by grant Nº RTI2018-093647-B-I00 to AC-A from Ministerio de Ciencia, Innovación e Universidades (MCIU), Agenda Estatal de Investigación (AEI), and Fondo Europeo de Desarrollo Regional (FEDER). AV-N is a recipient of an FPI fellowship (PRE2019-090341) from the Spanish Ministry of Science, Innovation, and Universities

Optimized Protocol for Characterization of Mouse Gut Innate Lymphoid Cells

Since their discovery, innate lymphoid cells (ILCs) have gradually been gaining greater relevance in the field of immunology due to their multiple functions in the innate immune response. They can mainly be found in mucosal and barrier organs like skin, gut, and lungs, and have been classified into five main types (NKs, ILC1s, ILC2s, ILC3s, and Lti cells) according to their function and development. They all play major roles in functions such as tissue homeostasis, early pathogen defense, regulation of inflammation, or tissue remodeling. ILCs are mostly tissue-resident cells tightly bound to the tissue structure, a fact that requires long and complex protocols that do not always provide sufficient yield for analysis. This suggests the need for optimized approaches aimed at ensuring that enriched and viable ILC samples are obtained, in order to furnish quality results. Herein a detailed protocol is established for obtaining a single-cell suspension highly enriched in lymphoid cells from mouse gut in order to identify the different subsets of ILCs by means of flow cytometry. The cell marker panel and flow cytometry gating strategies for identification and quantification of all the different ILC populations are provided for simultaneous analysis. Moreover, the protocol described includes a procedure for studying the different cytokines produced by ILC3s involved in maintaining the integrity of the gut barrier and defending against extracellular pathogens. As a result, herein an efficient method is presented for studying mouse ILCs within the lamina propria of the small intestine and colon; this can constitute a useful tool for future investigations in the field.Ministerio de Ciencia, Innovación e UniversidadesAgenda Estatal de InvestigaciónFondo Europeo de Desarrollo RegionalDepto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

The immunological functions of the Appendix: An example of redundancy?

Biological redundancy ensures robustness in living organisms at several levels, from genes to organs. In this review, we explore the concept of redundancy and robustness through an analysis of the caecal appendix, an organ that is often considered to be a redundant remnant of evolution. However, phylogenic data show that the Appendix was selected during evolution and is unlikely to disappear once it appeared. In humans, it is highly conserved and malformations are extremely rare, suggesting a role for that structure. The Appendix could perform a dual role. First, it is a concentrate of lymphoid tissue resembling Peyer's patches and is the primary site for immunoglobulin A production which is crucial to regulate the density and quality of the intestinal flora. Second, given its shape and position, the Appendix could be a unique niche for commensal bacteria in the body. It is extremely rich in biofilms that continuously shed bacteria into the intestinal lumen. The Appendix contains a microbiota as diverse as that found in the colon and could replenish the large intestine with healthy flora after a diarrhea episode. In conditions of modern medicine hygiene, and people live healthy without their appendix. However, several reports suggest that the effects of appendectomy could be subtler and associated with the development of inflammatory conditions such as inflammatory bowel disease (IBD), heart disease but also in less expected disorders such as Parkinson's disease. Lack of an Appendix also predicts a worsen outcome for recurrent Clostridium difficile infection, which is the first nosocomial infection in hospitals. Here, we review the literature and in combination with our own data, we suggest that the Appendix might be redundant in its immunological function but unique as a reservoir of microbiota

Transforming growth factor-beta and Notch ligands act as opposing environmental cues in regulating the plasticity of type 3 innate lymphoid cells

International audienceGroup 3 innate lymphoid cells (ILC3s) are composed of subsets that are either positive or negative for the natural cytotoxicity receptor (NCR) NKp46 (encoded by Ncr1). ILC3s are located at mucosal sites, such as in the intestine and lung, where they are exposed to billions of commensal microbes and potentially harmful pathogens. Together with T cells, the various ILC3 subsets maintain the balance between homeostasis and immune activation. Through genetic mapping, we identified a previously uncharacterized subset of NCR- ILC3s in mice that transiently express Ncr1, demonstrating previously undescribed heterogeneity within the ILC3 population. In addition, we showed that sustained Notch signaling was required for the maintenance of the NCR+ phenotype and that the cytokine transforming growth factor-beta (TGF-beta) impaired the development of NCR+ ILC3s. Thus, the plasticity of ILC3s is regulated by the balance between the opposing effects of Notch and TGF-beta signaling, maintaining homeostasis in the face of continual challenges

IL-10 control of CD11c+ myeloid cells is essential to maintain immune homeostasis in the small and large intestine

Although IL-10 promotes a regulatory phenotype of CD11c(+) dendritic cells and macrophages in vitro, the role of IL-10 signaling in CD11c(+) cells to maintain intestinal tolerance in vivo remains elusive. To this aim, we generated mice with a CD11c-specific deletion of the IL-10 receptor alpha (Cd11c(cre)Il10ra(fl/fl)). In contrast to the colon, the small intestine of Cd11c(cre)Il10ra(fl/fl) mice exhibited spontaneous crypt hyperplasia, increased numbers of intraepithelial lymphocytes and lamina propria T cells, associated with elevated levels of T cell-derived IFN gamma and IL-17A. Whereas naive mucosal T-cell priming was not affected and oral tolerance to ovalbumin was intact, augmented T-cell function in the lamina propria was associated with elevated numbers of locally dividing T cells, expression of T-cell attracting chemokines and reduced T-cell apoptosis. Upon stimulation, intestinal IL-10Ra deficient CD11c(+) cells exhibited increased activation associated with enhanced IL-6 and TNFa production. Following colonization with Helicobacter hepaticus Cd11c(cre)Il10ra(fl/fl) mice developed severe large intestinal inflammation characterized by infiltrating T cells and increased levels of Il17a, Ifng, and Il12p40. Altogether these findings demonstrate a critical role of IL-10 signaling in CD11c(+) cells to control small intestinal immune homeostasis by limiting reactivation of local memory T cells and to protect against Helicobacter hepaticus-induced colitis

IL-10 differentially affects MCMV-specific CD4 vs CD8 T cell responses.

<p>B6 or <i>Il10</i>^−/− mice were infected i.v. with 5×10⁶ PFU <i>Δm157</i> MCMV. Lymphocytes from spleen, lungs, liver and salivary gland (A) or lung (B) were isolated at day 14 post infection and <i>ex vivo</i> restimulated with appropriate peptides. A, B) CD4 T cells were restimulated with a pool of M14, m18, M25, M112, m139 and m142 peptides (CD4 peptide pool, A, B), or with M25 and m142 peptide alone (B). Fold increase in percentage of IFN-γ⁺ TNF-α⁺ peptide-specific CD4 T cells between <i>Il10</i>^−/− and B6 mice (A, right panel, (n = 3), data are representative for at least 3 experiments, error bars indicate the standard deviation). (B) Total numbers (lower row) and percentages (upper row) of IFN-γ ⁺ TNF-α⁺ peptide-specific CD4 T cells from B6 and <i>Il10</i>^−/− mice. C) Lung lymphocytes were isolated from infected mice and M45- or M38-specific CD8 T cells were quantified by tetramer staining (n = 3, data are representative from at least 3 experiments, error bars indicate the standard deviation). Statistical analysis was performed by 2-tailed unpaired student's t-test (* p<0.05, ** p<0.01, *** p<0.001).</p

<i>Il10</i>^−/− mice show reduced virus loads and increased body weight loss during acute MCMV infection.

<p>B6 and <i>Il10</i>^−/− mice were infected i.v. with 5×10⁶ PFU <i>Δm157</i> MCMV. A) Virus titers on different days post infection were determined in lungs and salivary glands. Each symbol represents one individual mouse, horizontal line indicates the mean (n = 3–6, dashed line indicates detection limit). Data are representative of 3 independent experiments. B) IFN-γ, TNF-α and IL-12 protein concentrations were determined in the sera of B6 and <i>Il10</i>^−/− mice at day 5.5 post infection by cytometric bead array (BD, Bioscience) and murine IL-12 ELISA Kit (Peprotech). Each symbol represents one individual mouse, horizontal line indicates the mean (n = 3). Data are representative of 2 independent experiments. C, E) Body weight change of B6 and <i>Il10</i>^−/− mice was measured up to 14 or 20 days post infection. Change in percentage of body weight relative to day 0 is shown. One group of <i>Il10</i>^−/− mice was treated with a neutralizing anti-TNF-α antibody (C) or depleted of CD4 T cells (E). Right panel in E indicates the same experiment showing significances for day 6 p.i. Data are representative of 3 independent experiments. Each symbol represents the mean of 3 mice per group, error bars indicate the standard deviation. D) Virus titers in lungs and salivary glands on day 14 post infection of B6 and <i>Il10</i>^−/− mice with or without depletion of CD4 or CD8 T cells. Each symbol represents one individual mouse, horizontal line indicates the mean (n = 5–7, dashed line indicates detection limit). Pooled data from 2 independent experiments are shown. Statistical analysis was performed by 2-tailed unpaired student's t-test (* p<0.05, ** p<0.01, *** p<0.001).</p

IL-10 is produced early upon MCMV infection.

<p>B6 or <i>Il10</i> GFP reporter (Tiger) mice were infected with 5×10⁶ PFU <i>Δm157</i> MCMV. A) IL-10 protein concentration was determined in the sera of infected B6 mice by IL-10 ELISA Set (BD, Biosciences) at indicated time points (n = 3, data are representative of 2 independent experiments, error bars indicate the standard deviation). B) Lung and spleen lymphocytes were isolated from infected <i>Il10</i> GFP reporter (Tiger) mice and control littermates at day 5.5 p.i. Percentages of GFP⁺ cells (after substraction of background fluorescence from littermate controls) are shown for the indicated cell populations: CD4⁺ cells, CD8⁺ cells, B220⁺ cells (B cells), NK1.1⁺CD3ε⁻ cells (NK cells), NK1.1⁺CD3ε⁺ cells (NK T cells), CD11b⁺CD11c⁻LyG/C⁻ cells (monocytes/macrophages) and splenic CD11c⁺CD11b⁺MHCII⁺B220⁻ cells (myeloid DCs). (n = 3, data are representative of 2 independent experiments, error bars indicate the standard deviation).</p