ABSTRACT
Gut-draining mesenteric and celiac lymph nodes (mLNs and celLNs) critically contribute to peripheral tolerance toward food and microbial antigens by supporting the de novo induction of regulatory T cells (Tregs). These tolerogenic properties of mLNs and celLNs are stably imprinted within stromal cells (SCs) by microbial signals and vitamin A (VA), respectively. Here, we report that a single, transient gastrointestinal infection in the neonatal, but not adult, period durably abrogates the efficient Treg-inducing capacity of celLNs by altering the subset composition and gene expression profile of celLNSCs. These cells carry information about the early-life pathogen encounter until adulthood and durably instruct migratory dendritic cells entering the celLN with reduced tolerogenic properties. Mechanistically, transiently reduced VA levels cause long-lasting celLN functional impairment, which can be rescued by early-life treatment with VA. Together, our data highlight the therapeutic potential of VA to prevent sequelae post gastrointestinal infections in infants.
Subject(s)
Lymph Nodes , T-Lymphocytes, Regulatory , Vitamin A , Animals , Lymph Nodes/immunology , Lymph Nodes/pathology , Lymph Nodes/drug effects , Vitamin A/pharmacology , Vitamin A/therapeutic use , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/drug effects , Mice , Animals, Newborn , Immune Tolerance/drug effects , Dendritic Cells/immunology , Mice, Inbred C57BL , FemaleABSTRACT
Mucosal-associated invariant T (MAIT) cells are innate-like T cells mainly found in the mucosa and peripheral blood. We have recently demonstrated that Clostridioides difficile activates MAIT cells in vitro. However, their role in the pathogenesis of C. difficile infection (CDI) in human patients remains elusive to date. In this study, we performed comprehensive immunophenotyping of MAIT cells derived from CDI patients and compared their phenotype to that of patients with inflammatory bowel diseases (IBD) and healthy controls. Our study revealed that blood MAIT cells from CDI patients exhibit an interleukin 17a (IL-17a)-dominated proinflammatory phenotype and an increased readiness to synthesize the proinflammatory cytokine interferon γ (IFN-γ) following in vitro re-stimulation. Moreover, the cytotoxic activity of MAIT cells, as measured by surface CD107a and intracellular granzyme B expression, was strongly increased in CDI. Multi epitope ligand cartography (MELC) analysis of intestinal biopsies from CDI patients revealed that MAIT cells exhibit an increased production of granzyme B and increased cytotoxicity compared to the control group. Together with previously published in vitro data from our group, our findings suggest that MAIT cells are functionally involved in the immune response against C. difficile and contribute to the pathogenesis of CDI.
Subject(s)
Antineoplastic Agents , Clostridioides difficile , Mucosal-Associated Invariant T Cells , Humans , Clostridioides difficile/metabolism , Granzymes/metabolism , Cytokines/metabolism , PhenotypeABSTRACT
DNA-binding protein A (DbpA) belongs to the Y-box family of cold shock domain proteins that exert transcriptional and translational activities in the cell via their ability to bind and regulate mRNA. To investigate the role of DbpA in kidney disease, we utilized the murine unilateral ureter obstruction (UUO) model, which recapitulates many features of obstructive nephropathy seen in humans. We observed that DbpA protein expression is induced within the renal interstitium following disease induction. Compared with wild-type animals, obstructed kidneys from Ybx3-deficient mice are protected from tissue injury, with a significant reduction in the number of infiltrating immune cells as well as in extracellular matrix deposition. RNAseq data from UUO kidneys show that Ybx3 is expressed by activated fibroblasts, which reside within the renal interstitium. Our data support a role for DbpA in orchestrating renal fibrosis and suggest that strategies targeting DbpA may be a therapeutic option to slow disease progression.
Subject(s)
Kidney Diseases , Ureteral Obstruction , Animals , Mice , Cold-Shock Response , DNA-Binding Proteins/metabolism , Fibrosis , Kidney Diseases/pathology , Kidney Tubules/pathology , Ureteral Obstruction/complications , Ureteral Obstruction/geneticsABSTRACT
In aging kidneys, a decline of function resulting from extracellular matrix (ECM) deposition and organ fibrosis is regarded as "physiological." Whether a direct link between high salt intake and fibrosis in aging kidney exists autonomously from arterial hypertension is unclear. This study explores kidney intrinsic changes (inflammation, ECM derangement) induced by a high-salt diet (HSD) in a murine model lacking arterial hypertension. The contribution of cold shock Y-box binding protein (YB-1) as a key orchestrator of organ fibrosis to the observed differences is determined by comparison with a knockout strain (Ybx1ΔRosaERT+TX). Comparisons of tissue from mice fed with normal-salt diet (NSD, standard chow) or high-salt diet (HSD, 4% NaCl in chow; 1% NaCl in water) for up to 16 mo revealed that with HSD tubular cell numbers decrease and tubulointerstitial scarring [periodic acid-Schiff (PAS), Masson's trichrome, Sirius red staining] prevails. In Ybx1ΔRosaERT+TX animals tubular cell damage, a loss of cell contacts with profound tubulointerstitial alterations, and tubular cell senescence was seen. A distinct tubulointerstitial distribution of fibrinogen, collagen type VI, and tenascin-C was detected under HSD, transcriptome analyses determined patterns of matrisome regulation. Temporal increase of immune cell infiltration was seen under HSD of wild type, but not Ybx1ΔRosaERT+TX animals. In vitro Ybx1ΔRosaERT+TX bone marrow-derived macrophages exhibited a defect in polarization (IL-4/IL-13) and abrogated response to sodium chloride. Taken together, HSD promotes progressive kidney fibrosis with premature cell aging, ECM deposition, and immune cell recruitment that is exacerbated in Ybx1ΔRosaERT+TX animals.NEW & NOTEWORTHY Short-term experimental studies link excessive sodium ingestion with extracellular matrix accumulation and inflammatory cell recruitment, yet long-term data are scarce. Our findings with a high-salt diet over 16 mo in aging mice pinpoints to a decisive tipping point after 12 mo with tubular stress response, skewed matrisome transcriptome, and immune cell infiltration. Cell senescence was aggravated in knockout animals for cold shock Y-box binding protein (YB-1), suggesting a novel protective protein function.
Subject(s)
Hypertension , Kidney Diseases , Mice , Animals , Sodium Chloride , Kidney/metabolism , Kidney Diseases/chemically induced , Kidney Diseases/genetics , Kidney Diseases/pathology , Inflammation/metabolism , Aging , Hypertension/metabolism , Sodium Chloride, Dietary/adverse effects , Fibrosis , EatingABSTRACT
Prolonged lung pathology has been associated with COVID-19, yet the cellular and molecular mechanisms behind this chronic inflammatory disease are poorly understood. In this study, we combine advanced imaging and spatial transcriptomics to shed light on the local immune response in severe COVID-19. We show that activated adventitial niches are crucial microenvironments contributing to the orchestration of prolonged lung immunopathology. Up-regulation of the chemokines CCL21 and CCL18 associates to endothelial-to-mesenchymal transition and tissue fibrosis within these niches. CCL21 over-expression additionally links to the local accumulation of T cells expressing the cognate receptor CCR7. These T cells are imprinted with an exhausted phenotype and form lymphoid aggregates that can organize in ectopic lymphoid structures. Our work proposes immune-stromal interaction mechanisms promoting a self-sustained and non-resolving local immune response that extends beyond active viral infection and perpetuates tissue remodeling.
Subject(s)
COVID-19 , Chemokine CCL21 , Chemokines, CC , Humans , COVID-19/immunology , Fibrosis , Lung , T-Lymphocytes/immunologyABSTRACT
Nitric oxide (NO) is an important antimicrobial effector but also prevents unnecessary tissue damage by shutting down the recruitment of monocyte-derived phagocytes. Intracellular pathogens such as Leishmania major can hijack these cells as a niche for replication. Thus, NO might exert containment by restricting the availability of the cellular niche required for efficient pathogen proliferation. However, such indirect modes of action remain to be established. By combining mathematical modeling with intravital 2-photon biosensors of pathogen viability and proliferation, we show that low L. major proliferation results not from direct NO impact on the pathogen but from reduced availability of proliferation-permissive host cells. Although inhibiting NO production increases recruitment of these cells, and thus pathogen proliferation, blocking cell recruitment uncouples the NO effect from pathogen proliferation. Therefore, NO fulfills two distinct functions for L. major containment: permitting direct killing and restricting the supply of proliferation-permissive host cells.
Subject(s)
Leishmania major/physiology , Leishmaniasis/immunology , Macrophages/immunology , Nitric Oxide/metabolism , Animals , Cell Growth Processes , Cell Movement , Cell Proliferation , Disease Models, Animal , Host-Pathogen Interactions , Humans , Intravital Microscopy , Mice , Mice, Inbred C57BL , Models, TheoreticalABSTRACT
Neutrophils represent one of the first immune cell types recruited to sites of infection, where they can control pathogens by phagocytosis and cytotoxic mechanisms. Intracellular pathogens such as Leishmania major can hijack neutrophils to establish an efficient infection. However the dynamic interactions of neutrophils with the pathogen and other cells at the site of the infection are incompletely understood. Here, we have investigated the role of Ly6G, a homolog of the human CD177 protein, which has been shown to interact with cell adhesion molecules, and serves as a bona fide marker for neutrophils in mice. We show that Ly6G deficiency decreases the initial infection rate of neutrophils recruited to the site of infection. Although the uptake of L. major by subsequently recruited monocytes was tightly linked with the concomitant uptake of neutrophil material, this process was not altered by Ly6G deficiency of the neutrophils. Instead, we observed by intravital 2-photon microscopy that Ly6G-deficient neutrophils entered the site of infection with delayed initial recruitment kinetics. Thus, we conclude that by promoting neutrophils' ability to efficiently enter the site of infection, Ly6G contributes to the early engagement of intracellular pathogens by the immune system.
Subject(s)
Antigens, Ly/blood , Leishmania major/genetics , Leishmaniasis, Cutaneous/blood , Neutrophils/metabolism , Animals , Disease Models, Animal , Humans , Leishmania major/pathogenicity , Leishmaniasis, Cutaneous/genetics , Leishmaniasis, Cutaneous/parasitology , Leishmaniasis, Cutaneous/pathology , Mice , Monocytes/parasitology , Neutrophil Infiltration/genetics , Neutrophils/parasitology , Neutrophils/pathology , Phagocytosis/genetics , Skin/parasitology , Skin/pathologyABSTRACT
Innate lymphoid cells (ILCs) emerge in the last few years as important regulators of immune responses and biological processes. Although ILCs are mainly known as tissue-resident cells, their precise localization and interactions with the microenvironment are still unclear. Here we combine a multiplexed immunofluorescence technique and a customized computational, open-source analysis pipeline to unambiguously identify CD127+ ILCs in situ and characterize these cells and their microenvironments. Moreover, we reveal the transcription factor IRF4 as a marker for tonsillar ILC3, and identify conserved stromal landmarks characteristic for ILC localization. We also show that CD127+ ILCs share tissue niches with plasma cells in the tonsil. Our works thus provide a platform for multiparametric histological analysis of ILCs to improve our understanding of ILC biology.
Subject(s)
Lymphocytes/immunology , Lymphocytes/pathology , Phenotype , Spatial Analysis , Algorithms , Cluster Analysis , Connective Tissue/diagnostic imaging , Connective Tissue/pathology , Humans , Image Processing, Computer-Assisted , Immunity, Innate , Interferon Regulatory Factors/metabolism , Interleukin-7 Receptor alpha Subunit/metabolism , Machine Learning , Palatine Tonsil/diagnostic imaging , Palatine Tonsil/pathologyABSTRACT
BACKGROUND: Kidney injuries that result in chronic inflammation initiate crosstalk between stressed resident cells and infiltrating immune cells. In animal models, whole-body receptor Notch3 deficiency protects from leukocyte infiltration and organ fibrosis. However, the relative contribution of Notch3 expression in tissue versus infiltrating immune cells is unknown. METHODS: Chimeric mice deficient for Notch3 in hematopoietic cells and/or resident tissue cells were generated, and kidney fibrosis and inflammation after unilateral ureteral obstruction (UUO) were analyzed. Adoptive transfer of labeled bone marrow-derived cells validated the results in a murine Leishmania ear infection model. In vitro adhesion assays, integrin activation, and extracellular matrix production were analyzed. RESULTS: Fibrosis follows UUO, but inflammatory cell infiltration mostly depends upon Notch3 expression in hematopoietic cells, which coincides with an enhanced proinflammatory milieu (e.g., CCL2 and CCL5 upregulation). Notch3 expression on CD45+ leukocytes plays a prominent role in efficient cell transmigration. Functionally, leukocyte adhesion and integrin activation are abrogated in the absence of receptor Notch3. Chimeric animal models also reveal that tubulointerstitial fibrosis develops, even in the absence of prominent leukocyte infiltrates after ureteral obstruction. Deleting Notch3 receptors on resident cells blunts kidney fibrosis, ablates NF-κB signaling, and lessens matrix deposition. CONCLUSIONS: Cell-specific receptor Notch3 signaling independently orchestrates leukocyte infiltration and organ fibrosis. Interference with Notch3 signaling may present a novel therapeutic approach in inflammatory as well as fibrotic diseases.
Subject(s)
Kidney/pathology , Leukocytes/physiology , Macrophages/physiology , Nephritis/pathology , Receptor, Notch3/genetics , Receptor, Notch3/metabolism , Animals , Bone Marrow Cells/metabolism , Bone Marrow Cells/physiology , Cell Adhesion , Cell Proliferation , Cells, Cultured , Chimera , Extracellular Matrix/metabolism , Female , Fibrosis , Integrins/metabolism , Leukocyte Common Antigens/metabolism , Leukocytes/metabolism , Macrophages/metabolism , Mice , NF-kappa B/metabolism , Nephritis/etiology , Signal Transduction , Transcriptome , Transendothelial and Transepithelial Migration , Ureteral Obstruction/complicationsABSTRACT
The survival of peripheral T cells is dependent on their access to peripheral LNs (pLNs) and stimulation by IL-7. In pLNs fibroblastic reticular cells (FRCs) and lymphatic endothelial cells (LECs) produce IL-7 suggesting their contribution to the IL-7-dependent survival of T cells. However, IL-7 production is detectable in multiple organs and is not restricted to pLNs. This raises the question whether pLN-derived IL-7 is required for the maintenance of peripheral T cell homeostasis. Here, we show that numbers of naive T cells (TN ) remain unaffected in pLNs and spleen of mice lacking Il7 gene activity in pLN FRCs, LECs, or both. In contrast, frequencies of central memory T cells (TCM ) are reduced in FRC-specific IL-7 KO mice. Thus, steady state IL-7 production by pLN FRCs is critical for the maintenance of TCM , but not TN , indicating that both T cell subsets colonize different ecological niches in vivo.
Subject(s)
Cell Survival , Fibroblasts/immunology , Immunologic Memory , Interleukin-7/immunology , Lymph Nodes/immunology , T-Lymphocytes/immunology , Animals , Fibroblasts/cytology , Interleukin-7/genetics , Lymph Nodes/cytology , Mice , Mice, Knockout , T-Lymphocytes/cytologyABSTRACT
BACKGROUND AND AIMS: In dialysis patients, vascular morbidities are highly prevalent and linked to leukocyte extravasation, especially of polarized monocytes. Experimental data demonstrate that phenotypic changes in monocytes require Y-box binding protein-1 (YB-1) upregulation. METHODS: We determined YB-1 expression in circulating and vessel-invading monocytes from healthy controls and dialysis patients to correlate results with intima plaque formation and systemic inflammation. RESULTS: Compared to healthy subjects, dialysis patients have fewer classical and more intermediate and non-classical monocytes. Post-translationally modified YB-1 (lysine 301/304 acetylation) is detected at high levels in the nucleus of adherent and invading CD14+CD68+ monocytes from umbilical cord and atherosclerosis-prone vessels. The content of non-acetylated YB-1 is significantly decreased (pâ¯<â¯0.001), whereas acetylated YB-1 is correspondingly increased (pâ¯<â¯0.001) throughout all monocyte subpopulations, such that the overall content remains unchanged. CONCLUSIONS: In dialysis patients the YB-1 acetylation status is higher with prevailing diabetes and intima plaque formation. Pro-inflammatory mediators TNFα, IL-6, uPAR, CCL2, M-CSF, progranulin, ANP, and midkine, as well as anti-inflammatory IL-10 are significantly increased in dialysis patients, emphasizing a systemic inflammatory milieu. Strong positive correlations of monocytic YB-1 content are seen with ANP, IP-10, IL-6, and IL-10 serum levels. This is the first study demonstrating an association of cold shock protein YB-1 expression with inflammation in hemodialysis patients.
Subject(s)
Inflammation/blood , Monocytes/metabolism , Vascular Diseases/blood , Y-Box-Binding Protein 1/metabolism , Acetylation , Adult , Aged , Aged, 80 and over , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Atherosclerosis , Carotid Intima-Media Thickness , Cold-Shock Response , Comorbidity , Cytokines/blood , Epitopes , Female , Humans , Ligands , Lipopolysaccharide Receptors/metabolism , Male , Middle Aged , Protein Processing, Post-Translational , Renal Dialysis/adverse effects , Tunica Intima/pathology , Umbilical Cord/metabolism , Young AdultABSTRACT
The virulence of intracellular pathogens such as Leishmania major (L. major) relies largely on their ability to undergo cycles of replication within phagocytes, release, and uptake into new host cells. While all these steps are critical for successful establishment of infection, neither the cellular niche of efficient proliferation, nor the spread to new host cells have been characterized in vivo. Here, using a biosensor for measuring pathogen proliferation in the living tissue, we found that monocyte-derived Ly6C+CCR2+ phagocytes expressing CD11c constituted the main cell type harboring rapidly proliferating L. major in the ongoing infection. Synchronization of host cell recruitment and intravital 2-photon imaging showed that these high proliferating parasites preferentially underwent cell-to-cell spread. However, newly recruited host cells were infected irrespectively of their cell type or maturation state. We propose that among these cells, CD11c-expressing monocytes are most permissive for pathogen proliferation, and thus mainly fuel the cycle of intracellular proliferation and cell-to-cell transfer during the acute infection. Thus, besides the well-described function for priming and activating T cell effector functions against L. major, CD11c-expressing monocyte-derived cells provide a reservoir for rapidly proliferating parasites that disseminate at the site of infection.
Subject(s)
Antigens, Ly/immunology , CD11c Antigen/metabolism , Cell Proliferation , Leishmania major/immunology , Leishmaniasis/parasitology , Monocytes/virology , Receptors, CCR2/immunology , Animals , Antigens, Ly/metabolism , Cells, Cultured , DNA Replication , Leishmania major/genetics , Leishmaniasis/immunology , Leishmaniasis/metabolism , Leishmaniasis/transmission , Mice , Mice, Inbred C57BL , Monocytes/immunology , Receptors, CCR2/metabolism , VirulenceABSTRACT
The bone marrow (BM) consists of multiple, structured micro-environmental entities-the so called niches, which contain hematopoietic cells as well as stromal cells. These niches fulfill a variety of functions, such as control of the hematopoietic stem cell pool, differentiation of hematopoietic cells, and maintenance of immunological memory. However, due to the molecular and cellular complexity and a lack of suitable histological multiplexing methods, the composition of the various BM niches is still elusive. In this study, we apply multiepitope-ligand-cartography (MELC) on bone sections from mice. We combine multiplexed immunofluorescence histology data with various object-based segmentation approaches in order to define irregularly shaped, net-like structures of stromal cells. We confirm MELC as a robust histological method and validate our automated segmentation algorithms using flow cytometry and manual evaluation. By means of MELC multiplexing, we reveal heterogeneous expression of leptin receptor (LpR), BP-1, and VCAM-1 in the stromal network. Moreover, we demonstrate by quantification a preferential contact of B cell subsets as well as of plasma cells to processes of CXCL12-expressing stromal cells, compared with stromal somata. In summary, our approach is suitable for spatial analysis of complex tissue structures.
Subject(s)
Bone Marrow Cells/cytology , Bone Marrow/physiology , Stromal Cells/cytology , Animals , Bone Marrow/metabolism , Bone Marrow Cells/metabolism , Cells, Cultured , Chemokine CXCL12/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence/methods , Receptors, Leptin/metabolism , Stromal Cells/metabolism , Transcription Factors/metabolism , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
JAK2-V617F-positive chronic myeloproliferative neoplasia (CMN) commonly displays dysfunction of integrins and adhesion molecules expressed on platelets, erythrocytes, and leukocytes. However, the mechanism by which the 2 major leukocyte integrin chains, ß1 and ß2, may contribute to CMN pathophysiology remained unclear. ß1 (α4ß1; VLA-4) and ß2 (αLß2; LFA-1) integrins are essential regulators for attachment of leukocytes to endothelial cells. We here showed enhanced adhesion of granulocytes from mice with JAK2-V617F knockin (JAK2+/VF mice) to vascular cell adhesion molecule 1- (VCAM1-) and intercellular adhesion molecule 1-coated (ICAM1-coated) surfaces. Soluble VCAM1 and ICAM1 ligand binding assays revealed increased affinity of ß1 and ß2 integrins for their respective ligands. For ß1 integrins, this correlated with a structural change from the low- to the high-affinity conformation induced by JAK2-V617F. JAK2-V617F triggered constitutive activation of the integrin inside-out signaling molecule Rap1, resulting in translocation toward the cell membrane. Employing a venous thrombosis model, we demonstrated that neutralizing anti-VLA-4 and anti-ß2 integrin antibodies suppress pathologic thrombosis as observed in JAK2+/VF mice. In addition, aberrant homing of JAK2+/VF leukocytes to the spleen was inhibited by neutralizing anti-ß2 antibodies and by pharmacologic inhibition of Rap1. Thus, our findings identified cross-talk between JAK2-V617F and integrin activation promoting pathologic thrombosis and abnormal trafficking of leukocytes to the spleen.
Subject(s)
CD18 Antigens/metabolism , Integrin beta1/metabolism , Janus Kinase 2/metabolism , Mutation, Missense , Venous Thrombosis/metabolism , Amino Acid Substitution , Animals , CD18 Antigens/genetics , Cell Adhesion , Integrin alpha4beta1/genetics , Integrin alpha4beta1/metabolism , Integrin beta1/genetics , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Janus Kinase 2/genetics , Leukocytes/metabolism , Leukocytes/pathology , Lymphocyte Function-Associated Antigen-1/genetics , Lymphocyte Function-Associated Antigen-1/metabolism , Mice , Mice, Mutant Strains , Spleen/metabolism , Spleen/pathology , Venous Thrombosis/genetics , Venous Thrombosis/pathology , rap1 GTP-Binding Proteins/genetics , rap1 GTP-Binding Proteins/metabolismABSTRACT
Regulatory T (Treg) cells require T-cell receptor (TCR) signalling to exert their immunosuppressive activity, but the precise organization of the TCR signalling network compared to conventional T (Tconv) cells remains elusive. By using accurate mass spectrometry and multi-epitope ligand cartography (MELC) we characterized TCR signalling and recruitment of TCR signalling components to the immunological synapse (IS) in Treg cells and Tconv cells. With the exception of Themis which we detected in lower amounts in Treg cells, other major TCR signalling components were found equally abundant, however, their phosphorylation-status notably discriminates Treg cells from Tconv cells. Overall, this study identified 121 Treg cell-specific phosphorylations. Short-term triggering of T cell subsets via CD3 and CD28 widely harmonized these variations with the exception of eleven TCR signalling components that mainly regulate cytoskeleton dynamics and molecular transport. Accordingly, conjugation with B cells indeed caused variant cellular morphology and revealed a Treg cell-specific recruitment of TCR signalling components such as PKCθ, PLCγ1 and ZAP70 as well as B cell-derived CD86 into the IS. Together, results from this study support the existence of a Treg cell-specific IS and suggest Treg cell-specific cytoskeleton dynamics as a novel determinant for the unique functional properties of Treg cells.
Subject(s)
Immunological Synapses/immunology , Receptors, Antigen, T-Cell/immunology , Signal Transduction/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Cells, Cultured , Female , Mice, Inbred BALB C , Microscopy, Fluorescence , Phosphorylation , Proteome/immunology , Proteome/metabolism , Proteomics/methods , Receptors, Antigen, T-Cell/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Regulatory/metabolism , ZAP-70 Protein-Tyrosine Kinase/immunology , ZAP-70 Protein-Tyrosine Kinase/metabolismABSTRACT
Tubular cells recruit monocytic cells in inflammatory tubulointerstitial kidney diseases. The cell-cell communication that establishes pro- or anti-inflammatory activities is mainly influenced by cytokines, reactive oxygen species, nitric oxide, and phagocytosis. Key proteins orchestrating these processes such as cold-shock proteins linked with chemoattraction and cell maturation have been identified. The prototypic member of the cold-shock protein family, Y-box binding protein (YB)-1, governs specific phenotypic alterations in monocytic cells and was explored in the present study. Following tubulointerstitial injury by unilateral ureteral obstruction, increased inflammatory cell infiltration and tubular cell CCL5 expression was found in conditional Ybx1 knockout animals with specific depletion in monocytes/macrophages (YB-1ΔLysM). Furthermore, YB-1ΔLysM mice exhibit enhanced tissue damage, myofibroblast activation, and fibrosis. To investigate relevant molecular mechanism(s), we utilized bone marrow-derived macrophage cultures and found that YB-1-deficient macrophages display defects in cell polarization and function, including reduced proliferation and nitric oxide production, loss of phagocytic activity, and failure to upregulate IL-10 and CCL5 expression in response to inflammatory stimuli. Co-culture with primary tubular cells confirmed these findings. Thus, monocytic YB-1 has prominent and distinct roles for cellular feed-forward crosstalk and resolution of inflammatory processes by its ability to regulate cell differentiation and cytokine/chemokine synthesis.
Subject(s)
Cell Differentiation , DNA-Binding Proteins/metabolism , Kidney Tubules/pathology , Monocytes/pathology , Nephritis, Interstitial/pathology , Animals , Cell Communication , Chemokine CCL5/metabolism , Coculture Techniques , DNA-Binding Proteins/genetics , Disease Models, Animal , Disease Progression , Female , Fibrosis , Humans , Interleukin-10/metabolism , Kidney Tubules/cytology , Macrophages , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Monocytes/metabolism , Myofibroblasts/metabolism , Myofibroblasts/pathology , Primary Cell CultureABSTRACT
Hepatotropic viruses such as hepatitis C virus cause life-threatening chronic liver infections in millions of people worldwide. Targeted in vivo antigen-delivery to cross-presenting dendritic cells (DCs) has proven to be extraordinarily efficient in stimulating antigen-specific T cell responses. To determine whether this approach would as well be suitable to induce local antiviral effector T cells in the liver we compared different vaccine formulations based on either the targeting of DEC-205 or TLR2/6 on cross-presenting DCs or formulations not involving in vivo DC targeting. As read-outs we used in vivo hepatotropic adenovirus challenge, histology and automated multidimensional fluorescence microscopy (MELC). We show that targeted in vivo antigen delivery to cross-presenting DCs is highly effective in inducing antiviral CTLs capable of eliminating virus-infected hepatocytes, while control vaccine formulation not involving DC targeting failed to induce immunity against hepatotropic virus. Moreover, we observed distinct patterns of CD8+ T cell interaction with virus-infected and apoptotic hepatocytes in the two DC-targeting groups suggesting that the different vaccine formulations may stimulate distinct types of effector functions. Our findings represent an important step toward the future development of vaccines against hepatotropic viruses and the treatment of patients with hepatic virus infection after liver transplantation to avoid reinfection.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Immunization/methods , Liver/immunology , Ovalbumin/immunology , Animals , Antigens, CD/metabolism , Cross-Priming , Female , Lectins, C-Type/metabolism , Liver/pathology , Mice, Inbred C57BL , Minor Histocompatibility Antigens/metabolism , Receptors, Cell Surface/metabolism , Toll-Like Receptors/metabolismABSTRACT
The enzymatic activity of the Src family tyrosine kinase p56Lck (Lck) is tightly controlled by differential phosphorylation of two tyrosine residues, Tyr394 and Tyr505 Phosphorylation of Tyr394 and the conformational opening of Lck are believed to activate the kinase, whereas Tyr505 phosphorylation is thought to generate a closed, inactive conformation of Lck. We investigated whether the conformation of Lck and its phosphorylation state act in concert to regulate the initiation of T cell receptor (TCR) signaling. With a sensitive biosensor, we used fluorescence lifetime imaging microscopy (FLIM) to investigate the conformations of wild-type Lck and its phosphorylation-deficient mutants Y394F and Y505F and the double mutant Y394F/Y505F in unstimulated T cells and after TCR stimulation. With this approach, we separated the conformational changes of Lck from the phosphorylation state of its regulatory tyrosines. We showed that the conformational opening of Lck alone was insufficient to initiate signaling events in T cells. Rather, Lck additionally required phosphorylation of Tyr394 to induce T cell activation. Consistent with the FLIM measurements, an optimized immunofluorescence microscopy protocol revealed that the TCR-stimulated phosphorylation of Lck at Tyr394 occurred preferentially at the plasma membrane of Jurkat cells and primary human T cells. Our study supports the hypothesis that T cell activation through the TCR complex is accompanied by the de novo activation of Lck and that phosphorylation of Tyr394 plays a role in Lck function that goes beyond inducing an open conformation of the kinase.
Subject(s)
Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , Blotting, Western , Cell Membrane/metabolism , Cells, Cultured , Humans , Jurkat Cells , Lymphocyte Activation , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/chemistry , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Microscopy, Confocal , Microscopy, Fluorescence , Mutation , Phosphorylation , Protein Conformation , T-Lymphocytes/metabolism , Tyrosine/chemistry , Tyrosine/genetics , Tyrosine/metabolismABSTRACT
The ß2-integrin lymphocyte function-associated antigen 1 (LFA-1) is needed for the T cell receptor (TCR)-induced activation of LFA-1 to promote T cell adhesion and interaction with antigen-presenting cells (APCs). LFA-1-mediated cell-cell interactions are critical for proper T cell differentiation and proliferation. The Src kinase-associated phosphoprotein of 55 kDa (SKAP55) is a key regulator of TCR-mediated LFA-1 signaling (inside-out/outside-in signaling). To gain an understanding of how SKAP55 controls TCR-mediated LFA-1 activation, we assessed the functional role of its pleckstrin homology (PH) domain. We identified two critical amino acid residues within the PH domain of SKAP55, aspartic acid 120 (D120) and lysine 152 (K152). D120 facilitates the retention of SKAP55 in the cytoplasm of nonstimulated T cells, while K152 promotes SKAP55 membrane recruitment via actin binding upon TCR triggering. Importantly, the K152-dependent interaction of the PH domain with actin promotes the binding of talin to LFA-1, thus facilitating LFA-1 activation. These data suggest that K152 and D120 within the PH domain of SKAP55 regulate plasma membrane targeting and TCR-mediated activation of LFA-1.
Subject(s)
Aspartic Acid/metabolism , Cell Membrane/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , Lysine/metabolism , Phosphoproteins/chemistry , Phosphoproteins/metabolism , T-Lymphocytes/metabolism , Actins/metabolism , Cell Adhesion , Humans , Jurkat Cells , Lipids/chemistry , Mutant Proteins/metabolism , Mutation/genetics , Phosphatidylinositol Phosphates/metabolism , Protein Domains , Receptors, Antigen, T-Cell/metabolism , Structure-Activity Relationship , Talin/metabolism , rap GTP-Binding Proteins/metabolismABSTRACT
The deposition of immune complexes is the causal factor in distinct renal pathologies, e.g., lupus nephritis and membranous nephritis. The location of these deposits within a tissue biopsy is often the key to establishing a diagnosis. However, how immune complexes come to be deposited below the vascular endothelium was, until now, a mystery, as was their contribution to inducing inflammation. A recent paper in Cell by Stamatiades et al. (Cell 164(4):991-1003, 2016) demonstrates the active transport of immune complexes by the vascular endothelial cells and an Fc receptor-dependent uptake by tissue-resident macrophages. This leads to the activation of these macrophages and the release of pro-inflammatory cytokines, which in turn recruits immune cells from the blood into the kidney. The identification of these mechanisms should lead to a better stratification of kidney diseases and hopefully to the development of specific therapies.