ABSTRACT
Asthma is a T helper 2 (Th2)-cell-mediated disease; however, recent findings implicate Th17 and innate lymphoid cells also in regulating airway inflammation. Herein, we have demonstrated profound interleukin-21 (IL-21) production after house dust mite (HDM)-driven asthma by using T cell receptor (TCR) transgenic mice reactive to Dermatophagoides pteronyssinus 1 and an IL-21GFP reporter mouse. IL-21-producing cells in the mediastinal lymph node (mLN) bore characteristics of T follicular helper (Tfh) cells, whereas IL-21(+) cells in the lung did not express CXCR5 (a chemokine receptor expressed by Tfh cells) and were distinct from effector Th2 or Th17 cells. Il21r(-/-) mice developed reduced type 2 responses and the IL-21 receptor (IL-21R) enhanced Th2 cell function in a cell-intrinsic manner. Finally, administration of recombinant IL-21 and IL-25 synergistically promoted airway eosinophilia primarily via effects on CD4(+) lymphocytes. This highlights an important Th2-cell-amplifying function of IL-21-producing CD4(+) T cells in allergic airway inflammation.
Subject(s)
Asthma/immunology , CD4-Positive T-Lymphocytes/immunology , Eosinophilia/immunology , Eosinophils/drug effects , Lung/immunology , Receptors, Interleukin-21/administration & dosage , Th2 Cells/immunology , Animals , Antigens, Dermatophagoides/immunology , Arthropod Proteins/immunology , Cells, Cultured , Cysteine Endopeptidases/immunology , Eosinophils/immunology , Immunity, Cellular , Interleukins/administration & dosage , Mice , Mice, Inbred C57BL , Mice, Knockout , Pyroglyphidae/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, CXCR5/metabolism , Receptors, Interleukin-21/geneticsABSTRACT
BACKGROUND: The emergence of IL-33 as a key molecular player in the development and propagation of widespread inflammatory diseases, including asthma and atopic dermatitis, has established the need for effective IL-33-neutralizing biologics. OBJECTIVE: Here we describe the development and validation of a new antagonist of IL-33, termed IL-33trap, which combines the extracellular domains of the IL-33 receptor (ST2) and its coreceptor, IL-1 receptor accessory protein, into a single fusion protein. METHODS: We produced and purified recombinant IL-33trap from human cells and analyzed its IL-33-binding affinity and IL-33 antagonistic activity in cultured cells and mice. IL-33trap activity was also benchmarked with a recombinant soluble ST2 corresponding to the naturally occurring IL-33 decoy receptor. Finally, we studied the effect of IL-33trap in the Alternaria alternata mouse model of allergic airway inflammation. RESULTS: In vitro IL-33trap binds IL-33 and inhibits IL-33 activity to a much stronger degree than soluble ST2. Furthermore, IL-33trap inhibits eosinophil infiltration, splenomegaly, and production of signature cytokines in splenic lymphocytes and lung tissue on IL-33 injection. Finally, administration of IL-33trap at the time of allergen challenge inhibits inflammatory responses in a preclinical mouse model of acute allergic airway inflammation. CONCLUSIONS: IL-33trap is a novel IL-33 antagonist that outperforms the natural IL-33 decoy receptor and shows anti-inflammatory activities in a preclinical mouse model of acute allergic airway inflammation when administered at the time of allergen challenge.
Subject(s)
Asthma/drug therapy , Biological Products/therapeutic use , Interleukin-33/antagonists & inhibitors , Alternaria/immunology , Animals , Asthma/immunology , Biological Products/pharmacology , Cells, Cultured , Eosinophils/drug effects , Eosinophils/immunology , HEK293 Cells , Humans , Interleukin-33/immunology , Lung/drug effects , Lung/immunology , Lymphocytes/drug effects , Lymphocytes/immunology , Mice , Mice, Inbred C57BL , RAW 264.7 Cells , Spleen/drug effects , Spleen/immunologyABSTRACT
RATIONALE: Chronic rhinosinusitis with nasal polyps is characterized by a T-helper cell type 2-skewed upper airway inflammation. Mucosal Staphylococcus aureus colonization is found in the majority of patients with nasal polyps. S. aureus is known to induce type 2 cytokine release via enterotoxins. OBJECTIVES: To investigate the impact of non-enterotoxin-producing S. aureus on type 2 cytokine release. METHODS: TSLP (thymic stromal lymphopoietin), IL-33, and type 2 cytokines were assessed in a human mucosal tissue model upon S. aureus infection. MEASUREMENTS AND MAIN RESULTS: S. aureus exposure increased the expression of IL-33, TSLP, IL-5, and IL-13 in nasal polyp tissue, accompanied by elevated expression levels of TSLP and IL-33 receptors, predominantly on CD3+ T cells. S. aureus infection led to the release of TSLP, but not IL-33, IL-5, or IL-13, from healthy inferior turbinate tissue. In contrast, S. epidermidis did not induce any epithelial cell-derived cytokines in nasal polyp or healthy tissue. S. aureus infection also increased the release of IL-33 and TSLP in BEAS-2B epithelial cells, accompanied by activation of NF-κB (nuclear factor κB) pathways. Incubation with CU-CPT22, a specific Toll-like receptor 2 antagonist, significantly reduced the S. aureus-induced release of TSLP and IL-33, and the activity of the NF-κB signal in BEAS-2B cells. CONCLUSIONS: This study demonstrates for the first time that S. aureus can directly induce epithelial cell-derived cytokine release via binding to Toll-like receptor 2, and may thereby propagate type 2 cytokine expression in nasal polyp tissue.
Subject(s)
Cytokines/immunology , Epithelial Cells/immunology , Nasal Mucosa/immunology , Nasal Polyps/immunology , Staphylococcal Infections/immunology , Staphylococcus aureus/immunology , Adolescent , Adult , Aged , Belgium , Female , Humans , Male , Middle Aged , Young AdultABSTRACT
BACKGROUND: Chronic airway inflammatory diseases, such as chronic rhinosinusitis with nasal polyps and asthma, show increased nasal Staphylococcus aureus colonization. Staphylococcus aureus-derived serine protease-like protein (Spl) D and other closely related proteases secreted by S aureus have recently been identified as inducers of allergic asthma in human subjects and mice, but their mechanism of action is largely unknown. OBJECTIVE: We investigated the role of recombinant SplD in driving TH2-biased responses and IgE formation in a murine model of allergic asthma. METHODS: Allergic asthma was induced in C57BL/6 J wild-type mice, Toll-like receptor (TLR) 4 knockout (Tlr4-/-) mice, and recombination-activating gene (Rag2) knockout (Rag2-/-) mice by means of repeated intratracheal applications of SplD. Inflammatory parameters in the airways were assessed by means of flow cytometry, ELISA, Luminex, and immunohistochemistry. Serum SplD-specific IgE levels were analyzed by using ELISA. RESULTS: We observed that repeated intratracheal exposure to SplD led to IL-33 and eotaxin production, eosinophilia, bronchial hyperreactivity, and goblet cell hyperplasia in the airways. Blocking IL-33 activity with a soluble ST2 receptor significantly decreased the numbers of eosinophils, IL-13+ type 2 innate lymphoid cells and IL-13+CD4+ T cells and IL-5 and IL-13 production by lymph node cells but had no effect on IgE production. SplD-induced airway inflammation and IgE production were largely dependent on the presence of the functional adaptive immune system and independent of TLR4 signaling. CONCLUSION: The S aureus-derived protein SplD is a potent allergen of S aureus and induces a TH2-biased inflammatory response in the airways in an IL-33-dependent but TRL4-independent manner. The soluble ST2 receptor could be an efficient strategy to interfere with SplD-induced TH2 inflammation but does not prevent the allergic sensitization.
Subject(s)
Asthma/immunology , Bacterial Proteins/toxicity , Interleukin-1 Receptor-Like 1 Protein/immunology , Interleukin-33/immunology , Serine Proteases/toxicity , Staphylococcus aureus/immunology , Animals , Asthma/chemically induced , Asthma/genetics , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , Disease Models, Animal , Interleukin-1 Receptor-Like 1 Protein/genetics , Interleukin-33/genetics , Mice , Mice, Knockout , Signal Transduction/drug effects , Signal Transduction/genetics , Staphylococcus aureus/pathogenicity , Th2 Cells/immunology , Th2 Cells/pathologyABSTRACT
BACKGROUND: Clinical and experimental studies have identified a crucial role for IL-33 and its receptor ST2 in allergic asthma. Inhalation of traffic-related pollutants, such as diesel exhaust particles (DEP), facilitates the development of asthma and can cause exacerbations of asthma. However, it is unknown whether IL-33/ST2 signalling contributes to the enhancing effects of air pollutants on allergic airway responses. OBJECTIVE: We aim to investigate the functional role of IL-33/ST2 signalling in DEP-enhanced allergic airway responses, using an established murine model. METHODS: C57BL/6J mice were exposed to saline, DEP alone, house dust mite (HDM) alone or combined DEP+HDM. To inhibit IL-33 signalling, recombinant soluble ST2 (r-sST2) was given prophylactically (ie, during the whole experimental protocol) or therapeutically (ie, at the end of the experimental protocol). Airway hyperresponsiveness and the airway inflammatory responses were assessed in bronchoalveolar lavage fluid (BALF) and lung. RESULTS: Combined exposure to DEP+HDM increased IL-33 and ST2 expression in lung, elevated inflammatory responses and bronchial hyperresponsiveness compared to saline, sole DEP or sole HDM exposure. Prophylactic interference with the IL-33/ST2 signalling pathway impaired the DEP-enhanced allergic airway inflammation in the BALF, whereas effects on lung inflammation and airway hyperresponsiveness were minimal. Treatment with r-sST2 at the end of the experimental protocol did not modulate the DEP-enhanced allergic airway responses. CONCLUSION: Our data suggest that the IL-33/ST2 pathway contributes to the onset of DEP-enhanced allergic airway inflammation.
Subject(s)
Air Pollutants/adverse effects , Interleukin-33/metabolism , Respiratory Hypersensitivity/etiology , Respiratory Hypersensitivity/metabolism , Signal Transduction , Allergens/immunology , Animals , Biomarkers , Disease Models, Animal , Female , Interleukin-1 Receptor-Like 1 Protein/metabolism , Leukocytes/immunology , Leukocytes/metabolism , Lymphocytes/immunology , Lymphocytes/metabolism , Mice , Particulate Matter/adverse effects , Pyroglyphidae/immunology , Recombinant Proteins/pharmacology , Respiratory Hypersensitivity/drug therapy , Respiratory Hypersensitivity/pathology , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolismABSTRACT
Caspase recruitment domain (CARD)-containing protein 14 (CARD14) is an intracellular protein that mediates nuclear factor-kappa B (NF-ĸB) signaling and proinflammatory gene expression in skin keratinocytes. Several hyperactivating CARD14 mutations have been associated with psoriasis and other inflammatory skin diseases. CARD14-induced NF-ĸB signaling is dependent on the formation of a CARD14-BCL10-MALT1 (CBM) signaling complex, but upstream receptors and molecular mechanisms that activate and regulate CARD14 signaling are still largely unclear. Using unbiased affinity purification and mass spectrometry (AP-MS) screening, we discover polo-like kinase 1 (PLK1) as a novel CARD14-binding protein. CARD14-PLK1 binding is independent of the CARD14 CARD domain but involves a consensus phospho-dependent PLK1-binding motif in the CARD14 linker region (LR). Expression of the psoriasis-associated CARD14(E138A) variant in human keratinocytes induces the recruitment of PLK1 to CARD14-containing signalosomes in interphase cells, but does not affect the specific location of PLK1 in mitotic cells. Finally, disruption of the PLK1-binding motif in CARD14(E138A) increases CARD14-induced proinflammatory signaling and gene expression. Together, our data identify PLK1 as a novel CARD14-binding protein and indicate a negative regulatory role for PLK1 in CARD14 signaling.
Subject(s)
CARD Signaling Adaptor Proteins , Cell Cycle Proteins , Keratinocytes , Polo-Like Kinase 1 , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins , Humans , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Keratinocytes/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , CARD Signaling Adaptor Proteins/metabolism , CARD Signaling Adaptor Proteins/genetics , Protein Binding/physiology , Guanylate Cyclase/metabolism , Guanylate Cyclase/genetics , HEK293 Cells , Membrane ProteinsABSTRACT
Background: Recent in vitro studies strongly implicated mast cell-derived proteases as regulators of IL-33 activity by enzymatic cleavage in its central domain. A better understanding of the role of mast cell proteases on IL-33 activity in vivo is needed. We aimed to compare the expression of mast cell proteases in C57BL/6 and BALB/c mice, their role in the cleavage of IL-33 cytokine, and their contribution to allergic airway inflammation. Results: In vitro, full-length IL-33 protein was efficiently degraded by mast cell supernatants of BALB/c mice in contrast to the mast cell supernatants from C57BL/6 mice. RNAseq analysis indicated major differences in the gene expression profiles of bone marrow-derived mast cells from C57BL/6 and BALB/c mice. In Alternaria alternata (Alt) - treated C57BL/6 mice the full-length form of IL-33 was mainly present, while in BALB/c mice, the processed shorter form of IL-33 was more prominent. The observed cleavage pattern of IL-33 was associated with a nearly complete lack of mast cells and their proteases in the lungs of C57BL/6 mice. While most inflammatory cells were similarly increased in Alt-treated C57BL/6 and BALB/c mice, C57BL/6 mice had significantly more eosinophils in the bronchoalveolar lavage fluid and IL-5 protein levels in their lungs than BALB/c mice. Conclusion: Our study demonstrates that lung mast cells differ in number and protease content between the two tested mouse strains and could affect the processing of IL-33 and inflammatory outcome of Alt -induced airway inflammation. We suggest that mast cells and their proteases play a regulatory role in IL-33-induced lung inflammation by limiting its proinflammatory effect via the IL-33/ST2 signaling pathway.
Subject(s)
Interleukin-33 , Peptide Hydrolases , Animals , Mice , Interleukin-33/metabolism , Peptide Hydrolases/metabolism , Mast Cells/metabolism , Mice, Inbred C57BL , Inflammation/metabolism , Endopeptidases/metabolismABSTRACT
PURPOSE: In simultaneous positron emission tomography/magnetic resonance (PET/MR) imaging, local receiver surface radiofrequency (RF) coils are positioned in the field-of-view (FOV) of the PET detector during PET/MR data acquisition and potentially attenuate the PET signal. For flexible body RF surface coils placed on top of the patient's body, MR-based attenuation correction (AC) is an unsolved problem since the RF coils are not inherently visible in MR images and their individual position in the FOV is patient specific and not known a priori. The aim of this work was to quantify the effect of local body RF coils used in the Biograph mMR hybrid PET/MR system on PET emission data and to present techniques for MR-based position determination of these specific local RF coils. METHODS: Acquisitions of a homogeneous phantom were performed on a whole-body PET/MRI scanner. Two different PET emission scans were performed, with and without the local body matrix RF coil placed on the top of the phantom. For position determination of the coil, two methods were applied. First, cod liver oil capsules were attached to the surface of the coil and second, an ultrashort echo time (UTE) sequence was used. PET images were reconstructed in five different ways: (1) PET reference scan without the coil, (2) PET scan with the coil, but omitting the coil in AC (PET/MR scanning conditions), (3) AC of the coil using a CT scan of the same phantom setup and registration via capsules, (4) same setup as 3, but registration was done using UTE images, neglecting the capsules, and (5) registration using the capsules, but the CT was performed with the coil placed flat on the CT table and the outer regions of the coil were cropped. The activity concentrations were then compared to the reference scan. For clinical evaluation of the concept, the presented methods were also evaluated on a patient. RESULTS: The oil capsules were visible in the MR and CT images and image registration was straightforward. The UTE images show only parts of the coil's plastic housing and image registration was more difficult. The overall loss of true counts due to the presence of the surface coil is 4.7%. However, a spatially dependent analysis shows larger deviation (10%-15% attenuation) of the activity concentration in the top part of the phantom close to the coil. When accounting for the RF coil for PET AC, attenuation due to the RF coil could mostly be corrected. These results of the phantom studies were confirmed by the patient measurements. CONCLUSIONS: Disregarding local coils in PET AC can lead to a bias of the AC PET images that is regional dependent. The closer the analyzed region is located to the coil, the higher the bias. Cod liver oil capsules or the UTE sequence can be used for RF coil position determination. The middle part of the examined RF coil hosting the preamplifiers and electronic components provides the highest attenuating part. Consequently, emphasis should be put on correcting for this portion of the RF coils with the suggested methods.
Subject(s)
Artifacts , Image Enhancement/instrumentation , Magnetic Resonance Imaging/instrumentation , Magnetics/instrumentation , Patient Positioning/methods , Positron-Emission Tomography/instrumentation , Subtraction Technique/instrumentation , Equipment Design , Equipment Failure Analysis , Humans , Magnetic Resonance Imaging/methods , Phantoms, Imaging , Positron-Emission Tomography/methods , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
PURPOSE: With the recent introduction of integrated whole-body hybrid positron emission tomography/magnetic resonance imaging (PET/MRI) scanners, the need for data collection strategies arises that provide time efficient, simultaneous, and easy acquisition of PET and MRI data. One approach is to develop acquisition protocols with continuous table motion. In this work, a reconstruction technique to allow for reconstruction of PET data that were acquired with continuous table motion on an integrated hybrid whole-body PET/MRI scanner is presented and evaluated. METHODS: PET and MRI data of two quality-control phantoms ((68)Ge-Cylinder and Jasczcak phantom), a custom-built large body phantom, and of a clinical patient were acquired on a Biograph mMR 3.0 Tesla whole-body PET/MRI system with continuous table motion and for comparison with the standard multistation acquisition approach. The data were postprocessed and reconstructed offline with custom software to allow for continuous table motion acquisition and analyzed with respect to noise, spatial resolution, and geometric accuracy as well as subjective image impression. RESULTS: It is shown that data acquisition with continuous table motion is equivalent and in some respects, superior to the traditional multistation approach. CONCLUSIONS: Continuous table motion can benefit the new hybrid modality PET/MRI by not being limited to several static bed positions, resulting in a better time efficiency, less continuity artifacts, and a faster and easier acquisition workflow.
Subject(s)
Magnetic Resonance Imaging/instrumentation , Motion , Positron-Emission Tomography/instrumentation , Artifacts , Female , Humans , Image Processing, Computer-Assisted , Phantoms, Imaging , Time Factors , Young AdultABSTRACT
BACKGROUND: In erythroblasts, the CoREST repressor complex is recruited to target promoters by the transcription factor Gfi1b, leading to repression of genes mainly involved in erythroid differentiation. Hmg20b is a subunit of CoREST, but its role in erythropoiesis has not yet been established. DESIGN AND METHODS: To study the role of Hmg20b in erythropoiesis, we performed knockdown experiments in a differentiation-competent mouse fetal liver cell line, and in primary mouse fetal liver cells. The effects on globin gene expression were determined. We used microarrays to investigate global gene expression changes induced by Hmg20b knockdown. Functional analysis was carried out on Hrasls3, an Hmg20b target gene. RESULTS: We show that Hmg20b depletion induces spontaneous differentiation. To identify the target genes of Hmg20b, microarray analysis was performed on Hmg20b knockdown cells and controls. In line with its association to the CoREST complex, we found that 85% (527 out of 620) of the deregulated genes are up-regulated when Hmg20b levels are reduced. Among the few down-regulated genes was Gfi1b, a known repressor of erythroid differentiation. Among the consistently up-regulated targets were embryonic ß-like globins and the phospholipase HRAS-like suppressor 3 (Hrasls3). We show that Hrasls3 expression is induced during erythroid differentiation and that knockdown of Hrasls3 inhibits terminal differentiation of proerythroblasts. CONCLUSIONS: We conclude that Hmg20b acts as an inhibitor of erythroid differentiation, through the down-regulation of genes involved in differentiation such as Hrasls3, and activation of repressors of differentiation such as Gfi1b. In addition, Hmg20b suppresses embryonic ß-like globins.
Subject(s)
Erythroid Cells/cytology , Erythroid Cells/metabolism , Erythropoiesis/genetics , High Mobility Group Proteins/metabolism , Repressor Proteins/metabolism , Animals , Cell Cycle Proteins , Cell Line, Tumor , Cells, Cultured , Co-Repressor Proteins , DNA-Binding Proteins , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , HeLa Cells , High Mobility Group Proteins/genetics , Humans , Mice , Multiprotein Complexes/metabolism , Nerve Tissue Proteins/metabolism , Phospholipases A2, Calcium-Independent/genetics , Proto-Oncogene Proteins/genetics , Repressor Proteins/geneticsABSTRACT
Future precision medicine requires further clarifying the mechanisms of inflammation in the severe endotypes of chronic airway diseases such as asthma and chronic rhinosinusitis (CRS). The presence of neutrophils in the airways is often associated with severe airway inflammation, while their precise contribution to the severe inflammation is largely unknown. We aimed to study the role of neutrophils in BALB/c and C57BL/6 mice exposed to Alternaria alternata (Alt). The mice were exposed to Alt extract for twelve hours or ten days to induce allergic airway inflammation. C57BL/6 mice exposed to Alt responded with eosinophilic infiltration and the characteristic IL-5 upregulation. In contrast, the inflammatory response to Alt extract in BALB/c mice was characterized by a neutrophilic response, high levels of G-CSF, and elastase in the lungs. The lack of neutrophils affected the processing of IL-33 in BALB/c mice, as was demonstrated by depletion of neutrophils through intraperitoneal injections of anti-Ly6G antibody. Our data identifies the key role of neutrophils in airway inflammation through IL-33 cleavage in the Alt-induced airway inflammation in mice, which could potentially underline the different endotypes in human disease.
Subject(s)
Allergens/immunology , Alternaria/immunology , Alternariosis/immunology , Asthma/immunology , Immunity, Innate , Interleukin-33/metabolism , Neutrophils/immunology , Rhinitis/immunology , Sinusitis/immunology , Alternariosis/microbiology , Animals , Asthma/microbiology , Disease Models, Animal , Female , Lung/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Rhinitis/microbiology , Sinusitis/microbiologyABSTRACT
Signal transduction typically displays a so-called bow-tie topology: Multiple receptors lead to multiple cellular responses but the signals all pass through a narrow waist of central signaling nodes. One such signaling node for several inflammatory and oncogenic signaling pathways is the CARD-CC/BCL10/MALT1 (CBM) complexes, which get activated by protein kinase C (PKC)-mediated phosphorylation of the caspase activation and recruitment domain (CARD)-coiled-coil domain (CC) component. In humans, there are four CARD-CC family proteins (CARD9, CARD10, CARD11, and CARD14) and 9 true PKC isozymes (α to ι). At this moment, less than a handful of PKC::CARD-CC relationships are known. In order to explore the biologically relevant combinatorial space out of all 36 potential permutations in this two-component signaling event, we made use of CARD10-deficient human embryonic kidney 293T cells for subsequent pairwise cotransfections of all CARD-CC family members and all activated PKCs. Upon analysis of NF-κB-dependent reporter gene expression, we could define specific PKC::CARD-CC relationships. Surprisingly, as many as 21 PKC::CARD-CC functional combinations were identified. CARD10 was responsive to most PKCs, while CARD14 was mainly activated by PKCδ. The CARD11 activation profile was most similar to that of CARD9. We also discovered the existence of mixed protein complexes between different CARD-CC proteins, which was shown to influence their PKC response profile. Finally, multiple PKCs were found to use a common phosphorylation site to activate CARD9, while additional phosphorylation sites contribute to CARD14 activation. Together, these data reveal the combinatorial space of PKC::CARD-CC signal transduction nodes, which will be valuable for future studies on the regulation of CBM signaling.
Subject(s)
B-Cell CLL-Lymphoma 10 Protein/genetics , CARD Signaling Adaptor Proteins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/genetics , NF-kappa B/genetics , Protein Kinase C/genetics , Amino Acid Sequence , Animals , B-Cell CLL-Lymphoma 10 Protein/metabolism , Binding Sites , CARD Signaling Adaptor Proteins/classification , CARD Signaling Adaptor Proteins/metabolism , Gene Expression Regulation , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein/metabolism , NF-kappa B/metabolism , Phosphorylation , Phylogeny , Plasmids/chemistry , Plasmids/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Isoforms/classification , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinase C/classification , Protein Kinase C/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , TransfectionABSTRACT
TAB2 and TAB3 bind to K63-linked polyubiquitin chains and recruit the critical kinase MAP3K7 (TAK1). The polyubiquitin-recruited TAK1/TAB2/TAB3 complex comes in close proximity with the IKK (IKKα/IKKß/IKKγ) complex, which is recruited to M1-linked polyubiquitin chains via the IKKγ (NEMO) component. Together, the two complexes activate the NF-κB family of transcription factors. NF-κB transcription factors are critical mediators of pro-inflammatory signals and must be tightly regulated at multiple levels. Recently, it was discovered that one such point of regulation occurs at the level of TAB2 and TAB3 protein stability by the deubiquitinase USP15. Comment on: https://doi.org/10.1111/febs.15202.
Subject(s)
Adaptor Proteins, Signal Transducing , NF-kappa B , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Gene Expression Regulation , I-kappa B Kinase , NF-kappa B/genetics , NF-kappa B/metabolism , Polyubiquitin/metabolism , Signal TransductionABSTRACT
Cytokines are small secreted proteins that among many functions also play key roles in the orchestration of inflammation in host defense and disease. Over the past years, a large number of biologics have been developed to target cytokines in disease, amongst which soluble receptor fusion proteins have shown some promise in pre-clinical studies. We have previously shown proof-of-concept for the therapeutic targeting of interleukin (IL)-33 in airway inflammation using a newly developed biologic, termed IL-33trap, comprising the ectodomains of the cognate receptor ST2 and the co-receptor IL-1RAcP fused into a single-chain recombinant fusion protein. Here we extend the biophysical and biological characterization of IL-33trap variants, and show that IL-33trap is a stable protein with a monomeric profile both at physiological temperatures and during liquid storage at 4°C. Reducing the N-glycan heterogeneity and complexity of IL-33trap via GlycoDelete engineering neither affects its stability nor its inhibitory activity against IL-33. We also report that IL-33trap specifically targets biologically active IL-33 splice variants. Finally, we document the generation and antagonistic activity of a single-chain IL-4/13trap, which inhibits both IL-4 and IL-13 signaling. Collectively, these results illustrate that single-chain soluble receptor fusion proteins against IL-4, IL-13, and IL-33 are novel biologics that might not only be of interest for research purposes and further interrogation of the role of their target cytokines in physiology and disease, but may also complement monoclonal antibodies for the treatment of allergic and other inflammatory diseases.
Subject(s)
Anti-Inflammatory Agents , Interleukin-33/antagonists & inhibitors , Recombinant Fusion Proteins , HEK293 Cells , Humans , Interleukin-13/antagonists & inhibitors , Interleukin-4/antagonists & inhibitorsABSTRACT
Immune checkpoint blockade immunotherapy delivers promising clinical results in colorectal cancer (CRC). However, only a fraction of cancer patients develop durable responses. The tumor microenvironment (TME) negatively impacts tumor immunity and subsequently clinical outcomes. Therefore, there is a need to identify other checkpoint targets associated with the TME. Early-onset factors secreted by stromal cells as well as tumor cells often help recruit immune cells to the TME, among which are alarmins such as IL-33. The only known receptor for IL-33 is stimulation 2 (ST2). Here we demonstrated that high ST2 expression is associated with poor survival and is correlated with low CD8+ T cell cytotoxicity in CRC patients. ST2 is particularly expressed in tumor-associated macrophages (TAMs). In preclinical models of CRC, we demonstrated that ST2-expressing TAMs (ST2+ TAMs) were recruited into the tumor via CXCR3 expression and exacerbated the immunosuppressive TME; and that combination of ST2 depletion using ST2-KO mice with anti-programmed death 1 treatment resulted in profound growth inhibition of CRC. Finally, using the IL-33trap fusion protein, we suppressed CRC tumor growth and decreased tumor-infiltrating ST2+ TAMs. Together, our findings suggest that ST2 could serve as a potential checkpoint target for CRC immunotherapy.
Subject(s)
Colorectal Neoplasms/immunology , Interleukin-1 Receptor-Like 1 Protein/immunology , Interleukin-33/immunology , Tumor Microenvironment/immunology , Tumor-Associated Macrophages/immunology , Animals , Cell Line, Tumor , Databases, Genetic , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Tumor-Associated Macrophages/cytologyABSTRACT
New monosaccharide-based lipid A analogues were rationally designed through MD-2 docking studies. A panel of compounds with two carboxylate groups as phosphates bioisosteres, was synthesized with the same glucosamine-bis-succinyl core linked to different unsaturated and saturated fatty acid chains. The binding of the synthetic compounds to purified, functional recombinant human MD-2 was studied by four independent methods. All compounds bound to MD-2 with similar affinities and inhibited in a concentration-dependent manner the LPS-stimulated TLR4 signaling in human and murine cells, while being inactive as TLR4 agonists when provided alone. A compound of the panel was tested in vivo and was not able to inhibit the production of proinflammatory cytokines in animals. This lack of activity is probably due to strong binding to serum albumin, as suggested by cell experiments in the presence of the serum. The interesting self-assembly property in solution of this type of compounds was investigated by computational methods and microscopy, and formation of large vesicles was observed by cryo-TEM microscopy.
Subject(s)
Glycolipids/chemistry , Lymphocyte Antigen 96/chemistry , Toll-Like Receptor 4/chemistry , Animals , Binding Sites , Glycolipids/metabolism , Glycolipids/pharmacology , Humans , Lymphocyte Antigen 96/metabolism , Macrophages/drug effects , Macrophages/metabolism , Mice , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , Protein Binding , Signal Transduction/drug effects , Structure-Activity Relationship , Toll-Like Receptor 4/antagonists & inhibitorsABSTRACT
We investigate the spectral properties of violet 405 nm (Al,In)GaN laser diodes (LDs). Depending on the substrate the LDs are grown on, the lasing spectra show significant differences. LDs grown on low dislocation GaN substrate have a broad spectrum with several longitudinal modes, while LDs grown on SiC substrate are lasing on a single longitudinal mode.With increasing current, the laser emission of LDs grown on SiC substrate jumps from one longitudinal mode to another (mode hopping), whereas GaN substrate LDs show a smooth but asymmetric mode comb. The different envelopes of these spectra can be understood by assuming a variation of the gain for each individual longitudinal mode. With a high spectral resolution setup, we measure the gain of each longitudinal mode, employing the Hakki-Paoli method. Measurements show a slightly fluctuating gain for the modes of GaN substrate LDs, but much larger fluctuations for LDs on SiC substrate. We carry out simulations of the longitudinal mode spectrum of (Al,In)GaN laser diodes using a rate equation model with nonlinear gain (self saturation, symmetric and asymmetric cross saturation) and including gain fluctuations. With a set of parameters which is largely identical for LDs on either substrate, the simulated spectra truly resemble those typical for LDs on GaN or SiC substrate.
ABSTRACT
(Al,In)GaN-based laser diodes with ridge widths broader than a few micrometer tend to show filamentation effects in the lateral direction. By time-resolved scanning near-field optical microscopy, we find different kinds of filaments depending on ridge width and lateral position. We investigate these effects systematically and compare them to the results of corresponding simulations, which are based on a simple rate equation model including the lateral dimension. By this comparison we find a consistent and reasonable set of material parameters that can describe the laser diode. Furthermore, we discuss several reasons for filamentation dynamics like ridge asymmetry or spatial hole-burning, as well as critical temperatures that induce filamentation.
ABSTRACT
Interleukin (IL)-33 is a cytokine that is released from epithelial and endothelial cells at barrier surfaces upon tissue stress or damage to operate as an alarmin. IL-33 has been primarily implicated in the induction of T helper (Th) 2 type immune responses. Therefore, IL-33 has attracted a lot of interest as a potential therapeutic target in asthma and other allergic diseases. Over the years, it has become clear that IL-33 has a much broader activity and also contributes to Th1 immunity, expanding the possibilities for therapeutic modulation of IL-33 activity to multiple inflammatory diseases. However, more recently IL-33 has also been shown to mediate immunosuppression and tissue repair by activating regulatory T cells (Treg) and promoting M2 macrophage polarization. These pleiotropic activities of IL-33 illustrate the need for a tight molecular regulation of IL-33 activity, and have to be taken into account when IL-33 or its receptor is targeted for therapeutic modulation. Here we review the multiple molecular mechanisms that regulate IL-33 activity and describe how IL-33 can shape innate and adaptive immune responses by promoting Th1, Th2 and Treg function. Finally, we will discuss the possibilities for therapeutic modulation of IL-33 signaling as well as possible safety issues.
Subject(s)
Hypersensitivity/metabolism , Inflammation/metabolism , Interleukin-33/metabolism , Drug Delivery Systems , Humans , Interleukin-33/antagonists & inhibitorsABSTRACT
Molecular mechanisms underlying the cancer stroma in metastasis need further exploration. Here, we discovered that cancer-associated fibroblasts (CAFs) produced high levels of IL-33 that acted on tumor-associated macrophages (TAMs), causing them to undergo the M1 to M2 transition. Genomic profiling of metastasis-related genes in the IL-33-stimulated TAMs showed a >200-fold increase of MMP9. Signaling analysis demonstrated the IL-33-ST2-NF-κB-MMP9-laminin pathway that governed tumor stroma-mediated metastasis. In mouse and human fibroblast-rich pancreatic cancers, genetic deletion of IL-33, ST2, or MMP9 markedly blocked metastasis. Pharmacological inhibition of NF-κB and MMP9 also blocked cancer metastasis. Deletion of IL-33, ST2, or MMP9 restored laminin, a key basement membrane component associated with tumor microvessels. Together, our data provide mechanistic insights on the IL-33-NF-κB-MMP9-laminin axis that mediates the CAF-TAM-committed cancer metastasis. Thus, targeting the CAF-TAM-vessel axis provides an outstanding therapeutic opportunity for cancer treatment.