| 1. |
- Lepzien, Rico, et al.
(författare)
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Pulmonary and blood dendritic cells from sarcoidosis patients more potently induce IFNγ-producing Th1 cells compared with monocytes
- 2022
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Ingår i: Journal of Leukocyte Biology. - 0741-5400 .- 1938-3673. ; 111:4, s. 857-866
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Tidskriftsartikel (refereegranskat)abstract
- Sarcoidosis is a systemic inflammatory disease mainly affecting the lungs. The hallmark of sarcoidosis are granulomas that are surrounded by activated T cells, likely targeting the disease-inducing antigen. IFNγ-producing Th1 and Th17.1 T cells are elevated in sarcoidosis and associate with disease progression. Monocytes and dendritic cells (DCs) are antigen-presenting cells (APCs) and required for T cell activation. Several subsets of monocytes and DCs with different functions were identified in sarcoidosis. However, to what extent different monocyte and DC subsets can support activation and skewing of T cells in sarcoidosis is still unclear. In this study, we performed a transcriptional and functional side-by-side comparison of sorted monocytes and DCs from matched blood and bronchoalveolar lavage (BAL) fluid of sarcoidosis patients. Transcriptomic analysis of all subsets showed upregulation of genes related to T cell activation and antigen presentation in DCs compared with monocytes. Allogeneic T cell proliferation was higher after coculture with monocytes and DCs from blood compared with BAL and DCs induced more T cell proliferation compared with monocytes. After coculture, proliferating T cells showed high expression of the transcription factor Tbet and IFNγ production. We also identified Tbet and RORγt coexpressing T cells that mainly produced IFNγ. Our data show that DCs rather than monocytes from sarcoidosis patients have the ability to activate and polarize T cells towards Th1 and Th17.1 cells. This study provides a useful in vitro tool to better understand the contribution of monocytes and DCs to T cell activation and immunopathology in sarcoidosis.
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| 2. |
- Maric, Jovana, et al.
(författare)
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Cytokine-induced endogenous production of prostaglandin D-2 is essential for human group 2 innate lymphoid cell activation
- 2019
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Ingår i: Journal of Allergy and Clinical Immunology. - : MOSBY-ELSEVIER. - 0091-6749 .- 1097-6825. ; 143:6, s. 2202-2214.e5
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Tidskriftsartikel (refereegranskat)abstract
- Objective: We set out to examine PG production in human ILC2s and the implications of such endogenous production on ILC2 function. Methods: The effects of the COX-1/2 inhibitor flurbiprofen, the hematopoietic prostaglandin D2 synthase (HPGDS) inhibitor KMN698, and the CRTH2 antagonist CAY10471 on human ILC2s were determined by assessing receptor and transcription factor expression, cytokine production, and gene expression with flow cytometry, ELISA, and quantitative RT-PCR, respectively. Concentrations of lipid mediators were measured by using liquid chromatography-tandem mass spectrometry and ELISA. Results: We show that ILC2s constitutively express HPGDS and upregulate COX-2 upon IL-2, IL-25, and IL-33 plus thymic stromal lymphopoietin stimulation. Consequently, PGD2 and its metabolites can be detected in ILC2 supernatants. We reveal that endogenously produced PGD2 is essential in cytokine-induced ILC2 activation because blocking of the COX-1/2 or HPGDS enzymes or the CRTH2 receptor abolishes ILC2 responses. Conclusion: PGD2 produced by ILC2s is, in a paracrine/autocrine manner, essential in cytokine-induced ILC2 activation. Hence we provide the detailed mechanism behind how CRTH2 antagonists represent promising therapeutic tools for allergic diseases by controlling ILC2 function.
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| 3. |
- Maric, Jovana, et al.
(författare)
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Prostaglandin E-2 suppresses human group 2 innate lymphoid cell function
- 2018
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Ingår i: Journal of Allergy and Clinical Immunology. - : MOSBY-ELSEVIER. - 0091-6749 .- 1097-6825. ; 141:5, s. 1761-1773.e6
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Tidskriftsartikel (refereegranskat)abstract
- Background: Group 2 innate lymphoid cells (ILC2s) are involved in the initial phase of type 2 inflammation and can amplify allergic immune responses by orchestrating other type 2 immune cells. Prostaglandin (PG) E-2 is a bioactive lipid that plays protective roles in the lung, particularly during allergic inflammation.Objective: We set out to investigate how PGE(2) regulates human ILC2 function.Methods: The effects of PGE(2) on human ILC2 proliferation and intracellular cytokine and transcription factor expression were assessed by means of flow cytometry. Cytokine production was measured by using ELISA, and real-time quantitative PCR was performed to detect PGE(2) receptor expression.Results: PGE(2) inhibited GATA-3 expression, as well as production of the type 2 cytokines IL-5 and IL-13, from human tonsillar and blood ILC2s in response to stimulation with a combination of IL-25, IL-33, thymic stromal lymphopoietin, and IL-2. Furthermore, PGE(2) downregulated the expression of IL-2 receptor alpha (CD25). In line with this observation, PGE(2) decreased ILC2 proliferation. These effects were mediated by the combined action of E-type prostanoid receptor (EP) 2 and EP4 receptors, which were specifically expressed on ILC2s.Conclusion: Our findings reveal that PGE(2) limits ILC2 activation and propose that selective EP2 and EP4 receptor agonists might serve as a promising therapeutic approach in treating allergic diseases by suppressing ILC2 function.
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| 4. |
- Mazzurana, Luca, et al.
(författare)
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Tissue-specific transcriptional imprinting and heterogeneity in human innate lymphoid cells revealed by full-length single-cell RNA-sequencing
- 2021
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Ingår i: Cell Research. - : Springer Science and Business Media LLC. - 1748-7838 .- 1001-0602. ; 31:5, s. 554-568
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Tidskriftsartikel (refereegranskat)abstract
- The impact of the microenvironment on innate lymphoid cell (ILC)-mediated immunity in humans remains largely unknown. Here we used full-length Smart-seq2 single-cell RNA-sequencing to unravel tissue-specific transcriptional profiles and heterogeneity of CD127+ ILCs across four human tissues. Correlation analysis identified gene modules characterizing the migratory properties of tonsil and blood ILCs, and signatures of tissue-residency, activation and modified metabolism in colon and lung ILCs. Trajectory analysis revealed potential differentiation pathways from circulating and tissue-resident naïve ILCs to a spectrum of mature ILC subsets. In the lung we identified both CRTH2+ and CRTH2− ILC2 with lung-specific signatures, which could be recapitulated by alarmin-exposure of circulating ILC2. Finally, we describe unique TCR-V(D)J-rearrangement patterns of blood ILC1-like cells, revealing a subset of potentially immature ILCs with TCR-δ rearrangement. Our study provides a useful resource for in-depth understanding of ILC-mediated immunity in humans, with implications for disease.
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| 5. |
- Ravindran, Avinash, et al.
(författare)
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An Optimized Protocol for the Isolation and Functional Analysis of Human Lung Mast Cells
- 2018
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Ingår i: Frontiers in Immunology. - : FRONTIERS MEDIA SA. - 1664-3224. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Background: Mast cells are tissue-resident inflammatory cells defined by their high granularity and surface expression of the high-affinity IgE receptor, Fc + RI, and CD117/KIT, the receptor for stem cell factor (SCF). There is a considerable heterogeneity among mast cells, both phenotypically and functionally. Human mast cells are generally divided into two main subtypes based on their protease content; the mucosa-associated MCT (tryptase positive and chymase negative mast cell) and the connective tissue associated-residing MCTC (tryptase and chymase positive mast cell). Human lung mast cells exhibit heterogeneity in terms of cellular size, expression of cell surface receptors, and secreted mediators. However, knowledge about human lung mast cell heterogeneity is restricted to studies using immunohistochemistry or purified mast cells. Whereas the former is limited by the number of cellular markers that can be analyzed simultaneously, the latter suffers from issues related to cell yield.Aim: To develop a protocol that enables isolation of human lung mast cells at high yields for analysis of functional properties and detailed analysis using single-cell based analyses of protein (flow cytometry) or RNA (RNA-sequencing) expression.Methods: Mast cells were isolated from human lung tissue by a sequential combination of washing, enzymatic digestion, mechanical disruption, and density centrifugation using Percoll (WEMP). As a comparison, we also isolated mast cells using a conventional enzyme-based protocol. The isolated cells were analyzed by flow cytometry.Results: We observed a significant increase in the yield of total human lung CD45(+) immune cells and an even more pronounced increase in the yield of CD117(+) mast cells with the WEMP protocol in comparison to the conventional protocols. In contrast, the frequency of the rare lymphocyte subset innate lymphoid cells group 2 (ILC2) did not differ between the two methods.Conclusion: The described WEMP protocol results in a significant increase in the yield of human lung mast cells compared to a conventional protocol. Additionally, the WEMP protocol enables simultaneous isolation of different immune cell populations such as lymphocytes, monocytes, and granulocytes while retaining their surface marker expression that can be used for advanced single-cell analyses including multi-color flow cytometry and RNA-sequencing.
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| 6. |
- Ravindran, Avinash
(författare)
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New insights on type 2 immunity key drivers : mast cells and group 2 innate lymphoid cells
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Type 2 immune responses are characterized by the production of cytokines IL-4, IL-5 and IL-13. They are known to have crucial physiological and pathophysiological functions in the body. Data from recent studies indicate their importance in the containment and clearance of helminth infections. They are induced to generate rapid response against helminth infections even before the elicitation of specific adaptive responses. On the other hand, Th2 responses are also involved in the promotion of inflammatory conditions such as allergy and asthma. Studies from the past have probed the mounting of type 2 immune responses and Th2 cytokine production by innate immune cells. In this thesis, we set out to study two innate cells, which are considered to be one of the key drivers of allergic conditions – mast cells and group 2 innate lymphoid cells. In paper I, an optimized protocol to efficiently isolate mast cells from human lung tissue with high yield and cell viability is described. Mast cells are heterogeneous, tissue-resident inflammatory cells characterized by the expression of high-affinity IgE receptor, FcεRI, and CD117/KIT, the receptor for stem cell factor (SCF). The isolation of mast cells was performed by a sequential combination of washing, enzymatic digestion, mechanical disruption and Percol centrifugation (WEMP). The yield from WEMP protocol was significantly high when compared to the conventional enzyme-based method. The isolated cells were further used for flow cytometry-based characterization studies and single cell RNA sequencing. Paper II is a study of human lung mast cells at the single cell level using RNA sequencing. The classic marker expression by mast cells revealed the integrity of the sort and the sequencing reactions. Following this, analyses of sequencing data was performed to depict highly variable genes, highly abundant genes and the presence of subpopulation. Preliminary data displayed homogeneity within the sorted population of the isolated lung mast cells with no clear subpopulations. In paper III, the fundamental aim was to determine the effects of prostaglandin D2 on ILC2. ILC2 play critical roles in the initiation and promotion of type 2 immune responses. The results indicated the constitutive expression of prostaglandin D2 synthase by ILC2 and the endogenous PGD2 production was involved in the activation of ILC2 through CRTH2 receptors in a para/autocrine fashion. In paper IV, the study was designed to elucidate the effect of PGE2 on ILC2. ILC2 were isolated, cultured and expanded under different treatment conditions to study the effect. PGE2 was found to have suppressive effect on GATA3 and IL-2 receptor (CD25) expression and cytokine production by ILC2. The suppressive effects were mediated by EP2 and EP4 receptors. The use of EP2 and EP4 agonists can therefore serve as potential therapeutic target in controlling ILC2 mediated inflammation. In conclusion, the results from our studies add to the already known information about initiation, maintenance and regulation of type 2 immune responses. They serve as basis for future mast cell-ILC2 interaction studies that could provide potential insights into their contribution in allergic and other TH2-driven conditions.
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| 7. |
- Ronnberg, Elin, et al.
(författare)
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Analysis of human lung mast cells by single cell RNA sequencing
- 2023
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Mast cells are tissue-resident cells playing major roles in homeostasis and disease conditions. Lung mast cells are particularly important in airway inflammatory diseases such as asthma. Human mast cells are classically divided into the subsets MCT and MCTC, where MCT express the mast cell protease tryptase and MCTC in addition express chymase, carboxypeptidase A3 (CPA3) and cathepsin G. Apart from the disctintion of the MCT and MCTC subsets, little is known about the heterogeniety of human lung mast cells and a deep analysis of their heterogeniety has previously not been performed. We therefore performed single cell RNA sequencing on sorted human lung mast cells using SmartSeq2. The mast cells showed high expression of classical mast cell markers. The expression of several individual genes varied considerably among the cells, however, no subpopulations were detected by unbiased clustering. Variable genes included the protease-encoding transcripts CMA1 (chymase) and CTSG (cathepsin G). Human lung mast cells are predominantly of the MCT subset and consistent with this, the expression of CMA1 was only detectable in a small proportion of the cells, and correlated moderately to CTSG. However, in contrast to established data for the protein, CPA3 mRNA was high in all cells and the correlation of CPA3 to CMA1 was weak.
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| 8. |
- Ronnberg, Elin, et al.
(författare)
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Immunoprofiling Reveals Novel Mast Cell Receptors and the Continuous Nature of Human Lung Mast Cell Heterogeneity
- 2022
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Background: Immunohistochemical analysis of granule-associated proteases has revealed that human lung mast cells constitute a heterogeneous population of cells, with distinct subpopulations identified. However, a systematic and comprehensive analysis of cell-surface markers to study human lung mast cell heterogeneity has yet to be performed.Methods: Human lung mast cells were obtained from lung lobectomies, and the expression of 332 cell-surface markers was analyzed using flow cytometry and the LEGENDScreen (TM) kit. Markers that exhibited high variance were selected for additional analyses to reveal whether they were correlated and whether discrete mast cell subpopulations were discernable.Results: We identified the expression of 102 surface markers on human lung mast cells, 23 previously not described on mast cells, of which several showed high continuous variation in their expression. Six of these markers were correlated: SUSD2, CD49a, CD326, CD34, CD66 and HLA-DR. The expression of these markers was also correlated with the size and granularity of mast cells. However, no marker produced an expression profile consistent with a bi- or multimodal distribution.Conclusions: LEGENDScreen analysis identified more than 100 cell-surface markers on mast cells, including 23 that, to the best of our knowledge, have not been previously described on human mast cells. The comprehensive expression profiling of the 332 surface markers did not identify distinct mast cell subpopulations. Instead, we demonstrate the continuous nature of human lung mast cell heterogeneity.
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