FIGURE 3 - uploaded by Hirohito Kita
Content may be subject to copyright.
ST2 expression on lineage low populations in the bone marrow. Bone marrow cells from 8-wk-old C57BL/6 mice were analyzed by flow cytometry. A , Gating strategy. Lineage low cells (low to no expression of B220, CD3 ε , CD4, CD8 a , DX5, CD11b, CD11c, Gr-1, and TER-119) were subdivided into four populations, based on their expression of Sca-1 and c-Kit. B , Expression of ST2 on the various lineage low subpopulations. Open histograms show anti-ST2 staining, and gray shaded histograms show isotype control Ab staining. Plots representative of stains from three different mice. C , ST2 and other cell surface markers expression on CD25 2 and CD25 + LSK 2 cells. Quadrants were drawn on the basis of isotype control Ab stains. All
Source publication
IL-33 promotes type 2 immune responses, both protective and pathogenic. Recently, targets of IL-33, including several newly discovered type 2 innate cells, have been characterized in the periphery. In this study, we report that bone marrow cells from wild-type C57BL/6 mice responded with IL-5 and IL-13 production when cultured with IL-33. IL-33 cul...
Contexts in source publication
Context 1
... we isolated bone marrow cells from a WT C57BL/6 mouse and tested for expression of ST2, the receptor for IL-33, in lineage-negative cell populations. After excluding Lin + cells (expressing B220, CD3ε, CD4, CD8a, DX5, CD11b, CD11c, Gr-1, or TER-119), we divided the Lin 2 cells into four populations, based on their expression of Sca-1 and c-Kit (Fig. 3A). Lin 2 Sca-1 + c-Kit + cells were ST2 2 , whereas the Lin 2 Sca-1 2 c-Kit + and the Lin 2 Sca-1 2 c-Kit 2 populations showed low-level ST2 staining (Fig. 3B). A distinctly ST2 + popula- tion was found among the Lin 2 Sca-1 + c-Kit 2 (LSK 2 ) cells (Fig. ...
Context 2
... excluding Lin + cells (expressing B220, CD3ε, CD4, CD8a, DX5, CD11b, CD11c, Gr-1, or TER-119), we divided the Lin 2 cells into four populations, based on their expression of Sca-1 and c-Kit (Fig. 3A). Lin 2 Sca-1 + c-Kit + cells were ST2 2 , whereas the Lin 2 Sca-1 2 c-Kit + and the Lin 2 Sca-1 2 c-Kit 2 populations showed low-level ST2 staining (Fig. 3B). A distinctly ST2 + popula- tion was found among the Lin 2 Sca-1 + c-Kit 2 (LSK 2 ) cells (Fig. ...
Context 3
... the Lin 2 cells into four populations, based on their expression of Sca-1 and c-Kit (Fig. 3A). Lin 2 Sca-1 + c-Kit + cells were ST2 2 , whereas the Lin 2 Sca-1 2 c-Kit + and the Lin 2 Sca-1 2 c-Kit 2 populations showed low-level ST2 staining (Fig. 3B). A distinctly ST2 + popula- tion was found among the Lin 2 Sca-1 + c-Kit 2 (LSK 2 ) cells (Fig. ...
Context 4
... based on their CD25 expression, and showed that LSK 2 CD25 2 cells have lymphoid differentiation potential. However, the function of LSK 2 CD25 + cells still remained un- known (27). Following this precedent, we used CD25 to further characterize the LSK 2 cells and found that the LSK 2 CD25 + cells, but not LSK 2 CD25 2 cells, are uniformly ST2 + (Fig. 3C). LSK 2 CD25 + cells, which made up 0.054 6 0.003% of the total nucleated bone marrow cells from 8-wk-old C57BL/6 mice (mean, n = 10), also express IL-7Ra, Thy1, CD44, and CD45 but are mutually ex- clusive with LSK 2 Flt3 + cells (Fig. 3C). These results suggest that LSK 2 CD25 + cells are potentially IL-33 ...
Context 5
... the LSK 2 cells and found that the LSK 2 CD25 + cells, but not LSK 2 CD25 2 cells, are uniformly ST2 + (Fig. 3C). LSK 2 CD25 + cells, which made up 0.054 6 0.003% of the total nucleated bone marrow cells from 8-wk-old C57BL/6 mice (mean, n = 10), also express IL-7Ra, Thy1, CD44, and CD45 but are mutually ex- clusive with LSK 2 Flt3 + cells (Fig. 3C). These results suggest that LSK 2 CD25 + cells are potentially IL-33 ...
Context 6
... + cells. Characterization of LSK 2 CD25 + cells revealed that they are ST2 + and that they are . LSK 2 CD25 + cells in mutant and knockout mice. A, Bone marrow cells were harvested from 8-to 11-wk-old WT C57BL/6, Rag-1 KO, IL-7Ra KO, IL-2Rg KO, and Kit W-sh/W-sh or from 8-to 9-wk-old WT C57BL/6 and Foxn1 nu/nu mice (B). Cells were stained as in Fig. 3 for LSK 2 CD25 + cells and for their expression of ST2 and IL-7Ra. Plots representative of two to three mice per mouse type. C, The frequency and absolute cell number of LSK 2 CD25 + cells in the bone marrow from two femurs and two tibias of each mouse. Error bars represent the SEM calculated from two to three mice per mouse type. ...
Context 7
... express varying level of c-Kit, and Ih-2 cells are FIGURE 6. LSK 2 CD25 + cells are responsive to IL-25 and IL-33 stimulation in vivo. PBS, 400 ng IL-25, or 400 ng IL-33 in PBS was injected i.p. into 8- wk-old WT C57BL/6 mice daily for 4 consecutive days. On day 5, nucleated bone marrow cells were harvested and stained for flow cytometry as in Fig. 3. A, Mean frequency and absolute cell number of LSK 2 CD25 2 and LSK 2 CD25 + cells per mouse in each treatment group. Error bars represent the SEM from four mice per treatment group (*p , 0.05, **p , 0.01 compared with PBS treatment by unpaired t test with Welch correction). B, ST2, IL-7Ra, and a 4 b 7 integrin expression on LSK 2 CD25 ...
Citations
... Blocking IL-1RL1 signalling in mice resulted in lower ILC2 numbers in the lung, which was associated with decreased lung function and loss of airway epithelial integrity, thereby indicating the importance of IL-33 activation of ILC2s in antiviral responses and airway epithelial repair (Monticelli et al., 2011). IL-1RL1 expression level in murine bone marrow ILC2s was significantly increased by IL-33 treatment (Brickshawana, Shapiro, Kita, & Pease, 2011), suggesting a positive feedback loop capable of amplifying ILC2 activation by IL-33 (Spooner et al., 2013) in mice. Likewise, another study indicated that thymic stromal lymphopoietin (TSLP) treatment of murine lung-derived ILC2s enhanced the expression of the IL-1RL1 receptor and enhanced phosphorylation of STAT5 upon IL-33 stimulation (Toki et al., 2020), indicating crosstalk between these epithelial alarmins. ...
Interleukin-33 (IL-33), a member of the IL-1 family, and its cognate receptor, Interleukin-1 receptor like-1 (IL-1RL1 or ST2), are susceptibility genes for childhood asthma. In response to cellular damage, IL-33 is released from barrier tissues as an 'alarmin' to activate the innate immune response. IL-33 drives type 2 responses by inducing signalling through its receptor IL-1RL1 in several immune and structural cells, thereby leading to type 2 cytokine and chemokine production. IL-1RL1 gene transcript encodes different isoforms generated through alternative splicing. Its soluble isoform, IL-1RL1-a or sST2, acts as a decoy receptor by sequestering IL-33, thereby inhibiting IL1RL1-b/IL-33 signalling. IL-33 and its receptor IL-1RL1 are therefore considered as putative biomarkers or targets for pharmacological intervention in asthma. This review will provide an overview of the genetics and biology of the IL-33/IL-1RL1 pathway in the context of asthma pathogenesis. It will discuss the potential and complexities of targeting the cytokine or its receptor, how genetics or biomarkers may inform precision medicine for asthma targeting this pathway, and the possible positioning of therapeutics targeting IL-33 or its receptor in the expanding landscape of novel biologicals applied in asthma management.
... IL-33, a member of the IL-1 family, is released from cells upon cellular damage (Lüthi et al., 2009). Additionally, it has been shown that cultured ILC2s become activated and produce type 2 cytokines including GM-CSF in response to IL-33 in vitro (Brickshawana et al., 2011;Hoyler et al., 2012), prompting us to analyze the expression of IL-33 in BM. Although ECs constitute a major source of IL-33 in some tissues (Moussion et al., 2008), our flow-cytometric analysis of IL-33-GFP mice showed that the IL-33 level in BM ECs was low ( Fig. S4 A). ...
The cell-cycle status of hematopoietic stem and progenitor cells (HSPCs) becomes activated following chemotherapy-induced stress, promoting bone marrow (BM) regeneration; however, the underlying molecular mechanism remains elusive. Here we show that BM-resident group 2 innate lymphoid cells (ILC2s) support the recovery of HSPCs from 5-fluorouracil (5-FU)–induced stress by secreting granulocyte-macrophage colony-stimulating factor (GM-CSF). Mechanistically, IL-33 released from chemo-sensitive B cell progenitors activates MyD88-mediated secretion of GM-CSF in ILC2, suggesting the existence of a B cell–ILC2 axis for maintaining hematopoietic homeostasis. GM-CSF knockout mice treated with 5-FU showed severe loss of myeloid lineage cells, causing lethality, which was rescued by transferring BM ILC2s from wild-type mice. Further, the adoptive transfer of ILC2s to 5-FU–treated mice accelerates hematopoietic recovery, while the reduction of ILC2s results in the opposite effect. Thus, ILC2s may function by “sensing” the damaged BM spaces and subsequently support hematopoietic recovery under stress conditions.
... Intracellular cytokine staining was performed as described previously [17]. Briefly, cells were harvested, washed and surface stained prior to intracellular staining using Fixation/Permeabilization kit (BD Biosciences) following the manufacturer's instructions. ...
Rationale: Aurora kinase A (Aurora-A), which is required for mitosis, is a therapeutic target in various tumors. Targeting Aurora-A led to an increase in the differentiation and polyploidization of megakaryocytes both in vivo and in vitro. However, the mechanisms involved in controlling megakaryocyte differentiation have not been fully elucidated.
Methods: Conditional Aurka knockout mice were generated. B cell development, platelet development and function were subsequently examined. Proplatelet formation, in vivo response to mTPO, post-transfusion experiment, colony assay, immunofluorescence staining and quantification, and ChIP assay were conducted to gain insights into the mechanisms of Aurka loss in megakaryocytopoiesis.
Results: Loss of Aurka in CD19⁺ B cells impaired B cell development in association with an increase in the number of platelets in peripheral blood (PB). Surprisingly, thrombopoietin (TPO) production and IL-6 were elevated in the plasma in parallel with an increase in the number of differentiated megakaryocytes in the bone marrow (BM) of Aurkaf/f;Cd19Cre/+ mice. Interestingly, compared with that of the Aurkaf/f mice, a higher number of CD19⁺ B cells close to megakaryocytes was observed in the BM of the Aurkaf/f;Cd19Cre/+ mice. Moreover, Aurka loss in CD19⁺ B cells induced signal transducer and activator of transcription-3 (STAT3) activation. Inhibition of STAT3 reduced the Tpo mRNA levels. ChIP assays revealed that STAT3 bound to the TPO promoter. Additionally, STAT3-mediated TPO transcription was an autocrine effect provoked by IL-6, at least partially.
Conclusions: Deletion of Aurka in CD19⁺ B cells led to an increase in IL-6 production, promoting STAT3 activation, which in turn contributed to TPO transcription and megakaryocytopoiesis.
... The mechanisms of the synergistic activation of ILC2 by IL-33 and TSLP are not fully understood. Previous studies reported that ST2 expression level was significantly increased in the bone marrow ILC2 by IL-33 treatment, 34 Development of human monoclonal antibodies is progressing as promising biological therapeutic agents for allergic diseases. 36 Tezepelumab (AMG 157/MEDI9929), an anti-human TSLP antibody, attenuated allergen-induced bronchoconstriction in both early and late asthmatic responses and decreased in levels of blood and sputum eosinophils. ...
Background
The epithelial cell‐derived danger signal mediators thymic stromal lymphopoietin (TSLP) and IL‐33 are consistently associated with adaptive Th2 immune responses in asthma. In addition, TSLP and IL‐33 synergistically promoted group 2 innate lymphoid cell (ILC2) activation to induce innate allergic inflammation. However, the mechanism of this synergistic ILC2 activation is unknown.
Methods
BALB/c WT and TSLP receptor‐deficient (TSLPR−/−) mice were challenged intranasally with Alternaria extract (Alt‐Ext) or PBS for 4 consecutive days to evaluate innate airway allergic inflammation. WT mice pre‐administered with rTSLP or vehicle, TSLPR−/− mice, and IL‐33 receptor‐deficient (ST2−/−) mice were challenged intranasally with Alt‐Ext or vehicle once or twice to evaluate IL‐33 release and TSLP expression in the lung. TSLPR and ST2 expression on lung ILC2 were measured by flow cytometry after treatment of rTSLP, rIL‐33, rTSLP + rIL‐33, or vehicle.
Results
Thymic stromal lymphopoietin receptor deficient mice had significantly decreased the number of lung ILC2 expressing IL‐5 and IL‐13 following Alt‐Ext‐challenge compared to WT mice. Further, eosinophilia, protein level of lung IL‐4, IL‐5, and IL‐13, and airway mucus score were also significantly decreased in TSLPR−/− mice compared to WT mice. Endogenous and exogenous TSLP increased Alt‐Ext‐induced IL‐33 release into BALF, and ST2 deficiency decreased Alt‐Ext‐induced TSLP expression in the lung. Further, rTSLP and rIL‐33 treatment reciprocally increased each other's receptor expression on lung ILC2 in vivo and in vitro.
Conclusion
Thymic stromal lymphopoietin and IL‐33 signaling reciprocally enhanced each other's protein release and expression in the lung following Alt‐Ext‐challenge and each other's receptor expression on lung ILC2 to enhance ILC2 activation.
... The IL-33 concentration in ascites from the ID8-IL33 tumor-bearing mice was significantly higher, whereas ascites from ID8-mock tumor-bearing mice were almost free of IL-33 (Fig. 2C). IL-33-responsive innate cells have been previously reported in mouse bone marrow (34). Therefore, we considered that IL-33 in ascites may directly inhibit the differentiation of lineage-negative progenitor bone marrow cells into CD11b + Gr-1 + cells. ...
Refractory peritoneal carcinomatosis is a common terminal feature of epithelial ovarian cancer (EOC). Previous reports have suggested that immunotherapy is a promising therapeutic strategy for EOC. Interleukin (IL)‑33 is a member of the IL‑1 superfamily of cytokines. The role of IL‑33 in tissue inflammation and promoting type 2 immune responses has been established, and recently, there is accumulating evidence to suggest the involvement of IL‑33 in carcinogenesis. In this study, we focused on the association between the tumor expression of IL‑33 and ovarian peritoneal carcinomatosis. We used an immunosufficient murine model of peritoneal carcinomatosis and human EOC samples. The overexpression of IL‑33 in the ID8 mouse EOC cell line tumors significantly prolonged the survival of immunocompetent mice in the peritoneal carcinomatosis setting, but not in the subcutaneous model. In addition, the silencing of IL‑33 in ID8‑T6 cells (subclone with high dissemination potential) significantly shortened the survival of the tumor‑bearing mice. This was likely due to the intratumoral accumulation of CD8+ and CD4+ T cells, and a decrease in CD11b+Gr1+ cells. Furthermore, IL‑33 induced the intraperitoneal microenvironment favoring tumor elimination through the inhibition of differentiation into CD11b+Gr1+ cells. On the whole, the findings of this study suggest IL‑33 to be a cytokine that reflects antitumor peritoneal conditions. Further investigation of the antitumorigenic role of IL‑33 may aid in the development of more effective therapeutic approaches for the treatment of EOC with peritoneal carcinomatosis.
... cell, natural-killer cell or other cell lineage markers, but express the IL-7 receptor α-chain (CD127), c-Kit, Sca-1, etc. 194,195,[197][198][199] ILC2s produce dramatic amounts of IL-5 and IL-13, and some IL-4 in response to the Th2 cell-stimulating cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) produced by epithelial cells. [200][201][202][203][204] The discovery of ILC2s puts forward a new challenge to the traditional opinions that T helper type 2 (Th2) cells play a dominant role in Th2-skewed allergic diseases. ...
Allergic rhinitis (AR) is a global health problem that causes major illnesses and disabilities worldwide. Epidemiologic studies have demonstrated that the prevalence of AR has increased progressively over the last few decades in more developed countries and currently affects up to 40% of the population worldwide. Likewise, a rising trend of AR has also been observed over the last 2-3 decades in developing countries including China, with the prevalence of AR varying widely in these countries. A survey of self-reported AR over a 6-year period in the general Chinese adult population reported that the standardized prevalence of adult AR increased from 11.1% in 2005 to 17.6% in 2011. An increasing number of Journal Articles and imporclinical trials on the epidemiology, pathophysiologic mechanisms, diagnosis, management and comorbidities of AR in Chinese subjects have been published in international peer-reviewed journals over the past 2 decades, and substantially added to our understanding of this disease as a global problem. Although guidelines for the diagnosis and treatment of AR in Chinese subjects have also been published, they have not been translated into English and therefore not generally accessible for reference to non-Chinese speaking international medical communities. Moreover, methods for the diagnosis and treatment of AR in China have not been standardized entirely and some patients are still treated according to regional preferences. Thus, the present guidelines have been developed by the Chinese Society of Allergy to be accessible to both national and international medical communities involved in the management of AR patients. These guidelines have been prepared in line with existing international guidelines to provide evidence-based recommendations for the diagnosis and management of AR in China.
... Congruent with our findings in human hematopoietic cells, immunophenotypic HSCs were not numerically affected by IL-33 ( Figures 7A and 7B). Interestingly, IL-33 also significantly expanded the population of Lin À Kit À Sca1 + BM cells, previously shown to contain early lymphoid-committed precursors with T cell, B cell, and natural killer (NK) cell potential (Kumar et al., 2008) and innate lymphoid cells (Brickshawana et al., 2011). Hematopoietic changes were accompanied by a relative increase, albeit not reaching statistical significance, in CD31 + CD105 + ECs as well as Lin À Ter119 À CD51 + Sca À cells (earlier shown to contain lineage-committed/osteoblastic cells) (Schepers et al., 2012) within the niche compartment ( Figures S7A and S7B). ...
Bone marrow formation requires an orchestrated interplay between osteogenesis, angiogenesis, and hematopoiesis that is thought to be mediated by endothelial cells. The nature of the endothelial cells and the molecular mechanisms underlying these events remain unclear in humans. Here, we identify a subset of endoglin-expressing endothelial cells enriched in human bone marrow during fetal ontogeny and upon regeneration after chemotherapeutic injury. Comprehensive transcriptional characterization by massive parallel RNA sequencing of these cells reveals a phenotypic and molecular similarity to murine type H endothelium and activation of angiocrine factors implicated in hematopoiesis, osteogenesis, and angiogenesis. Interleukin-33 (IL-33) was significantly overexpressed in these endothelial cells and promoted the expansion of distinct subsets of hematopoietic precursor cells, endothelial cells, as well as osteogenic differentiation. The identification and molecular characterization of these human regeneration-associated endothelial cells is thus anticipated to instruct the discovery of angiocrine factors driving bone marrow formation and recovery after injury.
... Interestingly, an ST2-expressing population of lineage À Sca1 + c-Kit À CD25 + cells has previously been described within the mouse bone marrow that, consistent with our findings, was reported to produce IL-5 when cultured in vitro in the presence of IL-33 and IL-7. 42 In our study, in addition to being ST2 + cells, the ILC2s expressed Sca-1 and ICOS but mostly negative for c-Kit and KLRG1 (see Supplementary material, Fig. S1b). Furthermore, the number of ILC2s remained unchanged in the bone marrow of IL-33-challenged mice compared with PBS-treated control mice, whereas the Th cells were found at significantly lower numbers after IL-33 challenge. ...
... Adult bone marrow contains ILC2-like cells (Brickshawana et al., 2011;Price et al., 2010). They were shown to be closely related to ILC2s by the genome-wide transcriptome analysis, have very low Table 1 Defining mouse ILCs ...
Group 2 Innate Lymphoid Cells (ILC2s) are a subset of the recently expanded family of innate lymphoid cells (ILCs). They produce type 2 cytokines and provide protective type 2 immunity during helminth infection and are implicated in the pathogenesis of allergy and asthma. In the adult stage, ILC2s arise from the common progenitor for all ILCs in the bone marrow, under the control of the transcription factors retinoic acid receptor–related orphan receptor (ROR)α, GATA binding protein-3 (GATA-3), T cell factor 1 (TCF-1), and B cell leukemia/lymphoma 11b (Bcl11b). Some of the transcription factors regulating ILC2 development also regulate T helper 2 (Th2) cell development suggesting their close developmental relationship. Further characterization of progenitors for ILCs and T cells as well as the transcription factors regulating their development will clarify the lineage relationship between ILCs and T cells.
... ST2-expressing ILC2 populations that produce IL-5 were previously been shown to regulate eosinophil homeostasis in the intestine and lung, but this production was spontaneous; moreover, these cells were shown to be largely absent from the bone marrow compartment (40). Interestingly, a Sca-1 + precursor population within the bone marrow that expresses ST2 and produces IL-5 in response to IL-33 has been reported (41), suggesting that both the expansion of the IL-5Rα + EoPre population and the elevation of IL-5 itself could occur locally within the bone marrow and be driven by IL-33. In our in vitro cultures, IL-5 was detected in the media after 3 days of culture with SCF, Flt3L, and IL-33 (54.6 ± 6.7 pg/ml) but was undetectable (less than 31.25 pg/ml) with SCF and Flt3L alone. ...
Eosinophils are important in the pathogenesis of many diseases, including asthma, eosinophilic esophagitis, and eczema. Whereas IL-5 is crucial for supporting mature eosinophils (EoMs), the signals that support earlier eosinophil lineage events are less defined. The IL-33R, ST2, is expressed on several inflammatory cells, including eosinophils, and is best characterized for its role during the initiation of allergic responses in peripheral tissues. Recently, ST2 expression was described on hematopoietic progenitor subsets, where its function remains controversial. Our findings demonstrate that IL-33 is required for basal eosinophil homeostasis, because both IL-33- and ST2-deficient mice exhibited diminished peripheral blood eosinophil numbers at baseline. Exogenous IL-33 administration increased EoMs in both the bone marrow and the periphery in wild-type and IL-33-deficient, but not ST2-deficient, mice. Systemic IL-5 was also increased under this treatment, and blocking IL-5 with a neutralizing Ab ablated the IL-33-induced EoM expansion. The homeostatic hypereosinophilia seen in IL-5-transgenic mice was significantly lower with ST2 deficiency despite similar elevations in systemic IL-5. Finally, in vitro treatment of bone marrow cells with IL-33, but not IL-5, led to specific early expansion of IL-5Rα-expressing precursor cells. In summary, our findings establish a basal defect in eosinophilopoiesis in IL-33- and ST2-deficient mice and a mechanism whereby IL-33 supports EoMs by driving both systemic IL-5 production and the expansion of IL-5Rα-expressing precursor cells.
