Abstract 118: Interleukin 4 And 13 Signaling In Macrophages Regulates Neonatal Cardiac Regeneration

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Santiago Alvarez ◽  
Caitlin Omeara
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Cardiac Regeneration ◽  
Capillary Density ◽  
Lymphoid Cells ◽  
Ischemic Myocardium ◽  
Current Therapy ◽  
Neonatal Mice

Introduction: Heart failure (HF) is a prevalent disease, projected to affect over 8 million Americans by 2030. Current therapy partially decreases progression; however, mortality and disease burden continue to be high. Therefore, there is an unmet need to develop new strategies that target HF progression. Our lab employs the neonatal mouse model of cardiac regeneration to identify pro-reparative pathways that can be applied to treating HF in humans. Previously we demonstrated that the anti-inflammatory cytokine, Interleukin 13 (IL13), promotes cardiac regeneration, however, the cell types mediating this response remain unknown. IL13 and the related cytokine, IL4, share a common receptor (IL4Rα) and both cytokines polarize macrophages into a reparative phenotype. Here, we hypothesize that IL4/13 signaling to macrophages promotes heart regeneration after cardiac injury and we explore the cell source of these cytokines during neonatal development and cardiac regeneration. Methods and Results: We generated a genetic model whereby IL4Rα is depleted in macrophages by crossing IL4Rα floxed (IL4Rα fl/fl ) mice with transgenic mice CX3Cr1 driven-Cre recombinase (CX3CR1 Cre ). Flow cytometry analysis confirmed depletion of IL4Rα in cardiac macrophages. We performed myocardial infarction (MI) on postnatal day 1 (P1) mice and assessed cardiac regeneration by ultrasonography 21 days post-injury (dpi). We found that IL4Rα fl/fl CX3CR1 Cre mice had lower ejection fraction compared to IL4Rα fl/fl littermate controls. Preliminary results suggest there is a reduced capillary density in peri-ischemic myocardium. In addition, we used fluorescent reporter mouse lines; IL4-enhanced green fluorescent protein (IL4-GFP) and IL13-yellow fluorescent protein (IL13-YFP), to assess the cellular source of IL4 and IL13 expression in the hearts of neonatal mice. In unoperated mice, we found no detectable expression of IL13 by any cell type in the heart, whereas IL4 was expressed in innate lymphoid cells (ILCs) and T cells. 4 days after MI in P1 mice IL13 was upregulated in ILCs and T cells and IL4 was expressed by ILCs and T cells and upregulated in myeloid cells. Conclusions and Discussion: We found that IL13 and IL4 are primarily expressed in ILCs and T cells following neonatal injury, suggesting a novel role for ILCs and T cells in the production of IL4 and 13 during the neonatal regeneration process. In addition, we found that lack of IL4/13 signaling in macrophages via depletion IL4Rα impairs cardiac regeneration after MI in neonatal mice, and results in reduced capillary density in peri-ischemic myocardium. We hypothesize that IL4Rα depletion in macrophages impairs reparative macrophage polarization after MI and promotes an inflammatory polarization. Future studies will be aimed to assess macrophage phenotypes in response to IL13/IL4 signaling by transcriptional profiling and flow cytometry.

scholarly journals T cell receptor ligation induces the formation of dynamically regulated signaling assemblies

2002 ◽  
Vol 158 (7) ◽  
pp. 1263-1275 ◽  
Author(s):  
Stephen C. Bunnell ◽  
David I. Hong ◽  
Julia R. Kardon ◽  
Tetsuo Yamazaki ◽  
C. Jane McGlade ◽  
...  
Keyword(s):  
T Cells ◽  
Lipid Raft ◽  
Synapse Formation ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Cell Receptor ◽  
Rich Cluster ◽  
Enhanced Yellow Fluorescent Protein ◽  
Immune Synapse ◽  
Signaling Complex

Tcell antigen receptor (TCR) ligation initiates tyrosine kinase activation, signaling complex assembly, and immune synapse formation. Here, we studied the kinetics and mechanics of signaling complex formation in live Jurkat leukemic T cells using signaling proteins fluorescently tagged with variants of enhanced GFP (EGFP). Within seconds of contacting coverslips coated with stimulatory antibodies, T cells developed small, dynamically regulated clusters which were enriched in the TCR, phosphotyrosine, ZAP-70, LAT, Grb2, Gads, and SLP-76, excluded the lipid raft marker enhanced yellow fluorescent protein–GPI, and were competent to induce calcium elevations. LAT, Grb2, and Gads were transiently associated with the TCR. Although ZAP-70–containing clusters persisted for more than 20 min, photobleaching studies revealed that ZAP-70 continuously dissociated from and returned to these complexes. Strikingly, SLP-76 translocated to a perinuclear structure after clustering with the TCR. Our results emphasize the dynamically changing composition of signaling complexes and indicate that these complexes can form within seconds of TCR engagement, in the absence of either lipid raft aggregation or the formation of a central TCR-rich cluster.

De novo generation of CD4 T cells against viruses present in the host during immune reconstitution

Blood ◽  
2005 ◽  
Vol 105 (6) ◽  
pp. 2410-2414 ◽  
Author(s):  
Tomas Kalina ◽  
Hailing Lu ◽  
Zhao Zhao ◽  
Earl Blewett ◽  
Dirk P. Dittmer ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Fluorescent Protein ◽  
De Novo ◽  
Yellow Fluorescent Protein ◽  
Autologous Transplantation ◽  
Elderly Persons ◽  
Barr Virus ◽  
Epstein Barr ◽  
Self Peptides

AbstractT cells recognizing self-peptides are typically deleted in the thymus by negative selection. It is not known whether T cells against persistent viruses (eg, herpesviruses) are generated by the thymus (de novo) after the onset of the infection. Peptides from such viruses might be considered by the thymus as self-peptides, and T cells specific for these peptides might be deleted (negatively selected). Here we demonstrate in baboons infected with baboon cytomegalovirus and baboon lymphocryptovirus (Epstein-Barr virus–like virus) that after autologous transplantation of yellow fluorescent protein (YFP)–marked hematopoietic cells, YFP+ CD4 T cells against these viruses were generated de novo. Thus the thymus generates CD4 T cells against not only pathogens absent from the host but also pathogens present in the host. This finding provides a strong rationale to improve thymopoiesis in recipients of hematopoietic cell transplants and, perhaps, in other persons lacking de novo–generated CD4 T cells, such as AIDS patients and elderly persons.

Integrin αE(CD103)β7 influences cellular shape and motility in a ligand-dependent fashion

Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 619-625 ◽  
Author(s):  
Stephanie Schlickum ◽  
Helga Sennefelder ◽  
Mike Friedrich ◽  
Gregory Harms ◽  
Martin J. Lohse ◽  
...  
Keyword(s):  
T Cells ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Current Data ◽  
Immune Functions ◽  
Wild Type ◽  
Deficient Mice ◽  
E Cadherin

Abstract While the extravasation cascade of lymphocytes is well characterized, data on their intraepithelial positioning and morphology are scant. However, the latter process is presumably crucial for many immune functions. Integrin αE(CD103)β7 has previously been implicated in epithelial retention of some T cells through binding to E-cadherin. Our current data suggest that αE(CD103)β7 also determines shape and motility of some lymphocytes. Time-lapse microscopy showed that wild-type αE(CD103)β7 conferred the ability to form cell protrusions/filopodia and to move in an amoeboid fashion on E-cadherin, an activity that was abrogated by αE(CD103)β7-directed antibodies or cytochalasin D. The αE-dependent motility was further increased (P < .001) when point-mutated αE(CD103) locked in a constitutively active conformation was expressed. Moreover, different yellow fluorescent protein–coupled αE(CD103) species demonstrated that the number and length of filopodia extended toward purified E-cadherin, cocultured keratinocytes, cryostat-cut skin sections, or epidermal sheets depended on functional αE(CD103). The in vivo relevance of these findings was demonstrated by wild-type dendritic epidermal T cells (DETCs), which showed significantly more dendrites and spanned larger epidermal areas as compared with DETCs of αE(CD103)-deficient mice (P < .001). Thus, integrin αE(CD103)β7 is not only involved in epithelial retention, but also in shaping and proper intraepithelial morphogenesis of some leukocytes.

ILC2-derived IL-9 inhibit s colorectal cancer progression by activating CD8+ T cells

2020 ◽  
Author(s):  
Jie Wan ◽  
Lan Huang ◽  
Yinqiu Wu ◽  
Xiaoyun Ji ◽  
Shun Yao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
Cancer Progression ◽  
Normal Tissue ◽  
Lymphoid Cells ◽  
Adjacent Normal Tissue

Abstract Background Type 2 innate lymphoid cells (ILC2s), characterized by secreting type 2 cytokines, regulate multiple immune responses. ILC2s are found in different tumor tissues and ILC2-derived interleukin (IL)-4, IL-5, and IL-13 act on the cells in tumor microenvironment to participate in tumor progression. ILC2s are abundant in colorectal cancer (CRC) tissue, but the role of ILC2s in CRC remains unclear. Methods ILC2s were sorted from the spleen using microbeads combined with flow cytometry and tumor infiltrating CD8+ T cells were isolated from tumor tissue by microbeads. Flow cytometry and immunofluorescence were used to detect the percentage of ILC2s and CD8+ T cells in the spleen and CRC tissue. Effects of IL-9 and IL-9-stimulated CD8+ T cells on CT26 cells were measured by proliferation, apoptosis, and migration assays in vitro. GEPIA was used to detect the ILC2s chemokines in CRC tissue and adjacent normal tissue. Results We found that ILC2s were increased in CRC tissue compared with the adjacent normal tissue. In vitro experiments showed that IL-9 could activate CD8+ T cells to promote the death of CT26 cells. ILC2s were the main IL-9-secreting cells in CRC tissue as shown by flow cytometry analysis. In vivo experiments showed that neutralizing ILC2s promoted the tumor growth, while tumor inhibition occurred by intravenous injection of IL-9. Conclusions Our results demonstrated that ILC2-derived IL-9 activated CD8+ T cells to promote anti-tumor effects in CRC.

Changes Identified by Flow Cytometry and WT1 Expression in Consecutive Bone Marrow Samples in Refractory Cytopenia of Childhood and Aplastic Anemia Before Start of the Therapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1342-1342
Author(s):  
Michaela Reiterova ◽  
Karolina Kramarzova ◽  
Martina Sukova ◽  
Vit Campr ◽  
Elena Vodickova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Aplastic Anemia ◽  
Conflicts Of Interest ◽  
Lymphoid Cells ◽  
Time Interval ◽  
Monosomy 7 ◽  
Time Points ◽  
Time Point

Abstract Abstract 1342 Introduction. Aplastic anemia (AA) and myelodysplastic syndrome (MDS) are rare diseases in childhood. The most common subtype of MDS is refractory cytopenia (RC). Both diseases typically exhibit with overlapping features and in both disorders dysregulation of immune system variably contributes to the degree of bone marrow (BM) failure. In the diagnostic algorithm plays role also analysis of consecutive BM samples by morphology. Patients and methods. Patients diagnosed between 2005 – 2011 with at least two BM samples analyzed by flow cytometry (FC) before treatment has started and with centrally evaluated BM biopsy according to EWOG MDS criteria were included into the study. We compared first and the last available sample before treatment (immunosupression or stem cell transplantation). By FC we analyzed following parameters: cell subsets (granulocytes, monocytes, lymphoid cells, erythroid precursors), BM precursors (CD34pos, CD117pos), T cells (CD3pos, CD3pos4pos, CD3pos8pos, CD3posHLA DRpos out of all cells, HLA DRpos out of CD3pos/CD3pos4pos/CD3pos8pos cells); B cells (CD19pos, CD19pos10pos, CD19pos45dim to neg, CD19pos34posout of all cells, CD10pos and CD20pos10neg out of CD19pos). In total 22 patients with AA (12 girls, 10 boys, mean age 11 years; 1.1–18 years) and 20 patients with RC (11 girls, 9 boys, mean age 11 years; 3.7–18) were included into the study. Median of time interval between both samples was 139 (1–1343) days in RC and 15 (1–56) days in AA. WT1 expression on mRNA level was analyzed in the sample before treatment with the highest number of CD34pos precursors to avoid blood contamination. All patients were screened by FISH for changes on chromosome 7 and 8. We asked following questions: Are there differences in the parameters in both bone marrow samples between SAA and RC? Is there any different pattern between d0 and before therapy sample between AA and RC? Are there any differences in WT1 expression between AA and RC group? Results. RC and AA significantly differ in both time points. AA patients have significantly decreased precursors (CD34, CD117); the difference is more pronounced at the later time point. More lymphocytes (both B and T) and less granulocytes are present at later time point in AA patients (p<0.05, Mann-Whitney test). Activation of CD8 cytotoxic T cells according to HLA DR expression is more distinct in AA patients at later time point. The most significant different parameter between RC and AA is a ratio CD19/CD34 also with the significant trend between two time points (Two way ANOVA, p<0.05). WT1 expression is statistically higher in RC patients; the higher expression is associated with presence of monosomy 7. Conclusion. By FC statistical differences can be identified in both samples (d0 and before treatment) between RC and AA. More pronounced differences are at later time point, which can be explained by further destruction of precursor and myeloid compartment more pronounced in AA patients compared to RC. WT1 expression is typically high in patients with RC and monosomy 7. Disclosures: No relevant conflicts of interest to declare.

High-Throughput Flow Cytometry–Based Assay to Identify Apoptosis-Inducing Proteins

2007 ◽  
Vol 12 (4) ◽  
pp. 510-520 ◽  
Author(s):  
Mamatha Sauermann ◽  
Florian Hahne ◽  
Christian Schmidt ◽  
Meher Majety ◽  
Heiko Rosenfelder ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Specific Antibody ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Nuclear Fragmentation ◽  
Protein Fluorescence ◽  
Transfected Cells ◽  
High Throughput Assay

After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry—based high-throughput assay to screen for apoptosis-activating proteins in transiently transfected cells. The assay is based on the detection of activated caspase-3 with a specific antibody, in cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent protein. Fluorescence intensities are measured using a flow cytometer integrated with a high-throughput autosampler. The applicability of this screen has been tested in a pilot screen with 200 proteins. The candidate proteins were all verified in an independent microscopy-based nuclear fragmentation assay, finally resulting in the identification of 6 apoptosis inducers. ( Journal of Biomolecular Screening 2007:510-520)

scholarly journals Parasite-Specific CD4+IFN-γ+IL-10+T Cells Distribute within Both Lymphoid and Nonlymphoid Compartments and Are Controlled Systemically by Interleukin-27 and ICOS during Blood-Stage Malaria Infection

2015 ◽  
Vol 84 (1) ◽  
pp. 34-46 ◽  
Author(s):  
Ana Villegas-Mendez ◽  
Tovah N. Shaw ◽  
Colette A. Inkson ◽  
Patrick Strangward ◽  
J. Brian de Souza ◽  
...  
Keyword(s):  
T Cells ◽  
Malaria Infection ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Interleukin 10 ◽  
Plasmodium Yoelii ◽  
Blood Stage ◽  
Content Type ◽  
Ifn Γ ◽  
Blood Stage Malaria

Immune-mediated pathology in interleukin-10 (IL-10)-deficient mice during blood-stage malaria infection typically manifests in nonlymphoid organs, such as the liver and lung. Thus, it is critical to define the cellular sources of IL-10 in these sensitive nonlymphoid compartments during infection. Moreover, it is important to determine if IL-10 production is controlled through conserved or disparate molecular programs in distinct anatomical locations during malaria infection, as this may enable spatiotemporal tuning of the regulatory immune response. In this study, using dual gamma interferon (IFN-γ)–yellow fluorescent protein (YFP) and IL-10–green fluorescent protein (GFP) reporter mice, we show that CD4+YFP+T cells are the major source of IL-10 in both lymphoid and nonlymphoid compartments throughout the course of blood-stagePlasmodium yoeliiinfection. Mature splenic CD4+YFP+GFP+T cells, which preferentially expressed high levels of CCR5, were capable of migrating to and seeding the nonlymphoid tissues, indicating that the systemically distributed host-protective cells have a common developmental history. Despite exhibiting comparable phenotypes, CD4+YFP+GFP+T cells from the liver and lung produced significantly larger quantities of IL-10 than their splenic counterparts, showing that the CD4+YFP+GFP+T cells exert graded functions in distinct tissue locations during infection. Unexpectedly, given the unique environmental conditions within discrete nonlymphoid and lymphoid organs, we show that IL-10 production by CD4+YFP+T cells is controlled systemically during malaria infection through IL-27 receptor signaling that is supported after CD4+T cell priming by ICOS signaling. The results in this study substantially improve our understanding of the systemic IL-10 response to malaria infection, particularly within sensitive nonlymphoid organs.

scholarly journals Identification of antigen-presenting dendritic cells in mouse aorta and cardiac valves

2009 ◽  
Vol 206 (3) ◽  
pp. 497-505 ◽  
Author(s):  
Jae-Hoon Choi ◽  
Yoonkyung Do ◽  
Cheolho Cheong ◽  
Hyein Koh ◽  
Silvia B. Boscardin ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Human Aorta ◽  
Protein Antigens ◽  
Cardiac Valves ◽  
Aortic Sinus ◽  
Enhanced Yellow Fluorescent Protein ◽  
Antigen Presenting

Presumptive dendritic cells (DCs) bearing the CD11c integrin and other markers have previously been identified in normal mouse and human aorta. We used CD11c promoter–enhanced yellow fluorescent protein (EYFP) transgenic mice to visualize aortic DCs and study their antigen-presenting capacity. Stellate EYFP+ cells were readily identified in the aorta and could be double labeled with antibodies to CD11c and antigen-presenting major histocompatability complex (MHC) II products. The DCs proved to be particularly abundant in the cardiac valves and aortic sinus. In all aortic locations, the CD11c+ cells localized to the subintimal space with occasional processes probing the vascular lumen. Aortic DCs expressed little CD40 but expressed low levels of CD1d, CD80, and CD86. In studies of antigen presentation, DCs selected on the basis of EYFP expression or binding of anti-CD11c antibody were as effective as DCs similarly selected from the spleen. In particular, the aortic DCs could cross-present two different protein antigens on MHC class I to CD8+ TCR transgenic T cells. In addition, after intravenous injection, aortic DCs could capture anti-CD11c antibody and cross-present ovalbumin to T cells. These results indicate that bona fide DCs are a constituent of the normal aorta and cardiac valves.

Role of RUNX1 in adult hematopoiesis: analysis of RUNX1-IRES-GFP knock-in mice reveals differential lineage expression

Blood ◽  
2004 ◽  
Vol 103 (7) ◽  
pp. 2522-2529 ◽  
Author(s):  
Robert B. Lorsbach ◽  
Jennifer Moore ◽  
Sonny O. Ang ◽  
Weili Sun ◽  
Noel Lenny ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Cell Development ◽  
B Cell Development ◽  
Lymphoid Cells ◽  
Maturation Stage ◽  
Hematopoietic System ◽  
Runx1 Expression

Abstract The Runx1/core binding factor-β (CBFβ) transcriptional complex is required for the establishment of hematopoiesis during development. Despite its critical role during development, a detailed analysis of Runx1 expression within specific lineages and developmental stages of the adult hematopoietic system is lacking. To address this, we have developed a Runx1—green fluorescent protein (GFP) knock-in mouse. We show that Runx1 is expressed in several hematopoietic lineages, including myeloid, B-lymphoid, and T-lymphoid cells. By contrast, Runx1 is weakly expressed in early erythroid cells, and its expression is rapidly extinguished during later stages of erythropoiesis. Runx1 expression is induced during early B-cell development and is expressed at a uniform level during all subsequent stages of B-cell development. Within the thymus, Runx1 is expressed at the highest level in CD4-CD8- double-negative thymocytes. In peripheral T cells, Runx1 is differentially expressed, with CD4+ T cells expressing 2- to 3-fold higher levels of Runx1 than CD8+ cells. Taken together, these findings indicate that although widely expressed in the hematopoietic system, the expression of Runx1 is regulated in a cell type— and maturation stage—specific manner. In addition, the Runx1-IRES-GFP knock-in mouse strain should prove valuable for investigation of Runx1 function in adult hematopoiesis.

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