Mycobacterium tuberculosis exploits various molecules on host cells to gain entry and establish a niche for survival and replication. We characterized the role of the glycoprotein CD43 in the pathogenesis of M. tuberculosis. Using gene-deleted mice (CD43-/-), we assessed association of the bacterium with macrophages and found that CD43 was required for optimal binding of M. tuberculosis strain Erdman by splenic, peritoneal, alveolar, and bone marrow-derived macrophages. Macrophages from heterozygote (CD43+/-) mice, which express 50% less CD43 than wild type (CD43+/+) mice, bound more bacteria than CD43-/- but less than CD43+/+ indicating that the surface expression of CD43 correlates with binding of M. tuberculosis. The role of CD43 in binding bacteria may be restricted to mycobacterial species as CD43-/- macrophages also bound less Mycobacterium avium and Mycobacterium tuberculosis H37Rv, but there was no observed role in the binding of Salmonella typhimurium or Listeria monocytogenes. Although absence of CD43 resulted in decreased binding of M. tuberculosis, the subsequent growth of the bacterium within CD43-/- macrophages was enhanced as illustrated by increased bacterial numbers and decreased doubling times, indicating that that the mechanism of entry may influence subsequent. To elucidate mechanisms by which CD43 controls of growth of M. tuberculosis, we examined the induction of antimycobacterial activities. In response to M. tuberculosis, CD43-/- macrophages were deficient in the production of nitric oxide, TNF-⍺, and IL-12. Furthermore, M. tuberculosis induced less apoptosis, but more necrosis, in CD43-/- macrophages compared to CD43+/+. The enhanced growth of M. tuberculosis was abrogated by IFN-Ɣ-stimulation with whereas addition of TNF-⍺ restored both the intracellular growth rates and amounts of apoptosis to wild type levels. To investigate the role of CD43 in vivo, we infected CD43-/- and CD43+/+ mice with M. tuberculosis and assessed bacterial loads and organ pathology. Absence of CD43 resulted in increased bacterial loads in lungs and spleens during both acute and chronic stages of infection, and formation of granulomas occurred more quickly in CD43-/- mice. These data point to a dual role for CD43 in the uptake and subsequent growth of M. tuberculosis in macrophages and mice.

Mycobacterium tuberculosis exploits various molecules on host cells to gain entry and establish a niche for survival and replication. We characterized the role of the glycoprotein CD43 in the pathogenesis of M. tuberculosis. Using gene-deleted mice (CD43-/-), we assessed association of the bacterium with macrophages and found that CD43 was required for optimal binding of M. tuberculosis strain Erdman by splenic, peritoneal, alveolar, and bone marrow-derived macrophages. Macrophages from heterozygote (CD43+/-) mice, which express 50% less CD43 than wild type (CD43+/+) mice, bound more bacteria than CD43-/- but less than CD43+/+ indicating that the surface expression of CD43 correlates with binding of M. tuberculosis. The role of CD43 in binding bacteria may be restricted to mycobacterial species as CD43-/- macrophages also bound less Mycobacterium avium and Mycobacterium tuberculosis H37Rv, but there was no observed role in the binding of Salmonella typhimurium or Listeria monocytogenes. Although absence of CD43 resulted in decreased binding of M. tuberculosis, the subsequent growth of the bacterium within CD43-/- macrophages was enhanced as illustrated by increased bacterial numbers and decreased doubling times, indicating that that the mechanism of entry may influence subsequent. To elucidate mechanisms by which CD43 controls of growth of M. tuberculosis, we examined the induction of antimycobacterial activities. In response to M. tuberculosis, CD43-/- macrophages were deficient in the production of nitric oxide, TNF-⍺, and IL-12. Furthermore, M. tuberculosis induced less apoptosis, but more necrosis, in CD43-/- macrophages compared to CD43+/+. The enhanced growth of M. tuberculosis was abrogated by IFN-Ɣ-stimulation with whereas addition of TNF-⍺ restored both the intracellular growth rates and amounts of apoptosis to wild type levels. To investigate the role of CD43 in vivo, we infected CD43-/- and CD43+/+ mice with M. tuberculosis and assessed bacterial loads and organ pathology. Absence of CD43 resulted in increased bacterial loads in lungs and spleens during both acute and chronic stages of infection, and formation of granulomas occurred more quickly in CD43-/- mice. These data point to a dual role for CD43 in the uptake and subsequent growth of M. tuberculosis in macrophages and mice.

Mycobacterium tuberculosis exploits various molecules on host cells to gain entry and establish a niche for survival and replication. We characterized the role of the glycoprotein CD43 in the pathogenesis of M. tuberculosis. Using gene-deleted mice (CD43-/-), we assessed association of the bacterium with macrophages and found that CD43 was required for optimal binding of M. tuberculosis strain Erdman by splenic, peritoneal, alveolar, and bone marrow-derived macrophages. Macrophages from heterozygote (CD43+/-) mice, which express 50% less CD43 than wild type (CD43+/+) mice, bound more bacteria than CD43-/- but less than CD43+/+ indicating that the surface expression of CD43 correlates with binding of M. tuberculosis. The role of CD43 in binding bacteria may be restricted to mycobacterial species as CD43-/- macrophages also bound less Mycobacterium avium and Mycobacterium tuberculosis H37Rv, but there was no observed role in the binding of Salmonella typhimurium or Listeria monocytogenes. Although absence of CD43 resulted in decreased binding of M. tuberculosis, the subsequent growth of the bacterium within CD43-/- macrophages was enhanced as illustrated by increased bacterial numbers and decreased doubling times, indicating that that the mechanism of entry may influence subsequent. To elucidate mechanisms by which CD43 controls of growth of M. tuberculosis, we examined the induction of antimycobacterial activities. In response to M. tuberculosis, CD43-/- macrophages were deficient in the production of nitric oxide, TNF-⍺, and IL-12. Furthermore, M. tuberculosis induced less apoptosis, but more necrosis, in CD43-/- macrophages compared to CD43+/+. The enhanced growth of M. tuberculosis was abrogated by IFN-Ɣ-stimulation with whereas addition of TNF-⍺ restored both the intracellular growth rates and amounts of apoptosis to wild type levels. To investigate the role of CD43 in vivo, we infected CD43-/- and CD43+/+ mice with M. tuberculosis and assessed bacterial loads and organ pathology. Absence of CD43 resulted in increased bacterial loads in lungs and spleens during both acute and chronic stages of infection, and formation of granulomas occurred more quickly in CD43-/- mice. These data point to a dual role for CD43 in the uptake and subsequent growth of M. tuberculosis in macrophages and mice. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

CD43, a representative of the leukocyte mucin family proteins, is a transmembrane protein highly expressed on most lymphohemopoietic cells and is believed to play a role in the regulation of leukocyte activation and/or migration. CD43 was shown to be proteolytically shed from human cells and high concentrations of soluble CD43 have been found in human plasma. The biological significance of CD43 shedding however remains enigmatic. To study the functional significance of CD43 shedding, we initiated our study by investigating whether CD43 shedding also occurs in the murine system and confirmed using flow cytometry, Western blot and ELISA techniques that murine CD43 is cleaved from the cell surface as is observed in the human system. To examine the biological significance of CD43 shedding, we designed and constructed non-sheddable forms of murine CD43. Ectopic expression of non-sheddable CD43 molecules in primary CD43 deficient bone marrow cells showed that these CD43 mutants have serious negative impacts on cell viability, revealing CD43 shedding as an essential process and implying that the CD43 mutants interfered with intracellular signaling processes. Our data support the hypothesis that CD43 ectodomain shedding is a requirement for release of the cytoplasmic domain and its translocation to the nucleus. In support of our hypothesis, we confirmed that the CD43 cytoplasmic domain is localized in the nucleus and is modified by SUMO (small ubiquitin-like modifier) peptides. In an attempt to determine the functional significance of CD43 nuclear translocation and SUMO modification, we examined nuclei from hemopoietic cells more closely and observed that the CD43 cytoplasmic tail is localized in a subnuclear structure called promyelocytic nuclear bodies, which control many nuclear functions including apoptosis. Consistent with this observation we find that leukocytes from CD43 deficient mice have an increased apoptotic response upon growth factor withdrawal. We conclude that nuclear translocation of the CD43 cytoplasmic tail serves to control the apoptotic response in leukocytes and that CD43 functions as an anti-apoptotic molecule.

CD43, a representative of the leukocyte mucin family proteins, is a transmembrane protein highly expressed on most lymphohemopoietic cells and is believed to play a role in the regulation of leukocyte activation and/or migration. CD43 was shown to be proteolytically shed from human cells and high concentrations of soluble CD43 have been found in human plasma. The biological significance of CD43 shedding however remains enigmatic. To study the functional significance of CD43 shedding, we initiated our study by investigating whether CD43 shedding also occurs in the murine system and confirmed using flow cytometry, Western blot and ELISA techniques that murine CD43 is cleaved from the cell surface as is observed in the human system. To examine the biological significance of CD43 shedding, we designed and constructed non-sheddable forms of murine CD43. Ectopic expression of non-sheddable CD43 molecules in primary CD43 deficient bone marrow cells showed that these CD43 mutants have serious negative impacts on cell viability, revealing CD43 shedding as an essential process and implying that the CD43 mutants interfered with intracellular signaling processes. Our data support the hypothesis that CD43 ectodomain shedding is a requirement for release of the cytoplasmic domain and its translocation to the nucleus. In support of our hypothesis, we confirmed that the CD43 cytoplasmic domain is localized in the nucleus and is modified by SUMO (small ubiquitin-like modifier) peptides. In an attempt to determine the functional significance of CD43 nuclear translocation and SUMO modification, we examined nuclei from hemopoietic cells more closely and observed that the CD43 cytoplasmic tail is localized in a subnuclear structure called promyelocytic nuclear bodies, which control many nuclear functions including apoptosis. Consistent with this observation we find that leukocytes from CD43 deficient mice have an increased apoptotic response upon growth factor withdrawal. We conclude that nuclear translocation of the CD43 cytoplasmic tail serves to control the apoptotic response in leukocytes and that CD43 functions as an anti-apoptotic molecule.

CD43, a representative of the leukocyte mucin family proteins, is a transmembrane protein highly expressed on most lymphohemopoietic cells and is believed to play a role in the regulation of leukocyte activation and/or migration. CD43 was shown to be proteolytically shed from human cells and high concentrations of soluble CD43 have been found in human plasma. The biological significance of CD43 shedding however remains enigmatic. To study the functional significance of CD43 shedding, we initiated our study by investigating whether CD43 shedding also occurs in the murine system and confirmed using flow cytometry, Western blot and ELISA techniques that murine CD43 is cleaved from the cell surface as is observed in the human system. To examine the biological significance of CD43 shedding, we designed and constructed non-sheddable forms of murine CD43. Ectopic expression of non-sheddable CD43 molecules in primary CD43 deficient bone marrow cells showed that these CD43 mutants have serious negative impacts on cell viability, revealing CD43 shedding as an essential process and implying that the CD43 mutants interfered with intracellular signaling processes. Our data support the hypothesis that CD43 ectodomain shedding is a requirement for release of the cytoplasmic domain and its translocation to the nucleus. In support of our hypothesis, we confirmed that the CD43 cytoplasmic domain is localized in the nucleus and is modified by SUMO (small ubiquitin-like modifier) peptides. In an attempt to determine the functional significance of CD43 nuclear translocation and SUMO modification, we examined nuclei from hemopoietic cells more closely and observed that the CD43 cytoplasmic tail is localized in a subnuclear structure called promyelocytic nuclear bodies, which control many nuclear functions including apoptosis. Consistent with this observation we find that leukocytes from CD43 deficient mice have an increased apoptotic response upon growth factor withdrawal. We conclude that nuclear translocation of the CD43 cytoplasmic tail serves to control the apoptotic response in leukocytes and that CD43 functions as an anti-apoptotic molecule. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Les métastases sont responsables de 90 % des décès causés par le cancer. Les métastases sont des foyers cancéreux secondaires qui se forment à distance de la tumeur d’origine. Des cellules cancéreuses quittent la tumeur primaire, rejoignent la circulation sanguine puis colonisent des organes voisins par migration à travers l’endothélium vasculaire. Ce phénomène d’adhésion à l’endothélium et de migration à travers l’endothélium appelé l’extravasation est une étape clé du processus métastatique. L’identification des molécules impliquées constitue une priorité dans le but d’élaborer de nouvelles drogues anticancéreuses. Nous avons précédemment montré que la molécule d’adhésion cellulaire InterCellular Adhesion Molecule-1 (ICAM-1) exprimée par les cellules endothéliales, est impliquée dans l’interaction des cellules de cancer de la vessie (BCs) avec l’endothélium. Cependant les ligands d’ICAM-1 n’ont pas été étudiés. Dans cette étude, nous utilisons des tests d'adhésion cellulaire et la microscopie à force atomique (AFM) afin d’identifier les ligands d’ICAM-1 et de mesurer les forces impliquées dans l’interaction ligand-ICAM-1. Nous avons identifié que les protéines MUC1 et CD43 exprimées par les BCs les plus invasives se lient à ICAM-1 en développant des forces d’intensité différente selon le couple considéré. Une analyse détaillée des événements de rupture suggère que CD43 est fortement lié au cytosquelette et que son interaction avec ICAM-1 correspond principalement à des sauts brusques. Au contraire, MUC1 semble être lié faiblement au cytosquelette et ses interactions avec ICAM-1 sont principalement associées à la formation de filaments membranaires ou « tethers ». Les forces mises en jeu lors de la migration des cellules cancéreuses à travers l'endothélium ont été étudiées par microscopie de forces de traction (TFM). Les résultats préliminaires montrent que les tractions exercées par les cellules cancéreuses lors de l’extravasation sont mesurables par TFM. / Cancer metastasis is associated with 90% cancer-associated deaths, when cancer cells escape from the primary tumor and form metastatic colonies in secondary sites. Extravasation is an important step in cancer metastasis, where cancer cells carried in blood, adhere and transmigrate through the endothelium. Therefore identifying the key molecules involved during the adhesion process could enable to develop new anticancer cancer drugs able to inhibit the adhesion of cancer cells to the endothelium. We have previously shown that InterCellular Adhesion Molecule-1 (ICAM-1) expressed by endothelial cells is involved in the interactions of bladder cancer cells (BCs) with the endothelium. However the ICAM-1 ligands have never been investigated. In this study, we combined adhesion assays and Atomic Force Microscopy (AFM) to identify the ligands involved and to quantify the forces relevant in such interactions. We report the expression of MUC1 and CD43 on BCs and demonstrate that these ligands interact with ICAM-1 to mediate cancer cell-endothelial cell adhesion in the case of the more invasive BCs. AFM experiments were performed to quantify the force ranges involved by MUC1 and CD43 during their interaction with ICAM-1. AFM measurements combined with a Gaussian Mixture Model showed distinct force ranges for the interaction of ICAM-1 with MUC1 and ICAM-1 with CD43. Furthermore, a detailed analysis of the rupture events suggests that CD43 is strongly connected to the cytoskeleton and that its interaction with ICAM-1 mainly corresponds to force ramps followed by sudden jumps. On the contrary, MUC1 seems to be weakly connected to the cytoskeleton as its interactions with ICAM-1 are mainly associated with the formation of tethers. The forces involved during the transmigration of cancer cells through the endothelium was investigated using Traction Force Microscopy (TFM). Preliminary results showed that tractions exerted by cancer cells during transmigration can be studied and quantified using TFM.

The first definitive haematopoietic stem cells (HSCs) produced during embryonic development are generated from a specialised subset of endothelial cells known as haemogenic endothelium. Recently, it was reported that Gata3 plays a dual role in the development of sympathetic nervous system and haematopoietic system. In fact, Gata3 has proven to be crucial for the production of HSCs through regulation of catecholamine production from the co-developing sympathetic nervous system. Also, it was recently shown that Gata3 is expressed in the haemogenic endothelium and haematopoietic progenitor cells. Here, I will specifically examine the role of Gata3 in the production of HSCs; if it is expressed and plays a role in the precursors from which HSCs arise. Using a Gata3-GFP reporter mouse line, we found that Gata3 is expressed in various cell types in the HSCs microenvironment, including mesenchymal cells, endothelial cells, haematopoietic cells and sympathetic nervous system, and this expression was stage dependant. In the endothelial cells, we have found that the haemogenic endothelium activity is enriched in Gata3 expressing cells. Within the haematopoietic cells, we have found that Gata3 marks a specific stage along the developmental pathway towards the generation of definitive haematopoietic stem cells, and that Gata3 expressing haematopoietic cells are enriched for the most immature and stem cell like progenitors. Moreover, Gata3 will be specifically knocked out in haemogenic endothelial cells to determine whether it plays an essential role in the production of HSCs from the endothelium using the Vec-Cre system. We found that Gata3 within the haemogenic endothelium plays a major role in haematopoietic progenitors formation, and possibly haematopoietic stem cell formation. Finally, we used molecular assay (RNA seq) to identify the role of Gata3 in the haematopoietic stem cell microenvironment and found that Gata3 plays a major role in the development and differentiation of various cells and systems, and implicated Gata3 as cell cycle regulator. In summary, we found that Gata3 expressing cells is enriched for haemogenic endothelium, crucial for the haematopoietic progenitors formation, plays and important role in endothelial to haematopoietic transition, and plays a key developmental role in both haematopoietic stem cell and its microenvironment.