Modulation of Neutrophil Function by a Secreted Mucinase of Escherichia coli O157∶H7

Escherichia coli O157∶H7 is a human enteric pathogen that causes hemorrhagic colitis which can progress to hemolytic uremic syndrome, a severe kidney disease with immune involvement. During infection, E. coli O157∶H7 secretes StcE, a metalloprotease that promotes the formation of attaching and effacing lesions and inhibits the complement cascade via cleavage of mucin-type glycoproteins. We found that StcE cleaved the mucin-like, immune cell-restricted glycoproteins CD43 and CD45 on the neutrophil surface and altered neutrophil function. Treatment of human neutrophils with StcE led to increased respiratory burst production and increased cell adhesion. StcE-treated neutrophils exhibited an elongated morphology with defective rear detachment and impaired migration, suggesting that removal of the anti-adhesive capability of CD43 by StcE impairs rear release. Use of zebrafish embryos to model neutrophil migration revealed that StcE induced neutrophil retention in the fin after tissue wounding, suggesting that StcE modulates neutrophil-mediated inflammation in vivo. Neutrophils are crucial innate effectors of the antibacterial immune response and can contribute to severe complications caused by infection with E. coli O157∶H7. Our data suggest that the StcE mucinase can play an immunomodulatory role by directly altering neutrophil function during infection. StcE may contribute to inflammation and tissue destruction by mediating inappropriate neutrophil adhesion and activation

ERK1 Regulates the Hematopoietic Stem Cell Niches

The mitogen-activated protein kinases (MAPK) ERK1 and ERK2 are among the major signal transduction molecules but little is known about their specific functions in vivo. ERK activity is provided by two isoforms, ERK1 and ERK2, which are ubiquitously expressed and share activators and substrates. However, there are not in vivo studies which have reported a role for ERK1 or ERK2 in HSCs and the bone marrow microenvironment. The present study shows that the ERK1-deficient mice present a mild osteopetrosis phenotype. The lodging and the homing abilities of the ERK1−/− HSC are impaired, suggesting that the ERK1−/−-defective environment may affect the engrafment of HSCs. Serial transplantations demonstrate that ERK1 is involved in the maintenance of an appropriate medullar microenvironment, but that the intrinsic properties of HSCs are not altered by the ERK1−/− defective microenvironment. Deletion of ERK1 impaired in vitro and in vivo osteoclastogenesis while osteoblasts were unaffected. As osteoclasts derive from precursors of the monocyte/macrophage lineage, investigation of the monocytic compartment was performed. In vivo analysis of the myeloid lineage progenitors revealed that the frequency of CMPs increased by approximately 1.3-fold, while the frequency of GMPs significantly decreased by almost 2-fold, compared with the respective WT compartments. The overall mononuclear-phagocyte lineage development was compromised in these mice due to a reduced expression of the M-CSF receptor on myeloid progenitors. These results show that the cellular targets of ERK1 are M-CSFR-responsive cells, upstream to osteoclasts. While ERK1 is well known to be activated by M-CSF, the present results are the first to point out an ERK1-dependent M-CSFR regulation on hematopoietic progenitors. This study reinforces the hypothesis of an active cross-talk between HSCs, their progeny and bone cells in the maintenance of the homeostasis of these compartments

The Mouse Cytomegalovirus Gene m42 Targets Surface Expression of the Protein Tyrosine Phosphatase CD45 in Infected Macrophages

The receptor-like protein tyrosine phosphatase CD45 is expressed on the surface of cells of hematopoietic origin and has a pivotal role for the function of these cells in the immune response. Here we report that following infection of macrophages with mouse cytomegalovirus (MCMV) the cell surface expression of CD45 is drastically diminished. Screening of a set of MCMV deletion mutants allowed us to identify the viral gene m42 of being responsible for CD45 down-modulation. Moreover, expression of m42 independent of viral infection upon retroviral transduction of the RAW264.7 macrophage cell line led to comparable regulation of CD45 expression. In immunocompetent mice infected with an m42 deletion mutant lower viral titers were observed in all tissues examined when compared to wildtype MCMV, indicating an important role of m42 for viral replication in vivo. The m42 gene product was identified as an 18 kDa protein expressed with early kinetics and is predicted to be a tailanchored membrane protein. Tracking of surface-resident CD45 molecules revealed that m42 induces internalization and degradation of CD45. The observation that the amounts of the E3 ubiquitin ligases Itch and Nedd4 were diminished in cells expressing m42 and that disruption of a PY motif in the N-terminal part of m42 resulted in loss of function, suggest that m42 acts as an activator or adaptor for these Nedd4-like ubiquitin ligases, which mark CD45 for lysosomal degradation. In conclusion, the down-modulation of CD45 expression in MCMV-infected myeloid cells represents a novel pathway of virus-host interaction

Overexpression of CXCR4 on human CD34(+) progenitors increases their proliferation, migration, and NOD/SCID repopulation

A major limitation to clinical stem cell-mediated gene therapy protocols is the low levels of engraftment by transduced progenitors. We report that CXCR4 overexpression on human CD34(+) progenitors using a lentiviral gene transfer technique helped navigate these cells to the murine bone marrow and spleen in response to stromal-derived factor 1 (SDF-1) signaling. Cells overexpressing CXCR4 exhibited significant increases in SDF-1-mediated chemotaxis and actin polymerization compared with control cells. A major advantage of CXCR4 overexpression was demonstrated by the ability of transduced CD34(+) cells to respond to lower, physiologic levels of SDF-1 when compared to control cells, leading to improved SDIF-1-induced migration and proliferation/survival, and finally resulting in significantly higher levels of in vivo repopulation of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice including primitive CD34(+)/CD38(-/low) cells. Importantly, no cellular transformation was observed following transduction with the CXCR4 vector. Unexpectedly, we documented lack of receptor internalization in response to high levels of SDF-1, which can also contribute to increased migration and proliferation by the transduced CD34(+) cells. Our results suggest CXCR4 overexpression for improved definitive human stem cell motility, retention, and multilineage repopulation, which could be beneficial for in vivo navigation and expansion of hematopoietic progenitors

Overexpression of CXCR4 on human CD34(+) progenitors increases their proliferation, migration, and NOD/SCID repopulation

A major limitation to clinical stem cell-mediated gene therapy protocols is the low levels of engraftment by transduced progenitors. We report that CXCR4 overexpression on human CD34(+) progenitors using a lentiviral gene transfer technique helped navigate these cells to the murine bone marrow and spleen in response to stromal-derived factor 1 (SDF-1) signaling. Cells overexpressing CXCR4 exhibited significant increases in SDF-1-mediated chemotaxis and actin polymerization compared with control cells. A major advantage of CXCR4 overexpression was demonstrated by the ability of transduced CD34(+) cells to respond to lower, physiologic levels of SDF-1 when compared to control cells, leading to improved SDIF-1-induced migration and proliferation/survival, and finally resulting in significantly higher levels of in vivo repopulation of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice including primitive CD34(+)/CD38(-/low) cells. Importantly, no cellular transformation was observed following transduction with the CXCR4 vector. Unexpectedly, we documented lack of receptor internalization in response to high levels of SDF-1, which can also contribute to increased migration and proliferation by the transduced CD34(+) cells. Our results suggest CXCR4 overexpression for improved definitive human stem cell motility, retention, and multilineage repopulation, which could be beneficial for in vivo navigation and expansion of hematopoietic progenitors