scholarly journals The role of dermis resident macrophages and their interaction with neutrophils in the early establishment of Leishmania major infection transmitted by sand fly bite

2020 ◽  
Author(s):  
Mariana M. Chaves ◽  
Sang Hun Lee ◽  
Olena Kamenyeva ◽  
Kashinath Ghosh ◽  
David Sacks
Keyword(s):  
Flow Cytometry ◽  
Tyrosine Kinases ◽  
Leishmania Major ◽  
Receptor Tyrosine Kinases ◽  
Sand Fly ◽  
Sand Flies ◽  
Cellular Interactions ◽  
Anti Inflammatory ◽  
Severe Pathology

AbstractThere is substantial experimental evidence to indicate that Leishmania infections that are transmitted naturally by the bites of infected sand flies differ in fundamental ways from the inflammatory and immune reactions initiated by needle inocula. We have used flow cytometry and intravital microscopy (IVM) to reveal the heterogeneity of sand fly transmission sites with respect to the subsets of phagocytes in the skin that harbor L. major within the first hours and days after infection. By flow cytometry analysis, dermis resident macrophages (TRMs) were on average the predominant infected cell type at 1 hr and 24 hr. By confocal IVM, the co-localization of L. major and neutrophils varied depending on the proximity of deposited parasites to the presumed site of vascular damage, defined by the highly localized swarming of neutrophils. Some of the dermal TRMs could be visualized acquiring their infections via transfer from or efferocytosis of parasitized neutrophils, providing direct evidence for the “Trojan Horse” model. The role of neutrophil engulfment by dermal TRMs and the involvement of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these interactions and in sustaining the anti-inflammatory program of dermal TRMs was supported by the effects observed in neutrophil depleted and in Axl-/-Mertk-/- mice. The Axl-/-Mertk-/- mice also displayed reduced parasite burdens but more severe pathology following L. major infection transmitted by sand fly bite.SummarySand flies transmit Leishmania major which causes cutaneous leishmaniasis in humans and in non-human hosts. Our analyses of sand fly transmission sites of L. major in the mouse skin revealed that dermis resident macrophages (TRM) were the predominant phagocytes to take up the parasite within the first 24 hr post-bite. The early involvement of neutrophils varied depending on the proximity of deposited parasites to the site of tissue damage around which the neutrophils coalesced. By intra-vital microscopy, some of the dermal TRMs could be visualized acquiring their infections by direct transfer from or phagocytosis of parasitized neutrophils. The involvement of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these cellular interactions and in sustaining the anti-inflammatory functions of dermal TRMs was supported by the reduced parasite burdens but more severe pathology observed in Axl-/-Mertk-/- mice. The heterogeneity of sand fly transmission sites with respect to the dose of parasites and the early cellular interactions described here likely contribute to the wide range of infection outcomes that are associated with natural transmission of L. major observed in mouse models and possibly humans.

scholarly journals The role of dermis resident macrophages and their interaction with neutrophils in the early establishment of Leishmania major infection transmitted by sand fly bite

2020 ◽  
Vol 16 (11) ◽  
pp. e1008674
Author(s):  
Mariana M. Chaves ◽  
Sang Hun Lee ◽  
Olena Kamenyeva ◽  
Kashinath Ghosh ◽  
Nathan C. Peters ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tyrosine Kinases ◽  
Leishmania Major ◽  
Direct Evidence ◽  
Sand Fly ◽  
Flow Cytometry Analysis ◽  
Cell Type ◽  
Severe Pathology ◽  
Early Establishment

There is substantial experimental evidence to indicate that Leishmania infections that are transmitted naturally by the bites of infected sand flies differ in fundamental ways from those initiated by needle inocula. We have used flow cytometry and intravital microscopy (IVM) to reveal the heterogeneity of sand fly transmission sites with respect to the subsets of phagocytes in the skin that harbor L. major within the first hours and days after infection. By flow cytometry analysis, dermis resident macrophages (TRMs) were on average the predominant infected cell type at 1 hr and 24 hr. By confocal IVM, the co-localization of L. major and neutrophils varied depending on the proximity of deposited parasites to the presumed site of vascular damage, defined by the highly localized swarming of neutrophils. Some of the dermal TRMs could be visualized acquiring their infections via transfer from or efferocytosis of parasitized neutrophils, providing direct evidence for the “Trojan Horse” model. The role of neutrophil engulfment by dermal TRMs and the involvement of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these interactions and in sustaining the anti-inflammatory program of dermal TRMs was supported by the effects observed in neutrophil depleted and in Axl-/-Mertk-/- mice. The Axl-/-Mertk-/- mice also displayed reduced parasite burdens but more severe pathology following L. major infection transmitted by sand fly bite.

scholarly journals The role of surface glycoconjugates in Leishmania midgut attachment examined by competitive binding assays and experimental development in sand flies

Parasitology ◽  
2013 ◽  
Vol 140 (8) ◽  
pp. 1026-1032 ◽  
Author(s):  
LUCIE JECNA ◽  
ANNA DOSTALOVA ◽  
RAY WILSON ◽  
VERONIKA SEBLOVA ◽  
KWANG-POO CHANG ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Critical Role ◽  
Competitive Binding ◽  
Sand Fly ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Binding Assays ◽  
Experimental Development ◽  
Competitive Binding Assays

SUMMARYBinding of promastigotes to the sand fly midgut epithelium is regarded as an essential part of the Leishmania life cycle in the vector. Among Leishmania surface molecules putatively involved in attachment to the sand fly midgut, two GPI-anchored molecules are the most prominent: lipophosphoglycan (LPG) and promastigote surface protease gp63. In this work, we examined midgut attachment of Leishmania lines mutated in GPI-anchored molecules and compared results from 2 different techniques: in vivo development in sand flies and in vitro competitive binding assays using fluorescently labelled parasites. In combination with previous studies, our data provide additional support for (1) an LPG-independent parasite-binding mechanism of Leishmania major within the midgut of the permissive vector Phlebotomus perniciosus, and provide strong support for (2) the crucial role of L. major LPG in specific vector Phlebotomus papatasi, and (3) a role for Leishmania amazonensis gp63 in Lutzomyia longipalpis midgut binding. Moreover, our results suggest a critical role for GPI-anchored proteins and gp63 in Leishmania mexicana attachment to L. longipalpis midguts, as the wild type (WT) line accounted for over 99% of bound parasites.

A novel role for the peritrophic matrix in protecting Leishmania from the hydrolytic activities of the sand fly midgut

Parasitology ◽  
1997 ◽  
Vol 115 (4) ◽  
pp. 359-369 ◽  
Author(s):  
P. F. P. PIMENTA ◽  
G. B. MODI ◽  
S. T. PEREIRA ◽  
M. SHAHABUDDIN ◽  
D. L. SACKS
Keyword(s):  
Digestive Enzymes ◽  
Leishmania Major ◽  
Specific Inhibitor ◽  
Sand Fly ◽  
Peritrophic Matrix ◽  
Phlebotomus Papatasi ◽  
Hydrolytic Activities ◽  
Natural Vector ◽  
Parasite Survival

The role of the peritrophic matrix (PM) in the development of Leishmania major infections in a natural vector, Phlebotomus papatasi, was investigated by addition of exogenous chitinase to the bloodmeal, which completely blocked PM formation. Surprisingly, the absence of the PM was associated with the loss of midgut infections. The chitinase was not directly toxic to the parasite, nor were midgut infections lost due to premature expulsion of the bloodmeal. Most parasites were killed in chitinase-treated flies within the first 4 h after feeding. Substantial early killing was also observed in control flies, suggesting that the lack of PM exacerbates lethal conditions which normally exist in the blood-fed midgut. Early parasite mortality was reversed by soybean trypsin inhibitor. Allosamadin, a specific inhibitor of chitinase, led to a thickening of the PM, and also prevented the early parasite mortality seen in infected flies. Susceptibility to gut proteases was extremely high in transitional-stage parasites, while amastigotes and fully transformed promastigotes were relatively resistant. A novel role for the PM in promoting parasite survival is suggested, in which the PM creates a barrier to the rapid diffusion of digestive enzymes, and limits the exposure of parasites to these enzymes during the time when they are especially vulnerable to proteolytic damage.

scholarly journals Receptor Tyrosine Kinases in Kidney Development

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Renfang Song ◽  
Samir S. El-Dahr ◽  
Ihor V. Yosypiv
Keyword(s):  
Tyrosine Kinases ◽  
Molecular Mechanisms ◽  
Kidney Development ◽  
Receptor Tyrosine Kinases ◽  
Adaptor Proteins ◽  
Downstream Signaling ◽  
Arterial Blood ◽  
Intracellular Signal ◽  
Human Birth

The kidney plays a fundamental role in the regulation of arterial blood pressure and fluid/electrolyte homeostasis. As congenital anomalies of the kidney and urinary tract (CAKUT) constitute one of the most common human birth defects, improved understanding of the cellular and molecular mechanisms that lead to CAKUT is critical. Accumulating evidence indicates that aberrant signaling via receptor tyrosine kinases (RTKs) is causally linked to CAKUT. Upon activation by their ligands, RTKs dimerize, undergo autophosphorylation on specific tyrosine residues, and interact with adaptor proteins to activate intracellular signal transduction pathways that regulate diverse cell behaviours such as cell proliferation, survival, and movement. Here, we review the current understanding of role of RTKs and their downstream signaling pathways in the pathogenesis of CAKUT.

scholarly journals The Role of Receptor Tyrosine Kinases in Lassa Virus Cell Entry

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 857 ◽  
Author(s):  
Chiara Fedeli ◽  
Hector Moreno ◽  
Stefan Kunz
Keyword(s):  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Complex Function ◽  
Hemorrhagic Fever ◽  
Receptor Expression ◽  
Cell Entry ◽  
Lassa Virus ◽  
Emerging Viruses ◽  
Extracellular Matrix Molecule

The zoonotic Old World mammarenavirus Lassa (LASV) causes severe hemorrhagic fever with high mortality and morbidity in humans in endemic regions. The development of effective strategies to combat LASV infections is of high priority, given the lack of a licensed vaccine and restriction on available treatment to off-label use of ribavirin. A better understanding of the fundamental aspects of the virus’s life cycle would help to improve the development of novel therapeutic approaches. Host cell entry and restriction factors represent major barriers for emerging viruses and are promising targets for therapeutic intervention. In addition to the LASV main receptor, the extracellular matrix molecule dystroglycan (DG), the phosphatidylserine-binding receptors of the Tyro3/Axl/Mer (TAM), and T cell immunoglobulin and mucin receptor (TIM) families are potential alternative receptors of LASV infection. Therefore, the relative contributions of candidate receptors to LASV entry into a particular human cell type are a complex function of receptor expression and functional DG availability. Here, we describe the role of two receptor tyrosine kinases (RTKs), Axl and hepatocyte growth factor receptor (HGFR), in the presence and absence of glycosylated DG for LASV entry. We found that both RTKs participated in the macropinocytosis-related LASV entry and, regardless of the presence or absence of functional DG, their inhibition resulted in a significant antiviral effect.

The Role of FGFR in the Progression of Waldenstrom's Macroglubulinemia and the Effect of Its Inhibition by TKI-258.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3737-3737
Author(s):  
Feda Azab ◽  
Abdel Kareem Azab ◽  
Aldo M. Roccaro ◽  
Antonio Sacco ◽  
Phong Quang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Cell Line ◽  
Dose Response ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Therapeutic Window ◽  
Low Grade ◽  
Pi3k Signaling ◽  
Healthy Donors

Abstract Abstract 3737 Poster Board III-673 INTRODUCTION Waldenström macroglobulinemia (WM) is a low grade non-Hodgkin lymphoma, characterized by the presence of abnormal lymphoplasmacytic cells producing high levels of IgM. Although indolent, WM remains incurable, and therefore, there is an urgent need for rationally designed therapy in WM. Receptor tyrosine kinases (RTKs) are cell surface receptors for growth factors, cytokines and hormones which have a critical role in the development and progression of many types of cancer. However, their role in WM was not identified. TKI-258 (Novartis, Basel, Switzerland) is an ATP-competitive inhibitor with activity against (multiple) receptor tyrosine kinases including FGFR and other RTKs. We hypothesized that FGFR is up-regulated in WM and plays a major role in its progression; and that TKI-258 would reduce tumor progression in WM. METHODS AND RESULTS We tested the expression of FGFR3 on WM cells and found overexpression of this RTK compared to CD19+ cells from healthy donors. The activation of FGFR3 by recombinant FGF induced MAPK signaling pathway in WM cells including phosphorylation of RAF, ERK and STAT3. Also it induced PI3K signaling including phosphorylation of AKT, S6R and GSK3. TKI-258 inhibited the FGF induced activation of the MAPK and PI3K signaling pathways in a dose- response manner. Using MTT assay we tested the effect of TKI-258 ( 0 to 2.5 uM) on the survival of WM cell line BCWM-1, on IgM secreting cell line MEC-1, and on CD19+ cells selected from WM patient sample. We found that the TKI-258 induced cell death in all sample tested with an IC50 ranging 0.8-1 uM. Testing the effect of TKI-258 on the survival of CD19+ cells selected from peripheral blood or mononuclear cell from healthy donors showed a minimal effect of less than 10% cell death. These results provide a wide therapeutic window for the use of TKI-258 in WM. Moreover, we tested the effect of TKI-258 on the apoptosis of WM cells by flow cytometry using the apoptosis marker APO-2.7, and found that TKI-258 induced apoptosis of WM cells in a dose-response manner at both 24 and 48 treatment. Moreover these results were confirmed by testing changes in the expression of apoptosis related proteins in response to TKI-258 by immunoblotting, including induction of PARP, and caspase-3 and caspase-9 cleavage. In correlation with these results, cell cycle analysis by PI staining and analysis by flow cytometry of WM cells treated with TKI-258 for 24 hrs showed induction of sub-G1 increase in a dose response manner with an IC50 about 1uM. To test the effect of TKI-258 on the interaction of WM cells with the microenvironment we examined the effect of TKI-258 on adhesion of WM cells to fibronectin and bone marrow stromal cells (BMSCs), and found that TKI-258 induced a 50% decrease of adhesion. Moreover, we found no effect on the chemotaxis of WM induce by stroma derived factor-1 (SDF1). To test the direct effect of TKI-258 on the interaction with the microenvironment, we examined the proliferation of WM when cultured alone of in co-culture with BMSCs by 3H-thymidine uptake assay. We showed that TKI-258 inhibited the proliferation of WM cells with an IC50 of 0.8 uM, in the presence or absence of BMSCs. CONCLUSION In conclusion, we found an overexpression of FGFR3 in WM cells compared to CD19+ cells from healthy donors, and that TKI-258 inhibited the activation of proliferative pathways induced by activation of FGFR3 and led to inhibition of proliferation and apoptosis of WM cells. Disclosures: Ghobrial: Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Evidence for Cooperation of Receptor Tyrosine Kinases and Activating NOTCH Mutations to Hyperactivate mTOR in T-Cell Leukemia: A Rationale Basis for Targeted Therapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1381-1381
Author(s):  
Adrian Schwarzer ◽  
Johann Meyer ◽  
Martijn Brugman ◽  
Axel Schambach ◽  
Martin Stanulla ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Microarray Analysis ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Akt Activation ◽  
Rtk Signaling

Abstract Abstract 1381 T-cell acute lymphoblastic leukemia (T-ALL) remains a therapeutic challenge. T-ALLs are characterized by recurring chromosomal rearrangements causing aberrant expression of transcription factors (Myb; TAL/SCL; HOX) dividing patients into different subgroups. Activating mutations in NOTCH, the master regulator of T-cell development, are found in more than 60% of T-ALLs independently of subtype. Most T-ALLs display a hyperactivation of the PI3K-AKT-mTOR pathway, a potential target for therapeutic intervention. The master regulator of PI3K-AKT signalling is PTEN, which is frequently inactivated in cancer. Recent data suggests that complete PTEN loss due to mutation is rare in primary human T-ALL, whereas PTEN-inhibiting posttranslational modifications are more common (Barata et al., J. Clin. Invest. 2008, 118). As these modifications decrease, but do not abolish the phosphatase activity of PTEN, we hypothesized that further input from tyrosine kinases, particularly receptor tyrosine kinases (RTK), may be needed to sustain PI3K-AKT-mTOR activation. In order to investigate how RTK-signaling may contribute to the pathogenesis of T-ALL we used an established murine bone marrow transplantation model (Li et al. Blood 2009, 113). To mimic tyrosine-kinase signaling we expressed δTrkA, a constitutively active TRKA receptor tyrosine kinase (TRK =tropomyosin-related kinase) from gammaretroviral or lentiviral vectors in c-kit+ Sca-1+ Lin− (KSL) cells. Intravenous injection of δTrkA-transduced hematopoietic cells in C57BL6 mice (n=10) induced transplantable T-ALL with a latency of about 120 days. The resulting T-ALLs could be propagated in culture as clonal cell lines. Signaling studies showed that δTRKA activates predominantly ERK upon expression in murine hematopoietic cell lines. However, the obtained δTRKA+ T-ALL lines (n=7) showed a profound shift in the use of downstream signaling cascades, displaying a very high activation of AKT-mTOR and absent ERK phosphorylation, resembling human T-ALL. High AKT activation was uniformly detected regardless of PTEN protein expression in all but one T-ALL (#003). To understand the rewired signaling network we looked for a potential contribution of insertional mutagenesis and chromosomal aberrations. Array-CGH showed homozygous deletions on chr14c2 involving the T-cell receptor alpha and delta genes in 3/3 cell lines and heterozygous deletions in Ikzf1 in 2/3 cell lines. Viral integration sites showed no common insertion pattern and no insertion in genes implicated in RTK-signaling. The expression of genes in proximity to viral integrations (±500 kb) appeared unaltered as determined by cDNA-microarray analysis of the T-ALL cell line #483 against wild type CD4+CD8+ thymocytes. Microarray analysis revealed enrichment of Notch1 target genes in the T-ALL cell line #483. Sequencing of Notch1 revealed both, PEST domain mutations and the recently described (Aster et al, Blood 2010, 116) RAG mediated 5'-deletions in cis, in all but one investigated T-ALL. Northern and Western Blots confirmed the expression of truncated Notch1 transcripts and protein, respectively. The one cell line (#003) which retained the original δTrkA signaling pattern had no Notch mutation and could only be cultured on OP9-Delta-like-1 stroma cells, highlighting the importance of Notch signaling. As this cell line was established from a mouse displaying an enlarged thymus, but no full manifestation of T-ALL, our data suggests that acquisition of Notch mutations is a late, but necessary step required for overt leukemia, whereas the initiating events may arise in kinase signaling pathways of prethymic progenitors. All T-ALL cell lines were sensitive to mTOR or Notch inhibition with Rapamycin or Compound E, respectively. Finally, we used phosphoprotein-arrays to monitor the phosphorylation of 42 RTK in childhood T-ALL samples with different activating NOTCH mutations (n=5) and detected several activated RTK (e.g. MSPR, FGFR, ErbB4, VEGFR) in the patient samples. Taken together, our findings suggest a cooperation of RTK and activating NOTCH mutations in mTOR activation seen in T-ALL and encourage further investigation of 1) aberrant RTK-signaling in T-ALL 2) the role of RTK activation in creating a preleukemic cell clone, 3) evaluation of combined therapy targeting RTKs and NOTCH, and 4) the role of activated NOTCH on mTORC2-AKT activation independently of PTEN. Disclosures: Baum: Patent office: Patents & Royalties.

Redox-dependent MAP kinase signaling by Ang II in vascular smooth muscle cells: role of receptor tyrosine kinase transactivation

2003 ◽  
Vol 81 (2) ◽  
pp. 159-167 ◽  
Author(s):  
Rhian M Touyz ◽  
Montserrat Cruzado ◽  
Fatiha Tabet ◽  
Guoying Yao ◽  
Steven Salomon ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Nadph Oxidase ◽  
Map Kinase ◽  
Vascular Smooth Muscle Cells ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
P38 Map Kinase ◽  
Ang Ii

We investigated the role of receptor tyrosine kinases in Ang II-stimulated generation of reactive oxygen species (ROS) and assessed whether MAP kinase signaling by Ang II is mediated via redox-sensitive pathways. Production of ROS and activation of NADPH oxidase were determined by DCFDA (dichlorodihydrofluorescein diacetate; 2 μmol/L) fluorescence and lucigenin (5 μmol/L) chemiluminescence, respectively, in rat vascular smooth muscle cells (VSMC). Phosphorylation of ERK1/2, p38MAP kinase and ERK5 was determined by immunoblotting. The role of insulin-like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) was assessed with the antagonists AG1024 and AG1478, respectively. ROS bioavailability was manipulated with Tiron (10–5 mol/L), an intra cellular scavanger, and diphenylene iodinium (DPI; 10–6 mol/L), an NADPH oxidase inhibitor. Ang II stimulated NADPH oxidase activity and dose-dependently increased ROS production (p < 0.05). These actions were reduced by AG1024 and AG1478. Ang II-induced ERK1/2 phosphorylation (276% of control) was decreased by AG1478 and AG1024. Neither DPI nor tiron influenced Ang II-stimulated ERK1/2 activity. Ang II increased phosphorylation of p38 MAP kinase (204% of control) and ERK5 (278% of control). These effects were reduced by AG1024 and AG1478 and almost abolished by DPI and tiron. Thus Ang II stimulates production of NADPH-inducible ROS partially through transactivation of IGF-1R and EGFR. Inhibition of receptor tyrosine kinases and reduced ROS bioavaliability attenuated Ang II-induced phosphorylation of p38 MAP kinase and ERK5, but not of ERK1/2. These findings suggest that Ang II activates p38MAP kinase and ERK5 via redox-dependent cascades that are regulated by IGF-1R and EGFR transactivation. ERK1/2 regulation by Ang II is via redox-insensitive pathways.Key words: ERK1/2, p38MAP kinase, EGFR, IGF-1R, signal transduction.

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