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1.
Immunity ; 54(3): 468-483.e5, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33484643

RESUMO

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrated that MC-derived tumor necrosis factor (TNF) was crucial for neutrophil extravasation to sites of contact hypersensitivity-induced skin inflammation by promoting intraluminal crawling. MC-derived TNF directly primed circulating neutrophils via TNF receptor-1 (TNFR1) while being dispensable for endothelial cell activation. The MC-derived TNF was infused into the bloodstream by directional degranulation of perivascular MCs that were part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules boosted neutrophil extravasation. Pronounced and rapid intravascular MC degranulation was also observed upon IgE crosslinking or LPs challenge indicating a universal MC potential. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.


Assuntos
Vasos Sanguíneos/imunologia , Dermatite de Contato/imunologia , Inflamação/imunologia , Mastócitos/imunologia , Neutrófilos/imunologia , Pele/patologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Circulação Sanguínea , Degranulação Celular , Células Cultivadas , Doenças do Sistema Imunitário , Transtornos Leucocíticos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação de Neutrófilo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Vesículas Secretórias/metabolismo , Fator de Necrose Tumoral alfa/genética
2.
Immunity ; 54(12): 2724-2739.e10, 2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34687607

RESUMO

Nitric oxide (NO) is an important antimicrobial effector but also prevents unnecessary tissue damage by shutting down the recruitment of monocyte-derived phagocytes. Intracellular pathogens such as Leishmania major can hijack these cells as a niche for replication. Thus, NO might exert containment by restricting the availability of the cellular niche required for efficient pathogen proliferation. However, such indirect modes of action remain to be established. By combining mathematical modeling with intravital 2-photon biosensors of pathogen viability and proliferation, we show that low L. major proliferation results not from direct NO impact on the pathogen but from reduced availability of proliferation-permissive host cells. Although inhibiting NO production increases recruitment of these cells, and thus pathogen proliferation, blocking cell recruitment uncouples the NO effect from pathogen proliferation. Therefore, NO fulfills two distinct functions for L. major containment: permitting direct killing and restricting the supply of proliferation-permissive host cells.


Assuntos
Leishmania major/fisiologia , Leishmaniose/imunologia , Macrófagos/imunologia , Óxido Nítrico/metabolismo , Animais , Processos de Crescimento Celular , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Humanos , Microscopia Intravital , Camundongos , Camundongos Endogâmicos C57BL , Modelos Teóricos
3.
Nature ; 618(7967): 1033-1040, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37316667

RESUMO

Most clinically applied cancer immunotherapies rely on the ability of CD8+ cytolytic T cells to directly recognize and kill tumour cells1-3. These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment4-6. The ability of CD4+ effector cells to contribute to antitumour immunity independently of CD8+ T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified7-10. Here, we describe a mechanism whereby a small number of CD4+ T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8+ T cell targeting. The CD4+ effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II+CD11c+ antigen-presenting cells. We show that T helper type 1 cell-directed CD4+ T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4+ T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4+ T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8+ T cells and natural killer cells and advance cancer immunotherapies.


Assuntos
Linfócitos T CD4-Positivos , Morte Celular , Imunoterapia , Inflamação , Neoplasias , Microambiente Tumoral , Humanos , Células Apresentadoras de Antígenos/imunologia , Antígeno CD11c/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Morte Celular/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunidade Inata , Inflamação/imunologia , Interferons/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Microambiente Tumoral/imunologia , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Células Mieloides/imunologia , Células Th1/citologia , Células Th1/imunologia
4.
Immunity ; 47(4): 789-802.e9, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-29045907

RESUMO

Inhibitors of the receptor tyrosine kinase c-MET are currently used in the clinic to target oncogenic signaling in tumor cells. We found that concomitant c-MET inhibition promoted adoptive T cell transfer and checkpoint immunotherapies in murine cancer models by increasing effector T cell infiltration in tumors. This therapeutic effect was independent of tumor cell-intrinsic c-MET dependence. Mechanistically, c-MET inhibition impaired the reactive mobilization and recruitment of neutrophils into tumors and draining lymph nodes in response to cytotoxic immunotherapies. In the absence of c-MET inhibition, neutrophils recruited to T cell-inflamed microenvironments rapidly acquired immunosuppressive properties, restraining T cell expansion and effector functions. In cancer patients, high serum levels of the c-MET ligand HGF correlated with increasing neutrophil counts and poor responses to checkpoint blockade therapies. Our findings reveal a role for the HGF/c-MET pathway in neutrophil recruitment and function and suggest that c-MET inhibitor co-treatment may improve responses to cancer immunotherapy in settings beyond c-MET-dependent tumors.


Assuntos
Imunoterapia/métodos , Neoplasias Experimentais/terapia , Neutrófilos/imunologia , Proteínas Proto-Oncogênicas c-met/imunologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Estimativa de Kaplan-Meier , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Neutrófilos/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
5.
Mol Microbiol ; 121(3): 453-469, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-37612878

RESUMO

Leishmania is the causative agent of the tropical neglected disease leishmaniasis and infects macrophages as its definitive host cell. In order to sustain and propagate infections, Leishmania parasites have to complete cycles of exit and re-infection. Yet, the mechanism driving the parasite spread to other cells remains unclear. Recent studies reported pro-inflammatory monocytes as replicative niche of Leishmania major and showed prolonged expression of IL-1ß at the site of infection, indicating an activation of the NLRP3 inflammasome and pointing toward pyroptosis as a possible mechanism of parasite spread. To address the species-specific inflammasome activation of human cells, we characterized the BLaER1 monocytes as a model for L. major infection. We found that BLaER1 monocytes support infection and activation by Leishmania parasites to the same extent as primary human macrophages. Harnessing the possibilities of this infection model, we first showed that BLaER1 GSDMD-/- cells, which carry a deletion of the pore-forming protein gasdermin D, are more resistant to pyroptotic cell death and, concomitantly, display a strongly delayed release of intracellular parasite. Using that knockout in a co-incubation assay in comparison with wild-type BLaER1 cells, we demonstrate that impairment of the pyroptosis pathway leads to lower rates of parasite spread to new host cells, thus, implicating pyroptotic cell death as a possible exit mechanism of L. major in pro-inflammatory microenvironments.


Assuntos
Inflamassomos , Leishmania , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Piroptose/fisiologia , Proteínas de Ligação a Fosfato/metabolismo , Macrófagos , Leishmania/metabolismo , Interleucina-1beta/metabolismo
6.
Eur J Immunol ; 50(6): 846-857, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32043573

RESUMO

The survival of peripheral T cells is dependent on their access to peripheral LNs (pLNs) and stimulation by IL-7. In pLNs fibroblastic reticular cells (FRCs) and lymphatic endothelial cells (LECs) produce IL-7 suggesting their contribution to the IL-7-dependent survival of T cells. However, IL-7 production is detectable in multiple organs and is not restricted to pLNs. This raises the question whether pLN-derived IL-7 is required for the maintenance of peripheral T cell homeostasis. Here, we show that numbers of naive T cells (TN ) remain unaffected in pLNs and spleen of mice lacking Il7 gene activity in pLN FRCs, LECs, or both. In contrast, frequencies of central memory T cells (TCM ) are reduced in FRC-specific IL-7 KO mice. Thus, steady state IL-7 production by pLN FRCs is critical for the maintenance of TCM , but not TN , indicating that both T cell subsets colonize different ecological niches in vivo.


Assuntos
Sobrevivência Celular , Fibroblastos/imunologia , Memória Imunológica , Interleucina-7/imunologia , Linfonodos/imunologia , Linfócitos T/imunologia , Animais , Fibroblastos/citologia , Interleucina-7/genética , Linfonodos/citologia , Camundongos , Camundongos Knockout , Linfócitos T/citologia
7.
Cytokine ; 148: 155699, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34530329

RESUMO

Interleukin-11 (IL-11) is an important member of the IL-6 family of cytokines. IL-11 activates its target cells via binding to a non-signaling α-receptor (IL-11R), which results in recruitment and activation of a gp130 homodimer. The cytokine was initially described as an anti-inflammatory protein, but has recently gained attention as a potent driver in certain types of cancer and different fibrotic conditions. Leishmania spp. are a group of eukaryotic parasites that cause the disease leishmaniasis. They infect phagocytes of their hosts, especially monocytes recruited to the site of infection, and are able to replicate within this rather harsh environment, often resulting in chronic infections of the patient. However, the molecular mechanisms underlying parasite and host cell interactions and factors of the immune cells that are crucial for Leishmania uptake are so far largely unspecified. Recently, increased IL-11 expression in the lesions of patients with cutaneous leishmaniasis has been reported, but the functional relevance is unknown. In this study, we show that monocytes express IL-11R on their cell surface. Furthermore, using an adoptive transfer model of IL-11R-/- monocytes, we analyze the contribution of IL-11 signaling on monocyte recruitment and monocyte infection in a mouse model of cutaneous leishmaniasis and find that IL-11 signaling is dispensable for monocyte recruitment and pathogen uptake during Leishmania major infection.


Assuntos
Leishmania major/metabolismo , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Monócitos/metabolismo , Monócitos/parasitologia , Receptores de Interleucina-11/metabolismo , Animais , Membrana Celular/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Transdução de Sinais
8.
Immunity ; 37(1): 147-57, 2012 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-22727490

RESUMO

Effector T cells are critical for clearance of pathogens from sites of infection. Like cytotoxic CD8(+) T cells, CD4(+) helper T cells have been shown to deliver effector molecules directionally toward the immunological synapse, suggesting that infected cells need to be engaged individually to receive effector signals. In contrast, we show here that CD4(+) T cells stably contacted a minority of infected cells, yet these interactions triggered intracellular defense mechanisms in bystander cells in vivo. By using a functional read-out, we provide evidence that this effector bystander activity extends via a gradient of IFN-γ more than 80 µm beyond the site of antigen presentation, promoting pathogen clearance in the absence of immunological synapse formation. Our results thus demonstrate that CD4(+) T cells can exert their protective activity by engaging a minority of infected cells.


Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Animais , Efeito Espectador/imunologia , Derme/imunologia , Derme/parasitologia , Interferon gama/imunologia , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/metabolismo
9.
J Am Soc Nephrol ; 31(11): 2589-2608, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32859670

RESUMO

BACKGROUND: Kidney injuries that result in chronic inflammation initiate crosstalk between stressed resident cells and infiltrating immune cells. In animal models, whole-body receptor Notch3 deficiency protects from leukocyte infiltration and organ fibrosis. However, the relative contribution of Notch3 expression in tissue versus infiltrating immune cells is unknown. METHODS: Chimeric mice deficient for Notch3 in hematopoietic cells and/or resident tissue cells were generated, and kidney fibrosis and inflammation after unilateral ureteral obstruction (UUO) were analyzed. Adoptive transfer of labeled bone marrow-derived cells validated the results in a murine Leishmania ear infection model. In vitro adhesion assays, integrin activation, and extracellular matrix production were analyzed. RESULTS: Fibrosis follows UUO, but inflammatory cell infiltration mostly depends upon Notch3 expression in hematopoietic cells, which coincides with an enhanced proinflammatory milieu (e.g., CCL2 and CCL5 upregulation). Notch3 expression on CD45+ leukocytes plays a prominent role in efficient cell transmigration. Functionally, leukocyte adhesion and integrin activation are abrogated in the absence of receptor Notch3. Chimeric animal models also reveal that tubulointerstitial fibrosis develops, even in the absence of prominent leukocyte infiltrates after ureteral obstruction. Deleting Notch3 receptors on resident cells blunts kidney fibrosis, ablates NF-κB signaling, and lessens matrix deposition. CONCLUSIONS: Cell-specific receptor Notch3 signaling independently orchestrates leukocyte infiltration and organ fibrosis. Interference with Notch3 signaling may present a novel therapeutic approach in inflammatory as well as fibrotic diseases.


Assuntos
Rim/patologia , Leucócitos/fisiologia , Macrófagos/fisiologia , Nefrite/patologia , Receptor Notch3/genética , Receptor Notch3/metabolismo , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Adesão Celular , Proliferação de Células , Células Cultivadas , Quimera , Matriz Extracelular/metabolismo , Feminino , Fibrose , Integrinas/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Leucócitos/metabolismo , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Nefrite/etiologia , Transdução de Sinais , Transcriptoma , Migração Transendotelial e Transepitelial , Obstrução Ureteral/complicações
10.
PLoS Pathog ; 14(10): e1007374, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30346994

RESUMO

The virulence of intracellular pathogens such as Leishmania major (L. major) relies largely on their ability to undergo cycles of replication within phagocytes, release, and uptake into new host cells. While all these steps are critical for successful establishment of infection, neither the cellular niche of efficient proliferation, nor the spread to new host cells have been characterized in vivo. Here, using a biosensor for measuring pathogen proliferation in the living tissue, we found that monocyte-derived Ly6C+CCR2+ phagocytes expressing CD11c constituted the main cell type harboring rapidly proliferating L. major in the ongoing infection. Synchronization of host cell recruitment and intravital 2-photon imaging showed that these high proliferating parasites preferentially underwent cell-to-cell spread. However, newly recruited host cells were infected irrespectively of their cell type or maturation state. We propose that among these cells, CD11c-expressing monocytes are most permissive for pathogen proliferation, and thus mainly fuel the cycle of intracellular proliferation and cell-to-cell transfer during the acute infection. Thus, besides the well-described function for priming and activating T cell effector functions against L. major, CD11c-expressing monocyte-derived cells provide a reservoir for rapidly proliferating parasites that disseminate at the site of infection.


Assuntos
Antígenos Ly/imunologia , Antígeno CD11c/metabolismo , Proliferação de Células , Leishmania major/imunologia , Leishmaniose/parasitologia , Monócitos/virologia , Receptores CCR2/imunologia , Animais , Antígenos Ly/metabolismo , Células Cultivadas , Replicação do DNA , Leishmania major/genética , Leishmaniose/imunologia , Leishmaniose/metabolismo , Leishmaniose/transmissão , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Receptores CCR2/metabolismo , Virulência
11.
Cytometry A ; 97(5): 458-470, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31777152

RESUMO

During infections, interactions between host immune cells and the pathogen occur in distinct anatomical locations and along defined time scales. This can best be assessed in the physiological context of an infection in the living tissue. Consequently, intravital imaging has enabled us to dissect the critical phases and events throughout an infection in real time in living tissues. Specifically, advances in visualizing specific cell types and individual pathogens permitted tracking the early events of tissue invasion of the pathogen, cellular interactions involved in the induction of the immune response as well the events implicated in clearance of the infection. In this respect, two vantage points have evolved since the initial employment of this technique in the field of infection biology. On the one hand, strategies acquired by the pathogen to establish within the host and circumvent or evade the immune defenses have been elucidated. On the other hand, analyzing infections from the immune system's perspective has led to insights into the dynamic cellular interactions that are involved in the initial recognition of the pathogen, immune induction as well as effector function delivery and immunopathology. Furthermore, an increasing interest in probing functional parameters in vivo has emerged, such as the analysis of pathogen reactivity to stress conditions imposed by the host organism in order to mediate clearance upon pathogen encounter. Here, we give an overview on recent intravital microscopy findings of host-pathogen interactions along the course of an infection, from both the immune system's and pathogen's perspectives. We also discuss recent developments and future perspectives in extracting intravital information beyond the localization of pathogens and their interaction with immune cells. Such reporter systems on the pathogen's physiological state and immune cell functions may prove useful in dissecting the functional dynamics of host-pathogen interactions. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.


Assuntos
Interações Hospedeiro-Patógeno , Microscopia Intravital
12.
Blood ; 130(12): 1445-1455, 2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28687614

RESUMO

Coagulation proteases have increasingly recognized functions beyond hemostasis and thrombosis. Disruption of activated protein C (aPC) or insulin signaling impair function of podocytes and ultimately cause dysfunction of the glomerular filtration barrier and diabetic kidney disease (DKD). We here show that insulin and aPC converge on a common spliced-X-box binding protein-1 (sXBP1) signaling pathway to maintain endoplasmic reticulum (ER) homeostasis. Analogous to insulin, physiological levels of aPC maintain ER proteostasis in DKD. Accordingly, genetically impaired protein C activation exacerbates maladaptive ER response, whereas genetic or pharmacological restoration of aPC maintains ER proteostasis in DKD models. Importantly, in mice with podocyte-specific deficiency of insulin receptor (INSR), aPC selectively restores the activity of the cytoprotective ER-transcription factor sXBP1 by temporally targeting INSR downstream signaling intermediates, the regulatory subunits of PI3Kinase, p85α and p85ß. Genome-wide mapping of condition-specific XBP1-transcriptional regulatory patterns confirmed that concordant unfolded protein response target genes are involved in maintenance of ER proteostasis by both insulin and aPC. Thus, aPC efficiently employs disengaged insulin signaling components to reconfigure ER signaling and restore proteostasis. These results identify ER reprogramming as a novel hormonelike function of coagulation proteases and demonstrate that targeting insulin signaling intermediates may be a feasible therapeutic approach ameliorating defective insulin signaling.


Assuntos
Coagulação Sanguínea , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Insulina/metabolismo , Peptídeo Hidrolases/metabolismo , Proteína C/metabolismo , Transdução de Sinais , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Nefropatias Diabéticas/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Homeostase , Humanos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Trombomodulina/metabolismo , Resposta a Proteínas não Dobradas/genética
13.
Eur J Immunol ; 47(12): 2043-2058, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28833060

RESUMO

Regulatory T (Treg) cells require T-cell receptor (TCR) signalling to exert their immunosuppressive activity, but the precise organization of the TCR signalling network compared to conventional T (Tconv) cells remains elusive. By using accurate mass spectrometry and multi-epitope ligand cartography (MELC) we characterized TCR signalling and recruitment of TCR signalling components to the immunological synapse (IS) in Treg cells and Tconv cells. With the exception of Themis which we detected in lower amounts in Treg cells, other major TCR signalling components were found equally abundant, however, their phosphorylation-status notably discriminates Treg cells from Tconv cells. Overall, this study identified 121 Treg cell-specific phosphorylations. Short-term triggering of T cell subsets via CD3 and CD28 widely harmonized these variations with the exception of eleven TCR signalling components that mainly regulate cytoskeleton dynamics and molecular transport. Accordingly, conjugation with B cells indeed caused variant cellular morphology and revealed a Treg cell-specific recruitment of TCR signalling components such as PKCθ, PLCγ1 and ZAP70 as well as B cell-derived CD86 into the IS. Together, results from this study support the existence of a Treg cell-specific IS and suggest Treg cell-specific cytoskeleton dynamics as a novel determinant for the unique functional properties of Treg cells.


Assuntos
Sinapses Imunológicas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Células Cultivadas , Feminino , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Fosforilação , Proteoma/imunologia , Proteoma/metabolismo , Proteômica/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Proteína-Tirosina Quinase ZAP-70/imunologia , Proteína-Tirosina Quinase ZAP-70/metabolismo
14.
PLoS Pathog ; 11(5): e1004929, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-26020515

RESUMO

The protozoan Leishmania mexicana parasite causes chronic non-healing cutaneous lesions in humans and mice with poor parasite control. The mechanisms preventing the development of a protective immune response against this parasite are unclear. Here we provide data demonstrating that parasite sequestration by neutrophils is responsible for disease progression in mice. Within hours of infection L. mexicana induced the local recruitment of neutrophils, which ingested parasites and formed extracellular traps without markedly impairing parasite survival. We further showed that the L. mexicana-induced recruitment of neutrophils impaired the early recruitment of dendritic cells at the site of infection as observed by intravital 2-photon microscopy and flow cytometry analysis. Indeed, infection of neutropenic Genista mice and of mice depleted of neutrophils at the onset of infection demonstrated a prominent role for neutrophils in this process. Furthermore, an increase in monocyte-derived dendritic cells was also observed in draining lymph nodes of neutropenic mice, correlating with subsequent increased frequency of IFNγ-secreting T helper cells, and better parasite control leading ultimately to complete healing of the lesion. Altogether, these findings show that L. mexicana exploits neutrophils to block the induction of a protective immune response and impairs the control of lesion development. Our data thus demonstrate an unanticipated negative role for these innate immune cells in host defense, suggesting that in certain forms of cutaneous leishmaniasis, regulating neutrophil recruitment could be a strategy to promote lesion healing.


Assuntos
Células Dendríticas/imunologia , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Monócitos/imunologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Neutrófilos/parasitologia , Animais , Células Cultivadas , Doença Crônica , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/parasitologia , Citometria de Fluxo , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Monócitos/parasitologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Blood ; 120(19): 3945-8, 2012 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-22995897

RESUMO

T-cell activation in lymph nodes relies on encounters with antigen (Ag)-bearing dendritic cells (DCs) but the number of DCs required to initiate an immune response is unknown. Here we have used a combination of flow cytometry, 2-photon imaging, and computational modeling to quantify the probability of T cell-DC encounters. We calculated that the chance for a T cell residing 24 hours in a murine popliteal lymph nodes to interact with a DC was 8%, 58%, and 99% in the presence of 10, 100, and 1000 Ag-bearing DCs, respectively. Our results reveal the existence of a threshold in DC numbers below which T-cell responses fail to be elicited for probabilistic reasons. In mice and probably humans, we estimate that a minimum of 85 DCs are required to initiate a T-cell response when starting from precursor frequency of 10(-6). Our results have implications for the rational design of DC-based vaccines.


Assuntos
Células Dendríticas/imunologia , Imunidade Celular/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Células Dendríticas/citologia , Feminino , Genes MHC da Classe II/imunologia , Imunidade Celular/genética , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Modelos Biológicos
16.
Cell Rep ; 43(5): 114153, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38687643

RESUMO

Gut-draining mesenteric and celiac lymph nodes (mLNs and celLNs) critically contribute to peripheral tolerance toward food and microbial antigens by supporting the de novo induction of regulatory T cells (Tregs). These tolerogenic properties of mLNs and celLNs are stably imprinted within stromal cells (SCs) by microbial signals and vitamin A (VA), respectively. Here, we report that a single, transient gastrointestinal infection in the neonatal, but not adult, period durably abrogates the efficient Treg-inducing capacity of celLNs by altering the subset composition and gene expression profile of celLNSCs. These cells carry information about the early-life pathogen encounter until adulthood and durably instruct migratory dendritic cells entering the celLN with reduced tolerogenic properties. Mechanistically, transiently reduced VA levels cause long-lasting celLN functional impairment, which can be rescued by early-life treatment with VA. Together, our data highlight the therapeutic potential of VA to prevent sequelae post gastrointestinal infections in infants.


Assuntos
Linfonodos , Linfócitos T Reguladores , Vitamina A , Animais , Linfonodos/imunologia , Linfonodos/patologia , Linfonodos/efeitos dos fármacos , Vitamina A/farmacologia , Vitamina A/uso terapêutico , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Camundongos , Animais Recém-Nascidos , Tolerância Imunológica/efeitos dos fármacos , Células Dendríticas/imunologia , Camundongos Endogâmicos C57BL , Feminino
17.
J Hematol Oncol ; 17(1): 43, 2024 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-38853260

RESUMO

BACKGROUND: Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils. METHODS: Ly6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1ß. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied. RESULTS: Strikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals. CONCLUSIONS: Taken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.


Assuntos
Inflamação , Janus Quinase 2 , Transtornos Mieloproliferativos , Neutrófilos , Animais , Neutrófilos/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Camundongos , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Transtornos Mieloproliferativos/metabolismo , Humanos , Inflamação/genética , Inflamação/patologia , Calreticulina/genética , Calreticulina/metabolismo , Camundongos Transgênicos , Camundongos Endogâmicos C57BL , Citocinas/metabolismo
18.
Proc Natl Acad Sci U S A ; 107(47): 20441-6, 2010 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-21048081

RESUMO

The protective effect of antibodies (Abs) is generally attributed to neutralization or complement activation. Using Legionella pneumophila and Mycobacterium bovis bacillus Calmette-Guérin as a model, we discovered an additional mechanism of Ab-mediated protection effective against intracellular pathogens that normally evade lysosomal fusion. We show that Fc receptor (FcR) engagement by Abs, which can be temporally and spatially separated from bacterial infection, renders the host cell nonpermissive for bacterial replication and targets the pathogens to lysosomes. This process is strictly dependent on kinases involved in FcR signaling but not on host cell protein synthesis or protease activation. Based on these findings, we propose a mechanism whereby Abs and FcR engagement subverts the strategies by which intracellular bacterial pathogens evade lysosomal degradation.


Assuntos
Anticorpos Antibacterianos/imunologia , Infecções Bacterianas/imunologia , Lisossomos/imunologia , Receptores Fc/imunologia , Transdução de Sinais/imunologia , Animais , Carga Bacteriana , Imunização , Legionella pneumophila , Camundongos , Microscopia de Fluorescência , Mycobacterium bovis
19.
JCI Insight ; 8(14)2023 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-37310793

RESUMO

The virulence of intracellular pathogens relies largely on the ability to survive and replicate within phagocytes but also on release and transfer into new host cells. Such cell-to-cell transfer could represent a target for counteracting microbial pathogenesis. However, our understanding of the underlying cellular and molecular processes remains woefully insufficient. Using intravital 2-photon microscopy of caspase-3 activation in the Leishmania major-infected (L. major-infected) live skin, we showed increased apoptosis in cells infected by the parasite. Also, transfer of the parasite to new host cells occurred directly without a detectable extracellular state and was associated with concomitant uptake of cellular material from the original host cell. These in vivo findings were fully recapitulated in infections of isolated human phagocytes. Furthermore, we observed that high pathogen proliferation increased cell death in infected cells, and long-term residency within an infected host cell was only possible for slowly proliferating parasites. Our results therefore suggest that L. major drives its own dissemination to new phagocytes by inducing host cell death in a proliferation-dependent manner.


Assuntos
Apoptose , Leishmania major , Fagócitos , Leishmania major/patogenicidade , Fagócitos/parasitologia , Humanos , Virulência , Camundongos Endogâmicos C57BL , Células Cultivadas , Camundongos , Animais
20.
Cells ; 11(22)2022 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-36429061

RESUMO

Our knowledge about respiratory virus spreading is mostly based on monolayer cultures that hardly reflect the complex organization of the airway epithelium. Thus, there is a strong demand for biologically relevant models. One possibility to study virus spreading at the cellular level is real-time imaging. In an attempt to visualize virus spreading under somewhat more physiological conditions, Calu-3 cells and human primary fibroblasts were co-cultured submerged or as air-liquid interface (ALI). An influenza A virus (IAV) replicating well in cell culture, and carrying a red fluorescent protein (RFP) reporter gene was used for real-time imaging. Our three-dimensional (3D) models exhibited important characteristics of native airway epithelium including a basement membrane, tight junctions and, in ALI models, strong mucus production. In submerged models, first fluorescence signals appeared between 9 and 12 h post infection (hpi) with a low multiplicity of infection of 0.01. Virus spreading further proceeded in the immediate vicinity of infected cells. In ALI models, RFP was found at 22 hpi and later. Consequently, the progression of infection was delayed, in contrast to the submerged model. With these features, we believe that our 3D airway models can deliver new insights in the spreading of IAV and other respiratory viruses.


Assuntos
Vírus da Influenza A , Microscopia , Humanos , Células Cultivadas , Células Epiteliais/metabolismo , Vírus da Influenza A/fisiologia , Técnicas de Cultura de Células
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