DH82 Canine and RAW264.7 Murine Macrophage Cell Lines Display Distinct Activation Profiles Upon Interaction With Leishmania infantum and Leishmania amazonensis

Nadaes, Natalia Rocha; Costa, Leandro Silva da; Santana, Raissa Couto; LaRocque-de-Freitas, Isabel Ferreira; Vivarini, Áislan de Carvalho; Soares, Deivid Costa; Wardini, Amanda Brito; Lopes, Ulisses Gazos; Saraiva, Elvira M.; Freire-de-Lima, Célio Geraldo; Decotè-Ricardo, Débora; Pinto-da-Silva, Lúcia Helena

doi:10.3389/fcimb.2020.00247

Cited by 10 publications

(9 citation statements)

References 43 publications

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“…To date, the DH82 canine macrophage cell line has primarily been utilized as a model of viral and protozoan infection [23][24][25], such as for the study of canine distemper and its oncolytic potential [55][56][57]. Although studies have described the expression of functional P2 receptors in human or rodent macrophages and macrophage cell lines [8,10,11,34], studies directly investigating P2 receptors in canine macrophages have been absent.…”

Section: Discussionmentioning

confidence: 99%

“…Studies have demonstrated that DH82 cells express a number of macrophage markers, such as CD11c and CD18 [ 21 ], and can secrete tumour necrosis factor (TNF)-α and interleukin (IL)-6 similar to that observed in lipopolysaccharide (LPS)-stimulated canine monocytes [ 22 ]. Despite its use as an in vitro model of viral and protozoan infection [ 23 , 24 , 25 ], knowledge regarding purinergic signalling in DH82 cells is limited to a single report describing ATP- and adenosine-induced cytokine release [ 26 ]. Although this study did not investigate any purinergic receptor per se, DH82 cells represent a possible model to study endogenous P2 receptors in canine macrophages for a number of reasons.…”

Section: Introductionmentioning

confidence: 99%

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P2Y2 and P2X4 Receptors Mediate Ca2+ Mobilization in DH82 Canine Macrophage Cells

Sophocleous

Miles

Ooi

et al. 2020

IJMS

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Purinergic receptors of the P2 subclass are commonly found in human and rodent macrophages where they can be activated by adenosine 5′-triphosphate (ATP) or uridine 5′-triphosphate (UTP) to mediate Ca2+ mobilization, resulting in downstream signalling to promote inflammation and pain. However, little is understood regarding these receptors in canine macrophages. To establish a macrophage model of canine P2 receptor signalling, the expression of these receptors in the DH82 canine macrophage cell line was determined by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. P2 receptor function in DH82 cells was pharmacologically characterised using nucleotide-induced measurements of Fura-2 AM-bound intracellular Ca2+. RT-PCR revealed predominant expression of P2X4 receptors, while immunocytochemistry confirmed predominant expression of P2Y2 receptors, with low levels of P2X4 receptor expression. ATP and UTP induced robust Ca2+ responses in the absence or presence of extracellular Ca2+. ATP-induced responses were only partially inhibited by the P2X4 receptor antagonists, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), paroxetine and 5-BDBD, but were strongly potentiated by ivermectin. UTP-induced responses were near completely inhibited by the P2Y2 receptor antagonists, suramin and AR-C118925. P2Y2 receptor-mediated Ca2+ mobilization was inhibited by U-73122 and 2-aminoethoxydiphenyl borate (2-APB), indicating P2Y2 receptor coupling to the phospholipase C and inositol triphosphate signal transduction pathway. Together this data demonstrates, for the first time, the expression of functional P2 receptors in DH82 canine macrophage cells and identifies a potential cell model for studying macrophage-mediated purinergic signalling in inflammation and pain in dogs.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

P2Y2 and P2X4 Receptors Mediate Ca2+ Mobilization in DH82 Canine Macrophage Cells

Sophocleous

Miles

Ooi

et al. 2020

IJMS

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“…Cofilin is one of the actin-binding proteins demonstrated to play roles in the actin cytoskeletal remodelling, migration directionality and phagosome formation of macrophages [54,55]. Interestingly, in the current study, we showed that the rTc-MUC-1 interacts with the actin binding protein CFL1 of the macrophage-like Abelson leukaemia virus-transformed cell line derived from BALB/c mice (RAW264.7) that has been commonly used to explore parasite-host interactions [56][57][58]. CFL1 was also predicted to associate with other proteins that were identified in the pull-down assay, including actin, cyclase associated actin cytoskeleton regulatory protein, coronin actin binding protein, lymphocyte cytosolic protein and Rho GDP dissociation inhibitor alpha, which are known to play roles in the myosin contraction and cofilin-mediated disassembly during macrophage migration [54,55].…”

Section: Plos Neglected Tropical Diseasesmentioning

confidence: 56%

The non-glycosylated protein of Toxocara canis MUC-1 interacts with proteins of murine macrophages

Zhou

Jia

et al. 2022

PLoS Negl Trop Dis

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Toxocariasis is a neglected parasitic disease caused predominantly by larvae of Toxocara canis. While this zoonotic disease is of major importance in humans and canids, it can also affect a range of other mammalian hosts. It is known that mucins secreted by larvae play key roles in immune recognition and evasion, but very little is understood about the molecular interactions between host cells and T. canis. Here, using an integrative approach (affinity pull-down, mass spectrometry, co-immunoprecipitation and bioinformatics), we identified 219 proteins expressed by a murine macrophage cell line (RAW264.7) that interact with prokaryotically-expressed recombinant protein (rTc-MUC-1) representing the mucin Tc-MUC-1 present in the surface coat of infective larvae of T. canis. Protein-protein interactions between rTc-MUC-1 and an actin binding protein CFL1 as well as the fatty acid binding protein FABP5 of RAW264.7 macrophages were also demonstrated in a human embryonic kidney cell line (HEK 293T). By combing predicted structural information on the protein-protein interaction and functional knowledge of the related protein association networks, we inferred roles for Tc-MUC-1 protein in the regulation of actin cytoskeletal remodelling, and the migration and phagosome formation of macrophage cells. These molecular interactions now require verification in vivo. The experimental approach taken here should be readily applicable to comparative studies of other ascaridoid nematodes (e.g. T. cati, Anisakis simplex, Ascaris suum and Baylisascaris procyonis) whose larvae undergo tissue migration in accidental hosts, including humans.

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“…In fact, a limited number of works have been conducted using the canine macrophage-like cell line DH82 as a cell model to study Leishmania infection. The DH82 cells can be considered an M2 subtype macrophage model for leishmaniasis [ 22 ], however, these cells were less permissive to L. infantum infection and showed a lower number of amastigotes per macrophages compared to mouse macrophages or human U937 cell line [ 23 ]. This was evident also in the present work, since the infection indexes were rather low compared to human or murine models used in our previous publications, where infection indexes were approximately 150–600 [ 11 , 16 ].…”

Section: Discussionmentioning

confidence: 99%

“…It is noteworthy, the ER stress markers induction with tunicamycin resulted mild compared to U937, THP1 or murine macrophages (in which for example, sXBP1 was 20–100 fold upregulated) [ 11 , 16 ]. In the same way, Nadaes et al showed general low responsiveness in DH82 stimulated by LPS compared with RAW264.7 cells in terms of reactive oxygen species (ROS) and nitric oxide (NO) production, underling a different activation mechanism in this cell line [ 22 ]. A significant induction of ER stress expression markers -including sXBP1 was observed after 48 h post-infection.…”

Section: Discussionmentioning

confidence: 99%

The host micro-RNA cfa-miR-346 is induced in canine leishmaniasis

et al. 2022

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Background Leishmaniases are a group of anthropo-zoonotic parasitic diseases caused by a protozoan of the Leishmania genus, affecting both humans and other vertebrates, including dogs. L. infantum is responsible for the visceral and occasionally cutaneous form of the disease in humans and canine leishmaniasis. Previously, we have shown that L. infantum induces a mild but significant increase in endoplasmic reticulum (ER) stress expression markers to promote parasites survival in human and murine infected macrophages. Moreover, we demonstrated that the miRNA hsa-miR-346, induced by the UPR-activated transcription factor sXBP1, was significantly upregulated in human macrophages infected with different L. infantum strains. However, the ER stress response in infected dogs, which represent an important reservoir for Leishmania parasite, was described once recently, whereas the miR-346 expression was not reported before. Therefore, this study aimed to investigate these pathways in the canine macrophage-like cell line DH82 infected by Leishmania spp. and to evaluate the presence of cfa-miR-346 in plasma of non-infected and infected dogs. The DH82 cells were infected with L. infantum and L. braziliensis parasites and the expression of cfa-mir-346 and several ER stress markers was evaluated by quantitative PCR (qPCR) at different time points. Furthermore, the cfa-miR-346 was monitored in plasma collected from non-infected dogs (n = 11) and dogs naturally infected by L. infantum (n = 18). Results The results in DH82 cells showed that cfa-mir-346 was induced at both 24 h and 48 h post-infection with all Leishmania strains but not with tunicamycin, accounting for a mechanism of induction independent from sXBP1, unlike what was previously observed in human cell lines. Moreover, the cfa-miR-346 expression analysis on plasma revealed a significant increase in infected dogs compared to non-infected dogs. Conclusions Here for the first time, we report the upregulation of cfa-miR-346 induced by Leishmania infection in canine macrophage-like cells and plasma samples of naturally infected dogs. According to our results, the cfa-miR-346 appears to be linked to infection, and understanding its role and identifying its target genes could contribute to elucidate the mechanisms underlying the host–pathogen interaction in leishmaniasis.

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DH82 Canine and RAW264.7 Murine Macrophage Cell Lines Display Distinct Activation Profiles Upon Interaction With Leishmania infantum and Leishmania amazonensis

Cited by 10 publications

References 43 publications

P2Y2 and P2X4 Receptors Mediate Ca2+ Mobilization in DH82 Canine Macrophage Cells

P2Y2 and P2X4 Receptors Mediate Ca2+ Mobilization in DH82 Canine Macrophage Cells

The non-glycosylated protein of Toxocara canis MUC-1 interacts with proteins of murine macrophages

The host micro-RNA cfa-miR-346 is induced in canine leishmaniasis

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