Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated &lt;em&gt;Plasmodium&lt;/em&gt; Sporozoites

Mac-Daniel, Laura; Buckwalter, Matthew R.; Gueirard, Pascale; Ménard, Robert

doi:10.3791/53796

Cited by 11 publications

(13 citation statements)

References 32 publications

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“…The last kind of sample was studied after sonication in order to confirm that biofilm aggregates were absent from the inoculum. Microdroplets of this sample were deposited on SEM Pore filters (DTM9305, Jeol) either with a 34G needle fitted to a NanoFil syringe (World Precision Instruments) (after micro-injection) [ 23 ] or a pipette (before micro-injection) and passively diffused (slowly/gently) through it. After absorption on the filters, the bacteria were fixed for 12 hours at 4°C in 0.2 mol/L sodium cacodylate buffer, pH 7.4, that contained 1.6% glutaraldehyde.…”

Section: Methodsmentioning

confidence: 99%

Biofilm-coated microbeads and the mouse ear skin: An innovative model for analysing anti-biofilm immune response in vivo

et al. 2020

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Owing to its ability to form biofilms, Staphylococcus aureus is responsible for an increasing number of infections on implantable medical devices. The aim of this study was to develop a mouse model using microbeads coated with S. aureus biofilm to simulate such infections and to analyse the dynamics of anti-biofilm inflammatory responses by intravital imaging. Scanning electron microscopy and flow cytometry were used in vitro to study the ability of an mCherry fluorescent strain of S. aureus to coat silica microbeads. Biofilm-coated microbeads were then inoculated intradermally into the ear tissue of LysM-EGFP transgenic mice (EGFP fluorescent immune cells). General and specific real-time inflammatory responses were studied in ear tissue by confocal microscopy at early (4-6h) and late time points (after 24h) after injection. The displacement properties of immune cells were analysed. The responses were compared with those obtained in control mice injected with only microbeads. In vitro, our protocol was capable of generating reproducible inocula of biofilm-coated microbeads verified by labelling matrix components, observing biofilm ultrastructure and confirmed in vivo and in situ with a matrix specific fluorescent probe. In vivo, a major inflammatory response was observed in the mouse ear pinna at both time points. Real-time observations of cell recruitment at injection sites showed that immune cells had difficulty in accessing biofilm bacteria and highlighted areas of direct interaction. The average speed of cells was lower in infected mice compared to control mice and in tissue areas where direct contact between immune cells and bacteria was observed, the average cell velocity and linearity were decreased in comparison to cells in areas where no bacteria were visible. This model provides an innovative way to analyse specific immune responses against biofilm infections on medical devices. It paves the way for live evaluation of the effectiveness of immunomodulatory therapies combined with antibiotics.

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Section: Methodsmentioning

confidence: 99%

Biofilm-coated microbeads and the mouse ear skin: An innovative model for analysing anti-biofilm immune response in vivo

et al. 2020

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“…Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (50 mg/kg) and xylazine (5 mg/kg). A small volume (3.8 μL) of planktonic or biofilm inocula or TS culture medium were injected into the dorsal ear dermis of anesthetized mice with a 34G needle fitted to a NanoFil syringe (World Precision Instruments) [25]. A characteristic papule was observable at the injection site, evidence of an intradermal injection.…”

Section: Inoculation Of Bacteria Into Micementioning

confidence: 99%

A mouse ear skin model to study the dynamics of innate immune responses against Staphylococcus aureus biofilms

Hamid¹,

Nakusi²,

Givskov³

et al. 2020

BMC Microbiol

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Background: Staphylococcus aureus is a human pathogen that is a common cause of nosocomial infections and infections on indwelling medical devices, mainly due to its ability to shift between the planktonic and the biofilm/ sessile lifestyle. Biofilm infections present a serious problem in human medicine as they often lead to bacterial persistence and thus to chronic infections. The immune responses elicited by biofilms have been described as specific and ineffective. In the few experiments performed in vivo, the importance of neutrophils and macrophages as a first line of defence against biofilm infections was clearly established. However, the bilateral interactions between biofilms and myeloid cells remain poorly studied and analysis of the dynamic processes at the cellular level in tissues inoculated with biofilm bacteria is still an unexplored field. It is urgent, therefore, to develop biologically sound experimental approaches in vivo designed to extract specific immune signatures from the planktonic and biofilm forms of bacteria. Results: We propose an in vivo transgenic mouse model, used in conjunction with intravital confocal microscopy to study the dynamics of host inflammatory responses to bacteria. Culture conditions were created to prepare calibrated inocula of fluorescent planktonic and biofilm forms of bacteria. A confocal imaging acquisition and analysis protocol was then drawn up to study the recruitment of innate immune cells in the skin of LysM-EGFP transgenic mice. Using the mouse ear pinna model, we showed that inflammatory responses to S. aureus can be quantified over time and that the dynamics of innate immune cells after injection of either the planktonic or biofilm form can be characterized. First results showed that the ability of phagocytic cells to infiltrate the injection site and their motility is not the same in planktonic and biofilm forms of bacteria despite the cells being considerably recruited in both cases. Conclusion:We developed a mouse model of infection to compare the dynamics of the inflammatory responses to planktonic and biofilm bacteria at the tissue and cellular levels. The mouse ear pinna model is a powerful imaging system to analyse the mechanisms of biofilm tolerance to immune attacks.

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“…The first series of experiments will be performed with Lysozyme-EGFP mice inoculated into the ear tissue with the same number of CFU of either planktonic or sessile bacteria (B). Inoculum will be micro-injected in two injection points in the dermis of the ear pinna tissue with a nanofil syringe ( Mac-Daniel et al, 2016 ). At early time-points, mice will be anesthetized and the cellular recruitment will be analyzed by confocal microscopy at the injection points (C1a and C1b).…”

Section: The Mouse Ear Skin Model To Study the Dynamics Of Innate Immmentioning

confidence: 99%

Unveiling and Characterizing Early Bilateral Interactions between Biofilm and the Mouse Innate Immune System

et al. 2017

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A very substantial progress has been made in our understanding of infectious diseases caused by invasive bacteria. Under their planktonic forms, bacteria transiently reside in the otherwise sterile mammal body tissues, as the physiological inflammation insures both their clearance and repair of any tissue damage. Yet, the bacteria prone to experience planktonic to biofilm developmental transition still need to be studied. Of note, sessile bacteria not only persist but also concur preventing the effectors and regulators of the physiological inflammation to operate. Thus, it is urgent to design biologically sound experimental approaches aimed to extract, at the earliest stage, immune signatures of mono-bacteria planktonic to biofilm developmental transition in vivo and ex vivo. Indeed, the transition is often the first event to which succeeds the “chronicization” process whereby classical bacteria-targeting therapies are no more efficacious. An in vivo model of micro-injection of Staphylococcus aureus planktonic or biofilm cells in the ear pinna dermis of laboratory transgenic mice with fluorescent immune cells is proposed. It allows visualizing, in real time, the range of the early interactions between the S. aureus and myeloid cell subsets- the resident macrophages and dendritic cells, the recruited neutrophil granulocytes/polymorphonuclear neutrophils, monocytes otherwise known to differentiate as macrophages or dendritic cells. One main objective is to extract contrasting immune signatures of the modulation of the physiological inflammation with respect to the two bacterial lifestyles.

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Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated <em>Plasmodium</em> Sporozoites

Cited by 11 publications

References 32 publications

Biofilm-coated microbeads and the mouse ear skin: An innovative model for analysing anti-biofilm immune response in vivo

Biofilm-coated microbeads and the mouse ear skin: An innovative model for analysing anti-biofilm immune response in vivo

A mouse ear skin model to study the dynamics of innate immune responses against Staphylococcus aureus biofilms

Unveiling and Characterizing Early Bilateral Interactions between Biofilm and the Mouse Innate Immune System

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