“INNATE IMMUNE RESPONSES IN EX VIVO SKIN EXPLANTS INFECTED WITH DENGUE VIRUS”
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1
CINVESTAV, Department of Molecular Biomedicine, Mexico
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2
National School of Biological Science IPN, Department of Morphology, Mexico
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3
CINVESTAV, Department of Physiology; Biophysics and Neurosciences;, Mexico
Dengue is an endemic disease in more than a 100 countries distributed throughout most continents. Studies made in 2013 estimate that there are more than 390 million people infected, with 96 millions of cases annually worldwide and an increased incidence in non-endemic regions, mainly because of the presence of the transmitter vector in this areas and to the increase in travelers who introduce the pathogen from one region to another.
Mosquitoes vector Aedes deliver Dengue virus into human skin when they are feeding, thus the first organ that comes in contact with the dengue virus is the skin; in consequence, the potential initial target cells for dengue infection should be localized in the dermis and epidermis, which are the tissues that form the first level of defense against pathogens and injuries via cells and molecular mediators of innate and adaptive immunity. This tissue is recognized as the skin immune system. A large number of different biologically active substances are synthetized in the skin, and they must play a relevant role when a pathogen inoculated by different vectors are introduced trough the skin, thus dendritic cells, immune cells and cells as keratinocytes and fibroblasts that are not typically cells from immune system but they are the most abundant cells at the inoculation site.
Although primary cultures of keratinocytes have been recently shown to be permissive to DENV infection, little is known about differences in the in situ immune response; thus fresh skin explants obtained from different donors. Could be a suitable strategy to address such issue. Furthermore, a previous study reported by Limon Flores Y (2005) in a non-cadaveric model of skin explants showed a negative strand DENV RNA; additionally positive signal was observed in both epidermis and dermis by using specific monoclonal antibodies against the NS1 non structural protein demonstrating productive infection.
Considering that the immunological events at the early contact of DENV remain not enough explored in the inoculation site. In this work, we examined some of the molecules produced by keratinocytes in human skin biopsies from healthy donors infected ex vivo by intradermal inoculation with 1x106 viral particles of DENV2. We determined the presence of productive infection by evaluating the expression of the non-structural proteins NS3 and NS5, and we also evaluated the potential proinflammatory mediators, chemokines and cytokines produced during the infection with dengue virus, which contribute to the development of the innate immune response in the skin.
The basal layer of epidermal keratinocytes was found to be infected with DENV2 at 48 hpi, as indicated by NS3 (viral protease) expression in the cytoplasm and NS5 (viral RNA polymerase) expression in the nucleus. Interestingly, after 24 h, the level of infection in keratinocytes was low; in contrast, most fibroblasts in the dermis were infected after 24 hours. These data indicate a higher permissiveness of fibroblasts in the first 24 hours of infection, followed by higher permissiveness of the basal layer keratinocytes one day later. These data are relevant, as our group previously reported the infection of fibroblasts in primary culture and their contribution to the early stages of infection with differential production of IFN-β among different skin donors.
To date, the expression of other important proinflammatory cytokines in the immune response that help control DENV infection, such as TNF-α and IL-1β, have been detected in primary culture fibroblasts but not in primary culture keratinocytes. We also established primary cultures of keratinocytes by treating the skin with collagenase/dispase and evaluated the expression of surface and cytoplasmic proteins using the antibodies Citokeratine+ and MHC-II+ for keratinocytes. Once the cultures were established, we evaluated keratinocyte permissiveness to DENV2 infection at up to 24 hpi at different multiplicities of infection from 1 to 10 by measuring the expression of NS3, a nonstructural protein only expressed during DENV viral replication. We found that keratinocytes are permissive to infection by DV2 in proportion to the multiplicity of infection (from 2.5-10 MOIs) and that this permissiveness changes over time.
To determine the time after inoculation with the highest rate of keratinocyte infection, we performed a kinetics analysis of the infection (MOI 10) at 12, 24, 48, 72 and 96 hours, and we evaluated the expression of the envelope protein (E) by immunofluorescence. The maximum rate of infection was observed at 48 h, after which the expression of E decreased significantly, probably due to accumulation of cytopathic effects in the culture. This infection rate was consistent after evaluation by flow cytometry and colony forming units (CFU), with 27% infection after 48 hours and twice as many colony forming units at 48 hpi compared to at 24 hpi. The expression levels of IL-6 and IL-8 were also determined by CBAs, with a titer of 200 pg for IL-6 and a titer of 400 pg for IL-8 at 48 hpi. The main function of IL-6 is regulation of the T cell response, and IL-8 is involved in neutrophil recruitment.
Keratinocytes produce high levels of antimicrobial peptides when there is damage to the skin; thus, we measured the production of cathelicidin in primary keratinocyte culture during infection. LL-37, were produced at different concentrations from 24 hpi and reached their highest concentrations at 48 hours. Importantly, these peptides were only produced by cells that were adjacent to the infected cells, not by the infected cells themselves; this finding could be due to viral inhibition of the production of such peptides in infected cells as a mechanism of immune system evasion. To date, the production of β-defensins 2 and 3 (hBD2 and hBD3) post-infection have been determined in keratinocyte cell lines but not in primary culture.
It is possible that the communication between both layers of the skin allows for the development of a effective antiviral response, which, depending on the active dialogue between different cell lines of the skin in the very early stages of infection, could help define whether the infected person will develop the serious or benign clinical form of dengue. Similarly, the expression of IFN types I and III and antimicrobial peptides greatly contribute to the control DENV infection.
Acknowledgements
This work was supported by Department of Molecular Biomedicine, Centre for Research and Advanced Studies (CINVESTAV-IPN) and CONACYT grant CB -2010 -01 154270, Posdoctoral internship in program PhD in Science in Molecular Biomedicine, grant CONACYT
References
Dengue virus inoculation to human skin explants: an effective approach to assess in situ the early infection and the effects on cutaneous dendritic cells. Limon-Flores AY1, Perez-Tapia M, Estrada-Garcia I, Vaughan G, Escobar-Gutierrez A, Calderon-Amador J, Herrera-Rodriguez SE, Brizuela-Garcia A, Heras-Chavarria M, Flores-Langarica A, Cedillo-Barron L, Flores-Romo L. Int J Exp Pathol. 2005 Oct;86(5):323-34.
Dengue virus replication in infected human keratinocytes leads to activation of antiviral innate immune responses.
Surasombatpattana P1, Hamel R, Patramool S, Luplertlop N, Thomas F, Desprès P, Briant L, Yssel H, Missé D. Infect Genet Evol. 2011 Oct;11(7):1664-73. doi: 10.1016/j.meegid.2011.06.009. Epub 2011 Jun 21.
Keywords:
Dengue Virus,
Skin explants,
ex vivo culture,
Keratinocyte culture,
innate immune response
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Oral Presentation
Topic:
Innate Immunity
Citation:
Meza Sánchez
DE,
Suárez Pérez
D,
Nava Dominguez
P and
Cedillo Barrón
L
(2015). “INNATE IMMUNE RESPONSES IN EX VIVO SKIN EXPLANTS INFECTED WITH DENGUE VIRUS”.
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00344
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Received:
30 May 2015;
Published Online:
15 Sep 2015.
*
Correspondence:
Dr. David E Meza Sánchez, CINVESTAV, Department of Molecular Biomedicine, Distrito Federal, 07360, Mexico, tunatitlalli@gmail.com