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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
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  Table of Contents  
BRIEF COMMUNICATION
Year : 2016  |  Volume : 34  |  Issue : 4  |  Page : 536-538
 

Internalisation of hepatitis C virus core protein by human conjunctival fibroblasts


1 L & T Microbiology Research Center, Department of Microbiology, Vision Research Foundation, Sankara Nethralaya; Department of Pathology (ARR), UT Southwestern Medical Center, Texas, USA
2 L & T Microbiology Research Center, Department of Microbiology, Vision Research Foundation, Sankara Nethralaya, Texas, USA
3 Department of Microbiology, Medical Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India

Date of Submission22-Jun-2015
Date of Acceptance10-Oct-2016
Date of Web Publication8-Dec-2016

Correspondence Address:
H N Madhavan
L & T Microbiology Research Center, Department of Microbiology, Vision Research Foundation, Sankara Nethralaya, Texas
USA
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.195358

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 ~ Abstract 

Recent studies indicate that hepatitis C virus (HCV) proteins can mediate innate immune response and inflammation in conjunctival fibroblasts which contributes to the pathology of dry eye condition associated with chronic HCV infection. The present study investigates the phagocytic potential of human conjunctival fibroblasts (HCFj) for HCV core protein. HCFj cells were incubated with HCV core antigen for different periods of time, and fluorescent micrographs were taken to observe protein internalisation. HCFj cells were capable of internalising HCV core antigen within 1 h; this gives an insight into another molecular mechanism which may contribute towards HCV-associated conjunctival inflammation.


Keywords: Conjunctiva, fibroblasts, hepatitis C virus core, inflammation, internalisation


How to cite this article:
Rajalakshmy A R, Malathi J, Madhavan H N, Bhaskar S, Iyer G K. Internalisation of hepatitis C virus core protein by human conjunctival fibroblasts. Indian J Med Microbiol 2016;34:536-8

How to cite this URL:
Rajalakshmy A R, Malathi J, Madhavan H N, Bhaskar S, Iyer G K. Internalisation of hepatitis C virus core protein by human conjunctival fibroblasts. Indian J Med Microbiol [serial online] 2016 [cited 2017 Jan 19];34:536-8. Available from: http://www.ijmm.org/text.asp?2016/34/4/536/195358



 ~ Introduction Top


Hepatitis C virus (HCV) belongs to the family Flaviviridae and primarily infects the liver.[1] About 70% of infections become chronic, and this arbitrate can involve multiple organs apart from the liver.[2] Dry eye is a multifactorial condition that is associated with HCV infection.[3] Viral RNA has been isolated from the tear fluid of patients with chronic HCV infection, and in vitro studies indicate that HCV core and NS3 proteins can induce conjunctival and corneal inflammation.[4],[5],[6] In this context, we studied the internalisation of HCV core antigen by human conjunctival fibroblasts (HCFj) cells as it may be a molecular mechanism involved in conjunctival inflammation seen in chronic HCV infection.


 ~ Materials and Methods Top


Establishment of primary human conjunctival fibroblasts

All experiments were performed as per the Declaration of Helsinki. Conjunctival tissue was collected with written consent from a 48-year-old male patient undergoing ocular surface reconstruction surgery after chemical injury. The study was approved by the Vision Research Foundation Institutional Ethical Committee Board of Sankara Nethralaya, Chennai, India. Primary HCFj culture was established according to the protocol established in our laboratory.[5] In brief, the tissue was washed thrice in 1× phosphate-buffered saline (PBS; Hi-Media, Mumbai, India) with 100 units/ml penicillin G and 100 units/ml streptomycin (Invitrogen, Carlsbad, CA, USA). The tissue was cut into 2-mm size pieces and placed in a 35-mm tissue culture dish. The dish was filled with dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (Gibco, Langley, OK, USA) to maintain the tissue at the air/liquid interface. Confluent cells were trypsinised and sub-cultured. We used cells from the second to fifth passages for our experiments.

Hepatitis C virus core treatment of human conjunctival fibroblasts cells

Approximately 1 × 105 cells were seeded onto 12-well plates. Twenty-four hours after seeding, the cells were incubated with 2 ng/ml of Cy3 is a fluorescein antigen (Sigma, St. Louis, MO, USA) for 1 and 3 h. The growth medium with viral protein was completely removed after incubation, and the cells were washed thrice with 1× PBS. Fluorescent micrographs were taken using a Zeiss AxioObserver Z1 inverted wide field microscope with digital CCD camera (1388 × 1040, 1.4 mega pixels, Axiocam MRm)


 ~ Results Top


We established primary HCFj from conjunctival tissue [Figure 1]a, and 100% of the cells were positive for the fibroblast marker, vimentin [Figure 1]b. The fibroblasts internalised HCV core protein within 1 h of exposure, and this was sustained up to 3 h [Figure 1]c.
Figure 1: Hepatitis C virus core protein internalisation by human conjunctival fibroblasts. (a) Cells (human conjunctival fibroblasts) were migrating from the human conjunctival tissue explant. (b) Cells stained positive for fibroblast marker, vimentin. (c) Hepatitis C virus core protein was localised near the nucleus of human conjunctival fibroblasts cells at 1 and 3 h time points. Data are representative of three independent experiments. Scale bar represents 50 μ

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 ~ Discussion Top


There is a well-known association between HCV infection and dry eye, and in vitro studies published from our laboratory have demonstrated the specific innate immune response via TLR pathways of corneal epithelium and conjunctival fibroblasts to HCV protein which can indirectly contribute to the pathogenesis of dry eye. In the present study, we find that conjunctival fibroblasts are capable of internalising one of the important antigens of HCV. Earlier studies have reported the involvement of toll-like receptors (TLRs) in HCV core-mediated ocular inflammation.[5],[6] TLR7 and TLR10 mRNA expression and then MyD88 expression were found to be significantly upregulated in conjunctival fibroblasts within 8 h of exposure to HCV core. TLR7-mediated recognition ligands require endosomal maturation.[7] TLRs and phagocytosis are linked with the clearance of bacterial pathogens.[8] Since HCV core was internalised within 1 h, we assume that phagocytosis could be the possible initial event before the recognition of viral protein by TLR. Our study provides insight into the possible molecular mechanisms involved in HCV core-mediated conjunctival inflammation. HCV core protein is found to be located mainly in the endoplasmic reticulum, mitochondria and inside lipid droplets in the cytoplasm in cell culture models.[9] Nuclear transport of the same requires specific nuclear localisation signals, and these differences in cellular targeting regulate the different biological roles of this viral protein. We have used 2 ng/ml of the core protein, which is compared with the serum concentration of this protein in HCV patients, for treating cell cultures.[10] In the current study, fluorescent images indicate that HCV core protein was located outside the nucleus and that the protein was unable to cross the nuclear membrane. We could observe the clustering of this protein around the nucleus, which suggests the possible translocation of HCV core inside endoplasmic reticulum. A recent study of ours has revealed the presence of HCV viral RNA in tear samples of dry eye patients without HCV infection; however, the source of viral RNA needs to be clarified.[10] This certainly suggests a role of the virus in the aetiology of the dry eye syndrome.[11] Infectious virus-like particles have also been isolated from the tear samples of chronic HCV patients.[12] These studies support the medical relevance of the present study, which has uncovered one of the molecular mechanisms behind HCV-mediated conjunctival inflammation in a primary cell culture model. In future, this can help in selecting drug targets to control the pathology of HCV-mediated dry eye syndrome with conjunctival involvement.

Acknowledgement

This work was supported by funding from the Department of Biotechnology, Government of India (BT/PR1028/med/29/303/2011). Ayilam Ramachandran Rajalakshmy is receiving fellowship from INSPIRE fellowship program (IF110775), Department of Science and Technology, Government of India.

Financial support and sponsorship

ARR is supported by INSPIRE fellowship from Department of Science and Technology, Government of India (IF 110775).

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

1.
Miller RH, Purcell RH. Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups. Proc Natl Acad Sci U S A 1990;87:2057-61.  Back to cited text no. 1
    
2.
Chen SL, Morgan TR. The natural history of hepatitis C virus (HCV) infection. Int J Med Sci 2006;3:47-52.  Back to cited text no. 2
    
3.
Palekar NA, Harrison SA. Extrahepatic manifestations of hepatitis C. South Med J 2005;98:1019-23.  Back to cited text no. 3
    
4.
Gumus K, Yurci A, Mirza E, Arda H, Oner A, Topaktas D, et al. Evaluation of ocular surface damage and dry eye status in chronic hepatitis C at different stages of hepatic fibrosis. Cornea 2009;28:997-1002.  Back to cited text no. 4
    
5.
Rajalakshmy AR, Malathi J, Madhavan HN, Srinivasan B, Iyer GK. Hepatitis C virus core and NS3 antigens induced conjunctival inflammation via toll-like receptor-mediated signaling. Mol Vis 2014;20:1388-97.  Back to cited text no. 5
    
6.
Rajalakshmy AR, Malathi J, Madhavan HN. HCV core and NS3 proteins mediate toll like receptor induced innate immune response in corneal epithelium. Exp Eye Res 2014;128:117-28.  Back to cited text no. 6
    
7.
Takeda K, Akira S. Toll-like receptors in innate immunity. Int Immunol 2005;17:1-14.  Back to cited text no. 7
    
8.
Blander JM, Medzhitov R. Regulation of phagosome maturation by signals from toll-like receptors. Science 2004;304:1014-8.  Back to cited text no. 8
    
9.
Suzuki R, Sakamoto S, Tsutsumi T, Rikimaru A, Tanaka K, Shimoike T, et al. Molecular determinants for subcellular localization of hepatitis C virus core protein. J Virol 2005;79:1271-81.  Back to cited text no. 9
    
10.
Rajalakshmy AR, Malathi J, Madhavan HN, Bhaskar S, Iyer GK. Patients with dry eye without hepatitis C virus infection possess the viral RNA in their tears. Cornea 2015;34:28-31.  Back to cited text no. 10
    
11.
Feucht HH, Zöllner B, Schröter M, Altrogge H, Laufs R. Tear fluid of hepatitis C virus carriers could be infectious. J Clin Microbiol 1995;33:2202-3.  Back to cited text no. 11
    
12.
Cividini A, Cerino A, Muzzi A, Furione M, Rebucci C, Segagni L, et al. Kinetics and significance of serum hepatitis C virus core antigen in patients with acute hepatitis C. J Clin Microbiol 2003;41:2144-6.  Back to cited text no. 12
    


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