|Year : 2016 | Volume
| Issue : 4 | Page : 536-538
Internalisation of hepatitis C virus core protein by human conjunctival fibroblasts
AR Rajalakshmy1, J Malathi2, HN Madhavan2, S Bhaskar3, GK Iyer3
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 Submission||22-Jun-2015|
|Date of Acceptance||10-Oct-2016|
|Date of Web Publication||8-Dec-2016|
H N Madhavan
L & T Microbiology Research Center, Department of Microbiology, Vision Research Foundation, Sankara Nethralaya, Texas
Source of Support: None, Conflict of Interest: None
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|| |
Hepatitis C virus (HCV) belongs to the family Flaviviridae and primarily infects the liver. About 70% of infections become chronic, and this arbitrate can involve multiple organs apart from the liver. Dry eye is a multifactorial condition that is associated with HCV infection. 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.,, 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|| |
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. 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|| |
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|| |
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., 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. TLRs and phagocytosis are linked with the clearance of bacterial pathogens. 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. 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. 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. This certainly suggests a role of the virus in the aetiology of the dry eye syndrome. Infectious virus-like particles have also been isolated from the tear samples of chronic HCV patients. 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.
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.
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