Activation of chemokine and inflammatory cytokine response in hepatitis C virus-infected hepatocytes depends on Toll-like receptor 3 sensing of hepatitis C virus double-stranded RNA intermediates.
Chemokines and inflammatory cytokines are key regulators of immunity and inflammation during viral infections. Hepatitis C virus (HCV) is a hepatotropic RNA virus frequently associated with chronic liver inflammation and hepatocellular carcinoma. Intrahepatic levels of chemokines and cytokines are elevated in chronic HCV infections, but the underlying mechanisms remain unclear. We found that Toll-like receptor-3 (TLR3) senses HCV infection in cultured hepatoma cells, leading to nuclear factor kappa B (NF-κB) activation and the production of numerous chemokines and inflammatory cytokines, such as regulated on activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, MIP-1β, IP-10, and interleukin-6. The chemokine/cytokine induction occurred late in HCV infection and was abrogated when HCV was ultraviolet-inactivated before infection, indicating a dependence on the cellular recognition of HCV replication products. Gel-shift and chromatin immunoprecipitation assays revealed that NF-κB plays a pivotal role in HCV-induced chemokine/cytokine transcription. Mutations specifically disrupting the double-stranded RNA (dsRNA)-binding activity of TLR3 ablated the chemokine/cytokine response to HCV infection, indicating that HCV dsRNA was the pathogen-associated molecular pattern triggering TLR3 signaling. In vitro synthesized HCV dsRNAs, with a minimal length of ∼80-100 base pairs, activated TLR3-dependent chemokine expression, regardless of the genome position from which they derived. In contrast, HCV single-stranded RNAs, including those derived from the structured 3'nontranslated region highly potent for RIG-I activation, failed to do so. Moreover, robust production of chemokines and inflammatory cytokines was also observed in primary human hepatocytes after stimulation with extracellular poly-I:C, a TLR3 ligand.


CONCLUSIONS
Our data suggest that TLR3-mediated chemokine and inflammatory cytokine responses may play an important role in host immune responses to HCV and the pathogenesis of HCV-associated liver diseases.
Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, TN 38163, USA. kli1@uthsc.edu
Kui
K
Li
Nan L
NL
Li
Dahai
D
Wei
Susan R
SR
Pfeffer
Meiyun
M
Fan
Lawrence M
LM
Pfeffer
D002460:Cell Line; D002478:Cells, Cultured; D018946:Chemokine CCL5 / Q000378:metabolism; D018925:Chemokines / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism*; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D019698:Hepatitis C, Chronic / Q000378:metabolism* / Q000473:pathology; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism* / Q000821:virology*; D006801:Humans; D019402:Macrophage Inflammatory Proteins / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D011070:Poly I-C / Q000494:pharmacology; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D051196:Toll-Like Receptor 3 / Q000378:metabolism*; D015854:Up-Regulation; D014779:Virus Replication / Q000502:physiology
D018946:Chemokine CCL5; D018925:Chemokines; D016207:Cytokines; D019402:Macrophage Inflammatory Proteins; D016328:NF-kappa B; D012330:RNA, Double-Stranded; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D011070:Poly I-C