Extracellular HCV-Core Protein Induces an Immature Regulatory Phenotype in NK Cells: Implications for Outcome of Acute Infection

<div><p>Background</p><p>Hepatitis C viral (HCV) proteins, including core, demonstrate immuno-modulatory properties; however, the effect of extracellular core on natural killer (NK) cells has not previously been investigated.</p><p>Aims</p><p>To characterise NKs in acute HCV infection over time, and, to examine the effect of exogenous HCV-core protein on NK cell phenotype and function.</p><p>Methods</p><p>Acute HCV patients (n = 22), including 10 subjects who spontaneously recovered, were prospectively studied. Flow-cytometry was used to measure natural cytotoxicity and to phenotype NKs directly <i>ex vivo</i> and after culture with HCV-core protein. Microarray analysis was used to identify pathways involved in the NK cell response to exogenous HCV-core.</p><p>Results</p><p>Direct <i>ex vivo</i> analysis demonstrated an increased frequency of immature/regulatory CD56^bright NKs early in acute HCV infection <i>per se</i> which normalized with viral clearance. Natural cytotoxicity was reduced and did not recover after viral clearance. There was a statistically significant correlation between the frequency of CD56^bright NKs and circulating serum levels of HCV core protein. <i>In vitro</i> culture of purified CD56^bright NK cells with HCV-core protein in the presence of IL-15 maintained a significant proportion of NKs in the CD56^bright state. The <i>in vitro</i> effect of core closely correlates with NK characteristics measured directly <i>ex vivo</i> in acute HCV infection. Pathway analysis suggests that HCV-core protein attenuates NK interferon type I responses.</p><p>Conclusions</p><p>Our data suggest that HCV-core protein alters NK cell maturation and may influence the outcome of acute infection.</p></div

Circulating HCV-core levels in acute HCV infection and effect of extracellular core on NK cell CD56 expression.

<p>Circulating HCV-core protein levels as measured by ELISA <b>(A)</b>. As expected, core levels correlated directly with viral levels in HCV-infected patients. There was also a direct statistically significant correlation between core and the levels of CD56^bright NK cells in these patients. Multiple time points were used for chronic patients <b>(B)</b>. Incubation of highly purified FACS sorted CD56^bright NK cells, from normal uninfected control subjects, in the presence of HCV-core or beta-galactosidase (β-Gal) control protein demonstrated inhibition of CD56 down-regulation by exogenous HCV-core protein. Histograms of CD56 expression levels after incubation are shown for four individual experiments <b>(C)</b>. The first three histograms represent individual normal control subjects and the fourth histogram is derived from an experiment in which PBMCs from five normal uninfected control subjects were pooled prior to sorting on CD56^bright NK cells and incubation with HCV-core.</p

Expression of NKRs CD94, NKG2A, CD161 and CD57 in patients maintaining or withdrawing therapy.

<p>Graphs illustrate (<b>A</b>) CD94 and NKG2A expression and (<b>B</b>) CD161 and CD57 expression at baseline (▴) and three months (○) in patients maintaining or withdrawing TNF inhibitor therapy. (C) Representative dot plots of two patients withdrawing TNF inhibitor therapy. Patient (I) remained in clinical remission for the study duration and maintained expansion of CD94 and NKG2A; Patient (II) had a flare in disease activity 6 months after withdrawal of therapy with loss of expansion of CD94 and NKG2A at 3 months (i.e. prior to clinical relapse). *p<0.05 significantly different.</p

Total CD3+, Total CD56+ and CD3+CD56− T cell populations in patients maintaining or withdrawing therapy.

<p>Graphs illustrate total CD3+, Total CD56+ and CD3+CD56− T cell populations (expressed as a percentage of the lymphogate) in patients maintaining or withdrawing therapy at baseline (▴) and three months (○). *p<0.05 significantly different.</p

Baseline characteristics of patients in remission following TNF inhibitor therapy and in patients with active rheumatoid arthritis.

<p>*p<0.05 remission cohort significantly different from active RA cohort.</p

T cell, NK and NKT cell populations in active RA, Remission RA and healthy controls.

<p>Graph illustrating T cell, NK and NKT cell populations in healthy controls (N = 15) (▴), patients with active RA (n = 18) (○) and patients in remission following TNF inhibitor (n = 15)(▪). *p<0.05 significantly different.</p

Expression of CD161, HLA-DR and CD57 in active RA, Remission RA and healthy controls.

<p>(A) Representative dot plots of CD161, HLA-DR and CD57 expression on healthy control, patient with active RA and patient in remission following TNF inhibitor therapy. (B) CD161, HLA-DR and CD57 expression on CD3+CD56− cells in healthy controls (▴), patients with active RA (○) and patients in remission following TNF inhibitor therapy (▪). *p<0.05 significantly different.</p

Hepatitis C Virus Pathogen Associated Molecular Pattern (PAMP) Triggers Production of Lambda-Interferons by Human Plasmacytoid Dendritic Cells

<div><p>Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell in the defense against viruses. Through pattern recognition receptors (PRRs), these cells detect viral pathogen associated molecular patterns (PAMPs) and initiate an Interferon (IFN) response. pDCs produce the antiviral IFNs including the well-studied Type I and the more recently described Type III. Recent genome wide association studies (GWAS) have implicated Type III IFNs in HCV clearance. We examined the IFN response induced in a pDC cell line and <i>ex vivo</i> human pDCs by a region of the HCV genome referred to as the HCV PAMP. This RNA has been shown previously to be immunogenic in hepatocytes, whereas the conserved X-region RNA is not. We show that in response to the HCV PAMP, pDC-GEN2.2 cells upregulate and secrete Type III (in addition to Type I) IFNs and upregulate PRR genes and proteins. We also demonstrate that the recognition of this RNA is dependent on RIG-I-like Receptors (RLRs) and Toll-like Receptors (TLRs), challenging the dogma that RLRs are dispensable in pDCs. The IFNs produced by these cells in response to the HCV PAMP also control HCV replication <i>in vitro</i>. These data are recapitulated in <i>ex vivo</i> pDCs isolated from healthy donors. Together, our data shows that pDCs respond robustly to HCV RNA to make Type III Interferons that control viral replication. This may represent a novel therapeutic strategy for the treatment of HCV.</p> </div

Expression of NKRs CD94 and NKG2A in active RA, Remission RA and healthy controls.

<p>(A) Representative dot plots of CD94 and NKG2A expression on healthy control, patient with active RA and patient in remission following TNF inhibitor therapy. (B) CD94 and NKG2A expression on CD3+CD56− cells in healthy controls (▴), patients with active RA (○) and patients in remission following TNF inhibitor therapy (▪). *p<0.05 significantly different.</p