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Recurrence of hepatitis C (HCV) postliver transplant is universal, with a subgroup developing rapid hepatic fibrosis. Toll-like receptors (TLRs) are critical to innate antiviral responses and HCV alters TLR function to evade immune clearance. Whether TLRs play a role in rapid HCV recurrence posttransplant is unknown. We stimulated peripheral blood mononuclear cells (PBMCs) from 70 patients with HCV postliver transplant with TLR subclass-specific ligands and measured cytokine production, TLR expression and NK cell function. Rate of fibrosis progression was calculated using posttransplant liver biopsies graded by Metavir scoring (F0–4; R = fibrosis stage/year posttransplant; rapid fibrosis defined as >0.4 units/year). Thirty of 70 (43%) patients had rapid fibrosis progression. PBMCs from HCV rapid-fibrosers produced less IFNα with TLR7/8 stimulation (p = 0.039), less IL-6 at baseline (p = 0.027) and with TLR3 stimulation (p = 0.008) and had lower TLR3-mediated monocyte IL-6 production (p = 0.028) compared with HCV slow fibrosers. TLR7/8-mediated NKCD56 dim cell secretion of IFNγ was impaired in HCV rapid fibrosis (p = 0.006) independently of IFNα secretion and TLR7/8 expression, while cytotoxicity remained preserved. Impaired TLR3 and TLR7/8-mediated cytokine responses may contribute to aggressive HCV recurrence postliver transplantation through impaired immune control of HCV and subsequent activation of fibrogenesis.
Hepatitis C infection (HCV) is the most common indication for liver transplantation globally, accounting for over 50% of liver transplants performed annually . However, HCV recurrence after transplantation is universal and follows an accelerated course compared with the natural history of HCV infection prior to transplantation . Clinical outcomes are highly variable, with around 10–30% developing aggressive HCV recurrence and rapid fibrosis progression, culminating in graft cirrhosis, liver failure and either death or retransplantation . Why some patients develop rapid fibrosis progression posttransplant remains poorly understood and there is currently no reliable method for predicting those patients most at risk of aggressive HCV recurrence.
Toll-like receptors (TLRs) form the cornerstone of the innate immune system and orchestrate the body's initial immune response against infection. TLRs bind to pathogen associated molecular patterns, or PAMPs, which are highly conserved molecules expressed by invading pathogens . When TLRs bind with their corresponding ligands, they initiate a downstream signaling cascade, culminating in production of type I interferons, antiviral proteins including interferon-stimulated genes and proinflammatory cytokines that have been shown to be critical in initiating antiviral immune responses, including those against hepatitis C infection .
There is a large body of evidence demonstrating the importance of TLRs in HCV infection. HCV core and nonstructural proteins are recognized by TLRs in both peripheral immune and liver cells . HCV is able to activate TLRs 2, 3, 4 and 9 [7, 8] and this triggers an inflammatory cascade leading to chronic inflammation and hepatic fibrosis. In particular, TLR stimulation of hepatocytes and hepatic stellate cells (HSCs) by HCV viral proteins has been shown to increase inflammatory cytokine release and therefore HSC activation, matrix production and collagen deposition leading to hepatic fibrosis and cirrhosis . HCV has also been shown to evade immune clearance through specifically targeting and impairing TLR 2, 3, 4, 7/8 and 9 signaling in T cells and plasmacytoid dendritic cells (pDCs) [10, 11]. Natural killer cells (NK cells) are also increasingly recognized to be essential in immune defenses against HCV infection [12, 13] and have been shown to express TLR3 and TLR7/8 functional receptors . NK cells have also been shown to have antifibrotic effects in HCV infection via inhibition of HSCs both directly through apoptosis or indirectly via IFNγ, which inhibits HSC activation . Therefore, both recipient peripheral immune cells and donor liver immune cells have key roles to play in HCV recurrence postliver transplantation.
Given the importance of TLRs in early viral infection (such as occurs in the grafted liver after transplantation), coupled with the proven importance of peripheral blood immune cell TLR function in chronic HCV infection, we hypothesized that abnormalities of TLR function may contribute to the development of HCV-mediated rapid fibrosis progression postliver transplantation. We therefore examined TLR function in peripheral blood mononuclear cells (PBMCs) in patients with hepatitis C posttransplant, comparing those with rapidly progressive and slowly progressive graft fibrosis. We identified several novel changes in TLR3 and TLR7/8 function that may contribute to more aggressive recurrence of HCV-mediated liver injury.
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HCV patients with rapid fibrosis progression have impaired TLR7/8-induced type I interferon and impaired TLR3 and TLR9-mediated proinflammatory cytokine production compared with those with slow fibrosis.
We compared type I interferon and proinflammatory cytokine production by PBMCs in response to TLR ligand stimulation in HCV patients with rapid fibrosis (n = 30) and slow fibrosis progression (n = 40). IFNα production was expressed as mean concentration, rather than a fold increase above baseline, as all IFNα production at baseline (unstimulated with TLR ligands) was zero pg/mL.
HCV patients with rapid fibrosis progression had impaired IFNα production in response to TLR7/8 stimulation with R848 compared with patients with slow fibrosis progression (p = 0.039, Figure 1).
Figure 1. Mean concentration of IFNα secretion (pg/mL) by PBMCs in response to TLR3, TLR7/8 and TLR9 stimulation in HCV patients with rapid fibrosis (n = 30) compared with slow fibrosis progression (n = 40) postliver transplant. (A) IFNα from TLR3 stimulation (PIC), p = 0.18. (B) IFNα from TLR7/8 stimulation (R848), p = 0.039. (C) IFNα from TLR9 stimulation (CpG), p = 0.23. *p-value < 0.05.
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HCV patients with rapid fibrosis also had impaired IL-6 production in PBMCs at baseline (p = 0.027) and in response to TLR3 stimulation with PIC (p = 0.008, Figure 2) and TLR9 stimulation with CpG (p = 0.038, Figure 3) compared to those with slow fibrosis. They also had reduced TNFα production at baseline (p = 0.060) and in response to TLR3 stimulation with PIC (p = 0.040, Figure 2). There was no significant difference in the fold increase above baseline production of these cytokines, suggesting that while there was impaired cytokine production at baseline and with TLR3 and TLR9 stimulation in HCV patients with rapid fibrosis compared with slow fibrosis, the incremental increase in cytokine with stimulation in patients with rapid fibrosis was not significantly different to that in patients with slow fibrosis.
Figure 2. Mean concentration (pg/mL) and fold increase over baseline of IL-6 and TNFα secretion by PBMCs in response to TLR3 stimulation in HCV patients with rapid fibrosis (n = 30) compared with slow fibrosis progression (n = 40) posttransplant. (A) Baseline unstimulated IL-6 production, p = 0.027. (B) IL-6 with TLR3 stimulation (PIC), p = 0.003. (C) Fold increase in IL-6 with TLR3 stimulation, p = 0.940. (D) Baseline unstimulated TNFα production, p = 0.06. (E) TNFα with TLR3 stimulation (PIC), p = 0.040. (F) Fold increase TNFα with TLR3 stimulation, p = 0.920. *p-value < 0.05.
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Figure 3. Mean concentration (pg/mL) and fold increase over baseline of IL-6 secretion by PBMCs in response to TLR9 stimulation in HCV patients with rapid fibrosis (n = 30) compared with slow fibrosis progression (n = 40) posttransplant. (A) Baseline unstimulated IL-6 production, p = 0.027. (B) IL-6 with TLR9 stimulation (CpG), p = 0.038. (C) Fold increase in IL-6 with TLR9 stimulation, p = 0.810. *p-value < 0.05.
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There were no other observed differences in cytokine production between HCV patients with rapid and slow fibrosis progression for any other TLR ligand.
These findings were confirmed in a subset of HCV patients evaluated at 1 year posttransplant
In order to determine whether our findings applied to the early posttransplant period and to avoid the potential confounding variable of the time posttransplant at which the blood samples were donated, we measured cytokine responses to TLR stimulation in a subgroup of 16 HCV patients early posttransplant. These patients all had PBMCs recovered and a liver biopsy to determine fibrosis stage between 6 and 12 months posttransplant. Rapid fibrosis progression in this smaller cohort was defined as the presence of the Metavir F1 stage or greater fibrosis on the year 1 liver biopsy, as the fibrosis rate equals the fibrosis stage measured at 1 year (fibrosis stage being divided by 1 year). Using this definition, nine HCV patients had slow fibrosis and seven patients had rapid fibrosis.
In this cohort, HCV patients with early rapid fibrosis also had impaired IFNα production in response to TLR7/8 stimulation (p = 0.010) and impaired a fold increase in IL-6 (p = 0.050) and TNFα production (p = 0.009) in response to TLR3 stimulation compared with HCV patients with slow fibrosis, as demonstrated in the larger cohort. However, there was no difference in TLR9-mediated IL-6 production between HCV patients with rapid fibrosis and slow fibrosis progression (data not shown). This suggests that early posttransplant there is impairment in cytokine responses to TLR3 and TLR7/8 stimulation in HCV patients with rapid fibrosis.
HCV patients with rapid fibrosis posttransplant have impaired TLR7/8-mediated IFNγ production by NK cells and impaired TLR3-mediated IL-6 and TNFα production by monocytes
In order to determine which cell types may be responsible for the differences in TLR3 and TLR7/8 function we had demonstrated in PBMCs, we next examined TLR3 and TLR7/8-mediated cytokine responses in individual cell types using flow cytometry.
In HCV patients with rapid fibrosis progression, NK CD56dim cells had significantly impaired IFNγ secretion in response to TLR7/8 stimulation with R848 (geometric mean fluorescence (GMF) of IFNγ p = 0.0057, fold increase in IFNγ GMF over baseline p = 0.036) compared with those with slow fibrosis progression). There was a similar trend also seen in NK CD56 bright cells in HCV patients with rapid fibrosis, which also had impaired GMF of IFNγ compared with those with slow fibrosis (p = 0.053, Figure 4). Furthermore, there was a significant correlation between fibrosis rate and NK CD56dim cell IFNγ secretion in response to TLR7/8 stimulation (p = 0.027, r = −0.4).
Figure 4. Geometric mean fluorescence and fold increase in fluorescence over baseline of IFNγ production with TLR7/8 stimulation (R848) in CD56dim and CD56bright NK cells, comparing HCV patients with rapid fibrosis (n = 16) and slow fibrosis progression (n = 16) posttransplant. (A) Baseline unstimulated IFNγ production by NK CD56dim cells, p = 0.214. (B) IFNγ with TLR7/8 stimulation in NK CD56dim cells, p = 0.006. (C) Fold increase IFNγ with TLR7/8 stimulation in NK CD56dim cells, p = 0.036. (D) Baseline unstimulated IFNγ production in NK CD56bright cells, p = 0.720. (E) IFNγ with TLR7/8 stimulation in NK CD56 bright cells, p = 0.053. (F) Fold increase IFNγ with TLR7/8 stimulation in NK CD56bright cells, p = 0.213. *p < 0.05.
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Monocytes from HCV patients with rapid fibrosis progression also produced less IL-6 with TLR3 stimulation with PIC (GMF IL-6 p = 0.028, Figure 5), with a trend to impaired IL-6 production by monocytes at baseline also in HCV patients with rapid fibrosis compared with slow fibrosis (GMF IL-6 p = 0.060).
Figure 5. Geometric mean fluorescence and fold increase in fluorescence over baseline of IL-6 production with TLR3 stimulation (PIC) in monocytes, comparing HCV patients with rapid fibrosis (n = 13) and slow fibrosis progression (n = 9) posttransplant. (A) Baseline unstimulated IL-6 production in monocytes, p = 0.06. (B) IL-6 with TLR3 stimulation in monocytes, p = 0.028. (C) Fold increase in IL-6 with TLR3 stimulation in monocytes, p = 0.616. *p-value < 0.05.
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NK CD56dim cell TLR7/8-mediated IFNγ secretion occurs through an IFNα-independent pathway in HCV rapid fibrosis
Type I interferon secretion by plasmacytoid dendritic cells (pDCs) is an important mechanism for NK cell activation and subsequent IFNγ secretion and cytotoxicity . We therefore wished to determine whether the impaired NK CD56dim cell TLR7/8-mediated IFNγ secretion in HCV rapid fibrosis we had identified using flow cytometry (Figure 3) was the downstream effect of the impaired IFNα secretion by PBMCs in response to TLR7/8 stimulation we had shown by ELISA (Figure 1). To do this, we cocultured PBMCs from patients with HCV patients with rapid fibrosis (n = 10) with TLR7/8 ligand R848, in the presence or absence of IFNα or an anti-IFNα neutralizing antibody. We then measured GMF of IFNγ produced by NK CD56dim cells using flow cytometry.
The addition of IFNα to PBMCs from HCV patients with rapid fibrosis did not restore NK CD56 dim cell IFNγ secretion in response to TLR7/8 stimulation to levels comparable to HCV patients with slow fibrosis (p = 0.89, Figure 6). Similarly, concomitant use of an anti-IFNα blocking antibody and TLR7/8 stimulation with R848 did not alter NK CD56dim cell IFNγ secretion.
Figure 6. Geometric mean fluorescence and fold increase in fluorescence over baseline of IFNγ production by NK CD56dim cells in HCV patients (n = 10), comparing stimulation with TLR7-specific agonist (loxoribine) and TLR7/8 agonist (R848), with and without IFNα. (A) IFNγ at baseline, with TLR7 agonist and TLR7/8 agonist in NK CD56dim cells, p = 0.049. (B) Fold increase in IFNγ at baseline, with TLR7 agonist and TLR7/8 agonist in NK CD56 dim cells, p = 0.009. (C) Geometric mean fluorescence of IFNγ with TLR7/8 stimulation in NK CD56dim cells, with and without the addition of IFNα, p = 0.89. (D) Fold increase in fluorescence of IFNγ with TLR7/8 stimulation in NK CD56dim cells, with and without addition of IFNα, p = 0.661. *p-value < 0.05.
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We then went on to determine whether NK cells express TLR7 and TLR8. We found that NK cells (including CD56 bright and dim subsets) express both TLR7 and TLR8 intracellular receptors. However, there was no difference in TLR7 or TLR8 expression on NK cells between HCV patients with rapid and slow fibrosis to explain the differences in IFNγ secretion (data not shown).
Impairment in IFNγ secretion by NK CD56dim cells in HCV rapid fibrosis is mediated through TLR8 rather than TLR7
In our experiments we had used a TLR7/8 agonist R848 as a reliable and powerful stimulant of cytokines through TLR7/8-mediated pathways. We therefore did not know whether the impairment in IFNγ secretion in NK CD56dim cells was mediated by TLR7 or TLR8. We next ascertained whether TLR7 or TLR8 were mediating the functional impairment we had demonstrated in NK cell secretion of IFNγ by comparing the effects of a TLR7-specific agonist loxoribine and TLR7/8 agonist R848 on NK CD56dim cell IFNγ production. In NK CD56dim cells from both HCV posttransplant patients and healthy controls, TLR7/8 ligand R848 produced a significantly greater fold increase in IFNγ production compared with the TLR7-specific agonist loxoribine. Furthermore, the IFNγ production ratio to baseline was clustered around 1.0 with loxoribine, suggesting that there was minimal TLR7 contribution to cytokine production in NK cells (Figure 6).
NK CD56dim cell degranulation is preserved in HCV rapid fibrosis despite impaired TLR7/8-mediated IFNγ secretion
Finally, we wished to determine whether NK CD56dim cell cytotoxicity was also affected given the impairment in NK CD56dim cell secretion of IFNγ we had demonstrated in HCV patients with rapid fibrosis. CD107a is a marker of NK cell degranulation, binding to the cell surface once degranulation has occurred. We therefore measured ex vivo NK CD56dim cell surface expression of NK degranulation marker CD107a via flow cytometry as a marker of cell cytotoxic degranulation that had occurred in-vivo, comparing HCV patients with rapid fibrosis to those with slow fibrosis.
We found that there was no difference in CD107a expression between HCV patients with rapid fibrosis progression in NK cells (p = 0.39), including NK CD56dim subsets (0.57). Interestingly, there was a trend toward increased CD107a expression in CD56bright NK cells in HCV patients with rapid fibrosis compared with slow fibrosis (p = 0.060), but this did not reach statistical significance (data not shown).
Time posttransplant was the only significant clinical factor that differed between HCV patients with rapid fibrosis and slow fibrosis
As there are well-recognized clinical factors which may influence both the risk of HCV rapid fibrosis, such as advanced donor age and HCV viral load, as well as factors that may theoretically influence TLR function posttransplant, such as immunosuppression level, we compared clinical characteristics of our HCV rapid and slow fibrosis cohorts (Table 1). We found that on univariate analysis, donor age (p = 0.003) and time posttransplant (p < 0.001) were the only significant differences between the two groups, which were otherwise well matched for clinical variables which may affect fibrosis outcomes. Of note, the number of patients with cirrhosis, HCV viral load and serum calcineurin inhibitor levels were similar between groups. Immunosuppressive regimen was also not statistically different between groups.
Table 1. Comparison of clinical factors in HCV patients posttransplant, comparing those with rapid fibrosis to those with slow fibrosis progression
| ||Slow fibrosis (n = 40)||Rapid fibrosis (n = 30)||p-Value|
|Univariate analysis|| || || |
|Age at transplant||49.1 + /− 1.1||51.2 + /−1.0||0.156|
|(mean +/−SEM)|| || || |
|Current age||55.0 + /− 1.0||54.0 + /− 0.9||0.474|
|(mean +/−SEM)|| || || |
|Donor age||33.9 + /− 2.5||45.1 + /− 2.7||0.003|
|(mean +/−SEM)|| || || |
|ETOH as a cofactor at time of transplant||12||9||1.00|
|HCV viral load at time of experiments (IU/mL) (mean+/−SEM)||1.79 × 106||1.00 × 106||0.399|
|Cold ischemia time (min, mean +/−SEM)||501.6 + /− 22.7||441.7 + /− 24.7||0.078|
|Cyclosporine level at time of experiments (mean +/−SEM)||404.6 + /− 42.7||449.4 + /− 55.0||0.520|
|Tacrolimus level at time of experiments||6.9+/−1.0||7.0+/−4.7||0.850|
|(mean +/−SEM)|| || || |
|F3 or F4 Metavir stage fibrosis within 5 years posttransplant||2||20||<0.0001|
|Presence of cirrhosis||4||11||0.085|
|Time posttransplant (years)||8.0+/−0.7||4.0+/−0.5||<0.0001|
|Multivariate analysis|| || || |
|Time posttransplant|| || ||0.002|
When we performed a multi-variate analysis, time posttransplant was the only significant difference between the two groups (p = 0.002, Table 1), with donor age no longer being significant.
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Despite rapid developments in our understanding of TLR function and its role in HCV infection, little is known of the role of TLR function in HCV recurrence posttransplantation. TLRs are the premier sentinel immune sensors in viral infection and have been linked with fibrosis development ; thus their role in HCV reinfection posttransplantation is theoretically of considerable importance to study. This work represents the first systematic exploration of TLR function in peripheral immune cells in hepatitis C recurrence postliver transplantation.
In patients with rapid fibrosis due to recurrent HCV posttransplant, we found evidence of impaired IFNα secretion by PBMCs in response to TLR7/8 stimulation. We also identified impaired secretion by PBMCs of TNFα and IL-6 at baseline and with TLR3 stimulation in those with rapid fibrosis progression. These findings were confirmed in a subgroup with liver biopsy and TLR function studies performed early posttransplant. For this reason, we chose to focus on TLR3 and TLR7/8-mediated cytokine production by cell subtypes using flow cytometry in order to determine which cell subsets may be responsible for these TLR functional changes.
Further studies demonstrated the impairment in TLR3-mediated IL-6 and TNFα production by PBMCs associated with rapid fibrosis was likely due to monocytes producing reduced amounts of proinflammatory cytokines in response to TLR3 stimulation. Monocytes have been shown to express TLR3 functional receptors  and TLR3 is a potent inducer of primary and secondary antiviral pathways . Monocyte-derived IL-6 and TNFα activate mDCs and pDCs, as well as recruiting inflammatory cells to the liver in HCV infection . Impaired secretion of TLR3-mediated IL-6 by monocytes has been described in HCV-HIV coinfection, a state where accelerated liver fibrosis is also seen . Furthermore, recent evidence suggests that IL-6 can have antifibrotic effects on hepatocytes through promoting hepatic regeneration without fibrosis [21, 22]. It is therefore conceivable that impairment of TLR3-mediated IL-6 and TNFα production by monocytes contributes to inappropriate activation of NK cells and dendritic cells and increased fibrosis.
TLR7/8-mediated IFNα secretion by PBMCs was reduced in HCV patients with rapid fibrosis progression. Almost all type I interferon is secreted by plasmacytoid dendritic cells  and these express functional TLR7 receptors . IFNα secreted by pDCs is critical for effective T cell activation and may contribute to poor T cell responses to HCV antigens, leading to reduced viral control and clearance in patients with rapid fibrosis . Our findings correspond well with the known importance of TLR7-mediated IFNα in the immune response to HCV, with substantial evidence for the efficacy of IFNα therapy for clearance of HCV infection and more specifically the efficacy of TLR7 agonist isotoribine in suppressing HCV viremia and achievement of sustained virological response . Further evidence for the importance of TLR7/8-mediated IFNα in HCV-mediated rapid fibrosis progression is that TLR7 polymorphisms have been linked to liver fibrosis progression .
Furthermore, we demonstrated impaired TLR7/8-mediated IFNγ secretion by NK CD56dim cells. NK CD56dim cells produce large amounts of antiviral IFNγ and initiate perforin-mediated cytotoxicity of viral-infected cells, but produce less proinflammatory cytokines compared with CD56bright NK cells . As failure of pDCs to secrete IFNα has been shown to reduce NK cell activation , we wished to determine whether this was a dependent effect. The addition of exogenous IFNα did not correct the deficit in IFNγ secretion by NK CD56dim cells, demonstrating that impairment in NKCD56 dim cell IFNγ secretion is independent of impaired TLR7/8-mediated IFNα secretion by PBMCs in HCV patients with rapid fibrosis progression.
We then wished to determine what other factors may be responsible for the impairment in TLR7/8-mediated NK CD56dim cell function. We determined that NK cells (including CD56dim) express TLR7 and TLR8 receptors and that NK CD56dim cell TLR7 and TLR8 expression was not different between HCV patients with rapid and slow fibrosis. In addition, we found that the TLR7/8 agonist R848 was more a more potent inducer of NK CD56dim cell IFNγ secretion than TLR7-specific agonist loxoribine, suggesting that NK CD56dim cell secretion of IFNγ is related to TLR8 stimulation, rather than TLR7 stimulation. We also demonstrated that CD107a expression was not different between HCV patients with rapid versus slow fibrosis. Taken together, these results suggest that NK cells are capable of being directly stimulated by TLR7/8 ligands and can produce IFNγ in an IFNα/plasmacytoid dendritic cell-independent manner. They also suggest that IFNγ secretion occurs separately to cytotoxicity. These findings are supported by evidence from other groups showing that NK cells express functional TLR7 and TLR8 receptors , NK cell cytotoxicity is more dependent upon pDC derived IFNα than IFNγ secretion  and that IFNγ secretion by NK cells can occur independently of IFNα . It has also been shown that CD107a expression is preserved in HCV infection despite reduction in IFNγ . Both NK and NKT cells have been shown to inhibit HCV viremia via both cytolytic and noncytolytic mechanisms  and this may explain why there is an effect of impaired NK cell-derived IFNγ without impaired cytotoxicity in patients with HCV-mediated rapid fibrosis posttransplant.
There are several potential mechanisms to explain how our findings may contribute to rapid fibrosis progression in HCV recurrence posttransplant. NK CD56dim cell IFNγ secretion activates dendritic cells and T cells and promotes anti-viral Th1 cytokine responses. Impairments in NK cell IFNγ secretion promote Th2 compared with Th1 cytokine responses , leading to impaired activation of T cell immune responses against HCV infection, particularly impairing the protective effect of CD4+ T cells against liver disease progression [25, 33]. Poor immune control of HCV viremia is well described in HCV rapid fibrosis progression  and leads to greater proinflammatory cytokine production and greater activation of profibrogenic pathways [35, 36]. NK CD56dim cells also have important antifibrotic effects via inhibition of hepatic stellate cells, either directly through induction of stellate cell apoptosis or indirectly through production of IFNγ, which inhibits hepatic stellate cell activation . Impaired NK cell IFNγ secretion as we have demonstrated leads to uncontrolled activation of hepatic stellate cells and increased fibrogenesis. In addition, the impairment in IFNγ secretion by NK CD56dim cells and subsequent promotion of Th2 cytokine responses in HCV infection leads to greater TGF-β secretion and IL-10 secretion , which both promote hepatic stellate cell survival through downregulation of CD95Ligand  and upregulation of liver fibrogenesis.
An important consideration for our data was whether the changes in TLR-mediated immune responses are merely a result of HCV viral load or immunosuppression levels, two key clinical determinants of HCV rapid fibrosis progression [34, 39]. It is well established that patients with rapid HCV fibrosis posttransplant have higher levels of HCV viremia . HCV has been shown to impair both TLR3[10, 11, 41, 42] and TLR7/8  signaling through various mechanisms, which may explain our findings in HCV rapid fibrosers. We demonstrated that there was no significant difference in either serum calcineurin inhibitor level or HCV viral load at the time of PBMC recovery between the HCV rapid and slow fibrosis cohorts (Table 1). Furthermore, we found no correlation between either HCV viral load or serum calcineurin inhibitor level and the TLR changes we identified (data not shown). This suggests that the TLR functional impairment we describe in HCV patients with rapid fibrosis development is independent of both HCV viral load and calcineurin inhibitor level.
Time posttransplant (in years) was the only clinical factor that significantly differed between the rapid fibrosis and slow fibrosis groups on multivariate analysis (Table 1). This likely reflects increasing rates of HCV progression postliver transplant in recent years, in part due to increased use of marginal grafts and greater immunosuppression [39, 44]. We endeavored to reduce the effect of time posttransplant in our study by analyzing a subset of patients who had a liver biopsy and TLR functional studies performed 6–12 months posttransplant and by ensuring time posttransplant was matched between rapid and slow fibrosis groups in the patients in whom flow cytometric analysis was performed (data not shown).
One difficulty with studies evaluating risk associations with rapid fibrosis progression in HCV recurrence postliver transplant is the choice of definition of rapid fibrosis progression, for which there is no current gold standard. In this study, we have used fibrosis rate and fibrosis progression on the first-year liver biopsy posttransplant as these have both been widely used in clinical studies previously [16, 17, 45-52]. Ideally, additional analyses using F2 stage or greater fibrosis on the year 1 liver biopsy and the development of cirrhosis within 5 years posttransplant would also have been performed, however in our study the numbers of patients developing F2 stage fibrosis on the year 1 biopsy or cirrhosis within 5 years posttransplant were small, meaning any analysis of the relationship between liver fibrosis and TLR function would be underpowered to detect a difference.
A key limitation to our study is that it is cross-sectional and retrospective and therefore precludes our ability to definitively establish cause and effect between HCV fibrosis and TLR function. Similarly, it provides no information about what happens to TLR function over time in HCV reinfection. Further prospective studies measuring TLR responses in PBMCs and fibrosis progression on liver biopsy at multiple time points posttransplant would help to answer these questions. However, our study still describes important novel relationships between TLR function and HCV fibrosis progression posttransplant that warrant further investigation.