There are nearly 170 million HCV-infected individuals worldwide (1
). Many studies have explored the mechanisms by which the immune system is capable of mediating viral clearance (2
). Although an HCV-specific humoral response is commonly seen, HCV-specific antibodies are not thought capable of conferring protection (3
). Instead, protection in chimpanzees has been shown to correspond with HCV-reactive CD8+
T cell responses (2
). Similarly, in humans, tetramer analysis of successful resolvers indicates the presence of a high frequency of functional HCV-specific CD8+
T cells (4
). In addition, there are genetic data to support a role for NK cells, based on the higher likelihood of KIR2DL3 and HLA-C1 individuals to clear infection (5
). A specific and unique issue for chronic infection is that cure is possible, as 40%–80% of chronically infected patients (numbers vary depending on viral genotype) that receive pegylated IFN-α2
)/RBV therapy clear the virus and achieve a sustained virologic response (SVR) (6
). Still for many, the virus manages to circumvent natural immunity and existing therapeutic strategies, thus resulting in significant morbidity and mortality.
To better define the distinct clinical outcomes of HCV infection, many investigators have performed candidate molecule screens or transcriptional profiling in order to identify correlates of viral clearance. One molecule that has gained significant attention is CXCL10 (also known as IFN-γ–induced protein–10, or IP-10), an IFN- and TNF-α–inducible chemokine that can be highly expressed by endothelial cells, keratinocytes, fibroblasts, mesangial cells, astrocytes, monocytes, neutrophils, and hepatocytes (7
). In addition to HCV disease, it has been shown to be expressed in many Th1-type inflammatory diseases, often correlating with target organ infiltration by T cells (8
). CXCL10 is part of a family of α-chemokines that bind CXCR3, which also includes CXCL9 (also known as monokine induced by IFN-γ, or MIG) and CXCL11 (also known as IFN-inducible T cell α chemoattractant, or I-TAC). While all three ligands are induced by IFN and bind the same receptor, there now exist substantial data to support their unique roles in disease pathogenesis. This is clearly evident in chronic HCV, where elevated levels of all three have been demonstrated but only CXCL10 is predictive of response to therapy (10
Regarding published data, at least four independent studies have demonstrated that baseline levels of CXCL10 are predictive of the failure to respond to HCV treatment (10
). Specifically, this has been demonstrated in patients with genotype 1 and 4 HCV; and in the largest study, a negative predictive value of 79% was reported for genotype 1 patients (12
). In addition, elevated levels of CXCL10 have been reported in patients coinfected by HCV/HIV (14
); and with HCV-associated cryoglobulinemia (16
). In several of these reports, CXCL10 levels correlated with necroinflammation, fibrosis stage, and/or HCV viral load. However, none of the reports have resolved the paradox of why a proinflammatory chemokine, responsible for recruiting activated lymphocytes to the liver, is a negative prognostic marker for response to therapy.
We hypothesized that CXCL10 may be present in an antagonist form in patients with chronic HCV, thus accounting for its correlation with treatment failure. To understand this prediction, some additional background is required. Briefly, chemokines act by binding to G protein–coupled receptors and mobilization of intracellular Ca2+
, which results in receptor internalization and the initiation of signaling pathways that facilitate chemotaxis. For the CXC class of chemokines, binding to and activation of the receptor is thought to be a two-step process. First, the core of the ligand binds the outer surface of the receptor; a second step involving the re-orientation of the flexible N-terminal tail of the protein allows binding to a distinct domain within the receptor (17
). Post-secretion modification of CXCL10 has been described, including C-terminal cleavage by MMP9 (or gelatinase B) and citrullination by peptidylarginine deiminase (PAD), both of which leave the protein in an agonist state (18
). Also reported is the N-terminal cleavage of 2 amino acids by members of the X-prolyl dipeptidyl peptidase (DPP) family, the most characterized being dipeptidyl peptidase IV (DPP4 or CD26) (20
). DPP4 has been shown to cleave several chemokines, including members of the α-chemokine family (21
). Importantly, DPP4 truncation of CXCL10 generates a dominant negative form of the protein, which is capable of binding CXCR3 but does not induce signaling. Pertinent in vivo examples of chemokine antagonism include the degradation of monocyte chemoattractant protein–3 (MCP3) by MMP2, reported to be relevant to the pathogenesis of spondyloarthritis (23
); and the cleavage of stromal cell–derived factor–1 (SDF1 or CXCL12) in HIV-associated neurodegeneration, also mediated by MMP2 (24
). To date, there has been no in vivo evidence to support a role for CXCL10 antagonism in disease pathogenesis. In part, the challenge in establishing in vivo evidence is due to the inability to discriminate the different forms of the analytes using sensitive immune-based assays. Furthermore, few studies take care to exclude the activity of proteolytic plasma enzymes that may act extracorporeally on protein biomarkers of interest.
Herein, we show a correlation between CXCL10 levels and the precursor frequency of CXCR3+ cells in circulation, suggesting that the chemokine gradient in vivo is nonfunctional. This initial observation prompted us to investigate MMP and DPP family members as mediators of the inactivation of CXCL10, and our data suggest a potential in vivo role for DPP4. To support such studies, we have generated antibody-based assays that have been Clinical Laboratory Improvement Amendments (CLIA) certified and help to provide direct evidence that the NH2-terminal residues of plasma CXCL10 are cleaved in patients with chronic HCV. Following from our observations, we believe we have begun to unravel what has been a paradox in the field for several years — CXCL10 is elevated in nonresponders (NRs) and is in an antagonist form. Furthermore, our data suggest an important link between the metabolic complications and the chronic inflammation seen in HCV patients.