The interplay between endogenous inflammatory mediators and microbial components in innate immunity

HCV is a hepatotropic virus with a global prevalence rate of approximately 70 million individuals worldwide. Following HCV infection, the majority of individuals develop a chronic infection that is characterized by the occurrence of hepatic fibrosis and HCC. While great strides have been made understanding and treating HCV infection, there remains much to be revealed. In this thesis, we were interested in determining the role of HCV proteins (HCV core protein, NS3 and glycoprotein E2) in the regulation of cytokine activity regarding their capacity to induce chemokine expression. As such, we carried out induction experiments in liver bystander cells (monocytes, fibroblasts and LSECs) where we measured chemokine expression in response to HCV proteins as a single stimulus. HCV core protein was a potent inducer of CCL2, CCL3 and CXCL8 in monocytes and a weak inducer of CCL2 and CXCL8 in endothelial cells. NS3 was considerably less active inducing modest levels of CCL3 and CXCL8 in monocytes only. We subsequently stimulated liver bystander cells with combinations of viral proteins and either IL-1β or IFN-γ. Interestingly, we observed a synergistic effect between the core protein and IL-1β or IFN-γ in the expression of chemokines (CCL2, CCL20, CXCL8 and CXCL10) in fibroblasts and LSECs. The synergistic effect of the core protein was mediated through its activation of TLR4. These findings reveal novel pathways by which HCV proteins may contribute towards HCV-induced hepatic injury.

SAA is both an apolipoprotein and APP protein whose expression is upregulated in response to inflammatory cytokines such as IL-1β, IL-6 and TNF-α. Hence, enhanced expression of SAA is to be anticipated during inflammatory conditions of variable underlying etiologies. Indeed, upregulated expression of SAA (>5 µg/ml) has been reported in patients with chronic HCV infection. SAA has been described to undergo post-translational modification by distinct proteases such as MMPs, cathepsins and neutrophil elastase. HCV and HCV-derived products have been shown to modulate the expression of such proteases. MMP-9 expression is enhanced during HCV infection. In fact, HCV core protein directly induces the expression of MMP-9 in hepatic stellate cells. Therefore, we were interested to determine the proteolytic processing of SAA in response to MMP-9. Following the incubation of human SAA1 with MMP-9, using mass spectrometry, we detected three cleavage sites of MMP-9 on SAA1 thereby producing SAA1(1-51), SAA1(52-104) and SAA1(58-104). These peptides were chemically synthesized using solid-phase peptide synthesis and subjected to biological characterization. Following cleavage, SAA1 C-terminal peptides retained their capacity to synergize with CXCL8 in the activation and recruitment of neutrophils via FPR2 activation.

An immense number of studies investigating the biological function of intact SAA have been carried out. Nevertheless, the true biological role of SAA has never been completely understood. Multiple functions have been attributed to SAA such as chemotaxis, induction of pro-inflammatory molecules (ROS, MMPs, cytokines and chemokines) and antimicrobial effects to name a few. To relay its function, SAA has been linked to multiple receptors including TLR2, TLR4, FPR2, RAGE, CD36, P2RX7, SR-BI and SR-BII. In order to investigate the biological function of SAA, researchers utilized recombinant forms of this APP derived from bacterial expression. Recently, a study attributed the TLR2-mediated functions of human SAA1, derived from recombinant bacterial expression, to bacterial lipoprotein contamination. Indeed, human SAA1 was found to bind lipophilic bacterial products such as lipoproteins and LPS. As such, we sought out to determine the inherent biological role of SAA. Following reversed-phase purification of recombinant forms of human SAA1 and murine SAA3, we observed the loss of multiple previously reported pro-inflammatory functions including induction of ROS, chemokine and MMP-9 expression in monocytes and in vitro macrophage polarization. On the other hand, purified homogenous human SAA1 and murine SAA3 retained the capacity to synergize with CXCL8 in the activation and recruitment of neutrophils via the activation of FPR2. Furthermore, human SAA1 induced in vivo neutrophil and mononuclear cell recruitment following intraarticular injection. In addition, homogenous human SAA1 displayed the capacity to promote monocyte survival. In light of these findings, we recommend that future researchers investigating the biological function of SAA utilize SAA derived from recombinant expression in eukaryotic cells or inflammatory plasma. Alternatively, animal models where SAA expression is KO or transgenically enhanced are also viable options to explore the biological nature of SAA.