Chronic hepatitis C virus (HCV) infection is a major public health issue (1), with approximately 58.6 million people living with chronic HCV and an estimated 1.5 million new infections each year (1, 2). HCV primarily infects liver hepatocytes causing inflammation and liver damage leading to morbidities including liver cirrhosis and hepatocellular carcinoma (3). HCV also causes extrahepatic manifestations including increased risk of non-hodgkins lymphoma, kidney and rheumatic disease, type 2 diabetes and cardiovascular disease (4, 5). These extrahepatic morbidities are likely mediated at least in part by systemic activation of circulating immune cells and inflammation in response to viral particles and antigens during viremia, as well as cellular damage resulting from virus infection. Similar to HCV, chronic HIV infection is also associated with increased risk of multiple inflammatory diseases (6) and this risk persists despite viral suppression with antiretroviral therapy, implying some impacts of chronic viral disease are not fully reversed upon suppression of viral replication.

HCV can now be cured with direct acting antiviral (DAA) therapy, which provides substantial reductions in both hepatic and non-hepatic sequelae of HCV (5). However, people treated for HCV with interferon-free DAA therapy continue to experience substantially higher rates of all-cause mortality after HCV cure, ranging from 3-14 times higher than the general population depending on the extent of liver disease at the time of treatment (7). It is thus unclear whether DAA therapy completely reverses HCV-related pathologies that may contribute to increased risk of both hepatic and extrahepatic morbidities. Understanding systemic HCV-associated changes which persist post-cure are highly relevant for the ongoing clinical management of individuals with a history of HCV infection.

Inflammation is a key pathogenic mechanism of liver damage during HCV infection and may also drive extrahepatic comorbidities such as cardiovascular disease which are more prevalent in people with HCV (reviewed in (5)). Chronic HCV infection is associated with systemic immune activation and elevated levels of interferons, inflammatory biomarkers including IL-6 and TNF/TNF receptors, chemokines such as CXCL10 and markers of myeloid cell activation including soluble (s) markers sCD163 and sCD14 (8–13). Conflicting data exist regarding the impact of HCV elimination on reversal of immune activation; whilst some biomarkers including CXCL10, IL-10 and sCD163 are consistently reduced following HCV elimination (8, 9, 13–18), others including IL-6, TNF and sCD14 are unaffected or inconsistently altered by DAA therapy (9, 13–16). Importantly, numerous studies suggest that DAA therapy does not restore levels of many parameters including sCD163 and CXCL10 to those found in comparable, HCV-seronegative populations (8, 13, 14, 17–19). Chronic inflammation is associated with increased risk of a number of chronic morbidities and mortality in both the general population and in the setting of HIV and HCV infection (20–23). Therefore, understanding whether elements of HCV-associated inflammation persist post DAA therapy is relevant for the long-term clinical management of individuals after HCV cure.

In most high-income countries, the HCV epidemic is concentrated in populations of people who inject drugs (PWID) who represent up to 80% of new HCV infections in many countries (24). PWID experience additional health risks associated with injecting drug use (25–27) and behavioural factors such as tobacco smoking, alcohol use and diet, which increase chronic inflammation (28, 29), may further contribute to poor health. The additional burden of HCV in PWID may therefore potentiate inflammatory diseases and/or compromise the restorative potential of DAA-mediated HCV cure on immune function. However, to date there is limited information regarding the discrete effects of HCV and substance use-related risks on inflammatory pathways in PWID populations who are disproportionately affected by HCV.

In this study we aimed to delineate the independent effects of both HCV infection and injecting drug use on soluble inflammatory and immune activation biomarkers among people with HCV who inject drugs. Furthermore, we investigated whether these biomarkers normalised to levels comparable to HCV-uninfected PWID after curative DAA therapy.

Curative DAA therapy for HCV prevents substantial morbidity and mortality, but it is unclear whether DAA therapy leads to a complete reversal of HCV-related immune alterations and thus related pathologies. We identified elevated levels of numerous soluble biomarkers associated with chronic disease risk in PWID, compared to those with no history of injecting drug use, including inflammatory and monocyte activation biomarkers. PWID with chronic HCV exhibited elevated levels of additional parameters including biomarkers of endothelial activation and only some of these parameters were normalised by curative DAA therapy. These data suggest PWID may be at increased risk and thus should be closely monitored for the development of inflammatory morbidities. Furthermore, PWID with a history of HCV infection may possess additional and unique immunological alterations which persist after HCV cure and may potentiate the development of chronic diseases.

The specific biomarkers identified to be elevated in PWID with no history of HCV included the inflammatory biomarkers sTNF-RII and IL-6 and markers of myeloid cell activation (sCD14 and sCD163). These findings are consistent with previous observations in people who use drugs (37–40) and suggest a persistent state of inflammation and myeloid cell activation. Elevated levels of these biomarkers are associated with increased risk and severity of cardiovascular disease and mortality in both the general population and in individuals with underlying chronic infections such as HIV (21–23, 41, 42), however their relationship with chronic disease risk and mortality in PWID with and without HCV is not known and warrants further investigation.

Elevated levels of these inflammatory markers in PWID may be driven by a number of behavioural and sociodemographic factors including, but not limited to, diet, tobacco and alcohol use (32% of PWID in the present study reported using alcohol ≥4 times/week). Tobacco use and moderate to heavy alcohol consumption are associated with heightened inflammation (28) and these risks are prevalent in PWID populations (43). Similarly, there is a well-established link between diet and systemic inflammation (29, 44) and substance use is known to affect diet and compromise nutrition, which is potentiated by factors including unstable housing (45). There are also mechanisms directly related to injecting drug use itself such as inflammatory and immunomodulatory effects of the drugs injected including methamphetamine, opioids and accompanying filler agents (46–50). Injury and infection related to injection site and injection practices can result from contaminated injecting equipment and/or the introduction of pathogens, foreign bodies and skin contaminants during the process of injecting (25, 27, 51). Over 60% of PWID report a history of skin and soft tissue infections with subcutaneous/intradermal injecting practices and vascular damage associated with injecting contributing to this risk (26). Injection frequency and duration correlate positively with markers of immune activation including d-dimer and sCD14 (39), whilst methamphetamine users with HIV who injected the drug exhibited elevated levels of numerous inflammatory biomarkers (including IL-6, sTNF-RI and sCD163) compared to those who did not inject (52). Together, these findings support a direct contribution of drug injection to systemic inflammation in PWID irrespective of HCV status, and more research is needed to characterise these risks and determine their long-term health implications.

PWID in the present study also exhibited substantially elevated levels of HbA1c, which is a well validated indicator of type 2 diabetes risk, diabetic complications and cardiovascular disease (53). These metabolic alterations are likely associated with heightened inflammation but may also be influenced by lifestyle-related differences such as diet which is often altered in PWID (45). These data are consistent with the well documented association of injecting drug use with poor health outcomes and suggest inflammatory and cardiometabolic alterations may predispose them to increased risk of chronic inflammatory diseases such as cardiovascular disease and diabetes. Future studies are warranted to better characterise diabetes prevalence in PWID and identify contributing risk factors.

Analysis of the impact of active HCV infection in PWID revealed a further elevation of sTNF-RII levels as compared to never-infected PWID, as well as significant increases in plasma levels of CXCL10, VCAM and LBP in PWID/HCV+, consistent with previous findings (8, 9, 12, 17, 54). We did not detect an increase in inflammatory factors previously associated with chronic HCV such as sCD163, sCD14 or IL-6 in our cohort of PWID with HCV, potentially due to the pre-existing elevation of these parameters observed in all PWID. However, we did observe a significant reduction in sCD163 levels following DAA initiation, implying viremic HCV was contributing to heightened sCD163 levels and by association heightened macrophage activation prior to DAA initiation.

DAA therapy was associated with normalisation of certain activation markers (e.g., CXCL10, VCAM) to levels comparable to uninfected PWID, suggesting these parameters are closely linked with HCV viremia and are thus largely reversed following HCV cure. Although DAA therapy initially reduced sTNF-RII, levels at 48 weeks post treatment were higher in PWID treated for HCV as compared to never-infected PWID. In contrast, LBP levels were not significantly reduced by DAA therapy and remained elevated as compared to HCV-uninfected PWID up to 48 weeks post HCV cure. LBP is primarily produced by hepatocytes and is involved in the inflammatory response to microbial products such as bacterial LPS. Elevated LBP observed here in PWID with current or past HCV infection may indicate impaired hepatocyte/liver function due to persistent tissue damage or may be due to enduring damage to the gut or other mucosal barriers, resulting in heightened translocation of bacterial products. The latter is consistent with elevated plasma LPS levels in untreated HCV (55) and is analogous to the compromised gut barrier integrity and heighten microbial translocation observed in chronic treated HIV infection, which is known to contribute to persistent immune activation and inflammatory comorbidities (56). Elevated LBP and LPS levels are associated with dietary glucose and diabetes (57), so the observed elevation in HbA1c and LBP in the present cohort may be indicative of metabolic alterations. Little is known regarding the clinical implications of elevated LBP levels in people with HCV, however in HIV/HCV co-infection it is associated with more advanced liver fibrosis (58) and in HIV, elevated LPS and LBP are linked with increased risk of cardiovascular disease and dementia (59, 60), supporting the clinical relevance of these findings. Follow-up studies are required to identify the relationship between persistently elevated levels of inflammatory factors identified here and clinical outcomes in individuals after HCV cure, especially in PWID.

Interestingly, we also observed a significant increase in sCD14 levels in PWID with HCV after 12 weeks of DAA therapy consistent with previous finding (13), although this was normalised after 48 weeks. This may be related to transient immune reconstitution induced by viral suppression and resultant increase in myeloid cell activity, however this remains to be explored. Consistent with the well-established relationship between inflammation and impaired liver function, all biomarker analysed here (except HbA1c) were significantly associated by multivariable regression analysis with indices of liver function including AST, ALT and the AST to platelet ratio index (APRI) score (data not shown), the latter being a surrogate marker of liver disease in HCV (61). People treated for HCV with DAA therapy are at heightened risk of mortality related not only to liver disease but also extrahepatic cancers and circulatory diseases, and the presence of comorbidities further increases mortality risk (7). The findings presented here of a persistent inflammatory state in individuals post HCV cure may therefore be relevant to these long term health outcomes and it would be of interest to determine whether these biomarkers were associated with morbidity and mortality post HCV cure.

The limitations of this study include the modest sample size due to the selection criteria and sample availability at the required timepoints and a lack of data pertaining to lifestyle factors such as body mass index, level of physical activity, as well as medical history and current medications. A longer duration of HCV viremia may heighten the immune changes measured here due to more extensive immune activation/exhaustion and a greater accumulation of cellular and tissue damage; this parameter could not be controlled for since time of infection is difficult to determine. Injection practices were not assessed in this analysis but would be useful to include in future studies. Finally, we had limited demographic data available in our never infected community control participants which precluded formal investigation of behavioural factors which may contribute to the observed biomarker differences. Given their influence on the parameters measured here, future studies are warranted to specifically assess the contribution of behavioural factors to chronic inflammatory disease risk.

These data add to a growing body of evidence that curative DAA therapy may not completely reverse immunological changes included by HCV infection, and biomarkers relevant to the development of chronic morbidities may remain elevated in some individuals. This study also highlights the particular vulnerability of PWID with HCV to these effects given the overlapping impact of injecting drug use and related lifestyle factors on immunological changes relevant to the development of chronic inflammatory diseases. These findings suggest ongoing clinical management of PWID with a history of HCV infection should include heightened vigilance for chronic inflammatory diseases even after curative HCV treatment.

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Written informed consent to participate in this study was obtained from all participants. This work was approved by the Alfred Hospital Research and Ethics Committee (Melbourne, Australia).

AH: Conceptualization, Formal analysis, Funding acquisition, Investigation, Resources, Writing – original draft. NV: Formal analysis, Writing – original draft. SE: Investigation, Writing – review & editing. BH: Data curation, Project administration, Validation, Writing – review & editing. IB: Resources, Writing – review & editing. LN: Data curation, Project administration, Writing – review & editing. DP: Formal analysis, Writing – review & editing. PD: Resources, Writing – review & editing. AD: Resources, Writing – review & editing. AT: Resources, Writing – review & editing. AJ: Conceptualization, Supervision, Writing – review & editing. MH: Resources, Writing – review & editing. RS-D: Data curation, Formal analysis, Supervision, Writing – original draft. JD: Conceptualization, Funding acquisition, Resources, Supervision, Writing – original draft.