HCV spontaneous clearers showed low senescence profile in people living with HIV under long ART

Coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) increases immune activation, inflammation, and oxidative stress that could lead to premature senescence. Different HCV infections, either acute or chronic infection, could lead to distinct premature cellular senescence in people living with HIV (PLWHIV). Observational study in 116 PLWHIV under antiretroviral treatment with different HCV status: (i) n = 45 chronically infected with HCV (CHC); (ii) n = 36 individuals who spontaneously clarify HCV (SC); (iii) n = 35 HIV controls. Oxidative stress biomarkers were analyzed at lipid, DNA, protein, and nitrates levels, as well as antioxidant capacity and glutathione reductase enzyme. Replicative senescence was evaluated by relative telomere length (RTL) measurement. Additionally, 26 markers of Senescence‐Associated Secretory Phenotype (SASP) were analyzed by multiplex immunoassays (Luminex xMAP technology). Differences were evaluated by generalized linear model (GLMs) adjusted by most significant covariates. The SC group had a senescence signature similar to the HIV control group and slightly lower SASP levels. However, significant differences were observed with respect to the CHC group, where an increase in the nitrate concentration [adjusted arithmetic mean ratio, aAMR = 1.73 (1.27–2.35), p < 0.001, q = 0.009] and the secretion of 13 SASP‐associated factors [granulocyte macrophage colony‐stimulating factor (GM‐CSF), interferon‐β, interleukin (IL)‐1β, IL‐2, IL‐8, IL‐13, tumor necrosis factor (TNF)‐α, IL‐1α, IL‐1RA, IL‐7, IL‐15, C‐X‐C motif chemokine ligand 10 (IP‐10), stem cell factor (SCF); q < 0.1)] was detected. The CHC group also showed higher values of IL‐1α, IP‐10, and placental growth factor 1 (PIGF‐1) than HIV controls. The SC group showed a slightly lower senescence profile than the HIV group, which could indicate a more efficient control of viral‐induced senescence due to their immune strengths. Chronic HCV infection in PLWHIV led to an increase in nitrate and elevated SASP biomarkers favoring the establishment of viral persistence.

After an acute infection, spontaneous clearance may occur in approximately 20% of patients within 6 months of infection without any treatment, 2 although it is a rare phenomenon in the context of coinfection with HIV. 3 Besides other coinfections, genetic background, clinical and virological factors also affect HCV spontaneous clearance. Thus, genetic variation at interferon lambda 3 gene (IFN-λ 3 ), formerly named as IL28B, is strongly associated with spontaneous clearance of HCV and also response to treatment. 4 Additionally, female gender also shows better prognosis of HCV infection, with lower HCV RNA viral loads and higher spontaneous clearance. 5 Although many studies have tried to unravel the factors that predispose to HCV spontaneous clearance, few studies have delved into their clinical consequences. Some studies have pointed out that patients that cleared the infection are at lower risk of mortality and liver-related death than patients who are chronically infected. 6 Conversely, HCV chronic infection showed similar impact on peripheral blood cells (PBMCs) than an acute infection followed by spontaneous resolution. 7 Therefore, more studies are essential to understand the potential immune consequences after HCV resolution.
Chronic HCV coinfected with HIV increases immune activation, which favors the progression of HIV infection and the development of liver disease leading to premature senescence. 8 Senescent cells often exhibit high levels of various forms of damage accumulation overtime in an old organism, including DNA damage and oxidative modifications. 9 In addition, coinfection can lead to T-cell exhaustion, mainly in chronic infections. 10 Progressive loss of immunological memory correlates with reduced proliferative capacity and telomere shortening in T cells. In fact, telomere shortening has been observed in circulating lymphocytes from both HCV+ 11 and HIV+ patients. 12 Telomeres are nucleoprotein structures located at the end of eukaryotic chromosomes formed by repeated DNA sequences (TTAGGG), which maintain the integrity of the chromosome. In each round of replication, telomeres become progressively shorter and when they reach a critical length, cell cycle arrest, cellular senescence, and apoptosis occur. 13 Thus, telomere length is a hallmark of cellular aging associated with the progression of several diseases 14,15 and an increased risk of infection. 16 HIV and HCV infections produce an indistinguishable form of cellular senescence that shares features of chronic inflammation in aging, 17 accompanied by a pro-inflammatory phenotype called the Senescence-Associated Secretory Phenotype (SASP). 18 This phenotype includes the production of cytokines, extracellular matrix factors, and coagulation mediators, 19 which acts as an antiviral mechanism of the senescent cell and allows limitation of viral replication. 20 However, whatever its primary function, SASP can have both beneficial effects and detrimental consequences. 19 Some of these parameters improve after HCV elimination, but there is increasing evidence that inflammatory markers are not restored after HCV elimination. 21 This may result in increased comorbidities and mortality in patients who have overcome chronic HCV infection after successful treatment, 22 but there is very little information on patients recovered from spontaneous clearance and even fewer studies focused on the assessment of senescence in these patients.
Therefore, we propose a comprehensive approach to assess inflammation, aging, and senescence-related markers in people living with HIV (PLWHIV) after HCV spontaneous clearance with respect to HCV chronically infected (CHC) and HIV controls.

| Biological material
Peripheral venous blood samples were collected in ethylenediaminetetraacetic acid tubes. PBMCs were isolated by density gradient centrifugation method with Lymphoprep™ and SepMate™ tubes (Stemcell Technologies) and stored at viability until use. Plasma was obtained after density gradient centrifugation, clarified, and storage at −80°C until use.

| Senescence biomarkers in plasma
The cellular senescence was assessed by reactive oxygen species (ROS) markers levels, total antioxidant capacity, glutathione, and SASP levels. We determined the reduced glutathione (GSH), and oxidized glutathione (GSSG) by the GSH Colorimetric Detection Kit (Thermo Fisher Scientific).

| SASP
We assessed a selection of 26 markers associated to SASP, including cytokines, chemokines, and growth factors 18,24 (complete list is available in Supporting Information: File 2). All markers were analyzed with a multiplex immunoassay (Luminex xMAP technology).

| Senescence biomarkers in PBMCs
2.6.1 | Replicative senescence DNA was extracted from PBMCs using the DNA Purification System Kit (Promega Wizard). We performed the quantification of replicative senescence by monochromatic multiplex real-time quantitative PCR (MMqPCR) as previously described. 25 To normalize and control the number of telomere copies/sample we used the single-copy gene (β-globin). Relative leukocyte telomere length (RTL) was expressed as the ratio of the telomere amplification product (T) to that of a single copy gene (S).

| Statistical analysis of epidemiological data
For the descriptive study of clinical and epidemiological data of the patients, continuous variables were summarized as median, and categorical as frequency and percentage. Concentration values are used for oxidative stress markers and raw fluorescence intensity as a relative quantification of the analyte abundances for SASP markers, as previously described. 26   Regarding metabolic characteristics, biochemical parameters related to liver function were significantly increased in CHC group (Table 2).

| Senescence profile analysis among different HCV statuses in PLWHIV
The senescence profile between groups was evaluated by a GLM model adjusted by age, sex and IFN-λ 3 genotype, which were selected by a stepwise procedure.

| Senescence-associated markers after HCV acute infection (SC) versus HIV
Overall, we did not find any differences in oxidative stress levels and replicative senescence between SC and HIV groups (    Virus-induced senescence has been previously described for a variety of viruses, 32 as part of an antiviral response mechanism to limit viral replication. 20 senescence contributes to the pathophysiology of respiratory viral infections, 34 and a recent report has observed that viruses may convergently contribute to the evolution of human aging. 35 Thus, we assumed that all patients suffered from viral infections in a similar proportion and all of them were recruited previous COVID-19
The SC group showed similar levels of oxidative stress markers to the HIV group, which indicates a normalization of oxidative stress after HCV spontaneous resolution. Oxidative stress is an important key in HCV pathogenesis during both acute and chronic stages of inflammation, but little is known about long-term effects after spontaneous resolution in either HCV monoinfected patients or PLWHIV. HCV-induced oxidative stress significantly contributes to hepatic disease, playing a critical role in hepatic fibrogenesis and carcinogenesis. 36 However, patients in our cohort did not show advanced liver fibrosis, and probably the HCV-related oxidative stress has not been fully activated. Previous studies showed that HCV acute infection is characterized by decreased GSH in erythrocytes and increased ROS, viral replication, and apoptotic death. 37 Our data show that these makers are restored after HCV resolution, achieving similar redox environment that those PLWHIV who have never been infected by HCV.
Oxidative stress is a hallmark of chronic HCV infection 38  In addition, oxidative stress and telomere shorting could lead to SASP associated with cellular aging. 51 Our data showed similar profile of SC and HIV groups, with a general tendency toward a reduction of plasma markers of immune senescence in the SC group. The more robust immune system of these subjects could explain the observed reduction of the inflammatory environment and suggest a more efficient resolution of HCV infection.
By contrast, when SASP plasma levels of SC and CHC patients were compared, we observed a highly different profile. The CHC group showed higher levels of several molecules associated with immune senescence, such as IL-6, IL-8, and TNF-α the most prominent interleukins within the SASP 24,52 and which are also known to induce ROS. 53 In particular, elevated concentrations of the pro-inflammatory cytokines IL-1β, IL-2, IL-6, IL-8, and TNF-α were found, cytokines that during normal healthy aging have been shown to be significantly increased compared with younger individuals. 54,55 This also agrees with previous studies, where increased cytokines of Th1 response 56,57 and IP-10 58 were also observed in CHC patients.
Likewise, the increase in factors associated with SASP has also been observed in other viral infections such as hepatitis B 59 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 60 The increase of these markers has been shown mainly in chronic viral hepatitis, increasing with fibrosis progression 30 and in hepatocellular carcinoma, 61 but there is no previous knowledge of these markers on patients without advanced fibrosis.
CHC patients also showed slightly higher levels of IL-1α, IP-10, and PIGF-1 than HIV group. It has been proposed that the level of some factors associated to SASP reflects the progression of liver fibrosis in patients with chronic HCV. 30 But the absence of large differences in the SASP between CHC and HIV groups could be due to the lack of advanced fibrosis present in our cohort, on contrary of previous studies. 50 To sum up, in SC individuals, short-term exposure to SASP factors drives the recruitment of immune cells to eliminate premalignant and senescent cells, thereby preventing tumorigenesis.
However, the long-term exposure to SASP experienced by CHC generates chronic inflammation and could contribute to tumorigenesis and age-associated pathologies. 62,63 Hence, the accumulation of senescence cells in these CHC individuals may significantly affect nonacquired immunodeficiency syndrome (AIDS) events via the SASP. Therefore, although nonadvanced fibrosis was present in CHC group, they could be prone to cancer development in the future.
To interpret our data correctly, we should bear in mind that some clinical and epidemiological variables were statistically different between groups, therefore, they were included in the full statistical model as adjustment variables for accounting their effect on senescence markers. The main limitation that should be considered is the limited sample size which could have restricted the possibility of finding statistical significance in some subgroups. Additional factors not considered, such as common respiratory viral infections may also play a role, but we assume that these events could impact in the same way in all study groups. Future studies on long-term F I G U R E 2 Quantification of senescence-associated secretory phenotype in people living with HIV under different HCV infectious status. Values are expressed as aAMR, obtained using a generalized linear model; *, p < 0.05 and q < 0.1. aAMR, adjusted arithmetic median rate; CHC, PLWHIV chronically infected with HCV; GM-CSF, granulocyte macrophage colony-stimulating factor; Gro-α, growth-regulated oncogenealpha; HCV, hepatitis C virus; HIV, human immunodeficiency virus monoinfected patients; IFN, Interferon; IL, interleukin; IP-10, C-X-C motif chemokine ligand 10; PIGF-1, placental growth factor 1; q, corrected level of significance by false discovery rate; SC, PLWHIV who spontaneously clarify HCV; SCF, stem cell factor; TNF, tumor necrosis factor.
follow-up would be essential to unravel the long-time effects of HCV-related SASP in liver-related comorbidities on PLWHIV. It also would be interesting to evaluate whether similar results are observed in non-HIV patients, to have a better understanding of the negative consequences of HCV infection and to address possible palliative strategies.

| CONCLUSION
In conclusion, PLWHIV who spontaneously resolved an HCV acute infection showed slightly lower senescence profile to monoinfected