Temporal Dynamics of SIM in Viral Infections
We employed the Quanterix HD SP-X Imaging and Analysis System™ to assess the impact of different viral infections and the dynamics of soluble inflammatory mediators (SIM). A heatmap was created to visualize the measured concentrations at various time points (Figure 1). At T1, baseline assessments for the HCV and SARS-CoV-2 cohorts showed elevated SIM activity (IL-6 and IL-22) compared to healthy donors. Following viral clearance at T2, all cohorts exhibited a reduction in SIM activity, indicating a decline in the immune response.
At T3 (six months), the SARS-CoV-2 cohort showed decreased SIM activity, particularly for MCP1, IP-10, and ITAC, compared to the median values of the selected SIMs. In contrast, the acute HCV and non-cirrhotic HCV cohorts displayed higher SIM responses than the control group and the SARS-CoV-2 cohort post-viral clearance. Notably, cirrhotic patients exhibited the highest SIM activity during long-term follow-up.
At T4 (nine months), SIM activity in the SARS-CoV-2 cohort remained decreased. The acute HCV and chronic non-cirrhotic HCV cohorts demonstrated a generalized decline in SIM activity, distinguishing them from the cirrhotic HCV group.
Comparative Analysis of SIM Dynamics in Viral Infections
Further insights into the dynamics of individual SIMs are presented in Figures 2-4. We observed a trend across most SIMs, with a significant increase in concentrations during acute SARS-CoV-2 infections compared to acute and chronic HCV infections. Notably, over time, the SIM levels in SARS-CoV-2 patients predominantly revert to those of healthy donors.
IL-10, IL-22, TNFα, IFNγ, and IL-6 are elevated during SARS-CoV-2 infection and return to baseline levels after viral clearance from follow-up T2 to T5 (Figure 2). However, in the cross-sectional cohort 1 of SARS-CoV-2, patients exhibit increased mean values at T4, despite individual SIM concentrations mostly aligning with healthy donor levels. The elevated mean values can be linked to outliers. Conversely, patients with acute and chronic HCV infections display different patterns. In non-cirrhotic patients with chronic HCV, SIM levels normalize to near-healthy levels, while cirrhotic patients maintain higher SIM expression. These values exceed those of healthy controls and also remain at the long-term follow-up (T5) after virus elimination (all p<0.0001) (Figure 2).
IL-8 and CXCL-10 (IP-10) exhibit elevated levels at baseline (T1) but show no statistically significant difference from healthy levels at T2 (after 3 months/SVR) in SARS-CoV-2, acute HCV, and chronic non-cirrhotic HCV patients (Figure 3). Persistent alterations are observed only in chronic HCV patients with cirrhosis, displaying a distinct long-term pattern compared to IL-10, IL-22, TNFα, IFNγ, and IL-6 (Figure 2). A notable decrease in concentration levels from T1 to T5, including IP-10/CXCL10 (T1: p<0.0001; T5: p<0.05) and IL-8 (T1: p<0.0001; T5 p<0.001), is noticeable (Figure 3).
Dynamics of Chemokines MCP1, ITAC and MIP-3β with prolonged elevation in SARS-CoV-2 and HCV
MCP1, ITAC, and MIP-3β concentrations exhibit dynamic changes, with SARS-CoV-2 cohort 2 showing an initial decline followed by a long-term increase. MIP-3β levels, in particular, significantly increase at follow-up T5 compared to T4, reaching almost 80% of baseline T1 levels. MCP1 levels show no long-term recovery, returning to T1 levels by T5 in SARS-CoV-2 cohort 2 (T1: p<0.0001; T5: p<0.0001). Similarly, ITAC concentrations in the long-term follow-up at T5 are elevated in SARS-CoV-2 cohort 2 (T1: p<0.0001; T5: p<0.01).
For all HCV cohorts, there is a general decrease in concentrations, although levels remain above those of healthy controls. Acute HCV patients exhibit the most significant decline in MIP-3β levels (T1: p<0.0001; T4: p>0.05). Chronic non-cirrhotic HCV patients also show a decrease in MIP-3β concentrations; however, this change is not statistically significant when compared to healthy donor levels. In contrast, chronic cirrhotic HCV patients have highly significant MIP-3β levels compared to healthy donors, indicating that their decrease at T5 is not reflected in the p-values.
ITAC concentrations decline in both acute HCV and chronic non-cirrhotic HCV patients at T4 and T5 (both p<0.01). Chronic cirrhotic HCV patients also exhibit a decrease in ITAC levels, although an outlier at 1406 pg/ml skews the mean value. Other concentrations ranged from 11.57 pg/ml to 123 pg/ml.
MCP1 levels remain elevated in both acute and chronic HCV patients, irrespective of cirrhosis. Despite small but significant changes, these levels are higher than those of healthy donors. For example, in chronic non-cirrhotic HCV patients, MCP1 levels change from p<0.001 to p<0.01 (Figure 4).
Comparative Analysis of SIM Dynamics Using Linear Models
We employed linear models to evaluate changes in SIM expression over time for each cohort. A negative estimate indicated a decrease in SIM expression, while a positive estimate suggested either an increase or stable expression. For almost all SIMs, the SARS-CoV-2 cohort 2 exhibited decreased density, while most SIMs in the HCV cohorts also showed a negative association over time (Figure 5).
Additionally, we fitted linear models for each time point to assess changing SIM expression in HCV cohorts compared to the SARS-CoV-2 cohort 2. At T1, SIM expression in all HCV cohorts mirrored that of the SARS-CoV-2 cohort. However, at T2 and T3, almost all SIMs in the HCV cohorts showed a positive association compared to SARS-CoV-2 cohort 2, indicating higher levels in HCV patients at these time points (Figure 6).
Association analysis of SIM and fatigue in SARS-CoV-2 cohorts
All SARS-CoV-2 patients underwent comprehensive fatigue screening and longitudinal evaluation (Supplementary figure S3). To identify associations between fatigue syndrome and the systemic inflammatory milieu (SIM), we performed a correlation analysis. Interestingly, none of the analyzed SIMs were directly associated with the fatigue scores collected during follow-up (p.adj > 0.05).