Inflammatory profiles are associated with long COVID up to 6 months after COVID-19 onset: A prospective cohort study of individuals with mild to critical COVID-19

Abstract

Background: After initial COVID-19, immune dysregulation may persist and drive post-acute sequelae of COVID-19 (PASC). We described longitudinal trajectories of cytokines in adults up to 6 months following SARS-CoV-2 infection and explored early predictors of PASC.

Methods: RECoVERED is a prospective cohort of individuals with laboratory-confirmed SARS-CoV-2 infection between May 2020 and June 2021 in Amsterdam, the Netherlands. Serum was collected at weeks 4, 12 and 24 of follow-up. Monthly symptom questionnaires were completed from month 2 after COVID-19 onset onwards; lung diffusion capacity (DLCO) was tested at 6 months. Cytokine concentrations were analysed by human magnetic Luminex screening assay. We used a linear mixed-effects model to study log-concentrations of cytokines over time, assessing their association with socio-demographic and clinical characteristics that were included in the model as fixed effects.

Results: 186/349 (53%) participants had ≥2 serum samples and were included in current analyses. Of these, 101/186 (54%: 45/101[45%] female, median age 55 years [IQR = 45-64]) reported PASC at 12 and 24 weeks after COVID-19 onset. We included 37 reference samples (17/37[46%] female, median age 49 years [IQR = 40-56]). In a multivariate model, PASC was associated with raised CRP and abnormal diffusion capacity with raised IL10, IL17, IL6, IP10 and TNFα at 24 weeks. Early (0-4 week) IL-1β and BMI at COVID-19 onset were predictive of PASC at 24 weeks.

Conclusions: Our findings indicate that immune dysregulation plays an important role in PASC pathogenesis, especially among individuals with reduced pulmonary function. Early IL-1β shows promise as a predictor of PASC.

Fig 1. Box and Whisker plots of serum cytokine levels in the study population at 9–12 and 21–24 weeks post-COVID.

Individuals were stratified by whether or not they reported post-acute sequelae of COVID-19 (PASC) at 12 (for measurements at 9–12 weeks) or 24 (for measurements at 21–24) weeks after COVID-19 onset. Each dot represents an individual coloured by initial severity of COVID-19. The mean value is plotted for each individual with multiple measurements within the binned time period. A Mann-Whitney U test was used to test if measurements between those with and without PASC were significantly different. Multiple testing correction was performed using the Bonferroni method and comparisons with family-wise error rate < 0.05 were marked with an “*”.

Fig 2. Multivariate linear regression analysis of factors associated with inflammatory marker concentrations at 9–12 weeks after COVID-19 onset.

For each inflammatory marker, we performed a mixed effects linear regression of various factors: characteristics present prior to COVID-19 onset, COVID-19-related factors, and post-COVID-19 related factors. Characteristics present prior to COVID-19 onset included sex, age (≥60 or <60 years old), body mass index (BMI) ≥ 30, and the presence of comorbidities (including cardiovascular disease, diabetes mellitus, chronic pulmonary disease or current cancer). COVID-19-related factors included the severity of initial COVID-19 disease, if dexamethasone was administered, and current (at 12 weeks) presence of post-acute sequelae of COVID-19 (PASC). Post-COVID-19 related factors included measurement of inflammatory markers within four weeks after SARS-CoV-2 vaccination. Statistically significant negative effects (associations with lower cytokine concentrations) are shown in red whilst positive effects (associations with higher cytokine levels) are shown in blue.

Fig 3. Multivariate linear regression analysis of factors associated with inflammatory marker concentrations at 21–24 weeks after COVID-19onset.

For each inflammatory marker, we performed a mixed effects linear regression of various factors: characteristics present prior to COVID-19 onset, COVID-19-related factors, and post-COVID-19 related factors. Characteristics present prior to COVID-19 onset included sex, age (≥60 or <60 years old), body mass index (BMI) ≥ 30, and the presence of comorbidities (including cardiovascular disease, diabetes mellitus, chronic pulmonary disease or current cancer). COVID-19-related factors included the severity of initial COVID-19 disease, if dexamethasone was administered, and current (at 24 weeks) presence of post-acute sequelae of COVID-19 (PASC). Post-COVID-19 related factors included measurement of inflammatory markers within four weeks after SARS-CoV-2 vaccination. Statistically significant negative effects (associations with lower cytokine concentrations) are shown in red whilst positive effects (associations with higher cytokine levels) are shown in blue.

Fig 4. Early (0–4 week) predictors of PASC and CRP/IL-6 levels at 21–24 weeks after COVID-19 onset.

Importance of different predictors (ordered from most [top] to least [bottom] important according to Shapley additive explanation (SHAP) values) on (a) individuals reporting PASC, level of (b) CRP and (c) IL-6 measurements at 21–24 weeks. Predictors include socio-demographic factors (i.e. age at infection, years, and sex), body mass index (BMI, kg/m²), presence/absence of comorbidities (i.e. cardiovascular disease, diabetes mellitus, chronic pulmonary disease and current cancer [malignancy]) and mean log-concentrations of inflammatory markers measured at 0–4 weeks after COVID-19 onset. For (a), each horizontal bar denotes the mean absolute SHAP value associated with the predictor. The larger the mean absolute SHAP value, the more important the covariate is in predicting the outcome of reporting PASC at 21–24 weeks. For (b) and (c), each point is the SHAP value for the corresponding predictor for each individual. The color of each point represents the value of the predictor for an individual. For instance, in (b), sCD14 levels at 0–4 weeks is the most important predictor for levels of CRP at 21–24 weeks, with high mean levels of sCD14 at 0–4 weeks expected to yield a higher level of CRP at 21–24 weeks.