Central nervous system and systemic inflammatory networks associated with acute neurological outcomes in COVID-19

Abstract

COVID-19 is associated with a wide spectrum of neurological alterations, ranging from headache and dizziness to severe encephalopathy and inflammatory neurological diseases (IND), and neuropathological findings suggest immune-mediated processes. Therefore, we sought to characterize profiles of cytokines, chemokines, growth factors, and markers of central nervous system (CNS) homeostasis in COVID-19 patients with neurological alterations to identify key factors and mechanisms underlying CNS disturbances in COVID-19. The study included a case series of 52 COVID-19 patients with neurological manifestations, which were categorized into three groups: isolated refractory headache (n = 14), encephalopathy (n = 24), and IND (n = 14). Individuals with non-inflammatory, non-infectious neurological conditions (n = 9) were included as negative controls. Paired CSF and serum samples were assessed for 56 biomarkers. Regardless of the neurological condition, COVID-19 patients exhibited elevated CSF levels of proinflammatory mediators, including IL-2, IL-3, IL-6, IL-15, IL-25, IFN-α2, CCL7, CCL11, and GM-CSF. Patients with encephalopathy and IND also showed increased IL-1β, IL-18, TNF-α, neopterin, IL-7, CXCL8, CXCL9, TGF-α, EGF, sTREM-2, and HMGB1, consistent with a CNS cytokine storm. In contrast, individuals with isolated refractory headache showed a modest inflammatory profile, compatible with the limited CNS involvement. COVID-19 patients showed elevated serum IL-13, IL-18, TNF-α, VILIP-1, TGF-α, and VEGF levels, indicating systemic inflammation and potential blood-brain barrier (BBB) disruption. β-NGF was increased in the CSF of patients with encephalopathy and IND, suggesting the activation of neuroprotective responses during patient recovery. Functional protein network analysis showed a significant enrichment of interactions between factors altered in the CSF of patients with encephalopathy and IND, many of them related to processes of neuroinflammation and microglial functions, and leukocyte chemotaxis, activation and proliferation. These findings support a model in which both systemic immune activation and localized neuroinflammation contribute to the diversity of neurological outcomes observed in COVID-19, and dysregulated cytokine production, glial activation, inflammasome activity and BBB disturbances represent key factors in neuro-COVID-19 pathogenesis.

Fig. 1

CSF analysis in COVID-19 patients with acute neurological disturbances. CSF samples from COVID-19 patients with isolated refractory headache (H) (n = 14), encephalopathy (E) (n = 24), and inflammatory neurological diseases (IND) (n = 14), and from a control group of individuals with non-inflammatory, non-infectious neurological diseases (NI) (n = 9) were assessed by Luminex (MILLIPLEX MAP Human Cytokine/Chemokine/Growth Factor Panel A, Merck Millipore), Cytometry bead array (LEGENDplex Human Neuroinflammation Panel 1, Biolegend), and ELISA. Concentration values in pg/mL were log₁₀-transformed and are presented as median and interquartile range (IQR). Comparative analysis was performed with the Kruskal–Wallis test and posthoc analysis using Dunn’s test with Benjamini–Hochberg method for adjusting p-values in multiple comparisons. Results of proinflammatory factors (IL-1β, IL-6, TNF-α, IL-15, IL-25, IL-18, IFN-α2, and neopterin), regulatory cytokines (IL-4 and IL-5), chemokines (MIP-1α/CCL3, MCP-3/CCL7, eotaxin/CCL11, IL-8/CXCL8, MIG/CXCL9, and fractalkine/CX3CL1), growth factors (IL-2, IL-3, IL-7, FLT3L, EGF, GM-CSF, β-NGF, TGF-α, and G-CSF), and markers of cellular stress response (HMGB1 and sTREM-2) that presented significant changes (Kruskal–Wallis p < 0.05) are shown. Summary data for all factors evaluated are presented in additional graphs and tables [see Additional file 1 and Additional file 2]. *, adj. p < 0.05; **, adj. p < 0.01.

Fig. 2

Serum analysis in COVID-19 patients with acute neurological disturbances. Paired serum samples from COVID-19 patients with isolated refractory headache (H) (n = 7), encephalopathy (E) (n = 13), and inflammatory neurological diseases (IND) (n = 11), and from a control group of individuals with non-inflammatory and non-infectious neurological diseases (NI) (n = 5) were assessed by Luminex (MILLIPLEX MAP Human Cytokine/Chemokine/Growth Factor Panel A, Merck Millipore), Cytometry bead array (LEGENDplex Human Neuroinflammation Panel 1, Biolegend), and ELISA. Concentration values in pg/mL were log₁₀-transformed and are presented as median and interquartile range (IQR). Comparative analysis was performed with the Kruskal–Wallis test and posthoc analysis using Dunn’s test with Benjamini–Hochberg method for adjusting p-values in multiple comparisons. Results of proinflammatory factors (IL-6, TNF-α, and IL-18), regulatory cytokines (IL-10, IL-13, and IL-27), growth factors (TGF-α, VEGF, and BDNF), and markers of cellular stress response (VILIP-1) that presented significant changes (Kruskal–Wallis p < 0.05) are shown. Summary data for all factors evaluated are presented in additional graphs and tables [see Additional file 1 and Additional file 2]. *, adj. p < 0.05; **, adj. p < 0.01.

Fig. 3

CSF and serum profiles of neuro-COVID-19 patients. Circular boxplots display data from CSF and serum analyses for proinflammatory and regulatory cytokines, chemokines, growth factors, and markers of cellular stress response in COVID-19 patients with isolated refractory headache (H), encephalopathy (E), and inflammatory neurological diseases (IND), and in a control group of individuals with non-inflammatory and non-infectious neurological diseases (NI). Each bar represents the proportion of individuals with concentrations above the median determined for each factor in the (a–d) CSF and (e–h) serum. Proportions are shown as gray (up to 50%), light gray (50–75%), and white (75–100%).

Fig. 4

Heatmap analysis of CSF factors differentially expressed in neuro-COVID-19 patients. Data from patients with isolated refractory headache (H), encephalopathy (E), and inflammatory neurological diseases (IND), and from a control group of individuals with non-inflammatory, non-infectious neurological diseases (NI) were log₁₀-transformed and normalized for each factor (columns). Reduced expression is depicted in shades of blue, increased expression in shades of red, with the median represented by white. Heatmaps were generated with the R software v.4.4.2 (https://cran.r-project.org/).

Fig. 5

Heatmap analysis of serum factors differentially expressed in neuro-COVID-19 patients. Data from patients with isolated refractory headache (H), encephalopathy (E), and inflammatory neurological diseases (IND), and from a control group of individuals with non-inflammatory, non-infectious neurological diseases (NI) were log₁₀-transformed and normalized for each factor (columns). Reduced expression is depicted in shades of blue, increased expression in shades of red, with the median represented by white. Heatmaps were generated with the R software v.4.4.2 (https://cran.r-project.org/).

Fig. 6

Correlation analysis of CSF factors differentially expressed in neuro-COVID-19 patients. Spearman’s rank correlation coefficients were calculated for factors that were significantly different in at least one study group compared to uninfected controls, and that had detectable levels in at least 50% of samples from patients with (a) isolated refractory headache, (b) encephalopathy, (c) inflammatory neurological diseases (IND), and (d) a control group of individuals with non-inflammatory, non-infectious neurological diseases. Clustering analysis delineated patterns associated with each neurological outcome. Reduced expression is depicted in shades of blue, increased expression in shades of red, with the median represented by white. Results were considered significant at p < 0.05. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 7

Functional protein network analysis of CSF factors differentially expressed in neuro-COVID-19 patients. Factors significantly altered in the cerebrospinal fluid (CSF) according to neurological outcomes—isolated refractory headache (H), encephalopathy (E), and inflammatory neurological diseases (IND)—were analyzed using the STRING database, with a high confidence interaction score (0.700). The analysis included Gene Ontology (GO) terms, subcellular localization data from the Compartments database (GOCC identifiers), Reactome pathways (HSA identifiers), and WikiPathways (WP identifiers). Biological functions and processes with significant enrichment (FDR test, p-value < 0.05) are shown. The protein ratio represents the proportion of proteins in the query relative to the total number associated with a given biological process.

Fig. 8

Functional protein network analysis of serum factors differentially expressed in neuro-COVID-19 patients. Serum factors significantly altered in patients according to neurological outcomes—isolated refractory headache (H), encephalopathy (E), and inflammatory neurological diseases (IND)—were analyzed using the STRING database, with a high confidence interaction score (0.700). The analysis included Gene Ontology (GO) terms, subcellular localization data from the Compartments database (GOCC identifiers), Reactome pathways (HSA identifiers), and WikiPathways (WP identifiers). Biological functions and processes with significant enrichment (FDR test, p-value < 0.05) are shown. The protein ratio represents the proportion of proteins in the query relative to the total number associated with a given biological process.