Central and peripheral neurological manifestations have been extensively reported in patients with COVID-19 since early stages of the pandemic^2,3,4,6 and even after established widespread immunity by mass vaccination and prior infections^9,13. Neurological alterations in COVID-19 were initially attributed to neuroinfection by SARS-CoV-2. However, viral RNA is rarely detected in the CSF¹⁸, and post-mortem analysis has shown that SARS-CoV-2 is present at low viral load in the brain^19,20. In contrast, CSF analysis, neuroimaging and neuropathologic findings, as well as patient’s responses to immunotherapies^6,20,21, support the notion of immune-mediated neuropathogenesis triggered by viral infection or antigens and/or systemic inflammation. Data in our study and from previous observations further support this hypothesis.

It this study, several biomarkers of inflammation were assessed in the CSF and serum of patients with COVID-19 and neurological manifestations to identify possible mechanisms of neuropathogenesis. In general, these patients had increased serum levels of IL-13, IL-18, TNF-α, VILIP-1, TGF-α, and VEGF, which were associated with the systemic inflammation induced by SARS-CoV-2 infection. In turn, the CSF analysis revealed an association between the neurological involvement and the activation, regulation and function of immune and glial cells, CNS cytokine storm, and inflammasome activity, particularly in cases of encephalopathy and IND. In contrast, patients with isolated refractory headache showed limited laboratory changes in the CSF, suggesting that physiopathological mechanisms other than neurological or systemic inflammation may also play a role. Most individuals (85.4%) with isolated refractory headache showed an altered intracranial opening pressure on lumbar puncture (≥ 200 mmH₂O) and half of them had intracranial hypertension (≥ 250 mmH₂O)¹⁵. Thus, disturbances in CSF production and absorption may contribute to the manifestation of isolated refractory headache in COVID-19 in the absence of evidence of meningitis or cerebrovascular disease.

The CSF of COVID-19 patients, regardless of their neurological outcomes, showed increased levels of IL-2, IL-3, IL-6, IL-15, IL-25, IFN-α2, MCP-3/CCL7, eotaxin/CCL11, and GM-CSF as a common feature. Functional protein network analysis indicated that these factors are involved in JAK-STAT-controlled signaling pathways, and changes in their levels can affect several CNS functions, including neuronal survival and glial activation. Functional enrichment analysis showed that these factors are also associated with the differentiation, proliferation, and recruitment of blood-derived leukocytes, such as monocytes, lymphocytes and NK cells in neuroinflammatory processes, and with adverse outcomes in COVID-19.

Notably, the CSF and serum of neuro-COVID-19 patients presented unrelated profiles. Indeed, no serum profile was specifically associated with a neurological condition, reflecting both the small number of differentially expressed factors and their independence from neurological outcomes. The limited changes in serum likely resulted from the predominance of patients with mild to moderate respiratory symptoms. In contrast, the correlation analysis of CSF factors in neuro-COVID-19 revealed a cluster of upregulated mediators, including IL-1β, IL-2, IL-25, IFN-α2, CCL7, CCL11, CX3CL1, TGF-α, and TNF-α. This pattern corroborates the activation of neuroimmune responses, both innate and adaptive responses, which was supported by results in functional protein enrichment analysis, which indicated microglial activation, recruitment of peripheral immune cells, including T-cells, monocytes/macrophages, and NK cells, disruption of the BBB, and tissue repair. Upregulation of CX3CL1 and TGF-α in this CNS response axis also highlights neuron-glia interactions. Neurons represent a major source of CX3CL1 in the CNS, leading to microglial activation²², and also secrete TGF-α, which is involved in tissue remodeling and gliosis²³, therefore potentially influencing the balance between neuronal regeneration and neurotoxicity. However, it is not possible to determine whether this resulted from a common event triggering CNS response or from intricate regulatory pathways activated by the neurological insult.

The serum of patients with neuro-COVID-19 exhibited an overall increase in IL-13, IL-18, TNF-α, TGF-α, VEGF, and VILIP-1 levels. VILIP-1 is a calcium-sensor protein primarily expressed in the brain that is involved in regulating neuronal growth, survival, and synaptic plasticity; its release into the extracellular space due to disturbed calcium homeostasis has led to its use as a biomarker of neuronal injury^24,25. However, VILIP-1 expression is also expressed in the heart, liver, kidney, and lung²⁶, and CSF and serum VILIP-1 levels correlate weakly²⁵. VILIP-1 was significantly increased in the serum of COVID-19 patients without corresponding changes in CSF, suggesting that elevated serum VILIP-1 was linked to systemic inflammation rather than CNS injury. In contrast, BDNF, another factor regulating neuronal homeostasis, was reduced in the serum of patients with encephalopathy compared to patients with isolated refractory headache and IND. However, BDNF and its receptor are also expressed by lung cells, and it is abundant in peripheral blood and can be secreted by lymphocytes and monocytes²⁷. In severe/moderate COVID-19, patients present a transient reduction in serum BDNF levels compared to individuals with mild disease, which are restored during recovery²⁸. Although consecutive serum samples were not available in our study to assess BDNF kinetics, lower serum BDNF levels in patients with encephalopathy may reflect differences in respiratory involvement rather than neurological manifestations.

Neuro-COVID-19 patients displayed elevated blood levels of TGF-α and VEGF, and high CSF levels of GM-CSF. Although VEGF has angiogenic and protective effects on endothelial cells, it can promote BBB leakage by transiently affecting cell–cell adhesion^29,30. GM-CSF also influences the BBB disruption by stimulating the expression of proinflammatory cytokines, such as IL-6 and TNF-α, that downregulate adhesion molecules in cerebral microvasculature³¹. Thus, both CSF and blood factors may contribute to neurological alterations in COVID-19 by inducing endothelial activation, increased vascular permeability, and BBB disruption.

Patients with IND and encephalopathy had elevated CSF neopterin, in agreement with the literature^32,33, and half of these patients had a neopterin (CSF/serum) ratio > 1.0 [see the Additional file 3], indicating active CNS production rather than peripheral diffusion. Neopterin is secreted by activated monocytes/macrophages in response to IFN-γ³⁴ and is a biomarker of immune activation, and associated with CNS disturbances triggered by infections, autoimmune disorders, and primary CNS lymphoma^35,36. High CSF neopterin in IND patients corroborated the mononuclear pleocytosis, whereas patients with encephalopathy did not exhibit CNS infiltration by immune cells. Resident CNS cells, such as microglia and astrocytes, can also secrete neopterin in response to IFN-γ^37,38. Edén et al.³² reported that CSF neopterin and IFN-γ levels correlated with SARS-CoV-2 nucleocapsid antigen concentrations, which was detected even in the absence of viral RNA. In our study population, encephalopathy and IND were associated with neuronal injury and elevated levels of proinflammatory factors that can disturb the BBB^6,14. Therefore, systemic inflammation in COVID-19 and/or the presence of SARS-CoV-2 antigens in CNS likely influenced the development of encephalopathy and IND, given that active viral replication in the CNS was not detected. However, we were not able to assess SARS-CoV-2 nucleocapsid protein levels in CSF and serum samples to confirm this.

Patients with encephalopathy and IND had elevated CSF levels of HMGB1 and IL-18, in addition to IL-1β in cases of encephalopathy. This supports the involvement of inflammasomes in COVID-19-associated neuroinflammation. HMGB1 can be passively released after cell death or actively secreted by stimulated cells upon triggering of NLRP3 inflammasomes, which finally results in IL-1β and IL-18 secretion^39,40. Although the exact source of elevated HMGB1 in the CSF of these patients cannot be specified, it is likely that neuronal damage has contributed to its increase. Patients with IND had elevated CSF NfL—a marker of injury to large myelinated axons—especially in cases with meningeal involvement⁶. Moreover, HMGB1 mediates acute and chronic inflammation by recruiting cells to sites of injury and infection and stimulating macrophages and endothelial cells to produce cytokines, particularly IL-1β, IL-6, TNF-α, and type I IFNs^40,41, which were elevated in the CSF of patients with encephalopathy and IND. Patients with IND also had higher levels of IL-2, IL-6, TNF-α, CXCL8, and CXCL10 in the CSF than in serum, indicating that neuroinflammation was driven by CNS-intrinsic events rather than a secondary response to systemic inflammation. In an experimental coronavirus neuroinfection model, microglia were identified as the main source of the CNS cytokine storm. Additionally, type I astrocytes can also contribute to neuroinflammation by upregulating IL-1α/β, IL-2, IL-7, IL-13, IL-15, IL-17, IL-18, and IFNs^42,43. Collectively, these findings indicate that CNS resident cells such as microglia and astrocytes play a major role in the development of acute neurological syndromes in COVID‑19, while infiltration by peripheral mononuclear immune cells exacerbates neuroinflammation. Results from functional protein network analysis also support this by indicating the enrichment of processes associated with microglial functions and chemotaxis, proliferation, activation, and cytokine production in various peripheral blood mononuclear cell types. Similar to findings in IND, individuals with encephalopathy exhibited a marked upregulation of multiple proinflammatory mediators, indicative of a cytokine storm and neuroinflammation. However, the lack of signs of acute brain inflammation, such as CSF pleocytosis and changes in brain magnetic resonance imaging (MRI), suggests a temporal sequence in which neurological manifestations arise from CNS response to preceding systemic inflammation, rather than from primary CNS involvement, which defined the IND.

Many factors upregulated in the CSF in encephalopathy and IND cases were associated with Th1 responses, which is typically induced by viral infections. However, the enrichment of factors associated with Th2 responses was also seen. In isolated headache and encephalopathy, IL-25, an anti-inflammatory cytokine of the IL-17 family, was elevated. In the CNS, IL-25 helps to prevent inflammatory cell infiltration and modulates BBB repair, and regulates Th2 cytokine production, as shown in murine models^44,45. This corroborates the self-limited neuroinflammation observed in patients in our study, who experienced spontaneous recovery or symptom resolution after corticotherapy or intravenous immunoglobulin⁶, as also reported elsewhere^2,3,32. Additionally, the CSF of patients with encephalopathy and IND had increased β-NGF, a growth factor promoting neuronal survival and plasticity⁴⁶, and encephalopathy patients also had elevated EGF, a factor supporting differentiation, maturation, and survival of multiple cell types, including neurons⁴⁷, further indicating ongoing neural recovery responses.

However, our findings may differ in the context of current SARS-CoV-2 variants or vaccinated populations, since study participants were enrolled in 2020, before the emergence of Omicron and COVID-19 vaccination. Omicron variant has been shown to be more transmissible and infectious than previous SARS-CoV-2 variants but causing milder unspecific and respiratory symptoms⁴⁸. In contrast, Omicron can also trigger new‐onset neurological conditions or worsen pre‐existing neurological diseases, and para-infectious cases of neuroimmune complications, including Guillain-Barré syndrome, spinal meningitis/myelitis, neuromyelitis optica, ADEM, autoimmune encephalitis, and encephalopathy have been described yet^49,50,51. Unfortunately, changes in the prevalence of acute neurological complications associated with Omicron and pre-Omicron variants are still unknown. However, the prevalence of post-infectious conditions such as long COVID showed a reduction after infection with Omicron lineages¹³. In addition, full vaccination has been also related to a reduced risk for cerebrovascular diseases⁵². Indeed, the humoral response (IgG and IgA) induced by a second booster dose of COVID-19 vaccines showed a protective effect on neurological symptoms, such as brain fog, sleep quality, impaired coordination, and physical pain⁵³, and at least one vaccine dose had a protective effect against long COVID⁵⁴. Protection against neurological manifestations conferred by immunization has been associated with short-term and long-term suppression of cytokine storm responses upon SARS-CoV-2 infection, with lower levels of inflammatory factors such as IL-2RA, IL-7, IL-8, IL-15, IL-29/IFN-λ, IP-10/CXCL10, CCL2/MCP-1, and TNF-α compared to unvaccinated counterparts⁵⁵. This highlights the importance of ongoing immunization programs in reducing the frequency and severity of neurological manifestations in COVID-19.

This study had limitations, including a small sample size, the lack of consecutive CSF and serum samples, and limited follow-up after discharge. Our study was also not sufficiently powered to formally adjust for confounding variables such as hypertension, diabetes, and cardiovascular disorders. These pre-existing conditions contribute to systemic inflammation, endothelial dysfunction, and the compromise of BBB, predisposing individuals to severe neurological manifestations in COVID-19 by exacerbating the cytokine storm and immune-mediated damage within the CNS^2,56. The exploratory design also limited the evaluation of biomarkers with potential prognostic value. However, strengths include a well-characterized cohort, comprehensive investigation of multiple factors related to inflammation, neuronal damage, and CNS homeostasis in paired CSF and serum samples, evaluation of a diverse range of neurological outcomes, and inclusion of non-hospitalized patients with mild COVID-19 as well as uninfected controls. Consequently, our data allowed us to identify mechanisms likely associated with distinct neurological manifestations in acute COVID-19.