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SARS-CoV-2 encephalitis is a cytokine release syndrome: evidences from cerebrospinal fluid analyses

Abstract Background Recent findings indicated that SARS-CoV-2 related neurological manifestations involve cytokine release syndrome along with endothelial activation, blood brain barrier dysfunction, and immune‐mediated mechanisms. Very few studies have fully investigated the CSF correlates of SARS-CoV-2 encephalitis. Methods Patients with PCR-confirmed SARS-CoV-2 infection and encephalitis (COV-Enc), encephalitis without SARS-CoV-2 infection (ENC) and healthy controls (HC) underwent an extended panel of CSF neuronal (NfL, T-tau), glial (GFAP, TREM2, YKL-40) and inflammatory biomarkers (IL-1β, IL-6, Il-8, TNF- α, CXCL-13 and β2-microglobulin). Results Thirteen COV-Enc, 21 ENC and 18 HC entered the study. In COV-Enc cases, CSF was negative for SARS-CoV-2 real-time PCR but exhibited increased IL-8 levels independently from presence of pleocytosis/hyperproteinorracchia. COV-Enc patients showed increased IL-6, TNF- α, and β2-microglobulin and glial markers (GFAP, sTREM-2, YKL-40) levels similar to ENC but normal CXCL13 levels. Neuronal markers NfL and T-Tau were abnormal only in severe cases. Conclusions SARS-CoV-2-related encephalitis were associated with prominent glial activation and neuroinflammatory markers, whereas neuronal markers were increased in severe cases only. The pattern of CSF alterations suggested a cytokine-release syndrome as the main inflammatory mechanism of SARS-CoV-2 related encephalitis.


INTRODUCTION
Coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is characterized predominantly by lower respiratory tract involvement. In addition to its pulmonary manifestations, growing evidence showed encephalitis as a possible manifestation of the disease [1][2][3]. While the number of reported cases of encephalitis in COVID-19 is rapidly increasing, the question whether SARS-CoV-2 may cause neurological manifestations through a direct neuropathic effect or by promoting a hyperinflammatory reaction in the host's immune system in the form cytokine release syndrome is still a theme of debate [1,4,5].
Cytokine release syndrome (CRS) is a potentially fatal complication of various infectious (e.g. influenza, SARS, Epstein−Barr virus) and noninfectious diseases (e.g. multiple organ dysfunction syndrome, multiple sclerosis) and is triggered by an initial release of cytokines able to activate bystander immune cells and endothelial cells to produce proinflammatory molecules. CRS-based neurological disturbances have been also recently described following CAR-T cell therapy and are termed Immune effector cell-associated neurotoxicity syndrome (ICANS).
Disproportionately high concentrations of IL-6 and IL-8 and TNF alpha have been found in the CSF of patients with severe CRS and ICANS neurotoxicity, thought to be due to the combination of increased barrier permeability and intrathecal production by activated myeloid, astrocyte, and/or endothelial cells. According to this, glial cell activation has been A c c e p t e d M a n u s c r i p t 6 recently pointed out as one of most sensitive alterations leading to neuroinflammation in ICANS and CRS [11][12][13].
According to these findings, we aimed to investigate CSF abnormalities in SARS-CoV-2 related encephalitis in order to confirm the hypothesis that neurologic involvement during COVID-19 is due to cytokine release syndrome [4,11,14].
To this end, we examined CSF samples from patients with SARS-CoV-2-related encephalitis (Cov-Enc) using an extensive panel of cytokines/chemokines and neuronal/glial biomarkers, contrasting them with healthy controls (HC) and encephalitis not associated with COVID-19 infection (ENC).

Patients
The study was carried out at ASST Spedali Civili and ASST Cremona hospitals between February 20 th and 30 th June 2020 including COVID-19 patients consecutively admitted fulfilling criteria for encephalitis according to a full screening protocol [15]. The case definition for COV-Enc included any person with confirmed SARS-CoV-2 infection aged 18 admitted to hospital with altered mental status lasting ≥24 hours and the presence of two or more of the following criteria: i) generalized or partial seizures not fully attributable to a pre-existing epilepsy ii) new onset of focal neurologic findings iii) CSF white blood cell count ≥5/cubic mm 3 iv) abnormality of brain parenchyma on neuroimaging suggestive of encephalitis that was either new from prior studies or appears acute in onset v) abnormality on electroencephalography consistent with encephalitis. Fever was not considered as supportive feature for encephalitis (as indicated by standard criteria [15], as it is highly prevalent in COVID-19 disease. Laboratory confirmation of SARS-CoV-2 infection was

Encephalitis assessment and diagnosis
First-line testing included all commonly recognized causes of encephalitis according to current guidelines [15,16] . Each COV-Enc underwent brain magnetic resonance imaging, standard serum at 37 °C with 5% CO2; used for the inoculation of samples [17].
The illness severity of COVID-19 was defined according to the Brescia COVID Respiratory Severity Scale (BCRSS) for COVID-19 [18] and according to the quick Sequential Organ Failure Assessment (qSOFA) [19]. The severity of neurological symptoms was defined according to the ICANS grading proposed by Lee and coauthors [13]. immunoassay on an Atellica instrument (Siemens Healthcare GmbH, Erlangen, Germany). CSF total tau (T-tau) concentration was measured by Lumipulse (Fujirebio, Ghent, Belgium). CSF NfL and GFAP concentrations were measured using in house enzyme-linked immunosorbent assays [20,21]. CSF sTREM2 concentration was measured using an in-house immunoassay with electrochemiluminescent detection, as previously described in detail [22]. CSF YKL-40 concentration was measured using the Human Chitinase 3-like 1 Quantikine kit (R&D Systems, Minneapolis, MN).
All analyses were performed by board-certified laboratory technicians who were blinded to clinical data.

Data availability
All clinical and CSF analyses data are available from authors upon reasonable request.

Clinical characteristics of COVID-19 related encephalitis
The study recruited thirteen cases of SARS-  showing generalized slow waves prominent on the frontal derivations in ten patients, while focal epileptic alteration was observed in three cases.
Brain imaging was normal in ten cases whereas three patients showed heterogeneous MRI alterations including multiple subcortical/cortical T2-hyperintensities, associated in one case to diffusion weighted imaging (DWI) hyperintensities (

CSF analyses
CSF biochemical standard analyses showed mild pleocytosis (5 to 26 cells) in nine patients and increased protein levels in eight COV-Enc patients (Tables 1 and 2).

RT-PCR for SARS-CoV-2 was negative in the CSF of all COVID-19 patients. In four additional samples
the virus culture showed no specific cytopathic effect after three days of inoculation (see methods for details).
Compared with HC, COV-Enc showed normal CXCL13 levels, significantly higher CSF levels of neuronal damage markers such as NfL and total tau, as well as glial-related markers such as GFAP, TREM2 and YKL-40, higher concentration of cytokines such as IL-1β, IL-6, IL-8, TNF-α (p<0.001 for all, Supplementary Table 1). COV-Enc exhibited comparable neuronal, glial and inflammatory markers but normal CXCL13 levels (p=0.026) compared to encephalitis not associated with SARS-CoV-2 infection (infectious and autoimmune, Figure   1 and supplementary Table 1).
All Cov-Enc cases showed increased IL-8 CSF levels, whereas eleven showed increased beta-2 microglobulin. Glial markers, namely GFAP and sTREM2, were abnormal in twelve and ten COV-Enc patients, respectively. Tau and NfL levels were abnormal in five and six A c c e p t e d M a n u s c r i p t 11 subjects with higher considering age-adjusted cut-offs [20]. Three out of four cases without pleocytosis exhibited increased glial markers, whereas all showed abnormal IL-8 CSF levels (table 2).

DISCUSSION
The present study demonstrated that SARS-CoV-2 encephalitis are associated with early IL-8 increases and glial alterations whereas neuronal damage markers were elevated in severe cases.
Furthermore, the pattern of neuroinflammatory markers assessed is highly suggestive for a cytokinerelease syndrome as driver of SARS-CoV-2 related encephalitis.

The sample included thirteen hospitalized patients affected by SARS-CoV-2 infection fulfilling
diagnostic criteria for probable encephalitis [15]. Clinically, most patients presented with early language disturbances associated with altered mental status, in line with clinical features exhibited by most ICANS cases [11]. The CSF biochemical analyses showed mild increased protein levels indicating a blood-brain barrier damage and also mild pleocytosis. Patients had negative infectious and neuronal antibodies screening and tested negative for the presence of SARS-CoV-2 virus in CSF.
Conversely, CSF neuronal damage markers appeared to be elevated in CSF compared to controls and similar to non COVID-19 encephalitis. ENC-Cov severe patients specifically exhibited very high levels of NfL, an axonal structural protein and a biomarker of neuronal injury, recently observed also in a subset of SARS-CoV-2 related encephalopathies [23]. In addition to this, COV-Enc showed increased CSF levels of markers of activation and damage of astrocytes and microglia, such as GFAP, TREM2 and YKL-40, arguing for a strong neuroinflammatory response similar to cases of non COVID-19 encephalitis. Specifically, we observed increased CSF levels in the membrane-bound receptor TREM-2, a known marker of microglia activation, and modulator of neuroinflammation in both chronic and acute CNS disorders [24] . COV-Enc showed also abnormal levels of YKL-40, a glycoprotein produced A c c e p t e d M a n u s c r i p t 12 by reactive astrocytes and microglia during neuroinflammation in different primary CNS diseases and encephalitis [25]. Of note, YKL-40 release could be activated by increased level of IL-6 and TNF-α [26], and thus plays an important role in CRS together with GFAP, an intermediate filament highly expressed in astrocytes [12]. Indeed, alterations within GFAP expression indicating involvement of astrocytes have been recently described widespread in subcortical and cortical regions in severe cases of SARS-CoV-2 infection [27]. GFAP increased levels have been also identified as early alterations of ICANS, when CRS was induced by T-cell therapy [11]. Of note, increased GFAP levels were presented by all but one COV-Enc patient, even in absence of pleocytosis or hyperproteinorrachia.
The analysis of CSF in COV-Enc patients additionally revealed increased level of several cytokines, consistent with an acute neuroinflammatory response and blood-brain barrier disruption [28].
Specifically, we observed a significant increase of CSF levels of IL-6, IL-8, β2 an NF-α *28, 29+ whereas levels of the chemokine CXCL13 were normal [31] at variance with other forms of encephalitis. In absence of CSF evidences of autoantibodies or SARS-CoV-2 virus, the normal levels of CXCL13 found in COV-ENC atients th s ar e a ainst a " lassi al" ire t in e tio s as ell as autoimmune based mechanisms in the pathogenesis of SARS-CoV-2-related encephalitis Additionally, COV-Enc exhibited very high levels of IL-8, a cytokine already associated with several inflammatory alterations but specifically high in both ICANS [11] and CRS complications in SARS-CoV-2 infection [32].
Thus, the biomarkers/cytokine alterations observed strongly support the claim of an inflammatoryinduced mechanism causing SARS-CoV-2 encephalitis. This fits well with the risk of cytokine-induced syndrome in COVID-19 disease [10] and with clinical presentations observed after immune effector associated treatment [14]. This pathophysiology, supported by our extensive CSF data, could fit with the majority of encephalitis reported in the literature with a disease onset concomitant the respiratory infection [1][2][3][4][5]9,10] A c c e p t e d M a n u s c r i p t

13
The close known relationship between CRS and neurotoxicity suggests that systemic inflammatory mediators act directly across the blood-brain barrier with neuroinflammation induction via glial and astrocyte activation [12]. This can lead to secondary neuroinflammatory-mediated encephalitis highlighted by positive axonal injury biomarkers and stronger cytokine/interleukin changes in CSF than serum.
These findings have deep implications for the management of patients presenting with encephalitis related to SARS-CoV-2 infection, as high-dose corticosteroid treatment (i.e. methylprednisolone 1 g/day for five days) is the first-line therapy for severe neurotoxicity in CRS cases [13] after exclusion of co-infections-whereas additional immunomodulatory treatment could be proposed according to single case-specific cytokine profile.
We acknowledge that this study entails several limitations. First, due to the absence of autopsy, our ases an be lassi ie as " robabl asso iate " ith S RS-CoV-2 infection   M a n u s c r i p t