key: cord-0964605-5cltw11t
authors: Remsik, J.; Wilcox, J. A.; Babady, N. E.; McMillen, T.; Vachha, B. A.; Halpern, N. A.; Dhawan, V.; Rosenblum, M.; Iacobuzio-Donahue, C. A.; Avila, E. K.; Santomasso, B.; Boire, A.
title: Inflammatory leptomeningeal cytokines mediate delayed COVID-19 encephalopathy
date: 2020-09-18
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2020.09.15.20195511
sha: 7b3833e01597ed84121ebe87d8d74e84552947f2
doc_id: 964605
cord_uid: 5cltw11t

SARS-CoV-2 infection induces a wide spectrum of neurologic dysfunction. Here we show that a particularly vulnerable population with neurologic manifestations of COVID-19 harbor an influx of inflammatory cytokines within the cerebrospinal fluid in the absence of viral neuro-invasion. The majority of these inflammatory mediators are driven by type 2 interferon and are known to induce neuronal injury in other disease models. Levels of matrix metalloproteinase-10 within the spinal fluid correlate with the degree of neurologic dysfunction. Furthermore, this neuroinflammatory process persists weeks following convalescence from the acute respiratory infection. These prolonged neurologic sequelae following a systemic cytokine release syndrome lead to long-term neurocognitive dysfunction with a wide range of phenotypes.

Emerging hospital series demonstrate that acute respiratory infection with SARS-CoV-2 is frequently associated with neurologic dysfunction. Mild neurologic symptoms, including headaches, early anosmia and dysgusia, occur in a large portion of the infected population and typically resolve 1 . More serious complications, such as protracted delirium, seizures, and meningoencephalitis, appear to afflict more critically ill patients with hypoxic respiratory failure and may be devastating to higly susceptible individuals 2 . Cancer patients, in particular, are at heightened risk of severe infections from COVID-19 due to their baseline immunocompromised state and poor functional reserve 3 . The mechanism by which COVID-19 impacts the CNS is unclear, with hypotheses including direct viral neuroinvasion, neurologic toxicity from the systemic cytokine release syndrome, or a combination of both. Investigations to date lack consensus regarding neuroinvasion potential of COVID-19, with detectable SARS-CoV-2 virus present within the CSF in only a small number of patients with neurologic toxicity 2,4-6 . Small case reports of 1 to 3 patients have reported an elevation in pro-inflammatory cytokines, such as IL-6, CXCL10 (also known as IFN-induced protein-10), and CCL2, in the spinal fluid of acutely infected individuals [6] [7] [8] Between May and July 2020, we prospectively evaluated 18 cancer patients with confirmed SARS-CoV-2 respiratory infection who subsequently developed moderate to severe neurologic symptoms ( Table 1 and Supplementary Table 1) . COVID-19 nasopharyngeal swab was positive in 16 patients; two additional patients were included on the basis of a positive serum COVID-19 antibody screen and recent respiratory illness consistent with COVID-19 (Supplementary Table 2 ). All patients presented with classic features of a COVID-19 respiratory infection including cough, dypsnea and fever; eleven patients experienced severe hypoxic respiratory failure and cytokine release syndrome requiring mechanical ventilatory support (Supplementary Table 3 ). Patients displayed a wide range of neurologic manifestations, including prolonged hyperactive or hypoactive delirium (n = 10), limbic encephalitis (n = 4), refractory headaches (n = 2), rhombencephalitis (n = 1), and large-territory infarctions (n = 1). There was a delay of 19 days (median, range 0 -77 days) between the onset of respiratory infection and clincal documentation of neurological symptoms.

In addition to bedside neurologic examination, standard neurologic testing included neuroimaging in the form of brain MRI (n = 14) or head CT (n = 4) ( Table 1 and Extended Data Fig. 1 ). Three patients demonstrated encephalitic changes in the form of non-enhancing T2hyperintense white matter and cortical changes afflicting the limbic or cerebellar structures.

Large territory infarcts, diffuse microhemorrhages, and increased subcortical or periventricular white disease were also evident. Electroencephalography (Table 1) Table 4 ). Notably, cell count, protein, and glucose levels were normal; only 2 patients had measurable pleocytosis due to metastatic disease to the CNS. CSF immune cell differentials were lymphocyte-predominant in 76.9% and monocyte-predominant in 23.1% of patients. Oligoclonal bands were detected in both the serum and CSF in 83% of tested patients, indicating systemic rather than intracerebral production of gammaglobulins. Only two patients had elevated intracranial pressure and abnormal protein levels, a sequela attributed to known brain metastases rather than COVID-19 neurologic toxicity.

To characterize COVID-19 neuroinvasion within this cancer population, we optimized a PCR-based assay for the CSF and used a commercially available ELISA to test for SARS-CoV-2 viral RNA and N and S structural proteins, respectively, in the CSF. No patients demonstrated detectable levels of viral PCR or structural proteins in the CSF, supporting prior reports 4, 9, 10 . We also tested the levels of ACE2, the SARS-CoV-2 receptor, within the CSF-brain and CSF-blood barriers in banked pre-pandemic cerebral tissues. We detected low levels of ACE2 in the intravascular monocytes and capillary endothelium within both the choroid plexus and leptomeninges, representing potential means of viral entry into the leptomeninges (Extended compare with other well-characterized neuro-inflammatory syndromes in cancer patients, the second group consisted of patients with CAR T cell-associated neurotoxicity, also known as ICANS 12 (Immune Effector Cell Associated Neurotoxicity Syndrome), and the third group with autoimmune encephalitis (idiopathic n = 3 and suspected immune checkpoint inhibitorassociated n = 3) 13, 14 . Correlative analysis across these 4 cohorts identified a significant accumulation of 12 inflammatory mediators in the CSF of COVID-19 patients, at levels approaching that of patients with severe CAR T-associated neurotoxicity ( Fig. 1A and Extended Data Fig. 3) . These included IL-6 and -8, IFN-γ, CXCL-1, -6, -9, -10, and -11, CCL-8 and -20, MMP-10 and 4E-BP1. Using an inflammatory signature comprising these 12 proteins, we also found a marked cumulative increase in inflammatory signalling relative to the non-COVID-19 cancer controls (p = 0.029, Fig. 1B) . These 12 factors associate with cytokine and chemokine signaling, immune cell function, senescence, and neuroinflammation (Extended Data Fig. 4) . We queried publicly available peripheral immune cell scRNA-sequencing data 15 from patients with mild and severe COVID-19 infections to demonstrate that the majority of these factors appear to be absent peripherally and are likely isolated to the CNS compartment and studies to date have not identified any obvious meningitic, ischemic, or ictal cause. This is the first series to demonstrate an increase in pro-inflammatory CSF cytokines in such patients in the weeks following respiratory illness. Similar to other encephalidities, these cytokines may result in neuronal damage in the absence of obvious radiographic abnormalities.

Dexamethasone, a potent steroid and immunosuppressant, is the only intervention that has been shown in large-scale studies to reduce the incidence of death in hospitalized patients with severe COVID-19 infections 24 . The impact of dexamethasone on COVID-19 related neurologic toxicity, specifically, has not been defined. Our data demonstrate that the neurologic toxicity associated with COVID-19 is biochemically similar to CAR T cell neurotoxicity. Clinical rubrics for the grading and management of CAR T cell neurotoxicity support both early diagnosis and use of anti-inflammatory agents including dexamethasone 25-27 .

In conclusion, our data indicates that neurologic syndromes associated with moderate and severe COVID-19 infections are associated with a wide range of intracranial inflammatory cytokines, a finding that may persist for weeks to months following convalescence of the respiratory syndrome. The global accumulation of inflammatory mediators in CSF and inability to detect COVID-19 in the CSF suggests that the identification of a single, universal COVID-19 CSF biomarker is unlikely to be successful. However, the correlation of MMP-10 with neurologic dysfunction marks a potential prognostic biomarker for future study. Moreover, our findings further justify the investigation of early neurologic assessments, such those used for monitoring ICANS 12 , and use of anti-inflammatory therapies in patients with severe or prolonged COVID-19 disease.

Abbreviations. ACE2 -angiotensin-converting enzyme 2; AIE -autoimmune encephalitis; CAR T -chimeric antigen receptor T cell; CCL -C-C motif chemokine ligand; CRP -C-reactive protein; CSF -cerebrospinal fluid; CXCL -C-X-C motif chemokine ligand; DRS -disability rating scale; EEG -electroencephalography; ELISA -enzyme-linked immunosorbent assay;

GRDA -generalized rhythmic delta activity; ICAN -Immune Effector Cell-Associated Neurotoxicity Syndrome; IFN -interferon; IL -interleukin; KPS -Karnofsky performance status; LP -lumbar puncture; MRI -magnetic resonance imaging; MMP-10 -matrix metallopeptidase-All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.15.20195511 doi: medRxiv preprint 10; MCP2 -monocyte chemoattractant protein-2; PCR -polymerase chain reaction; PDRposterior dominant rhythm.

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The copyright holder for this this version posted September 18, 2020. were detected and negative if both targets were not detected. N and S (S2 subunit) SARS-CoV-2 structural proteins were detected using commercially available ELISA kits (ELV-All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.15.20195511 doi: medRxiv preprint COVID19N-1 and ELV-COVID19S2-1, RayBiotech), as recommended by the manufacturer. All ELISA samples were run in technical replicates.

Imaging studies were conducted either on 1.5 or 3 Tesla MRI.

Images were obtained as part of the patient's routine standard of care imaging, which did not allow standardization of sequences. The most frequently sequences performed were diffusionweighted imaging (DWI), susceptibility-weighted angiography (SWAN), 2D fluid-attenuated inversion recovery (FLAIR), T2-weighted fast spin-echo and T1-weighted fast spin-echo MRI before and after administration of gadolinium-based contrast agent. MRI parameters of the most commonly used sequences in this study are tabulated in Supplementary Table 7 .

Electroencephalography. Long-term and routine electroencephalography monitoring with video was performed using a Natus 32 channel computerized EEG system with digital analysis of video-EEG for spike detection and analysis, computerized automated seizure detection algorithms, and patient "event marker." Electrode set up of 8 channels or greater was performed by the EEG technologist. Standard 10-20 system montages were employed for EEG review.

Clinical laboratory analyses. Levels of CSF protein and glucose and serum D-dimer, ferritin, CRP and IL-6 were determined per standard laboratory methods using FDA-approved, in vitro diagnostics (IVD) kits.

Clinical IgG test against SARS-CoV-2 was performed using FDA EUA kit from Abbott (6R86-20). Experimental IgG tests against SARS-CoV-2 N and S1RBD proteins were detected in plasma and CSF using quantitative ELISA kits (IEQ-CoVN-IgG1 and IEQ-CoVS1RBD-IgG1, RayBiotech). Samples were analyzed as recommended by manufacturer, except that the plasma was diluted 1,500x and CSF 750x in 1x sample buffer. IgM and IgA against SARS-CoV-2 N protein were detected in plasma and CSF using semi-quantitative ELISA kits (IE-CoVN-IgM-1 and IE-CoVN-IgA-1, RayBiotech), as recommended by the manufacturer. All samples were run in technical replicates. These kits were for research use only and did not have FDA approval at the time of initial submission. and Hochberg correction for FDR. Proteins with P and q values lower than 0.05 were considered significant. Differences in inflammatory score between patient cohorts were determined with Mann-Whitney U test (unpaired, two-tailed). Inflammatory score was computed All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. . preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. . All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. Reactome All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. . preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 18, 2020. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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