key: cord-0856394-klbxajzl
authors: Hirzel, Cédric; Grandgirard, Denis; Surial, Bernard; Wider, Manon F.; Leppert, David; Kuhle, Jens; Walti, Laura N.; Schefold, Joerg C.; Spinetti, Thibaud; Suter-Riniker, Franziska; Dijkman, Ronald; Leib, Stephen L.
title: Neuro-axonal injury in COVID-19: the role of systemic inflammation and SARS-CoV-2 specific immune response
date: 2022-03-12
journal: Ther Adv Neurol Disord
DOI: 10.1177/17562864221080528
sha: fb69b45afd428a73a84df96cccdd868cff220368
doc_id: 856394
cord_uid: klbxajzl

BACKGROUND: In coronavirus disease-2019 (COVID-19) patients, there is increasing evidence of neuronal injury by the means of elevated serum neurofilament light chain (sNfL) levels. However, the role of systemic inflammation and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific immune response with regard to neuronal injury has not yet been investigated. METHODS: In a prospective cohort study, we recruited patients with mild–moderate (n = 39) and severe (n = 14) COVID-19 and measured sNfL levels, cytokine concentrations, SARS-CoV-2-specific antibodies including neutralizing antibody titers, and cell-mediated immune responses at enrollment and at 28(±7) days. We explored the association of neuro-axonal injury as by the means of sNfL measurements with disease severity, cytokine levels, and virus-specific immune responses. RESULTS: sNfL levels, as an indicator for neuronal injury, were higher at enrollment and increased during follow-up in severely ill patients, whereas during mild–moderate COVID-19, sNfL levels remained unchanged. Severe COVID-19 was associated with increased concentrations of cytokines assessed [interleukin (IL)-6, IL-8, interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α)], higher anti-spike IgG and anti-nucleocapsid IgG concentrations, and increased neutralizing antibody titers compared with mild–moderate disease. Patients with more severe disease had higher counts of defined SARS-CoV-2-specific T cells. Increases in sNfL concentrations from baseline to day 28(±7) positively correlated with anti-spike protein IgG antibody levels and with titers of neutralizing antibodies. CONCLUSION: Severe COVID-19 is associated with increased serum concentration of cytokines and subsequent neuronal injury as reflected by increased levels of sNfL. Patients with more severe disease developed higher neutralizing antibody titers and higher counts of SARS-CoV-2-specific T cells during the course of COVID-19 disease. Mounting a pronounced virus-specific humoral and cell-mediated immune response upon SARS-CoV-2 infection did not protect from neuro-axonal damage as by the means of sNfL levels.

Furthermore, there is increasing evidence for persistent neurologic symptoms such as chronic fatigue, memory impairment, and depression after acute coronavirus disease-2019 (COVID- 19) . 2 Neuropathological studies reveal evidence for central nervous system (CNS) damage in fatal COVID-19 cases. [3] [4] [5] A common finding in these studies was cerebral cell loss, either attributed to hypoxia 3, 4 or inflammation 5 either with or without immunohistochemical evidence of viral CNS invasion. 3, 4, [6] [7] [8] Members of the betacoronavirus genus are potent pathogens, usually associated with respiratory tract disease in humans [e.g. SARS-CoV-2, severe acute respiratory syndrome coronavirus (SARS-CoV), or Middle East respiratory syndrome (MERS)] and gastrointestinal, hepatic, and neurologic disease in animals. 9 Mouse hepatitis virus (MHV) belongs to the betacoronavirus genus and shows neurotropic properties. MHV-JHM and MHV-A59 have been studied for ages as a mouse model of virus-induced encephalitis, myelitis, and chronic progressive demyelination concurrent with axonal loss of the CNS. 9 The importance of the humoral immune response for clearance of betacoronaviruses (MHV-59 and MHV-JHM) from CNS in mice is well established. [10] [11] [12] In humans, humoral immune deficiencies have been associated with prolonged SARS-CoV-2 shedding and severe disease, [13] [14] [15] [16] [17] but the role of the SARS-CoV-2 specific immune response with regard to neuronal injury in COVID-19 patients has not yet been investigated.

Neurofilament light chain (NfL) is an intraaxonal cytoskeleton protein highly expressed in large caliber myelinated axons. 18 Serum neurofilament light chain (sNfL) measurements are used to detect and monitor CNS injury in various neurodegenerative conditions, including multiple sclerosis, Alzheimer's disease, and amyotrophic lateral sclerosis. 18 In moderate to severely ill COVID-19 patients, there is increasing evidence for neuronal injury by the means of elevated sNfL levels. [19] [20] [21] [22] [23] [24] [25] The aim of this study was to examine in COVID-19 patients the association of neuro-axonal injury as by the means of sNfL measurements with disease severity and virus-specific immune response. We explored whether an insufficient immunological control of the virus might play a role in promoting neuro-axonal injury.

We prospectively included patients with confirmed SARS-CoV-2 infection from 5 March 2020 to 16 July 2020. Serum samples were collected at the time of enrollment in the study (baseline) and at follow-up 28 ± 7 days later. Patients with preexisting neuroinflammatory disorders were excluded from the analysis. COVID-19 disease severity was categorized according to the COVID-19 WHO Ordinal Scale for Clinical Improvement, ranging from 1 (no symptoms) to [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . 26 For analysis, patients with a score of 1-4 (patients without need for high-flow oxygen) were defined to have mild/moderate disease, and patients with a score of 5-8 (patients with need for high-flow oxygen, noninvasive ventilation, or mechanical ventilation) were defined to have severe disease. The present study is part of a larger COVID project (NCT04510012), and data of some patients were previously investigated regarding immune functionality in COVID-19. 27 The trial was approved by the Ethics Commission of the Canton of Bern, Bern, Switzerland, Nr. 2020-00877 and registered at ClinicalTrials.gov (NCT04510012). Patients were included after provision of informed consent. In case of lack of capacity and/or inability to provide consent, enrollment followed the procedures for research projects in emergency situations according to Swiss law.

Patient nasopharyngeal sample was obtained using Copan FLOQSwabs and Copan UTM Viral Transport Medium (Copan, Brescia, Italy). For polymerase chain reaction (PCR) testing, three different methodologies were used: a laboratory developed workflow based on a published protocol, 28 and two commercial workflows, the Seegene Allplex 2019-nCoV Assay (Seegene, Seoul, Korea) and the Roche Cobas ® SARS-CoV-2 Assay (Roche Diagnostics, Rotkreuz, Switzerland).

Cytokines [interleukin (IL)-6, IL-8, interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNFα)] and NfL were quantified in serum samples using an automated enzyme-linked immunosorbent assay (ELISA)-based microfluidic system (ELLA®, ProteinSimple, San Jose, CA, USA) journals.sagepub.com/home/tan 3 with dedicated cartridges according to the manufacturer's instructions. Samples were separated into triplicates that were automatically and independently processed. Raw data were analyzed using the manufacturer's software. A mean value derived from the three replicates was generated.

Humoral and cell-mediated immune response Anti-spike IgG antibody concentrations in serum were measured using the DiaSorin LIAISON ® SARS-CoV-2 S1/S2 

Demographics were analyzed using descriptive statistics (Fisher's exact test). Mann-Whitney U tests were used to assess the associations between dichotomous COVID-19 severity (mild-moderate and severe), cytokine levels, antibody concentrations, CMI (SFU/250,000 cells), and sNfL concentrations. To account for the potential for confounding by age, corticosteroid use, and time from symptom onset to sample measurement, we repeated the analyses using multiple linear regression adjusted for those covariates. Variables of interest were log-transformed where appropriate to approximate a normal distribution. We compared outcomes between individuals with severe and mild-moderate COVID-19 using adjusted mean values, and calculated mean differences for untransformed and ratio of means for log-transformed variables. 31 Cytokine levels, antibody concentrations, CMI (SFU/250,000 cells), and sNfL concentrations in paired samples over time were compared using Wilcoxon signed-rank test.

Because sNfL levels increase with age, 18 which is also a major risk factor for severe COVID-19, we further refined our analyses by associating sNfL changes (ΔsNfL: sNfL at follow-up minus sNfL at baseline; sNfL fold-change: sNfL at follow-up divided by sNfL at baseline) with the immune response. We used univariable and multivariable linear regression adjusted for age, corticosteroid use, and time from symptom onset to sample measurement to estimate the association between changes in sNfL levels and cytokine concentrations, antibody levels, and SFUs. sNfL fold-changes were modeled using log-transformation, and absolute sNfL differences were modeled using a cube-root transformation to account for the long tails in addition to positive and negative values. 32 Statistical significance was defined as a p value <.05. In this exploratory study, p values and widths of 95% confidence intervals were not adjusted for multiplicity. 33 

We enrolled 55 patients with symptomatic COVID-19 and excluded two patients from analysis due to the presence of an active neuroinflammatory disorder (one patient with Guillain-Barré syndrome; one patient with a facial palsy due to a suppurative otitis). Patients with mild to moderate disease (n = 39) were younger and had less comorbidities compared with severe COVID-19 cases (n = 14) ( Figure 1 (a)). After adjusting for age, time from symptom onset to sampling, and use of immunomodulatory drugs, IL-6, IL-8, and TNFα levels at baseline remained significantly higher in patients with severe disease ( (Figure 1(b) ). In unadjusted analysis, anti-spike and anti-nucleocapsid IgG levels along with neutralizing antibody titers were significantly higher in follow-up samples of patients with severe COVID-19 ( Figure 1(b) ), without any changes in the adjusted analyses (Table 2) .

PBMCs were available for 84.9% (45/53) of patients at baseline and for 86.8% (46/53) of patients at follow-up. Dichotomized disease severity (mild-moderate versus severe) was not associated with the number of IFN-γ positive SFUs (unadjusted analyses: Figure 2 (a) and (b); adjusted analyses: Table 2 ).

IL-2 SFUs significantly increased from baseline to follow-up upon stimulation with the S2N and SNMO peptide pool (Figure 2(a) and (b) ). Upon cell stimulation with the SNMO peptide pool, patients with severe COVID-19 had significantly more IL-2-positive SFUs compared with individuals with mild-moderate disease at follow-up (unadjusted analyses: Figure 2 (a); adjusted analyses: Table 2 ).

IFN-γ/IL-2 double-positive spots significantly increased from baseline to follow-up after cell stimulation with the SNMO peptide pool and in unadjusted analyses; patients with severe disease had more IFN-γ/IL-2 double-positive SFU at follow-up compared with those with mild-moderate disease ( Figure 2(a) ). Adjusted differences in IFN-γ/IL-2 double-positive SFUs at follow-up were no longer statistically significant (Table 2) . Upon stimulation with the S2N peptide pool, IFN-γ/ IL-2 double-positive spots increased from baseline to follow-up, but changes were only statistically significant in the larger mild-moderate COVID-19 group (Figure 2(b) ). There was no statistically significant difference in IFN-γ/IL-2 double-positive spots at follow-up between mild/ moderate and severe COVID-19 patients in unadjusted (Figure 2(b) ) and adjusted (Table 2) analyses.

Serum NfL measurements were available for 98.1% (52/53) at baseline and for 92.5% (49/53) at day 28(±7).

In unadjusted analysis, median sNfL concentrations at baseline were higher in patients with The results remained unchanged when excluding the patient who suffered a cerebral hemorrhage after the first and before the second serum sample was drawn (data not shown).

To further explore the association between neuroaxonal damage and the SARS-CoV-2-specific immune response, we correlated changes in sNfL (irrespective of COVID-19 disease severity scores) with cytokine levels, antibody concentrations, and the CMI response.

In unadjusted and adjusted analyses, IL-6, IL-8, and IL-1β concentrations at baseline significantly correlated with log10 sNfL fold-changes (Supplementary Figure 1A) and cube-root transformed ΔsNfL (Supplementary Figure 1B) .

Increases in sNfL concentrations positively correlated with anti-spike IgG concentrations and neutralizing antibody titers in unadjusted and adjusted analyses (Figure 4(a) and (b) ). There was a positive correlation for changes in sNfL concentrations and anti-nucleocapsid levels in the unadjusted analyses, but not in adjusted analyses (Figure 4(b) ).

We did not observe significant correlations between CMI responses and sNfL changes (Supplementary Figure 2 ).

We examined the association of COVID-19 disease severity, cytokine levels, humoral and CMI response, and biochemical evidence for neuroaxonal injury. The major findings of our study were as follows: (a) We observed that elevated IL-6, IL-8, IL-1β, and TNF-α serum cytokine levels are a characteristic feature of severe COVID-19; (b) patients with severe COVID-19 elicit more pronounced anti-spike, anti-nucleocapsid, and neutralizing antibody responses; (c) some SARS-CoV-2-specific T-cell subsets are elevated in severely ill individuals; (d) severe COVID-19 is associated with subsequent neuronal injury as reflected by increased levels of sNfL; and (e) neuronal injury is not associated with inadequate SARS-CoV-2-specific humoral or CMI responses.

In accordance with previous findings, we observed elevated levels of pro-inflammatory cytokines in patients with severe COVID-19 compared with individuals with mild-moderate disease. 35, 36 The degree of cytokinemia in our cohort was consistent with previous reports. 37 IL-6 levels of patients with severe COVID-19 lay within the reported range of individuals with severe bacterial pneumonia [38] [39] [40] but are markedly lower compared with patients with chimeric antigen receptor (CAR)-T-cellinduced cytokine release syndrome. 37 Our findings corroborate the results of other groups, who reported elevated sNfL levels in patients with severe COVID-19 compared with patients with mild and moderate disease. 19, 20, 25 Our observations suggest effects of the systemic inflammatory response, as potential drivers of neuro-axonal injury in severe COVID-19. This hypothesis is also supported by the results of a recent study, which examined sNfL levels in patients with septic shock without primary CNS infection. 41 In this study, sNfL levels increased in sepsis patients but remained stable in patients without sepsis. 41 It has been suggested that neurologic damage in sepsis develops along with an activation of the cerebral endothelium and an increase in the permeability of the blood-brain barrier (BBB). 42 Cytokines, which are known to increase the BBB permeability [e.g. IL-6, 43 IL-8, 44 interleukin-1b (IL-1b) 43 ], are elevated in both severe SARS-CoV-2 infection and bacterial sepsis. Therefore, our findings might not be COVID-19 specific but rather reflect neuro-axonal injury seen in severely ill patients with systemic inflammation. However, without cerebrospinal fluid (CSF) analysis, it remains unclear to which extent sNfL levels originate from the CNS or from the peripheral nervous system as sNfL concentrations are also elevated in individuals with critical illness polyneuropathy. 45 We used the ELLA platform (ProteinSimple) for quantification of sNfL concentrations in serum.

The current reference method for sNfL quantification is the Single Molecular Array (Simoa, Quanterix Corp., Boston, MA, USA). However, both assays are using the same antibody for NfL detection, and a recent thorough validation study showed that the two platforms are equivalent. 46 We are therefore confident that our results can be reliably interpreted in the context of previous studies that used the Simoa technology for sNfL measurement in COVID-19 patients. [19] [20] [21] [22] [23] [24] [25] Serum NfL shows distinct kinetics, with a delayed increase in serum levels and a peak 2 weeks after brain injury, followed by a slow decrease of serum concentrations for 3-9 months. 18 Based on the knowledge of sNfL dynamics, it is not surprising that the difference in sNfL levels among mildmoderate and severe COVID-19 patients was most evident in the follow-up serum sample after 28(±7) days.

Severe COVID-19 was associated with higher antibody production and neutralizing titers in our cohort. This phenomenon has also been described in other cohorts. 35, 47, 48 One possible explanation for this finding could be that severe disease caused by hyper-inflammation or uncontrolled viral replication induces excess antibody production as surrogate marker of disease severity. This is supported by our finding that the group of severely ill COVID-19 patients had not only the highest neutralizing antibody titers but also the highest levels of pro-inflammatory cytokines. We observed a similar phenomenon for a subset of IL-2-specific and IL-2/IFN-γ double-positive T-cells (SNMO peptide pool stimulated only). There is conflicting evidence about the neuroinvasive capacity of SARS-CoV-2. 3, 4, [6] [7] [8] 49 However, in mice, members of the betacoronavirus genus (MHV-JHM and MHV-A59) are clearly neurotropic and induce CNS infection. [10] [11] [12] In humans, humoral immune deficiencies have been associated with prolonged SARS-CoV-2 shedding and severe disease, [13] [14] [15] [16] [17] but the role of the SARS-CoV-2 antibody response with regard to neuronal injury in COVID-19 patients has not been investigated. In contrast to humans, the importance of the humoral and CMI response for clearance of betacoronaviruses (MHV-59 and MHV-JHM) from CNS in mice is well established. [10] [11] [12] 50, 51 A recent in vitro study showed that anti-viral antibodies from human CSF block SARS-CoV-2 infection of human brain organoids. 49 We therefore hypothesized that low neutralizing antibody titers or decreased CMI response might contribute to neuro-axonal injury in COVID-19. However, when correlating the humoral or cell-mediated SARS-CoV-2-specific immune response with sNfL increases over time, we did not find evidence to support this hypothesis.

A unique aspect of our study was the prospective design with collection of paired samples at uniform time points, which allows to analyze the dynamic change of sNfL concentration in the development of the disease. Here, we demonstrate for the first time an association between the systemic inflammatory response in severe COVID-19 and neuronal injury, as postulated previously. 19 Our cohort covered the whole spectrum of COVID-19 disease severity, from outpatients to mechanically ventilated patients on the ICU. Since sNfL levels highly depend on age, 18 which is also a risk factor for severe COVID-19, our study design allows to analyze the dynamic change of sNfL concentration, which is most likely age independent. In addition, we performed adjusted analyses corrected for age, time from symptom onset to sampling, and use of immunomodulatory drugs. One strength of our study includes the measurement of neutralizing antibody titers by using an authentic SARS-CoV-2 isolate for serum neutralization assays instead of using pseudovirus-based neutralization assays or solely ELISA-based methods. In contrast to surrogate methods, this approach ensures that neutralizing capacities of antibodies represent real findings. Our study has important limitations that require discussion. Systematic clinical neurological and/or neurocognitive evaluation was not performed due to resource limitations and restrictions associated with the pandemic. Therefore, we cannot provide data on the correlation of sNfL levels and post-illness neurocognitive disorders. In addition, we did not systematically carry out neuroradiological imaging of our patients to correlate radiologic findings with sNfL measurements. Also, in this observational study, we did not perform lumbar punctures and cannot provide data on CNS inflammatory parameters or evidence for viral CNS invasion. Therefore, these aspects will have to be included in follow-up studies to assess their role in the association of neuro-axonal damage. Furthermore, we acknowledge that this is a small cohort. Including an independent validation cohort and a comparison group of healthy volunteers (without COVID-19) would have strengthened our findings.

In summary, we provide novel information indicating that systemic inflammation in severe COVID-19 disease is associated with ensuing neuro-axonal damage. Patients with more severe disease developed higher neutralizing antibody titers and higher counts of SARS-CoV-2-specific T cells during the course of COVID-19 disease. Mounting a pronounced virus-specific humoral and CMI response upon SARS-CoV-2 infection did not protect from neuro-axonal damage as by the means of sNfL levels.

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We thank Daniela Hirter, Manuela Manz, and Pia Scherler (Department of Infectious Diseases, Bern University Hospital) for logistic support.