Utility of CSF Cytokine/Chemokines as Markers of Active Intrathecal Inflammation: Comparison of Demyelinating, Anti-NMDAR and Enteroviral Encephalitis

Kavitha Kothur, Louise Wienholt, Shekeeb S Mohammad, Esther M Tantsis, Sekhar Pillai, Philip N Britton, Cheryl A Jones, Rajeshwar R Angiti, Elizabeth H Barnes, Timothy Schlub, Sushil Bandodkar, Fabienne Brilot, Russell C Dale, Kavitha Kothur, Louise Wienholt, Shekeeb S Mohammad, Esther M Tantsis, Sekhar Pillai, Philip N Britton, Cheryl A Jones, Rajeshwar R Angiti, Elizabeth H Barnes, Timothy Schlub, Sushil Bandodkar, Fabienne Brilot, Russell C Dale

Abstract

Background: Despite the discovery of CSF and serum diagnostic autoantibodies in autoimmune encephalitis, there are still very limited CSF biomarkers for diagnostic and monitoring purposes in children with inflammatory or autoimmune brain disease. The cause of encephalitis is unknown in up to a third of encephalitis cohorts, and it is important to differentiate infective from autoimmune encephalitis given the therapeutic implications.

Aim: To study CSF cytokines and chemokines as diagnostic biomarkers of active neuroinflammation, and assess their role in differentiating demyelinating, autoimmune, and viral encephalitis.

Methods: We measured and compared 32 cytokine/chemokines using multiplex immunoassay and APRIL and BAFF using ELISA in CSF collected prior to commencing treatment from paediatric patients with confirmed acute disseminated encephalomyelitis (ADEM, n = 16), anti-NMDAR encephalitis (anti-NMDAR E, n = 11), and enteroviral encephalitis (EVE, n = 16). We generated normative data using CSF from 20 non-inflammatory neurological controls. The sensitivity of CSF cytokine/chemokines to diagnose encephalitis cases was calculated using 95th centile of control values as cut off. We correlated CSF cytokine/chemokines with disease severity and follow up outcome based on modified Rankin scale. One-way hierarchical correlational cluster analysis of molecules was performed in different encephalitis and outcome groups.

Results: In descending order, CSF TNF-α, IL-10, IFN-α, IL-6, CXCL13 and CXCL10 had the best sensitivity (>79.1%) when all encephalitis patients were included. The combination of IL-6 and IFN-α was most predictive of inflammation on multiple logistic regression with area under the ROC curve 0.99 (CI 0.97-1.00). There were no differences in CSF cytokine concentrations between EVE and anti-NMDAR E, whereas ADEM showed more pronounced elevation of Th17 related (IL-17, IL-21) and Th2 (IL-4, CCL17) related cytokine/chemokines. Unlike EVE, heat map analysis showed similar clustering of cytokine/chemokine molecules in immune mediated encephalitis (ADEM and anti-NMDAR E). Th1 and B cell (CXCL13 and CXCL10) molecules clustered together in patients with severe encephalopathy at admission and worse disability at follow up in all encephalitis. There was no correlation between CSF neopterin and IFN-γ or IFN-α.

Conclusion: A combination panel of cytokine/chemokines consisting of CSF TNF-α, IL-10, IFN-α, IL-6, CXCL13 and CXCL10 measured using multiplex immunoassay may be used to diagnose and monitor intrathecal inflammation in the brain. Given their association with worse outcome, certain key chemokines (CXCL13, CXCL10) could represent potential therapeutic targets in encephalitis.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. CSF concentrations of cytokine/chemokines with…
Fig 1. CSF concentrations of cytokine/chemokines with >75% sensitivity to detect intrathecal inflammation in all children with acute encephalitis- acute disseminated encephalomyelitis (ADEM), anti-NMDAR encephalitis (anti-NMDAR E) and enteroviral encephalitis (EVE).
Dotted lines represent medians. The statistical analysis was performed using Kruskal Wallis test. The 95% centile of the control values are presented (P95 control).
Fig 2. Heat map of elevated CSF…
Fig 2. Heat map of elevated CSF cytokine/ chemokines# in encephalitis groups compared to controls presented according to T and B cell subsets.
There is a broad elevation of cytokine/chemokines related to all Th helper subsets (Th1, Th2, T reg, Th17, B cell and other cytokines and chemokines) in ADEM patients unlike anti-NMDAR E and EVE. # Cytokine/chemokines were shaded only if these molecules were statistically significantly elevated in different encephalitis groups compared to controls (p<0.05).
Fig 3. CSF cytokine/chemokine levels that were…
Fig 3. CSF cytokine/chemokine levels that were more elevated in ADEM compared to EVE and anti-NMDAR E.
Th17 (IL-21, IL-17A) and Th2 (CCL17, and IL-4) related cytokine/chemokines showed statistically significant elevation in ADEM compared to both EVE and anti-NMDAR E. CXCL12 showed paradoxical decrease in EVE and anti-NMDAR E, in contrast to elevation noted in ADEM.
Fig 4. Hierarchical cluster analysis heat-map showing…
Fig 4. Hierarchical cluster analysis heat-map showing nearest-neighbour correlations of cytokines and chemokines in ADEM, anti-NMDAR E, EVE and controls.
Cytokines with positive correlations are represented in graded shades of black and negative correlations in graded shades of red. The same order of the analytes along axis is used for all the three heatmaps to allow comparisons. The clustering pattern showed some similarities (cluster A) in immune mediated encephalitis (ADEM and anti-NMDAR E), which was less observed in viral encephalitis and controls.
Fig 5. Heat map representation of cytokine/chemokine…
Fig 5. Heat map representation of cytokine/chemokine molecule interaction in the CSF of patients with all encephalitis with severe encephalopathy at admission (modified Rankin scale, MRS 5) and worse disability at follow up (MRS >2).
The cluster B molecules showed similar positive correlations between those with higher severity of encephalopathy and those with disability in follow up.

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