Circulating CD4 and CD8 T cells expressing pro-inflammatory cytokines in a cohort of mesial temporal lobe epilepsy patients with hippocampal sclerosis

Highlights

• mTLE patients showed reduced frequency of CD8⁺ T lymphocytes expressing IFN-γ, TNF-α.

• mTLE patients showed reduced frequency of CD8⁺ T lymphocytes expressing IL-17, IL-4.

• mTLE patients presented increased frequency of CD4⁺ T lymphocytes expressing IL-6.

• No difference was observed on the frequency of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells.

Abstract

Objective

To compare the proinflammatory and anti-inflammatory cytokine expression profile of CD4⁺ and CD8⁺ T lymphocytes between drug resistant mesial Temporal Lobe Epilepsy (mTLE) patients and healthy subjects.

Methods

mTLE patients were enrolled at the Neurology Center of Santa Casa de Misericórdia de Belo Horizonte (SCM-BH) and healthy volunteers were selected at Universidade Federal de Minas Gerais. Individuals from both groups accepted to participate in this study and signed an informed consent. Peripheral venous blood samples were collected using sodium heparin vacuum tubes on the day before the surgery and in the interictal period, isolated from whole blood using Ficoll/Hypaque followed by flow cytometry analysis. Data analysis was performed using FlowJo.

Results

Compared to healthy individuals, mTLE patients showed reduced frequency of CD8⁺ T lymphocytes expressing IFN-γ, TNF-α, IL-17 and IL-4. Moreover, mTLE patients presented increased frequency of CD4⁺ T lymphocytes expressing IL-6 when compared to healthy volunteers.

Discussion

Epilepsy is the third most common chronic brain disorder. Mesial temporal lobe epilepsy (mTLE) is a major and severe form of epilepsy and 30% of the mTLE patients do not respond to conventional medications. Our data suggest that mTLE patients have distinct immunological profiles that are related to disease pathophysiology.

Introduction

Epilepsy is the third most common chronic brain disorder, which consists of recurrent seizures that affect 0.8–1% of the world population (Vezzani et al., 2011). Nearly 90% of patients with epilepsy are found in developing areas (Fabene et al., 2013). This higher prevalence in developing nations is attributed to an increased chance to suffer pro-epileptogenic events such as head trauma, stroke, viral infection, febrile seizures, status epilepticus occurring either in infancy or during lifetime (Herman, 2002).

Mesial temporal lobe epilepsy (mTLE) presents with seizures initiated in hippocampus and is the most frequent form of epilepsy (Chang and Lowenstein, 2003, Spencer, 1998). About 30% of individuals with mTLE do not respond to conventional medications. Treatment-resistant epilepsy is a huge social and individual burden due to impaired quality of life and increased mortality rate (Malkki, 2014). mTLE is often associated with a characteristic pattern of selective and extensive hippocampal atrophy, referred as hippocampal sclerosis (HS). The surgical removal of HS produces clinical improvement (Falconer and Taylor, 1968, Falconer, 1974). However, whether hippocampal sclerosis is the consequence of repeated seizures or whether it plays a role in the development of the epileptic focus is still under debate (Jefferys, 1999). Both clinical and preclinical data suggest that HS can be associated, but not necessary, for a long-lasting epileptic condition (Fabene et al., 2010).

For many years, preclinical and clinical studies in epilepsy have been almost uniquely focused on neuronal alterations (Fabene et al., 2013). However, other cell types, including glial, vascular and immune cells, clearly emerged as new targets to be considered in a broader integrated picture of epileptic pathogenesis. A consistent body of data has suggested in recent years that epilepsy might be associated with non-neuronal alterations. For example, immune and blood–brain-barrier (BBB) factors seems to play a crucial role in the initiation and maintenance of epileptic activity (Zattoni et al., 2011).

While neuroinflammation directly affects neurovascular and glial function, the effects of systemic inflammation are mediated or facilitated by loss of BBB function (Rivest, 2010; Friedman, 2011). BBB disruption (BBBD) can be triggered by a direct insult to the endothelium (Marchi et al., 2007) or by systemic factors, including activation of circulating leukocytes (Fabene et al., 2008, Bauer et al., 2008; Marchi et al., 2010; Ravizza et al., 2008, Zattoni et al., 2011) and release of molecular mediators that increase vascular permeability (Librizzi et al., 2012).

In addition to direct immunological effects, the blood–brain-barrier impairment might also play a role (Friedman, 2011). Recently, it was reported that CD8⁺ T lymphocytes lead to BBB dysfunction through a perforin-dependent process (Li et al., 2013). Marchi et al. (2011) tested the hypothesis that immunosupression by splenectomy or lack of perforin could reduce seizure onset. Onset of status epilepticus (SE) and mortality were significantly decreased in perforin-deficient mice. The induction of BBB disruption may establish a link between the systemic immune system and brain dysfunction.

Ong et al. (2014) performed an analysis of a sample of more than two million individuals and identified the co-occurrence of auto-immune conditions and epilepsy in 17.5%, suggesting that inflammation might be linked to some epilepsy cases. Inflammatory mediators such as cytokines, as well as leukocyte–endothelial interaction factors, can cause blood–brain barrier damage and the subsequent leakage of serum proteins into the brain (Fabene et al., 2008, Oby and Janigro, 2006, Shlosberg et al., 2010). Furthermore, inflammatory mediators could be also released by macrophages and granulocytes entering the brain from blood vessels during the epileptogenic phase (Fabene et al., 2008, Ravizza et al., 2008, Zattoni et al., 2011).

Among immune mediators, cytokines are soluble peptides that mediate response in the periphery and the central nervous system. After the seizure, long lasting CNS inflammation promotes neuronal death in cortical and subcortical areas generating cell loss, synaptic reorganization and neuronal circuit remodeling (Engel, 2001, Vezzani et al., 2011). In the brain, astrocytes overexpress pro-inflammatory cytokines, such as TNF-α, IL-1 or IL-6, that are implicated in the genesis of mTLE (Akassoglou et al., 1997, Stalder et al., 1998).

Bauer et al. (2008) showed that CD4⁺T cells were decreased while total lymphocytes, including natural killer cells, were increased in patients with temporal lobe epilepsy. Comparing some antiepileptic drugs, treatment with lamotrigine was associated with a higher percentage of B lymphocytes and valproate with an increased percentage of CD4⁺ T lymphocytes. Therapy with levetiracetam showed a trend towards decreased CD8⁺ T cell counts. No significant differences in these measures were seen between focal and generalized epilepsies, and between temporal and extratemporal lobe epilepsies (Nowak et al., 2011).

In this study, we compared the frequency of expression of proinflammatory and anti-inflammatory cytokines by CD4+ and CD8+ T lymphocytes derived from drug-resistant mTLE patients and healthy controls. We found that mTLE patients present a distinct immunological profile that may be related to disease pathophysiology.