Low IL-35 expression in CSF is associated with Neuro-Behcet Disease: Comparative analysis between parenchymal and...

IL-35 is a recently discovered immunoregulatory cytokine that inhibits inflammatory cytokines by suppressing their lineage-specific transcription factors. The objective of this study was to investigate the expression of IL-35 in the cerebrospinal fluid (CSF) of patients with Neuro-Behçet Disease (NBD). An immuno-comparative analysis was performed between parenchymal NBD (pNBD) and non-parenchymal NBD (npNBD).

We are investigating CSF IL-35 levels in 45 patients with (NBD), comprising 25 patients with pNBD and 20 with npNBD, compared to 27 patients with multiple sclerosis (MS) and 20 patients with non-inflammatory neurological diseases (NIND). We assessed the inflammatory cytokines (IL-1α, IL-18, IL-33, IL-36), Foxp3 and CD4⁺ CD25⁺ Foxp3⁺ regulatory Treg T cells (Tregs). The following methodologies were employed: flow cytometry, ELISA, and real-time polymerase chain reaction (RT-PCR). For RT-PCR analysis, we calculated relative gene expression in target genes using the comparative CT method with the equation 2^-ΔΔCt. We employed a receiver operating characteristic (ROC) curve to investigate the predictive value of IL-35 levels.

Protein and relative mRNA expression of IL-35 were significantly decreased in NBD and MS patients compared to the NIND group. Significantly lower CSF IL-35 mRNA (p= 0.0001) and protein (p= 0.0004) were observed in patients with pNBD compared to npNBD. The study revealed that NBD patients exhibited low Treg counts, and a significant positive correlation was identified between Treg numbers and CSF IL-35 (r = 0.554, p= 0.0001). Negative associations were observed between Tregs and CRP (r =- 0.518; p= 0.0001) and ESR (r = -0.571; p= 0.0001) in NBD. Levels of the pro-inflammatory mediators were found to be elevated in contrast to a low Foxp3 level in NBD, which was more reduced in pNBD compared to npNBD. In vitro cultured memory T cells from pNBD patients stimulated with LPS showed high levels of IL-1α, IL-18, IL-33, IL-36 and low levels of Foxp3 and IL-35 measured in the culture medium. After the addition of recombinant human IL-35 (rhIL-35), Foxp3 and IL-35 were significantly increased and inflammatory cytokine levels were reduced. These results suggest that rhIL-35 may induce a regulatory effect on Foxp3 and IL-35.

These findings imply a critical reduction of IL-35 in pNBD patients. The combined protein and gene expression of the tested inflammatory cytokines suggest that there are distinct inflammatory mechanisms governing the central nervous system in pNBD. Further work is essential for the development of targeted interventions for the effective treatment of patients.