Peripheral inflammatory markers and clinical correlations in patients with frontotemporal lobar degeneration with and without the C9orf72 repeat expansion

In this study, our aim was to evaluate potential peripheral inflammatory changes in frontotemporal lobar degeneration (FTLD) patients carrying or not the C9orf72 repeat expansion. To this end, levels of several inflammatory markers (MCP-1, RANTES, IL-10, IL-17A, IL-12p, IFN-γ, IL-1β, IL-8, and hs-CRP) and blood cells counts in plasma and/or serum of FTLD patients (N = 98) with or without the C9orf72 repeat expansion were analyzed. In addition, we evaluated whether the analyzed peripheral inflammatory markers correlated with disease progression or distinct clinical phenotypes under the heterogenous FTLD spectrum. Elevated levels of pro-inflammatory RANTES or MCP-1 and decreased levels of anti-inflammatory IL-10 were found to associate with Parkinsonism and a more rapid disease progression, indicated by longitudinal measurements of either MMSE or ADCS-ADL decline. These findings were observed in the total cohort in general, whereas the C9orf72 repeat expansion carriers showed only slight differences in IL-10 and hemoglobin levels compared to non-carriers. Furthermore, these C9orf72 repeat expansion-associated differences were observed mostly in male subjects. The females in general showed elevated levels of several pro-inflammatory markers compared to males regardless of the C9orf72 genotype. Our study suggests that pro-inflammatory changes observed in the early symptomatic phase of FTLD are associated with distinct clinical profiles and a more rapid disease progression, and that the C9orf72 repeat expansion and gender may also affect the inflammatory profile in FTLD. Electronic supplementary material The online version of this article (10.1007/s00415-019-09552-1) contains supplementary material, which is available to authorized users.


Profiling the clinical features and disease progression
Data for presence or absence of several FTLD-related clinical features (psychotic symptoms, parkinsonism and motoneuron symptoms) was collected as dichotomous variables. To be included in the group of FTLD with psychotic symptoms, the patient must have had at least one evident psychotic symptom, i.e. either delusion or hallucination. FTLD with Parkinsonism was defined when at least two symptoms of the following were observed: resting tremor, bradykinesia, rigidity, prominent hypomimia, postural instability or loss of automatic movements. FTLD with motoneuron symptoms required at least "clinically possible" diagnosis according to the revised El Escorial criteria [1]. All of these clinical features were included if they were observed either at the disease onset or during the follow-up. The mean follow-up time was 33.8 months (range 3-123 months, median 24 months).

IL-12p, IFN-γ)
Cytokine concentrations were determined using BD CBA Flex Set system for human samples (BD Biosciences, San Jose, CA, USA). A total of 50 FTLD sample aliquots were thawed before the analysis. The samples were incubated for three hours with kit components (beads and detection reagent) according to manufacturer's instructions. After three washes with Wash Buffer, the samples were run with FACS Aria flow cytometer (BD Biosciences) and converted to Excel file by using FCAP Array 2.0.0 Software (Soft Flow Hungary Ltd, Pecs, Hungary). All analytes were quantified separately and the sample location on plate was randomized. All samples were analyzed as duplicates, and mean concentration calculated from the duplicates was used.

Single Molecule Array (Simoa) analyses (serum IL-1β, IL-8, IL-10 and MCP-1)
A total of 91 FTLD serum samples were thawed, divided in to four aliquots and frozen for future analysis.
After a second thaw, the aliquots were mixed, centrifuged (10, 000 x g 5 min, + 22 ºC) and transferred to 96well plates with calibrators and quality control samples. Serum IL-1β and IL-8 were quantified using Simoa HD-1 two-step Advantage digital immunoassays. The quantification of IL-10 and MCP-1 utilized three-step advantage digital immunoassays [2]. All analytes were quantified separately and the sample location on plate was randomized. All samples were analyzed as duplicates, and mean concentration calculated from the duplicates was used. Samples having a coefficient of variation higher than 15 % were excluded. The highest concentration of the assay`s quantification range was used for samples with both duplicate values exceeding the quantification range. Values below the quantification range were excluded (only IL-1β showed levels below the quantification range).

High-sensitive C-Reactive Protein analyses (plasma CRP)
A total of 44 plasma samples were thawed, divided into aliquots, and frozen before the analysis. After a second thaw, the aliquots were centrifuged and analyzed with particle enhanced immunoturbidimetric assay by Cobas 6000 (c 501) -analyzer, Hitachi High Technology Co, Tokyo, Japan. Measuring range was 0.15 -20 mg/L. Samples with concentration below the measuring range (N=8) were considered as non-detectable and excluded from the analyses. One sample with a concentration of 28 mg/L (FTLD C9orf72 HRE carrier) was excluded as such a high concentration likely reflects another inflammatory state, such as infection.