Inflammatory state exists in familial amyloid polyneuropathy that may be triggered by mutated transthyretin

The relationship between familial amyloid polyneuropathy (FAP), which is caused by mutated transthyretin (TTR), and inflammation has only recently been noted. To determine whether inflammation is present in FAP carriers and patients, serum interleukin (IL)−6 concentration in 57 healthy donors (HD), 21 FAP carriers, and 66 FAP patients was examined, with the relationship between IL-6 and TTR assessed in each group by multiple regression analysis and structural equation models (SEM). Compared with HD, IL-6 concentration was elevated in FAP carriers (p = 0.001, 95% CI 0.398–1.571) and patients (p = 0.002, 95% CI 0.362–1.521). Further, SEM indicated a positive relationship between IL-6 and TTR in FAP carriers (p = 0.010, 95% CI 0.019–0.140), but not in HD and FAP patients. In addition, we determined whether TTR induces production of pro-inflammatory cytokines ex vivo. HD-derived CD14 + monocytes and induced pluripotent stem cell-derived myeloid lineage cells from a HD and FAP patient dose-dependently produced IL-6 under mutated and aggregated TTR conditions, compared with wild-type TTR. In conclusion, FAP carriers and patients are in an inflammatory state, with the presence of mutated TTR being a trigger of inflammation, especially in FAP carriers.


Results
Elevated serum IL-6 concentration in FAP carriers and patients. To determine the presence of inflammation, serum IL-6 and high-sensitivity C-reactive protein (hs-CRP) concentration were cross-sectionally analysed in HD (n = 57), as well as FAP carriers (n = 21) and patients (n = 66) (Supplementary Table S1). Concentration of IL-6 but not hs-CRP was higher in FAP carriers and patients than HD. However, because older age increases IL-6 levels 8 , we used multiple regression analysis adjusted by age to determine any differences in serum IL-6 concentration between the three groups (Table 1). We found that IL-6 was related to age (p = 0.046, 95% CI 0.000-0.030), with significantly elevated levels in FAP carriers (p = 0.001, 95% CI 0.398-1.571) and patients (p = 0.002, 95% CI 0.362-1.521) compared with HD. In contrast, there were no differences in hs-CRP concentration between the three groups (data not shown). To confirm this regression model, we used bootstrap testing (n = 2000) and found the same tendency (Supplementary Table S2). Next, we compared the V30M phenotype (n = 39) and other phenotypes (n = 27) in FAP patients (Supplementary Table S3). Our results show elevated IL-6 concentration in both groups, with no significant difference in between them.
Bootstrap testing (n = 2000) in SEM showed a similar tendency (Supplementary Table S5). Next, we examined differences in pathway parameters between HD and FAP carriers and patients ( Table 2). The relationship between IL-6 and TTR was significantly different in FAP carriers compared with HD (p = 0.003, 95% CI −0.199-−0.042) and FAP patients (p = 0.009, 95% CI −0.170-−0.025), while there was no difference between HD and FAP patients (p = 0.49, 95% CI −0.088-0.042). Estimated differences between hs-CRP and IL-6 were not significant in HD compared with FAP carriers or patients, or between FAP carriers and patients. We examined SEM with covariates, in which the effect of age and sex were adjusted accordingly, as previously reported 8,9 . This showed that the relationship between IL-6 and TTR manifested a similar tendency as in SEM without covariates analysis (Supplementary Figure 1, Supplementary Tables S6 and S7).

Mutated TTR increases IL-6 production in myeloid cells ex vivo.
As shown, IL-6 concentration was elevated in FAP carriers and patients compared with HD. In addition, the relationship between IL-6 and TTR in FAP carriers was different compared with HD and FAP patients. Because FAP carriers and patients both have mutated TTR, we determined if mutated TTR affects increased IL-6 concentration ex vivo. HD-derived CD4 + and CD8 + T cells, CD14 + monocytes, and induced pluripotent stem cell-derived myeloid lineage cells (iPS-MLs) originating from HD and FAP patients were cultured in the presence of native wild-type or V30M mutated TTR and aggregated TTR. Cytokines in culture supernatants were quantified using the Bio-Plex system. In native TTR culture conditions, IL-6 increased in CD14 + monocytes and iPS-MLs in the presence of V30M mutated TTR, compared with wild-type TTR, in a TTR-dose-dependent manner ( Fig. 2a and Supplementary Figure 2). In contrast, although IL-6 concentration increased in CD4 + T cells and CD8 + T cells in a TTR-dose-dependent manner, there was no difference between V30M mutated and wild-type TTR (Fig. 2a).
In aggregated TTR culture conditions, IL-6 concentration was elevated in the presence of V30M mutated TTR compared with wild-type TTR in CD14 + monocytes, iPS-MLs, and CD4 + T cells (but not CD8 + T cells) in a TTR-dose-dependent manner ( Fig. 2a and Supplementary Figure 2). Further, the pro-inflammatory cytokines, IL-1β and TNF-α, and inhibitory cytokine, IL-10, increased in a TTR-dose-dependent manner in native and aggregated V30M mutated TTR conditions in CD14 + monocytes but not CD4 + T cells, CD8 + T cells, or iPS-MLs ( Fig. 2b and c, Supplementary Figures 2 and 3a). Other cytokines, namely interferon (IFN)-γ and IL-15 (in all cell subsets), and IL-4, IL-7, and IL-12 (in CD14 + monocytes) were not dependent on the type nor dose of TTR ( Supplementary Figures 2 and 3b-f).

Discussion
We sought to determine if FAP carriers and patients are in an inflammatory state, and additionally, if presence of mutated TTR is involved in the inflammation. IL-6 is a pro-inflammatory cytokine that has been reported in chronic inflammatory diseases such as cancer, arteriosclerosis, and advancing age 8, 10-12 . Moreover, V122I mutated TTR affects IL-6 expression in chondrocytes 6 . Therefore, we focused on the pro-inflammatory cytokine, IL-6. We found increased serum concentration of IL-6 in FAP carriers and patients. Indeed, regardless of preclinical stage, FAP carriers were in an inflammatory state. Consistently, upregulation of inflammatory genes in peripheral blood cells from male FAP patients was recently reported 13 . Next, we examined the relationship between TTR and IL-6 using SEM, which are multivariate regression models that can incorporate multiple regression equations 14 . For our purposes (i.e., to explore TTR and IL-6 involvement), hs-CRP (as an IL-6 related molecule) was added into the model. Although IL-6 positively regulated hs-CRP in HD, as previously reported 15 , this effect was weak in FAP patients and not confirmed in FAP carriers. In addition, differences in pathway parameters between HD and FAP carriers or patients were not significant. These results suggest that although IL-6 positively regulates hs-CRP in all groups, high IL-6 quantity in FAP carriers and patients induces uncertain correlation. For IL-6 and TTR involvement, only FAP carriers exerted a significant positive effect, with this pathway parameter also differentiated from the other two groups. These findings suggest that native mutated TTR may induce IL-6 in FAP carriers. Consequently, we determined whether IL-6 production was affected by mutated, mainly native TTR ex vivo. Accordingly, we confirmed that native V30M  , native V30M mutated TTR, wild-type-derived aggregated TTR, and V30M mutated-derived aggregated TTR for 5 days. HD-derived CD14 + monocytes were cultured with each type of TTR for 2 days. The Bio-Plex system was used to examine interleukin (IL)−1β (a) and IL-6 (b) concentration in culture supernatants of CD4 + T cells, CD8 + T cells, and CD14 + monocytes. Tumor necrosis factor (TNF)-α (c) concentration was also analysed in CD14 + monocytes. mutated TTR dose-dependently increased IL-6 concentration in CD14 + monocytes. In addition, instead of cell subsets from FAP patients, we used FAP patient-derived iPS-MLs (which function like macrophages 16 and show a similar result in IL-6 concentration), as well as iPS-MLs derived from HD. However, although dose-dependency of native TTR was observed in IL-6 production in CD4 + and CD8 + T cells, there was no difference between V30M mutated and wild-type TTR. In aggregated TTR culture conditions, IL-6 production in CD14 + monocytes, iPS-MLs, and CD4 + T cells was elevated with V30M mutated TTR in comparison to wild-type TTR. In contrast, IL-6 concentration was higher in the presence of native and aggregated mutated TTR in CD14 + monocytes and iPS-MLs than CD4 + and CD8 + T cell conditions. We selected the concentration of recombinant TTR in culture (1000 nM). Although culture conditions do not completely replicate in vivo conditions, as TTR circulates constantly in the blood 1, 2 , the dose of recombinant TTR used here is likely to influence IL-6 production in a physiologically realistic manner.
Wild-type TTR is usually present as a tetramer in healthy subjects. However, wild-type and mutated TTR heterotetramers are unstable in FAP carriers and patients, dissociating easily into wild-type and mutant monomers, with the latter being particularly susceptible to misfolding. Therefore, our ex vivo experiments of native TTR may reflect the in vivo occurrence of TTR monomers.
Megalin and the receptor for advanced glycation end products (RAGE) are known TTR receptors [17][18][19] . Although megalin expression in immune cells remains unknown, membranous expression of RAGE has been reported in human monocytes and T cells [20][21][22] . Moreover, intracellular RAGE expression is detected in human T cells following T cell receptor activation, and RAGE ligands may enhance RAGE expression via mechanisms such as endosomes 23,24 . RAGE expression levels in monocytes were higher than in T cells, and the site of expression differed, suggesting that our observations may relate to differences in RAGE expression levels or locations in different cell subsets.
Wild-type TTR inhibits amyloid formation in Alzheimer's disease 25 , and has an inhibitory effect on IL-1β production in vitro 26 . However, using two types of TTR (wild-type and mutated TTR), increased IL-1β was found in FAP nerve and mouse models 5,27 . Moreover, mutated TTR upregulates IL-6 expression in chondrocytes 6 . These results suggest that unlike wild-type TTR, mutated TTR easily initiates a pro-inflammatory state, and this phenomenon in FAP carriers may be a potential risk for FAP onset. Despite our result of elevated IL-6 concentration in FAP patients by multiple regression analysis, SEM did not show a significant and positive relationship between IL-6 and TTR. Considering our finding that aggregated TTR also produces IL-6 ex vivo, and the fact that FAP patients show amyloid fibril deposition originated from both wild-type and mutated TTR in several organs, it is possible that deposited amyloid, rather than mutated TTR, is the main inducer of pro-inflammatory cytokines such as IL-6. Consequently, the relationship might not be confirmed by SEM.
Our study has limitations with respect to the number of carriers and longitudinal data. Additionally, our FAP carriers and patients largely had a V30M phenotype. Thus, although our data shows that IL-6 concentration was also elevated in other phenotypes compared with HD, the number of these phenotypes was small and the IL-6 state in unexamined phenotypes is unknown. Therefore, whether elevated IL-6 in FAP carriers and patients is a common phenomenon for all phenotypes is not known. Besides, high IL-6 concentration in FAP may be attributed to causes other than existence of native mutated TTR. Further studies are needed to assess temporal changes in IL-6 concentration and the relationship between IL-6 and FAP onset in increased numbers of FAP carriers (including a wider variety of phenotypes). Nonetheless, we believe that mutated TTR may increase the risk of inflammation involving IL-6.

Materials and Methods
Blood samples. Serum samples were collected from 66 non-liver transplantation FAP patients (39 V30M, two V30M/V30M, one V30M/V50M, one F33V, one A36D, two G47R, two G47V, one T49I, two S50I, one S50R, one G53E, one L55P, one T59R, one T60A, one Q61K, one S77Y, one K80R, two E89K, two I107V, and three Y114C), 21 FAP carriers (14 V30M, three S50I, one I107V, and three Y114C), and 57 HD. FAP carrier diagnosis was determined by genetic analysis. FAP diagnosis was confirmed based on clinical phenomena, amyloid deposition in tissue, and genetic diagnosis. More clinical information on FAP carriers and patients is provided in Table 3 and 4. Samples were stored at −80 °C until the time of assay at Kumamoto University Hospital between 2010 and 2016. Written informed consent was obtained from all participants after the procedure had been fully explained. A cross-sectional study was performed using these serum samples, with further detailed information shown in Supplementary Tables S1 and S3. To prepare human CD4 + and CD8 + T cells, and CD14 + monocytes, blood samples from HD were collected at Kumamoto University Hospital. All experiments using human samples were performed in accordance with the Declaration of Helsinki and the approval of the Institutional Review Board of Kumamoto University (Permit Number: 1087).
ELISA for serum samples. Stored serum samples were centrifuged for 15 minutes at 1,000× g before assays.
Serum IL-6 levels were measured using human IL-6 Quantikine immunoassays (R&D Systems, Minneapolis, MN, USA). Serum TTR and hs-CRP concentration were determined at a central clinical laboratory in Kumamoto University Hospital.

Statistical analysis.
Because FAP is a rare neurodegenerative disease, sample size was determined with consideration of the number of outpatients to Kumamoto University hospital during the survey period. To ascertain a normal distribution of variables, Shapiro-Wilk's test was performed. For univariate analysis, one-way analysis of variance or the Kruskal-Wallis test were used. Additionally, the pairwise t or Wilcoxon rank sum test with Bonferroni correction were used for continuous variables. For categorical variables, pairwise Fisher's exact test with Bonferroni correction was performed. Multiple regression analysis was used to confirm differences in serum IL-6 concentration between groups. These analyses were performed using R version 3.3.1 (The R Foundation for Statistical Computing, Vienna, Austria). To investigate the significance and similarity of pathways between HD and FAP carriers and patients, SEM with observed measurements were used. The effect of age and sex were  adjusted accordingly in the model 8,9 . Some variables were log-transformed to approximate a normal distribution after visual investigation of a measurement's distribution. STATA version 14.1 (Stata Corp., College Station, TX, USA) was used to fit the above models, with two-sided tests performed and the level of statistical significance set at p < 0.05.