An Altered Relationship Between Soluble TREM2 and Inflammatory Markers in Young Adults with Down Syndrome

An Altered Relationship between Soluble TREM2 and Inflammatory Markers in Young Adults with Down Syndrome: A Preliminary Report This information is current as of September 25, 2021. Grace E. Weber, Katherine A. Koenig, Maria Khrestian, Yvonne Shao, Elizabeth D. Tuason, Marie Gramm, Dennis Lal, James B. Leverenz and Lynn M. Bekris J Immunol published online 20 January 2020 http://www.jimmunol.org/content/early/2020/01/17/jimmun Downloaded from ol.1901166

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 20, 2020, doi:10.4049/jimmunol.1901166 The Journal of Immunology

An Altered Relationship between Soluble TREM2 and Inﬂammatory Markers in Young Adults with Down Syndrome: A Preliminary Report

Grace E. Weber,*,1 Katherine A. Koenig,†,1 Maria Khrestian,* Yvonne Shao,* Elizabeth D. Tuason,* Marie Gramm,*,‡ Dennis Lal,* James B. Leverenz,x and Lynn M. Bekris*

Individuals with Down syndrome (DS) develop Alzheimer’s disease (AD)–related neuropathology, characterized by amyloid plaques with amyloid b (Ab) and neurofibrillary tangles with tau accumulation. Peripheral inflammation and the innate immune response are elevated in DS. Triggering receptor expressed in myeloid cells 2 (TREM2) genetic variants are risk factors for AD and other neurodegenerative diseases. Soluble TREM2 (sTREM2), a soluble cleavage product of TREM2, is elevated in AD cerebro- Downloaded from spinal fluid and positively correlates with cognitive decline. There is relatively little information about TREM2 in DS. Our objective was to examine the relationship between sTREM2 and inflammatory markers in young adults with DS, prior to the development of dementia symptoms. Because TREM2 plays a role in the innate immune response and has been associated with dementia, the hypothesis of this exploratory study was that young adults with DS predementia (n = 15, mean age = 29.5 y) would exhibit a different relationship between sTREM2 and inflammatory markers in plasma, compared with neurotypical, age-matched controls (n = 16, mean age = 29.6 y). Indeed, young adults with DS had significantly elevated plasma sTREM2 and inflammatory http://www.jimmunol.org/ markers. Additionally, in young adults with DS, sTREM2 correlated positively with 24 of the measured cytokines, whereas there were no significant correlations in the control group. Hierarchical clustering of sTREM2 and cytokine concentrations also differed between the groups, supporting the hypothesis that its function is altered in people with DS predementia. This preliminary report of human plasma provides a basis for future studies investigating the relationship between TREM2 and the broader immune response predementia. The Journal of Immunology, 2020, 204: 000–000.

n the last half of the 20th century, the life expectancy of resulting from three copies of APP leads to Ab plaque depo- individuals with Down syndrome (DS) has increased to sition in ∼50% of people with DS over the age of 30 and by guest on September 25, 2021 I 60 years (1–3), highlighting the importance of understanding subsequently increases their risk for dementia in middle age age-related disease in this population (4). Most individuals with (around age 40) (5, 6). The study of early adulthood in DS DS carry three full copies of chromosome 21. The amyloid potentially provides a unique model for the early biology of precursor protein (APP) gene, located on chromosome 21, is Alzheimer’s disease (AD) in the general population. processedintoamyloidb (Ab). The overabundance of Ab Assessment of subtle cognitive changes is challenging in DS, partly because of the presence and variability of intellectual *Genomic Medicine Institute, Cleveland Clinic, Cleveland, OH 44195; †Imaging Insti- disability (7, 8). Therefore, defining biomarkers of dementia tute, Cleveland Clinic, Cleveland, OH 44195; ‡Cologne Center for Genomics, Univer- x progression is critical in this population of individuals. AD sity of Cologne, 50931 Cologne, Germany; and Cleveland Clinic Lou Ruvo Center for neuropathology, amyloid, and tau accumulate early in DS, and Brain Health, Neurological Institute, Clevland Clinic, Cleveland, OH 44195 plasma Ab and tau represent potential biomarker candidates 1G.E.W. and K.A.K. contributed equally. (9–11). In DS, the variable severity of Ab deposition within age ORCIDs: 0000-0002-4273-7911 (G.E.W.); 0000-0002-1504-7130 (K.A.K.); 0000- 0002-1621-4711 (M.K.); 0000-0003-4125-1351 (E.D.T.). groups and age of onset of dementia suggest that factors other than the presence of triplication of the APP gene contribute to Received for publication September 26, 2019. Accepted for publication December 13, 2019. the timing and severity of amyloid deposition and therefore the This work was supported by U.S. Department of Defense Grant AZ160058, National onset of AD (5, 12–14). Institute on Aging (NIA) Grants P30AG062428, R01 AG046543, and RF1 AG051495, Biomarkers of peripheral inflammation and the innate im- National Institute of Neurological Disorders and Stroke Grant U01 NS100610, NIA mune response are elevated in DS (15–18). Triggering receptor Grants R01 AG057552 and R01 AG022304, the Alzheimer’s Association (2016-NIRG- 395867), and the Jane and Lee Seidman Fund. expressed in myeloid cells 2 (TREM2) is a pattern recognition Address correspondence and reprint requests to Dr. Lynn M. Bekris, Cleveland receptor that can be activated by both pathogen- and danger- Lerner College of Medicine of Case Western Reserve University, Genomic Medicine associated molecular patterns in the innate immune response Institute, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave- (19, 20). Loss-of-function genetic variants in TREM2, located nue, R4, Cleveland, OH 44195. E-mail address: [email protected] on chromosome 6, are risk factors for AD and other neurode- The online version of this article contains supplemental material. generative diseases (21–23). Soluble TREM2 (sTREM2), a solu- Abbreviations used in this article: Ab, amyloid b; AD, Alzheimer’s disease; APP, amyloid precursor protein; CRP, C-reactive protein; DS, Down syndrome; FDR, false ble cleavage product of TREM2, is elevated in AD cerebrospinal discovery rate; Flt-3L, FMS-like tyrosine kinase 3 ligand; GRO, growth-regulated fluid and positively correlates with Ab and cognitive decline oncogene; MDC, macrophage-derived chemokine; sCD40L, soluble CD40-ligand; (24–32). There is relatively little information about TREM2 in sTREM2, soluble TREM2; TREM2, triggering receptor expressed in myeloid cells 2. DS, although one group describes elevated TREM2 protein Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 levels in young adults and declining levels with aging (33, 34).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901166 2 ALTERED SOLUBLE TREM2 AND CYTOKINES IN DOWN SYNDROME

The hypothesis of this investigation was that there is a rela- CCL7), IL-12 40 kDa (IL-12p40), macrophage-derived chemokine (MDC), tionship between sTREM2 and inflammatory markers in DS, prior IL-12 70 kDa (IL-12P70), IL-13, IL-15, soluble CD40-ligand (sCD40L), to the development of dementia symptoms. We found that young IL-17A, IL-1 receptor agonist (IL-1RA), IL-1a,IL-9,IL-1b,IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IFN-g inducible protein 10 kDa (IP-10), adults with DS displayed an altered immune profile compared with MCP-1 (also known as CCL2), macrophage inflammatory protein (MIP) neurotypical controls, with increased levels of sTREM2 and in- 1a (MIP-1a, also known as CCL3), MIP-1b (also known as CCL4), flammatory markers in plasma. In young adults with DS, sTREM2 TNF-a, TNF-b, and vascular endothelial growth factor (VEGF). correlated positively with 24 of the 38 measured cytokines, whereas Statistical analyses there were no significant correlations in the control group. In addition, sTREM2 clustered with different cytokines in the two DS and control demographics were compared using unpaired t test for age and Fisher exact test for APOE4 status and sex distribution. Plasma groups. The results of this preliminary report implicate an al- sTREM2andinflammatorymarkersweremeasuredinplasmaonplates tered relationship between sTREM2 and inflammatory markers that also contained buffer-alone background wells (containing 0 pg/ml of in young adults with DS predementia. each protein) and standard curve wells containing known concentrations of each protein measured. Concentrations of analytes were determined Materials and Methods based on fluorescence of the standard curves for the respective proteins. Concentration values were log10 transformed for comparison. Values Study design below fluorescence levels of the background wells were considered to be Fifteen young adults with DS (mean age: 29.5 y, range: 26–34 y; five physiologically 0 pg/ml and set equal to 0.001 pg/ml to be able to log females) and 16 neurotypical age-matched controls (mean age: 29.6 y, transform the data. Individual plasma samples were run in duplicate on range: 25–36 y; four females) were enrolled in the study under a protocol two different plates, and the overall mean of the four replicates was determined. Replicates with a high coefficient of variation (.30%) were approved by the Cleveland Clinic Institutional Review Board. We have Downloaded from reviewed the medical histories of the participants and found that none have analyzed to identify outliers, which were then removed from the anal- reported systemic inflammatory diseases or known infections at the time of ysis. Complete blood count, biomarker, and inflammatory marker data enrollment. No participants reported use of nonsteroidal anti-inflammatory were compared between groups using unpaired t tests, and false dis- drugs, which could have impacted their cytokine profiles. For participants covery rates (FDR) were determined using the original FDR method of with DS, inclusion criteria included a medical diagnosis of DS, age of Benjamini and Hochberg, with Q = 1%. Linear regression was used to 25–35 y, and the ability to assent to participation and complete the study determine correlations between analytes using SPSS version 22 (IBM, procedures. Participants were excluded for a history of major neurologic or Armonk, NY). Correlograms were made using the “corrplot” function in psychiatric illness other than DS or documented symptoms of dementia. the corrplot package (39) and arranged according to hierarchical clustering http://www.jimmunol.org/ Participants with DS provided verbal or written informed assent, as ap- using the Ward method in R version 3.6.1 (R Foundation for Statistical propriate, and their guardians provided written informed consent. Control Computing, Vienna, Austria). Heat maps, including hierarchical clustering participants provided written informed consent. Biospecimens were with the Ward method, were produced using the “heatmap.2” function in collected and processed through the Lou Ruvo Center for Brain Health the gplots package (40) in R 3.6.1. Data were scaled using the “scale” Aging and Neurodegenerative Disease Biobank. Prior to additional function in R prior to input into the corrplot and heatmap.2 functions. All study interventions, participants with DS underwent a comprehensive other data analysis used GraphPad Prism version 8.1.1 (GraphPad Soft- clinical assessment to minimize the chance of including participants ware, San Diego, CA). that were showing subtle signs of dementia. This assessment was completed by a psychiatrist specializing in developmental disabilities and Results included a psychiatric diagnostic interview with participant and care- by guest on September 25, 2021 giver, medical history, and mental status examination (35). Participant Cohort characteristics caregivers also completed the Dementia Screening Questionnaire for Young adults with DS did not differ significantly from neuro- Individuals with Intellectual Disabilities (36) and the Dementia Ques- tionnaire for People with Learning Disabilities (37). No participants were typical controls in terms of age (mean: 29.5 y versus 29.6 y, judged to be showing signs of dementia. p = 0.89), distribution of sex (33.3% versus 25.0%, p =0.70),or presence of the AD risk allele APOE4 (13.3% versus 18.8%, Complete blood counts p . 0.99) (Table I). Venipunctures were performed for the collection of whole blood, and plasma was isolated from lavender-top EDTA tubes. Complete blood counts Complete blood counts and inflammatory markers were performed on most participants (n = 14 DS and n = 15 controls) via Plasma sTREM2 was significantly elevated in DS compared microscopy by trained technicians blinded to sample group. with neurotypical controls (p = 0.000966) (Fig. 1A). Markers of APOE4 genotyping inflammation, C-reactive protein (CRP) and erythrocyte sedimentation rate, were compared; CRP was elevated in DS APOE4 genotyping was performed from blood samples using the 7500 Real-Time PCR System and TaqMan SNP Genotyping Assays (rs429358, (p = 0.0169), and erythrocyte sedimentation rate trended to- rs7412) (Thermo Fisher Scientific), as previously described (38). ward being elevated in DS (p = 0.05098) compared with controls (Fig. 1B, 1C). Participants with DS had significantly higher Plasma biomarkers percentages of basophils (p = 0.000185) and trended toward Plasma sTREM2 levels were measured using a Luminex 200 3.1 xPONENT higher percentages of neutrophils (p = 0.087) compared with System (EMD Millipore, Chicago, IL) and a custom detection method controls, whereas percentages of lymphocytes and mono- designed to capture the soluble portion of TREM2 protein, as previously described (27). Briefly, a capture Ab bound to MagPlex beads binds cytes did not differ between groups (Fig. 1D). Participants with sTREM2 (human TREM2 Ab MAB1828 [R&D Systems], monoclonal DS had fewer lymphocytes (p = 0.037) and more basophils mouse IgG2B clone 263602 [His19-Ser174; Immunogen]). A biotinylated (p = 0.001) compared with controls (Fig. 1E) when comparing Ab with a streptavidin/phycoerythrin conjugate was used for detection cell counts. (human TREM2 biotinylated Ab BAF1828 [R&D Systems], Ag affinity- purified polyclonal goat IgG [His19-Ser174; Immunogen]). Inflamma- Levels of plasma sTREM2 and plasma inflammatory markers in tory markers were measured with a human cytokine/chemokine panel DS predementia using Luminex 200 xMap technology and the MILLIPLEX MAP multiplex kits (HCYTMAG60PMX41BK; EMD Millipore) following Given prior observations of elevated peripheral immune factors the manufacturer’s instructions for analyte detection in human plasma. (15, 16, 18, 41) and our own finding of elevated sTREM2 in DS, The inflammatory markers in the panel were as follows: epidermal we addressed the question of whether other plasma inflammatory growth factor (EGF), fibroblast growth factor 2 (FGF-2), eotaxin, TGF-a, G-CSF, FMS-like tyrosine kinase 3 ligand (Flt-3L), GM-CSF, fractalkine markers (cytokines, chemokines, and growth factors) were elevated (also known as CX3CL1), IFN-a2, IFN-g, growth-regulated oncogene in our DS cohort using an immune profiling panel. Inflammatory (GRO), IL-10, monocyte chemotactic protein-3 (MCP-3, also known as markers were grouped based on known function into the general The Journal of Immunology 3

Table I. Population description The relationship between peripheral sTREM2 and peripheral inflammatory markers Neurotypical DS p Value Pearson correlations were plotted, and the correlation matrix was n 16 15 — clustered based on the Ward method of least variance. Importantly, Age, mean (SD) 29.6 (3.6) 29.5 (2.5) 0.8901 sTREM2 showed statistically significant correlations only in the % female 25.0 33.3 0.7043 % APOE ε4+ 18.8 13.3 .0.9999 group with DS, and in each instance, the correlations were positive. We observed significant positive correlations with sTREM2 and 24 Characteristics of the study populations were compared between neurotypical controls (n = 16) and individuals with DS (n = 15). There were no statis- out of 38 measured cytokines in the DS group (MDC, sCD40L, tically significant differences in age (unpaired t test), gender, or APOE4 status TNF-a, TGF-a, IFN-a2, IL-6, Flt-3L, GM-CSF, IL-1b, IL-12p70, (Fisher exact test) between groups. IFN-g, MIP-1b, IL-17A, VEGF, IL-5, eotaxin, IL-8, IL-3, IL-4, IL-12p40, IL-9, IL-7, Il-10, and IL-15) and 0 out of 38 measured categories “proinflammatory,” “immunoregulatory/pleiotropic,” cytokines in the control group (Fig. 3, Pearson correlation co- and “anti-inflammatory” (42–51). Values below assay back- efficients [r]andp values are shown in Supplemental Table I). ground levels were determined to be physiologically 0 pg/ml Additionally, the clustering showed distinctly different patterns and set as 0.001 pg/ml for the purpose of log transformation in neurotypical controls compared with the DS group (Fig. 3). and analysis. Out of the 38 factors tested, 32 were significantly To further investigate this relationship, hierarchical clustering higher in DS compared with age-matched controls (Fig. 2); of the scaled log10-transformed immune factor concentration theexceptionswereIL-4,FGF-2,MDC,IL-17A,GRO,and values was performed on each group. Lower sTREM2 levels in Downloaded from MCP-1. To determine if it was valid to set values below neurotypical controls (Fig. 1A) clustered closest to GRO, Flt-3L, background equal to 0.001, we repeated the analysis after re- MDC, and IP-10, whereas higher sTREM2 in the DS group moving all values that had been below background detection (Fig. 1A) clustered with MDC, sCD40L, and TNF-a (Fig. 4). levels (Supplemental Fig. 1A). We found that 30 out of 38 cytokines were significantly higher in DS compared with con- Discussion trols; the exceptions were IL-13,G-CSF,IL-6,MDC,IL-17A, In this preliminary report, we found evidence of peripheral in- GRO, sCD40L, and MCP-1 (Supplemental Fig. 1A, 1B). flammation and elevated sTREM2 in a group of young adults with http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. Immune markers and peripheral blood cell types. Significantly higher plasma sTREM2 levels in young adults with DS predementia (n = 15), compared with neurotypical controls (n = 16) (p = 0.000966; 1% FDR significant) (A). Inflammatory markers CRP (B) and erythrocyte sedimentation rate (C) were elevated in DS compared with controls. WBC subset percentages (D) and counts (E) were compared between groups. Unpaired t tests were performed between groups and p values are shown. 4 ALTERED SOLUBLE TREM2 AND CYTOKINES IN DOWN SYNDROME Downloaded from http://www.jimmunol.org/

FIGURE 2. Plasma inflammatory markers in DS predementia. Significantly higher plasma inflammatory markers in young adults with DS predementia by guest on September 25, 2021 (n = 15, dark gray bars), compared with neurotypical controls (n = 16, light gray bars) for 32 out of 38 of the inflammatory markers (A). Significance as indicated by unpaired t test p values (asterisk denotes 1% FDR significance) (B). Inflammatory markers are separated by known functions into the general categories of anti-inflammatory, immunoregulatory/pleiotropic, and proinflammatory. Individual data points that had calculated concentrations below background levels for the assay were considered physiological 0s and set equal to 0.001 prior to log transformation for analysis purposes. Lines indicate mean levels. Minimum and maximum levels are indicated.

DS without symptoms of dementia. These participants with DS had immunity in the variable age-at-onset of dementia in DS. Addi- increased levels of anti-inflammatory, proinflammatory, and im- tionally, it would be helpful to include other neurotypical control munoregulatory cytokines compared with neurotypical controls, groups, such as APOE ε4 noncarriers at low risk for AD or in- which cannot be explained by known acute or autoimmune ill- dividuals with systemic inflammatory diseases that are not asso- nesses in our cohort. In fact, the DS participants had lower levels ciated with dementia. of lymphocytes compared with controls. Although the observed We observed elevated cytokines in the group with DS compared basophilia has been reported in DS (52), significantly decreased with the neurotypical group. Our findings were similar to another lymphocyte count and elevated (or trending toward elevated) study that found elevated TNF-a, IL-6, and IL-10 in adults with neutrophils have been reported in AD (53, 54). DS predementia (15). Interestingly, this previous report predicted Plasma sTREM2 was significantly elevated in DS; interestingly, AD development when these inflammatory markers were com- sTREM2 is elevated in cerebrospinal fluid in the mild cognitive bined with measures of Ab (15). In further support of our findings, impairment stage of AD and in early-onset forms of AD but not in another report describes increased IL-6, VEGF-A, MCP-1, IL-22, peripheral biofluids, such as plasma (24, 25, 27, 28, 55). The el- and TNF-a in adults aged 20–65 with DS (56). In contrast, a evation of sTREM2 in young DS predementia in plasma (Fig. 1A) previous study of adults (mean age 30 y in DS and controls) suggests that peripheral sTREM2 may increase very early in found elevated serum MIP-1a but not IFN-g,TNF-a, MIP-1b, individuals with elevated Ab. Our previous work showed a RANTES, or IL-6 (57). The similarities across these studies and relationship between plasma sTREM2 and CRP in AD-related our own preliminary report support the notion that, in general, mild cognitive impairment (predementia) (27). This was not ob- the immune response is altered in DS, which may contribute to served in this DS predementia cohort. This discrepancy may be the development of AD. related to differing stages of AD-related progression in DS pre- Our findings demonstrated that sTREM2 differed in its dementia compared with AD-related mild cognitive impairment. clustering and correlation patterns between neurotypical and An important next study would be to compare peripheral sTREM2 DS groups. Hierarchical clustering linked sTREM2 to different in adults with DS predementia to adults with DS and dementia. inflammatory markers within the control and DS groups. Because These future analyses would help elucidate the role of the innate the controls had lower levels of sTREM2, a logical conclusion is The Journal of Immunology 5 Downloaded from

FIGURE 3. sTREM2 correlates positively with many inflammatory markers in DS. Pearson correlations of sTREM2 and inflammatory markers in plasma from neurotypical controls (A) versus young adults with DS (B). Color gradient shows Pearson correlation coefficients (r), with dark blue = 1, indicating a perfect positive correlation, and red = 21, indicating a perfect negative correlation. The clustering patterns were determined by Ward method of least http://www.jimmunol.org/ variance and differ between groups; negative correlations were seen only in the control group. sTREM2 is highlighted with a red arrow. Red asterisks next to inflammatory marker labels in the vertical orientation indicate significant correlation with sTREM2 of p , 0.05. Asterisks within the boxes indicate significance. *p , 0.05, **p , 0.01, ***p , 0.001. that they had more functional, membrane-bound TREM2, an MDC in the context of AD, but one study found that levels of important immune factor for normal innate immunity function MDC in cerebrospinal fluid decreased after 1 y of resveratrol (19, 20). This remains to be determined but may have important treatment in AD compared with the placebo group (71), which implications for the cluster analysis results observed. The clus- may implicate MDC in neuroinflammatory pathways. Together, tering of sTREM2 with Flt-3L and GRO in neurotypical controls it was clear that people with DS had an altered relationship by guest on September 25, 2021 (Fig. 4A) is novel (to our knowledge) information and interesting between sTREM2 and inflammatory markers, which could be because Flt-3L is important for proliferation of hematopoietic contributing to the accelerated onset of AD in this population. stem cells. Knockout mice models have shown that it is crucial This is a preliminary report that is limited by the small sample for the development of hematopoietic progenitor cells, B cells, size and potential for false-negative results. DS biomarker studies, and dendritic cells (58, 59). Flt-3L was elevated in our DS including our study, are limited by sample size and should be predementia cohort, suggesting that normally the role of TREM2 considered with caution until replicated in larger populations cleavage and consequent production of sTREM2 may be related (10, 15, 72, 73). Covariates such as age and APOE4 status did to Flt-3L activity. Although GRO grouped with sTREM2 in not differ between our two groups but are important to control controls, it was not significantly elevated in our DS cohort, for in larger future studies. Another limitation of this study is suggesting a weaker role in DS than Flt-3L. GRO is expressed that karyotypes were not available for the DS participants. in monocytes and neutrophils and has proinflammatory and mi- Although unlikely, given the unique clinical presentation of togenic functions (60, 61) and may have an important TREM2- these two types of patients, it is possible that some of the DS related role in normal innate immunity function. TREM2 is participants had mosaic DS or chromosome 21 translocations, similarly involved in cell proliferation, specifically of micro- rather than full trisomy 21. This could lead to heterogeneity of glia and macrophages through an interaction with the adapter the subjects. Additionally, we were not able to collect cere- protein DNAX activating protein of 12 kDa (DAP12) (62). brospinal fluid from these subjects. Cerebrospinal fluid has been In contrast, elevated sTREM2 in the DS group clustered with studied more in depth in AD studies compared with plasma and TNF-a, sCD40L, and MDC (Fig. 4B). Whereas MDC had a could lend additional evidence of the contribution of sTREM2 similar concentration in both the DS and control groups, TNF-a and the observed immune state in DS. Furthermore, inclusion and sCD40L were significantly elevated in DS. TNF-a is an acute- of DS with dementia was beyond the limits of this preliminary phase cytokine secreted by cells including activated macrophages report. However, future studies of progressive changes in inflam- and brain microglial, which also express TREM2 (58, 63, 64). matory markers in not only young adults with DS predementia but TNF-a has been implicated in AD pathology numerous times also DS of multiple ages, including children with DS and DS with and is reportedly associated with AD progression (65, 66). dementia, will greatly enhance our understanding of the role of sCD40L (CD154) is an inflammatory cytokine secreted by acti- inflammation in progressive neurodegeneration. vated platelets. It has been shown to be elevated in early AD, and The results of this preliminary report strongly suggest a rela- there is evidence that it contributes to disease progression (67–69). tionship between plasma sTREM2 and inflammatory markers in MDC (or CCL22) is expressed by activated T cells, NK cells, DS predementia. We observed significantly elevated inflammatory macrophages, and monocytes and has chemoattractant and in- markers in DS, in agreement with previous reports (16, 18, 41, 74). flammatory properties (70). Few studies to date have looked at Notably, we observed a strong positive correlation between many 6 ALTERED SOLUBLE TREM2 AND CYTOKINES IN DOWN SYNDROME Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Plasma sTREM2 clusters with MDC, sCD40L, and TNF-a in adults with DS, not controls. Hierarchical clustering of sTREM2 and inflammatory markers in the two groups. sTREM2 clustered with Flt-3L and GRO in the neurotypical control group (A) and with MDC, sCD40L, and TNF-a in the group with DS (B). Participants in each group, shown in the rows, were arranged based on hierarchical clustering. Inflammatory markers are separated by known functions into the general categories of anti-inflammatory (blue), immunoregulatory/pleiotropic (lavender), and proinflammatory (red). Individual data points that had calculated concentrations below background levels for the assay were considered physiological 0s and set equal to 0.001 prior to log transformation for analysis purposes. Heat map values shown were scaled, log-transformed cytokine concentrations. Yellow indicates higher levels compared with red. of the tested immune factors and sTREM2 in the group with DS 4. Moran, J. A., M. S. Rafii, S. M. Keller, B. K. Singh, and M. P. Janicki, American Academy of Developmental Medicine and Dentistry, Rehabilitation Research and that was not observed in controls. Given that our cohort of young Training Center on Aging With Developmental Disabilities, University of Illinois at adults with DS were all predementia, these observed correlations Chicago, American Association on Intellectual and Developmental Disabilities. with sTREM2 may be early predictors of dementia onset. To 2013. The National Task Group on Intellectual Disabilities and Dementia Practices consensus recommendations for the evaluation and management of dementia in our knowledge, this is the first report of a correlation between adults with intellectual disabilities. Mayo Clin. Proc. 88: 831–840. sTREM2 and multiple inflammatory markers in DS. This finding 5. Wisniewski, K. E., H. M. Wisniewski, and G. Y. Wen. 1985. Occurrence of is supported by previously described dysregulation of the immune neuropathological changes and dementia of Alzheimer’s disease in Down’s syndrome. Ann. Neurol. 17: 278–282. response in DS (16, 18, 41, 74) and suggests that TREM2 plays a 6. Schupf, N., and G. H. Sergievsky. 2002. Genetic and host factors for dementia in role in the peripheral immune response in DS predementia. Down’s syndrome. Br. J. Psychiatry 180: 405–410. 7. Rosser, T. C., J. O. Edgin, G. T. Capone, D. R. Hamilton, E. G. Allen, K. J. Dooley, P. Anand, J. F. Strang, A. C. Armour, M. A. Frank-Crawford, et al. Disclosures 2018. Associations between medical history, cognition, and behavior in youth The authors have no financial conflicts of interest. with Down syndrome: a report from the down syndrome cognition project. Am. J. Intellect. Dev. Disabil. 123: 514–528. 8. Sabat, C., M. Tasse´, and M. Tenorio. 2019. Adaptive behavior and intelligence in adolescents with down syndrome: an exploratory investigation. Intellect. Dev. References Disabil. 57: 79–94. 1. Bittles, A. H., and E. J. Glasson. 2004. Clinical, social, and ethical implications 9. Englund, H., G. Annereń, J. Gustafsson, U. Wester, J. Wiltfang, L. Lannfelt, of changing life expectancy in Down syndrome. Dev. Med. Child Neurol. 46: K. Blennow, and K. Ho¨glund. 2007. Increase in beta-amyloid levels in cere- 282–286. brospinal fluid of children with Down syndrome. Dement. Geriatr. Cogn. Disord. 2. Carfı`, A., M. Antocicco, V. Brandi, C. Cipriani, F. Fiore, D. Mascia, 24: 369–374. S. Settanni, D. L. Vetrano, R. Bernabei, and G. Onder. 2014. Characteristics 10. Lee, N. C., S. Y. Yang, J. J. Chieh, P. T. Huang, L. M. Chang, Y. N. Chiu, of adults with down syndrome: prevalence of age-related conditions. Front. A. C. Huang, Y. H. Chien, W. L. Hwu, and M. J. Chiu. 2017. Blood beta-amyloid Med. (Lausanne) 1: 51. and tau in Down syndrome: a comparison with Alzheimer’s disease. Front. 3. Bayen, E., K. L. Possin, Y. Chen, L. Cleret de Langavant, and K. Yaffe. 2018. Aging Neurosci. 8: 316. Prevalence of aging, dementia, and multimorbidity in older adults with down 11. Fortea, J., M. Carmona-Iragui, B. Benejam, S. Fernańdez, L. Videla, I. Barroeta, syndrome. JAMA Neurol. 75: 1399–1406. D. Alcolea, J. Pegueroles, L. Munõz, O. Belbin, et al. 2018. Plasma and CSF The Journal of Immunology 7

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Neurotypical Controls Down Syndrome

Inflammatory Correlation coefficient Correlation coefficient Marker (r) p-value (r) p-value

IL-1RA -0.11938 0.659661 0.373011 0.170883 IL-4 0.282265 0.289507 0.636508 0.010732 IL-5 0.188979 0.483332 0.589516 0.020732 IL-10 0.03364 0.901569 0.53105 0.041655

inflammatory

- IL-13 0.163148 0.546027 0.387818 0.153195 FGF-2 0.183802 0.495612 0.359753 0.187817

Anti G-CSF 0.021051 0.938321 0.463081 0.082149

/ IL-6 -0.45213 0.078701 0.653647 0.00822

IL-7 -0.36549 0.163896 0.601404 0.017712 IL-9 -0.23644 0.377982 0.539851 0.037781 TGF-α -0.05808 0.830823 0.612446 0.015221 Pleiotropic Fractalkine 0.031534 0.907708 0.341302 0.213137

Immunoregulatory MDC -0.09112 0.737152 0.720114 0.002465 IL-1a 0.196437 0.465903 0.434714 0.105383 IL-1B 0.094974 0.726434 0.643217 0.009686 IL-2 0.207458 0.440728 0.700044 0.003663 IL-3 0.187139 0.487678 0.743038 0.001503 IL-8 0.345878 0.189443 0.561442 0.02942 IL-12P40 -0.10373 0.702234 0.519073 0.047391 IL-12P70 -0.33975 0.197932 0.584502 0.022118 IL-15 0.143349 0.596368 0.647416 0.009073 IL-17A 0.246765 0.356866 0.733969 0.001839

IP-10 -0.2239 0.404497 0.366394 0.179205

ry EGF 0.181262 0.50169 0.335067 0.222159 Eotaxin 0.375012 0.152364 0.620537 0.013575 Flt-3L 0.418974 0.106247 0.635159 0.010952

inflammato GM-CSF -0.26334 0.324399 0.668044 0.00649

- IFNa2 -0.01583 0.9536 0.539287 0.038021

Pro IFNy -0.13981 0.605549 0.708296 0.003124 GRO 0.332197 0.208726 0.335196 0.22197 MCP-1 0.186366 0.489512 0.409892 0.129171 MCP-3 0.114938 0.671661 0.416814 0.1222 sCD40L -0.20058 0.456354 0.77983 0.000607 MIP-1α -0.16915 0.531151 0.376438 0.166675 MIP-1β -0.02389 0.930033 0.685127 0.004823 TNFα -0.22831 0.395057 0.815007 0.000214 TNFβ 0.153127 0.571271 0.445747 0.09586 VEGF -0.0756 0.780819 0.758205 0.001054

1 There were significant correlations (Pearson correlation not corrected for multiple comparisons) between plasma sTREM2 and plasma inflammatory markers in young adults with DS but not neurotypical controls. P-values <0.05 are bolded. A

Supplemental Figure 1. Plasma Inflammatory Markers in B Down Syndrome Pre-dementia (removing values below background detection). Significantly higher plasma inflammatory markers in young adults with Down Syndrome pre-dementia (dark grey bars), compared to neurotypical controls (light grey bars) for 30/38 of the inflammatory markers on the panel (A), significance as indicated by unpaired t-test p-values (asterisk denotes 1% FDR significance) (B). Inflammatory markers are separated by known functions into the general categories of anti- inflammatory, immunoregulatory/pleiotropic, and proinflammatory. Individual data points that had calculated concentrations below background levels for the assay were considered physiological 0s and removed from the analysis. Participants analyzed per group differed based on cytokine due to the number of values below background, as noted in B. Lines indicate mean levels. Minimum and maximum levels are indicated.