ORIGINAL RESEARCH published: 12 July 2021 doi: 10.3389/fimmu.2021.685139 Identification of the Immunological Changes Appearing in the CSF During the Early Immunosenescence Process Occurring in Multiple Sclerosis Edited by: Deborah K. Dunn-Walters, Carmen Picón 1,2†, Amalia Tejeda-Velarde 1†, José Ignacio Fernández-Velasco 1, University of Surrey, United Kingdom Manuel Comabella 3, Roberto Álvarez-Lafuente 4, Ester Quintana 5, Susana Sainz de la Maza 6, Reviewed by: Enric Monreal 6, Noelia Villarrubia 1, José Carlos Álvarez-Cermeño 6, Daming Zuo, Marı́a Inmaculada Domı́nguez-Mozo 4, Lluı́s Ramió-Torrentà 5, Eulalia Rodrı́guez-Martı́n 1, Southern Medical University, China Ernesto Roldán 1, Yolanda Aladro 7, Silvia Medina 1, Mercedes Espiño 1, Jaime Masjuan 6, Xiaoming Zhang, Clara Matute-Blanch 3, Marta Muñoz-San Martı́n 5, Carmen Espejo 3, Carmen Guaza 8, Institut Pasteur of Shanghai (CAS), Alfonso Muriel 9, Lucienne Costa-Frossard 6 and Luisa Marı́a Villar 1* China 1 Department of Immunology, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigacón Sanitaria (IRYCIS), *Correspondence: Luisa Marı´a Villar Red Española de Esclerosis Múltiple (REEM), Madrid, Spain, 2 Department of Brain Science, Imperial College London, London, luisamaria.villar@salud.madrid.org United Kingdom, 3 Servei de Neurologia-Neuroimmunologia, Centre d’ Esclerosi Múltiple de Catalunya (Cemcat), Vall d’ Hebron Institut de Recerca, Hospital Universitari Vall d’ Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain, 4 Department of † These authors have contributed Neurology, Hospital Clı´nico San Carlos, Madrid, Instituto de Investigación Sanitaria del Hospital Clı´nico San Carlos (IdISSC), equally to this work REEM, Madrid, Spain, 5 Neuroimmunology and Multiple Sclerosis Unit, Department of Neurology, Hospital Dr. Josep Trueta, Institut d’Investigació Biomèdica de Girona (IDIBGI), Girona, Medical Sciences Department, Universitat de Girona, REEM, Girona, Specialty section: Spain, 6 Department of Neurology, Hospital Universitario Ramón y Cajal, IRYCIS, REEM, Madrid, Spain, 7 Department of This article was submitted to Neurology, Hospital Universitario de Getafe, REEM, Madrid, Spain, 8 Neuroimmunology Group, Functional and Systems B Cell Biology, Neurobiology Department, Instituto Cajal, CSIC, Madrid, Spain, 9 Clinical Biostatistics Unit, Hospital Universitario Ramón y Cajal, a section of the journal IRYCIS, CIBERESP, Nursing Department, Universidad de Alcalá, Madrid, Spain Frontiers in Immunology Received: 24 March 2021 Patients with multiple sclerosis (MS) suffer with age an early immunosenescence process, Accepted: 24 June 2021 Published: 12 July 2021 which influence the treatment response and increase the risk of infections. We explored Citation: whether lipid-specific oligoclonal IgM bands (LS-OCMB) associated with highly Picón C, Tejeda-Velarde A, inflammatory MS modify the immunological profile induced by age in MS. This cross- Fernández-Velasco JI, Comabella M, Álvarez-Lafuente R, Quintana E, sectional study included 263 MS patients who were classified according to the presence Sainz de la Maza S, Monreal E, (M+, n=72) and absence (M-, n=191) of LS-OCMB. CSF cellular subsets and molecules Villarrubia N, Álvarez-Cermeño JC, implicated in immunosenescence were explored. In M- patients, aging induced Domı´nguez-Mozo MI, Ramió-Torrentà L, Rodrı´guez-Martı´n E, remarkable decreases in absolute CSF counts of CD4+ and CD8+ T lymphocytes, Roldán E, Aladro Y, Medina S, including Th1 and Th17 cells, and of B cells, including those secreting TNF-alpha. It Espiño M, Masjuan J, Matute-Blanch C, Muñoz-San also increased serum anti-CMV IgG antibody titers (indicative of immunosenescence) and Martı´n M, Espejo C, Guaza C, Muriel A, CSF CHI3L1 levels (related to astrocyte activation). In contrast, M+ patients showed an Costa-Frossard L and Villar LM (2021) age-associated increase of TIM-3 (a biomarker of T cell exhaustion) and increased values Identification of the Immunological Changes Appearing in the CSF During of CHI3L1, independently of age. Finally, in both groups, age induced an increase in CSF the Early Immunosenescence Process levels of PD-L1 (an inductor of T cell tolerance) and activin A (part of the senescence- Occurring in Multiple Sclerosis. Front. Immunol. 12:685139. associated secretome and related to inflammaging). These changes were independent of doi: 10.3389/fimmu.2021.685139 the disease duration. Finally, this resulted in augmented disability. In summary, all MS Frontiers in Immunology | www.frontiersin.org 1 July 2021 | Volume 12 | Article 685139 Picón et al. IgM and Immunosenescence in MS patients experience with age a modest induction of T-cell tolerance and an activation of the innate immunity, resulting in increased disability. Additionally, M- patients show clear decreases in CSF lymphocyte numbers, which could increase the risk of infections. Thus, age and immunological status are important for tailoring effective therapies in MS. Keywords: multiple sclerosis, aging, innate immunity, adaptive immunity, inflammation INTRODUCTION MATERIALS AND METHODS Multiple sclerosis (MS) is an autoimmune disease that is Study Approval characterized by demyelination, chronic inflammation, and This study was approved by the Ethical Committee of Ramó n y neuronal loss, causing irreversible damage to the central Cajal University Hospital (Madrid, Spain). Written informed nervous system (CNS). Accumulating evidence suggests that consent was obtained from every patient before inclusion in aging is a risk factor for the progression of MS, and late onset the study. of the disease (40-50 years of age) is associated with earlier conversion to progressive phases (1). Additionally, age Patients reduces the capability of the CNS to remyelinate (2) and In this cross-sectional prospective study, we included 263 increases extracellular accumulation of iron (3), consecutive patients (149 females/114 males) who were diagnosed mitochondrial dysfunction (4), and chronic microglia with MS at Ramó n y Cajal University Hospital (Madrid, Spain) activation (5). according to modified McDonald criteria (18). Patients did not Age-related changes in the immune system have been receive any disease-modifying treatment before inclusion. Patient extensively studied (6, 7). Aging is generally considered to be characteristics are shown in Table 1. The expanded disability status associated with chronic low-grade inflammation, which affects scale (EDSS) score and the MS severity score (MSSS) were evaluated the innate and adaptive immune systems in a phenomenon at lumbar puncture, or in case the patient was in a relapse at that known as “immunosenescence” (8). The innate immune moment, one month after when the clinical situation was stabilized. system increases the secretion of pro-inflammatory cytokines EDSS score was measured in the whole patient cohort, MSSS in the and proteases, including Tumor Necrosis Factor (TNF)-a, 161 patients with more than six months of disease duration at interleukin (IL)-6, and metalloproteases. This is associated with lumbar puncture (41 M+ and 120 M-), and the number of relapses reduced tissue clearance and phagocytosis capacity by myeloid in the first year in the 119 patients with RRMS and more than six cells (9). On the other hand, the adaptive immune system months of disease duration (34 M+ and 101 M-). displays a reduction in the number of naïve T cells due to thymic involution, which reduces their ability to react against Samples new antigens (10). Paired serum and CSF samples were always obtained for clinical Importantly, the number of memory T cells in the purposes. Fresh CSF samples were centrifuged at 500 g for 10 min. bloodstream increases, especially the CD8 subset (11). In The cellular pellet was resuspended and analyzed for subsequent particular, an increase in memory T cells and antibodies flow cytometry studies as described below. After centrifugation, CSF specific for cytomegalovirus (CMV) has been reported, which and serum samples were aliquoted and stored at -80°C is broadly considered to be a hallmark of immunosenescence until assessment. (12). Remarkably, this process that usually occurs in people older than 65 years arises about 20 years earlier in different Immunoglobulin G and M Oligoclonal inflammatory diseases including MS. This phenomenon is Band Detection named as premature or early immunosenescence (13–15). Serum and CFS IgG, IgM, and albumin were quantified by Investigating the influence of a highly inflammatory disease in nephelometry in an Immage nephelometer (Beckman Coulter, the immunosenescence process could help to identify whether Brea, CA). Oligoclonal IgG and IgM bands were studied in paired age-related changes are a uniform process in MS or depend on CSF and serum samples by isoelectric focusing and immunoblotting patient idiosyncrasies. as described previously (16, 19). The presence of intrathecal IgG or To explore this, we studied the effect of aging in the adaptive IgM synthesis was demonstrated by the appearance of two or more and innate immune responses in the CSF of MS patients. We also oligoclonal bands in CSF, not present in paired serum sample. Lipid explored whether the intrathecal synthesis of lipid-specific specific IgM bands were assessed in the CSF of patients showing oligoclonal IgM bands (LS-OCMB), a well-established marker intrathecal IgM synthesis as previously described (16). Briefly, of a high inflammatory disease course in MS (16, 17), plays a role oligoclonal IgM bands are separated by isoelectrofocusing and in this process. Understanding the age-related alterations in the transferred to nitrocellulose membranes coated with different immune system of MS patients is critical for the development of lipids and blocked with Polypep (Merck). A membrane blocked targeted therapeutic approaches and the discovery of novel with polypep is used as negative control. The presence of anti-lipid potential markers of the progression of the disease. IgM antibodies restricted to the CSF is then evidenced by Frontiers in Immunology | www.frontiersin.org 2 July 2021 | Volume 12 | Article 685139 Picón et al. IgM and Immunosenescence in MS TABLE 1 | Clinical and demographic data of patients included in the study. Total patients (n=263) M- patients (n=191) M+ patients (n=72) P Age (years). Median (range) 39.0 (16-65) 40.0 (16-65) 35.0 (18-62) ns Disease duration (months) (*) 7.4 (3.2-32.1) 8.4 (3.7-45.4) 6.5 (2.0-15.5) ns Sex (male/female) 114/149 80/111 34/38 ns Disease onset (RR/PP) 231/32 171/20 60/12 ns EDSS score (*) 1.5 (1.5-2.0) 1.5 (1.0-2.0) 2.0 (1.5-3.0) 0.004 MSSS score (**) 4.3 (2.4-5.9) 4.3 (2.4-5.2) 5.9 (4.3-7.9) <0.0001 N. of Relapses in the previous year (*)(**) 1 (1-2) 1 (1-1) 2 (1-2) <0.0001 CSF CHI3L1 (ng/ml) (*) 209.5 (159.5-284.8) 200.0 (156.0-266.2) 246.5 (183.1-395.7) 0.017 CSF NfL (pg/ml) (*) 924.4 (567.3-2058.7) 842.6 (466.0-1364.5) 1558.9 (759.7-4053.1) 0.0002 CSF C3 (ng/ml) (*) 9586 (6808-13744) 8172 (6256-10856) 10498 (8305-15151) 0.018 IgG Index (*) 0.91 (0.72-1.41) 0.91 (0.69-1.42) 0.76 (0.54-0.98) ns IgM Index (*) 0.16 (0.11-0.27) 0.14 (0.10-0.22) 0.24 (0.15-0.42) <0.0001 Alb Index (*) 4.24 (3.10-5.65) 4.24 (3.03-5.43) 4.24 (3.36-5.89) ns (*): Data shown as Median (25-75% IQR); (**): Explored in patients with at least six months of disease duration. Alb, Albumin; CHI3L1, Chitinase 3-like 1; C3, C3 complement component; CSF, cerebrospinal fluid; EDSS, Expanded Disability Status Scale; IQR, Interquartile range; LP, lumbar puncture; M+ patients, Those showing lipid-specific oligoclonal IgM bands; M- patients, Those lacking lipid-specific oligoclonal IgM bands; MSSS, Multiple Sclerosis Severity Score; N., number; NfL, neurofilament light chain; ns, not significant; PP, Progressive onset; RR, relapsing remitting onset. P values are referred to M+ vs M- patient comparisons. Continuous variables were analyzed using Mann-Whitney U test and categorical ones by Chi-square tests. immunoblotting with anti-human IgM antibodies labeled with antibodies recognizing different cytokines. Then, cells were biotin and with streptavidin labeled with alkaline phosphatase. A washed and analyzed in a FACSCanto II flow cytometer. Data representative image of anti-lipid IgM bands is shown in analysis was performed using the software FACSDiva V.8.0 (BD Supplementary Figure 1. Biosciences) and the gating strategy shown in Supplementary Figures 2, 3. All labeled cells were acquired to calculate total CSF Leukocyte Subpopulations cell numbers. The following monoclonal antibodies were used in the study: CD14- FITC, IFN-g-FITC, GM-CSF-PE, CD3-PerCP, TNF-a-PerCP- Detection of Soluble Molecules in CSF Cy5.5, CD16-PE-Cy7, CD19-PE-Cy7, CD56-APC, CD8-APC-H7, We used ELISA to explore the CSF values of the following CD3-BV421, and CD45-V500 (BD Biosciences, San Diego, CA). IL- molecules: activin A (R&D Systems, MN), chitinase 3-like 1 17-APC was obtained from R&D Systems, Minneapolis, MN. For (CHI3L1; Quidel Corporation, San Diego, CA), C3 complement patients whose CSF samples were 3-5 ml (36 M+ and 86 M- component (Abcam; Cambridge, UK), neurofilament light chains patients), only a tube measuring surface antigens was studied. In (NfL; Uman Diagnostics, Sweden), programmed death-ligand 1 cases of 5-8 ml of CSF (36 M+ and 105 M- patients), the samples (PD-L1; R&D Systems), and T-cell immunoglobulin and mucin were divided into two identical aliquots, and surface antigens and domain 3 (TIM-3; Bio-Techne, R&D Systems). All assays were run intracellular cytokine production were studied as detailed below. according to the manufacturer’s instructions with the exception of The precise CSF volume used in every case was recorded to calculate NfL, for which 10 and 50 µl of CSF were assayed for every patient. total cell numbers. Anti-CMV IgG ELISA Flow Cytometry Analysis Serum anti-CMV IgG antibodies were measured by ELISA (Zeus For surface antigen identification, cellular pellets obtained after CSF Scientific, USA) according to the manufacturer’s instructions. The centrifugation were resuspended in the residual volume (about 100 results were expressed as an index value (IV) that was calculated as µl), stained with the appropriate amounts of monoclonal antibodies follows: 10 x sample absorbance/cut-off value. Samples were for 30 minutes at 4°C in the dark, washed twice with PBS, and analyzed in duplicate for each test. analyzed in a FACSCanto II flow cytometer (BD Biosciences). For intracellular cytokine detection, cellular pellets were stimulated and Statistical Analyses stained for flow cytometry analysis as described previously (20). In Statistical analyses were done using the software GraphPad brief, cellular pellets were incubated for 4 hours at 37°C in 5% CO2 Prism 6.0 (GraphPad Prism Inc., La Jolla, CA) and Stata 16 with 50 ng/ml Phorbol 12-myristate 13-acetate (PMA) (Sigma- (StataCorp LLC, Lakeway, TX). For continuous variables, we Aldrich, St. Louis, MO) and 750 ng/ml Ionomycin (Sigma-Aldrich) used the Mann-Whitney U-test with Bonferroni post-hoc in presence of 2 µg/ml Brefeldin A (GolgiPlug, BD Biosciences) and correction or the Kruskal-Wallis test with Dunn’s multiple 2.1 µM Monensin (Golgi Stop, BD Biosciences). After incubation, comparison post-test when comparing 3 or more groups. The cells were washed and stained with the monoclonal antibodies chi-squared test was used to compare categorical variables. P-values recognizing the surface antigens. Then, cells were washed, fixed and below 0.05 were considered as significant. Spearman correlation was permeabilized with Cytofix/Cytoperm Kit (BD Biosciences), washed used to test for associations between groups, and the Spearman r twice and subjected to intracellular staining with monoclonal and p values are reported in each instance. Multivariate regression Frontiers in Immunology | www.frontiersin.org 3 July 2021 | Volume 12 | Article 685139 Picón et al. IgM and Immunosenescence in MS analysis was used to explore the relationship between age and remarkable decrease of mononuclear cell numbers (p<0.0001) due immunological factors and between age and disability scores while to a drop-in lymphocytes (p<0.0001) and natural killer cells (NK) adjusting for disease duration. (p=0.0001). A similar reduction was found in CD4+ (p<0.0001) and CD8+ (p<0.0001) T cells and in B cells (p<0.0001). In contrast, monocyte numbers did not show any significant RESULTS changes (Table 2). We also explored the intracellular production of pro-inflammatory cytokines by CSF lymphocytes. In the M- Patient Characteristics group, age induced significant decreases of CD4+ and CD8+ T We investigated the association between aging and intrathecal cells producing IFN-g, TNF-a, and GM-CSF, as well as CD4+ T immune response in MS by studying a large cohort of treatment- cells producing IL-17. A reduction in B cells producing TNF-a naive MS patients (n=263). We stratified them in two groups was observed in both groups of patients (Table 2). according to the absence (M-, n=191) and presence (M+, n=72) To rule out the effect of disease duration on these correlations, of LS-OCMB. Clinical, demographic, and laboratory data are shown we performed a multivariable linear regression analysis between age in Table 1. M+ patients showed higher number of relapses in the and leukocyte numbers adjusting for disease duration. Most previous year (p<0.0001), and higher values of the EDSS) (p=0.004) associations remained significant with the only exceptions of and the MSSS (p<0.0001) scores. In addition, they showed increased CD4+ T cells producing IL-17 in M- patients and B cells CSF values of CHI3L1 (p=0.017), neurofilament light chains (NfL) producing TNF-a in M+ patients that were lost (Figures 1A, B (p=0.0002), C3 complement factor (p=0.018) and of the IgM and Supplementary Table 1). index (p<0.0001). To investigate the age at which intrathecal leukocyte decline occurs, we classified patients according to their age in subgroups Aging Diminishes Numbers of Intrathecal of five years (i.e., ≤25, 26-30, 31-35 years, and so on). The highest Lymphocytes and NK Cells in M- Patients changes in lymphocyte numbers were observed in M- patients older Next, we studied age-related changes in the main leukocyte subsets than 45 years (Figure 2A). The same results were observed when in the CSF. The results are shown in Table 2. M- patients showed a studied CSF CD4+ and CD8+ T cell numbers (Figures 2B, C). TABLE 2 | Correlations between age and variables related to immune response or axonal damage. Total patients (n=263) M- patients (n=191) M+ patients (n=72) Variable r p r p r p Leukocyte subsets (cells/ml) Total Mononuclear cells -0.40 <0.0001 -0.46 <0.0001 0.02 0.886 Total Lymphocytes -0.40 <0.0001 -0.47 <0.0001 0.02 0.889 CD4+ T cells -0.37 <0.0001 -0.45 <0.0001 0.07 0.597 CD4+T cells IFN-gamma+ -0.31 0.0002 -0.33 0.0007 -0.15 0.364 CD4+T cells TNF-alpha+ -0.32 0.0001 -0.35 0.0003 -0.14 0.411 CD4+T cells IL-17+ -0.34 <0.0001 -0.31 0.0012 -0.34 0.042 CD4+ GM-CSF+ -0.41 0.002 -0.44 0.003 -0.28 0.378 CD8+ T cells -0.34 <0.0001 -0.41 <0.0001 0.02 0.852 CD8+T cells IFN-gamma+ -0.30 0.0003 -0.32 0.0008 -0.11 0.510 CD8+T cells TNF-alpha+ -0.27 0.0014 -0.29 0.003 -0.08 0.614 CD8+T cells IL-17+ -0.21 0.0136 -0.15 0.121 -0.23 0.172 CD8+ GM-CSF+ -0.43 0.001 -0.49 0.001 -0.23 0.477 CD19+ B cells -0.41 <0.0001 -0.49 <0.0001 0.02 0.847 CD19+ TNF-alpha+ -0.41 0.002 -0.36 0.02 -0.68 0.01 CD19+ GM-CSF+ -0.31 0.02 -0.25 0.103 -0.65 0.02 Total NK cells -0.31 0.0002 -0.39 0.0001 -0.08 0.590 Total Monocytes -0.09 0.142 -0.13 0.070 0.03 0.795 Soluble factors PD-L1 (pg/ml) 0.34 <0.0001 0.30 0.0011 0.56 0.0005 TIM-3 (pg/ml) 0.30 0.0012 0.27 0.04 0.41 0.002 IgG anti-CMV (IV) 0.28 0.0003 0.31 0.0006 0.17 0.247 CHI3L1 (ng/ml) 0.41 <0.0001 0.49 <0.0001 0.29 0.057 Activin A (pg/ml) 0.46 <0.0001 0.47 <0.0001 0.44 0.003 NfL (pg/ml) -0.07 0.368 0.06 0.518 -0.28 0.051 C3 (ng/ml) 0.18 0.098 0.18 0.220 0.27 0.085 C3, C3 complement component; CHI3L1, Chitinase 3-like 1; CMV, Cytomegalovirus; CSF, Cerebrospinal fluid; GM-CSF, Granulocyte/macrophage-colony stimulating factor; IV, Index Value; NK, Natural Killer; IFN, Interferon; PD-L1, Programmed Death-ligand 1; TIM-3, T-cell immunoglobulin and mucin domain-3; TNF, Tumor necrosis factor; IL, Interleukin; M+ patients, those showing lipid-specific oligoclonal IgM bands; M- patients, those lacking lipid-specific oligoclonal IgM bands. All variables were quantified in CSF with the exception of IgG anti-CMV, quantified in serum. r and p values were determined by Spearman correlation. Frontiers in Immunology | www.frontiersin.org 4 July 2021 | Volume 12 | Article 685139 Picón et al. IgM and Immunosenescence in MS A B C D E F FIGURE 1 | Multivariate regression analysis to explore the effect of age on immune response and disability in M- and M+ patients. We explored influence of age in different leukocyte subsets [(A, B) cells/ml] and in soluble factors levels (C, D) related to adaptive and innate immune responses in patients lacking [M-, n=191 (A, C)] and showing [M+, n=72, B, (D)] lipid-specific oligoclonal IgM bands. All variables were quantified in CSF with the exception of IgG anti-CMV, quantified in serum. The effect of age on disability in M- (E) and M+ (F) patients was also studied. All coefficients (black circles) and 95% confidence intervals were adjusted by disease duration. CHI3L1, Chitinase 3-like 1; CMV, Cytomegalovirus; CSF, cerebrospinal fluid; EDSS, Expanded Disability Status Scale; IFN, Interferon; IL, Interleukin; IV, Index Value; GM-CSF, Granulocyte/macrophage-colony stimulating factor; MSSS, MSSS, Multiple Sclerosis Severity Score. NK, Natural Killer; PD-L1, Programmed Death-ligand 1; TIM-3, T-cell immunoglobulin and mucin domain-3; TNF, Tumor necrosis factor. Remarkably, no differences were observed in the M+ group, even in producing IFN-gamma, TNF-alpha, and GM-CSF (Figure 3). patients older than 50 years. M- patients older than 45 years showed lower CSF numbers of In view of these results, we classified M+ and M- patients in CD4 and CD8 T cells producing IFN-gamma (p=0.004 and two groups (Age ≤45 or > 45 years) and further explored the p=0.003 respectively), TNF-alpha (p=0.002 and p=0.004 influence of age in the numbers of CD4+ and CD8+ T cells respectively), and GM-CSF (p=0.029 and 0.005 respectively). Frontiers in Immunology | www.frontiersin.org 5 July 2021 | Volume 12 | Article 685139 Picón et al. IgM and Immunosenescence in MS A B C FIGURE 2 | Changes in CSF lymphocyte counts in MS patients classified according to age and to the absence or presence of lipid-specific oligoclonal IgM bands (LS-OCMB). Absolute cell numbers (N.) of CSF total lymphocytes (A), CD4+ (B) and CD8+ (C) T lymphocytes were studied in multiple sclerosis patients lacking (M-, grey bars, n=191) and showing (M+, white bars, n=72) LS-OCMB, classified according to their age: ≤25, 26-30, 31-35, 36-40, 41-45, 46-50 and ≥ 51 years. ns, not significant. ****p < 0.0001. A B C D E F FIGURE 3 | Changes in CSF CD4+ and CD8+ T lymphocytes producing cytokines in M- and M+ patients according to age. Absolute cell numbers (N.) of CD4+ (A–C) and CD8+ (D–F) T lymphocytes producing Interferon-gamma (IFNg), Tumor Necrosis Factor-alpha (TNFa) and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) in patients lacking (M-, n=105) and showing (M+, n=36) lipid-specific oligoclonal IgM bands, and classified according to their age in ≤ 45 years (white bars) and > 45 years (grey bars). ns, not significant; y, years. *p < 0.05; **p < 0.01. Frontiers in Immunology | www.frontiersin.org 6 July 2021 | Volume 12 | Article 685139 Picón et al. IgM and Immunosenescence in MS However, none of these T cell subsets changed in M+ patients duration (Figures 1C, D and Supplementary Table 1). By with age. Representative images of intracellular cytokine contrast, CSF NfL and C3 complement component levels did production by CD4+ and CD8+ T lymphocytes from M- not change with age in M- or M+ patients (Table 2), but were patients are shown in Figure 4. higher in M+ patients independently of age (Table 1). Changes in Soluble Factors Associated With Age and Disability Progression Immunosenescence and T Cell Exhaustion Finally, we explored the EDSS and MSSS scores. The first variable To gain insight into the regulation of adaptive immune cell activity, was studied in the total patient cohort, and the second one was we analyzed CSF levels of PD-L1 and TIM-3, which are markers of studied in the 161 patients with at least six months of disease T cell tolerance and exhaustion, respectively. We also explored duration at lumbar puncture (120 M- and 41 M+). As reported serum titers of anti-CMV IgG, a marker of immunosenescence. above, M+ patients showed higher values of EDSS and MSSS PD-L1 and TIM-3 levels increased with age in M- and M+ patients scores (Table 1). When exploring the effect of age after adjusting (Table 2). The association with TIM-3 was lost in M- patients after for disease duration, we found increases in both scores in M- and adjusting for disease duration (Figures 1C, D and Supplementary M+ patients (Figures 1E, F and Supplementary Table 1). Table 1). By contrast, serum anti-CMV IgG, a marker of immunosenescence, increased with age only in the M- group (Figures 1C, D and Supplementary Table 1). DISCUSSION Soluble Factors Related to Innate Immunosenescence is characterized by a chronic activation of Immune Response the innate immune response and reduced effectiveness of the To further examine age-related differences between M+ and M- adaptive response, which commonly occur after the age of 65 patients, we assessed CSF values of CHI3L1 and activin A, which years (6). Converging evidence suggests that this process can are soluble factors related to the innate response. Activin A occur earlier in patients with chronic immune-system activation, increased with age in both M- and M+ patients, while CHI3L1 such as those with acquired immunodeficiency syndrome or levels only increased in the M- group. Notably, M+ patients had rheumatoid arthritis (21, 22). In MS, older age affects the higher levels of this protein independently of age (Table 1). No response to treatments and increases the risk of side effects significant correlations were lost after adjusting for disease (23, 24). A B C D E F FIGURE 4 | Representative flow cytometry images of CSF T lymphocytes producing cytokines in M- patients. Representative dot plots showing intracellular cytokine production by CSF CD4+ and CD8+ T lymphocytes from a young M- patient [34 years old; (A–C)] and from an old one [57 years; (D–F)]. Percentages over total CSF CD45+ lymphocytes are indicated on plots. GM-CSF, Granulocyte-Macrophage Colony Stimulating Factor; IFNg, Interferon-gamma; TNFa, Tumor necrosis factor-alpha. Frontiers in Immunology | www.frontiersin.org 7 July 2021 | Volume 12 | Article 685139 Picón et al. IgM and Immunosenescence in MS Pathological studies involving patient necropsies have adaptive immune response described for aged MS patients (38), provided recent reports on the molecular mechanisms involved and M+ status may protect from opportunistic infections in in these changes came (25). These studies found an increase in older patients (39, 40). innate immune-cell activation restricted to the CNS and a Regarding the innate immune response, M+ patients showed decrease in lymphocyte influx into the CNS in progressive higher values of the C3 component of the complement and of forms of the disease (26). However, MS is a heterogeneous CHI3L1 in CSF, independently of age. IgM antibodies can fix disease, and this process could not be uniform in all patients. complement much more efficiently than IgG antibodies, so M+ Along these lines, it was shown that LS-OCMB (which is patients can have an early activation of the innate immune associated with a worse course of MS (16, 26) and higher system mediated by the complement activation. CHI3L1 is inflammation demonstrated by augmented NfL values (27) expressed in demyelinated lesions by activated astrocytes and have a protective effect on PML risk in patients receiving is related to the innate immune response (41). This may account natalizumab treatment, independently of age (17, 28). This for the early worsening of disability shown by these patients. could imply that M+ status can modulate the effect of age in MS. The M- group only showed an increase in CHI3L1 levels We aimed to explore this idea in 263 consecutive MS patients with aging, which suggests an additional activation of the who had not previously received any disease-modifying innate immune system occurring with age. This was treatment. M- patients showed a remarkable reduction in the confirmed by the age-associated increase of activin A numbers of B, T, and NK cells in the CSF, which was not experienced by M+ and M- patients. This molecule is observed in M+ patients. M- patients also showed a clear expressed by activated microglia (42), which is an important reduction in the numbers of B and T cells producing pro- part of the senescence-associated secretoma, which induces inflammatory cytokines. These data are interesting since they inflammaging in older individuals (43). This indicates that MS show that highly inflammatory MS can counteract the effect of patients enter an inflammaging process at a much younger age age in the inflammation of the adaptive immune system. than healthy individuals. This could be at least partly To explore the origin of these differences, we studied soluble responsible for the increased worsening of disability shown factors associated with immune cell exhaustion and by MS patients at ages beyond 45 years (44). In view of these immunosenescence. All MS patients showed a modest age- results, activin A levels could be a biomarker of early associated increase of PD-L1, a check point molecule capable inflammaging in MS. Further exploration of the cytokine of inducing T cell tolerization that rises in old individuals (29). profile of innate immune cells in the CSF of older MS This confirms the appearance of an early immunosenescence patients and its association with disability could provide tools process in MS patients, since this study was performed in for identifying new effective treatments for older MS patients. individuals aged between 16 and 65 years, an age at which most healthy individuals did not enter in the immunosenescence process yet. In addition, age induced in M+ patients an elevation in DATA AVAILABILITY STATEMENT the CSF values of TIM-3, a co-receptor that frequently increases upon repeated T cell activation (30–33). TIM- favors T cell Anonymized data supporting the findings of this study will be exhaustion in junction with other factors associated with shared by reasonable request from any qualified investigator senescence but its expression alone does not seem to correlate during three years after the publication of the study. with this condition (34). Despite the increases of CSF levels of PD- L1 and TIM-3, M+ patients did not experience a decrease in the CSF levels the numbers of pro-inflammatory lymphocytes with age. This ETHICS STATEMENT may be occasioned by the lack of other molecules involved in the immunosenescence process, or by the up-regulation of other pro- The studies involving human participants were reviewed and inflammatory signals. In this line, we described the association of approved by Ethical Committee of Ramó n y Cajal University the M+ status with polymorphisms in the TNF-alpha promoter Hospital (Madrid, Spain). The patients/participants provided associated with sustained inflammation (35). their written informed consent to participate in this study. Moreover, M- but not M+ patients showed an age-related increase of the titers of anti CMV antibodies. The increase of clonally expanded CD8 T cells and of anti-CMV antibodies AUTHOR CONTRIBUTIONS associate with immunosenescence (12). In addition, CMV infection occurs more frequently in older individuals in MS CP, AT-V, JF-V, SM, and NV collected CSF samples and and associates with lower production of pro-inflammatory performed the flow cytometry and ELISA experiments. CP, cytokines and (36, 37). These data show that M- patients show AT-V, NV, and LV drafted the manuscript. ER and ER-M an early immunosenescence process associated with a down- supervised flow cytometry studies. MC and CM-B quantified regulation of the pro-inflammatory adaptive immune response. CHI3L1 in CSF samples. EQ, MM-S, and LR-T contributed by The M- phenotype is twice as frequent as the M+ phenotype in sending samples. RA ́ -L and MD-M performed IgG anti-CMV MS. Thus, the reduction of pro-inflammatory adaptive immune quantification. SSM, EM, LC-F, JA ́ -C, JM, and YA visited MS cells shown by these patients may account for the changes in patients and collected clinical data. CE and CG contributed to Frontiers in Immunology | www.frontiersin.org 8 July 2021 | Volume 12 | Article 685139 Picón et al. IgM and Immunosenescence in MS study the innate immune system. LV designed and supervised ACKNOWLEDGMENTS the study. All authors contributed to the article and approved the submitted version. Authors acknowledge MA Ferná ndez de Pablos, AI Pé rez Macı́as and S Ortega for their excellent technical support. FUNDING SUPPLEMENTARY MATERIAL This work was supported by grants FIS-PI15/00513, FIS-PI18/ 00572 and RD16/0015/0001 from the Instituto de Salud Carlos The Supplementary Material for this article can be found online III. Ministerio de Ciencia e Innovació n, Spain and FEDER: "Una at: https://www.frontiersin.org/articles/10.3389/fimmu.2021. manera de hacer Europa". 685139/full#supplementary-material REFERENCES Predicts an Aggressive Disease Course in MS. J Clin Invest (2005) 115:187–94. doi: 10.1172/JCI22833 1. Tutuncu M, Tang J, Zeid NA, Kale N, Crusan DJ, Atkinson EJ, et al. Onset of 17. Villar LM, Costa-Frossard L, Masterman T, Fernandez O, Montalban X, Progressive Phase Is an Age-Dependent Clinical Milestone in Multiple Casanova B, et al. Lipid-Specific Immunoglobulin M Bands in Cerebrospinal Sclerosis. Mult Scler (2013) 19:188–98. doi: 10.1177/1352458512451510 Fluid are Associated With a Reduced Risk of Developing Progressive 2. Rist JM, Franklin RJM. 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Ageing Res This is an open-access article distributed under the terms of the Creative Commons Rev (2018) 41:64–81. doi: 10.1016/j.arr.2017.10.005 Attribution License (CC BY). The use, distribution or reproduction in other forums 39. Grebenciucova E, Berger JR. Immunosenescence: The Role of Aging in the is permitted, provided the original author(s) and the copyright owner(s) are credited Predisposition to Neuro-Infectious Complications Arising From the and that the original publication in this journal is cited, in accordance with accepted Treatment of Multiple Sclerosis. Curr Neurol Neurosci Rep (2017) 17:61. academic practice. No use, distribution or reproduction is permitted which does not doi: 10.1007/s11910-017-0771-9 comply with these terms. Frontiers in Immunology | www.frontiersin.org 10 July 2021 | Volume 12 | Article 685139