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Tissue microarray methodology identifies complement pathway activation and dysregulation in progressive multiple sclerosis.

ABSTRACT: The complement pathway has potential contributions to both white (WM) and grey matter (GM) pathology in Multiple Sclerosis (MS). A quantitative assessment of complement involvement is lacking. Here we describe the use of Tissue MicroArray (TMA) methodology in conjunction with immunohistochemistry to investigate the localization of complement pathway proteins in progressive MS cortical GM and subcortical WM. Antibodies targeting complement proteins C1q, C3b, regulatory proteins C1 inhibitor (C1INH, complement receptor 1 (CR1), clusterin, factor H (FH) and the C5a anaphylatoxin receptor (C5aR) were utilised alongside standard markers of tissue pathology. All stained slides were digitised for quantitative analysis. We found that numbers of cells immunolabelled for HLA-DR, GFAP, C5aR, C1q and C3b were increased in WM lesions (WML) and GM lesions (GML) compared to normal appearing WM (NAWM) and GM (NAGM), respectively. The complement regulators C1INH, CR1, FH and clusterin were more abundant in WM lesions, while the number of C1q+ neurons were increased and the number of C1INH+, clusterin+, FH+ and CR1+ neurons decreased in GM lesions. The number of complement component positive cells (C1q, C3b) correlated with complement regulator expression in WM, but there was no statistical association between complement activation and regulator expression in the GM. We conclude that TMA methodology and quantitative analysis provides evidence of complement dysregulation in MS GML, including an association of the numerical density of C1q+ cells with tissue lesions. Our work confirms that complement activation and dysregulation occur in all cases of progressive MS and suggest that complement may provide potential biomarkers of the disease.

SUBMITTER: Loveless S

PROVIDER: S-EPMC8028318 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMultiple sclerosis (MS) is an immune-mediated, chronic inflammatory, and demyelinating disease of the central nervous system (CNS). Several cytokines are thought to be involved in the regulation of MS pathogenesis. We recently identified interleukin (IL)-9 as a cytokine reducing inflammation and protecting from neurodegeneration in relapsing-remitting MS patients. However, the expression of IL-9 in CNS, and the mechanisms underlying the effect of IL-9 on CNS infiltrating immune cells have never been investigated.MethodsTo address this question, we first analyzed the expression levels of IL-9 in post-mortem cerebrospinal fluid of MS patients and the in situ expression of IL-9 in post-mortem MS brain samples by immunohistochemistry. A complementary investigation focused on identifying which immune cells express IL-9 receptor (IL-9R) by flow cytometry, western blot, and immunohistochemistry. Finally, we explored the effect of IL-9 on IL-9-responsive cells, analyzing the induced signaling pathways and functional properties.ResultsWe found that macrophages, microglia, and CD4 T lymphocytes were the cells expressing the highest levels of IL-9 in the MS brain. Of the immune cells circulating in the blood, monocytes/macrophages were the most responsive to IL-9. We validated the expression of IL-9R by macrophages/microglia in post-mortem brain sections of MS patients. IL-9 induced activation of signal transducer and activator of transcription (STAT)1, STAT3, and STAT5 and reduced the expression of activation markers, such as CD45, CD14, CD68, and CD11b in inflammatory macrophages stimulated in vitro with lipopolysaccharide and interferon (IFN)-γ. Similarly, in situ the number of activated CD68+ macrophages was significantly reduced in areas with high levels of IL-9. Moreover, in the same conditions, IL-9 increased the secretion of the anti-inflammatory cytokine, transforming growth factor (TGF)-β.ConclusionsThese results reveal a new cytokine expressed in the CNS, with a role in the context of MS. We have demonstrated that IL-9 and its receptor are both expressed in CNS. Moreover, we found that IL-9 decreases the activation state and promotes the anti-inflammatory properties of human macrophages. This mechanism may contribute to the beneficial effects of IL-9 that are observed in MS, and may be therapeutically potentiated by modulating IL-9 expression in MS.

Project description:IntroductionMultiple sclerosis (MS) is a demyelinating disease of the central nervous system with an underlying immune-mediated and inflammatory pathogenesis. Innate immunity, in addition to the adaptive immune system, plays a relevant role in MS pathogenesis. It represents the immediate non-specific defense against infections through the intrinsic effector mechanism "immunothrombosis" linking inflammation and coagulation. Moreover, decreased cerebral blood volume (CBV), cerebral blood flow (CBF), and prolonged mean transit time (MTT) have been widely demonstrated by MRI in MS patients. We hypothesized that coagulation/complement and platelet activation during MS relapse, likely during viral infections, could be related to CBF decrease. Our specific aims are to evaluate whether there are differences in serum/plasma levels of coagulation/complement factors between relapsing-remitting (RR) MS patients (RRMS) in relapse and those in remission and healthy controls as well as to assess whether brain hemodynamic changes detected by MRI occur in relapse compared with remission. This will allow us to correlate coagulation status with perfusion and demographic/clinical features in MS patients.Materials and methodsThis is a multi-center, prospective, controlled study. RRMS patients (1° group: 30 patients in relapse; 2° group: 30 patients in remission) and age/sex-matched controls (3° group: 30 subjects) will be enrolled in the study. Patients and controls will be tested for either coagulation/complement (C3, C4, C4a, C9, PT, aPTT, fibrinogen, factor II, VIII, and X, D-dimer, antithrombin, protein C, protein S, von-Willebrand factor), soluble markers of endothelial damage (thrombomodulin, Endothelial Protein C Receptor), antiphospholipid antibodies, lupus anticoagulant, complete blood count, viral serological assays, or microRNA microarray. Patients will undergo dynamic susceptibility contrast-enhanced MRI using a 3.0-T scanner to evaluate CBF, CBV, MTT, lesion number, and volume.Statistical analysisANOVA and unpaired t-tests will be used. The level of significance was set at p ≤ 0.05.DiscussionIdentifying a link between activation of coagulation/complement system and cerebral hypoperfusion could improve the identification of novel molecular and/or imaging biomarkers and targets, leading to the development of new effective therapeutic strategies in MS.Clinical trial registrationClinicaltrials.gov, identifier NCT04380220.

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Tissue microarray methodology identifies complement pathway activation and dysregulation in progressive multiple sclerosis.

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