Elevated cytokine levels in the central nervous system of cluster headache patients in bout and in remission

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To further investigate inflammation in cluster headache, inflammatory markers were analysed in patients with cluster headache and controls. Methods: We performed a case-control study, collecting cerebrospinal fluid and serum samples from healthy controls, cluster headache patients in remission, active bout, and during an attack to cover the dynamic range of the cluster headache phenotype. Inflammatory markers were quantified using Target 48 OLINK cytokine panels. Results: Altered levels of several cytokines were found in patients with cluster headache compared to controls. CCL8, CCL13, CCL11, CXCL10, CXCL11, HGF, MMP1, TNFSF10 and TNFSF12 levels in cerebrospinal fluid were comparable in active bout and remission, though significantly higher than in controls. In serum samples, CCL11 and CXCL11 displayed decreased levels in patients. Only one cytokine, IL-13 was differentially expressed in serum during attacks. Conclusion and Interpretation: Our data shows signs of possible neuroinflammation occurring in biological samples from cluster headache patients. Increased cerebrospinal fluid cytokine levels are detectable in active bout and during remission, indicating neuroinflammation could be considered a marker for cluster headache and is unrelated to the different phases of the disorder. Inflammation neuroinflammation headache chemokines Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Cluster headache (CH) is a trigeminal autonomic cephalalgia characterized by recurring unilateral headache attacks of extreme severity. Patients with CH typically experience active bouts where they can have up to eight attacks per day, followed by symptom free remission periods[ 1 ]. The trigeminovascular pathway and the trigeminal–autonomic reflex are activated during CH attacks, resulting in severe pain in the periorbital region as well as the autonomic symptoms manifesting during CH attacks. Nevertheless, the underlying mechanisms are unclear and CH pathophysiology remains to be fully elucidated[ 2 ]. Inflammatory reactions have been suggested in the past to cause CH, in particular those of the cavernous sinus, and the corticosteroide Prednisolone, a powerful anti-inflammatory drug, is commonly used as an interim treatment for CH[ 3 ]. Meanwhile, there is no evidence of systemic inflammation in CH patients[ 4 ]. Targeted analysis of inflammatory markers in CH has yielded inconclusive results, often because of small sample sizes and usage of different methodologies. In a large microarray panel screening for differentially expressed genes in peripheral blood, inflammation related genes were found to be upregulated in CH patients as compared to controls (major histocompatibility complex, class II (HLA) -DQA1 and HLA-DQB1), or specifically during active bouts (S100 calcium binding protein (S100) A8 and S100A12)[ 5 ]. In another study, a trend for interleukin (IL) -1β and nuclear factor-κB (NF-κB) upregulation was found in blood cells from patients with CH in active bout[ 6 ]. Interestingly, the authors found a concurrent reduction in inflammasome component NLR family pyrin domain containing 3 (NLRP3) expression levels, required for IL-1β release[ 6 ]. In another global gene expression analysis in peripheral blood cells, inflammatory processes were highlighted both by pathway analysis (IL-4 and sialic acid binding Ig like lectin 7 (CD328)) and by expression network analysis as potentially involved in CH[ 7 ]. Increased gene expression of IL-2 has further been reported in CH patients, specifically in active bout in between their CH attacks, while mRNA levels were normalized to control levels during attacks[ 8 ]. Reports on differentially expressed cytokines in CH are scarce, IL-2, and IL-1β have been suggested to be increased in CH patients, but a recent meta-analysis could not confirm elevated IL-1β[ 9 – 11 ]. The same meta-analysis summarized findings on the more common primary headaches migraine and tension type headache (TTH) and confirmed higher levels of IL-6, IL-8 and tumour necrosis factor alpha (TNF-α) in migraine patients and of TNF-α and transforming growth factor beta (TGF-β) in TTH compared to controls[ 10 ]. Even less is known regarding inflammation of the nervous system in CH. Due to recent developments in CH genetics there have been speculations of microglia involvement in the pathogenesis of CH. Microglia have been linked to CH through genome wide association studies (GWASs) highlighting the gene MER proto-oncogene, tyrosine kinase ( MERTK ), a gene highly expressed in microglia and macrophages involved in phagocytosis of apoptotic cells[ 12 ]. In this study we aimed to perform inflammatory profiling of CH patients in remission period and in active bout in order to get a better understanding of the importance of inflammation in CH and if inflammatory reactions may constitute a hallmark of the phenotypic switch occurring in CH patients when they go into active bout. Methods Material Study participants were recruited at the Neurology clinic at Karolinska University Hospital, 34 patients diagnosed with CH and 40 control subjects. CH patients were diagnosed by a neurologist (co-author A.S.) according to the international classification of headache disorders (ICHD) 3rd edition[ 1 ]. Informed consent was obtained from all study participants prior to inclusion. Ethical permit to perform the study was obtained from the Swedish Ethical Review Authority (diary number 217/02) and all experiments were conducted in accordance with the declaration of Helsinki for research involving human subjects. Study participants were asked to give a sample of cerebrospinal fluid (CSF) or serum (blood) or both. Tissue from patients was sampled at several timepoints if possible; in remission, in active bout, and during a CH attack. In total we collected and analysed 120 samples: 21 CSF samples and 27 serum samples from control individuals, 43 CSF samples from CH patients, 18 in remission and 25 in active bout, and 29 serum samples from CH patients, 11 in remission, 15 in active bout and 3 during an attack. Samples were acquired using standard procedures and kept at -80°C until analysis. OLINK cytokine panels Samples (CSF and serum) were analysed, and quality control and calibrator normalization were performed at SciLifeLab Affinity Proteomics Uppsala on OLINK® Proximity Extension Assay (PEA) Target 48 cytokine panels, (Affinity Proteomics Uppsala, SciLifeLab, Uppsala University, SE-751 85 Uppsala). The panel contains 45 assays, 89% (n = 40) of which passed quality control (assays failing quality control: C-X-C motif chemokine ligand (CXCL) 12, IL-1β, IL-15, IL-17A, and IL-4). 99% (n = 119) of the analysed samples passed quality control. Data is reported as normalized protein expression (NPX) unit in Log2 scale and was analysed separately for CSF and serum samples in R(v4.1.3) using the OlinkAnalyze package. Statistical analysis PCA analysis confirmed that one CSF sample did not pass the quality control and also revealed a cluster of serum samples which may represent outliers (n = 7), Supplementary Figs. 1A and 1B. Comparison of group averages between the flagged samples and the remaining controls showed highly heterogenous values, justifying their removal from the analysis. Plotting all samples in a histogram and performing the Shapiro-wilks test showed that data was non normally distributed, Supplementary Figs. 1C and 1D. Two samples were run in duplicate and removed from the analysis. Assays were removed from analysis if they did not pass quality control and if the number of samples with NPX values below the limit of detection (LOD) was higher than 75%; 11 CSF assays (C-C motif chemokine ligand (CCL) 7, colony stimulating factor 3 (CSF3), interferon gamma (IFN-γ), IL-2, IL-10, IL-13, IL-17F, IL-27, IL-33, TNF-α, thymic stromal lymphopoietin (TSLP)) and 3 serum assays (IL-33, IL-17F, TSLP). Group comparisons were made with one-way ANOVA or Wilcoxon rank-sum test using the Benjamini & Hochberg method for correction for multiple testing. A network analysis of associated cytokines was performed using the STRING database v.12.0[ 13 ]. Results The final analysis comprised of 49 serum samples (20 from controls and 29 from CH patients; 11 in remission, 15 in active bout and 3 during an attack) and 61 CSF samples (20 from controls and 41 from CH patients; 17 in remission and 24 in active bout) from 39 control individuals and 34 CH patients in total. Analysis of CSF cytokine levels in controls and CH patients showed differences in nine cytokines; CCL8, CCL13, CCL11, CXCL10, CXCL11, hepatocyte growth factor (HGF), matrix metallopeptidase (MMP) 1, TNF superfamily member (TNFSF) 10 and TNFSF12 (Fig. 1, Supplementary Table 1). All nine cytokines were found to be elevated in CSF in CH patients as compared to controls but did not differ between CH patients in remission and in active bout (Fig. 1, Table 1 ). Network analysis further showed strong interactions between all upregulated chemokines; CCL8, CCL13, CCL11, CXCL10, CXCL11, with a protein-protein interaction enrichment p-value < 1.0e-16 (Fig. 2). [ Figure 1 ] [ Figure 2 ] Table 1 Results from comparison of normalized cytokine expression in controls and cluster headache patients. Remission Active Attack Cytokine Sample Ref Estimate Adj. P Estimate Adj. P Estimate Adj. P CCL11 CSF C -0.56 0.016 -0.73 < 0.001 NA NA Serum C 0.98 0.031 1.16 0.003 -0.14 0.995 CCL13 CSF C -0.76 0.002 -0.81 < 0.001 NA NA CCL8 CSF C -0.97 < 0.001 -0.90 < 0.001 NA NA CXCL10 CSF C -0.78 0.029 -0.77 0.018 NA NA CXCL11 CSF C -0.76 0.028 -0.70 0.028 NA NA Serum C 1.95 0.028 2.42 0.001 -0.23 0.997 HGF CSF C -1.11 < 0.001 -1.00 < 0.001 NA NA MMP1 CSF C -1.78 < 0.001 -1.61 < 0.001 NA NA TNFSF10 CSF C -0.55 0.013 -0.52 0.010 NA NA TNFSF12 CSF C -0.75 0.003 -0.62 0.007 NA NA CCL11 CSF Rem - - 0.17 0.644 NA NA Serum Rem - - -0.18 0.961 1.12 0.248 CCL13 CSF Rem - - -0.04 0.976 NA NA CCL8 CSF Rem - - -0.07 0.947 NA NA CXCL10 CSF Rem - - 0.01 0.999 NA NA CXCL11 CSF Rem - - -0.06 0.972 NA NA Serum Rem - - 0.47 0.913 -2.18 0.255 HGF CSF Rem - - 0.11 0.817 NA NA MMP1 CSF Rem - - 0.16 0.861 NA NA TNFSF10 CSF Rem - - 0.03 0.985 NA NA TNFSF12 CSF Rem - - 0.12 0.817 NA NA CCL11 Serum Active - - - - -1.30 0.125 CXCL11 Serum Active - - - - -2.65 0.104 CSF: cerebrospinal fluid; Ref: Reference group in analysis; C: Control; Rem: Cluster headache patients in remission; Act: Cluster headache patients in active bout; Adj. P: adjusted P-value; NA: Not Available. The analysis of cytokine levels in serum from controls and CH patients in remission, active bout and during a CH attack showed less variation between groups (Supplementary Table 2). Only two cytokines were found to differ between controls and CH patients; CCL11 and CXCL11, both of which were found at lower levels in patients than controls during remission phase and in active bout (Fig. 3). Although lower at baseline, CCL11 and CXCL11 levels in patients were found to raise to control levels during CH attacks. The difference observed between CH patients in remission, or active bout and patients during an attack was not significant, possibly because of a low number of samples (n = 3) (Table 1 ). [ Figure 3 ] Post hoc analysis of serum cytokines As the number of patients leaving a sample during a CH attack were few (serum n = 3, no CSF samples), the data was revisited comparing assays below significance to understand trends in cytokine levels in serum. Cytokines with significant p-values before correction for multiple testing, or a change in NPX values bigger or smaller than 10% comparing CH patients in active bout with controls and CH patients during attack with CH patients in active bout were included, resulting in a post hoc analysis of 22 cytokines. Data revealed sub threshold associations for several cytokines in serum, variations in four cytokines were of the same direction as the associations detected in the primary analysis (showing decreased levels of CXCL11 and CCL11). Namely CCL13, CCL19, IL-17C and CCL7 which were all displaying trends of decreased levels in CH patients in remission (Fig. 4). Interestingly, these cytokines showed trends of opposite direction in serum as compared to CSF. NPX levels of HGF, CSF3, MMP1, oncostatin M (OSM), IL6 and vascular endothelial growth factor A (VEGFA) were elevated in CH patients in active bout as compared to controls (Fig. 4). Cytokine levels in samples from CH patients during an attack were consistently comparable with those of controls rather than patients in active bout or remission, with the exception of IL-13, which was detected at higher levels during an attack than in controls and CH patients in remission and in active bout. [ Figure 4 ] Discussion We have conducted a large screening of cytokine levels in CH patients and controls using serum and CSF samples in order to get an overview of the inflammatory state in the periphery and in the central nervous system (CNS). Nine cytokines were found to be higher in CSF from patients as compared to controls, particularly cytokines with chemoattractant properties on leukocytes. Interestingly, major differences in cytokine levels were detected between CSF and serum. In the discovery analysis two of the cytokines found at higher levels in CSF were lower in serum. Previous studies on cytokines in CH have suggested an increase of IL-2 and IL-1β in blood of CH patients although data are conflicting for IL-1β[ 9 , 10 ]. IL-2 gene expression and protein levels in blood was found to be increased in CH active bout compared to controls, while gene expression levels decreased to control levels during attacks[ 8 , 11 ]. In our study, the assay for IL-1β did not pass quality control and we could not replicate the findings of an increase for IL-2 in our material. These differences could potentially be due to discrepancies between methodology, quantitative Real-time PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) in previous studies while we used PEA, an ELISA based array that increases the sensitivity coupling the detection to qPCR. In this report we found evidence of a possible ongoing inflammation in the CNS of CH patients with elevated levels of several cytokines in the CSF of CH patients as compared to controls. To our knowledge, this is the first reported screening for cytokines in CSF from CH patients. There was very little difference between patients in remission phase and active bout for all the differentially expressed cytokines, which indicates that the neuroinflammatory state in CH patients is not exclusive to the active bout. The cytokines showing the largest increase in CSF from patients were HGF, MMP1, TNSF10 and 12 and several chemokines, all of which were found to have strong interactions in a network analysis. CCL8, CCL11, and CCL13 belong to the CC chemokine family and are primarily involved in attracting leukocytes to sites of inflammation as part of the innate immune system[ 14 ]. CCL8, along with other proinflammatory cytokines, have previously been found to be elevated in calvarial periosteum tissue of chronic migraine patients[ 15 ]. Experiments on rats have further shown that an inflammatory stimulation of the trigeminal ganglion afferent nerves innervating the calvarial periosteum results in periorbital hypersensitivity, a mechanism potentially relevant for CH, although we found increased CCL8 in CSF and not in serum[ 16 ]. Another chemokine, CCL2 has also been found to be elevated in CSF from patients with other primary headache disorders[ 17 ]. CXCL10 and CXCL11 are cytokines of the CXC chemokine family, CXC chemokines have chemoattractant properties on leukocytes like the CC chemokines described above, but mainly attract T lymphocytes[ 18 ]. Both CXCL10 and 11 bind to the CXCR3 receptor leading to activation of the phospholipase C-dependent pathway, which drives actin rearrangement and increase of intracellular calcium[ 19 , 20 ]. CXCL10 further plays a role in activation of microglia and migration to sites of lesions or inflammation in the CNS[ 21 , 22 ]. Considering the broad range of functions and substrates of these proteins, their potential role in CH remains to be clarified. Nociception, intracellular calcium signalling, and microglia activation are all potentially relevant for CH pathophysiology; the first line preventative CH treatment, being a calcium channel blocker, and the top GWAS loci identified for CH, MERTK , being primarily expressed in microglia and other glial cells[ 12 , 23 ]. It is noteworthy that two of the cytokines that were higher in CSF from CH patients, CCL11 and CXCL11, inversely were lower in serum from CH patients compared to controls. A negative correlation between CXCL11 and galectin-3 has previously been found in serum from individuals with ulcerative colitis[ 24 ]. Similarly, we found increased concentrations of galectin-3 in serum from CH patients[ 25 ]. We also observed a trend for lower levels of CCL13 and CCL7 in serum in the post-hoc analysis, suggesting an overall downregulation of chemoattractant cytokines in serum from CH patients. The opposite direction of regulation of chemokines between CSF and serum is interesting and does not correlate with what is reported for example in multiple sclerosis[ 26 ]. Though most elevated cytokines in the CSF of CH patients had a proinflammatory profile, some, such as HGF, are considered as having anti-inflammatory effects[ 27 ]. HGF drives cell survival in various cell types and has been known to dampen proinflammatory cytokine release from macrophages[ 28 ]. A mendelian randomization study concluded HGF to be a potential causative factor for migraine onset[ 29 ]. The HGF-Met pathway has been known to drive differentiation of peptidergic neurons which are especially important in calcitonin gene-related peptide (CGRP) signalling[ 30 ]. The importance of CGRP in both migraine and CH pathophysiology suggests a similar mechanism may occur in CH. TNFSF10/tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and TNFSF12/TNF-related weak inducer of apoptosis (TWEAK), two proapoptotic cytokines in the TNF ligand superfamily were also elevated in CSF in patients. Higher levels of the TNFSF10 receptor, TNFRSF10C, has been found in CH patients during an attack, supporting a role for TNF signalling in CH[ 5 ]. The link between TNFSF12 and CH is vague, experimental data supports a potential role for the TNFSF12 receptor, fibroblast growth factor-inducible-14 (Fn14), in neuropathic pain in rodents[ 31 ]. MMP1 is part of the matrix metalloproteinase family and are considered immune modulators. Their main function is to break down extracellular matrix, but they also play a role in cytokine release and generation of chemokine gradients[ 32 ]. Elevated levels of another member of the MMP family, MMP9, has previously been shown in migraine patients[ 33 ]. HGF and MMP1 among other cytokines were also found at higher levels in CH patients in active bout than controls in a posthoc analysis of serum samples. Several of the cytokines identified in the sub-threshold serum analysis are part of the IL-6 superfamily; IL-6, OSM and CSF3. VEGFA was slightly elevated in serum of CH patients and is an interesting candidate as it has repeatedly been shown to have a pro-nociceptive effect[ 34 , 35 ]. Higher levels of VEGFA have also been found in migraine patients together with elevated CGRP and nitric oxide (NO).[ 36 ] The differences observed between CSF and serum could point towards a greater importance of inflammation in the CNS in the pathophysiology of CH while the peripheral mechanisms have a weaker connection to the immune response. In particular, factors involved in the recruitment of immune cells were upregulated in CSF and we hypothesize that there is a chemoattractant gradient present in these patients, recruiting immune cells and concentrating the inflammation to the CNS. In concordance with our data, previous studies have shown that there is no systemic inflammation in CH patients[ 4 , 37 ]. However, current data does not provide information on potential local inflammatory reactions that may occur in the peripheral nervous system during CH attacks such as in the trigeminal ganglion. Initially our hypothesis stated that inflammatory reactions would be specifically upregulated during active bout and may even constitute a hallmark of the phenotypic switch occurring in CH patients when they transition between these two phases. Our results now show a completely different view, demonstrating very similar levels of cytokines between active bout and remission phase. CH patients are typically considered healthy in between bouts, but these data suggest that CH may be considered a chronic disorder, manifesting physiological changes also when patients are in remission, this is in line with several studies investigating biomarkers for CH not finding differences between active bout and remission[ 38 – 40 ]. It would be of interest to analyse if inflammatory markers normalize with time in elderly patients experiencing long-time/complete remission. The only cytokine specifically related to attacks, IL-13, displayed a trend for increased levels in serum during an attack. IL-13 is typically considered an anti-inflammatory cytokine and is commonly involved in asthma and allergies[ 41 ]. It is interesting to note that cultured primary microglia driven to an M1 phenotype have been found to increase their MERTK and galectin-3 gene expression in response to IL-13 stimulation, knowing that patients with CH have elevated MERTK and galectin-3 in peripheral blood[ 25 , 42 ]. Visual inspection of the data revealed that serum cytokine levels during attacks were highly similar to those of controls, even when there was a difference in patients in active bout or during remission. This normalisation of cytokine levels during attacks has been described for IL-2 in an earlier study[ 8 ], and raises questions regarding the underlying mechanisms of the attacks, the cellular origin of the cytokines, and the activation and migration of immune cells in the different phases of the disease. One possibility may be that the CH attack somehow disturbs the chemokine gradient observed between serum and CSF. Unfortunately, we did not have access to CSF from patients during an attack to verify this hypothesis. OLINK screening of cytokines gives the benefit of performing an unbiased search for immunological markers that may relate to the pathophysiology of CH. Another strength of the study is the large number of markers investigated maximizing the chances to reveal markers as compared to previous studies on CH investigating specific cytokines. Furthermore, we had the possibility to study cytokines both peripherally and in CSF, so our study provides a more complete view of the immune pathways involved than was previously available. This study also has limitations, including little information on the controls, which implies that factors that can influence the results such as age, sex and comorbidities may be overlooked. Also, immune cells have a variety of functions all of which are not completely covered by this OLINK array. In addition, the array does not include any indications of the underlying mechanisms or reveal which cells are responsible for the release of the detected cytokines. Future studies should comprise a broader panel of cytokines and preferably include a larger cohort to increase the power of detecting differences in cytokine levels. Conclusion This study provides the first comprehensive overview of differences in inflammatory markers in CSF and serum of CH patients. Our data clearly show an increase in several inflammatory markers in CSF from CH patients which is indicative of an inflammation in the CNS. Moreover, differences in chemokines between serum and CSF are indicative of a chemotactic gradient concentrating the inflammation to the nervous system. The inflammation is ongoing regardless of disease status, active bout or remission. Abbreviations CCL: C-C motif chemokine ligand CD328: sialic acid binding Ig like lectin 7 CGRP: calcitonin gene-related peptide CH: cluster headache CNS: central nervous system CSF: cerebrospinal fluid CSF3: colony stimulating factor 3 CXCL: C-X-C motif chemokine ligand ELISA: enzyme-linked immunosorbent assay Fn14: fibroblast growth factor-inducible-14 GWAS: genome wide association study HGF: hepatocyte growth factor HLA-DQA1: major histocompatibility complex, class II, DQ alpha 1 HLA-DQB1: major histocompatibility complex, class II, DQ beta 1 ICHD: international classification of headache disorders IFN-γ: interferon gamma IL: interleukin LOD: limit of detection MERTK: MER proto-oncogene, tyrosine kinase MMP: matrix metallopeptidase NF-κB: nuclear factor- κB NLRP3: NLR family pyrin domain containing 3 NO: nitric oxide NPX: normalized protein expression OSM: oncostatin M PEA: proximity extension assay qPCR: quantitative real-time PCR S100: S100 calcium binding protein TGF-β: transforming growth factor beta TNF-α: tumour necrosis factor alpha TNFSF: TNF superfamily member TRAIL: tumour necrosis factor-related apoptosis-inducing ligand TSLP: thymic stromal lymphopoietin TTH: tension type headache TWEAK: TNF-related weak inducer of apoptosis VEGFA: vascular endothelial growth factor A Declarations Ethics approval and consent to participate Ethical permit was obtained from the Swedish Ethical Review Authority prior to the study (diary number 217/02). Informed consent was obtained from all study participants and experiments were conducted in accordance with the declaration of Helsinki for research involving human subjects. Consent for publication Not applicable. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Declaration of Conflicting Interests The authors declare that there is no conflict of interest. Funding The study was funded by the Swedish Brain Foundation (FO2023-0223) and the Mellby Gård Foundation. Contributions CR, AS and ACB designed the study, AS diagnosed and sampled patients and healthy study participants, KW handled and prepared all samples for analysis, CR performed data analysis statistics and visualization, CR, FJO and ACB drafted the manuscript. All authors have read and revised the final manuscript. 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Headache: The Journal of Head and Face Pain 48:135–139. https://doi.org/10.1111/J.1526-4610.2007.00958.X Lim TKY, Shi XQ, Martin HC, et al (2014) Blood-nerve barrier dysfunction contributes to the generation of neuropathic pain and allows targeting of injured nerves for pain relief. Pain 155:954–967. https://doi.org/10.1016/J.PAIN.2014.01.026 Hulse RP, Beazley-Long N, Hua J, et al (2014) Regulation of alternative VEGF-A mRNA splicing is a therapeutic target for analgesia. Neurobiol Dis 71:245–259. https://doi.org/10.1016/J.NBD.2014.08.012 Rodríguez-Osorio X, Sobrino T, Brea D, et al (2012) Endothelial progenitor cells: A new key for endothelial dysfunction in migraine. Neurology 79:474–479. https://doi.org/10.1212/WNL.0B013E31826170CE/SUPPL_FILE/RODRIQUEZ.PDF Søborg MLK, Jensen RH, Barloese M, Petersen AS (2024) Biomarkers in cluster headache: A systematic review. Headache: The Journal of Head and Face Pain 64:98–116. https://doi.org/10.1111/HEAD.14641 Snoer A, Vollesen ALH, Beske RP, et al (2019) Calcitonin-gene related peptide and disease activity in cluster headache. Cephalalgia 39:575–584. https://doi.org/10.1177/0333102419837154 Neeb L, Anders L, Euskirchen P, et al (2015) Corticosteroids alter CGRP and melatonin release in cluster headache episodes. Cephalalgia 35:317–326. https://doi.org/10.1177/0333102414539057 Pellesi L, Chaudhry BA, Vollesen ALH, et al (2022) PACAP38- and VIP-induced cluster headache attacks are not associated with changes of plasma CGRP or markers of mast cell activation. Cephalalgia 42:687–695. https://doi.org/10.1177/03331024211056248 Opal SM, DePalo VA (2000) Anti-Inflammatory Cytokines. Chest 117:1162–1172. https://doi.org/10.1378/CHEST.117.4.1162 Kolosowska N, Keuters MH, Wojciechowski S, et al (2019) Peripheral Administration of IL-13 Induces Anti-inflammatory Microglial/Macrophage Responses and Provides Neuroprotection in Ischemic Stroke. Neurotherapeutics 16:1304. https://doi.org/10.1007/S13311-019-00761-0 Additional Declarations No competing interests reported. Supplementary Files ElevatedcytokinelevelsinthecentralnervoussystemofclusterheadachepatientsinboutandinremissionSUPPLEMENTARY.pdf Cite Share Download PDF Status: Published Journal Publication published 22 Jul, 2024 Read the published version in The Journal of Headache and Pain → Version 1 posted Editorial decision: Revision requested 06 Jul, 2024 Reviews received at journal 03 Jul, 2024 Reviewers agreed at journal 25 Jun, 2024 Reviewers agreed at journal 22 Jun, 2024 Reviews received at journal 21 Jun, 2024 Reviewers agreed at journal 21 Jun, 2024 Reviewers invited by journal 21 Jun, 2024 Editor assigned by journal 21 Jun, 2024 Submission checks completed at journal 21 Jun, 2024 First submitted to journal 20 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4610858","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":323525561,"identity":"17a2c7f2-0b36-44ce-b3e9-e103234979d4","order_by":0,"name":"Caroline Ran","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYLCCBwZQxocDEkRqSYBqYZxBvBYozcxzgAjV/O1nHz5IKLgjxyB9+NljmzMWDPLtDfi1SJxJNzZIMHhmzMCXZm6cc0OCweAMAasMGNLYJBIMDic28DCYSed8AGqRSCCghf8ZWEt9Aw/7N2kLoBb5+Q8IaJGA2JLAwMNjJs0AdBjDDfw6GCRuPGMG+uWwYRsPT5lkzxkJHoMzBBzG35/G+ODDn8Py/Dzs2yR+HKuTk28/QMAaGGCD0jxEqh8Fo2AUjIJRgA8AAC6cOgAh+NDVAAAAAElFTkSuQmCC","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":true,"prefix":"","firstName":"Caroline","middleName":"","lastName":"Ran","suffix":""},{"id":323525562,"identity":"f5c22d56-4eaa-42a8-849c-c440b737b2d1","order_by":1,"name":"Felicia Jennysdotter Olofsgård","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Felicia","middleName":"Jennysdotter","lastName":"Olofsgård","suffix":""},{"id":323525563,"identity":"862461df-2eae-49f7-aeb0-d2d2932fd431","order_by":2,"name":"Katrin Wellfelt","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Katrin","middleName":"","lastName":"Wellfelt","suffix":""},{"id":323525564,"identity":"526b3aa8-2790-42c8-a72d-2535448b9473","order_by":3,"name":"Anna Steinberg","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Steinberg","suffix":""},{"id":323525565,"identity":"ce55f3fb-d9b1-4a18-9efe-66f02e35d7e8","order_by":4,"name":"Andrea Carmine Belin","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"Carmine","lastName":"Belin","suffix":""}],"badges":[],"createdAt":"2024-06-20 09:48:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4610858/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4610858/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s10194-024-01829-9","type":"published","date":"2024-07-23T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60600902,"identity":"7ceb6d58-6fcc-491c-b898-c4af96f2c024","added_by":"auto","created_at":"2024-07-18 16:03:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":54349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNormalized cytokine expression in CSF from controls and cluster headache patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData are represented as NPX, which are normalized protein expression values on a Log2 scale. Analysis comprises 20 controls and 41 CH patients: 17 in remission and 24 in active bout. *: p-value \u0026lt;0.05, **: p-value \u0026lt;0.01, ***: p-value \u0026lt;0.005.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Binder11.png","url":"https://assets-eu.researchsquare.com/files/rs-4610858/v1/648df49d28353f6d5cbbf909.png"},{"id":60600903,"identity":"557f92de-5975-4122-ae89-3107cbd11ebc","added_by":"auto","created_at":"2024-07-18 16:03:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteractions of cytokines differentially expressed in CSF.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSTRING network analysis shows known interactions;\u003c/em\u003e \u003cem\u003eblue lines represent interactions from curated databases, pink lines represent experimentally determined interactions. Number of nodes: 9, number of edges: 11, average node degree: 2.44, avg. local clustering coefficient: 0.778, protein-protein interaction enrichment p-value: \u0026lt; 1.0e-16\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Binder12.png","url":"https://assets-eu.researchsquare.com/files/rs-4610858/v1/8edf9c2dfc14f4eebca8bdd2.png"},{"id":60602275,"identity":"eb9de1ae-2423-4349-b457-054bc04ee51c","added_by":"auto","created_at":"2024-07-18 16:11:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":20644,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNormalized cytokine expression in serum from controls and cluster headache patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCH Attack: CH patients in Active Bout sampled during an attack. Data are represented as NPX, which are normalized protein expression values on a Log2 scale. Analysis comprises 20 controls and 29 CH patients: 11 in remission, 15 in active bout and 3 during an attack. *: p-value \u0026lt;0.05, **: p-value \u0026lt;0.01, ***: p-value \u0026lt;0.005.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Binder13.png","url":"https://assets-eu.researchsquare.com/files/rs-4610858/v1/223f0dffbb5fe0d778f4a9be.png"},{"id":60600907,"identity":"deb72270-5326-49d8-9733-7264fd5d990e","added_by":"auto","created_at":"2024-07-18 16:03:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":109379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSub threshold association analysis of cytokines in serum from cluster headache patients and controls.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCH Attack: CH patients in Active Bout sampled during an attack. Data are represented as NPX, which are normalized protein expression values on a Log2 scale. Analysis comprises 20 controls, 11 CH patients in remission, 15 CH patients in active bout and 3 CH patients during an attack. P-values significant after correction for multiple testing in the initial analysis are labelled as follows *: p-value \u0026lt;0.05, **: p-value \u0026lt;0.01, ***: p-value. Sub-threshold analysis included 22 cytokines,\u003c/em\u003e \u003cem\u003ep-values significant after correction for multiple testing in the subthreshold analysis are labelled as follows \u003c/em\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e: subthreshold association p-value \u0026lt;0.05, \u003c/em\u003e\u003csup\u003e\u003cem\u003e# #\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e: subthreshold association p-value \u0026lt;0.01, \u003c/em\u003e\u003csup\u003e\u003cem\u003e# # #\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e: subthreshold association p-value \u0026lt;0.005,\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Binder14.png","url":"https://assets-eu.researchsquare.com/files/rs-4610858/v1/162779cc5aab5ce7ad9a696e.png"},{"id":61095912,"identity":"ddb769b7-d9f1-4619-8d94-fa7161649a34","added_by":"auto","created_at":"2024-07-25 14:03:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":862536,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4610858/v1/97e99c12-ed4b-4e07-bf09-42cee59646c2.pdf"},{"id":60600904,"identity":"849ee2d0-6402-4ae4-b2f0-b21f3d23437b","added_by":"auto","created_at":"2024-07-18 16:03:19","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":430472,"visible":true,"origin":"","legend":"","description":"","filename":"ElevatedcytokinelevelsinthecentralnervoussystemofclusterheadachepatientsinboutandinremissionSUPPLEMENTARY.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4610858/v1/9259140a4991319bb420a097.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Elevated cytokine levels in the central nervous system of cluster headache patients in bout and in remission","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCluster headache (CH) is a trigeminal autonomic cephalalgia characterized by recurring unilateral headache attacks of extreme severity. Patients with CH typically experience active bouts where they can have up to eight attacks per day, followed by symptom free remission periods[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The trigeminovascular pathway and the trigeminal\u0026ndash;autonomic reflex are activated during CH attacks, resulting in severe pain in the periorbital region as well as the autonomic symptoms manifesting during CH attacks. Nevertheless, the underlying mechanisms are unclear and CH pathophysiology remains to be fully elucidated[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInflammatory reactions have been suggested in the past to cause CH, in particular those of the cavernous sinus, and the corticosteroide Prednisolone, a powerful anti-inflammatory drug, is commonly used as an interim treatment for CH[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Meanwhile, there is no evidence of systemic inflammation in CH patients[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Targeted analysis of inflammatory markers in CH has yielded inconclusive results, often because of small sample sizes and usage of different methodologies. In a large microarray panel screening for differentially expressed genes in peripheral blood, inflammation related genes were found to be upregulated in CH patients as compared to controls (major histocompatibility complex, class II (HLA) -DQA1 and HLA-DQB1), or specifically during active bouts (S100 calcium binding protein (S100) A8 and S100A12)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In another study, a trend for interleukin (IL) -1β and nuclear factor-κB (NF-κB) upregulation was found in blood cells from patients with CH in active bout[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Interestingly, the authors found a concurrent reduction in inflammasome component NLR family pyrin domain containing 3 (NLRP3) expression levels, required for IL-1β release[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In another global gene expression analysis in peripheral blood cells, inflammatory processes were highlighted both by pathway analysis (IL-4 and sialic acid binding Ig like lectin 7 (CD328)) and by expression network analysis as potentially involved in CH[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Increased gene expression of IL-2 has further been reported in CH patients, specifically in active bout in between their CH attacks, while mRNA levels were normalized to control levels during attacks[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Reports on differentially expressed cytokines in CH are scarce, IL-2, and IL-1β have been suggested to be increased in CH patients, but a recent meta-analysis could not confirm elevated IL-1β[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The same meta-analysis summarized findings on the more common primary headaches migraine and tension type headache (TTH) and confirmed higher levels of IL-6, IL-8 and tumour necrosis factor alpha (TNF-α) in migraine patients and of TNF-α and transforming growth factor beta (TGF-β) in TTH compared to controls[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEven less is known regarding inflammation of the nervous system in CH. Due to recent developments in CH genetics there have been speculations of microglia involvement in the pathogenesis of CH. Microglia have been linked to CH through genome wide association studies (GWASs) highlighting the gene MER proto-oncogene, tyrosine kinase (\u003cem\u003eMERTK\u003c/em\u003e), a gene highly expressed in microglia and macrophages involved in phagocytosis of apoptotic cells[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In this study we aimed to perform inflammatory profiling of CH patients in remission period and in active bout in order to get a better understanding of the importance of inflammation in CH and if inflammatory reactions may constitute a hallmark of the phenotypic switch occurring in CH patients when they go into active bout.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterial\u003c/h2\u003e \u003cp\u003eStudy participants were recruited at the Neurology clinic at Karolinska University Hospital, 34 patients diagnosed with CH and 40 control subjects. CH patients were diagnosed by a neurologist (co-author A.S.) according to the international classification of headache disorders (ICHD) 3rd edition[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Informed consent was obtained from all study participants prior to inclusion. Ethical permit to perform the study was obtained from the Swedish Ethical Review Authority (diary number 217/02) and all experiments were conducted in accordance with the declaration of Helsinki for research involving human subjects.\u003c/p\u003e \u003cp\u003e Study participants were asked to give a sample of cerebrospinal fluid (CSF) or serum (blood) or both. Tissue from patients was sampled at several timepoints if possible; in remission, in active bout, and during a CH attack. In total we collected and analysed 120 samples: 21 CSF samples and 27 serum samples from control individuals, 43 CSF samples from CH patients, 18 in remission and 25 in active bout, and 29 serum samples from CH patients, 11 in remission, 15 in active bout and 3 during an attack. Samples were acquired using standard procedures and kept at -80\u0026deg;C until analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eOLINK cytokine panels\u003c/h2\u003e \u003cp\u003eSamples (CSF and serum) were analysed, and quality control and calibrator normalization were performed at SciLifeLab Affinity Proteomics Uppsala on OLINK\u0026reg; Proximity Extension Assay (PEA) Target 48 cytokine panels, (Affinity Proteomics Uppsala, SciLifeLab, Uppsala University, SE-751 85 Uppsala). The panel contains 45 assays, 89% (n\u0026thinsp;=\u0026thinsp;40) of which passed quality control (assays failing quality control: C-X-C motif chemokine ligand (CXCL) 12, IL-1β, IL-15, IL-17A, and IL-4). 99% (n\u0026thinsp;=\u0026thinsp;119) of the analysed samples passed quality control. Data is reported as normalized protein expression (NPX) unit in Log2 scale and was analysed separately for CSF and serum samples in R(v4.1.3) using the OlinkAnalyze package.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003ePCA analysis confirmed that one CSF sample did not pass the quality control and also revealed a cluster of serum samples which may represent outliers (n\u0026thinsp;=\u0026thinsp;7), Supplementary Figs.\u0026nbsp;1A and 1B. Comparison of group averages between the flagged samples and the remaining controls showed highly heterogenous values, justifying their removal from the analysis. Plotting all samples in a histogram and performing the Shapiro-wilks test showed that data was non normally distributed, Supplementary Figs.\u0026nbsp;1C and 1D. Two samples were run in duplicate and removed from the analysis. Assays were removed from analysis if they did not pass quality control and if the number of samples with NPX values below the limit of detection (LOD) was higher than 75%; 11 CSF assays (C-C motif chemokine ligand (CCL) 7, colony stimulating factor 3 (CSF3), interferon gamma (IFN-γ), IL-2, IL-10, IL-13, IL-17F, IL-27, IL-33, TNF-α, thymic stromal lymphopoietin (TSLP)) and 3 serum assays (IL-33, IL-17F, TSLP). Group comparisons were made with one-way ANOVA or Wilcoxon rank-sum test using the Benjamini \u0026amp; Hochberg method for correction for multiple testing. A network analysis of associated cytokines was performed using the STRING database v.12.0[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe final analysis comprised of 49 serum samples (20 from controls and 29 from CH patients; 11 in remission, 15 in active bout and 3 during an attack) and 61 CSF samples (20 from controls and 41 from CH patients; 17 in remission and 24 in active bout) from 39 control individuals and 34 CH patients in total.\u003c/p\u003e \u003cp\u003eAnalysis of CSF cytokine levels in controls and CH patients showed differences in nine cytokines; CCL8, CCL13, CCL11, CXCL10, CXCL11, hepatocyte growth factor (HGF), matrix metallopeptidase (MMP) 1, TNF superfamily member (TNFSF) 10 and TNFSF12 (Fig.\u0026nbsp;1, Supplementary Table\u0026nbsp;1). All nine cytokines were found to be elevated in CSF in CH patients as compared to controls but did not differ between CH patients in remission and in active bout (Fig.\u0026nbsp;1, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Network analysis further showed strong interactions between all upregulated chemokines; CCL8, CCL13, CCL11, CXCL10, CXCL11, with a protein-protein interaction enrichment p-value\u0026thinsp;\u0026lt;\u0026thinsp;1.0e-16 (Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e[\u003cb\u003eFigure 1\u003c/b\u003e]\u003c/p\u003e \u003cp\u003e[\u003cb\u003eFigure 2\u003c/b\u003e]\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults from comparison of normalized cytokine expression in controls and cluster headache patients.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eRemission\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eActive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eAttack\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCytokine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEstimate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAdj. P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEstimate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAdj. P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eEstimate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAdj. P\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCL11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.016\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.031\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.003\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.995\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCL13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCL8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCXCL10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.029\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.018\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCXCL11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.028\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.028\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.028\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.997\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-1.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNFSF10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.013\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.010\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNFSF12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.003\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.007\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCL11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.644\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.961\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.248\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCL13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.976\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCL8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.947\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCXCL10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCXCL11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.972\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.913\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-2.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.255\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.817\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMMP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.861\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNFSF10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.985\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNFSF12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.817\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCL11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eActive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-1.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.125\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCXCL11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eActive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-2.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.104\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eCSF: cerebrospinal fluid; Ref: Reference group in analysis; C: Control; Rem: Cluster headache patients in remission; Act: Cluster headache patients in active bout; Adj. P: adjusted P-value; NA: Not Available.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe analysis of cytokine levels in serum from controls and CH patients in remission, active bout and during a CH attack showed less variation between groups (Supplementary Table\u0026nbsp;2). Only two cytokines were found to differ between controls and CH patients; CCL11 and CXCL11, both of which were found at lower levels in patients than controls during remission phase and in active bout (Fig.\u0026nbsp;3). Although lower at baseline, CCL11 and CXCL11 levels in patients were found to raise to control levels during CH attacks. The difference observed between CH patients in remission, or active bout and patients during an attack was not significant, possibly because of a low number of samples (n\u0026thinsp;=\u0026thinsp;3) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e[\u003cb\u003eFigure 3\u003c/b\u003e]\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePost hoc analysis of serum cytokines\u003c/h2\u003e \u003cp\u003eAs the number of patients leaving a sample during a CH attack were few (serum n\u0026thinsp;=\u0026thinsp;3, no CSF samples), the data was revisited comparing assays below significance to understand trends in cytokine levels in serum. Cytokines with significant p-values before correction for multiple testing, or a change in NPX values bigger or smaller than 10% comparing CH patients in active bout with controls and CH patients during attack with CH patients in active bout were included, resulting in a post hoc analysis of 22 cytokines.\u003c/p\u003e \u003cp\u003eData revealed sub threshold associations for several cytokines in serum, variations in four cytokines were of the same direction as the associations detected in the primary analysis (showing decreased levels of CXCL11 and CCL11). Namely CCL13, CCL19, IL-17C and CCL7 which were all displaying trends of decreased levels in CH patients in remission (Fig.\u0026nbsp;4). Interestingly, these cytokines showed trends of opposite direction in serum as compared to CSF. NPX levels of HGF, CSF3, MMP1, oncostatin M (OSM), IL6 and vascular endothelial growth factor A (VEGFA) were elevated in CH patients in active bout as compared to controls (Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003eCytokine levels in samples from CH patients during an attack were consistently comparable with those of controls rather than patients in active bout or remission, with the exception of IL-13, which was detected at higher levels during an attack than in controls and CH patients in remission and in active bout.\u003c/p\u003e \u003cp\u003e[\u003cb\u003eFigure 4\u003c/b\u003e]\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have conducted a large screening of cytokine levels in CH patients and controls using serum and CSF samples in order to get an overview of the inflammatory state in the periphery and in the central nervous system (CNS). Nine cytokines were found to be higher in CSF from patients as compared to controls, particularly cytokines with chemoattractant properties on leukocytes. Interestingly, major differences in cytokine levels were detected between CSF and serum. In the discovery analysis two of the cytokines found at higher levels in CSF were lower in serum.\u003c/p\u003e \u003cp\u003ePrevious studies on cytokines in CH have suggested an increase of IL-2 and IL-1β in blood of CH patients although data are conflicting for IL-1β[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. IL-2 gene expression and protein levels in blood was found to be increased in CH active bout compared to controls, while gene expression levels decreased to control levels during attacks[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In our study, the assay for IL-1β did not pass quality control and we could not replicate the findings of an increase for IL-2 in our material. These differences could potentially be due to discrepancies between methodology, quantitative Real-time PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) in previous studies while we used PEA, an ELISA based array that increases the sensitivity coupling the detection to qPCR.\u003c/p\u003e \u003cp\u003eIn this report we found evidence of a possible ongoing inflammation in the CNS of CH patients with elevated levels of several cytokines in the CSF of CH patients as compared to controls. To our knowledge, this is the first reported screening for cytokines in CSF from CH patients. There was very little difference between patients in remission phase and active bout for all the differentially expressed cytokines, which indicates that the neuroinflammatory state in CH patients is not exclusive to the active bout. The cytokines showing the largest increase in CSF from patients were HGF, MMP1, TNSF10 and 12 and several chemokines, all of which were found to have strong interactions in a network analysis. CCL8, CCL11, and CCL13 belong to the CC chemokine family and are primarily involved in attracting leukocytes to sites of inflammation as part of the innate immune system[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. CCL8, along with other proinflammatory cytokines, have previously been found to be elevated in calvarial periosteum tissue of chronic migraine patients[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Experiments on rats have further shown that an inflammatory stimulation of the trigeminal ganglion afferent nerves innervating the calvarial periosteum results in periorbital hypersensitivity, a mechanism potentially relevant for CH, although we found increased CCL8 in CSF and not in serum[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Another chemokine, CCL2 has also been found to be elevated in CSF from patients with other primary headache disorders[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. CXCL10 and CXCL11 are cytokines of the CXC chemokine family, CXC chemokines have chemoattractant properties on leukocytes like the CC chemokines described above, but mainly attract T lymphocytes[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Both CXCL10 and 11 bind to the CXCR3 receptor leading to activation of the phospholipase C-dependent pathway, which drives actin rearrangement and increase of intracellular calcium[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. CXCL10 further plays a role in activation of microglia and migration to sites of lesions or inflammation in the CNS[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Considering the broad range of functions and substrates of these proteins, their potential role in CH remains to be clarified. Nociception, intracellular calcium signalling, and microglia activation are all potentially relevant for CH pathophysiology; the first line preventative CH treatment, being a calcium channel blocker, and the top GWAS loci identified for CH, \u003cem\u003eMERTK\u003c/em\u003e, being primarily expressed in microglia and other glial cells[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is noteworthy that two of the cytokines that were higher in CSF from CH patients, CCL11 and CXCL11, inversely were lower in serum from CH patients compared to controls. A negative correlation between CXCL11 and galectin-3 has previously been found in serum from individuals with ulcerative colitis[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Similarly, we found increased concentrations of galectin-3 in serum from CH patients[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. We also observed a trend for lower levels of CCL13 and CCL7 in serum in the post-hoc analysis, suggesting an overall downregulation of chemoattractant cytokines in serum from CH patients. The opposite direction of regulation of chemokines between CSF and serum is interesting and does not correlate with what is reported for example in multiple sclerosis[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThough most elevated cytokines in the CSF of CH patients had a proinflammatory profile, some, such as HGF, are considered as having anti-inflammatory effects[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. HGF drives cell survival in various cell types and has been known to dampen proinflammatory cytokine release from macrophages[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A mendelian randomization study concluded HGF to be a potential causative factor for migraine onset[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The HGF-Met pathway has been known to drive differentiation of peptidergic neurons which are especially important in calcitonin gene-related peptide (CGRP) signalling[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The importance of CGRP in both migraine and CH pathophysiology suggests a similar mechanism may occur in CH. TNFSF10/tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and TNFSF12/TNF-related weak inducer of apoptosis (TWEAK), two proapoptotic cytokines in the TNF ligand superfamily were also elevated in CSF in patients. Higher levels of the TNFSF10 receptor, TNFRSF10C, has been found in CH patients during an attack, supporting a role for TNF signalling in CH[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The link between TNFSF12 and CH is vague, experimental data supports a potential role for the TNFSF12 receptor, fibroblast growth factor-inducible-14 (Fn14), in neuropathic pain in rodents[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. MMP1 is part of the matrix metalloproteinase family and are considered immune modulators. Their main function is to break down extracellular matrix, but they also play a role in cytokine release and generation of chemokine gradients[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Elevated levels of another member of the MMP family, MMP9, has previously been shown in migraine patients[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. HGF and MMP1 among other cytokines were also found at higher levels in CH patients in active bout than controls in a posthoc analysis of serum samples. Several of the cytokines identified in the sub-threshold serum analysis are part of the IL-6 superfamily; IL-6, OSM and CSF3. VEGFA was slightly elevated in serum of CH patients and is an interesting candidate as it has repeatedly been shown to have a pro-nociceptive effect[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Higher levels of VEGFA have also been found in migraine patients together with elevated CGRP and nitric oxide (NO).[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe differences observed between CSF and serum could point towards a greater importance of inflammation in the CNS in the pathophysiology of CH while the peripheral mechanisms have a weaker connection to the immune response. In particular, factors involved in the recruitment of immune cells were upregulated in CSF and we hypothesize that there is a chemoattractant gradient present in these patients, recruiting immune cells and concentrating the inflammation to the CNS. In concordance with our data, previous studies have shown that there is no systemic inflammation in CH patients[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, current data does not provide information on potential local inflammatory reactions that may occur in the peripheral nervous system during CH attacks such as in the trigeminal ganglion.\u003c/p\u003e \u003cp\u003eInitially our hypothesis stated that inflammatory reactions would be specifically upregulated during active bout and may even constitute a hallmark of the phenotypic switch occurring in CH patients when they transition between these two phases. Our results now show a completely different view, demonstrating very similar levels of cytokines between active bout and remission phase. CH patients are typically considered healthy in between bouts, but these data suggest that CH may be considered a chronic disorder, manifesting physiological changes also when patients are in remission, this is in line with several studies investigating biomarkers for CH not finding differences between active bout and remission[\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. It would be of interest to analyse if inflammatory markers normalize with time in elderly patients experiencing long-time/complete remission. The only cytokine specifically related to attacks, IL-13, displayed a trend for increased levels in serum during an attack. IL-13 is typically considered an anti-inflammatory cytokine and is commonly involved in asthma and allergies[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. It is interesting to note that cultured primary microglia driven to an M1 phenotype have been found to increase their \u003cem\u003eMERTK\u003c/em\u003e and galectin-3 gene expression in response to IL-13 stimulation, knowing that patients with CH have elevated \u003cem\u003eMERTK\u003c/em\u003e and galectin-3 in peripheral blood[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Visual inspection of the data revealed that serum cytokine levels during attacks were highly similar to those of controls, even when there was a difference in patients in active bout or during remission. This normalisation of cytokine levels during attacks has been described for IL-2 in an earlier study[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and raises questions regarding the underlying mechanisms of the attacks, the cellular origin of the cytokines, and the activation and migration of immune cells in the different phases of the disease. One possibility may be that the CH attack somehow disturbs the chemokine gradient observed between serum and CSF. Unfortunately, we did not have access to CSF from patients during an attack to verify this hypothesis.\u003c/p\u003e \u003cp\u003eOLINK screening of cytokines gives the benefit of performing an unbiased search for immunological markers that may relate to the pathophysiology of CH. Another strength of the study is the large number of markers investigated maximizing the chances to reveal markers as compared to previous studies on CH investigating specific cytokines. Furthermore, we had the possibility to study cytokines both peripherally and in CSF, so our study provides a more complete view of the immune pathways involved than was previously available. This study also has limitations, including little information on the controls, which implies that factors that can influence the results such as age, sex and comorbidities may be overlooked. Also, immune cells have a variety of functions all of which are not completely covered by this OLINK array. In addition, the array does not include any indications of the underlying mechanisms or reveal which cells are responsible for the release of the detected cytokines. Future studies should comprise a broader panel of cytokines and preferably include a larger cohort to increase the power of detecting differences in cytokine levels.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides the first comprehensive overview of differences in inflammatory markers in CSF and serum of CH patients. Our data clearly show an increase in several inflammatory markers in CSF from CH patients which is indicative of an inflammation in the CNS. Moreover, differences in chemokines between serum and CSF are indicative of a chemotactic gradient concentrating the inflammation to the nervous system. The inflammation is ongoing regardless of disease status, active bout or remission.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCCL: C-C motif chemokine ligand\u003c/p\u003e\n\u003cp\u003eCD328: sialic acid binding Ig like lectin 7 \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCGRP: calcitonin gene-related peptide\u003c/p\u003e\n\u003cp\u003eCH: cluster headache \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCNS: central nervous system\u003c/p\u003e\n\u003cp\u003eCSF: cerebrospinal fluid\u003c/p\u003e\n\u003cp\u003eCSF3: colony stimulating factor 3\u003c/p\u003e\n\u003cp\u003eCXCL: C-X-C motif chemokine ligand\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eELISA: enzyme-linked immunosorbent assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFn14: fibroblast growth factor-inducible-14\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGWAS: genome wide association study\u003c/p\u003e\n\u003cp\u003eHGF: hepatocyte growth factor\u003c/p\u003e\n\u003cp\u003eHLA-DQA1: major histocompatibility complex, class II, DQ alpha 1\u003c/p\u003e\n\u003cp\u003eHLA-DQB1: major histocompatibility complex, class II, DQ beta 1\u003c/p\u003e\n\u003cp\u003eICHD: international classification of headache disorders\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIFN-γ: interferon gamma\u003c/p\u003e\n\u003cp\u003eIL: interleukin\u003c/p\u003e\n\u003cp\u003eLOD: limit of detection\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMERTK: MER proto-oncogene, tyrosine kinase \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMMP: matrix metallopeptidase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNF-κB: nuclear factor- κB\u003c/p\u003e\n\u003cp\u003eNLRP3: NLR family pyrin domain containing 3\u003c/p\u003e\n\u003cp\u003eNO: nitric oxide \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNPX: normalized protein expression\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOSM: oncostatin M\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePEA: proximity extension assay \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eqPCR: quantitative real-time PCR \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eS100: S100 calcium binding protein\u003c/p\u003e\n\u003cp\u003eTGF-β: transforming growth factor beta \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTNF-α: tumour necrosis factor alpha\u003c/p\u003e\n\u003cp\u003eTNFSF: TNF superfamily member\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTRAIL: tumour necrosis factor-related apoptosis-inducing ligand\u003c/p\u003e\n\u003cp\u003eTSLP: thymic stromal lymphopoietin\u003c/p\u003e\n\u003cp\u003eTTH: tension type headache \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTWEAK: TNF-related weak inducer of apoptosis\u003c/p\u003e\n\u003cp\u003eVEGFA: vascular endothelial growth factor A\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical permit was obtained from the Swedish Ethical Review Authority prior to the study (diary number 217/02). Informed consent was obtained from all study participants and experiments were conducted in accordance with the declaration of Helsinki for research involving human subjects.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated\u0026nbsp;during\u0026nbsp;and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Conflicting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was funded by the Swedish Brain Foundation (FO2023-0223) and the Mellby G\u0026aring;rd Foundation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCR, AS and ACB designed the study, AS diagnosed and sampled patients and healthy study participants, KW handled and prepared all samples for analysis, CR performed data analysis statistics and visualization, CR, FJO and ACB drafted the manuscript. All authors have read and revised the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge support of SciLifeLab Affinity Proteomics at Uppsala University, Sweden for providing assistance in protein analyses.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eThe International Headache Society (2018) Headache Classification Committee of the International Headache Society (IHS) The International Classification of Headache Disorders, 3rd edition. 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Circ Res 67:469\u0026ndash;480. https://doi.org/10.1161/01.RES.67.2.469\u003c/li\u003e\n \u003cli\u003eVolarevic V, Zdravkovic N, Harrell CR, et al (2019) Galectin-3 Regulates Indoleamine-2,3-dioxygenase-Dependent Cross-Talk between Colon-Infiltrating Dendritic Cells and T Regulatory Cells and May Represent a Valuable Biomarker for Monitoring the Progression of Ulcerative Colitis. Cells 2019, Vol 8, Page 709 8:709. https://doi.org/10.3390/CELLS8070709\u003c/li\u003e\n \u003cli\u003eEdvinsson JCA, Ran C, Olofsg\u0026aring;rd FJ, et al (2024) MERTK in the rat trigeminal system: a potential novel target for cluster headache? J Headache Pain 25:. https://doi.org/10.1186/S10194-024-01791-6\u003c/li\u003e\n \u003cli\u003eMartynova E, Goyal M, Johri S, et al (2020) Serum and Cerebrospinal Fluid Cytokine Biomarkers for Diagnosis of Multiple Sclerosis. https://doi.org/10.1155/2020/2727042\u003c/li\u003e\n \u003cli\u003eImamura R, Matsumoto K (2017) Hepatocyte growth factor in physiology and infectious diseases. Cytokine 98:97\u0026ndash;106. https://doi.org/10.1016/J.CYTO.2016.12.025\u003c/li\u003e\n \u003cli\u003eKusunoki H, Taniyama Y, Otsu R, et al (2014) Anti-inflammatory effects of hepatocyte growth factor on the vicious cycle of macrophages and adipocytes. Hypertension Research 37:500\u0026ndash;506. https://doi.org/10.1038/hr.2014.41\u003c/li\u003e\n \u003cli\u003eFu C, Chen Y, Xu W, Zhang Y (2023) Exploring the causal relationship between inflammatory cytokines and migraine: a bidirectional, two-sample Mendelian randomization study. 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Neurology 79:474\u0026ndash;479. https://doi.org/10.1212/WNL.0B013E31826170CE/SUPPL_FILE/RODRIQUEZ.PDF\u003c/li\u003e\n \u003cli\u003eS\u0026oslash;borg MLK, Jensen RH, Barloese M, Petersen AS (2024) Biomarkers in cluster headache: A systematic review. Headache: The Journal of Head and Face Pain 64:98\u0026ndash;116. https://doi.org/10.1111/HEAD.14641\u003c/li\u003e\n \u003cli\u003eSnoer A, Vollesen ALH, Beske RP, et al (2019) Calcitonin-gene related peptide and disease activity in cluster headache. Cephalalgia 39:575\u0026ndash;584. https://doi.org/10.1177/0333102419837154\u003c/li\u003e\n \u003cli\u003eNeeb L, Anders L, Euskirchen P, et al (2015) Corticosteroids alter CGRP and melatonin release in cluster headache episodes. Cephalalgia 35:317\u0026ndash;326. https://doi.org/10.1177/0333102414539057\u003c/li\u003e\n \u003cli\u003ePellesi L, Chaudhry BA, Vollesen ALH, et al (2022) PACAP38- and VIP-induced cluster headache attacks are not associated with changes of plasma CGRP or markers of mast cell activation. Cephalalgia 42:687\u0026ndash;695. https://doi.org/10.1177/03331024211056248\u003c/li\u003e\n \u003cli\u003eOpal SM, DePalo VA (2000) Anti-Inflammatory Cytokines. Chest 117:1162\u0026ndash;1172. https://doi.org/10.1378/CHEST.117.4.1162\u003c/li\u003e\n \u003cli\u003eKolosowska N, Keuters MH, Wojciechowski S, et al (2019) Peripheral Administration of IL-13 Induces Anti-inflammatory Microglial/Macrophage Responses and Provides Neuroprotection in Ischemic Stroke. Neurotherapeutics 16:1304. https://doi.org/10.1007/S13311-019-00761-0\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-headache-and-pain","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tjhp","sideBox":"Learn more about [The Journal of Headache and Pain](https://thejournalofheadacheandpain.biomedcentral.com/)","snPcode":"10194","submissionUrl":"https://submission.nature.com/new-submission/10194/3","title":"The Journal of Headache and Pain","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Inflammation, neuroinflammation, headache, chemokines","lastPublishedDoi":"10.21203/rs.3.rs-4610858/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4610858/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eCluster headache\u003cstrong\u003e \u003c/strong\u003eis characterized by activation of the trigeminovascular pathway with subsequent pain signalling in the meningeal vessels, and inflammation has been suggested to play a role in the pathophysiology. To further investigate inflammation in cluster headache, inflammatory markers were analysed in patients with cluster headache and controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e We performed a case-control study, collecting cerebrospinal fluid and serum samples from healthy controls, cluster headache patients in remission, active bout, and during an attack to cover the dynamic range of the cluster headache phenotype. Inflammatory markers were quantified using Target 48 OLINK cytokine panels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Altered levels of several cytokines were found in patients with cluster headache compared to controls. CCL8, CCL13, CCL11, CXCL10, CXCL11, HGF, MMP1, TNFSF10 and TNFSF12 levels in cerebrospinal fluid were comparable in active bout and remission, though significantly higher than in controls. In serum samples, CCL11 and CXCL11 displayed decreased levels in patients. Only one cytokine, IL-13 was differentially expressed in serum during attacks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion and Interpretation: \u003c/strong\u003eOur data shows signs of possible neuroinflammation occurring in biological samples from cluster headache patients. Increased cerebrospinal fluid cytokine levels are detectable in active bout and during remission, indicating neuroinflammation could be considered a marker for cluster headache and is unrelated to the different phases of the disorder.\u003c/p\u003e","manuscriptTitle":"Elevated cytokine levels in the central nervous system of cluster headache patients in bout and in remission","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 16:03:14","doi":"10.21203/rs.3.rs-4610858/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-06T15:18:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-03T05:32:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120789195309400746832908763224585611732","date":"2024-06-26T03:12:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45150647493302173539555115832542868393","date":"2024-06-22T08:14:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-21T11:35:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252382779119598267636323534010853528235","date":"2024-06-21T09:05:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-21T09:01:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-21T06:50:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-21T06:49:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Journal of Headache and Pain","date":"2024-06-20T09:47:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-headache-and-pain","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tjhp","sideBox":"Learn more about [The Journal of Headache and Pain](https://thejournalofheadacheandpain.biomedcentral.com/)","snPcode":"10194","submissionUrl":"https://submission.nature.com/new-submission/10194/3","title":"The Journal of Headache and Pain","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ce76f789-e794-49ae-9fe6-4c06cc1e1ff6","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-07-25T14:03:24+00:00","versionOfRecord":{"articleIdentity":"rs-4610858","link":"https://doi.org/10.1186/s10194-024-01829-9","journal":{"identity":"the-journal-of-headache-and-pain","isVorOnly":false,"title":"The Journal of Headache and Pain"},"publishedOn":"2024-07-23 00:00:00","publishedOnDateReadable":"July 23rd, 2024"},"versionCreatedAt":"2024-07-18 16:03:14","video":"","vorDoi":"10.1186/s10194-024-01829-9","vorDoiUrl":"https://doi.org/10.1186/s10194-024-01829-9","workflowStages":[]},"version":"v1","identity":"rs-4610858","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4610858","identity":"rs-4610858","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}