Expression of Suppressor of Cytokine Signalling (SOCS) 1 and 3 in Behcet's Disease
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1
Queen Mary's Schools of Medicine and Dentistry, Centre for Clinical and Diagnostic Oral Sciences, United Kingdom
Abstract: Behçet’s disease (BD) is an autoinflammatory disease of unknown aetiology where a pro-inflammatory cytokine profile is a key feature. Suppressor of Cytokine Signalling molecules (SOCS) 1 and 3 belong to a family of eight intracellular proteins which have been shown to negatively regulate cytokine signals through the JAK-STAT pathway and have profound effects on many immunological processes. SOCS 1 deficient mice develop severe skin and eye lesions, reminiscent of BD, through recruitment of TH1 and TH17 cells into these non-lymphoid tissues. Therefore, we have investigated the expression of SOCS1, 2 and 3 along with TH1 and TH17 cytokines and the STATs and Transcription factors associated with these pathways in BD.
Introduction: Behçet’s disease (BD) is a chronic, multi systemic, recurrent vasculitis disease of unknown aetiology with relapsing episodes of recurrent oral ulcers, uveitis, skin lesions and genital ulcers. The aetiopathogenesis of BD remains unknown but an autoimmune reaction triggered by an infectious or environmental agent in a genetically susceptible individual is the most widely accepted hypothesis. One of the key immunopathological features of BD is the high level of pro-inflammatory and Th1 cytokines which play important role in onset and perpetuation of disease. The elevated levels of typical Th1 cytokines: IL-12 and IFN g, have been reported in BD patients with uveitis (Pineton de Chambrun et al 2012; Guenane et al, 2006; Belguendouz et al, 2008).Up regulation of Th17 cytokines including IL-23 and IL-17 in PBMCs and sera of Behçet’s patients with active uveitis have also been reported (Chi et al, 2008). Immunogenetic studies demonstrated the association of SNPs on several cytokines i.e. IL-1, TNFa, IFNg, IL-12, IL-10 and IL23R- IL12RB2 region with BD disease (Alayli et al, 2007; Remmers et al, 2010; Mizuki et al, 2010; Karasneh et al, 2003).Cytokines implicated in BD (Zhou et al 2012) such as IFNg, IL-12, IL-23 and IL-6 exert their effect through activation of JAK-STAT signalling pathways which are negatively regulated by the Suppressor of Cytokine Signalling molecules (SOCS).SOCS proteins are a family of 8 proteins (CIS and SOCS1-7) which regulate cytokine signalling by inactivation of the JAK, blocking access of the STATs to receptor binding sites, and ubiquintination of signalling proteins (reviewed by Yoshimura et al 2012 and Linossi et al 2013). Furthermore both STATs and SOCS are key regulators of CD4+ T-cell differentiation (Knosp et al 2012). In this respect any disregulation in this balance can affect cellular responsiveness to cytokines and cause subsequent pathological effects. Studies of a SOCS1- deficient mouse have revealed that dysfunction of SOCS proteins leads to development of skin and eyes lesions both of which are characteristic of Behçet’s disease (Yu et al 2008). At present there is little data regarding expression of SOCS family in BD. This study was designed to investigate the expression of SOCS1, SOCS2 and SOCS3 in Behçet’s disease.
Materials and Methods: Samples from a total of four study groups were collected: BD patients (as diagnosed by the International Study Group 1990), recurrent aphthous stomatitis (RAS) and healthy controls (HC) and a small group of patients with oro-genital ulceration unrelated to BD. Peripheral blood mononuclear cells were separated using Ficoll density gradient centrifugation and Neutrophils were separated using the Ammonium Chloride lysis method. Buccal mucosal brush biopsies (BMBB) were collected using a nylon cytology brush (Seoudi et al 2013 In Press). Unstimulated saliva was also collected. SOSC1-3 mRNA in Peripheral blood mononuclear cells (PBMCs), Neutrophils and BMBB from BD patients (relapsed and quite phases of the disease) were compared with samples from recurrent aphthous stomatitis (RAS) patients and healthy controls (HC) using RT-PCR. SOCS1-3 protein was studied by western blot. Cytokine profiles of serum and saliva from BD were investigated using multiplex bead analysis (11 cytokines representing Th1, TH2 and Th17) and cytokine secreting cells were enumerated by ELISPOT. Cytokine secretion from PBMC’s following in vitro stimulation was also measured. 46 kinase phosphorylation sites in PBMCs were investigated using human phospho-kinase arrays. Stats 1, 3 and 5 and the transcription factors T-bet and ROR g t were assayed by PhosFlow FACS analysis.
Statistical analysis was performed using analysis of variance (ANOVA) and non-parametric Mann–Whitney U-test.
Results: Protein and mRNA expression SOCS1, SOCS2 and SOCS3
There was a significant difference in the expression of both SOCS1 and 3 at both the protein and mRNA level. SOCS1 protein in the Neutrophils of relapsed BD patients was greater compared that in quiet BD (p=0.0338) or with HC (p=0.0317). Interestingly SOCS3 protein was elevated in the Neutrophils of quiet BD compared with relapsed BD (p=0.0393). There was no significant difference in the protein levels in the PBMCs for either SOCS1 or SOCS3. There was no significant difference in the expression of SOCS2 in the groups investigated for either Neutrophils (ANOVA: P=0.9464) or PBMCs (ANOVA: P=0.1217). Message for both SOCS1 and SOCS3 were elevated in BD patients compared with both HC and RAS. SOCS1 and 3 were also significantly upregulated in Buccal Mucosal brush Biopsies (BMBB), with discrete differences in ulcerated and non-ulcerated mucosal sites.
Cytokines: 79 serum samples from BD patient, HC, OGU (Oro Genital Ulcer) and RAS were analysed (Table1). The results were compared with disease activity: quiet and relapsed. The most abundant cytokines in the serum of BD patients were IL-8(detected in 56 out of 58 patient sample), TNF-a (39 /58), IL-1β (23 /58) and IL-4(21/58) respectively.IL-6 was undetectable (only 2 out of 79) also IL-5 and TNF-β was found only in a few samples. The level of detectable cytokines were analysed in all 4 groups plus quiet and relapsed BD subgroups. The results showed that IL-8, IFN-g,TNF-a and IL-1β were raised in BD patient compare to HC but the differences were not significant. Our results are different from other studies; however, there is considerable variation from study to study which might be due to differences in cohort: ethnic origins, age, patient medication and organ involvement which may bias the results.
Table 1: The number of positive cytokine expression of serum samples (n = 79) in the range of detectable levels
IL-6, IL-1β and TNF-a were significantly higher in the saliva of BD patients compared with HC (p=0.0280; p=0.0451 and p=0.0177 respectively). Cytokine secretion from PBMCs following stimulation was carried out to evaluate the capacity of the cells produce key cytokines. IL-10 and IL12p70 secretion was induced by stimulation of PBMC with Human HSP70 in HC but not BD while IL-6, IL-23 and IL-17 ELISPOTS were detected after short tern (24 or 48 hours) stimulation (Fig 2).
Fig 2: Cytokine secretion from PBMC’s following stimulation with Human HSP70. Results expressed as stimulation index compared with medium controls
PhosphFlow analysis of Transcription factors: FACS analysis was carried out to examine the levels of activation of the STATS and transcription factors T-bet and ROR T in the PBMCs of BD compare with HC. There was no significant difference in the groups for T-bet (ANOVA P=0.2724). However, there was a highly significant difference in the levels of this factor in unstimulated cells when BD patients, who showed activity in the mouth only, were compared with fully relapsed patients (Mann-Whitney, p=0.0011). STAT3 showed some differences between the groups, just failing to reach significance (ANOVA:P=0.0854). There were significant differences between the sub-groups for example BD quiet (but mouth active) compared with BD-R (fully relapsed according to the International Criteria 1990) where p=0.0079 (Fig 3). There were also differences in the expression of STAT 5 in unstimualted PBMCs from BD quiet compared with BD quiet but mouth active (p= 0.0337).
Fig 3: PhophoFlow analysis of STAT 3 in PBMCs
PhosphoKinase Microarray analysis: Microarray analysis of 46 Phosphokinases supported the FACs analysis and suggested that there were significant phosphorylation in STATS 1-5, but not STAT 6 in BD compared with HC. This analysis also revealed several unexpected differences in the profile of BD compared with health controls and has provided several targets for further investigation.
Discussio: We have been able to show significant differences in the expression of SOCS protein and mRNA between the groups studied in this investigation. Of considerable interest is the subtle difference in expression of these molecules in BD patients who are fully relapsed compared to those who show activity only in the mouth. This may be a reflection of the cells infiltrating ulcerated compared to non-ulcerated sites and reinforces the importance of the oral mucosa as an indicator tissue (Dalghous et al 2006; Kose et al 2007). There is considerable anecdotal evidence to suggest that controlling the oral symptoms of the disease has a considerable impact on the ability to control the other more challenging symptoms of the disease. The involvement of the Th1 and Th17 pathways in this disease has received considerable attention of late and again the differences observed in the cytokine profile of the saliva in BD patients are worthy of note. It should be remembered that when sampling the serum or plasma the values represent an accumulation over time in an enclosed system. In saliva, the constant production and flow of this fluid reflects a huge biochemical activity as we swallow up to 1 litre of saliva a day so that cytokines secreted into saliva do so at a rate that may be far greater than that found in serum.
References
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Keywords:
SOCS,
Behcets,
pro-inflammatory,
STAT,
Pathology
Conference:
15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.
Presentation Type:
Abstract
Topic:
Innate immunity
Citation:
Hamedi
M,
Bergmeier
LA,
Hagi-Pavli
E and
Fortune
F
(2013). Expression of Suppressor of Cytokine Signalling (SOCS) 1 and 3 in Behcet's Disease.
Front. Immunol.
Conference Abstract:
15th International Congress of Immunology (ICI).
doi: 10.3389/conf.fimmu.2013.02.01035
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Received:
29 Jun 2013;
Published Online:
22 Aug 2013.
*
Correspondence:
Dr. Lesley A Bergmeier, Queen Mary's Schools of Medicine and Dentistry, Centre for Clinical and Diagnostic Oral Sciences, London, E1 2AT, United Kingdom, l.a.bergmeier@qmul.ac.uk