Mass spectrometry-based analysis of cerebrospinal fluid from arthritis patients-immune-related candidate proteins affected by TNF blocking treatment.

BACKGROUND: Signs of inflammation in cerebrospinal fluid (CSF) of rheumatoid arthritis patients correlate positively with fatigue, a central nervous system (CNS)-related symptom that can be partially suppressed by TNF blockade. This suggests a possible role for CNS inflammation in arthritis that may be affected by TNF blockade. We therefore investigated the effects of TNF blockade on the arthritis CSF proteome and how candidate proteins related to clinical measures of disease activity and inflammation. METHODS: Mass spectrometry-based quantitative proteomic analysis was performed on CSF from seven polyarthritis patients before and during infliximab treatment. Treatment-associated proteins were identified using univariate (Wilcoxon signed rank test) and multivariate (partial least squares discriminant analysis (PLS-DA)) strategies. Relations between selected candidate proteins and clinical measures were investigated using the Spearman correlations. Additionally, selected proteins were cross-referenced to other studies investigating human CSF in a thorough literature search to ensure feasibility of our results. RESULTS: Univariate analysis of arthritis CSF proteome revealed a decrease of 35 proteins, predominantly involved in inflammatory processes, following TNF blockade. Seven candidate proteins, Contactin-1 (CNTN1), fibrinogen gamma chain (FGG), hemopexin (HPX), cell adhesion molecule-3 (CADM3), alpha-1B-glycoprotein (A1BG), complement factor B (CFB), and beta-2-microglobulin (B2M), were selected for further studies based on identification by both univariate and multivariate analyses and reported detection in human CSF and known associations to arthritis. Decreased levels of FGG and CFB in CSF after treatment showed strong correlations with both erythrocyte sedimentation rate and disability scores, while CNTN1 and CADM3 were associated with pain. CONCLUSION: Several immune-related proteins in the CSF of arthritis patients decreased during TNF blockade, including FGG and CFB that both correlated strongly with systemic inflammation. Our findings stress that also intrathecal inflammatory pathways are related to arthritis symptoms and may be affected by TNF blockade.

Exploring the transcriptome of resident spinal microglia after collagen antibody-induced arthritis.

Recent studies have suggested a sexually dimorphic role of spinal glial cells in the maintenance of mechanical hypersensitivity in rodent models of chronic pain. We have used the collagen antibody-induced arthritis (CAIA) mouse model to examine differences between males and females in the context of spinal regulation of arthritis-induced pain. We have focused on the late phase of this model when joint inflammation has resolved, but mechanical hypersensitivity persists. Although the intensity of substance P, calcitonin gene-related peptide, and galanin immunoreactivity in the spinal cord was not different from controls, the intensity of microglia (Iba-1) and astrocyte (glial fibrillary acidic protein) markers was elevated in both males and females. Intrathecal administration of the glial inhibitors minocycline and pentoxifylline reversed mechanical thresholds in male, but not in female mice. We isolated resident microglia from the lumbar dorsal horns and observed a significantly lower number of microglial cells in females by flow cytometry analysis. However, although genome-wide RNA sequencing results pointed to several transcriptional differences between male and female microglia, no convincing differences were identified between control and CAIA groups. Taken together, these findings suggest that there are subtle sex differences in microglial expression profiles independent of arthritis. Our experiments failed to identify the underlying mRNA correlates of microglial actions in the late phase of the CAIA model. It is likely that transcriptional changes are either subtle and highly localised and therefore difficult to identify with bulk isolation techniques or that other factors, such as changes in protein expression or epigenetic modifications, are at play.

Correction: Microsomal prostaglandin E synthase-1 gene deletion impairs neuro-immune circuitry of the cholinergic anti-inflammatory pathway in endotoxaemic mouse spleen.

[This corrects the article DOI: 10.1371/journal.pone.0193210.].

Microsomal prostaglandin E synthase-1 gene deletion impairs neuro-immune circuitry of the cholinergic anti-inflammatory pathway in endotoxaemic mouse spleen.

The cholinergic anti-inflammatory pathway (CAP) is an innate neural reflex where parasympathetic and sympathetic nerves work jointly to control inflammation. Activation of CAP by vagus nerve stimulation (VNS) has paved way for novel therapeutic strategies in treating inflammatory diseases. Recently, we discovered that VNS mediated splenic acetylcholine (ACh) release and subsequent immunosuppression in response to LPS associated inflammation is impaired in mice lacking microsomal prostaglandin E synthase-1 (mPGES-1) expression, a key enzyme responsible for prostaglandin E2 synthesis. Here, we have further investigated the consequences of mPGES-1 deficiency on various molecular/cellular events in the spleen which is critical for the optimal functioning of VNS in endotoxaemic mice. First, VNS induced splenic norepinephrine (NE) release in both mPGES-1 (+/+) and (-/-) mice. Compared to mPGES-1 (+/+), immunomodulatory effects of NE on cytokines were strongly compromised in mPGES-1 (-/-) splenocytes. Interestingly, while LPS increased choline acetyltransferase (ChAT) protein level in mPGES-1 (+/+) splenocytes, it failed to exert similar effects in mPGES-1 (-/-) splenocytes despite unaltered ß2 AR protein expression. In addition, nicotine inhibited TNFα release by LPS activated mPGES-1 (+/+) splenocytes in vitro. However, such immunosuppressive effects of nicotine were reversed both in mPGES-1 (-/-) mouse splenocytes and human PBMC treated with mPGES-1 inhibitor. In summary, our data implicate PGE2 as an important mediator of ACh synthesis and noradrenergic/cholinergic molecular events in the spleen that constitute a crucial part of the CAP immune regulation. Our results suggest a possible link between cholinergic and PG system of CAP that may be of clinical significance in VNS treatment.

GM1 ganglioside-independent intoxication by Cholera toxin.

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (LeX) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the LeX glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to LeX. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.

Evidence of fatigue, disordered sleep and peripheral inflammation, but not increased brain TSPO expression, in seasonal allergy: A [11C]PBR28 PET study.

Allergy is associated with non-specific symptoms such as fatigue, sleep problems and impaired cognition. One explanation could be that the allergic inflammatory state includes activation of immune cells in the brain, but this hypothesis has not been tested in humans. The aim of the present study was therefore to investigate seasonal changes in the glial cell marker translocator protein (TSPO), and to relate this to peripheral inflammation, fatigue and sleep, in allergy. We examined 18 patients with severe seasonal allergy, and 13 healthy subjects in and out-of pollen season using positron emission tomography (n = 15/13) and the TSPO radioligand [11C]PBR28. In addition, TNF-α, IL-5, IL-6, IL-8 and IFN-γ were measured in peripheral blood, and subjective ratings of fatigue and sleepiness as well as objective and subjective sleep were investigated. No difference in levels of TSPO was seen between patients and healthy subjects, nor in relation to pollen season. However, allergic subjects displayed both increased fatigue, sleepiness and increased percentage of deep sleep, as well as increased levels of IL-5 and TNF-α during pollen season, compared to healthy subjects. Allergic subjects also had shorter total sleep time, regardless of season. In conclusion, allergic subjects are indicated to respond to allergen exposure during pollen season with a clear pattern of behavioral disruption and peripheral inflammatory activation, but not with changes in brain TSPO levels. This underscores a need for development and use of more specific markers to understand brain consequences of peripheral inflammation that will be applicable in human subjects.

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses.

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.