Neuroimmune mechanisms in chronic inflammation : translational studies of the inflammatory reflex

A functional immune system is crucial for our survival from the pathogens and toxins we are constantly subjected to. For reasons only partially understood, in some individuals the immune response instead target self antigens, as suggested in rheumatoid arthritis (RA), or environmental non-pathogenic antigens, as suggested in allergy, leading to a failure of resolution and development of a state of chronic inflammation. Although current treatment strategies are largely effective at treating the peripheral inflammation, symptoms that can be attributed to the central nervous system (CNS) such as pain sensitization or fatigue often persist, causing considerable distress for the patient. A growing amount of evidence point towards that chronic inflammatory diseases are accompanied by central inflammation which could then be involved in driving CNS related symptoms. In general, the immune response may result in damage not only to the pathogen but also to healthy tissues in the vicinity. Therefore it is essential that an inflammatory process is concluded as soon as the threat is cleared. Recently, the cholinergic anti-inflammatory pathway (CAP) was described promoting a fast vagus mediated control of systemic inflammation. The anti-inflammatory potential of CAP initiated clinical trials exploring the use of vagal stimulation as an alternative treatment strategy for immune suppression in human chronic inflammatory diseases such as rheumatoid arthritis. Even so, much remain to be understood regarding the mechanism of CAP and its anti-inflammatory extent. In this thesis, work has been undertaken to explore the role of central nervous mechanisms in RA and seasonal allergy. Furthermore, CNS involvement in RA and other arthropaties was studied by mapping the cerebrospinal fluid proteome and its treatment associated changes. Additionally, CAP mechanisms were studied using animal models of endotoxaemia in a translational fashion. Whereas we could not detect an increased brain microglial activity in either RA or Allergy, we further confirm a state of autonomic dysregulation in RA with close associations to peripheral inflammation. We demonstrate for the first time that modern treatment strategies not only exert effects peripherally, but also lead to a central nervous reduction of inflammatory related proteins. We additionally identify several of these proteins as potentially important players to study further in the context of neuro immune responses. Furthermore, we provide evidence that the dependence of prostaglandins for a functional CAP is located to splenic events, demonstrating that prostaglandin E2 is important for acetylcholine production as well as immunosuppressive function in splenocytes. In addition, an activated CAP is shown to exert effects on additional immune cells and immune compartments than previously known. Taken together, this thesis has contributed to further our understanding of CNS involvement in chronic inflammatory conditions, of effects exerted by commonly used as well as experimental treatment strategies and neural regulation of inflammation. In the future, the results here presented may hopefully benefit the patient by contributing to the development of improved treatment strategies and better understanding of disease pathology

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

<div><p>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 β₂ AR protein expression. In addition, nicotine inhibited TNFα release by LPS activated mPGES-1 (+/+) splenocytes <i>in vitro</i>. 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.</p></div

Cytokine profiling of activated mPGES-1 (-/-) splenocytes in response to NE stimulation.

<p>Primary splenocyte cultures established from mPGES-1 (+/+) and (-/-) mice were pretreated with norepinephrine (NE) at 1μM concentration for 30 mins and then activated with the endotoxin, LPS (100ng/ml). Cell supernatants were analyzed for cytokine production following 3 hours of treatment. (*p<0.05; LPS versus LPS+NE). (^# p<0.05; mPGES-1(+/+) versus (-/-) within LPS+NE treatment; student’s T-test). Each sample was run as duplicates during the assay and values are represented as mean ±SEM from 3 independent experiments. Statistical analysis was done using One-way ANOVA unless otherwise indicated.</p

beta 2 Adrenergic receptor expression analysis by flow cytometry on CD4+ T cells isolated from mPGES-1 KO or WT mouse splenocytes

Flow cytometric data from five experiments analyzing beta-2-adrenergic receptor expression on CD4+ T cells (MACS positive selection from spleen) from mPGES-1 ko (-/-) or WT (+/+) mice subjected to vagus nerve stimulation (VNS) and intraperitoneal LPS injection 30 min prior to sample collection.<br><br><u>Panel</u> (<i>more detailed info can be found in the "FCS file identification list" document</i>):<br>FITC-Viability<br>PE-b2AR<br>PCPCy5.5-CD69<br>PECy7-CD62L<br>APC-CD44<br>APCCy7-CD3<br>BV421-CD25<br>BV510-CD4<br><br>The protocol for staining, cell extraction VNS etc. is described in the publication "<u>Microsomal prostaglandin E synthase-1 gene deletion impairs neuro-immune circuitry of the cholinergic anti-inflammatory pathway in endotoxaemic mpuse spleen</u>" published in <b>PLOS ONE 2017</b> by the authors: Priya Revathikumar, Johanna Estelius, Utsa Karmakar, Erwan LeMaître, Marina Korotkova, Per-Johan Jakobsson and Jon Lampa. <br><br><br

Lipopolysaccharide activated splenocytes lacking mPGES-1 gene expression display an altered response to NE stimulation.

<p>Primary splenocyte cultures established from mPGES-1 (+/+) and (-/-) mice were pretreated with norepinephrine (NE) at 1, 10 and 100 μM concentration for 30 mins and then activated with the endotoxin, LPS (100ng/ml). Cell supernatants were analyzed for cytokine production following 3 hours of treatment. ^a,b p<0.0001; LPS versus LPS+NE within WT or KO; One-way ANOVA. ^# p<0.05; mPGES-1(+/+) versus mPGES-1 (-/-) within LPS+NE treatment; student’s T-test. Each sample was run as duplicates during the assay and values are represented as mean ±SEM from 3 independent experiments.</p

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

Abstract 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

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

Abstract 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

mPGES-1 deletion has no effect on spleen T cell subset relative numbers or their β2 AR expression following VNS in LPS treated mice.

<p>Both mPGES-1(+/+) and (-/-) DBA/1lacJ mice were subjected to VNS (n = 5) following intraperitoneal LPS (2mg/kg) injection 30 min before spleen collection. (a) Single cell suspensions of splenocytes were analyzed by flow cytometry in the T cell subsets CD44^lo CD62L^hi (naïve), CD44^lo CD62L^lo (effector), CD44^hi CD62L^hi (central memory) and CD44^hi CD62L^lo (effector memory) as identified according to figure A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193210#pone.0193210.s001" target="_blank">S1 File</a>. The proportions of the different T cell subsets are represented as % of viable CD4+ CD3+ lymphocytes. (b) The relative number of β₂ AR+ cells among naïve, effector, central memory and effector memory T cell subsets were determined by flow cytometry. The proportions of β₂ AR+ cells are reported as % of viable T cell subset. (c) β₂ AR protein levels on the cell surface of naïve, effector, central memory and effector memory T cell subsets were determined by flow cytometry as (geometric) MFI. (d) Representative β₂ AR histograms. Values are represented as mean ±SEM from 5 individual animals per treatment group.</p

<i>In vitro</i> mPGES-1 blockade impairs nicotine’s limiting effects on TNFα production by LPS activated human peripheral blood mononuclear cells (PBMCs).

<p>Human PBMC cultures were freshly prepared from healthy blood donors using ficoll density gradient separation. (a) TNFα levels measured by ELISA in culture supernatants after treatment with endotoxin, LPS (100ng/ml) for 6, 14 and 20 hours respectively. Untreated cells served as control. TNFα levels in the culture supernatants were measured by ELISA (*p>0.05, **p<0.01; control versus LPS; One-way ANOVA, *p>0.05; LPS versus LPS+mPGES-1 inhibitor; One-way ANOVA). (b) TNFα levels measured by ELISA in culture supernatants after treatment with nicotine (100 μM) for 14 hours. (*p>0.05; LPS versus LPS+ Nicotine; One-way ANOVA, **p>0.05; LPS+ Nicotine versus LPS+Nicotine+mPGES-1 inhibitor; One-way ANOVA). Each sample was run as duplicates during ELISA and values are represented as mean ±SEM from (a) 3 and (b) 4 independent experiments.</p