The infiltration of immune cells into the central nervous system mediates the development of autoimmune neuroinflammatory diseases. We previously showed that the loss of either Fabp5 or calnexin causes resistance to the induction of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of multiple sclerosis (MS). Here we show that brain endothelial cells lacking either Fabp5 or calnexin have an increased abundance of cell surface CD200 and soluble CD200 (sCD200) as well as decreased T-cell adhesion. In a tissue culture model of the blood–brain barrier, antagonizing the interaction of CD200 and sCD200 with T-cell CD200 receptor (CD200R1) via anti-CD200 blocking antibodies or the RNAi-mediated inhibition of CD200 production by endothelial cells increased T-cell adhesion and transmigration across monolayers of endothelial cells. Our findings demonstrate that sCD200 produced by brain endothelial cells regulates immune cell trafficking through the blood–brain barrier and is primarily responsible for preventing activated T-cells from entering the brain.

A homologue of binding immunoglobulin protein/BiP—IRL201805 alters the function of immune cells in pre-clinical in vivo and in vitro studies. The aim of the study was to select biomarkers that clearly delineate between RA patients who respond to IRL201805 and placebo patients and reveal the immunological mode of action of IRL201805 driving the extended pharmacodynamics observed in responding patients. Biomarkers that distinguished between responding patients and placebo patients included downregulation of serum interferon-γ and IL-1β; upregulation of anti-inflammatory mediators, serum soluble CTLA-4, and intracellular monocyte expression of IDO; and sustained increased CD39 expression on CD3+CD4+CD25hi CD127lo regulatory T cells. In the responding patients, selected biomarkers verified that the therapeutic effect could be continuous for at least 12 weeks post-infusion. In secondary co-culture, pre-infusion PBMCs cultured 1:1 with autologous PBMCs, isolated at later time-points during the trial, showed significantly inhibited IL-6 and IL-1β production upon anti-CD3/CD28 stimulation demonstrating IRL201805 alters the function of immune cells leading to prolonged pharmacodynamics confirmed by biomarker differences. IRL201805 may be the first of a new class of biologic drug providing long-term drug-free therapy in RA.

Despite the considerable social, medical, and economic impact of food allergy, there is currently a clear lack of therapeutic options to treat this disease. IRL201104 is a novel clinical stage immunomodulatory peptide that despite a very short half-life (∼10-15min across species), shows a long-lasting effect in different preclinical models of allergic inflammation. Our aim was to explore the therapeutic potential of IRL201104 in a model of food allergy driven by ovalbumin (OVA).

Background: Some neurodegenerative diseases have an element of neuroinflammation that is triggered by viral nucleic acids, resulting in the generation of type I interferons. In the cGAS-STING pathway, microbial and host-derived DNA bind and activate the DNA sensor cGAS, and the resulting cyclic dinucleotide, 2′3-cGAMP, binds to a critical adaptor protein, stimulator of interferon genes (STING), which leads to activation of downstream pathway components. However, there is limited work demonstrating the activation of the cGAS-STING pathway in human neurodegenerative diseases. Methods: Post-mortem CNS tissue from donors with multiple sclerosis (n = 4), Alzheimer’s disease (n = 6), Parkinson’s disease (n = 3), amyotrophic lateral sclerosis (n = 3) and non-neurodegenerative controls (n = 11) were screened by immunohistochemistry for STING and relevant protein aggregates (e.g., amyloid-β, α-synuclein, TDP-43). Human brain endothelial cells were cultured and stimulated with the STING agonist palmitic acid (1–400 μM) and assessed for mitochondrial stress (release of mitochondrial DNA into cytosol, increased oxygen consumption), downstream regulator factors, TBK-1/pIRF3 and inflammatory biomarker interferon-β release and changes in ICAM-1 integrin expression. Results: In neurodegenerative brain diseases, elevated STING protein was observed mainly in brain endothelial cells and neurons, compared to non-neurodegenerative control tissues where STING protein staining was weaker. Interestingly, a higher STING presence was associated with toxic protein aggregates (e.g., in neurons). Similarly high STING protein levels were observed within acute demyelinating lesions in multiple sclerosis subjects. To understand non-microbial/metabolic stress activation of the cGAS-STING pathway, brain endothelial cells were treated with palmitic acid. This evoked mitochondrial respiratory stress up to a ~2.5-fold increase in cellular oxygen consumption. Palmitic acid induced a statistically significant increase in cytosolic DNA leakage from endothelial cell mitochondria (Mander’s coefficient; p < 0.05) and a significant increase in TBK-1, phosphorylated transcription factor IFN regulatory factor 3, cGAS and cell surface ICAM. In addition, a dose response in the secretion of interferon-β was observed, but it failed to reach statistical significance. Conclusions: The histological evidence shows that the common cGAS-STING pathway appears to be activated in endothelial and neural cells in all four neurodegenerative diseases examined. Together with the in vitro data, this suggests that the STING pathway might be activated via perturbation of mitochondrial stress and DNA leakage, resulting in downstream neuroinflammation; hence, this pathway may be a target for future STING therapeutics.

Two major chaperones, calreticulin (CRT) and binding immunoglobulin protein (GRP78/BiP) dependent on their location, have immunoregulatory or anti-inflammatory functions respectively. CRT induces pro-inflammatory cytokines, dendritic cell (DC) maturation and activates cytotoxic T cells against tumours. By contrast, GRP78/BiP induces anti-inflammatory cytokines, inhibits DC maturation and heightens T-regulatory cell responses. These latter functions rebalance immune homeostasis in inflammatory diseases, such as rheumatoid arthritis. Both chaperones are therapeutically relevant agents acting primarily on monocytes/DCs. Endogenous exposure of CRT on cancer cell surfaces acts as an 'eat-me' signal and facilitates improved elimination of stressed and dying tumour cells by DCs. Therefore, therapeutics that promote endogenous CRT translocation to the cell surface can improve the removal of cancer cells. However, infused recombinant CRT dampens this cancer cell eradication by binding directly to the DCs. Low levels of endogenous BiP appear as a surface biomarker of endoplasmic reticulum (ER) stress in some types of tumour cells, a reflection of cells undergoing proliferation, in which resulting hypoxia and nutrient deprivation perturb ER homeostasis triggering the unfolded protein response, leading to increased expression of GRP78/BiP and altered cellular location. Conversely, infusion of an analogue of GRP78/BiP (IRL201805) can lead to long-term immune resetting and restoration of immune homeostasis. The therapeutic potential of both chaperones relies on them being relocated from their intracellular ER environment. Ongoing clinical trials are employing therapeutic interventions to either enhance endogenous cell surface CRT or infuse IRL201805, thereby triggering several disease-relevant immune responses leading to a beneficial clinical outcome.

Background Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone, but can appear surface bound on cancers cells, including ovarian cancers (OC). We investigated at what stage of cell viability, CRT appeared associated with surface of human OC cells. CRT on pre-apoptotic tumour cells is thought to initiate their eradication via a process termed immunogenic cell death (ICD). Methods We treated OC cells with the chemotherapeutic—doxorubicin (DX) known to induce translocation of CRT to some tumour cell surfaces, with and without the ER stressor—thapsigargin (TG)—and/or an ER stress inhibitor—TUDCA. We monitored translocation/release of CRT in pre-apoptotic cells by flow cytometry, immunoblotting and ELISA. We investigated the difference in binding of FITC-CRT to pre-apoptotic, apoptotic and necrotic cells and the ability of extracellular CRT to generate immature dendritic cells from THP-1 monocytes. Results Dx-treatment increased endogenously released CRT and extracellular FITC_CRT binding to human pre-apoptotic OC cells. DX and TG also promoted cell death in OC cells which also increased CRT release. These cellular responses were significantly inhibited by TUDCA, suggesting that ER stress is partially responsible for the changes in CRT cellular distribution. Extracellular CRT induces maturation of THP-1 towards a imDC phenotype, an important component of ICD. Conclusion Collectively, these cellular responses suggest that ER stress is partially responsible for the changes in CRT cellular distribution. ER-stress regulates in part the release and binding of CRT to human OC cells where it may play a role in ICD.

We previously showed that calnexin (Canx)-deficient mice are desensitized to experimental autoimmune encephalomyelitis (EAE) induction, a model that is frequently used to study inflammatory demyelinating diseases, due to increased resistance of the blood-brain barrier to immune cell transmigration. We also discovered that Fabp5, an abundant cytoplasmic lipid-binding protein found in brain endothelial cells, makes protein-protein contact with the cytoplasmic C-tail domain of Canx. Remarkably, both Canx-deficient and Fabp5-deficient mice commonly manifest resistance to EAE induction. Here, we evaluated the importance of Fabp5/Canx interactions on EAE pathogenesis and on the patency of a model blood-brain barrier to T-cell transcellular migration. The results demonstrate that formation of a complex comprised of Fabp5 and the C-tail domain of Canx dictates the permeability of the model blood-brain barrier to immune cells and is also a prerequisite for EAE pathogenesis.

Background: Thousands of people with multiple sclerosis (MS) have used self-administered oxygen therapy in the UK. Clinical trials have been performed, with scant evidence that people with MS have been consulted to explore how they benefit from or how to optimize this treatment. The conventional MS disease disability scores used in trials seldom reflect the effects individuals report when using oxygen therapy to treat their symptoms. Methods: Three people with MS and the manager of an MS Centre formed a public involvement group and collaborated with clinicians and scientists to inform a lab-based study to investigate the physiological effects of oxygen therapy on microvascular brain endothelial cells. Results: People with MS often use oxygen therapy at a later stage when their symptoms worsen and only after using other treatments. The frequency of oxygen therapy sessions and hyperbaric pressure is individualized and varies for people with MS. Despite direct comparisons of efficacy proving difficult, most individuals are exposed to 100% O-2 at 1.5 atmosphere absolute (ATA; 1140 mmHg absolute) for 60 min. In a laboratory-based study human brain endothelial cells were exposed in vitro to 152 mmHg O-2 for 60 min with and without pressure, as this equates to 20% O-2 achievable via hyperbarics, which was then replicated at atmospheric pressure. A significant reduction in endothelial cells ICAM-1 (CD54) implicated in inflammatory cell margination across the blood brain barrier was observed under oxygen treatment. Conclusions: By collaborating with people living with MS, we were able to design laboratory-based experimental protocols that replicate their treatment regimens to advance our understanding of the physiological effects of hyperbaric oxygen treatment on brain cells and their role in neuroinflammation.

The aetiology of rheumatoid arthritis (RA) is unknown, but citrullination of proteins is thought to be an initiating event. In addition, it is increasingly evident that the lung can be a potential site for the generation of autoimmune triggers before the development of joint disease. Here, we identified that serum levels of galectin-9 (Gal-9), a pleiotropic immunomodulatory protein, are elevated in RA patients, and are even further increased in patients with comorbid bronchiectasis, a lung disease caused by chronic inflammation. The serum concentrations of Gal-9 correlate with C-reactive protein levels and DAS-28 score. Gal-9 activated polymorphonuclear leukocytes (granulocytes) in vitro, which was characterized by increased cytokine secretion, migration, and survival. Further, granulocytes treated with Gal-9 upregulated expression of peptidyl arginine deiminase 4 (PAD-4), a key enzyme required for RA-associated citrullination of proteins. Correspondingly, treatment with Gal-9 triggered citrullination of intracellular granulocyte proteins that are known contributors to RA pathogenesis (i.e., myeloperoxidase, alpha-enolase, MMP-9, lactoferrin). In conclusion, this study identifies for the first time an immunomodulatory protein, Gal-9, that triggers activation of granulocytes leading to increased PAD-4 expression and generation of citrullinated autoantigens. This pathway may represent a potentially important mechanism for development of RA.

The endoplasmic reticulum (ER) chaperone protein, calreticulin (CRT), is essential for proper glycoprotein folding and maintaining cellular calcium homeostasis. During ER stress, CRT is overexpressed as part of the unfolded protein response (UPR). In addition, CRT can be released as a damage-associated molecular pattern (DAMP) molecule that may interact with pathogen-associated molecular patterns (PAMPs) during the innate immune response. One such PAMP is lipopolysaccharide (LPS), a component of the gram-negative bacterial cell wall. In this report, we show that recombinant and native human placental CRT strongly interacts with LPS in solution, solid phase, and the surface of gram-negative and gram-positive bacteria. Furthermore, LPS induces oilgomerization of CRT with a disappearance of the monomeric form. The application of recombinant CRT (rCRT) to size exclusion and anion exchange chromatography shows an atypical heterogeneous elution profile, indicating that LPS affects the conformation and ionic charge of CRT. Interestingly, LPS bound to CRT is detected in sera of bronchiectasis patients with chronic bacterial infections. By ELISA, rCRT dose-dependently bound to solid phase LPS via the N- and C-domain globular head region of CRT and the C-domain alone. The specific interaction of CRT with LPS may be important in PAMP innate immunity.

In multiple sclerosis (MS), a demyelinating inflammatory disease of the CNS, and its animal model (experimental autoimmune encephalomyelitis; EAE), circulating immune cells gain access to the CNS across the blood-brain barrier to cause inflammation, myelin destruction, and neuronal damage. Here, we discovered that calnexin, an ER chaperone, is highly abundant in human brain endothelial cells of MS patients. Conversely, mice lacking calnexin exhibited resistance to EAE induction, no evidence of immune cell infiltration into the CNS, and no induction of inflammation markers within the CNS. Furthermore, calnexin deficiency in mice did not alter the development or function of the immune system. Instead, the loss of calnexin led to a defect in brain endothelial cell function that resulted in reduced T cell trafficking across the blood-brain barrier. These findings identify calnexin in brain endothelial cells as a potentially novel target for developing strategies aimed at managing or preventing the pathogenic cascade that drives neuroinflammation and destruction of the myelin sheath in MS.

AimPatients with rheumatoid arthritis (RA) and co-existent bronchiectasis (BRRA) have a five-fold increased mortality compared to rheumatoid arthritis alone. Yet previous studies have found no difference in clinical and serological markers of RA disease severity between BRRA patients and RA alone. However, RA disease activity measures such as Disease Activity Score of 28 joints - C-reactive protein (DAS28-CRP) and anti-cyclic citrullinated peptide antibodies (anti-CCP) have not been studied, so we assessed these parameters in patients with BRRA and RA alone. MethodsBRRA patients (n=53) had high-resolution computed tomography proven bronchiectasis without any interstitial lung disease and 2 respiratory infections/year. RA alone patients (n=50) had no clinical or radiological evidence of lung disease. DAS28-CRP, rheumatoid factor (immunoglobulin M) and anti-CCP were measured in all patients, together with detailed clinical and radiology records. ResultsIn BRRA, bronchiectasis predated RA in 58% of patients. BRRA patients had higher DAS28 scores (3.51 vs. 2.59), higher levels of anti-CCP (89% vs. 46%) and rheumatoid factor (79% vs. 52%) (P=0.003) compared to RA alone. Where hand and foot radiology findings were recorded, 29/37 BRRA (78%) and 13/30 (43%) RA alone had evidence of erosive change (P=0.003). There were no significant differences between groups in smoking history or disease-modifying anti-rheumatic drug/biologic therapy. ConclusionsIncreased levels of RA disease activity, severity and RA autoantibodies are demonstrated in patients with RA and co-existent bronchiectasis compared to patients with RA alone, despite lower tobacco exposure. This study demonstrates that BRRA is a more severe systemic disease than RA alone.

Bronchiectasis (BR) and smoking are risk factors for rheumatoid arthritis (RA) development. The mechanisms by which smoking and BR trigger RA are unknown, but are associated with concurrent rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody (anti-CCP) positivity. Anti-carbamylated protein antibodies (anti-CarP) have also been observed in BR patients and can be induced by smoking. Given that RF only has one antigen, immunoglobulin G (IgG) we have suggested that post-translational modifications to the Fc region of the heavy chain of IgG (IgGH) are a potential explanation for the clustering of the RA-associated autoantibodies in RA. Protein analysis was undertaken on 22 individuals. Four of the individuals had a diagnosis of BR at the time of protein analysis and subsequently developed RA up to 18 months following blood sampling. Four smoking RA patients and 4 patients with both BR and RA and 10 healthy controls were also studied. We identified modified arginines (Arg) frequently in the variable region and CH3 domains of IgG in patients and control subjects alike, but only observed carbamylated Lys and/or citrullinated Arg modifications in the RF binding site of the IgG CH2 domain of 5/12 (41.7%) patients investigated (1 BR, 2 RA and 2 BRRA), but in no control subjects (0/10, 0%) p=0.02. This is the first report of citrullination and carbamylation at the RF binding site of IgG in RA. These results point towards the concept of a universal antigen in RA, an antigen that is post-translationally modified at the Fc region of IgGH.

Background: Endoplasmic reticulum (ER) stress is a hallmark of neurodegenerative diseases such as multiple sclerosis (MS). However, this physiological mechanism has multiple manifestations that range from impaired clearance of unfolded proteins to altered mitochondrial dynamics and apoptosis. While connections between the triggering of the unfolded protein response (UPR) and downstream mitochondrial dysfunction are poorly understood, the membranous contacts between the ER and mitochondria, called the mitochondria-associated membrane (MAM), could provide a functional link between these two mechanisms. Therefore, we investigated whether the guanosine triphosphatase (GTPase) Rab32, a known regulator of the MAM, mitochondrial dynamics, and apoptosis, could be associated with ER stress as well as mitochondrial dysfunction.Methods: We assessed Rab32 expression in MS patient and experimental autoimmune encephalomyelitis (EAE) tissue, via observation of mitochondria in primary neurons and via monitoring of survival of neuronal cells upon increased Rab32 expression.Results: We found that the induction of Rab32 and other MAM proteins correlates with ER stress proteins in MS brain, as well as in EAE, and occurs in multiple central nervous system (CNS) cell types. We identify Rab32, known to increase in response to acute brain inflammation, as a novel unfolded protein response (UPR) target. High Rab32 expression shortens neurite length, alters mitochondria morphology, and accelerates apoptosis/necroptosis of human primary neurons and cell lines.Conclusions: ER stress is strongly associated with Rab32 upregulation in the progression of MS, leading to mitochondrial dysfunction and neuronal death.

Multiple sclerosis (MS) is normally considered a chronic inflammatory disease of the central nervous system (CNS), where T-cells breaching the blood brain barrier react against proteins of the axonal myelin sheaths, leading to focal plaques and demyelination in the brain and spinal cord. Many current therapies are immunosuppressive in nature and are designed to target the immune system at an early stage of the disease. But there is no cure and MS may evolve into a neurodegenerative disease, where immunomodulatory treatments appear less effective. Neurodegeneration is influenced by oxidative and endoplasmic reticulum (ER) mediated stress which can be induced independently of immune processes. Since 1970, MS patients have been self-managing their long term symptoms using hyperbaric oxygen and reporting improvement in their symptoms, especially bladder control. In contrast, the majority of clinical trial evidence does not support the views of patients. Therefore does oxygen under pressure affect brain tissue by modulating oxidative or ER stress at the cellular level resulting in CNS tissue repair or deterioration? This chapter reviews our understanding and the role of oxidative and ER stress in the context of employing hyperoxia treatments to treat MS and evaluate its effects on neural cells.

We read with interest the paper “Genetic variation of the alpha-1-antitrypsin (AT) gene is associated with increased autoantibody production in rheumatoid arthritis” by McCarthy et al. A strong association between mutant alpha-1-antitrypsin Z (ATZ) and ACPA production was noted in RA (1). The authors suggest that alpha-1-antitrypsin deficiency (ATD) may give rise to a tendency towards ACPA production. We suggest that it is the abnormal mutant protein ATZ itself that induces autophagy resulting in ACPA production in inflammatory cells such as macrophages rather than as a consequence of ATD per se. We feel this is likely to occur in the context of established synovitis rather than preceding the development of RA. Chronic obstructive pulmonary disease (COPD) is the most prevalent disease manifestation of ATD (2). Given the evidence that ACPA+ve RA arises as a consequence of chronic lung disease (3), it is important to consider whether ATD as a consequence of its strong observed association with COPD confers an increased risk of ACPA+ve RA. This article is protected by copyright. All rights reserved.