Human Macrophages Activate Bystander Neutrophils' Metabolism and Effector Functions When Challenged with Mycobacterium tuberculosis.

Neutrophils are dynamic cells, playing a critical role in pathogen clearance; however, neutrophil infiltration into the tissue can act as a double-edged sword. They are one of the primary sources of excessive inflammation during infection, which has been observed in many infectious diseases including pneumonia and active tuberculosis (TB). Neutrophil function is influenced by interactions with other immune cells within the inflammatory lung milieu; however, how these interactions affect neutrophil function is unclear. Our study examined the macrophage-neutrophil axis by assessing the effects of conditioned medium (MΦ-CM) from primary human monocyte-derived macrophages (hMDMs) stimulated with LPS or a whole bacterium (Mycobacterium tuberculosis) on neutrophil function. Stimulated hMDM-derived MΦ-CM boosts neutrophil activation, heightening oxidative and glycolytic metabolism, but diminishes migratory potential. These neutrophils exhibit increased ROS production, elevated NET formation, and heightened CXCL8, IL-13, and IL-6 compared to untreated or unstimulated hMDM-treated neutrophils. Collectively, these data show that MΦ-CM from stimulated hMDMs activates neutrophils, bolsters their energetic profile, increase effector and inflammatory functions, and sequester them at sites of infection by decreasing their migratory capacity. These data may aid in the design of novel immunotherapies for severe pneumonia, active tuberculosis and other diseases driven by pathological inflammation mediated by the macrophage-neutrophil axis.

Trained immunity is induced in humans after immunization with an adenoviral vector COVID-19 vaccine.

BackgroundHeterologous effects of vaccines are mediated by "trained immunity," whereby myeloid cells are metabolically and epigenetically reprogrammed, resulting in heightened responses to subsequent insults. Adenovirus vaccine vector has been reported to induce trained immunity in mice. Therefore, we sought to determine whether the ChAdOx1 nCoV-19 vaccine (AZD1222), which uses an adenoviral vector, could induce trained immunity in vivo in humans.MethodsTen healthy volunteers donated blood on the day before receiving the ChAdOx1 nCoV-19 vaccine and on days 14, 56, and 83 after vaccination. Monocytes were purified from PBMCs, cell phenotype was determined by flow cytometry, expression of metabolic enzymes was quantified by RT-qPCR, and production of cytokines and chemokines in response to stimulation ex vivo was analyzed by multiplex ELISA.ResultsMonocyte frequency and count were increased in peripheral blood up to 3 months after vaccination compared with their own prevaccine controls. Expression of HLA-DR, CD40, and CD80 was enhanced on monocytes for up to 3 months following vaccination. Moreover, monocytes had increased expression of glycolysis-associated enzymes 2 months after vaccination. Upon stimulation ex vivo with unrelated antigens, monocytes produced increased IL-1ß, IL-6, IL-10, CXCL1, and MIP-1α and decreased TNF, compared with prevaccine controls. Resting monocytes produced more IFN-γ, IL-18, and MCP-1 up to 3 months after vaccination compared with prevaccine controls.ConclusionThese data provide evidence for the induction of trained immunity following a single dose of the ChAdOx1 nCoV-19 vaccine.FundingThis work was funded by the Health Research Board (EIA-2019-010) and Science Foundation Ireland Strategic Partnership Programme (proposal ID 20/SPP/3685).

The Effect of Tuberculosis Antimicrobials on the Immunometabolic Profiles of Primary Human Macrophages Stimulated with Mycobacterium tuberculosis.

Tuberculosis (TB) remains a global health challenge. Patients with drug-sensitive and drug-resistant TB undergo long, arduous, and complex treatment regimens, often involving multiple antimicrobials. While these drugs were initially implemented based on their bactericidal effects, some studies show that TB antimicrobials can also directly affect cells of the immune system, altering their immune function. As use of these antimicrobials has been the mainstay of TB therapy for over fifty years now, it is more important than ever to understand how these antimicrobials affect key pathways of the immune system. One such central pathway, which underpins the immune response to a variety of infections, is immunometabolism, namely glycolysis and oxidative phosphorylation (OXPHOS). We hypothesise that in addition to their direct bactericidal effect on Mycobacterium tuberculosis (Mtb), current TB antimicrobials can modulate immunometabolic profiles and alter mitochondrial function in primary human macrophages. Human monocyte-derived macrophages (hMDMs) were differentiated from PBMCs isolated from healthy blood donors, and treated with four first-line and six second-line TB antimicrobials three hours post stimulation with either iH37Rv-Mtb or lipopolysaccharide (LPS). 24 h post stimulation, baseline metabolism and mitochondrial function were determined using the Seahorse Extracellular Flux Analyser. The effect of these antimicrobials on cytokine and chemokine production was also assayed using Meso Scale Discovery Multi-Array technology. We show that some of the TB antimicrobials tested can significantly alter OXPHOS and glycolysis in uninfected, iH37Rv-Mtb, and LPS-stimulated hMDMs. We also demonstrate how these antimicrobial-induced immunometabolic effects are linked with alterations in mitochondrial function. Our results show that TB antimicrobials, specifically clofazimine, can modify host immunometabolism and mitochondrial function. Moreover, clofazimine significantly increased the production of IL-6 in human macrophages that were stimulated with iH37Rv-Mtb. This provides further insight into the use of some of these TB antimicrobials as potential host-directed therapies in patients with early and active disease, which could help to inform TB treatment strategies in the future.

Lactate Alters Metabolism in Human Macrophages and Improves Their Ability to Kill Mycobacterium tuberculosis.

In order to mount an appropriate immune response to infection, the macrophage must alter its metabolism by increasing aerobic glycolysis and concomitantly decreasing oxidative phosphorylation; a process known as the Warburg effect. Consequently, lactate, the end-product of glycolysis, accumulates in the extracellular environment. The subsequent effect of lactate on surrounding macrophages is poorly understood. Mycobacterium tuberculosis (Mtb), the causative organism of Tuberculosis (TB), is phagocytosed by macrophages in the airways. Mtb infected macrophages upregulate aerobic glycolysis and effector functions to try to kill the bacteria. Our lab has previously shown that human macrophages produce lactate in response to infection with Mtb. Although lactate has largely been considered a waste product of aerobic glycolysis, we hypothesised that the presence of extracellular lactate would impact subsequent immunometabolic responses and modulate macrophage function. We demonstrate that the presence of exogenous lactate has an immediate effect on the cellular metabolism of resting human macrophages; causing a decrease in extracellular acidification rate (ECAR; analogous to the rate of glycolysis) and an increase in the oxygen consumption rate (OCR; analogous to oxidative phosphorylation). When lactate-treated macrophages were stimulated with Mtb or LPS, glycolysis proceeds to increase immediately upon stimulation but oxidative phosphorylation remains stable compared with untreated cells that display a decrease in OCR. This resulted in a significantly reduced ECAR/OCR ratio early in response to stimulation. Since altered metabolism is intrinsically linked to macrophage function, we examined the effect of lactate on macrophage cytokine production and ability to kill Mtb. Lactate significantly reduced the concentrations of TNF and IL-1ß produced by human macrophages in response to Mtb but did not alter IL-10 and IL-6 production. In addition, lactate significantly improved bacillary clearance in human macrophages infected with Mtb, through a mechanism that is, at least in part, mediated by promoting autophagy. These data indicate that lactate, the product of glycolysis, has a negative feedback effect on macrophages resulting in an attenuated glycolytic shift upon subsequent stimulation and reduced pro-inflammatory cytokine production. Interestingly, this pro-resolution effect of lactate is associated with increased capacity to kill Mtb.

The Effects of Trained Innate Immunity on T Cell Responses; Clinical Implications and Knowledge Gaps for Future Research.

The burgeoning field of innate immune training, also called trained immunity, has given immunologists new insights into the role of innate responses in protection against infection and in modulating inflammation. Moreover, it has led to a paradigm shift in the way we think about immune memory and the interplay between innate and adaptive immune systems in conferring immunity against pathogens. Trained immunity is the term used to describe the medium-term epigenetic and metabolic reprogramming of innate immune cells in peripheral tissues or in the bone marrow stem cell niche. It is elicited by an initial challenge, followed by a significant period of rest that results in an altered response to a subsequent, unrelated challenge. Trained immunity can be associated with increased production of proinflammatory mediators, such as IL-1ß, TNF and IL-6, and increased expression of markers on innate immune cells associated with antigen presentation to T cells. The microenvironment created by trained innate immune cells during the secondary challenge may have profound effects on T cell responses, such as altering the differentiation, polarisation and function of T cell subtypes, including Th17 cells. In addition, the Th1 cytokine IFN-γ plays a critical role in establishing trained immunity. In this review, we discuss the evidence that trained immunity impacts on or can be impacted by T cells. Understanding the interplay between innate immune training and how it effects adaptive immunity will give insights into how this phenomenon may affect the development or progression of disease and how it could be exploited for therapeutic interventions or to enhance vaccine efficacy.

The Iron Chelator Desferrioxamine Increases the Efficacy of Bedaquiline in Primary Human Macrophages Infected with BCG.

For over 50 years, patients with drug-sensitive and drug-resistant tuberculosis have undergone long, arduous, and complex treatment processes with several antimicrobials. With the prevalence of drug-resistant strains on the rise and new therapies for tuberculosis urgently required, we assessed whether manipulating iron levels in macrophages infected with mycobacteria offered some insight into improving current antimicrobials that are used to treat drug-resistant tuberculosis. We investigated if the iron chelator, desferrioxamine, can support the function of human macrophages treated with an array of second-line antimicrobials, including moxifloxacin, bedaquiline, amikacin, clofazimine, linezolid and cycloserine. Primary human monocyte-derived macrophages were infected with Bacillus Calmette-Guérin (BCG), which is pyrazinamide-resistant, and concomitantly treated for 5 days with desferrioxamine in combination with each one of the second-line tuberculosis antimicrobials. Our data indicate that desferrioxamine used as an adjunctive treatment to bedaquiline significantly reduced the bacterial load in human macrophages infected with BCG. Our findings also reveal a link between enhanced bactericidal activity and increases in specific cytokines, as the addition of desferrioxamine increased levels of IFN-γ, IL-6, and IL-1ß in BCG-infected human monocyte-derived macrophages (hMDMs) treated with bedaquiline. These results provide insight, and an in vitro proof-of-concept, that iron chelators may prove an effective adjunctive therapy in combination with current tuberculosis antimicrobials.

The Warburg Effect Occurs Rapidly in Stimulated Human Adult but Not Umbilical Cord Blood Derived Macrophages.

The Warburg effect, defined as increased glycolysis and decreased oxidative phosphorylation, occurs in murine macrophages following LPS stimulation and is required for activation. There are differences between human and murine macrophage metabolic responses to stimulation, with peak metabolite concentrations occurring earlier in humans than mice. Complex changes occur in the human immune system with age, resulting in the very young and the very old being more susceptible to infections. Anti-bacterial immune responses in umbilical cord immune cells are considered deficient but there is a paucity of data on the role that metabolism plays. We hypothesized that metabolic responses in human macrophages occur early during activation. In addition, we hypothesized that umbilical cord derived macrophages have an altered immunometabolic response compared with adult macrophages. We demonstrate that adult and cord blood monocyte derived macrophages (MDM) immediately increase glycolysis in response to stimulation with LPS or Mycobacterium tuberculosis (Mtb), however only adult MDM decrease oxidative phosphorylation. At 24 hours post stimulation, glycolysis remains elevated in both adult and cord blood MDM, oxidative phosphorylation remains unchanged in the cord blood MDM and has normalized in the adult MDM stimulated with Mtb. However, LPS stimulated adult MDM have increased oxidative phosphorylation at 24 hours, illustrating differences in metabolic responses to different stimuli, time-dependent variation in responses and differences in macrophage metabolism in adults compared with umbilical cord blood. We compared the phenotype and function of macrophages derived from adult or cord blood. Cord blood MDM secreted less TNF following Mtb stimulation and more IL-6 following LPS stimulation compared with adult MDM. Our findings demonstrate that whilst cord blood MDM exhibit an immediate increase in glycolytic flux in response to stimulation, similar to adult MDM, cord blood MDM do not concomitantly decrease oxygen consumption. This indicates that adult macrophages shift to Warburg metabolism immediately after stimulation, but cord blood macrophages do not. Understanding the differences in the metabolic profiles of macrophages over a human lifetime will enable the translation of immunometabolism into effective immuno-supportive therapies that could potentially be targeted at vulnerable populations, such as the very old and the very young.

Inhibiting Histone Deacetylases in Human Macrophages Promotes Glycolysis, IL-1ß, and T Helper Cell Responses to Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading infectious killer in the world. Mycobacterium tuberculosis (Mtb), the bacteria that causes the disease, is phagocytosed by alveolar macrophages (AM) and infiltrating monocyte-derived macrophages (MDM) in the lung. Infected macrophages then upregulate effector functions through epigenetic modifications to make DNA accessible for transcription. The metabolic switch to glycolysis and the production of proinflammatory cytokines are key effector functions, governed by epigenetic changes, that are integral to the ability of the macrophage to mount an effective immune response against Mtb. We hypothesised that suberanilohydroxamic acid (SAHA), an FDA-approved histone deacetylase inhibitor (HDACi), can modulate epigenetic changes upstream of the metabolic switch and support immune responses during Mtb infection. The rate of glycolysis in human MDM, infected with Mtb and treated with SAHA, was tracked in real time on the Seahorse XFe24 Analyzer. SAHA promoted glycolysis early in the response to Mtb. This was associated with significantly increased production of IL-1ß and significantly reduced IL-10 in human MDM and AM. Since innate immune function directs downstream adaptive immune responses, we used SAHA-treated Mtb-infected AM or MDM in a co-culture system to stimulate T cells. Mtb-infected macrophages that had previously been treated with SAHA promoted IFN-γ, GM-CSF, and TNF co-production in responding T helper cells but did not affect cytotoxic T cells. These results indicate that SAHA promoted the early switch to glycolysis, increased IL-1ß, and reduced IL-10 production in human macrophages infected with Mtb. Moreover, the elevated proinflammatory function of SAHA-treated macrophages resulted in enhanced T helper cell cytokine polyfunctionality. These data provide an in vitro proof-of-concept for the use of HDACi to modulate human immunometabolic processes in macrophages to promote innate and subsequent adaptive proinflammatory responses.

Burnout in the emergency department: Randomized controlled trial of an attention-based training program.

BACKGROUND: Burnout (encompassing emotional exhaustion, depersonalization and personal accomplishment) in healthcare professionals is a major issue worldwide. Emergency medicine physicians are particularly affected, potentially impacting on quality of care and attrition from the specialty. OBJECTIVE: The aim of this study was to apply an attention-based training (ABT) program to reduce burnout among emergency multidisciplinary team (MDT) members from a large urban hospital. DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: Emergency MDT members were randomized to either a no-treatment control or an intervention group. Intervention group participants engaged in a four session (4 h/session) ABT program over 7 weeks with a practice target of 20 min twice-daily. Practice adherence was measured using a smart phone application together with a wearable Charge 2 device. MAIN OUTCOME MEASURES: The primary outcome was a change in burnout, comprising emotional exhaustion, depersonalization and personal achievement. The secondary outcomes were changes in other psychological and biometric parameters. RESULTS: The ABT program resulted in a significant reduction (P < 0.05; T1 [one week before intervention] vs T3 [follow-up at two months after intervention]) in burnout, specifically, emotional exhaustion, with an effect size (probability of superiority) of 59%. Similar reductions were observed for stress (P < 0.05) and anxiety (P < 0.05). Furthermore, ABT group participants demonstrated significant improvements in heart rate variability, resting heart rate, sleep as well as an increase in pro-inflammatory cytokine expression. CONCLUSION: This study describes a positive impact of ABT on emergency department staff burnout compared to a no-treatment control group. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT02887300.

Modulating Iron for Metabolic Support of TB Host Defense.

Tuberculosis (TB) is the world's biggest infectious disease killer. The increasing prevalence of multidrug-resistant and extensively drug-resistant TB demonstrates that current treatments are inadequate and there is an urgent need for novel therapies. Research is now focused on the development of host-directed therapies (HDTs) which can be used in combination with existing antimicrobials, with a special focus on promoting host defense. Immunometabolic reprogramming is integral to TB host defense, therefore, understanding and supporting the immunometabolic pathways that are altered after infection will be important for the development of new HDTs. Moreover, TB pathophysiology is interconnected with iron metabolism. Iron is essential for the survival of Mycobacterium tuberculosis (Mtb), the bacteria that causes TB disease. Mtb struggles to replicate and persist in low iron environments. Iron chelation has therefore been suggested as a HDT. In addition to its direct effects on iron availability, iron chelators modulate immunometabolism through the stabilization of HIF1α. This review examines immunometabolism in the context of Mtb and its links to iron metabolism. We suggest that iron chelation, and subsequent stabilization of HIF1α, will have multifaceted effects on immunometabolic function and holds potential to be utilized as a HDT to boost the host immune response to Mtb infection.

All-trans Retinoic Acid Augments Autophagy during Intracellular Bacterial Infection.

Vitamin A deficiency strongly predicts the risk of developing tuberculosis (TB) in individuals exposed to Mycobacterium tuberculosis (Mtb). The burden of antibiotic-resistant TB is increasing globally; therefore, there is an urgent need to develop host-directed adjunctive therapies to treat TB. Alveolar macrophages, the niche cell for Mtb, metabolize vitamin A to all-trans retinoic acid (atRA), which influences host immune responses. We sought to determine the mechanistic effects of atRA on the host immune response to intracellular bacterial infection in primary human and murine macrophages. In this study, atRA promoted autophagy resulting in a reduced bacterial burden in human macrophages infected with Mtb and Bordetella pertussis, but not bacillus Calmette-Guérin (BCG). Autophagy is induced by cytosolic sensing of double-stranded DNA via the STING/TBK1/IRF3 axis; however, BCG is known to evade cytosolic DNA sensors. atRA enhanced colocalization of Mtb, but not BCG, with autophagic vesicles and acidified lysosomes. This enhancement was inhibited by blocking TBK1. Our data indicate that atRA augments the autophagy of intracellular bacteria that trigger cytosolic DNA-sensing pathways but does not affect bacteria that evade these sensors. The finding that BCG evades the beneficial effects of atRA has implications for vaccine design and global health nutritional supplementation strategies. The ability of atRA to promote autophagy and aid bacterial clearance of Mtb and B. pertussis highlights a potential role for atRA as a host-directed adjunctive therapy.

Mindfulness-based cognitive therapy in COPD: a cluster randomised controlled trial.

A considerable proportion of patients with chronic obstructive pulmonary disease (COPD) entering pulmonary rehabilitation (PR) report psychological distress, which is often accompanied by poor physical health status. Mindfulness-based cognitive therapy (MBCT) has been shown to improve psychological and physical outcomes in other chronic diseases. We therefore evaluated the efficacy of MBCT as an add-on to a standard PR programme in COPD.COPD patients eligible for PR were cluster randomised to receive either an 8-week, group-based MBCT programme as an add-on to an 8-week PR programme (n=39), or PR alone (n=45). The primary outcomes of psychological distress and physical health status impairment were measured with the Hospital Anxiety and Depression Scale (HADS) and the COPD Assessment Test (CAT) before randomisation (T1), mid- (T2) and post-intervention (T3), and at 3 (T4) and 6 (T5) months' follow-up .A statistically significant time×arm effect was found for the HADS (Cohen's d=0.62, 95% CIs (d)=0.18-1.06, p=0.010). The treatment effect on the CAT failed to reach statistical significance (d=0.42, 95% CIs (d)=-0.06-0.90, p=0.061).MBCT showed a statistically significant and durable effect on psychological distress, indicating that MBCT may be an efficacious add-on to standard PR programmes in COPD.

Effects of vitamin D3 in clinically isolated syndrome and healthy control participants: A double-blind randomised controlled trial.

BACKGROUND: Lowserum vitamin D levels are associated with susceptibility to, and severity of, multiple sclerosis. High dose vitamin D has been proposed as a potential immunomodulator in multiple sclerosis. OBJECTIVES: We performed a single centre, investigator-led, exploratory, double-blind, randomised, placebo controlled, trial of vitamin D3 in clinically isolated syndrome and healthy control participants to assess its immunological effects. Secondary end-points included clinical and magnetic resonance imaging outcomes and safety. METHODS: Clinically isolated syndrome patients and healthy control participants were randomised to: placebo, 5000 IU or 10,000 IU vitamin D3/day (Vigantol oil). Study duration was 24 weeks. RESULTS: The trial did not meet its primary end point, with no difference in the frequency of pro-inflammatory CD4+ T cells (interleukin (IL)-17+/interferon (IFN)-γ+) seen. A higher level of disease freedom (67% versus 50%) was seen in those with serum 1,25 (OH) vitamin D levels>100 nmol/l but this did not reach significance. High dose vitamin D3 was well tolerated with no safety signal. CONCLUSIONS: High dose vitamin D3 over 24 weeks was well tolerated but without immunological, magnetic resonance imaging or clinical evidence of benefit. The hypothesised therapeutic effects in clinically isolated syndrome or multiple sclerosis patients may require longer periods of administration or may only be seen in patients treated with vitamin D3 as an adjunct to established disease modifying therapies.

Ex-Th17 (Nonclassical Th1) Cells Are Functionally Distinct from Classical Th1 and Th17 Cells and Are Not Constrained by Regulatory T Cells.

Th17 cells are an important therapeutic target in autoimmunity. However, it is known that Th17 cells exhibit considerable plasticity, particularly at sites of autoimmune inflammation. Th17 cells can switch to become ex-Th17 cells that no longer produce IL-17 but produce IFN-γ. These ex-Th17 cells are also called nonclassical Th1 cells because of their ability to produce IFN-γ, similar to Th1 cells; however, it is unclear whether they resemble Th1 or Th17 cells in terms of their function and regulation, and whether they have a pathogenic role in autoimmunity. We compared the phenotypic and functional features of human Th17, Th1, and ex-Th17 cell populations. Our data showed that despite their loss of IL-17 expression, ex-Th17 cells were more polyfunctional in terms of cytokine production than either Th1 or bona fide Th17 cells, and produced increased amounts of proinflammatory cytokines. The proliferative brake on Th17 cells appeared to be lifted because ex-Th17 cells proliferated more than Th17 cells after stimulation. In contrast with Th1 and Th17 cells, ex-Th17 cells were highly resistant to suppression of proliferation and cytokines by regulatory T cells. Finally, we showed that ex-Th17 cells accumulated in the joints of rheumatoid arthritis patients. Taken together, these data indicate that human ex-Th17 cells are functionally distinct from Th1 and Th17 cells, and suggest that they may play a pathogenic role at sites of autoimmunity, such as the rheumatoid arthritis joint where they accumulate. These findings have implications for therapeutic strategies that target IL-17, because these may not inhibit pathogenic ex-Th17 cells.

Suppression of human alloreactive T cells by linear tetrapyrroles; relevance for transplantation.

The main limitation to successful transplantation is the antigraft response developed by the recipient immune system, and the adverse side effects of immunosuppressive agents which are associated with significant toxicity and counter indications such as infection and cancer. Furthermore, immunosuppressants do little to prevent ischemia-reperfusion injury during the transplantation procedure itself hence there is a growing need to develop novel immunosuppressive drugs specifically aimed at prolonging graft survival. Linear tetrapyrroles derived from the breakdown of mammalian heme have been shown in numerous studies to play a protective role in allograft transplantation and ischemia-reperfusion injury; however, commercial sources of these products have not been approved for use in humans. Plants and algae produce equivalent linear tetrapyrroles called bilins that serve as chromophores in light-sensing. One such marine-derived tetrapyrrole, phycocyanobilin (PCB), shows significant structural similarity to mammalian biliverdin (BV) and may prove to be a safer alternative for use in the clinic if it can exert direct effects on human immune cells. Using a mixed lymphocyte reaction, we quantified the allogeneic responses of recipient cells to donor cells and found that PCB, like BV, effectively suppressed proliferation and proinflammatory cytokine production. In addition, we found that BV and PCB can directly downregulate the proinflammatory responses of both innate dendritic cells and adaptive T cells. We therefore propose that PCB may be an effective therapeutic drug in the clinical setting of transplantation and may also have wider applications in regulating inappropriate inflammation.

Increased expression of Tbet in CD4(+) T cells from clinically isolated syndrome patients at high risk of conversion to clinically definite MS.

BACKGROUND: The ability to identify clinically isolated syndrome (CIS) patients at high risk of progression to clinically definite multiple sclerosis (CDMS) would be clinically beneficial. The initiation of T cell mediated autoimmune diseases such as multiple sclerosis (MS) requires the initial inappropriate activation and differentiation of auto-reactive CD4(+) T cells. The quiescence of naive T cells is actively maintained by molecules such as TOB1, which control the threshold of activation. Upon activation, CD4(+) T cells can differentiate into various subsets depending on the milieu present. Th1 and Th17 cells are strongly implicated in MS, while regulatory T (Treg) cells constrain autoimmune inflammation and prevent autoimmunity. FINDINGS: We therefore investigated the expression of TOB1, CD44 and Treg, Th1 and Th17 transcription factors in relation to CIS progression. The expression of TOB1, CD44, FOXP3, TBX21 and RORC genes were measured in CD4(+) T cells from 10 healthy controls, 20 CIS patients within 3 months of initial clinical presentation and 10 relapsing remitting MS patients sampled within 2 months of relapse. CIS patients were subsequently grouped into those who converted to CDMS within 1 year and those who remained CIS. No differences in the expression of TOB1, CD44, FOXP3 and RORC were observed. There was a significant increase in the expression of the Th1 transcription factor Tbet, encoded by TBX21, in CIS patients that converted within 1 year compared with those who did not. CONCLUSION: This pilot data suggests a role for Th1 cells in CIS progression and warrants further evaluation in a larger cohort.

Polyfunctional, Pathogenic CD161+ Th17 Lineage Cells Are Resistant to Regulatory T Cell-Mediated Suppression in the Context of Autoimmunity.

In autoimmune diseases such as rheumatoid arthritis (RA), regulatory T cells (Tregs) fail to constrain autoimmune inflammation; however, the reasons for this are unclear. We investigated T cell regulation in the RA joint. Tregs from RA synovial fluid suppressed autologous responder T cells; however, when compared with Tregs from healthy control peripheral blood, they were significantly less suppressive. Despite their reduced suppressive activity, Tregs in the RA joint were highly proliferative and expressed FOXP3, CD39, and CTLA-4, which are markers of functional Tregs. This suggested that the reduced suppression is due to resistance of RA synovial fluid responder T cells to Treg inhibition. CD161(+) Th17 lineage cells were significantly enriched in the RA joint; we therefore investigated their relative susceptibility to Treg-mediated suppression. Peripheral blood CD161(+) Th cells from healthy controls were significantly more resistant to Treg-mediated suppression, when compared with CD161(-) Th cells, and this was mediated through a STAT3-dependant mechanism. Furthermore, depletion of CD161(+) Th cells from the responder T cell population in RA synovial fluid restored Treg-mediated suppression. In addition, CD161(+) Th cells exhibited pathogenic features, including polyfunctional proinflammatory cytokine production, an ability to activate synovial fibroblasts, and to survive and persist in the inflamed and hypoxic joint. Because CD161(+) Th cells are known to be enriched at sites of autoinflammation, our finding that they are highly proinflammatory and resistant to Treg-mediated suppression suggests an important pathogenic role in RA and other autoimmune diseases.

A pilot study of the immunological effects of high-dose vitamin D in healthy volunteers.

Although vitamin D deficiency is considered an environmental factor in multiple sclerosis (MS), the immunological and clinical effects of vitamin D supplementation remain unclear. We performed a pilot study of the immunomodulatory effects of vitamin D in healthy individuals (n=4), who took 5000-10,000 IU/day of vitamin D over 15 weeks. After 15 weeks of vitamin D supplementation, serum 25(OH) vitamin D levels rose significantly from baseline, with a corresponding increase in IL-10 production by peripheral blood mononuclear cells and a reduced frequency of Th17 cells. These data provide a strong rationale for randomised trials to assess the clinical effects of vitamin D supplementation in MS.

Therapeutic use of vitamin D and its analogues in autoimmunity.

In recent years, there has been great interest in the role of vitamin D in a number of diverse human diseases including autoimmunity, allergy, infection, cardiovascular disease, chronic lung disease, transplantation and cancer. Vitamin D is best known for its role in calcium metabolism; however it also has potent immunomodulatory effects. Epidemiological studies suggest that vitamin D deficiency may be a significant risk factor for many diseases. Furthermore, there is accumulating evidence from experimental studies that vitamin D has anti-inflammatory effects. Recent studies have indicated that a surprisingly high proportion of people are vitamin D deficient, suggesting that vitamin D supplementation may be of benefit to human health. This review will focus on the role of vitamin D in autoimmune diseases, including multiple sclerosis, rheumatoid arthritis and diabetes. We will review the epidemiological and experimental evidence for the protective effects of vitamin D in autoimmunity, as well as the preliminary vitamin D intervention studies and the most recent patented vitamin D analogues.

Caspase-1-processed cytokines IL-1beta and IL-18 promote IL-17 production by gammadelta and CD4 T cells that mediate autoimmunity.

IL-1ß plays a critical role in promoting IL-17 production by γδ and CD4 T cells. However, IL-1-targeted drugs, although effective against autoinflammatory diseases, are less effective against autoimmune diseases. Conversely, gain-of-function mutations in the NLRP3 inflammasome complex are associated with enhanced IL-1ß and IL-18 production and Th17 responses. In this study, we examined the role of caspase-1-processed cytokines in IL-17 production and in induction of experimental autoimmune encephalomyelitis (EAE). Killed Mycobacterium tuberculosis, the immunostimulatory component in CFA used for inducing EAE, stimulated IL-1ß and IL-18 production by dendritic cells through activation of the inflammasome complex and caspase-1. Dendritic cells stimulated with M. tuberculosis and myelin oligodendrocyte glycoprotein promoted IL-17 production by T cells and induced EAE following transfer to naive mice, and this was suppressed by a caspase-1 inhibitor and reversed by administration of IL-1ß or IL-18. Direct injection of the caspase-1 inhibitor suppressed IL-17 production by CD4 T cells and γδ T cells in vivo and attenuated the clinical signs of EAE. γδ T cells expressed high levels of IL-18R and the combination of IL-18 and IL-23, as with IL-1ß and IL-23, stimulated IL-17 production by γδ T cells, but also from CD4 T cells, in the absence of TCR engagement. Our findings demonstrate that caspase-1-processed cytokines IL-1ß and IL-18 not only promote autoimmunity by stimulating innate IL-17 production by T cells but also reveal redundancy in the functions of IL-1ß and IL-18, suggesting that caspase-1 or the inflammasome may be an important drug target for autoimmune diseases.