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Exploiting Bioenergetic Differences to Stop GVHD

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Image: Flickr - Junichiro Aoyama

Bz-423, a mitochondrial F1F0-ATP synthase inhibitor, that has previously shown promise for the treatment of autoimmune disorders such as lupus, arthritis and psoriasis has now been shown to halt the progression of established graft-versus-host disease (GVHD) in mouse models of allogeneic bone marrow transplantation. GVHD, in which functional immune cells in the transplanted marrow mount an immunological attack on the recipient, is a common complication of allogeneic bone marrow transplantation that can cause severe organ damage and even be life-threatening. As well as demonstrating that Bz-423 can reduce GVHD clinical scores and improve survival in mice, researchers at the University of Michigan and the University of Florida have shed new light on the metabolism of alloreactive T cells.

Bz-423 structure

Bz-423

Resting lymphocytes meet their minimal demand for ATP using low rates of oxidative phosphorylation. Upon activation, normal lymphocytes meet the increased demand for ATP by dramatically increasing their rate of aerobic glycolysis which also helps to maintain high levels of antioxidants in activated cells. Data from the present study suggest that alloreactive T cells, on the other hand, rely heavily on increased oxidative phosphorylation to generate more ATP. This difference in energy generation is also seen in pathogenic T cells involved in autoimmune diseases and provides a mechanistic basis for the specific elimination of pathogenic cells by Bz-423, whilst preserving normal immune function.

The study is published in Science Translational Medicine.

Lycera Corporation has a program to develop orally bioavailable F1F0-ATP synthase inhibitors that exploit bioenergetic abnormalities in pathologically activated lymphocytes and result in the selective silencing of these cells.


Trauma: the Enemy Within

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Image: Flickr - Torley

Systemic inflammatory response syndrome (SIRS) is an exaggerated host inflammatory response to infection (sepsis) or to physical insults such as trauma. SIRS can lead to multiple organ dysfunction syndrome (MODS) and, amongst the under-35s, trauma is the leading cause of death in the United States. Although the pathways leading from infection to sepsis are relatively well understood, it has been much less clear why physical insults lead to SIRS. A study led by researchers at Beth Israel Deaconess Medical Center has now suggested a link between sepsis and SIRS that is caused by trauma. The team propose that mitochondria are released into the bloodstream after physical injury and, because mitochondria closely resemble the symbiotic bacteria from which they are believed to originate, they elicit a sepsis-like response.

Pathogen-associated molecular patterns (PAMPs) are molecules such as bacterial endotoxins which are recognised by pattern recognition receptors (PRRs) as non-self, and so trigger an innate immune response. Injured or necrotic tissue generates molecules known as damage-associated molecular pattern molecules (DAMPs) that can also initiate and perpetuate an immune response. Many DAMPs are molecules that are usually found exclusively within cells and, when released into the bloodstream, are not recognised as self and trigger an immune response. The team found blood samples from patients who had suffered multiple trauma contained high levels of mitochondrial DNA – often thousands-of-fold higher than normal levels – and that this DNA activates immune cells via toll-like receptor 9 which normally recognises bacterial or viral DNA. Mitochondrial peptides were also found to elicit an immune response via the formyl peptide receptor 1 (FPR1) which also plays a role in the immune response to bacterial infections. Mitochondrial DDA and peptides were found to act synergistically to activate neutrophils via downstream kinase pathways. In further experiments, injection of mitochondria into rats caused peritonitis and reproduced the pulmonary and hepatic inflammation typical of traumatic SIRS.

The study, which is published in the journal Nature, shows that trauma can initiate innate immune pathways identical to those activated in sepsis and may lead to new strategies for treating trauma patients as well as re-evaluation of patients believed to be suffering from sepsis.


Immune Response to Swine Flu May Explain Disease Severity

The pandemic swine flu (H1N1) virus has proved to be less lethal than originally feared but, although most infected individuals experience relatively mild and self-limiting symptoms, some patients with no previous underlying medical condition have died. An international team of researchers has now found a possible explanation of why some people develop severe pneumonia when infected with the H1N1 virus. The team profiled immune mediators in 20 patients with severe symptoms, 15 patients with mild symptoms and 15 healthy controls. A typical innate antiviral response with increased levels of chemokines IP-10, MCP-1 and MIP-1β and an absence of anti-H1N1 antibodies characterised the early response in all infected individuals, but elevated levels of IFN-γ and mediators that stimulate Th17 and Th1 responses were found only in hospitalised patients. Both critical and non-critical hospitalised patients had increased levels of IFN-γ, Th17 mediators (IL-8, IL-9, IL17 and IL6) and Th1 mediators (TNF-α, IL-15 and IL-12p70) compared to outpatients. All hospitalised patients showed higher levels of IL-17 and TNF-α than controls but only the non-critical patients showed significant higher levels of IL-17 and TNF-α than those with mild symptoms. Levels of IL-15, IL-12p70 increased exclusively in critical patients who also showed the highest levels of IL-6.

Around half of hospitalised patients and nearly all outpatients tested positive for virus, with all those who tested positive having similar viral loads. Significantly higher levels of IL-13 and IL-17 were found in hospitalised patients with undetectable virus. IL6 was found to show a significant inverse association with arterial blood oxygen pressure in hospitalised patients and a similar inverse relationship was found for IL-8 in the critically ill patients.

Th1 and Th17 cells form an important part of host defence against pathogens but TH17 cells have also been linked to the pathogenesis of autoimmune and inflammatory diseases. It is presently unclear whether the increase in Th1 and Th17 responses reflects a vigorous antiviral defence necessary to clear lower respiratory infection or whether the inflammatory response contributes to disease severity. Although the ability of influenza viruses to evoke an inflammatory response is well known, this is the first study to link a Th17 response to severe influenza disease in humans. The authors suggest that immunomodulatory drugs which down-modulate Th1 and Th17 responses could be used to clarify the role of these pathways in the pathogenesis of the acute respiratory symptoms shown by patients with severe H1N1 disease. “Hypercytokinemia” of specific chemokines and cytokines has previously been shown to be associated with severe and often fatal cases of human H5N1 infections.

The study is published in the journal Critical Care.


P-glycoprotein Opens Gate to Neuroinflammation

P-glycoprotein (P-gp, ABCB1) was originally characterized by its ability to confer a multidrug-resistant phenotype to cancer cells. It was also the first drug efflux transporter to be detected on blood-brain barrier endothelial cells and is now recognized to be involved in the transport of a wide variety of substrates and drug molecules. P-gp is also expressed on cells of the immune system where it is believed to play a role in the efflux of inflammatory mediators such as steroids, prostaglandins and cytokines.

Scientists from the Netherlands have now shown that P-gp may play a key role in multiple sclerosis (MS). In MS, autoreactive myelin-specific T helper (Th) cells – which are primed in the periphery by antigen-presenting dendritic cells (DCs) – cause extensive destruction of myelin sheaths and axonal loss. The team found that both DC maturation and T-cell stimulatory capacity were severely impaired in P-gp knockout mice (Mdr1a/1b-/-) which showed less severe clinical symptoms of experimental autoimmune encephalomyelitis (EAE) following administration of recombinant myelin oligodendrocyte glycoprotein (rMOG) than wild type animals. The observed differences in clinical symptoms were associated with decreased demyelination in the brains of the knockout animals and reduced brain inflammation. Following immunisation with rMOG, secretion of Th1 cytokines, IFN-γ and TNF-α and Th2 cytokines, IL10 and IL5, by lymph node cells was significantly reduced in the Mdr1a/1b-/- mice compared with wild type animals whereas no changes were observed in the secretion of the Th17 cytokine, IL-17. Since P-gp did not affect the ability of T-cells to become activated, the authors propose that the observed differences in T-cell responses are likely mediated by regulation of cytokine secretion by DCs.

The study, which is published in PLoSone, highlights a novel immunomodulatory role for P-gp and may provide new opportunities to treat immune-related or inflammatory diseases.


Toll-Like Receptor 2 Linked to Atherosclerotic Damage

Atherosclerosis is caused by a build up of lipids, cholesterol, calcium, and cellular debris within the artery, resulting in plaque formation. This restricts the flow of blood and decreases oxygen supply to target organs, increasing the risk of cardiovascular diseases including heart attacks and stroke. The true incidence of atherosclerosis is difficult, if not impossible, to determine since it is predominantly asymptomatic, but the ensuing cardiovascular diseases are the leading cause of death in many Western societies. Risk factors for atherosclerosis include high blood pressure and a high fat diet and current non-surgical treatments, such as antihypertensive medicines and statins, focus on reducing these risks.

The precise mechanisms underlying atherogenesis are unclear but scientists at Imperial College London have now identified a pathway that plays a key role in the inflammation and matrix degradation characteristic of human atherosclerosis.

The researchers studied sections of carotid artery taken from 58 stroke patients and found that toll-like receptor 2 (TLR-2) was unusually active in the plaques. TLRs are expressed on immune cells and play a fundamental role in pathogen recognition and innate immunity, mediating release of cytokines and other inflammatory mediators. One arm of the TLR-induced inflammatory response is dependent on a signalling pathway that is mediated by the adaptor molecule, myeloid differentiation primary response gene 88 (MyD88), and the study showed that a dominant-negative form of MyD88 decreased the production of MCP-1, IL-8, IL-6, MMP-1 and MMP-3 as well as NF-κB activation in cell cultures prepared from the carotid arteries. TLR-2 neutralizing antibodies were also shown to inhibit NF-κB activation and significantly reduce MCP-1, IL-8, IL-6, MMP-1, MMP-2, MMP-3, and MMP-9 production. In contrast, an IL-1R antagonist, TLR-4 blocking antibodies, or overexpression of a dominant-negative form of the TLR-4 signalling adaptor, TRIF-related adaptor molecule, reduced NF-κB activity but did not have a broad impact on the production of the inflammatory mediators studied.

The authors hope that TLR-2 blockers might be developed to prevent or treat atherosclerosis and the resulting cardiovascular disease without compromising the ability to fight infection.

The study is published in the journal Circulation.


Reflux Esophagitis May Not Be Caused Directly by Acid Burn

Gastroesophageal reflux (GER), or acid reflux, which occurs when the lower esophageal sphincter opens spontaneously or doesn’t close properly, is experienced intermittently by most people. Gastroesophageal reflux disease (GERD) occurs when the amount of gastric juice that refluxes into the esophagus exceeds the normal limit and the esophageal mucosa is damaged, causing esophagitis. It has generally been thought that damage to the esophagus is the direct result of chemical burns by the acid in refluxed gastric juice but researchers at UT Southwestern Medical Center have now suggested that this may not be the case. The team created GERD in rats by connecting the duodenum to the esophagus, which allows stomach acid and bile to enter the esophagus, but were surprised to find that esophagitis didn’t develop for a number of weeks after the operation. If GERD is really caused by acid burns, the damage would be expected to appear much more quickly. In earlier studies, perfusion with highly concentrated acid did cause rapid damage to the esophagus, but early events in animal models designed to more closely resemble human GERD had not been investigated in detail.

In the present study, the researchers expected to see death of surface cells followed by injury to the deeper layers of the esophagus but found just the opposite. Reflux esophagitis started at postoperative day 3; at this stage there was no damage to surface epithelial cells, but lymphocytes had begun to infiltrate the submucosa and later progressed to the epithelial surface. Since damage to the deeper layers of the esophagus preceded surface erosions, the team suggest that it is infiltrating lymphocytes, rather than direct chemical burns, that cause the damage. Exposure of esophageal epithelial cells to acidified bile salts was shown to increase the secretion of cytokines interleukin-8 and interleukin-1β, and conditioned media from these cells was found to cause significant increases in the migration rates of T cells and neutrophils. Current treatments for GERD focus on reducing acid production by the stomach but the new study provides a more complex picture and suggests that treatments that modulate the immune response could also be of benefit.

The study is published in Gastroenterology.


Antibody Recruiting Molecules Target HIV, Cancer

hiv virus cartoon Both HIV and cancer cells have found ways to evade the body’s immune system but researchers at Yale University have now found a way to boost the body’s ability to fight HIV and cancer. The team have identified bifunctional small molecules, termed “antibody-recruiting molecule targeting HIV” (ARM-H) and “antibody-recruiting molecule targeting prostate cancer” (ARM-P), which bind simultaneously to antibodies and to proteins on HIV, HIV-infected cells or cancer cells.

ARM-H molecules bind to gp120, a component of the Env glycoprotein on the surface of HIV and virus-infected cells and to anti-2,4-dinitrophenyl antibodies already present in the bloodstream. The ternary complex formed between the antibody, ARM-H, and gp120 is immunologically active, and leads to complement-mediated destruction of Env-expressing cells. ARM-H also prevents virus entry into human T-cells and so has the potential to inhibit viral replication by two mutually reinforcing mechanisms.

ARM-P molecules bind with high affinity to prostate-specific membrane antigen (PSMA) and, by inducing complexes of anti-2,4-dinitrophenyl antibodies with prostate cancer cells, mediate antibody-dependent killing of the cancer cells.

The team has begun to evaluate the ARM molecules in mice, and hope that the strategy of using antibody-recruiting small molecules to boost the immune response will prove useful for treating HIV, cancer, and other diseases.

Both studies are published in the Journal of the American Chemical Society (ARM-H and ARM-P).


New Role for Immune System in Osteoporosis

Many reports have linked high cholesterol with osteoporosis and HMG-CoA reductase inhibitors (statins) have been proposed to reduce the risk of fractures, although clinical data have been more equivocal than results from animal studies. osteoporosisMechanisms for the beneficial actions of statins on bone density have been proposed, but a new study by researchers at UCLA has focused on the link between bone loss and the immune system. Recent evidence has pointed to a role for RANKL (Receptor Activator for Nuclear Factor κB Ligand) in bone metabolism by activating osteoclasts and increasing bone resorption. RANKL is produced by activated T-cells and is involved in mediating interactions with dendritic cells. Since products of lipid oxidation are known to affect T-cell function and to contribute to inflammation and metabolic disorders, the UCLA team hypothesised that production of RANKL might be modulated by oxidised lipids. Both unstimulated and activated human T-cells significantly increased production of RANKL and expression of the lectin-like oxidized LDL receptor-1 (LOX-1) on short term exposure to minimally oxidised LDL but not on exposure to native LDL. The effect was shown to be mediated by via the NFκB pathway and involve increased RANKL mRNA expression.

In follow-up studies in hyperlipidaemic mice, bone loss was found to be associated with increased RANKL mRNA in T lymphocytes as well as elevated RANKL serum levels. The finding that oxidised lipids contribute to bone changes by increasing RANKL production by T-cells may explain the link between cardiovascular disease and osteoporosis and also suggests the possibility of new immune-based therapies for osteoporosis.

The study was published on August 20th in the journal Clinical Immunology.


Complement-ary Treatment for Alzheimer’s Disease?

dominoesThe complement system is a complex cascade of reactions forming a central component of the innate immune system which assists in the removal of invading pathogens and cellular debris, and in the processing of immune complexes. There is substantial evidence that complement activation is associated with amyloid plaques in the brains of Alzheimer’s disease patients, although whether this is beneficial or detrimental has been unclear.

PMX-205 structureWriting in the Journal of Immunology, US and Australian scientists have now described the effect of administration of an antagonist of the receptor for the complement activation product, C5a, in animal models of Alzheimer’s disease. Oral administration of PMX205 in drinking water for 2-3 months resulted in substantial reductions in disease markers such as fibrillar amyloid deposits and activated glia in two mouse models of Alzheimer’s disease. The reduction in pathological markers correlated with improved performance in a passive avoidance task in Tg2576 mice. In 3xTg mice, PMX205 also significantly reduced hyperphosphorylated tau.

PMX205 is a cyclic hexapeptide derivative that was developed by Promics as a second generation C5a receptor antagonist for the treatment of inflammatory disorders, including inflammatory bowel disease. An earlier compound, PMX53, was found to be well tolerated in phase I clinical trials for rheumatoid arthritis and psoriasis.

The new study shows for the first time that antagonists of the C5a receptor interfere with inflammation and neurodegeneration in mouse models of Alzheimer’s disease and could, one day, lead to new treatments for human patients.


Long-Life Immunity

Tree of LifeThe primary function of the thymus is to produce mature T-cells and to implement controls to prevent auto-immunity. Lymphocyte precursors migrate from the bone-marrow to the thymus, where they become thymocytes and subsequently mature into T-cells. Since the T-cell repertoire is generated relatively early in life, the thymus is most active during childhood, begins to atrophy around puberty, and is barely detectable in the elderly.

A team led by researchers at the Children’s Hospital of Pittsburgh have now reported knock-out mice that live 30% longer than their wild-type counterparts and in which the thymus remains intact throughout life. The scientists knocked out the gene encoding pregnancy-associated plasma protein A (PAPPA), a recently identified zinc metalloprotease that degrades insulin-like growth factor binding proteins (IGFBP). The knockout mice exhibit proportional dwarfism, similar to IGF1, IGF2 and IGF-receptor (IGFR) knockouts (although IGF1 and IGFR knockout mice are not viable).

The team suggests that the PAPPA-knockout mice benefit from reduced IGF signalling in tissues as a consequence of increased levels of IGFBPs. Indeed, maintenance of thymic structure with ageing correlated with lower steady-state levels of IGF1 in the thymus. Elderly knockout mice continued to generate new T-cells and maintained a robust immune system. Whilst the subtleties of the model require further elucidation, the work so far suggests that manipulation of PAPPA may be a route to modulation of immune competence and healthy ageing.

The study is published in full in the Proceedings of the National Academy of Sciences.