The immune system responds to pathogens by a variety of pattern recognition molecules such as the Toll-like receptors (TLRs), which promote recognition of dangerous foreign pathogens. However, recent evidence indicates that normal intestinal microbiota might also positively influence immune responses, and protect against the development of inflammatory diseases1,2. One of these elements may be short-chain fatty acids (SCFAs), which are produced by fermentation of dietary fibre by intestinal microbiota. A feature of human ulcerative colitis and other colitic diseases is a change in ‘healthy’ microbiota such as Bifidobacterium and Bacteriodes3, and a concurrent reduction in SCFAs4. Moreover, increased intake of fermentable dietary fibre, or SCFAs, seems to be clinically beneficial in the treatment of colitis5-9. SCFAs bind the G-protein-coupled receptor 43 (GPR43, also known as FFAR2)10,11, and here we show that SCFA–GPR43 interactions profoundly affect inflammatory responses. Stimulation of GPR43 by SCFAs was necessary for the normal resolution of certain inflammatory responses, because GPR43-deficient (Gpr43−/−) mice showed exacerbated or unresolving inflammation in models of colitis, arthritis and asthma. This seemed to relate to increased production of inflammatory mediators by Gpr43−/− immune cells, and increased immune cell recruitment. Germ-free mice, which are devoid of bacteria and express little or no SCFAs, showed a similar dysregulation of certain inflammatory responses. GPR43 binding of SCFAs potentially provides a molecular link between diet, gastrointestinal bacterial metabolism, and immune and inflammatory responses.

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Influenza A virus (IAV) infection is associated with outcomes ranging from subclinical infection to severe pneumonia. In this study, we compared IAV strains BJx109 (H3N2), HKx31 (H3N2), and PR8 (H1N1), for their ability to elicit innate immune responses from mouse airway cells in vitro and their virulence in mice. The viruses differed markedly in their ability to induce disease in mice (PR8 > HKx31 > BJx109). In particular, PR8 infection was associated with high levels of virus replication and pulmonary inflammation. We next compared the ability of each virus strain to infect and induce inflammatory mediators from mouse airway cells. First, major differences were observed in the ability of viruses to infect and induce chemokines and cytokines from mouse alveolar macrophages (BJx109 > HKx31 > PR8), but not from airway epithelial cells (AEC) in vitro. Second, C-type lectins of the innate immune system in mouse lung fluids blocked the ability of BJx109, but not PR8, to infect mouse macrophages and AEC. The failure of the virulent PR8 virus to elicit responses from airway macrophages, combined with resistance to antiviral proteins in mouse airway fluids, likely contribute to virulence in mice. These findings provide insight into the mechanisms underlying disease severity in the mouse model of influenza infection.

Fibrosis is characterized by the excessive deposition of extracellular matrix and crosslinked proteins, in particular collagen and elastin, leading to tissue stiffening and disrupted organ function. Lysyl oxidases are key players during this process, as they initiate collagen crosslinking through the oxidation of the ε‐amino group of lysine or hydroxylysine on collagen side‐chains, which subsequently dimerize to form immature, or trimerize to form mature, collagen crosslinks. The role of LOXL2 in fibrosis and cancer is well documented, however the specific enzymatic function of LOXL2 and LOXL3 during disease is less clear. Herein, we describe the development of PXS‐5153A, a novel mechanism based, fast‐acting, dual LOXL2/LOXL3 inhibitor, which was used to interrogate the role of these enzymes in models of collagen crosslinking and fibrosis. PXS‐5153A dose‐dependently reduced LOXL2‐mediated collagen oxidation and collagen crosslinking in vitro. In two liver fibrosis models, carbon tetrachloride or streptozotocin/high fat diet‐induced, PXS‐5153A reduced disease severity and improved liver function by diminishing collagen content and collagen crosslinks. In myocardial infarction, PXS‐5153A improved cardiac output. Taken together these results demonstrate that, due to their crucial role in collagen crosslinking, inhibition of the enzymatic activities of LOXL2/LOXL3 represents an innovative therapeutic approach for the treatment of fibrosis.

Semicarbazide-sensitive amine oxidase (SSAO), also known as vascular adhesion protein-1 (VAP-1), is a member of the copperdependent amine oxidase family that is associated with various forms of inflammation and fibrosis. To investigate the therapeutic potential of SSAO/VAP-1 inhibition, potent and selective inhibitors with drug-like properties are required. PXS-4681A [(Z)-4-(2-(aminomethyl)-3-fluoroallyloxy)benzenesulfonamide hydrochloride] is a mechanism-based inhibitor of enzyme function with a pharmacokinetic and pharmacodynamic profile that ensures complete, longlasting inhibition of the enzyme after a single low dose in vivo.PXS-4681A irreversibly inhibits the enzyme with an apparent K i of 37 nM and a k inact of 0.26 min 21 with no observed turnover in vitro. It is highly selective for SSAO/VAP-1 when profiled against related amine oxidases, ion channels, and seven-transmembrane domain receptors, and is superior to previously reported inhibitors. In mouse models of lung inflammation and localized inflammation, dosing of this molecule at 2 mg/kg attenuates neutrophil migration, tumor necrosis factor-a, and interleukin-6 levels. These results demonstrate the drug-like properties of PXS-4681A and its potential use in the treatment of inflammation.

Background and purposeThe persistent influx of neutrophils into the lung and subsequent tissue damage are characteristics of COPD, cystic fibrosis and acute lung inflammation. VAP-1/SSAO is an endothelial bound adhesion molecule with amine oxidase activity that is reported to be involved in neutrophil egress from the microvasculature during inflammation. This study explored the role of VAP-1/SSAO in neutrophilic lung mediated diseases and examined the therapeutic potential of the selective inhibitor PXS-4728A.MethodsMice treated with PXS-4728A underwent intra-vital microscopy visualization of the cremaster muscle upon CXCL1/KC stimulation. LPS inflammation, Klebsiella pneumoniae infection, cecal ligation and puncture as well as rhinovirus exacerbated asthma models were also assessed using PXS-4728A.ResultsSelective VAP-1/SSAO inhibition by PXS-4728A diminished leukocyte rolling and adherence induced by CXCL1/KC. Inhibition of VAP-1/SSAO also dampened the migration of neutrophils to the lungs in response to LPS, Klebsiella pneumoniae lung infection and CLP induced sepsis; whilst still allowing for normal neutrophil defense function, resulting in increased survival. The functional effects of this inhibition were demonstrated in the RV exacerbated asthma model, with a reduction in cellular infiltrate correlating with a reduction in airways hyperractivity.Conclusions and implicationsThis study demonstrates that the endothelial cell ligand VAP-1/SSAO contributes to the migration of neutrophils during acute lung inflammation, pulmonary infection and airway hyperractivity. These results highlight the potential of inhibiting of VAP-1/SSAO enzymatic function, by PXS-4728A, as a novel therapeutic approach in lung diseases that are characterized by neutrophilic pattern of inflammation.

BACKGROUND AND PURPOSEChronic obstructive pulmonary disease (COPD) is a major cause of illness and death, often induced by cigarette smoking (CS). It is characterized by pulmonary inflammation and fibrosis that impairs lung function. Existing treatments aim to control symptoms but have low efficacy, and there are no broadly effective treatments. A new potential target is the ectoenzyme, semicarbazidesensitive mono-amine oxidase (SSAO; also known as vascular adhesion protein-1). SSAO is elevated in smokers' serum and is a pro-inflammatory enzyme facilitating adhesion and transmigration of leukocytes from the vasculature to sites of inflammation. EXPERIMENTAL APPROACHPXS-4728A was developed as a low MW inhibitor of SSAO. A model of COPD induced by CS in mice reproduces key aspects of human COPD, including chronic airway inflammation, fibrosis and impaired lung function. This model was used to assess suppression of SSAO activity and amelioration of inflammation and other characteristic features of COPD. KEY RESULTSTreatment with PXS-4728A completely inhibited lung and systemic SSAO activity induced by acute and chronic CS-exposure. Daily oral treatment inhibited airway inflammation (immune cell influx and inflammatory factors) induced by acute CS-exposure. Therapeutic treatment during chronic CS-exposure, when the key features of experimental COPD develop and progress, substantially suppressed inflammatory cell influx and fibrosis in the airways and improved lung function. CONCLUSIONS AND IMPLICATIONSTreatment with a low MW inhibitor of SSAO, PXS-4728A, suppressed airway inflammation and fibrosis and improved lung function in experimental COPD, demonstrating the therapeutic potential of PXS-4728A for this debilitating disease.Abbreviations BALF, bronchoalveolar lavage fluid; COPD, chronic obstructive pulmonary disease; CS, cigarette smoke; EM, extracellular matrix; G-CSF, granulocyte-colony stimulating factor; MLI, mean linear intercept; SSAO, semicarbazide-sensitive amine oxidase IntroductionChronic obstructive pulmonary disease (COPD) is the third leading cause of chronic morbidity and death worldwide, and its prevalence is increasing (Lozano et al., 2012). It is a heterogeneous disease variously comprising debilitating and complex pathological features including chronic pulmonary inflammation (bronchitis), fibrosis and emphysema (Keely et al., 2011;Fricker et al., 2014). These features combine to impair lung function and result in reduced oxygen transfer leading to breathlessness. Cigarette smoke (CS) exposure is the primary etiological agent in Western countries, accounting for more than 90% of cases. Once induced, the patients' condition often continues to deteriorate, even after cessation of smoking. Wood and cooking smoke and pollution are also important risk factors particularly in developing nations (Fabbri et al., 2004;Eisner et al., 2010;Ko and Hui, 2012).There are no cures for COPD, and current treatments have substantial limitations. High doses of corticosteroids, long-acting β-adrenoceptor agonists, ...

The present study was carried out to investigate the immune response against Strongyloides venezuelensis infection in Balb/c mice previously immunized with larva-antigens or primed with live-larvae. Our results indicate that all primed mice developed a strong protection against challenge infection that remained active for 45 days. In mice primed with live-larvae the challenge infection resulted in great reduction of migrating larvae and the worms were completely eliminated from the small intestine before maturation. The protection pattern did not alter when the primary infection was aborted by drug treatment. In these experimental groups, the challenge infection was accompanied by a type-2 predominant immune response, intense IgE and reactive IgG1 production, and granulocyte infiltration in skin, lungs and intestine. The challenge infection in antigen-immunized mice also resulted in great reduction of migrating larvae. However, the worms that reached the host intestine matured, produced eggs and were eliminated similarly to the ones from nonimmunized mice. Protective mechanisms after immunization with larva antigen were migrating larva-specific and associated with a strong and mixed Th1 and Th2 response, without tissue granulocyte infiltration. In conclusion, protective immunity induced by a previous infection or antigen-immunization are stage-specific and operate through different effector mechanisms.