Jean-Michel Sallenave

SLPI and elafin: one glove, many fingers

Elafin and SLPI (secretory leucocyte protease inhibitor) have multiple important roles both in normal homoeostasis and at sites of inflammation. These include antiprotease and antimicrobial activity as well as modulation of the response to LPS (lipopolysaccharide) stimulation. Elafin and SLPI are members of larger families of proteins secreted predominantly at mucosal sites, and have been shown to be modulated in multiple pathological conditions. We believe that elafin and SLPI are important molecules in the controlled functioning of the innate immune system, and may have further importance in the integration of this system with the adaptive immune response. Recent interest has focused on the influence of inflamed tissues on the recruitment and phenotypic modulation of cells of the adaptive immune system and, indeed, the local production of elafin and SLPI indicate that they are ideally placed in this regard. Functionally related proteins, such as the defensins and cathelicidins, have been shown to have direct effects upon dendritic cells with potential alteration of their phenotype towards type I or II immune responses. This review addresses the multiple functions of elafin and SLPI in the inflammatory response and discusses further their roles in the development of the adaptive immune response.

IL-33 Enhances Lipopolysaccharide-Induced Inflammatory Cytokine Production from Mouse Macrophages by Regulating Lipopolysaccharide Receptor Complex

Bacterial LPS triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. However, once exposed to LPS, they become hyporesponsive to a subsequent endotoxin challenge. This phenomenon is defined as LPS desensitization or tolerance. Previous studies have identified some components of the biochemical pathways involved in negative modulation of LPS responses. In particular, it has been shown that the IL-1R-related protein ST2 could be implicated in LPS tolerance. The natural ligand of ST2 was recently identified as IL-33, a new member of the IL-1 family. In this study, we investigated whether IL-33 triggering of ST2 was able to induce LPS desensitization of mouse macrophages. We found that IL-33 actually enhances the LPS response of macrophages and does not induce LPS desensitization. We demonstrate that this IL-33 enhancing effect of LPS response is mediated by the ST2 receptor because it is not found in ST2 knockout mice. The biochemical consequences of IL-33 pretreatment of mouse macrophages were investigated. Our results show that IL-33 increases the expression of the LPS receptor components MD2 (myeloid differentiation protein 2) and TLR-4, the soluble form of CD14 and the MyD88 adaptor molecule. In addition, IL-33 pretreatment of macrophages enhances the cytokine response to TLR-2 but not to TLR-3 ligands. Thus, IL-33 treatment preferentially affects the MyD88-dependent pathway activated by the TLR.

Food-Grade Bacteria Expressing Elafin Protect Against Inflammation and Restore Colon Homeostasis

Lactic acid–producing bacteria engineered to produce the antiprotease Elafin restore colon homeostasis in mice with colitis and protect human tissue from inflammation. Bugs Deliver Drug and Keep the Gut Happy Elafin is a natural protease inhibitor that is normally expressed by the human intestine and protects the gut from insults. Elafin expression is lost in patients suffering from inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis. In a new study, Motta et al. tested whether delivery of human Elafin directly into the gut would protect from inflammatory insults and restore gut homeostasis. They commandeered helpful, safe bacteria that are commonly present in the gut and in food products and genetically modified the bacteria so that they would produce Elafin. They introduced the human Elafin gene into Lactococcus lactis and Lactobacillus casei, two bacteria present in dairy food. When given orally to mice, the two strains of genetically modified bacteria were detected a few hours later at the surface of the intestine, where they produced human Elafin. In different mouse models of acute or chronic intestinal inflammation, oral treatment with Elafin-expressing food-grade bacteria protected the gut from inflammatory damage. Elafin-expressing bacteria were also able to protect cultured human intestinal cells from inflammatory insults and to restore homeostasis and physiological functions. This approach may offer a safe, cost-effective long-term treatment for inflammatory bowel diseases. Elafin, a natural protease inhibitor expressed in healthy intestinal mucosa, has pleiotropic anti-inflammatory properties in vitro and in animal models. We found that mucosal expression of Elafin is diminished in patients with inflammatory bowel disease (IBD). This defect is associated with increased elastolytic activity (elastase-like proteolysis) in colon tissue. We engineered two food-grade strains of lactic acid bacteria (LAB) to express and deliver Elafin to the site of inflammation in the colon to assess the potential therapeutic benefits of the Elafin-expressing LAB. In mouse models of acute and chronic colitis, oral administration of Elafin-expressing LAB decreased elastolytic activity and inflammation and restored intestinal homeostasis. Furthermore, when cultures of human intestinal epithelial cells were treated with LAB secreting Elafin, the inflamed epithelium was protected from increased intestinal permeability and from the release of cytokines and chemokines, both of which are characteristic of intestinal dysfunction associated with IBD. Together, these results suggest that oral delivery of LAB secreting Elafin may be useful for treating IBD in humans.

Gene transfer for cytokine functional studies in the lung: the multifunctional role of GM-CSF in pulmonary inflammation.

Using adenoviral‐mediated gene transfer techniques, the murine granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) transgene is efficiently targeted to and highly expressed by the respiratory epithelium of rat lung. This lung tissue‐directed expression of GM‐CSF induces accumulation of both eosinophils and macrophages at early stages and an irreversible fibrotic reaction at later stages. These tissue responses to GM‐CSF appear to be distinct from those induced by other proinflammatory cytokines, interleukin (IL) ‐5, IL‐6, macrophage inflammatory protein‐2 (MIP‐2), or RANTES overexpressed in the lung. These findings clearly demonstrate that GM‐CSF is more than a hematopoietic cytokine in the lung and may play a pivotal role in the multiple pathological processes underlying numerous respiratory illnesses, including asthma. In this overview, the differences in tissue responses induced by GM‐CSF and other individual cytokines are highlighted. In addition, the mechanisms by which GM‐CSF contributes to the development of eosinophilia, macrophage granuloma, and fibrosis are discussed in conjunction with the recent findings from us and others.

Intranasal Mucosal Boosting with an Adenovirus- Vectored Vaccine Markedly Enhances the Protection of BCG-Primed Guinea Pigs against Pulmonary Tuberculosis

Background Recombinant adenovirus-vectored (Ad) tuberculosis (TB) vaccine platform has demonstrated great potential to be used either as a stand-alone or a boost vaccine in murine models. However, Ad TB vaccine remains to be evaluated in a more relevant and sensitive guinea pig model of pulmonary TB. Many vaccine candidates shown to be effective in murine models have subsequently failed to pass the test in guinea pig models. Methods and Findings Specific pathogen-free guinea pigs were immunized with BCG, AdAg85A intranasally (i.n), AdAg85A intramuscularly (i.m), BCG boosted with AdAg85A i.n, BCG boosted with AdAg85A i.m, or treated only with saline. The animals were then infected by a low-dose aerosol of M. tuberculosis (M.tb). At the specified times, the animals were sacrificed and the levels of infection in the lung and spleen were assessed. In separate studies, the long-term disease outcome of infected animals was monitored until the termination of this study. Immunization with Ad vaccine alone had minimal beneficial effects. Immunization with BCG alone and BCG prime-Ad vaccine boost regimens significantly reduced the level of M.tb infection in the tissues to a similar extent. However, while BCG alone prolonged the survival of infected guinea pigs, the majority of BCG-immunized animals succumbed by 53 weeks post-M.tb challenge. In contrast, intranasal or intramuscular Ad vaccine boosting of BCG-primed animals markedly improved the survival rate with 60% of BCG/Ad i.n- and 40% of BCG/Ad i.m-immunized guinea pigs still surviving by 74 weeks post-aerosol challenge. Conclusions Boosting, particularly via the intranasal mucosal route, with AdAg85A vaccine is able to significantly enhance the long-term survival of BCG-primed guinea pigs following pulmonary M.tb challenge. Our results thus support further evaluation of this viral-vectored TB vaccine in clinical trials.

Secretory leukocyte protease inhibitor and elafin/trappin-2: versatile mucosal antimicrobials and regulators of immunity.

Elafin and secretory leukocyte protease inhibitor (SLPI) are pleiotropic molecules chiefly synthesized at the mucosal surface that have a fundamental role in the surveillance against microbial infections. Their initial discovery as anti-proteases present in the inflammatory milieu in chronic pathologies such as those of the lung suggested that they may play a role in keeping in check extracellular proteases released during the excessive activation of innate immune cells such as neutrophils. This soon proved to be a simplistic explanation, as other functions were also soon ascribed to these molecules (antimicrobial, modulation of innate and adaptive immunity, regulation of tissue repair). Data emanating from patients with chronic pathologies (in the lung and elsewhere) have shown that SLPI and elafin are often inactivated in inflammatory secretions, either through the action of host or microbial products, justifying attempts at antiprotease supplementation in clinical protocols. Although these have been sparse, proof of principle has been demonstrated, and future challenges will undoubtedly rest with improvements in methods of delivery in the context of tissue inflammation and in careful selection of patients more likely to benefit from SLPI/elafin augmentation.

Overexpressing mouse model demonstrates the protective role of Muc5ac in the lungs

MUC5AC, a major gel-forming mucin expressed in the lungs, is secreted at increased rates in response to infectious agents, implying that mucins exert a protective role against inhaled pathogens. However, epidemiological and pathological studies suggest that excessive mucin secretion causes airways obstruction and inflammation. To determine whether increased MUC5AC secretion alone produces airway obstruction and/or inflammation, we generated a mouse model overexpressing Muc5ac mRNA ∼20-fold in the lungs, using the rCCSP promoter. The Muc5ac cDNA was cloned from mouse lungs and tagged internally with GFP. Bronchoalveolar lavage fluid (BALF) analysis demonstrated an approximate 18-fold increase in Muc5ac protein, which formed high-molecular-weight polymers. Histopathological studies and cell counts revealed no airway mucus obstruction or inflammation in the lungs of Muc5ac-transgenic (Muc5ac-Tg) mice. Mucus clearance was preserved, implying that the excess Muc5ac secretion produced an “expanded” rather than more concentrated mucus layer, a prediction confirmed by electron microscopy. To test whether the larger mucus barrier conferred increased protection against pathogens, Muc5ac-Tg animals were challenged with PR8/H1N1 influenza viruses and showed significant decreases in infection and neutrophilic responses. Plaque assay experiments demonstrated that Muc5ac-Tg BALF and purified Muc5ac reduced infection, likely via binding to α2,3-linked sialic acids, consistent with influenza protection in vivo. In conclusion, the normal mucus transport and absence of a pulmonary phenotype in Muc5ac-Tg mice suggests that mucin hypersecretion alone is not sufficient to trigger luminal mucus plugging or airways inflammation/goblet cell hyperplasia. In contrast, increased Muc5ac secretion appears to exhibit a protective role against influenza infection.

C5a Mediates Peripheral Blood Neutrophil Dysfunction in Critically Ill Patients

RATIONALE Critically ill patients are highly susceptible to hospital-acquired infection. Neutrophil function in critical illness remains poorly understood. OBJECTIVES To characterize and define mechanisms of peripheral blood neutrophil (PBN) dysfunction in critically ill patients. To determine whether the inflamed lung contributes additional phagocytic impairment. METHODS Prospective collection of blood and bronchoalveolar lavage fluid from patients with suspected ventilator-associated pneumonia and from age- and sex-matched volunteers; laboratory analysis of neutrophil functions. MEASUREMENTS AND MAIN RESULTS Seventy-two patients and 21 volunteers were included. Phagocytic capacity of PBNs was 36% lower in patients than in volunteers (P < 0.0001). From several biologically plausible candidates only activated complement was significantly associated with impaired PBN phagocytosis (P < 0.0001). Phagocytosis was negatively correlated with serum C3a and positively correlated with expression of C5a receptor type 1 (CD88) on PBNs. C5a recapitulated impaired PBN phagocytosis and significantly down-regulated CD88 expression in vitro. C5a-mediated phagocytic impairment was prevented by blocking either CD88 or phosphoinositide 3-kinase, and completely reversed by granulocyte-macrophage colony-stimulating factor. C5a also impaired killing of Pseudomonas aeruginosa by, and migration of, PBNs, indicating that effects were not restricted to phagocytosis. Bronchoalveolar lavage fluid leukocytes from patients also demonstrated significantly impaired function, and lavage supernatant reduced phagocytosis in healthy neutrophils by 43% (P = 0.0001). However, lavage fluid did not affect CD88 expression and lavage-mediated impairment of phagocytosis was not blocked by anti-CD88 antibody. CONCLUSIONS Critically ill patients have significant dysfunction of PBNs, which is mediated predominantly by activated complement. Further, profound complement-independent neutrophil dysfunction occurs in the inflamed lung.

Human neutrophil elastase: Mediator and therapeutic target in atherosclerosis

Human neutrophil elastase (HNE) is present within atherosclerotic plaques where it contributes to matrix degradation and weakening of the vessel wall associated with the complications of aneurysm formation and plaque rupture. It is joined by other extracellular proteases in these actions but the broad range of substrates and potency of HNE coupled with the potential for rapid increases in HNE activity associated with neutrophil degranulation in acute coronary syndromes single this disruptive protease out as therapeutic target in atherosclerotic disease. This review summarises the role of HNE in neutrophil-mediated endothelial injury and the evidence for HNE as a mediator of atherosclerotic plaque development. The therapeutic potential of HNE neutralising antiproteases, alpha-1-antitrypsin and elafin, in atherosclerosis, is discussed.

Diagnostic importance of pulmonary interleukin-1β and interleukin-8 in ventilator-associated pneumonia

Background Ventilator-associated pneumonia (VAP) is the most commonly fatal nosocomial infection. Clinical diagnosis of VAP remains notoriously inaccurate. The hypothesis was tested that significantly augmented inflammatory markers distinguish VAP from conditions closely mimicking VAP. Methods A prospective, observational cohort study was carried out in two university hospital intensive care units recruiting 73 patients with clinically suspected VAP, and a semi-urban primary care practice recruiting a reference group of 21 age- and sex-matched volunteers. Growth of pathogens at >104 colony-forming units (cfu)/ml of bronchoalveolar lavage fluid (BALF) distinguished VAP from “non-VAP”. Inflammatory mediators were quantified in BALF and serum. Mediators showing significant differences between patients with and without VAP were analysed for diagnostic utility by receiver operator characteristic (ROC) curves. Results Seventy-two patients had recoverable lavage—24% had VAP. BALF interleukin-1β (IL-1β), IL-8, granulocyte colony-stimulating factor and macrophage inflammatory protein-1α were significantly higher in the VAP group (all p<0.005). Using a cut-off of 10 pg/ml, BALF IL-1β generated negative likelihood ratios for VAP of 0.09. In patients with BALF IL-1β <10 pg/ml the post-test probability of VAP was 2.8%. Using a cut-off value for IL-8 of 2 ng/ml, the positive likelihood ratio was 5.03. There was no difference in cytokine levels between patients with sterile BALF and those with growth of <104 cfu/ml. Conclusions BALF IL-1β and IL-8 are amongst the strongest markers yet identified for accurately demarcating VAP within the larger population of patients with suspected VAP. These findings have potential implications for reduction in unnecessary antibiotic use but require further validation in larger populations.