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Background-Lipopolysaccharide (LPS) decreases hepatic CETP (cholesteryl ester transfer protein) expression albeit that the underlying mechanism is disputed. We recently showed that plasma CETP is mainly derived from Kupffer cells (KCs). In this study, we investigated the role of KC subsets in the mechanism by which LPS reduces CETP expression. Methods and Results-In CETP-transgenic mice, LPS markedly decreased hepatic CETP expression and plasma CETP concentration without affecting hepatic macrophage number. This was paralleled by decreased expression of the resting KC markers C-type lectin domain family 4, member f (Clec4f) and V-set and immunoglobulin domain containing 4 (Vsig4), while expression of the infiltrating monocyte marker lymphocyte antigen 6 complex locus C (Ly6C) was increased. Simultaneously, the ratio of plasma high-density lipoprotein-cholesterol over non-high-density lipoprotein-cholesterol transiently increased. After ablation hepatic macrophages via injection with liposomal clodronate, the reappearance of hepatic gene and protein expression of CETP coincided with Clec4f and Vsig4, but not Ly6C. Double-immunofluorescence staining showed that CETP co-localized with Clec4f(+)KCs and not Ly6C(+) monocytes. In humans, microarray gene-expression analysis of liver biopsies revealed that hepatic expression and plasma level of CETP both correlated with hepatic VSIG4 expression. LPS administration decreased the plasma CETP concentration in humans. In vitro experiments showed that LPS reduced liver X receptor-mediated CETP expression. Conclusions-Hepatic expression of CETP is exclusively confined to the resting KC subset (ie, F4/80(+)Clec4f(+)Vsig4(+)Ly6C(-)). LPS activated resting KCs, leading to reduction of Clec4f and Vsig4 expression and reduction of hepatic CETP expression, consequently decreasing plasma CETP and raising high-density lipoprotein (HDL)-cholesterol. This sequence of events is consistent with the anti-inflammatory role of HDL in the response to LPS and may be relevant as a defense mechanism against bacterial infections.

Contains fulltext : 172589.pdf (Publisher’s version ) (Closed access) Despite the importance of immune variation for the symptoms and outcome of Lyme disease, the factors influencing cytokine production during infection with the causal pathogen Borrelia burgdorferi remain poorly understood. Borrelia infection-induced monocyte- and T cell-derived cytokines were profiled in peripheral blood from two healthy human cohorts of Western Europeans from the Human Functional Genomics Project. Both non-genetic and genetic host factors were found to influence Borrelia-induced cytokine responses. Age strongly impaired IL-22 responses, and genetic studies identified several independent QTLs that impact Borrelia-induced cytokine production. Genetic, transcriptomic, and functional validation studies revealed an important role for HIF-1alpha-mediated glycolysis in the cytokine response to Borrelia. HIF-1alpha pathway activation and increase in glycolysis-derived lactate was confirmed in Lyme disease patients. In conclusion, functional genomics approaches reveal the architecture of cytokine production induced by Borrelia infection of human primary leukocytes and suggest a connection between cellular glucose metabolism and Borrelia-induced cytokine production.

Septins, cytoskeletal proteins with well‐characterised roles in cytokinesis, form cage‐like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single‐cell analysis, we show that the septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneri‐infected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin‐related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin‐polymerising Shigella fragment mitochondria to escape from septin caging. Our results demonstrate a role for mitochondria in anti‐Shigella autophagy and uncover a fundamental link between septin assembly and mitochondria.

Macrophages are multifunctional cells that perform diverse roles in health and disease. Emerging evidence has suggested that these innate immune cells might also be capable of developing immunological memory, a trait previously associated with the adaptive system alone. While recent studies have focused on the dramatic macrophage reprogramming that follows infection and protects against secondary microbial attack, can macrophages also develop memory in response to other cues? Here, we show that apoptotic corpse engulfment by Drosophila macrophages is an essential primer for their inflammatory response to tissue damage and infection in vivo. Priming is triggered via calcium-induced JNK signaling, which leads to upregulation of the damage receptor Draper, thus providing a molecular memory that allows the cell to rapidly respond to subsequent injury or infection. This remarkable plasticity and capacity for memory places macrophages as key therapeutic targets for treatment of inflammatory disorders.

BACKGROUND: Chronic rhinosinusitis with nasal polyposis (CRSwNP) in Western countries is characterized by eosinophilia, IgE production, and TH2 cytokine expression. Type 2 innate lymphoid cells from polyps produce IL-5 and IL-13 in response to IL-25 and IL-33, although the relevance of this axis to local mucosal T-cell responses is unknown.OBJECTIVE: We sought to investigate the role of the IL-25/IL-33 axis in local mucosal T-cell responses in patients with CRSwNP.METHODS: Polyp tissue and blood were obtained from patients undergoing nasal polypectomy. Control nasal biopsy specimens and blood were obtained from healthy volunteers. Tissue was cultured in a short-term explant model. T-cell surface phenotype/intracellular cytokines were assessed by means of flow cytometry. T-cell receptor variable β-chain analysis was performed with the immunoSEQ assay. Microarrays were performed for gene expression analysis.RESULTS: IL-25 receptor (IL-17RB)-expressing TH2 effector cells were identified in nasal polyp tissue but not the healthy nasal mucosa or periphery. IL-17RB(+)CD4(+) polyp-derived TH2 cells coexpressed ST2 (IL-33 receptor) and responded to IL-25 and IL-33 with enhanced IL-5 and IL-13 production. Within IL-17RB(+)CD4(+) T cells, several identical T-cell receptor variable β-chain complementarity-determining region 3 sequences were identified in different subjects, suggesting clonal expansion driven by a common antigen. Abundant IL-17-producing T cells were observed in both healthy nasal mucosal and polyp populations, with TH17-related genes the most overexpressed compared with peripheral blood T cells.CONCLUSION: IL-25 and IL-33 can interact locally with IL-17RB(+)ST2(+) polyp T cells to augment TH2 responses in patients with CRSwNP. A local TH17 response might be important in healthy nasal mucosal immune homeostasis.

Pulmonary aspergillosis is an opportunistic fungal infection affecting immunocompromised individuals. Increasing understanding of natural killer (NK) cell immunobiology has aroused considerable interest around the role of NK cells in pulmonary aspergillosis in the immunocompromised host. Murine studies indicate that NK cells play a critical role in pulmonary clearance of A. fumigatus. We show that the in vitro interaction between NK cells and A. fumigatus induces partial activation of NK cell immune response, characterised by low-level production of IFN-γ, TNF-α, MIP-1α, MIP-1β, and RANTES, polarisation of lytic granules and release of fungal DNA. We observed a contact-dependent down-regulation of activatory receptors NKG2D and NKp46 on the NK cell surface, and a failure of full granule release. Furthermore, the NK cell cytokine-mediated response to leukaemic cells was impaired in the presence of A. fumigatus. These observations suggest that A. fumigatus-mediated NK cell immunoparesis may represent an important mechanism of immune evasion during pulmonary aspergillosis.

Respiratory syncytial virus (RSV) is a leading cause of respiratory tract infection in infants, causing significant morbidity and mortality. No vaccine or specific, effective treatment is currently available. A more complete understanding of the key components of effective host response to RSV and novel preventative and therapeutic interventions are urgently required. Cathelicidins are host defense peptides, expressed in the inflamed lung, with key microbicidal and modulatory roles in innate host defense against infection. In this article, we demonstrate that the human cathelicidin LL-37 mediates an antiviral effect on RSV by inducing direct damage to the viral envelope, disrupting viral particles and decreasing virus binding to, and infection of, human epithelial cells in vitro. In addition, exogenously applied LL-37 is protective against RSV-mediated disease in vivo, in a murine model of pulmonary RSV infection, demonstrating maximal efficacy when applied concomitantly with virus. Furthermore, endogenous murine cathelicidin, induced by infection, has a fundamental role in protection against disease in vivo postinfection with RSV. Finally, higher nasal levels of LL-37 are associated with protection in a healthy human adult RSV infection model. These data lead us to propose that cathelicidins are a key, nonredundant component of host defense against pulmonary infection with RSV, functioning as a first point of contact antiviral shield and having additional later-phase roles in minimizing the severity of disease outcome. Consequently, cathelicidins represent an inducible target for preventative strategies against RSV infection and may inform the design of novel therapeutic analogs for use in established infection.

Many pathologies of the respiratory tract are inadequately treated with existing small molecule-based therapies. The emergence of RNA interference (RNAi) enables the post-transcriptional silencing of key molecular disease factors that cannot readily be targeted with conventional small molecule drugs. Pulmonary administration of RNAi effectors, such as small interfering RNA (siRNA), allows direct delivery into the lung tissue, hence reducing systemic exposure. Unfortunately, the clinical translation of RNAi is severely hampered by inefficient delivery of siRNA therapeutics towards the cytoplasm of the target cells. In order to have a better control of the siRNA delivery process, both extra- and intracellular, siRNAs are typically formulated in nanosized delivery vehicles (nanoparticles, NPs). In the lower airways, which are the targeted sites of action for multiple pulmonary disorders, these siRNA-loaded NPs will encounter the pulmonary surfactant (PS) layer, covering the entire alveolar surface. The interaction between the instilled siRNA-loaded NPs and the PS at this nano-bio interface results in the adsorption of PS components onto the surface of the NPs. The formation of this so-called biomolecular corona conceals the original NP surface and will therefore profoundly determine the biological efficacy of the NP. Though this interplay has initially been regarded as a barrier towards efficient siRNA delivery to the respiratory target cell, recent reports have illustrated that the interaction with PS might also be beneficial for local pulmonary siRNA delivery.

The inflammasome adaptor ASC contributes to innate immunity through the activation of caspase-1. Here we found that signaling pathways dependent on the kinases Syk and Jnk were required for the activation of caspase-1 via the ASC-dependent inflammasomes NLRP3 and AIM2. Inhibition of Syk or Jnk abolished the formation of ASC specks without affecting the interaction of ASC with NLRP3. ASC was phosphorylated during inflammasome activation in a Syk- and Jnk-dependent manner, which suggested that Syk and Jnk are upstream of ASC phosphorylation. Moreover, phosphorylation of Tyr144 in mouse ASC was critical for speck formation and caspase-1 activation. Our results suggest that phosphorylation of ASC controls inflammasome activity through the formation of ASC specks., 炎症応答を制御する新たな仕組みを解明 -慢性炎症や自己炎症性疾患の病態理解に貢献-. 京都大学プレスリリース. 2013-11-04.

Microfluidics-based nanoLC-MS/MS (chipLC-MS/MS) was used to identify and quantify proteins in epithelial lining fluid (ELF), collected during bronchoscopy from the main bronchi of chronic obstructive pulmonary disease (COPD) patients and healthy controls using microprobes. ELF is a biofluid that is well suited to study pathophysiological processes in the lung, because it contains high concentrations of biologically active molecules. 1D-PAGE followed by in-gel tryptic digestion and chipLC-MS/MS resulted in identification of approximately 300 proteins. A comparative study of ELF from COPD patients and non-COPD controls using chemical stable isotope labeling (iTRAQ (R)-8Plex) showed that the levels of lactotransferrin, high-mobility group protein B1 (HMGB 1), alpha 1-antichymotrypsin and cofilin-1 differed significantly in ELF from COPD patients and non-COPD controls (p-values

Chemoattractant-induced reactive oxygen species (ROS) generation by adherent neutrophils occurs in two phases: the first is very rapid and transient, and the second one is delayed and lasts up to 30–40 min. We examined the role of phosphoinositide 3-kinases (PI3Ks) and Src-family kinases (SFKs) in these responses using human neutrophils treated with inhibitory compounds or murine neutrophils deficient of PI3Kγ or Hck, Fgr, and Lyn. Our studies show that PI3Kγ is indispensable for the early, fMLF-induced ROS generation and AKT and ERK phosphorylation, but is dispensable for the late response to fMLF. Additionally, the response to TNF, an agonist triggering only the delayed phase of ROS generation, was also unaffected in PI3Kγ-deficient neutrophils. In contrast, inhibition of SFKs by a selective inhibitor in human, or SFK deficiency in murine, neutrophils resulted in the inhibition of both the early and late phase of ROS generation, without affecting the early phase of AKT phosphorylation, but inhibiting the late one. Selective inhibitors of PI3Kα and PI3Kδ markedly reduced both the early and late response to fMLF and TNF in human neutrophils. These findings suggest that class IA PI3Ks may be activated by PI3Kγ via Ras in the early phase of the response and by SFKs in the late phase. The evidence that inhibition of SFKs in human, or SFK deficiency in murine, neutrophils results in suppression of Vav phosphorylation at all time points of the response to fMLF or TNF suggests that SFKs are indispensable for Vav phosphorylation.

Although obesity is implicated in numerous health complications leading to increased mortality, the relationship between obesity and outcomes for critically ill patients appears paradoxical. Recent studies have reported better outcomes and lower levels of inflammatory cytokines in obese patients with acute lung injury (ALI)/acute respiratory distress syndrome, suggesting that obesity may ameliorate the effects of this disease. We investigated the effects of obesity in leptin-resistant db/db obese and diet-induced obese mice using an inhaled LPS model of ALI. Obesity-associated effects on neutrophil chemoattractant response were examined in bone marrow neutrophils using chemotaxis and adoptive transfer; neutrophil surface levels of chemokine receptor CXCR2 were determined by flow cytometry. Airspace neutrophilia, capillary leak, and plasma IL-6 were all decreased in obese relative to lean mice in established lung injury (24 h). No difference in airspace inflammatory cytokine levels was found between obese and lean mice in both obesity models during the early phase of neutrophil recruitment (2–6 h), but early airspace neutrophilia was reduced in db/db obese mice. Neutrophils from uninjured obese mice demonstrated diminished chemotaxis to the chemokine keratinocyte cytokine compared with lean control mice, and adoptive transfer of obese mouse neutrophils into injured lean mice revealed a defect in airspace migration of these cells. Possibly contributing to this defect, neutrophil CXCR2 expression was significantly lower in obese db/db mice, and a similar but nonsignificant decrease was seen in diet-induced obese mice. ALI is attenuated in obese mice, and this blunted response is in part attributable to an obesity-associated abnormal neutrophil chemoattractant response.

SummaryPeptidoglycan recognition proteins (PGRPs) are key regulators of insect immune responses. In addition to recognition PGRPs, which activate the Toll and Imd pathways, the Drosophila genome encodes six catalytic PGRPs with the capacity to scavenge peptidoglycan. We have performed a systematic analysis of catalytic PGRP function using deletions, separately and in combination. Our findings support the role of PGRP-LB as a negative regulator of the Imd pathway and brought to light a synergy of PGRP-SCs with PGRP-LB in the systemic response. Flies lacking all six catalytic PGRPs were still viable but exhibited deleterious immune responses to innocuous gut infections. Together with recent studies on mammalian PGRPs, our study uncovers a conserved role for PGRPs in gut homeostasis. Analysis of the immune phenotype of flies lacking all catalytic PGRPs and the Imd regulator Pirk reveals that the Imd-mediated immune response is highly constrained by the existence of multiple negative feedbacks.

Staphylococcus aureus is a pathogen that often causes severe nosocomial infections including pneumonia. The present study was designed to examine innate phagocyte mediated immune mechanisms using a previously described murine S. aureus Newman pneumonia model. We found that BALB/c mice represent a more susceptible mouse strain compared to C57BL/6 mice after intranasal S. aureus Newman challenge. Depletion experiments revealed that neutrophils are a crucial determinant for resistance whereas depletion of alveolar macrophages protected mice to some degree from acute pulmonary S. aureus challenge. C57BL/6 mice lacking the subunit gp91phox of the NADPH-oxidase (gp91phox-/- mice) proved to be highly susceptible against the pathogen. In contrast, C57BL/6 inducible nitric oxidase synthase deficient (iNOS-/-) mice did not differ in their clinical outcome after infection. Neither bone marrow macrophages from iNOS-/- nor from gp91phox-/- mice were impaired in controlling intracellular persistence of S. aureus. Our data suggest that neutrophil and NADPH-oxidase mediated mechanisms are essential components in protecting the host against pulmonary S. aureus Newman challenge. On contrary, macrophages as well as NO mediated mechanisms do not seem to play a critical role for resistance in this model.

Adjuvants are essential components of most clinically used vaccines. This is because the majority of nonliving vaccines are relatively poor inducers of adaptive immunity unless effective adjuvants are co-administered. Aluminum salts (alum) have been used as adjuvants with great success for almost a century and have been particularly effective at promoting protective humoral immunity. However, alum is not optimally effective for diseases where cell-mediated immunity is required for protection. Furthermore, adjuvants including oil-in-water emulsions have shown improved efficacy for avian influenza protection suggesting that even for diseases where humoral immunity can confer protection, there is scope for developing improved adjuvants. There have been major developments in antigen discovery over the past decade, which has accelerated the vaccine development process for new indications and this demands a new generation of adjuvants that can drive and specifically direct the desired immune responses. A number of systems are under investigation that combine different types of adjuvants into specific formulations with greater activity. Additionally, targeting of vaccines to specific immune cells shows great promise. In the case of cancer and chronic infectious diseases, it may be difficult to develop effective vaccines without blocking immune regulatory pathways, which impede cell-mediated responses. However, increased understanding of immunology and particularly the innate immune system is informing vaccine adjuvant research and consequently driving the development of novel and specifically directed vaccine adjuvant strategies. In this article we address the importance of adjuvants in vaccine development, the known mode of action of specific adjuvants and recent developments in this important field.

The peritrophic matrix (PM) forms a layer composed of chitin and glycoproteins that lines the insect intestinal lumen. This physical barrier plays a role analogous to that of mucous secretions of the vertebrate digestive tract and is thought to protect the midgut epithelium from abrasive food particles and microbes. Almost nothing is known about PM functions in Drosophila , and its function as an immune barrier has never been addressed by a genetic approach. Here we show that the Drosocrystallin (Dcy) protein, a putative component of the eye lens of Drosophila , contributes to adult PM formation. A loss-of-function mutation in the dcy gene results in a reduction of PM width and an increase of its permeability. Upon bacterial ingestion a higher level of expression of antibacterial peptides was observed in dcy mutants, pointing to an influence of this matrix on bacteria sensing by the Imd immune pathway. Moreover, dcy -deficient flies show an increased susceptibility to oral infections with the entomopathogenic bacteria Pseudomonas entomophila and Serratia marcescens . Dcy mutant flies also succumb faster than wild type upon ingestion of a P. entomophila toxic extract. We show that this lethality is due in part to an increased deleterious action of Monalysin, a pore-forming toxin produced by P. entomophila . Collectively, our analysis of the dcy immune phenotype indicates that the PM plays an important role in Drosophila host defense against enteric pathogens, preventing the damaging action of pore-forming toxins on intestinal cells.

Plant defensins are small cysteine-rich peptides that inhibit the growth of a broad range of microbes. In this article, we describe NmDef02, a novel cDNA encoding a putative defensin isolated from Nicotiana megalosiphon upon inoculation with the tobacco blue mould pathogen Peronospora hyoscyami f.sp. tabacina. NmDef02 was heterologously expressed in the yeast Pichia pastoris, and the purified recombinant protein was found to display antimicrobial activity in vitro against important plant pathogens. Constitutive expression of NmDef02 gene in transgenic tobacco and potato plants enhanced resistance against various plant microbial pathogens, including the oomycete Phytophthora infestans, causal agent of the economically important potato late blight disease, under greenhouse and field conditions

Wathes DC, Cheng Z, Chowdhury W, Fenwick MA, Fitzpatrick R, Morris DG, Patton J, Murphy JJ. Negative energy balance alters global gene expression and immune responses in the uterus of postpartum dairy cows. Physiol Genomics 39: 1-13, 2009. First published June 30, 2009; doi: 10.1152/physiolgenomics.00064.2009.-Most dairy cows suffer uterine microbial contamination postpartum. Persistent endometritis often develops, associated with reduced fertility. We used a model of differential feeding and milking regimes to produce cows in differing negative energy balance status in early lactation (mild or severe, MNEB or SNEB). Blood hematology was assessed preslaughter at 2 wk postpartum. RNA expression in endometrial samples was compared using bovine Affymetrix arrays. Data were mapped using Ingenuity Pathway Analysis. Circulating concentrations of IGF-I remained lower in the SNEB group, whereas blood nonesterified fatty acid and beta-hydroxybutyrate concentrations were raised. White blood cell count and lymphocyte number were reduced in SNEB cows. Array analysis of endometrial samples identified 274 differentially expressed probes representing 197 recognized genes between the energy balance groups. The main canonical pathways affected related to immunological and inflammatory disease and connective tissue disorders. Inflammatory response genes with major upregulation in SNEB cows included matrix metalloproteinases, chemokines, cytokines, and calgranulins. Expression of several interferon-inducible genes including ISG20, IFIH1, MX1, and MX2 were also significantly increased in the SNEB cows. These results provide evidence that cows in SNEB were still undergoing an active uterine inflammatory response 2 wk postpartum, whereas MNEB cows had more fully recovered from their energy deficit, with their endometrium reaching a more advanced stage of repair. SNEB may therefore prevent cows from mounting an effective immune response to the microbial challenge experienced after calving, prolonging the time required for uterine recovery and compromising subsequent fertility.

We analyzed interleukin (IL) 12 and IL-23 production by monocyte-derived dendritic cells (mono-DCs). Mycobacterium tuberculosis H37Rv and zymosan preferentially induced IL-23. IL-23 but not IL-12 was efficiently induced by the combination of nucleotide-binding oligodimerization domain and Toll-like receptor (TLR) 2 ligands, which mimics activation by M. tuberculosis, or by the human dectin-1 ligand beta-glucan alone or in combination with TLR2 ligands, mimicking induction by zymosan. TLR2 ligands inhibited IL-12 and increased IL-23 production. DC priming with interferon (IFN) gamma strongly increased IL-12 production, but was not required for IL-23 production and inhibited IL-23 production induced by beta-glucan. The pattern of IL-12 and IL-23 induction was reflected in accumulation of the IL-12p35 and IL-23p19 transcripts, respectively, but not IL-12/23p40. Although IL-23, transforming growth factor beta, and IL-6 contained in the supernatants of activated mono-DCs played a role in the induction of IL-17 by human CD4(+) T cells, IL-1beta, in combination with one or more of those factors, was required for IL-17 production, and its production determined the differential ability of the stimuli used to elicit mono-DCs to produce soluble factors directing IL-17 production. Thus, the differential ability of pathogens to induce antigen-presenting cells to produce cytokines regulates the immune response to infection.

The enteropathogen Salmonella Typhimurium (S. Typhimurium) is the most commonly non-typhoideal serotype isolated in pig worldwide. Currently, one of the main sources of human infection is by consumption of pork meat. Therefore, prevention and control of salmonellosis in pigs is crucial for minimizing risks to public health. The aim of the present study was to use isobaric tags for relative and absolute quantification (iTRAQ) to explore differences in the response to Salmonella in two segment of the porcine gut (ileum and colon) along a time course of 1, 2, and 6 days post infection (dpi) with S. Typhimurium. A total of 298 proteins were identified in the infected ileum samples of which, 112 displayed significant expression differences due to Salmonella infection. In colon, 184 proteins were detected in the infected samples of which 46 resulted differentially expressed with respect to the controls. The higher number of changes in protein expression was quantified in ileum at 2 dpi. Further biological interpretation of proteomics data using bioinformatics tools demonstrated that the expression changes in colon were found in proteins involved in cell death and survival, tissue morphology or molecular transport at the early stages and tissue regeneration at 6 dpi. In ileum, however, changes in protein expression were mainly related to immunological and infection diseases, inflammatory response or connective tissue disorders at 1 and 2 dpi. iTRAQ has proved to be a proteomic robust approach allowing us to identify ileum as the earliest response focus upon S. Typhimurium in the porcine gut. In addition, new functions involved in the response to bacteria such as eIF2 signaling, free radical scavengers or antimicrobial peptides (AMP) expression have been identified. Finally, the impairment at of the enterohepatic circulation of bile acids and lipid metabolism by means the under regulation of FABP6 protein and FXR/RXR and LXR/RXR signaling pathway in ileum has been established for the first time in pigs. Taken together, our results provide a better understanding of the porcine response to Salmonella infection and the molecular mechanisms underlying Salmonella-host interactions., This research was supported by the National R&D Program of the Spanish Ministry of Science and Innovation (AGL2011-28904 and AGL2014-54089-R).

T helper 1 driven immune responses facilitate host defence during viral infections. Because interleukin-18 (IL-18) mediates T helper 1 driven immune responses, and since mature IL-18 is up-regulated in human macrophages after influenza virus infection in vitro, it has been suggested that IL-18 plays an important role in the immune response to influenza. To determine the role of IL-18 in respiratory tract infection with influenza, IL-18 gene-deficient (IL-18(-/-)) and normal wildtype mice were intranasally inoculated with influenza A virus. Influenza resulted in an increase in constitutively expressed IL-18 in the lungs of wildtype mice. The clearance of influenza A was inhibited by IL-18, as indicated by reduced viral loads on day 8 and day 12 after infection in IL-18(-/-) mice. This enhanced viral clearance correlated with increased CD4(+) T-cell activation in the lungs as reflected by CD69 expression on the cell surface. Surprisingly, interferon-gamma (IFN-gamma) levels were similar in the lungs of IL-18(-/-) mice and wildtype mice. Intracellular IFN-gamma staining revealed similar expression levels in lung-derived natural killer cells, CD4(+) and CD8(+) T cells, indicating that IFN-gamma production is IL-18-independent during influenza virus infection. Tumour necrosis factor-alpha production by CD4(+) T cells was significantly lower in IL-18(-/-) mice than in wildtype mice. Our data indicate that endogenous IL-18 impairs viral clearance during influenza A infection.

According to the hitherto accepted view, neutrophils kill ingested microorganisms by subjecting them to high concentrations of highly toxic reactive oxygen species (ROS) and bringing about myeloperoxidase-catalysed halogenation. We show here that this simple scheme, which for many years has served as a satisfactory working hypothesis, is inadequate. We find that mice made deficient in neutrophil-granule proteases but normal in respect of superoxide production and iodinating capacity, are unable to resist staphylococcal and candidal infections. We also show that activation provokes the influx of an enormous concentration of ROS into the endocytic vacuole. The resulting accumulation of anionic charge is compensated for by a surge of K+ ions that cross the membrane in a pH-dependent manner. The consequent rise in ionic strength engenders the release of cationic granule proteins, including elastase and cathepsin G, from the anionic sulphated proteoglycan matrix. We show that it is the proteases, thus activated, that are primarily responsible for the destruction of the bacteria.

Mucositis, also referred to as mucosal barrier injury, is one of the most debilitating side effects of radiotherapy and chemotherapy treatment. Clinically, mucositis is associated with pain, bacteremia, and malnutrition. Furthermore, mucositis is a frequent reason to postpone chemotherapy treatment, ultimately leading towards a higher mortality in cancer patients. According to the model introduced by Sonis, both inflammation and apoptosis of the mucosal barrier result in its discontinuity, thereby promoting bacterial translocation. According to this five-phase model, the intestinal microbiota plays no role in the pathophysiology of mucositis. However, research has implicated a prominent role for the commensal intestinal microbiota in the development of several inflammatory diseases like inflammatory bowel disease, pouchitis, and radiotherapy-induced diarrhea. Furthermore, chemotherapeutics have a detrimental effect on the intestinal microbial composition (strongly decreasing the numbers of anaerobic bacteria), coinciding in time with the development of chemotherapy-induced mucositis. We hypothesize that the commensal intestinal microbiota might play a pivotal role in chemotherapy-induced mucositis. In this review, we propose and discuss five pathways in the development of mucositis that are potentially influenced by the commensal intestinal microbiota: 1) the inflammatory process and oxidative stress, 2) intestinal permeability, 3) the composition of the mucus layer, 4) the resistance to harmful stimuli and epithelial repair mechanisms, and 5) the activation and release of immune effector molecules. Via these pathways, the commensal intestinal microbiota might influence all phases in the Sonis model of the pathogenesis of mucositis. Further research is needed to show the clinical relevance of restoring dysbiosis, thereby possibly decreasing the degree of intestinal mucositis.

Anti-myeloperoxidase (anti-MPO) antibodies have been implicated in the pathogenesis of small-vessel vasculitis, but the molecular mechanisms by which these antibodies contribute to disease are unknown. For determination of how anti-MPO antibodies affect inflammatory cell recruitment in small-vessel vasculitis, intravital microscopy was used to monitor leukocyte behavior in the accessible cremasteric microvessels under various experimental conditions. After local pretreatment of the cremaster muscle with cytokines (TNF-alpha, IL-1 beta, or keratinocyte-derived chemokine), administration of anti-MPO IgG to wild-type mice reduced leukocyte rolling in favor of augmented adhesion to and transmigration across the endothelium. This led to a decrease in the number of systemic circulating leukocytes and, similar to the early events in the development of vasculitic lesions, an increase in leukocyte recruitment to renal and pulmonary tissue. TNF-a led to the greatest recruitment of inflammatory cells, and IL-1 beta led to the least. When anti-CD18 was co-administered, anti-MPO IgG did not affect leukocyte rolling, adhesion, or transmigration; similarly, anti-MPO IgG did not produce these effects in Fc receptor gamma chain(-/-) mice. This study provides direct in vivo evidence of enhanced leukocyte-endothelial cell interactions in the presence of anti-MPO IgG and highlights the critical roles of Fc gamma receptors and beta(2) integrins in mediating these interactions. In addition, it suggests that neutrophils primed by cytokines in the presence of anti-MPO IgG can have systemic effects and target specific vascular beds.

To the Editor: Vitamin D insufficiency is an environmental factor that has been strongly associated with asthma and its severity.1 The genes IL33 and IL1RL1 have been repeatedly identified as predisposing to asthma risk in genome-wide association studies.2 IL-33 is an alarmin cytokine that acts on multiple pulmonary cell types, including TH2 lymphocytes, mast cells, and innate lymphoid cells, to promote TH2-type cytokine secretion and airway inflammatory responses of the kind observed in asthmatic patients.3,4 The receptor for IL-33 is encoded by IL1RL1; differential splicing of the gene can produce a functional membrane-bound receptor (ST2L) or a soluble decoy receptor (sST2).5 Therefore we investigated whether IL1RL1 is regulated by vitamin D in cells relevant to asthma. We did this first by measuring gene expression by means of quantitative real-time PCR with 2 different TaqMan probe sets: Hs01073300, which detects mRNA splice variants encoding both the membrane-bound and soluble receptors (the total mRNA for IL1RL1), and Hs00249389, which detects mRNA only for the splice variant encoding the membrane-bound receptor ST2L (Fig 1, A). Methods are provided in this article's Online Repository at www.jacionline.org. Fig 1 Vitamin D induces expression of sST2. A, Schematic for IL1RL1 mRNA: the 2 splice variants are differentially detected in PCR by using the probe sets Hs01073300 and Hs00249389. B-D,IL1RL1 transcripts detected by each probe set in cultures of HBECs at 24 ... Both probe sets display similar efficiency of amplification (data not shown). Human primary bronchial epithelial cells (HBECs), CD4 lymphocytes, CD8 lymphocytes, eosinophils, and LUVA mast cells were cultured in the presence or absence of 1α,25-dihydroxyvitamin D3 (1,25[OH]D3), the active form of vitamin D (Fig 1, B-D, and see Fig E1, A and B, in this article's Online Repository at www.jacionline.org). Addition of 1,25(OH)D3 significantly increased the total number of IL1RL1 mRNA transcripts expressed by HBECs and CD4 and CD8 lymphocytes, as measured by using the Hs01073300 probe set. However, 1,25(OH)D3 did not significantly increase expression of IL1RL1 mRNA transcripts by primary eosinophils and LUVA mast cells, despite it significantly increasing expression of the gene cathelicidin antimicrobial peptide (CAMP), which is known to be induced by vitamin D (see Fig E1, A and B). Higher IL1RL1 expression levels were detected with the Hs01073300 probe set than with the Hs00249389 probe set in HBEC and lymphocyte cultures. Although 1,25(OH)D3 increased the total copies of IL1RL1 mRNA measured by using the Hs01073300 probe set in these cell types, it did not increase the number of transcripts encoding ST2L detected with Hs00249389, indicating that vitamin D selectively upregulates the expression of mRNA for the soluble decoy receptor sST2. sST2 concentrations in culture supernatants were measured by means of ELISA to confirm the findings of gene expression studies at the protein level. sST2 concentrations were significantly increased by 1,25(OH)D3 in both HBEC and CD4 lymphocyte cultures (Fig 1, E and F). In contrast, CD8 lymphocytes produced less than 200 pg/mL sST2 in all conditions, and there was no evidence of upregulation by 1,25(OH)D3 (data not shown). Vitamin D circulates primarily as the inactive precursor 25-hydroxyvitamin D3 (25[OH]D3); however, epithelial cells constitutively express CYP27B1, the enzyme that converts 25(OH)D3 to 1,25(OH)D3.6 HBECs cultured with 100 nmol/L 25(OH)D3, a physiologic concentration, increased expression of IL1RL1 (see Fig E1, C) and produced significantly greater amounts of sST2 (Fig 1, G). Primary human nasal epithelial cells (HNECs) cultured with a similar concentration range of 25(OH)D3 also showed a clear concentration-dependent increase in sST2 production (Fig 1, H). CD4 lymphocytes did not respond to 25(OH)D3, which is consistent with their lower expression of CYP27B1 (see Fig E1, D). An IL-33 sensitive bioassay was developed using the LUVA mast cell line to test the biological activity of sST2. Twenty-four hours of exposure of LUVA cell cultures to IL-33 resulted in marked homotypic aggregation, which was associated with increased expression of CD54 (intercellular adhesion molecule 1; see Fig E2, A and B, in this article's Online Repository at www.jacionline.org). IL-33 increased CD54 expression on LUVA cells in a concentration-dependent manner (Fig 2, A). Other stimuli can also induce mast cell CD54 expression, and a similar concentration-dependent induction of CD54 expression was achieved with a combination of IFN-γ and IL-4 (Fig 2, A). Treatment of LUVA cells with a recombinant sST2-Fc chimera significantly inhibited IL-33–induced CD54 expression (P = .0076; Fig 2, B, and see Fig E2, C) but not IFN-γ plus IL-4–induced CD54 expression (see Fig E2, D). Addition of 1,25(OH)D3 itself to LUVA cell cultures did not significantly alter constitutive or induced CD54 expression (P > .05; see Fig E2, E), as previously reported for another mast cell line.7 Fig 2 Conditioned medium from vitamin D–treated epithelial cultures inhibits IL-33–induced expression of CD54 by LUVA cells. A, Induction of CD54 on LUVA cells above that of unstimulated cells by different stimuli in the presence of conditioned ... To examine the effect of vitamin D–enhanced epithelial sST2 production on the IL-33 bioassay, HNECs were cultured in the presence or absence of 25(OH)D3 for 48 hours, and then the supernatants were added to cultures of LUVA cells stimulated with concentrations of IL-33 or IFN-γ plus IL-4 that induced comparable CD54 expression. Conditioned medium from vitamin D–treated epithelial cells significantly reduced IL-33–induced CD54 expression on LUVA cells compared with conditioned medium from matched epithelial cell cultures not treated with vitamin D (P = .015; Fig 2, C and E). In contrast, there was no significant difference in IFN-γ plus IL-4–induced CD54 expression between LUVA cells cultured with the different epithelial cell–conditioned media (P > .05; Fig 2, D and E). Although mediators in the epithelial cell–conditioned medium other than sST2 might conceivably have affected LUVA cell CD54 expression, the selective effect of vitamin D treatment on IL-33–induced CD54 expression suggests that the sST2 in the vitamin D–treated epithelial cell supernatants is biologically active. The capacity of IL-33 to induce production of TH2-type proinflammatory cytokines by multiple cell types likely underpins the reported genetic associations of IL33 and IL1RL1 with asthma. Here we report the novel finding that 100 nmol/L vitamin D is able to augment expression by epithelial cells and lymphocytes of the soluble decoy receptor sST2, which in turn inhibits the actions of IL-33. Importantly, this effect occurs at physiologic concentrations (vitamin D sufficiency is defined as a serum 25[OH]D level of 75 to 150 nmol/L), and similar concentrations of 1,25(OH)D3 have been shown to be able to be generated from 25(OH)D3 in culture.1 We hypothesize that the capacity of vitamin D to augment the synthesis of an inhibitor of IL-33 in situ in the airways mucosa is of potential benefit in the limitation of asthmatic mucosal inflammation. Furthermore, this might in part account for the paradox that epidemiologic studies have repeatedly revealed associations between vitamin D insufficiency and both the risk of more severe asthma1 and increased serum IgE concentrations,8 whereas other studies have reported that in culture vitamin D can act directly on TH2 lymphocytes to promote TH2 cytokine secretion (eg, Boonstra et al9). The apparent beneficial association of vitamin D in asthma in vivo, which in many patients is a TH2-type cytokine pathology, suggests that the direct action of vitamin D in promoting TH2 lymphocyte responses is less important in vivo than other vitamin D–mediated mechanisms (eg, vitamin D upregulated production of sST2 and the induction of regulatory mechanisms) in inhibiting TH2-type cytokine responses. Because the enhancement by vitamin D of sST2 production is concentration dependent, this supports therapeutic strategies to boost pulmonary vitamin D levels to reduce asthmatic inflammation.