Antimicrobial Immunity Research Articles

212: Innate neutrophil function is regulated locally through IL-6 control of prostaglandins

Interleukin-6 (IL-6) is vital for competent anti-microbial host defense. Mice deficient in IL-6 showed an inability to appropriately control bacterial peritonitis (5 × 108 cfu Staphylococcus epidermidis) and displayed both increased dissemination and reduced bacterial clearance. This response was reversed through local reconstitution of IL-6 trans-signaling using a chimeric sIL-6R-IL-6 fusion protein (termed HDS). Administration of HDS (10–1000 ng/ml) to infected IL-6−/− mice promoted bacterial clearance and reduced bacteremia in a dose-dependent fashion. This response was associated with improved resolution of the acute phase response and a more rapid normalization of body temperature. To define the mechanisms governing IL-6 involvement in anti-microbial immunity, we first developed an ex vivo flow cytometric assay to simultaneously monitor neutrophil respiratory burst and phagocytosis. Bacterial stimulation of circulating blood neutrophils from IL-6−/− and IL-6R−/− mice showed comparable effector function to that displayed by WT controls. Thus, IL-6 deficiency is not associated with an inherent defect in neutrophil function. However, evaluation of infiltrating neutrophils isolated by lavage from the peritoneal cavity of infected IL-6−/− and IL-6R−/− mice showed significantly reduced respiratory burst and phagocytic activity. Interleukin-6 therefore controls responses local to the site of infection and is consistent with the activation of non-hematopoietic stromal cells by IL-6 and sIL-6R. In human peritoneal mesothelial cells and peritoneal membranes from infected mice, IL-6 trans-signaling promoted cyclooxygenase-2 (COX2) expression. Blockade of IL-6 signaling in infected WT mice with the antagonist sgp130 or anti-IL-6R antibodies inhibited peritoneal COX2 expression. Mass spectrometry for the COX2 products, prostaglandin-E2 (PGE2) prostaglandin-D2 (PGD2) in lavage samples from infected mice showed that the generation of PGD2, but not PGE2, was impaired in IL-6 deficiency. Here, PGD2 levels correlated with the number of infiltrating neutrophils undergoing respiratory burst. These studies suggest IL-6 signaling regulates the effector characteristics of infiltrating neutrophils through local control of immuno-regulatory prostaglandin production.

Genome-wide association study of lung function phenotypes in a founder population

Lung function is a long-term predictor of mortality and morbidity. We sought to identify single nucleotide polymorphisms (SNPs) associated with lung function. We performed a genome-wide association study (GWAS) of FEV1, forced vital capacity (FVC), and FEV1/FVC in 1144 Hutterites aged 6 to 89 years, who are members of a founder population of European descent. We performed least absolute shrinkage and selection operation regression to select the minimum set of SNPs that best predict FEV1/FVC in the Hutterites and used the GRAIL algorithm to mine the Gene Ontology database for evidence of functional connections between genes near the predictive SNPs. Our GWAS identified significant associations between FEV1/FVC and SNPs at the THSD4-UACA-TLE3 locus on chromosome 15q23 (P = 5.7 × 10(-8) to 3.4 × 10(-9)). Nine SNPs at or near 4 additional loci had P < 10(-5) with FEV1/FVC. Only 2 SNPs were found with P < 10(-5) for FEV1 or FVC. We found nominal levels of significance with SNPs at 9 of the 27 previously reported loci associated with lung function measures. Among a predictive set of 80 SNPs, 6 loci were identified that had a significant degree of functional connectivity (GRAIL P < .05), including 3 clusters of β-defensin genes, 2 chemokine genes (CCL18 and CXCL12), and TNFRSF13B. This study identifies genome-wide significant associations and replicates results of previous GWASs. Multimarker modeling implicated for the first time common variation in genes involved in antimicrobial immunity in airway mucosa that influences lung function.

Clostridium difficile Modulates Host Innate Immunity via Toxin-Independent and Dependent Mechanism(s)

Clostridium difficile infection (CDI) is the leading cause of hospital and community-acquired antibiotic-associated diarrhoea and currently represents a significant health burden. Although the role and contribution of C. difficile toxins to disease pathogenesis is being increasingly understood, at present other facets of C. difficile-host interactions, in particular, bacterial-driven effects on host immunity remain less studied. Using an ex-vivo model of infection, we report that the human gastrointestinal mucosa elicits a rapid and significant cytokine response to C. difficile. Marked increase in IFN-γ with modest increase in IL-22 and IL-17A was noted. Significant increase in IL-8 suggested potential for neutrophil influx while presence of IL-12, IL-23, IL-1β and IL-6 was indicative of a cytokine milieu that may modulate subsequent T cell immunity. Majority of C. difficile-driven effects on murine bone-marrow-derived dendritic cell (BMDC) activation were toxin-independent; the toxins were however responsible for BMDC inflammasome activation. In contrast, human monocyte-derived DCs (mDCs) released IL-1β even in the absence of toxins suggesting host-specific mediation. Infected DC-T cell crosstalk revealed the ability of R20291 and 630 WT strains to elicit a differential DC IL-12 family cytokine milieu which culminated in significantly greater Th1 immunity in response to R20291. Interestingly, both strains induced a similar Th17 response. Elicitation of mucosal IFN-γ/IL-17A and Th1/Th17 immunity to C. difficile indicates a central role for this dual cytokine axis in establishing antimicrobial immunity to CDI.

Signalling C-type lectins in antimicrobial immunity.

Since it was first proposed that the innate immune system could recognise conserved microbial-associated molecular patterns (or PAMPs) through inherited receptors expressed by the host (termed pattern recognition receptors, or PRRs), several families of PRRs have been discovered and characterised. The most famous of these are the Toll-like receptors (TLRs), but there is growing appreciation that another large family of PRRs, known as the C-type lectin receptors (CLRs), also play a major role in antimicrobial immunity. CLRs have one or more carbohydrate recognition domains (CRDs) that recognise a wide variety of carbohydrate ligands. Other members of the CLR family, which do not recognise carbohydrate ligands but contain similar protein folds called C-type lectin-like domains (CTLD), have also been discovered and are included in this large family whose members are divided into 17 groups relating to phylogeny and structure. Upon ligand binding, some CLRs (such as Dectin-1, Dectin-2, and Mincle) undergo intracellular signalling to drive cellular responses. Here, we outline the signalling pathways downstream of these receptors and discuss how they, and some other CLRs (including the Mannose Receptor, CLEC5A, CLEC9A, and DC-SIGN), contribute to immunity against fungi, bacteria, viruses, and parasites.

Hepatoprotective and anti‐fibrotic functions of interleukin‐22: Therapeutic potential for the treatment of alcoholic liver disease

Interleukin-22 (IL-22) plays a key role in promoting antimicrobial immunity and tissue repair at barrier surfaces by binding to the receptors IL-22R1, which is generally thought to be expressed exclusively in epithelial cells, and IL-10R2. Our laboratory previously demonstrated that IL-22 plays an important role in ameliorating liver injury in many rodent models by targeting hepatocytes that express high levels of IL-22R1 and IL-10R2. Recently, we have identified high expression levels of IL-22R1 and IL-10R2 in liver progenitor cells and hepatic stellate cells (HSCs). Overexpression of IL-22 in vivo or treatment with IL-22 in vitro promotes proliferation of liver progenitor cells via a signal transducer and activator of transcription 3 (STAT3)-dependent mechanism. IL-22 treatment also prevents HSC apoptosis in vitro and in vivo. Surprisingly, overexpression of IL-22, via either gene targeting or exogenous administration of adenovirus expressing IL-22, reduces liver fibrosis and accelerates the resolution of liver fibrosis during recovery. The anti-fibrotic effects of IL-22 are mediated via the activation of STAT3 in HSCs and subsequent induction of suppressor of cytokine signaling 3, which induces HSC senescence. Taken together, the hepatoprotective, mitogenic, and anti-fibrotic effects of IL-22 are beneficial in ameliorating alcoholic liver injury. Importantly, due to the restricted expression of IL-22R1, IL-22 therapy is expected to have few side effects, thus making IL-22 a potential candidate for treatment of alcoholic liver disease.

Innate and acquired bacteriophage-mediated immunity

We recently described a novel, non-host-derived, phage-mediated immunity active at mucosal surfaces, the main site of pathogen entry in metazoans. In that work, we showed that phage T4 adheres to mucus glycoproteins via immunoglobulin-like domains displayed on its capsid. This adherence positions the phage in mucus surfaces where they are more likely to encounter and kill bacteria, thereby benefiting both the phage and its metazoan host. We presented this phage-metazoan symbiosis based on an exclusively lytic model of phage infection. Here we extend our bacteriophage adherence to mucus (BAM) model to consider the undoubtedly more complex dynamics in vivo. We hypothesize how mucus-adherent phages, both lytic and temperate, might impact the commensal microbiota as well as protect the metazoan epithelium from bacterial invasion. We suggest that BAM may provide both an innate and an acquired antimicrobial immunity.

Structural Insights into the Functions of TBK1 in Innate Antimicrobial Immunity

Tank-binding kinase 1 (TBK1) is a serine/threonine protein-kinase mediating innate antimicrobial immunity. TBK1 is involved in the signaling of TLRs, RLRs, and STING-mediated sensing of cytosolic DNA. Stimulation of these receptors results in the activation of TBK1, which phosphorylates interferon regulatory factor (IRF)-3. Phosphorylated IRF-3 translocates into the nucleus to initiate the transcription of the interferon (IFN)-β gene. Here, we show that TBK1 is activated by autophosphorylation at residue Ser172. Structures of TBK1 bound to two inhibitors showed that TBK1 has the IκB kinase fold with three distinct domains: the kinase domain, the ubiquitin-like domain, and the scaffold and dimerization domain. However, the overall structures of the TBK1 monomer and its dimer are different from IKKβ in the arrangements of the three domains and in dimer formation. Phosphorylation of IRF-3 by TBK1 invitro results in its oligomerization, and phosphorylation of residue Ser386 plays a key role in IRF-3 activation.

SP0111 What are innate lympoid cells and what do they do

Innate lymphoid cells (ILCs) are immune cells that lack a specific antigen receptor yet can produce an array of effector cytokines that in variety match those of T helper cell...

Inflammatory DCs fail to present antigen but provide a crucial source of IL-12 in priming Th1 responses to CpG based vaccines. (P4514)

Abstract DCs govern the induction of antigen specific adaptive immunity and thus constitute the main targets of novel vaccine approaches. Classical steady state DCs consist of multiple subsets, differing in their capacity to respond to TLR stimulation and to present antigen to CD4 and CD8 T cells. Under inflammatory conditions, an additional subset of monocyte derived inflammatory DCs (iDCs) arises. Although the innate role of iDCs in antimicrobial immunity is now clearly established, their role in directing the adaptive response is less evident. Immunization with OVA combined with the TLR9 agonist CpG resulted in the fast recruitment of CCR2 CD64 Ly6Chi CD11bhi monocytes to the draining lymph nodes, where they gradually adopt a CD11cint MHCIIint phenotype. Although a large fraction of these iDCs were OVA+, sorted iDCs failed to present the antigen to OT-I and OT-II T cells. Instead, antigen presentation was limited to migratory DCs. iDCs however proved to be the major source the Th1 polarising cytokine IL12. To investigate the role of iDC-derived IL12 in priming Th1 responses, mixed CCR2-/- /IL12-/- bone marrow chimaeras were immunized with OVA/CpG. In these chimaeras, solely IL12-/- iDCs can reach the lymph node, while classical lymph node DCs are fully competent of producing IL12. When compared to control WT/IL12-/- chimaeras, CCR2 -/-/IL12-/- mice displayed dramatically reduced Th1 responses, establishing the crucial role of iDCs in priming Th1 responses.

Regulation of neutrophil-mediated anti-microbial immunity by type 2 cytokines (P4253)

Abstract Neutrophils are innate immune effectors primarily acting as a first line of defense against bacterial infections. Interestingly, acute type 2 inflammation, as seen in allergic disorders or during parasitic infections, displays a striking reduction in neutrophils. Moreover, allergic patients show an increased susceptibility towards certain bacteria usually contained by neutrophils. Here, we show that both elevated and physiologic levels of distinct type 2 cytokines dominantly inhibit several functional aspects of murine and human neutrophils, thus impairing neutrophil-mediated bacterial immunity. In particular, type 2 cytokines were found to inhibit egress of neutrophils from bone marrow as well as their anti-bacterial cytotoxicity and migration. Notably, mice lacking type 2 cytokine receptor signaling or receiving blocking type 2 cytokine antibodies survived a lethal infection with different bacteria. Based on the afore-mentioned results, we postulate a prototypic role of certain type 2 cytokines in regulating neutrophil-mediated anti-bacterial immunity.

Engineering DNA nanoparticles as immunomodulatory reagents (P1076)

Abstract Cytoplasmic DNA sensing triggers anti-microbial immunity by inducing interferon type I (IFNαβ) via STING-TBK1 activation but sustained STING activation incites autoimmunity. Systemic DNA nanoparticle (DNP) treatment triggered rapid and potent tolerogenic responses in mice via selective activation of the STING-IFNαβ pathway in myeloid dendritic cells (DCs) to induce indoleamine 2, 3 dioxygenase (IDO) activity. DNPs associated rapidly with splenic marginal zone (MZ) macrophages (MΦs) and some DCs, and IFNαβ and IL-1β were induced soon after DNP injection. IFNβ1 was primarily induced in myeloid (CD11b+) DCs and this response was STING dependent, while IL-1β was induced primarily in granulocytes and was STING independent. IFNαβ-mediated IDO induction caused DCs and regulatory T cells (Tregs) to acquire potent tolerogenic phenotypes. Physiologic depletion of (CD11c+) DCs and STING ablation abolished IDO induction and tolerogenic responses to DNPs, but MZ (CD169+) MΦ depletion did not. Moreover, systemic treatment with the cyclic dinucleotide (c-diGMP) to activate STING directly induced IDO and suppressed antigen-specific Th1 responses. Thus myeloid DCs sense circulating cargo DNA and c-diGMP to induce selective IFNαβ release that promotes dominant tolerogenic responses via IDO. Targeting this distinctive tolerogenic pathway may be an effective strategy to suppress hyper-immune syndromes, including autoimmune diseases incited by excessive IFNαβ release.

Modulation of mucosal immune responses to Clostridium difficile by probiotic bacteria (P3073)

Abstract Commensal bacteria play a critical role in maintaining intestinal epithelium and mucosal immune homeostasis. Alterations in intestinal microflora predispose mammalian hosts to infection with pathogens such as Clostridium difficile, which has recently re-emerged as a pathogen causing nosocomial diarrhea, colitis and death. We investigated the mechanisms by which the probiotic VSL#3 ameliorates C. difficile-associated disease (CDAD) in mice. Oral VSL#3 administration reduced disease activity, body weight loss, and colonic inflammatory lesions after infection. Probiotic treatment also decreased Th17 and IL-17-producing TCRγδ cell populations at the colonic lamina propria as well as production of IL-17 and IL-1β in the colonic mucosa. Increased numbers of regulatory T cells (Treg) were seen in spleens of VSL#3-treated mice, which correlated to increased peroxisome proliferator-activated receptor γ (PPARγ) activity. PPARγ is a transcription factor and nuclear receptor that modulates immune responses and whose activation is essential for the maintenance of innate antimicrobial immunity in the colon. Mice treated with VSL#3 had an upregulation of calprotectin antimicrobial peptides S100A8 and S100A9 in the colon correlating to decreased fecal clostrial burden at day 4 post-infection. VSL#3 probiotic bacteria regulate colitis by increasing PPARγ activity and decreasing IL-17 responses and increase clostridial clearance by upregulating antimicrobial peptides in mice with CDAD.

IL-1β Promotes Antimicrobial Immunity in Macrophages by Regulating TNFR Signaling and Caspase-3 Activation

In vivo control of Mycobacterium tuberculosis reflects the balance between host immunity and bacterial evasion strategies. Effector Th1 cells that mediate protective immunity by depriving the bacterium of its intracellular niche are regulated to prevent overexuberant inflammation. One key immunoregulatory molecule is Tim3. Although Tim3 is generally recognized to downregulate Th1 responses, we recently described that its interaction with Galectin-9 expressed by M. tuberculosis-infected macrophages stimulates IL-1β secretion, which is essential for survival in the mouse model. Why IL-1β is required for host resistance to M. tuberculosis infection is unknown. In this article, we show that IL-1β directly kills M. tuberculosis in murine and human macrophages and does so through the recruitment of other antimicrobial effector molecules. IL-1β directly augments TNF signaling in macrophages through the upregulation of TNF secretion and TNFR1 cell surface expression, and results in activation of caspase-3. Thus, IL-1β and downstream TNF production lead to caspase-dependent restriction of intracellular M. tuberculosis growth.

The inflammasome as a target of modulation by DNA viruses.

The cellular innate immune response represents the initial reaction of a host against infecting pathogens. Host cells detect incoming microbes by way of a large and expanding array of receptors that react with evolutionarily conserved molecular patterns exhibited by microbial intruders. These receptors are responsible for initiating signaling that leads to both transcriptional activation of immunologically important genes as well as protease-dependent processing of cellular proteins. The inflammasome refers to a protein complex that functions as an activation platform for the cysteine protease caspase-1, which then processes inflammatory molecules such as IL-1β and IL-18 into functional forms. Assembly of this complex is triggered following receptor-mediated detection of pathogen-associated molecules. Receptors have been identified that are essential to inflammasome activation in response to numerous molecular patterns including virus-associated molecules such as DNA. In fact, the importance of cytoplasmic DNA as an immune stimulus is exemplified by the existence of at least nine distinct cellular receptors capable of initiating innate reactivity in response to this molecule. Viruses that employ DNA as genomic material include herpesviruses, poxviruses and adenoviruses. Each has been described as capable of inducing inflammasome-mediated activity. Interestingly, however, the cellular molecules responsible for these responses appear to vary according to host species, cell type and even viral strain. Secretion of IL-1β and IL-18 are important components of antimicrobial immunity and, as a result, pathogens have evolved factors to evade or counteract this response. This includes DNA-based viruses, many of which encode multiple redundant counteractive molecules. However, it is clear that such phenotypes are only beginning to be uncovered. The purpose of this review is to describe what is known regarding the activation of inflammasome-mediated processes in response to infection with well-examined families of DNA viruses and to discuss characterized mechanisms of manipulation and neutralization of inflammasome-dependent activity. This review aims to shed light on the biologically important phenomena regarding this virus-host interaction and to highlight key areas where important information is lacking.

White button, portabella, and shiitake mushroom supplementation up-regulates interleukin-23 secretion in acute dextran sodium sulfate colitis C57BL/6 mice and murine macrophage J.744.1 cell line

Interleukin-23 (IL-23), a cytokine produced primarily by dendritic cells, is involved in host defense against gut pathogens and promotes innate immunity and inflammatory responses through the IL-23/interleukin-17 axis. We previously reported that extracts from edible mushrooms enhanced antimicrobial α-defensin production n HL60 cells. Because IL-23 is involved in defensin production, we hypothesized that edible mushrooms may modulate its secretion and gut inflammation. Eight-week-old C57BL/6 mice were fed the AIN76 diet or the same diet supplemented with 5% white button (WBM), portabella, or shiitake mushrooms. To assess in vivo and in vitro cytokine secretion, 7 to 8 mice per group received 3% dextran sodium sulfate (DSS) in drinking water during the last 5 days of the 6-week feeding period. To delineate the mechanisms by which mushrooms alter IL-23 secretion, J.744.1 cells were incubated with (100 μg/mL) WBM, portabella, and shiitake extracts without and with 100 μg/mL curdlan (a dectin-1 agonist) or 1 mg/mL laminarin (a dectin-1 antagonist). The dectin-1 receptor is a pattern-recognition receptor found in phagocytes, and its activation promotes antimicrobial innate immunity and inflammatory responses. In DSS-untreated mice, mushrooms significantly increased IL-23 plasma levels but decreased those of interleukin-6 (IL-6) (P < .05). In DSS-treated mice, mushroom-supplemented diets increased IL-6 and IL-23 levels (P < .05). Mushroom extracts potentiated curdlan-induced IL-23 secretion, and mushroom-induced IL-23 secretion was not blocked by laminarin in vitro, suggesting the involvement of both dectin-1–dependent and dectin-1–independent pathways. Although all mushrooms tended to increase IL-6 in the colon, only WBM and shiitake tended to increase IL-23 levels. These data suggest that edible mushrooms may enhance gut immunity through IL-23.

Thalidomide attenuates excessive inflammation without interrupting lipopolysaccharide-driven inflammatory cytokine production in chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare inherited disorder characterized by an inability to produce reactive oxygen species, resulting in recurrent life-threatening infections. Curiously, half of the patients with CGD suffer from aseptic bowel inflammation (CGD colitis) due to dysregulated inflammation induced by TNF-α and IL-1β. Thus, developing therapies that regulate excessive inflammatory responses without interrupting antimicrobial immunity would benefit CGD colitis patients. Here, we show that thalidomide suppressed TNF-α-induced NF-κB activation and ATP-induced IL-1β secretion, but did not interrupt the production of IL-1β, IL-6, IL-8, and TNF-α in response to lipopolysaccharide in CGD monocytes. We report on a CGD colitis patient that showed decreased bowel inflammation characterized by reduced serum levels of inflammatory cytokines without evidence of progression of fungal and bacterial infections present at initiation of thalidomide therapy. Our results suggest that thalidomide could be an efficacious therapeutic option for patients with CGD colitis suffering from serious infections.

MDA5

Myocarditis is an inflammation of the heart muscle, which frequently follows microbial infections. Picornaviruses and adenoviruses are the most commonly identified viral etiologic agents.1 Cardiac injury results from both virus infection/replication and host inflammatory responses, but in either case, elimination of the virus from the heart can have a beneficial effect on disease outcome as is evident from clinical trials treating biopsy-proven, virus-positive patients with interferon β (IFNβ).2 The success of type I IFN therapy in myocarditis may partly depend on the initiating etiologic agent, as virus clearance and cardiac improvement was greater in patients with enteroviruses than with parvovirus B19 or human herpesvirus 6.2 This finding is not surprising as it is well known that virus families vary dramatically in their sensitivity to type I IFN, in part because many viruses have developed immune evasion mechanisms targeting this innate host defense mechanism.3 Enteroviruses of the Picornavirus family are small, nonenveloped, positive-sensed, single-stranded RNA viruses, which are classically among the most sensitive to type I IFN, with encephalomyocarditis virus (EMCV) being one of the most IFN sensitive of the enteroviruses.4 Article see p 326 Type I IFN are induced through virus-sensing pattern recognition receptors (PRR) of the innate immunity system, which recognize shared pathogen-associated molecular patterns between many infectious agents.5 PRRs include Toll-like, retinoic acid-inducible gene I (RIG-I)-like, nucleotide-binding oligomerization domain receptors (NOD)-like, C-type lectin and possibly absent in melanoma 2 (AIM2)-like receptors; a diverse subcellular compartmentalization of the pattern recognition receptors allows optimal recognition of their relevant pathogen-associated molecular patterns. Toll-like receptors (TLR) are primarily implicated in reacting to extracellular bacterial pathogen-derived molecules (TLR5 [flagellin], TLR4 [lipopolysaccharide], TLR2/1, or TLR2/6 [lipoprotein]) and are located in the plasma membrane; in contrast, RIG-I-like (single-stranded/double-stranded RNA) and NOD-like (muramyl tripeptide/muramyl dipeptide) receptors are located in the …

Molecular Basis of DNA Recognition in the Immune System

Recognition of microbial nucleic acids is one strategy by which mammalian hosts respond to infectious agents. Intracellular DNA that is introduced into cells during infection elicits potent inflammatory responses by triggering the induction of antiviral type I IFNs and the maturation and secretion of inflammatory cytokines, such as TNF-α, IL-1β, and IL-18. In addition, if nucleases, such as DNase II or DNase III (Trex1), fail to clear self-DNA, accumulated DNA gains access to intracellular compartments where it drives inflammatory responses leading to autoimmune disease. In this review, we discuss a rapidly evolving view of how cytosolic DNA-sensing machineries coordinate antimicrobial immunity and, if unchecked, lead to autoimmune disease.

Bacterial lipopolysaccharides stimulate production of XCL1, a calcium-dependent lipopolysaccharide-binding serum lectin, in Xenopus laevis

Xenopus laevis serum lectin XCL1 is a newly identified molecule of the XCGL (or X-lectin) family, a unique group of Ca2+-dependent lectins that have a fibrinogen-like domain. The XCL1 protein was purified from lipopolysaccharide (LPS)-stimulated frog sera by sequential affinity chromatography on heparin-acrylic beads and galactose–Sepharose. XCL1 comprises multiple oligomeric proteins consisting of 37-kDa subunit polypeptides, as revealed by sodium dodecyl sulfate–polyacrylamide electrophoresis (SDS–PAGE) and Western blot analyses using the monoclonal antibody (mAb) produced against the recombinant XCL1 polypeptide. In the presence of Ca2+, the protein bound to Escherichia coli, Staphylococcus aureus, LPS and galactose and the bound XCL1 was competitively eluted using ribose and xylose, and the elution was as efficient as that using EDTA, whereas elution using hexoses, GalNAc or GlcNAc was less effective. In reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses, XCL1 expression was ubiquitously detected in frog tissues, with relatively high levels in hematopoietic tissues including the spleen, liver and kidney. Intraperitoneal injection of E. coli, S. aureus or 100–300μg S-type LPS from various bacteria induced several-fold increases in serum XCL1 concentrations on day 3, and the elevated levels retained up to day 12. It also caused a remarkable increase of the splenic XCL1 expression on day 3, followed by a rapid decline to nearly nonstimulated control levels by day 7. The R-type LPS with shortened polysaccharide chains was less effective in inducing the serum XCL1 response, indicating that the sugar chains of LPS were important, if not essential, for the stimulation of XCL1 production. These results suggest that XCL1 is a pathogen recognition molecule involved in antimicrobial innate immunity in Xenopus.

Cigarette Smoke Decreases Airway Epithelial FABP5 Expression and Promotes Pseudomonas aeruginosa Infection

Cigarette smoking is the primary cause of Chronic Obstructive Pulmonary Disease (COPD), which is characterized by chronic inflammation of the airways and destruction of lung parenchyma. Repeated and sustained bacterial infections are clearly linked to disease pathogenesis (e.g., exacerbations) and a huge burden on health care costs. The airway epithelium constitutes the first line of host defense against infection and our previous study indicated that Fatty Acid Binding Protein 5 (FABP5) is down regulated in airway epithelial cells of smokers with COPD as compared to smokers without COPD. We hypothesized that cigarette smoke (CS) exposure down regulates FABP5, thus, contributing to a more sustained inflammation in response to bacterial infection. In this report, we show that FABP5 is increased following bacterial infection but decreased following CS exposure of primary normal human bronchial epithelial (NHBE) cells. The goal of this study was to address FABP5 function by knocking down or overexpressing FABP5 in primary NHBE cells exposed to CS. Our data indicate that FABP5 down regulation results in increased P. aeruginosa bacterial load and inflammatory cytokine levels (e.g., IL-8) and decreased expression of the anti-bacterial peptide, β defensin-2. On the contrary, FABP5 overexpression exerts a protective function in airway epithelial cells against P. aeruginosa infection by limiting the production of IL-8 and increasing the expression of β defensin-2. Our study indicates that FABP5 exerts immunomodulatory functions in the airway epithelium against CS exposure and subsequent bacterial infection through its modulation of the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-γ activity. These findings support the development of FABP5/PPAR-γ-targeted therapeutic approach to prevent airway inflammation by restoring antimicrobial immunity during COPD exacerbations.