Lung Mucosal Immunity Research Articles

Lung mucosal immunity to NTHi vaccine antigens: Antibodies in sputum of chronic obstructive pulmonary disease patients

ABSTRACT Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory illness in older adults. A major cause of COPD-related morbidity and mortality is acute exacerbation of COPD (AECOPD). Bacteria in the lungs play a role in exacerbation development, and the most common pathogen is non-typeable Haemophilus influenzae (NTHi). A vaccine to prevent AECOPD containing NTHi surface antigens was tested in a clinical trial. This study measured IgG and IgA against NTHi vaccine antigens in sputum. Sputum samples from 40 COPD patients vaccinated with the NTHi vaccine were collected at baseline and 30 days after the second dose. IgG and IgA antibodies against the target antigens and albumin were analyzed in the sputum. We compared antibody signals before and after vaccination, analyzed correlation with disease severity and between sputum and serum samples, and assessed transudation. Antigen-specific IgG were absent before vaccination and present with high titers after vaccination. Antigen-specific IgA before and after vaccination were low but significantly different for two antigens. IgG correlated between sputum and serum, and between sputum and disease severity. Sputum albumin was higher in patients with severe COPD than in those with moderate COPD, suggesting changes in transudation played a role. We demonstrated that immunization with the NTHi vaccine induces antigen-specific antibodies in sputum. The correlation between IgG from sputum and serum and the presence of albumin in the sputum of severe COPD patients suggested transudation of antibodies from the serum to the lungs, although local IgG production could not be excluded. Clinical Trial Registration: NCT02075541

Respiratory mucosal immune memory to SARS-CoV-2 after infection and vaccination

Respiratory mucosal immunity induced by vaccination is vital for protection from coronavirus infection in animal models. In humans, the capacity of peripheral vaccination to generate sustained immunity in the lung mucosa, and how this is influenced by prior SARS-CoV-2 infection, is unknown. Here we show using bronchoalveolar lavage samples that donors with history of both infection and vaccination have more airway mucosal SARS-CoV-2 antibodies and memory B cells than those only vaccinated. Infection also induces populations of airway spike-specific memory CD4+ and CD8+ T cells that are not expanded by vaccination alone. Airway mucosal T cells induced by infection have a distinct hierarchy of antigen specificity compared to the periphery. Spike-specific T cells persist in the lung mucosa for 7 months after the last immunising event. Thus, peripheral vaccination alone does not appear to induce durable lung mucosal immunity against SARS-CoV-2, supporting an argument for the need for vaccines targeting the airways.

Frontline workers: Mediators of mucosal immunity in community acquired pneumonia and COVID-19.

The current COVID-19 pandemic has highlighted a need to further understand lung mucosal immunity to reduce the burden of community acquired pneumonia, including that caused by the SARS-CoV-2 virus. Local mucosal immunity provides the first line of defence against respiratory pathogens, however very little is known about the mechanisms involved, with a majority of literature on respiratory infections based on the examination of peripheral blood. The mortality for severe community acquired pneumonia has been rising annually, even prior to the current pandemic, highlighting a significant need to increase knowledge, understanding and research in this field. In this review we profile key mediators of lung mucosal immunity, the dysfunction that occurs in the diseased lung microenvironment including the imbalance of inflammatory mediators and dysbiosis of the local microbiome. A greater understanding of lung tissue-based immunity may lead to improved diagnostic and prognostic procedures and novel treatment strategies aimed at reducing the disease burden of community acquired pneumonia, avoiding the systemic manifestations of infection and excess morbidity and mortality.

The Role of the Gut-Lung Axis in COVID-19 Infections and Its Modulation to Improve Clinical Outcomes.

The main entry point of SARS-CoV-2 is the respiratory tract and as such immune defence in this site determines if the virus will spill-over to the systemic circulation and circulate and infect other major organs. The first line of mucosal immune defence is composed of mucins, an epithelial barrier, and immune cells in the nasal cavity. The lung immune defence is carried out by numerous alveoli. The lung microbiota is a key factor in determining the efficacy of lung mucosal immunity protection. The intestinal microbiota has been demonstrated to affect the severity of COVID-19. Gut dysbiosis is involved in hyperinflammation and multiple organ failure through communications with multiple organs. The gut lung axis could be the earliest axis affected in COVID-19. Through the gut-lung axis, gut dysbiosis can affect the pathogenesis of the lung in COVID-19. In this review, we summarise the effects that gut dysbiosis can progress on the lung, and the lung microbiota. The possible mechanisms and approaches for modulation are discussed.

Cyclic-di-AMP Phosphodiesterase Elicits Protective Immune Responses Against Mycobacterium tuberculosis H37Ra Infection in Mice.

Many antigens from Mycobacterium tuberculosis (M. tuberculosis) have been demonstrated as strong immunogens and proved to have application potential as vaccine candidate antigens. Cyclic di-AMP (c-di-AMP) as a bacterial second messenger regulates various bacterial processes as well as the host immune responses. Rv2837c, the c-di-AMP phosphodiesterase (CnpB), was found to be relative to virulence of M. tuberculosis and interference with host innate immune response. In this study, recombinant CnpB was administered subcutaneously to mice. We found that CnpB had strong immunogenicity and induced high levels of humoral response and lung mucosal immunity after M. tuberculosis intranasally infection. CnpB immunization stimulated splenocyte proliferation and the increasing number of activated NK cells but had little effects on Th1/Th2 cellular immune responses in spleens. However, CnpB induced significant Th1/Th2 cellular immune responses with a decreased number of T and B cells in the lungs, and significantly recruits of CD4+ and CD8+ T cells after M. tuberculosis attenuated strain H37Ra infection. Besides, we first reported that CnpB could stimulate IFN-β expression transitorily and inhibit the autophagy of macrophages in vitro. In mice intranasally infection model, CnpB immunization alleviated pathological changes and reduced M. tuberculosis H37Ra loads in the lungs. Thus, our results suggested that CnpB interferes with host innate and adaptive immune responses and confers protection against M. tuberculosis respiratory infection, which should be considered in vaccine development as well as a drug target.

Potentiating Lung Mucosal Immunity Through Intranasal Vaccination.

Yearly administration of influenza vaccines is our best available tool for controlling influenza virus spread. However, both practical and immunological factors sometimes result in sub-optimal vaccine efficacy. The call for improved, or even universal, influenza vaccines within the field has led to development of pre-clinical and clinical vaccine candidates that aim to address limitations of current influenza vaccine approaches. Here, we consider the route of immunization as a critical factor in eliciting tissue resident memory (Trm) populations that are not a target of current licensed intramuscular vaccines. Intranasal vaccination has the potential to boost tissue resident B and T cell populations that reside within specific niches of the upper and lower respiratory tract. Within these niches, Trm cells are poised to respond rapidly to pathogen re-encounter by nature of their anatomic localization and their ability to rapidly deliver anti-pathogen effector functions. Unique features of mucosal immunity in the upper and lower respiratory tracts suggest that antigen localized to these regions is required for the elicitation of protective B and T cell immunity at these sites and will need to be considered as an important attribute of a rationally designed intranasal vaccine. Finally, we discuss outstanding questions and areas of future inquiry in the field of lung mucosal immunity.

TIPE polarity proteins are required for mucosal deployment of T lymphocytes and mucosal defense against bacterial infection

Mucosal surfaces are continuously exposed to, and challenged by, numerous commensal and pathogenic organisms. To guard against infections, a majority of the thymus-derived T lymphocytes are deployed at the mucosa. Although chemokines are known to be involved in the mucosal lymphocyte deployment, it is not clear whether lymphocytes enter the mucosa through directed migration or enhanced random migration. Here we report that TIPE (tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like) proteins mediate directed migration of T lymphocytes into lung mucosa, and they are crucial for mucosal immune defense against Streptococcus pneumoniae infection. Knockout of both Tnfaip8 and Tipe2, which encode polarity proteins that control the directionality of lymphocyte migration, significantly reduced the numbers of T lymphocytes in the lung of mice. Compared with wild-type mice, Tnfaip8−/−Tipe2−/− mice also developed more severe infection with more pathogens entering blood circulation upon nasal Streptococcus pneumoniae challenge. Single-cell RNA-sequencing analysis revealed that TIPE proteins selectively affected mucosal homing of a unique subpopulation of T cells, called “T cells-2”, which expressed high levels of Ccr9, Tcf7, and Rag1/2 genes. TNFAIP8 and TIPE2 appeared to have overlapping functions since deficiency in both yielded the strongest phenotype. These data demonstrate that TIPE family of proteins are crucial for lung mucosal immunity. Strategies targeting TIPE proteins may help develop mucosal vaccines or treat inflammatory diseases of the lung.

Pseudomonas aeruginosa Induced Host Epithelial Cell Mitochondrial Dysfunction

The pathogenicity of P. aeruginosa is dependent on quorum sensing (QS), an inter-bacterial communication system that can also modulate host biology. The innate immune function of the lung mucosal barrier is dependent on proper mitochondrial function. The purpose of this study was to define the mechanism by which bacterial factors modulate host lung epithelial cell mitochondrial function and to investigate novel therapies that ameliorate this effect. 3-oxo-C12-HSL disrupts mitochondrial morphology, attenuates mitochondrial bioenergetics, and induces mitochondrial DNA oxidative injury. Mechanistically, we show that 3-oxo-C12-HSL attenuates expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, antioxidant defense, and cellular respiration, and its downstream effectors in both BEAS-2B and primary lung epithelial cells. Overexpression of PGC-1α attenuates the inhibition in cellular respiration caused by 3-oxo-C12-HSL. Pharmacologic activation of PGC-1α restores barrier integrity in cells treated with 3-oxo-C12-HSL. These data demonstrate that the P. aeruginosa QS molecule, 3-oxo-C12-HSL, alters mitochondrial pathways critical for lung mucosal immunity. Genetic and pharmacologic strategies that activate the PGC-1α pathway enhance host epithelial cell mitochondrial function and improve the epithelial innate response to P. aeruginosa. Therapies that rescue PGC-1α function may provide a complementary approach in the treatment of P. aeruginosa infection.

Lung mucosal immunity from secretory IgA may be impaired by MAA-adducted protein stimulation of epithelial cell TGF-β

Lung mucosal immunity from secretory IgA may be impaired by MAA-adducted protein stimulation of epithelial cell TGF-β

Post-exposure immunization by capsid-modified AdC7 vector expressing Pseudomonas aeruginosa OprF clears P. aeruginosa respiratory infection.

Respiratory infections with Pseudomonas aeruginosa are major health problems, particularly in patients with cystic fibrosis (CF). No vaccine against P. aeruginosa is yet available. A vaccine that controls colonization of the respiratory tract with P. aeruginosa could be useful to prevent chronic infection and exacerbations. Replication-deficient adenoviral (Ad) vectors based on non-human serotypes are attractive vaccine platforms as they can circumvent the problem of pre-existing anti-Ad immunity in humans. The primate-based AdC7 vector AdC7OprF.RGD that expresses the outer membrane protein F (OprF) of P. aeruginosa (AdC7OprF) and that displays an integrin-binding arginine-glycine-aspartic acid (RGD) sequence is a potent inducer of lung mucosal and protective immunity. Here, we investigated the efficacy of immunization with AdC7OprF.RGD to clear an already established P. aeruginosa respiratory infection in mice (wild-type and CF) and rats. Intratracheal administration of the clinical P. aeruginosa strain RP73 embedded in agar beads was used to establish persistent infection. Subsequent intranasal immunization with AdC7OprF.RGD induced robust P. aeruginosa-specific systemic and mucosal, humoral and cellular immune responses. Importantly, the AdC7OprF.RGD immunized mice effectively cleared P. aeruginosa from the lungs. Likewise, immunization with AdC7OprF.RGD of CF mice and Sprague Dawley rats with established P. aeruginosa respiratory infection showed enhanced anti-Pseudomonas immune responses and increased clearance of P. aeruginosa from the lungs. These data suggest that AdC7OprF.RGD can be effective as a post-exposure vaccine and may be useful in clinical settings in particular for patients with CF who frequently harbor the bacteria over prolonged periods.

IL-36\u03b3 is a crucial proximal component of protective type-1-mediated lung mucosal immunity in Gram-positive and -negative bacterial pneumonia

Interleukin-36γ (IL-36γ) is a member of novel IL-1-like proinflammatory cytokine family that are highly expressed in epithelial tissues and several myeloid-derived cell types. Little is known about the role of the IL-36 family in mucosal immunity, including lung anti-bacterial responses. We used murine models of IL-36γ deficiency to assess the contribution of IL-36γ in the lung during experimental pneumonia. Induction of IL-36γ was observed in the lung in response to Streptococcus pneumoniae (Sp) infection, and mature IL-36γ protein was secreted primarily in microparticles. IL-36γ-deficient mice challenged with Sp demonstrated increased mortality, decreased lung bacterial clearance and increased bacterial dissemination, in association with reduced local expression of type-1 cytokines, and impaired lung macrophage M1 polarization. IL-36γ directly stimulated type-1 cytokine induction from dendritic cells in vitro in a MyD88-dependent manner. Similar protective effects of IL-36γ were observed in a Gram-negative pneumonia model (Klebsiella pneumoniae). Intrapulmonary delivery of IL-36γ-containing microparticles reconstituted immunity in IL-36γ−/− mice. Enhanced expression of IL-36γ was also observed in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome because of pneumonia. These studies indicate that IL-36γ assumes a vital proximal role in the lung innate mucosal immunity during bacterial pneumonia by driving protective type-1 responses and classical macrophage activation.

Intestinal commensal bacteria mediate lung mucosal immunity and promote resistance of newborn mice to infection.

Immature mucosal defenses contribute to increased susceptibility of newborn infants to pathogens. Sparse knowledge of age-dependent changes in mucosal immunity has hampered improvements in neonatal morbidity because of infections. We report that exposure of neonatal mice to commensal bacteria immediately after birth is required for a robust host defense against bacterial pneumonia, the leading cause of death in newborn infants. This crucial window was characterized by an abrupt influx of interleukin-22 (IL-22)-producing group 3 innate lymphoid cells (IL-22+ILC3) into the lungs of newborn mice. This influx was dependent on sensing of commensal bacteria by intestinal mucosal dendritic cells. Disruption of postnatal commensal colonization or selective depletion of dendritic cells interrupted the migratory program of lung IL-22+ILC3 and made the newborn mice more susceptible to pneumonia, which was reversed by transfer of commensal bacteria after birth. Thus, the resistance of newborn mice to pneumonia relied on commensal bacteria-directed ILC3 influx into the lungs, which mediated IL-22-dependent host resistance to pneumonia during this developmental window. These data establish that postnatal colonization by intestinal commensal bacteria is pivotal in the development of the lung defenses of newborns.

22. Post-Exposure Vaccination by Capsid-Modified AdC7 Vector Expressing Pseudomonas aeruginosa OprF in Chronic P. aeruginosa Lung Infection

Chronic infections by Pseudomonas aeruginosa are a common problem in cystic fibrosis and other chronic lung diseases associated with bronchiectasis. With antimicrobial resistance of P. aeruginosa becoming more common, alternate prophylactic and therapeutic approaches are needed. Despite extensive efforts, a vaccine against P. aeruginosa is not yet available. A post-exposure vaccine that eliminates already established P. aeruginosa from the respiratory tract, could be useful in the management of chronic P. aeruginosa colonization. Replication-deficient adenoviral (Ad) vectors are an attractive platform for vaccines against respiratory pathogens. We have previously found that, in addition to circumvent pre-existing anti-human Ad immunity, a non-human primate-based AdC7 vector expressing outer membrane protein F (OprF) of P. aeruginosa (AdC7OprF) was more potent in inducing lung mucosal and protective immunity compared to a human Ad5-based vector. In addition, genetic modification of the AdC7 fiber to display an integrin-binding arginine-glycine-aspartic acid (RGD) sequence can further enhance mucosal protective immunogenicity of AdC7OprF. In this study we investigated if post-exposure vaccination by AdC7OprF. RGD can clear the already established P. aeruginosa in a mouse model. Intratracheal inoculation of P. aeruginosa (clinical strain RP73) encapsulated in agar beads (10^6 cfu/mouse) was used to establish persistent infection. Intranasal immunization of P. aeruginosa infected mice with AdC7OprF. RGD (10^10 pu/mouse) induced significantly high serum anti-OprF IgG antibodies as early as 1 week of immunization that further increased to higher levels after 2 week of immunization compared to AdC7Null or PBS inoculated mice (p<0.05; all comparisons). In addition to robust humoral response, immunization with AdC7OprF. RGD induced OprF-specific T-cell responses, as indicated by the higher secretion of IFN-γ or IL-4 from the OprF-stimulated cultured splenocytes compared to AdNull or PBS inoculated mice (p<0.05; all comparisons). Importantly, the AdC7OprF. RGD immunized mice showed significantly higher clearance of P. aeruginosa from the infected lungs after 1 week or 2 weeks of immunization (p<0.05; all comparisons). In fact, after 2 weeks of immunization, 50% of AdC7OprF. RGD immunized mice (3 out of 6) completely cleared the P. aeruginosa from the infected lungs. These data suggest that immunization with AdC7OprF. RGD induced robust humoral and cellular anti-P. aeruginosa immunity that could clear established pulmonary P. aeruginosa infections.

DEFB1 gene polymorphisms and tuberculosis in a Northeastern Brazilian population.

β-Defensin-1, an antimicrobial peptide encoded by the DEFB1 gene, is known to play an important role in lung mucosal immunity. In our association study we analyzed three DEFB1 functional polymorphisms −52G>A (rs1799946), −44C>G (rs1800972) and −20G>A (rs11362) in 92 tuberculosis patients and 286 healthy controls, both from Northeast Brazil: no association was found between the studied DEFB1 polymorphisms and the disease. However we cannot exclude that this lack of association could be due to the low number of subjects analyzed, as suggested by the low statistical power achieved for the three analyzed SNPs (values between 0.16 and 0.50).

Redundant and Cooperative Interactions between TLR5 and NLRC4 in Protective Lung Mucosal Immunity against Pseudomonas aeruginosa

Flagellin is the major structural component of flagella expressed by Pseudomonas aeruginosa (PA) and other bacteria. This protein has been shown to activate the Toll-like receptor TLR5 and the Nod-like receptor Nlrc4/Ipaf, culminating in the expression of innate cytokines and antimicrobial molecules. In this study, we tested the hypothesis that TLR5 and Nlrc4 in combination are required for maximal protective lung innate mucosal immunity against PA. To test this hypothesis, we compared innate immune responses in wild-type (WT) C57B6 mice challenged with PA intratracheally to those observed in mice genetically deficient in TLR5 (TLR5^-/-) or Nlrc4 (Nlrc4^-/-) alone or in combination (TLR5/Nlrc4^-/-). As compared to WT, TLR5^-/- and Nlrc4^-/- mice, we observed a significant increase in mortality in TLR5/Nlrc4^-/- mice, which was associated with a >5,000-fold increase in lung PA colony-forming units and systemic bacterial dissemination. The increased mortality observed in double-deficient mice was not attributable to differences in lung leukocyte influx or lung injury responses. Levels of biologically active IL-1β and IL-18 were reduced in the bronchoalveolar lavage fluid from PA-infected Nlrc4^-/- and TLR5/Nlrc4^-/- but not TLR5^-/- mice, indicating the requirement for Nlrc4-dependent caspase-1 activation. Similarly, decreased production of biologically active IL-1β and activation of caspase-1 was observed in PA-stimulated pulmonary macrophages isolated from Nlrc4^-/- and TLR5/Nlrc4^-/- but not TLR5^-/- mice, whereas the expression of iNOS and the production of NO were significantly reduced in cells from double-mutant but not single-mutant mice. Collectively, our findings indicate that TLR5 and Nlrc4 have both unique and redundant roles in lung antibacterial mucosal immunity, and the absence of both pathogen recognition receptors results in an increase in susceptibility to invasive lung infection.

Activation-induced cell death drives profound lung CD4(+) T-cell depletion in HIV-associated chronic obstructive pulmonary disease.

As overall survival improves, individuals with HIV infection become susceptible to other chronic diseases, including accelerated chronic obstructive pulmonary disease (COPD). To determine whether individuals with HIV-associated COPD exhibit dysregulated lung mucosal T-cell immunity compared with control subjects. Using flow cytometry, we evaluated peripheral blood and lung mucosal T-cell immunity in 14 HIV(+)COPD(+), 13 HIV(+)COPD(-), and 7 HIV(-)COPD(+) individuals. HIV(+)COPD(+) individuals demonstrated profound CD4(+) T-cell depletion with reduced CD4/CD8 T-cell ratios in bronchoalveolar lavage-derived lung mononuclear cells, not observed in peripheral blood mononuclear cells, and diminished CD4(+) T cell absolute numbers, compared with control subjects. Furthermore, HIV(+)COPD(+) individuals demonstrated decreased pulmonary HIV-specific and staphylococcal enterotoxin B-reactive CD4(+) memory responses, including loss of multifunctionality, compared with HIV(+)COPD(-) control subjects. In contrast, lung mucosal HIV-specific CD8(+) T-cell responses were preserved. Lung CD4(+) T cells from HIV(+)COPD(+) individuals expressed increased surface Fas death receptor (CD95) and programmed death-1, but similar bronchoalveolar lavage viral loads as control subjects. However, programmed death-1 expression inversely correlated with HIV-specific lung CD4(+)IFN-γ(+) T-cell responses, suggesting functional exhaustion. Moreover, lung CD4(+) T cells from HIV(+)COPD(+) patients demonstrated increased basal and HIV antigen-induced expression of the early apoptosis marker annexin V compared with control subjects, which was significantly attenuated with anti-Fas blockade. Lastly, lung mucosal, but not blood, CD4(+)/CD8(+) ratios from HIV(+) patients significantly correlated with the FEV1, but not in HIV(-)COPD(+) patients. Together, our results provide evidence for profound lung mucosal CD4(+) T-cell depletion via a Fas-dependent activation-induced cell death mechanism, along with impaired HIV-specific CD4(+) immunity as immunologic features of HIV-associated COPD.

RGD capsid modification enhances mucosal protective immunity of a non-human primate adenovirus vector expressing Pseudomonas aeruginosa OprF

Replication-deficient adenoviral (Ad) vectors of non-human serotypes can serve as Ad vaccine platforms to circumvent pre-existing anti-human Ad immunity. We found previously that, in addition to that feature, a non-human primate-based AdC7 vector expressing outer membrane protein F of P. aeruginosa (AdC7OprF) was more potent in inducing lung mucosal and protective immunity compared to a human Ad5-based vector. In this study we analysed if genetic modification of the AdC7 fibre to display an integrin-binding arginine-glycine-aspartic acid (RGD) sequence can further enhance lung mucosal immunogenicity of AdC7OprF. Intratracheal immunization of mice with either AdC7OprF.RGD or AdC7OprF induced robust serum levels of anti-OprF immunoglobulin (Ig)G up to 12 weeks that were higher compared to immunization with the human vectors Ad5OprF or Ad5OprF.RGD. OprF-specific cellular responses in lung T cells isolated from mice immunized with AdC7OprF.RGD and AdC7OprF were similar for T helper type 1 (Th1) [interferon (IFN)-γ in CD8(+) and interleukin (IL)-12 in CD4(+)], Th2 (IL-4, IL-5 and IL-13 in CD4(+)) and Th17 (IL-17 in CD4(+)). Interestingly, AdC7OprF.RGD induced more robust protective immunity against pulmonary infection with P. aeruginosa compared to AdC7OprF or the control Ad5 vectors. The enhanced protective immunity induced by AdC7OprF.RGD was maintained in the absence of alveolar macrophages (AM) or CD1d natural killer T cells. Together, the data suggest that addition of RGD to the fibre of an AdC7-based vaccine is useful to enhance its mucosal protective immunogenicity.

Flagellin Administration Protects Respiratory Tract from Burkholderia cepacia Infection

Burkholderia cepacia is an important pathogen that often causes pneumonia in immunocompromised individuals. Here, it was demonstrated that the TLR5 agonist flagellin could locally activate innate immunity. This was characterized by rapid expressions of IL-1beta, TNF-alpha, and iNOS mRNA and a delay in the expression of IL-10 mRNA. A significant elevation in the IL-1beta, TNF-alpha, and nitric oxide levels was also noted. In the respiratory tract, flagellin induced neutrophil infiltration into the airways, which was observed by histopathological examination and confirmed by the neutrophil count and level of myeloperoxidase activity. This was concomitant with a high activity of alveolar macrophages that engulfed and killed B. cepacia in vitro. The flagellin mucosal treatment improved the B. cepacia clearance in the mouse lung. Thus, the present findings illustrate the profound stimulatory effect of flagellin on the lung mucosal innate immunity, a response that needs to be exploited therapeutically to prevent the development of respiratory tract infection by B. cepacia.

Cathelicidin-Related Antimicrobial Peptide Is Required for Effective Lung Mucosal Immunity in Gram-Negative Bacterial Pneumonia

Cathelicidins are a family of endogenous antimicrobial peptides that exert diverse immune functions, including both direct bacterial killing and immunomodulatory effects. In this study, we examined the contribution of the murine cathelicidin, cathelicidin-related antimicrobial peptide (CRAMP), to innate mucosal immunity in a mouse model of Gram-negative pneumonia. CRAMP expression is induced in the lung in response to infection with Klebsiella pneumoniae. Mice deficient in the gene encoding CRAMP (Cnlp(-/-)) demonstrate impaired lung bacterial clearance, increased bacterial dissemination, and reduced survival in response to intratracheal K. pneumoniae administration. Neutrophil influx into the alveolar space during K. pneumoniae infection was delayed early but increased by 48 h in CRAMP-deficient mice, which was associated with enhanced expression of inflammatory cytokines and increased lung injury. Bone marrow chimera experiments indicated that CRAMP derived from bone marrow cells rather than structural cells was responsible for antimicrobial effects in the lung. Additionally, CRAMP exerted bactericidal activity against K. pneumoniae in vitro. Similar defects in lung bacterial clearance and delayed early neutrophil influx were observed in CRAMP-deficient mice infected with Pseudomonas aeruginosa, although this did not result in increased bacterial dissemination, increased lung injury, or changes in lethality. Taken together, our findings demonstrate that CRAMP is an important contributor to effective host mucosal immunity in the lung in response to Gram-negative bacterial pneumonia.

Alternative Spliced CD1D Transcripts in Human Bronchial Epithelial Cells

CD1d is a MHC I like molecule which presents glycolipid to natural killer T (NKT) cells, a group of cells with diverse but critical immune regulatory functions in the immune system. These cells are required for optimal defence against bacterial, viral, protozoan, and fungal infections, and control of immune-pathology and autoimmune diseases. CD1d is expressed on antigen presenting cells but also found on some non-haematopoietic cells. However, it has not been observed on bronchial epithelium, a site of active host defence in the lungs. Here, we identify for the first time, CD1D mRNA variants and CD1d protein expression on human bronchial epithelial cells, describe six alternatively spliced transcripts of this gene in these cells; and show that these variants are specific to epithelial cells. These findings provide the basis for investigations into a role for CD1d in lung mucosal immunity.