Interaction and cellular localization of the human host defense peptide LL-37 with lung epithelial cells

LL-37 is a human cationic host defense peptide that is an essential component of innate immunity. In addition to its modest antimicrobial activity, LL-37 affects the gene expression and behavior of effector cells involved in the innate immune response, although its mode of interaction with eukaryotic cells remains unclear. The interaction of LL-37 with epithelial cells was characterized in tissue culture by using biotinylated LL-37 and confocal microscopy. It was demonstrated that LL-37 was actively taken up into A549 epithelial cells and eventually localized to the perinuclear region. Specific inhibitors were used to demonstrate that the uptake process was not mediated by actin but required elements normally involved in endocytosis and that trafficking to the perinuclear region was dependent on microtubules. By using nonlinear regression analysis, it was revealed that A549 epithelial cells have two receptors for LL-37B, with high and low affinity for LL-37, respectively. These results indicate the mode of interaction of LL-37 with epithelial cells and further our understanding of its role in modulating the innate immune response.     

The human cationic host defense peptide LL-37 mediates contrasting effects on apoptotic pathways in different primary cells of the innate immune system

The human cathelicidin LL-37 is a cationic host defense peptide (antimicrobial peptide) expressed primarily by neutrophils and epithelial cells. This peptide, up-regulated under conditions of inflammation, has immunomodulatory and antimicrobial functions. We demonstrate that LL-37 is a potent inhibitor of human neutrophil apoptosis, signaling through P2X(7) receptors and G-protein-coupled receptors other than the formyl peptide receptor-like-1 molecule. This process involved modulation of Mcl-1 expression, inhibition of BID and procaspase-3 cleavage, and the activation of phosphatidylinositol-3 kinase but not the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase pathway. In contrast to the inhibition of neutrophil apoptosis, LL-37 induced apoptosis in primary airway epithelial cells, demonstrating alternate consequences of LL-37-mediated modulation of apoptotic pathways in different human primary cells. We propose that these novel immunomodulatory properties of LL-37 contribute to peptide-mediated enhancement of innate host defenses against acute infection and are of considerable significance in the development of such peptides and their synthetic analogs as potential therapeutics for use against multiple antibiotic-resistant infectious diseases.     

Human host defense peptide LL-37 facilitates double-stranded RNA pro-inflammatory signaling through up-regulation of TLR3 expression in vascular smooth muscle cells

Inflammation Research - The importance of human host defense peptide LL-37 in vascular innate immunity is not understood. Here, we assess the impact of LL-37 on double-stranded RNA (dsRNA)...     

The antimicrobial peptide LL‐37 modulates the inflammatory and host defense response of human neutrophils

The human cathelicidin antimicrobial peptide acts as an effector molecule of the innate immune system with direct antimicrobial and immunomodulatory effects. The aim of this study was to test whether the cathelicidin LL‐37 modulates the response of neutrophils to microbial stimulation. Human neutrophils were exposed to LPS, Staphylococcus aureus and Pseudomonas aeruginosa subsequent to incubation with LL‐37 and cytokine release was measured by ELISA. The incubation with LL‐37 significantly decreased the release of proinflammatory cytokines from stimulated human neutrophils. ROS production of neutrophils was determined by a luminometric and a flow cytometry method. The peptide induced the production of ROS and the engulfment of bacteria into neutrophils. Peritoneal mouse neutrophils isolated from CRAMP‐deficient and WT animals were treated with LPS and TNF‐α in the supernatant was measured by ELISA. Antimicrobial activity of neutrophils was detected by incubating neutrophils isolated from CRAMP‐knockout and WT mice with bacteria. Neutrophils from CRAMP‐deficient mice released significantly more TNF‐α after bacterial stimulation and showed decreased antimicrobial activity as compared to cells from WT animals. In conclusion, LL‐37 modulates the response of neutrophils to bacterial activation. Cathelicidin controls the release of inflammatory mediators while increasing antimicrobial activity of neutrophils.     

The role of the Src family kinase Lyn in the immunomodulatory activities of cathelicidin peptide LL-37 on monocytic cells

Cathelicidin LL-37 is a multifunctional, immunomodulatory and antimicrobial host-defense peptide of the human immune system. Here, we identified the role of SFKs in mediating the chemokine induction activity of LL-37 in monocytic cells. LL-37 induced SFK phosphorylation; and chemical inhibitors of SFKs suppressed chemokine production in response to LL-37 stimulation. SFKs were required for the downstream activation of AKT, but Ca(2+)-flux and MAPK induction were SFK-independent. Through systematic siRNA knockdown of SFK members, a requirement for Lyn in mediating LL-37 activity was identified. The involvement of Lyn in cathelicidin activities was further confirmed using Lyn-knockout mouse BMDMs. The role of SFKs and Lyn was also demonstrated in the activities of the synthetic cationic IDR peptides, developed as novel, immunomodulatory therapeutics. These findings elucidate the common molecular mechanisms mediating the chemokine induction activity of natural and synthetic cationic peptides in monocytic cells and identify SFKs as a potential target for modulating peptide responses.     

Neurogenic inflammation and the peripheral nervous system in host defense and immunopathology

The peripheral nervous and immune systems are traditionally thought of as serving separate functions. The line between them is, however, becoming increasingly blurred by new insights into neurogenic inflammation. Nociceptor neurons possess many of the same molecular recognition pathways for danger as immune cells, and, in response to danger, the peripheral nervous system directly communicates with the immune system, forming an integrated protective mechanism. The dense innervation network of sensory and autonomic fibers in peripheral tissues and high speed of neural transduction allows rapid local and systemic neurogenic modulation of immunity. Peripheral neurons also seem to contribute to immune dysfunction in autoimmune and allergic diseases. Therefore, understanding the coordinated interaction of peripheral neurons with immune cells may advance therapeutic approaches to increase host defense and suppress immunopathology.     

Priming of the neutrophil respiratory burst: role in host defense and inflammation

Neutrophils are the major circulating white blood cells in humans. They play an essential role in host defense against pathogens. In healthy individuals, circulating neutrophils are in a dormant state with very low efficiency of capture and arrest on the quiescent endothelium. Upon infection and subsequent release of pro-inflammatory mediators, the vascular endothelium signals to circulating neutrophils to roll, adhere, and cross the endothelial barrier. Neutrophils migrate toward the infection site along a gradient of chemo-attractants, then recognize and engulf the pathogen. To kill this pathogen entrapped inside the vacuole, neutrophils produce and release high quantities of antibacterial peptides, proteases, and reactive oxygen species (ROS). The robust ROS production is also called ‘the respiratory burst’, and the NADPH oxidase or NOX2 is the enzyme responsible for the production of superoxide anion, leading to other ROS. In vitro, several soluble and particulate agonists induce neutrophil ROS production. This process can be enhanced by prior neutrophil treatment with ‘priming’ agents, which alone do not induce a respiratory burst. In this review, we will describe the priming process and discuss the beneficial role of controlled neutrophil priming in host defense and the detrimental effect of excessive neutrophil priming in inflammatory diseases.     

Expression of cathelicidin LL-37 during Mycobacterium tuberculosis infection in human alveolar macrophages, monocytes, neutrophils, and epithelial cells

The innate immune response in human tuberculosis is not completely understood. To improve our knowledge regarding the role of cathelicidin hCAP-18/LL37 in the innate immune response to tuberculosis infection, we used immunohistochemistry, immunoelectron microscopy, and gene expression to study the induction and production of the antimicrobial peptide in A549 epithelial cells, alveolar macrophages (AM), neutrophils, and monocyte-derived macrophages (MDM) after infection with Mycobacterium tuberculosis. We demonstrated that mycobacterial infection induced the expression and production of LL-37 in all cells studied, with AM being the most efficient. We did not detect peptide expression in tuberculous granulomas, suggesting that LL-37 participates only during early infection. Through the study of Toll-like receptors (TLR) in MDM, we showed that LL-37 can be induced by stimulation through TLR-2, TLR-4, and TLR-9. This last TLR was strongly stimulated by M. tuberculosis DNA. We concluded that LL-37 may have an important role in the innate immune response against M. tuberculosis.     

Factor H-binding protein is important for meningococcal survival in human whole blood and serum and in the presence of the antimicrobial peptide LL-37

Factor H-binding protein (fHBP; GNA1870) is one of the antigens of the recombinant vaccine against serogroup B Neisseria meningitidis, which has been developed using reverse vaccinology and is the basis of a meningococcal B vaccine entering phase III clinical trials. Binding of factor H (fH), an inhibitor of the complement alternative pathway, to fHBP enables N. meningitidis to evade killing by the innate immune system. All fHBP null mutant strains analyzed were sensitive to killing in ex vivo human whole blood and serum models of meningococcal bacteremia with respect to the isogenic wild-type strains. The fHBP mutant strains of MC58 and BZ83 (high fHBP expressors) survived in human blood and serum for less than 60 min (decrease of >2 log(10) CFU), while NZ98/254 (intermediate fHBP expressor) and 67/00 (low fHBP expressor) showed decreases of >1 log(10) CFU after 60 to 120 min of incubation. In addition, fHBP is important for survival in the presence of the antimicrobial peptide LL-37 (decrease of >3 log(10) CFU after 2 h of incubation), most likely due to electrostatic interactions between fHBP and the cationic LL-37 molecule. Hence, the expression of fHBP by N. meningitidis strains is important for survival in human blood and human serum and in the presence of LL-37, even at low levels. The functional significance of fHBP in mediating resistance to the human immune response, in addition to its widespread distribution and its ability to induce bactericidal antibodies, indicates that it is an important component of the serogroup B meningococcal vaccine.     

The progression of the inflammation in established collagen-induced arthritis can be altered by treatments with immunological or pharmacological agents which inhibit T cell activities

Alterations in the progression of the inflammatory disease in mice with established collagen-induced arthritis (CIA) by in vivo treatments with either anti-T cell antibodies or an immunosuppressive drug, whose main targets of action are T cells, were studied. It was demonstrated that the in vivo administration of either monoclonal anti-L3T4, anti-Ly-2 or the combination of anti-L3T4 and anti-Ly-2 failed to modify the inflammatory disease after the arthritis had been initiated. Nevertheless, the progression of disease was decreased by treatments with rabbit anti-mouse lymphocyte sera (ALS) in mice with established CIA. While there was a substantial reduction in the proliferative responses to the T cell mitogen concanavalin A (Con A) in these ALS-treated mice, proliferation to a B cell mitogen, lipopolysaccharide, was unaffected. Furthermore, treatments with anti-Thy-1.2 monoclonal antibody (mAb) by itself did not alter the progression of the ongoing inflammatory disease, but combined treatments with both anti-Thy-1.2 and anti-L3T4 mAb prevented the further advancement of the arthritic disease. Although mice receiving either anti-Thy-1.2 alone or both anti-Thy-1.2 and anti-L3T4 exhibited complete absence of Thy-1+ cells within their inguinal lymph nodes, the proliferative responses to Con A by LN cells from mice treated with the combination of anti-Thy-1.2 and anti-L3T4 were drastically reduced compared to the responses of either the anti-Thy-1.2 or anti-L3T4-treated groups. Finally, daily treatments with cyclosporin A, an immunosuppressive drug which preferentially acts on T cells, were also effective in modifying the clinical course of the arthritic disease in mice with established CIA. These results suggest that immunocompetent cells which express the Thy-1 surface marker, most likely Thy-1+ T cells, may play a role in maintaining and perpetuating the inflammatory disease of established CIA.     

Improved antibacterial host defense and altered peripheral granulocyte homeostasis in mice lacking the adhesion class G protein receptor CD97

CD97 is a member of the adhesion family of G protein-coupled receptors. Alternatively spliced forms of CD97 bind integrins alpha5beta1 and alphavbeta3, decay accelerating factor, or dermatan sulfate. CD97 is expressed on myeloid cells at high levels and a variety of other cell types at lower levels. Little is known about the physiological function of CD97. To begin dissecting the function of CD97, we evaluated the immune response of CD97 null mice to systemic infection by Listeria monocytogenes. CD97 null mice were significantly more resistant to listeriosis than matched wild-type mice. A major determinant of the difference in survival appeared to be the comparatively more robust accumulation of granulocytes in the blood and in infected livers of CD97 null mice within 18 h of inoculation, correlating with a decrease in the number of bacteria. CD97 null mice also displayed a mild granulocytosis in the nonchallenged state. Because there is a strong suggestion that CD97 functions in an adhesive capacity, we examined the migratory properties of granulocytes in CD97 null mice. In chimeric animals, CD97 null and wild-type granulocytes migrated similarly, as determined by inflammation-induced emigration from the bone marrow and accumulation in the peritoneum. Granulocyte development in the bone marrow of CD97 null mice was comparable to that of wild-type mice, and CD97 deficiency did not appear to stimulate granulocytosis secondary to peripheral inflammation and resultant granulocyte colony-stimulating factor induction, unlike various other models of adhesion deficiencies. Our results suggest that CD97 plays a role in peripheral granulocyte homeostasis.     

Prekallikrein inhibits innate immune signaling in the lung and impairs host defense during pneumosepsis in mice

Prekallikrein (PKK, also known as Fletcher factor and encoded by the gene KLKB1 in humans) is a component of the contact system. Activation of the contact system has been implicated in lethality in fulminant sepsis models. Pneumonia is the most frequent cause of sepsis. We sought to determine the role of PKK in host defense during pneumosepsis. To this end, mice were infected with the common human pathogen Klebsiella pneumoniae via the airways, causing an initially localized infection of the lungs with subsequent bacterial dissemination and sepsis. Mice were treated with a selective PKK-directed antisense oligonucleotide (ASO) or a scrambled control ASO for 3 weeks prior to infection. Host response readouts were determined at 12 or 36 h post-infection, including genome-wide messenger RNA profiling of lungs, or mice were followed for survival. PKK ASO treatment inhibited constitutive hepatic Klkb1 mRNA expression by >80% and almost completely abolished plasma PKK activity. Klkb1 mRNA could not be detected in lungs. Pneumonia was associated with a progressive decline in PKK expression in mice treated with control ASO. PKK ASO administration was associated with a delayed mortality, reduced bacterial burdens, and diminished distant organ injury. While PKK depletion did not influence lung pathology or neutrophil recruitment, it was associated with an upregulation of multiple innate immune signaling pathways in the lungs already prior to infection. Activation of the contact system could not be detected, either during infection in vivo or at the surface of Klebsiella in vitro. These data suggest that circulating PKK confines pro-inflammatory signaling in the lung by a mechanism that does not involve contact system activation, which in the case of respiratory tract infection may impede early protective innate immunity. © 2019 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

The anti-microbial peptide LL-37 modulates immune responses in the palatine tonsils where it is exclusively expressed by neutrophils and a subset of dendritic cells

Antimicrobial peptides are essential elements of epithelial defense against invading micro-organisms. The palatine tonsils are positioned at the entry of the airway and the gut and as such are ideally situated to act as immune sentinels in the pharynx protecting against microbial invasion. Tonsils express a number of antimicrobial peptides including hCAP18/LL-37. Here we clearly define the expression of hCAP18/LL-37 in the tonsils showing unequivocally that hCAP18/LL-37 is mainly expressed by infiltrating neutrophils and follicular CD11c+CD13+HLA-DR+ dendritic cells, rarely by macrophages, and never by the epithelium itself. To explore possible functions for follicle-derived LL-37, we stimulated tonsil mononuclear cells with LL-37 in vitro and observed the secretion of the proinflammatory cytokines CCL5 and CXCL9, expression of IFN-γ and MX-1 and down-regulation of chemokine receptors CCR4 and CCR6 which are involved in tissue-selective T cell trafficking. Taken together, these data illustrate new potential immunoregulatory functions for hCAP18/LL-37 in the tonsils.     

Kinetics and mode of peptide delivery via the respiratory mucosa determine the outcome of activation versus TH2 immunity in allergic inflammation of the airways

BACKGROUND: Specific immunotherapy involving systemic injection of allergen, though highly effective, can cause severe side effects due to IgE-mediated activation of effector cells. Allergen-derived peptides might provide a safer alternative. We have investigated the use of mucosally delivered peptide to induce CD4(+) T(H)2 cell tolerance and thus protect against allergen-induced airway inflammation. OBJECTIVE: The purpose of this study was to investigate whether intranasal administration of an allergen-derived peptide, either alone or adsorbed to chitosan, can prevent the induction of T(H)2-mediated pulmonary inflammation after sensitization and challenge of the airways with allergen. METHODS: Mice were given (intranasally) a peptide containing an immunodominant epitope of the Dermatophagoides pteronyssinus (Der p) 1 allergen, either as soluble antigen or adsorbed to chitosan, before sensitization and allergen challenge. Pulmonary inflammation, antigen-specific CD4(+) T-cell responses, and antibody levels in sera were then determined. RESULTS: Mice given peptide adsorbed to chitosan had significant reductions in airway eosinophilia, which correlated with reduced levels of IL-4 and IL-5 in the bronchoalveolar lavage fluid. There was decreased recruitment of activated CD4(+) T cells into the airways after allergen challenge, which correlated with a loss of Der p 1-specific T-cell cytokine responses in the periphery and the localized production of IL-10 by antigen-specific T cells in bronchial lymph nodes. Induction of peripheral T-cell tolerance was preceded by transient T-cell activation and IFN-gamma production. CONCLUSION: Our data demonstrate that suppression of airway inflammation by intranasal administration of peptide antigen adsorbed to chitosan is initiated by transient T-cell activation and maintained by the production of IL-10 by antigen-specific T cells in the draining lymph nodes.     

Endothelial cells, megakaryoblasts, platelets and alveolar epithelial cells express abundant levels of the mouse AA4 antigen, a C-type lectin-like receptor involved in homing activities and innate immune host defense

Phagocytic complement C1q receptor (C1qRp) and thrombomodulin (TM, CD141), are two key members of a novel family of lectin receptors involved in cell-cell interactions and innate immune host defense. We report here that the mouse homologue of human C1qRp (AA4) and TM are derived from a common ancestor on the basis that both genes co-localized to the same region of the chromosome 2 and also because they share similar domain composition and organization. Moreover, we found that mouse AA4 was localized to sites known to express TM. Mouse AA4 was identified by Western blot as a heavily glycosylated protein (110 kDa) expressed in all tissues tested. Brain and spleen expressed an additional 130-kDa band. Immunoperoxidase and double-immunofluorescence experiments showed that, surprisingly, the major site of AA4 expression in all tissue tested is on endothelial cells and that despite the apparent involvement of AA4 in the phagocytic response, it is not expressed by tissue macrophages. In contrast, megakaryoblasts (in bone marrow and spleen) and platelets expressed abundant levels of AA4 stored in cytoplasmic vesicles. Interestingly, alveolar epithelial cells (lung) but not other epithelia (e.g. uterus) were strongly stained for AA4. Although AA4 has been described on all hematopoietic progenitors, we found that only circulating immature B cells, monocytes and NK cells but not T cells and neutrophils expressed AA4.     

Defective generalized extracellular matrix in the neoplasm bearing host: decreased inflammation, immunity and resistance

Neoplasms have extensive qualitative and quantitative changes in their extracellular matrix. Lytic factors from neoplasms breakdown extracellular matrix of the areas adjacent to the neoplasms and fibroblasts in these areas produce increased extracellular matrix. Some of the extracellular matrix fractions are normally present in serum. Multiple fractions derived from extracellular matrix are present in the serum of tumor patients. Tumor patients often have decreased early cellular inflammation and immunity. These reactions depend on a normal extracellular matrix. A number of known facts about the tumor bearing host can be explained by the concept that the host's generalized extracellular matrix is altered by circulating fractions of extracellular matrix. These could be qualitative or quantitative defects as well as an abundance of depolymerized fractions. The beneficial effects of cytokines, streptococcal infections and pyridoxine deficiency on tumors can be explained by this concept.     

Association of polymorphisms in the BDNF,DRD1 and DRD3 genes with tobacco smoking in schizophrenia

Emerging evidence indicates that the DRD1‐BDNF‐DRD3 cluster plays an important role in nicotine addiction. We have performed an association analysis of 42 SNPs within these genes with cigarette consumption in a group of 341 schizophrenia patients. The ACCG haplotype consisting of four BDNF markers (Val66Met (rs6265), rs11030104, rs2049045 and rs7103411) showed an association with the risk of smoking (p = 0.0002). Both DRD1 markers tested (rs4532 and rs686) and the DRD3 marker (rs1025398) showed association with quantity of tobacco smoked (p = 0.01, 0.005 and 0.002, respectively). Our findings are preliminary; however, they support the involvement of the DRD1, BDNF and DRD3 genes in smoking behaviour.