Nades Palaniyar

NET balancing: a problem in inflammatory lung diseases.

Neutrophil extracellular traps (NETs) are beneficial antimicrobial defense structures that can help fight against invading pathogens in the host. However, recent studies reveal that NETs exert adverse effects in a number of diseases including those of the lung. Many inflammatory lung diseases are characterized with a massive influx of neutrophils into the airways. Neutrophils contribute to the pathology of these diseases. To date, NETs have been identified in the lungs of cystic fibrosis (CF), acute lung injury (ALI), allergic asthma, and lungs infected with bacteria, virus, or fungi. These microbes and several host factors can stimulate NET formation, or NETosis. Different forms of NETosis have been identified and are dependent on varying types of stimuli. All of these pathways however appear to result in the formation of NETs that contain DNA, modified extracellular histones, proteases, and cytotoxic enzymes. Some of the NET components are immunogenic and damaging to host tissue. Innate immune collectins, such as pulmonary surfactant protein D (SP-D), bind NETs, and enhance the clearance of dying cells and DNA by alveolar macrophages. In many inflammatory lung diseases, bronchoalveolar SP-D levels are altered and its deficiency results in the accumulation of DNA in the lungs. Some of the other therapeutic molecules under consideration for treating NET-related diseases include DNases, antiproteases, myeloperoxidase (MPO) inhibitors, peptidylarginine deiminase-4 inhibitors, and anti-histone antibodies. NETs could provide important biological advantage for the host to fight against certain microbial infections. However, too much of a good thing can be a bad thing. Maintaining the right balance of NET formation and reducing the amount of NETs that accumulate in tissues are essential for harnessing the power of NETs with minimal damage to the hosts.

Surfactant Protein D Reduces Alveolar Macrophage Apoptosis In Vivo

Surfactant protein D (SP-D) is a molecule of the innate immune system that recognizes the patterns of surface carbohydrate on pathogens and targets them for phagocytosis and killing. SP-D-deficient mice show an increased number of macrophages in the alveolar space, excess surfactant phospholipid, overproduction of reactive oxygen species, and the development of emphysema. We report here that SP-D-deficient mice have a 5- to 10-fold increase in the number of apoptotic and necrotic alveolar macrophages, as defined by annexin V and propidium iodine staining, respectively. Intrapulmonary administration of a truncated 60-kDa fragment of human recombinant SP-D reduces the number of apoptotic and necrotic alveolar macrophages and partially corrects the lipid accumulation in SP-D-deficient mice. The same SP-D fragment binds preferentially to apoptotic and necrotic alveolar macrophages in vitro, suggesting that SP-D contributes to immune homeostasis in the lung by recognizing and promoting removal of necrotic and apoptotic cells.

SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx

Significance Formation of neutrophil extracellular traps (NETs) is a recently described process by which neutrophils combat microbial pathogens. Recent studies demonstrate causative relationships between NETs and debilitating disorders such as rheumatoid arthritis, vasculitis, thrombosis, cystic fibrosis, and acute respiratory distress syndrome. However, the understanding of signaling pathways governing the process termed “NETosis” remains elusive. Two major types of NETosis have been reported; however, the mechanistic differences between these two types are not clearly established. Here we describe that NETosis induced by calcium ionophores is fast, NADPH-oxidase independent, and is mediated by mitochondrial reactive oxygen species (ROS) and a calcium-activated small conductance potassium channel. Thus, drugs that target mitochondrial ROS production or the potassium channels may provide previously unidentified therapeutic approaches for combating disorders with unregulated NETosis. Neutrophils cast neutrophil extracellular traps (NETs) to defend the host against invading pathogens. Although effective against microbial pathogens, a growing body of literature now suggests that NETs have negative impacts on many inflammatory and autoimmune diseases. Identifying mechanisms that regulate the process termed “NETosis” is important for treating these diseases. Although two major types of NETosis have been described to date, mechanisms regulating these forms of cell death are not clearly established. NADPH oxidase 2 (NOX2) generates large amounts of reactive oxygen species (ROS), which is essential for NOX-dependent NETosis. However, major regulators of NOX-independent NETosis are largely unknown. Here we show that calcium activated NOX-independent NETosis is fast and mediated by a calcium-activated small conductance potassium (SK) channel member SK3 and mitochondrial ROS. Although mitochondrial ROS is needed for NOX-independent NETosis, it is not important for NOX-dependent NETosis. We further demonstrate that the activation of the calcium-activated potassium channel is sufficient to induce NOX-independent NETosis. Unlike NOX-dependent NETosis, NOX-independent NETosis is accompanied by a substantially lower level of activation of ERK and moderate level of activation of Akt, whereas the activation of p38 is similar in both pathways. ERK activation is essential for the NOX-dependent pathway, whereas its activation is not essential for the NOX-independent pathway. Despite the differential activation, both NOX-dependent and -independent NETosis require Akt activity. Collectively, this study highlights key differences in these two major NETosis pathways and provides an insight into previously unknown mechanisms for NOX-independent NETosis.

Collectin 11 (CL-11, CL-K1) Is a MASP-1/3–Associated Plasma Collectin with Microbial-Binding Activity

Collectins play important roles in the innate immune defense against microorganisms. Recently, a new collectin, collectin 11 (CL-11 or CL-K1), was identified via database searches. In present work, we characterize the structural and functional properties of CL-11. Under nonreducing conditions, in gel permeation chromatography recombinant CL-11 forms disulfide-linked oligomers of 100 and 200 kDa. A mAb-based ELISA estimates the concentration of CL-11 in plasma to be 2.1 μg/ml, and the presence of CL-11 in plasma was further verified by Western blotting and mass spectrometry. Mannan-binding lectin-associated serine protease 1 (MASP-1) copurified with CL-11 and the interaction in plasma with MASP-1 and/or MASP-3 was further demonstrated using ELISA. We identified the adrenal glands, the kidneys, and the liver as primary sites of expression. CL-11 lectin activity was demonstrated by ELISA and showed that CL-11 has preference for l-fucose and d-mannose. We finally show that CL-11 binds to intact bacteria, fungi, and viruses and that CL-11 decreases influenza A virus infectivity and forms complexes with DNA. On the basis of the significant concentration of CL-11 in circulation and CL-11’s interaction with various microorganisms and MASP-1 and/or MASP-3, it is conceivable that CL-11 plays a role in activation of the complement system and in the defense against invading microorganisms.

Innate Immune Collectin Surfactant Protein D Simultaneously Binds Both Neutrophil Extracellular Traps and Carbohydrate Ligands and Promotes Bacterial Trapping

Neutrophils release DNA-based extracellular traps to capture and kill bacteria. The mechanism(s) and proteins that promote neutrophil extracellular trap (NET)-mediated bacterial trapping are not clearly established. Surfactant protein D (SP-D) is an innate immune collectin present in many mucosal surfaces. We hypothesized that SP-D can bind both the pathogens and NETs to augment NET-mediated bacterial trapping. To test this hypothesis, we used LPS and Pseudomonas aeruginosa pneumonia mouse models and performed in vivo and ex vivo assays. In this study, we show that NETs are produced by the neutrophils recruited to the airways in response to the bacterial ligand. Notably, NETs are detected as short fragments of DNA–protein complexes in the airways as opposed to the long stringlike structures seen in ex vivo cultures. SP-D recognizes both the short NET fragments and the long NET DNA structures. SP-D–NET copurification studies further show that SP-D can simultaneously recognize NETs and carbohydrate ligands in vivo. Similar to the LPS model, soluble DNA–protein complexes and increased amounts of SP-D are detected in the murine model of P. aeruginosa pneumonia. We then tested the effect of SP-D on NET-mediated trapping of P. aeruginosa by means of Western blots, fluorescence microscopy, and scanning electron microscopy. Results of these experiments show that SP-D microagglutinates P. aeruginosa and allows an efficient bacterial trapping by NETs. Collectively, these findings provide a unique biological relevance for SP-D–DNA interactions and places SP-D as an important innate immune protein that promotes bacterial trapping by NETs during neutrophil-mediated host defense.

Mannose-Binding Lectin Recognizes Peptidoglycan via the N-Acetyl Glucosamine Moiety, and Inhibits Ligand-Induced Proinflammatory Effect and Promotes Chemokine Production by Macrophages

Peptidoglycan (PGN) is the major cell wall component (90%, w/w) of Gram-positive bacteria and consists of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharide repeating arrays that are cross-linked by short peptides. We hypothesized that PGN is a ligand for pathogen-associated pattern-recognition proteins. Mannose-binding lectin (MBL) and serum amyloid component P are two carbohydrate-binding innate immune proteins present in the blood. In this study we show that human MBL, but not serum amyloid component P, binds significantly to PGN via its C-type lectin domains, and that the interaction can be more effectively competed by GlcNAc than by MurNAc. Surface plasmon resonance analyses show that native MBL binds immobilized PGN with high avidity. Competition experiments also show that both native MBL and MBL(n/CRD), a 48-kDa recombinant trimeric fragment of MBL containing neck and carbohydrate recognition domains, have higher affinity for GlcNAc than for MurNAc. Protein arrays and ELISA show that PGN increases the secretion of TNF-α, IL-8, IL-10, MCP-2, and RANTES from PMA-stimulated human monocytic U937 cells. Interestingly, the presence of MBL together with PGN increases the production of IL-8 and RANTES, but reduces that of TNF-α. Our results indicate that Gram-positive bacterial is a biologically relevant ligand for MBL, and that the collectin preferentially binds to the GlcNAc moiety of the PGN via its C-type lectin domains. MBL inhibits PGN-induced production of proinflammatory cytokines while enhancing the production of chemokines by macrophages, which suggests that MBL may down-regulate macrophage-mediated inflammation while enhancing phagocyte recruitment.

Review: Soluble innate immune pattern-recognition proteins for clearing dying cells and cellular components: implications on exacerbating or resolving inflammation

Soluble innate immune pattern-recognition proteins (sPRPs) identify non-self or altered-self molecular patterns. Dying cells often display altered-self arrays of molecules on their surfaces. Hence, sPRPs are ideal for recognizing these cells and their components. Dying cell surfaces often contain, or allow the access to different lipids, intracellular glycoproteins and nucleic acids such as DNA at different stages of cell death. These are considered as ‘eat me’ signals that replace the native ‘don’t eat me’ signals such as CD31, CD47 present on the live cells. A programmed cell death process such as apoptosis also generates cell surface blebs that contain intracellular components. These blebs are easily released for effective clearance or signalling. During late stages of cell death, soluble components are also released that act as ‘find me’ signal (e.g. LysoPC, nucleotides). The sPRPs such as collectins, ficolins, pentraxins, sCD14, MFG-E8, natural IgM and C1q can effectively identify some of these specific molecular patterns. The biological end-point is different depending on sPRP, tissue, stage of apoptosis and the type of cell death. The sPRPs that reside in the immune-privileged surfaces such as lungs often act as opsonins and enhance a silent clearance of dying cells and cellular material by macrophages and other phagocytic cells. Although the recognition of these materials by complement-activating proteins could amplify the opsonic signal, this pathway may aggravate inflammation. Clear understanding of the involvement of specific sPRPs in cell death and subsequent clearance of dying cell and their components is essential for devising appropriate treatment strategies for diseases involving infection, inflammation and auto-antibody generation.

Pulmonary Innate Immune Proteins and Receptors that Interact with Gram-positive Bacterial Ligands

The two major gram-positive bacterial (GPB) ligands are peptidoglycan (PGN) and lipoteichoic acid (LTA). These polymeric LTA and highly organized PGN contain repeating carbohydrate moieties, which are potential targets for pattern recognition molecules. The major pattern recognition proteins and receptors, which bind GPB, either have a lectin, PGN recognition, collagen or leucine-rich repeat (LRR) domain. The soluble innate immune proteins (IIPs) that bind to PGN and LTA include pulmonary collectins surfactant-associated proteins (SP-) A and D, lectin-like pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP), and sCD14. Membrane-anchored lectin or lectin-like group members include macrophage mannose receptor (MR), complement receptor 3 (CR3, or Mac-1, or integrin CD11b/CD18), scavenger receptor A (SRCL-1), lectin-like oxidized LDL receptor 1 (LOX-1), and GPI-anchored CD14. Although Toll-like receptor (TLR) 2 and 4, and CD14 contain extracellular LRR domains, only TLRs have a cytoplasmic domain for signal transduction. Three of the four recently discovered human PGN recognition proteins (PGRP) have a transmembrane domain, and hence, considered as true receptors for GPB. Since lysozyme is the only known pulmonary enzyme that can lyse bacterial cell wall PGN, other innate immune molecules appear to be responsible for signalling and enhancing the clearance of GPB infection from the lung. Interestingly, pulmonary collectins bind not only to GPB ligands but also to the receptors, CD14 and TLR, and antigen processing cells such as dentritic cells. These complex interactions appear to play major roles in linking innate and adaptive immunity, and maintaining a pathogen-free lung with minimal, or no inflammation.

Innate Immune Collectin Surfactant Protein D Enhances the Clearance of DNA by Macrophages and Minimizes Anti-DNA Antibody Generation

Dying microbes and necrotic cells release highly viscous DNA that induces inflammation and septic shock, and apoptotic cells display DNA, a potential autoantigen, on their surfaces. However, innate immune proteins that mediate the clearance of free DNA and surface DNA-containing cells are not clearly established. Pulmonary surfactant proteins (SP-) A and D are innate immune pattern recognition collectins that contain fibrillar collagen-like regions and globular carbohydrate recognition domains (CRDs). We have recently shown that collectins SP-A, SP-D, and mannose binding lectin recognize DNA and RNA via their collagen-like regions and CRDs. Here we show that SP-D enhances the uptake of Cy3-labeled fragments of DNA and DNA-coated beads by U937 human monocytic cells, in vitro. Analysis of DNA uptake by freshly isolated mouse alveolar macrophages shows that SP-D, but not SP-A, deficiency results in reduced clearance of DNA, ex vivo. Analysis of bronchoalveolar lavage fluid shows that SP-D- but not SP-A-deficient mice are defective in clearing free DNA from the lung. Additionally, both SP-A- and SP-D-deficient mice accumulate anti-DNA Abs in sera in an age-dependent manner. Thus, we conclude that collectins such as SP-A and SP-D reduce the generation of anti-DNA autoantibody, which may be explained in part by the defective clearance of DNA from the lungs in the absence of these proteins. Our findings establish two new roles for these innate immune proteins and that SP-D enhances efficient pinocytosis and phagocytosis of DNA by macrophages and minimizes anti-DNA Ab generation.

Surfactant protein D binds genomic DNA and apoptotic cells, and enhances their clearance, in vivo.

Abstract: Collectins, such as surfactant protein D (SP‐D), bind apoptotic cells; however, the ligands that they recognize on these cells are unknown. We hypothesized that SP‐D binds to the DNA present on these cells. We show that SP‐D binds and aggregates mouse alveolar macrophage DNA effectively. Alveolar macrophages of SP‐D(−/−) mice contained more nicked DNA than those of SP‐A(−/−) and wild type mice. Our results also suggest that carbohydrate recognition domains of SP‐D may recognize DNA present on the apoptotic cells. Therefore, cell‐surface DNA could be a ligand for recognition of apoptotic cells by collectins.