Protect Host Tissues Research Articles

Ebselen improves fungal keratitis through exerting anti-inflammation, anti-oxidative stress, and antifungal effects

Fungal keratitis is a severely vision-threatening corneal infection, where the prognosis depends on both fungal virulence and host immune defense. Inappropriate host responses can induce substantial inflammatory damage to the cornea. Therefore, in the treatment of fungal keratitis, it is important to concurrently regulate the immune response while efforts are made to eliminate the pathogen. Ebselen is a widely studied organo-selenium compound and has been demonstrated to have antifungal, antibacterial, anti-inflammatory, and oxidative stress-regulatory properties. The effectiveness of ebselen for the treatment of fungal keratitis remains unknown. In this study, ebselen was demonstrated to produce a marked inhibitory effect on Aspergillus fumigatus (A. fumigatus), including spore germination inhibition, mycelial growth reduction, and fungal biofilm disruption. The antifungal activity of ebselen was related to the cell membrane damage caused by thioredoxin (Trx) system inhibition-mediated oxidative stress. On the contrary, ebselen enhanced the antioxidation of Trx system in mammalian cells. Further, ebselen was proven to suppress the expressions of inflammatory mediators (IL-1β, IL-6, TNF-α, COX-2, iNOS, and CCL2) and reduce the production of oxidative stress-associated indicators (ROS, NO, and MDA) in fungi-stimulated RAW264.7 cells. In addition, ebselen regulated PI3K/Akt/Nrf2 and p38 MAPK signaling pathways, which contributed to the improvement of inflammation and oxidative stress. Finally, we verified the therapeutic effect of ebselen on mouse fungal keratitis. Ebselen improved the prognosis and reduced the fungal burden in mouse corneas. Expressions of inflammatory mediators, as well as the infiltration of macrophages and neutrophils in the cornea were also obviously decreased by ebselen. In summary, ebselen exerted therapeutic effects by reducing fungal load and protecting host tissues in fungal keratitis, making it a promising treatment for fungal infections.

Inside the phagosome: A bacterial perspective.

The neutrophil phagosome is one of the most hostile environments that bacteria must face and overcome if they are to succeed as pathogens. Targeting bacterial defense mechanisms should lead to new therapies that assist neutrophils to kill pathogens, but this has not yet come to fruition. One of the limiting factors in this effort has been our incomplete knowledge of the complex biochemistry that occurs within the rapidly changing environment of the phagosome. The same compartmentalization that protects host tissue also limits our ability to measure events within the phagosome. In this review, we highlight the limitations in our knowledge, and how the contribution of bacteria to the phagosomal environment is often ignored. There appears to be significant heterogeneity among phagosomes, and it is important to determine whether survivors have more efficient defenses or whether they are ingested into less threatening environments than other bacteria. As part of these efforts, we discuss how monitoring or recovering bacteria from phagosomes can provide insight into the conditions they have faced. We also encourage the use of unbiased screening approaches to identify bacterial genes that are essential for survival inside neutrophil phagosomes.

Does the epithelial barrier hypothesis explain the increase in allergy, autoimmunity and other chronic conditions?

There has been a steep increase in allergic and autoimmune diseases, reaching epidemic proportions and now affecting more than one billion people worldwide. These diseases are more common in industrialized countries, and their prevalence continues to rise in developing countries in parallel to urbanization and industrialization. Intact skin and mucosal barriers are crucial for the maintenance of tissue homeostasis as they protect host tissues from infections, environmental toxins, pollutants and allergens. A defective epithelial barrier has been demonstrated in allergic and autoimmune conditions such as asthma, atopic dermatitis, allergic rhinitis, chronic rhinosinusitis, eosinophilic esophagitis, coeliac disease and inflammatory bowel disease. In addition, leakiness of the gut epithelium is also implicated in systemic autoimmune and metabolic conditions such as diabetes, obesity, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis and autoimmune hepatitis. Finally, distant inflammatory responses due to a 'leaky gut' and microbiome changes are suspected in Alzheimer disease, Parkinson disease, chronic depression and autism spectrum disorders. This article introduces an extended 'epithelial barrier hypothesis', which proposes that the increase in epithelial barrier-damaging agents linked to industrialization, urbanization and modern life underlies the rise in allergic, autoimmune and other chronic conditions. Furthermore, it discusses how the immune responses to dysbiotic microbiota that cross the damaged barrier may be involved in the development of these diseases.

Targeted Amino Acid Substitutions in a Trichoderma Peptaibol Confer Activity against Fungal Plant Pathogens and Protect Host Tissues from Botrytis cinerea Infection.

Fungal species belonging to the Trichoderma genus are commonly used as biocontrol agents against several crop pathogens. Among their secondary metabolites, peptaibols are helical, antimicrobial peptides, which are structurally stable even under extreme pH and temperature conditions. The promise of peptaibols as agrochemicals is, however, hampered by poor water solubility, which inhibits efficient delivery for practical use in crop protection. Using a versatile synthetic strategy, based on green chemistry procedures, we produced water-soluble analogs of the short-length peptaibol trichogin. Although natural trichogin was inactive against the tested fungal plant pathogens (Botrytis cinerea, Bipolaris sorokiniana, Fusarium graminearum, and Penicillium expansum), three analogs completely inhibited fungal growth at low micromolar concentrations. The most effective peptides significantly reduced disease symptoms by B. cinerea on common bean and grapevine leaves and ripe grape berries without visible phytotoxic effects. An in-depth conformational analysis featuring a 3D-structure–activity relationship study indicated that the relative spatial position of cationic residues is crucial for increasing peptide fungicidal activity.

Mobilization of Tissue-Resident Memory CD4+ T Lymphocytes and Their Contribution to a Systemic Secondary Immune Reaction

While it is generally accepted that tissue-resident memory T lymphocytes protect host tissues from secondary immune challenges, it is unclear whether, and if so, how they contribute to systemic secondary immune responses. Here we show that when individuals with an established immunological memory to measles, mumps and rubella viruses are re-challenged with the measles-mumps-rubella (MMR) vaccine, tissue-resident memory CD4+ T cells are mobilized into the blood within 16 to 48 hours after vaccination before disappearing again soon after. The mobilization of tissue-resident memory T cells was found to be cognate: memory CD4+ T lymphocytes that recognize other antigens, e.g. tetanus toxin, are not mobilized, unless they cross-react with antigens present in the vaccine. Post immunization, mobilized T-cell receptor Vβ clonotypes from day 1 were represented among the newly generated circulating memory CD4+ T-cell repertoire from day 14. These data demonstrate that tissue-resident memory T cells can leave their tissue and contribute significantly to secondary systemic immune responses.

Renal Purge of Hemolymphatic Lipids Prevents the Accumulation of ROS-Induced Inflammatory Oxidized Lipids and Protects Drosophila from Tissue Damage

Animals require complex metabolic and physiological adaptations to maintain the function of vital organs in response to environmental stresses and infection. Here, we found that infection or injury in Drosophila induced the excretion of hemolymphatic lipids by Malpighian tubules, the insect kidney. This lipid purge was mediated by a stress-induced lipid-binding protein, Materazzi, which was enriched in Malpighian tubules. Flies lacking materazzi had higher hemolymph concentrations of reactive oxygen species (ROS) and increased lipid peroxidation. These flies also displayed Malpighian tubule dysfunction and were susceptible to infections and environmental stress. Feeding flies with antioxidants rescued the materazzi phenotype, indicating that the main role of Materazzi is to protect the organism from damage caused by stress-induced ROS. Our findings suggest that purging hemolymphatic lipids presents a physiological adaptation to protect host tissues from excessive ROS during immune and stress responses, a process that is likely to apply to other organisms.

Mucociliary Defense: Emerging Cellular, Molecular, and Animal Models.

Respiratory tissues are bombarded by billions of particles daily. If allowed to accumulate, these particles can cause injury, inflammation, or infection, and thus may significantly disrupt airflow and gas exchange. Mucociliary defense, a primary mechanism for protecting host tissues, operates through the coordinated functions of mucus and cilia that trap and eliminate inhaled materials. Mucociliary function is also required for the elimination of endogenous cells and debris. Although defense is necessarily robust, it is also tightly regulated to minimize physiologic disruption of the host. Indeed, mucociliary dysfunction contributes to the pathogenesis of many lung diseases-including asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, and cystic fibrosis-in which airflow limitation, inflammation, persistent tissue injury, and structural remodeling occur. Here, we highlight recent advances in cilia and mucin biology, the importance of well-controlled mucociliary interactions, and the need to better understand how these regulate innate barrier and immune defense.

Roles of DNA repair enzyme OGG1 in innate immunity and its significance for lung cancer.

Cytokines are pivotal mediators of the immune response, and their coordinated expression protects host tissue from excessive damage and oxidant stress. Nevertheless, the development of lung pathology, including asthma, chronic obstructive pulmonary disease, and ozone-induced lung injury, is associated with oxidant stress; as evidence, there is a significant increase in levels of the modified guanine base 7,8-dihydro-8-oxoguanine (8-oxoG) in the genome. 8-OxoG is primarily recognized by 8-oxoguanine glycosylase 1 (OGG1), which catalyzes the first step in the DNA base excision repair pathway. However, oxidant stress in the cell transiently halts enzymatic activity of substrate-bound OGG1. The stalled OGG1 facilitates DNA binding of transactivators, including NF-κB, to their cognate sites to enable expression of cytokines and chemokines, with ensuing recruitments of inflammatory cells. Hence, defective OGG1 will modulate the coordination between innate and adaptive immunity through excessive oxidant stress and cytokine dysregulation. Both oxidant stress and cytokine dysregulation constitute key elements of oncogenesis by KRAS, which is mechanistically coupled to OGG1. Thus, analysis of the mechanism by which OGG1 modulates gene expression helps discern between beneficial and detrimental effects of oxidant stress, exposes a missing functional link as a marker, and yields a novel target for lung cancer.

Variation in Macrophage Phagocytosis of Streptococcus agalactiae Does Not Reflect Bacterial Capsular Serotype, Multilocus Sequence Type or Association with Invasive Infection

Background:Group B Streptococcus (GBS) is an encapsulated Gram-positive coccus that is an important cause of infections in adults with chronic medical conditions, pregnant women, and neonates. GBS causes a range of clinical syndromes, from asymptomatic colonization to deep-seated invasive and highly lethal infections. Macrophages are important sentinels of innate immunity, protecting host tissues from infection when bacteria advance beyond cutaneous or mucosal barriers. We hypothesized that the capacity for macrophages to phagocytose unopsonized GBS would vary across distinct clinical strains, and such differences would reflect serotype diversity.Methods:A high-throughput screen using the phorbol ester-differentiated THP-1 macrophage-like human cell line was used to quantify phagocytosis of a diverse group of 35 different human clinical isolates of GBS representing a wide variety of capsular serotypes. Validation studies were conducted using human primary phagocytes.Results:Phagocytosis of GBS differed widely across clinical isolates but this was not related to capsular serotype, genetic sequence type, pilus type, or clinical source of the GBS isolate (colonizing or invasive strain).Conclusions:Structural and/or biochemical differences among diverse GBS strains are reflected in a diverse capacity for macrophages to ingest them through non-opsonic phagocytosis. Mechanisms explaining these differences are not clear.

Immunometabolic Phenotype Alterations Associated with the Induction of Disease Tolerance and Persistent Asymptomatic Infection of Salmonella in the Chicken Intestine.

The adaptation of Salmonella enterica to the eukaryotic host is a key process that enables the bacterium to survive in a hostile environment. Salmonella have evolved an intimate relationship with its host that extends to their cellular and molecular levels. Colonization, invasion, and replication of the bacteria in an appropriate host suggest that modification of host functions is central to pathogenesis. Intuitively, this subversion of the cell must be a complex process, since hosts are not inherently programmed to provide an environment conducive to pathogens. Hosts have evolved countermeasures to pathogen invasion, establishment, and replication through two types of defenses: resistance and tolerance. Resistance functions to control pathogen invasion and reduce or eliminate the invading pathogen. Research has primarily concentrated on resistance mechanisms that are mediated by the immune system. On the other hand, tolerance is mediated by different mechanisms that limit the damage caused by a pathogen’s growth without affecting or reducing pathogen numbers or loads. The mechanisms of tolerance appear to be separated into those that protect host tissues from the virulence factors of a pathogen and those that limit or reduce the damage caused by the host immune and inflammatory responses to the pathogen. Some pathogens, such as Salmonella, have evolved the capacity to survive the initial robust immune response and persist. The persistent phase of a Salmonella infection in the avian host usually involves a complex balance of protective immunity and immunopathology. Salmonella is able to stay in the avian ceca for months without triggering clinical signs. Chronic colonization of the intestinal tract is an important aspect of persistent Salmonella infection because it results in a silent propagation of bacteria in poultry stocks due to the impossibility to isolate contaminated animals. Data from our lab promote the hypothesis that Salmonella have evolved a unique survival strategy in poultry that minimizes host defenses (disease resistance) during the initial infection and then exploits and/or induces a dramatic immunometabolic reprogramming in the cecum that alters the host defense to disease tolerance. Unfortunately, this disease tolerance results in the ongoing human food safety dilemma.

Recruitment of Factor H as a Novel Complement Evasion Strategy for Blood-Stage Plasmodium falciparum Infection.

The human complement system is the frontline defense mechanism against invading pathogens. The coexistence of humans and microbes throughout evolution has produced ingenious molecular mechanisms by which microorganisms escape complement attack. A common evasion strategy used by diverse pathogens is the hijacking of soluble human complement regulators to their surfaces to afford protection from complement activation. One such host regulator is factor H (FH), which acts as a negative regulator of complement to protect host tissues from aberrant complement activation. In this report, we show that Plasmodium falciparum merozoites, the invasive form of the malaria parasites, actively recruit FH and its alternative spliced form FH-like protein 1 when exposed to human serum. We have mapped the binding site in FH that recognizes merozoites and identified Pf92, a member of the six-cysteine family of Plasmodium surface proteins, as its direct interaction partner. When bound to merozoites, FH retains cofactor activity, a key function that allows it to downregulate the alternative pathway of complement. In P. falciparum parasites that lack Pf92, we observed changes in the pattern of C3b cleavage that are consistent with decreased regulation of complement activation. These results also show that recruitment of FH affords P. falciparum merozoites protection from complement-mediated lysis. Our study provides new insights on mechanisms of immune evasion of malaria parasites and highlights the important function of surface coat proteins in the interplay between complement regulation and successful infection of the host.

Blimp-1-Dependent IL-10 Production by Tr1 Cells Regulates TNF-Mediated Tissue Pathology.

Tumor necrosis factor (TNF) is critical for controlling many intracellular infections, but can also contribute to inflammation. It can promote the destruction of important cell populations and trigger dramatic tissue remodeling following establishment of chronic disease. Therefore, a better understanding of TNF regulation is needed to allow pathogen control without causing or exacerbating disease. IL-10 is an important regulatory cytokine with broad activities, including the suppression of inflammation. IL-10 is produced by different immune cells; however, its regulation and function appears to be cell-specific and context-dependent. Recently, IL-10 produced by Th1 (Tr1) cells was shown to protect host tissues from inflammation induced following infection. Here, we identify a novel pathway of TNF regulation by IL-10 from Tr1 cells during parasitic infection. We report elevated Blimp-1 mRNA levels in CD4+ T cells from visceral leishmaniasis (VL) patients, and demonstrate IL-12 was essential for Blimp-1 expression and Tr1 cell development in experimental VL. Critically, we show Blimp-1-dependent IL-10 production by Tr1 cells prevents tissue damage caused by IFNγ-dependent TNF production. Therefore, we identify Blimp-1-dependent IL-10 produced by Tr1 cells as a key regulator of TNF-mediated pathology and identify Tr1 cells as potential therapeutic tools to control inflammation.

Induced CD8+FoxP3+ Treg cells in rheumatoid arthritis are modulated by p38 phosphorylation and monocytes expressing membrane tumor necrosis factor α and CD86.

Limiting the severity of inflammation and promoting its eventual resolution are vital for protecting host tissues both in autoimmunity and chronic infection. The aim of this study was to determine the suitability of repurposing anti-CD3 monoclonal antibody (mAb) therapy for rheumatoid arthritis (RA) by analyzing its ability to induce CD8+FoxP3+ Treg cells from peripheral blood mononuclear cells (PBMCs). Anti-CD3 mAb was cultured with RA PBMCs to induce CD8+FoxP3+ Treg cells, which were analyzed by flow cytometry to determine their phenotype. Treg cell induction was investigated via neutralization or blocking antibodies, cellular depletion, or ImageStream technology. Blotting was used to determine the signaling pathways involved in CD8+FoxP3+ Treg cell induction. Suppression of CD4+ T cell effector responses was assessed by Treg cell suppression assays and Mosaic enzyme-linked immunosorbent assay. Potent CD8+FoxP3+ Treg cells were induced from RA PBMCs by anti-CD3 mAb. Unlike their CD4+ counterparts, CD8+FoxP3+ Treg cells inhibited Th17 responses in a contact-dependent manner, thereby functioning to limit a wider range of inflammatory pathways. CD8+FoxP3+ Treg cell induction was supported both by p38 phosphorylation intrinsic to naive CD8+ T cells and by monocytes via CD86 and membrane tumor necrosis factor α (TNFα). Artificially increasing monocyte membrane TNFα or inhibiting CD8+ T cell p38 phosphorylation drove FoxP3 expression in a subset of initially unresponsive CD8+ T cells. These data define an unknown mechanism of CD8+FoxP3+ Treg cell induction by anti-CD3 mAb, which could be combined with a p38 inhibitor to improve therapeutic efficacy in RA patients and resolve chronic inflammation via the restoration of tolerance.

Liver Disease in Alpha-1 Antitrypsin Deficiency: Current Understanding and Future Therapy

Alpha-1 antitrypsin Deficiency (AATD) is a common, but under recognized metabolic genetic disease. Although many mutations in the alpha-1 antitrypsin (AAT) gene are described, the Z variant is the allele overwhelmingly associated with liver disease. PI*ZZ homozygotes occur in approximately 1 in 2,000-5,000 births in North American and European populations. The AAT protein is synthesized in large quantities by the liver, and then secreted into serum. Its physiologic function is to inhibit neutrophil proteases in order to protect host tissues from non-specific injury during periods of inflammation. The mutant Z gene of AAT directs the synthesis of a mutant protein which folds abnormally during biogenesis in the endoplasmic reticulum of hepatocytes and is retained intracellularly, rather than efficiently secreted. Intracellular proteolysis pathways, including the proteasome and autophagy, are activated as a response to the intracellular burden of misfolded protein. The lack of circulating anti-protease activity leaves the lung vulnerable to injury and the development of emphysema. The intracellular accumulation of AAT mutant Z protein within hepatocytes can cause liver injury, cirrhosis and hepatocellular carcinoma by triggering a cascade of chronic hepatocellular apoptosis, regeneration, and end organ injury. There is no specific treatment for PI*ZZ associated liver disease, other than standard liver supportive care and liver transplantation. There is a high degree of variability in the clinical manifestations among PI*ZZ homozygous patients, suggesting a strong influence of as yet poorly characterized, genetic and environmental disease modifiers. Studies of the processes of intracellular injury have led to a new era of rational therapeutic development.

Dermal exposure to immunostimulants induces changes in activity and proliferation of coelomocytes of Eisenia andrei.

Due to the specific habitat conditions in which they live, earthworms are constantly exposed to pathogens. Consequently, they have evolved various immuno-defense mechanisms, including cellular (coelomocytes) and humoral responses, which may help to eliminate deleterious micro-organisms but also repair and/or protect host cells and tissues. Similar to mammalian phagocytes, coelomocytes can kill ingested pathogens with reactive oxygen species (ROS) and nitric oxide. In the present work, we studied the effects of the dermal exposure of Eisenia andrei earthworms to different immuno-stimulants: phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS) or concanavalin A (ConA). After 3 days of treatment with all immuno-stimulants, decreased numbers and changed composition of the coelomocytes were observed. The immuno-stimulants also induced numerous changes in bactericidal activity, including ROS production. Furthermore, all stimulants increased cell proliferation while only LPS-treatment significantly elevated apoptosis of coelomocytes. These results demonstrate that in vivo treatment of earthworms with immuno-stimulants induces various changes in their coelomocyte response.

Crystallographic determination of the disease-associated T1184R variant of complement regulator factor H

The soluble 155 kDa glycoprotein factor H (FH) protects host tissue from damage by the human complement system. It accelerates decay of the alternative-pathway C3 convertase, C3bBb, and is a cofactor for factor I-mediated cleavage of theopsonin C3b. Numerous mutations and single-nucleotide polymorphisms (SNPs) occur in the gene encoding FH and the resulting missense mutations and truncation products result in altered functionality that predisposes to the development of the serious renal condition atypical haemolytic uraemic syndrome (aHUS). Other polymorphisms are linked to membranoproliferative glomerulonephritis and macular degeneration. The two C-terminal modules of FH (FH19-20) harbour numerous aHUS-associated mutations that disrupt the ability of factor H to protect host cells from complement-mediated damage. In this work, the crystal structure of an aHUS-associated T1184R variant of FH19-20 at a resolution of 1.52 Å is described. It is shown that this mutation has negligible structural effects but causes a significant change in the electrostatic surface of these two domains. Mechanisms are discussed by which this mutation may alter FH-ligand interactions, particularly with regard to the extension of a region of this molecule within module 20 that has been associated with the binding of glycosaminoglycans (GAGs) or sialic acid residues.

Alpha-1-Antitrypsin Deficiency and Mechanisms of Liver Disease

Alpha-1-antitrypsin (a1AT) Deficiency is a metabolic genetic disease in which individuals homozygous for the mutant Z a1AT gene are at risk for liver and lung disease. Homozygotes, called PIZZ in World Health Organization nomenclature, occur in approximately 1 in 2000 births in North American and European populations. A1AT is a protein synthesized in large quantities by the liver, leukocytes, and other tissues and then secreted into serum and extracellular fluid. Its physiologic function is to inhibit neutrophil proteases during periods of inflammation in order to protect host tissues from non-specific inflammatory injury. The mutant Z gene of a1AT directs the synthesis of a mutant protein which folds abnormally in the endoplasmic reticulum of hepatocytes during biogenesis and is retained intracellularly rather than being efficiently secreted. This intracellular accumulation of a1AT mutant Z protein within hepatocytes can cause liver injury, cirrhosis and hepatocellular carcinoma, and the lack of circulating anti-protease activity leaves the lung vulnerable to injury and the development of emphysema. There is a high degree of variability in the clinical manifestations among PIZZ patients, suggesting a strong influence of genetic and environmental disease modifiers. Cigarette smoking has been identified as an especially strong risk factor for the development of PIZZ associated emphysema. The accumulation of the a1AT mutant Z protein within hepatocytes triggers an injury cascade which includes aspects of ER stress, caspase activation, and apoptosis which appears to lead to liver injury, fibrosis and cirrhosis. There is no specific treatment for PIZZ associated liver disease, other than standard liver disease supportive care and liver transplantation. Studies of the processes of intracellular injury, and of new therapies for this disease, are areas of intense and ongoing investigation.

Hypertonic saline increases γδT cell-mediated killing of activated neutrophils

Hypertonic saline fluids used to resuscitate trauma patients can prevent neutrophil-mediated lung tissue damage, making them attractive alternatives to conventional resuscitation fluids. We have previously shown that gammadeltaT cells, a small T lymphocyte subset, reduce acute inflammatory lung damage by eliminating activated neutrophils that express heat shock protein 72 on the cell surface. Here, we studied whether these protective effects of hypertonic saline are related to improved gammadeltaT cell-mediated neutrophil killing. Laboratory investigation. University research laboratory. Human peripheral blood from healthy subjects--isolated gammadeltaT lymphocytes and neutrophils. Isolated blood cells were treated with different concentrations of hypertonic saline and endotoxin of Escherichia coli O111:B4 (lipopolysaccharide). In some experiments, gammadeltaT cells were activated by CD3 cross-linking or by phorbol-myristate acetate and ionomycin, or by phytohemagglutinin. Clinically relevant concentrations of hypertonic saline (20 mM) significantly augmented CD69 expression of gammadeltaT cells that were stimulated with 100 ng/mL lipopolysaccharide. Additionally, lipopolysaccharide induced a three- to five-fold increase in tumor necrosis factor-alpha and interleukin-10 expression by gammadeltaT cells. This response was completely abrogated by hypertonic saline. These data indicate that hypertonic saline can modulate gammadeltaT cell functions. Stimulation of neutrophils with 1-1,000 ng/mL lipopolysaccharide caused a greater than 3-fold increase in heat shock protein-72 expression on the cell surface, which was significantly augmented by hypertonic saline. In cocultures of gammadeltaT cells with autologous neutrophils, 15.6 +/- 3.4% of all neutrophils were killed within 120 min. In the presence of lipopolysaccharide (1 microg/mL), this percentage increased to 23.7 +/- 2.1%, and it was further increased to 31.8 +/- 3.1% when 20 mM hypertonic saline was added with lipopolysaccharide. Our findings suggest that hypertonic saline enhances the elimination of inflammatory neutrophils by gammadeltaT cells by augmenting heat shock protein-72 expression on the cell surface of neutrophils. Hypertonic saline resuscitation may therefore protect host tissues by enhancing neutrophil clearance from the lungs.

Complement-Dependent Enhancement of CD8+ T Cell Immunity to Lymphocytic Choriomeningitis Virus Infection in Decay-Accelerating Factor-Deficient Mice

Decay-accelerating factor (DAF, CD55) is a GPI-anchored membrane protein that regulates complement activation on autologous cells. In addition to protecting host tissues from complement attack, DAF has been shown to inhibit CD4+ T cell immunity in the setting of model Ag immunization. However, whether DAF regulates natural T cell immune response during pathogenic infection is not known. We describe in this study a striking regulatory effect of DAF on the CD8+ T cell response to lymphocytic choriomeningitis virus (LCMV) infection. Compared with wild-type mice, DAF knockout (Daf-1(-/-)) mice had markedly increased expansion in the spleen of total and viral Ag-specific CD8+ T cells after acute or chronic LCMV infection. Splenocytes from LCMV-infected Daf-1(-/-) mice also displayed significantly higher killing activity than cells from wild-type mice toward viral Ag-loaded target cells, and Daf-1(-/-) mice cleared LCMV more efficiently. Importantly, deletion of the complement protein C3 or the receptor for the anaphylatoxin C5a (C5aR) from Daf-1(-/-) mice reversed the enhanced CD8+ T cell immunity phenotype. These results demonstrate that DAF is an important regulator of CD8+ T cell immunity in viral infection and that it fulfills this role by acting as a complement inhibitor to prevent virus-triggered complement activation and C5aR signaling. This mode of action of DAF contrasts with that of CD59 in viral infection and suggests that GPI-anchored membrane complement inhibitors can regulate T cell immunity to viral infection via either a complement-dependent or -independent mechanism.

The Factor H Variant Associated with Age-related Macular Degeneration (His-384) and the Non-disease-associated Form Bind Differentially to C-reactive Protein, Fibromodulin, DNA, and Necrotic Cells

Recently, a polymorphism in the complement regulator factor H (FH) gene has been associated with age-related macular degeneration. When histidine instead of tyrosine is present at position 384 in the seventh complement control protein (CCP) domain of FH, the risk for age-related macular degeneration is increased. It was recently shown that these allotypic variants of FH, in the context of a recombinant construct corresponding to CCPs 6-8, recognize polyanionic structures differently, which may lead to altered regulation of the alternative pathway of complement. We show now that His-384, corresponding to the risk allele, binds C-reactive protein (CRP) poorly compared with the Tyr-384 form. We also found that C1q and phosphorylcholine do not compete with FH for binding to C-reactive protein. The interaction with extracellular matrix protein fibromodulin, which we now show to be mediated, at least in part, by CCP6-8 of FH, occurs via the polypeptide of fibromodulin and not through its glycosaminoglycan modifications. The Tyr-384 variant of FH bound fibromodulin better than the His-384 form. Furthermore, we find that CCP6-8 is able to interact with DNA and necrotic cells, but in contrast the His-384 allotype binds these ligands more strongly than the Tyr-384 variant. The variations in binding affinity of the two alleles indicate that complement activation and local inflammation in response to different targets will differ between His/His and Tyr/Tyr homozygotes.