Line Of Innate Immunity Research Articles

Zoledronic acid affects the process of Porphyromonas gingivalis infecting oral mucosal epithelial barrier: An in-vivo and in-vitro study.

Zoledronic acid (ZA), one of the commonly used bisphosphonates, is mainly used for bone-metabolic diseases. Studies proved that ZA has adverse effects on oral soft tissues. As the first line of innate immunity, the gingival epithelium could be infected by periodontal pathogens, which is a key process of the initiation of periodontal diseases. Yet, how ZA affects the periodontal pathogens infecting the epithelial barrier remains unclear. This study aimed to investigate the influences of ZA on the process of Porphyromonas gingivalis (P. gingivalis) infecting the gingival epithelial barrier via in-vitro and in-vivo experiments. In the in-vitro experiments, under the condition of different concentrations of ZA (0, 1, 10, and 100 μM), P. gingivalis was used to infect human gingival epithelial cells (HGECs). The infections were detected by transmission electron microscope and confocal laser scanning microscope. Besides, the internalization assay was applied to quantify the P. gingivalis, which infected the HGECs, in the different groups. To evaluate the expression levels of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, and IL-8, by infected HGECs, real-time quantitative reverse transcription-polymerase chain reactions were applied. In the in-vivo experiments, rats were given ZA solution (ZA group) or saline (control group) by tail intravenous injection for 8 weeks. Subsequently, we put ligatures around the maxillary second molars of all the rats and inoculated P. gingivalis to the gingiva every other day from day 1 to day 13. The rats were sacrificed on days 3, 7, and 14 for micro-CT and histological analyses. The in-vitro results manifested that the quantity of P. gingivalis that had infected HGECs increased with the ZA concentrations. Pro-inflammatory cytokines expression by HGECs were significantly increased by 100 μM ZA. In the in-vivo study, compared to the control group, more P. gingivalis was detected in the superficial layer of gingival epithelium in the ZA group. Besides, ZA significantly increased the expression level of IL-1β on day 14 and IL-6 on days 7 and 14 in gingival tissues. These findings suggest that the oral epithelial tissues of patients who receive high-dose ZA treatment may be more susceptible to periodontal infections, resulting in severe inflammatory conditions.

Macrophage Stimulation as a Useful Approach for Immunoscreening of Potential Vaccine Candidates Against Toxoplasma gondii and Neospora caninum Infections.

Toxoplasmosis and neosporosis are protozoan diseases that adversely affect the medical and additionally veterinary sectors, respectively. Toxoplasmosis is caused by Toxoplasma gondii which infects almost all warm-blooded animals including humans. While, neosporosis is caused by Neospora caninum, which induces infection in many animal species particularly in cattle. Currently, control measures for both infections are defective because of no effective vaccine or treatment. Macrophages constitute the first line of innate immunity, which contributes to the effective elimination of T. gondii or N. caninum. This action is mediated by IL-12, which is critical for the secretion of interferon gamma (IFN-γ). Successful vaccine candidates against both protozoan parasites should possess the ability to induce the cellular immune response and IFN-γ production. In this chapter, we will focus on an efficient immunological approach for discovery of potential vaccine candidates against above-mentioned parasites. Our previous studies revealed a strong correlation between vaccine antigens that enhanced the macrophage secretion of IL-12 and their efficacy as potential vaccine candidates in murine model. In case of T. gondii, peroxiredoxin 1 (TgPrx1) and peroxiredoxin 3 stimulated the production of IL-12 from murine peritoneal macrophages and conferred strong to moderate protection in C57BL/6 mice, respectively. At the same context, Neospora antigens of dense granule protein 6 (NcGRA6) and cyclophilin entrapped with oligo-mannose coated-liposomes stimulated macrophage IL-12 secretion and substantially protected immunized BALB/c mice. Therefore, we can deduce that macrophage stimulation evidenced in IL-12 production can be used as a useful approach for judgment of vaccine efficacy before further evaluation using in vivo experiments. Methods of vaccine preparation and macrophage stimulation will be fully described for TgPrx1 and NcGRA6 as potential vaccine candidates against toxoplasmosis and neosporosis, respectively.

Developmental and Tissue Patterns of the Basal Expression of Chicken Avian β-Defensins.

Defensins are a class of antimicrobial peptides in vertebrates that function as the first line of innate immunity with potent antimicrobial and immunomodulatory activities. Fourteen defensins, namely, avian β-defensin 1 to 14 (AvBD1-14), have been identified in chickens. Before characterizing the role of AvBDs in innate immunity during the early development of chickens, we collected tissue segments from the liver, spleen, and gastrointestinal (GI) tract including the esophagus, crop, proventriculus, gizzard, duodenum, jejunum, ileum, cecum, and colon from broilers at days 1, 3, 7, 14, and 28. After RNA isolation and reverse transcription, we determined the expression levels of the 14 AvBD genes in these tissues during the first 28 days after hatching by real-time PCR. The results suggested the AvBDs were widely expressed in the chicken liver, spleen, and gastrointestinal (GI) tract. Interestingly, we did not detect AvBD11 expressed in the GI tract, even in the liver and spleen. Additionally, AvBDs were differentially expressed in the chicken GI tract. AvBD5 and AvBD14 were expressed most abundantly in the proximal GI tract, especially the esophagus and crop. Moreover, AvBD5, AvBD7, AvBD9, and AvBD14 were expressed in an inverted-V pattern with the peak being the observed expression at days 3, 7, or 14 in the chicken spleen, esophagus, duodenum, and cecum. Other AvBDs presented biphasic or inverted-V expression patterns in different tissues. The expression levels of all detected AvBDs were strengthened after hatching rather than decreasing steadily. Therefore, AvBDs were found to be expressed widely in the chicken liver, spleen, and GI tract and their expression levels were primarily up regulated during the early development of chicken, implying the potential essential roles of AvBDs in early innate defense and infection resistance of chickens.

Endothelial-Derived Interleukin-1α Activates Innate Immunity by Promoting the Bactericidal Activity of Transendothelial Neutrophils.

Migration of neutrophils across endothelial barriers to capture and eliminate bacteria is served as the first line of innate immunity. Bacterial virulence factors damage endothelium to produce inflammatory cytokines interacts with neutrophils. However, the mechanisms that behind endothelial-neutrophil interaction impact on the bactericidal activity remain unclear. Therefore, we aimed to find the target proteins on endothelial cells that triggered the bactericidal activity of transendothelial neutrophils. Herein, we built the infected models on rats and endothelial-neutrophil co-cultural system (Transwell) and discovered that endothelial-derived IL-1α promoted the survival of rats under Escherichia coli infection and enhanced the bactericidal activity of transendothelial neutrophils in vivo and in vitro. Results further showed that IL-1α was inhibited by lipopolysaccharide (LPS) in the endothelial-neutrophil interaction. We found that LPS mainly damaged cell membrane and induced cell necrosis to interrupt neutrophil migration from endothelial barrier. Thus, we used the isobaric tags for relative and absolute quantification (iTRAQ) method to identify different proteins of endothelial cells. Results showed that IL-1α targeted cellular plasma membrane, endoplasmic reticulum and mitochondrial envelope and triggered eleven common proteins to persistently regulate. During the early phase, IL-1α triggered the upregulation of cell adhesion molecules (CAMs) to promote neutrophil adhesion, while oxidative phosphorylation was involved in long time regulation to induce transmigration of neutrophils against bacteria. Our results highlight the critical mechanism of endothelial-derived IL-1α on promoting bactericidal activity of transendothelial neutrophils and the findings of IL-1α triggered proteins provide the potentially important targets on the regulation of innate immunity.

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

An in vitro generated variant of Tephrosia villosa defensin (α-TvD1) enhances biotic stress tolerance in transgenic tobacco

Plant defensins are small, cationic, cysteine-rich peptides with well conserved four disulphide bridges between an α-helix and three anti-parallel β-strands, produced by plants as a first line of innate immunity against the invading fungal pathogens. Some of the plant defensins exhibit strong antibacterial and anti-insect activities in vitro as well as in vivo. TvD1, a defensin from Tephrosia villosa was characterized in our previous studies with demonstrated inhibitory effect against many plant pathogenic fungi and some insects. To further enhance its activity, α-TvD1, a peptide variant of TvD1, was generated through site directed mutagenesis. It showed enhanced antifungal and Tenebrio molitor α-amylase inhibitory activity under in vitro condition. In the present study, we compared the antifungal and anti-insect activities of α-TvD1 with the native TvD1 in transgenic tobacco plants. α-TvD1 transgenic plants showed enhanced tolerance against the oomycetes pathogen Phytophthora parasitica var. nicotianae and the fungal pathogens Alternaria alternata and Rhizoctonia solani. Also, it showed enhanced anti-insect activity against the 1st and 2nd instar larvae of the generalist herbivore, Spodoptera litura. These results demonstrated that α-TvD1 appears to be a potent antimicrobial gene that can be used for generating superior transgenic crops with enhanced resistance against many phytopathogens and insect pests.

Potential roles of neutrophils in maintaining the health and productivity of dairy cows during various physiological and physiopathological conditions: a review.

Neutrophils represent the first line of innate immunity and are the most prominent line of cellular defence against invading microorganisms. On stimulation, they can quickly move through the walls of veins and into the tissues of the body to immediately attack or monitor the foreign antigens. Neutrophils are highly versatile and sophisticated cells which are endowed with highly sensitive receptor-based perception systems. They were traditionally classified as short-lived phagocytes actively involved during infection and inflammation, but recently, it has been seen that neutrophils are capable of detecting the presence of sperms during insemination as well as an implanting embryo in the female reproductive tract. These specialised phagocytes play a major role in tissue remodelling and wound healing, and maintain homeostasis during parturition, expulsion of placenta, folliculogenesis, corpus luteum formation and luteolysis. Here, we review the role played by neutrophils in maintaining homeostasis during normal and inflammatory conditions of dairy cattle. We have summarised the alteration in the expression of some cell adhesion molecules and cytokines on bovine neutrophils during different physiological and physiopathological conditions. Some emerging issues in the field of neutrophil biology and the possible strategies to strengthen their activity during the period of immunosuppression have also been discussed.

The inflammatory response of neutrophils in an in vitro model that approximates the postcardiac arrest state.

PurposePostcardiac arrest syndrome (PCAS) shares many features with sepsis including plasma cytokine elevation with dysregulation of cytokine production, and the presence of endotoxin in plasma. PCAS is closely related to ischemia-reperfusion injury. During ischemia-reperfusion injury, neutrophil, which is the first line of innate immunity, plays a major role. In this study, we investigated the inflammatory response of human neutrophils in an in vitro model which we simulated with hypoxia-normoxia and hypoxia-hyperoxia environments.MethodsAfter separation of neutrophils from the whole blood, they were divided into 3 experimental groups: normoxia-normoxia, hypoxia-normoxia, and hypoxia-hyperoxia groups. The production of H2O2, the expression of Toll-like receptor 4 (TLR4) receptor, and the extent of apoptosis of the neutrophils were checked.ResultsThe in vitro hypoxia-normoxia and -hyperoxia models, which simulated the PCAS, showed initiation of the neutrophils' inflammatory reaction by hypoxia insult. Lipopolysaccharide amplifies such inflammation; therefore, prevention of secondary infection may be critical in postresuscitation patients. Temporary hyperoxia following hypoxic insult showed no difference in inflammatory reaction compared with hypoxia-normoxia. Rather, temporary hyperoxia may suppress or minimize inflammation by attenuation of TLR4 receptor.ConclusionIt is well known that continuous hyperoxygenation after successful cardiac arrest harms patients, but temporary hyperoxygenation with 100% O2 in a clinical situation may be helpful.

Neutrophils from chronic lymphocytic leukemia patients exhibit an increased capacity to release extracellular traps (NETs).

Chronic lymphocytic leukemia (CLL) is characterized by immune defects that contribute to a high rate of infections and autoimmune cytopenias. Neutrophils are the first line of innate immunity and respond to pathogens through multiple mechanisms, including the release of neutrophil extracellular traps (NETs). These web-like structures composed of DNA, histones, and granular proteins are also produced under sterile conditions and play important roles in thrombosis and autoimmune disorders. Here we show that neutrophils from CLL patients are more prone to release NETs compared to those from age-matched healthy donors (HD). Increased generation of NETs was not due to higher levels of elastase, myeloperoxidase, or reactive oxygen species production. Instead, we found that plasma from CLL patients was able to prime neutrophils from HD to generate higher amounts of NETs upon activation. Plasmatic IL-8 was involved in the priming effect since its depletion reduced plasma capacity to enhance NETs release. Finally, we found that culture with NETs delayed spontaneous apoptosis and increased the expression of activation markers on leukemic B cells. Our study provides new insights into the immune dysregulation in CLL and suggests that the chronic inflammatory environment typical of CLL probably underlies this inappropriate neutrophil priming.

Melanocytes and melanin represent a first line of innate immunity against Candida albicans.

Melanocytes are dendritic cells located in the skin and mucosae that synthesize melanin. Some infections induce hypo- or hyperpigmentation, which is associated with the activation of Toll-like receptors (TLRs), especially TLR4. Candida albicans is an opportunist pathogen that can switch between blastoconidia and hyphae forms; the latter is associated with invasion. Our objectives in this study were to ascertain whether C. albicans induces pigmentation in melanocytes and whether this process is dependent on TLR activation, as well as relating this with the antifungal activity of melanin as a first line of innate immunity against fungal infections. Normal human melanocytes were stimulated with C. albicans supernatants or with crude extracts of the blastoconidia or hyphae forms, and pigmentation and TLR2/TLR4 expression were measured. Expression of the melanosomal antigens Melan-A and gp100 was examined for any correlation with increased melanin levels or antifungal activity in melanocyte lysates. Melanosomal antigens were induced earlier than cell pigmentation, and hyphae induced stronger melanization than blastoconidia. Notably, when melanocytes were stimulated with crude extracts of C. albicans, the cell surface expression of TLR2/TLR4 began at 48 h post-stimulation and peaked at 72 h. At this time, blastoconidia induced both TLR2 and TLR4 expression, whereas hyphae only induced TLR4 expression. Taken together, these results suggest that melanocytes play a key role in innate immune responses against C. albicans infections by recognizing pathogenic forms of C. albicans via TLR4, resulting in increased melanin content and inhibition of infection.

Interaction of Antimicrobial Peptides with Bacterial Membrane

The antimicrobial peptides AMPs represent abundant and diverse group of molecules, which are evolved from many tissues and cells in the plant and animal kingdoms as protective reaction against infection organisms. They build a very strong first line of innate immunity. Despite their enormous variety, most AMPs work directly against microbes, through a mechanism involving membrane disintegration or/and formation of pores, which allows easier passage of the ions and the basic nutrients. Molecular mechanisms, and the way of penetration through the membrane can be varied to various peptides, depending on the sequence of amino acids, the lipid composition of the membrane and the concentration of peptides, but for the beginning of their action, they must first be attracted to the bacterial surface. The objective of the present examination is namely the so called S-state, or an inactive state in which an AMP is oriented parallel before binding to the membrane. To this end we have conducted molecular dynamics studies. The conformational changes in the quaternary structure of one of the most popular antimicrobial peptides - magainin 2 (alpha-helical positively charged amphiphilic peptide) near to a charged lipid bilayer were examined. We have analyzed the behavior of peptides at their connection to the bilayers and their impact on the lateral organization of charged lipids. The system for MD simulation consists of a charged, equilibrated membrane (five uncharged POPE lipid molecules to three charged POPG lipid molecules) and eight copies of AMPs magainin 2 (located four on each side of bilayers), submerged in water.

OP06 Annexin A1 (AnxA1) mediates hydrogen sulfide (H2S) effects in the control of inflammation

Hydrogen sulfide (H2S), a gaseous mediator synthesized in several mammalian tissues by two main enzymes CBS and CSE, increases under inflammatory conditions or sepsis [1] , [2] . Inflammation per se does not represent an unwanted event, since it also provides a physiological response to an injury, keeping inflammation under control [3] . Annexin A1 (AnxA1) represent the front line of innate immunity as it is externalized on leukocytes membrane, preventing their adhesion to vascular endothelium and their transmigration into inflamed tissues [4] . Since H2S affords potent inhibitory properties on the process of leukocyte trafficking [5] , we tested whether endogenous AnxA1 could display intermediary functions. In order to achieve our aim we used different approaches: isolated human polymorphonuclear cells (PMNs), murine bone marrow-derived macrophages (BMDM) and intravital microscopy on mesenteric microcirculation in AnxA1−/− mice. Treatment of human PMNs with the H2S donor NaHS (10–100 μM) provoked prompted and intense mobilization (>50%) of AnxA1 from the cytosolic pool to the cell surface. Such in vitro actions could be translated in the analysis of the inflamed microcirculation, where NaHS (100 μmol/kg s.c., −30 min) afforded marked inhibition of IL1β-induced cell adhesion and emigration in the mesenteric vessels of wild type, but not AnxA1−/−, mice. Next, we investigated whether endogenous AnxA1 could link to H2S pathway, indicating existence of a positive circuit. To this end, a marked increase in CBS and/or CSE expression in a variety of tissues (aorta, kidney, spleen) tested from AnxA1−/− mice, as compared to wild type animals, was quantified by qPCR. Moreover, NaHS was able to counteract the increase in expression in iNOS and COX2 (4-fold and 7-fold reduction, respectively) upon LPS-stimulation of BMDM, though it was totally inactive in cells prepared from AnxA1−/− mice. Taken together, these data strongly suggest – for the first time – the existence of a positive loop between AnxA1 and the H2S pathway, providing a novel mechanistic explanation for the exquisite properties of H2S in the control of experimental inflammation. These findings may be relevant to innovative discovery programmes aiming at harnessing the biological properties of H2S.

208: Understanding the mechanisms of interferon induction by a large DNA virus and its application to vaccine development

African swine fever virus (ASFV) is a complex DNA virus and the aetiological agent of a domestic pig disease that can assume different clinical forms, ranging from an acute haemorrhagic disease to unapparent infections. The complexity of the immune response has impaired the development of an effective vaccine against ASFV infection. Previous work has established that immunization of pigs with attenuated replication competent ASFV strains can induce good levels of protection against lethal challenge with virulent strains. However in some pigs adverse clinical signs occur following immunization. On the other hand, due to the highly acute nature of the disease following infection with the virulent strains, it is clear that the porcine innate response is insufficient to prolong the life of the animal until the serological and cellular adaptive immune responses provide immediate protection and, importantly, the generation of the immunological memory crucial to vaccine development. The type I interferon (IFN) response is the first line of innate immunity against viral infection. Most, if not all viruses have to overcome this host defense before they can establish productive infection. We are currently characterizing the induction of type I IFN in primary porcine macrophages infected with strains of different virulence. A better understating of how the IFN response is modulated by these different viruses, will provide a rational basis for the development of novel vaccine strategies, for example, further attenuation of the virus by deleting genes involved in the evasion of the IFN host system is in progress.

Natural killer cells: versatile roles in autoimmune and infectious diseases

Natural killer (NK) cells are essential members of innate immunity and they rapidly respond to a variety of insults via cytokine secretion and cytolytic activity. Effector functions of NK cells form an important first line of innate immunity against viral, bacterial and parasitic infections, as well as an important bridge for the activation of adaptive immune responses. The control of NK-cell activation and killing is now understood to be a highly complex system of diverse inhibitory and activatory receptor–ligand interactions, sensing changes in MHC expression. NK cells have a functional role in innate immunity as the primary source of NK-cell-derived immunoregulatory cytokines, which have been identified in target organs of patients suffering from autoimmune diseases, and play a critical role in early defense against infectious agents. This review focuses on recent research of NK cells, summarizing their potential immunoregulatory role in modulating autoimmunity and infectious diseases.