Differential induction of innate immune responses by synthetic lipid a derivatives.
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ABSTRACT:
SUBMITTER: Gaekwad J
PROVIDER: S-EPMC2937970 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The lipid A moiety of lipopolysaccharides (LPS) initiates innate immune responses by interacting with Toll-like receptor 4 (TLR4), which results in the production of a wide range of cytokines. Derivatives of lipid A show potential for use as immuno-modulators for the treatment of a wide range of diseases and as adjuvants for vaccinations. Development to these ends requires a detailed knowledge of patterns of cytokines induced by a wide range of derivatives. This information is difficult to obtain by using isolated compounds due to structural heterogeneity and possible contaminations with other inflammatory components. To address this problem, we have developed a synthetic approach that provides easy access to a wide range of lipid A's by employing a common disaccharide building block functionalized with a versatile set of protecting groups. The strategy was employed for the preparation of lipid A's derived from E. coli and S. typhimurium. Mouse macrophages were exposed to the synthetic compounds and E. coli 055:B5 LPS, and the resulting supernatants were examined for tumor necrosis factor alpha (TNF-alpha), interferon beta (IFN-beta), interleukin 6 (IL-6), interferon-inducible protein 10 (IP-10), RANTES, and IL-1beta. It was found that for each compound, the potencies (EC50 values) for the various cytokines differed by as much as 100-fold. These differences did not follow a bias toward a MyD88- or TRIF-dependent response. Instead, it was established that the observed differences in potencies of secreted TNF-alpha and IL-1beta were due to differences in the processing of respective pro-proteins. Examination of the efficacies (maximum responses) of the various cytokines showed that each synthetic compound and E. coli 055:B5 LPS induced similar efficacies for the production of IFN-beta and IP-10. However, lipid A's 1-4 gave lower efficacies for the production of RANTES and IL-6 as compared to LPS. Collectively, the presented results demonstrate that cytokine secretion induced by LPS and lipid A is complex, which can be exploited for the development of immuno-modulating therapies.
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Project description:Differences in the pattern and chemical nature of fatty acids of lipid A of Neisseria meningitides lipooligosaccharides (LOS) and Escherichia coli lipopolysaccharides (LPS) may account for differences in inflammatory properties. Furthermore, there are indications that dimeric 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) moieties of LOS and LPS enhance biological activities. Heterogeneity in the structure of lipid A and possible contaminations with other inflammatory components have made it difficult to confirm these observations. To address these problems, a highly convergent approach for the synthesis of a lipid A derivative containing KDO has been developed, which relies on the ability to selectively remove or unmask in a sequential manner an isopropylidene acetal, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonate (Alloc), azide, and thexyldimethylsilyl (TDS) ether. The strategy was employed for the synthesis of N. meningitidis lipid A containing KDO (3). Mouse macrophages were exposed to the synthetic compound and its parent LOS, E. coli lipid A (2), and a hybrid derivative (4) that has the asymmetrical acylation pattern of E. coli lipid A, but the shorter lipids of meningococcal lipid A. The resulting supernatants were examined for tumor necrosis factor alpha (TNF-alpha) and interferon beta (IFN-beta) production. The lipid A derivative containing KDO was much more active than lipid A alone and just slightly less active than its parent LOS, indicating that one KDO moiety is sufficient for full activity of TNF-alpha and IFN-beta induction. The lipid A of N. meningitidis was a significantly more potent inducer of TNF-alpha and IFN-beta than E. coli lipid A, which is due to a number of shorter fatty acids. The compounds did not demonstrate a bias towards a MyD88- or TRIF-dependent response.
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Project description:The sophisticated adaptive immune system of vertebrates overlies an ancient set of innate immune-response pathways, which have been genetically dissected in Drosophila. Although conserved regulatory pathways have been defined, calcineurin, a Ca(2+)-dependent phosphatase, has not been previously implicated in Drosophila immunity. Calcineurin activates mammalian immune responses by activating the nuclear translocation of the vertebrate-specific transcription factors NFAT1-4. In Drosophila, infection with gram-negative bacteria promotes the activation of the Relish transcription factor through the Imd pathway. The activity of this pathway in the larva is modulated by nitric oxide (NO). Here, we show that the input by NO is mediated by calcineurin. Pharmacological inhibition of calcineurin suppressed the Relish-dependent gene expression that occurs in response to gram-negative bacteria or NO. One of the three calcineurin genes in Drosophila, CanA1, mediated NO-induced nuclear translocation of Relish in a cell-culture assay. A CanA1 RNA interference (RNAi) transgene suppressed immune induction in larvae upon infection or upon treatment with NO donors, whereas a gain-of-function CanA1 transgene activated immune responses in untreated larvae. Interestingly, CanA1 RNAi in hemocytes but not the fat body was sufficient to block immune induction in the fat body. Thus, CanA1 provides an additional input into Relish-promoted immune responses and functions in hemocytes to promote a tissue-to-tissue signaling cascade required for robust immune response.
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Project description:The ability of the central nervous system (CNS) to generate innate immune responses was investigated in an in vitro model of CNS infection. Cultures containing CNS cells were infected with mouse hepatitis virus-JHM, which causes fatal encephalitis in mice. Immunostaining indicated that viral infection had a limited effect on culture characteristics, overall cell survival, or cell morphology at the early postinfection times studied. Results from Affymetrix gene array analysis, assessed on RNA isolated from virally and sham-infected cultures, were compared with parallel protein assays for cytokine, chemokine, and cell surface markers. Of the 126 transcripts found to be differentially expressed between viral and sham infections, the majority were related to immunological responses. Virally induced increases in interleukin-6 and tumor necrosis factor alpha mRNA and protein expression correlated with the genomic induction of acute-phase proteins. Genomic and protein analysis indicated that viral infection resulted in prominent expression of neutrophil and macrophage chemotactic proteins. In addition, mRNA expression of nonclassical class I molecules H2-T10, -T17, -M2, and -Q10, were enhanced three- to fivefold in virus-infected cells compared to sham-infected cells. Thus, upon infection, resident brain cells induced a breadth of innate immune responses that could be vital in directing the outcome of the infection and, in vivo, would provide signals which would summon the peripheral immune system to respond to the infection. Further understanding of how these innate responses participate in immune protection or immunopathology in the CNS will be critical in efforts to intervene in severe encephalitis.
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Project description:BACKGROUND:Wolbachia has been reported to suppress a variety of pathogen infections in mosquitoes, but the mechanism is undefined. Two possibilities have been proposed. One is that Wolbachia activates host immune responses, and the other one is that Wolbachia competes with pathogens for limited nutrients. METHODOLOGY/PRINCIPAL FINDINGS:In this study, we compared host immune responses and the densities of two different strains of Wolbachia in naturally occurring parental and artificially created hybrid host genetic backgrounds. No significant difference in Wolbachia density was found between these hosts. We found that Wolbachia could activate host innate immune responses when the host genetic profile was different from that of its natural host. When these hosts were challenged with pathogenic bacteria, mosquitoes in new host-Wolbachia symbioses had a higher survival rate than in old host-Wolbachia symbioses. CONCLUSIONS/SIGNIFICANCE:The presence of Wolbachia per se does not necessarily affect pathogen infections, suggesting that a competition for limited nutrients is not the main reason for Wolbachia-mediated pathogen suppression. Instead, host immune responses are responsible for it. The elucidation of an immunity nature of PI is important to guide future practice: Wolbachia may be genetically engineered to be more immunogenic, it is desired to search and isolate more strains of Wolbachia, and test more host-Wolbachia symbioses for future applications. Our results also suggest Wolbachia-based PI may be applied to naturally Wolbachia-infected mosquito populations, and extend to the control of a broader range of mosquito-borne diseases.
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Project description:In recent years, several newly discovered tick-borne viruses causing a wide spectrum of diseases in humans have been ascribed to the Phlebovirus genus of the Bunyaviridae family. The nonstructural protein (NSs) of bunyaviruses is the main virulence factor and interferon (IFN) antagonist. We studied the molecular mechanisms of IFN antagonism employed by the NSs proteins of human apathogenic Uukuniemi virus (UUKV) and those of Heartland virus (HRTV) and severe fever with thrombocytopenia syndrome virus (SFTSV), both of which cause severe disease. Using reporter assays, we found that UUKV NSs weakly inhibited the activation of the beta interferon (IFN-β) promoter and response elements. UUKV NSs weakly antagonized human IFN-β promoter activation through a novel interaction with mitochondrial antiviral-signaling protein (MAVS), confirmed by coimmunoprecipitation and confocal microscopy studies. HRTV NSs efficiently antagonized both IFN-β promoter activation and type I IFN signaling pathways through interactions with TBK1, preventing its phosphorylation. HRTV NSs exhibited diffused cytoplasmic localization. This is in comparison to the inclusion bodies formed by SFTSV NSs. HRTV NSs also efficiently interacted with STAT2 and impaired IFN-β-induced phosphorylation but did not affect STAT1 or its translocation to the nucleus. Our results suggest that a weak interaction between STAT1 and HRTV or SFTSV NSs may explain their inability to block type II IFN signaling efficiently, thus enabling the activation of proinflammatory responses that lead to severe disease. Our findings offer insights into how pathogenicity may be linked to the capacity of NSs proteins to block the innate immune system and illustrate the plethora of viral immune evasion strategies utilized by emerging phleboviruses. IMPORTANCE Since 2011, there has been a large expansion in the number of emerging tick-borne viruses that have been assigned to the Phlebovirus genus. Heartland virus (HRTV) and SFTS virus (SFTSV) were found to cause severe disease in humans, unlike other documented tick-borne phleboviruses such as Uukuniemi virus (UUKV). Phleboviruses encode nonstructural proteins (NSs) that enable them to counteract the human innate antiviral defenses. We assessed how these proteins interacted with the innate immune system. We found that UUKV NSs engaged with innate immune factors only weakly, at one early step. However, the viruses that cause more severe disease efficiently disabled the antiviral response by targeting multiple components at several stages across the innate immune induction and signaling pathways. Our results suggest a correlation between the efficiency of the virus protein/host interaction and severity of disease.
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