Endotoxins (E) are a unique and abundant family of glycolipids located in the outer leaflet of the outer membrane of Gram-negative bacteria. Host immune responses to endotoxin depend on ordered endotoxin-host protein interactions, resulting in delivery of an endotoxin monomer to MD-2 which acts as a potent agonist of Toll-Like Receptor (TLR) 4. Activated TLR4 is unique among TLRs in its ability to mobilize two distinct intracellular signaling pathways: the MyD88- and TRIF-dependent pathways. The regulated action of both pathways is likely important for optimal host immune responses to Gram-negative bacterial infection, but how this is achieved is not well understood Recent studies have indicated an essential role for host CD14 in TRIF-dependent signaling by activated TLR4 but the extent to which these observations reflect a general role of CD14 in endotoxin-triggered TRIF signaling or one more narrowly restricted to the specific endotoxins and/or cell types used is uncertain. We have addressed this question by identifying a novel CD14-independent mechanism for efficient delivery of E monomer to MD-2 and TLR4 activation, that is mediated by endotoxin.albumin complexes. We have used these complexes to demonstrate CD14-independent activation of MD-2⋅TLR4 by a wider range of endotoxin species than previously thought possible and activation of both MyD88- and TRIF-dependent pathways. Taken together, the findings in this thesis indicate that the molecular structure and physical presentation of endotoxin as well as CD14-independent properties of the host cell help determine the extent to which CD14 is required for TRIF-dependent signaling by activated TLR4.

Thèse de doctorat : Biologie cellulaire et moléculaire. Immunologie : Orléans : 2009. / Titre provenant de l'écran-titre.

No description available.

Background During the first stages of development human infants are either fed human milk or human milk substitutes (infant formulas). The composition of infant formulas and human milk differ drastically, including a difference in protein constituents and bacterial load. Due to the high global frequency of infant formula use, the humanization of infant formulas to better reflect the complex nature of human milk is warranted. To better understand the role of human milk components, the fate and function of a key bacterial sensor in human milk, soluble CD14, was determined. Additionally, the microbiome of human milk was analyzed from a metagenomic standpoint in an attempt to determine which types of bacteria are present in human milk and what their potential biological function might be. Results In rodent models, ingested sCD14 persisted in the gastrointestinal tract and was transferred intact into the blood stream. Once transferred to the blood, ingested sCD14 retained its ability to recognize lipopolysaccharide and initiate an immune response in pups. This transfer of sCD14 across the epithelial barrier was also observed in human cells in vitro, where it appears to be dependent on Toll-like receptor 4. Using Illumina sequencing and the MG-RAST pipeline, the human milk metagenome of ten mothers was sequenced. DNA from human milk aligned to over 360 prokaryotic genera, and contained 30,128 open reading frames assigned to various functional categories. The DNA from human milk was also found to harbor immune-modulatory DNA motifs that may play a significant role in immune development of the infant. Conclusions Given the complex nature of human milk in comparison to its bovine or plant based substitutes, the results presented in this thesis warrant future modification of infant formulas to include non-nutritive bioactive components. Current human milk components not yet present in infant formulas include the diverse microbiome of human milk, the immune-modulatory DNAs which those microbes harbor, and bioactive human proteins such as sCD14.

Endotoxin recognition by the innate immune receptor CD14 is a critical part of the innate immune system’s early detection and activation of the inflammatory response during microbial invasion. The differential recognition and high affinity binding of endotoxins from gram-negative and gram-positive bacteria is performed by the innate immune receptor CD14. Upon endotoxin binding, CD14 transfers the specific endotoxins to a Toll-like receptor signaling complex, which is responsible for initiating the intracellular signaling cascade. In the presence of overwhelming infection, the effects of CD14 lead to the over-activation of the inflammatory response, which results in the life threatening condition known as sepsis. Preparation of a 15N isotopically labeled truncated version of soluble CD14, using Pichia pastoris, allowed direct structural observation of the binding interaction between CD14 and two endotoxin ligands, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), from gram-negative and gram-positive bacteria, respectively using solution NMR spectroscopy. These studies revealed that CD14 uses both a common set of residues, and endotoxin specific subsets of residues, to bind LPS and LTA. To further investigate the structural features of each endotoxin recognized by CD14, 13C 15N isotopically labeled Kdo2–Lipid A, a fully active chemically defined gram-negative endotoxin, and LTA lipid anchor, the minimal unit of LTA, were produced. This allowed detailed NMR spectral mapping of these agonist ligands bound to sCD14 which identified, for the first time, structural regions and features in each that are strongly affected during complex formation with sCD14. Additionally, the presence of differential dynamic behavior was seen in both CD14 and the ligands upon complexation. This behavior suggests a likely role for dynamics in the mechanism of pattern recognition by CD14, which uses the dynamic ability of specific residue combinations to differentially affect endotoxin binding. Using NMR, the dynamic behavior of CD14 was further investigated using temperature and pH-dependence studies of isotopically labeled CD14. These studies clearly demonstrated the presence of multiple conformations for several residues, and may provide a possible explanation for the broad specificity of ligand binding by CD14. In addition, the spin-labeling of isotopically labeled lipid A enabled the collection of intermolecular distances on CD14 bound lipid A.

Tübingen, Univ., Diss., 2005.

Made available in DSpace on 2015-07-22T20:50:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-27. Added 1 bitstream(s) on 2015-08-11T03:25:43Z : No. of bitstreams: 1 Publico-10767a.pdf: 1296672 bytes, checksum: 6f8ba558bac665a25fad4ca2e64a288c (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:43Z : No. of bitstreams: 2 Publico-10767a.pdf: 1296672 bytes, checksum: 6f8ba558bac665a25fad4ca2e64a288c (MD5) Publico-10767b.pdf: 1513875 bytes, checksum: b0bbfd84ceaf858aad2bc6c9a3aaa160 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Introducao: O LPS induz a ativacao celular apos ligacao com CD14 e TLR4. A forma R de LPS (sem o polissacarideo O) pode ativar de forma independente de CD14. Neutrofilos e monocitos diferem de forma expressiva em sua expressao de CD14 na superficie. Objetivos: Comparar a acao biologica de formas R e S de LPS em neutrofilos e monocitos do sangue periferico de humanos. Metodos: A modulacao de receptores de superficie celular foi observada em monocitos e neutrofilos, e producao de especies reativas de oxigenios (EROs) e de oxido nitrico (NO), alem de ativacao de p38 e NFƒÈB foram avaliadas em neutrofilos por citometria de fluxo, apos incubacao com r e sLPS. Resultados: Houve uma pequena alteracao de TLR4 em monocitos com 6 h de incubacao, enquanto nenhuma modulacao foi vista para TLR2 em ambas as celulas. A modulacao de CD14 na superficie de monocitos mostrou-se bifasica, havendo incremento inicial na primeira hora seguida de queda com 6 h. Nao foi observada modulacao de CD14 na superficie de neutrofilos. Ambas as formas de LPS levaram a inducao de CD66b na superficie de neutrofilos, efeito que permanece pelo menos ate 24 h de incubacao. CD11b foi aumentado em neutrofilos e monocitos por r e sLPS, chegando ate 4 vezes a expressao depois de 3h, enquanto que a expressao CXCR2 cai em poucos minutos e permanece reduzida em neutrofilos. A expressao de HLADR foi aumentada nos monocitos no tempo de 3h. O efeito do sLPS na expressao de CD11b, CD66b e CXCR2 em neutrofilos mostrou-se um pouco mais precoce. Ambas as formas de LPS induziram pequena producao de NO e de EROs, quando comparadas com outros estimulos, assim como a translocacao de NFƒÈB p65 e p50 e ativacao de p38. Conclusoes: as formas R e S de LPS ativam similarmente os neutrofilos, apesar de sua baixa expressao de CD14 na superficie. Enquanto proteinas do soro podem otimizar a acao do LPS e sCD14 pode suprir a falta de CD14 em neutrofilos, e interacoes com monocitos podem ter seu papel na resposta dessas celulas ao LPS, e provavel que outros receptores estejam envolvidos na sinalizacao ao LPS em neutrofilos. Uma modulacao complexa de receptores de superficie foi vista em ambas as celulas com aumento ou decrescimo dependendo do receptor avaliado. / Objective: To compare the biological action of R form and S form LPS in human peripheral blood neutrophils and monocytes. Methods: Cell surface receptors modulation, ROS and NO production were evaluated on monocytes and neutrophils in whole blood, and NF-êB activation was evaluated in isolated neutrophils by flow cytometry after incubation with R and S-LPS. Results: A small enhancement of TLR4 expression was observed on monocytes with 6 h stimulation whereas no modulation of TLR2 was seen on either cell. Modulation of CD14 on monocytes was biphasic, with initial increment in the first hour followed by decreased expression at 6 h. Both LPS increased CD66b expression on neutrophils. Expression of CD11b was rapidly up-regulated on monocytes and neutrophils by both LPS, while down regulation of CXCR2 expression on neutrophils was observed after a few minutes, lasting for 6 h. S-LPS effect on neutrophils expression of CD11b, CD66b and CXCR2 receptors was a little earlier than the R-LPS effect. Both LPS induced low production of ROS and NO compared to other stimuli, as well as NF-êB translocation. Conclusions: A complex LPS-induced cell surface receptors modulation was seen on monocytes and neutrophils in human blood, with up and down regulation depending on the receptors evaluated. R for and S form LPS have similarly activated human neutrophils, despite the low expression of CD14 in those cells. While the presence of LBP and of sCD14 could provide the requirements for S-form LPS induction of neutrophils activity in whole blood, other receptors are also likely to be involved in LPS cell signaling in neutrophils. / FAPESP: 04/15584-2 / FAPESP: 05/58015-7 / TEDE / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2014-06-11T19:26:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-08Bitstream added on 2014-06-13T19:33:16Z : No. of bitstreams: 1 prioli_ra_me_jabo.pdf: 689132 bytes, checksum: 17eaeca002f2374370af36bb9266bcc4 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os objetivos deste trabalho foram a padronização de metodologia alternativa de genotipagem do SNP AY_731081:g.1900T>C do gene CD14 equino por PCR-RFLP, bem como a caracterização em equinos da raça Mangalarga deste e de outros dois polimorfismos, o AY_005808: c.1530A>G do TLR4 e o AX_463789: g.133T>C do Cε, a fim de promover o embasamento necessário para futuras pesquisas visando associação entre marcadores de DNA e características relacionadas à fisiologia do sistema imune na raça. Para tanto, foram utilizados 151 animais Mangalarga, de ambos os sexos e de idades variadas, representativos da população do estado de São Paulo. O método de PCR-RFLP mostrou-se adequado para a genotipagem do SNP AY_731081: g.1900T>C do gene CD14 equino. Entretanto, tal polimorfismo provavelmente não ocorre em equinos Mangalarga, impossibilitando estudos de associação com o marcador na raça. Os parâmetros genético-populacionais obtidos para os polimorfismos AY_005808:c.1530A>G do gene TLR4 e o AX_463789:g.133T>C do gene Cε demonstraram a possibilidade de realização de pesquisas visando a associação entre os marcadores e características relacionadas ao sistema imune na raça Mangalarga / The objective of this work was to contribute to the molecular characterization of the equine Mangalarga, aiming at future studies on the association of DNA polymorphisms and traits associated with the immune system physiology of this equine breed. An alternative PCR-RFLP genotyping method was developed for the SNP AY_731081:g.1900T>C, in the gene CD14. Furthermore, this SNP plus the AY_005808: c.1530A>G, in the gene TLR4, and the AX_463789: g.133T>C, in the gene Cε, were used to analyze 151 Mangalarga individuals. The analyzed horses are representative of the São Paulo State population and consisted of male and female animals of several ages. The PCR-RLP method has been demonstrated to be suitable for equine genotyping using the SNP AY_731081: g.1900T>C of the gene CD14. However, this polymorphism is apparently absent in the Mangalarga breed. The population genetic data obtained for the polymorphisms AY_005808:c.1530A>G, of the gene TLR4, and the AX_463789:g.133T>C, in the gene Cε, indicated the feasibility of these SNPs for further studies aiming at the association of molecular markers with traits related to the immune system of the Mangalarga horse breed

Abstract Successful pregnancy is characterized by a shift toward Th2 type immune response and suppression of adaptive immune responses to ensure acceptance of the semi-allogenic fetal graft. Also the innate immune system plays a major role during pregnancy. Recurrent miscarriage is defined as three or more consecutive pregnancy losses. About 1% of all women will suffer recurrent miscarriage. The causes of recurrent miscarriage remain unexplained in half (50%) of the cases. Susceptibility to recurrent miscarriage is probably mediated by Th1 type immune response with pronounced expression and secretion of pro-inflammatory cytokines (e.g. TNFα and IFNγ) paralleled with decreased production of anti-inflammatory cytokines (e.g. IL-10). Factors that regulate immune response during pregnancy include hormonal factors (e.g. hCG and progesterone). Immunogenetic factors also contribute to this regulation. Several functionally important polymorphisms in various immunomodulatory genes have been identified during recent years. Some of these polymorphisms may be important in regulating the Th1/Th2 balance during pregnancy. Putative immune dysregulation caused by these polymorphisms has been researched intensively. Conflicting results have been published about associations between several of these polymorphisms and recurrent miscarriage. In this study, HLA-G (exon 2 and 3), IL-10 (-1082A/G), IL-1RA (intron 2 VNTR) and CD14 (-159C/T) polymorphisms were studied in 38 Finnish women with RM. All of these polymorphisms have been associated with altered gene expression. Distribution of HLA-G*I, II, III and IV were 0.577, 0.375, 0 and 0.048 respectively in the studied Finnish population. According to the present classification the G*I allele group mostly consists of the allele 010101, while G*II covers the combination of 010102, 010401 and 0105N, as well as some other rare alleles. There were no associations between recurrent miscarriage and the HLA-G, IL-10 and CD14 polymorphisms. However, in IL-1RA polymorphism, the rare IL1RN*3 allele was increased in women with recurrent miscarriage. It is not known, if this particular allele is associated with differences in IL-1RA or IL-1 production. Although the study population was small, it may be supposed that quantitative differences in the production of single immunomodulatory molecules due to normal genetic variation may not be grossly harmful to the fetal allograft. This indicates the robustness and flexibility of the reproduction system. For survival, it is essential that minor variations are tolerated. Thus, large-scale studies focusing on the effect of a pro-inflammatory genetic profile based on the presence of several pro/anti-inflammatory genetic markers are needed to discover if immunogenetic factors predispose women to recurrent miscarriage.

Background During the first stages of development human infants are either fed human milk or human milk substitutes (infant formulas). The composition of infant formulas and human milk differ drastically, including a difference in protein constituents and bacterial load. Due to the high global frequency of infant formula use, the humanization of infant formulas to better reflect the complex nature of human milk is warranted. To better understand the role of human milk components, the fate and function of a key bacterial sensor in human milk, soluble CD14, was determined. Additionally, the microbiome of human milk was analyzed from a metagenomic standpoint in an attempt to determine which types of bacteria are present in human milk and what their potential biological function might be. Results In rodent models, ingested sCD14 persisted in the gastrointestinal tract and was transferred intact into the blood stream. Once transferred to the blood, ingested sCD14 retained its ability to recognize lipopolysaccharide and initiate an immune response in pups. This transfer of sCD14 across the epithelial barrier was also observed in human cells in vitro, where it appears to be dependent on Toll-like receptor 4. Using Illumina sequencing and the MG-RAST pipeline, the human milk metagenome of ten mothers was sequenced. DNA from human milk aligned to over 360 prokaryotic genera, and contained 30,128 open reading frames assigned to various functional categories. The DNA from human milk was also found to harbor immune-modulatory DNA motifs that may play a significant role in immune development of the infant. Conclusions Given the complex nature of human milk in comparison to its bovine or plant based substitutes, the results presented in this thesis warrant future modification of infant formulas to include non-nutritive bioactive components. Current human milk components not yet present in infant formulas include the diverse microbiome of human milk, the immune-modulatory DNAs which those microbes harbor, and bioactive human proteins such as sCD14.