O fator de transcrição nuclear kappa B (NFKB), central na resposta inflamatória, é constitutivamente expresso em glândulas pineais de rato. A inibição da translocação nuclear deste fator em pineais de rato por corticosterona potencia, enquanto que a inibição pela citocina fator de necrose tumoral (TNF) inibe a síntese de melatonina por inibição da transcrição da Aa-nat. Esta redução da produção noturna de melatonina está implicada em favorecer a montagem da resposta inflamatória. Embora dados da literatura sugerirem redução da produção de melatonina durante processos infecciosos, não há evidências diretas da habilidade da glândula pineal em reconhecer o lipopolissacarídeo (LPS), a endotoxina da membrana de bactérias gram-negativas. Esta dissertação investigou se a glândula pineal de ratos expressa receptores para o reconhecimento do LPS e estabeleceu possíveis mecanismos de ação desta endotoxina na glândula pineal de ratos. Nossos resultados demonstram que a glândula pineal expressa de maneira constitutiva os receptores CD14 e o TLR4. LPS induz a translocação nuclear dos dímeros p50/p50 e p50/RelA e a síntese de TNF em glândulas cultivadas. A máxima produção de TNF no meio de cultura é coincidente com a máxima expressão do receptor TNFR1 em pinealócitos. Além disso, LPS inibe a síntese de N-acetilserotonina e melatonina. Em conclusão, neste estudo, demonstramos que a pineal é alvo para o componente de bactérias gram-negativas LPS, reforçando a proposta de que esta glândula reconhece e gera respostas a moléculas que sinalizam a montagem da resposta inflamatória. / Nuclear factor-kappa B (NFKB), a pivotal player in inflammatory responses, is constitutively expressed in the pineal gland. Corticosterone inhibits pineal NFKB leading to an enhancement of melatonin production, while tumor necrosis factor (TNF) leads to inhibition of Aa-nat transcription and the production of N-acetylserotonin (NAS) in cultured glands. The reduction of nocturnal melatonin surge favors the mounting of the inflammatory response. Despite these data, there is no clear evidence of the ability of the pineal gland to recognize molecules that signal infection. This study investigated whether the rat pineal gland expresses receptors for lipopolysaccharide (LPS), the endotoxin from the membranes of gram-negative bacteria, and to establish the mechanism of action of LPS. Here we show that pineal glands possesses both CD14 and toll-like receptor 4 (TLR4), membrane proteins that bind LPS and trigger the NFKB pathway. LPS induced the nuclear translocation of p50/p50 and p50/RELA dimers and the synthesis of TNF. The maximal expression of TNF in cultured glands coincides with an increase in the expression of TNF receptor 1 (TNFR1) in isolated pinealocytes. In addition, LPS inhibited the synthesis of N-acetylserotonin and melatonin. Therefore, the pineal gland transduces gram-negative endotoxin stimulation by producing TNF and inhibiting melatonin synthesis. Here we provide evidence to reinforce the idea of an immune-pineal axis, showing that the pineal gland is a constitutive player in the innate immune response.

The increasing emergence and spread of antibiotic-resistant bacteria is a serious threat to public health. Of particular concern are Gram-negative bacteria such as Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa. Some of these strains are resistant to a large number of antibiotics and thus our treatment options are rapidly declining. In addition to the increasing number of antibiotic-resistant bacteria, a major problem is that many of the antibiotics at our disposal are ineffective against Gram-negative bacteria. This is partly due to the properties of the outer membrane (OM) which prevents efficient uptake. The overarching goal of this thesis was to investigate how the OM of the Gram-negative bacterium E. coli could be weakened to improve the activity of antibiotics. In the first two papers of my thesis (paper I + II), I investigated the periplasmic chaperone network which consists of the two parallel pathways SurA and Skp/DegP. This network is essential for the integrity of the OM and strains lacking either SurA or Skp are defective in the assembly of the OM, which results in an increased sensitivity towards vancomycin and other antimicrobials. We identified a novel component of the periplasmic chaperone network, namely YfgM, and showed that it operates in the same network as Skp and SurA/DegP. In particular, we demonstrated that deletion of YfgM in strains with either a ΔsurA or Δskp background further compromised the integrity of the OM, as evidenced by an increased sensitivity towards vancomycin. In the remaining two papers of my thesis (paper III + IV), the goal was to characterize small molecules that permeabilize the OM and thus could be used to improve the activity of antibiotics. Towards this goal, we performed a high-throughput screen and identified an inhibitor of the periplasmic chaperone LolA, namely MAC-13243, and showed that it can be used to permeabilize the OM of E. coli (paper III). We further demonstrated that MAC-13243 can be used to potentiate the activity of antibiotics which are normally ineffective against E. coli. In the last paper of my thesis (paper IV), we undertook a more specific approach and wanted to identify an inhibitor against the glycosyltransferase WaaG. This enzyme is involved in the synthesis of LPS and genetic inactivation of WaaG results in a defect in the OM, which leads to an increased sensitivity to various antibiotics. In this paper, we identified a small molecular fragment (compound L1) and showed that it can be used to inhibit the activity of WaaG in vitro. To summarize, this thesis provides novel insights into how the OM of the Gram-negative bacterium E. coli can be weakened by using small molecules. We believe that the two identified small molecules represent important first steps towards the design of more potent inhibitors that could be used in clinics to enhance the activity of antibiotics. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Les plaquettes jouent un rôle majeur dans l’hémostase primaire ainsi que dans l’inflammation. Elles contiennent et sécrètent une grande variété de facteurs solubles et parmi les nombreux récepteurs qu’elles expriment à leur surface, les plaquettes expriment les « Toll-Like Receptor » (TLR), récepteurs clés de l’interaction entre l’immunité innée et adaptative. En réponse à un stimulus infectieux, comme le lipopolysaccharide (LPS) des bactéries Gram-négative, ligand naturel du TLR4, ou des peptides issus d’une partie de la protéine d’enveloppe du VIH (gp41), les plaquettes vont s’activer de manière différentielle. L’activation plaquettaire est variable en fonction de leur activation par à un stimulus hémostatique (exemple : la thrombine) vs. infectieux (exemple : le LPS) ; le panel de cytokines libérées dans le surnageant plaquettaire semble en fait finement régulé. De plus, nous avons démontré la présence intra-plaquettaire de la majorité des protéines composant les voies de signalisation du TLR4 eucaryote. Nous avons ensuite montré que ces voies pouvaient être modulées. L’engagement du TLR4 plaquettaire par deux types biochimiques de LPS entraîne un relargage différentiel des facteurs solubles immunomodulateurs dans le surnageant de culture et que ce surnageant dernier génère une activation différentielle des cellules cibles, comme les cellules mononucléées du sang circulant. Ces travaux montrent que la réponse inflammatoire plaquettaire est régulée en fonction du stimulus. Ainsi, mes travaux s’inscrivent dans la ré-exploration de la fonction inflammatoire des plaquettes sanguines et l’étude du rôle des plaquettes comme cellules de l’immunité innée et inflammatoire / Blood platelets are anucleated cells which play a major role on primary hemostasis and well demonstrated other functions in inflammation. Platelets store and secrete a great variety of soluble factors, with immunomodulatory functions. They also contain transcription factors that exert non-genomic activities. Among numerous receptors expressed at the surface of platelets, they display Toll-Like Receptors (TLR) that are key molecules for the interaction between innate and adaptive immunity. Platelets can be activated in response to infectious stimulation, such as with a bacterial gram-negative Lipopolysaccharide (LPS) - the natural ligand of the TLR4, or peptides from the gp41, part of the HIV envelope. Moreover, stimulation with hemostatic or infectious agonists results in the differential secretion of panels of immunomodulatory products, that seems to be finely regulated. To further understand this regulatory process, we have studied the presence in the platelet cytosol of the majority of eukaryotic TLR4 pathways proteins. The engagement of the platelet TLR4 with two biochemically distinct LPS (smooth vs. rough) leads to a differential release of immunomodulatory products in platelet supernatants; those supernatants can then differently activate target cells such as peripheral blood mononuclear cells. These results demonstrate that the inflammatory response of human platelets is regulated by the nature of the stimulus, showing new evidence on the sentry role of these cells. Thus, my work is part of a novel study of the inflammatory function of blood platelets and the role of these cells as immune cells, essentially in the innate and inflammatory branch

As vacinas pertussis celulares apresentam certa reatogenicidade e as acelulares, menos reatogênicas, têm seu uso restrito, devido a seu alto custo. O Instituto Butantan desenvolveu uma vacina pertussis celular (Plow), com baixo teor de lipopolissacarídeo e outra acelular (Pa), por metodologia simples e econômica. Essas preparações, combinadas aos toxóides diftérico e tetânico (DTPlow e DTPa, respectivamente), foram comparadas à DTP tradicional, com diferentes adjuvantes: vitamina A, surfactante pulmonar, BCG, monofosforil lipídeo A (MPL) e Al(OH)3. A resposta humoral em camundongos foi semelhante para as diferentes formulações e independente do adjuvante utilizado. As vacinas induziram níveis equilibrados de IgG1/IgG2a anti-pertussis e a DTPlow mostrou-se menos reatogênica, induzindo níveis significativamente menores de IL-6 sérica. A adição de MPL sugeriu tendência de proteção contra a colonização nasotraqueal no grupo imunizado com DTPa e levou à indução de IFN-g nos grupos imunizados com DTP e DTPa, sugerindo possível atividade imunomodulatória para Th1. / The whole cell pertussis vaccines present some reactogenicity and the acellular, less reactogenic, have prohibitive use due to its high cost. Instituto Butantan developed a less reactogenic whole cell pertussis vaccine (Plow), with low lipopolysaccharide content and an acellular vaccine (Pa), by simple and economic methodology. These preparations, combined to diphtheria and tetanus toxoids (DTPlow and DTPa, respectively), were compared with the traditional DTP, with different adjuvants: vitamin A, pulmonary surfactant, BCG, monophosphoryl lipid A (MPL) and Al(OH)3. The humoral immune response induced in mice by the different vaccine formulations, was similar and independent of the adjuvant used. The vaccines induced balanced levels of IgG1/IgG2a anti-pertussis and DTPlow showed to be less rectogenic, inducing lower levels of serum IL-6. The use of MPL suggested to induce higher protection against nasotracheal colonization in DTPa group and induced IFN-g in the DTP and DTPa groups, suggesting a possible immunemodulatory activity for Th1.

A sinalização por endocanabinóides é finalizada por meio de hidrólise enzimática; um processo que para o endocanabinóide 2-Arachidonoylglycerol (2-AG) é mediado pela lipase monoacilglicerol (MAGL). O JZL184, é um fármaco que apresenta alta seletividade na inibição da MAGL. Assim, o JZL184 aumenta os níveis de 2-AG que, por sua vez, atua sobre os receptores canabinóides CB1 e CB2 produzindo diversos efeitos como, por exemplo, o anti-inflamatório. A inflamação pulmonar aguda (ALI) e a sua forma mais grave, a síndrome do desconforto respiratório agudo (SDRA), em humanos, são doenças pulmonares, caracterizadas por infiltrado pulmonar bilateral com acúmulo de neutrófilos. A sepse é a causa mais comum da ALI/SDRA; aproximadamente 40% de pacientes com sépsis, também apresentam ALI ou ARDS e a ALI/ARDS são síndromes graves associadas com mortalidade superior a 40%. Considerando que não há cura para a ARDS / ALI, foi utilizado um modelo murino de ALI para averiguar se a inibição da MAGL seria capaz de aliviar os sintomas inflamatórios ou, até mesmo, promover a cura do processo. Para isso, foram analisados fatores que promovem a migração de leucócitos para o pulmão e o dano tecidual. Ainda, para avaliar se os LPS e/ou o JZL184 promoveram mudanças no sistema nervoso central, foram avaliados a atividade locomotora no campo aberto (CA), a ansiedade no labirinto (LCE), a capacidade de adaptação em CA e os níveis de glicocorticóides séricos, assim como os níveis hipotalâmicos de citocinas. Assim, o JZL184, foi administrado por via intraperitoneal (i.p.) e 60 minutos depois o LPS foi instilado por via intranasal. As análises foram realizadas 6, 24 e/ou 48 horas após a indução da ALI. Observou-se que a inibição MAGL diminuiu a migração de leucócitos para os pulmões, bem como a permeabilidade vascular e o dano tecidual. O JZL184 também reduziu os níveis de citocinas e quimiocinas e o extravasamento vascular no lavado bronco alveolar (LBA), a atividade de MPO no tecido pulmonar e a expressão da molécula de adesão no sangue e no LBA. Os receptores CB1 e CB2 foram considerados como envolvidos nos efeitos anti-inflamatórios produzidos pelo JZL184 porque o AM281, um antagonista seletivo do receptor CB1, e o AM630, um antagonista seletivo do receptor CB2, reduziram ou bloquearam os efeitos anti-inflamatórios para JZL184. O LPS e o JZL184 não promoveram comportamento doentio e tampouco alteraram os parâmetros de ansiedade. Entretanto, o LPS e/ou o JZL184 aumentaram a expressão gênica de citocinas hipotalâmicas. Concluiu-se que a inibição MAGL produziu efeitos anti-inflamatórios no modelo murino de ALI induzida por LPS, uma descoberta que foi considerada uma consequência da ativação dos receptores canabinóide CB1 e CB2. A inibição da MAGL pode ser, no futuro, uma ferramenta terapêutica relevante para o tratamento de inflamações pulmonares / Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that, for 2-Arachidonoylglycerol (2-AG), is mediated by monoacylglycerol lipase (MAGL). The JZL184, is a drug that inhibits MAGL and presents high potency and selectivity. Thus, JZL184 increases the levels of 2-AG, an endocannabinoid that acts on the CB1 and CB2 cannabinoid receptors, and has shown anti-inflammatory effects. Acute lung injury (ALI) and its most severe form the acute respiratory distress syndrome (ARDS), in humans, are lung diseases, characterized by bilateral pulmonary infiltrate with neutrophils accumulation. The sepsis is the most common cause of ALI / ARDS; approximately 40% of patients with sepsis have also ALI or ARDS. ALI and ARDS are severe syndromes associated with mortality 40% exceeding rates. Considering that there is no cure for ARDS/ALI, we used a ALI murine model to evaluate if the MAGL inhibition was able to alleviating the inflammatory symptoms or even promote the cure. For this, factors that promote migration of leukocytes into the lungs and the tissue damage were analyzed. Still, to assess whether LPS and / or JZL184 promoted changes in the central nervous system, the locomotor activity and ability to adapt were evaluated in the open field end the anxiety in the plus maze. Were also evaluated the glucocorticoid levels in the serum, and the hypothalamic levels of cytokines. Thus, the JZL184 was used intraperitoneally, 60 minutes after LPS was intranasally instilled and 6, 24 and/or 48 hours, after induction of ALI, analyzes were performed. It was observed that the MAGL inhibition decreased the leukocyte migration into the lungs as well as the vascular permeability and the lung damage. JZL184 also reduced the cytokine and chemokine levels and the vascular extravasation in the BAL, the MPO activity in the lungs and adhesion molecule expression in the blood and BAL. The CB1 and CB2 receptors were considered involved in the anti-inflammatory effects of JZL184 because the AM281, a selective CB1 receptor antagonist, and the AM630, a selective CB2 receptor antagonist, reduced or blocked the anti-inflammatory effects previously described for JZL184. The LPS and the JZL184 did not promote unhealthy behavior and did not change the parameters of anxiety. However, LPS and/orJZL184 increased gene expression of hypothalamic cytokines. It was concluded that MAGL inhibition produced anti-inflammatory effects in a murine model of LPS-induced ALI, a finding that was considered a consequence of the activation of the CB1 and CB2 cannabinoid receptors. The MAGL inhibition in the future may be a therapeutic tool for the pulmonary inflammation treatment

Dans le cadre de l’évaluation du risque immunotoxique de composés glycolypidiques d’origine bactérienne, cette thèse a portée sur l’évaluation de toxicité d’endotoxines d’E.coli de deux sérotype différents : LPS O55:B5 utilisé couramment en comme immunostimulant et le LPS O157:H7 dont la réalité environnementale a soulevé notre questionnement scientifique quant à son impact sur le système immunitaire du poisson et son caractère potentiellement pro-inflammatoire. Les différents procédés employés ont compris l’évaluation des paramètres cellulaires (production d’espèces réactives d’oxygène et phagocytose) ainsi que la caractérisation et la quantification de l’expression de gènes de cytokines (TGFβ et IL-10) et facteurs immuno-associés (MARCO, HSP60 et vitellogénine) chez le modèle gardon (Rutilus rutilus).Les approches expérimentales se sont déroulées tout d’abord en ex vivo sur des leucocytes isolés d’organes lymphoïdes (Rein antérieur, rate et sang) de gardon et ont montré une tolérance endotoxique vis-à-vis de ces LPS à de 1µg/mL même combinés au diclofénac à 0,1 µM. ce travaill a été suivi par une évaluation du risque potentiel d’autres composés glycolipidiques d’origine bactérienne (rhamnolipides).Les approches in vivo qui ont suivies ont été réalisées sur : (i) sur modèle poisson-zèbre (Danio rerio) au laboratoire et (ii) sur modèle gardon par encagement sur terrain. Les résultats obtenus sur danios au laboratoire ont montré une toxicité du sérotype O157 :H7 et une influence sur les paramètres comportementaux par les LPS (Sickness behaviour). Sur terrain, l’approche in vivo par encagement a révélé – au niveau de la rate et du rein antérieur – des réponses cellulaires et moléculaires, sérotype, organe et sexe dépendante avec une immunomodulation prédominante chez les mâles d’autant plus que la période d’étude s’est déroulée durant la maturation sexuelle des gardons. Ce travail fait état du caractère inflammatoire et toxique du LPS peu étudié d’E.coli O157 :H7, évalué par des immunomarqueurs cellulaires bien maitrisés et moléculaires néo-développés. / In the context of immunotoxic risk evaluation of glycolypidic compounds of bacterial origin, this thesis focused on the evaluation of E.coli endotoxin toxicity of two different serotypes: LPS O55: B5 commonly used as an immunostimulant et LPS O157: H7, whose environmental reality has raised our scientific questioning about its impact on the fish's immune system et its potentially pro-inflammatory nature. The various methods used included the evaluation of cellular parameters (production of reactive oxygen species et phagocytosis) as well as the characterization of cytokines (TGFβ et IL-10) et immune-realted factors (MARCO, HSP60 et vitellogenin) genes et the quantification of their expression in the roach model (Rutilus rutilus).The experimental approaches were first carried out ex vivo on leukocytes isolated from lymphoid organs (anterior kidney, spleen et blood) roach et showed an endotoxic tolerance at 1μg / mL even combined with 0.1 μM diclofenac. This work was followed by an evaluation of the potential risk of other glycolipidic compounds of bacterial origin (rhamnolipids).The in vivo approaches that followed were performed on: (i) zebrafish (Danio rerio) model in the laboratory et (ii) on roach model by field caging. The results obtained on danios in the laboratory showed a toxicity of the serotype O157: H7 et an influence on the behavioral parameters by the LPS (Sickness behavior). On the field, the caging approach revealed - at spleen et the anterior kidney level - cellular et molecular responses, serotype, organ et sex-dependent with a predominant immunomodulation in males, especially since the study period took place during the sexual maturation of roaches. This work reports the inflammatory et toxic nature of the less studied E.coli O157: H7 LPS serotype, evaluated by well-mastered cellular et neo-developed molecular immunomarkers.

Puberty is a critical period of development characterized by rapid physiological changes and significant brain reorganizing and remodeling. These rapid changes render the developing brain particularly vulnerable to stress and immune challenge. In mice, exposure to an immune challenge (lipopolysaccharide; LPS) during puberty causes enduring effects on stress reactivity, cognitive functioning, and depression- and anxiety-like behaviors later in life. However, the mechanisms underlying these effects are unknown. The gut microbiome can profoundly influence the immune system. There is also close bidirectional communication between the gut microbiome and the central nervous system (CNS) through neural, endocrine and immune signaling pathways, which can alter brain chemistry and emotional behaviour. Thus, we hypothesized that altering microbial composition during puberty could mitigate acute immune responses and prevent enduring outcomes later in life. The current thesis examined the effect of gut manipulation with probiotics during puberty on LPS-induced immune responses and enduring anxiety- and depression-like behaviours, and stress-reactivity in adulthood, in male and female CD1 mice (Article 1). Next, we examined age and sex differences in gut microbial composition before and after exposure to an immune challenge. We also examined the effects of consuming a single strain probiotic bacterium (Lactobacillus Reuteri) during puberty on the immune response and the long-term changes in memory, anxiety-like behavior, and stress reactivity in adulthood (Article 2). Lastly, we examined how microbial colonization between pubertal and adult mice can alter acute peripheral and central inflammatory responses to LPS (Article 3). The current dissertation has addressed sex-specific vulnerabilities to an immune challenge during pubertal development and the moderating influence of the gut microbiome. These studies have demonstrated that manipulating the gut microbiome during puberty can mitigate acute immune responses and prevent enduring neurobehavioural outcomes later in life.

Heat shock protein A12B (HSPA12B) is a newly discovered member of the HSP70 protein family. This study investigated the effects of HSPA12B on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs) and the possible mechanisms involved. A HUVECs inflammatory model was induced by LPS. Overexpression of HSPA12B in HUVECs was achieved by infection with recombinant adenoviruses encoding green fluorescence protein-HSPA12B. Knockdown of HSPA12B was achieved by siRNA technique. Twenty four hours after virus infection or siRNA transfection, HUVECs were stimulated with 1 μg/ml LPS for 4 hrs. Endothelial cell permeability ability was determined by transwell permeability assay. The binding rate of human neutrophilic polymorphonuclear leucocytes (PMN) with HUVECs was examined using myeloperoxidase assay. Cell migrating ability was determined by the wound-healing assay. The mRNA and protein expression levels of interested genes were analyzed by RT-qPCR and Western blot, respectively. The release of cytokines interleukin-6 and tumour necrosis factor-α was measured by ELISA. HSPA12B suppressed LPS-induced HUVEC permeability and reduced PMN adhesion to HUVECs. HSPA12B also inhibited LPS-induced up-regulation of adhesion molecules and inflammatory cytokine expression. By contrast, knockdown of HSPA12B enhanced LPS-induced increases in the expression of adhesion molecules and inflammatory cytokines. Moreover, HSPA12B activated PI3K/Akt signalling pathway and pharmacological inhibition of this pathway by Wortmannin completely abrogated the protection of HSPA12B against inflammatory response in HUVECs. Our results suggest that HSPA12B attenuates LPS-induced inflammatory responses in HUVECs via activation of PI3K/Akt signalling pathway.

Objective: The mechanisms by which lipopolysaccharide (LPS) pretreatment induces cardioprotection following ischaemia/reperfusion (I/R) have not been fully elucidated. We hypothesized that activation of phosphoinositide 3-kinase (PI3K)/Akt and high mobility group box 1 (HMGBx1) signaling plays an important role in LPS-induced cardioprotection. Methods: In in vivo experiments, age- and weight-matched male C57BL/10Sc wild type mice were pretreated with LPS before ligation of the left anterior descending coronary followed by reperfusion. Infarction size was examined by triphenyltetrazolium chloride (TTC) staining. Akt, phospho-Akt, and HMGBx1 were assessed by immunoblotting with appropriate primary antibodies. In situ cardiac myocyte apoptosis was examined by the TdT-mediated dUTP nick-end labeling (TUNEL) assay. In an in vitro study, rat cardiac myoblasts (H9c2) were subdivided into two groups, and only one was pretreated with LPS. After pretreatment, the cells were transferred into a hypoxic chamber under 0.5% O2. Levels of HMGBx1 were assessed by immunoblot. Results: In the in vivo experiment, pretreatment with LPS reduced the at risk infarct size by 70.6% and the left ventricle infarct size by 64.93% respectively. Pretreatment with LPS also reduced cardiac myocytes apoptosis by 39.1% after ischemia and reperfusion. The mechanisms of LPS induced cardioprotection involved increasing PI3K/Akt activity and decreasing expression of HMGBx1. In the in vitro study, pretreatment with LPS reduced the level of HMGBx1 in H9c2 cell cytoplasm following hypoxia. Conclusion: The results suggest that the cardioprotection following I/R induced by LPS pretreatment involves PI3K/Akt and HMGBx1 pathways.

Confinement of the obligate intracellular bacterium Chlamydia trachomatis to a membrane-bound vacuole, termed an inclusion, within infected epithelial cells neither prevents secretion of chlamydial antigens into the host cytosol nor protects chlamydiae from innate immune detection. However, the details leading to chlamydial antigen presentation are not clear. By immunoelectron microscopy of infected endometrial epithelial cells and in isolated cell secretory compartments, chlamydial major outer membrane protein (MOMP), lipopolysaccharide (LPS) and the inclusion membrane protein A (IncA) were localized to the endoplasmic reticulum (ER) and co-localized with multiple ER markers, but not with markers of the endosomes, lysosomes, Golgi nor mitochondria. Chlamydial LPS was also co-localized with CD1d in the ER. Since the chlamydial antigens, contained in everted inclusion membrane vesicles, were found within the host cell ER, these data raise additional implications for antigen processing by infected uterine epithelial cells for classical and non-classical T cell antigen presentation.