These studies were aimed at investigating the potential application of nanoparticles engineered from oil-in-water microemulsion precursors for enhancing immune responses to HIV-1 Tat and Gag p24 proteins. Both of the HIV-1 proteins have been reported to be critical in the virus life cycle and are being evaluated in clinical trials as vaccine candidates. Anionic nanoparticles were prepared using emulsifying wax as the oil phase and Brij 78 and sodium dodecyl sulfate as the surfactants. The resulting nanoparticles were coated with Tat and were demonstrated to produce superior immune responses after administration to BALB/c mice compared to Tat adjuvanted with Alum. Similarly, cationic nanoparticles were prepared using emulsifying wax and Brij 78 and cetyl trimethyl ammonium bromide as the surfactants. The cationic nanoparticles were investigated for delivery of immunostimulatory adjuvants, namely three Toll-like receptor ligands, for obtaining synergistic enhancements in immune responses to a model antigen, Ovalbumin (OVA). In vitro and in vivo studies were carried out to elucidate possible mechanisms by which nanoparticles may result in enhancements in immune responses. In vitro studies were carried out to evaluate the uptake of nanoparticles into dendritic cells and to assess the release of pro-inflammatory cytokines from dendritic cells in the presence of nanoparicles. In vivo studies were carried out using a MHC class I restricted transgenic mouse model to investigate the potential for nanoparticles coated with OVA to enhance presentation of the protein to CD8+ T cells compared to OVA alone. Finally, the preparation of nanoparticles with a low amount of surface chelated nickel for high affinity binding to histidine-tagged (his-tag) proteins was investigated. It was hypothesized that this strengthened interaction of his-tag protein to the nickel chelated nanoparticles (Ni-NPs) would result in a greater uptake of antigen in vivo; therefore, enhanced immune responses compared to protein bound to anionic nanoparticles. In vivo evaluation of his-tag HIV-1 Gag p24 bound to Ni-NPs resulted in enhanced immune responses compared to protein either adjuvanted with Alum or coated on the surface of nanoparticles.

The blood-brain barrier (BBB) is a dynamic interface, mainly consisting of highly specialized brain microvascular endothelial cells (BMECs) that segregate the central nervous system (CNS) from the peripheral circulation. Impairment of the BBB, due to disruption of tight junction (TJ) proteins and inflammatory responses, may initiate and/or contribute to the progress of CNS disorders, including stroke. Stroke is the second leading cause of death worldwide. It has been shown that aging and environmental pollutants can induce brain endothelium dysfunction, and are considered as risk factors for stroke. Deficiency of telomerase is highly linked with aging-associated vascular diseases. Evidence indicates that patients with shorter telomere length are at higher risk of heart disease or stroke. Results in this dissertation address the influence of telomerase reverse transcriptase (TERT), a key component of telomerase, on the BBB integrity in the context of ischemic stroke induced brain injury. Our results indicate that aging-related BBB alterations aggregate the stroke outcomes by inducing oxidative stress and stimulating proinflammatory responses on the brain microvessels. The ability of the BBB to protect the brain from harmful compounds indicates that the BBB may be targeted by chemical toxicants in the peripheral circulation. Polychlorinated biphenyls (PCBs) are persistent organic pollutants that frequently bind to nanoparticles (NPs) in the environment. Our results demonstrate that binding PCB153, one of the most abundant PCB congeners in the environment, to silica nanoparticles (PCB153-NPs) potentiates cerebrovascular toxicity and stroke outcomes via stimulation of inflammatory responses and disruption of BBB integrity. These events are mediated by activation of toll-like receptor 4 (TLR4), which subsequently recruits tumor necrosis factor-associated factor 6 (TRAF6) and initiates the production of multiple inflammatory mediators. Research presented in this dissertation demonstrates that aging and environmental pollutants play crucial roles in modifying the function of the BBB through alterations of inflammatory responses and TJ protein expression, which further contribute to the progression of stroke-induced cerebral ischemic injury.

AIM: to determine if a relationship exists between TLR-2, TLR-4, TLR-9, or CD14 polymorphisms and risk for developing chronic periodontal disease in an African-American population. This is the first study conducted to determine role of SNPs in TLR genes and CD14 gene in a periodontally-diseased African-American population. Additionally, this is the first study to assess the role of TLR-9 polymorphism in periodontitis patients. METHODS: A total of 130 subjects were involved in the study. The chronic periodontitis (CP) group contained 73 subjects, and the healthy control (NP) group 57subjects. Genotyping was performed in TLR2 (G2408A), TLR4 (A896G),TLR9 (T1486C) and CD14 (C260T) genes by TaqMan® allelic discrimination using Assay-by-DesignSM SNP Genotyping Assays (Applied Biosystems). Accuracy of genotyping was confirmed by known DNA samples of each genotype and by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analyses on selected samples. Fisher’s exact test and chi-square analyses were performed to compare genotype and allele frequencies. Within disease groups, we investigated whether SNPs were related to disease severity by step-wise logistic regression adjusted for age, gender, and smoking status. RESULTS: There was a significant difference in the distribution of specific TLR9 (T1486C) genotypes between the periodontally diseased group versus the control group. Expression of TT genotype was more prevelant in periodontally-diseased individuals compared to periodontally-healthy subjects (p<0.0001) whereas individuals expressing C allele of the TLR9 SNP (CC&CT) were more frequently found in the control group after adjusting for age, gender, and smoking status (p<0.0001) There was no statistically significant difference in the distribution of genotypes between groups for any other TLRs or CD14 polymorphism. CONCLUSION: Based on findings of this study, homozygocity for the T allele of TLR 9 polymorphism was related to chronic periodontal disease susceptibility in African Americans. Additionally, presence of the C allele at TLR-9 appeared to confer resistance to periodontal destruction. Our results showed that specific SNPs in TLR-2, -4 and CD14 genes are not related to periodontitis in African Americans.

Os tecidos periodontais estão em confronto continuo com microorganismos capazes de disparar mecanismos da resposta imune inata, dando origem ao infiltrado inflamatório. Estudos recentes mostraram a importancia dos receptores do tipo Toll (TLRs) na fase inicial de reconhecimento de diferentes patogenos. A participação de receptores tipo Toll (TLRs) na resposta de neutrófilos e macrófagos frente a periodontopatógenos precisa ser determinada. Nesse estudo procuramos caracterizar o infiltrado inflamatório presente no peritônio de animais deficientes de TLR4-/-, avaliar a atividade fagocítica, bem como a produção de óxido nítrico (NO) e a atividade de mieloperoxidase (MPO) no curso da infecção por Aggregatibacter actinomycetemcomitans. A ausência de TLR4 não influenciou a quimiotaxia de neutrófilos e macrófagos para o local da infecção, a produção de óxido nítrico, a atividade de MPO e a viabilidade celular. No entanto, neutrófilos e macrófagos de animais TLR4-/- apresentaram menor atividade fagocítica quando comparado ao grupo controle (camundongos WT). Em relação a doença periodontal induzida experimentalmente com Aggregatibacter actinomycetemcomitans em camundongos deficientes de TLR4, os resultados mostraram que 100% dos animais deficientes de TLR4 sobreviveram a infecção durante o período de observação. Em relação a análise de perda óssea, os dados revelaram uma menor perda progressiva de osso alveolar na região dos molares de animais deficientes de TLR4. A ausência do receptor interferiu na disseminação da bactéria, uma vez que se observou um grande número de bacilos no linfonodo e baco dos animais que não expressaram TLR4, diferente do observado para os animais selvagens (WT). Os resultados indicam a importância da sinalização via TLR4 durante a resposta imune contra Aggregatibacter actinomycetemcomitans. / Aggregatibacter actinomycetemcomitans is an oral gram negative bacteria that contributes to periodontitis progression. Isolated antigens from A. actinomycetemcomitans could be activating innate immune cells through Toll-like receptors (TLRs), molecules that recognize structural components conserved among microorganisms. In this study, we evaluated the role of TLR4 in the recognition of Aggregatibacter actinomycetemcomitans. Neutrophils and macrophage from TLR4 deficient mice and WT mice were collected and used for the subsequent assays. The phagocytosis of leukocytes against A. actinomycetemcomitans and the presence of apoptotic cells were determined by flow cytometry. The in vivo and in vitro production of NO and MPO was evaluated 24h after A. actinomycetemcomitans challenge. In addition, we examined the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR4-/- mice. The results show that inflammatory cells influx in peritoneal cavity of TLR4-/- mice was similar to that observed into their littermate controls. The phagocytic activity was diminished by cells from TLR4-/- mice. In addition, we did not observe difference in NO and MPO production and the frequency of apoptotic cells between cells from TLR4-/- and WT mice. The results showed that TLR4-deficient mice developed less severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly lower bone loss and inflammatory cell migration to periodontal tissues. Together, these data demonstrate the role TLR4 signals for neutrophils activation after A. actinomycetemcomitans infection and development of periodontal disease.

Seres humanos dependem incessantemente de um sistema de reconhecimento efetivo contra infecções para sobreviver. Dentre as diversas proteínas que compõem a resposta imune inata estão os receptores do tipo Toll (TLR Toll-like Receptors), que possuem a função de reconhecer padrões moleculares associados a patógenos e dar início a uma resposta imune adequada. O carcinoma do colo uterino é uma das principais causas de morte de mulheres por câncer mundialmente, sendo o terceiro tipo de câncer mais comum entre mulheres. Este tipo de neoplasia é vinculada etiologicamente à infecção pelo Papilomavírus humano (HPV). Dentre as principais proteínas virais, E6 e E7 são responsáveis pela manipulação dos processos celulares para promover ciclo viral, sendo essenciais no processo de transformação celular. Nesse contexto, o objetivo deste trabalho foi investigar a importância da via de sinalização de TLRs sobre a infecção por HPV. O polimorfismo rs5743836, na região promotora de TLR9, capaz de alterar a expressão deste receptor, foi estudado quanto à influência sobre a história natural da infecção por HPV em uma coorte de mulheres brasileiras; nenhuma associação relevante foi encontrada, indicando que este polimorfismo não interfere significativamente na resposta à infecção e risco de desenvolvimento de lesões no colo do útero causadas por HPV. Proteínas componentes da via de TLRs demonstraram serem alvos de interação com E6 de HPV16; dentre elas, o notável adaptador MyD88 e IKK&#949;, enzima ativadora de importantes transfatores do sistema imune. Estas interações foram aqui estudadas. A interação de E6 com MyD88 resultou em estabilização da proteína viral, o que parece não depender do sítio LxxLL presente em MyD88, como ocorre com outros parceiros moleculares de E6. O sítio de interação de E6 com IKK&#949; coincide com a região onde se localiza o sítio catalítico desta enzima, sugerindo a ação de E6 na ativação de proteínas alvo de IKK&#949;. Esta interação foi observada em queratinócitos, células alvo das infecções por HPV. A produção de citocinas foi afetada por E6 de HPV16, resultando num aumento da quantidade de IL-8 e IL-6; a indução desta citocina poderia ser explicada pela ativação de IKK&#949;. Estes resultados apontam para a capacidade do HPV16 de interferir com o sistema imune, contribuindo para o processo de carcinogênese. / Humans constantly rely on an effective recognition system against infections in order to survive. Among various proteins that compose the innate immune response, Toll-like Receptors (TLRs) have the role to recognize pathogen associated molecular patterns and initiate a proper immune response. The cervical cancer is one of the main causes of women death worldwide, being the third most common cancer type among women. This type of neoplasia is etiologically associated with the Human papillomavirus (HPV) infection. E6 and E7, two main viral proteins, are responsible for manipulating the cellular processes to promote the virus\' life-cycle, being essential to the cellular transformation process. In the context, the objective of this work was to investigate the relevance of the TLR signaling pathway on the HPV infection. The rs5743836 polymorphism, in the TLR9 promoter region, capable of altering this receptor\'s expression, was studied regarding its influence on the natural history of HPV infection in a Brazilian women cohort; no relevant association was found, indicating that this polymorphism does not interfere significantly in the infection response and risk of developing cervix lesions caused by HPV. Component proteins of TLR pathway were shown to be interaction targets of HPV16 E6; among them, the notable adaptor MyD88 and IKK&#949;, enzyme that activates important immune system transfactors. These interactions were studied in this work. The interaction of E6 with MyD88 resulted in the stabilization of the viral protein, which seems independent of the LxxLL site present on MyD88, as in other E6 molecular partners. The interaction site on IKK with E6 matches with the region containing the enzyme\'s catalytic site, suggesting an influence of E6 in the activation of IKK&#949; target proteins. This interaction was observed in keratinocytes, natural targets of HPV infections. The cytokines production was altered by HPV16 E6, resulting in an increase of IL-8 and IL-6 concentration; the induction of the latter could be explained by the activation of IKK&#949;. These results point to the ability of HPV16 of interfering with the immune system, contributing to the carcinogenesis process.

Les nouveau-nés (enfants, ruminants) sont particulièrement sensibles à l’infection intestinale par le parasite Cryptosporidium parvum car leur système immunitaire est encore en cours de développement. Peu de solutions de contrôle existent à ce jour. Il n’existe pas de vaccin et seule une molécule l’Halocur™ possède une AMM pour les veaux mais l’utilisation du traitement est contraignante et il peut présenter une toxicité pour l’animal. Le développement de nouvelles alternatives immunoprophylactiques requiert de mieux comprendre les mécanismes immunitaires mis en jeux lors de l’infection. L’immunité innée joue un rôle prépondérant pour le contrôle de la phase aigüe de l’infection et nous avions montré au laboratoire que les phagocytes mononucléés CD11c+ sont des acteurs déterminant dans le processus protection. Lors de cette thèse nous avons confirmé le rôle des cellules dendritiques (DC) CD103+ en utilisant des souriceaux BatF3-/- chez qui le développement des deux sous-populations CD103+CD11b+ et CD103+CD11b- est altéré au niveau intestinal ce qui rend les animaux beaucoup plus sensibles à l’infection. / Newborns (children, ruminants) are particularly susceptible to intestinal infection by the parasite Cryptosporidium parvum because their immune system is still developing. To date, parasite control methods are limited. There is no vaccine and the only molecule which possess a marketing authorization for calves, Halocur ™, presents toxicity at 2 times the therapeutic dose. The development of new immunoprophylactic methods requires better understanding of the immune mechanisms occurring during infection. Innate immunity plays a major role in controlling the acute phase of infection and we previously demonstrated in the laboratory that intestinal mononuclear phagocytes CD11c+ are key players in the protection process. In this thesis, we confirmed the role of dendritic cells (DC) CD103+ using mice BatF3-/- in which the development of the two DC subsets CD103+CD11b+ and CD103+CD11b- is altered in the intestine making these animals more susceptible to infection. This high susceptibility can be partially mitigated by preventive administration of IL-12 to Batf3-/- neonatal mice. Batf3-/- adult mice which are only deficient for the CD103+CD11b- DC subset were transiently susceptible to infection in contrast to conventional mice that are highly resistant.

O Zika vírus (ZIKV) pertence à família flaviviridae que inclui espécies transmitidas principalmente por mosquitos Aedes sp. No Brasil, casos de microcefalia fetal foram associados à infecção congênita por ZIKV. Vários mecanismos de imunorregulação operantes durante a gestação como a inflamação gerada e resposta antiviral são vitais para o entendimento da infecção congênita do ZIKV. Desta forma, é proposto avaliar se a inflamação via ativação do receptor Toll-like 4 (TLR4-LPS) em vilos placentários humanos e em modelo murino interfere na infecção por ZIKV. Para isso, explantes de vilos placentários humanos de 3o trimestre foram ativados com LPS e posteriormente infectados com ZIKV. Nossos achados mostram que a ativação com LPS é capaz de aumentar a replicação de ZIKV em vilos placentários e de elevar a atividade enzimática da lactato desidrogenase, indicativo de ativação celular. A replicação viral foi inibida com o tratamento dos explantes com um antioxidante, Naringenina, mostrando potencial terapêutico. A ativação placentária com LPS inibiu a via IRF-3, diminuindo a atividade antiviral e aumentando a clivagem proteica do componente da via de autofagia LC3, sendo que estes mecanismos podem estar relacionados com o aumento da replicação viral. Em paralelo, foi detectado aumento da produção de citocinas inflamatórias e hiperplasia das células de Hofbauer nos explantes inflamados e infectados, indicando que estes macrófagos fetais podem ser um nicho de replicação viral. Em modelo murino, evidenciamos que a inoculação intravaginal de LPS em fêmeas prenhas previamente à infecção com ZIKV é capaz de promover o aumento de carga viral no cérebro das mães e da prole, com agravamento do quadro de microcefalia nos fetos. Em conjunto, os dados mostram que em modelo de explante placentário humano e, in vivo , em camundongos, a inflamação é fator predisponente da replicação do ZIKV. As estratégias imunomoduladoras mostram potencial terapêutico, atenuando a resposta inflamatória e a diminuição da replicação viral. / The Zika virus (ZIKV) belongs to the flaviviridae family that includes species transmitted mainly by mosquitoes Aedes sp. In Brazil, cases of fetal microcephaly were associated with ZIKV congenital infection. Several mechanisms of immunoregulation operative during gestation as the inflammation generated and antiviral response are vital for the understanding of congenital ZIKV infection. Thus, it is proposed to evaluate whether inflammation via Toll-like receptor 4 (TLR4-LPS) activation in human and murine placental villi interferes with ZIKV infection. For this, human placental explants of 3rd trimester were activated with LPS and later infected with ZIKV. Our findings show that LPS activation is capable of increasing ZIKV replication in placental villi and elevating the enzymatic activity of lactate dehydrogenase, indicative of cellular activation. Viral replication was inhibited with the treatment of explants with an antioxidant, Naringenin, showing therapeutic potential. LPS placental activation inhibited the IRF-3 pathway, decreasing antiviral activity and increasing protein cleavage of the autophagy pathway component LC3, and these mechanisms may be related to increased viral replication. In parallel, increased production of inflammatory cytokines and Hofbauer cell hyperplasia were detected in inflamed and infected explants, indicating that these fetal macrophages may be a niche for viral replication. In the murine model, we demonstrated that the intravaginal inoculation of LPS in pregnant females prior to infection with ZIKV is able to promote the increase of viral load in the brains of mothers and offspring, with worsening of the microcephaly in fetuses. Together, the data show that in the model of human placental explant and, in vivo, in mice, inflammation is a predisposing factor of ZIKV replication. Immunomodulatory strategies show therapeutic potential, attenuating the inflammatory response and decreasing viral replication.

Occupational exposure to endotoxin-rich swine barn air induces respiratory diseases and loss of lung function. Barn exposure induces recruitment of pulmonary intravascular monocytes/macrophages (PIMMs) and subsequent increased host sensitivity to <i>Escherichia coli</i> LPS challenge. Therefore, to further clarify the biology of PIMMs we examined the role of recruited PIMMs in a rat <i>Escherichia coli</i>-induced lung inflammation model. Following sepsis, lung inflammation was induced with recruitment of PIMMs and subsequently, <i>Escherichia coli</i> LPS challenge exacerbated the lung inflammation with localization of multiple inflammatory cytokines in PIMMs to suggest their possible involvement in modulating lung inflammation in this model.<p> In order to delineate mechanisms of barn air induced lung dysfunction, a rat model of occupational exposure was characterized to show that one and five exposures to the barn environment induced acute lung inflammation and increased airway hyperresponsiveness (AHR). Following 20 exposures, AHR was dampened to indicate adaptive responses. Barn air contains high levels of endotoxin which led us to investigate its role in lung inflammation and AHR. Exposure of mice with either a functional TLR4 (WT) or non-functional TLR4 (mutants) to barn air revealed dependence of lung inflammation but not AHR on a functional TLR4.<p> I investigated whether exposure to barn air alters host responses to a subsequent microbial challenge. Following one day barn exposure and <i>Escherichia coli</i> LPS challenge, lung inflammation was exacerbated with increased granulocytes and IL-1β levels compared to one day barn exposed rats without <i>Escherichia coli</i> LPS challenge. However, increased granulocytes and IL-1β levels in barn exposed and <i>Escherichia coli</i> LPS challenged rats were not different from control rats treated with <i>Escherichia coli</i> LPS indicating a lack of priming effect of barn exposure. However, above results are suggestive of an underlying risk of increased lung inflammation following secondary microbial infection in naïve barn workers.<p> Lastly, I investigated the expression and activity of novel signalling molecules called <i>N</i>-myristoyltransferase and calcineurin in barn air and <i>E. coli</i> LPS induced lung inflammation models. Following one day barn exposure, increased protein expression but not activity of <i>N</i>-myristoyltransferase and calcineurin was shown. However, there is a need to identify the specific role of these two molecules in barn air induced lung inflammation. To conclude, animal models of barn exposure are useful tools to understand mechanisms of lung inflammation and AHR. However, there is still a need to examine endotoxin-independent nature of AHR and roles of other molecules of the innate immune system in regulating barn air induced effects.

River buffalo are economically important to many countries and only recently has their genome been explored for the purpose of mapping genetic variation in traits of economic and biologic interest. The purpose of this research is to characterize the genetic and evolutionary profile of Toll-like receptor 5 (TLR5), which mediates the mammalian innate immune response to bacterial flagellin. This study is comprised of three parts: 1) generating a radiation hybrid (RH) map of river buffalo chromosome 5 (BBU5) where the TLR5 gene is located and building a comparative map with homologous cattle chromosomes; 2) conducting a single-nucleotide polymorphism (SNP) survey of the TLR5 gene to reveal variation within river buffalo and other species; and 3) performing an evolutionary study by inferring phylogenetic trees of TLR5 across multiple taxa and determining the possible evolutionary constraints within the TLR5 coding region. River buffalo chromosome 5 is a bi-armed chromosome with arms corresponding to cattle chromosomes 16 and 29. A BBU5 RH map was developed using the previously published river buffalo RH mapping panel and cattle-derived markers. The RH map developed in this study became an integral part of the first river buffalo whole genome RH map. Genetic variation of the TLR5 gene was evaluated in a small domestic herd of river buffalo. Sequencing of the TLR5 coding region and partial associated 5'- and 3'-untranslated regions yielded 16 novel SNPs. Six SNPs were identified as non-synonymous with one predicted to potentially code for a functionally altered product. For the evolutionary study of the TLR5 coding region, phylogenetic trees were inferred based on TLR5 variation across multiple orders and another for artiodactyla. Species that are closely related to river buffalo appear to have undergone negative selection in TLR5 while those that diverged from river buffalo earlier may be retaining alleles that river buffalo are removing from the population. In conclusion, putative chromosomal rearrangements were identified between river buffalo and cattle, the variation that was uncovered in the TLR5 coding region could potentially lead to differential immunity across species, and there appears be some evolutionary flexibility in the DNA sequence of the TLR5 coding region.

Occupational exposure to endotoxin-rich swine barn air induces respiratory diseases and loss of lung function. Barn exposure induces recruitment of pulmonary intravascular monocytes/macrophages (PIMMs) and subsequent increased host sensitivity to <i>Escherichia coli</i> LPS challenge. Therefore, to further clarify the biology of PIMMs we examined the role of recruited PIMMs in a rat <i>Escherichia coli</i>-induced lung inflammation model. Following sepsis, lung inflammation was induced with recruitment of PIMMs and subsequently, <i>Escherichia coli</i> LPS challenge exacerbated the lung inflammation with localization of multiple inflammatory cytokines in PIMMs to suggest their possible involvement in modulating lung inflammation in this model.<p> In order to delineate mechanisms of barn air induced lung dysfunction, a rat model of occupational exposure was characterized to show that one and five exposures to the barn environment induced acute lung inflammation and increased airway hyperresponsiveness (AHR). Following 20 exposures, AHR was dampened to indicate adaptive responses. Barn air contains high levels of endotoxin which led us to investigate its role in lung inflammation and AHR. Exposure of mice with either a functional TLR4 (WT) or non-functional TLR4 (mutants) to barn air revealed dependence of lung inflammation but not AHR on a functional TLR4.<p> I investigated whether exposure to barn air alters host responses to a subsequent microbial challenge. Following one day barn exposure and <i>Escherichia coli</i> LPS challenge, lung inflammation was exacerbated with increased granulocytes and IL-1β levels compared to one day barn exposed rats without <i>Escherichia coli</i> LPS challenge. However, increased granulocytes and IL-1β levels in barn exposed and <i>Escherichia coli</i> LPS challenged rats were not different from control rats treated with <i>Escherichia coli</i> LPS indicating a lack of priming effect of barn exposure. However, above results are suggestive of an underlying risk of increased lung inflammation following secondary microbial infection in naïve barn workers.<p> Lastly, I investigated the expression and activity of novel signalling molecules called <i>N</i>-myristoyltransferase and calcineurin in barn air and <i>E. coli</i> LPS induced lung inflammation models. Following one day barn exposure, increased protein expression but not activity of <i>N</i>-myristoyltransferase and calcineurin was shown. However, there is a need to identify the specific role of these two molecules in barn air induced lung inflammation. To conclude, animal models of barn exposure are useful tools to understand mechanisms of lung inflammation and AHR. However, there is still a need to examine endotoxin-independent nature of AHR and roles of other molecules of the innate immune system in regulating barn air induced effects.