Studies examining the immune response upon exposure to chitin microparticles in living models have reached drastically differing conclusions, and the reason remains unclear. One notable issue between the experiments is that they have not characterized their chitin preparations for degree of acetylation. They all use different chitin processing methods prior to administration, which could potentially be the source of the variance between studies. Chitin and chitosan preparations specified in the literature and several novel preparations were analyzed for degree acetylation (DA) using High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). It was found that autoclaving and sonication processing steps do not have a significant influence on degree of acetylation. Chitin and chitosan preparations were used to create a dose-response curve of DA compared to cytokine elicitation from THP-1 monocytes, and it was found that the initial response was dominated by TNF (similar to previous studies), though after 12 hours showed a tip toward the start of an IL-1β-dominated Th17 effector response. This study also confirmed that immunostimulatory effects can occur from chitin and chitosan particles at orparticles, which would have long residence times in air, might be implicated in initiating allergic or asthmatic processes.

<p> Mammalian sialidases are hydrolytic enzymes that initiate the removal of terminal a2-3, a2-6 and a2-8 sialic acid residues from various sialylated glycoconjugates. Sialidases are reportedly involved in numerous cellular functions involving proliferation, differentiation, antigenic expression, inflammation and the tumorigenicity of malignant cells. Recently, sialidase has been implicated in various immune signaling pathways, involving immune effector cells, such as activated lymphocytes and macrophages. The human lysosomal sialidase gene encodes a 46 kD glycoprotein which exists in a multienzyme complex with β-galactosidase and PPCA. Neurodegenerative diseases such as Tay-Sachs and Sandhoff are characterized by the progressive storage of glycoproteins and sialylated oligosaccharides in the nervous system. The induction of inflammatory mediators is a critical step in the pathogenesis of neurodegeneration that remains largely undefined. As such, an in vitro model of Tay-Sachs disease was used to identify potential mediators involved in disease progression. In addition, we have used the THP-1 monocytic cell line as a model of human macrophages which play a key role in potentiating a variety of immune responses. </p> <p> Translocation of neul from lysosomes to the cell surface and the resulting interaction with signaling molecules suggests neul is involved in the regulation of immune activities. We have investigated the role of sialidase on CD44 expression, an inflammation-associated glycoprotein found on the cell surface. Our data indicate that sialidase interacts with CD44 on the cell surface which may contribute to disease progression in Tay-Sachs disease. We illustrate that overexpression of sialidase stimulates interleukin-6 (IL-6) secretion in both human Tay-Sachs neuroglia and THP-1 derived macrophages. Moreover, the sialidase inhibitor 2-deoxy-2, 3-dehydro-N-acetylneuraminic acid (DANA) was found to attenuate IL-6 secretion and sialidase expression in THP-1 derived macrophages. </p> / Thesis / Master of Science (MSc)

The system of mononuclear phagocytes includes macrophages that modulate their phenotype based on microenvironmental signals. Their properties vary considerably in a differentiated stage. M1 macrophages, which are classically activated (typically by IFN-γ), are involved in phagocytosis and produce some pro-inflammatory cytokines, that can stimulate other immune cells. A phenotypically different cell population are M2 macrophages, which are alternatively activated by exposure by Th2 cytokines. M2 macrophages produce preferentially anti-inflammatory cytokines IL-10, TGF-β and participate in repair and tissue healing. The main aim of this study was to standardize a model of differentiation of THP-1 cells and human monocytes towards the M2 phenotyp using an in vitro model. This is represented in particular by increased expression of CD163 and CD206 molecules. The second aim was to assess the dynamics of expression (and co-expression) of CD163 and CD206 molecules in monocytes of patients after kidney transplantation. Expression of surface markers was determined by flow cytometry. Both THP-1 cells and human monocytes, isolated from buffy coat fraction, were stimulated by IL-4, TNF-α, TGF-β and IL-10. Changes in CD163 and CD206 expression were measured after day 1, day 3 and day 6 of stimulation. The most...

En dehors de son rôle de stockage de l’énergie cellulaire, l’adénosine-triphosphate (ATP) est également une molécule de signalisation extracellulaire qui agit sur deux familles de récepteurs, les récepteurs métabotropiques P2Y et les récepteurs ionotropiques P2X. Dans le système nerveux central, les récepteurs P2X et la signalisation purinergique sont impliqués dans de nombreuses fonctions physiologiques comme la modulation de la transmission synaptique et la communication neurone-glie ainsi que dans diverses pathologies telles que les douleurs chroniques, l’épilepsie ou les maladies neurodégénératives. Enregistrer l’activité des récepteurs P2X in situ reste aujourd’hui encore difficile de par la paucité des outils pharmacologiques et de par les propriétés biophysiques particulières de ces récepteurs canaux. De surcroit, les mécanismes de libération de l’ATP sont encore mal caractérisés et la détection de cette molécule dans l’espace extracellulaire est limitée par la faible résolution spatio-temporelle des techniques disponibles. A ce jour, il n’existe aucune méthode satisfaisante permettant de suivre l’activité des récepteurs P2X ou détecter la libération d’ATP en temps réel. Pour pallier à ces manques, nous avons développé de nouveaux outils fluorescents basés sur la fusion du rapporteur calcique GCaMP6s aux récepteurs P2X.Notre étude montre d’abord que ces biosenseurs P2X-GCaMP6s sont capables de rapporter spécifiquement et dynamiquement, en fluorescence, l’activité des récepteurs P2X dans différentes lignées cellulaires (HEK, astrocytes, macrophages), ainsi que dans des neurones hippocampiques en culture. Dans un deuxième temps, l’outil P2X2-GCaMP6s a été modifié afin de créer un biosenseur de haute affinité pour l’ATP extracellulaire. Deux mutants, dont l’affinité apparente est de l’ordre de la centaine de nanomolaire, ont permis de détecter et de quantifier une libération d’ATP endogène. En combinant l’utilisation de ces biosenseurs avec des approches pharmacologiques et génétiques, nous avons montré que lors d’un choc hypotonique l’activation du canal VRAC LRRC8 contribue à la libération d’ATP par les cellules HEK et par des monocytes humains différenciés en macrophages. Enfin, nous avons montré que la libération d’ATP lors du gonflement des cellules déclenchait le phénomène de régulation du volume cellulaire, permettant aux cellules de retrouver leur volume initial.Ces biosenseurs fluorescents permettent donc de visualiser de façon dynamique l’activité des récepteurs P2X et la libération d’ATP. Ces outils étant compatibles avec des approches in vivo, ils devraient permettre une meilleure caractérisation des mécanismes moléculaires de la communication purinergique. / Adenosine 5'-triphosphate (ATP) is an extracellular signaling molecule acting on two major classes of membrane receptors, metabotropic P2Y receptors and ionotropic P2X receptors. In the central nervous system, P2X receptors are involved in diverse functions such as modulation of synaptic transmission or neuron-glia communication and are implicated in different pathologies including chronic pain, epilepsy or neurodegenerative diseases. Recording P2X receptor activity is difficult because of the paucity of pharmacological tools and because P2X receptors are prone to desensitization. In addition, measuring extracellular ATP concentration is challenging since the mechanisms and the source of ATP are still poorly characterized. In addition, classical ATP detecting approaches have clear spatial and temporal limitations. As a consequence, following P2X activity and visualizing or quantifying ATP release in real time remains challenging. To overcome these issues, we developed new fluorescent biosensors based on the fusion of the fluorescent calcium reporter GCaMP6s to P2X receptors.We first determined that fluorescence specifically reports on the activity of the P2X2 channel in different cell line (HEK, astrocytes, macrophages) and in primary culture of hippocampal neurons. We next engineered P2X2 receptor to create high affinity ATP biosensors. We identified two mutants with EC50s for ATP in the 100 nanomolar range that allow for the detection and the quantification of endogenous ATP release evoked by cell swelling. Using pharmacological approaches and knock-out cells, we demonstrated the implication in ATP release of the recently identify volume-regulated anion channel, LRRC8A in HEK cells and differentiated human macrophages. Finally, we provided evidence that the LRRC8-dependant ATP release is necessary for the cellular regulation of volume decrease after swelling.Our results show that these fluorescent ATP biosensors can be used to dynamically track P2X channel activity and can be used in vivo to decipher the molecular mechanisms involved in purinergic signaling.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Alžbeta Horvátová Supervisor: prof. PharmDr. Petr Pávek PhD. Title of diploma thesis: Anti-inflammatory effects of ursodeoxycholyl lysophosphatidylethanolamide on THP-1 human macrophages via Toll-like receptor 4 Nonalcoholic steatohepatitis (NASH) became the most common liver disease in developed countries. It is well-known that the level of protectant phosphatidylcholine (PC) is decreased in NASH. The bile acid-phospholipid conjugate ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE) was designed in order to specifically deliver PC to hepatocytes. However, previous studies have proved that UDCA-LPE possesses its proper hepatoprotectant capacity and exhibits anti-apoptotic, anti-inflammatory, anti-fibrotic properties and also improved steatosis and hyperlipidaemia in various models in vivo. These effects may be mediated secondary through modulation of immune system. Therefore, in order to dissect if UDCA-LPE directly influences immune cells in vitro, release of pro-inflammatory cytokines TNFα, IL-6 and IL-1β in LPS-induced THP-1-derived human macrophages was measured by ELISA. Moreover, effects of UDCA-LPE on MAPK signalling pathways and nuclear translocation of NFκB were...

A diferenciação das células THP-1 em macrófagos, induzida por PMA, é associada ao aumento da expressão de PPAR. A UTR 5` de PPAR regula negativamente sua síntese, porém, o mecanismo molecular envolvido não foi esclarecido. Neste estudo, o estado traducional das células THP-1 diferenciadas por PMA foi investigado em associação à superprodução de PPAR. A presença de uORFs no transcrito de PPAR, contendo códons de iniciação compatíveis com seqüências de Kosak, poderia ser a causa do efeito inibitório da UTR 5`. A incorporação reduzida de L-[U-14C]leucina revelou que a superprodução de PPAR ocorre durante inibição global da tradução, confirmada pela redução dos polissomos. Além disso, desfosforilação de 4E-BP1 foi observada após tratamento com PMA e é associada a inibição da iniciação da tradução e estimulação da tradução dependente de IRES. De fato, a estrutura da UTR 5` de PPAR apresenta características de transcritos que formam IRES. Assim, a produção de PPAR pode ser regulada por IRES e ocorre concomitantemente com a inibição da tradução dependente de cap / The differentiation of THP-1 cells in macrophages, induced by PMA, is associated to overexpression of PPARb. Previous studies have shown that the PPARb 5\' UTR negatively regulates its expression. In our study the translational status of PMA-differentiated THP-1 cells was investigated in association to PPARb overexpression. Putative compatible Kosak initiation codons were identified in the PPARb uORFs and could be involved in the inhibitory effect of 5\' UTR. Decreased incorporation of L-[U-14C]leucine in proteins revealed that the overproduction of PPARb in PMA-differentiated THP-1 cells coincides with a global decrease in the protein synthesis process. Translation impairment was confirmed by polysome profile assay. An intense dephosphorylation of 4E-BP by PMA treatment was observed. Dephosphorylated 4E-BP causes inhibition of eIF4E cap-dependent translation initiation and favors IRES-dependent translation. The PPARb 5\' UTR structure has some characteristics that resemble the one described for IRES. Therefore, the PPARb production may be controlled by IRES

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-14T11:53:05Z No. of bitstreams: 1 danilo-parmera.pdf: 3155055 bytes, checksum: ac63cd2b7c2719955b83925c310aabbc (MD5) / Made available in DSpace on 2012-11-14T11:53:05Z (GMT). No. of bitstreams: 1 danilo-parmera.pdf: 3155055 bytes, checksum: ac63cd2b7c2719955b83925c310aabbc (MD5) Previous issue date: 2007 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / As culturas de células vêm sendo utilizadas extensivamente no desenvolvimento e na produção de uma variedade de produtos terapêuticos e profiláticos, tornando-se uma ferramenta indispensável para geneticistas, imunologistas, vacinologistas e a indústria farmacêutica. A adoção de sistemas de ensaios celulares in vitrotem sido aplicada como um método alternativo para a substituição ou diminuição do uso de animais nas fases de desenvolvimento, produção e testes de vacinas, demonstrando resultados promissores. Da mesma forma, sistemas computacionais podem ampliar a utilização desta ferramenta para etapas do desenvolvimento de vacinas candidatas na fase pré-clínica. O presente trabalho teve como objetivo o desenvolvimento de umprotocolo in vitroutilizando culturas de células humanas - a linhagem de monócitos THP-1, para a seleção de um protótipo vacinal - a cepa Pasteur de Mycobacterium bovisBCG expressando o antígeno Sm14 de Schistossoma mansoni(BCG/sm14). Para isso foram empregadas duas metodologias – citometria de fluxo e imunocitoquímica, para a identificação do perfil da expressão das citocinas IL-10, IL-12 e TNF-α. Foram utilizados como controles a cepa Pasteur do M. bovisBCG e a construção da cepa Pasteur do M. bovisBCG contendo o vetor de expressão pAU5 (BCG/pAU5).Após padronização, a multiplicidade de infecção utilizada para os experimentos foi de 10 bacilos para 1 célula THP-1 (10MOI). A expressão daproteína Sm14 foi detectada em todos os protótipos vacinais. Para assegurar queo protótipo BCG/sm14 era capaz de diferenciar a linhagem de monócitos THP-1 em macrófagos, avaliamos a taxa de crescimento celular e foi possível observarque após a infecção estas células apresentavam o índice de proliferação diminuído em 2 logs em relação à célula não infectada. O protótipo vacinal BCG/sm14não mostrou diferenças quanto a capacidade de infecção, a taxa de persistência intracelular e a estabilidade da construção plasmidial. As duas metodologias empregadas mostraram resultados discrepantes em relação ao percentual de citocinas expressas após a infecção com o protótipo vacinal ou os BCG controles, mostrando um maior percentual de células positivas quando avaliados por citometria de fluxo.Contudo, mesmo com esta diferença observamos que o protótipo vacinal BCG/sm14 não alterou o perfil de expressão de IL-10, IL-12 e TNF-α. O fato do plasmídeo pAU5 ser um vetor de expressão citoplasmática, sugere que a proteína Sm14 não foi capaz de estimular a mudança de citocinas em monócitos humanos. Experimentos futuros devem investigar o papel do BCG/sm14em macrófagos maduros, quanto a indução de citocinas pró e anti-inflamatórias, TLR, bem como na indução da resposta imune adaptativa, como a apresentação antigênica, na tentativa de melhor entender a resposta imune a esta vacina candidata. / Cell cultures has been used extensively in the development of a broad range of therapeutic and prophylactic products, and are an important tool for geneticists, immunologists, vaccinologists and the pharmaceuticsindustry. In vitrocell assay has been applied as an alternative method to replace ordiminish the use of animal model in the developmental, production in vaccine test phases, with promising results. Otherwise, computational methods should amplify theuse of cell cultures tools to test candidate vaccines. The mean goal of this work was to develop an in vitro protocol using human cell line – monocytic cell THP-1, to select a vaccine prototype – strain Pasteur Mycobacterium bovisBCG expressing Schistosoma mansoniSm14-antigen (BCG/sm14). Two methodologies were employed – a flow cytometry and immunocytochemistry, to quantify the expression of IL-10, IL-12 e TNF-α. Pasteur M. bovisBCG and the strain Pasteur do M. bovisBCG containing the expression vector pAU5 (BCG/pAU5) were used as controls. Multiplicityof infection (MOI) was determined and showed a better 10 bacilli to 1 cells ratios, regarding the expression of intracellular cytokines. Sm14 protein expressionwas detected in all vaccine prototype before use. To assure that BCG/sm14was able to differentiate monocytic THP1 cell line in mature macrophage, we evaluated the proliferative ration after BCGs infection and all strain showed the ability toinduce monocytic THP1 cells maturation, diminishing in 2.0 logs the cell proliferation. No differences were seen in the uptake, intracellular persistence and plasmidial stability. Interestingly, we observe discrepant results regarding the amount of positivecells expressing cytokines detected by the two methods used, independent of which BCG was tested. The overall results obtained by the cytometric method was high than immunocytochemistry. However, beside these ambiguous results, no alteration in the IL-10, IL-12 and TNF-alfa profile was observed whenBCG/sm14was compared with BCG. These results point to the plamidial BCG construction pAU5 and its intracellular expression, suggesting no modification in the cytokine profile in human monocytic cell lines. Further experiments should be addressedto identify the role of BCG/sm14 in modulate mature macrophage and, the induction ofadaptive immune response, as antigen presentation, pro- and anti-inflammatory cytokines, TLR expression and activation, to better understating the vaccine prototype immune response.

A diferenciação das células THP-1 em macrófagos, induzida por PMA, é associada ao aumento da expressão de PPAR. A UTR 5` de PPAR regula negativamente sua síntese, porém, o mecanismo molecular envolvido não foi esclarecido. Neste estudo, o estado traducional das células THP-1 diferenciadas por PMA foi investigado em associação à superprodução de PPAR. A presença de uORFs no transcrito de PPAR, contendo códons de iniciação compatíveis com seqüências de Kosak, poderia ser a causa do efeito inibitório da UTR 5`. A incorporação reduzida de L-[U-14C]leucina revelou que a superprodução de PPAR ocorre durante inibição global da tradução, confirmada pela redução dos polissomos. Além disso, desfosforilação de 4E-BP1 foi observada após tratamento com PMA e é associada a inibição da iniciação da tradução e estimulação da tradução dependente de IRES. De fato, a estrutura da UTR 5` de PPAR apresenta características de transcritos que formam IRES. Assim, a produção de PPAR pode ser regulada por IRES e ocorre concomitantemente com a inibição da tradução dependente de cap / The differentiation of THP-1 cells in macrophages, induced by PMA, is associated to overexpression of PPARb. Previous studies have shown that the PPARb 5\' UTR negatively regulates its expression. In our study the translational status of PMA-differentiated THP-1 cells was investigated in association to PPARb overexpression. Putative compatible Kosak initiation codons were identified in the PPARb uORFs and could be involved in the inhibitory effect of 5\' UTR. Decreased incorporation of L-[U-14C]leucine in proteins revealed that the overproduction of PPARb in PMA-differentiated THP-1 cells coincides with a global decrease in the protein synthesis process. Translation impairment was confirmed by polysome profile assay. An intense dephosphorylation of 4E-BP by PMA treatment was observed. Dephosphorylated 4E-BP causes inhibition of eIF4E cap-dependent translation initiation and favors IRES-dependent translation. The PPARb 5\' UTR structure has some characteristics that resemble the one described for IRES. Therefore, the PPARb production may be controlled by IRES

During normal cell development, many genes are activated and repressed, usually through epigenetic mechanisms. These are modifications of the DNA and protein within the nucleus that result in changes in gene expression without alteration in DNA sequence. Key proteins for epigenetic modifications are the histone proteins bound to DNA in the nucleus. The best-characterised epigenetic complexes that modify histone proteins are the polycomb group proteins (PcG), comprising polycomb repressive complexes 1 (PRC1) and 2 (PRC2). The repressive modifications generated by these complexes can be removed, and the blocked genes reactivated, by enzymes that are the subject of this project. PRC1 repressive marks are removed by deubiquitinases USP12, USP16 and BAP1, whereas PRC2 marks are removed by demethylases KDM6A, KDM6B and potentially UTY. During the development of cancer, the regulation of many genes becomes abnormal, allowing the cells to escape normal growth restrictions. In this thesis, the expression of this set of chromatin-modifying enzymes in a leukaemia cell line was investigated. The FANTOM consortium has been helping to understand patterns of gene expression for over 10 years. The FANTOM4 dataset described changes in gene expression and promoter usage during differentiation of the THP-1 acute monocytic leukaemia cell line, using CAGE (Cap Analysis of Gene Expression) technology. This human monocyte-like cancer cell line can be stimulated with phorbol esters to halt proliferation and differentiate into macrophages. However, the FANTOM4 time course did not capture detailed mechanisms of regulatory factors in macrophage differentiation due to sparse time points and low read coverage. The main aim of this project was therefore to repeat the time course with tighter time points and deeper sequencing of the transcriptome to develop a very precise picture of sequential activation of gene expression, transcription start site (TSS) usage and the activity of enhancers during the transition from proliferating monocytes to differentiated macrophage phenotype of the THP-1 leukaemia cell line, using CAGE. The focus of this research was on the chromatin-modifying enzymes, but other key cell cycle and macrophage genes have also been examined. The differentiation time course was repeated in triplicate. RNA was extracted and CAGE libraries generated for 18 time points, including the 6 originally studied in FANTOM4. Sequencing results were analysed and normalised using bioinformatics tools. It was shown that analysing 8 samples on one Illumina HiSeq 2500 lane yielded enough read coverage to detect activity from even low expression TSSs, such as those associated with enhancer activity. Clusters of genes which were up- and downregulated at different time points during the differentiation process were identified and characterised. CAGE results for key genes encoding chromatin modifying enzymes and macrophage markers were validated by qRT-PCR. There was a rapid increase of histone demethylase KDM6B mRNA once differentiation was initiated. Histone deubiquitinase USP12 mRNA was also upregulated early in the process. Histone deubiquitinase BAP1 mRNA shows an interesting cyclic regulation pattern which was not seen in the more limited samples of FANTOM4. These interesting chromatin-modifying enzymes and their close paralogues (deubiquitinases USP12, USP16 and BAP1, together with demethylases KDM6A, KDM6B and UTY) were investigated by bioinformatics and genetic tools. USP16 knockout THP-1 cell line was successfully created using CRISPR-Cas9 and its ability to differentiate into macrophages was examined using cell cycle analysis and CAGE sequencing. The USP16 knockout cell line, along with siRNA knock downs of USP12, USP16 and BAP1, was also compared to wildtype THP-1 differentiation using CAGE. Unfortunately, creating other mutant THP-1 cell lines was unsuccessful due to low THP-1 viability after single cell sorting. Investigating KDM6A, KDM6B and UTY using bioinformatics showed that UTY and KDM6A gene expression is positively correlated and this is disrupted in cancer samples. Gene expression and sequence comparison suggested that KDM6A and UTY are coregulated and may act in a similar way in histone demethylation. In summary, the results in this thesis show the transcriptomic changes as the leukaemia cell line ceases proliferation and commences differentiation. Detailed examination suggests that histone modifications are important in the transition between proliferation and differentiation and provide better understanding of regulatory factors in macrophage differentiation and leukaemia.

The interaction between epithelial cells and macrophages is integral to mucosal immune fate: determining the decision between tolerance and immune activation/inflammation. Endotoxin tolerisation (ET) is a circumstance where cells go through a hypo-responsive state, unable to respond to further endotoxin-LPS challenge. Mucosal macrophages (MΦs) have a dual functionality that determines tolerance to commensal organisms or immune response to entropathogens such as E. coli. In the case of mucosal inflammatory pathologies, such as Crohn’s disease, this state of tolerance is broken, resulting in destruction of gut mucosal tissue where the macrophage phenotype has been altered from a regulatory M2-like subset phenotype to an inflammatory M1-like subset phenotype, responding to both pathogenic and commensal bacteria. Chronic inflammation by bacterial pathogen related molecular patterns (PAMPs), such as LPS, is well established to induce tolerisation. The aims of this project were firstly, to characterise the control of macrophage differentiation in a mucosal setting by investigating the immunomodulatory effects of PAMPs, such as LPS in presence or absence of TNFα and to investigate ET mechanisms associated with MΦ subsets responding to the entropathogen E. coli K12-LPS. Secondly, to investigate the effect of epithelial cells on macrophage subsets behaviour upon inflammation and ET. M1- and M2-like MΦs were generated in vitro from the THP-1 monocyte cell line by differentiation with PMA and vitamin D3, respectively, whereas differentiated epithelial cells (Caco-2) were obtained by long term culturing for 21 days. A transwell co-culture system of Caco2 cells and MΦ subsets was developed to mimic the cell-to-cell cross-talk between epithelial cells and immune cells. Mono- and co-culture models were pre-treated with either LPS, TNFα or IL-1β prior to stimulation by PAMPs. TNFα, IL-1β, IL-18, IL-6 and IL-10 were qualified by ELISA. Cytokines, PRRs and endogenous negative regulatory molecules were detected by RT-PCR and WB and epithelial barrier function was measured by trans epithelial electrical resistance (TEER). ET induced by K12-LPS failed to demonstrate a differential subset-specific response in MΦ mono-culture system whereas, LPS differentially suppress LPS induced cytokine expression in MΦ co-culture system. Tolerised M1- and M2-like MΦs exhibited a significant reduction in expression and secretion of pro-inflammatory cytokines and comparable levels of anti-inflammatory cytokine, IL-10. The suppression of pro-inflammatory cytokine in these MΦs appeared to be linked to the differential TLR4 expression and up-regulation of negative regulators, such as IRAK-M and Tollip. In addition, MΦ subsets differentially responded to inflammation induced by pro-inflammatory cytokines, TNFα and IL-1β in mono- and co-culture models. In conclusion, tolerisation induced in MΦs is presented by the suppression of pro-inflammatory cytokine, which is associated with corresponding up-regulation of IL-10, TLR4 receptor and the negative regulators, in a subset-independent manner. In the case of cross-talk between epithelial cells and macrophages however, a differential sensitivities to ET was displayed. These findings allow more understanding of MΦ subsets functions and ET mechanisms, which may be beneficial for the development of in-vitro models of MΦ subsets and therapeutics targeting Crohn’s diseases.