O mastocitoma é o tumor cutâneo mais comum dos cães, representando 7% a 21% dos tumores da pele e tecidos moles, 11% a 27% dos tumores malignos cutâneos nessa espécie. Eles possuem uma grande variedade de aparência e comportamento, o qual o torna um desafio seu tratamento. Os retinóides são uma promessa na luta contra o câncer. Entretanto, há poucos estudos sobre os efeitos dos retinóides em neoplasias caninas. O presente trabalho teve como objetivo caracterizar a cultura primária de mastocitomas caninos assim como investigar a quimiosensibilidade deste tumor ao ácido retinóico todo-trans (ATRA). A cultura primária de mastocitomas caninos foi realizada em co-cultivo com fibroblastos, que demonstrou uma interação favorável entre mastócitos e fibroblastos, com uma sobrevida média de 30 dias. A quimiosensibilidade dos mastocitomas caninos ao ATRA não mostrou diferenças entre os graus de mastocitomas, ou seja, tanto um mastocitoma grau II ou III respondem igualmente ao ATRA nas doses estudadas. Foi constatado também que o mastocitoma é mais sensível na concentração 10-4M de ATRA (p < 0,002). Existe também um efeito já nas primeiras 24h, mas esse não se altera em 48h, entretanto se intensifica após 72h. Podemos inferir, então, que a maior quimiosensibilidade de mastocitomas caninos ao ATRA se dá após 72h de exposição na dose de 10-4M. Podemos concluir que o ATRA apresenta efeitos sobre as células de mastocitomas caninos e pode ser usado como potencial adjuvante no tratamento desta neoplasia. / Mast cell tumor (MCT) is one of the most frequent neoplasms that affect the skin and soft tissue of the dog, representing about 7% a 21% of all skin tumors and 11% a 27% of malignant skin tumors in this specie. They present a great variety of appearance and behavior, which becomes a challenge to the treatment. The retinoids are well recognized as promising antitumor agents. However, there have only been a few reports about the effect of retinoids in canine cancers. The aim of this study was to characterize the primary mast cell tumor culture and to investigate the chemosensitivity of this tumor to all trans retinoic acid (ATRA). The primary cell culture of MCT was performed as co-cultive with fibroblasts, showing a positive interaction between mast cells and fibroblasts, with a lifetime of 30 days. The chemosensitivity of MCT to ATRA showed no difference between grade II or III, thus either a MCT grade II or grade III has the same response with ATRA at the doses studied. It has been shown that the MCT is more sensible at the dose 10-4M (p < 0,002). There is also an effect on first 24h untill 48h, changing after 72h. According to these results, it is possible to state that the great chemosensitivity of MCT to ATRA is after 72h of exposition at 10-4M. We can conclude that ATRA may be a potential adjunctive chemotherapeutic agent for the treatment of canine mast cell tumor.

O mastocitoma é o tumor cutâneo mais comum dos cães, representando 7% a 21% dos tumores da pele e tecidos moles, 11% a 27% dos tumores malignos cutâneos nessa espécie. Eles possuem uma grande variedade de aparência e comportamento, o qual o torna um desafio seu tratamento. Os retinóides são uma promessa na luta contra o câncer. Entretanto, há poucos estudos sobre os efeitos dos retinóides em neoplasias caninas. O presente trabalho teve como objetivo caracterizar a cultura primária de mastocitomas caninos assim como investigar a quimiosensibilidade deste tumor ao ácido retinóico todo-trans (ATRA). A cultura primária de mastocitomas caninos foi realizada em co-cultivo com fibroblastos, que demonstrou uma interação favorável entre mastócitos e fibroblastos, com uma sobrevida média de 30 dias. A quimiosensibilidade dos mastocitomas caninos ao ATRA não mostrou diferenças entre os graus de mastocitomas, ou seja, tanto um mastocitoma grau II ou III respondem igualmente ao ATRA nas doses estudadas. Foi constatado também que o mastocitoma é mais sensível na concentração 10-4M de ATRA (p < 0,002). Existe também um efeito já nas primeiras 24h, mas esse não se altera em 48h, entretanto se intensifica após 72h. Podemos inferir, então, que a maior quimiosensibilidade de mastocitomas caninos ao ATRA se dá após 72h de exposição na dose de 10-4M. Podemos concluir que o ATRA apresenta efeitos sobre as células de mastocitomas caninos e pode ser usado como potencial adjuvante no tratamento desta neoplasia. / Mast cell tumor (MCT) is one of the most frequent neoplasms that affect the skin and soft tissue of the dog, representing about 7% a 21% of all skin tumors and 11% a 27% of malignant skin tumors in this specie. They present a great variety of appearance and behavior, which becomes a challenge to the treatment. The retinoids are well recognized as promising antitumor agents. However, there have only been a few reports about the effect of retinoids in canine cancers. The aim of this study was to characterize the primary mast cell tumor culture and to investigate the chemosensitivity of this tumor to all trans retinoic acid (ATRA). The primary cell culture of MCT was performed as co-cultive with fibroblasts, showing a positive interaction between mast cells and fibroblasts, with a lifetime of 30 days. The chemosensitivity of MCT to ATRA showed no difference between grade II or III, thus either a MCT grade II or grade III has the same response with ATRA at the doses studied. It has been shown that the MCT is more sensible at the dose 10-4M (p < 0,002). There is also an effect on first 24h untill 48h, changing after 72h. According to these results, it is possible to state that the great chemosensitivity of MCT to ATRA is after 72h of exposition at 10-4M. We can conclude that ATRA may be a potential adjunctive chemotherapeutic agent for the treatment of canine mast cell tumor.

Le couple télomère/télomérase apparaît comme une cible prometteuse pour de potentiels agents anticancéreux qui seraient actifs sur un large éventail de tumeurs. Le laboratoire d’accueil a montré dans un modèle de leucémie aiguë promyélocytaire (LAP), qu'un agent utilisé en clinique, l'acide rétinoïque (ATRA), exerce une activité anti-tumorale en réprimant la transcription de la sous-unité catalytique hTERT indépendamment de la différenciation. Ce modèle (NB4) avec ses variants cellulaires résistant (NB4-LR1SFD) ou non à la répression de hTERT (NB4-LR1) par l’ATRA constitue un outil de choix pour l’identification de facteurs régulateurs de hTERT et la recherche des bases moléculaires de sa réactivation.Une approche transcriptomique a été utilisée afin d’identifier de nouveaux gènes et/ou réseaux de signalisation induits par l’ATRA et régulateurs de hTERT. L’analyse bioinformatique nous a permis de construire des profils d’expression différentielle entre les 2 lignées et des réseaux d’interaction. Parmi les candidats, H19, un ARN long de 2.5Kb, polyadénylé et non codant. H19 est classé parmi les gènes supresseurs de tumeurs : en son absence il y a développement de cancer (cas de la tumeur de Wilms, rhabdomyosarcome embryonnaire, Syndrome Beekwith-Wiedman) ; sa réintroduction par transfection conduit à une perte de tumoriginicité. Cependant H19 est reconnu de plus en plus comme un oncogène vu que son expression est élevée dans plusieurs types de cancers solides. Par contre peu d’études s’intéressent au rôle de H19 dans les leucémies, d’où notre intérêt pour l’étudier dans le modèle LAP que nous avons développé.Nous avons mis au point la mesure d’expression de H19 par RT-PCR quantitative, validé les données obtenues dans l’analyse transcriptomique et montré que le traitement ATRA induit l’expression de H19 dans les cellules NB4-LR1 alors que cette expression est plutôt diminuée dans les cellules NB4-LR1SFD. L’induction observée dans les cellules NB4-LR1 existe indépendamment de la différenciation. Par contre, cette induction peut être observée associée à la différenciation ou à l’apoptose dans la lignée cellulaire NB4-LR1SFD parallèlement à une diminution importante de l’expression de hTERT. Ce résultat important montre que la lignée NB4-LR1SFD ne présente pas de défaut général d’induction de H19. Ces données suggèrent l’existence d’une corrélation inverse entre le niveau d’expression de hTERT et celui de H19 dans ce modèle cellulaire. De façon importante, l’analyse des banques de données issues de patients LAP publiquement accessibles retrouve cette corrélation inverse.Une diminution d’activité télomérasique est observée dans des extraits cellulaires incubés en présence de l’ARN H19 transcrit in vitro. Cette diminution d’activité est observée aussi après surexpression de H19 in cellulo. Les expériences de RIP (RNA immunoprecipitation) ont montré une diminution de la quantité de hTR lié à hTERT suite à une augmentation d’expression de H19 après traitement ATRA in vitro ou après surexpression de H19 in cellulo. Une hypothèse serait que H19 induirait un déplacement de hTR du complexe hTR-hTERT. Cependant, les expériences de « pull-down » n’ont pas réussi à confirmer l’hypothèse d’une interaction possible entre l’ARN H19 et la protéine TERT.Mon travail de thèse identifie pour la première fois H19, un ARN long non codant, comme facteur régulateur potentiel de hTERT pouvant modifier son activité. Ce travail proposerait non seulement un mécanisme nouveau de régulation de l’activité télomérase mais aussi une fonction nouvelle pour H19 dans ce type de cancer. / The telomere / telomerase pair appears to be a promising target for potential anticancer agents that would be active on a wide range of tumors. The host laboratory has shown in a model of acute promyelocytic leukemia (APL), that a clinically used agent, retinoic acid (ATRA), exerts anti-tumor activity by repressing the transcription of the catalytic subunit hTERT regardless of differentiation. This model (NB4) with its resistant cell variants (NB4-LR1SFD) or not to the repression of hTERT (NB4-LR1) by ATRA is a tool of choice for the identification of hTERT regulatory factors and the search for molecular bases of its reactivation.A "microarray" approach has been used to identify new ATRA-mediated genes and / or signaling networks and potential hTERT regulators. Bioinformatic analysis allowed us to build differential expression profiles between the 2 lineages and interaction networks. Among the candidates, H19, a 2.5Kb long, polyadenylated and non-coding RNA. H19 is classified as a tumor suppressor gene: in its absence there is cancer development (case of Wilms tumor, embryonic rhabdomyosarcoma, Beckwith-Wiedman syndrome); its reintroduction by transfection leads to a loss of tumorigenicity. However H19 is increasingly recognized as an oncogene as its expression is elevated in several types of solid cancers. However, few studies are interested in the role of H19 in leukemias, hence our interest in studying it in the APL model that we have developed.We developed the H19 expression measurement by quantitative RT-PCR, validated the data obtained in the "microarray" analysis and showed that the ATRA treatment induces the expression of H19 in NB4-LR1 cells whereas this expression is rather diminished in NB4-LR1SFD cells. The induction observed in NB4-LR1 cells exists independently of differentiation. On the other hand, this induction can be observed associated with the differentiation or apoptosis in the NB4-LR1SFD cell line in parallel with a significant decrease in the expression of hTERT. This important result shows that the NB4-LR1SFD line does not have a general H19 induction defect. These data suggest the existence of an inverse correlation between the expression level of hTERT and that of H19 in this cellular model. Importantly, the analysis of publicly accessible APL patients’ databases finds this inverse correlation as well.We observed a decrease in telomerase activity in cellular extracts incubated in the presence of in vitro transcribed H19 RNA. This decrease in activity was also observed after overexpression of H19 in cellulo. The RIP (RNA immunoprecipitation) experiments showed a decrease in hTR amount bound to hTERT following an increase in H19 expression after ATRA treatment in vitro or after overexpression of H19 in cellulo. We hypothesize that H19 induces a displacement of hTR from the hTR-hTERT complex. However, the "pull-down" experiments failed to confirm the hypothesis of a possible interaction between H19 RNA and TERT protein.My thesis work identifies, for the first time, the long non-coding RNA H19, as a potential regulator of hTERT that can modify its activity. This work would propose not only a new mechanism of regulation of telomerase activity but also a new function for H19 in this type of cancer.

<p>Epigenetic modifications were shown to play an essential role in tumorigenesis. Epigenetic mechanisms can alter transcription in several ways, through DNA methylation and/or through histone modification. DNA methylation at the TSS (transcriptional start site) has been implicated in tumor development and gene silencing. However, several examples of atypical methylation were shown. In Paper I we present the ICSBP/IRF8 gene that belongs to the IRF family and has characteristics of a tumor suppressor gene. The ICSBP/IRF8 is fully methylated in the promoter and TSS regions in U-937 and despite high expression of the gene. Presence of positive histone marks suggests that methylated DNA can be overridden by histone modification.</p><p>In Paper II a panel of 13 MM (multiple myeloma) cell lines and 9 primary patient tumors were analysed for methylation status of the ICSBP/IRF8 gene. In most cell lines (8/13) the gene was partially or fully methylated and partial methylation was also observed in 1/9 primary tumors. In vitro methylation analysis and treatment with 5-aza-2’deoxycytidine (DAC) proved that the ICSBP/IRF8 gene is silenced by methylation and may be associated with the malignant phenotype.</p><p>In Paper III and IV the NFκB signalling pathway was analysed and the role of ATRA and TNFα induction. In Paper III the data shows that activation of the NFκB pathway is essential in ATRA-induced terminal differentiation in the U-937 cell line and IκBα (S32A/S36A) inhibits ATRA-induced differentiation and G1 cell cycle arrest. This was accompanied by delayed down-regulation of several cyclins (A and E) and up-regulation of p21^WAF1/CIP1 (CDKN1A) and p27^KIP1 (CDKN1B).</p><p>TNFα alone did not induce expression of RA-induced genes analysed in Paper IV. However, ATRA in combination with TNFα showed enhanced activation of RA-induced genes. TNFα triggers demethylation of H3K9me3/H3K9me2 and H3K4me3 at RAR/RXR target genes, which were not accompanied by changes in the level of H3K9-ac. This decrease in H3 methylation by TNFα may pave way for the later ATRA-induced gene transcription.</p>

<p>All-trans retinoic acid (ATRA), a biologically active metabolite of vitamin A, is a powerful inducer of terminal differentiation and growth arrest of several myeloid cell lines <i>in vitro</i>. Although the efficacy of ATRA as an anti-cancer drug has been demonstrated by the successful treatment of acute promyelocytic leukemia (APL), knowledge concerning the molecular mechanisms directing ATRA-induced differentiation and cell cycle arrest of myeloid cells is lacking. Our results show, for the first time, that the complex regulation of cell cycle proteins and myeloid-specific transcription factors induced by ATRA relies on functional Stat1. We found that Stat1 is activated by both tyrosine-701 and serine-727 phosphorylation upon ATRA-induced differentiation of the human monoblastic cell line U-937. Expression of phosphorylation deficient mutants of Stat1 (Stat1Y701F or Stat1S727A) inhibited both ATRA-induced differentiation and cell cycle arrest of U-937 cells, pointing to a requirement of active Stat1 in these processes. </p><p>Detailed analysis of the molecular mechanism of ATRA-induced cell cycle arrest and differentiation showed that the onset of cell cycle arrest was associated with a decrease in c-Myc and cyclin E levels and upregulation of p27^Kip1 and p21^WAF1/CIP1. This was followed by a rapid fall in cyclin A and B and a coordinate dephosphorylation of the retinoblastoma protein (pRb). The inhibition of ATRA-induced cell-cycle arrest by constitutive expression of Stat1Y701F or Stat1S727A was associated with impaired regulation of these cyclins and p27^Kip1, positioning Stat1 activation upstream of these events. To further understand the process of ATRA-induced differentiation, the regulation of myeloid-specific transcription factors was investigated during ATRA-treatment. Notably, ATRA-induced upregulation of Stat2, ICSBP and C/EBP-ε was selectively impaired in sublines expressing Stat1Y701F or Stat1S727A, suggesting an important function of these factors downstream Stat1. Taken together, the work in this thesis clearly demonstrates that Stat1 plays a key role in ATRA-induced terminal differentiation of myeloid cells, through regulation of cell cycle proteins and myeloid-specific transcription factors. </p>

All-trans retinoic acid (ATRA), a biologically active metabolite of vitamin A, is a powerful inducer of terminal differentiation and growth arrest of several myeloid cell lines in vitro. Although the efficacy of ATRA as an anti-cancer drug has been demonstrated by the successful treatment of acute promyelocytic leukemia (APL), knowledge concerning the molecular mechanisms directing ATRA-induced differentiation and cell cycle arrest of myeloid cells is lacking. Our results show, for the first time, that the complex regulation of cell cycle proteins and myeloid-specific transcription factors induced by ATRA relies on functional Stat1. We found that Stat1 is activated by both tyrosine-701 and serine-727 phosphorylation upon ATRA-induced differentiation of the human monoblastic cell line U-937. Expression of phosphorylation deficient mutants of Stat1 (Stat1Y701F or Stat1S727A) inhibited both ATRA-induced differentiation and cell cycle arrest of U-937 cells, pointing to a requirement of active Stat1 in these processes. Detailed analysis of the molecular mechanism of ATRA-induced cell cycle arrest and differentiation showed that the onset of cell cycle arrest was associated with a decrease in c-Myc and cyclin E levels and upregulation of p27Kip1 and p21WAF1/CIP1. This was followed by a rapid fall in cyclin A and B and a coordinate dephosphorylation of the retinoblastoma protein (pRb). The inhibition of ATRA-induced cell-cycle arrest by constitutive expression of Stat1Y701F or Stat1S727A was associated with impaired regulation of these cyclins and p27Kip1, positioning Stat1 activation upstream of these events. To further understand the process of ATRA-induced differentiation, the regulation of myeloid-specific transcription factors was investigated during ATRA-treatment. Notably, ATRA-induced upregulation of Stat2, ICSBP and C/EBP-ε was selectively impaired in sublines expressing Stat1Y701F or Stat1S727A, suggesting an important function of these factors downstream Stat1. Taken together, the work in this thesis clearly demonstrates that Stat1 plays a key role in ATRA-induced terminal differentiation of myeloid cells, through regulation of cell cycle proteins and myeloid-specific transcription factors.

Epigenetic modifications were shown to play an essential role in tumorigenesis. Epigenetic mechanisms can alter transcription in several ways, through DNA methylation and/or through histone modification. DNA methylation at the TSS (transcriptional start site) has been implicated in tumor development and gene silencing. However, several examples of atypical methylation were shown. In Paper I we present the ICSBP/IRF8 gene that belongs to the IRF family and has characteristics of a tumor suppressor gene. The ICSBP/IRF8 is fully methylated in the promoter and TSS regions in U-937 and despite high expression of the gene. Presence of positive histone marks suggests that methylated DNA can be overridden by histone modification. In Paper II a panel of 13 MM (multiple myeloma) cell lines and 9 primary patient tumors were analysed for methylation status of the ICSBP/IRF8 gene. In most cell lines (8/13) the gene was partially or fully methylated and partial methylation was also observed in 1/9 primary tumors. In vitro methylation analysis and treatment with 5-aza-2’deoxycytidine (DAC) proved that the ICSBP/IRF8 gene is silenced by methylation and may be associated with the malignant phenotype. In Paper III and IV the NFκB signalling pathway was analysed and the role of ATRA and TNFα induction. In Paper III the data shows that activation of the NFκB pathway is essential in ATRA-induced terminal differentiation in the U-937 cell line and IκBα (S32A/S36A) inhibits ATRA-induced differentiation and G1 cell cycle arrest. This was accompanied by delayed down-regulation of several cyclins (A and E) and up-regulation of p21WAF1/CIP1 (CDKN1A) and p27KIP1 (CDKN1B). TNFα alone did not induce expression of RA-induced genes analysed in Paper IV. However, ATRA in combination with TNFα showed enhanced activation of RA-induced genes. TNFα triggers demethylation of H3K9me3/H3K9me2 and H3K4me3 at RAR/RXR target genes, which were not accompanied by changes in the level of H3K9-ac. This decrease in H3 methylation by TNFα may pave way for the later ATRA-induced gene transcription.

Acute promyelocytic leukemia (APL) is a clinically distinct and rare type of acute myeloid leukemia and represents an oncologic emergency. Even rarer is the incidence of APL in pregnancy with less than 60 cases described in the literature. A 33-year-old pregnant female at 34 week gestation presented to hospital with reports of abdominal pain. On admission she was found to have acute onset pancytopenia with a WBC count of 1.2, Hemoglobin of 9.7g/dl, and platelet count of 26000. Initial history, exam, and investigations including a peripheral smear, coagulation panel, liver function, vitamin b12 and folate levels did not reveal possible etiology of pancytopenia. Given worsening pancytopenia, bone marrow biopsy was done which showed 58% promyelocytes and 11% blasts with numerous Auer rods present. Cytogenetics showed abnormal female karyotype with t(15:17) and FISH analysis revealed PML/RARA fusion in 76.5% of analyzed cells. The above findings were diagnostic of APL. After multidisciplinary discussion with high risk obstetrics physician, it was decided to immediately induce labor for immediate initiation of treatment of APL. She had a prolonged labor requiring aggressive blood product support and initiation of All trans retinoic acid (ATRA) before delivery given concerns of coagulopathy. Induction treatment with Arsenic trioxide (ATO) was started the day after her delivery. Repeat bone marrow biopsy on day 24 showed complete morphologic remission. Shortly thereafter, she started cycle 1 of consolidation with ATRA and arsenic trioxide. APL is characterized by a translocation between chromosome 15 and 17. Coagulopathy is a pathognomonic feature of this leukemia and often the reason for high mortality in early course of disease. APL when treated with ATRA and ATO, has excellent remission rate and 99% overall survival at 2 years. APL in pregnancy is associated with increased risk of preterm delivery, perinatal mortality, and miscarriage. Following pregnancy, there is an increased risk of bleeding, infection, or placental abruption. ATRA, one of the pillars around which treatment of APL revolves, is highly teratogenic during the first trimester and has low risk later in pregnancy. Treatment is directed by the trimester of pregnancy. Termination of pregnancy or treatment with single agent conventional chemotherapy is preferred in the first trimester whereas treatment with ATRA prior to delivery and use of chemotherapy after delivery is the preferred approach in the 2nd and 3rd trimester. This case is an example of individualized approach with a multidisciplinary team need in the setting of scarce data.

Retinoids are a class of compounds derived from the metabolism of vitamin A and β-carotene. Retinoid signaling has vital functions in both vertebrate and invertebrate embryogenesis such as the formation of body axes and the control of organogenesis. Genetic evidence suggests a role of retinoids in cell fate decision, maturation and homeostasis of the prostate epithelium. Knockout (KO) of the retinoic acid receptor gamma (RARG) gene in mice leads to growth deficits and male sterility due to squamous metaplasia and keratinization of the seminal vesicles and prostate. Noteworthy, synergistic antitumor effects of retinoids and vitamin D have been described in prostate cancer cell lines, although a mechanistic link between retinoic acid (RA) signaling and prostate epithelium differentiation and tumorigenesis has not yet been elucidated. Here, taking advantage of mouse prostate organoids (mPrOs), we report an essential role for RA in the differentiation and integrity of the periurethral and proximal luminal compartments of the prostate epithelium. Mechanistically, RA, through the activation of RARγ, promotes the expression of Foxa1, a pioneer transcription factor that cooperates with androgen receptor (AR) in directing progenitor cells towards the luminal lineage. Reduced RA signaling in organoids leads to downregulation of key structural and polarity proteins along with a loss of luminal identity, a phenotype that is fully rescued by constitutive expression of exogenous Foxa1. Overall, our study demonstrates the importance of RA signaling in prostate epithelium differentiation and homeostasis. In addition to the tumorigenic role of Foxa1 mutations recently described in several human cancers, alteration in RA pathway due to altered uptake/absorption/metabolism of vitamin A and β-carotene, or depending on specific molecular dysfunctions (e.g., epigenetic RARB silencing), could represent a critical rheostat for prostate tumorigenesis.

Macrophages can be activated by a variety of extracellular signals to polarize to either the M1 (inflammatory and antimicrobial) or to the M2 (wound repair and inflammation resolution) phenotype. Expression of arginase 1 in macrophages is a key marker of the M2 phenotype. Arginase 1 expression is induced by interleukin 4 (IL-4), a cytokine secreted by Th2 helper cells. All-trans retinoic acid (ATRA) is a product of metabolism of dietary retinol (vitamin A). In a manner analogous to hormones, ATRA binds to nuclear receptors in cells and influences gene expression and cell physiology. ATRA is important in the resolution of inflammation systemically and on the cellular level, however it has not been linked to M2 activation or arginase 1 expression. Testing the hypothesis that ATRA can induce arginase 1 in macrophages either directly or indirectly, it was found that ATRA alone cannot cause murine bone marrow-derived macrophages (BMDM) to activate in the M2 phenotype (as indicated by arginase 1 expression), however it can dramatically potentiate induction of arginase 1 expression and activity by IL-4. This is the first observation positively linking ATRA to arginase 1. Lipopolysaccharide (LPS), is a conserved structural component of the outer membrane of Gram negative bacteria, and a potent pyrogen. In metabolic endotoxemia, LPS concentration in the blood is slightly elevated, and over the long term this contributes to diverse inflammatory diseases such as atherosclerosis, obesity, and diabetes. LPS promotes the M1 phenotype and suppresses the M2 phenotype, but its contribution at low doses such as those found in metabolic endotoxemia are not well studied. In order to investigate mechanisms of LPS suppression at low doses, mice deficient in IRAK1 and tollip, key mediators or proinflammatory LPS signaling, were used to study IL-4, ATRA, and LPS crosstalk. LPS suppression of arginase 1 was found to be dependent on IRAK1 and tollip, but only at low doses of LPS. / Ph. D.