Interleukin (IL)-1α and IL-1β are pivotal to the initiation and orchestration of inflammation. Unlike most cytokines, IL-1 does not have a signal peptide and therefore secretion requires 2 independent processes; an initial signal to induce the up-regulation of the inactive precursor (pro-IL-1) and a second signal to drive cleavage and subsequent secretion. Whereas many previous studies have focused on the mechanisms that drive IL-1 secretion, the aims of this thesis were to investigate the processes that regulate the intracellular precursors of IL-1 (pro-IL-1α and pro-IL-1β). The hypothesis here was that regulation of these precursors may serve to control the vigour of IL-1 secretion and, ultimately, may influence the potency of pro-inflammatory responses. Post-translational modifications were of particular interest in this thesis, as these modifications are becoming increasingly important to immune system function. Ubiquitination is an important post-translational modification whereby ubiquitin, an 8.5kDa protein, is covalently bound to lysine residues on substrate proteins. In chapter 2, evidence was provided to show that in murine DC, IL-1α and IL-1β are polyubiquitinated and that, in both DC and macrophages, this polyubiquitination drives the proteasomal degradation of IL-1. In addition, these data demonstrated that in the presence of a second signal, polyubiquitinated IL-1 is still available for secretion. Overall, these investigations highlight that the polyubiquitination and proteasomal degradation of IL-1 serves as an essential process in the regulation of IL-1 and, therefore, should be considered as an extra dimension to the current two-signal paradigm of IL-1 release. To support this work, an immortalized bone marrow derived murine macrophage cell line and a human monocyte cell line that both stably express fluorescent IL-1β were employed to measure the rate of IL-1β degradation. In these investigations, it was shown that fluorescence is a reliable readout for measuring IL-1β degradation in these cell lines. In addition, it was demonstrated that that TLR-stimulation leads to an inhibition in IL-1β ubiquitination and degradation. Together, the work presented herein highlights that ubiquitination actively regulates the vigour of IL-1β protein expression and thus may be an important regulator of inflammation. To complement this work, a broader approach was taken, whereby the interactome of pro-IL-1β was explored using a human protein microarray. In these investigations, a human proteome microarray containing 19,951 unique proteins was used to identify proteins that bind human recombinant pro-IL-1β. In these analyses, calmodulin was identified as a particularly strong hit, with a SNR of ~11. Using an ELISA-based protein-binding assay, the interaction of recombinant calmodulin with pro-IL-1β, but not mature IL-1β, was confirmed and shown to be calcium dependent. Finally, using small molecule inhibitors it was demonstrated that both calcium and calmodulin were required for nigericin induced IL-1β secretion in human monocytes. Collectively, the evidence presented in these investigations suggests that following calcium influx, pro-IL-1β interacts with intracellular calmodulin and that this interaction is central for IL-1β processing and release. In addition, a number of other potentially important pro-IL-1β-interacting proteins were also identified in this work, including IL22RA2 and PLCXD3. Overall, the work presented in this thesis serves to highlight that IL-1 is regulated by a broad range of potentially important intracellular processes. We postulate that these processes may be pivotal in the regulation of inflammation and thus the maintenance of homeostasis.

A tuberculose (TB) transmitida pelo Mycobacterium tuberculosis está entre as doenças que mais matam no mundo. O excesso de inflamação é deletério na TB e está presente em quadros de comorbidade. Nossa hipótese de estudo foi fundamentada em dados da literatura que mostram: 1- a obesidade pode agravar o desenvolvimento de infecções no trato respiratório; 2- a obesidade induz inflamação sistêmica de baixo grau; 3- nos países de baixa e média renda, onde são notificados o maior número de casos de tuberculose, desnutrição e obesidade coexistem, atualmente. Nosso objetivo foi investigar se a inflamação decorrente de alimentação com dieta hiperlipídica acentuava a inflamação pulmonar dependente do eixo IL-1/IL-1R (IL-1 Receptor) na tuberculose experimental. Nossa hipótese foi embasada no fato de que o eixo IL-1/IL-1R participa da inflamação decorrente da obesidade e confere proteção contra a tuberculose experimental. Porém, a ativação excessiva do IL-1R pode causar imunopatologia na tuberculose. Resultados prévios do laboratório mostram que a obesidade induzida por dieta hiperlipídica (HFD - High Fat Diet) acentua a inflamação pulmonar e a suscetibilidade à infecção. Os resultados mostram que na ausência do receptor para IL-1 (IL-1R-/-) ou na ausência de IL-1? (IL-1?-/-), camundongos alimentados com HFD tiveram aumento de peso corporal, aumento de peso dos tecidos adiposos, hipertrofia dos adipócitos e hiperglicemia quando comparados aos respectivos grupos IL-1R-/- ou IL-1?-/- alimentados com dieta padrão (LFD - Low Fat Diet). A ausência do receptor para IL-1 (IL-1R-/-) em animais obesos reduziu a inflamação pulmonar, e a frequência de neutrófilos e de células CD4+IL-17+, porém não afetou a suscetibilidade à infecção quando comparados ao grupo WT (Wild Type) obeso. Sabendo que o IL-1R interage com IL-1? e com IL-1?, mostramos que o tratamento com anticorpo contra IL-1? reduziu a inflamação pulmonar e aumentou a resistência dos animais obesos comparados aos animais obesos que não foram tratados. Entretanto, animais deficientes para IL-1? tiveram exacerbação da inflamação pulmonar, acompanhada de redução de células CD4+IL-17+ e foram mais suscetíveis à infecção do que os animais WT obesos. Do contrário, o tratamento de animais IL-1?-/- obesos com IL-1? recombinante reduziu a inflamação, aumentou as células CD4+IL-17+ e a resistência à infecção. Em síntese, os resultados mostram que na comorbidade obesidade e tuberculose, o eixo IL-1/IL-1R participa da indução da inflamação pulmonar, sendo a IL- 1? associada com aumento da inflamação e da suscetibilidade, e a IL-1? com redução da inflamação e com resistência à infecção por M. tuberculosis / Tuberculosis (TB) transmitted by Mycobacterium tuberculosis is among the most deadly diseases in the world. Excess inflammation is deleterious in TB and is present in comorbidities. Our hypothesis was based on data from the literature that show: 1- obesity can aggravate the development of infections in the respiratory tract; 2- obesity induces low-grade systemic inflammation; 3- in low- and middle-income countries, where they are reported the highest number of cases of tuberculosis, malnutrition and obesity coexist, currently. Our objective was to investigate whether inflammation due to hyperlipidic diet accentuated the IL-1/IL-1R (IL-1 Receptor) axis dependent pulmonary inflammation in experimental tuberculosis. Our hypothesis was based on the fact that the IL-1/IL-1R axis participates in inflammation due to obesity and also provides protection against experimental tuberculosis. However, excessive activation of IL-1R may cause immunopathology in tuberculosis. Previous laboratory results show that obesity induced by a high-fat diet (HFD) accentuates pulmonary inflammation and susceptibility to infection. The results of the present study show that in the absence of the IL-1 receptor (IL-1R-/-) or in the absence of IL-1? (IL-1?-/-), HFD- weight gain of adipose tissues, adipocyte hypertrophy and hyperglycemia when compared to the respective IL-1R-/- or IL-1?-/- groups fed with standard diet (LFD). The absence of the IL-1 (IL-1R-/-) receptor in obese animals reduced lung inflammation, and the frequency of neutrophils and CD4+ IL-17+ cells, but did not affect the susceptibility to infection when compared to the group WT (Wild Type) obese. Knowing that IL-1R interacts with IL-1? and IL-1?, we show that IL-1? antibody treatment reduced lung inflammation and increased the resistance of obese animals compared to obese animals that were not treated. However, IL-1? deficient animals had an exacerbation of lung inflammation, accompanied by a reduction of CD4+ IL-17+ cells and were more susceptible to infection than obese WT animals. Otherwise, treatment of obese IL-1?-/- animals with recombinant IL-1? reduced inflammation, increased CD4+ IL-17+ cells and resistance to infection. In summary, the results show that in the comorbidity obesity and tuberculosis, the IL-1/IL-1R axis participates in the induction of pulmonary inflammation, IL-1? is associated with increased inflammation and susceptibility, and IL-1? with reduced inflammation and resistance to M. tuberculosis infection

A tuberculose (TB) transmitida pelo Mycobacterium tuberculosis está entre as doenças que mais matam no mundo. O excesso de inflamação é deletério na TB e está presente em quadros de comorbidade. Nossa hipótese de estudo foi fundamentada em dados da literatura que mostram: 1- a obesidade pode agravar o desenvolvimento de infecções no trato respiratório; 2- a obesidade induz inflamação sistêmica de baixo grau; 3- nos países de baixa e média renda, onde são notificados o maior número de casos de tuberculose, desnutrição e obesidade coexistem, atualmente. Nosso objetivo foi investigar se a inflamação decorrente de alimentação com dieta hiperlipídica acentuava a inflamação pulmonar dependente do eixo IL-1/IL-1R (IL-1 Receptor) na tuberculose experimental. Nossa hipótese foi embasada no fato de que o eixo IL-1/IL-1R participa da inflamação decorrente da obesidade e confere proteção contra a tuberculose experimental. Porém, a ativação excessiva do IL-1R pode causar imunopatologia na tuberculose. Resultados prévios do laboratório mostram que a obesidade induzida por dieta hiperlipídica (HFD - High Fat Diet) acentua a inflamação pulmonar e a suscetibilidade à infecção. Os resultados mostram que na ausência do receptor para IL-1 (IL-1R-/-) ou na ausência de IL-1? (IL-1?-/-), camundongos alimentados com HFD tiveram aumento de peso corporal, aumento de peso dos tecidos adiposos, hipertrofia dos adipócitos e hiperglicemia quando comparados aos respectivos grupos IL-1R-/- ou IL-1?-/- alimentados com dieta padrão (LFD - Low Fat Diet). A ausência do receptor para IL-1 (IL-1R-/-) em animais obesos reduziu a inflamação pulmonar, e a frequência de neutrófilos e de células CD4+IL-17+, porém não afetou a suscetibilidade à infecção quando comparados ao grupo WT (Wild Type) obeso. Sabendo que o IL-1R interage com IL-1? e com IL-1?, mostramos que o tratamento com anticorpo contra IL-1? reduziu a inflamação pulmonar e aumentou a resistência dos animais obesos comparados aos animais obesos que não foram tratados. Entretanto, animais deficientes para IL-1? tiveram exacerbação da inflamação pulmonar, acompanhada de redução de células CD4+IL-17+ e foram mais suscetíveis à infecção do que os animais WT obesos. Do contrário, o tratamento de animais IL-1?-/- obesos com IL-1? recombinante reduziu a inflamação, aumentou as células CD4+IL-17+ e a resistência à infecção. Em síntese, os resultados mostram que na comorbidade obesidade e tuberculose, o eixo IL-1/IL-1R participa da indução da inflamação pulmonar, sendo a IL- 1? associada com aumento da inflamação e da suscetibilidade, e a IL-1? com redução da inflamação e com resistência à infecção por M. tuberculosis / Tuberculosis (TB) transmitted by Mycobacterium tuberculosis is among the most deadly diseases in the world. Excess inflammation is deleterious in TB and is present in comorbidities. Our hypothesis was based on data from the literature that show: 1- obesity can aggravate the development of infections in the respiratory tract; 2- obesity induces low-grade systemic inflammation; 3- in low- and middle-income countries, where they are reported the highest number of cases of tuberculosis, malnutrition and obesity coexist, currently. Our objective was to investigate whether inflammation due to hyperlipidic diet accentuated the IL-1/IL-1R (IL-1 Receptor) axis dependent pulmonary inflammation in experimental tuberculosis. Our hypothesis was based on the fact that the IL-1/IL-1R axis participates in inflammation due to obesity and also provides protection against experimental tuberculosis. However, excessive activation of IL-1R may cause immunopathology in tuberculosis. Previous laboratory results show that obesity induced by a high-fat diet (HFD) accentuates pulmonary inflammation and susceptibility to infection. The results of the present study show that in the absence of the IL-1 receptor (IL-1R-/-) or in the absence of IL-1? (IL-1?-/-), HFD- weight gain of adipose tissues, adipocyte hypertrophy and hyperglycemia when compared to the respective IL-1R-/- or IL-1?-/- groups fed with standard diet (LFD). The absence of the IL-1 (IL-1R-/-) receptor in obese animals reduced lung inflammation, and the frequency of neutrophils and CD4+ IL-17+ cells, but did not affect the susceptibility to infection when compared to the group WT (Wild Type) obese. Knowing that IL-1R interacts with IL-1? and IL-1?, we show that IL-1? antibody treatment reduced lung inflammation and increased the resistance of obese animals compared to obese animals that were not treated. However, IL-1? deficient animals had an exacerbation of lung inflammation, accompanied by a reduction of CD4+ IL-17+ cells and were more susceptible to infection than obese WT animals. Otherwise, treatment of obese IL-1?-/- animals with recombinant IL-1? reduced inflammation, increased CD4+ IL-17+ cells and resistance to infection. In summary, the results show that in the comorbidity obesity and tuberculosis, the IL-1/IL-1R axis participates in the induction of pulmonary inflammation, IL-1? is associated with increased inflammation and susceptibility, and IL-1? with reduced inflammation and resistance to M. tuberculosis infection

Previous investigations have indicated that shoulder stiffness was related with the degree of inflammation and the number of myofibroblasts in subacromial bursa tissue of rotator cuff lesions. But the interaction mechanism between inflammatory cytokines and myofibroblasts induced shoulder stiffness has not been fully explored. The purpose of this research is to study the effect of IL-1 £] on myofibroblasts in rotator cuff lesions with shoulder stiffness. Myofibroblasts from subacromial bursa tissue of rotator cuff lesions were cultured with or without IL-1 £]. The gene and protein expression of £\-smooth muscle actin (£\-SMA) and prolyl 4-hydroxylase (PH-4) were determined. The results showed that high levels of PH-4 and low levels of £\-SMA were expressed in myofibroblasts cultured with IL-1 £]. PH-4 gene had higher expression in tissue of stiff patients than non-stiff patients, but £\-SMA gene expression had no significant difference. According to the results, myofibroblasts cultured with extra IL-1 £] showed increased PH-4 expression and decreased £\-SMA expression. This indicates myofibroblasts might transdifferentiate into fibroblasts via IL-1 £] effect.

No description available.

Burn induces the inflammation response, and causes the intestinal barrier failure. The failure of intestinal barrier may cause organ damage. Pervious studies have shown that the increase of iNOS activity is closely related to the organ damage after burn. The expression of iNOS is regulated by the activation of NF-£eB, and that is regulated by MAPKs. The pro-inflammatory cytokines play important roles to promote the inflammation through activating a series of signal transduction cascade, via binding to their receptors on cell membrane. The signal transduction cascades are turn on, MAPKs and NF-£eB are activated and the expression of iNOS is promoted. In this study, the role of pro-inflammatory cytokine interleukin-1 receptor (IL-1R) in burn induced intestinal damage was focused on. In experiments, the animals (C57BL/6 mice) were undergone 30~35 % total body surface area (TBSA) burn. The change of intestinal permeability was examined, and intestinal mucosa was assayed for the activation of iNOS and MAPKs by immunoblotting, and the activation of NF-£eB was detected by EMSA. The results reveal that activation of NF-£eB, intestinal permeability and expression of iNOS were increased after burn in wild type mice (WT). ERK MAPK plays an important role to regulate the activation of NF-£eB and expression of iNOS. Surprisingly, the permeability had no change after burn in IL-1R knock out mice (KO). The activation of ERK, NF-£eB and the expression of iNOS were also measured in KO. The levels of p-ERK, NF-£eB activation and iNOS expression were low in KO. When WT mice were treated with U0126 (5 mg/kg i.p.) right after burn to block the activation of ERK, the activation of ERK and NF-£eB, the expression of iNOS, and the intestinal permeability were all decreased significantly. To sum up, the changes in iNOS expression, NF-£eB activation, and intestinal permeability increase are mostly related to the activation of ERK after burn. IL-1 R plays a promotion role in ERK, NF-£eB activation, and iNOS expression that lead to the increase in intestinal permeability and promote damage in intestine.

No description available.

A flagelina é um agonista natural do sensor TLR5 e do inflamassoma NAIP/NLRC4 que é responsável pela secreção de IL-1β e IL-18 e pela indução de morte celular necrótica, via ativação da caspase-1. Entretanto, nós observamos que a inserção da flagelina de B. subtilis no citosol celular por meio de vesículas lipídicas, induz um processo atípico de morte nos macrófagos peritoneais (PMs) deficientes em NLRC4, ASC e caspase-1/11. A morte dos PMs manteve seu resultado antimicrobiano, sendo acompanhada da liberação de IL-1α. A morte celular e a secreção das citocinas IL-1α e IL-1β, foi mediada por catepsinas lisossomais, sugerindo uma cooperação entre a via lisossomal e os inflamassomas nas respostas induzidas pela flagelina. Além disso, a flagelina de S. typhimurium foi capaz de induzir dano lisossomal e secreção de IL-1α e IL-1β mediada pelo eixo caspase-catepsinas, na ausência de carreadores, e estas citocinas tiveram um impacto na imunidade adaptativa induzida pela flagelina, no modelo de ativação de linfócitos T específicos por células dendríticas, in vitro. / Flagellin is a natural agonist of TLR5 and NAIP/NLRC4 inflammasome that is responsible for IL-1β and IL-18 secretion and for the induction of a necrotic cell death, both mediated by caspase-1. However, we observed that flagellin from B. subtilis inserted into lipid vesicles, induced an atypical cell death in peritoneal macrophages (PMs) in the absence of NLRC4, ASC and caspase-1/11. This inflammasome-independent cell death retained its antimicrobial outcome, being accompanied with IL-1α secretion. Importantly, cell death and caspase-1-dependent IL-1α and IL-1β secretion were regulated by lysosomal cathepsins, suggesting a cooperation between the inflammasome and lysosomal pathway in response to flagellin. We also observed that flagellin from S. typhimurium is able to induce lysosomal damage and IL-1α and IL-1β secretion by PMs in the absence of a carrier, through a caspase-catepsins-dependent manner, and that cytokines were important to the ability of flagellin in to induce adaptive immune response by antigen-specific T cells.

A flagelina é um agonista natural do sensor TLR5 e do inflamassoma NAIP/NLRC4 que é responsável pela secreção de IL-1β e IL-18 e pela indução de morte celular necrótica, via ativação da caspase-1. Entretanto, nós observamos que a inserção da flagelina de B. subtilis no citosol celular por meio de vesículas lipídicas, induz um processo atípico de morte nos macrófagos peritoneais (PMs) deficientes em NLRC4, ASC e caspase-1/11. A morte dos PMs manteve seu resultado antimicrobiano, sendo acompanhada da liberação de IL-1α. A morte celular e a secreção das citocinas IL-1α e IL-1β, foi mediada por catepsinas lisossomais, sugerindo uma cooperação entre a via lisossomal e os inflamassomas nas respostas induzidas pela flagelina. Além disso, a flagelina de S. typhimurium foi capaz de induzir dano lisossomal e secreção de IL-1α e IL-1β mediada pelo eixo caspase-catepsinas, na ausência de carreadores, e estas citocinas tiveram um impacto na imunidade adaptativa induzida pela flagelina, no modelo de ativação de linfócitos T específicos por células dendríticas, in vitro. / Flagellin is a natural agonist of TLR5 and NAIP/NLRC4 inflammasome that is responsible for IL-1β and IL-18 secretion and for the induction of a necrotic cell death, both mediated by caspase-1. However, we observed that flagellin from B. subtilis inserted into lipid vesicles, induced an atypical cell death in peritoneal macrophages (PMs) in the absence of NLRC4, ASC and caspase-1/11. This inflammasome-independent cell death retained its antimicrobial outcome, being accompanied with IL-1α secretion. Importantly, cell death and caspase-1-dependent IL-1α and IL-1β secretion were regulated by lysosomal cathepsins, suggesting a cooperation between the inflammasome and lysosomal pathway in response to flagellin. We also observed that flagellin from S. typhimurium is able to induce lysosomal damage and IL-1α and IL-1β secretion by PMs in the absence of a carrier, through a caspase-catepsins-dependent manner, and that cytokines were important to the ability of flagellin in to induce adaptive immune response by antigen-specific T cells.

No description available.