A malária causa complicações envolvendo diversos órgãos, inclusive o fígado, onde se sabe que ocorrem inflamação e dano hepáticos, os quais contribuem para a severidade da doença. A interleucina (IL)-1 α é uma citocina pró-inflamatória que pertence à família da IL-1 e que pode ser produzida e liberada tanto por células não-hematopoiéticas, como hematopoiéticas em contextos como dano tecidual, infecção e doenças autoimunes em diversos órgãos. O presente estudo tem como objetivo avaliar a produção e o papel da IL-1 na imunopatogênese e na proteção durante a malária experimental. A produção da IL-1 foi investigada durante a inflamação e necrose no fígado em camundongos C57BL/6 infectados com o estágio eritrocítico do Plasmodium chabaudi. A fonte de IL-1 no fígado dos camundongos infectados foi determinada por imunofluorescência e citometria de fluxo. Camundongos IL1A-/- foram utilizados para avaliar o papel da IL-1 neste contexto. Durante a infecção aguda com o P. chabaudi, a inflamação hepática e o desenvolvimento das lesões necróticas são acompanhados pelo aumento nos níveis de IL-1 no fígado, que ocorre de forma independente do inflamassoma NLRP3 (Nod-like receptor protein 3). Os neutrófilos foram identificados como sendo a fonte de IL-1 α no fígado dos camundongos C57BL/6 infectados. De forma sistêmica, a deficiência em IL-1 α resultou numa diminuição da perda de peso e da hipotermia causados pela malária, mas teve um efeito menos significativo no controle da parasitemia. No fígado, a ausência de IL-1 reduziu o número de células TUNEL+, atenuando a necrose induzida pela infecção. A melhora no dano hepático nos camundongos IL1A-/- infectados está associada com uma menor resposta inflamatória em comparação aos camundongos C57BL/6 infectados, incluindo uma menor produção do TNF- α (Tumor necrosis factor alpha), citocina associada à apoptose de hepatócitos e, consequentemente, à necrose do tecido hepático durante a malária causada pelo P. chabaudi. Apesar de exercer um importante papel na imunopatogênese do dano e inflamação do fígado durante a fase eritrocítica da infecção pelo P. chabaudi, a ausência da IL-1 α não impediu a proteção conferida pela presença das formas eritrocíticas contra a reinfecção com esporozoítos. Em conclusão, neutrófilos produzem IL-1 α no fígado durante a fase aguda da malária causada pelo P. chabaudi. Esta citocina amplifica a resposta inflamatória à infecção e promove a necrose hepática, assim como exacerba a perda de peso e a hipotermia. / Malaria causes complications involving several organs, including the liver, where there are inflammation and damage that contribute to the disease severity. Interleukin (IL)-1 is a pro-inflammatory cytokine from the IL-1 family that can be released by non-hematopoietic or hematopoietic cells during tissue damage, infection and autoimmune diseases in different organs. The present study aims to evaluate the production and the role of IL-1 in the immunopathogenesis and in the protection during experimental malaria. IL-1 production was assessed during hepatic inflammation and necrosis in C57BL/6 mice infected with blood stages of Plasmodium chabaudi. The source of IL-1 in the liver of infected mice was determined by immunofluorescence and flow cytometry analyses. IL1A-/- mice were used to assess the role of IL-1 in this context. During acute P. chabaudi infection, hepatic inflammation and development of necrotic lesions were accompanied by an increase in IL-1 levels in the liver, which occurred independently of the Nod-like receptor protein 3 (NLRP3) inflammasome. Neutrophils were identified as the source of IL-1 α in the liver of infected C57BL/6 mice. Systemically, IL-1 deficiency resulted in reduction of weight loss and hypothermia caused by P. chabaudi malaria, but had minor effect on parasitemia control. In the liver, the absence of IL-1 reduced the number of TUNEL+ cells and attenuated the necrotic process induced by infection. The amelioration of liver damage in infected IL1A-/- mice was associated with lower inflammatory response compared to infected C57BL/6 mice, in particular with a decrease in tumor necrosis factor alpha (TNF- α) production, which has been directly implicated in the apoptosis of hepatocytes and, in consequence, the necrosis of the liver tissue during P. chabaudi malaria. Despite the important role in liver damage and inflammation immunopathogenesis during the blood-stage P. chabaudi infection, the absence of IL-1 α did not impair the protection conferred by blood stages against sporozoite reinfection. This study shows that neutrophils produce IL-1 α in the liver during acute P. chabaudi malaria. This cytokine amplifies the inflammatory response to infection and promotes liver necrosis, as well as exacerbates the weight loss and hypothermia.

The P2X7R is a functionally distinct member of the P2X non-selective cation channels and has been implicated in the initiation of immune responses. One of the most extensively characterised immune responses of the receptor is to signal the rapid aggregation of the inflammasome complex and signal the release of IL-1β. These investigations have focused in providing direct comparisons of P2X7R-driven IL-1 responses between DC and mouse macrophages (peritoneal macrophages [PMΦ] and bone marrow derived macrophages [BM-MΦ]). Expression of the P2X7R has been identified in all three populations both at the transcriptional (P2X7A variant) and protein levels. Activation with lipopolysaccharide (LPS) (2h) induced a rapid dose dependent release of IL-6 but not of IL-1β in BM-DC. Rapid (2h) IL-1β release required both LPS priming and ATP activation. Both signals were also required for IL- 1β release in mouse ΒΜ-ΜΦ and PMΦ, however, at comparatively markedly lower levels. Furthermore, like with IL-1β, LPS did not induce IL-1α release in BM-DC. Interestingly, subsequent challenge with ATP evoked IL-1α release in BM-DC alone, with little or no detectable levels observed in activated BM-MΦ. This rapid IL-1β release (but not IL-6) was potently inhibited in both macrophages and DC with a P2X7R-specific inhibitor (A-740003) providing evidence that is predominantly a P2X7R-driven process. Treatment with A-740003 also potently inhibited IL-1α release from BM-DC suggesting that the ATP-P2X7R and caspase-1 activation might have a role in the release of the cytokine. Expression of gain-of-function P2X7K and loss-of- function P2X7J splice variants has been identified in both BM-DC and BM-MΦ, at the level of transcription. The possibility that a differential baseline or LPS-induced expression (at the transcriptional level) of P2X7J and P2X7K variants accounts for the diverse cytokine responses observed in BM-DC and BM-MΦ was also explored. However, the levels of expression for the various splice variants of interest (P2X7K and P2X7J) were found to be similar between the two cell types. The results of these investigations identify some subtle but intriguing differences in the mechanism of P2X7R activation and IL-1 release between DCs and macrophages. Purinergic signalling is increasing being implicated in the regulation of immune responses both in potentiating or suppressing inflammation. However, further work is required to decipher how the dynamic interplay between different purines can influence the immune activation of different cell types and indeed different cell subsets.

Pancreatic cancer is one of the most lethal cancers and despite all research efforts the last 50 years, there are still no effective therapy for this terrible disease. Until quite recently most research in the field of pancreatic duct adenocarcinoma (PDAC) was focused on the tumor cells and mechanisms essential for their proliferation and survival. However, the tumor does not only consist of tumor cells, rather a combination of tumor cells and numerous stroma cell types, i.e. the tumor microenvironment. The tumor cells have developed the ability to activate the surrounding cells to produce factors important for the progression of the tumor. Cancer associated fibroblasts (CAFs) are the major stroma component and as much as 70% of the total PDAC tumor mass consists of these cells. In this thesis I have investigated the mechanisms involved in the cross-talk between tumor cells and CAFs and distinguished the local and systemic effects of this communication. Tumor derived IL-1α was identified as an important factor creating the inflammatory profile seen in CAFs. In PDAC patients, IL-1α was detected in 90% of the tumors and high expression was associated with poor clinical outcome. Moreover, the PDAC tumors had elevated expression levels of many inflammatory factors that were induced in CAFs by the tumor derived IL-1α in vitro. Consequently, this high expression of inflammatory factors in CAFs will attract immune cells including tumor associated macrophages (TAMs), dendritic cells (DCs), and CD8+ T cells. This indicates an immune suppressive role of CAFs, protecting the tumor cells by acting as decoy targets for immune cells homing into the tumor. The inflammatory factors produced in the PDAC microenvironment did not only affect the infiltrating immune cells, but had also systemic effects that included decreased levels of blood DCs in PDAC patients. Furthermore, these myeloid and plasmacytoid DCs were partly activated and had a semi mature phenotype and impaired immunostimulatory function. Low levels of blood DCs were direct associated with poor patient prognosis and the same was seen for low expression of ICOSL by the DCs. The findings presented in this thesis indicate an essential role for the cross-talk between tumor cells and stroma in the production of tumor  promoting factors. Treatment of PDAC patients with drugs that target the IL-1α signaling pathway could prevent the communication between these cells, thus reduce the amount of inflammatory factors both locally and systemically. Altogether, our findings support the idea that neutralization of the IL-1α signaling molecule could be a promising therapy for pancreatic cancer. The findings presented in this thesis indicate an essential role for the cross-talk between tumor cells and stroma in the production of tumor promoting factors. Treatment of PDAC patients with drugs that target the IL-1α signaling pathway could prevent the communication between these cells, thus reduce the amount of inflammatory factors both locally and systemically. Altogether, our findings support the idea that neutralization of the IL-1α signaling molecule could be a promising therapy for pancreatic cancer. / Mindre än 5% av patienterna som drabbas av cancer i bukspottkörteln förväntas överleva i mer än fem år. De typiska symtomen kommer sent och sjukdomen framskrider snabbt. Några av de riskfaktorer som identifierats är tobaksrökning, fetma och typ 2 diabetes. Forskningen har hittills siktat in sig på tumörcellerna och de mekanismer de använder för att överleva och föröka sig. Men en tumör innehåller också normala kroppsceller och vid bukspottkörtelcancer kan så mycket som 70 procent bestå av i sig ofarliga bindvävsceller. Miljön i tumören skapas av samspelet mellan dessa celltyper. De cancerceller som är bäst på att utnyttja omgivningen för sin tillväxt fortlever och för sina egenskaper vidare. En sådan egenskap är att kunna manipulera bindvävsceller till att producera signalsubstanser och tillväxtfaktorer som gynnar tumören. Mekanismerna bakom denna kommunikation har studerats och ett viktigt fynd var att tumörcellerna producerar signalämnet interleukin 1-alpha (IL-1a). Detta protein upptäcktes i 90 procent av de undersökta tumörerna, och var kopplat till dålig prognos hos patienterna. Signalen via IL-1a sätter igång tillverkningen av substanser som behövs för nybildning och tillväxt av blodkärl, i sin tur en förutsättning för att tumören ska leva vidare och växa. Proteinet stimulerar också celldelning i tumören, bidrar till att lura kroppens immunförsvar och underlättar spridning av dottertumörer till andra delar av kroppen. När vi slår ut signaleringen kan tumörcellerna inte längre påverka bindvävscellerna lika effektivt, och således minskar förekomsten av flera faktorer som gynnar tumörtillväxten. IL-1a kan därför vara en lovande kandidat att utforska vidare för framtida som ett läkemedel mot bukspottkörtelcancer.

IL-1α and IL-1β are two IL-1 agonists which signals at the same receptor complex composed of IL-1R1/IL-1RAcP. However, IL-1α and IL-1β exert differential actions. A recent CNS-specific IL-1 receptor accessory protein, called IL-1RAcPb, has been characterised but its actions are unknown. In T cell line, over expression of IL-1RAcPb negatively regulate IL-1 action (Smith et al, 2009), but over-expression of IL-1RAcPb in HEK cell line induces IL-1 signaling (Lu et al, 2008). The role of IL-1RAcPb has not been studied in primary cells. The aim of this project was to investigate the role of IL-1RAcPb in IL-1-induced actions in neurones and glia, and to determine IL-1α and IL-1β differential actions in these two cell types. The role of IL-1RAcPb in IL-1-induced protein expression and IL 1α and IL-1β differential effects were investigated by treating WT and IL 1RAcPb-/- neurones and glia with IL-1α or IL-1β in the presence or absence of IL-1RA for 24 h followed by assessment of IL-6 induction by ELISA. The mechanism of IL-1RAcPb actions were studied by examining the effects of IL-1α or IL-1β on p38, ERK1/2 and Src kinase activation in neurones and glia by Western blot analysis. SB203580 (p38 inhibitor), UO126 (ERK1/2 inhibitor), and PP2 (Src kinase inhibitor) were used to determine the contribution of p38, ERK1/2 and Src kinase activation to IL-1-induced IL-6 synthesis in neuronal cultures. In WT neurones, IL-1α and IL-1β were equipotent at inducing IL-6 synthesis and p38 activation, whilst both ligands failed to induce ERK1/2 or Src kinase activation. In IL-1RAcPb-/- neurones, IL-1α and IL-1β induced similar levels of IL-6, but IL-1β was more potent than IL-1α at inducing p38 activation. IL-1α-induced p38 activation was reduced in IL-1RAcPb-/- neurones compared to WT neurones. In contrast to WT neurones, ERK1/2 was activated in IL-1RAcPb-/- neurones in response to IL-1α, whilst Src kinase was not activated by IL-1α or IL 1β. IL-1-induced IL-6 synthesis was abolished by IL-1RA, SB203580, UO126 and PP2. Interestingly PP2, a specific Src kinase inhibitor also partially inhibited basal ERK1/2 activity. In WT glial cells, IL-1α was more potent than IL-1β at inducing IL-6 synthesis but both cytokines induced ERK1/2 activation with equal potency. In IL-1RAcPb-/- glia, IL-1α and IL-1β were equally potent at inducing IL-6 synthesis and ERK1/2 activation. However, IL-α-induced-IL-6 synthesis was reduced in IL 1RAcPb-/- glia compared to WT glia. In both WT and IL-1RAcPb-/- glia, IL-1α and IL-1β induced p38 activation but not Src kinase activation . In conclusion, this study showed that in neurones, IL-1RAcPb may contribute to IL-1α-induced p38 activation but negatively regulates IL-1-induced ERK1/2 activation, therefore IL-1RAcPb may have specific effects on different signalling pathways. The effect of IL-1RAcPb could also be cell specific, as IL 1RAcPb contributed to IL-1α-induced p38 signalling in neurones but IL-6 production in glia. The role of IL-1RAcPb remains largely unknown and more investigations are required to elucidate its role in IL-1 signalling in the brain.