Pneumonia is considered to be one of the leading causes of death worldwide. The outcome depends on both, proper antibiotic treatment and the effectivity of the immune response of the host. However, due to the complexity of the immunologic cascade initiated during infection, the latter cannot be predicted easily. We construct a biomathematical model of the murine immune response during infection with pneumococcus aiming at predicting the outcome of antibiotic treatment. The model consists of a number of non-linear ordinary differential equations describing dynamics of pneumococcal population, the inflammatory cytokine IL-6, neutrophils and macrophages fighting the infection and destruction of alveolar tissue due to pneumococcus. Equations were derived by translating known biological mechanisms and assuming certain response kinetics. Antibiotic therapy is modelled by a transient depletion of bacteria. Unknown model parameters were determined by fitting the predictions of the model to data sets derived from mice experiments of pneumococcal lung infection with and without antibiotic treatment. Time series of pneumococcal population, debris, neutrophils, activated epithelial cells, macrophages, monocytes and IL-6 serum concentrations were available for this purpose. The antibiotics Ampicillin and Moxifloxacin were considered. Parameter fittings resulted in a good agreement of model and data for all experimental scenarios. Identifiability of parameters is also estimated. The model can be used to predict the performance of alternative schedules of antibiotic treatment. We conclude that we established a biomathematical model of pneumococcal lung infection in mice allowing predictions regarding the outcome of different schedules of antibiotic treatment. We aim at translating the model to the human situation in the near future.

Pneumonia is considered to be one of the leading causes of death worldwide. The outcome depends on both, proper antibiotic treatment and the effectivity of the immune response of the host. However, due to the complexity of the immunologic cascade initiated during infection, the latter cannot be predicted easily. We construct a biomathematical model of the murine immune response during infection with pneumococcus aiming at predicting the outcome of antibiotic treatment. The model consists of a number of non-linear ordinary differential equations describing dynamics of pneumococcal population, the inflammatory cytokine IL-6, neutrophils and macrophages fighting the infection and destruction of alveolar tissue due to pneumococcus. Equations were derived by translating known biological mechanisms and assuming certain response kinetics. Antibiotic therapy is modelled by a transient depletion of bacteria. Unknown model parameters were determined by fitting the predictions of the model to data sets derived from mice experiments of pneumococcal lung infection with and without antibiotic treatment. Time series of pneumococcal population, debris, neutrophils, activated epithelial cells, macrophages, monocytes and IL-6 serum concentrations were available for this purpose. The antibiotics Ampicillin and Moxifloxacin were considered. Parameter fittings resulted in a good agreement of model and data for all experimental scenarios. Identifiability of parameters is also estimated. The model can be used to predict the performance of alternative schedules of antibiotic treatment. We conclude that we established a biomathematical model of pneumococcal lung infection in mice allowing predictions regarding the outcome of different schedules of antibiotic treatment. We aim at translating the model to the human situation in the near future.

Die Ergebnisse der Arbeit weisen mehrfach auf eine defizitäre IL-10 Produktion in Alveolarmakrophagen von Asthmatikern hin. Die reduzierte IL-10 Expression auf Protein- und Genebene korrelierte mit einer erhöhten Produktion proinflammatorischer Zytokine (TNF-?, MIP1-?, GM-CSF). Diese Beobachtung impliziert einen Defekt in der IL-10 Synthese, der in einer verstärkten und prolongierten pulmonalen Entzündungsantwort resultiert. Daraus läßt sich schlußfolgern, dass beim Asthma bronchiale eine Dysbalance zwischen pro- und antiinflammatorischen Zytokinen pathogenetisch von Bedeutung ist. Die verringerte Sensitivität von Alveolarmakrophagen auf die inhibitorischen Effekte von exogenem IL-10 im Vergleich zu Blutmonozyten ist durch Unterschiede in den Mechanismen der Signaltransduktion bedingt (37, 54). Der Nachweis der Expression von proinflammatorischen Zytokinen in Bronchialmyozyten (RANTES, IL-8) führte zu einer Neubewertung dieser Zellen als Immuneffektorzellen in der Pathogenese des Asthma bronchiale. Neben der Kontraktilität sind Myozyten auch aktiv an der Aufrechterhaltung der Atemwegsentzündung beteiligt. Die inhibitorischen Effekte von IL-10 und IL-13 auf die Synthese proinflammatorischer Chemokine (RANTES, IL-8, MIP-1() in migrierten Entzündungszellen und residenten Bronchialmyozyten konnten in verschiedenen Arbeiten gut dokumentiert werden. Die Vielzahl antiinflammatorischer Effekte von IL-10, die sich auf unterschiedliche Zellsysteme wie Monozyten, Makrophagen und Bronchialmyozyten erstrecken, unterstreicht die pathogenetische Bedeutung dieses Zytokins. Der molekulare Mechanismus, welcher die IL-10 Wirkung vermittelt, ist derzeit noch nicht vollständig aufgeklärt. Angenommen wird eine rezeptorvermittelte Inhibition von Transkriptionsfaktoren des Stat Systems und NF-(B (76). Zukünftige molekularbiologische und klinische Studien sind jedoch notwendig, um den Kenntnisstand der Effekte antiinflammatorischer Zytokine zu vertiefen, und die Gabe von rekombinantem IL-10 als möglichen Ansatz zur Therapie chronisch entzündlicher Lungenerkrankungen zu evaluieren (81). / The results of this present thesis show a deficiency of IL-10 production in alveolar macrophages in asthma. The reduced IL-10 expression on protein and m-RNA level correlated with an increased production of pro-inflammatory cytokines such as TNF-(, MIP1- ( and GM-CSF. These observations implicate an impaired IL-10 synthesis in asthma with a subsequent prolongation of the inflammatory response. This leads to the conclusion that a dysbalance between pro- and anti-inflammatory cytokines is present in asthma and may be therefore of pathogenetic importance. The reduced sensitivity of alveolar macrophages to the inhibitory effects of exogenous IL-10 compared to peripheral blood monocytes may be caused by different signal transduction mechanisms. The expression of the proinflammatory cytokines RANTES and IL-8 in cultured human airway smooth muscle cells led to the conclusion that airway smooth muscle cells may act beside their contractile function as immunomodulatory cells in the pathogenesis of asthma. The inhibitory effects of IL-10 and IL-13 on the synthesis of proinflammatory cytokines (RANTES, IL-8, MIP1-() in immigrated inflammatory cells and resident cells such as airway smooth muscle cells have been shown in several publications that are part of the present thesis. The numerous antiinflammatory effects of IL-10 on different inflammatory cell systems such as monocytes/macrophages and smooth muscle cells underline the pathogenetic importance of this cytokine. The molecular mechanisms that mediate the IL-10 effects involve the transcription factors NF-(B and the Stat-System. Future studies are needed to determine the molecular mechanisms of the anti-inflammatory effects of IL-10 and IL-13 more deeply and to evaluate their application for the therapy of chronic inflammatory pulmonary diseases.

Alveolar macrophages comprising up to 95% of the pulmonary alveoli, are the gate-keepers of homeostasis by ensuring efficient tissue function through metabolizing excessive surfactant and phagocyting inhaled day-to-day and innocuous pathogens and particles, without triggering an immune response. Despite that, they are capable of orchestrating a very well-balanced immune response upon invasion of pathogens. These embryonic-derived cells are capable of self-renewal and therefore maintain themselves in the lungs throughout adult life, with minimal contribution from the circulating monocytes. This self-renewal capacity is attained intrinsically by maintaining low levels of transcription factors MafB and cMaf, and extrinsically through two main cytokines, namely GM-CSF secreted by alveolar epithelial type II cells, and TGFb secreted by AMs themselves in an autocrine manner. However, in inflammatory conditions such as acute respiratory distress syndrome (ARDS), depletion of AM pool and upregulation of MAFB among lung macrophages have been reported. Keeping in mind the role of transcription factors MafB and cMaf in inhibiting proliferative capacity of macrophages; we hypothesized that this depletion is due to upregulation of MafB and hence the suppression of enhancer regions of self-renewal genes in AMs. To investigate the role of MafB and its compensatory partner cMaf in ARDS, we have established a mouse model of ARDS using oropharyngeal instillation of LPS in WT and MafB/cMaf double-knockout (Maf-DKO) mice. Alongside, the molecular mechanisms of the effect of LPS on AMs was investigated ex-vivo. The obtained results have clearly shown that ex-vivo, LPS inhibits proliferation of AMs in a dose dependent manner, and induces apoptosis significantly. Regain of proliferative potential of LPS-stimulated AMs was evident upon TLR4 inhibition, and MyD-88 was shown to be the dominant adaptor downstream of TLR4 (as opposed to TRIF). Both WT and DKO AMs responded to LPS stimulation within 2 hours, by switching from OXPHOS to glycolysis, which accounts for their efficient pro-inflammatory phenotype once activated. Upon activation, MafB and cMaf were upregulated after 48 hours and the inhibition of AM proliferation was shown to be Maf-independent. Similarly, depletion of AM pool was shown to be Maf independent invivo, evident by similar kinetics of AM numbers in WT and DKO at different timepoints upon LPS stimulation. However, several findings indicated potential advantage of Maf-deficiency in tissue regeneration; this includes: 1) higher number of Ly6C+ monocytes and their earlier differentiation into resident AMs, 2) lower degree of tissue damage revealed by H&E staining, 3) higher number of alveolar epithelial type II cells, 4) significantly higher levels of cytotoxicity pointing towards cellular turnover, and 5) significantly higher levels of SP-D and thus its antiinflammatory effects. In a quest for investigating factors which could enhance proliferative potential of AMs and ultimately neutralize the inhibitory effect of LPS, the impact of TGFb and ActivinA was studied. I have shown that TGFb and to a higher extend ActivinA boost the proliferation rate of AMs, ex-vivo. The autocrine effect of these cytokines was validated by blocking signal transduction through inhibition of SMAD2/3, which resulted in a significant increase in doubling time of AMs. Interestingly, Inhba was shown to be significantly upregulated in AMs, as opposed to TGFb. The importance of ActivinA was further demonstrated by its direct inhibition and the resulting reduction in growth rate of AMs. On the contrary to the significant role of these cytokines in enhancing the growth rate of AMs ex-vivo, they could rescue AM proliferation under the effect of LPS. In conclusion, I have demonstrated that LPS inhibits AM proliferation in a dose dependent and Maf-independent manner. Furthermore, neither TGFb nor ActivinA could rescue proliferation of LPS-stimulated AMs. Although Maf-deficiency was not shown to be beneficial during the inflammatory phase of ARDS, due to the fact that both WT and DKO AMs were equally depleted at the peak of inflammation, multiple data indicated potential advantage of Maf deficiency during resolution phase.

Alveolar macrophages (AMs) are lung tissue-resident macrophages that can be expanded in culture, but it is unknown to what extent culture affects their in vivo identity. Here we show that mouse long-term ex vivo expanded AMs (exAMs) maintained a core AM gene expression program, but showed culture adaptations related to adhesion, metabolism and proliferation. Upon transplantation into the lung, exAMs reacquired full transcriptional and epigenetic AM identity, even after several months in culture and could self-maintain long-term in the alveolar niche. Changes in open chromatin regions observed in culture were fully reversible in transplanted exAMs and resulted in a gene expression profile indistinguishable from resident AMs. Our results indicate that long-term proliferation of AMs in culture did not compromise cellular identity in vivo. The robustness of exAM identity provides new opportunities for mechanistic analysis and highlights the therapeutic potential of exAMs.