Scaffolds are proteins with high conformational stability, allowing us to implement multiple mutations into specific parts of the protein. Even with these mutations, the structural integrity of the protein is maintained as well as its physical-chemical properties. These mutations give the specific scaffold new properties. In most cases it is the binding specificity towards previously chosen target. The biggest advantages of scaffolds are their small size, stability, low-cost manufacturing, and easiness of preparation. Scaffold utilized in this thesis is unique for having two binging surfaces designed on which it can be mutated. Each of those two surfaces can be separately mutated to develop a binging site for two different proteins. In our case these mutations led to binding two nonidentical receptors of a human cytokine. Mutations are made with a use of yeast display, one of the methods of directed evolution. The main focus of this thesis is changing an expression system of the binding proteins from the yeast system to a bacterial one, their production and purification followed by characterization of those binding proteins using biophysical methods. These methods were used to evaluate structural and thermal stability, and binding affinity to both receptors of the beforementioned binding proteins....

Adrenal gland insufficiency – the clinical manifestation of deficient production or action of adrenal steroids – is a life-threatening disorder. Among many factors which can predispose to primary adrenal failure, an autoimmune adrenalitis and infectious agents play a major role. The initial host defense against bacterial infections is executed primarily by the pattern recognition receptors, e.g. Toll-like receptors (TLRs), expressed in cells from the innate immune system. Upon activation, TLRs have been found to regulate various levels of innate and adaptive immunity as well as control tissue inflammation. TLRs are implicated in adrenal cell turnover and steroidogenesis during inflammation. Therefore, TLRs play a crucial role in the activation of adrenal inflammation mediating adrenal gland dysfunction during septicemia. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The different strategies of tissue engineering for functional reconstruction of critical-size bone defects require a thorough knowledge of physiological mechanisms of bone repair. Bone healing is a complex process affected by various mediators. Several investigations have studied the gene expression 1 to 3 days after an acute or experimental fracture. Little is known about the humoral and cellular in vivo reaction in the early stages of bone healing. In contrast to other methods of molecule sampling and detection, which usually lead to the inhibition of the biological activity following complex sample preparation and quantification, microdialysis is a real-time monitoring technique which can be applied in living tissues providing a strong link between analytical methodology and biochemistry. In this study, the optimal conditions for microdialysis in a critical size rat long bone defect model for both in vivo and in vitro analyses were developed. Mediators and components of the extracellular matrix occurring in the first 24 to 48 hours of bone healing locally and systemically were monitored via microdialysis and blood sampling, respectively. Furthermore, novel proteins and their modulation were explored during this time frame. In vitro microdialysis was used to optimize the condition for protein recovery. Addition of bovine serum albumin (BSA) resulted in an enhanced recovery of interleukin (IL)-6. The maximal relative recovery (RR) was from 15.0% without BSA and 23.6% with BSA, while the maximal RR of transforming growth factor (TGF)-β1 was 11.2% with BSA and the concentration of TGF-β1 was below the detection limit of enzyme-linked immunosorbent assay (ELISA) without BSA. Using in vivo microdialysis, total protein concentrations varied between 0.20±0.12 mg/mL and 0.44±0.18 mg/mL. Among the mediators produced in the fracture hematoma within 24 h after the injury, IL-6 was secreted with the highest concentration of 309.1 pg/mL between 12 and 15 h after creation of the critical size bone defect. Meanwhile, the detectable concentrations of TGF-β1 in microdialysates ranged from 3.6 to 44.0 pg/mL and in blood plasma TGF-β1 was constantly producted ranging from 656.3 to 8398.2 pg/mL for 24 h after bone defct. Moreover, another constant producted growth factor in blood plasma was PDGF-BB and the concentration ranged from 222.1 to 589.4 pg/mL for 8 h after bone defect. Using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), 36 proteins were identified in the microdialysates over 8 h, and 884 proteins were identified on probes which were implanted into the bone defect over 24 h. Among the proteins identified in the hematoma, only a minority originated from the extracellular space. Protein analysis indicated five pathways associated with bone healing that were overrepresented after creating soft tissue and bone defects, of which FGF signaling was specific for bone defects. Furthermore, C-X-C motif ligands CXCL-1, CXCL-2, CXCL-3, CXCL-4, CXCL-5, CXCL-7, rodent bone protein (RoBo-1), insulin-like growth factor (IGF)-I, and chitinase-3-like protein 1 were detected in the fracture hematoma. These proteins are potentially associated to early bone healing. As seen by histological analysis, polymorphonuclear leukocytes (PMNs) and lymphocytes penetrated into the fracture hematoma immediately after surgery and peaked at 24 h. This study for the first time presents data from both the local and systemic acute response to bone and soft tissue injury in a small animal model. The results of mcrodialysis sampling may serve as a baseline for future investigations on different models and time frames. Several proteins and pathways have been identifeid as potentially important for early bone regeneration warranting in depth analysis in further studies.:I. Table of content II. List of abbreviations 1 Summary 2 Introduction 2.1 The process of bone healing 2.1.1 Stages of fracture healing 2.1.2 Early stage of inflammation 2.2 Clinical challenges 2.3 Microdialysis 2.3.1 The principle of Microdialysis 2.3.2 Parameters influencing the recovery 2.4 Aim of this study 3 Materials 3.1 Materials, devices and animals 3.2 Chemicals 3.3 Buffers and solutions 4 Methods 4.1 Background 4.2 In vitro microdialysis 4.2.1 Preparation of the protein solution 4.2.2 Microdialysis sampling procedure 4.3 In vivo microdialysis 4.3.1 Surgical procedure 4.3.2 Sample collection 4.4 Plasma samples 4.5 Determination of the fluid recovery 4.6 Determination of the relative recovery 4.7 Total protein measurement 4.8 Cytokine and growth factor analysis 4.8.1 IL-1β, IL-6, TNF-α and PDGF-BB ELISA 4.8.2 VEGF ELISA 4.8.3 TGF-β1 ELISA 4.8.4 BMP-2 ELISA 4.8.5 Proteome profilerTM array 4.9 Proteomic analysis 4.10 Histological analysis 4.11 Statistical analysis 5 Results 5.1 Protein selection 5.2 Determination of fluid recovery in vitro and in vivo 5.3 Determination of relative recovery (RR) in vitro 5.4 Determination of total protein concentration in vivo 5.5 Determination of cytokine and growth factor concentration in the microdialysate in vivo 5.5.1 IL-6 concentration 5.5.2 TGF-β1 concentration 5.5.3 IL-1β concentration 5.5.4 TNF-α concentration 5.5.5 PDGF-BB, BMP-2 and VEGF concentration 5.6 Determination of further cytokines and chemokines in the microdialysate in vivo 5.7 Protein determination using HPLC-MS/MS analysis 5.7.1 Proteins in the microdialysate 5.7.2 Proteins on the surface of the probe 5.8 Protein annotation 5.9 Determination of cytokines and growth factors in the blood plasma 5.9.1 Determination of IL-6 in the blood plasma 5.9.2 Determination of TGF-β1 in the blood plasma 5.9.3 Determination of PDGF-BB in the blood plasma 5.10 Histological analysis of the hematoma 6 Discussion 6.1 Fluid recovery 6.2 Influence of the crystalloid perfusate on relative recovery 6.3 Relative recovery of cytokines and growth factors in vitro 6.4 In vivo microdialysis 6.4.1 Total protein concentration 6.4.2 Annotation of proteins in hematoma identified by HPLC-MS/MS 6.4.3 Identification of cytokines and bone related proteins 6.5 The humoral inflammatory response 6.6 Cellular response 7 Conclusions 8 References 9 Appendix 9.1 Figure index 9.2 Table index III. Eidesstattliche Erklärung IV. Selbständigkeitserklärung V. Acknowledgements / Zur Entwicklung neuer Strategien der Geweberegenerierung in kritischen Knochendefekten, die sich durch Selbstheilungsprozesse nicht schließen, ist das Verständnis der beteiligten physiologischen Prozesse essentiell. Der Wiederaufbau von Gewebe, wie etwa während Knochenheilungsprozesse ist komplex reguliert und erfordert das koordinierte Zusammenspiel einer Vielzahl von Zellen und Mediatoren. Obwohl bereits in zahlreichen Studien die Veränderungen in der Genexpression in den ersten 3 Tagen nach einer akuten oder experimentell induzierten Fraktur untersucht wurden, ist noch immer wenig über die zellulären und humoralen Vorgänge in den frühen Phasen der Knochenheilung in vivo bekannt. Gebräuchliche Analysemethoden erfordern komplexe Verfahren zur Probenentnahme und Nachweisreaktionen währenddessen die biologische Aktivität der untersuchten Mediatoren häufig graduell verloren geht. Die Mikrodialyse hingegen kann in Echtzeit am lebenden Objekt und am Ort der Verletzung durchgeführt werden und bildet somit eine erfolgsversprechende Plattform um die Probengewinnung noch enger mit der anschließenden biochemischen Nachweistechnik zu verbinden. Im Rahmen dieser Arbeit wurden die optimalen Konditionen zur Mikrodialyse erstmals an einem kritischen Defektmodell eines Ratten-Röhrenknochens zur in vivo und in vitro Applikation ermittelt. Dazu wurde das Vorkommen verschiedener Komponenten der extrazellulären Matrix und ausgewählter Mediatoren während der ersten 24 bis 48 Stunden der Knochenheilung überwacht. Neben der durch Mikrodialyse gewonnenen Proben wurden auch Blutproben verarbeitet um sowohl die lokale, als auch systemische Konzentration der untersuchten Proteine zu erfassen. Durch eine Proteomanalyse konnten zudem bislang in diesem Prozess unbekannte Moleküle identifiziert und verfolgt werden. Zur Optimierung der Mikrodialyse wurden zunächst die Bedingungen hinsichtlich der Proteinrückgewinnung verbessert. Durch den Zusatz von Rinderserumalbumin (BSA) konnte die Rückgewinnung von Interleukin (IL)-6 erhöht werden. Die maximale relative Rückgewinnung (RR) konnte von 15.0% ohne BSA auf 23.6% mit BSA gesteigert werden. Noch dramatischer war dieser Effekt für den transforming growth factor (TGF)-β1 von dessen eingesetzter Menge in vitro 11.2% detektiert werden konnte, während in der BSA-freien Dialyselösung kein TGF-β1 nachgewiesen wurde. Die RR blieb stets unter der Detektionsgrenze des verwendeten enzyme-linked immunosorbent assay (ELISA). In vivo-Dialysate enthielten totale Proteinkonzentrationen zwischen 0,20±0,12 mg/mL und 0,44±0,18 mg/mL. Von den innerhalb von 24 h nach Verletzung im Frakturhämatom produzierten Mediatoren wurde IL-6 am stärksten exprimiert. Die höchsten Konzentrationen (309,1pg/mL) konnten hierfür nach 12 bis 15 Stunden nach Einführung des Defekts gemessen werden. Die Konzentrationslevel von TGF-β1 hingegegen betrug nur 3,6 bis 44,0 pg/mL.Mittels high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), konnten 36 Proteine in den über 8 Stunden gewonnenen Mikrodialysaten, und 884 Proteine von Explantaten, die 24 h im Knochendefekt integriert waren, identifiziert werden. Von den im Frakturhämatom identifizierten Proteinen war nur eine Minderheit extrazellulären Ursprungs. Durch die Proteomanalyse konnten fünf Signalwegskaskaden identifiziert werden. Von diesen trat „FGF (fibroblast growth factor) signaling“ ausschließlich in Knochendefekten, nicht jedoch in den zur Kontrolle mitgeführten reinen Weichgewebedefekten auf. Im Frakturhämatom konnten die, C-X-C motif-Liganden CXCL-1, CXCL-2,CXCL-3, CXCL-4, CXCL-5, CXCL-7, rodent bone protein (RoBo-1), insulin-like growth factor (IGF)-I, und das chitinase-3-like protein 1 nachgewiesen werden. Die identifizierten Proteine könnten von Bedeutung für die Steuerung früher Knochenheilungsprozesse sein. Histologische Untersuchungen zeigten, dass polymorphkernige Leukozyten (PMNs) und Lymphozyten sofort nach der Operation in das Frakturhämatom einwandern und ihre Anzahl nach etwa 24 h ihr Maximum erreicht. Diese Studie präsentiert erstmals Daten der lokal und systemisch ablaufenden zellulären und humoralen Vorgänge als Antwort auf einen Weichgewebs-bzw. Knochendefekt in einem Nagetier-Kleintiermodell. Die Mikrodialyse-Resultate stellen eine vielversprechende Grundlage für zukünftige Untersuchungen in anderen Modellen dar. Außerdem bilden die hier identifizierten Proteine und Signalwege eine Gruppe potenter Kandidaten für weiterführende Untersuchungen zur Knochenregeration.:I. Table of content II. List of abbreviations 1 Summary 2 Introduction 2.1 The process of bone healing 2.1.1 Stages of fracture healing 2.1.2 Early stage of inflammation 2.2 Clinical challenges 2.3 Microdialysis 2.3.1 The principle of Microdialysis 2.3.2 Parameters influencing the recovery 2.4 Aim of this study 3 Materials 3.1 Materials, devices and animals 3.2 Chemicals 3.3 Buffers and solutions 4 Methods 4.1 Background 4.2 In vitro microdialysis 4.2.1 Preparation of the protein solution 4.2.2 Microdialysis sampling procedure 4.3 In vivo microdialysis 4.3.1 Surgical procedure 4.3.2 Sample collection 4.4 Plasma samples 4.5 Determination of the fluid recovery 4.6 Determination of the relative recovery 4.7 Total protein measurement 4.8 Cytokine and growth factor analysis 4.8.1 IL-1β, IL-6, TNF-α and PDGF-BB ELISA 4.8.2 VEGF ELISA 4.8.3 TGF-β1 ELISA 4.8.4 BMP-2 ELISA 4.8.5 Proteome profilerTM array 4.9 Proteomic analysis 4.10 Histological analysis 4.11 Statistical analysis 5 Results 5.1 Protein selection 5.2 Determination of fluid recovery in vitro and in vivo 5.3 Determination of relative recovery (RR) in vitro 5.4 Determination of total protein concentration in vivo 5.5 Determination of cytokine and growth factor concentration in the microdialysate in vivo 5.5.1 IL-6 concentration 5.5.2 TGF-β1 concentration 5.5.3 IL-1β concentration 5.5.4 TNF-α concentration 5.5.5 PDGF-BB, BMP-2 and VEGF concentration 5.6 Determination of further cytokines and chemokines in the microdialysate in vivo 5.7 Protein determination using HPLC-MS/MS analysis 5.7.1 Proteins in the microdialysate 5.7.2 Proteins on the surface of the probe 5.8 Protein annotation 5.9 Determination of cytokines and growth factors in the blood plasma 5.9.1 Determination of IL-6 in the blood plasma 5.9.2 Determination of TGF-β1 in the blood plasma 5.9.3 Determination of PDGF-BB in the blood plasma 5.10 Histological analysis of the hematoma 6 Discussion 6.1 Fluid recovery 6.2 Influence of the crystalloid perfusate on relative recovery 6.3 Relative recovery of cytokines and growth factors in vitro 6.4 In vivo microdialysis 6.4.1 Total protein concentration 6.4.2 Annotation of proteins in hematoma identified by HPLC-MS/MS 6.4.3 Identification of cytokines and bone related proteins 6.5 The humoral inflammatory response 6.6 Cellular response 7 Conclusions 8 References 9 Appendix 9.1 Figure index 9.2 Table index III. Eidesstattliche Erklärung IV. Selbständigkeitserklärung V. Acknowledgements

C1q TNF Related Protein 3 (CTRP3), is a cytokine that is primarily secreted from adipose tissue, which classifies it as an adipokine. Our previous research has shown that CTRP3 prevents alcoholic fatty liver disease (ALD) in male mice. However, even when accounting for confounding factors such as absolute and relative alcohol intake, females are more sensitive to the effects of consumption compared to male mice. Therefore, the goal of this project was to determine whether CTRP3 prevented ALD in female mice. Methods: Female wild type (WT) and female CTRP3 transgenic over expressing (Tg) mice were fed an ethanol containing liquid diet (5% v/v) for 6 weeks. Daily weight and food intake measurements were taken and external heat-pads were placed under a portion of the cage to facilitate thermoregulation. Hepatic steatosis was determined by total triglyceride quantification and lipid droplet quantitation in liver sections. Data were analyzed by repeated measures ANOVA, t-test, or Log-rank (Mantel-Cox) test as appropriate. Results: There was no difference between WT and Tg mice in food intake or body weight. There was no difference in survival between WT and Tg mice, however, Tg mice trended towards a reduced rate of survival compared with WT mice (78% in WT versus 44% in Tg, p=0.13). Stereological analysis indicated no difference in the percent of lipid liver volume between the two groups (WT 7.2±3.6 vs Tg 5.1±4.1%). This finding was consistent with no difference in total hepatic triglyceride accumulation observed between WT and Tg mice (12.7±4.4 vs. 13.1±6.8 mg triglycerides/gram liver protein). Conclusion: Combined these data indicate that unlike previous studies with male mice, CTRP3 is not protective against alcohol-induced hepatic steatosis in female mice. Combined, these data indicate that the adipokines such as CTRP3 contribute to physiology in a sex-specific manner.

I december 2019 rapporterades fall av oidentifierad lunginflammation, senare känd som Covid-19. Antalet fall ökar fortfarande snabbt i världen och därmed utgör Covid-19 ett extraordinärt hot mot folkhälsan. Studier har visat en ökad mängd proinflammatoriska cytokiner TNF-α, associerade med andningssvårigheter samt en ökad kortisolproduktion. Utsöndring av kortisol och TNF-α är associerade med fysiska- och psykiska hälsoeffekter, sjukdomsgrad samt dödsfall av Covid-19. Syftet med studien var att undersöka utsöndringen av biomarkörerna kortisol och TNF-α i saliv hos individer tidigare diagnostiserade med Covid-19 samt hos individer utan diagnostiserad Covid-19 (kontrollgrupp). Det för att undersöka huruvida kvarvarande symtom kunde associeras till biomarkörerna. Studien inkluderade 40 individer, varav 20 individer tidigare diagnostiserade med Covid-19 samt 20 individer utan diagnostiserad Covid-19. Saliv och ett frågeformulär om deras allmänna hälsotillstånd och symtom kopplade till Covid-19 infektion samlades in och analyserades. Salivprover tagna på morgon analyserades med ELISA och resultatet jämfördes mellan grupperna. Inga signifikanta skillnader visade mellan grupperna gällande biomarkörer. Resultaten visade dessutom inga korrelation mellan symtom och biomarkörer samt att inga förhöjda nivåer av analyserade biomarkörer kan associeras med kvarvarande symtom efter en Covid-19 infektion. / A cluster of unidentified pneumonia cases, later known as Covid-19, were reported in December 2019. The number of Covid-19 cases is still rapidly increasing and poses an extraordinary threat to our public health. Studies have shown an increased level of the cytokine TNF-α, associated with severe respiratory symptoms as well as an increased cortisol production. Together associated with physical and mental health effects, disease severity and increased mortality of Covid-19. This study aimed to evaluate the secretion of cortisol and TNF-α in the saliva of individuals previously diagnosed with Covid-19 and individuals not diagnosed with Covid-19 (control group). This to examine whether the residual symptoms can be associated with the analyzed biomarkers. The study included 40 participants, 20 individuals diagnosed with Covid-19 and 20 individuals not diagnosed with Covid -19. Saliva and a questionnaire concerning their overall health and possible symptoms associated with Covid-19 infection were collected and analyzed. Saliva morning samples were analyzed with ELISA and results were compared between the groups. No significant differences were shown between the groups regarding the biomarkers. Also, the results showed no correlations between symptoms and biomarkers, nor elevated levels of analyzed biomarkers could be associated with residual symptoms after a Covid-19 infection.

Diese Arbeit beschreibt Ergebnisse einer in-vitro Untersuchung der Antipsychotika Chlorpromazin, Haloperidol, Clozapin, N-Desmethylclozapin und Quetiapin bezüglich ihrer Wirkung auf die Zytokinproduktion. Dafür wurde Vollblut von gesunden Probandinnen invitro mit dem Immunmodulator Toxic-Shock-Syndrome-Toxin-1 (TSST-1) stimuliert. Dabei wurden die Konzentrationen der Zytokine Interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-17 und Tumornekrosefaktor-α (TNF-α) im unstimulierten Blut und im stimulierten Blut, jeweils mit und ohne Zusatz der Antipsychotika gemessen. Es zeigte sich, dass TSST-1 eine signifikante Stimulation der Produktion aller getesteten Zytokine bewirkte und dass es über diese Stimulation mit TSST-1 hinaus zu einer Erhöhung von IL-17 unter allen getesteten Antipsychotika kam. Aufgrund dieser Ergebnisse ist es denkbar, dass Antipsychotika, in Ergänzung zu ihrer Wirkung an Dopaminrezeptoren, auch über diese immunologische Eigenschaft Wirkungen und Nebenwirkungen entfalten können. Weiterhin könnte die IL-17-Produktion ein Biomarker in der Behandlung mit Antipsychotika sein, der wiederum zur individuellen Vorhersage von Wirkungen und Nebenwirkungen beitragen könnte.

The paradigm shift in cardiovascular biology has been the understanding that atherosclerosis involves not just a mechanical deposition of lipids in the vessel wall, but a dynamic process involving the inflammatory response with cellular infiltration and inflammatory mediator expression. Typical cellular elements that have been studied include endothelial cells, vascular smooth muscle, T lymphocyte and the macrophage. Recent data suggests a role for the human mast cell. The human mast cell is a tissuedwelling cell, typically perivascular in distribution. This multifunctional cell responds rapidly to challenge with the release of inflammatory mediators that can orchestrate an immune response and may have relevance to atherogenesis. Mast cells have been shown to modulate various aspects of cardiovascular disease such as atherogenesis (endothelial activation, cytokine generation and foam cell formation) as well as rupture of an unstable atheromatous plaque. Mast cell activation in the context of cardiovascular disease may occurby cognate cell-cell interactions (interactions with macrophages, T cells, endothelial cells or smooth muscle) or by non-cognate means (such as lipoproteins and other proatherogenic components). More studies are required in order to better understand the molecular role of mast cells in vascular inflammatory disease.

Chronic hepatitis C virus (HCV) infection is associated with cirrhosis, autoimmunity and lymphoproliferative disorders. We have previously reported a differential regulation of T and B lymphocytes by HCV core protein in vitro. In this report, we employed a translational approach to characterize the activation status of peripheral B cells from individuals with chronic HCV infection and to explore potential mechanisms for B-cell dysregulation in the setting of HCV infection. In contrast to the T-cell suppression observed in HCV-infected individuals, B cells exhibit a non-specific polyclonal activation phenotype, characterized by significantly higher levels of (1) the early activation marker, CD69, (2) the costimulatory molecule, CD86, and (3) the CCR5 chemokine receptor, CD195, when compared with B cells from healthy donors in response to phytohaemagglutinin (PHA) stimulation. Importantly, tumour necrosis factor- and Apo-L-related leucocyte-expressed ligand-1 (TALL-1), also known as B-lymphocyte stimulator (BLYS), was found to be up-regulated on the surface of B cells from HCV patients in response to PHA as well as HCV core antigen stimulation. This up-regulation of TALL-1 was associated with vigorous memory B-cell responses to viral antigenic stimulation. Additionally, suppressor of cytokine signalling-1 (SOCS-1), a negative feedback immunoregulator that is inhibited in B lymphocytes by HCV core in vitro, was also inhibited in B cells from HCV patients when compared with healthy donors. These findings suggest that TALL-1 over-expression and SOCS-1 suppression are associated with aberrant B-cell activation, providing a plausible basis for the B-cell clonal expansion underlying the lymphoproliferative disorders and autoimmune phenomena observed during chronic HCV infection.

Alteration of gut microbiota has been associated with development of immune mediated diseases, such as allergy. In part, this could be due to the influence of microbes in shaping the immune response. In paper I, we investigated the association of early-life gut colonization with bacteria, and numbers of IL-4, IL-10 and IFN-γ producing cells at two years of age in response to PBMC stimulation with phytohemagglutinin (PHA) in vitro. Early Staphylococcus (S) aureus colonization was directly proportional to increased numbers of IL-4 and IL-10 secreting cells, while early co-colonization with lactobacilli and S. aureus associated with a decrease in IL-4, IL-10 and IFN-γ secreting cells compared to S. aureus alone. This was also confirmed in in vitro stimulations of PBMC with Lactobacillus and/or S. aureus strains, where S. aureus-induced IFN-γ production by Th cells was down regulated by co-stimulation with Lactobacillus. In paper II, we investigated the effects of UV-killed and/or culture supernatant (sn) of Lactobacillus strains and S. aureus strains on IEC and immune cell responses. IEC exposed to S. aureus-sn produced CXCL-1/GRO-α and CXCL-8/IL-8, while UV-killed bacteria had no effect. Further, PBMC from healthy donors exposed to Lactobacillus-sn and S. aureus-sn were able to produce a plethora of cytokines, but only S. aureus induced the T-cell associated cytokines: IL-2, IL-17, IFN-γ and TNF-α; which were down regulated by co-stimulation with any of the different Lactobacillus strains. Intracellular staining verified S. aureus-induced IFN-γ and IL-17 production by Th cells, and increased CTLA-4 expression and IL-10 production by T reg cells. In conclusion, we show that colonization with gut microbiota at early age modulates the cytokine response in infancy. In addition, bacterial species influence cytokine response in a species-specific manner and we demonstrate that lactobacilli modulate S. aureus-induced immune response away from an inflammatory phenotype.

Alcohol is a commonly consumed beverage, a drug of abuse and an important molecule affecting nearly every organ-system in the body. This project seeks to investigate the interplay between alcohol’s effects on critical organ-systems making up gut-liver-brain axis. Alcohol initially interacts with the gastrointestinal tract. Our research describes the alterations seen in intestinal microbiota following alcohol consumption in an acute-on-chronic model of alcoholic hepatitis and indicates that reducing intestinal bacteria using antibiotics protects from alcohol-induced intestinal cytokine expression, alcoholic liver disease and from inflammation in the brain. Alcohol-induced liver injury can occur due to direct hepatocyte metabolic dysregulation and from leakage of bacterial products from the intestine that initiates an immune response. Here, we will highlight the importance of this immune response, focusing on the role of infiltrating immune cells in human patients with alcoholic hepatitis and alcoholic cirrhosis. Using a small molecule inhibitor of CCR2/CCR5 chemokine receptor signaling in mice, we can protect the liver from damage and alcohol-induced inflammation. In the brain, we observe that chronic alcohol leads to the infiltration of macrophages in a region-specific manner. CCR2/CCR5 inhibition reduced macrophage infiltration, alcohol-induced inflammation and microglial changes. We also report that chronic alcohol shifts excitatory/inhibitory synapses in the hippocampus, possibly through complement-mediated remodeling. Finally, we show that anti-inflammasome inhibitors altered behavior by reducing alcohol consumption in female mice. Together, these data advance our understanding of the gut-liver-brain axis in alcoholism and suggest novel avenues of therapeutic intervention to inhibit organ pathology associated with alcohol consumption and reduce drinking.