Sarcoidosis is a systemic inflammatory disease mostly affecting the lungs, marked by the presence of granulomas and the accumulation of interferon gamma (IFNγ)-producing T cells in the affected organs. Extracellular vesicles (EVs) are membranous vesicles with a size range of 50 nm to 5000 nm, with exosomes ranging from 50 to 150 nm. They are major players in intercellular communication and have been found in all body fluids including plasma. In this study, we wanted to characterize the exosomes from sarcoidosis patients’ plasma and try to detect differences in their surface marker expression. Additionally, we aimed to investigate the cellular origin of these exosomes in the blood circulation of mice. We used a combination of Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), Western Blot and Flow cytometry as characterization methods. Moreover, we took advantage of immune cell-specific knock-out mice (Rag KO, Ly6G-Mcl-1 KO) and examined the effect on the number of vesicles and on the marker expression levels on the vesicle surface. We showed that in Rag KO mice, that lack mature B cells and T cells, the levels of T cell markers CD4 and CD8 were decreased. No difference was detected between wild type and neutrophil KO mice on neither of the neutrophil-specific markers Ly6G and CD11b. Sarcoidosis patient plasma EVs showed lower levels of CD9 expression compared to healthy subjects. Differences were observed in a subgroup of patients, namely Löfgren syndrome patients that exhibited higher CD31 expression than the non-Löfgren patients. Overall, our data indicate a possible contribution of the immune cell-derived exosomes in the blood. Furthermore, differences detected in the sarcoidosis plasma EVs could give a better insight on the role of those exosomes during lung inflammation and provide grounds for their use as liquid tissue biomarkers.

<p>The maternal immune system must be able to adjust during pregnancy and accept the foetus that expresses paternal antigens. These changes are found both in placenta and circulation, including a mild inflammatory response. NK cells are abundant during the early part of pregnancy in placenta and are thought to be important for placental development. During preeclampsia the placenta is poorly developed, together with an escalated pro-inflammatory profile noticed in both placenta and circulation. We wanted to study NK cells in placenta and circulation from preeclamptic cases as well as levels of cytokines. HMGB1, an alarmin involved in inflammation, was also measured in preeclamptic placentae.</p><p>When studying preeclamptic placentae in third trimester we found higher numbers of NK cells as well as a higher expression of CD94+ NK cells. We also found slightly elevated levels of HMGB1 together with significantly lower expression of IL-12 in preeclamptic placentae. Further, the NK cell activating cytokines IL-12/IL-23p40 and IL-15 in sera from preeclamptic women were increased compared to healthy pregnancies. The elevated levels of NK cell activating IL-12/IL-23p40 and IL-15 found in preeclamptic sera, made us investigate the circulating NK cells in preeclampsia. However, no differences were seen between healthy and preeclamptic pregnancies.</p><p>The main immunological alterations in third trimester preeclamptic pregnancies with regard to NK cells were found in placenta. Altered maternal cytokine levels in placenta could influence decidual NK cells in preeclampsia, noticed by their higher numbers and altered receptor expression. If these alterations also exist during early pregnancy it could result in a poorly developed and dysfunctional placenta.</p>

The maternal immune system must be able to adjust during pregnancy and accept the foetus that expresses paternal antigens. These changes are found both in placenta and circulation, including a mild inflammatory response. NK cells are abundant during the early part of pregnancy in placenta and are thought to be important for placental development. During preeclampsia the placenta is poorly developed, together with an escalated pro-inflammatory profile noticed in both placenta and circulation. We wanted to study NK cells in placenta and circulation from preeclamptic cases as well as levels of cytokines. HMGB1, an alarmin involved in inflammation, was also measured in preeclamptic placentae. When studying preeclamptic placentae in third trimester we found higher numbers of NK cells as well as a higher expression of CD94+ NK cells. We also found slightly elevated levels of HMGB1 together with significantly lower expression of IL-12 in preeclamptic placentae. Further, the NK cell activating cytokines IL-12/IL-23p40 and IL-15 in sera from preeclamptic women were increased compared to healthy pregnancies. The elevated levels of NK cell activating IL-12/IL-23p40 and IL-15 found in preeclamptic sera, made us investigate the circulating NK cells in preeclampsia. However, no differences were seen between healthy and preeclamptic pregnancies. The main immunological alterations in third trimester preeclamptic pregnancies with regard to NK cells were found in placenta. Altered maternal cytokine levels in placenta could influence decidual NK cells in preeclampsia, noticed by their higher numbers and altered receptor expression. If these alterations also exist during early pregnancy it could result in a poorly developed and dysfunctional placenta.

There are many aspects of the immunology of P. falciparum infection that are not understood. Genetic approaches are of great value for dissecting the complexity of immune responses to malaria in natura by providing new insights into molecular interactions between the parasite and the host. The work presented in this thesis had two major aims: to investigate the role of IFN-γ signalling in susceptibility to malaria; and to understand the biological basis of the low susceptibility to malaria shown by the Fula people of West Africa. We conducted genetic association studies to investigate the role of four candidate loci: IFNG, IFNGR1, IFNGR2 and IRF1. We observed significant associations between common genetic variation at the IRF1 locus and the ability to control P. falciparum infection. Our studies did not provide evidence for a major role of this gene in determining susceptibility to severe malaria. Using allele-specific expression assays we obtained preliminary results suggesting the existence of a regulatory element(s) in the 5’ upstream region of the IRF1 locus. IRF1 polymorphisms regulating gene expression could affect the production of inflammatory cytokines and the control of infection, but not the immune-based pathogenesis of severe disease. To understand the biological basis of the resistance to malaria shown by the Fula, we analysed HLA class II polymorphism and confirmed previous data showing that the Fula from Burkina Faso are genetically distinct from sympatric Mossi and Rimaibé. We then compared the expression profiles of PBMCs and CD4+CD25+ cells isolated from healthy adults of Fula and Mossi ethnicity. In the Fula we observed higher expression of several genes related to Th1 and Th2 function and reduced expression of important genes related to immune tolerance: FOXP3, CTLA4, TGFB and TGFBRs. These results suggest a functional deficit of T regulatory cells in the Fula and identify key genes as good candidates for future genetic association studies.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2007. / Härtill 4 uppsatser.

Cancer is a major health burden in modern society, costing millions of lives worldwide and negatively impacting many more. With increasing rates of cancer, there is a need for new approaches to its treatment. This is where immunotherapies step in, this a relatively new approach to cancer treatment which caught public’s attention only in recent years. The main goal of these therapies is to enhance and help immune cells to identify and kill tumor cells, thereby initiating the cycle of cancer immunity. In this project LOAd platform viruses were evaluated and compared for their ability to induce oncolysis in cancer cells and ability to produce immunostimulatory molecules. Established LOAd703 virus armed with CD40L and 4-1BBL transgenes was compared to new constructs LOAd732, LOAd780 and LOAd786. All three new viruses are armed with CD40L and 4-1BBL, but also have additional transgenes X, Y and Z, respectively. Specific molecules coded by these transgenes cannot be disclosed at this moment. All viruses demonstrated high competence in oncolysis of A549-lung, T24-bladder and 526-mel melanoma cancer cell lines and were able to express transgenes coding for CD40L and 4-1BBL in all cell lines. New viruses were able to induce expression of new transgenes in infected cells, except for LOAd780 infected cell which had low concentration of protein Y in their supernatants. Also dendritic cells matured using LOAd viruses were able to induce expansion of CMV-specific T cells and a major expansion of natural killer cells.

Mosquitoes are constantly under threats of infections by different kinds of pathogens. Due to a lack of adaptive immune system unlike higher animals, they defend themselves by using their innate immune system. In our project, we assessed the implications of the two flaviviruses (named Lammi virus and Usutu virus) on the mosquitoes’ innate immune system as well as how these flaviviruses manipulated their innate immune system during infection. First, the mosquito cell lines were examined at five different time points to identify changes of cellular structure after USUV and LAMV infections. Then, the USUV infection was evaluated by using immunofluorescence Assay (IFA staining). In this present study, we also investigated the virus load from supernatant of single and double infections separately with the help of RT-qPCR. For immune gene studies, the transcript-levels of our selected twenty-three immune genes were analyzed by using the 2-ΔΔCq Livak method from single and double infected samples with three biological replicates. After examination of the cellular structure of infected cells, all characteristics of cytopathic effects were unclear compared with the mock infection. In the supernatant samples, the USUTU virus replication was decreased but Lammi virus load was higher in the double infection that was proven to be statistically significant at five different time points. After that, RT-qPCR data of infected cells demonstrated different expressions of certain immune genes due to the presence of USUV and LAMV in the single as well as double infections. Out of 23 genes, fold changes of nine genes’ expression were verified as statistically significant. For example, the fold changes of differentially expressed REL1A and unchar_ncRNA genes were significant in the LAMV infected U4.4 cells compared with the double infected cells (USUV and LAMV together). Concurrently, some genes were up-regulated and some were down-regulated significantly in different infections at the 24 hr and 48 hr time points compared with the mock infection. In conclusion, the mosquito cell line U4.4 was infected by single (e.g. LAMV or USUV) and double infections (USUV and LAMV) separately to see the effects of these two flaviviruses and our selected immune genes revealed fold changes  of differential gene expressions in both single and double infections compared with the mock infection. Furthermore, this investigation demonstrates how mosquitoes’ immune signaling pathways were manipulated by these flavivirus infections. / <p>Due to pandemic, it was a zoom presentation.</p>

Chronic Obstructive Pulmonary Disease (COPD) is a common lung disease characterized by progressive and irreversible airway obstruction, and mainly caused by a chronic exposure to lung irritants. As of 2010, 384 million people suffered from COPD worldwide. It is widely accepted that a chronic inflammatory response is integral to COPD pathogenesis and linked to disease progression. The cellular mediators and molecular mechanisms of COPD-associated inflammation are not completely understood and are difficult to emulate in animal models, which hinders the development of better treatments. In this study, experimental COPD and its associated inflammation were induced in mice using a 4-week protocol involving intranasal administration of LPS and elastase. Model validation on wild-type mice yielded COPD-like disease judging from flow cytometric analyses with and pulmonary function testing. After 4 weeks of exposure to LPS and elastase, mice developed classic aspects of COPD such an increase in lung-infiltrating cells, (e.g. neutrophils, CD4+and CD8+ T-cells). Acute inflammation in the form of substantial neutrophilia was due to the last LPS administration, whereas the observed eosinophilia and elevated counts of mast cell populations, CD4+ and CD8+ T-cells were due to the cumulative effects of LPS and elastase. The nature of COPD-associated inflammation in mast cell deficient mice was investigated in two experiments. Our first experiment suggested a mild protective role of mast cells, a finding not reproduced in the second experiment possibly due to expired elastase. Our study suggests that mast cells are not required for COPD-associated inflammation.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, a disease that for years was considered to belong of the past, but tuberculosis is back causing over 2 million deaths per year. The infection can be dormant for decades and an active immune response can prevent the infection from progressing into active disease. However, the HIV/AIDS epidemic has caused an alarming rise in tuberculosis cases. The main infectious route for Mtb is through the airways into the lungs, where they encounter alveolar macrophages. Mtb are phagocytosed by these macrophages, but instead of being killing within the phagosome, Mtb modulates the cell to become a host in which the bacteria thrive. The lack of capacity to eradicate the infection stimulate cells of the immune system to gather around infected macrophages and form a granuloma that walls off the infection. Within this granuloma, Mtb can wait silently and later progress into active disease. However, only a fraction of exposed individuals develop disease, indicating that initial eradication of Mtb infections is possible. Such immediate response must be directed by the innate immunity comprised of phagocytes such as neutrophils (PMNs) and non-activated macrophages. Upon Mtb infection, macrophages become anergic and PMNs enter apoptosis. PMNs have a short lifespan and are cleared by neighbouring phagocytes, a mechanism described to resolve the inflammation and modulate tissue regeneration. We found that Mtb-induced apoptosis in PMNs was not dependent on phagocytosis of the bacteria, indicating that Mtb have the capacity to induce apoptosis in multiple PMNs. Complement-mediated phagocytosis induce survival signals such as Akt in PMNs, but despite this, complement-opsonized Mtb was able to override the anti-apoptotic activation in the cells. Since phagocytes clear apoptotic cells, we investigated how clearance of Mtb-induced apoptotic PMNs affected macrophages. We found that Mtb-induced apoptotic PMNs inflicted pro-inflammatory activation of the macrophages that cleared them. In addition, this activation was mediated by Hsp72 released from the Mtb-induced apoptotic PMNs. Furthermore, apoptotic PMNs can work in synergy with phagocytosed Mtb to activate macrophages and enhance intracellular killing of Mtb. Since dendritic cells are important for the regulation of immunity, we investigated whether Mtb-induced apoptotic PMNs affected the inflammatory response and maturation of dendritic cells. We found that Mtb-induced apoptotic PMNs trigger dendritic cells to enter a mature state able to activate naïve T-cell proliferation. We propose that infected apoptotic PMNs is a potent activator of the inflammatory response during infections. Taken together, PMNs not only kill their share of pathogens but also modulate other immune cells, thereby forming a link between the early innate and the adaptive immune response during microbial challenge with Mtb.