Activation of the Inflammatory Response by Fungal Components

Fungi are associated with a wide range of diseases from superficial skin syndromes to invasive life-threating conditions. Furthermore, exposure to non-infectious fungal components in the context of agricultural work or in water-damaged houses has been associated to illnesses in the respiratory tract. The inadequate knowledge of the immune mechanisms behind these illnesses has triggered an intense research effort attempting to understand how fungi can activate the defense mechanisms of immune system. This thesis focused on the inflammation triggered by the fungal components. The inflammatory response and related mechanisms were studied in vitro in the key defense cell of innate immunity, the macrophage, which were treated with a central cell wall component of fungi, (1,3)- β-glucan. One major outcome of this thesis was the finding that β-glucan evokes a strong pro-inflammatory response via the IL-1 family cytokines in human macrophages. These cytokines are crucial mediators of inflammation, thus their secretion is highly regulated. Our study showed that β-glucan, on its own, could cause the efficient secretion of functional IL-1β and other mediators of inflammation via unconventional secretion pathways by activating both dectin-1 signaling pathway and NLRP3 inflammasome. This β-glucan-induced protein secretion via unconventional pathways was suppressed by inhibition of inflammasome activity or by preventing the process of autophagy. Furthermore, the well-known myeloid cell growth factor, granulocyte-macrophage colony-stimulating factor seems to be one of the factors boosting the β-glucan-triggered inflammatory response of macrophages. In an attempt to obtain direct information about conditions caused by the exposure to fungal and other microbial particles, we characterized the proteomic changes present in the bronchoalveolar lavage obtained from patients suffering illnesses associated with exposure to inhaled microbial particles. The proteomic profiles of hypersensitivity pneumonitis (HP) were different from the profile of damp building-related illness (DBRI), indicating that these conditions are not closely associated. However, the increase in the levels of two well-known markers of inflammation was observed in both HP and DBRI, evidence for the activation of inflammatory mechanisms in both of these conditions. This thesis provides novel knowledge concerning the inflammatory response and related mechanisms triggered by fungal components. These results may help to clarify the mechanisms behind the inflammatory symptoms experienced by individuals with fungal infections or exposure to fungal particles and will provide future tools for the treatment of fungal-related disorders.Sienet aiheuttavat monenlaisia tauteja vaihdellen ihon pintainfektioista hengenvaarallisiin syviin sieni-infektioihin. Sienialtistuksesta johtuvaa oireilua tavataan myös henkilöillä, jotka ovat altistuneet sienen rakennekomponenteille tehdessään maataloustöitä homeisen heinän parissa, tai oleskellessaan rakennuksissa, joissa on todettu kosteusvaurioista johtuvaa sienikasvustoa. Jotta sienten aiheuttamien tautien hoitoa ja ennaltaehkäisyä voitaisiin parantaa on ensiarvoisen tärkeää ymmärtää sienen käynnistämä immmuunipuolustusreaktio ja siihen vaikuttavat tekijät elimistössämme. Tässä väitöskirjassa tutkittiin sienen seinämäkomponentin, (1,3)-β-glukaanin, aiheuttamaa tulehdusvastetta luontaisen immuunipuolustuksemme keskeisimmässä valkosolussa, makrofagissa. IL-1 perheen sytokiinit ovat tulehdusta edistäviä soluvälittäjäaineita, minkä takia niiden tuotanto on soluissa tiukasti säädeltyä. B-glukaanin aktivoima voimakas IL-1 -sytokiinivaste on tämän väitöskirjan yksi keskeisimmistä tuloksista. B-glukaani käynnisti myös muiden tulehdusproteiinien erityksen ihmisen makrofagissa, sekä klassista, että niin kutsuttuja epätyypillisiä proteiinieritysreittejä pitkin. B-glukaani pystyi yksistään aktivoimaan sekä dektiini-1 reseptorin, että NLRP3 inflammasomin, mikä johti biologisesti aktiivisen IL-1β tulehdussytokiinin ja muiden epätyypillisiä proteiinieristysreittejä hyödyntävien tulehdusproteiinien eritykseen. Lisäksi tähän proteiinieritykseen vaikuttivat sekä β-glukaanin käynnistämä autofagiaprosessi, että valkosolujen kasvua ja erilaistumista edistävät kasvutekijät. Saadaksemme informaatiota mikrobikomponenttialtistumisen aihettamista tautiloista karakterisoimme keuhkohuuhtelunesteessä tapahtuneita proteiinimuutoksia sairausten kuten allergisen alveoliitin tai kosteusvauriorakennuksiin liittyvän sairastelun (damp building-related illness) yhteydessä. Näiden kahden sairauden proteiiniprofiili erosi huomattavasti toisistaan viitaten siihen, että kyseessä on erilaiset tautiprosessit. Kummassakin tautitilassa havaittiin kuitenkin kahden tulehdusproteiinimarkkerin nousu, mikä viittaa tulehdusmekanismien käynnistymiseen molemmissa sairauksissa. Tämän väitöskirjatyön tulokset tuovat meille ymmärrystä immuunipuolustusreaktiosta, joka käynnistyy sieni-infektioiden ja sienikomponenttialtistumisten yhteydessä. Tämä edesauttaa myös hoitojen ja diagnostiikan kehittymistä

Regulation of the complement system and immunological tolerance in pregnancy

Preeclampsia is a serious vascular complication of the human pregnancy, whose etiology is still poorly understood. In preeclampsia, exacerbated apoptosis and fragmentation of the placental tissue occurs due to developmental qualities of the placental trophoblast cells and/or mechanical and oxidative distress to the syncytiotrophoblast, which lines the placental villi. Dysregulation of the complement system is recognized as one of the mechanisms of the disease pathology. Complement has the ability to promote inflammation and facilitate phagocytosis of placenta-derived particles and apoptotic cells by macrophages. In preeclampsia, an overload of placental cell damage or dysregulated complement system may lead to insufficient clearance of apoptotic particles and placenta-derived debris. Excess placental damage may lead to sequestration of microparticles, such as placental vesicles, to capillaries in the glomeruli of the kidney and other vulnerable tissues. This phenomenon could contribute to the manifestations of typical diagnostic symptoms of preeclampsia: proteinuria and new-onset hypertension. In this review we propose that the complement system may serve as a regulator of the complex tolerance and clearance processes that are fundamental in healthy pregnancy. It is therefore recommended that further research be conducted to elucidate the interactions between components of the complement system and immune responses in the context of complicated and healthy pregnancy.Peer reviewe

Dysfunction of complement receptors CR3 (CD11b/18) and CR4 (CD11c/18) in pre-eclampsia : a genetic and functional study

Objective To study genetic variants and their function within genes coding for complement receptors in pre-eclampsia. Design A case-control study. Setting Pre-eclampsia is a common vascular disease of pregnancy. The clearance of placenta-derived material is one of the functions of the complement system in pregnancy. Population We genotyped 500 women with pre-eclamptic pregnancies and 190 pregnant women without pre-eclampsia, as controls, from the FINNPEC cohort, and 122 women with pre-eclamptic pregnancies and 1905 controls from the national FINRISK cohort. Methods The functional consequences of genotypes discovered by targeted exomic sequencing were explored by analysing the binding of the main ligand iC3b to mutated CR3 or CR4, which were transiently expressed on the surface of COS-1 cells. Main outcome measures Allele frequencies were compared between pre-eclamptic pregnancies and controls in genetic studies. The functional consequences of selected variants were measured by binding assays. Results The most significantly pre-eclampsia-linked CR3 variant M441K (P = 4.27E-4, OR = 1.401, 95% CI = 1.167-1.682) displayed a trend of increased adhesion to iC3b (P = 0.051). The CR4 variant A251T was found to enhance the adhesion of CR4 to iC3b, whereas W48R resulted in a decrease of the binding of CR4 to iC3b. Conclusions Results suggest that changes in complement-facilitated phagocytosis are associated with pre-eclampsia. Further studies are needed to ascertain whether aberrant CR3 and CR4 activity leads to altered pro- and anti-inflammatory cytokine responses in individuals carrying the associated variants, and the role of these receptors in pre-eclampsia pathogenesis. Tweetable abstract Genetic variants of complement receptors CR3 and CR4 have functional consequences that are associated with pre-eclampsia.Peer reviewe

Proteomic Changes of Alveolar Lining Fluid in Illnesses Associated with Exposure to Inhaled Non-Infectious Microbial Particles

Peer reviewe

Major symptoms and HRCT findings.

a<p>before BAL procedure.</p>b<p>HRCT = high-resolution computed tomography.</p>c<p>Hypersensitivity pneumonitis.</p

Immunoblot validation of protein markers in BAL.

<p>(A) Differential expression analysis based on DeCyder data for chosen protein markers. Every dot indicates the identified protein spot and its expression level in that particular study group. The difference in protein expression (average expression) between the groups is visualized with a continuous curve. Histone and semenogelin plots contains more than one curve due to several protein spot identification for histones H4, H2B (n = 3) and semenogelin I (n = 2) as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102624#pone.0102624.s005" target="_blank">Table S1</a>. (B) Images of immunoblot membranes and (C) immunoblot based validation results for protein markers. The number of samples in validation: CTR n = 19, AME n = 7–9, DBRI n = 17, HP n = 9, SARC n = 8. The means (black lines) are shown in scatter plots. *Indicates statistical significance, which is shown between experimental and control group, at the level of p≤0.05, **p≤0.01 and, ***p≤0.001.</p

The false color image of two-dimensional DIGE gel of BAL.

<p>The gel image represents the Cy3 labeled (red), Cy5 labeled (blue) and Cy2 labeled (yellow) patient samples. The latter is a pooled sample, which served as an internal standard. Spot abbreviations refer to the identified proteins listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102624#pone.0102624.s005" target="_blank">Table S1</a>. Molecular weights are shown on the right edge of the gel and the pI range on the top part of the SDS-PAGE.</p

Patient characteristics.

<p>*Data available for ≥80% of samples.</p><p>**Data available for ≥75% of samples.</p>a<p>Damp building-related illness.</p>b<p>Agricultural type of microbial exposure.</p>c<p>Hypersensitivity pneumonitis.</p>d<p>Sarcoidosis.</p