A systems genetics approach to the characterization of differential low dose radiation responses in BXD recombinant inbred mice

High doses of radiation (HDR) are clearly detrimental to human health, but relatively little is known about the health consequences following exposure to low doses of radiation (LDR, \u3c10cGy). Understanding the risks associated with LDR is of great importance to the general public due to the recent dramatic increase in diagnostic radiological imaging. While HDR clearly suppress immune function, there is evidence that LDR can be immunostimulatory. Within the organism, defining the consequences of LDR is further complicated by the impact of genetic background, particularly in systems such as the immune system for which both radiosensitivity and genetic effects are profound. We addressed the issue of genetic susceptibility to LDR using the immune system as a target system and treated the LDR response as a complex trait analyzed using a systems genetics framework. Using the BXD recombinant inbred strain mouse panel as a genetic reference population allowed us to address the radiation response within the context of natural genetic variation. Our overarching hypothesis is that, within a population, the immunological effects of LDR exposure depend in part on the individual’s baseline immunoprofile and gene expression which are ultimately dependent upon genetic background. We began by establishing the immunophenotypic variation (i.e., T:B cell ratio, CD4:CD8 ratio) within the BXD panel and used baseline spleen transcriptome profiling to identify putative candidate genes controlling these traits, specifically Acp1 and Ptprk for CD4:CD8 ratio. The same set of BXD strains was exposed to LDR (10cGy gamma radiation) to determine effects on immune function and oxidative stress. LDR significantly enhanced neutrophil phagocytosis in a manner that was independent of genetic background. In contrast, genetic background significantly impacted LDR-induced changes in spleen superoxide dismutase activity. By integrating these results with our previous analyses of BXD RI strains, we have demonstrated that baseline expression of Sod2 correlates with LDR-induced SOD activity, and baseline CD4:CD8 ratio is inversely correlated with LDR-induced neutrophil phagocytosis. In addition, spleen transcriptomic data from the BXD parental strains further highlighted the impact of genetic background on LDR responses. These data provide the groundwork for predicting LDR responses using baseline expression, immunophenotypes, and/or genotype

Crippling Rapid Evolution of Metastasis and Drug Resistance in A549 Non-Small Cell Lung Cancer Cells with the Clinically Relevant HSP90 Inhibitor AUY922

The ability for species to evolve new features in response to changing circumstances in order to survive and propagate is a ubiquitous observation on both the macroscopic and microscopic levels of living systems. It should be no surprise, then, that diseases such as cancer utilize their own forms of adaptation to perpetuate themselves when exposed to external threats. Indeed, concepts drawn from Darwinian evolution are now widely accepted to help explain certain aspects of carcinogenesis and malignant progression, the sum of which have come to be known as the theory of tumor evolution. Since metastasis and drug resistance are features that manifest toward the late stages in the disease after withstanding numerous selective pressures, cancer cells harboring these features can be viewed as the most evolutionarily fit. Just as many forms of life rely on common adaptive mechanisms to promote their survival during dramatic shifts in their environment, metastatic and drug resistant cancers may rely upon common cellular mechanisms to promote their survival when faced with untenable circumstances. We hypothesize that one of the oldest genes in the human genome, HSP90, functions as a link between metastatic and drug resistant behavior of cancer. We believe this occurs through HSP90’s relationship in supporting the function of gene products that define the cancer hallmarks and clinical evidence suggesting HSP90 is important in progressing cancer into advanced stages. In the following chapters we discuss HSP90 and its role in orchestrating evolution of metastatic and drug resistant phenotypes. We use the clinically relevant HSP90 inhibitor, AUY922, to explore our assertions in vitro in the context of non-small cell lung cancer (NSCLC), which is prone to evolving metastatic and drug resistant phenotypes. We examine the implications for our findings, future directions, and new possibilities for utilizing HSP90 inhibitors to treat cancer

Oppstalling av laboratoriemus i et naturlig habitat : virkning på immunsystem, tarmmikrobiota og utvikling av kolorektal kreft

The natural habitat for the house mouse is on the ground, typically close to humans and their livestock and hence surrounded by a rich microbial diversity that throughout evolutionary history has driven the adaptation of the house mouse. It is thus paradoxical that almost without exception, experimental disease studies using this mammalian model take place in perfect isolation from the outer microbial world. The end goal for preclinical research, humans, rarely live in microbial isolation, although lifestyles can arguably be said to vary on a scale. To develop a preclinical model that better resemble realistic lifestyles of mammals, we have established a system where laboratory mice are raised under a full set of environmental conditions present in a typical farmyard habitat for the house mouse. We call the process feralization, and the first paper covered by this thesis show the resulting mammal display more functionally mature states of immune cells and a diverse gut microbiota, likely surpassing conventional laboratory mice in resembling responses of free-living mice. Furthermore, we demonstrated the use of this animal modelling approach that recapitulates realistic disease responses in a naturalized mammal. We first established a protocol of the AOM/DSS model for colorectal cancer (CRC) induction in mice using a lower-than-usual dose of DSS that is presented in paper II. In paper III, we employed the AOM/DSS model, as well as a previously established genetic Min/+ model of CRC, in a feralization system. We showed that the mice feralized in a farmyard-type habitat were protected against colorectal carcinogenesis compared to conventionally reared laboratory mice. Moreover, our feralization model allows for full control of the timing of microbial exposure. We took advantage of this by including groups of mice that were either born in the farmyard habitat or introduced to it in later life, demonstrating that neonatal microbial exposure was not essential for the CRC protection. The findings were supported by changes in gut microbiota profiles, as well as immunophenotypes indicative of antigenic experience in the feralized mice. In currently unpublished work, we aimed to narrow in on mechanisms for the protective effects conveyed by feralization in the intestines. Assays investigating mucus layer properties showed no differences following feralization, yet a few genes in the colon mucosa related to barrier function were found to be significantly upregulated between feralized and conventional laboratory mice. Further assessments are needed to elaborate on the mechanisms underlying the beneficial effects of feralization.Det naturlige habitatet for husmus er typisk nær mennesker og deres husdyr, og husmusa er gjennom evolusjon tilpasset et slikt rikt mikrobielt levemiljø. Det er derfor paradoksalt at eksperimentelle studier der mus brukes for å studere sykdomsmekanismer, omtrent uten unntak foregår i perfekt isolasjon fra den ytre, mikrobielle verden. Målet for prekliniske studier ved bruk av forsøksmus er typisk å overføre funnene til relevans for mennesker, men mennesker lever sjeldent i mikrobiell isolasjon, selv om individuelle livsstiler varierer stort. For å utvikle en preklinisk musemodell som mer realistisk representerer naturlige livstiler hos pattedyr, har vi etablert et system der laboratoriemus fostres opp i en mer naturlig situasjon, der habitatet deres er beriket med elementer som er tilstede i et typisk gårdsmiljø. Vi har kalt denne prosessen «feralisering», og den første artikkelen som omfattes av denne avhandlingen demonstrerer at de «feraliserte» musene viser tegn til mer funksjonelt modne immunceller og en rikere tarmmikrobiota sammenliknet med laboratoriemus oppstallet under tradisjonelle, rene forhold. Videre har vi demonstrert bruken av feraliserings-systemet som kan benyttes til å studere realistiske sykdomsresponser i et naturalisert pattedyr. Først etablerte vi en protokoll for kjemisk induksjon av kolorektalkreft hos mus, ved å bruke en lavere-enn-normal dose av DSS i en AOM/DSS modell. Dette arbeidet er presentert i artikkel II. I artikkel III benyttet vi AOM/DSS modellen, så vel som en tidligere etablert genetisk Min/+ musemodell for kolorektalkreft, i feraliseringssystemet. Vi demonstrerte at musene som ble feralisert i et naturalistisk gårdsmiljø var beskyttet mot tykktarmskreft, sammenliknet med laboratoriemus oppstallet under tradisjonelle, rene forhold. Videre tillater feraliserings-systemet full kontroll av timingen for mikrobiell eksponering. Vi utnyttet dette ved å inkludere en gruppe av mus som enten var født i gårdsmiljøet, eller introdusert dit senere i livet. Med dette viste vi at neonatal mikrobiell eksponering ikke var essensiell for beskyttelse mot kolorektal kreft. Funnene ble støttet av endringer i tarmmikrobiotaprofiler, samt immunofenotyper som kan indikere at de har blitt eksponert for antigener. I nåværende upublisert arbeid hadde vi som mål å undersøke mulige mekanismer for hvordan feralisering i et gårdsmiljø kunne gi beskyttelse mot kolorektal kreft. Undersøkelser av slimlaget i tarm viste ingen forskjeller etter feralisering. Likevel var det none få gener i tykktarmsslimhinnen relatert til barrierefunksjon som var oppregulert i feraliserte mus sammenliknet med konvensjonelt oppstallede laboratoriemus. Videre undersøkelser er nødvendig for å kunne utdype mer når det gjelder mekanismene bak feraliserings beskyttende effekt

Immune Activation by Novel Allobaculum Species Reveals Reciprocal Epistasis Among Human Gut Commensals

Gut commensal microbes that elicit human immune responses are noteworthy for their ability to influence both local mucosal inflammation and, more rarely, systemic antibody responses. Here we isolated and characterized novel strains belonging to genus Allobaculum from inflammatory bowel disease (IBD) stool samples. In defined gnotobiotic mouse models we recapitulated the inflammatory effects of Allobaculum sps. and their notable induction of systemic immune responses at baseline. A microbial ecology screen revealed that this taxon is inversely correlated with Akkermansia muciniphila, and co-colonization experiments uncovered microbe-dependent redirection of immune phenotypes, which we term reciprocal epistasis. These immunostimulatory gut commensal strains exemplify the remarkable effects microbial ecology can have upon inflammation and immunity, as well as present a framework for unraveling the complexity of the gut microbiota with more mechanistic insight

A study on variation in immune responses and protective mechanisms to Ostertagia circumcincta and their relationship with parasitological parameters in Scottish blackface sheep

The aim of this study was to examine the variation in immunological control of Ostertagia circumcincta, following natural exposure of sheep by grazing contaminated pasture, and relate this variation to that in parasitological parameters. Selection of sheep by faecal egg count resulted in different results in two consecutive years, in the first year those sheep with higher faecal egg counts had more adult female worms, in the second those sheep with higher faecal egg counts had longer, more fecund worms. These observations are consistent with earlier studies showing that faecal egg count is a function of worm burden and worm fecundity. Immunophenotyping of peripheral blood revealed that lymphocyte subset percentages are repeatable and that lymphocyte numbers were greater in grazing lambs. A negative association between worm burden and B cell percentage representation of peripheral blood lymphocytes was observed, possibly as a result of immune modulation, whilst immunophenotyping of cells from lambs given a deliberate challenge provided further evidence of immune-modulation by O. circumcincta. Following natural infection those sheep with a larger abomasal nodes had shorter worms, suggesting that the magnitude of the immune response was important. Proliferation assays emphasised the importance of the local immune response by showing that responses to antigen were greater in lymphocytes from local tissues. Lymphocyte responses were dependent on previous exposure. Negative associations were observed between worm burden and proliferative responses, and because there was no evidence of active control of worm burden the results also provided further evidence of immune modulation. Enumeration of mast cells and globule leucocytes showed that there was a marked increase in the density of these cells following exposure to O. circumcincta, and although there was no evidence that these cells were involved in the control of worm burden there was a relationship between the globule leucocyte density and worm length that suggest this cell type may have a direct role in restricting or controlling worm length. The observation that lambs were able to mount a vigorous mast cell and globule leucocyte response although there was no evidence that they were able to control worm burden raises the possibility that these cell types may have a direct role in the control of the O. circumcincta burden. Parasite specific IgA responses were investigated and shown to increase with exposure and a positive association was observed between worm burden and local IgA activity following deliberate infection. The study provided further evidence that parasite-specific IgA activity has a role in regulating worm length. In conclusion the work in this thesis demonstrated considerable individual variation in the mechanisms of immunological control and host protection. The results provided further evidence of the role of limiting worm length and fecundity in the genetic control of O. circumcincta. Many of the findings could best be explained by suggesting immune modulation of the host by the parasite. It is likely that further studies on how O. circumcincta attempts to modulate the immune response, and whether or not the host can resist these attempts, will greatly increase our understanding of the host-parasite interface

Learning cell states from high-dimensional single-cell data

Recent developments in single-cell measurement technologies have yielded dramatic increases in throughput (measured cells per experiment) and dimensionality (measured features per cell). In particular, the introduction of mass cytometry has made possible the simultaneous quantification of dozens of protein species in millions of individual cells in a single experiment. The raw data produced by such high-dimensional single-cell measurements provide unprecedented potential to reveal the phenotypic heterogeneity of cellular systems. In order to realize this potential, novel computational techniques are required to extract knowledge from these complex data. Analysis of single-cell data is a new challenge for computational biology, as early development in the field was tailored to technologies that sacrifice single-cell resolution, such as DNA microarrays. The challenges for single-cell data are quite distinct and require multidimensional modeling of complex population structure. Particular challenges include nonlinear relationships between measured features and non-convex subpopulations. This thesis integrates methods from computational geometry and network analysis to develop a framework for identifying the population structure in high-dimensional single-cell data. At the center of this framework is PhenoGraph, and algorithmic approach to defining subpopulations, which when applied to healthy bone marrow data was shown to reconstruct known immune cell types automatically without prior information. PhenoGraph demonstrated superior accuracy, robustness, and efficiency, compared to other methods. The data-driven approach becomes truly powerful when applied to less characterized systems, such as malignancies, in which the tissue diverges from its healthy population composition. Applying PhenoGraph to bone marrow samples from a cohort of acute myeloid leukemia (AML) patients, the thesis presents several insights into the pathophysiology of AML, which were extracted by virtue of the computational isolation of leukemic subpopulations. For example, it is shown that leukemic subpopulations diverge from healthy bone marrow but not without bound: Leukemic cells are apparently free to explore only a restricted phenotypic space that mimics normal myeloid development. Further, the phenotypic composition of a sample is associated with its cytogenetics, demonstrating a genetic influence on the population structure of leukemic bone marrow. The thesis goes on to show that functional heterogeneity of leukemic samples can be computationally inferred from molecular perturbation data. Using a variety of methods that build on PhenoGraph's foundations, the thesis presents a characterization of leukemic subpopulations based on an inferred stem-like signaling pattern. Through this analysis, it is shown that surface phenotypes often fail to reflect the true underlying functional state of the subpopulation, and that this functional stem-like state is in fact a powerful predictor of survival in large, independent cohorts. Altogether, the thesis takes the existence and importance of cellular heterogeneity as its starting point and presents a mathematical framework and computational toolkit for analyzing samples from this perspective. It is shown that phenotypic and functional heterogeneity are robust characteristics of acute myeloid leukemia with clinically significant ramifications

Osteoartriidi patogenees: immunoloogiline kulg sünoviaalmembraan- sünoviaalvedelik teljel

Väitekirja elektrooniline versioon ei sisalda publikatsiooneOsteoartriit (OA) on kõige levinum liigesehaigus, millel ei ole tänaseni patogeneetilist ravi. Tõhusate ravimeetodite arendamise peamised väljakutsed on OA heterogeensus ja ebaselge patoloogia. OA-d, mida tavapäraselt tuntakse kui „vananemishaigust“, peetakse järjest enam krooniliseks põletikuliseks haiguseks. Käesoleva doktoritöö eesmärk oli hinnata sünoviaalvedelikuga seotud põletikulist mehhanismi, mis on OA patoloogias üks põhilisi põletikukohti. Seda silmas pidades piirdub sünoviaalvedeliku analüüs sageli tema molekulaarse koostise uurimisega, kuid funktsionaalse ulatuse hindamiseks on vaja laiemat tausta. Seetõttu püüti molekulaarse signaali dekodeerimisega uurida sünoviaalvedeliku laiemat kaasatust haiguse patogeneesis. Viidi läbi mitmetasandiline analüüs, kasutades OA-st mõjutatud sünoviaalmembraani ja osteofüütide biopsiaid ning sünoviaalvedeliku proove. Lisaks tehti rakupõhiseid analüüse, kasutades inimese monotsüütide rakuliine (THP1 ja U37) ning inimese vereloome tüvirakke. Bioloogiliste testide tulemuste toetamiseks viidi läbi proteoomi analüüs ja sünoviaalvedeliku immuno-fenotüüpimine. Sünoviaalmembraani rakkude geeniekspressiooni uuring näitas püsivat põletikku OA liigestes. Suurem põletik haiguse varases staadiumis annab vältimatu tõuke kõhrkoe kiiremaks lagunemiseks haiguse hilisemates staadiumites. Kõigi OA staadiumide sünoviaalvedeliku proovid, inkubeerides neid THP1 ja U937 rakkudega, suutsid rakkudes esile kutsuda põletikku. Samas katses näidati, et OA sünoviaalvedelik võib indutseerida immuunrakkude eristumist ja toimida nišina, pakkudes olulist mikrokeskkonda, mis toetab immuunrakkude funktsionaalset aktiivsust ning põhjustab põletiku süvenemist ja kestmist. Sünoviaalvedeliku proteoomianalüüs paljastas mitmeid suures koguses esinevaid valke, mis võivad, nagu täheldati, indutseerida immuunrakkude eristumist. Lõpuks viidi läbi osteofüütide RNA-seq analüüs, et uurida selles toimuvaid molekulaarseid ja rakulisi protsesse, eriti OA patoloogia kontekstis. Geeniekspressiooni mustri funktsionaalne analüüs näitas progresseeruvat aktiivset luu ümberkujunemist ja nuumrakkude tihedat seotust selles protsessis. See teadmine on OA patoloogias uus ja peaks olema arvesse võetud sobivate ravimeetodite kavandamisel.Osteoarthritis (OA) is the most common joint disease, which has no cure to date. The major challenges in developing effective therapies are, heterogenous nature and obscure pathology of OA. Conventionally known as ‘aging disease’, OA is increasingly accepted as a complex disease with an implication of chronic-low-grade inflammation. More illumination is needed on the involved inflammatory mechanism. The present thesis was aimed to evaluate inflammatory mechanism revolving around the synovium-synovial fluid (SF) axis, which are the main inflammation sites in OA pathology. In this regard, particularly SF analysis is limited to understand a molecular make-up of the fluid and there is a wider scope to assess its functional involvement. Therefore, the attempt was to investigate a ‘performance-based involvement’ of SF by decoding the molecular signalling. A multilevel analysis was done using OA affected synovium biopsies, SF and osteophyte samples. Also, cell-based assays were performed using human monocyte cell lines (THP1 and U937) and human hematopoietic stem cells. Proteome analysis and SF immunophenotyping was performed to support the outcomes from the biological assays. Synovium gene expression study showed a persistent nature of inflammation in OA joints. Higher inflammation in early stage provides a necessary impetus for accelerated cartilage-loss at later stages. SF samples of all OA stages, when incubated with THP1 and U937 cells, was able to induce inflammation in the cells. In the same experimental design, we showed that OA SF can induce immune cell differentiation and act as a niche by providing an essential microenvironment, which enable immune cells to be functionally active and for contributing to aggravate and maintain inflammation. Proteome analysis of SF revealed many proteins in abundance, which can induce immune cell differentiation as was observed. Finally, RNA-seq analysis of osteophytes was performed to investigate molecular and cellular events in it, especially in the context of OA pathology. Functional analysis of the gene expression patten, revealed an on-going active bone remodelling and close involvement of mast cells in the process, which is a new dimension of OA pathology and should prove worthy for taking into consideration for designing appropriate therapies.https://www.ester.ee/record=b551832

Activation of Fas/FasL pathway and the role of c-FLIP in primary culture of human cholangiocarcinoma cells

Intrahepatic cholangiocarcinoma (iCCA) represents a heterogeneous group of malignancies emerging from the biliary tree, often in the context of chronic bile ducts inflammation. The immunological features of iCCA cells and their capability to control the lymphocytes response have not yet been investigated. The aims of the present study were to evaluate the interaction between iCCA cells and human peripheral blood mononuclear cells (PBMCs) and the role of Fas/FasL in modulating T-cells and NK-cells response after direct co-culture. iCCA cells express high levels of Fas and FasL that increase after co-culture with PBMCs inducing apoptosis in CD4(+), CD8(+) T-cells and in CD56(+) NK-cells. In vitro, c-FLIP is expressed in iCCA cells and the co-culture with PBMCs induces an increase of c-FLIP in both iCCA cells and biliary tree stem cells. This c-FLIP increase does not trigger the caspase cascade, thus hindering apoptotis of iCCA cells which, instead, underwent proliferation. The increased expression of Fas, FasL and c-FLIP is confirmed in situ, in human CCA and in primary sclerosing cholangitis. In conclusion our data indicated that iCCA cells have immune-modulatory properties by which they induce apoptosis of T and NK cells, via Fas/FasL pathway, and escape inflammatory response by up-regulating c-FLIP system