Functional characterization of genetic risk factors in autoimmune Addison’s disease

Autoimmune diseases occur when the immune system attacks and damages the body’s own tissue. Why people develop these diseases, and how the autoimmune reaction develops are unanswered questions. Autoimmune Addison's disease (AAD) is an organ-specific autoimmune disorder characterized by an immunological attack of the adrenal cortex. The complex genetic architecture underlying AAD has not been entirely established, and the overall aim of this project was therefore to identify and functionally characterize genetic risk factors in AAD. We discovered several rare and damaging inborn errors of antiviral immunity in AAD patients. Among them, variants in the gene encoding Toll-like receptor 3 (TLR3), which recognizes double-stranded RNAs (dsRNAs) upon viral infection. Functional characterization of the TLR3 variants revealed a partial loss of function effect on the receptor’s signaling activity, leading to impaired interferon (IFN) responses ex vivo. Next, we identified a homozygous stop-gain variant in the gene encoding 3βhydroxysteroid dehydrogenase type 2 (3βHSD2), causing a rare form of congenital adrenal hyperplasia (CAH). The mutation was carried by an AAD patient with circulating antibodies against the major AAD autoantigen 21-hydroxylase (21OH). To our knowledge, this combination represents a novel disease etiology. Finally, we wanted to identify HLA-specific immunodominant epitopes of 21OH, targeted by autoreactive T cells. We discovered a new immunodominant epitope, ARLELFVVL (21OH434-442), presented by HLA-C*0701. This is the first HLAC*0701 restricted epitope described for a self-antigen in an autoimmune disease. We also confirmed the presence of autoreactive CD8+ T cells responses to the previously proposed epitope LLNATIAEV (21OH342-350), restricted to HLA-A2. Altogether, the work in this doctoral dissertation has provided new insights into why certain individuals might be more genetically susceptible to develop AAD, and partly how the autoimmune reaction progresses

21-Hydroxylase-Specific CD8+ T Cells in Autoimmune Addison’s Disease Are Restricted by HLA-A2 and HLA-C7 Molecules

Objectives: CD8+ T cells targeting 21-hydroxylase (21OH) are presumed to play a central role in the destruction of adrenocortical cells in autoimmune Addison’s disease (AAD). Earlier reports have suggested two immunodominant CD8+ T cell epitopes within 21OH: LLNATIAEV (21OH342-350), restricted by HLA-A2, and EPLARLEL (21OH431-438), restricted by HLA-B8. We aimed to characterize polyclonal CD8+ T cell responses to the proposed epitopes in a larger patient cohort with AAD. Methods: Recombinant fluorescent HLA-peptide multimer reagents were used to quantify antigen-specific CD8+ T cells by flow cytometry. Interferon-gamma (IFNγ) Elispot and biochemical assays were used to functionally investigate the 21OH-specific T cells, and to map the exactly defined epitopes of 21OH. Results: We found a significantly higher frequency of HLA-A2 restricted LLNATIAEV-specific cells in patients with AAD than in controls. These cells could also be expanded in vitro in an antigen specific manner and displayed a robust antigen-specific IFNγ production. In contrast, only negligible frequencies of EPLARLEL-specific T cells were detected in both patients and controls with limited IFNγ response. However, significant IFNγ production was observed in response to a longer peptide encompassing EPLARLEL, 21OH430-447, suggesting alternative dominant epitopes. Accordingly, we discovered that the slightly offset ARLELFVVL (21OH434-442) peptide is a novel dominant epitope restricted by HLA-C7 and not by HLA-B8 as initially postulated. Conclusion: We have identified two dominant 21OH epitopes targeted by CD8+ T cells in AAD, restricted by HLA-A2 and HLA-C7, respectively. To our knowledge, this is the first HLA-C7 restricted epitope described for an autoimmune disease.publishedVersio

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.publishedVersio

Investigation of the WNT and AKT/mTOR signaling pathways in early differentiation in embryonic stem cells

Embryonic stem cells have a huge potential in the field of tissue engineering, biomedical research and regenerative medicine as they possess the capacity to generate every type of cell and tissue in the body. The ability to control and manipulate embryonic stem cells to differentiate into the cell type of interest requires knowledge about the molecular principles governing early differentiation events. Several regulating pathways have been identified to have strong effects on cell fate commitment, such as the pleiotropic WNT signaling pathway and the major anabolic pathway AKT/mTOR. However, more research is needed to fully understand how the molecular mechanisms behind these two pathways and their signaling crosstalk integrate in early differentiation of embryonic stem cells. This study demonstrates that the critical proteins, involved in these two pathways, are regulated biphasically during endodermal differentiation of human embryonic stem cells. Notably, the changes in the activity of AMPK, the major activator of catabolic processes, are clearly inversely related to the changes in the activities of mTOR and AKT throughout the differentiation process. Moreover, AMPK appeared to be highly activated towards the end of the endodermal differentiation process, concomitantly with reduced activity of AKT/mTOR, thus revealing that there is a stimulation of catabolic processes during stem cell fate commitment. Development of endoderm requires high activity of the WNT signaling pathway. Using a pharmacological inhibitor of TNKS1 and 2, this study demonstrates that GSK-3β inhibition-mediated stimulation of WNT signaling and the effects of TNKS1 and 2 on downstream components during endodermal differentiation, can be efficiently reversed. As a result, the endoderm differentiation program becomes blocked, raising the possibility that the activity of TNKS1 and 2 is crucial for a proper endodermal differentiation. Combined, this study has expanded our understanding of the molecular mechanisms underlying early differentiation steps of embryonic stem cells.Embryonale stamceller har vist seg å ha et stort potensial innen biomedisinsk forskning og medisinsk behandling fordi de har evnen til å differensiere til alle celletyper. For å kunnekontrollere og manipulere embryonale stamceller i den tidlige differensieringen, er kunnskap om de molekylære prinsippene som ligger bak de første trinnene viktig. Flere cellesignaliseringsveier har blitt funnet å være viktige for reguleringen av denne differensieringsprosessen, blant annet den pleiotropiske WNT signalveien og den viktige anabolske signalveien AKT/mTOR. Forståelsen av hvordan de molekylære mekanismene bak disse signalveiene er involvert i tidlig differensiering er ufullstendig og mer forskning er påkrevd. Denne studien viser at proteinene, involvert i WNT og AKT/mTOR signalveiene, reguleres i et bifasisk mønster under utviklingen av endoderm generert fra humane embryonale stamceller. I tillegg ble det funnet at det er en motsatt sammenheng mellom aktiveringen av AMPK (viktig aktivator av katabolske prosesser) og aktiveringen av AKT/mTOR signalveien i løpet av differensieringsprosessen. Mot slutten av prosessen var AMPK kraftig aktivert samtidig som aktiviteten til AKT/mTOR signalveien var redusert. Dette tyder på at humane embryonale stamceller får økt aktivering av katabolske prosesser under differensieringsforløpet mot endoderm. Tidligere studier har vist at utviklingen av endoderm er sterkt knyttet til stimulert aktivitet av WNT signalveien. Ved å inhibere aktiviteten til TNKS1 og 2 i de humane embryonale stamcellene, ble det demonstrert at CHIR99021-mediert stimulering av WNT signalisering og effektene av TNKS1 og 2 på deres nedstrømskomponenter, effektivt kan reverseres. Dette førte dermed til en blokkert endodermutvikling, noe som kan tyde på at differensiering mot endoderm er avhengig av aktiviteten til TNKS1 og 2. Samlet sett har denne studien utvidet vår forståelse av de molekylære mekanismene som ligger bak tidlig differensiering av embryonale stamceller.M-BIOTE

Functional characterization of genetic risk factors in autoimmune Addison’s disease

Autoimmune diseases occur when the immune system attacks and damages the body’s own tissue. Why people develop these diseases, and how the autoimmune reaction develops are unanswered questions. Autoimmune Addison's disease (AAD) is an organ-specific autoimmune disorder characterized by an immunological attack of the adrenal cortex. The complex genetic architecture underlying AAD has not been entirely established, and the overall aim of this project was therefore to identify and functionally characterize genetic risk factors in AAD. We discovered several rare and damaging inborn errors of antiviral immunity in AAD patients. Among them, variants in the gene encoding Toll-like receptor 3 (TLR3), which recognizes double-stranded RNAs (dsRNAs) upon viral infection. Functional characterization of the TLR3 variants revealed a partial loss of function effect on the receptor’s signaling activity, leading to impaired interferon (IFN) responses ex vivo. Next, we identified a homozygous stop-gain variant in the gene encoding 3βhydroxysteroid dehydrogenase type 2 (3βHSD2), causing a rare form of congenital adrenal hyperplasia (CAH). The mutation was carried by an AAD patient with circulating antibodies against the major AAD autoantigen 21-hydroxylase (21OH). To our knowledge, this combination represents a novel disease etiology. Finally, we wanted to identify HLA-specific immunodominant epitopes of 21OH, targeted by autoreactive T cells. We discovered a new immunodominant epitope, ARLELFVVL (21OH434-442), presented by HLA-C*0701. This is the first HLAC*0701 restricted epitope described for a self-antigen in an autoimmune disease. We also confirmed the presence of autoreactive CD8+ T cells responses to the previously proposed epitope LLNATIAEV (21OH342-350), restricted to HLA-A2. Altogether, the work in this doctoral dissertation has provided new insights into why certain individuals might be more genetically susceptible to develop AAD, and partly how the autoimmune reaction progresses

Versatile, sensitive liquid chromatography mass spectrometry–Implementation of 10 μm OT columns suitable for small molecules, peptides and proteins

We have designed a versatile and sensitive liquid chromatographic (LC) system, featuring a monolithic trap column and a very narrow (10 μm ID) fused silica open tubular liquid chromatography (OTLC) separation column functionalized with C(18)-groups, for separating a wide range of molecules (from small metabolites to intact proteins). Compared to today’s capillary/nanoLC approaches, our system provides significantly enhanced sensitivity (up to several orders) with matching or improved separation efficiency, and highly repeatable chromatographic performance. The chemical properties of the trap column and the analytical column were fine-tuned to obtain practical sample loading capacities (above 2 μg), an earlier bottleneck of OTLC. Using the OTLC system (combined with Orbitrap mass spectrometry), we could perform targeted metabolomics of sub-μg amounts of exosomes with 25 attogram detection limit of a breast cancer-related hydroxylated cholesterol. With the same set-up, sensitive bottom-up proteomics (targeted and untargeted) was possible, and high-resolving intact protein analysis. In contrast to state-of-the-art packed columns, our platform performs chromatography with very little dilution and is “fit-for-all”, well suited for comprehensive analysis of limited samples, and has potential as a tool for challenges in diagnostics

21-Hydroxylase-Specific CD8+ T Cells in Autoimmune Addison’s Disease Are Restricted by HLA-A2 and HLA-C7 Molecules

Objectives: CD8+ T cells targeting 21-hydroxylase (21OH) are presumed to play a central role in the destruction of adrenocortical cells in autoimmune Addison’s disease (AAD). Earlier reports have suggested two immunodominant CD8+ T cell epitopes within 21OH: LLNATIAEV (21OH342-350), restricted by HLA-A2, and EPLARLEL (21OH431-438), restricted by HLA-B8. We aimed to characterize polyclonal CD8+ T cell responses to the proposed epitopes in a larger patient cohort with AAD. Methods: Recombinant fluorescent HLA-peptide multimer reagents were used to quantify antigen-specific CD8+ T cells by flow cytometry. Interferon-gamma (IFNγ) Elispot and biochemical assays were used to functionally investigate the 21OH-specific T cells, and to map the exactly defined epitopes of 21OH. Results: We found a significantly higher frequency of HLA-A2 restricted LLNATIAEV-specific cells in patients with AAD than in controls. These cells could also be expanded in vitro in an antigen specific manner and displayed a robust antigen-specific IFNγ production. In contrast, only negligible frequencies of EPLARLEL-specific T cells were detected in both patients and controls with limited IFNγ response. However, significant IFNγ production was observed in response to a longer peptide encompassing EPLARLEL, 21OH430-447, suggesting alternative dominant epitopes. Accordingly, we discovered that the slightly offset ARLELFVVL (21OH434-442) peptide is a novel dominant epitope restricted by HLA-C7 and not by HLA-B8 as initially postulated. Conclusion: We have identified two dominant 21OH epitopes targeted by CD8+ T cells in AAD, restricted by HLA-A2 and HLA-C7, respectively. To our knowledge, this is the first HLA-C7 restricted epitope described for an autoimmune disease

Identification and characterization of rare Toll-like receptor 3 variants in patients with autoimmune Addison’s disease

Autoimmune Addison's disease (AAD) is a classic organ-specific autoimmune disease characterized by an immune-mediated attack on the adrenal cortex. As most autoimmune diseases, AAD is believed to be caused by a combination of genetic and environmental factors, and probably interactions between the two. Persistent viral infections have been suggested to play a triggering role, by invoking inflammation and autoimmune destruction. The inability of clearing infections can be due to aberrations in innate immunity, including mutations in genes involved in the recognition of conserved microbial patterns. In a whole exome sequencing study of anonymized AAD patients, we discovered several rare variants predicted to be damaging in the gene encoding Toll-like receptor 3 (TLR3). TLR3 recognizes double stranded RNAs, and is therefore a major factor in antiviral defense. We here report the occurrence and functional characterization of five rare missense variants in TLR3 of patients with AAD. Most of these variants occurred together with a common TLR3 variant that has been associated with a wide range of immunopathologies. The biological implications of these variants on TLR3 function were evaluated in a cell-based assay, revealing a partial loss-of-function effect of three of the rare variants. In addition, rare mutations in other members of the TLR3-interferon (IFN) signaling pathway were detected in the AAD patients. Together, these findings indicate a potential role for TLR3 and downstream signaling proteins in the pathogenesis in a subset of AAD patients

Coexistence of Congenital Adrenal Hyperplasia and Autoimmune Addison's Disease

Background: Underlying causes of adrenal insufficiency include congenital adrenal hyperplasia (CAH) and autoimmune adrenocortical destruction leading to autoimmune Addison's disease (AAD). Here, we report a patient with a homozygous stop-gain mutation in 3β-hydroxysteroid dehydrogenase type 2 (3βHSD2), in addition to impaired steroidogenesis due to AAD. Case Report: Whole exome sequencing revealed an extremely rare homozygous nonsense mutation in exon 2 of the HSD3B2 gene, leading to a premature stop codon (NM_000198.3: c.15C>A, p.Cys5Ter) in a patient with AAD and premature ovarian insufficiency. Scrutiny of old medical records revealed that the patient was initially diagnosed with CAH with hyperandrogenism and severe salt-wasting shortly after birth. However, the current steroid profile show complete adrenal insufficiency including low production of pregnenolone, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S), without signs of overtreatment with steroids. Conclusion: To the best of our knowledge, this is the first description of autoimmune adrenalitis in a patient with 3βHSD2 deficiency and suggests a possible association between AAD and inborn errors of the steroidogenesis

Coexistence of Congenital Adrenal Hyperplasia and Autoimmune Addison's Disease

Background: Underlying causes of adrenal insufficiency include congenital adrenal hyperplasia (CAH) and autoimmune adrenocortical destruction leading to autoimmune Addison's disease (AAD). Here, we report a patient with a homozygous stop-gain mutation in 3β-hydroxysteroid dehydrogenase type 2 (3βHSD2), in addition to impaired steroidogenesis due to AAD. Case Report: Whole exome sequencing revealed an extremely rare homozygous nonsense mutation in exon 2 of the HSD3B2 gene, leading to a premature stop codon (NM_000198.3: c.15C>A, p.Cys5Ter) in a patient with AAD and premature ovarian insufficiency. Scrutiny of old medical records revealed that the patient was initially diagnosed with CAH with hyperandrogenism and severe salt-wasting shortly after birth. However, the current steroid profile show complete adrenal insufficiency including low production of pregnenolone, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S), without signs of overtreatment with steroids. Conclusion: To the best of our knowledge, this is the first description of autoimmune adrenalitis in a patient with 3βHSD2 deficiency and suggests a possible association between AAD and inborn errors of the steroidogenesis