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Dissecting the regulation of cell fate decisions in humoral immune responses
No full textCell fate decisions made by B cells upon activation determine the outcome of responses to pathogens and vaccines. In this thesis, we first examined the cell fate decisions that take place early upon B cell activation and identified the transcription factor Bhlhe40 as a crucial cell-intrinsic negative regulator affecting both the B and T cell sides of the GC reaction by limiting TFH cell proliferation and restricting early GCBC differentiation while not interfering with EMBC and EPC differentiation. Bhlhe40-deficient mice with age developed B cell lymphoma, highlighting the role of this transcription factor as a negative regulator of the GC reaction.Subsequently, using single-cell RNA sequencing and fate-mapping approaches, we revealed that upon activation, a surprisingly large fraction of B cells rapidly exits the cell cycle, giving rise to non-GC-derived EMBCs that make a prominent contribution to the overall MBC pool, a process that was evolutionary conserved between rodents and primates. This phenomenon was controlled by the rapid decline of antigen availability, and the provision of antigen excess precluded the cell cycle exit and induced a new wave of EPCs, indicating that a reservoir of EMBCs may enable the rapid readjustment of the immune response when failure to contain a threat is manifested by increased antigen availability.Next, focusing on MBC differentiation, we investigated the origin and function of an MBC population that resembles phenotypically MZB cells. We revealed that antigen-specific MZ-memory B cells (MZ-MBCs) generated upon immunization can be longer-lived relative to follicular memory B cells (FO-MBCs) residing in the spleen or lymph nodes. These MBCs, due to their strategic positioning in the MZ of the spleen, could provide the first line of defense in secondary immune responses to bloodborne challenges.Overall, the data presented in this thesis reveal complex dynamics regarding early B cell activation and memory formation, with findings that can be relevant for future vaccine design.List of scientific papersI. René Rauschmeier*, Annika Reinhardt*, Charlotte Gustafsson, Vassilis Glaros, Artem V. Artemov, Josefine Dunst, Reshma Taneja, Igor Adameyko, Robert Månsson, Meinrad Busslinger, and Taras Kreslavsky.Bhlhe40 function in activated B and TFH cells restrains the GC reaction and prevents lymphomagenesis.Journal of Experimental Medicine. 2022 Feb 7;219(2):e20211406. *Equal contribution. https://doi.org/10.1084/jem.20211406II. Vassilis Glaros*, René Rauschmeier*, Artem V. Artemov*, Annika Reinhardt*, Sebastian Ols*, Aikaterini Emmanouilidi, Charlotte Gustafsson, Yuanyuan You, Claudio Mirabello, Asa K. Björklund, Laurent Perez, Neil P. King, Robert Månsson, Davide Angeletti, Karin Loré, Igor Adameyko, Meinrad Busslinger, and Taras Kreslavsky.Limited access to antigen drives generation of early B cell memory while restraining the plasmablast response.Immunity. 2021 Sep 14;54(9):2005-2023.e10. * Equal contribution. https://doi.org/10.1016/j.immuni.2021.08.017III. Vassilis Glaros, Nimmy Francis, Gretchen Harms Pritchard, Julia Hauenstein, Kewei Ye, Gustavo Monasterio, William R. Schief, Eduardo J. Villablanca, Stephan P. Rosshart, Jonathan M. Coquet, Robert Månsson, Marion Pepper, E. Ashley Moseman and Taras Kreslavsky.The marginal zone B cell molecular program is a niche-induced state associated with long-term persistence of B cell memory.[Manuscript].</p
Genetic and epigenetic diagnostics of viral susceptibility and inborn errors of immunity
No full textIn inborn errors of immunity (IEI), as in other monogenic rare diseases, rate of diagnosis has failed to improve beyond 35% despite accessibility of whole genome sequencing for many patients. Here, efforts to improve diagnostics of patients with severe inflammatory diseases in different contexts are presented.We performed three studies on groups of patients who suffered critical COVID- 19 between 2019-2022. In Paper I, we described several patients with an IEI of type I interferon (IFN) which presented unexpectedly late in life after infection with SARS-CoV-2. We diagnosed two patients with biallelic deficiency of IRF7, and characterized the clinical and immunologic phenotypes of seven patients in total. This marks the first time this has been possible in more than an orphan case for this IEI. We further suggested that, based on investigations in two patients, enhanced T cell responses may offer some compensatory immunity for the reduction of type I interferon response. In Paper II, we evaluated a cohort from a single intensive care unit who suffered from critical COVID-19 despite relative health and youth prior to infection. We identified two cases caused by presence of autoantibodies to type I IFN, preventing IFN signalling. We investigated incidence of rare variants in the type I IFN production and signalling pathways compared to controls, as well as screening for rare variants in other IEI genes. We followed up with immunological assays to functionally validate identified variants, and noting a trend towards lower responses in type I IFN signalling in three of six individuals carrying heterozygous very rare, or homozygous rare variants. Paper III investigated genetics of hyperinflammation in COVID-19 patients. Haemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome with some clinical features similar to the cytokine storm experienced by many critical COVID-19 patients, suggesting that variation in cytotoxicity genes associated with HLH may predispose to cytokine storm if triggered by viral infection. Here, we took advantage of large datasets of critical patients and asymptomatic infected provided by the COVID Human Genetic Effort. With thousands of genomes available, we established that variants in genes associated with HLH were not more frequent in critical disease, arguing against a strong pathogenetic overlap.Paper IV described the increased efficacy of a set of diagnostic flow cytometry assays piloted by our lab and reported previously. By evaluating our improved assays in almost 500 donors, including 92 with genetically diagnosed primary HLH, we confirmed that our assays reliably separate data generated from patient samples from control healthy donors or patients with other IEIs not affecting lymphocyte cytotoxicity. Our assays also showed improvement in separating degranulation defects from controls compared to the assays which are the current standard in most laboratories.Finally, Paper V details MAGNET, a novel algorithm for approaching patients who have a predicted rare disease, but for whom whole genome sequencing in a clinical context has been unsuccessful. Using integrated ATAC-seq with patient and parental genomes, and supported by RNA-seq, we analysed two proof-of- concept patients previously diagnosed with HLH and were able to prioritise the variants outside of protein coding regions which cause their disease from genome-wide screening. We optimised this workflow with studies of allelic chromatin accessibility in patients and controls, and identified a strong candidate variant in an IEI patient still unexplained in the clinic.Taken together, these studies have increased our understanding of the genetic aetiology of IEIs, and the speed and rate with which we can diagnose patients suffering these conditions.List of scientific papersI. Respiratory viral infections in otherwise healthy humans with inherited IRF7 deficiency. Campbell TM, Liu Z, Zhang Q, Moncada-Velez M, Covill LE, Zhang P, Darazam IA, Bastard P, Bizien L, Bucciol G, Lind Enoksson S, Jouanguy E, Karabela SN, Khan T, Kendir-Demirkol Y, Arias AA, Mansouri D, Marits P, Marr N, Migeotte I, Moens L, Ozcelik T, Pellier I, Sendel A, Şenoğlu S, Shahrooei M, Smith CIE, Vandernoot I, Willekens K, Yaşar KK, Bergman P, Abel L, Cobat A, Casanova JL, Meyts I, Bryceson YT J Exp Med., 219(7):e20220202 (2022). https://doi.org/10.1084/jem.20220202II. Evaluation of genetic or cellular impairments in Type I IFN immunity in a cohort of young adults with critical COVID-19. Covill LE*, Sendel A*, Campbell TM*, Piiroinen I, Lind Enoksson S, Wahren Borgström E, Hansen S, Ma K, Marits P, Norlin AC, Smith CIE, Kåhlin J, Eriksson LI, Bergman PS, Bryceson YTS J Clin Immunol. 44(2):50 (2024). https://doi.org/10.1007/s10875-023-01641-1III. No association between HLH-associated gene variants and life- threatening COVID-19. Covill LE, Cobat A, COVID Human Genetic Effort, Zhang Q, Bryceson YT. [Manuscript]IV. Efficacy of T cell exocytosis assays for the diagnosis of primary defects in cytotoxic lymphocyte exocytosis. Chiang SCC, Covill LE*, Tesi B*, Campbell TM, Schlums H, Nejati- Zendegani J, Mördrup K, Wood S, Theorell J, Sekine T, Al-Herz W, Akar HH, Belen FB, Chan MY, Devecioglu O, Aksu T, Ifversen M, Malinowska I, Sabel M, Unal E, Unal S, Introne W, Krzewski K, Gilmour KC, Ehl S, Ljunggren HG, Nordenskjold M, Horne AC, Henter JI, Meeths M, Bryceson YT Blood. 144(8):873-887 (2024). https://doi.org/10.1182/blood.2024024499V. ATAC-seq from affected cell types robustly prioritizes pathogenic non-coding variants in patients with monogenic disease. Covill LE, Campbell TM, Holmes TD, Frengen NS, Gustafsson C, Winroth A, Hauenstein J, Vinay Pandey R, Erichsen HC, Redzic D, Yoke CM, Vonlanthen S, Månsson R, Wedell A, Meeths M, Sundin M, Bryceson YT. [Manuscript]</p
Cardiovascular comorbidity in rheumatoid arthritis : studies on venous thromboembolism
No full textIndividuals with rheumatoid arthritis (RA) are at around 50-100% increased risk of venous thromboembolism (VTE). In 2019, signals emerged suggesting an increased risk of VTE in RA patients treated with JAK inhibitors (JAKi), a new and highly efficacious class of disease- modifying antirheumatic drug (DMARD). In this thesis, we aimed to further disentangle the associations between RA, its treatment, and VTE risk in a series of observational studies. We used data from the Swedish rheumatology quality register and other national registers, thereby including a majority of all Swedish patients with RA, as well as a general population comparator cohort.Study I was a study validating International classification of diseases (ICD) 10 codes for VTE registered in the National patient register specifically in patients with RA, through manual review of medical records. Based on review of 269 registered VTE events at hospitals in Region Stockholm from 2009 through 2018, 235 were confirmed as incident VTE. This resulted in a positive predictive value (PPV) of 87% for incident VTE, which ranged from 80% to 98% when we applied various restrictions to our definition of VTE. The majority of the confirmed events were diagnosed independently of RA (only 17 cases involved initial work- up by a rheumatologist, and in 3 cases there was an initial suspicion af an RA-related symptom). This study demonstrates a high validity for registered ICD-10 codes for VTE specifically in patients with RA, and suggests a low degree of surveillance or diagnostic bias.Study II was a cohort study on the association between RA disease activity, measured by the Disease activity score 28 (DAS28), and the risk of VTE. Based on 322,601 rheumatologist visits in 46,316 patients with RA from 2006 through 2018, we identified 2,241 incident VTE events during the year following the rheumatologist visit. This resulted in a cumulative 1- year incidence of 0.52% for DAS28 remission, and 1.08% for high disease activity, with a corresponding adjusted risk ratio of 2.03 (95% CI 1.73-2.38) for high DAS28 vs. remission. Compared to the general population, the risk ratio of VTE for the overall RA population was 1.88 (95% CI 1.65-2.05). The study demonstrates a significant association between RA disease activity and VTE risk, thereby underscoring the importance of VTE risk stratification in patients with RA, and that treating RA to remission is important to minimize VTE risk.Study III was an active comparator, new user design cohort study on the risk of VTE in patients with RA treated with JAKi, TNFi and other biologic DMARDs (bDMARDs) from 2010 through 2021. Based on 32,737 treatment episodes and 559 incident VTE events, the adjusted hazard ratio (HR) for the risk of VTE for JAKi vs. TNFi was 1.73 (95% CI 1.24-2.42). Regarding VTE subtypes, the corresponding HR was 3.21 (95% CI 2.11-4.88) for pulmonary embolism and 0.83 (95% CI 0.47-1.45) for deep vein thrombosis. The incidence of VTE was doubled with use of JAKi compared to the entire RA population, and tripled compared to the general population. The study demonstrates an increased risk of VTE for patients with RA treated with JAKi in clinical practice, an increase confined to pulmonary embolism rather than deep vein thrombosis.Study IV was a cohort study on the potential impact of channeling bias for biologic and targeted synthetic DMARD (b/tsDMARD) drug classes when new on the market, compared to when established, in patients with RA from 2006 through 2022. We identified 33,550 b/tsDMARD initiations and 5,862 composite safety events (first of: major adverse cardiovascular event, VTE, cancer or serious infection). b/tsDMARD treatments initiated >5 years since market entry of the drug class in question (vs. List of scientific papersI. Validation and characterization of venous thromboembolism diagnoses in the Swedish National Patient Register among patients with rheumatoid arthritis. Viktor Molander, Hannah Bower, Johan Askling. Scand J Rheumatol 2023;52:111-117. https://doi.org/10.1080/03009742.2021.2001907II. Risk of venous thromboembolism in rheumatoid arthritis, and its association with disease activity: a nationwide cohort study from Sweden. Viktor Molander, Hannah Bower, Thomas Frisell, Johan Askling. Annals of the Rheumatic Diseases 2021;80:169-175. https://doi.org/10.1136/annrheumdis-2020-218419III. Risk of venous thromboembolism with JAK inhibitors and other immune- modulatory drugs: a Swedish comparative safety study among patients with rheumatoid arthritis. Viktor Molander, Hannah Bower, Thomas Frisell, Bénédicte Delcoigne, Daniela Di Giuseppe, Johan Askling. Annals of the Rheumatic Diseases 2023;82:189-197. https://doi.org/10.1136/ard-2022-223050IV. Do newly approved drugs have a worse observed safety profile than once established? A study on time-trends in observed risks of key safety outcomes with immune-modulatory drugs against rheumatoid arthritis. Viktor Molander, Hannah Bower, Thomas Frisell, Johan Askling. [Manuscript]</p
New insights in sepsis epidemiology and long-term consequences of critical illness
No full textSepsis and critical illness are global health challenges and impact both short- and long-term outcomes for patients. This thesis investigates different aspects of sepsis and critical illness, in a short- and long-term perspective.Study I analyzes in a retrospective single-center observational setting, the risk of developing post-injury sepsis in intensive care admitted trauma patients. The incidence of sepsis after trauma was high and associated with 30-day mortality. Several risk factors were independently associated with post-injury sepsis. Post- injury sepsis patients had also a more complicated clinical course. The study emphasizes the need for early awareness and personalized care for trauma patients who developed post-injury sepsis.Study II analyzes in a nationwide case-control study outcomes and risk factors for intensive care patients with community-acquired sepsis. Sepsis survivors had substantial excess mortality compared to controls. Elevated mortality was also notable in the subgroup analysis of cases without somatic comorbidity at admission. Risk factors for developing sepsis were low socioeconomic status, psychiatric illness, substance abuse, metastatic malignancy, renal disease, liver disease, and congestive heart failure. The findings underscore the impact of sepsis on short-term outcomes and highlight who is at risk.Study III was a nationwide cohort design investigating long-term outcomes after community-acquired sepsis. Exploring sepsis patients, we found they had a high morbidity burden before admission, followed by a distinct rise in the post- discharge phase. Patients with sepsis had a sustained excess mortality up until three years. Furthermore, for one-year survivors, sepsis remained associated with increased mortality during the period one to three years after admission. These findings address the multifaceted health challenges of sepsis survivors beyond their initial acute illness.Study IV examined prolonged high-potency benzodiazepine use after intensive care. We identified that 4% of benzodiazepine-naïve patients continued long- term use post-discharge. Key risk factors included older age, female sex, and pre-existing psychiatric and somatic conditions. Prolonged benzodiazepine use was linked to increased mortality. The findings call for careful monitoring of medication use in intensive care survivors to prevent dependency and adverse long-term outcomes.List of scientific papersThis thesis is based on the following papers, which will be referred to by Roman numerals as indicated below:I. Post-injury Sepsis-Associations with Risk Factors, Impact on Clinical Course, and Mortality: A Retrospective Observational StudyJesper Eriksson*, Ann-Charlotte Lindström*, Elisabeth Hellgren, Ola Friman, Emma Larsson, Mikael Eriksson, Anders Oldner *Shared first authorshipCritical Care Explorations, 2021 Aug 2;3(8):e0495. https://doi.org/10.1097/cce.0000000000000495Il. Nationwide case-control study of risk factors and outcomes for community-acquired sepsisAnn-Charlotte Lindström, Mikael Eriksson, Johan Mårtensson, Anders Oldner, Emma LarssonScientific Reports, 2021 Jul 23;11(1):15118. https://doi.org/10.1038/s41598-021-94558-xIII. The impact of critical community-acquired sepsis on long-term mortality and morbidity-a nationwide cohort studyAnn-Charlotte Lindström*, Jesper Eriksson*, Mikael Eriksson, Johan Mårtensson, Emma Larsson ** , Anders Oldner ** *Equal author contribution, ** Equal author contribution[Submitted]IV. Onset of prolonged high-potency benzodiazepine use among ICU survivors: a nationwide cohort studyAnn-Charlotte Lindström, Erik von Oelreich, Jesper Eriksson, Mikael Eriksson, Johan Mårtensson, Emma Larsson, Anders OldnerCritical Care Explorations, 2024 Jul 8;6(7):e1124. https://doi.org/10.1097/cce.0000000000001124</p
Surgical management of gallstone disease and complications from gallstone surgery
No full textIntroductionSurgery for gallstone disease is one of the most frequently performed surgical procedures in the world. Complications occur in 6-11% of these cases, amounting to much suffering and large expenses for healthcare systems worldwide. The aim of this thesis was to find strategies to optimize the surgical management of gallstone disease, focusing mostly on ways to avoid complications.MethodsPAPER I and II are retrospective register-based cohort studies, using data from the Swedish register for Gallstone Surgery and ERCP, GallRiks. In PAPER I, data about all cholecystectomies performed between 2008 and 2019 were used. Multivariable logistic regression analyses were performed, with post operative abscess as outcome and intraoperative gallbladder perforation as predictor, adjusting for ASA-classification, age, sex, surgical approach, and presence of acute cholecystitis. In PAPER II, all cholecystectomies performed 2006-2020 were identified and stratified in three groups: surgeries carried out by surgeons that use FFLC in less than 20% of the cases, in 20-79% of the cases and in 80% or more of the cases. The groups were compared with logistic regression, adjusting for sex, age, surgical experience, year of surgery and history of acute cholecystitis. All surgical complications were included as outcome. A separate analysis was done with regards to operation time.PAPER III is a questionnaire-based study, using data from GallRiks to select a case group (all patients in GallRiks 2007-2018 with registered suspicion of retained gallstones) and a control group without intraoperative gallbladder perforation matched 1:2. Cases and controls were sent a validated questionnaire including twenty-one questions about persisting abdominal pain and inflammatory symptoms. The questions were divided into four groups: abdominal pain, consequences of pain e.g., fatigue and loss of sleep, gastrointestinal symptoms e.g., bloating and nausea, and repeated operation. Simple weighted linear regression was performed on the sum of all answers within the group and mean numerical values between cases and controls were compared.PAPER IV is a matched cohort study, using data from GallRiks to identify patients who had undergone a cholecystectomy at Södersjukhuset 2008-2022 and had registered retained gallstones in the abdominal cavity. These patients were matched 1:2 with a control group without an intraoperatively perforated gallbladder. Preoperative, intraoperative, and postoperative variables were collected. The cases and controls were compared with chi-square test and data was analyzed using Cox regression with stone spillage as exposure, adjusted for ASA-classification and antibiotic treatment.ResultsPAPER I: Data from 108 714 cholecystectomies was analyzed. Intraoperative perforation was recorded in 33.8% of the cases, and in 2.4% of procedures there was a record of retained gallstones. There was a significantly increased risk for postoperative abscess in patients with gallbladder perforation (OR 1.53 [1.36- 1.71]) and stone spillage (OR 1.75 [1.35.2.27]) when adjusting for age, sex, antibiotic prophylaxis, presence of acute cholecystitis and surgical approach. Antibiotic prophylaxis yielded an OR of 0.99 [0.86-1.16] in case of gallbladder perforation and 1.07 [CI 0.92-1.24] in the case of retained stones.PAPER II: No difference in incidence of all surgical complications were seen between groups. Operation time was shorter (OR 0.76 [0.69 - 0.83]), and gallbladder perforation (OR 0.61 [0.45 - 0.82]) and bleeding complications (OR 0.34 [0.14 - 0.86]) were significantly fewer in patients operated by the "Fundus first > 80%" group.PAPER III: No significant differences between cases and controls were found in any of the groups of questions. We saw a tendency towards an increased re- operation rate among the patients with retained gallstones, 7.1% in the study group and 5.3 % in the control group underwent a repeated operation, but this difference was not significant (p= 0.057).PAPER IV: No significant associations between retained gallstones and abdominal pain, nor abscess development, were found. There was however a significant association between retained gallstones and SSI with a hazard ratio of 6.18 [1.63 - 23.4]. There was no significant effect of antibiotics on the hazard ratio for any of the outcomes.ConclusionsIntraoperative gallbladder perforation increases the risk for abscess and should ideally be avoided. However, if perforation does occur, antibiotic prophylaxis or treatment do not decrease the risk for abscess and should only be used in patients that have an infection. If the gallbladder perforation leads to spilled gallstones, these should be retrieved, if possible. However, since so few patients develop complications from retained stones, the surgery should not be extended for too long, and not converted to open surgery to retrieve the stones. The standard method of dissection as well as fundus first dissection are safe methods, none resulting in more post operative complications than the other. Surgeons should try to learn both methods in a controlled way, so they are able to choose the most appropriate method for each individual case.List of scientific papersThe thesis is based on the following papers, which will be referred to in the text by their Roman numerals.I. Intraoperative gallbladder perforation and risk of postoperative abscess with or without antibiotics: a national cohort study of more than 108 000 cholecystectomies. Åsa Edergren, Gabriel Sandblom, Thorhallur Agustsson and Gona Jaafar. British Journal of Surgery. 2023 Jul 17;110(8):896-900. https://doi.org/10.1093/bjs/znac351II. Safety of cholecystectomy performed by surgeons who prefer fundus first versus surgeons who prefer a standard laparoscopic approach. Åsa Edergren, Gabriel Sandblom, Mikael Franko, Thorhallur Agustsson, Yucel Cengiz and Gona Jaafar. Surgery Open Science. 2024 Jun; 19: 141-145. https://doi.org/10.1016/j.sopen.2024.04.004III. No significant persistent symptoms from gallstones left in the abdomen after cholecystectomy. Åsa Edergren, Gabriel Sandblom, Henrik Renlund, Thorhallur Agustsson and Gona Jaafar. The surgeon. 2024 Aug 14; online ahead of print. https://doi.org/10.1016/j.surge.2024.08.002IV. Don't cry over spilled gallstones. Sequalae from gallstones left in the abdominal cavity, a matched cohort study. Åsa Edergren, Gabriel Sandblom, Omar Ali, Alva von Gerber, Thorhallur Agustsson and Gona Jaafar. [Manuscript]</p
Hematoma expansion and hemostasis in traumatic brain injury
No full textWhen a traumatic brain injury (TBI) causes an intracranial hematoma, follow-up scans often reveal hematoma expansion - a common secondary injury believed to worsen outcomes. This thesis explores the measurement, impact, and underlying mechanisms of hematoma expansion in TBI.In Study I, we compared the ABC/2 method, which estimates hematoma volume based on ellipsoid geometry, with computer-assisted volumetric analysis. We found that ABC/2 consistently overestimated hematoma volume, particularly for irregularly shaped lesions. Volumetric analysis offered more precise measurements and is recommended for studies where volume accuracy is critical.Study II focused on identifying the best method for measuring hematoma expansion. We found that absolute expansion (measured in milliliters) was a better predictor of outcomes than relative expansion (measured as a percentage), suggesting that absolute expansion should be the standard in future studies.In Study III, we mapped the time course of hematoma expansion after injury, finding that the majority of expansion occurs within the first six hours, underscoring this as a critical window for intervention. Contusions also expanded more and over a longer period of time compared to extra-axial hematomas. Additionally, hematoma expansion was identified as an independent predictor of outcomes, supporting its potential as a therapeutic target.Finally, Study IV examined how post-admission disruptions in coagulation and fibrinolysis contribute to contusion expansion. We observed early changes in fibrinogen levels, activated partial thromboplastin time, prothrombin time, and platelet count. However, these alterations were relatively modest and did not correlate with contusion volume, raising questions about the necessity of targeting these hemostatic changes, and that alternative markers may be needed to measure the body's hemostatic response to TBI.Together, these studies shed light on the clinical significance of hematoma expansion in TBI and provide guidance for its measurement and potential treatment.List of scientific papersI. Fletcher-Sandersjöö A, Lewén A, Hånell A, Nelson DW, Maegele M, Svensson M, Bellander BM, Enblad P, Thelin EP, Svedung Wettervik T. Volumetric assessment of traumatic intracranial hematomas: is ABC/2 reliable? Journal of Neurotrauma. 2024 online ahead of print. https://doi.org/10.1089/neu.2024.0248II. Fletcher-Sandersjöö A*, Svedung Wettervik T*, Tatter C, Tjerkaski J, Nelson DW, Maegele M, Svensson M, Lewén A, Enblad P, Bellander BM, Thelin EP. Absolute Contusion Expansion Is Superior to Relative Expansion in Predicting Traumatic Brain Injury Outcomes: A Multi- Center Observational Cohort Study. Journal of Neurotrauma. 2024 Mar;41(5-6):705-713. https://doi.org/10.1089/neu.2023.0274III. Fletcher-Sandersjöö A, Tatter C, Tjerkaski J, Bartek J Jr, Maegele M, Nelson DW, Svensson M, Thelin EP, Bellander BM. Time Course and Clinical Significance of Hematoma Expansion in Moderate-to- Severe Traumatic Brain Injury: An Observational Cohort Study. Neurocritical Care. 2023 Feb;38(1):60-70. https://doi.org/10.1007/s12028-022-01609-wIV. Fletcher-Sandersjoo A*, Hammarlund E*, Lindblad C, Froese L, Maegele M, Svensson M, Bellander BM, Nelson DW, Thelin EP. Relationship between post-traumatic hemostatic alterations and contusion volume in isolated traumatic brain injury: an observational cohort study. [Manuscript]* shared first authorship</p
Detection, visualization and quantification of protein complexes in human Alzheimer's disease brains using proximity ligation assay
No full textExamination of healthy and diseased human brain is essential to translational neuroscience. Protein-protein interactions play a pivotal role in physiological and pathological processes, but their detection is difficult, especially in aged and fixed human brain tissue. We used the in-situ proximity ligation assay (PLA) to broaden the range of molecular interactions assessable in-situ in the human neuropathology. We adapted fluorescent in-situ PLA to detect ubiquitin-modified proteins in human brains with Alzheimer's disease (AD), including approaches for the management of autofluorescence and quantification using a high-content image analysis system. We confirmed that phosphorylated microtubule-associated protein tau (Serine202, Threonine205) aggregates were modified by ubiquitin and that phospho-tau-ubiquitin complexes were increased in hippocampal and frontal cortex regions in AD compared to non-AD brains. Overall, we refined PLA for use in human neuropathology, which has revealed a profound change in the distribution of ubiquitin in AD brain and its association with characteristic tau pathologies.</p
Pathogenic mitochondrial DNA point mutations in the immune system : ‘A tale of two cities’
No full textStudy I: The Effects of High Heteroplasmic Pathogenic mt-tRNA Mutations on Immunity. High levels of heteroplasmic pathogenic mtDNA mutations in mitochondrial tRNA (mt-tRNA) genes can severely disrupt mitochondrial translation and oxidative phosphorylation, leading to multisystemic disorders. However, the detailed impact of these mutations on the immune system at a molecular level is not fully understood.In this study, we investigated the immune systems of mice with the m.5024 C>T mutation in mt-tRNA^Ala and human patients with MELAS syndrome, or carriers of the m.3243 A>G mutation in mt-tRNA^Leu. Our findings reveal that myeloid cells tend to maintain a higher mutation burden compared to lymphoid cells, despite the impairment of macrophage mitochondrial translation and oxidative phosphorylation by the m.5024 C>T mutation. Additionally, we observed that naturally accumulated memory T and B cells carry lower burdens of pathogenic mttRNA mutations compared to their naive counterparts. This difference was further highlighted during well-controlled immune challenges with vaccines, which confirmed the disparity in an antigen-specific manner.Mechanistically, our study uncovered that antigen receptor activation induces proliferation in T and B cells, which rapidly dilutes the burden of the m.5024 C>T mutation. This selection process is further accelerated under conditions of metabolic stress and most likely executed at the cell population level. Moreover, post-activation, the high burden m.5024 C>T mutation disrupts CD8+ T cell metabolic remodelling and interferon-gamma (IFN-_) production. These findings suggest that the generation of memory lymphocytes modifies the mtDNA mutation landscape, impacting the regulation of immune responses by pathogenic mt-tRNA variants.Study II: Investigating the Immunological Impact of the Homoplasmic m.11778 G>A Mutation in mt-ND4 of Complex I. The m.11778 G>A mutation in MT-ND4 leads to a substitution of arginine with histidine at position 340, mildly impairing Complex I activity and altering reactive oxygen species (ROS) production. This mutation is the primary cause of Leber's Hereditary Optic Neuropathy (LHON), a condition characterized by degeneration of retinal ganglion cells, mainly impacting the vision of young adult males.Building on insights from Study I, which highlighted how high levels of mt-tRNA mutations can impair mitochondrial protein synthesisŃgiven that mtDNA encodes seven critical subunits of Complex IŃthis study further investigates the impact of such mutations, specifically in LHON. The homoplasmic nature of the m.11778 G>A mutation, found in about 85% of LHON patients, provides an ideal cohort disease to study the influence of mtDNA mutations in complex I on systemic immunity.In Study II, we examined the immune profiles of 45 LHON patients or carriers, comparing them to 45 healthy individuals from the Stockholm region. We observed a tendency for LHON patients or carriers to accumulate more effector/memory cells, suggesting a shift towards an immunosenescence profile in the overall immune cell composition. Additionally, the homoplasmic m.11778 G>A mutation was found to disrupt T cell activation at the transcriptome level during short-term stimulation.Mechanistically, this study delves into the enigmatic hyperactivation and 'memory' phenotype tendency of LHON T cells by exploring: 1. The intricate patterns of calcium influx triggered by antigen receptor-mediated T cell activation; 2. The dynamic phosphorylation signaling cascades, both proximal and distal, initiated by T cell receptor activation; and 3. The short and long-term proliferation of T cells and the potential metabolic rewiring that accompanies this process.Study III: Role of I_BNS and Oxidative Phosphorylation in B Cell Activation and Plasma Cell Differentiation. In Study III, we investigate the role of I_BNS and oxidative phosphorylation in B cell activation and plasma cell differentiation using an I_BNS-deficient mouse model. I_BNS is a nuclear immune related gene, its deficiency offers valuable insights into metabolic processes influencing immune cell function. LPS-stimulated I_BNSdeficient B cells exhibited increased mTOR activation, higher OXPHOS activity, greater mitochondrial biogenesis or accumulation, and elevated mitochondrial reactive oxygen species production compared to wildtype B cells, along with reduced autophagic capacity. Accelerated Blimp-1 upregulation in these cells correlated with more pre-plasmablasts and plasmablasts but lower CD138 levels and impaired PC differentiation. These findings highlight I_BNS's crucial role in B cell metabolism and differentiation, emphasizing the impact of metabolic dysregulation on the immune system.List of scientific papersI. Zhang J, Koolmeister C, Han J, Filograna R, Hanke L, Ëdori M, Sheward DJ, Teifel S, Gopalakrishna S, Shao Q, Liu Y, Zhu K, Harris RA, McInerney G, Murrell B, Aoun M, BŠckdahl L, Holmdahl R, Pekalski M, Wedell A, Engvall M, Wredenberg A, Karlsson Hedestam GB, Castro Dopico X*, Rorbach J*. Antigen receptor stimulation induces purifying selection against pathogenic mitochondrial tRNA mutations. JCI Insight. 2023 Sep 8;8(17): e167656. *Co-senior authors. https://doi.org/10.1172/jci.insight.167656 II. Jingdian Zhang, Qi Chen, Jinming Han, Qiuya Shao, Lakshmikanth Tadepally, Constantin Habimana Mugabo, Hugo Barcenilla, Marcin Pekalski, Anna Wredenberg, Martin Engvall, Petter Brodin , Xaquin Castro Dopico*, Joanna Rorbach*. Ancestral mitochondrial complex I mutations drive T cell exhaustion. *Co-senior authors. [Manuscript]III. Erikson E, çdori M, Khoenkhoen S, Zhang J, Rorbach J, Castro Dopico X, Karlsson Hedestam GB. Impaired plasma cell differentiation associates with increased oxidative metabolism in I_BNS-deficient B cells. Cell Immunol. 2022 May;375:104516. (In this study, I contributed to the molecular metabolic characterization of I_BNS-deficient B cells following activation and proliferation). https://doi.org/10.1016/j.cellimm.2022.104516 </p
A clear picture of the perivascular cell landscape and its implication in tissue functions
No full textThe vasculature of every organ is adapted to the specific tissue functions with activity-dependent blood flow control. While the lumen of blood vessels is formed by endothelial cells (ECs), their abluminal surface is covered by morphologically and functionally distinct perivascular cells (PVCs). Currently known PVCs encompass vascular smooth muscle cells (vSMCs), pericytes, perivascular fibroblasts (PVFs) and perivascular macrophages (PVMs). However, their distribution in relation to each other along the arteriovenous (AV) axis remains poorly characterized. Determining the distribution of PVCs along the AV axis would help to better understand the vascular architecture to infer their functions.During my doctoral studies, I developed a tissue-clearing based pipeline enabling multi-scale 3D imaging of diverse PVCs to probe their organization along the AV axis. Moreover, I designed an open-source volumetric image handling framework to systematically store and analyze the data. Using this workflow, I characterized the distribution and morphology of vMSCs, pericytes, PVFs and PMVs throughout the central nervous system (CNS). Probing the distributional difference of PVFs and pericytes along the AV axis in the spinal cord, I contributed to the understanding on the heterogenous origin of the fibrotic scar. Furthermore, I analyzed how penile erection alters the contact between PVFs and other cells and the results help to understand the role of PVFs in penile blood flow regulation.In paper I, I presented the tissue-clearing based pipeline and the volumetric image handling framework. Utilizing various transgenic mouse lines and antibody labeling, we probed the distribution and organization of vSMCs, pericytes, PVFs, and PVMs along the AV axis. Being able to visualize the vasculature landscape deep within the brain and spinal cord, we could determine the major arterial and venous network in various CNS regions to act as reference for PVC mapping. We found that morphological heterogeneity along AV axis was conserved throughout the CNS. In the brain, PVFs and PVMs were predominately located along arterioles and only a few large venules but in the spinal cord, they were found in virtually all arterioles and venules. We also found distinct morphological differences of PVFs and PVMs along the AV axis and described a novel Ôthin-strandedŐ PVF morphology at the entry or exit of the capillary network which extended long processes along multiple capillary branches. This study provides a comprehensive analysis of the perivascular landscape along the AV axis which infers functional specialization.Previous work from our lab has shown that scar-forming stromal fibroblasts in the injured spinal cord and brain originate from a small population of glutamate aspartate transporter (GLAST)-expressing PVCs. In paper II, we investigated the heterogeneity of these scar-forming PVCs in mice. Based on single-cell RNA sequencing analysis, we discovered that GLAST-expressing PVCs in the spinal cord encompassed two types of cells that could be transcriptionally defined as PVFs and pericytes. Based on strong and specific expression of COL1A1 in PVFs, we selected a COL1A1-CreERT2 mouse line to target PVFs. Using 3D imaging analysis, we compared the distribution of GLAST+ and COL1A1+ PVCs using GLAST- CreERT2;R26-tdTomato and COL1A1-CreERT2;R26-tdTomato mouse lines and found that GLAST+COL1A1- PVFs distributed along larger penetrating vessels enriched in white matter regions, while GLAST+COL1A1- pericytes were in the microvasculature enriched in grey matter regions. Upon injuries, both populations were recruited locally, proliferated, and contributed to fibrotic scar formation. The contribution to scar forming stromal fibroblasts was dependent on the anatomical location of the lesion. The results of this study highlight the functional relevance of the spatial distribution of PVCs in response to injury.In paper III, we examined the role of PVFs in penile blood flow regulation which is important for erectile function. This study resulted in the discovery of a novel function of corpora cavernosa PVFs in supporting vasodilation by reducing norepinephrine availability. We found that more frequent erections led to higher number of PVFs and improved blood flow, and vice versa. Employing volumetric analysis of cleared tissue, we determined that tissue structural changes led to altered contacts between PVFs and other cells in the corpora cavernosa which prompted us to investigate the Notch signaling pathway which involved physical contact between cells. Our results demonstrated that downregulation of Notch signaling in PVFs increased the numbers of PVFs. The study highlights the coupling of PVC organization and their physiological functions.In summary, I presented a pipeline to interrogate the morphology and organization of diverse PVCs along the 3D vascular network. Using this pipeline, we refined the organization of PVCs along the AV axis in the CNS. Furthermore, we revealed the linkage between 3D PVC organization and (patho)physiological responses as demonstrated in the case of fibrotic scar formation and penile erection.List of scientific papersI. Wing Fung Hau, Rosa Mar’a L—pez Cabezas and Christian Gšritz. Multi-scale 3D imaging reveals vessel-specific perivascular landscape in the central nervous system. [Manuscript]II. Daniel Holl, Wing Fung Hau, Anais Julien, Shervin Banitalebi, Jannis Kalkitsas, Soniya Savant, Enric Llorens-Bobadilla, Yann Herault, Guillaume Pavlovic, Mahmood Amiry-Moghaddam, David Dias and Christian Gšritz. Distinct origin and region-dependent contribution of stromal fibroblasts to fibrosis following traumatic injury in mice. Nature Neuroscience. 2024, 27, 1285-1298. https://doi.org/10.1038/s41593-024-01678-4 III. Eduardo Linck Guimaraes, David Oliveira Dias, Wing Fung Hau, Anais Julien, Daniel Holl, Maria Garcia-Collado, Soniya Savant, Evelina VŚgesjš, Mia Phillipson, Lars Jakobsson and Christian Gšritz. Corpora cavernosa fibroblasts mediate penile erection. Science. 2024, 383, issue 6683. https://doi.org/10.1126/science.ade8064 </p
Cancer risk, autoimmune comorbidity and autoantibodies in primary sclerosing cholangitis
No full textPrimary sclerosing cholangitis (PSC) is a rare cholestatic liver disease affecting the bile ducts primarily, and the liver parenchyma secondarily. The disease is progressive but highly variable between diseased. Some progress quickly to liver cirrhosis and end-stage liver disease, with need of liver transplantation (LTX), while in others the disease may stay quiescent for years. There is a close association to inflammatory bowel disease (IBD), and especially ulcerative colitis (UC). Comorbidity with autoimmune disease (AID) together with elevated levels of autoantibodies (AAB) is also commonly described. Pathogenesis in PSC is ambiguous, where genetic studies link PSC closer to other AIDs than IBD. The importance of the comorbidities and AABs seen in PSC is, despite numerous studies, not well understood.The most devastating threat in PSC is the high and unpredictable risk of developing cancer, especially hepatobiliary cancer (HBC), but also colorectal cancer (CRC) for those with a concomitant IBD. Cholangiocarcinoma (CCA) has a poor prognosis with a low estimated survival. All individuals with PSC are recommended yearly surveillance. Better understanding of risks and pathogenesis in PSC may help in, clinical evaluation, and prognostication. This thesis aimed to contribute to the knowledge on both cancer risk and comorbidity in PSC, along with an assessment on the role of the colon for disease development. Evaluation on family autoimmune inheritance and a complete proteomic analysis on autoantibodies linked to different phenotypes of the disease may contribute to a better understanding the pathogenesis.A large cohort of individuals with a well-defined diagnosis of PSC (n=1 768) in Sweden were matched on sex, age, and municipality to a random reference cohort 10:1 from the general population in Sweden. Data on first-degree relatives, diagnoses of cancer, AID, LTX and death were retrieved from the Swedish national registers. This cohort may have comprised the prevalent population of individuals with PSC in Sweden which together with high coverage in Swedish registers are a stable fundament for these studies. This cohort was used for Studies I and III and serum from a smaller part of the cohort (n=500) was used for Study IV.In Study I we estimated risk of cancer in PSC, with the hypothesis that PSC increases the risk for other than gastrointestinal cancers. Start of follow up commenced at date of diagnosis and the cohort was followed until December 31, 2016, or any of the following events: cancer diagnosis, LTX, first emigration date or death. We reproduced previous findings with high rates of HBC and CRC. Higher risks for pancreatic cancer and lymphoma were also detected. Analyzing the potential confounder of IBD in Study I led us to the aim of Study II; to evaluate the role of the bowel for development of future PSC. We evaluated all UC patients in Sweden (n=61 993) between 1990-2018 where detection of UC was performed according to a validated algorithm. In the cohort, 5 577 colectomies were performed, at which time point matching was performed and follow-up began. Matched comparators were achieved from the remaining noncolectomized UC cohort. The total cohort was followed over a median time of 13 years covering a period where great medical evolvement in IBD emerged. Overall, the incidence of PSC remained the same over the years, and we detected no difference between those with and without remaining colon. In a subgroup analysis discrepancies on incidence of PSC, related to age at UC diagnosis and colectomy as well as period of colectomy and time to colectomy, were detected. Another factor that we speculated may have influenced risk of cancer in Study I was the autoimmune linkage. In Studies III and IV we intended to tackle the longstanding idea of PSC as an AID by evaluating autoimmune comorbidity in both individuals with PSC and their first-degree relatives. For a part of the PSC cohort, a full-genome screening of AABs was performed. High odds for several AIDs were seen in PSC and in their first-degree relatives, which remained in first-degree relatives also when removing cases with the disease of interest. We quantified 1 153 selected autoantibodies, in 500 PSC and 220 controls as an explorative initiative. AABs were present in 5-10% of the cohort with a large variability. No single AAB marker was detected but rather clusters of AABs associated to different phenotypes and severity of disease.In conclusion we confirmed previous known high risks of HBC and CRC along with an increased risk for lymphoma which has not been presented previously. Removal of the colon was not seen to prevent development of PSC on a group level, but the importance of colectomy for subgroups shall not be ruled out. Autoimmune comorbidity in PSC is common but does not seem to influence severe outcome. Both autoimmune heredity in PSC and occurrence of AABs support pathogenetic findings where some clusters of AAB may be of prognostic value.List of scientific papersI. Aiva Lundberg Båve, Annika Bergquist, Matteo Bottai, Anna Warnqvist, Erik von Seth, Caroline Nordenvall. Increased risk of cancer in patients with primary sclerosing cholangitis. Hepatology International. (2021) 15:1174–1182. https://doi.org/10.1007/s12072-021-10214-6 II. Aiva Lundberg Båve, Ola Olén, Jonas Söderling, SWIBREG study Group, Jonas F. Ludvigsson, Annika Bergquist, Caroline Nordenvall. Colectomy in patients with ulcerative colitis is not associated to future diagnosis of primary sclerosing cholangitis. United European Gastroenterol J. (2023) 11:471–481. https://doi.org/10.1002/ueg2.12388 III. Aiva Lundberg Båve, Erik von Seth, Michael Ingre, Caroline Nordenvall, Annika Bergquist. Autoimmune diseases in primary sclerosing cholangitis and their first-degree relatives. Hepatology. 2024 Mar 5. Online ahead of print. https://doi.org/10.1097/HEP.0000000000000823 IV. Martin Cornillet, Aiva Lundberg Båve, Dan Sun, Christina Villard, Aristeidis Grigoriadis, Erik von Seth, Hannes Jansson, Per Stål, SweHep, Ernesto Sparrelid, Niklas Björkström, Jonas Halfvarsson, Annika Bergquist. Genome-scale autoantibody profiling in primary sclerosing cholangitis provides an ATLAS of autoimmune traits associated with clinical phenotypes and disease progression. [Manuscript]</p