Towards the identification of environmental exposures and epigenetic marks related to the etiology of Autism

Autism is a complex disorder with possible genetic, epigenetic and environmental components. As the etiology remains uncertain and an increase in incidence is suspected, the involvement of possible environmental risk factors has gained increasing attention. With this thesis, I aim to provide tools for assessing such risk factors. Firstly, I aim to construct a questionnaire for the analysis of an environmental component in the etiology of autism. Secondly, I aim to assess the importance of prenatal exposure to metals in certain diseases and thirdly I aim to construct a methodology enabling the analysis of the mitochondrial epigenome, which is especially interesting in relation to autism as mitochondrial diseases occur more frequently in an autistic population than in the general population. For the creation of the questionnaire the scientific literature was reviewed. The resulting questionnaire contains general, prenatal, neonatal and paternal risk factors. The metal analysis was conducted on the cord blood of patients who later developed autism, antinuclear antibodies positive rheumatoid arthritis or diabetes, which were then compared to healthy control subjects. My findings propose a link between elevated levels of cord blood cadmium or aluminum and rheumatic arthritis. In addition, elevated aluminum levels might be associated with autism. In regards to the analysis of the mitochondrial epigenome, to my knowledge, no standard protocol exists with frozen human whole blood as a source. In this thesis, I succeeded in creating the basis for such a protocol, however still needing several small modifications for an increased overall yield

Identification of potentially relevant metals for the etiology of autism by using a Bayesian multivariate approach for partially censored values

Heavy metals are known to be able to cross the placental and blood brain barriers to affect critical neurodevelopmental processes in the fetus. We measured metal levels (Al, Cd, Hg, Li, Pb and Zn) in the cord blood of newborns and in the serum of the same children at 5 years of age, and compared between individuals with or without (controls) autism spectrum disorder (ASD) diagnosis. The samples were from a biobank associated with the All Babies in Southeast Sweden (ABIS) registry. We proposed a Bayesian multivariate log-normal model for partially censored values to identify potentially relevant metals for the etiology of ASD. Our results in cord blood suggest prenatal Al levels could be indicative of later ASD incidence, which could also be related to an increased possibility of a high, potentially toxic, exposure to Al and Li during pregnancy. In addition, a larger possibility of a high, potentially beneficial, exposure to Zn could occur during pregnancy in controls. Finally, we found decisive evidence for an average increase of Hg in 5-year-old ASD children compared to only weak evidence for controls. This is concordant with previous research showing an impaired ability for eliminating Hg in the ASD group

GBS-MeDIP: A protocol for parallel identification of genetic and epigenetic variation in the same reduced fraction of genomes across individuals

Summary:The GBS-MeDIP protocol combines two previously described techniques, Genotype-by-Sequencing (GBS) and Methylated-DNA-Immunoprecipitation (MeDIP). Our method allows for parallel and cost-efficient interrogation of genetic and methylomic variants in the DNA of many reduced genomes, taking advantage of the barcoding of DNA samples performed in the GBS and the subsequent creation of DNA pools, then used as an input for the MeDIP. The GBS-MeDIP is particularly suitable to identify genetic and methylomic biomarkers when resources for whole genome interrogation are lacking

Increased SARS-CoV-2 Infection, Protease, and Inflammatory Responses in Chronic Obstructive Pulmonary Disease Primary Bronchial Epithelial Cells Defined with Single-Cell RNA Sequencing

RATIONALE: Patients with chronic obstructive pulmonary disease (COPD) develop more severe coronavirus disease (COVID-19); however, it is unclear whether they are more susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and what mechanisms are responsible for severe disease. OBJECTIVES: To determine whether SARS-CoV-2 inoculated primary bronchial epithelial cells (pBECs) from patients with COPD support greater infection and elucidate the effects and mechanisms involved. METHODS: We performed single-cell RNA sequencing analysis on differentiated pBECs from healthy subjects and patients with COPD 7 days after SARS-CoV-2 inoculation. We correlated changes with viral titers, proinflammatory responses, and IFN production. MEASUREMENTS AND MAIN RESULTS: Single-cell RNA sequencing revealed that COPD pBECs had 24-fold greater infection than healthy cells, which was supported by plaque assays. Club/goblet and basal cells were the predominant populations infected and expressed mRNAs involved in viral replication. Proteases involved in SARS-CoV-2 entry/infection (TMPRSS2 and CTSB) were increased, and protease inhibitors (serpins) were downregulated more so in COPD. Inflammatory cytokines linked to COPD exacerbations and severe COVID-19 were increased, whereas IFN responses were blunted. Coexpression analysis revealed a prominent population of club/goblet cells with high type 1/2 IFN responses that were important drivers of immune responses to infection in both healthy and COPD pBECs. Therapeutic inhibition of proteases and inflammatory imbalances reduced viral titers and cytokine responses, particularly in COPD pBECs. CONCLUSIONS: COPD pBECs are more susceptible to SARS-CoV-2 infection because of increases in coreceptor expression and protease imbalances and have greater inflammatory responses. A prominent cluster of IFN-responsive club/goblet cells emerges during infection, which may be important drivers of immunity. Therapeutic interventions suppress SARS-CoV-2 replication and consequent inflammation

Mucosal TLR2-activating protein-based vaccination induces potent pulmonary immunity and protection against SARS-CoV-2 in mice

Current vaccines against SARS-CoV-2 substantially reduce mortality, but protection against infection is less effective. Enhancing immunity in the respiratory tract, via mucosal vaccination, may provide protection against infection and minimise viral spread. We tested a novel subunit vaccine in mice, consisting of SARS-CoV-2 Spike protein with a TLR2-stimulating adjuvant, delivered to mice parenterally or mucosally. Both routes of vaccination induced substantial neutralising antibody (nAb) titres, however, mucosal vaccination uniquely generated anti-Spike IgA, increased nAb in the serum and airways, and increased lung CD4+ T-cell responses. TLR2 is expressed by respiratory epithelia and immune cells. Using TLR2 deficient chimeric mice, we determined that TLR2 expression in either compartment facilitated early innate responses to mucosal vaccination. By contrast, TLR2 on hematopoietic cells was essential for optimal lung-localised, antigen-specific responses. In a K18-hACE2 mice, vaccination provided complete protection against disease and sterilising lung immunity against SARS-CoV-2. These data support mucosal vaccination as a strategy to improve protection in the respiratory tract against SARS-CoV-2 and other respiratory viruses

Pharmacological inhibition of TBK1/IKKε blunts immunopathology in a murine model of SARS-CoV-2 infection

Abstract TANK-binding kinase 1 (TBK1) is a key signalling component in the production of type-I interferons, which have essential antiviral activities, including against SARS-CoV-2. TBK1, and its homologue IκB kinase-ε (IKKε), can also induce pro-inflammatory responses that contribute to pathogen clearance. While initially protective, sustained engagement of type-I interferons is associated with damaging hyper-inflammation found in severe COVID-19 patients. The contribution of TBK1/IKKε signalling to these responses is unknown. Here we find that the small molecule idronoxil inhibits TBK1/IKKε signalling through destabilisation of TBK1/IKKε protein complexes. Treatment with idronoxil, or the small molecule inhibitor MRT67307, suppresses TBK1/IKKε signalling and attenuates cellular and molecular lung inflammation in SARS-CoV-2-challenged mice. Our findings additionally demonstrate that engagement of STING is not the major driver of these inflammatory responses and establish a critical role for TBK1/IKKε signalling in SARS-CoV-2 hyper-inflammation

Dimethyl fumarate and 4-octyl itaconate are anticoagulants that suppress Tissue Factor in macrophages via inhibition of Type I Interferon

Abstract Excessive inflammation-associated coagulation is a feature of infectious diseases, occurring in such conditions as bacterial sepsis and COVID-19. It can lead to disseminated intravascular coagulation, one of the leading causes of mortality worldwide. Recently, type I interferon (IFN) signaling has been shown to be required for tissue factor (TF; gene name F3) release from macrophages, a critical initiator of coagulation, providing an important mechanistic link between innate immunity and coagulation. The mechanism of release involves type I IFN-induced caspase-11 which promotes macrophage pyroptosis. Here we find that F3 is a type I IFN-stimulated gene. Furthermore, F3 induction by lipopolysaccharide (LPS) is inhibited by the anti-inflammatory agents dimethyl fumarate (DMF) and 4-octyl itaconate (4-OI). Mechanistically, inhibition of F3 by DMF and 4-OI involves suppression of Ifnb1 expression. Additionally, they block type I IFN- and caspase-11-mediated macrophage pyroptosis, and subsequent TF release. Thereby, DMF and 4-OI inhibit TF-dependent thrombin generation. In vivo, DMF and 4-OI suppress TF-dependent thrombin generation, pulmonary thromboinflammation, and lethality induced by LPS, E. coli, and S. aureus, with 4-OI additionally attenuating inflammation-associated coagulation in a model of SARS-CoV-2 infection. Our results identify the clinically approved drug DMF and the pre-clinical tool compound 4-OI as anticoagulants that inhibit TF-mediated coagulopathy via inhibition of the macrophage type I IFN-TF axis