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Genetic variants in α-actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut presents a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray, and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2-associated cardiomyopathies.
BCL11B, an essential transcription factor for thymopoiesis, regulates also vital processes in post-thymic lymphocytes. Increased expression of BCL11B was recently correlated with the maturation of NK cells, whereas reduced BCL11B levels were observed in native and induced T cell subsets displaying NK cell features. We show that BCL11B-depleted CD8+ T cells stimulated with IL-15 acquired remarkable innate characteristics. These induced innate CD8+ (iiT8) cells expressed multiple innate receptors like NKp30, CD161, and CD16 as well as factors regulating migration and tissue homing while maintaining their T cell phenotype. The iiT8 cells effectively killed leukemic cells spontaneously and neuroblastoma spheroids in the presence of a tumor-specific monoclonal antibody mediated by CD16 receptor activation. These iiT8 cells integrate the innate natural killer cell activity with adaptive T cell longevity, promising an interesting therapeutic potential. Our study demonstrates that innate T cells, albeit of limited clinical applicability given their low frequency, can be efficiently generated from peripheral blood and applied for adoptive transfer, CAR therapy, or combined with therapeutic antibodies.
Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549), and human embryonic kidney cells (HEK 293). Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen's proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2 × 106 bacteria, roughly 1450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants.
This thesis contains results from transcriptome studies on different aspects of host-pathogen interactions. First, liver gene expression profiles from a murine chronic stress model served to elucidate aspects of the influence of stress on metabolism and immune response state. Chronic stress in female BALB/c mice was shown to lead to a hypermetabolic syndrome including induction of gluconeogenesis, hypercholesteremia, and loss of essential amino acids, to the induction of the acute phase response, but also of immune suppressive pathways and to the repression of hepatic antigen presentation. Increased leukocyte trafficking, increased oxidative stress together with counter-regulatory gene expression changes, and an induction of apoptosis were detected. The influence of intra-venous infection on the host kidney gene expression was analyzed in another murine model using the wild type strain Staphylococcus aureus RN1HG and its isogenic sigB mutant. Gene expression profiling indicated a highly reproducible host kidney response to infection. The comparison of infected with non-infected samples revealed a strong inflammatory reaction of kidney tissue, e. g. Toll-like receptor signaling, complement system, antigen presentation, interferon and IL-6 signaling. However, the results of this study did not provide any hints for differences in the pathomechanism of the S. aureus strains RN1HG and ΔsigB, since the host response did not differ between infections with the two strains analyzed. Effects of SigB might be transient, only apparent at earlier time points, or might also be compensated for in the in vivo infection by the interlaced pattern of other regulators. SigB might possess only to a lesser extent characteristics attributed to virulence factors and might act in vivo more like a virulence modulator and fine tune bacterial reactions. In addition to the analysis of tissue samples, different in vitro models were furthermore studied. The third part of this thesis focuses on bone-marrow derived macrophages (BMM) of the two mouse strains BALB/c and C57BL/6, which are described in literature to exhibit genetically determined differences in their reaction to infection. Expression profiling was performed on control and IFN-γ treated samples from a serum-free cultivation system and revealed mainly induction of gene expression after treatment of BMM with IFN-γ. Gene expression changes confirmed known IFN-γ effects like induction of immunoproteasome, antigen presentation, interferon signaling related genes, GTPase/GBPs, and inducible NO synthase. IFN-γ dependent gene expression changes were highly similar in BALB/c and C57BL/6 BMM. Considering gene expression differences between BMM of both strains, a similar expression trend was visible on the level of untreated controls as well as after IFN-γ treatment. Differentially expressed genes between BMM of both strains included immune-relevant genes as well as genes linked to cell death, but the coverage of functional groups was limited. The bronchial epithelial cell line S9 was used as an in vitro model system for the infection with S. aureus RN1HG. The fourth chapter in this thesis includes S9 cell gene expression signatures 2.5 h and 6.5 h after start of infection. At the early time point, only 40 genes were differentially expressed, which nevertheless indicated a beginning pro-inflammatory response, e. g. induction of cytokines (IL-6, IFN-β, LIF) or prostaglandin-endoperoxide synthase 2 (PTGS2), but also counter-regulatory processes, e. g. induction of CD274. The host cell response was dramatically aggravated at the later 6.5 h time point. Differential expression was detected for 1196 genes. These included induced cytokines, pattern recognition receptor signaling, antigen presentation, and genes involved in immune defense (e. g. GBPs, MX, APOL). Negative effects on growth and proliferation were even more enhanced in comparison to the early time point, and signs for apoptotic processes were revealed. Finally, the last chapter addresses amongst others the pathogen’s expression profile in the S9 cell in vitro infection model at the two time points 2.5 h and 6.5 h after start of infection by tiling array gene expression analysis. The pathogen expression profiling revealed the activity of the SaeRS two-component system in internalized staphylococci. Partly dependent on SaeRS, the induction of adhesins (e. g. fnbAB, clfAB), toxins (hlgBC, lukDE, hla), and immune evasion genes (e. g. chp, eap) was observed. Furthermore, expression changes of metabolic genes were recorded (gene induction of amino acid biosynthesis, TCA cycle, gluconeogenesis; gene repression of glycolysis, purine biosynthesis, tRNA synthetases). Expression analysis recorded a distinct bacterial expression program, which supported literature results of a specific, bacterial strain and host cell line dependent transcriptional adaptation of the pathogen.