Modeling MyD88 deficiency

Abstract: Toll-like receptors (TLRs) are important pathogen recognition receptors, allowing the immune system to recognize and respond to conserved pathogen components. Myeloid differentiation primary response protein 88 (MyD88), is a TLR downstream adaptor protein that is essential for proper functioning of the immune system. MyD88-deficiency is an autosomal recessive disease displaying defective TLR- signaling resulting in high mortality (~50%) due to invasive bacterial disease; disease-causing pathogens are restricted to a small range of bacteria with Streptococcus pneumoniae and Staphylococcus aureus accounting for roughly 2/3 of all serious infections. The phenotype of MyD88-deficiency in mice is surprisingly different: mice are markedly more susceptible to a wider range of pathogens including viruses such as Cytomegalovirus and Herpes simplex virus-1; bacteria such as Listeria monocytogenese and Mycobacteria; parasites such as Toxoplasma gondii; as well as fungal species such as Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus. These significant differences between mouse and human are not well understood and suggest additional redundancies in the innate immune system of humans that are not found in mice. Interrogation of the innate immune system has lagged that of the adaptive immune system for several important reasons including lack of clonality and dramatically increased number of proteins of interest (large number of unique pattern recognition receptors compared to a single T- or B-cell-receptor). These significant differences between humans and mice indicate the need for a disease model in humans. An appropriate disease model requires the use of specialized human immune cells, in this case macrophages, which can be precisely genetically modified. Few existing models of the innate immune system use human primary cell macrophages that are easily genetically modifiable.

Such a disease model to further elucidate the role of the central TLR adapter protein, MyD88, was established: fibroblasts were removed from a child with MyD88-deficiency and reprogrammed into induced pluripotent stem cells (iPS cells). A transgenic copy of MyD88 was stably integrated into the genome of the MyD88-deficient iPS cells using the Sleeping Beauty transposon system, creating several iPS cell lines. There are two important splice variants of MyD88—a long version hereafter referred to as MyD88-L, and a short version hereafter referred to as MyD88-S. Signaling through MyD88-L results in NF-kB activation with resulting inflammation, whereas MyD88-S does not. Therefore, iPS cell lines containing MyD88-L, MyD88-S, MyD88E53del (uncorrected), as well as cells derived from a healthy donor, were created. These iPS cells were then differentiated into macrophages, hereafter referred to as iMacs (iPS-derived macrophages), for further analysis. No differences were seen between clones morphologically and by cell marker analysis as well as by several functional tests including phagocytosis and stimulation of reactive oxygen species. Upon stimulation with heat-inactivated Staphylococcus aureus dramatic differences between the clones emerged: the MyD88-deficient macrophages had a significantly reduced cytokine response; the corrected macrophages, however, produced a very similar profile compared to macrophages from a healthy donor. The model shows that a principal problem in MyD88-deficiency is in cytokine signaling. Further, having created this model showed additional insights: iPS cells can be genetically modified using the Sleeping Beauty transposon system. Gene transfer was titrated to allow for only 2-3 integration events, which when combined with the EF1α (elongation factor 1 alpha) promoter results in physiological transcription levels. The modified iPS cells could be successfully differentiated into iMacs without silencing of the transgene. This shows both the success of the genetic alteration as well as well as the ability to use the system to answer biological questions: no obvious defects in differentiation in cells with a genetic lesion in MyD88 were seen. Surprisingly, MyD88-deficient iMacs produced more IL-8 compared to other cell lines.

While the scientific possibilities of this model are fascinating, the long-term goal is to eventually use gene therapy in the clinic. This thesis shows that it is technically feasible to remove fibroblasts from a patient, reprogram them into iPS cells, repair the genetic lesion, and differentiate the corrected iPS cells into biologically relevant effector cells. In summary, a disease model of MyD88-deficiency led to interesting biological surprises as well as laying the groundwork for bringing this technology into the clinic

Angaben zum Objekt

• Standort: Deutsche Nationalbibliothek Frankfurt am Main
• Umfang: Online-Ressource
• Sprache: Englisch
• Anmerkungen: Universität Freiburg, Dissertation, 2023

Klassifikation und Themen

• Schlagwort: Immunreaktion ; Toll-like-Rezeptoren ; Immunsystem ; Angeborene Immunität ; Makrophage

Beteiligte, Orts- und Zeitangaben

• Ereignis: Veröffentlichung
• (wo): Freiburg
• (wer): Universität
• (wann): 2023
• Urheber: Craig-Müller, Nils
• Beteiligte Personen und Organisationen: Cathomen, Anton ; Cathomen, Anton ; Henneke, Philipp ; Centrum für Chronische Immundefizienz ; Zentrum Translationale Zellforschung ; Albert-Ludwigs-Universität Freiburg. Medizinische Fakultät

Weitere Informationen

• DOI: 10.6094/UNIFR/235937
• URN: urn:nbn:de:bsz:25-freidok-2359372
• Rechteinformation: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Letzte Aktualisierung: 25.03.2025, 13:48 MEZ