Sensing of nucleic acids by endolysosomal Toll-like receptors (TLRs) is essential for triggering host defense responses. Upon ligand engagement, TLR7-9 recruits adaptor proteins to activate NF-κB, MAPK, and IRF pathways, leading to the production of pro-inflammatory cytokines and type I interferon (IFN). Dysregulation of TLR7/9 signaling, leading to IRF5 overactivation, has been implicated in inflammatory and autoimmune diseases such as systemic lupus erythematosus (SLE). Despite the established importance of TLR signaling for immune responses and SLE, the detailed regulatory network controlling TLR7/9-induced responses remains incompletely understood. Therefore, this thesis aims to better define the molecular mechanisms regulating the TLR7/9-IRF5 signaling pathway. We previously identified the TASL protein as the fourth innate immune adaptor interacting with SLC15A4 at the endolysosomes, required for TLR7/9-induced IRF5 activation. While the SLC15A4-TASL complex plays a crucial role in this pathway, several unresolved questions regarding its regulation and biological relevance remain and were addressed in the first part of this thesis. Here, we demonstrate that the main function of SLC15A4 is to act as a scaffold to recruit TASL, whilst its transporter function is dispensable to anchor it to endolysosomes. We further show that SLC15A4 binds TASL when it adopts a cytosol-open conformation. Importantly, this interaction can be chemically targeted. We identified the compound C5/Feeblin, which binds and stabilizes SLC15A4 in an outward-facing conformation incompatible with TASL binding. As a result, TASL is degraded, leading to specific impairment of IRF5 activation and downstream cytokine production. The potential SLC15A4-TASL inhibition is particularly relevant, as we also demonstrate that the complex is essential across immune cell types for TLR7/9 responses in vitro and in vivo, as well as for antiviral immunity and the pathogenesis of SLE. Additionally, we provide insights into how phosphorylation and interaction with co-chaperones modulate TASL stability and activity. In the second part of the thesis, we explore additional regulatory mechanisms of TLR7/9 signaling independent from the SLC15A4-TASL complex. We identify CCDC134 as a novel regulator of TLR biogenesis and responses. Located in the ER, CCDC134 binds and stabilizes the TLR chaperone Gp96, ensuring its proper folding and trafficking. Deletion of CCDC134 leads to Gp96 hyperglycosylation and subsequent ER-associated protein degradation (ERAD)-mediated clearance, impairing the folding, trafficking and signaling of both plasma membrane and endolysosomal TLRs. Together, these findings uncover important regulators of TLR biogenesis and signaling, providing a deeper molecular understanding of innate immune responses and offering valuable insights for the development of targeted therapies in SLE and other disorders.