Mutant Huntingtin affects toll-like receptor 4 intracellular trafficking and cytokine production in mast cells

Abstract

Background: Huntington's disease (HD) is caused by the expression of a mutated variant of Huntingtin (mHtt), which results in the complex pathology characterized by a defective function of the nervous system and altered inflammatory responses. While the neuronal effects of mHtt expression have been extensively studied, its effects on the physiology of immune cells have not been fully described. Mast cells (MCs) are unique tissue-resident immune cells whose activation has been linked to protective responses against parasites and bacteria, but also to deleterious inflammatory allergic reactions and, recently, to neurodegenerative diseases. Methods: Bone marrow-derived mast cells (BMMCs) were obtained from wild-type (WT-) and mHtt-expressing (R6/ 1) mice to evaluate the main activation parameters triggered by the high-affinity IgE receptor (FcεRI) and the Tolllike receptor (TLR) 4. Degranulation was assessed by measuring the secretion of β-hexosaminidase, MAP kinase activation was detected by Western blot, and cytokine production was determined by RT-PCR and ELISA. TLR-4 receptor and Htt vesicular trafficking was analyzed by confocal microscopy. In vivo, MC-deficient mice (c-Kit Wsh/Wsh) were intraperitonally reconstituted with WT or R6/1 BMMCs and the TLR4-induced production of the tumor necrosis factor (TNF) was determined by ELISA. A survival curve of mice treated with a sub-lethal dose of bacterial lipopolysaccharide (LPS) was constructed. Results: R6/1 BMMCs showed normal β-hexosaminidase release levels in response to FcεRI, but lower cytokine production upon LPS stimulus. Impaired TLR4-induced TNF production was associated to the lack of intracellular dynamin-dependent TLR-4 receptor trafficking to perinuclear regions in BMMCs, a diminished ERK1/2 and ELK-1 phosphorylation, and a decrease in c-fos and TNF mRNA accumulation. R6/1 BMMCs also failed to produce TLR4induced anti-inflammatory cytokines (like IL-10 and TGF-β). The detected defects were also observed in vivo, in a MCs-dependent model of endotoxemia. R6/1 and c-Kit Wsh/Wsh mice reconstituted with R6/1 BMMCs showed a decreased TLR4-induced TNF production and lower survival rates to LPS challenge than WT mice.

TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling

“…Only one human conventional (myeloid) DC subtype, cDC2 (also called CD1C +), expresses TLR4 among them only subsets CD5 low/high expresses CD14, whereas murine conventional DC express both Tlr4 and Cd14 [29,124,[167][168][169][170][171][172]. In addition, in human DC and also monocytes, TLR4 seems to be mainly intracellular [95,173]. This suggests that in human DC, details of CD14-TLR4 signaling and trafficking can differ from those in their murine counterparts or in human macrophages.…”

“…This suggests that in human DC, details of CD14-TLR4 signaling and trafficking can differ from those in their murine counterparts or in human macrophages. In addition, recent studies of murine mast cells have revealed that TLR4 trafficking in these cells can be regulated by huntingtin [ 174 ].…”

Responses of Mast Cells to Pathogens: Beneficial and Detrimental Roles

“…Also, Ca 2+ mobilization and activation of Lyn and Fyn kinases occurred in BMMCs after LPS-dependent TLR4 triggering ( 154 , 189 , 192 ). Finally, recent evidence indicated that the multifunctional protein Huntingtin was required for the activation of the ERK1/2-AP-1 axis after TLR4 triggering in BMMCs, contributing to the accumulation of TNF-α, IL-6, IL-10 and transforming growth factor (TGF)-β mRNAs and secretion of those cytokines ( 194 ).…”

Anti-inflammatory and pro-resolving role of mast cells during resolution of local inflammation.

“…Meanwhile proposed that the lack of IFN-β produced by mast cells in response to non-viral TLR induction is due to their inability to internalize TLR-receptor ligand complexes. This is in contrast to other studies, demonstrating the ability of mast cells to internalize TLR receptors, for example after LPS induction(Pérez-Rodríguez et al, 2020),…”

“…Eine der Studien begründete die fehlende Induktion der Typ I Interferon Antwort in Mastzellen auf den TLR4 Agonisten LPS damit, dass Mastzellen den Rezeptor-Pathogen Komplex nicht aktiv internalisieren können. Dies ist jedoch im Widerspruch mit mehreren Studien, die die Internalisierung des TLR4 Rezeptors in Mastzellen zeigen konnten (Meng et al, 2013;Okumura et al, 2003;Pérez-Rodríguez et al, 2020). In Übereinstimmung damit konnte die Freisetzung von IFN-β sowohl nach Zymosan- Makrophagen zur Phagozytose und Efferozytose von Neutrophilen erhöhen kann (Daseke et al, 2020;Wainszelbaum et al, 2006).…”