Gngt2 promotes inflammatory dendritic cell programming through an ATG5-NF-κB signaling axis in neutrophilic asthma

Background and objective: Type 2 (T2)-low neutrophilic asthma represents a severe asthma endotype characterized by corticosteroid resistance and persistent airway inflammation driven by dendritic cells (DCs)-mediated T helper (Th)17 responses. Although immunogenic DC programming plays a central role in this process, the intracellular mechanisms that couple inflammatory signaling to DC functional polarization remain poorly understood. This study investigated whether the G protein subunit gamma transducin 2 (Gngt2) regulates Autophagy related protein-dependent inflammatory programming in DCs and thereby contributes to Th17-driven neutrophilic airway inflammation.

Methods: Ovalbumin (OVA)-induced eosinophilic asthma and OVA/lipopolysaccharide (LPS)-induced neutrophilic asthma models were established in BALB/c mice to compare endotype-specific inflammatory responses. Gngt2 was silenced in vivo by intratracheal delivery of lentiviral short hairpin RNA. In vitro, bone marrow-derived dendritic cells (BMDCs) were stimulated with OVA or OVA/LPS and then manipulated by Gngt2 knockdown, ATG5 knockdown, or ATG5 overexpression. In selected experiments, the NF-κB nuclear translocation inhibitor JSH-23 was used to assess pathway dependence. BMDCs were co-cultured with naïve CD4 + T cells to evaluate Th17 differentiation. Airway inflammation, cytokine production, autophagy-related protein expression, and NF-κB activation were assessed by histological, immunological, and molecular analyses.

Results: Gngt2 expression was selectively upregulated in lung DCs from mice with neutrophilic asthma and was accompanied by a phenotype with altered Autophagy related protein, as indicated by reduced ATG5 abundance, decreased microtubule-associated protein 1 light chain 3 beta (LC3B) expression, and a lower LC3B-II/I ratio. In vivo silencing of Gngt2 markedly attenuated neutrophilic airway inflammation and decreased bronchoalveolar lavage fluid (BALF) levels of interleukin (IL)-6, IL-23, and IL-17, without significantly affecting IL-4. In OVA/LPS-stimulated BMDCs, Gngt2 knockdown restored ATG5/LC3B-associated autophagy-related markers, reduced NF-κB p65 phosphorylation, suppressed IL-6 and IL-23 production, and impaired DC-mediated Th17 differentiation. Rescue experiments further demonstrated that ATG5 acted downstream of Gngt2 to regulate NF-κB activation and inflammatory cytokine production, thereby controlling Th17 polarization.

Conclusion: Our findings identify a previously unrecognized DC-intrinsic Gngt2-ATG5-NF-κB signaling axis that links G protein-coupled receptor (GPCR)- signaling with immune regulation of altered Autophagy related protein in neutrophilic asthma. By suppressing ATG5-mediated signaling, Gngt2 promotes NF-κB activation and contributes to Th17-driven neutrophilic airway inflammation. This pathway provides mechanistic insight into DC-driven immune dysregulation in type 2-low asthma and suggests Gngt2 as a potential therapeutic target for neutrophilic asthma.