In Vitro Characterization of Heat-Stress and Leaky-Gut-Associated Immune Dysfunction in Bovine Dendritic Cells and Modulation by Alpha-Lipoic Acid in Vrindavani Cattle

Escalating environmental heat poses a major threat to livestock health, yet its effects on professional antigen-presenting cells and their modulation by nutritional interventions remain incompletely defined. This study characterized heat-stress- and leaky-gut-associated inflammatory responses in bovine monocyte-derived dendritic cells (BoMDCs) and evaluated the immunomodulatory potential of alpha-lipoic acid (ALA; 250 µM). BoMDCs generated from PBMC were morphologically validated by phase-contrast microscopy and Giemsa staining, and thermal stress was standardized using viability and cytotoxicity kinetics across 37-42°C for 1-24 h. Exposure to 40°C for 24 h was selected as the sublethal heat stress condition (75.08% viability), while 42°C produced marked cytotoxicity (1.81% viability). BoMDCs were subsequently exposed to heat stress alone or in combination with heat-stress plasma (HSpl) and/or LPS to model compounded thermal, systemic and endotoxin-associated stress. ALA supplementation improved cellular survival, with viability increasing from 52.20% to 64.06% in the HS+HSpl+LPS group and cytotoxicity decreasing from 49.60% to 36.84% (P < 0.001). Morphological preservation and increased cell counts were also observed following ALA treatment. Fluorescence-based analyses demonstrated reduced intracellular HSP70 and DCFDA-associated ROS fluorescence following ALA supplementation, with the greatest attenuation observed under combined stress. At the transcript level, ALA significantly modulated HSP70, HSP90, IL-1β, IL-12p40, CD80, CD86, and MHC II expression, with changes in HSP70, HSP90, IL-1β and IL-12p40 reaching P < 0.0001 and alterations in CD80, CD86 and MHC II reaching P < 0.01. Functionally, ALA reduced BoMDC-mediated lymphocyte proliferation, particularly in HS+LPS at the 1:10 ratio (P < 0.01) and in HS and HS+LPS groups at the 1:20 ratio (P < 0.001). Overall, ALA attenuated cellular, oxidative and inflammatory perturbations induced by combined heat and leaky-gut-associated stressors and modulated dendritic-cell functional responses. These findings provide proof-of-concept evidence for ALA as a potential nutritional immunomodulator for improving immune-cell resilience under heat stress-associated inflammatory conditions in ruminants. Keywords: Alpha-lipoic acid; Heat stress; Bovine dendritic cells; Immunomodulation.