Modulation of local and systemic immune responses by fermented garlic extract

Abstract

Fermented garlic (FG), prepared by fermenting garlic with <em>Bacillus subtilis</em> for 3‑15 days at 20‑40˚C has been linked to immune‑boosting properties. In the present study, FG was utilized to investigate its immunomodulatory effects using RAW264.7 cells, as well as BALB/c and C3H/HeN mice. Nitric oxide (NO) and pro‑inflammatory cytokine levels were quantified in RAW264.7 cells treated with or without FG (0.016, 0.08, 0.4, 2 and 10 mg/ml), garlic extract (GE) (0.016, 0.08, 0.4, 2 and 10 mg/ml) and lipopolysaccharide (1 µg/ml). Peritoneal macrophages and splenocytes were obtained from BALB/c mice for <em>ex vivo</em> analyses. MTT and enzyme‑linked immunosorbent assays were used to determine splenocyte proliferation, natural killer (NK) cell activity and cytokine production. The intestinal immunostimulatory <em>in vitro</em> effects of FG were also investigated. In a concentration‑dependent manner, FG treatment markedly accelerated the proliferation of RAW264.7 cells, murine peritoneal macrophages and splenocytes. Moreover, FG initiated macrophage activation by significantly increasing the NO, tumor necrosis factor‑α, interleukin (IL)‑12 and IL‑6 levels in RAW264.7 cells and peritoneal macrophages. In contrast to FG, GE promoted RAW264.7 cell proliferation at lower concentrations and toxicity at higher concentrations, while no detectable NO synthesis was observed at the concentrations tested. Likewise, the FG extract at larger concentrations fortified the intestinal immune system and improved bone marrow cell proliferation by significantly elevating IL‑6 and granulocyte‑macrophage colony‑stimulating factor production in splenocytes and Peyer's patch cells of mice. In addition, the NK cell activities of splenocytes from FG‑treated mice at lower doses against YAC‑1 cells were significantly increased, which must have been fostered and activated by interferon‑γ generated by splenocytes. On the whole, these findings provide relevant <em>in vitro</em> and <em>ex vivo</em> information that describes the mechanisms through which FG can strengthen a compromised immune system by influencing local and systemic gut immune responses directly or indirectly.