The Antiviral Effects of Heat-Killed Lactococcus lactis Strain Plasma Against Dengue, Chikungunya, and Zika Viruses in Humans by Upregulating the IFN-α Signaling Pathway

Abstract

The growing risk of contracting viral infections due to high-density populations and ecological disruptions, such as climate change and increased population mobility, has highlighted the necessity for effective antiviral treatment and preventive measures against Dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Recently, there has been increasing attention on the use of probiotics as a potential antiviral option to reduce virus infections. The present study aimed to assess the immunomodulatory effects of heat-killed Lactococcus lactis strain plasma (LC-Plasma) on peripheral blood mononuclear cells (PBMCs) and its subsequent antiviral response against DENV, CHIKV, and ZIKV. To evaluate the immunomodulatory effects of LC-Plasma on PBMCs isolated from healthy individuals, PBMCs were cultured at a density of 2 × 10⁵ cells/well and stimulated with 10 µg/mL of LC-Plasma. LC-plasma-stimulated PBMCs demonstrated elevated interferon-alpha (IFN-α) production and cluster of differentiation 86 (CD86) and human leukocyte antigen-DR isotype (HLA-DR) upregulation, potentially linked to plasmacytoid dendritic cell (pDC) activation. The replication of DENV, CHIKV, and ZIKV was dose-dependently inhibited when Huh-7 cells were stimulated with LC-Plasma-stimulated PBMC supernatant (LCP Sup). IFN-stimulated gene (ISG) expression, including IFN-stimulated gene 15 (ISG15), IFN-stimulated exonuclease gene 20 (ISG20), IFN-induced transmembrane protein 1 (IFITM-1), myxovirus resistance protein A (MxA), and radical S-adenosyl methionine domain-containing protein 2 (RSAD2), was significantly upregulated in LCP Sup-stimulated Huh-7 cells. Findings from this study indicate that LC-Plasma has the potential to induce IFN-α production, leading to an enhancement in the expression of ISGs and contributing to a broad-spectrum antiviral response. Thus, LC-Plasma may serve as a rational adjunctive treatment to ameliorate viral diseases, warranting future clinical trials.

Keywords: LC-Plasma; antiviral; arboviruses; interferon; probiotics.

Figure 1

The concentration of IFN-α in the supernatant of PBMCs stimulated with CpG ODN2216 (1 µM) and LC-Plasma (10 µg/mL). Box and violin graph illustrating the median ± SD (N = 5) of IFN-α concentration in the PBMC supernatant determined using ELISA. Neg Ctl = negative control, indicating PBMCs without treatment. * p < 0.05 (nonparametric Kruskal–Wallis and Steel test).

Figure 2

Fold increase in expression of CD86 and HLA-DR of pDCs after 24 h of CpG ODN 2216 (1 µM) and LC-Plasma (10 µg/mL) stimulation. Box and violin graph illustrating the median ± SD (N = 5) fold increase in expression of HLR-DR (left) and CD86 (right) of pDCs without stimulation (Neg Ctl) and after CpG ODN 2216 and LC-Plasma stimulation. * p < 0.05, ns= non-significant difference (nonparametric Kruskal–Wallis and Steel test).

Figure 3

Antiviral effects of supernatant from PBMCs stimulated with LC-Plasma against DENV, CHIKV, and ZIKV replication. Comparison of DENV, CHIKV, and ZIKV titers from 10-fold diluted supernatant of nonstimulated PBMC (Neg Sup 1:10), 10-fold diluted supernatants of LC-Plasma-stimulated PBMC (LCP Sup 1:10), and 10-fold diluted supernatants of CpG ODN 2216-stimulated PBMC (CpG Sup 1:10). The antiviral effects of LCP Sup 1:10 against DENV, CHIKV, and ZIKV replication were observed via qRT-PCR (a–c) and the infectious virus reduction assay (plaque/foci assay) (d–f) The Box and violin graphs represent the median ± SD (N = 5). * p < 0.05 indicates a significant difference, while # p < 0.1 indicates no significant difference but showed a tendency toward reduction (nonparametric Kruskal–Wallis and Steel test).

Figure 4

Induction of IFN-stimulated gene (ISG) expression in Huh-7 cells. Huh-7 cells were treated with recombinant IFN-α (100u), CpG Sup 1:10, LCP Sup 1:10, or Neg Sup 1:10, and the expression of ISGs was measured using qRT-PCR. The comparison included IFITM, ISG15, ISG20, MxA, OAS-1, RSAD2, and RyDEN expression in Huh-7 cells following treatments with recombinant IFN-α, CpG Sup 1:10, and LCP Sup 1:10. The gene expression levels were referenced to the untreated Huh-7 cells (cell control) and normalized using the housekeeping gene beta-actin. The Box and violin graph represents median ± SD (N = 5). * p < 0.05 indicates statistical significance (nonparametric Kruskal–Wallis and Steel tests).

Figure 5

Effects of blocking Type I IFN signaling. To further elucidate the mechanism of the anti-DENV effect of LC-Plasma, Huh-7 cells were treated with blocking antibodies against IFNAR2 for 1 h before stimulation with 10-fold diluted LCP Sup (LCP Sup 1:10). The Box and violin graph represents median ± SD (N = 5). * p < 0.05 indicates statistical significance (nonparametric Kruskal–Wallis and Steel tests).