Elsevier

Antiviral Research

Volume 75, Issue 3, September 2007, Pages 266-274
Antiviral Research

Interactions of macrophages with probiotic bacteria lead to increased antiviral response against vesicular stomatitis virus

https://doi.org/10.1016/j.antiviral.2007.03.013Get rights and content

Abstract

Macrophages are an important cellular component of the innate immune system and are normally rapidly recruited and/or activated at the site of virus infection. They can participate in the antiviral response by killing infected cells, by producing antiviral cytokines such as nitric oxide and by producing chemokines and immunoregulatory cytokines that enable the adaptive immune response to recognize infected cells and perform antiviral effector functions. Probiotics, as a part of the normal gut intestinal flora, are important in supporting a functional yet balanced immune system. Improving our understanding of their role in the activation of macrophages and their stimulation of proinflammatory cytokine production in early viral infection was the main goal of this study. Our in vitro model study showed that probiotic bacteria, either from the species Lactobacillus or Bifidobacteria have the ability to decrease viral infection by establishing the antiviral state in macrophages, by production of NO and inflammatory cytokines such as interleukin 6 and interferon-gamma. These effects correlated with the mitochondrial activity of infected macrophages, therefore, the measurements of mitochondrial dehydrogenases activity could be implied as the first indicator of potential inhibitory effects of the probiotics on virus replication. The interactions between probiotic bacteria, macrophages and vesicular stomatitis virus (VSV), markedly depended on the bacterial strain studied.

Introduction

In host–pathogen interactions, nonspecific and highly specific adaptive mechanisms of immune response have been developed by the host to combat pathogens. Macrophages and dendritic cells are first barrier against infections, located in tissue throughout the body where they sense signals via a variety of receptors for molecules including lipopolisacharide (LPS), mannose, CpG dinucleotides and lipotechoic acid that are conserved in bacteria, including intestinal microflora (Miettinen et al., 2000). These signals promote the secretion of a variety of cytokines that mobilise innate immune reactions and signal T cells to initiate specific responses against pathogens. Feedback communication is additionally directed from T cells to macrophages in the form of interferon-gamma (INF-γ)—a major stimulatory signal for macrophages. It became evident that, under some physiological and pathological conditions, macrophages themselves are an important source of INF-γ (Gessani et al., 1989, Morita et al., 2002). INF-γ activates several macrophage functions including antimicrobial activity that lead to increased eradication of intracellular pathogens. Activation of macrophages is an important step in impeding viral replication in the infected host, as shown for a variety of viruses, including herpes simplex virus (HSV), human immunodeficiency virus (HIV) and vesicular stomatitis virus (VSV) (Born et al., 1993, Ellermann-Eriksen, 2005, Pohjavuori et al., 2004, Bi and Reiss, 1995). Antigen processing and presentation to lymphocytes induces Th1-driven mediated cell response to infection but inhibits Th2 cell differentiation and production of anti-inflammatory cytokines such as IL-4, IL-5 and IL-13 (Gessani and Belardelli, 1998). In macrophages exposed to inflammatory stimuli, nitric oxide synthase (iNOS) is induced, which eventually results in production of nitric oxide (NO) around 18 h post-infection. The production of NO and other reactive oxygen species (ROS) is considered to be the second barrier of innate defense mechanisms (Kidd, 2003, Nathan, 1992). NO carries out a variety of tasks for the innate immune system such as the killing of virus-infected cells, tumor cells and parasitic pathogens (Hibbs et al., 1998). The antiviral effects of NO have been well documented in several viral infections (Ellermann-Eriksen, 2005, Paludan et al., 1998, Bi and Reiss, 1995). However, NO causes damage to DNA, proteins and lipids in cells and tissues and could thus be deleterious for the host (Henry et al., 1993, Szabo et al., 1996). Therefore, the final effect of NO should be viewed as the balance between antiviral versus toxic effects.

Recent decades have witnessed intense research interest in probiotic lactic acid bacteria – as defined by Metchnikoff (1908) – such as lactobacilli and bifidobacteria, which are most commonly present in fermented food. Several studies have pointed out that orally delivered probiotics can transiently colonize the gastrointestinal (GI) tract and actively communicate with immune system cells, at the local as well as at the systemic level (Acheson and Luccioli, 2004, Cross, 2002, Dugas et al., 1999, Gill, 1998). Recent reports have additionally indicated that surveillance cells of the GI system respond to these de novo colonizers by the release of proinflammatory cytokines such as tumor necrosis factor (TNF-α), interleukin (IL)-12, IL-6 and interferon (INF-γ) or the production of anti-inflammatory/regulatory cytokines (such as the transforming growth factors TGF-β and IL-10) with the specific tumor necrosis factor or transforming growth factors being dependent on the strain of bacterium (Cross et al., 2004, Pohjavuori et al., 2004). Various strains of probiotic lactobacilli, for instance Lb. rhamnosus (strain GG), Lb. reuteri, Lb. casei Shirota and others, have been well characterized in terms of their ability to induce cytokine production following contact with macrophages (Cross et al., 2004, Morita et al., 2002).

Probiotics are undoubtedly important in supporting a functional yet balanced immune systems and further employment of immunomodulatory bacteria in health care can be seen in combating microbial pathogens, including viruses. Several studies involving Lb. rhamnosus (strain GG) as a probiotic strain showed a positive effect in treatment of rotavirus gastroenteritis in infants and children admitted to hospital. Immunological analyses of blood samples have shown that probiotic treatment can be associated with significant increases of rotavirus specific IgA titers (Kaila et al., 1995, Majamaa et al., 1995). However, understanding the role of probiotic and protective cultures against viral infections must be investigated in more detail.

This study was, to our knowledge, the first study that aimed to investigate the interactions between macrophages, pathogens and potentially protective probiotic cultures in early immune system defenses against viral infections in cell cultures of pig macrophages, using VSV as a model virus.

Section snippets

Cells

The 3D4/21 pig alveolar macrophage derived cell line was obtained as previously described by Weingartl et al. (2002).

Cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Sigma–Aldrich), supplemented with 10% Fetal Calf Serum (Biowhittaker, Europe), l-glutamine (2 mmol/l), penicillin (100 units/ml) and streptomycin (1 mg/ml) at 37 °C in a humidified 5% CO2 atmosphere in tissue culture flasks until confluent. The cell culture medium was regularly changed. To perform biological assays, the

The inhibition of VSV infection by pre-treatment of cell monolayer is stimulated by lactobacilli and bifidobacteria

All bacteria tested were able to attach to macrophage cell line 3D4/21, but at different rates (Fig. 1). The highest rate of attachment was shown for Lb. paracasei F19 (27%) followed equally by Lb. paracasei rhamnosus Q85, Lb. paracasei A14 (14%) and B. longum Q46 (9%). Increased cell survival (protective effect of probiotics) in the 3D4/21 cell line upon VSV challenge was observed upon treatment with live bacteria (Fig. 2) and was dependent on strain and bacterial number applied. Notably

Discussion

Several studies (Dugas et al., 1999, Gill et al., 2000, Miettinen et al., 1996) have reported the ex vivo/in vitro cytokine response when co-culturing cells of the innate immune defense system with probiotic bacteria. Moreover, induction of host immunity by such orally derived stimuli has been shown to play an important role in the defense against rapidly growing pathogenic bacteria (Reid and Burton, 2002). In contrast, little is known about the impact of interactions of probiotic bacteria with

Acknowledgments

This work was supported by funding from the EU-FP6 project PathogenCombat-FOOD-CT-2005-007081. The authors thank Lidija Gradišnik for her excellent technical assistance and Steven Boyne from Scottish Agriculture College for English revision of the manuscript.

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