• lactobacilli;
  • polyinosinic:polycytidylic acid;
  • pro-inflammatory cytokine;
  • Toll-like receptor


  1. Top of page
  2. Abstract

Lactobacilli are frequently used as probiotics due to their beneficial effects on health. Lactobacillus casei Zhang (LcZ), which has favorable probiotic properties, was first isolated from koumiss. In this study, the immunomodulating effects of LcZ on cytokine and toll-like receptor expression in RAW264.7 macrophages was assessed and it was found that live LcZ promotes production of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6 and interferon (IFN)-β. Transcription of inducible nitric oxide synthase (iNOS) was also enhanced by viable LcZ. The immunostimulating effects of live LcZ are significantly attenuated in heat-killed LcZ. Live LcZ promotes TLR2 mRNA transcription, whereas heat-killed LcZ enhances transcription of TLR2, TLR3, TLR4 and TLR9. Furthermore, live LcZ significantly suppresses polyinosinic:polycytidylic acid (poly I:C)-stimulated NO, iNOS and TNF-α expression while enhancing expression of IFN-β. It was also found that poly I:C-induced interferon regulatory factor 3 (IRF-3) reporter gene activity was significantly up-regulated by live LcZ. These results suggest that LcZ keeps the innate immune system alert by increasing transcription of Toll-like receptors and enhancing production of pro-inflammatory mediators and type I IFN in macrophages. The synergistic effect of live LcZ with poly I:C on IFN-β expression is associated with increased activity of IRF-3. LcZ has the potential to be used as an adjuvant against viral infections.

List of Abbreviations:

colony forming unit


double-stranded RNA






inducible nitric oxide synthase


interferon regulatory factor 3


lactic acid bacteria


Lactobacillus casei Zhang




nuclear factor-κ light chain enhancer of activated B cells


nitric oxide


optical density


pathogen associated molecular patterns

poly I:C

polyinosinic:polycytidylic acid


pattern recognition receptor


standard error of the mean


Toll-like receptor


tumor necrosis factor-α

A vast community of commensal bacteria belonging to hundreds of species lives in the human gastrointestinal tract. Interactions between the normal gut microbiota and host not only maintain gastrointestinal homeostasis but also prevent enteritis caused by pathogenic microorganisms ([1][3]).

Lactic acid bacteria, a group of commensal bacteria that includes lactobacilli, are widely used by humans as probiotics and the potential health benefits of consuming them are receiving increasing recognition [4], [5]. LAB reportedly exert therapeutic effects not only on gut-related diseases but also on diseases outside the gut, including, for example, cancer, asthma, diabetes and viral infections ([6][9]). The mechanisms of action of probiotics include improved efficiency of digestion and nutrient absorption and modulation of intestinal immunity. A growing volume of recent data has reported that consuming probiotic species induces immunomodulatory functions both locally (within the intestines) and systemically ([10][13]). However, the effects of probiotics on the immune system vary between strains and safety is a relevant issue when assessing live probiotics in clinical trials.

Innate immune responses are initiated in the gut through PRRs expressed on immune and epithelial cells [14]. TLRs are one such group of PRRs; they recognize PAMPs, including bacterial cell wall structures, genome DNA or viral DNA/RNA intermediates [15]. TLR3 is a receptor for dsRNA and can also be activated by poly I:C, a synthetic dsRNA analogue. Upon recognition of its ligand, TLR3 transmits signals to up-regulate expression of pro-inflammatory mediators through activation of NF-κB. It also induces production of type I IFN through activation of the transcription factor IRF-3. Production of type I IFN induced by poly I:C or viral dsRNA plays a key role in antiviral immune responses [16].

In fact, induction of cytokine production following contact with mononuclear phagocytic or accessory cells characteristically mediates many probiotic effects, a fact which adds to our understanding of the in vivo immunomodulatory effects of probiotics ([17][19]). However, the exact molecular mechanisms of probiotic action remain elusive.

Lactobacillus casei Zhang is a novel LAB that our lab recently isolated from koumiss. This probiotic candidate exhibits several favorable properties. Our in vivo experiments have shown that LcZ enhances immunity by increasing production of IgA and IFN-γ [20], [21]. In this study, we aimed to assess the effect of LcZ on the innate immune system. We focused on expression of cytokines and TLRs in RAW264.7 cells upon challenge with live or heat-killed LcZ. Furthermore, we paid particular attention to assessing the anti-viral capacity of LcZ by using poly I:C-activated macrophages as a model.


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  2. Abstract

Bacteria strain and culture conditions

Lactobacillus casei Zhang was isolated from koumiss; its probiotic properties have been described previously [20]. This strain was inoculated at 1% (vol/vol) and propagated in trypticase peptone yeast broth (Difco, Magog, QC, Canada) at 37°C for 24 hrs. Each culture was then activated twice in trypticase peptone yeast broth before use. Serial dilutions of freshly prepared culture were grown in de Man, Rogosa and Sharpe broth (Hopebio, Qingdao, China) and cultured anaerobically for 36 hrs prior to enumeration. The bacterial pellets were harvested by centrifugation, washed twice in PBS (pH 7.4) and adjusted to 109 CFU/mL. A heat-killed bacterial preparation was obtained by exposing freshly cultured bacterial samples to heat at 70°C for 30mins. Complete loss of cell viability was confirmed by the absence of bacterial growth on appropriate agar plates. The concentration of heat-killed LcZ was adjusted to correspond to live plate counts and used in further experiments.

Cell culture and exposure to bacteria

The murine macrophage cell line RAW264.7 was obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained in Dulbecco's modified Eagle medium (Gibco, NY, USA) containing 10% (v/v) FCS (Gibco) as described previously [22]. RAW264.7 cells (1 × 105 cells /well) were plated in fresh medium in 6-well plates overnight, and then exposed to live or heated-killed LcZ (1 × 106 CFU/mL) for 2, 6, 12, and 24 hrs. The supernatants were collected to measure production of NO, IL-6, TNF-α and IFN-β. The cells were washed with ice-cold PBS and used for RNA extraction. To assess the extent to which LcZ modulates cytokine expression by poly I:C (Sigma Aldrich, St. Louis, MO, USA)-stimulated macrophages, the bacteria (1 × 106 CFU/mL) were added 3 hrs prior to stimulation with poly I:C (10 μg/mL) for the indicated periods. The subsequent procedures were similar to those described above.

Detection of nitric oxide and cytokine expression

The concentration of NO in the supernatants of cell culture was determined using Griess reagent (Sigma Aldrich). Briefly, a mixture of Griess reagent, composed of equal amounts of 1% sulfanilamide and 0.1% naphtylethylenediamide in 2.5% phosphoric acid was prepared just before use. 100 μL of cell supernatants were mixed with equal amounts of the mixture of Griess reagent and incubated at room temperature for 10 mins. The OD at 570 nm was measured using a microplate reader (MK3, Thermo Scientific, Pittsburgh, PA, USA). The concentration of NO was calculated according to the sodium nitrite standard curve. The concentrations of IL-6, TNF-α and IFN-β in the supernatants were determined using commercially available ELISA kits (R & D Systems, Minneapolis, MN, USA).

RNA extraction and real-time polymerase chain reaction

RAW264.7 cells were harvested at various time points after stimulation and total RNA extracted with TRIzol reagent (Takara, Dalian, China) according to the manufacturer's instructions. RNA quality was verified by assessing the optical density at 260 nm (OD260) and 280 nm (OD280). 1 μg total RNA with OD260/OD280 ratios between 1.8 and 2.0 was used for reverse transcription to cDNA. Real time-PCR analysis for iNOS, IL-6, TNF-α, IFN-β, TLR2, TLR3, TLR4, and TLR9 mRNA was performed on ABI 7300 (Applied Biosystems, Foster, CA, USA) with a SYBR RT-PCR kit (Takara). The primer sequences for each gene are listed in Table 1. The cycling parameters were initiated by 5 mins at 95°C, followed by 40 cycles of 5 s at 95°C and 1 min at 60°C. Expression of target genes was normalized by the degree of endogenous control β-actin expression in each sample. All data from real-time PCR were obtained in duplicate in at least three independent experiments.

Table 1. The sequences of primers used in this study

Assay of interferon regulatory factor 3 luciferase reporter gene expression

RAW264.7 cells were co-transfected with a mixture of IRF3 luciferase reporter plasmid and pRL-TK Renilla luciferase plasmid. After 36 hrs of transfection, the cells were treated with poly I:C alone or in the presence of live or heat-killed LcZ (1 × 106 CFU/mL) for 6 hrs. Reporter gene activity was detected by the dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer's instructions. Data were normalized for transfection efficiency by dividing firefly luciferase activity by that of Renilla luciferase. The relative values are shown as fold increases against indicated controls [22]. Data were obtained in duplicate in four independent experiments.

Statistical data analysis

Experimental data are expressed as mean ± SEM. Statistical analysis (one-way analysis of variance) was performed using SPSS17.0 software, with the Scheffe formula for post hoc multiple comparisons. Values of P < 0.05 were considered statistically significant.


  1. Top of page
  2. Abstract

Live Lactobacillus casei Zhang increases production of nitric oxide, interleukin-6, tumor necrosis factor-α and interferon-β in RAW264.7 cells

We assessed the immunomodulating capacity of LcZ on murine RAW264.7 macrophages by examining its effect on expression of pro-inflammatory mediators. We co-incubated live or heat-killed LcZ with RAW264.7 cells for varying durations and the concentrations of cytokines and amount of NO measured. As shown in Figure 1, live LcZ time-dependently increased production of NO. The amounts of NO production at 12 hrs (61.22 ± 2.96 μM) and 24 hrs (71.13 ± 4.44 μM) were significantly higher in cells stimulated with live LcZ than in those stimulated with heat-killed LcZ (20.79 ± 2.55 μM at 12 hrs, 18.17 ± 2.43 μM at 24 hrs, P < 0.01). Compared with the effect of live LcZ, heat-killed LcZ only increased production of NO from 0 to 6 hrs. Nitric oxide synthases from L-arginine catalyze production of NO and iNOS is involved in immune responses [23]. We then compared iNOS transcription in viable and nonviable LcZ cultures. As shown in Figure 1b, live LcZ continuously increased transcription of iNOS, significantly stronger iNOS responses occurring at 12 and 24 hrs than with heat-killed LcZ (P < 0.01). Heat-killed LcZ induced significantly greater iNOS transcription than did live LcZ-treated macrophages at 6 hrs (P < 0.01). As to expression of IL-6, live LcZ also enhanced expression of IL-6 in RAW264.7 cells. We also found early induction of IL-6 transcription in heat-killed LcZ stimulated cells with more than a 16-fold increase in transcription of IL-6 after 6 hrs co-culture with RAW264.7 cells (Fig.1c, d). Only live LcZ significantly increased production of TNF-α (Fig. 1e, f). Expression of IFN-β increased significantly with treatment with live LcZ compared with nonviable LcZ (Fig. 1g, h, P < 0.01).


Figure 1. Expression of pro-inflammatory mediators and type I IFN production in macrophages stimulated by live and heat-killed LcZ. (a) Production of NO according to Griess assay. (b), (c), (e) and (g) Transcription of iNOS, IL-6, TNF-α, and IFN-β quantitated by real-time PCR. (d), (f) and (h) Concentrations of IL-6, TNF-α, and IFN-β quantitated by ELISA. Data are shown as mean ± SEM of three independent experiments and each experiment was done in duplicate. *, P < 0.05; **, P < 0.01.

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Effects of live and heat-killed Lactobacillus casei Zhang treatment on Toll-like receptor expression of murine macrophages

In addition to assessing expression of cytokines, we were also interested in determining whether LcZ affects expression of TLRs. As shown in Figure 2a, TLR2 gene transcription was increased by treatment with live LcZ, with sevenfold (7.63 ± 1.02) and tenfold (11.53 ± 0.92) increases in transcription of TLR2 mRNA as recorded by quantitative-PCR after 12 and 24 hrs treatment, respectively (2b). TLR4 mRNA expression peaked after 2 hrs of treatment with live LcZ. Live LcZ did not increase transcription of TLR3 (2a). We found no significant change in transcription of TLR9 in RAW264.7 cells stimulated with live LcZ (data not shown). Heat-killed LcZ had a different pattern of modulation of expression of Toll-like receptors than did live LcZ. Transcription of TLR2, TLR3, TLR4 and TLR9 significantly increased in RAW264.7 cells after 6 hrs when challenged with non-viable LcZ (Fig. 2c–f).


Figure 2. TLR expression induced by live and heat-killed LcZ. (a) Live LcZ were co-cultured with RAW264.7 cells for the indicated times and expression of TLR2, TLR3 and TLR4 assessed by RT-PCR. Results of one typical experiment of three are shown. (b) Real-time PCR analysis of TLR2 and TLR4 mRNA expression. (c–f) Transcription of TLR2, TLR3, TLR4, and TLR9 in macrophages induced by heat-killed LcZ quantitated by real-time PCR. In b–f, data are shown as mean ± SEM. Similar results were obtained from three independent experiments and each experiment was done in duplicate. *, P < 0.05; **, P < 0.01.

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Effects of Lactobacillus casei Zhang on polyinosinic:polycytidylic acid-induced pro-inflammatory cytokines, nitric oxide and type 1 interferon production in RAW264.7 cells

The above results show live LcZ modulates cytokine and TLR expression by RAW264.7 cells. Furthermore, we wanted to clarify the effect of live LcZ on macrophages upon infection. In this experiment, we used poly I:C (ligand for TLR3) to mimic a viral infection. As shown in Figure 3a and b, treatment with poly:IC for 6 and 12 hrs induced increased transcription of iNOS and production of NO, suggesting successful activation of RAW264.7 macrophages. Live LcZ significantly inhibited poly I:C-induced iNOS mRNA expression (P < 0.01). As shown in Figure 3c, live LcZ significantly inhibited poly I:C-induced TNF-α mRNA expression. Similarly, pretreatment with live LcZ also suppressed poly I:C-induced TNF-α protein expression as verified by ELISA (3d). Live LcZ also inhibited poly I:C-induced IL-6 production, although this difference was not statistically significant (data not shown). However, treatment with live LcZ together with poly I:C significantly increased production of IFN-β compared to cells treated with poly I:C alone (Fig. 3e, f). In summary, live LcZ suppressed poly I:C-induced NO and TNF-α expression, while promoting poly I:C-induced IFN-β expression.


Figure 3. Dual effects of live LcZ on Poly I:C-activated macrophages. RAW264.7 cells were pretreated with live LcZ for 3 hrs and then stimulated with poly I:C for the indicated times. (a) Real-time PCR quantitation of iNOS mRNA expression. (b) Production of NO according to Griess assay. (c, e) Real-time PCR quantitation of expression of TNF-α mRNA and IFN-β mRNA. (d, f) Production of TNF-α and IFN-β quantitated by ELISA. Bars represent mean ± SEM. The results were obtained in duplicate in three independent experiments. **P < 0.01.

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Heat-killed Lactobacillus casei Zhang inhibits polyinosinic:polycytidylic acid-triggered nitric oxide and interferon-β production in murine macrophages

As shown in Figure 4a, b, heat-killed LcZ significantly inhibited poly I:C-induced iNOS transcription and NO production. Heat-killed LcZ also inhibited poly I:C-induced TNF-α expression (Fig. 4c, d). We did not find suppression of poly I:C-induced IL-6 production by heat-killed LcZ (data not shown). Heat-killed LcZ also down-regulated expression of IFN-β in RAW264.7 cells as shown in Figure 4e, f.


Figure 4. Heat-killed LcZ suppresses production of pro-inflammatory cytokine, NO and type I IFN in poly I:C-activated macrophages. (a) Real-time PCR quantitation of iNOS mRNA. (b) Production of NO according to Griess assay. (c) Real-time PCR quantitation of TNF-α mRNA expression. (d) Production of TNF-α quantitated by ELISA. (e) Real-time PCR quantitation of IFN-β mRNA expression. (f) Production of IFN-β quantitated by ELISA. The values are shown as mean ± SEM. The data were obtained in duplicate in three independent experiments. *, P < 0.05; **, P < 0.01.

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Effects of live and heat-killed Lactobacillus casei Zhang on polyinosinic:polycytidylic acid-induced regulatory factor 3 activation in RAW264.7 macrophages

Finally, we studied the underlying mechanism for the synergistic effects of LcZ and poly I:C on expression of IFN-β by assessing poly I:C-triggered IRF-3 activity in the presence of viable or nonviable LcZ. As shown in Figure 5, live LcZ significantly increased poly I:C-induced IRF3 reporter gene activity, whereas heat-killed LcZ decreased it (P < 0.01).


Figure 5. Live and heat-killed LcZ different modulate the activity of IRF3 in poly I:C-activated macrophages. Macrophages were transfected with 100 ng IRF3 luciferase reporter plasmid and 20 ng pRL-TK Renilla luciferase. After 36 hrs of transfection, the cells were stimulated with poly I:C for 6 hrs. Luciferase activity was measured and normalized by Renilla luciferase activity. Data are shown as mean ± SEM. Similar results were obtained in four independent experiments and each experiment was done in duplicate. **, P < 0.01.

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  1. Top of page
  2. Abstract

The immunomodulatory effects of probiotic lactic acid bacteria, most notably the lactobacilli and bifidobacteria, have been widely studied. Multiple studies have shown that viable bacterial cells and heat-killed lactobacilli are capable of inducing production of NO, IL-6 and TNF-α in macrophage-like cell lines ([24][26]). However, some researchers have demonstrated that viable bacterial cells also induce higher concentrations of cytokines than d heat-killed cells [27], [28]. In this study, we showed that Lactobacillus casei Zhang exerts immunomodulatory effects on RAW264.7 macrophages in a unique way.

We found that live LcZ induces time-dependently increased secretion of NO in RAW264.7 macrophages. Furthermore, continuously increased expression of iNOS mRNA occurred in live LcZ treated cells. Our results indicate that modulation of expression of iNOS may mediate the NO-enhancing ability of LcZ. Notably, we found that live LcZ induced greater amounts of NO than did heat-killed LcZ 12 and 24 hrs after stimulation (1a). However, 6 hsr after stimulation iNOS transcription was significantly greater in heat-killed LcZ cultures than in live LcZ cultures (1b). Concerning the above results, we cannot simply conclude that live LcZ induces greater NO production and iNOS transcription than heat-killed LcZ. The earlier induction of iNOS transcription by heat-killed LcZ suggests that heat-killed LcZ function directly through the cell wall components rather than by colonizing the cells.

We saw similar kinetics in the expression of IL-6 induced by live and heat-killed LcZ. We noticed that live LcZ induced significantly greater IL-6 production than did heat-killed LcZ after 16 hrs co-culture with RAW264.7 cells. Our data are in line with a previous study in which viable Lactobacillus plantarum KFCC11389P reportedly induced greater concentrations of IL-6 and NO production than did heat-killed cells after 24 hrs co-culture with RAW264.7 cells [29].

Viable Lactobacillus casei Shirota reportedly induce higher concentrations of TNF-α in J774.1 macrophages than do heat-killed cells [28]. Our study confirmed this observation because live LcZ induced greater expression of TNF-α in RAW264.7 cells than did heat-killed LcZ (Fig. 1e, f). This indicates that the stimulatory effect of LcZ on TNF-α production is heat sensitive. This study is the first to note stimulatory effects of both live and heat-killed LcZ on IFN-β expression. We found that viable LcZ induces significantly greater IFN-β expression than does heat-killed LcZ. These findings suggest that the ability of LcZ to stimulate IFN-β expression is heat sensitive. Weiss et al. reported that Lactobacillus acidophilus NCFM triggers stronger expression of IFN-β, IL-2 and IL-10 in dendritic cells than does poly I:C, indicating the strong antiviral ability of Lactobacillus acidophilus NCFM [30]. Our results provide a plausible explanation for the adjuvant effect of peptidoglycan isolated from LcZ in avian influenza vaccination [31].

Because activation of TLRs plays a vital role in cytokine production, we went further and measured expression of TLRs after treatment with live or heat-killed LcZ. We found that both live and heat-killed LcZ enhance expression of TLR2. We also found early increases in TLR4 transcription in cells treated with live and heat-killed LcZ. Our results are consistent with those reported by Galdeano et al. [32]. Similarly, another study showed that administration of Lactobacillus casei to BALB/c mice increases expression of TLR2, TLR4 and TLR9 and enhances production of TNF-α, IFN-γ, and IL-10 in the inductor sites of the gut immune response [12]. The enhancement of TLR2 and TLR4 transcription suggests that LcZ may function through both receptors. Further studies are required to verify this possibility. The explanation for heat-killed LcZ inducing significant increases in TLR3 transcription is not yet known. Because unmethylated CpG motifs are frequently present in the genomes of many bacteria and viruses, the increased transcription of TLR9 in heat-killed LcZ-treated cells may be related to the stimulating effects of unmethylated CpG motif in the bacteria.

We are interested in clarifying the role of LcZ during viral infection. In our experiment, Poly I:C was used as a dsRNA virus analogue to mimic the conditions of viral infection. Our results showed that live LcZ boosts IFN-β production while inhibiting the inflammatory responses induced by poly I:C. There is a paradoxical aspect to the effects of LcZ. On one hand, it enhances production of pro-inflammatory cytokines and NO in RAW264.7 cells. On the other hand, it inhibits poly I:C-induced inflammatory responses. We believe that such bacteria both keep the innate system alert to potential pathogens and have anti-inflammatory effects when there is pathogen infection. The synergistic effect of Lactobacillus casei Zhang and poly I:C on IFN-β expression is unexpected. Because IRF3 activation leads to transcription of genes such as IFN-β [33], we then studied IRF3 reporter gene activity. Our results suggest that live LcZ enhancement of production of IFN-β in poly I:C–activated RAW264.7 cells correlates with increased activity of IRF3.

Because poly I:C is often used as an adjuvant for vaccines, we propose that live LcZ is also a good candidate adjuvant for promoting antiviral response. Further in vivo studies are required to verify the role of live LcZ during viral infections.

In summary, we found that both live and heat-killed LcZ improve the innate response of macrophages by enhancing the expression of pro-inflammatory cytokines and transcription of Toll-like receptors. In addition, live LcZ suppresses poly I:C-induced inflammatory responses while enhancing production of IFN-β. Furthermore, our results suggest there is a synergistic effect between live LcZ and poly I:C on IFN-β production. Our study indicates that LcZ may have clinical application. Live LcZ has the potential for use as an adjuvant against viral infections.


  1. Top of page
  2. Abstract

This study was supported by the National Natural Science Foundation of China (No.30801001, 31270922, 81260622), China Postdoctoral Science Foundation (No. 20110491553), Hi-Tech Research and Development Program of China (863 Planning, No.2011AA100901, 2011AA100902) and the Earmarked Fund for Modern Agro-industry Technology Research System (No. nycytx-0501).


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  2. Abstract

The authors declare that they have no conflicting interests.


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  2. Abstract
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