Intranasal pretreatments with heat-killed DK128 confers protection against influenza H3N2 virus

In our previous study, we have reported that intranasal pretreatments with live Lactobacillus plantarum DK119 could develop resistance to influenza virus H1N1 infection in mice despite a certain degree of morbidity20. LAB DK128, a new isolate from fermented vegetables, was suggested to be a promising probiotic25. To determine whether pretreatments with heat-killed LAB endows mice with resistance to influenza virus, mock and heat-killed DK128-treated mice (BALB/c) were infected with a lethal dose of A/Philippines/82 (H3N2) virus (Fig. 1). BALB/c mice that were treated with heat-killed DK128 at a low dose, 1 × 107 CFU or 1 × 108 CFU showed approximately 12% to 10% weight loss (Fig. 1a,b) but all survived the lethal infection with H3N2 virus. In contrast, mice treated with heat-killed DK128 at a higher dose (1 × 109 CFU) prior to infection did not show weight loss, whereas mock-treated mice displayed severe weight loss reaching to the endpoint by day 8–9 post infection and all died (Fig. 1d,e,f). Thus, the efficacy of protection against influenza virus via heat-killed DK128 appeared to be dependent on the LAB doses of pretreatments. These results are highly significant because mice with heat-killed DK128 pretreatments can be protected against lethal influenza virus infection, resulting in 100% survival and prevention of weight loss.

Figure 1 Pretreatment of mice with heat-killed DK128 confers protection against H3N2 influenza virus (A/Phil/82) infection. BALB/c mice (n = 5/group) were intranasally (IN) pretreated with different doses of heat-killed DK128 at 4-day and 1-day prior to H3N2 influenza virus (2 LD 50 , A/Philippines/2/1982) infection. (a) Body weight change after H3N2 virus infection of mice with heat-killed DK128 (1 × 107 CFU/50 ul/mouse) pretreatment. (b) Body weight change after H3N2 virus infection of mice with heat-killed DK128 (1 × 108 CFU/50 ul/mouse) pretreatment. (c) Body weight change after H3N2 virus infection of mice with heat-killed DK128 (1 × 109 CFU/50 ul/mouse) pretreatment. (d) Survival rates after H3N2 virus infection of mice with heat-killed DK128 (1 × 107 CFU/50 ul/mouse) pretreatment. (e) Survival rate after H3N2 virus infection of mice with heat-killed DK128 (1 × 108 CFU/50 ul/mouse) pretreatment. (f) Survival rate after H3N2 virus infection of mice with heat-killed DK128 (1 × 109 CFU/50 ul/mouse) pretreatment. PBS.H3N2: mock-treated mice infected with H3N2 influenza virus. DK128.H3N2: mice with heat-killed DK128 pretreatment prior to H3N2 virus infection. Full size image

Mice with heat-killed DK128 pretreatment control lung viral loads and virus-induced pro-inflammatory cytokines to lower levels

In an independent set of experiments, we determined the efficacy of clearing lung viral loads, which is an important parameter for assessing protective efficacy. At 7 days after infection, mice were sacrificed and lung extract samples were diluted to determine egg infectious titers (Fig. 2a). Heat-killed DK128 (109 CFU) treated mice showed approximately 18-fold lower levels of viral titers than those in mock-treated mice after infection. Infection with a pathogenic influenza virus can cause excessive production of proinflammatory cytokines. IL-6 and TNF-α inflammatory cytokines were determined in BALF and lung samples. At 7 days after infection, IL-6 was detected at significantly lower levels in BALF and lung samples from heat-killed DK128 pretreated mice than those in mock-treated mice after infection (Fig. 2b,c). TNF-α was also significantly lower in BALF from heat-killed DK128 pretreated mice compared to naïve mice (Fig. 2d) despite no statistical difference in the lung TNF-α levels between the two groups (Fig. 2e). Therefore, the induction of proinflammatory cytokines due to viral infection was substantially reduced as a result of heat-killed DK128 treatment.

Figure 2 Pretreatment of BALB/c mice with heat-killed DK128 lowers lung viral loads and inflammatory cytokines upon H3N2 influenza virus infection. BALB/c mice (n = 5/group) were intranasally (IN) pre-treated with heat-killed DK128 (1 × 109 CFU/50 μl/mouse) at 4-day and 1-day prior to infection with H3N2 virus (2 LD 50 , A/Philippines/2/1982). Mice were sacrificed at 7 days after infection to determine lung virus titers and cytokines in bronchoalveolar lavage fluids (BALF) and lung extracts. (a) Lung virus titers (Log 10 EID 50 /0.2 ml). (b) IL-6 (pg/ml) in BALF. (c) IL-6 (pg/ml) in lung. (d) TNF-α (pg/ml) in BALF. (e) TNF-α (pg/ml) in lung. PBS: uninfected mouse control. PBS.H3N2: mock-treated mice infected with H3N2 influenza virus. DK128.H3N2: heat-killed DK128 pretreatment prior to H3N2 virus infection. ND indicates not detected. Cytokine levels were described as mean ± SEM. Symbols *, *** denote p < 0.05 and 0.001 respectively by Two-tailed Student’s paired t test. The viral loads in the lungs was described as mean ± SEM. ** denotes p < 0.01 by Two-tailed Student’s paired t test. Full size image

Recruitment of virus-induced innate immune cells is differentially modulated by heat-killed DK128 pretreatment

Pathogenic influenza virus can cause severe inflammatory disease in lower respiratory tracts and lungs. Thus, the analysis of immune cell populations recruited to the site of virus infection would provide insight into the possible protective mechanisms mediated by heat-killed DK128 pretreatment. At 7 days after infection with lethal A/Philippines (H3N2) virus, we collected lung and BALF samples to analyze phenotypes of immune cells by multi-color flow cytometry. Alveolar macrophage phenotypic (CD11C+CD11b−F4/80+) cells were observed at higher levels in BALF from mice with heat-killed DK128 pretreatment than those in mock-treated mice with severe weight loss (Fig. 3a).

Figure 3 BALB/c mice with heat-killed DK128 pretreatment differentially modulate infiltrating innate immune cells in bronchoalveolar lavage fluids (BALF) and lungs upon H3N2 influenza virus infection. BALB/c mice (n = 5/group) were intranasally (IN) pretreated with heat-killed DK128 (1 × 109 CFU/50 μl/mouse) at 4-day and 1-day prior to H3N2 virus (2 LD 50 , A/Philippines/2/1982) infection. Mice were sacrificed day 7 post-infection to determine cell phenotypes and cellularity of infiltrating immune cells in BALF and lungs. (a–d) Infiltrated immune cells in BALF (cellularity/mouse); (a) Alveolar macrophages (CD11c+CD11b−F4/80+), (b) Monocytes (CD11c−CD11b+siglecF-Ly6chiF4/80+), (c) NK cells (F4/80-DX5+), (d) Activated NK cells (F4/80−DX5+CD69+). (e–h) Infiltrating immune cells in the lungs (cellularity/mouse); (e) Alveolar macrophages (CD11c+CD11b−F4/80+), (f) Monocytes (CD11c−CD11b+siglecF-Ly6chiF4/80+), (g) NK cells (F4/80-DX5+), (h) Activated NK cells (F4/80−DX5+CD69+). PBS: uninfected mouse control. PBS.H3N2: mock-treated mice infected with H3N2 influenza virus. DK128.H3N2: heat-killed DK128 pretreatment prior to H3N2 virus infection. The numbers of immune cells (per mouse in BALF or in the lung) were described as mean ± SEM. *, **, and *** denote p < 0.05, 0.1, and 0.001 respectively by One-way ANOVA and Tukey’s multiple comparison test. Full size image

In contrast, BALF and lung samples from mice with heat-killed DK128 pretreatment showed lower levels of monocytes (F4/80+CD11c−CD11b+Ly6ChiSiglecF−), natural killer cells (F4/80−DX5+CD69+), and activated natural killer cells (F4/80−DX5+CD69+) (Fig. 3b–d,f–h), while naïve untreated-mice showed significantly enhanced recruitment of monocytes, natural killer cells, and activated natural killer cells in response to the lethal infection with A/Philippines (H3N2) virus (Fig. 3b–d,f–h). Cellular analysis of BALF and lungs suggest that LAB DK128-mediated modulation of innate immune cells may prevent pulmonary inflammation, contributing to protection against influenza virus infection.

To further detail the possible role of alveolar macrophages, we applied clodronate-liposome to deplete airway macrophages in mice with heat-killed LAB treatment prior to influenza virus lethal infection (Supplementary Fig. 1). We confirmed that over 90% alveolar macrophages were depleted in the lungs after clodronate-liposome treatment prior to influenza virus infection (Supplementary Fig. 1a). Upon lethal infection with influenza virus, the clodronate-treated LAB DK128 mice displayed severe weight loss of over 20% and a survival rate of 25% whereas control LAB DK128 mice (100%) survived lethal infection and prevented weight loss (DK.H3N2, Supplementary Fig. 1b,c). All mice without heat-killed LAB treatment died of infection (data not shown). These data indicate the protective roles of alveolar macrophages that were elevated by prior treatment with heat-killed LAB.

Heat-killed DK128 pretreatment of mice mediates earlier induction of virus specific antibodies upon infection

B cells are responsible for producing antibodies that play a critical role in establishing long-lived adaptive immunity. To better understand the possible roles of antibodies in conferring protection in the heat-killed DK128 pretreated mice, we compared virus specific antibody responses in heat-killed DK128 (108 CFU) treated and untreated BALB/c mice at 6 days after infection of with a low dose (1.5 x LD 50 ) of H3N2 virus (Fig. 4). Heat-killed DK128 pretreated mice displayed a moderate level of weight loss (6–9%) whereas mock control mice exhibited severe weight loss (20–25%) resulting in partial survival rates upon H3N2 virus infection (Fig. 4a,e). Interestingly, the mice with heat-killed DK128 pretreatment raised significantly higher levels of IgG, IgG1, and IgG2a antibodies at an earlier time point of day 6 post infection compared to those in mock untreated mice with infection (Fig. 4b,c,d). At a later time point of 14 days after infection, H3N2 virus infected naïve untreated mice that exhibited severe weight loss reached the high levels of IgG, IgG1, and IgG2a antibodies comparable to those from heat-killed DK128 treated mice (Fig. 4f,g,h). These results suggest that mice with heat-killed DK128 pretreatment are effective in inducing early IgG antibodies, possibly contributing to controlling viral replication as well as reducing weight loss whereas the untreated mice are ineffective in producing IgG antibodies at early times post infection, resulting in high viral replication and severe weight loss, and likely to die.

Figure 4 Heat-killed DK128 pretreatment mediates early induction of IgG antibodies after H3N2 virus infection. BALB/c mice (n = 5/group) were intranasally (IN) pretreated with heat-killed DK128 (1 × 108 CFU/50 μl/mouse) at 4-day and 1-day prior to infection with H3N2 virus (1.5 LD 50 , A/Philippines/2/1982). At 6 and 14 days after H3N2 virus infection, serum was collected and the levels of IgG and IgG isotypes specific for H3N2 virus were measured by enzyme-linked immunosorbent assay (ELISA). (a) Body weight changes in mice after H3N2 virus infection. (b–d) Antibody responses at 6 days after H3N2 virus infection; (b) IgG, (c) IgG1, (d) IgG2a. (e) Survival rates of mice after H3N2 virus infection. (f–h) Antibody responses at 14 days after H3N2 virus infection; (e) IgG, (f) IgG1, (g) IgG2a. PBS.H3N2: untreated mice with H3N2 virus infection, DK128.H3N2: mice with heat-killed DK128 pretreatment prior to H3N2 virus infection. Full size image

Protected BALB/c mice against H3N2 virus primary infection by heat-killed DK128 pretreatment acquire heterosubtypic immunity against secondary infection later

It is highly desirable for the hosts to acquire immunity against primary and secondary heterosubtypic viral infections without morbidity (weight loss). To determine cross-protective immunity against subsequent secondary infection, BALB/c mice that were protected against H3N2 virus primary infection as a result of the heat-killed DK128 pretreatment (Fig. 5a,c) were lethally challenged with heterosubtypic H1N1 (A/California/2009) pandemic virus at 4 weeks later (Fig. 5b). Naïve mice showed over 20% weight loss and 0% survival rates after infection with H1N1 pandemic virus (Fig. 5d). Mice that were protected against H3N2 A/Philippines/2/1982 virus primary infection as a result of heat-killed DK128 pretreatment displayed only transient 2–5% weight loss and 100% protection after secondary infection with heterosubtypic H1N1 virus (Fig. 5b,d).

Figure 5 Mice with heat-killed DK128 mediated protection against H3N2 virus primary infection show immunity to heterosubtypic H1N1 virus secondary infection. BALB/c mice (n = 5/group) were intranasally (IN) pretreated with heat-killed DK128 (109 CFU/50 μl/mouse) at 4-day and 1-day prior to infection with H3N2 virus (2 LD 50 , A/Philippines/2/1982). Body weight change and survival rate were monitored for 14 days. After primary virus (H3N2) infection, the protected mice were secondary infected with heterosubtypic virus (H1N1, 5 LD 50 , A/California/04/2009) and then weight loss and survival rate were monitored for 14 days. (a) Body weight changes after primary infection of BALB/c mice with H3N2 virus. (b) Body weight changes after secondary infection of BALB/c mice with H1N1 virus. (c) Survival rates after primary infection of BALB/c mice with H3N2 virus. (d) Survival rates after secondary infection of BALB/c mice with H1N1 virus. PBS.H3N2: H3N2 virus infection, DK128.H3N2: mice with heat-killed DK128 pretreatment prior to H3N2 virus infection, PBS.H1N1: H1N1 virus infection, DK128.H3N2.H1N1: The protected-mice against primary H3N2 virus via heat-killed DK128 pretreatment were then infected with secondary H1N1 virus. Full size image

To better understand the potential immune correlates conferring protection against secondary infection, an assay of hemagglutination inhibition (HI) was performed to determine cross protective HA antibodies. Consistent with IgG antibody levels (Fig. 4), sera from mice survived H3N2 primary infection regardless of LAB treatment showed high titers of HI activity against the homologous H3N2 virus (Supplementary Fig. 2a). Interestingly, sera from mice that were protected against H3N2 infection via prior LAB treatment showed substantial levels of HI activity against heterosubtypic H1N1 virus at a 2-fold higher level when compared to sera from mice survived H3N2 infection without prior LAB treatment (Supplementary Fig. 2b). Therefore, mice that were protected from primary infection (H3N2) via heat-killed DK128 pretreatment have acquired heterosubtypic immunity against secondary heterosubtypic infection (H1N1), most likely due to the elevated host immune responses during primary infection.

C57BL/6 mice with heat-killed DK128 pretreatment acquire immunity against primary H1N1 and secondary rgH5N1 virus

It is highly desirable if the protective effects of heat-killed LAB treatment would be expected in a strain non-specific pattern. We tested whether heat-killed DK128 pretreatment would mediate protection against a different subtype of influenza viruses in a different mouse strain. C57BL/6 mice (n = 5) were treated with heat-killed DK128 (1 × 109 CFU) at days -4 and -1 prior to infection with H1N1 virus (A/California/2009). As we observed with BALB/c mice, heat-killed DK128 treated C57BL/6 mice showed protection against lethal H1N1 virus infection with minimal weight loss of approximately 5% (Fig. 6a,d). In contrast, the mock control group did not survive viral infection and all died by day 9 post infection (Fig. 6a,d). Additionally, we determined whether heat-killed DK128-treated C57BL/6 mice that were protected against H1N1 virus primary infection without weight loss would be also protected against secondary infection with rgH5N1 virus (Fig. 6b,e). C57BL/6 mice that were protected against primary H1N1 virus via heat-killed DK128 pretreatment were completely protected against lethal infection with the secondary rgH5N1 virus which was lethal for all naïve mice (Fig. 6b,e). These results suggest that heat-killed DK128 pretreatment can equip the mice with the capacity to confer protective immunity against a broader range of influenza A virus primary and secondary infections. Sera from mice survived H1N1 primary infection showed high titers of HI activity against the homologous H1N1 virus (Supplementary Fig. 2c) as well as significant levels of HI activity against heterosubtypic rgH5N1 virus, which are higher than those of naïve serum control (Supplementary Fig. 2b). Also, sera of H1N1 infection showed cross reactive binding antibodies against rgH5N1 virus (data not shown). Thus, cross-reactive immune responses developed during primary infection appear to be partially responsible for conferring protection against secondary infection.

Figure 6 B cells are required to establish heat-killed DK128 mediated long-lasting immunity. Wilde type (C57BL/6) and B cell deficient (µMT) mice (n = 5/group) received intranasal pretreatment of heat-killed DK128 (109 CFU/50 μl/mouse) at 4-day and 1-day prior to H1N1 (2 LD 50 , A/California/04/2009) virus for primary infection. Body weight change and survival rate were monitored for 14 days. The protected-mice against primary H1N1 virus via heat-killed DK128 pretreatment were then infected with secondary heterosubtypic virus rgH5N1 (5 LD 50 , reassortant with HA from A/Indonesia/05/2005), and then weight changes and survival rates were monitored for 14 days. (a) Body weight changes after primary infection of C57BL/6 mice with H1N1 virus. (b) Body weight changes after secondary infection of C57BL/6 mice with rgH5N1 virus. (c) Body weight changes in µMT mice with H1N1 virus infection. (d) Survival rates of C57BL/6 mice with H1N1 virus infection. (e) Survival rates of C57BL/6 mice with rgH5N1 secondary virus infection. (f) Survival rate of µMT mice with H1N1 primary virus infection. PBS.H1N1: H1N1 virus infection, DK128.H1N1: mice with heat-killed DK128 pretreatment prior to H1N1 virus infection. PBS.H5N1: rgH5N1 virus infection, DK128.H1N1.H5N1: The protected-mice against primary H1N1 virus via heat-killed DK128 pretreatment were then infected with secondary rgH5N1 heterosubtypic virus. Full size image

B cells are required for establishing protective immunity in mice with heat-killed DK128 treatment

We observed that heat-killed DK128 pretreated mice elicited earlier induction of IgG isotype-switched antibodies specific for the infecting virus (Fig. 4), indicating the important roles of B cells. Using a B-cell deficient (µMT) mouse model, we further determined whether B-cells would play a critical role in establishing protective immunity against influenza virus infection via heat-killed DK128 pretreatments. B-cell deficient (µMT) mice (n = 5) were treated with heat-killed DK128 (109 CFU per mouse) at day 4 and day 1 prior to infection with H1N1 pandemic virus. Compared to the mock-treated µMT mice after infection, heat-killed DK128 pretreated µMT mice showed a delay of 3 to 5 days in displaying severe weight loss (Fig. 6c,f). Despite a prolonged delay in weight loss, the heat-killed DK128 treated µMT mice were deemed to progressive morbidity and did not survive H1N1 virus infection. These results suggest that B cells are essential for establishing sustained long-lasting protection although a significant delay in weight loss might be due to innate immunity mediated by DK128 pretreatment.

T cells are not required for protection against primary infection by heat-killed DK128 pretreatment but CD4 T cells partially contribute to preventing severe weight loss

In contrast to B cell-deficient mice, CD8 T cell-deficient (CD8KO) mice that received heat-killed DK128 pretreatment showed complete protection against H1N1 virus primary infection without weight loss (Fig. 7a,e). The CD4 T cell-deficient (CD4KO) mice that were pretreated with heat-killed DK128 were protected against H1N1 virus primary infection, displaying approximately 8% body weight loss and all CD4KO mice (100%) survived lethal infection (Fig. 7c,g). However, all mice died by day 9 post infection in the untreated mock control CD4KO and CD8KO groups (Fig. 7e,g).

Figure 7 CD4 T cells contribute to preventing weight loss in heat-killed DK128 pretreated mice after primary or secondary infection. CD8-deficient (CD8KO) and CD4-deficient (CD4KO) mice (n = 5/group) received intranasal pretreatment of heat-killed DK128 (109 CFU/50 μl/mouse) prior to H1N1 (2 LD 50 , A/California/04/2009) virus for primary infection, and then weight changes and survival rates were monitored for 14 days. The protected-mice against primary H1N1 virus via heat-killed DK128 pretreatment were then infected with secondary heterosubtypic virus rgH5N1 (5 LD 50 , A/Indonesia/05/2005), and then weight changes and survival rates were monitored for 14 days. (a) Body weight changes of CD8KO mice after primary infection with H1N1 virus. (b) Body weight changes of protected CD8KO mice against H1N1 virus after secondary infection with rgH5N1 virus. (c) Body weight changes of CD4KO mice after primary infection with H1N1 virus. (d) Body weight changes of CD4KO mice after secondary infection with rgH5N1 virus. (e) Survival rates of CD8KO mice after primary infection with H1N1 virus. (f) Survival rates of CD8KO mice after secondary infection with rgH5N1. (g) Survival rates of CD4KO mice after primary infection with H1N1 virus. (h) Survival rates of CD4KO mice after secondary infection with rgH5N1. PBS.H1N1: H1N1 virus infection, DK128.H1N1: mice with heat-killed DK128 pretreatment prior to H1N1 virus infection, PBS.H5N1: H5N1 virus infection, DK128.H1N1.H5N1: Protected mice against primary H1N1 virus via heat-killed DK128 pretreatment were exposed to secondary infection with rgH5N1. Full size image

Next, we determined the efficacy of subsequent protection against rgH5N1(A/Indonesia/05/2005) virus secondary infection in T cell-deficient mice that were well protected against H1N1 virus primary infection via heat-killed DK128 pretreatment (Fig. 7b,d). The CD8KO mice that were protected against H1N1 virus primary infection via heat-killed DK128 pretreatment without weight loss were well protected against rgH5N1 secondary virus, accompanying only a slight weight loss of 2–3% (Fig. 7b) under a condition with lethal infection (Fig. 7f). Whereas the CD4KO mice that survived H1N1 virus primary infection via killed-DK128 exhibited substantial weight loss of approximately 15–18% after secondary lethal infection with rgH5N1 virus under lethal infection (Fig. 7d,h). These results suggest that T cells are not needed for survival protection via heat-killed DK128 pretreatment against primary infection. However, CD4 T cells are required for establishing sufficient immunity to prevent severe weight loss during primary and secondary viral infections.