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Targeting of a Fixed Bacterial Immunogen to Fc Receptors Reverses the Anti-Inflammatory Properties of the Gram-Negative Bacterium, Francisella tularensis, during the Early Stages of Infection.

Babadjanova Z, Wiedinger K, Gosselin EJ, Bitsaktsis C - PLoS ONE (2015)

Bottom Line: It is well established that IL-10 has the ability to inhibit robust antigen presentation by dendritic cells and macrophages, thus suppressing the generation of protective immunity.The pathogenesis of F. tularensis is not fully understood, and research has failed to develop an effective vaccine to this date.These results provide important insight for further understanding the protective immune mechanisms generated when targeting immunogens to Fc receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Seton Hall University, South Orange, New Jersey, United States of America.

ABSTRACT
Production of pro-inflammatory cytokines by innate immune cells at the early stages of bacterial infection is important for host protection against the pathogen. Many intracellular bacteria, including Francisella tularensis, the agent of tularemia, utilize the anti-inflammatory cytokine IL-10, to evade the host immune response. It is well established that IL-10 has the ability to inhibit robust antigen presentation by dendritic cells and macrophages, thus suppressing the generation of protective immunity. The pathogenesis of F. tularensis is not fully understood, and research has failed to develop an effective vaccine to this date. In the current study, we hypothesized that F. tularensis polarizes antigen presenting cells during the early stages of infection towards an anti-inflammatory status characterized by increased synthesis of IL-10 and decreased production of IL-12p70 and TNF-α in an IFN-ɣ-dependent fashion. In addition, F. tularensis drives an alternative activation of alveolar macrophages within the first 48 hours post-infection, thus allowing the bacterium to avoid protective immunity. Furthermore, we demonstrate that targeting inactivated F. tularensis (iFt) to Fcγ receptors (FcɣRs) via intranasal immunization with mAb-iFt complexes, a proven vaccine strategy in our laboratories, reverses the anti-inflammatory effects of the bacterium on macrophages by down-regulating production of IL-10. More specifically, we observed that targeting of iFt to FcγRs enhances the classical activation of macrophages not only within the respiratory mucosa, but also systemically, at the early stages of infection. These results provide important insight for further understanding the protective immune mechanisms generated when targeting immunogens to Fc receptors.

No MeSH data available.


Related in: MedlinePlus

FcγR targeting drives polarization of mouse macrophages towards the AM1 phenotype at the early stages of LVS infection.Lungs of immunized mice were harvested 24, 48 and 96 hours post-infection. For cell surface marker staining, cells were fluorescently labeled with antibodies against CD11b, F4/80, MHC class II, B7.1, B7.2, CCR7, or their corresponding isotype controls were added. Cells were then analyzed by flow cytometry on an LSRII flow cytometer (BD Biosciences). Results are representative of three independent experiments. (*) P-value < 0.1; (**) P-value < 0.05; bars represent the SD.
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pone.0129981.g006: FcγR targeting drives polarization of mouse macrophages towards the AM1 phenotype at the early stages of LVS infection.Lungs of immunized mice were harvested 24, 48 and 96 hours post-infection. For cell surface marker staining, cells were fluorescently labeled with antibodies against CD11b, F4/80, MHC class II, B7.1, B7.2, CCR7, or their corresponding isotype controls were added. Cells were then analyzed by flow cytometry on an LSRII flow cytometer (BD Biosciences). Results are representative of three independent experiments. (*) P-value < 0.1; (**) P-value < 0.05; bars represent the SD.

Mentions: Our results have suggested that one of the immune evasion mechanisms exploited by F. tularensis is the reduction of pro-inflammatory cytokines during the early stages of infection. To further investigate possible mechanisms responsible for this shift in the innate immune response, we analyzed the effect of FcγR targeting on macrophage activation and phenotype in the lungs of immunized mice. Previous research showed that one of the ways of F. tularensis survival and replication within the host cell is its ability to alter the macrophage activation from classically activated alveolar macrophages (AM1) to alternatively activated alveolar macrophages (AM2) [34]. The AM1 macrophages are characterized by high levels of pro-inflammatory cytokines and thus, play an essential role in anti-bacterial innate immune response. In contrast, AM2 macrophages are associated with high levels of anti-inflammatory cytokines, in particular IL-10 [35, 36]. Therefore, we performed flow cytometric analysis to assess the number of AM1 and AM2 present upon immunization with mAb-iFt. C57BL/6 mice were immunized with PBS, iFt, or mAb-iFt, boosted on day 21 and infected with a lethal dose of LVS on day 35 post-immunization. The lungs of euthanized mice were harvested 24, 48 and 96 hours post-challenge and the levels of F4/80, CD11b, CCR7, MHC class II and B7.2 marker expression on white blood cells was analyzed by flow cytometry. Classically activated AM1 cells are characterized as F4/80+/CD11bint/CCR7+/MHC class II+/B7.2+ cells, while AM2 cells were identified as F4/80+/CD11bint/CCR7-/ MHC class II-/B7.2- [35, 37]. Our results indicate that the frequency and number of AM1 cells was significantly higher in mAb-iFt immunized group of mice compared to mice immunized with iFt alone at 24, 48, and 96 hours post challenge (Fig 6). AM that were CCR7+ were also positive for MHC class II and B7.2 and thus considered classically activated macrophages (AM1) while AM that were CCR7- were also negative for the MHC class II and B7.2 markers, indicative of an alternative activated macrophage (AM2) (data not shown). Interestingly, the levels of AM1 cells were comparable among the different immunized groups at 96 hours of post challenge. In addition, our data showed a significantly lower number of AM2 cells in the lungs of mAb-iFt immunized mice relative to mice immunized with iFt alone at all three time points post-infection. It is also of interest that mice immunized with PBS had the highest frequency of AM2 cells, proposing that AM2 polarization in vivo may be an additional immune evasion strategy of F. tularensis, especially at the early stages of infection.


Targeting of a Fixed Bacterial Immunogen to Fc Receptors Reverses the Anti-Inflammatory Properties of the Gram-Negative Bacterium, Francisella tularensis, during the Early Stages of Infection.

Babadjanova Z, Wiedinger K, Gosselin EJ, Bitsaktsis C - PLoS ONE (2015)

FcγR targeting drives polarization of mouse macrophages towards the AM1 phenotype at the early stages of LVS infection.Lungs of immunized mice were harvested 24, 48 and 96 hours post-infection. For cell surface marker staining, cells were fluorescently labeled with antibodies against CD11b, F4/80, MHC class II, B7.1, B7.2, CCR7, or their corresponding isotype controls were added. Cells were then analyzed by flow cytometry on an LSRII flow cytometer (BD Biosciences). Results are representative of three independent experiments. (*) P-value < 0.1; (**) P-value < 0.05; bars represent the SD.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482730&req=5

pone.0129981.g006: FcγR targeting drives polarization of mouse macrophages towards the AM1 phenotype at the early stages of LVS infection.Lungs of immunized mice were harvested 24, 48 and 96 hours post-infection. For cell surface marker staining, cells were fluorescently labeled with antibodies against CD11b, F4/80, MHC class II, B7.1, B7.2, CCR7, or their corresponding isotype controls were added. Cells were then analyzed by flow cytometry on an LSRII flow cytometer (BD Biosciences). Results are representative of three independent experiments. (*) P-value < 0.1; (**) P-value < 0.05; bars represent the SD.
Mentions: Our results have suggested that one of the immune evasion mechanisms exploited by F. tularensis is the reduction of pro-inflammatory cytokines during the early stages of infection. To further investigate possible mechanisms responsible for this shift in the innate immune response, we analyzed the effect of FcγR targeting on macrophage activation and phenotype in the lungs of immunized mice. Previous research showed that one of the ways of F. tularensis survival and replication within the host cell is its ability to alter the macrophage activation from classically activated alveolar macrophages (AM1) to alternatively activated alveolar macrophages (AM2) [34]. The AM1 macrophages are characterized by high levels of pro-inflammatory cytokines and thus, play an essential role in anti-bacterial innate immune response. In contrast, AM2 macrophages are associated with high levels of anti-inflammatory cytokines, in particular IL-10 [35, 36]. Therefore, we performed flow cytometric analysis to assess the number of AM1 and AM2 present upon immunization with mAb-iFt. C57BL/6 mice were immunized with PBS, iFt, or mAb-iFt, boosted on day 21 and infected with a lethal dose of LVS on day 35 post-immunization. The lungs of euthanized mice were harvested 24, 48 and 96 hours post-challenge and the levels of F4/80, CD11b, CCR7, MHC class II and B7.2 marker expression on white blood cells was analyzed by flow cytometry. Classically activated AM1 cells are characterized as F4/80+/CD11bint/CCR7+/MHC class II+/B7.2+ cells, while AM2 cells were identified as F4/80+/CD11bint/CCR7-/ MHC class II-/B7.2- [35, 37]. Our results indicate that the frequency and number of AM1 cells was significantly higher in mAb-iFt immunized group of mice compared to mice immunized with iFt alone at 24, 48, and 96 hours post challenge (Fig 6). AM that were CCR7+ were also positive for MHC class II and B7.2 and thus considered classically activated macrophages (AM1) while AM that were CCR7- were also negative for the MHC class II and B7.2 markers, indicative of an alternative activated macrophage (AM2) (data not shown). Interestingly, the levels of AM1 cells were comparable among the different immunized groups at 96 hours of post challenge. In addition, our data showed a significantly lower number of AM2 cells in the lungs of mAb-iFt immunized mice relative to mice immunized with iFt alone at all three time points post-infection. It is also of interest that mice immunized with PBS had the highest frequency of AM2 cells, proposing that AM2 polarization in vivo may be an additional immune evasion strategy of F. tularensis, especially at the early stages of infection.

Bottom Line: It is well established that IL-10 has the ability to inhibit robust antigen presentation by dendritic cells and macrophages, thus suppressing the generation of protective immunity.The pathogenesis of F. tularensis is not fully understood, and research has failed to develop an effective vaccine to this date.These results provide important insight for further understanding the protective immune mechanisms generated when targeting immunogens to Fc receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Seton Hall University, South Orange, New Jersey, United States of America.

ABSTRACT
Production of pro-inflammatory cytokines by innate immune cells at the early stages of bacterial infection is important for host protection against the pathogen. Many intracellular bacteria, including Francisella tularensis, the agent of tularemia, utilize the anti-inflammatory cytokine IL-10, to evade the host immune response. It is well established that IL-10 has the ability to inhibit robust antigen presentation by dendritic cells and macrophages, thus suppressing the generation of protective immunity. The pathogenesis of F. tularensis is not fully understood, and research has failed to develop an effective vaccine to this date. In the current study, we hypothesized that F. tularensis polarizes antigen presenting cells during the early stages of infection towards an anti-inflammatory status characterized by increased synthesis of IL-10 and decreased production of IL-12p70 and TNF-α in an IFN-ɣ-dependent fashion. In addition, F. tularensis drives an alternative activation of alveolar macrophages within the first 48 hours post-infection, thus allowing the bacterium to avoid protective immunity. Furthermore, we demonstrate that targeting inactivated F. tularensis (iFt) to Fcγ receptors (FcɣRs) via intranasal immunization with mAb-iFt complexes, a proven vaccine strategy in our laboratories, reverses the anti-inflammatory effects of the bacterium on macrophages by down-regulating production of IL-10. More specifically, we observed that targeting of iFt to FcγRs enhances the classical activation of macrophages not only within the respiratory mucosa, but also systemically, at the early stages of infection. These results provide important insight for further understanding the protective immune mechanisms generated when targeting immunogens to Fc receptors.

No MeSH data available.


Related in: MedlinePlus