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Characterization of the interferon-producing cell in mice infected with Listeria monocytogenes.

Stockinger S, Kastner R, Kernbauer E, Pilz A, Westermayer S, Reutterer B, Soulat D, Stengl G, Vogl C, Frenz T, Waibler Z, Taniguchi T, Rülicke T, Kalinke U, Müller M, Decker T - PLoS Pathog. (2009)

Bottom Line: Synthesis of serum IFN-I did not require TLR9.In contrast, in vitro-differentiated pDC infected with L. monocytogenes needed TLR9 to transcribe IFN-I mRNA.Based on these data, we propose that the engagement of pDC, the mode of IFN-I mobilization, as well as the shaping of the antimicrobial innate immune response by IFN-I differ between intracellular pathogens.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, University of Vienna, Vienna, Austria.

ABSTRACT
Production of type I interferons (IFN-I, mainly IFNalpha and IFNbeta) is a hallmark of innate immune responses to all classes of pathogens. When viral infection spreads to lymphoid organs, the majority of systemic IFN-I is produced by a specialized "interferon-producing cell" (IPC) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pDC). It is unclear whether production of systemic IFN-I is generally attributable to pDC irrespective of the nature of the infecting pathogen. We have addressed this question by studying infections of mice with the intracellular bacterium Listeria monocytogenes. Protective innate immunity against this pathogen is weakened by IFN-I activity. In mice infected with L. monocytogenes, systemic IFN-I was amplified via IFN-beta, the IFN-I receptor (IFNAR), and transcription factor interferon regulatory factor 7 (IRF7), a molecular circuitry usually characteristic of non-pDC producers. Synthesis of serum IFN-I did not require TLR9. In contrast, in vitro-differentiated pDC infected with L. monocytogenes needed TLR9 to transcribe IFN-I mRNA. Consistent with the assumption that pDC are not the producers of systemic IFN-I, conditional ablation of the IFN-I receptor in mice showed that most systemic IFN-I is produced by myeloid cells. Furthermore, results obtained with FACS-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pDC is responsible for bulk IFN-I synthesis. The amount of IFN-I produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. Based on these data, we propose that the engagement of pDC, the mode of IFN-I mobilization, as well as the shaping of the antimicrobial innate immune response by IFN-I differ between intracellular pathogens.

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The role of TLR9 in mice and cells infected with L. monocytogenes.(A–C) C57BL/6 wt or TLR9−/− mice were injected i.p. with 5×106 L. monocytogenes (four mice per genotype) or PBS as a control (data not shown). After 24 h of infection, serum was collected and ELISAs for IFNβ (A) and IFNα (B) were performed. Data representing IFNβ (A) and IFNα (B) concentrations were log-transformed (after adding one) to achieve approximate normality. Linear models with genotype as fixed effect were fitted using SPSS. Means plus/minus standard errors of wt and mutant genotypes are plotted (after back-transformation), n.s.: not significant (p>0.05). (C) After 24 h of infection, mice were killed and spleens isolated. RNA was extracted from spleens of three infected mice per genotype (1–3) or from mice injected with PBS (−), and RT-PCR for the indicated genes was performed. As a normalization control, the housekeeping gene L32 was measured. (D–H) pDCs of the indicated genotypes were infected with L. monocytogenes at a MOI of 10. At the indicated time points, total RNA was prepared. The isolated RNA was reverse-transcribed, and induction of the IFNβ (D,E,G) or IFNα (F,H) genes was measured by real-time PCR. For normalization to a housekeeping gene, GAPDH was measured. (I) C57BL/6 wt or TLR9−/− mice (13 mice per genotype, data represent a summary of two experiments) were injected i.p. with 5×106 L. monocytogenes. Survival was monitored for ten days.
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ppat-1000355-g005: The role of TLR9 in mice and cells infected with L. monocytogenes.(A–C) C57BL/6 wt or TLR9−/− mice were injected i.p. with 5×106 L. monocytogenes (four mice per genotype) or PBS as a control (data not shown). After 24 h of infection, serum was collected and ELISAs for IFNβ (A) and IFNα (B) were performed. Data representing IFNβ (A) and IFNα (B) concentrations were log-transformed (after adding one) to achieve approximate normality. Linear models with genotype as fixed effect were fitted using SPSS. Means plus/minus standard errors of wt and mutant genotypes are plotted (after back-transformation), n.s.: not significant (p>0.05). (C) After 24 h of infection, mice were killed and spleens isolated. RNA was extracted from spleens of three infected mice per genotype (1–3) or from mice injected with PBS (−), and RT-PCR for the indicated genes was performed. As a normalization control, the housekeeping gene L32 was measured. (D–H) pDCs of the indicated genotypes were infected with L. monocytogenes at a MOI of 10. At the indicated time points, total RNA was prepared. The isolated RNA was reverse-transcribed, and induction of the IFNβ (D,E,G) or IFNα (F,H) genes was measured by real-time PCR. For normalization to a housekeeping gene, GAPDH was measured. (I) C57BL/6 wt or TLR9−/− mice (13 mice per genotype, data represent a summary of two experiments) were injected i.p. with 5×106 L. monocytogenes. Survival was monitored for ten days.

Mentions: The data collected so far suggest that the majority of IFN-I is produced by a splenic cell type that employs an IFNβ/IFNAR/IRF7 pathway for feed-forward amplification of IFNα synthesis. This profile does not match IFN-I production by pDC where expression of IFN-I genes is regulated by a pathway originating from endosomal TLRs [34]. To further exclude the relevance of this pathway for IFN-I production during Listeria infection, TLR9-deficient mice were analyzed. The data summarized in Figure 5 show that neither serum IFNβ or IFNα (Figure 5A and 5B), nor splenic IFN-I mRNA synthesis or response were reduced in the absence of TLR9 (Figure 5C, Figure S4A). By contrast, production of IFN-I mRNA in bone-marrow-derived pDCs exposed to L. monocytogenes showed strong (IFNβ) or absolute (IFNα) dependence on TLR9 and/or MyD88 (Figure 5D–5F). Both IFNβ and IFNα mRNA synthesis were strongly affected by IRF7 deficiency and, surprisingly, also the absence of IRF3 (Figure 5G and 5H). Unlike pDC, mDC produced IFNβ independently of the TLR9 pathway, resembling macrophages in this regard (Figure S4B). Consistent with IFN-I production (Figure 5A–5C), the absence of TLR9 did not increase survival following infection with L. monocytogenes (Figure 5I). Rather, TLR9 deficiency slightly enhanced the lethality of L. monocytogenes infection, suggesting that the TLR9 pathway plays a protective role.


Characterization of the interferon-producing cell in mice infected with Listeria monocytogenes.

Stockinger S, Kastner R, Kernbauer E, Pilz A, Westermayer S, Reutterer B, Soulat D, Stengl G, Vogl C, Frenz T, Waibler Z, Taniguchi T, Rülicke T, Kalinke U, Müller M, Decker T - PLoS Pathog. (2009)

The role of TLR9 in mice and cells infected with L. monocytogenes.(A–C) C57BL/6 wt or TLR9−/− mice were injected i.p. with 5×106 L. monocytogenes (four mice per genotype) or PBS as a control (data not shown). After 24 h of infection, serum was collected and ELISAs for IFNβ (A) and IFNα (B) were performed. Data representing IFNβ (A) and IFNα (B) concentrations were log-transformed (after adding one) to achieve approximate normality. Linear models with genotype as fixed effect were fitted using SPSS. Means plus/minus standard errors of wt and mutant genotypes are plotted (after back-transformation), n.s.: not significant (p>0.05). (C) After 24 h of infection, mice were killed and spleens isolated. RNA was extracted from spleens of three infected mice per genotype (1–3) or from mice injected with PBS (−), and RT-PCR for the indicated genes was performed. As a normalization control, the housekeeping gene L32 was measured. (D–H) pDCs of the indicated genotypes were infected with L. monocytogenes at a MOI of 10. At the indicated time points, total RNA was prepared. The isolated RNA was reverse-transcribed, and induction of the IFNβ (D,E,G) or IFNα (F,H) genes was measured by real-time PCR. For normalization to a housekeeping gene, GAPDH was measured. (I) C57BL/6 wt or TLR9−/− mice (13 mice per genotype, data represent a summary of two experiments) were injected i.p. with 5×106 L. monocytogenes. Survival was monitored for ten days.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654726&req=5

ppat-1000355-g005: The role of TLR9 in mice and cells infected with L. monocytogenes.(A–C) C57BL/6 wt or TLR9−/− mice were injected i.p. with 5×106 L. monocytogenes (four mice per genotype) or PBS as a control (data not shown). After 24 h of infection, serum was collected and ELISAs for IFNβ (A) and IFNα (B) were performed. Data representing IFNβ (A) and IFNα (B) concentrations were log-transformed (after adding one) to achieve approximate normality. Linear models with genotype as fixed effect were fitted using SPSS. Means plus/minus standard errors of wt and mutant genotypes are plotted (after back-transformation), n.s.: not significant (p>0.05). (C) After 24 h of infection, mice were killed and spleens isolated. RNA was extracted from spleens of three infected mice per genotype (1–3) or from mice injected with PBS (−), and RT-PCR for the indicated genes was performed. As a normalization control, the housekeeping gene L32 was measured. (D–H) pDCs of the indicated genotypes were infected with L. monocytogenes at a MOI of 10. At the indicated time points, total RNA was prepared. The isolated RNA was reverse-transcribed, and induction of the IFNβ (D,E,G) or IFNα (F,H) genes was measured by real-time PCR. For normalization to a housekeeping gene, GAPDH was measured. (I) C57BL/6 wt or TLR9−/− mice (13 mice per genotype, data represent a summary of two experiments) were injected i.p. with 5×106 L. monocytogenes. Survival was monitored for ten days.
Mentions: The data collected so far suggest that the majority of IFN-I is produced by a splenic cell type that employs an IFNβ/IFNAR/IRF7 pathway for feed-forward amplification of IFNα synthesis. This profile does not match IFN-I production by pDC where expression of IFN-I genes is regulated by a pathway originating from endosomal TLRs [34]. To further exclude the relevance of this pathway for IFN-I production during Listeria infection, TLR9-deficient mice were analyzed. The data summarized in Figure 5 show that neither serum IFNβ or IFNα (Figure 5A and 5B), nor splenic IFN-I mRNA synthesis or response were reduced in the absence of TLR9 (Figure 5C, Figure S4A). By contrast, production of IFN-I mRNA in bone-marrow-derived pDCs exposed to L. monocytogenes showed strong (IFNβ) or absolute (IFNα) dependence on TLR9 and/or MyD88 (Figure 5D–5F). Both IFNβ and IFNα mRNA synthesis were strongly affected by IRF7 deficiency and, surprisingly, also the absence of IRF3 (Figure 5G and 5H). Unlike pDC, mDC produced IFNβ independently of the TLR9 pathway, resembling macrophages in this regard (Figure S4B). Consistent with IFN-I production (Figure 5A–5C), the absence of TLR9 did not increase survival following infection with L. monocytogenes (Figure 5I). Rather, TLR9 deficiency slightly enhanced the lethality of L. monocytogenes infection, suggesting that the TLR9 pathway plays a protective role.

Bottom Line: Synthesis of serum IFN-I did not require TLR9.In contrast, in vitro-differentiated pDC infected with L. monocytogenes needed TLR9 to transcribe IFN-I mRNA.Based on these data, we propose that the engagement of pDC, the mode of IFN-I mobilization, as well as the shaping of the antimicrobial innate immune response by IFN-I differ between intracellular pathogens.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, University of Vienna, Vienna, Austria.

ABSTRACT
Production of type I interferons (IFN-I, mainly IFNalpha and IFNbeta) is a hallmark of innate immune responses to all classes of pathogens. When viral infection spreads to lymphoid organs, the majority of systemic IFN-I is produced by a specialized "interferon-producing cell" (IPC) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pDC). It is unclear whether production of systemic IFN-I is generally attributable to pDC irrespective of the nature of the infecting pathogen. We have addressed this question by studying infections of mice with the intracellular bacterium Listeria monocytogenes. Protective innate immunity against this pathogen is weakened by IFN-I activity. In mice infected with L. monocytogenes, systemic IFN-I was amplified via IFN-beta, the IFN-I receptor (IFNAR), and transcription factor interferon regulatory factor 7 (IRF7), a molecular circuitry usually characteristic of non-pDC producers. Synthesis of serum IFN-I did not require TLR9. In contrast, in vitro-differentiated pDC infected with L. monocytogenes needed TLR9 to transcribe IFN-I mRNA. Consistent with the assumption that pDC are not the producers of systemic IFN-I, conditional ablation of the IFN-I receptor in mice showed that most systemic IFN-I is produced by myeloid cells. Furthermore, results obtained with FACS-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pDC is responsible for bulk IFN-I synthesis. The amount of IFN-I produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. Based on these data, we propose that the engagement of pDC, the mode of IFN-I mobilization, as well as the shaping of the antimicrobial innate immune response by IFN-I differ between intracellular pathogens.

Show MeSH
Related in: MedlinePlus