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Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection.

Liu Z, Reba S, Chen WD, Porwal SK, Boom WH, Petersen RB, Rojas R, Viswanathan R, Devireddy L - J. Exp. Med. (2014)

Bottom Line: Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron.In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro.Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.

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Affiliation: Case Comprehensive Cancer Center; Department of Pathology; Department of Medicine, Tuberculosis Research Institute and Division of Infectious Diseases; and Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106Case Comprehensive Cancer Center; Department of Pathology; Department of Medicine, Tuberculosis Research Institute and Division of Infectious Diseases; and Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106.

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Bdh2 production is repressed by TLRs. (A) Quantitative analysis of bdh2 mRNA 4 h after treatment of RAW264.7 macrophages with the indicated TLR ligands. Expression of levels of bdh2 in naive cells was set at 1. The relative mRNA levels in each sample were normalized to actin mRNA. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05, **, P < 0.01, ***, P < 0.001. (B) Quantitative analysis of bdh2 mRNA 4 h after treatment of primary bone marrow macrophages with LPS. Analysis was as described in A. Results are the means of three independent experiments ± SD. Statistical analysis by two-tailed unpaired Student’s t test: **, P < 0.01. (C) Time-course analysis of bdh2 mRNA at the indicated time points after LPS treatment of RAW264.7 macrophages. The expression level of bdh2 in naive cells was set at 1. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: *, P < 0.05; **, P < 0.01. (D) Analysis of bdh2 mRNA in LPS-treated RAW264.7 macrophages with or without cycloheximide. The expression level of bdh2 in cycloheximide only treated cells was set at 1. Inset, IL-6 levels in culture supernatants from the same cells determined by ELISA. The results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: *, P < 0.05. (E) Effect of cytokines on bdh2 mRNA level in RAW264.7 macrophages. Cells were treated with the indicated amounts of recombinant cytokines for 12 h and bdh2 mRNA levels were determined by qRT-PCR. The expression level of bdh2 mRNA in untreated cells was set at 1. The results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ns, not significant; *, P < 0.05. (F) Quantitative analysis of bdh2 mRNA in liver and spleen samples of 8-wk old female C57BL/6 WT mice after poly (I:C) or LPS injection. Values relative to the mRNA from PBS-injected mice. Analysis was as described in A. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001. (G) Quantitative analysis of bdh2 mRNA in liver samples of 8-wk-old female C57BL/6 WT mice after E. coli or S. aureus infection. Values relative to the mRNA from PBS-injected mice. Analysis was as described in A. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001. (H) Immunoblot analysis of Bdh2 in C57BL/6 WT mice after E. coli or S. aureus infection. Actin was used as a loading control. Molecular weight markers are indicated on the right. (I) Quantitative analysis of 24p3 in serum samples of WT and bdh2- mice after E. coli H9049 or S. aureus infection. Symbols represent individual mice. Bars represent the mean values. Data are representative of two independent experiments, each with at least three to four mice per group. Statistical analysis by two-tailed unpaired t test: *, P < 0.05; ***, P < 0.001. (J) Assessment of 2,5-DHBA levels in urine samples of control and bdh2- mice challenged with E. coli H9049 or S. aureus. Mass spectrometry analysis of cold ethanol precipitated urine samples. 2,5-DHBA levels were quantitated by comparing to known amounts of 2,5-DHBA. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: **, P < 0.01; ***, P < 0.001. (K) Quantitative analysis of bdh2 mRNA in liver and spleen samples of LPS-injected TLR4- mice. Values relative to the mRNA from PBS-injected mice. Analysis was as described in A. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by two-tailed unpaired Student’s t test: P < 0.05 was considered significant. (L) The bdh2 promoter is down-regulated by LPS stimulation in RAW264.7 macrophages. Progressive deletions in 5′ flanking region of the bdh2 gene. (left) Luciferase activity in naive RAW264.7 cells. (right) Luciferase activity in LPS-stimulated RAW264.7 cells. Bottom, predicted transcription factor binding sites. The DNA binding site for Blimp-1 is shown. Results are means of three independent experiments ± SD. (M) Mutations in Blimp-1 binding sites in murine bdh2 promoter blunts LPS-mediated repression. Base substitutions are indicated below. RAW264.7 cells were transfected with WT (−1.5 to +0.5 kb) or mutant bdh2-luc reporter plasmids containing base substitutions and luciferase activity was determined as in G. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001. (N) Blimp-1 silencing abrogates TLR4-mediated repression of bdh2 in RAW264.7 macrophages. Bdh2 message was quantified in LPS treated and Blimp-1 deficient RAW264.7 cells. The relative mRNA levels in each sample were normalized to actin mRNA. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: **, P < 0.01. (O) Luciferase activity in Blimp-1 repressed cells. RAW264.7 cells were co-transfected with the bdh2-luc reporter (−1.5 to +0.5 kb) and with either increasing amounts of blimp-1 siRNA or control siRNA and the luciferase activity was determined after LPS stimulation as in G. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001.
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fig6: Bdh2 production is repressed by TLRs. (A) Quantitative analysis of bdh2 mRNA 4 h after treatment of RAW264.7 macrophages with the indicated TLR ligands. Expression of levels of bdh2 in naive cells was set at 1. The relative mRNA levels in each sample were normalized to actin mRNA. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05, **, P < 0.01, ***, P < 0.001. (B) Quantitative analysis of bdh2 mRNA 4 h after treatment of primary bone marrow macrophages with LPS. Analysis was as described in A. Results are the means of three independent experiments ± SD. Statistical analysis by two-tailed unpaired Student’s t test: **, P < 0.01. (C) Time-course analysis of bdh2 mRNA at the indicated time points after LPS treatment of RAW264.7 macrophages. The expression level of bdh2 in naive cells was set at 1. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: *, P < 0.05; **, P < 0.01. (D) Analysis of bdh2 mRNA in LPS-treated RAW264.7 macrophages with or without cycloheximide. The expression level of bdh2 in cycloheximide only treated cells was set at 1. Inset, IL-6 levels in culture supernatants from the same cells determined by ELISA. The results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: *, P < 0.05. (E) Effect of cytokines on bdh2 mRNA level in RAW264.7 macrophages. Cells were treated with the indicated amounts of recombinant cytokines for 12 h and bdh2 mRNA levels were determined by qRT-PCR. The expression level of bdh2 mRNA in untreated cells was set at 1. The results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ns, not significant; *, P < 0.05. (F) Quantitative analysis of bdh2 mRNA in liver and spleen samples of 8-wk old female C57BL/6 WT mice after poly (I:C) or LPS injection. Values relative to the mRNA from PBS-injected mice. Analysis was as described in A. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001. (G) Quantitative analysis of bdh2 mRNA in liver samples of 8-wk-old female C57BL/6 WT mice after E. coli or S. aureus infection. Values relative to the mRNA from PBS-injected mice. Analysis was as described in A. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001. (H) Immunoblot analysis of Bdh2 in C57BL/6 WT mice after E. coli or S. aureus infection. Actin was used as a loading control. Molecular weight markers are indicated on the right. (I) Quantitative analysis of 24p3 in serum samples of WT and bdh2- mice after E. coli H9049 or S. aureus infection. Symbols represent individual mice. Bars represent the mean values. Data are representative of two independent experiments, each with at least three to four mice per group. Statistical analysis by two-tailed unpaired t test: *, P < 0.05; ***, P < 0.001. (J) Assessment of 2,5-DHBA levels in urine samples of control and bdh2- mice challenged with E. coli H9049 or S. aureus. Mass spectrometry analysis of cold ethanol precipitated urine samples. 2,5-DHBA levels were quantitated by comparing to known amounts of 2,5-DHBA. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: **, P < 0.01; ***, P < 0.001. (K) Quantitative analysis of bdh2 mRNA in liver and spleen samples of LPS-injected TLR4- mice. Values relative to the mRNA from PBS-injected mice. Analysis was as described in A. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by two-tailed unpaired Student’s t test: P < 0.05 was considered significant. (L) The bdh2 promoter is down-regulated by LPS stimulation in RAW264.7 macrophages. Progressive deletions in 5′ flanking region of the bdh2 gene. (left) Luciferase activity in naive RAW264.7 cells. (right) Luciferase activity in LPS-stimulated RAW264.7 cells. Bottom, predicted transcription factor binding sites. The DNA binding site for Blimp-1 is shown. Results are means of three independent experiments ± SD. (M) Mutations in Blimp-1 binding sites in murine bdh2 promoter blunts LPS-mediated repression. Base substitutions are indicated below. RAW264.7 cells were transfected with WT (−1.5 to +0.5 kb) or mutant bdh2-luc reporter plasmids containing base substitutions and luciferase activity was determined as in G. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001. (N) Blimp-1 silencing abrogates TLR4-mediated repression of bdh2 in RAW264.7 macrophages. Bdh2 message was quantified in LPS treated and Blimp-1 deficient RAW264.7 cells. The relative mRNA levels in each sample were normalized to actin mRNA. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: **, P < 0.01. (O) Luciferase activity in Blimp-1 repressed cells. RAW264.7 cells were co-transfected with the bdh2-luc reporter (−1.5 to +0.5 kb) and with either increasing amounts of blimp-1 siRNA or control siRNA and the luciferase activity was determined after LPS stimulation as in G. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001.

Mentions: In mammals, distinct combinations of at least 10 toll-like receptors (TLRs) discriminate between a large number of microbial components (Beutler, 2009; Kawai and Akira, 2010). Engagement of TLRs by cognate ligands leads to inflammatory and innate immune responses. Mammalian siderophore-deficient mice are resistant to bacterial infections suggesting that the siderophore participates in the innate immune response. We assessed TLR-mediated mammalian siderophore expression in immune cells by determining levels of bdh2 mRNA and protein in cultured naive macrophages (RAW264.7), as well as in macrophages stimulated with TLR agonists. As expected, cultured naive macrophages showed abundant transcription of bdh2 mRNA (Fig. 6 A). However, bdh2 mRNA was reduced upon stimulation with TLR agonists, except agonists for TLRs 5 and 7 (Fig. 6 A). Interestingly, among all TLR ligands, treatment with LPS showed the greatest repression of bdh2 expression (Fig. 6 A). This observation corroborates the finding that bdh2- mice are resistant to LPS producing E. coli. In primary macrophages derived from mouse bone marrow treatment with LPS also repressed bdh2 message (Fig. 6 B).


Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection.

Liu Z, Reba S, Chen WD, Porwal SK, Boom WH, Petersen RB, Rojas R, Viswanathan R, Devireddy L - J. Exp. Med. (2014)

Bdh2 production is repressed by TLRs. (A) Quantitative analysis of bdh2 mRNA 4 h after treatment of RAW264.7 macrophages with the indicated TLR ligands. Expression of levels of bdh2 in naive cells was set at 1. The relative mRNA levels in each sample were normalized to actin mRNA. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05, **, P < 0.01, ***, P < 0.001. (B) Quantitative analysis of bdh2 mRNA 4 h after treatment of primary bone marrow macrophages with LPS. Analysis was as described in A. Results are the means of three independent experiments ± SD. Statistical analysis by two-tailed unpaired Student’s t test: **, P < 0.01. (C) Time-course analysis of bdh2 mRNA at the indicated time points after LPS treatment of RAW264.7 macrophages. The expression level of bdh2 in naive cells was set at 1. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: *, P < 0.05; **, P < 0.01. (D) Analysis of bdh2 mRNA in LPS-treated RAW264.7 macrophages with or without cycloheximide. The expression level of bdh2 in cycloheximide only treated cells was set at 1. Inset, IL-6 levels in culture supernatants from the same cells determined by ELISA. The results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: *, P < 0.05. (E) Effect of cytokines on bdh2 mRNA level in RAW264.7 macrophages. Cells were treated with the indicated amounts of recombinant cytokines for 12 h and bdh2 mRNA levels were determined by qRT-PCR. The expression level of bdh2 mRNA in untreated cells was set at 1. The results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ns, not significant; *, P < 0.05. (F) Quantitative analysis of bdh2 mRNA in liver and spleen samples of 8-wk old female C57BL/6 WT mice after poly (I:C) or LPS injection. Values relative to the mRNA from PBS-injected mice. Analysis was as described in A. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001. (G) Quantitative analysis of bdh2 mRNA in liver samples of 8-wk-old female C57BL/6 WT mice after E. coli or S. aureus infection. Values relative to the mRNA from PBS-injected mice. Analysis was as described in A. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001. (H) Immunoblot analysis of Bdh2 in C57BL/6 WT mice after E. coli or S. aureus infection. Actin was used as a loading control. Molecular weight markers are indicated on the right. (I) Quantitative analysis of 24p3 in serum samples of WT and bdh2- mice after E. coli H9049 or S. aureus infection. Symbols represent individual mice. Bars represent the mean values. Data are representative of two independent experiments, each with at least three to four mice per group. Statistical analysis by two-tailed unpaired t test: *, P < 0.05; ***, P < 0.001. (J) Assessment of 2,5-DHBA levels in urine samples of control and bdh2- mice challenged with E. coli H9049 or S. aureus. Mass spectrometry analysis of cold ethanol precipitated urine samples. 2,5-DHBA levels were quantitated by comparing to known amounts of 2,5-DHBA. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: **, P < 0.01; ***, P < 0.001. (K) Quantitative analysis of bdh2 mRNA in liver and spleen samples of LPS-injected TLR4- mice. Values relative to the mRNA from PBS-injected mice. Analysis was as described in A. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by two-tailed unpaired Student’s t test: P < 0.05 was considered significant. (L) The bdh2 promoter is down-regulated by LPS stimulation in RAW264.7 macrophages. Progressive deletions in 5′ flanking region of the bdh2 gene. (left) Luciferase activity in naive RAW264.7 cells. (right) Luciferase activity in LPS-stimulated RAW264.7 cells. Bottom, predicted transcription factor binding sites. The DNA binding site for Blimp-1 is shown. Results are means of three independent experiments ± SD. (M) Mutations in Blimp-1 binding sites in murine bdh2 promoter blunts LPS-mediated repression. Base substitutions are indicated below. RAW264.7 cells were transfected with WT (−1.5 to +0.5 kb) or mutant bdh2-luc reporter plasmids containing base substitutions and luciferase activity was determined as in G. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001. (N) Blimp-1 silencing abrogates TLR4-mediated repression of bdh2 in RAW264.7 macrophages. Bdh2 message was quantified in LPS treated and Blimp-1 deficient RAW264.7 cells. The relative mRNA levels in each sample were normalized to actin mRNA. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: **, P < 0.01. (O) Luciferase activity in Blimp-1 repressed cells. RAW264.7 cells were co-transfected with the bdh2-luc reporter (−1.5 to +0.5 kb) and with either increasing amounts of blimp-1 siRNA or control siRNA and the luciferase activity was determined after LPS stimulation as in G. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001.
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fig6: Bdh2 production is repressed by TLRs. (A) Quantitative analysis of bdh2 mRNA 4 h after treatment of RAW264.7 macrophages with the indicated TLR ligands. Expression of levels of bdh2 in naive cells was set at 1. The relative mRNA levels in each sample were normalized to actin mRNA. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05, **, P < 0.01, ***, P < 0.001. (B) Quantitative analysis of bdh2 mRNA 4 h after treatment of primary bone marrow macrophages with LPS. Analysis was as described in A. Results are the means of three independent experiments ± SD. Statistical analysis by two-tailed unpaired Student’s t test: **, P < 0.01. (C) Time-course analysis of bdh2 mRNA at the indicated time points after LPS treatment of RAW264.7 macrophages. The expression level of bdh2 in naive cells was set at 1. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: *, P < 0.05; **, P < 0.01. (D) Analysis of bdh2 mRNA in LPS-treated RAW264.7 macrophages with or without cycloheximide. The expression level of bdh2 in cycloheximide only treated cells was set at 1. Inset, IL-6 levels in culture supernatants from the same cells determined by ELISA. The results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: *, P < 0.05. (E) Effect of cytokines on bdh2 mRNA level in RAW264.7 macrophages. Cells were treated with the indicated amounts of recombinant cytokines for 12 h and bdh2 mRNA levels were determined by qRT-PCR. The expression level of bdh2 mRNA in untreated cells was set at 1. The results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ns, not significant; *, P < 0.05. (F) Quantitative analysis of bdh2 mRNA in liver and spleen samples of 8-wk old female C57BL/6 WT mice after poly (I:C) or LPS injection. Values relative to the mRNA from PBS-injected mice. Analysis was as described in A. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001. (G) Quantitative analysis of bdh2 mRNA in liver samples of 8-wk-old female C57BL/6 WT mice after E. coli or S. aureus infection. Values relative to the mRNA from PBS-injected mice. Analysis was as described in A. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001. (H) Immunoblot analysis of Bdh2 in C57BL/6 WT mice after E. coli or S. aureus infection. Actin was used as a loading control. Molecular weight markers are indicated on the right. (I) Quantitative analysis of 24p3 in serum samples of WT and bdh2- mice after E. coli H9049 or S. aureus infection. Symbols represent individual mice. Bars represent the mean values. Data are representative of two independent experiments, each with at least three to four mice per group. Statistical analysis by two-tailed unpaired t test: *, P < 0.05; ***, P < 0.001. (J) Assessment of 2,5-DHBA levels in urine samples of control and bdh2- mice challenged with E. coli H9049 or S. aureus. Mass spectrometry analysis of cold ethanol precipitated urine samples. 2,5-DHBA levels were quantitated by comparing to known amounts of 2,5-DHBA. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: **, P < 0.01; ***, P < 0.001. (K) Quantitative analysis of bdh2 mRNA in liver and spleen samples of LPS-injected TLR4- mice. Values relative to the mRNA from PBS-injected mice. Analysis was as described in A. Results show pooled data from two independent experiments, each with at least three mice per group. Statistical analysis by two-tailed unpaired Student’s t test: P < 0.05 was considered significant. (L) The bdh2 promoter is down-regulated by LPS stimulation in RAW264.7 macrophages. Progressive deletions in 5′ flanking region of the bdh2 gene. (left) Luciferase activity in naive RAW264.7 cells. (right) Luciferase activity in LPS-stimulated RAW264.7 cells. Bottom, predicted transcription factor binding sites. The DNA binding site for Blimp-1 is shown. Results are means of three independent experiments ± SD. (M) Mutations in Blimp-1 binding sites in murine bdh2 promoter blunts LPS-mediated repression. Base substitutions are indicated below. RAW264.7 cells were transfected with WT (−1.5 to +0.5 kb) or mutant bdh2-luc reporter plasmids containing base substitutions and luciferase activity was determined as in G. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001. (N) Blimp-1 silencing abrogates TLR4-mediated repression of bdh2 in RAW264.7 macrophages. Bdh2 message was quantified in LPS treated and Blimp-1 deficient RAW264.7 cells. The relative mRNA levels in each sample were normalized to actin mRNA. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: **, P < 0.01. (O) Luciferase activity in Blimp-1 repressed cells. RAW264.7 cells were co-transfected with the bdh2-luc reporter (−1.5 to +0.5 kb) and with either increasing amounts of blimp-1 siRNA or control siRNA and the luciferase activity was determined after LPS stimulation as in G. Results are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test: ***, P < 0.001.
Mentions: In mammals, distinct combinations of at least 10 toll-like receptors (TLRs) discriminate between a large number of microbial components (Beutler, 2009; Kawai and Akira, 2010). Engagement of TLRs by cognate ligands leads to inflammatory and innate immune responses. Mammalian siderophore-deficient mice are resistant to bacterial infections suggesting that the siderophore participates in the innate immune response. We assessed TLR-mediated mammalian siderophore expression in immune cells by determining levels of bdh2 mRNA and protein in cultured naive macrophages (RAW264.7), as well as in macrophages stimulated with TLR agonists. As expected, cultured naive macrophages showed abundant transcription of bdh2 mRNA (Fig. 6 A). However, bdh2 mRNA was reduced upon stimulation with TLR agonists, except agonists for TLRs 5 and 7 (Fig. 6 A). Interestingly, among all TLR ligands, treatment with LPS showed the greatest repression of bdh2 expression (Fig. 6 A). This observation corroborates the finding that bdh2- mice are resistant to LPS producing E. coli. In primary macrophages derived from mouse bone marrow treatment with LPS also repressed bdh2 message (Fig. 6 B).

Bottom Line: Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron.In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro.Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.

View Article: PubMed Central - HTML - PubMed

Affiliation: Case Comprehensive Cancer Center; Department of Pathology; Department of Medicine, Tuberculosis Research Institute and Division of Infectious Diseases; and Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106Case Comprehensive Cancer Center; Department of Pathology; Department of Medicine, Tuberculosis Research Institute and Division of Infectious Diseases; and Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106.

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