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Activation of HIF-1α and LL-37 by commensal bacteria inhibits Candida albicans colonization.

Fan D, Coughlin LA, Neubauer MM, Kim J, Kim MS, Zhan X, Simms-Waldrip TR, Xie Y, Hooper LV, Koh AY - Nat. Med. (2015)

Bottom Line: Candida albicans colonization is required for invasive disease.Although antibiotic treatment enables C. albicans colonization, pharmacologic activation of colonic Hif1a induces CRAMP expression and results in a significant reduction of C. albicans GI colonization and a 50% decrease in mortality from invasive disease.Thus, modulating C. albicans GI colonization by activation of gut mucosal immune effectors may represent a novel therapeutic approach for preventing invasive fungal disease in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas.

ABSTRACT
Candida albicans colonization is required for invasive disease. Unlike humans, adult mice with mature intact gut microbiota are resistant to C. albicans gastrointestinal (GI) colonization, but the factors that promote C. albicans colonization resistance are unknown. Here we demonstrate that commensal anaerobic bacteria-specifically clostridial Firmicutes (clusters IV and XIVa) and Bacteroidetes-are critical for maintaining C. albicans colonization resistance in mice. Using Bacteroides thetaiotamicron as a model organism, we find that hypoxia-inducible factor-1α (HIF-1α), a transcription factor important for activating innate immune effectors, and the antimicrobial peptide LL-37 (CRAMP in mice) are key determinants of C. albicans colonization resistance. Although antibiotic treatment enables C. albicans colonization, pharmacologic activation of colonic Hif1a induces CRAMP expression and results in a significant reduction of C. albicans GI colonization and a 50% decrease in mortality from invasive disease. In the setting of antibiotics, Hif1a and Camp (which encodes CRAMP) are required for B. thetaiotamicron-induced protection against C. albicans colonization of the gut. Thus, modulating C. albicans GI colonization by activation of gut mucosal immune effectors may represent a novel therapeutic approach for preventing invasive fungal disease in humans.

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Bacteroidetes thetaiotamicron induces Hif1a and Cramp in mouse colons(a) Hif1a and (b) Cramp mRNA expression in colons resected from CA colonization resistant mice (WT or STREP) and CA susceptible mice (PCN or PS). n=4. (c) HIF1a-Hif1a and (d) LL-37-Cramp mRNA expression measured in cultured human colonocytes exposed to ± CA and colons of antibiotic-treated mice ± CA colonization. n=4. (e) Hif1a and (f) Cramp mRNA expression measured in the colons of antibiotic treated mice ± oral gavage with commensal bacteria. n=4 (g) A representative western blot using an anti-CRAMP antibody (amino acids 135-173) against protein extracts from the distal colon of wild-type, Cramp KO, and antibiotic-treated mice ± oral gavage with commensal bacteria. Synthetic CRAMP peptide (5 ng, amino acids 135-173, 4.419 kDa) was used as a positive control. Cramp KO protein extracts were used as a negative control. Actin used as a loading control. Mouse CRAMP (amino acids 28-173) has a molecular mass of 16.422 kDa. A non-specific band (&) ~25kDa was detected in all lanes loaded with mouse colon protein extract. (f) Quantitative western blot analysis. Values obtained for CRAMP immunoblots were normalized to the optical density of corresponding immunoblots for actin. n=4. (i) Hif1a and (j) Cramp mRNA expression measured in the distal colon of germ-free mice colonized with CA, B. theta, or B. theta and CA. For all experiments, n=4. All data shown are means ± SEM. Statistical analysis by Mann-Whitney test. * p< 0.05; ** p<0.01; ns, not significant.
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Figure 3: Bacteroidetes thetaiotamicron induces Hif1a and Cramp in mouse colons(a) Hif1a and (b) Cramp mRNA expression in colons resected from CA colonization resistant mice (WT or STREP) and CA susceptible mice (PCN or PS). n=4. (c) HIF1a-Hif1a and (d) LL-37-Cramp mRNA expression measured in cultured human colonocytes exposed to ± CA and colons of antibiotic-treated mice ± CA colonization. n=4. (e) Hif1a and (f) Cramp mRNA expression measured in the colons of antibiotic treated mice ± oral gavage with commensal bacteria. n=4 (g) A representative western blot using an anti-CRAMP antibody (amino acids 135-173) against protein extracts from the distal colon of wild-type, Cramp KO, and antibiotic-treated mice ± oral gavage with commensal bacteria. Synthetic CRAMP peptide (5 ng, amino acids 135-173, 4.419 kDa) was used as a positive control. Cramp KO protein extracts were used as a negative control. Actin used as a loading control. Mouse CRAMP (amino acids 28-173) has a molecular mass of 16.422 kDa. A non-specific band (&) ~25kDa was detected in all lanes loaded with mouse colon protein extract. (f) Quantitative western blot analysis. Values obtained for CRAMP immunoblots were normalized to the optical density of corresponding immunoblots for actin. n=4. (i) Hif1a and (j) Cramp mRNA expression measured in the distal colon of germ-free mice colonized with CA, B. theta, or B. theta and CA. For all experiments, n=4. All data shown are means ± SEM. Statistical analysis by Mann-Whitney test. * p< 0.05; ** p<0.01; ns, not significant.

Mentions: We postulated that B. theta induces host immune effectors critical for maintaining colonization resistance and focused on the transcription factor, hypoxia-inducible factor (HIF)-1α, and the antimicrobial peptide LL-37-CRAMP. HIF-1α is an essential regulator of mammalian innate defense16 and increases expression of antimicrobial cathelicidin peptides17 in myeloid cells. Cathelicidin-related antimicrobial peptides are a family of polypeptides that serve a critical role in mammalian innate immune defense against bacterial infection16,17. The human cathelicidin LL-37 has been shown to have anti-Candida activity18 and inhibits CA adhesion to epithelial surfaces19. Interestingly, Hif1a and Cramp (cathelicidin-related antimicrobial peptide, LL-37 ortholog) expression significantly increased in colonization resistant mice compared to CA colonized mice (Fig. 3a-b). Both HIF1a-Hif1a and LL-37-Cramp expression significantly increased in both human colonocytes (HT-29) exposed to CA and in the colon of CA-colonized, antibiotic-treated mice (Fig. 3c-d). The colon had the highest concentration of fungi (Supplementary Fig. 3), thus all of our experiments utilize colons. Of note, other Candida spp. also induced mouse colonic expression of Hif1a and Cramp (Supplementary Fig. 4). Interestingly, B. theta and B. producta induced a significantly greater degree of mouse colonic Hif1a (mRNA) and Cramp-CRAMP (mRNA and protein) expression (Fig. 3e-h) compared to other commensal bacteria that we tested in the bacterial add-back experiments. In germ-free mice, co-colonization with B. theta and CA induced greater colonic Hif1a and Cramp expression (Fig. 3i-j) compared to mono-colonization with either B. theta or CA. We concluded that this additive mucosal immune stimulatory effect might explain how B theta facilitates CA colonization reduction in germ-free mice. Our findings suggest that Hif1a and Cramp may be critical immune effectors for maintaining CA colonization resistance.


Activation of HIF-1α and LL-37 by commensal bacteria inhibits Candida albicans colonization.

Fan D, Coughlin LA, Neubauer MM, Kim J, Kim MS, Zhan X, Simms-Waldrip TR, Xie Y, Hooper LV, Koh AY - Nat. Med. (2015)

Bacteroidetes thetaiotamicron induces Hif1a and Cramp in mouse colons(a) Hif1a and (b) Cramp mRNA expression in colons resected from CA colonization resistant mice (WT or STREP) and CA susceptible mice (PCN or PS). n=4. (c) HIF1a-Hif1a and (d) LL-37-Cramp mRNA expression measured in cultured human colonocytes exposed to ± CA and colons of antibiotic-treated mice ± CA colonization. n=4. (e) Hif1a and (f) Cramp mRNA expression measured in the colons of antibiotic treated mice ± oral gavage with commensal bacteria. n=4 (g) A representative western blot using an anti-CRAMP antibody (amino acids 135-173) against protein extracts from the distal colon of wild-type, Cramp KO, and antibiotic-treated mice ± oral gavage with commensal bacteria. Synthetic CRAMP peptide (5 ng, amino acids 135-173, 4.419 kDa) was used as a positive control. Cramp KO protein extracts were used as a negative control. Actin used as a loading control. Mouse CRAMP (amino acids 28-173) has a molecular mass of 16.422 kDa. A non-specific band (&) ~25kDa was detected in all lanes loaded with mouse colon protein extract. (f) Quantitative western blot analysis. Values obtained for CRAMP immunoblots were normalized to the optical density of corresponding immunoblots for actin. n=4. (i) Hif1a and (j) Cramp mRNA expression measured in the distal colon of germ-free mice colonized with CA, B. theta, or B. theta and CA. For all experiments, n=4. All data shown are means ± SEM. Statistical analysis by Mann-Whitney test. * p< 0.05; ** p<0.01; ns, not significant.
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Figure 3: Bacteroidetes thetaiotamicron induces Hif1a and Cramp in mouse colons(a) Hif1a and (b) Cramp mRNA expression in colons resected from CA colonization resistant mice (WT or STREP) and CA susceptible mice (PCN or PS). n=4. (c) HIF1a-Hif1a and (d) LL-37-Cramp mRNA expression measured in cultured human colonocytes exposed to ± CA and colons of antibiotic-treated mice ± CA colonization. n=4. (e) Hif1a and (f) Cramp mRNA expression measured in the colons of antibiotic treated mice ± oral gavage with commensal bacteria. n=4 (g) A representative western blot using an anti-CRAMP antibody (amino acids 135-173) against protein extracts from the distal colon of wild-type, Cramp KO, and antibiotic-treated mice ± oral gavage with commensal bacteria. Synthetic CRAMP peptide (5 ng, amino acids 135-173, 4.419 kDa) was used as a positive control. Cramp KO protein extracts were used as a negative control. Actin used as a loading control. Mouse CRAMP (amino acids 28-173) has a molecular mass of 16.422 kDa. A non-specific band (&) ~25kDa was detected in all lanes loaded with mouse colon protein extract. (f) Quantitative western blot analysis. Values obtained for CRAMP immunoblots were normalized to the optical density of corresponding immunoblots for actin. n=4. (i) Hif1a and (j) Cramp mRNA expression measured in the distal colon of germ-free mice colonized with CA, B. theta, or B. theta and CA. For all experiments, n=4. All data shown are means ± SEM. Statistical analysis by Mann-Whitney test. * p< 0.05; ** p<0.01; ns, not significant.
Mentions: We postulated that B. theta induces host immune effectors critical for maintaining colonization resistance and focused on the transcription factor, hypoxia-inducible factor (HIF)-1α, and the antimicrobial peptide LL-37-CRAMP. HIF-1α is an essential regulator of mammalian innate defense16 and increases expression of antimicrobial cathelicidin peptides17 in myeloid cells. Cathelicidin-related antimicrobial peptides are a family of polypeptides that serve a critical role in mammalian innate immune defense against bacterial infection16,17. The human cathelicidin LL-37 has been shown to have anti-Candida activity18 and inhibits CA adhesion to epithelial surfaces19. Interestingly, Hif1a and Cramp (cathelicidin-related antimicrobial peptide, LL-37 ortholog) expression significantly increased in colonization resistant mice compared to CA colonized mice (Fig. 3a-b). Both HIF1a-Hif1a and LL-37-Cramp expression significantly increased in both human colonocytes (HT-29) exposed to CA and in the colon of CA-colonized, antibiotic-treated mice (Fig. 3c-d). The colon had the highest concentration of fungi (Supplementary Fig. 3), thus all of our experiments utilize colons. Of note, other Candida spp. also induced mouse colonic expression of Hif1a and Cramp (Supplementary Fig. 4). Interestingly, B. theta and B. producta induced a significantly greater degree of mouse colonic Hif1a (mRNA) and Cramp-CRAMP (mRNA and protein) expression (Fig. 3e-h) compared to other commensal bacteria that we tested in the bacterial add-back experiments. In germ-free mice, co-colonization with B. theta and CA induced greater colonic Hif1a and Cramp expression (Fig. 3i-j) compared to mono-colonization with either B. theta or CA. We concluded that this additive mucosal immune stimulatory effect might explain how B theta facilitates CA colonization reduction in germ-free mice. Our findings suggest that Hif1a and Cramp may be critical immune effectors for maintaining CA colonization resistance.

Bottom Line: Candida albicans colonization is required for invasive disease.Although antibiotic treatment enables C. albicans colonization, pharmacologic activation of colonic Hif1a induces CRAMP expression and results in a significant reduction of C. albicans GI colonization and a 50% decrease in mortality from invasive disease.Thus, modulating C. albicans GI colonization by activation of gut mucosal immune effectors may represent a novel therapeutic approach for preventing invasive fungal disease in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas.

ABSTRACT
Candida albicans colonization is required for invasive disease. Unlike humans, adult mice with mature intact gut microbiota are resistant to C. albicans gastrointestinal (GI) colonization, but the factors that promote C. albicans colonization resistance are unknown. Here we demonstrate that commensal anaerobic bacteria-specifically clostridial Firmicutes (clusters IV and XIVa) and Bacteroidetes-are critical for maintaining C. albicans colonization resistance in mice. Using Bacteroides thetaiotamicron as a model organism, we find that hypoxia-inducible factor-1α (HIF-1α), a transcription factor important for activating innate immune effectors, and the antimicrobial peptide LL-37 (CRAMP in mice) are key determinants of C. albicans colonization resistance. Although antibiotic treatment enables C. albicans colonization, pharmacologic activation of colonic Hif1a induces CRAMP expression and results in a significant reduction of C. albicans GI colonization and a 50% decrease in mortality from invasive disease. In the setting of antibiotics, Hif1a and Camp (which encodes CRAMP) are required for B. thetaiotamicron-induced protection against C. albicans colonization of the gut. Thus, modulating C. albicans GI colonization by activation of gut mucosal immune effectors may represent a novel therapeutic approach for preventing invasive fungal disease in humans.

Show MeSH
Related in: MedlinePlus