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Siglec-5 and Siglec-14 are polymorphic paired receptors that modulate neutrophil and amnion signaling responses to group B Streptococcus.

Ali SR, Fong JJ, Carlin AF, Busch TD, Linden R, Angata T, Areschoug T, Parast M, Varki N, Murray J, Nizet V, Varki A - J. Exp. Med. (2014)

Bottom Line: GBS amnion immune activation was likewise influenced by the SIGLEC14- polymorphism.We provide initial evidence that the polymorphism could influence the risk of prematurity among human fetuses of mothers colonized with GBS.This first functionally proven example of a paired receptor system in the Siglec family has multiple implications for regulation of host immunity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Glycobiology Research and Training Center, Department of Cellular and Molecular Medicine, Department of Pediatrics, Department of Pathology, Department of Medicine, and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093Glycobiology Research and Training Center, Department of Cellular and Molecular Medicine, Department of Pediatrics, Department of Pathology, Department of Medicine, and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093Glycobiology Research and Training Center, Department of Cellular and Molecular Medicine, Department of Pediatrics, Department of Pathology, Department of Medicine, and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093.

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The unusual ectopic expression of Siglec-5 and Siglec-14 on human AMs influences inflammatory responses to GBS. (A) Human AMs were stained for Siglec-5 and Siglec-14 expression using a human Siglec-5– and Siglec-14–recognizing antibody and Siglec-14 antibody by immunohistochemistry. (B) Human AMs were incubated either with FITC-labeled GBS or FITC-labeled GBSΔβ, and bacterial binding to the membrane was analyzed by fluorescence microscopy. (A and B) Bars, 50 µm. (C) Sig-14−/− AMs were cut into small pieces of similar size and infected either with GBS or GBSΔβ; IL-6 mRNA was analyzed by Q-RT-PCR after 2 h of infection. Results were normalized to GAPDH mRNA. Results are means ± SD; *, P < 0.05. (D and E) Sig-14−/− AMs were infected either with GBS or GBSΔβ as above. At the indicated times, cell lysates were prepared and analyzed for p38 phosphorylation and IκB-α degradation (D) and phosphorylation of AKT and S6 (E) by immunoblotting. (F) AMs of the indicated genotypes were infected either with GBS or GBSΔβ. At the indicated times, cell lysates were prepared and analyzed for phosphorylation of AKT protein by immunoblotting. The p-AKT/tubulin (×10) densitometry ratio is shown on the blot. p-AKT/tubulin, IκB-α/tubulin, and p-S6/tubulin (×10) densitometry values are shown on corresponding blots. Data in A–F are representative of two to five independent experiments with two to three different amnions per genotype in each experiment. (G–L) Human amnion genotyping: Association of the SIGLEC14- allele with preterm birth in infants of GBS-positive pregnancies. (G and H) Tables showing distribution of WT and  alleles in term and preterm groups in infants of GBS+ (G) and GBS− (H) pregnancies. Fisher’s exact test was performed using data computing infant allele × term/preterm. (I and J) Percentage of infants with various genotypes (Sig-14+/+, Sig-14+/−, or Sig-14−/−) in preterm and term group; infants from GBS-positive and -negative pregnancies were analyzed in I and J, respectively. (K) Table showing the ratio of preterm to term genotype percentage obtained from I and J. (L) Association of SIGLEC14/5 alleles with GBS colonization in preterm infants. The table shows the distribution of WT and  alleles in GBS-positive and -negative infants in the preterm group only. Fisher’s exact test was performed using data computing infant allele × term/preterm.
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fig5: The unusual ectopic expression of Siglec-5 and Siglec-14 on human AMs influences inflammatory responses to GBS. (A) Human AMs were stained for Siglec-5 and Siglec-14 expression using a human Siglec-5– and Siglec-14–recognizing antibody and Siglec-14 antibody by immunohistochemistry. (B) Human AMs were incubated either with FITC-labeled GBS or FITC-labeled GBSΔβ, and bacterial binding to the membrane was analyzed by fluorescence microscopy. (A and B) Bars, 50 µm. (C) Sig-14−/− AMs were cut into small pieces of similar size and infected either with GBS or GBSΔβ; IL-6 mRNA was analyzed by Q-RT-PCR after 2 h of infection. Results were normalized to GAPDH mRNA. Results are means ± SD; *, P < 0.05. (D and E) Sig-14−/− AMs were infected either with GBS or GBSΔβ as above. At the indicated times, cell lysates were prepared and analyzed for p38 phosphorylation and IκB-α degradation (D) and phosphorylation of AKT and S6 (E) by immunoblotting. (F) AMs of the indicated genotypes were infected either with GBS or GBSΔβ. At the indicated times, cell lysates were prepared and analyzed for phosphorylation of AKT protein by immunoblotting. The p-AKT/tubulin (×10) densitometry ratio is shown on the blot. p-AKT/tubulin, IκB-α/tubulin, and p-S6/tubulin (×10) densitometry values are shown on corresponding blots. Data in A–F are representative of two to five independent experiments with two to three different amnions per genotype in each experiment. (G–L) Human amnion genotyping: Association of the SIGLEC14- allele with preterm birth in infants of GBS-positive pregnancies. (G and H) Tables showing distribution of WT and alleles in term and preterm groups in infants of GBS+ (G) and GBS− (H) pregnancies. Fisher’s exact test was performed using data computing infant allele × term/preterm. (I and J) Percentage of infants with various genotypes (Sig-14+/+, Sig-14+/−, or Sig-14−/−) in preterm and term group; infants from GBS-positive and -negative pregnancies were analyzed in I and J, respectively. (K) Table showing the ratio of preterm to term genotype percentage obtained from I and J. (L) Association of SIGLEC14/5 alleles with GBS colonization in preterm infants. The table shows the distribution of WT and alleles in GBS-positive and -negative infants in the preterm group only. Fisher’s exact test was performed using data computing infant allele × term/preterm.

Mentions: CD33-related Siglecs are mainly present on immune cells (Crocker et al., 2007). One exception is Siglec-6, which was unexpectedly found on the placental trophoblast (Brinkman-Van der Linden et al., 2007). While screening for other Siglecs in fetal tissues, we were surprised to find positive staining on the amniotic epithelial membrane (AM) of human placental sections with the antibody that recognizes Siglec-5 and Siglec-14 (Fig. 5 A). This staining is apparently unique to the human amnion, as it was not found in amnions from the closely related great apes (not depicted). Siglec-5 and Siglec-14 protein expression on human AM was confirmed by Western blot (not depicted). This unusual tissue site of Siglec human-specific expression is particularly intriguing because the human-specific pathogen GBS produces ascending infections of the placental membranes, gaining access to the amniotic fluid and fetus and leading to the potentially life-threatening early-onset pneumonia and sepsis (Edwards et al., 2011). The ability of fluorescently labeled GBS to bind to AM was established, and the key contribution of the surface-anchored Siglec-binding β-protein was confirmed (Fig. 5 B).


Siglec-5 and Siglec-14 are polymorphic paired receptors that modulate neutrophil and amnion signaling responses to group B Streptococcus.

Ali SR, Fong JJ, Carlin AF, Busch TD, Linden R, Angata T, Areschoug T, Parast M, Varki N, Murray J, Nizet V, Varki A - J. Exp. Med. (2014)

The unusual ectopic expression of Siglec-5 and Siglec-14 on human AMs influences inflammatory responses to GBS. (A) Human AMs were stained for Siglec-5 and Siglec-14 expression using a human Siglec-5– and Siglec-14–recognizing antibody and Siglec-14 antibody by immunohistochemistry. (B) Human AMs were incubated either with FITC-labeled GBS or FITC-labeled GBSΔβ, and bacterial binding to the membrane was analyzed by fluorescence microscopy. (A and B) Bars, 50 µm. (C) Sig-14−/− AMs were cut into small pieces of similar size and infected either with GBS or GBSΔβ; IL-6 mRNA was analyzed by Q-RT-PCR after 2 h of infection. Results were normalized to GAPDH mRNA. Results are means ± SD; *, P < 0.05. (D and E) Sig-14−/− AMs were infected either with GBS or GBSΔβ as above. At the indicated times, cell lysates were prepared and analyzed for p38 phosphorylation and IκB-α degradation (D) and phosphorylation of AKT and S6 (E) by immunoblotting. (F) AMs of the indicated genotypes were infected either with GBS or GBSΔβ. At the indicated times, cell lysates were prepared and analyzed for phosphorylation of AKT protein by immunoblotting. The p-AKT/tubulin (×10) densitometry ratio is shown on the blot. p-AKT/tubulin, IκB-α/tubulin, and p-S6/tubulin (×10) densitometry values are shown on corresponding blots. Data in A–F are representative of two to five independent experiments with two to three different amnions per genotype in each experiment. (G–L) Human amnion genotyping: Association of the SIGLEC14- allele with preterm birth in infants of GBS-positive pregnancies. (G and H) Tables showing distribution of WT and  alleles in term and preterm groups in infants of GBS+ (G) and GBS− (H) pregnancies. Fisher’s exact test was performed using data computing infant allele × term/preterm. (I and J) Percentage of infants with various genotypes (Sig-14+/+, Sig-14+/−, or Sig-14−/−) in preterm and term group; infants from GBS-positive and -negative pregnancies were analyzed in I and J, respectively. (K) Table showing the ratio of preterm to term genotype percentage obtained from I and J. (L) Association of SIGLEC14/5 alleles with GBS colonization in preterm infants. The table shows the distribution of WT and  alleles in GBS-positive and -negative infants in the preterm group only. Fisher’s exact test was performed using data computing infant allele × term/preterm.
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fig5: The unusual ectopic expression of Siglec-5 and Siglec-14 on human AMs influences inflammatory responses to GBS. (A) Human AMs were stained for Siglec-5 and Siglec-14 expression using a human Siglec-5– and Siglec-14–recognizing antibody and Siglec-14 antibody by immunohistochemistry. (B) Human AMs were incubated either with FITC-labeled GBS or FITC-labeled GBSΔβ, and bacterial binding to the membrane was analyzed by fluorescence microscopy. (A and B) Bars, 50 µm. (C) Sig-14−/− AMs were cut into small pieces of similar size and infected either with GBS or GBSΔβ; IL-6 mRNA was analyzed by Q-RT-PCR after 2 h of infection. Results were normalized to GAPDH mRNA. Results are means ± SD; *, P < 0.05. (D and E) Sig-14−/− AMs were infected either with GBS or GBSΔβ as above. At the indicated times, cell lysates were prepared and analyzed for p38 phosphorylation and IκB-α degradation (D) and phosphorylation of AKT and S6 (E) by immunoblotting. (F) AMs of the indicated genotypes were infected either with GBS or GBSΔβ. At the indicated times, cell lysates were prepared and analyzed for phosphorylation of AKT protein by immunoblotting. The p-AKT/tubulin (×10) densitometry ratio is shown on the blot. p-AKT/tubulin, IκB-α/tubulin, and p-S6/tubulin (×10) densitometry values are shown on corresponding blots. Data in A–F are representative of two to five independent experiments with two to three different amnions per genotype in each experiment. (G–L) Human amnion genotyping: Association of the SIGLEC14- allele with preterm birth in infants of GBS-positive pregnancies. (G and H) Tables showing distribution of WT and alleles in term and preterm groups in infants of GBS+ (G) and GBS− (H) pregnancies. Fisher’s exact test was performed using data computing infant allele × term/preterm. (I and J) Percentage of infants with various genotypes (Sig-14+/+, Sig-14+/−, or Sig-14−/−) in preterm and term group; infants from GBS-positive and -negative pregnancies were analyzed in I and J, respectively. (K) Table showing the ratio of preterm to term genotype percentage obtained from I and J. (L) Association of SIGLEC14/5 alleles with GBS colonization in preterm infants. The table shows the distribution of WT and alleles in GBS-positive and -negative infants in the preterm group only. Fisher’s exact test was performed using data computing infant allele × term/preterm.
Mentions: CD33-related Siglecs are mainly present on immune cells (Crocker et al., 2007). One exception is Siglec-6, which was unexpectedly found on the placental trophoblast (Brinkman-Van der Linden et al., 2007). While screening for other Siglecs in fetal tissues, we were surprised to find positive staining on the amniotic epithelial membrane (AM) of human placental sections with the antibody that recognizes Siglec-5 and Siglec-14 (Fig. 5 A). This staining is apparently unique to the human amnion, as it was not found in amnions from the closely related great apes (not depicted). Siglec-5 and Siglec-14 protein expression on human AM was confirmed by Western blot (not depicted). This unusual tissue site of Siglec human-specific expression is particularly intriguing because the human-specific pathogen GBS produces ascending infections of the placental membranes, gaining access to the amniotic fluid and fetus and leading to the potentially life-threatening early-onset pneumonia and sepsis (Edwards et al., 2011). The ability of fluorescently labeled GBS to bind to AM was established, and the key contribution of the surface-anchored Siglec-binding β-protein was confirmed (Fig. 5 B).

Bottom Line: GBS amnion immune activation was likewise influenced by the SIGLEC14- polymorphism.We provide initial evidence that the polymorphism could influence the risk of prematurity among human fetuses of mothers colonized with GBS.This first functionally proven example of a paired receptor system in the Siglec family has multiple implications for regulation of host immunity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Glycobiology Research and Training Center, Department of Cellular and Molecular Medicine, Department of Pediatrics, Department of Pathology, Department of Medicine, and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093Glycobiology Research and Training Center, Department of Cellular and Molecular Medicine, Department of Pediatrics, Department of Pathology, Department of Medicine, and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093Glycobiology Research and Training Center, Department of Cellular and Molecular Medicine, Department of Pediatrics, Department of Pathology, Department of Medicine, and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093.

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