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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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TLR priming increases GBS suppression of Sig-14−/− but not Sig-14+/+ neutrophil responses. (A) Human neutrophils of the indicated genotypes were left unstimulated or stimulated with LPS for 6 h, incubated with H2DFCA for 30 min, and then infected with GBS (MOI = 10); ROS production was measured 20 min after infection by FACS. The histogram shows fluorescence of ROS indicator H2DCFDA. (B) Human neutrophils of the indicated genotypes were left unstimulated or stimulated with LPS for 6 h and infected with GBS (MOI = 10), and NET formation was visualized by myeloperoxidase and DAPI staining 30 min after infection. Bar, 50 µm. (C) Quantification of NETs shown in B. (D) Human neutrophils of the indicated genotypes were left unstimulated or stimulated with LPS for 8 h; expression of Siglec-5 and Siglec-14 was analyzed by FACS using antibody recognizing both human Siglec-5 and Siglec-14. The histogram depicts combined surface expression of Siglec-5 and Siglec-14. (E) Human neutrophils of the indicated genotypes were stimulated or not with LPS for 6 h and infected with GBS (MOI = 10), and bacterial killing was assayed 20 min after infection. (F) Human neutrophils of the indicated genotypes were stimulated with LPS for 6 h and infected with GBS (MOI = 10), and cell lysates prepared at the indicated times were immunoprecipitated with Siglec-5– and Siglec-14–recognizing antibody. SHP-1 recruitment and total IgG were analyzed by immunoblot. The SHP-1/IgG (×10) densitometry value is shown on the immunoblot. Data in this figure are representative of two to four independent experiments with one to three different donors per genotype in each experiment. Results are means ± SD; *, P < 0.05, **, P < 0.01.
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fig4: TLR priming increases GBS suppression of Sig-14−/− but not Sig-14+/+ neutrophil responses. (A) Human neutrophils of the indicated genotypes were left unstimulated or stimulated with LPS for 6 h, incubated with H2DFCA for 30 min, and then infected with GBS (MOI = 10); ROS production was measured 20 min after infection by FACS. The histogram shows fluorescence of ROS indicator H2DCFDA. (B) Human neutrophils of the indicated genotypes were left unstimulated or stimulated with LPS for 6 h and infected with GBS (MOI = 10), and NET formation was visualized by myeloperoxidase and DAPI staining 30 min after infection. Bar, 50 µm. (C) Quantification of NETs shown in B. (D) Human neutrophils of the indicated genotypes were left unstimulated or stimulated with LPS for 8 h; expression of Siglec-5 and Siglec-14 was analyzed by FACS using antibody recognizing both human Siglec-5 and Siglec-14. The histogram depicts combined surface expression of Siglec-5 and Siglec-14. (E) Human neutrophils of the indicated genotypes were stimulated or not with LPS for 6 h and infected with GBS (MOI = 10), and bacterial killing was assayed 20 min after infection. (F) Human neutrophils of the indicated genotypes were stimulated with LPS for 6 h and infected with GBS (MOI = 10), and cell lysates prepared at the indicated times were immunoprecipitated with Siglec-5– and Siglec-14–recognizing antibody. SHP-1 recruitment and total IgG were analyzed by immunoblot. The SHP-1/IgG (×10) densitometry value is shown on the immunoblot. Data in this figure are representative of two to four independent experiments with one to three different donors per genotype in each experiment. Results are means ± SD; *, P < 0.05, **, P < 0.01.

Mentions: Because TLR-4 activation by LPS reduced the expression of Siglec-5 in Sig-14+/+ neutrophils but increased expression in Sig-14−/− neutrophils (Fig. 3 E), we hypothesized that preactivation of TLR-4 signaling in Sig-14−/− neutrophils would significantly increase their susceptibility to GBS-mediated immune suppression. Two phenotypes that are suppressed upon GBS Siglec-5 engagement are reactive oxygen species (ROS) generation and formation of DNA-based NET (Carlin et al., 2009a). To test our hypothesis, neutrophils of the indicated genotypes were pretreated with LPS or media control for 8 h, followed by low-dose GBS infection for 30 min. No significant increase in ROS or NET formation was observed in response to GBS stimulation alone after 30 min. However, LPS priming induced significant ROS and NET production in GBS-infected Sig-14+/+ but not Sig-14−/− neutrophils (Fig. 4, A–C). Surface expression of Siglec-5 and Siglec-14 was determined by flow cytometry using an antibody that recognizes both Siglec-5 and Siglec-14 (currently no specific antibody exists that is specific for Siglec-5 only). As indicated in Fig. 4 D, an LPS-mediated increase in mean fluorescence intensity (MFI) from 32 (control) to 78 (LPS) in Sig-14+/+ corresponds to Siglec-5/14 expression, whereas the observed increase from 30 to 135 in Sig-14−/− neutrophils corresponds to Siglec-5 expression alone. The increase of Siglec-5 expression in Sig-14−/− neutrophils may contribute, along with the absence of the activating Siglec-14 receptor, to the enhanced susceptibility of Sig-14−/− neutrophils to immune suppression by GBS. Consistent with this model, neutrophil killing of GBS was significantly reduced in LPS-treated Sig-14−/− compared with Sig-14+/+ neutrophils (Fig. 4 E). In contrast, GBSΔβ survival was similar between the two genotypes (not depicted). To analyze the molecular events associated with Siglec receptor activation in LPS + GBS–treated neutrophils, cells were LPS pretreated or not for 8 h, followed by GBS infection for 20 min. When neutrophil lysates were immunoprecipitated by antibody recognizing human Siglec-5 and Siglec-14, followed by immunoblotting for SHP-1, we found that LPS priming + GBS infection increased recruitment of inhibitory SHP-1 in Sig-14−/− compared with Sig-14+/+ neutrophils (Fig. 4 F), consistent with the initiation of inhibitory signaling through Siglec-5 (Carlin et al., 2009a).


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)

TLR priming increases GBS suppression of Sig-14−/− but not Sig-14+/+ neutrophil responses. (A) Human neutrophils of the indicated genotypes were left unstimulated or stimulated with LPS for 6 h, incubated with H2DFCA for 30 min, and then infected with GBS (MOI = 10); ROS production was measured 20 min after infection by FACS. The histogram shows fluorescence of ROS indicator H2DCFDA. (B) Human neutrophils of the indicated genotypes were left unstimulated or stimulated with LPS for 6 h and infected with GBS (MOI = 10), and NET formation was visualized by myeloperoxidase and DAPI staining 30 min after infection. Bar, 50 µm. (C) Quantification of NETs shown in B. (D) Human neutrophils of the indicated genotypes were left unstimulated or stimulated with LPS for 8 h; expression of Siglec-5 and Siglec-14 was analyzed by FACS using antibody recognizing both human Siglec-5 and Siglec-14. The histogram depicts combined surface expression of Siglec-5 and Siglec-14. (E) Human neutrophils of the indicated genotypes were stimulated or not with LPS for 6 h and infected with GBS (MOI = 10), and bacterial killing was assayed 20 min after infection. (F) Human neutrophils of the indicated genotypes were stimulated with LPS for 6 h and infected with GBS (MOI = 10), and cell lysates prepared at the indicated times were immunoprecipitated with Siglec-5– and Siglec-14–recognizing antibody. SHP-1 recruitment and total IgG were analyzed by immunoblot. The SHP-1/IgG (×10) densitometry value is shown on the immunoblot. Data in this figure are representative of two to four independent experiments with one to three different donors per genotype in each experiment. Results are means ± SD; *, P < 0.05, **, P < 0.01.
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fig4: TLR priming increases GBS suppression of Sig-14−/− but not Sig-14+/+ neutrophil responses. (A) Human neutrophils of the indicated genotypes were left unstimulated or stimulated with LPS for 6 h, incubated with H2DFCA for 30 min, and then infected with GBS (MOI = 10); ROS production was measured 20 min after infection by FACS. The histogram shows fluorescence of ROS indicator H2DCFDA. (B) Human neutrophils of the indicated genotypes were left unstimulated or stimulated with LPS for 6 h and infected with GBS (MOI = 10), and NET formation was visualized by myeloperoxidase and DAPI staining 30 min after infection. Bar, 50 µm. (C) Quantification of NETs shown in B. (D) Human neutrophils of the indicated genotypes were left unstimulated or stimulated with LPS for 8 h; expression of Siglec-5 and Siglec-14 was analyzed by FACS using antibody recognizing both human Siglec-5 and Siglec-14. The histogram depicts combined surface expression of Siglec-5 and Siglec-14. (E) Human neutrophils of the indicated genotypes were stimulated or not with LPS for 6 h and infected with GBS (MOI = 10), and bacterial killing was assayed 20 min after infection. (F) Human neutrophils of the indicated genotypes were stimulated with LPS for 6 h and infected with GBS (MOI = 10), and cell lysates prepared at the indicated times were immunoprecipitated with Siglec-5– and Siglec-14–recognizing antibody. SHP-1 recruitment and total IgG were analyzed by immunoblot. The SHP-1/IgG (×10) densitometry value is shown on the immunoblot. Data in this figure are representative of two to four independent experiments with one to three different donors per genotype in each experiment. Results are means ± SD; *, P < 0.05, **, P < 0.01.
Mentions: Because TLR-4 activation by LPS reduced the expression of Siglec-5 in Sig-14+/+ neutrophils but increased expression in Sig-14−/− neutrophils (Fig. 3 E), we hypothesized that preactivation of TLR-4 signaling in Sig-14−/− neutrophils would significantly increase their susceptibility to GBS-mediated immune suppression. Two phenotypes that are suppressed upon GBS Siglec-5 engagement are reactive oxygen species (ROS) generation and formation of DNA-based NET (Carlin et al., 2009a). To test our hypothesis, neutrophils of the indicated genotypes were pretreated with LPS or media control for 8 h, followed by low-dose GBS infection for 30 min. No significant increase in ROS or NET formation was observed in response to GBS stimulation alone after 30 min. However, LPS priming induced significant ROS and NET production in GBS-infected Sig-14+/+ but not Sig-14−/− neutrophils (Fig. 4, A–C). Surface expression of Siglec-5 and Siglec-14 was determined by flow cytometry using an antibody that recognizes both Siglec-5 and Siglec-14 (currently no specific antibody exists that is specific for Siglec-5 only). As indicated in Fig. 4 D, an LPS-mediated increase in mean fluorescence intensity (MFI) from 32 (control) to 78 (LPS) in Sig-14+/+ corresponds to Siglec-5/14 expression, whereas the observed increase from 30 to 135 in Sig-14−/− neutrophils corresponds to Siglec-5 expression alone. The increase of Siglec-5 expression in Sig-14−/− neutrophils may contribute, along with the absence of the activating Siglec-14 receptor, to the enhanced susceptibility of Sig-14−/− neutrophils to immune suppression by GBS. Consistent with this model, neutrophil killing of GBS was significantly reduced in LPS-treated Sig-14−/− compared with Sig-14+/+ neutrophils (Fig. 4 E). In contrast, GBSΔβ survival was similar between the two genotypes (not depicted). To analyze the molecular events associated with Siglec receptor activation in LPS + GBS–treated neutrophils, cells were LPS pretreated or not for 8 h, followed by GBS infection for 20 min. When neutrophil lysates were immunoprecipitated by antibody recognizing human Siglec-5 and Siglec-14, followed by immunoblotting for SHP-1, we found that LPS priming + GBS infection increased recruitment of inhibitory SHP-1 in Sig-14−/− compared with Sig-14+/+ neutrophils (Fig. 4 F), consistent with the initiation of inhibitory signaling through Siglec-5 (Carlin et al., 2009a).

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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Related in: MedlinePlus