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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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Expression of Siglec-14 on THP-1 monocytes increases responsiveness to LPS and GBS. (A) THP-1 cells expressing Siglec-5 (THP-1–Sig-5), Siglec-14 (THP-1–Sig-14), or empty vector (THP-1–EV) were infected with FITC-labeled GBS or with GBSΔβ at 4oC, and then bacterial binding to neutrophils at the 20-min time point was analyzed by FACS. The graph depicts MFI of GFP fluorescence on neutrophils. (B) The indicated THP-1 cell variants were stimulated with 10 ng/ml LPS or media control for 2 h, and TNF mRNA was measured by Q-RT-PCR and normalized to GAPDH mRNA. (C) The indicated THP-1 cell variants were stimulated with or without 10 ng/ml LPS for the indicated times, lysed, and analyzed for AKT phosphorylation by immunoblot. The p-AKT/actin (×10) densitometry value is shown on the immunoblot. (D–F) IL-8 protein was measured in the supernatant of uninfected THP-1 cell variants and those infected with GBS or GBSΔβ (MOI = 10) for 6 h. (G) The indicated THP-1 cell variants were infected or not with GBS, and cell lysates were prepared and analyzed for p38 phosphorylation and IκB-α degradation by immunoblotting. The p-p38/actin and IκB-α/actin (×10) densitometry value is shown on corresponding blots. All data are representative of two to three independent experiments. Results are means ± SD; *, P < 0.05, **, P < 0.01.
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fig1: Expression of Siglec-14 on THP-1 monocytes increases responsiveness to LPS and GBS. (A) THP-1 cells expressing Siglec-5 (THP-1–Sig-5), Siglec-14 (THP-1–Sig-14), or empty vector (THP-1–EV) were infected with FITC-labeled GBS or with GBSΔβ at 4oC, and then bacterial binding to neutrophils at the 20-min time point was analyzed by FACS. The graph depicts MFI of GFP fluorescence on neutrophils. (B) The indicated THP-1 cell variants were stimulated with 10 ng/ml LPS or media control for 2 h, and TNF mRNA was measured by Q-RT-PCR and normalized to GAPDH mRNA. (C) The indicated THP-1 cell variants were stimulated with or without 10 ng/ml LPS for the indicated times, lysed, and analyzed for AKT phosphorylation by immunoblot. The p-AKT/actin (×10) densitometry value is shown on the immunoblot. (D–F) IL-8 protein was measured in the supernatant of uninfected THP-1 cell variants and those infected with GBS or GBSΔβ (MOI = 10) for 6 h. (G) The indicated THP-1 cell variants were infected or not with GBS, and cell lysates were prepared and analyzed for p38 phosphorylation and IκB-α degradation by immunoblotting. The p-p38/actin and IκB-α/actin (×10) densitometry value is shown on corresponding blots. All data are representative of two to three independent experiments. Results are means ± SD; *, P < 0.05, **, P < 0.01.

Mentions: It has been suggested but not proven that Siglec-5 and Siglec-14 are paired receptors that could mediate opposing effects on leukocyte responses to bacterial products or bacterial receptor engagement. Our prior finding demonstrates that type 1a GBS β-protein can engage Siglec-5 and deliver an inhibitory signal to leukocytes; however, its interaction with Siglec-14 remained unknown (Carlin et al., 2009a). In keeping with the nearly identical ligand-binding domains of the two Siglecs, we found that GBS β-protein bound equally strongly to Siglec-14 Fc-chimera and Siglec-5 Fc-chimera (not depicted). To conduct an initial in vitro analysis, we used THP-1 (human monocyte like) cells that express only low levels of endogenous Siglec-5 and lack Siglec-14 protein (not depicted). As described earlier, these cells were stably transfected with vectors expressing Siglec-5 (THP-1–Siglec-5), Siglec-14 (THP-1–Siglec-14), or empty vector control (THP-1–EV; Yamanaka et al., 2009). We observed an increased GBS binding to THP-1–Siglec-5 and THP-1–Siglec-14 cells compared with THP-1–EV control cells (Fig. 1 A). Previous work has shown that LPS-triggered cell activation is altered by the surface expression of Siglec-5, -7, -9, -11, and -14 by unknown mechanisms (Lock et al., 2004; Wang and Neumann, 2010; Pillai et al., 2012). Consistent with prior findings (Yamanaka et al., 2009), LPS administration stimulated more TNF mRNA production from THP-1–Siglec-14 compared with THP-1–Siglec-5 and empty vector control (Fig. 1 B). Siglec-14 is associated with DAP12 (DNAX activation protein of 12 kD), which is known to signal through phosphoinositide-3-kinase (PI3K)/Akt pathways (Angata et al., 2006). Enhanced AKT activation, as indicated by immunoblot for AKT S473 phosphorylation, was observed in THP-1–Siglec-14 compared with THP-1–Siglec-5 cells (Fig. 1 C). To investigate the role of Siglec-5 and Siglec-14 in the innate immune response to GBS, THP-1 cell variants were infected with GBS at a multiplicity of infection (MOI) of 10; GBSΔβ infections served as negative control for β-protein–mediated effects. Interestingly, THP-1 cells expressing Siglec-14 (THP-1–Sig-14) exhibited increased IL-8 protein secretion when infected with WT versus mutant GBS (P = 0.06). In contrast, THP-1–Sig-5 cells produced less IL-8 protein when infected with WT versus mutant GBS (Fig. 1 D). Importantly, IL-8 production was significantly enhanced in THP-1–Sig-14 compared with THP-1–Sig-5 cells (Fig. 1 D). The enhanced IL-8 protein production by THP-1–EV cells infected with WT versus mutant GBS was likely caused by endogenous Siglec-5 expression. Moreover, THP-1–Siglec-5/14 (THP-1 cells overexpressing Siglec-5 and Siglec-14; Angata et al., 2013) produced elevated IL-8 compared with THP-1–Sig-5 but reduced IL-8 compared with THP-1–Sig-14 cells, in response to GBS but not GBSΔβ (Fig. 1, E and F). Thus, our results demonstrate an activatory role of Siglec-14 in GBS β-protein–mediated responses.


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)

Expression of Siglec-14 on THP-1 monocytes increases responsiveness to LPS and GBS. (A) THP-1 cells expressing Siglec-5 (THP-1–Sig-5), Siglec-14 (THP-1–Sig-14), or empty vector (THP-1–EV) were infected with FITC-labeled GBS or with GBSΔβ at 4oC, and then bacterial binding to neutrophils at the 20-min time point was analyzed by FACS. The graph depicts MFI of GFP fluorescence on neutrophils. (B) The indicated THP-1 cell variants were stimulated with 10 ng/ml LPS or media control for 2 h, and TNF mRNA was measured by Q-RT-PCR and normalized to GAPDH mRNA. (C) The indicated THP-1 cell variants were stimulated with or without 10 ng/ml LPS for the indicated times, lysed, and analyzed for AKT phosphorylation by immunoblot. The p-AKT/actin (×10) densitometry value is shown on the immunoblot. (D–F) IL-8 protein was measured in the supernatant of uninfected THP-1 cell variants and those infected with GBS or GBSΔβ (MOI = 10) for 6 h. (G) The indicated THP-1 cell variants were infected or not with GBS, and cell lysates were prepared and analyzed for p38 phosphorylation and IκB-α degradation by immunoblotting. The p-p38/actin and IκB-α/actin (×10) densitometry value is shown on corresponding blots. All data are representative of two to three independent experiments. Results are means ± SD; *, P < 0.05, **, P < 0.01.
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fig1: Expression of Siglec-14 on THP-1 monocytes increases responsiveness to LPS and GBS. (A) THP-1 cells expressing Siglec-5 (THP-1–Sig-5), Siglec-14 (THP-1–Sig-14), or empty vector (THP-1–EV) were infected with FITC-labeled GBS or with GBSΔβ at 4oC, and then bacterial binding to neutrophils at the 20-min time point was analyzed by FACS. The graph depicts MFI of GFP fluorescence on neutrophils. (B) The indicated THP-1 cell variants were stimulated with 10 ng/ml LPS or media control for 2 h, and TNF mRNA was measured by Q-RT-PCR and normalized to GAPDH mRNA. (C) The indicated THP-1 cell variants were stimulated with or without 10 ng/ml LPS for the indicated times, lysed, and analyzed for AKT phosphorylation by immunoblot. The p-AKT/actin (×10) densitometry value is shown on the immunoblot. (D–F) IL-8 protein was measured in the supernatant of uninfected THP-1 cell variants and those infected with GBS or GBSΔβ (MOI = 10) for 6 h. (G) The indicated THP-1 cell variants were infected or not with GBS, and cell lysates were prepared and analyzed for p38 phosphorylation and IκB-α degradation by immunoblotting. The p-p38/actin and IκB-α/actin (×10) densitometry value is shown on corresponding blots. All data are representative of two to three independent experiments. Results are means ± SD; *, P < 0.05, **, P < 0.01.
Mentions: It has been suggested but not proven that Siglec-5 and Siglec-14 are paired receptors that could mediate opposing effects on leukocyte responses to bacterial products or bacterial receptor engagement. Our prior finding demonstrates that type 1a GBS β-protein can engage Siglec-5 and deliver an inhibitory signal to leukocytes; however, its interaction with Siglec-14 remained unknown (Carlin et al., 2009a). In keeping with the nearly identical ligand-binding domains of the two Siglecs, we found that GBS β-protein bound equally strongly to Siglec-14 Fc-chimera and Siglec-5 Fc-chimera (not depicted). To conduct an initial in vitro analysis, we used THP-1 (human monocyte like) cells that express only low levels of endogenous Siglec-5 and lack Siglec-14 protein (not depicted). As described earlier, these cells were stably transfected with vectors expressing Siglec-5 (THP-1–Siglec-5), Siglec-14 (THP-1–Siglec-14), or empty vector control (THP-1–EV; Yamanaka et al., 2009). We observed an increased GBS binding to THP-1–Siglec-5 and THP-1–Siglec-14 cells compared with THP-1–EV control cells (Fig. 1 A). Previous work has shown that LPS-triggered cell activation is altered by the surface expression of Siglec-5, -7, -9, -11, and -14 by unknown mechanisms (Lock et al., 2004; Wang and Neumann, 2010; Pillai et al., 2012). Consistent with prior findings (Yamanaka et al., 2009), LPS administration stimulated more TNF mRNA production from THP-1–Siglec-14 compared with THP-1–Siglec-5 and empty vector control (Fig. 1 B). Siglec-14 is associated with DAP12 (DNAX activation protein of 12 kD), which is known to signal through phosphoinositide-3-kinase (PI3K)/Akt pathways (Angata et al., 2006). Enhanced AKT activation, as indicated by immunoblot for AKT S473 phosphorylation, was observed in THP-1–Siglec-14 compared with THP-1–Siglec-5 cells (Fig. 1 C). To investigate the role of Siglec-5 and Siglec-14 in the innate immune response to GBS, THP-1 cell variants were infected with GBS at a multiplicity of infection (MOI) of 10; GBSΔβ infections served as negative control for β-protein–mediated effects. Interestingly, THP-1 cells expressing Siglec-14 (THP-1–Sig-14) exhibited increased IL-8 protein secretion when infected with WT versus mutant GBS (P = 0.06). In contrast, THP-1–Sig-5 cells produced less IL-8 protein when infected with WT versus mutant GBS (Fig. 1 D). Importantly, IL-8 production was significantly enhanced in THP-1–Sig-14 compared with THP-1–Sig-5 cells (Fig. 1 D). The enhanced IL-8 protein production by THP-1–EV cells infected with WT versus mutant GBS was likely caused by endogenous Siglec-5 expression. Moreover, THP-1–Siglec-5/14 (THP-1 cells overexpressing Siglec-5 and Siglec-14; Angata et al., 2013) produced elevated IL-8 compared with THP-1–Sig-5 but reduced IL-8 compared with THP-1–Sig-14 cells, in response to GBS but not GBSΔβ (Fig. 1, E and F). Thus, our results demonstrate an activatory role of Siglec-14 in GBS β-protein–mediated responses.

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