Loss of Siglec-14 reduces the risk of chronic obstructive pulmonary disease exacerbation

Abstract

Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality worldwide. COPD exacerbation, or episodic worsening of symptoms, often results in hospitalization and increased mortality rates. Airway infections by new bacterial strains, such as nontypeable Haemophilus influenzae (NTHi), are a major cause of COPD exacerbation. NTHi express lipooligosaccharides that contain sialic acids, and may interact with Siglec-14, a sialic acid recognition protein on myeloid cells that serves as an activating signal transduction receptor. A null allele polymorphism in SIGLEC14 may attenuate the inflammatory responses to NTHi by eliminating Siglec-14 expression. We asked if the loss of Siglec-14 attenuates the inflammatory response by myeloid cells against NTHi, and if the SIGLEC14-null polymorphism has any effect on COPD exacerbation. We found that NTHi interacts with Siglec-14 to enhance proinflammatory cytokine production in a tissue culture model. Inhibitors of the Syk tyrosine kinase suppress this response. Loss of Siglec-14, due to SIGLEC14-null allele homozygosity, is associated with a reduced risk of COPD exacerbation in a Japanese patient population. Taken together, Siglec-14 and its downstream signaling pathway facilitate the "infection-inflammation-exacerbation" axis of COPD disease progression, and may represent promising targets for therapeutic intervention.

Fig. 1

Expression of SIGLEC14/5 fusion gene product. Peripheral blood leukocytes from homozygous SIGLEC14-null individuals were analyzed by flow cytometry. Gray shaded areas cells stained with a negative control antibody (IC002A, R&D Systems). Solid lines cells stained with antibody recognizing both Siglec-5 and Siglec-14 (FAB10721A, R&D Systems). SIGLEC14/5 fusion gene product, which is identical to Siglec-5 in amino acid sequence, is expressed on granulocytes, monocytes, and B lymphocytes

Fig. 2

Influence of Siglec-14 and Siglec-5 expression on inflammatory responses. a Binding of NTHi to human myeloid Siglecs. Both sialic acid-positive (solid bars) and sialic acid-negative (open bars) NTHi bound to Siglec-5 and Siglec-14, whereas only sialic acid-positive NTHi bound to Siglec-9. Background fluorescence (autofluorescence of assay buffer) was subtracted. *p < 0.05 by one-way ANOVA followed by Dunnett’s multiple comparison test, vs. “none” within each group. b Binding of NTHi to Siglec-14 wildtype and R119A mutant. Both sialic acid-positive NTHi (solid bars) and sialic acid-negative NTHi (open bars) showed binding to both wildtype Siglec-14 and the R119A mutant. Background fluorescence (autofluorescence of assay buffer) was subtracted. *p < 0.05 by two-way ANOVA followed by Bonferroni multiple comparison test (four pairwise comparisons: Siglec-14 wildtype, sialic acid-negative vs. sialic acid-positive NTHi; Siglec-14 R119A, sialic acid-negative vs. sialic acid-positive NTHi; sialic acid-negative NTHi, Siglec-14 wildtype vs. R119A; sialic acid-positive NTHi, Siglec-14 wildtype vs. R119A). a, b Measurements performed in triplicate wells and repeated at least three times independently. Consistent results were obtained, and representative data are shown. Values shown are means ± standard error of the mean (SEM). c, d IL-8 production (c) and TNF-α production (d) from THP-1 sublines stimulated with NTHi (solid bars THP-1 cells mimicking homozygous wildtype monocytes, gray bars THP-1 cells mimicking heterozygous monocytes, open bars THP-1 cells mimicking homozygous SIGLEC14-null monocytes). *p < 0.05 by one-way ANOVA followed by Bonferroni’s multiple comparison test (three pairwise comparisons). e, f IL-8 production (e) and TNF-α production (f) from Siglec-14⁺5⁻ THP-1 subline stimulated with NTHi in the presence of R406 (open circles) or BAY61-3606 (closed circles). *p < 0.05 by one-way ANOVA followed by Dunnett’s multiple comparison test (vs. vehicle control = “0 μM”, within each treatment group). c–f Measurements were performed in quadruplicate wells and repeated four times independently with consistent results, and representative data are shown. All values shown are means ± SEM

Fig. 3

Effect of SIGLEC14 genotype on the frequency of COPD exacerbations. a SIGLEC14 genotype and the incidence of bacterial exacerbations using the definition of Anthonisen et al. [32] (without EX patients without bacterial exacerbations, with EX patients with one or more incidences). b SIGLEC14 genotype and the frequency of all exacerbations using the definition of Rodriguez-Roisin. Values are means ± standard deviation. c SIGLEC14 genotype and severity of airflow obstruction (post-bronchodilator FEV1 %). Each point represents an individual patient (Wild homozygous wildtype, Hetero heterozygous, Null homozygous SIGLEC14-null)

Fig. 4

Correlation between SIGLEC14 genotype, serum Siglec-14, and COPD exacerbations. a Siglec-14-Fc (solid circles) and Siglec-5-Fc (open circles), serially diluted from 100 to 1.5625 ng/ml, were analyzed using a sandwich ELISA specific for Siglec-14 as described in Materials and Methods. Values are means ± SEM. b Correlations between serum Siglec-14 and SIGLEC14 genotype. Siglec-14 serum concentrations on average reflect the number of wildtype (SIGLEC14-positive) alleles. c The Siglec-14 serum concentrations in COPD patient sera before and during exacerbation. The lines connect Siglec-14 concentrations in samples collected before and during exacerbations (stable and exacerbation, respectively) from the same individuals (n = 21; homozygous wildtype and heterozygous patients only). d Correlations between Siglec-14 serum concentrations and frequency of exacerbations (using the definition of Rodriguez-Roisin). e Correlation between Siglec-14 serum concentrations and post-bronchodilator FEV1 %

Fig. 5

Possible mechanism behind the correlation between SIGLEC14 genotype and COPD exacerbation-prone phenotype. The wildtype (SIGLEC14-positive) allele confers expression of Siglec-14 on monocytes, while the SIGLEC14-null (SIGLEC14/5 fusion) allele confers expression of Siglec-5. Monocytes of homozygous wildtype individuals express Siglec-14 only, those of heterozygous individuals both Siglec-14 and Siglec-5, and those of homozygous SIGLEC14-null individuals Siglec-5 only. Siglec-14 associates with adapter protein DAP12 and the tyrosine kinase Syk to transduce cell-activating signal upon engagement. Siglec-5, on the other hand, associates with tyrosine phosphatase SHP-1 to transduce a cell-inhibitory signal. The myeloid cells of those who carry the wildtype allele respond strongly to infectious cues, while those of homozygous SIGLEC14-null individuals respond weakly. Excessive proinflammatory responses in COPD patients lead to exacerbation