ASGR-mediated KC capture of serotype-7F and -14 pneumococci. (A) Strategy for screening CPS14-binding proteins of mouse KCs. (B) Plot of membrane-associated proteins significantly enriched by CPS14 beads. Solid line represents the threshold of twofold enrichment by CPS14 compared with CPS8. The top five enriched proteins with potential ligand-binding activities are labeled. (C) TH87014 adhesion to CHO cells expressing mASGR and other CPS14-binding candidates is expressed as the percentage of adherent bacteria out of the input. n = 3. (D) Adhesion of 15 selected serotypes to CHO-mASGR cells was measured with the TH870 derivatives as in C. n = 3. (E) Blocking of mASGR-mediated TH87014 adhesion by CPS14 and known ASGR ligands (galactose or GalNAc). n = 3. (F) Cross-blocking of mASGR-mediated pneumococcal adherence by CPS7F and CPS14. n = 3. (G) TH87014 adhesion to CHO cells expressing hASGR was measured as in C. n = 3. (H) Blocking of mouse (left) and human (right) ASGR-mediated TH87014 adhesion by ASGR antibodies. The antibodies and isotype IgG were added at 5 μg/ml at the same time with bacteria. n = 3. (I) Clearance of TH87014 in ASGR1−/− mice infected i.v. with 107 CFU. This dose was used because the mice showed no deficiency in TH87014 clearance at 106 CFU. n = 3–6. (J) Survival of ASGR1−/− mice after i.v. infection with 108 CFU of TH87014. n = 9. All experiments were performed with C57BL/6 mice. Data were representative (C–H) or pooled (B, I, and J) results from two to three independent experiments. Ordinary one-way ANOVA with Tukey’s (C, E, and F) multiple comparisons test, two-way ANOVA with Sidak’s (D and G) or Tukey’s (I) multiple comparisons test, unpaired t test (H), log-rank test (J), * P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001.
