Loss of C5aR reduces the levels of inflammatory mediators in the middle ear.

<p>Concentrations of IL-6, mKC, and MCP-1 in the middle ear lavage samples on days 1, 3, and 7 post Spn infection (corresponding to days 8, 9 and 10 post-viral infection) in coinfected WT and <i>C5ar1^−/−</i> mice or mice infected with Spn or IAV alone. Results are the mean concentrations (±S.E.M.) from two separate experiments. *; <i>P</i><0.01 for the comparison of <i>C5ar1^−/−</i> with WT mice infected with IAV and Spn. ^#; <i>P</i><0.05 for the comparison of Spn alone infected <i>C5ar1^−/−</i> with WT mice. +; <i>P</i><0.05 for the comparison of <i>C5ar1^−/−</i> mice infected with IAV alone with WT mice.</p

Representative H&E-stained sections of mid-portion of eustachian tube (ET).

<p>The structural changes on day 3 after Spn infection in WT, <i>C1qa^−/−</i>, and <i>Bf ^−/−</i> mice infected with influenza virus or either pathogen alone. The tissue damages of ETs in complement deficient mice infected with IAV and Spn were evident (magnification, ×200). Eustachian tube lumen is labeled with “L”. Open arrowheads indicate ciliated mucosa. Filled arrowheads indicate epithelial denudation in IAV infected cohorts, and thickened mucosal epithelium with goblet cell hyperplasia in Spn and coinfected cohorts.</p

Spn type 6A titers in the middle ear lavage and blood samples.

<p>Survival of Spn type 6A in the middle ear of WT (A), <i>C1qa^−/−</i> (B), and <i>Bf^−/−</i> (C) mice infected with Spn with or without a prior influenza virus infection and in the blood sample of mice infected with Spn and IAV(D). Each data point represents the geometric mean of CFU of Spn (± SEM) per milliliter of the middle ear lavage fluid and blood samples. All samples were collected from a total of 6 to 8 animals combined from two separate experiments. Relative <i>P</i> values are indicated for each pairwise comparison. The dashed line represents the detection limit of the assay.</p

Representative H&E-stained middle ear sections.

<p>Middle ear inflammation on day 3 after Spn infection in WT, <i>C1qa^−/−</i>, and <i>Bf ^−/−</i> mice infected with IAV or either pathogen alone. The enhanced inflammatory infiltrates and mucosa thickness in the middle ear epithelium were evident in <i>C1qa^−/−</i> and <i>Bf ^−/−</i> mice infected with IAV and Spn. Open arrowheads indicate inflammatory cell infiltrates, and arrows indicate the middle ear mucosa (magnification, ×200).</p

Representative immunofluorescence staining for complement component deposition in the middle ear mucosa.

<p>(A) C3 (green) and C5aR (red) deposition in the middle ear mucosa at 24 h post Spn infection. Nucleic acids were stained with DAPI (blue). Faint staining was observed in sham control mice. Increased C3 and C5aR immunofluorescence staining in the middle ear epithelium of mice infected with IAV and Spn was evident. (B). Increased C5b-9 immunofluorescence staining (green) in the middle ear epithelium in mice at 24 after transtympanical inoculation with Spn with a prior IAV infection compared with the sham control and single pathogen infected cohorts. Arrows indicate the middle ear mucosa. Representative imagines from three mice are shown.</p

Viral titers in tracheonasal and middle ear lavage samples.

<p>Viral clearance from the nasopharynx of WT, <i>C1qa^−/−</i>, and <i>Bf^−/−</i> mice (A) and viral titers on day 7 in middle ear (B). Each data point represents the geometric mean of PFU of IAV (± SEM) per milliliter of the tracheonasal and middle ear lavage fluid. All samples were collected from a total of 10 animals combined from two separate experiments. Relative <i>P</i> values are indicated for each comparison with WT mice. The dashed line represents the detection limit of the assay.</p

Deletion of C5aR did not affect the complement activation during acute pneumococcal OM.

<p>Concentrations of C3, C3a, and C5a in the middle ear lavage samples on days 1, 3, and 7 post Spn infection (corresponding to days 8, 9 and 10 post-viral infection) in WT and <i>C5ar1^−/−</i> mice infected with Spn alone, or Spn with prior IAV, or IAV alone. Results are the mean concentrations (± SEM) in the middle ear lavage pooled from five mice from two separate experiments. There were no significant differences in the concentrations of C3, C3a and C5a between <i>C5ar1^−/−</i> and WT mice.</p

Complement activation during OM.

<p>Concentrations of C3, C3a, and C5a in the middle ear lavage fluid samples (A) and serum samples (B) from WT mice infected with a combination of IAV and Spn or either pathogen alone. Results are the mean concentrations (± SEM) in the middle ear lavage and serum samples from 5 mice from two separate experiments. *<i>P</i><0.05 for the comparison of the combined infection with each single pathogen alone. #, <i>P</i><0.05 for the comparison of mice infected with Spn alone with IAV alone.</p

Deficiency in C5aR enhances bacterial clearance and reduces the severity of acute pneumococcal OM.

<p>(A). Accumulation of inflammatory cells in the middle ears of mice following transtympanical inoculation of Spn. Each data point represents the mean concentration of inflammatory cells (± SEM) per cubic millimeter of the middle ear lavage fluid. These results were obtained from a total of 6 to 11 animals combined from two separate experiments. *, <i>P</i><0.05 for the comparison of the WT with <i>C5ar1^−/−</i> mice infected with IAV and Spn or WT mice infected with Spn alone. #. <i>P</i><0.05 for the comparison of the <i>C5ar1^−/−</i> mice infection with both pathogens with Spn alone infected C5ar1<i>^−/−</i> mice. +. <i>P</i><0.05 for the comparison of <i>C5ar1^−/−</i> mice infected with Spn alone iwith WT mice. (B) and (C). Representative H&E-stained middle ear sections of the mice at 24 h post Spn infection from coinfected WT (B) and <i>C5ar1^−/−</i> mice (C). Arrows indicate the middle ear mucosa. (D) Survival of Spn type 6A in the middle ear of WT and <i>C5ar1^−/−</i> mice coinfected with influenza virus or mice infected with Spn alone. Each data point represents the mean of CFU of Spn (± SEM) per milliliter of the middle ear lavage fluid samples from a total of 6 to 8 animals combined from two separate experiments. Relative <i>P</i> values are indicated for each pairwise comparison. The dashed line represents the detection limit of the assay.</p

Deposition of C4 breakdown products on <i>S. pneumoniae</i>.

<p>ELSIA plates were coated with <i>S. pneumoniae</i> D39, <i>E. coli</i> or mannan (as positive controls) and incubated with NHS for 1 h at 37°C. Bound C4 was detected using an antibody against C4c (which also detects C4b) (<b>A</b>) or an antibody against C4dg, the last breakdown product of C4 that remains covalently attached to the activating surface (<b>B</b>). <b>C</b> and <b>D</b>: Detection of C4c (<b>C</b>) and C4dg (<b>D</b>) on live <i>S. pneumoniae</i> D39 opsonised with NHS. Green trace shows anti-C4 antibodies; the purple shading, an isotype control Ab.</p