Deletion of the Complement C5a Receptor Alleviates the Severity of Acute Pneumococcal Otitis Media following Influenza A Virus Infection in Mice

There is considerable evidence that influenza A virus (IAV) promotes adherence, colonization, and superinfection by S. pneumoniae (Spn) and contributes to the pathogenesis of otitis media (OM). The complement system is a critical innate immune defense against both pathogens. To assess the role of the complement system in the host defense and the pathogenesis of acute pneumococcal OM following IAV infection, we employed a well-established transtympanically-induced mouse model of acute pneumococcal OM. We found that antecedent IAV infection enhanced the severity of acute pneumococcal OM. Mice deficient in complement C1qa (C1qa−/−) or factor B (Bf −/−) exhibited delayed viral and bacterial clearance from the middle ear and developed significant mucosal damage in the eustachian tube and middle ear. This indicates that both the classical and alternative complement pathways are critical for the oto-immune defense against acute pneumococcal OM following influenza infection. We also found that Spn increased complement activation following IAV infection. This was characterized by sustained increased levels of anaphylatoxins C3a and C5a in serum and middle ear lavage samples. In contrast, mice deficient in the complement C5a receptor (C5aR) demonstrated enhanced bacterial clearance and reduced severity of OM. Our data support the concept that C5a-C5aR interactions play a significant role in the pathogenesis of acute pneumococcal OM following IAV infection. It is possible that targeting the C5a-C5aR axis might prove useful in attenuating acute pneumococcal OM in patients with influenza infection

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 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

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

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

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

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