Lethal coinfection of influenza virus and Streptococcus pneumoniae lowers antibody response to influenza virus in lung and reduces numbers of germinal center B cells, T follicular helper cells, and plasma cells in mediastinal lymph Node

Abstract

Secondary Streptococcus pneumoniae infection after influenza is a significant clinical complication resulting in morbidity and sometimes mortality. Prior influenza virus infection has been demonstrated to impair the macrophage and neutrophil response to the subsequent pneumococcal infection. In contrast, how a secondary pneumococcal infection after influenza can affect the adaptive immune response to the initial influenza virus infection is less well understood. Therefore, this study focuses on how secondary pneumococcal infection after influenza may impact the humoral immune response to the initial influenza virus infection in a lethal coinfection mouse model. Compared to mice infected with influenza virus alone, mice coinfected with influenza virus followed by pneumococcus had significant body weight loss and 100% mortality. In the lung, lethal coinfection significantly increased virus titers and bacterial cell counts and decreased the level of virus-specific IgG, IgM, and IgA, as well as the number of B cells, CD4 T cells, and plasma cells. Lethal coinfection significantly reduced the size and weight of spleen, as well as the number of B cells along the follicular developmental lineage. In mediastinal lymph nodes, lethal coinfection significantly decreased germinal center B cells, T follicular helper cells, and plasma cells. Adoptive transfer of influenza virus-specific immune serum to coinfected mice improved survival, suggesting the protective functions of anti-influenza virus antibodies. In conclusion, coinfection reduced the B cell response to influenza virus. This study helps us to understand the modulation of the B cell response to influenza virus during a lethal coinfection.

Importance: Secondary pneumococcal infection after influenza virus infection is an important clinical issue that often results in excess mortality. Since antibodies are key mediators of protection, this study aims to examine the antibody response to influenza virus and demonstrates that lethal coinfection reduced the B cell response to influenza virus. This study helps to highlight the complexity of the modulation of the B cell response in the context of coinfection.

FIG 1

Coinfection enhanced influenza virus replication in the lung. Mice were infected with either influenza virus (PR8), or Streptococcus pneumoniae (Sp), or coinfected with influenza virus and then pneumococcus (PR8+Sp). At the indicated time points after infection, survival and weight loss (A and B), lung virus titer as measured in TCID₅₀ (C), and pneumococcal cell count as measured in CFU in lung homogenate and bronchoalveolar lavage (BAL) fluid (D) were recorded. (E and F) Survival and weight loss of mice coinfected with live or UV-irradiated (UV) or heat-killed (HK) PR8 and live or heat-killed pneumococcus. Results ± standard deviations are representative of four to nine (A and B), three (C), or two (E and F) independent experiments. *, P < 0.05; ***, P < 0.001, unpaired t test.

FIG 2

Coinfection reduced PR8-specific immunoglobulins. Mice were infected with influenza virus (PR8) or coinfected (PR8+Sp). On day 8 after PR8 infection, whole-lung homogenate (A), BAL fluid (B), and serum (C) were collected to determine the relative levels of PR8-specific IgG, IgM, and IgA by ELISA. (D) Serum hemagglutinin inhibition (HAI) titer was measured on day 8 after PR8 infection. Results ± standard deviations are representative of five (A and C) and two (B) independent experiments and grouped from three independent experiments (D). *, P < 0.05; **, P < 0.01; ***, P < 0.001, by two-way ANOVA (A to C) and unpaired t test (D). OD, optical density.

FIG 3

Coinfection reduced B cells, CD4 T cells, and plasma cells in the lung. Mice were infected with influenza virus (PR8) or coinfected (PR8+Sp). On day 8 after PR8 infection, whole lung was collected to determine total numbers and percentages of CD19⁺ B cells, CD3⁺ CD4⁺ T cells, and CD138⁺ plasma cells (A). Percentages of CD19⁺ B cells and CD3⁺ CD4⁺ T cells in peripheral blood were determined on day 8 after PR8 infection (B). Results ± standard deviations were grouped from three independent experiments. ***, P < 0.001, unpaired t test.

FIG 4

Coinfection reduced the cells in spleen. Mice were infected with influenza virus (PR8) or coinfected (PR8+Sp). On day 4 and day 8 after PR8 infection, spleen was collected to examine different cell populations. (A) Splenic size and weight. (B) Numbers of total splenic cells, B220⁺ B cells and CD3⁺ CD4⁺ T cells. (C) Numbers in different B cell subpopulations. (D) Percentages of different B cell subpopulations. (E) Numbers of germinal center B cells, Tfh cells, and plasma cells on day 8 post-PR8 infection. (F) B cell surface marker expression on day 4 post-PR8 infection. (G) Germinal center B cell and Tfh cell surface marker expression on day 8 post-PR8 infection. Results ± standard deviations are representative of three independent experiments (A) and grouped from five independent experiments (B to G). *, P < 0.05; **, P < 0.01; ***, P < 0.001, by one-way ANOVA (A to D) or unpaired t test (E to G). For panels B to G, on day 4 post-PR8 infection, n = 16 for PR8 and n = 15 for PR8+Sp; on day 8 post-PR8 infection, n = 17 for PR8 and n = 14 for PR8+Sp. Cell populations are as follows: T1-NF, T1-newly formed B (CD23⁻ CD21⁻ IgM^hi IgD^lo); T2-FP, T2-follicular B precursor (CD23⁺ CD21^int IgM^hi IgD^hi); FOB, follicular B (CD23⁺ CD21^int IgM^lo IgD^hi); T2-MZP, T2-marginal zone precursor (CD23⁻ CD21^hi IgM^hi IgD^hi); MZB, marginal zone B (CD23⁻ CD21^hi IgM^hi IgD^lo);GCB, germinal center B (B220⁺ GL7⁺ Fas⁺); CD138, plasma cells (CD138⁺ B220^neg/low); Tfh, T follicular helper cells (CD3⁺ CD4⁺ CD44⁺ CXCR5⁺ PD1⁺ ICOS⁺ CCR7^neg). MFI, mean fluorescence intensity.

FIG 5

Coinfection reduced the numbers of cells in mLN. Mice were infected with influenza virus (PR8) or coinfected (PR8+Sp). On day 4 and day 8 after PR8 infection, mLN was collected to examine different cell populations. (A) Numbers of total lymph node cells, B220⁺ B cells, B220⁺ CD23⁺ CD21^int FOB cells, and CD3⁺ CD4⁺ T cells. (B) B cell surface marker expression on day 4 post-PR8 infection. (C) Percentage of FOB cells in B220⁺ cells. (D) Numbers of germinal center B cells, Tfh cells, and plasma cells on day 8 post-PR8 infection. (E) Germinal center B cell and Tfh cell surface marker expression on day 8 post-PR8 infection. (F) Total IgG and PR8-specific IgG ELISpot assays were performed using cells isolated from mLN and spleen on day 8 post-PR8 infection. Results ± standard deviations (A to E) are grouped from five independent experiments. Panel C shows a representative gating of FOB cells from B220⁺ B cells. Panel F shows a representative ELISpot photo. For panels A to E, on day 4 post-PR8 infection, n = 16 for PR8 and n = 14 for PR8+Sp; on day 8 post-PR8 infection, n = 17 for PR8 and n = 14 for PR8+Sp. For panel F, n = 4 for each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001, by one-way ANOVA (A and B) or unpaired t test (C to F).

FIG 6

PR8-specific immune serum treatment after coinfection improved survival. (A) Relative level of PR8-specific IgG in mock and PR8-specific immune serum with IgG depleted or mock depleted. (B) Survival of coinfected mice receiving the serum treatments. (C) On day 5 to day 8 post-PR8 infection, whole-lung homogenate from coinfected mice receiving mock or PR8-specific immune serum was collected to determine virus titer. Results ± standard deviations are grouped from two experiments (B) or are representative of two independent experiments (C). **, P < 0.01; ***, P < 0.001, by an unpaired t test.

FIG 7

Lung inflammatory response after coinfection. Mice were infected with either influenza virus (PR8), or Streptococcus pneumoniae (Sp), or coinfected with influenza virus and then pneumococcus (PR8+Sp). (A) On day 8 post-PR8 infection, whole lung was collected to examine the number of Ly6G⁺ neutrophils and F480⁺ macrophages. (B) Whole-lung homogenate was collected on day 4 and day 8 post-PR8 infection to measure the levels of inflammatory cytokines and chemokines. Results ± standard deviations are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, by one-way ANOVA. IL-1β, interleukin-1β; IL-6, interleukin-6; MIP-1α/1β, macrophage inflammatory protein 1α/1β; MCP-1, monocyte chemotactic protein 1; GMCSF, granulocyte-macrophage colony-stimulating factor.