Specific pathogen-free, BALB/c WT, Rag2 ^-/- Il2rg ^-/-, and hCsf2/Il3 KI mice, and C57BL/6 WT, Ifngr1^-/- and Csf2rb^-/- mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred at University of Texas Medical Branch (UTMB) following Animal Care and Use Committee (IACUC) guidelines. All animal experiments were approved by UTMB, and all experiments were carried out in accordance with UTMB Assurance of Compliance with PHS Policy on Humane Care and Use of Laboratory Animals, which is on file with the Office of Protection from Research Risks, NIH.

Viral challenge was performed with a low dose (0.01 LD₅₀) of X31 (~5×10³ PFU/mouse) or PR8 (~10 PFU/mouse, ~0.02 LD₅₀) administered intranasally (i.n.) to anesthetized mice in 50 µl of sterile PBS. Titers of virus stocks and viral levels in the bronchoalveolar lavage fluids (BALF) and lungs of infected mice were determined by plaque assays on MDCK cell monolayers.

To induce bacterial pneumonia, anesthetized mice were inoculated i.n. with 50 μl of PBS containing 2×10⁴ CFU of serotype 14 strain TJO983 or 1×10⁵ CFU of serotype 2 strain D39 (26). Bacterial burdens in the BALF and lungs were measured by sacrificing infected mice at the indicated time points, and plating serial 10-fold dilutions of each sample onto blood agar plates.

BALF samples were collected by making an incision in the trachea and lavaging the lung twice with 0.8 ml PBS, pH 7.4. Total leukocyte counts were determined using a hemacytometer.

For flow cytometric analysis, BALF cells were incubated with 2.4G2 mAb against FcγRII/III, and stained with BV510- or FITC-conjugated anti-CD45, Allophycocyanin (APC)-conjugated anti-CD11c, APC-Cy7-conjugated anti-MHC II (I-A/I-E), BV510- or PE-Cy7-conjugated anti-CD11b, PE-Cy7- or FITC-conjugated anti-Ly6G (clone 1A8), PerCp-Cy5.5-conjugated anti-Ly6C, BV421-conjugated anti-Siglec-F, and PE- or PerCp-Cy5.5-conjugated anti-TCRβ mAbs ((H57-59, BioLegend) for myeloid cell analysis. The stained cells were analyzed on a MACSQuant analyzer. Data analysis was performed with FlowJo software.

BALF were harvested and assayed for TNF-α, IL-1β, IL-6, IFN-γ, MCP-1 and KC by ELISA using commercially available kits from BD Biosciences and R&D Systems (Minneapolis, MN).

Neutrophils were depleted using anti-Gr1 mAb RB6-8C5 or anti-Ly6G mAb 1A8 (BioXCell). Specifically, starting at one day before bacterial infection, mice were injected intraperitoneally (i.p.) with anti-Gr1 or anti-Ly6G mAb (0.1 mg/day) to deplete neutrophils or with rat IgG as a control. The efficiency of neutrophil depletion in bacterial-infected mice was confirmed by flow cytometry.

For T cell depletion, BALB/c WT mice were injected i.p. with 20 μg/mouse of hamster anti-murine CD3e mAb (145-2C11, BioXCell) every 5 days beginning 10 days before X31 infection, and then 100 μg/mouse every three days following PR8 infection. Control mice were treated with hamster IgG (HIgG). The efficiency of T cell depletion was confirmed by flow cytometry analysis of TCRβ⁺ cells.

Significant differences between experimental groups were determined using a two-tailed Student t-test (to compare two samples), an ANOVA analysis followed by Tukey’s multiple comparisons test (to compare multiple samples) or Mann Whitney test (nonparametric test) in GraphPad Prism 6 (La Jolla, CA). Survival analyses were performed using the log-rank test. For all analyses, a P value <0.05 was considered to be significant.

To determine whether host genetic differences contribute to the risk of secondary bacterial pneumonia, we performed side-by-side comparisons of B6 and BALB/c WT mice in their susceptibility to post-IAV SPn infection. Specifically, B6 and BALB/c WT mice were infected either with low-virulent X31 or high-virulent PR8 and seven days later super-challenged with a SPn serotype 14 strain TJO983 (SPn14). A low dose of X31 (0.01 LD₅₀) or PR8 (0.02 LD₅₀) infection alone did not induce apparent weight loss in B6 or BALB/c WT mice. However, at 7 days after PR8 infection, both B6 and BALB/c WT mice were highly susceptible to SPn14 superinfection ( Figure 1A ). Compared with PR8/SPn14 coinfection, B6 mice exhibited reduced bacterial outgrowth after X31/SPn14 coinfection. Nonetheless, X31/SPn14 coinfection resulted in ~10³-fold increased bacterial burdens in B6 lungs as compared with SPn14 infection alone. In sharp contrast, prior X31 infection in BALB/c mice did not impair lung bacterial control. In fact, lung bacterial burden was significantly reduced in X31/SPn14 coinfected BALB/c mice compared with SPn14 infection alone. There were barely any detectable bacterial and viral burdens in BALB/c mice at 3 days post-coinfection (dpc), i.e, 10 days after X31 infection ( Figures 1B, C ). These striking differences between B6 and BALB/c mice suggest that host genetic traits play a significant role in susceptibility to secondary pneumococcal pneumonia after mild X31 infection.

We have previously shown in both B6 and BALB/c mouse models that IFN-γ inhibits innate antibacterial immunity during recovery from PR8 infection (7, 21). In fact, PR8 infection alone is sufficient to induce prominent IFN-γ production by T cells (27). Indeed, PR8/SPn14 coinfection resulted in prominent IFN-γ response in B6 and BALB/c mice, in agreement with their high susceptibility ( Figure 2A ). In contrast, X31/SPn14 coinfection induced significant IFN-γ production in the susceptible B6 but not resistant BALB/c mice, indicating that IAV-induced susceptibility to SPn14 superinfection is correlated with IFN-γ response. We next determined whether T cells are responsible for IFN-γ production in BALB/c mice after PR8 infection. T cell depletion with anti-CD3 antibodies (28) diminished IFN-γ response and completely restored initial bacterial clearance in BALB/c mice 24 h after PR8/SPn14 coinfection ( Figures 2B, C ). It suggests that like that in B6 mice (21), PR8-activated T cells are responsible for driving susceptibility to secondary pneumococcal infection in BALB/c mice.

Phagocytes, particularly AM and neutrophils, are essential for acute clearance of pneumococcal infection. Our previous studies in B6 mice have shown that T cell-derived IFN-γ impairs AM antibacterial function (21). Thus, we wanted to examine whether this is also the case for the BALB/c mice. Rag2 ^-/- Il2rg ^-/- (also known as Rag2 ^-/- γc ^-/-) mice are deficient in T, B and innate lymphoid cells. In the absence of IAV infection, both BALB/c WT and Rag2 ^-/- Il2rg ^-/- mice were capable of efficient SPn14 clearance ( Figures 3A, B ). Conversely, prior PR8 infection impaired acute bacterial clearance in BALB/c WT but not Rag2 ^-/- Il2rg ^-/- mice ( Figure 3B ). These results agree with findings that PR8-induced T cell activation impairs innate antibacterial immunity in the lung.

Due to knock-in (KI) replacement of mouse csf2 gene in Rag2 ^-/- Il2rg ^-/- mice, human (h) Csf2Il3 double KI (DKI) mice (29) are also devoid of AM ( Figure 3C ). hCsf2/Il3 DKI mice exhibited ~10³-fold increased lung CFUs 24 h after SPn14 infection alone, as compared with Rag2^-/- Il2rg ^-/- controls. These results indicate that AM are also required for acute clearance of SPn14 in BALB/c mice. Note that after X31/SPn14 coinfection, hCsf2/Il3 DKI mice exhibited substantial monocyte and neutrophil recruitment. Importantly, prior X31 infection had no additional impact on lung bacterial control in BALB/c WT, T cell-deficient Rag2^-/- Il2rg ^-/-, or AM-deficient hCsf2/Il3 DKI mice, despite defective viral clearance in the latter two groups ( Figures 3D, E ). These findings, particularly the similar bacterial CFUs in hCsf2/Il3 DKI mice after SPn14 single- or super-infection, suggest that prior X31 infection has no significant impact on the AM-independent bacterial clearance mechanisms in BALB/c hCsf2/Il3 DKI mice.

In the absence of IAV infection, SPn14 is innocuous to mice even after a high dose of systemic infection (7, 26). In line with that, we have shown that although X31/SPn14 coinfection leads to lung bacterial outgrowth in B6 WT mice, it does not have significant impact on animal survival (30). We wanted to determine whether the resistance of BALB/c mice is limited to a low dose (2×10⁴ CFU) of SPn14 superinfection. Accordingly, we used a higher dose (1×10⁵ CFU/mouse) of bacteremia strain D39 to determine the effect of genetic differences between B6 and BALB/c mice on the progression and severity of X31/SPn coinfection.

We first examined whether the window of susceptibility to SPn superinfection differs between B6 and BALB/c mice following X31 infection. Accordingly, B6 and BALB/c mice were challenged with ~10⁵ CFU of D39 bacteria at various days after X31 infection. Mice were sacrificed 24 h later for determination of lung viral and bacterial burdens, as well as cytokine responses ( Figure 4A ). Of note, a 0.01 LD₅₀ dose of X31 infection alone did not induce obvious weight loss in either B6 or BALB/c mice ( Figure 4B ). Nonetheless, compared to respective D39 single-infected controls, B6 mice exhibited defective bacterial clearance starting at 6 d after X31 infection (6d_X31/D39), whereas BALB/c mice were relatively competent in their ability to clear bacteria ( Figure 4C ). Although B6 and BALB/c mice were distinct in SPn susceptibility at days 6 and 7 after X31 infection, D39 superinfection did not appear to interfere with lung viral clearance in two strains of mice ( Figure 4D ). Of note, both mouse strains were resistant to D39 superinfection 4 d after X31 infection (4d_X31/D39), despite high viral replication at this early time point. Lung viral titers were also comparable between two groups after 6d_X31/D39 coinfection, despite the bacterial outgrowth in B6 mice. These results verify that rather than direct viral pathogenicity, the resistance versus susceptibility of BALB/c and B6 mice to SPn superinfection results from their differential immune responses during recovery from IAV infection.

In line with that, B6 mice exhibited significantly enhanced IFN-γ response after 6d_X31/D39 coinfection, compared with X31 or D39 single infection ( Figure 5 ). Interestingly, B6 mice tended to enhance monocyte chemoattractant protein-1 (MCP-1) response, whereas BALB/c mice exhibited significantly increased neutrophil chemokine KC production after 4d_X31/D39 coinfection ( Figure 5C ). Nonetheless, D39 superinfection at this innate phase of X31 infection did not induce significant IFN-γ production in B6 or BALB/c mice ( Figure 5A ).

To determine the role of IFN-γ in the susceptible B6 mice, we examined the pathogenicity of X31/D39 coinfection in B6 WT and IFN-γ receptor gene-deficient (Ifngr1 ^-/-) mice. Ifngr1 ^-/- mice exhibited increased neutrophil recruitment and bacterial clearance compared with B6 WT controls, in agreement with their significantly improved survival after 6d_X31/D39 coinfection ( Figure 6 ). Thus, the susceptibility of B6 mice to SPn superinfection at least in part results from IFN-γ-biased immune response.

We next analyzed myeloid cell profiles in B6 and BALB/c mice to determine whether neutrophils are responsible for their differential susceptibility to D39 superinfection ( Figure 7A ). Compared with corresponding BALB/c mice, neutrophil counts were increased in B6 mice during X31 infection alone ( Figure 7B ) but decreased with D39 single infection ( Figure 7C ). Nonetheless, B6 mice exhibited significantly increased neutrophil recruitment after 6d_X31/D39 coinfection than BALB/c mice ( Figure 7D ). Interestingly, neutrophils in BALB/c mice exhibited increased Ly6G surface expression than those in B6 animals after coinfection ( Figure 7A ). Conversely, AM numbers were comparable between two groups under different infectious conditions. Thus, it agrees with findings in the X31/SPn14 coinfection model that rather than regulating AM or neutrophil numbers, prior X31 infection has a differential impact on the phagocyte function in BALB/c and B6 mice.

We have previously shown in B6 mice that AM are essential and sufficient for acute clearance of SPn14 in the airway, whereas neutrophils are required for protection against SPn14 systemic infection (7). Similarly, we found that AM were essential for acute clearance of D39 in B6 mice, as evidenced by ~10³-fold increased lung CFUs in Csf2rb ^-/- mice after 10⁵ CFU D39 infection alone ( Figure 7E ). On the other hand, antibody-mediated neutrophil depletion had no significant impact on bacterial control in B6 lungs ( Figure 7F ), suggesting that neutrophils are not essential for acute clearance of D39 in the absence of IAV infection. Similar to B6 mice, anti-Ly6G antibody treatment of BALB/c mice had no significant effect on acute bacterial clearance during 10⁵ CFU D39 infection alone. However, neutrophil depletion resulted in >100-fold increased bacterial burdens in BALB/c mice after X31/D39 coinfection ( Figure 7G ). These results suggest that neutrophils are essential for maintaining antibacterial immunity in BALB/c mice after X31 infection. Furthermore, the differential requirement for neutrophils before and after X31 infection indicates that the antibacterial function of AM is also impaired in BALB/c mice after X31 infection.

We further investigated the progression of X31/D39 coinfection in resistant BALB/c and susceptible B6 mice. At 3 days after 6d_X31/D39 coinfection ( Figure 8A ), B6 mice not only exhibited heightened bacterial outgrowth in the lungs but also widespread bacterial invasion into the bloodstream ( Figures 8B, C ). At the same time, BALB/c mice started to resolve coinfection, as indicated by diminished bacterial burden and neutrophil accumulation in the lungs ( Figures 8B–D ). In accordance with that, B6 mice exhibited significantly increased mortality rates after X31/D39 coinfection, as compared with BALB/c animals ( Figure 8E, F ). Collectively, these data establish that antibacterial immunity is profoundly inhibited in B6 mice during recovery from X31 infection, resulting in the lethal outcome of D39 superinfection.