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. Author manuscript; available in PMC: 2023 Jul 1.
Published in final edited form as: J Immunol. 2022 Jun 15;209(1):128–135. doi: 10.4049/jimmunol.2101135

Type I IFN Signaling is Essential for Preventing IFN-γ Hyperproduction and Subsequent Deterioration of Antibacterial Immunity during Post-Influenza Pneumococcal Infection

Sunil Palani *, Shruti Bansal †, Atul K Verma *, Christopher Bauer †, Shengjun Shao *, Bashir Uddin *, Keer Sun *,†
PMCID: PMC9247018  NIHMSID: NIHMS1803120  PMID: 35705254

Abstract

Post-influenza bacterial pneumonia is a significant cause of hospitalization and death in humans. The mechanisms underlying this viral and bacterial synergy remain incompletely understood. Recent evidence indicates that influenza-induced interferons (IFN), particularly type I IFN (IFN-I) and IFN-γ, suppress antibacterial defenses. Here we have investigated the relative importance and interplay of IFN-I and IFN-γ pathways in influenza-induced susceptibility to Streptococcus pneumoniae infection. Using gene-deficient mouse models as well as in vivo blocking antibodies, we show that both IFN-I and IFN-γ signaling pathways contribute to the initial suppression of antibacterial immunity; however, IFN-γ plays a dominant role in the disease deterioration, in association with increased TNF-α production and alveolar macrophage (AM) depletion. We have previously shown that IFN-γ impairs AM antibacterial function and thereby acute bacterial clearance. The findings in this study indicate that IFN-γ signaling also impairs AM viability and αβ T cell recruitment during the progression of influenza/S. pneumoniae coinfection. Macrophages insensitive to IFN-γ (MIIG) mice express a dominant negative mutant IFN-γ receptor in mononuclear phagocytes. Interestingly, MIIG mice exhibited significantly improved recovery and survival from coinfection, despite delayed bacterial clearance. Importantly, we demonstrate that IFN-I receptor signaling is essential for preventing IFN-γ hyperproduction and animal death during the progression of post-influenza pneumococcal pneumonia.

Introduction

Secondary Streptococcus pneumoniae infection is a significant cause of morbidity and mortality after influenza (1–4). Recent evidence suggests that influenza-induced immune changes suppress innate antibacterial defense and thereby cause susceptibility to secondary bacterial infection (5–13). However, the fundamental pathogenic mechanisms remain incompletely understood.

Acute respiratory viral infection typically activates IFN responses in the lung. Type I IFNs (IFN-I), i.e., IFN-α/β, are often recognized as key innate cytokines in limiting virus replication at the early stage of infection. It has also been shown that IFN-I signaling attenuates inflammatory lung damage during influenza A virus (IAV) infection (14–16). However, multiple recent studies have suggested that IFN-I signaling inhibits neutrophil recruitment and thereby increases host susceptibility to secondary bacterial infection (10, 17–19). On the other hand, IFN-γ is mainly produced by T cells at the recovery phase of IAV infection (20). It has been suggested that IFN-γ is not required for protective antiviral immunity but limits lung inflammation during IAV infection (21). Conversely, we have shown that influenza-induced IFN-γ impairs alveolar macrophage (AM)-mediated bacterial killing and thereby increases host susceptibility to pneumococcal infection (5, 22). Although these reported studies have demonstrated detrimental impacts of IFN-I and IFN-γ on innate antibacterial immunity, the interplay between these two signaling pathways remains unclear during post-influenza bacterial infection.

In this study, we have investigated the relative importance of IFN-I and IFN-γ pathways in the pathogenesis of post-influenza pneumococcal infection side-by-side, using gene-deficient mouse models as well as in vivo blocking antibodies. The results indicate that IFN-I and IFN-γ each employs distinct regulatory impacts on the inflammatory cytokine and cell response during coinfection. While both IFN pathways are involved in the initial suppression of bacterial clearance, IFN-γ plays a predominant role in the deterioration of immune defense at the later stage of coinfection. Importantly, our study demonstrates that IFN-I signaling is required for inhibiting IFN-γ hyperproduction and preventing animal death during the progression of post-influenza pneumococcal pneumonia.

Materials and Methods

Murine model of viral and bacterial infection

Specific pathogen-free WT, Ifnar1−/−, Ifng−/− and Ifngr1−/− mice on the C57BL/6 background were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred at University of Nebraska Medical Center (UNMC) and University of Texas Medical Branch (UTMB) following Animal Care and Use Committee (IACUC) guidelines. C57BL/6 MIIG mice were originally generated at Cincinnati Children’s Hospital Medical Center (23) and maintained at UNMC. All animal experiments were approved by UNMC and UTMB, and all experiments were carried out in accordance with UNMC and 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 sublethal dose of A/Puerto Rico/8/1934 (PR8), i.e., ~10 plaque-forming unit (PFU)/mouse, administered intranasally (i.n.) to anesthetized, sex and age-matched adult 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 S. pneumoniae infection, anesthetized mice were inoculated i.n. with 50 μl of PBS containing 104 colony-forming unit (CFU) of serotype 14 strain TJO983 (24). Bacterial burdens in the BALF and lungs were measured by sacrificing infected mice at indicated time points after infection, and plating serial 10-fold dilutions of BALF and lung samples onto blood agar plates. Mortality was monitored twice daily until day 15 after bacterial infection.

Bronchoalveolar lavage (BAL) cell analysis

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 cytometry analysis, BALF cells were incubated with 2.4G2 mAb against FcγRII/III and stained with APC conjugated anti-CD11c (Biolegend), BUV395-conjugated anti-CD11b (BD Biosciences), FITC-conjugated or PE-Cy7-conjugated anti-Ly6G (clone 1A8, Biolegend), PerCP-Cy5.5-conjugated (eBiosciences) or PE-conjugated anti-Ly6C (Biolegend), and BV421-conjugated or PE-conjugated anti-Siglec-F (BD Biosciences) mAbs. The stained cells were analyzed on a BD LSRII-green or LSRFortessa using BD FACSDiva and FlowJo software analysis.

Determination of cytokine/chemokine production by ELISA

BALF were harvested and assayed for TNF-α, IL-1β, IL-6, IFN-γ, KC and MIP-2 by ELISA using commercially available kits from BD Biosciences and R&D Systems (Minneapolis, MN).

Evaluation of airway damage

BALF were harvested and assayed for albumin by ELISA using a commercially available kit from Bethyl Laboratories (Montgomery, TX). Total protein levels and lactic acid dehydrogenase (LDH) activities in BALF were analyzed by a Micro bicinchoninic acid assay (BCA) protein assay kit (Thermo Scientific) and a LDH cytotoxicity assay kit (Thermo Scientific), respectively.

In vivo labeling of resident alveolar macrophages.

We inoculated anesthetized mice i.n. with 100 μl of 10 μM PKH26-PCL (Sigma) 7 d before IAV infection (5). We then collected BALF or lung cells after PR8 and/or SPn infection.

In vivo cytokine blockage

For blocking IFNAR1, mice were administrated intraperitoneally (i.p.) with 500 μg/day of MAR1-5A3 (mouse anti-murine IFNAR1 mAb) for six doses beginning on the day of PR8 infection. For IFN-γ neutralization, mice were injected i.p. with 300 μg/day of XMG1.2 (rat anti-murine IFN-γ mAb) for five doses beginning 5 d after PR8 infection. Other groups of mice were treated with mouse or rat IgG as controls accordingly.

Statistics

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 9. Survival analyses were performed using the log-rank test. For all analyses, a P value <0.05 was considered to be significant.

Results

IFN-γ plays a dominant role in driving lethal susceptibility to post-influenza pneumococcal pneumonia

Influenza-induced cytokines IFN-γ and IFN-I have both been shown to suppress innate antibacterial immunity. In this study, we evaluated their relative importance in the pathogenic process side-by-side during post-influenza pneumococcal infection. Specifically, C57BL/6 WT, Ifnar1−/−, and Ifng−/− mice were infected with ~10 PFU/mouse of PR8 (H1N1), and seven days later super-challenged with a ~104 CFU/mouse of S. pneumoniae serotype 14 strain TJO983 (SPn). In the absence of prior IAV infection, 90% of bacteria were cleared from lungs within 24 h, as indicated by <103 CFU/lung in all SPn single-infected mice (Fig. 1A). IAV/SPn coinfection resulted in bacterial outgrowth in all groups of animals. Nonetheless, both Ifnar1−/− and Ifng−/− mice showed 10-fold reduced bacterial burdens at 24 h as compared with WT controls. These results indicate a suppressive effect of IFN-I and IFN-γ on initial bacterial clearance in WT mice, where both IFN pathways are intact (Fig. 1B). Interestingly, only Ifng−/− mice exhibited persistent lung bacterial control at 3 d post SPn super-infection (dps), as evidenced by >100-fold reduced bacterial burdens compared with Ifnar1−/− and WT animals. Consistent with that, only Ifng−/− animals were protected from an otherwise lethal coinfection in Ifnar1−/− and WT mice (Fig. 1C). These results suggest that IFN-γ plays a dominant role in deterioration of antibacterial immunity during the progression of IAV/SPn coinfection.

Figure 1. IFN-γ plays a dominant role in causing lethal susceptibility to post-influenza pneumococcal pneumonia.

Figure 1.

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(A) Lung bacterial burdens at 24 h after challenge of naïve B6 WT, Ifnar1−/− and Ifng−/− mice with 104 SPn. (B) Lung bacterial burdens at 1 and 3 d, and (C) animal survival after super-challenge of WT, Ifnar1−/−, and Ifng−/− mice with 104 SPn on day seven after PR8 infection (Flu/SPn). The median survival of WT and Ifnar1−/− mice were 5.5 and 8 d, respectively. **P<0.01, ***P<0.001, Mann Whitney test (A-B) or log-rank test (C). Data shown are representative two independent experiments.

IFN-I signaling inhibits IFN-γ hyperproduction during IAV/SPn coinfection

It has been shown that IFN-I signaling impairs CXC chemokine production and thereby decreases neutrophil recruitment during secondary pneumococcal infection (10). We thus examined the regulatory effect of IFN-I and IFN-γ on the key inflammatory cytokine and chemokine responses during IAV/SPn coinfection. IAV infection alone induced IFN-γ production in WT mice, which was not significantly affected by SPn super-infection (Fig. 2A). Conversely, IAV/SPn coinfection resulted in increased productions of pro-inflammatory cytokines TNF-α and IL-6 in BALF, but IL-1β levels were decreased compared with SPn single-infection (Fig. 2A). The levels of neutrophil chemoattractant KC and MIP-2 were also significantly elevated in coinfected Ifnar1−/− and WT mice at 3 dps (Fig. 2). At the same time, these inflammatory cytokine and chemokine responses were diminished in coinfected Ifng−/− mice (Fig. 2B). These observations indicate that IFN-γ not only impairs bacterial clearance but also promotes inflammatory cytokine response during IAV/SPn coinfection. Of particular interest, IFN-γ levels were significantly elevated in coinfected Ifnar1−/− mice as compared with WT controls, indicating an inhibitory effect of IFN-I signaling on IFN-γ production.

Figure 2. IFN-γ promotes inflammatory cytokine and chemokine production after influenza and pneumococcal coinfection.

Figure 2.

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(A) Airway production of inflammatory cytokines TNF-α, IL-1β, IL-6, IFN-γ, and CXC chemokines KC and MIP-2 (mean±SE, 5~12 mice/group) at 1 and 3 d after challenge of naïve (SPn), day seven PR8-infected (Flu/SPn) B6 WT mice with SPn or PBS control (Flu). (B) Airway inflammatory cytokine and chemokine levels (mean±SE, 9~14 mice/group) at 1 and 3 d after super-challenge of day seven PR8-infected WT, Ifnar1−/−, Ifng−/− mice with SPn. *P< 0.05, **P< 0.01, ***P<0.001, t test. Data shown are representative of two independent experiments.

IFN-γ induces AM depletion during IAV/SPn coinfection

Phagocytes are essential effector cells for immune clearance of S. pneumoniae. It has been shown that IFN-I and IFN-γ synergistically inhibit inflammatory cell recruitment during IAV infection alone (20). During IAV/SPn coinfection, we found that the actual numbers of airway CD11b+Ly6G+ neutrophils were comparable between WT and Ifnar1−/− mice, but CD11b+Ly6C+ monocytes were significantly decreased in the latter (Fig. 3A–B). Conversely, the actual numbers of neutrophils were increased in Ifng−/− mice at 24 h (Fig. 3A–B), suggesting an inhibitory effect of IFN-γ on neutrophil accumulation in the airway.

Figure 3. IFN-γ impairs AM survival after influenza and pneumococcal coinfection.

Figure 3.

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(A) Airway myeloid cell profiles (mean of 5 mice/group) at 1 d, and (B) the actual numbers (mean±SE) of AM, inflammatory monocytes (Mo), and neutrophils (PMN) at 1 and 3 d after super-challenge of day seven PR8-infected WT, Ifnar1−/−, Ifng−/− mice with SPn. Data shown are representative of two independent experiments.

Of particular interest, Ifng−/− mice exhibited significantly increased numbers of CD11c+Siglec-F+ AM as compared with Ifnar1−/− and WT animals after IAV/SPn coinfection (Fig. 3B). Considering the critical role of AM in innate defense against pneumococcal infection (22, 25–27), we next investigated the impact IAV/SPn coinfection on AM self-maintenance. Resident phagocytic cells were labeled by PKH26-PCL dye 7 d before PR8 infection (Fig. 4). At 3 d after SPn super-infection, we found that the numbers of PKH26+ AM in the BALF were significantly decreased compared with IAV or SPn single-infected WT controls (Fig. 4A). While lung Ly6C+ monocytes were comparable between IAV single-infected and IAV/SPn coinfected mice, the actual numbers of neutrophils were 5~10 fold increased after coinfection (Fig. 4B). In agreement with findings in the airway (Fig. 4A), the total numbers of lung PKH26+ AM were 5~7-fold decreased in coinfected WT mice at 3 dps (Fig. 4C–D). These findings suggest that IFN-γ is involved in the AM depletion during IAV/SPn coinfection.

Figure 4. Resident AM are depleted during the progression of influenza and pneumococcal coinfection.

Figure 4.

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Naïve mice were inoculated i.n. with PKH26-PCL dye seven days before PR8 infection. (A) the actual numbers (mean±SE, 4–9 mice/group) of PKH26+ AM in BALF at 1 and 3 d after challenge of naïve (SPn), day seven PR8-infected (Flu/SPn) WT mice with SPn or PBS control (Flu). (B) Total numbers (mean±SE) of lung inflammatory monocytes (Mo) and neutrophils (PMN), (C) flow cytometry analysis of AM and other inflammatory cells, and (D) total numbers of PKH26+ AM in WT mice at 1 and 3 d after challenge of naïve, PR8-infected WT mice with SPn or PBS control. **P< 0.01, ***P<0.001, Mann Whitney test. Data shown are representative of two independent experiments.

Blocking IFN-I signaling improves bacterial clearance but is not sufficient to prevent lethal IAV/SPn coinfection

We next examined whether temporarily blocking IFN-I and IFN-γ signaling replicates the findings in the constitutive gene-deficient mice. It is well-established that IAV induces innate IFN-I and adaptive IFN-γ production at the early and later stage of infection, respectively (20). Based on this information, specific anti-IFNAR1 and anti-IFN-γ monoclonal antibodies (mAb) were administered daily to WT mice (5, 10, 28), from 0–5 days after PR8 infection or 5–9 days later, respectively (Fig. 5). Indeed, blocking IFN-I or IFN-γ pathway significantly improved bacterial clearance after coinfection (Fig. 5A). In agreement with findings in Ifnar1−/− mice, airway Ly6C+ monocytes were reduced but IFN-γ production was highly elevated after IFNAR1 blockage. Conversely, the actual numbers of neutrophils were increased after IFN-γ neutralization (Fig. 5B). Interestingly, lung viral clearance was also significantly improved after anti-IFN-γ treatments (Fig. 5C). Furthermore, compared with rat IgG-treated controls, IFN-γ neutralization reduced acute TNF-α production and significantly improved protection against AM depletion and animal death (Fig. 5B–E). These results further confirm that IFN-γ plays a dominant role in the deterioration process of IAV/SPn coinfection. In line with heightened IFN-γ production, anti-IFNAR1 treatment was not sufficient to rescue WT mice from lethal coinfection (Fig. 5E). These results suggest that IFN-I signaling prevents IFN-γ hyperproduction and its associated detrimental effect at the later stage of IAV/SPn coinfection.

Figure 5. Temporarily blocking IFNAR1 signaling improves bacterial clearance but not animal survival.

Figure 5.

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WT mice were infected with influenza PR8 and seven days later super-challenged with SPn. (A) Lung bacterial burdens at 1 and 3 d, (B) BALF numbers (mean±SE) of AM, inflammatory monocytes (Mo), and neutrophils (PMN) at 1 and 3 d, (C) lung viral burdens at 3 d, (D) BALF TNF-α, IL-6 and IFN-γ levels (mean±SE) at 3 d, and (E) animal survivals after PR8/SPn coinfection. Mice were treated daily with anti-IFNAR1 and anti-IFN-γ mAb from 0–5 days after PR8 infection or 5–9 days later, respectively. Control mice were administrated with mouse or rat IgG. The median survival of mouse or rat IgG-treated mice were 5 d, while the median survival of anti-IFNAR1 and anti-IFN-γ-treated mice were 4 and 7 d, respectively, *P<0.05, **P< 0.01, ***P<0.001, Mann Whitney test (A-D) and log-rank test (E). Data shown are representative of two independent experiments.

Defective viral clearance is not primarily responsible for the lethality of IAV/SPn coinfection

As shown above, IAV/SPn coinfection was lethal to the majority of Ifnar1−/− mice, even though their bacterial clearance was initially improved compared with WT controls. It has been reported that IFN-I signaling improves antiviral immunity and attenuates inflammatory lung damage during IAV infection (14, 16). Thus, we examined whether the defect in antiviral immunity is responsible for the lethality of IAV/SPn coinfection in Ifnar1−/− mice. Compared with IAV infection alone, IAV/SPn coinfection led to similarly decreased AM numbers in both WT and Ifnar1−/− mice at 3 dps (Fig. 6A). Conversely, compared with WT controls, Ifnar1−/− mice exhibited reduced airway TCRβ+ cells after IAV infection alone. Interestingly, airway TCRβ+ cells were significantly decreased in WT mice after SPn super-infection, implying an impairment in αβ T cell recruitment. Furthermore, compared with IAV infection alone, IAV/SPn coinfection resulted in significantly increased activities of lactate dehydrogenase (LDH) in the bronchoalveolar lavage fluid (BALF) of both WT and Ifnar1−/− mice (Fig. 6B), suggesting aggravated lung tissue damage. However, there are no significant differences in BALF total protein, LDH and albumin levels between Ifnar1−/− and WT mice, during IAV infection alone or IAV/SPn coinfection, suggesting a limited impact of IFN-I on acute lung damage (Fig. 6B).

Figure 6. IFNAR1 deficiency delays viral clearance but does not cause animal death after IAV infection alone.

Figure 6.

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(A) The numbers (mean±SE, 5 mice/group) of AM, inflammatory monocytes (Mo), neutrophils (PMN), and TCRβ+ cells in the BALF, (B) airway protein, lactate dehydrogenase (LDH), and albumin levels (mean±SE, 9~10 mice/group), and (C) lung viral titers 3 d after super-challenge of day seven PR8-infected WT and Ifnar1−/− mice with SPn or PBS control. (D) Animal survival after super-challenge of day seven PR8-infected Ifnar1−/− mice with SPn or PBS control. The median survival of IAV/SPn coinfected Ifnar1−/− mice were 4 d. *P<0.05, **P<0.01, Tukey’s multiple comparisons test. Data shown are representative two independent experiments.

In agreement with reduced αβ T cells, Ifnar1−/− mice tended (P = 0.08) to have increased viral titers as compared with WT controls during IAV infection alone (Fig. 6C). Furthermore, compared with respective IAV-infected controls, IAV/SPn coinfection tended (P = 0.10) to increase lung viral titers in both WT and Ifnar1−/− mice (Fig. 6C). As a result, lung viral titers became comparable between IAV-infected Ifnar1−/− and coinfected WT mice at 3 dps. Nonetheless, all Ifnar1−/− mice survived the same dose of IAV infection alone (Fig. 6D). These data suggest that although the antiviral immunity is also impaired during the deterioration of IAV/SPn coinfection, the defect in viral control is not the primary cause of animal lethality.

IFN-γ signaling in mononuclear myeloid cells induces AM depletion and animal death

We have previously shown that SPn super-infection does not have an immediate (24 h) impact on lung viral clearance (22, 25). On the other hand, our results shown above suggest that SPn super-infection impairs the resolution of viral infection at a later stage. We thus examined the role of IFN-γ receptor (IFN-γR) signaling in this process. Like Ifng1−/− mice, coinfected Ifngr1−/− exhibited improved bacterial clearance as compared with WT controls (Fig. 7A). Interestingly, although viral titers were comparable between WT and Ifngr1−/− lungs during IAV infection alone, Ifngr1−/− mice exhibited significantly reduced viral titers after coinfection (Fig. 7B). Furthermore, TNF-α production was diminished in Ifngr1−/− mice at 3 dps, even though their IFN-γ levels were highly elevated during IAV infection alone or IAV/SPn coinfection, likely due to the compensatory IFN-γ production (Fig. 7C).

Figure 7. IFN-γR signaling impairs both antiviral and antibacterial immunity during the progression of IAV/SPn coinfection.

Figure 7.

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(A) Lung bacterial and (B) viral burdens (mean±SE, 5 mice/group), (C) TNF-α and IFN-γ levels, and (D) the numbers of AM, inflammatory monocytes (Mo), neutrophils (PMN), and TCRβ+ cells in the BALF 3 d after super-challenge of day seven PR8-infected WT and Ifngr1−/− mice with SPn or PBS control. *P<0.05, **P<0.01, ***P<0.001, Tukey’s multiple comparisons test. Data shown are representative two independent experiments.

Although IAV/SPn coinfection reduced AM in both WT and Ifngr1−/− mice, coinfected Ifngr1−/− mice indeed exhibited increased AM numbers as compared with WT controls (Fig. 7D). Conversely, monocyte recruitment was reduced in Ifngr1−/− mice during IAV infection alone and IAV/SPn coinfection. Neutrophils were also reduced in coinfected Ifngr1−/− mice at 3 dps. Of particular interest, distinct from WT controls, IAV/SPn coinfection had no significant impact on the number of airway TCRβ+ cells in Ifngr1−/− mice, implying a detrimental role of IFN-γR signaling on αβ T cell recruitment in coinfected WT mice (Fig. 7D).

We have shown that IFN-γ inhibits AM phagocytic function and thereby impairs acute bacterial clearance in the lung (5). The results in this study suggest that IFN-γ also induces AM depletion and disease deterioration during the progression of coinfection. Thus, we determined whether this detrimental role of IFN-γ at the later stage of coinfection is dependent on the initial suppression of bacterial clearance. “Macrophages insensitive to IFN-gamma” (MIIG) mice express a dominant negative mutant IFN-γ receptor in CD68+ cells (23). Macrophages and monocytes in these mice are unable to respond to IFN-γ, whereas neutrophils respond to this cytokine normally. Interestingly, while lung bacterial burdens were comparable between WT and MIIG mice 24 h after IAV/SPn coinfection (Fig. 8A), TNF-α production was reduced in MIIG mice (Fig. 8B). In a sharp contrast to Ifngr1−/− mice, the level of IFN-γ in BALF was also reduced in coinfected MIIG mice, probably due to the binding of IFN-γ to decoy receptors expressed by all CD68+ cells (Fig. 8B). Nonetheless, compared with as WT controls, MIIG mice exhibited significantly improved bacterial clearance at 3 dps (Fig. 8A), despite similar neutrophil recruitment (Fig. 8C). Importantly, MIIG mice displayed significantly increased AM numbers and animal survival as compared with WT littermates (Fig. 8C–D). Together, these findings suggest that IFN-γ signaling in mononuclear phagocytes promotes TNF-α production, AM depletion, and subsequent deterioration of immune defense, independent of initial suppression of antibacterial immunity.

Figure 8. IFN-γ signaling in mononuclear myeloid cells promotes deterioration of antibacterial immunity after influenza and pneumococcal coinfection.

Figure 8.

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(A) Lung bacterial burdens, (B) BALF levels (mean±SE) of TNF-α and IL-6, (C) numbers (mean±SE) of AM, inflammatory monocytes (Mo), and neutrophils (PMN) at 1 and 3 d, and (D) animal survivals after super-challenge of day seven PR8-infected WT and MIIG with SPn. The median survival of WT mice was 5.5 d. *P<0.05, **P< 0.01, ***P<0.001, Mann Whitney test (A-C) and log-rank test (D). Data shown are representative of two independent experiments.

Discussion

Recent evidence indicates that influenza-induced IFN responses, particularly IFN-I and IFN- γ signaling, suppress antibacterial immunity, thereby increasing host susceptibility to secondary bacterial pneumonia. Using a comparative model of IAV and S. pneumoniae coinfection, here we evaluated the relative importance and interplay of IFN-I and IFN-γ signaling pathways in the pathogenic process. Our findings indicate that during host response to IAV/SPn coinfection, IFN-γR signaling plays a dominant role in the disease deterioration, by delaying the resolution of both viral and bacterial infection. On the other hand, although IFN-I signaling plays a significant role in initial suppression of antibacterial immunity, it is also essential for preventing IFN-γ hyperproduction and subsequent animal death after IAV/SPn coinfection.

Influenza typically induces Th1/IFN-biased type 1 cytokine response. Recent evidence indicates that IFN responses during IAV infection, including IFN-γ, IFN-λ, IFN-I, and IFNAR signaling-dependent IL-27 production, suppress antibacterial immunity, thereby increasing host susceptibility to secondary bacterial pneumonia (10, 17, 18, 29–32). However, the crosstalk among these IFN signaling pathways remains unclear during the pathogenesis process of IAV/bacterial coinfection.

We have previously demonstrated that AM are not only essential but also sufficient for airway clearance of S. pneumoniae serotype 14 strain TJO983. However, IAV infection induces AM dysfunction and thereby enables this otherwise noninvasive S. pneumoniae to cause deadly pneumonia (22). Furthermore, we have shown that IFN-γ inhibits AM-mediated bacterial killing at the initial phase of IAV/SPn coinfection (5). In the current study, we focused on the detrimental role of IFN-γ during the progression of coinfection. Using both Ifng−/− and Ifngr1−/− mice as well as IFN-γ neutralizing antibodies, we show, for the first time in our studies, that IFN-γ signaling induces AM depletion at the later stage of coinfection. These findings suggest that IFN-γ not only impairs AM antibacterial function but also their viability during the coinfection process.

A mathematical model predicts that AM dysfunction is primarily responsible for bacterial outgrowth after influenza and pneumococcal coinfection (27). Moreover, it has been shown in BALB/c mice that IAV infection depletes AM and thereby leads to increased susceptibility to pneumococcal super-infection (26). However, the direct effects of IAV infection on AM survival appear to be dependent on mouse genetic strain. As such, AM levels in B6 mice are maintained throughout the course of IAV infection (33). Here we show that after a low dose of PR8 infection, the numbers of AM were largely unaltered in B6 WT mice within 24 h after SPn super-infection (Fig. 3B). Thus, we believe that IFN-γ suppresses initial bacterial clearance primarily by impairing AM antibacterial function.

In contrast, MIIG mice did not exhibit enhanced bacterial clearance at 24 h after SPn super-infection. It is likely that despite the expression of dominant negative IFN-γR, the low level of IFN-γ signaling is sufficient to inhibit AM-mediated acute bacterial clearance in MIIG mice (23). As a result, the initial bacterial burden was comparable between coinfected MIIG and WT mice. This transgenic mouse model allowed us to determine the role of IFN-γ at the later stage of coinfection, distinct from its initial suppression of antibacterial immunity. Indeed, MIIG mice exhibited significantly improved AM maintenance and bacterial control at 3 dps, in association with increased animal survival. These findings indicate that IFN-γ signaling in mononuclear cells directly induces AM depletion and deterioration of antibacterial immunity at the later stage of IAV/SPn coinfection.

It has been shown that IAV infection inhibits Toll-like receptor signaling in AM, and thereby reduces neutrophil recruitment and enhances susceptibility to secondary bacterial infection (11). In line with that, studies from other groups have suggested neutrophil suppression as the key pathogenic mechanism of IAV-induced susceptibility to secondary bacterial infection (7, 8). Particularly, this suppression has been attributed to activation of the IFN-I signaling pathway after IAV infection (10, 32). Indeed, we found that blocking IFNAR1 improves acute bacterial clearance during IAV/SPn coinfection, despite no apparent effect on neutrophils. However, IFNAR1 blockage was not effective in protection against animal death in our coinfection model. This inefficacy is not due to the short-term of antibody treatments, as indicated by ~90% mortality rate in coinfected Ifnar1−/− mice.

Of note, although Ifnar1−/− mice are more susceptible to IAV infection (16), we did not detect a significant impact of temporary IFNAR1 blockage on lung viral control (Fig. 5C). During IAV infection alone, it has been shown that rather than regulating IFN-γ production, IFN-I reduces IFN-γR expression and thereby protects Ly6Clo monocytes/macrophages from IFN-γ-induced activation (20). In the current study, we found that IFN-I signaling inhibits IFN-γ expression during IAV/SPn coinfection. Thus, it is likely that IFN-γ hyperproduction, together with IFN-γR upregulation (34), offsets the beneficial impact of IFNAR blockage on the initial bacterial clearance, thereby resulting in the inefficacy of anti-IFNAR1 treatment for protection against lethal IAV/S. pneumoniae coinfection.

It has been shown that IFN-I in the upper respiratory tract promotes S. pneumoniae colonization and transmission during S. pneumoniae-influenza coinfection (35, 36). Interestingly, using this nasopharyngeal carriage model of S. pneumoniae followed by influenza super-infection, Barman et al have recently demonstrated that sequential neutralization of IFN-I and IFN-γ pathways provides optimal protection against animal death (28). Additionally, it has been shown that IFNAR1 blockage improved bacterial clearance and animal survival when WT mice were super-challenged with SPn on day 5 after IAV infection (10). On the other hand, we have shown that influenza-induced peak susceptibility to bacterial super-infection coincides with T cell recruitment and IFN-γ production, i.e., day 7 after IAV infection. Thus, it appears that the relative contribution of IFN-I and IFN-γ to coinfection process is dependent on the virulence mechanism of viral and bacterial strains, and importantly, the timing of bacterial infection in relation to influenza disease stage. Specially, when SPn infection occurs before or at the innate phase of IAV infection (i.e. before T cell activation), IFN-I plays a key role in the suppression of antibacterial immunity by inhibiting neutrophil recruitment (10, 28). Conversely, at the adaptive phase of IAV infection, T cell-derived IFN-γ plays a predominant role in inducing host susceptibility to SPn super-infection (Fig. 9). Furthermore, we show in the current study that intact IFN-I receptor signaling is essential for preventing IFN-γ hyperproduction and its associated deterioration of antiviral and antibacterial immunity at this later stage.

Figure 9. The relative contributions of IFN-I and IFN-γ to the pathogenesis of IAV/SPn coinfection.

Figure 9.

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Schematic model of myeloid cell changes and influence of IFN-I and IFN-γ signaling on antibacterial immunity at the innate and adaptive phase of IAV infection. In WT mice, IFN-I produced at the innate phase of IAV infection promotes Ly6C+ monocytes but inhibits neutrophil recruitment, and thereby impairs the capability of bacterial control in the lung. In Ifnar1−/− mice, neutrophil recruitment is enhanced, leading to increased bacterial killing in the lung. After the onset of the adaptive immune response (~ 7 d after IAV infection), T cell IFN-γ production in WT mice plays a central role in the suppression of antibacterial immunity, including AM dysfunction and depletion; IFN-I limits IFN-γ hyper-production at this adaptive phase of IAV infection. As a result, Ifnar1−/− mice become susceptible to bacterial super-infection like WT animals. In Ifngr1−/− mice, AM retain their capacity in bacterial control, which in turn attenuates inflammatory response and improves animal survival from post-influenza bacterial infection.

In association with bacterial outgrowth, IAV/SPn coinfection resulted in increased TNF-α but decreased IL-1β production in the lung. Of note, although both anti-IFN-γ and anti-IFNAR1 mAb treatments improved bacterial clearance, only anti-IFN-γ-treated mice exhibited decreased TNF-α production. Similarly, TNF-α levels were reduced in MIIG mice as compared with WT littermates, despite their comparable lung bacterial burdens at 24 h. These observations suggest that IFN-γ signaling directly enhances TNF-α production during coinfection. In agreement, our recent studies in an acute lung damage model show that IFN-γ drives TNF-α hyperproduction and thereby lethal lung inflammation (37). On the other hand, we have previously shown that IL-1 signaling prevents AM depletion during IAV/SPn coinfection (25). However, we did not detect a direct effect of IFN-γ on IL-1β production. In line with that, it has been recently reported that TNF-α and IFN-γ synergistically trigger inflammatory cell death, tissue damage, and mortality in SARS-CoV-2 infection (38). Thus, it will be interesting to determine whether IFN-γ promotes TNF-α hyperproduction, which in turn induces inflammatory lung damage and AM depletion during the progression of IAV/SPn coinfection in future studies.

In summary, our current study demonstrates that while both IFN-I and IFN-γ pathways are involved in the initial suppression of antibacterial immunity, IFN-γ plays a dominant role during the deterioration process of IAV/SPn coinfection. Importantly, we demonstrate that IFN-I receptor signaling is essential for preventing IFN-γ hyperproduction and animal death during the progression of post-influenza pneumococcal pneumonia.

Key points.

  • IFN-γ plays a dominant role in the deterioration of IAV/bacterial coinfection

  • IFN-γ induces alveolar macrophage depletion during IAV/bacterial coinfection

  • IFN-α/β signaling prevents IFN-γ hyperproduction during IAV/bacterial coinfection

Footnote:

This work was funded by NIH grants R01 HL118408 and R21 AI128527 to K.S.

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