Secondary Bacterial Pneumonia Research Articles

Novel influenza A antibodies reduce severity of secondary pneumococcal pneumonia after influenza infection in mice

Secondary bacterial pneumonia after influenza infection can cause severe disease with a high mortality. Recently, a new group 2 influenza A antibody (AT10_002) has been developed, which binds to multiple H3 and H7 subtypes. In a mouse model of primary influenza infection, treatment with AT10_002 as a fusion antibody protects against lethal infection, and reduces loss of bodyweight [1]. We hypothesized that treatment with AT10_002 reduces weight loss, lung injury and bacterial outgrowth, in a mouse model of viral infection followed by secondary pneumococcal infection.

Influenza viral neuraminidase primes bacterial coinfection through TGF-β–mediated expression of host cell receptors

Influenza infection predisposes the host to secondary bacterial pneumonia, which is a major cause of mortality during influenza epidemics. The molecular mechanisms underlying the bacterial coinfection remain elusive. Neuraminidase (NA) of influenza A virus (IAV) enhances bacterial adherence and also activates TGF-β. Because TGF-β can up-regulate host adhesion molecules such as fibronectin and integrins for bacterial binding, we hypothesized that activated TGF-β during IAV infection contributes to secondary bacterial infection by up-regulating these host adhesion molecules. Flow cytometric analyses of a human lung epithelial cell line indicated that the expression of fibronectin and α5 integrin was up-regulated after IAV infection or treatment with recombinant NA and was reversed through the inhibition of TGF-β signaling. IAV-promoted adherence of group A Streptococcus (GAS) and other coinfective pathogens that require fibronectin for binding was prevented significantly by the inhibition of TGF-β. However, IAV did not promote the adherence of Lactococcus lactis unless this bacterium expressed the fibronectin-binding protein of GAS. Mouse experiments showed that IAV infection enhanced GAS colonization in the lungs of wild-type animals but not in the lungs of mice deficient in TGF-β signaling. Taken together, these results reveal a previously unrecognized mechanism: IAV NA enhances the expression of cellular adhesins through the activation of TGF-β, leading to increased bacterial loading in the lungs. Our results suggest that TGF-β and cellular adhesins may be potential pharmaceutical targets for the prevention of coinfection.

Antipneumococcal activity of neuraminidase inhibiting artocarpin

Streptococcus (S.) pneumoniae is a major cause of secondary bacterial pneumonia during influenza epidemics. Neuraminidase (NA) is a virulence factor of both pneumococci and influenza viruses. Bacterial neuraminidases (NAs) are structurally related to viral NA and susceptible to oseltamivir, an inhibitor designed to target viral NA. This prompted us to evaluate the antipneumococcal potential of two NA inhibiting natural compounds, the diarylheptanoid katsumadain A and the isoprenylated flavone artocarpin. Chemiluminescence, fluorescence-, and hemagglutination-based enzyme assays were applied to determine the inhibitory efficiency (IC50 value) of the tested compounds towards pneumococcal NAs. The mechanism of inhibition was studied via enzyme kinetics with recombinant NanA NA. Unlike oseltamivir, which competes with the natural substrate of NA, artocarpin exhibits a mixed-type inhibition with a Ki value of 9.70μM. Remarkably, artocarpin was the only NA inhibitor (NAI) for which an inhibitory effect on pneumococcal growth (MIC: 0.99–5.75μM) and biofilm formation (MBIC: 1.15–2.97μM) was observable. In addition, we discovered that the bactericidal effect of artocarpin can reduce the viability of pneumococci by a factor of >1000, without obvious harm to lung epithelial cells. This renders artocarpin a promising natural product for further investigations.

Interleukin 4 Deficiency Reverses Development of Secondary Pseudomonas aeruginosa Pneumonia During Sepsis-Associated Immunosuppression.

Interleukin 4 (IL-4) is an important cytokine that may modulate development of secondary bacterial pneumonia during sepsis-induced immunosuppression. We established an experimental model of cecal ligation and puncture (CLP)-induced sublethal polymicrobial sepsis followed by secondary Pseudomonas aeruginosa pulmonary infection, IL-4-deficient mice that underwent CLP were resistant to secondary pulmonary P. aeruginosa infection. As compared to wild-type mice, IL-4 knockout (KO) mice displayed improved survival and better bacterial clearance. Furthermore, IL-4 KO mice exhibited enhanced lung inflammation, neutrophil recruitment to airspaces, and elevated pulmonary cytokine production, with significantly increased tumor necrosis factor α (TNF-α) production. Neutralization of TNF-α could reverse the enhanced protection against secondary P. aeruginosa pneumonia in septic IL-4 KO mice, indicating that the resistance of septic IL-4 KO mice to secondary bacterial pneumonia was partially mediated by TNF-α. In addition, IL-4 priming displayed marked impairment of the ability of alveolar macrophages to phagocytose and kill P. aeruginosa in vitro, and this defect was associated with decreased activation of Akt, JNK, p38MAPK, and ERK intracellular signaling pathways by IL-4. Finally, neutralization of IL-4 in septic mice could improve survival and clearance of bacteria from the lungs of septic mice infected with P. aeruginosa. Our findings provide new insight for immunopathologic mechanisms of sepsis-induced secondary bacterial pneumonia.

123Influenza-induced Susceptibility to Secondary Bacterial Pneumonia Is Rescued in STAT2-/- Mice Via an IL-23 Independent Mechanism

123Influenza-induced Susceptibility to Secondary Bacterial Pneumonia Is Rescued in STAT2-/- Mice Via an IL-23 Independent Mechanism

Nonstructural protein 1 (NS1)-mediated inhibition of c-Abl results in acute lung injury and priming for bacterial co-infections: insights into 1918 H1N1 pandemic?

Nonstructural protein 1 (NS1) proteins from avian influenza viruses like the 1918 pandemic NS1 are capable of inhibiting the key signaling integrator c-Abl (Abl1), resulting in massive cytopathic cell alterations. In the current study, we addressed the consequences of NS1-mediated alteration of c-Abl on acute lung injury and pathogenicity in an in vivo mouse model. Comparing isogenic strains that differ only in their ability to inhibit c-Abl, we observed elevated pathogenicity for the c-Abl-inhibiting virus. NS1-mediated blockade of c-Abl resulted in severe lung pathology and massive edema formation and facilitated secondary bacterial pneumonia. This phenotype was independent of differences in replication and immune responses, defining it as an NS1 virulence mechanism distinct from its canonical functions. Microarray analysis revealed extensive downregulation of genes involved in cell integrity and vascular endothelial regulation. NS1 protein-mediated blockade of c-Abl signaling drives acute lung injury and primes for bacterial coinfections revealing potential insights into the pathogenicity of the 1918 pandemic virus.

Female resistance to pneumonia identifies lung macrophage nitric oxide synthase-3 as a therapeutic target.

To identify new approaches to enhance innate immunity to bacterial pneumonia, we investigated the natural experiment of gender differences in resistance to infections. Female and estrogen-treated male mice show greater resistance to pneumococcal pneumonia, seen as greater bacterial clearance, diminished lung inflammation, and better survival. In vitro, lung macrophages from female mice and humans show better killing of ingested bacteria. Inhibitors and genetically altered mice identify a critical role for estrogen-mediated activation of lung macrophage nitric oxide synthase-3 (NOS3). Epidemiologic data show decreased hospitalization for pneumonia in women receiving estrogen or statins (known to activate NOS3). Pharmacologic targeting of NOS3 with statins or another small-molecule compound (AVE3085) enhanced macrophage bacterial killing, improved bacterial clearance, and increased host survival in both primary and secondary (post-influenza) pneumonia. The data identify a novel mechanism for host defense via NOS3 and suggest a potential therapeutic strategy to reduce secondary bacterial pneumonia after influenza.

Antibiotics for bronchiolitis in children under two years of age.

Bronchiolitis is a serious, potentially life-threatening respiratory illness commonly affecting babies. It is often caused by respiratory syncytial virus (RSV). Antibiotics are not recommended for bronchiolitis unless there is concern about complications such as secondary bacterial pneumonia or respiratory failure. Nevertheless, they are often used. To evaluate the effectiveness of antibiotics for bronchiolitis in children under two years of age compared to placebo or other interventions. We searched the Cochrane Central Register of Controlled Trials (CENTRAL 2014, Issue 6), which includes the Cochrane Acute Respiratory Infection Group's Specialised Register, and the Database of Abstracts of Reviews of Effects, MEDLINE (1966 to June 2014), EMBASE (1990 to June 2014) and Current Contents (2001 to June 2014). Randomised controlled trials (RCTs) comparing antibiotics to placebo in children under two years diagnosed with bronchiolitis, using clinical criteria (including respiratory distress preceded by coryzal symptoms with or without fever). Primary clinical outcomes included time to resolution of signs or symptoms (pulmonary markers included respiratory distress, wheeze, crepitations, oxygen saturation and fever). Secondary outcomes included hospital admissions, length of hospital stay, readmissions, complications or adverse events and radiological findings. Two review authors independently analysed the search results. We included seven studies with a total of 824 participants. The results of these seven included studies were often heterogeneous, which generally precluded meta-analysis, except for deaths, length of supplemental oxygen use and length of hospital admission.In this update, we included two new studies (281 participants), both comparing azithromycin with placebo. They found no significant difference for length of hospital stay, duration of oxygen requirement and readmission. These results were similar to an older study (52 participants) that demonstrated no significant difference comparing ampicillin and placebo for length of illness.One small study (21 participants) with higher risk of bias randomised children with proven RSV infection to clarithromycin or placebo and found a trend towards a reduction in hospital readmission with clarithromycin.The three studies providing adequate data for days of supplementary oxygen showed no difference between antibiotics and placebo (pooled mean difference (MD) (days) -0.20; 95% confidence interval (CI) -0.72 to 0.33). The three studies providing adequate data for length of hospital stay, similarly showed no difference between antibiotics (azithromycin) and placebo (pooled MD (days) -0.58; 95% CI -1.18 to 0.02).Two studies randomised children to intravenous ampicillin, oral erythromycin and control and found no difference for most symptom measures.There were no deaths reported in any of the arms of the seven included studies. No other adverse effects were reported. This review did not find sufficient evidence to support the use of antibiotics for bronchiolitis, although research may be justified to identify a subgroup of patients who may benefit from antibiotics. Further research may be better focused on determining the reasons that clinicians use antibiotics so readily for bronchiolitis, how to reduce their use and how to reduce clinician anxiety about not using antibiotics.

Influenza Promotes Pneumococcal Growth during Coinfection by Providing Host Sialylated Substrates as a Nutrient Source

Much of the mortality attributed to influenza virus is due to secondary bacterial pneumonia, particularly from Streptococcus pneumoniae. However, mechanisms underlying this coinfection are incompletely understood. We find that prior influenza infection enhances pneumococcal colonization of the murine nasopharynx, which in turn promotes bacterial spread to the lungs. Influenza accelerates bacterial replication in vivo, and sialic acid, a major component of airway glycoconjugates, is identified as the host-derived metabolite that stimulates pneumococcal proliferation. Influenza infection increases sialic acid and sialylated mucin availability and enhances desialylation of host glycoconjugates. Pneumococcal genes for sialic acid catabolism are required for influenza to promote bacterial growth. Decreasing sialic acid availability in vivo by genetic deletion of the major airway mucin Muc5ac or mucolytic treatment limits influenza-induced pneumococcal replication. Our findings suggest that higher rates of disease during coinfection could stem from influenza-provided sialic acid, which increases pneumococcal proliferation, colonization, and aspiration.

Sulforaphane improves MARCO expression, bacterial clearance and survival in post‐influenza bacterial pneumonia (145.1)

Mechanisms that increase susceptibility for post‐influenza bacterial pneumonia include down‐regulation of the alveolar macrophage (AM) scavenger receptor MARCO by interferon‐γ (IFN‐γ), leading to diminished bacterial clearance. Since MARCO expression can be increased by NRF2 signaling, we tested whether the NRF2‐inducer sulforaphane (SFN) can up‐regulate MARCO expression and improve bacterial clearance after influenza. Human AM‐like monocyte‐derived macrophages (HMDMs) were treated with IFN‐γ (10‐50 IU/ml, 24h) to simulate influenza infection, followed by sulforaphane (SFN, 10µM) for 24h before quantitation of MARCO expression or challenge with green fluorescence protein (GFP)‐expressing S. aureus. Scanning cytometry was used to quantitate MARCO surface labeling and uptake of bacteria. C57/BL6 mice were inoculated i.n. on day 0 with 3500PFU of PR8 influenza virus, and on day 7 with 500 CFU of S. pneumoniae serotype 3. Mice were injected with SFN 25 or 50 mg/kg, or PBS daily from day 6 to 9 after influenza infection. Bacterial clearance was measured on day 1 after S. pneumoniae infection using lung lavage samples. IFN‐γ down‐regulated MARCO expression (p<0.001) of HMDMs and impaired phagocytosis of S. aureus (p<0.001, n=4). SFN added after IFN‐γ significantly improved MARCO expression (p=0.003) and S. aureus phagocytosis (p=0.05), an effect that was inhibited by scavenger receptor blockers poly(I) (p=0.001) or MARCO blocking peptide (P<0.001). In vivo, sulforaphane‐treated mice showed improved bacterial clearance (e.g. 84%, 95% reduction in SFN‐treated mice in 2 trials) and a significantly higher survival rate compared to PBS‐treated mice (75.0% vs. 12.5%, n = 8/grp, p=0.022). Therapeutic targeting of the NRF2 pathway may be beneficial to reduce risk or severity of secondary bacterial pneumonia after influenza.Grant Funding Source: Supported by ES00002

Protecting Against Post-influenza Bacterial Pneumonia by Increasing Phagocyte Recruitment and ROS Production

Seasonal and especially pandemic influenza predispose patients to secondary bacterial pneumonias, which are a major cause of deaths and morbidity. Staphylococcus aureus is a particularly common and deadly form of post-influenza pneumonia, and increasing staphylococcal drug resistance makes the development of new therapies urgent. We explored an innate immune-mediated model of the lung to define novel mechanisms by which the host can be protected against secondary staphylococcal pneumonia after sub-lethal influenza infection. We found that stimulating the innate immunity in the lung by overexpression of GM-CSF will result in resistance to S. aureus pneumonia after sublethal influenza infection. Resistance was mediated by alveolar macrophages and neutrophils, and was associated with increased production of reactive oxygen species (ROS) by alveolar macrophages. Resistance was abrogated by treatment with agents that scavenged ROS. We conclude that stimulating innate immunity in the lung markedly reduces susceptibility to post-influenza staphylococcal pneumonia and that this may represent a novel immunomodulatory strategy for prevention and treatment of secondary bacterial pneumonia after influenza.

Influence of prior pandemic A(H1N1)2009 virus infection on invasion of MDCK cells by community-associated methicillin-resistant Staphylococcus aureus

Secondary bacterial pneumonia due to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a highly publicized cause of death associated with influenza. In this study, we performed the gentamicin-killing assay using Madin-Darby canine kidney (MDCK) cells and MRSA strains to investigate whether prior infection from pandemic A(H1N1)2009 virus (A[H1N1]pdm09) lead to increased invasion of MDCK cells by MRSA. We found that the invasion rate of two MRSA strains (ATCC BAA-1680 [USA 300] and ATCC BAA-1699 [USA 100]) into intact MDCK cell monolayers was 0.29±0.15% and 0.007±0.002%, respectively (p<0.01, n≥3). In addition, the relative invasion rate of both ATCC BAA-1680 and ATCC BAA-1699 was significantly increased by prior A(H1N1)pdm09 infection of MDCK monolayers from 1±0.28 to 1.38±0.02 and from 1±0.24 to 1.73±0.29, respectively (p<0.01). These results indicate that ATCC BAA-1680 displays much stronger invasiveness of MDCK cells than ATCC BAA-1699, although invasion of both strains was increased by prior A(H1N1)pdm09 infection. In conclusion, this study provided the first evidence that prior A(H1N1)pdm09 infection facilitates the invasion of MDCK cells by MRSA, presumably due to cellular injury caused by the virus.

Measles Epidemics of Variable Lethality in the Early 20th Century

Until the mid-20th century, mortality rates were often very high during measles epidemics, particularly among previously isolated populations (e.g., islanders), refugees/internees who were forcibly crowded into camps, and military recruits. Searching for insights regarding measles mortality rates, we reviewed historical records of measles epidemics on the Polynesian island of Rotuma (in 1911), in Boer War concentration camps (in 1900-1902), and in US Army mobilization camps during the First World War (in 1917-1918). Records classified measles deaths by date and clinical causes; by demographic characteristics, family relationships (for Rotuma islanders and Boer camp internees), and prior residences; and by camp (for Boer internees and US Army recruits). During the Rotuman and Boer War epidemics, measles-related mortality rates were high (up to 40%); however, mortality rates differed more than 10-fold across camps/districts, even though conditions were similar. During measles epidemics, most deaths among camp internees/military recruits were due to secondary bacterial pneumonias; in contrast, most deaths among Rotuman islanders were due to gastrointestinal complications. The clinical expressions, courses, and outcomes of measles during first-contact epidemics differ from those during camp epidemics. The degree of isolation from respiratory pathogens other than measles may significantly determine measles-related mortality risk.

Adjunctive Corticosteroid Therapy Improves Lung Immunopathology and Survival During Severe Secondary Pneumococcal Pneumonia in Mice

Secondary bacterial pneumonia is a significant cause of morbidity and mortality during influenza, despite routine use of standard antibiotics. Antibiotic-induced immunopathology associated with bacterial cell wall lysis has been suggested to contribute to these poor outcomes. Using Streptococcus pneumoniae in a well-established murine model of secondary bacterial pneumonia (SBP) following influenza, we stratified disease severity based on pneumococcal load in the lungs via in vivo bioluminescence imaging. Ampicillin treatment cured mice with mild pneumonia but was ineffective against severely pneumonic mice, despite effective bacterial killing. Adjunctive dexamethasone therapy improved ampicillin-induced immunopathology and improved outcomes in mice with severe SBP. However, early dexamethasone therapy during primary influenza infection impaired lung adaptive immunity as manifest by increased viral titers, with an associated loss of its protective functions in SBP. These data support adjunctive clinical use of corticosteroids in severe cases of community-acquired pneumonia.

Infants with severe respiratory syncytial virus needed less ventilator time with nasal continuous airways pressure then invasive mechanical ventilation

Nasal continuous positive airway pressure (NCPAP) has been proposed as an early first-line support for infants with severe respiratory syncytial virus (RSV) infection. We hypothesised that infants <6months with severe RSV would require shorter ventilator support on NCPAP than invasive mechanical ventilation (IMV). Retrospective cohort analysis of infants admitted to two paediatric intensive care units, one primarily using NCPAP and one exclusively using IMV, between January 2008 and February 2010. We studied 133 (NCPAP n=89, IMV n=46) consecutively admitted infants. On admission, disease severity [i.e. Paediatric RISk of Mortality (PRISM) II score (NCPAP 5.1±2.8 vs. IMV 12.2±6.0, p<0.001) and SpO2 /Fi O2 ratio (NCPAP 309±81 vs. IMV 135±98, p<0.001)] was higher in the IMV group. NCPAP remained independently associated with shorter ventilatory support (hazard ratio 2.3, 95% CI 1.1-4.7, p=0.022) after adjusting for PRISM II score, PCO2 , SpO2 /Fi O2 ratio, bronchopulmonary dysplasia and occurrence of clinically suspected secondary bacterial pneumonia. Nasal continuous positive airway pressure was independently associated with a shorter duration of ventilatory support. Differences in baseline disease severity mandate a randomised trial before the routine use of NCPAP can be recommended.

Influenza A Virus Exacerbates Staphylococcus aureus Pneumonia in Mice by Attenuating Antimicrobial Peptide Production

Influenza A represents a significant cause of morbidity and mortality worldwide. Bacterial complications of influenza A confer the greatest risk to patients. TH17 pathway inhibition has been implicated as a mechanism by which influenza A alters bacterial host defense. Here we show that preceding influenza causes persistent Staphylococcus aureus infection and suppression of TH17 pathway activation in mice. Influenza does not inhibit S. aureus binding and uptake by phagocytic cells but instead attenuates S. aureus induced TH17 related antimicrobial peptides necessary for bacterial clearance in the lung. Importantly, exogenous lipocalin 2 rescued viral exacerbation of S. aureus infection and decreased free iron levels in the bronchoalveolar lavage from mice coinfected with S. aureus and influenza. These findings indicate a novel mechanism by which influenza A inhibits TH17 immunity and increases susceptibility to secondary bacterial pneumonia. Identification of new mechanisms in the pathogenesis of bacterial pneumonia could lead to future therapeutic targets.

Genome-wide fitness profiling reveals adaptations required by Haemophilus in coinfection with influenza A virus in the murine lung

Bacterial coinfection represents a major cause of morbidity and mortality in epidemics of influenza A virus (IAV). The bacterium Haemophilus influenzae typically colonizes the human upper respiratory tract without causing disease, and yet in individuals infected with IAV, it can cause debilitating or lethal secondary pneumonia. Studies in murine models have detected immune components involved in susceptibility and pathology, and yet few studies have examined bacterial factors contributing to coinfection. We conducted genome-wide profiling of the H. influenzae genes that promote its fitness in a murine model of coinfection with IAV. Application of direct, high-throughput sequencing of transposon insertion sites revealed fitness phenotypes of a bank of H. influenzae mutants in viral coinfection in comparison with bacterial infection alone. One set of virulence genes was required in nonvirally infected mice but not in coinfection, consistent with a defect in anti-bacterial defenses during coinfection. Nevertheless, a core set of genes required in both in vivo conditions indicated that many bacterial countermeasures against host defenses remain critical for coinfection. The results also revealed a subset of genes required in coinfection but not in bacterial infection alone, including the iron-sulfur cluster regulator gene, iscR, which was required for oxidative stress resistance. Overexpression of the antioxidant protein Dps in the iscR mutant restored oxidative stress resistance and ability to colonize in coinfection. The results identify bacterial stress and metabolic adaptations required in an IAV coinfection model, revealing potential targets for treatment or prevention of secondary bacterial pneumonia after viral infection.

Linezolid Decreases Susceptibility to Secondary Bacterial Pneumonia Postinfluenza Infection in Mice Through its Effects on IFN-γ

Influenza infection predisposes patients to secondary bacterial pneumonia that contributes significantly to morbidity and mortality. Although this association is well documented, the mechanisms that govern this synergism are poorly understood. A window of hyporesponsiveness following influenza infection has been associated with a substantial increase in local and systemic IFN-γ concentrations. Recent data suggest that the oxazolidinone antibiotic linezolid decreases IFN-γ and TNF-α production in vitro from stimulated PBMCs. We therefore sought to determine whether linezolid would reverse immune hyporesponsiveness after influenza infection in mice through its effects on IFN-γ. In vivo dose-response studies demonstrated that oral linezolid administration sufficiently decreased bronchoalveolar lavage fluid levels of IFN-γ at day 7 postinfluenza infection in a dose-dependent manner. The drug also decreased morbidity as measured by weight loss compared with vehicle-treated controls. When mice were challenged intranasally with Streptococcus pneumoniae 7 d postinfection with influenza, linezolid pretreatment led to decreased IFN-γ and TNF-α production, decreased weight loss, and lower bacterial burdens at 24 h postbacterial infection in comparison with vehicle-treated controls. To determine whether these effects were due to suppression of IFN-γ, linezolid-treated animals were given intranasal instillations of rIFN-γ before challenge with S. pneumoniae. This partially reversed the protective effects observed in the linezolid-treated mice, suggesting that the modulatory effects of linezolid are mediated partially by its ability to blunt IFN-γ production. These results suggest that IFN-γ, and potentially TNF-α, may be useful drug targets for prophylaxis against secondary bacterial pneumonia following influenza infection.

Clinical, Virological, and Histopathological Manifestations of Fatal Human Infections by Avian Influenza A(H7N9) Virus

Systematic analysis of histopathological and serial virological changes of fatal influenza A(H7N9) cases is lacking. Patients with A(H7N9) infection admitted to our intensive care unit during 10-23 April 2013 were included. Viral loads in the respiratory tract, as inferred from the cycle threshold (Ct) value of reverse transcription polymerase chain reaction (RT-PCR), and the serum hemagglutination inhibition (HAI) antibody titer, were analyzed. Postmortem biopsies of the lung, liver, kidney, spleen, bone marrow, and heart were examined. Twelve patients (6 deaths, 6 survivors) were included. Median viral load was higher in sputa than the nasopharyngeal swabs for fatal cases (median Ct, 23 vs 30.5; P = .08). RT-PCR for A(H7N9) was positive in stool samples (4/6 [67%]) of fatal cases and (2/6 [33%]) of survivors, but was negative in the cerebrospinal fluid, urine, or blood of all patients. Nosocomial bacterial infections were more common in patients who died than in survivors (83% vs 50%). HAI titers increased by ≥4-fold in those with convalescent sera. Postmortem biopsy for 3 patients showed acute diffuse alveolar damage. Patient 1, who died 8 days after symptom onset, had intra-alveolar hemorrhage. Patients 2 and 3, who died 11 days after symptom onset, had pulmonary fibroproliferative changes. Reactive hemophagocytosis in the bone marrow and lymphoid atrophy in splenic tissues were compatible with laboratory findings of leukopenia, lymphopenia, and thrombocytopenia. Hypoxic and fatty changes of kidney and liver tissues are compatible with impaired renal or liver function. Fatal A(H7N9) infection was characterized by viral and secondary bacterial pneumonia with 67% having positive RT-PCR in stool.

Depletion of Alveolar Macrophages during Influenza Infection Facilitates Bacterial Superinfections

Viruses such as influenza suppress host immune function by a variety of methods. This may result in significant morbidity through several pathways, including facilitation of secondary bacterial pneumonia from pathogens such as Streptococcus pneumoniae. PKH26-phagocytic cell labeling dye was administered intranasally to label resident alveolar macrophages (AMs) in a well-established murine model before influenza infection to determine turnover kinetics during the course of infection. More than 90% of resident AMs were lost in the first week after influenza, whereas the remaining cells had a necrotic phenotype. To establish the impact of this innate immune defect, influenza-infected mice were challenged with S. pneumoniae. Early AM-mediated bacterial clearance was significantly impaired in influenza-infected mice: ~50% of the initial bacterial inoculum could be harvested from the alveolar airspace 3 h later. In mock-infected mice, by contrast, >95% of inocula up to 50-fold higher was efficiently cleared. Coinfection during the AM depletion phase caused significant body weight loss and mortality. Two weeks after influenza, the AM population was fully replenished with successful re-establishment of early innate host protection. Local GM-CSF treatment partially restored the impaired early bacterial clearance with efficient protection against secondary pneumococcal pneumonia. We conclude that resident AM depletion occurs during influenza infection. Among other potential effects, this establishes a niche for secondary pneumococcal infection by altering early cellular innate immunity in the lungs, resulting in pneumococcal outgrowth and lethal pneumonia. This novel mechanism will inform development of novel therapeutic approaches to restore lung innate immunity against bacterial superinfections.