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Melanin concentrating hormone (MCH) controls many brain functions, such as sleep/wake cycle and memory, and modulates the inflammation response. Previous studies have shown that influenza A virus (IAV) infection-induced neuroinflammation leads to central nervous damage. This study investigated the potential effects of MCH against neuroinflammation induced by IAV infection and its mechanism. MCH (1 and 2 mg/ml) was administrated for 5 consecutive days before IAV infection. Pentobarbital-induced sleep tests, an open-field test, and a Morris water maze were performed to measure sleep quality, spatial learning and memory ability. Neuronal loss and microglial activation were observed with Nissl staining and immunofluorescence assay. The levels of inflammatory cytokines and the expression of the JNK/ERK signaling pathway were examined by ELISA and western blot. IAV infection led to poor sleep quality, impaired the ability of spatial learning and memory, caused neuronal loss and microglial activation in mice's hippocampus and cortex. Meanwhile the level of inflammatory cytokines increased, and the JNK/ERK signaling pathway was activated after IAV infection. MCH administration significantly alleviated IAV-induced neuroinflammation, cognitive impairment, and sleep disorder, decreased the levels of inflammatory cytokines, and inhibited neuronal loss and microglial activation in the hippocampus and cortex by regulating the JNK/ERK signaling pathway. Therefore, MCH alleviated the neuroinflammation, spatial learning and memory impairment, and sleep disorder in IAV-infected mice by regulating the JNK/ERK signaling pathway., (© 2024. The Author(s).)

Background: Influenza-associated pulmonary aspergillosis (IAPA) is a severe fungal superinfection in critically ill influenza patients that is of incompletely understood pathogenesis. Despite the use of contemporary therapies with antifungal and antivirals, mortality rates remain unacceptably high. We aimed to unravel the IAPA immunopathogenesis as a means to develop adjunctive immunomodulatory therapies., Methods: We used a murine model of IAPA to investigate how influenza predisposes to the development of invasive pulmonary aspergillosis. Immunocompetent mice were challenged with an intranasal instillation of influenza on day 0 followed by an orotracheal inoculation with Aspergillus 4 days later. Mice were monitored daily for overall health status, lung pathology with micro-computed tomography (μCT) and fungal burden with bioluminescence imaging (BLI). At endpoint, high parameter immunophenotyping, spatial transcriptomics, histopathology, dynamic phagosome biogenesis assays with live imaging, immunofluorescence staining, specialized functional phagocytosis and killing assays were performed., Findings: We uncovered an early exuberant influenza-induced interferon-gamma (IFN-γ) production as the major driver of immunopathology in IAPA and delineated the molecular mechanisms. Specifically, excessive IFN-γ production resulted in a defective Th17-immune response, depletion of macrophages, and impaired killing of Aspergillus conidia by macrophages due to the inhibition of NADPH oxidase-dependent activation of LC3-associated phagocytosis (LAP). Markedly, mice with partial or complete genetic ablation of IFN-γ had a restored Th17-immune response, LAP-dependent mechanism of killing and were fully protected from invasive fungal infection., Interpretation: Together, these results identify exuberant viral induced IFN-γ production as a major driver of immune dysfunction in IAPA, paving the way to explore the use of excessive viral-induced IFN-γ as a biomarker and new immunotherapeutic target in IAPA., Funding: This research was funded by the Research Foundation Flanders (FWO), project funding under Grant G053121N to JW, SHB and GVV; G057721N, G0G4820N to GVV; 1506114 N to KL and GVV; KU Leuven internal funds (C24/17/061) to GVV, clinical research funding to JW, Research Foundation Flanders (FWO) aspirant mandate under Grant 1186121N/1186123 N to LS, 11B5520N to FS, 1SF2222N to EV and 11M6922N/11M6924N to SF, travel grants V428023N, K103723N, K217722N to LS. FLvdV was supported by a Vidi grant of the Netherlands Association for Scientific Research. FLvdV, JW, AC and GC were supported by the Europeans Union's Horizon 2020 research and innovation program under grant agreement no 847507 HDM-FUN. AC was also supported by the Fundação para a Ciência e a Tecnologia (FCT), with the references UIDB/50026/2020, UIDP/50026/2020, PTDC/MED-OUT/1112/2021 (https://doi.org/10.54499/PTDC/MED-OUT/1112/2021), and 2022.06674.PTDC (http://doi.org/10.54499/2022.06674.PTDC); and the "la Caixa" Foundation under the agreement LCF/PR/HR22/52420003 (MICROFUN)., Competing Interests: Declaration of interests SF declares having received travel support for conference attendance by Pfizer and Gilead, and a speaker fee from The Healthbook Company Ltd. JW declares having received investigator-initiated grants, speakers’ fees and travel support by Pfizer and Gilead, outside the scope of this manuscript. The other authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

Epidemiological studies have shown that smoking is associated with increased incidence of severe viral infections leading to hospitalization. Moreover, studies in experimental models have identified impaired antiviral responses and altered inflammatory responses, yet it is unclear how the effects of smoke exposure and influenza A infection interact and how this varies over the course of infection. We hypothesized that smoking would exacerbate innate immune responses against influenza. To test this, female BALB/c mice were exposed to cigarette smoke or air twice a day for 24-28 days and (mock) infected with H3N2 influenza A on day 21 while smoking continued. About 3 and 7 days after infection, changes in immune cell populations, the transcriptome, and viral clearance in lung tissue were analyzed. After influenza A infection, smoke-exposed mice lost significantly more weight than air-exposed controls, indicating that smoking resulted in more severe disease. Immune cell and lung tissue transcriptome analysis revealed that neutrophil infiltration was prolonged and macrophage activation dysregulated after infection of smoke-exposed mice compared with air-exposed controls. Expression of genes in IL-6 and interferon pathways was similarly longer active. In parallel, we observed slower clearance of influenza virus in smoke-exposed mice after infection compared with air-exposed controls, indicating ineffective antiviral responses. Altogether, the data from our mouse model indicate that cigarette smoke exposure prolongs innate immune responses against influenza A. The results from this study help to explain the susceptibility of current smokers to severe influenza A disease. NEW & NOTEWORTHY In this study, we describe how cigarette smoke exposure modulates immune responses against influenza in mice over time. Using a unique model that continues smoke exposure after infection, we demonstrate that inflammation is prolonged and viral clearance is delayed. This clinically relevant model for smokers that contract influenza is well positioned to investigate interactions between smoke and influenza infection.

Immune activation is essential for lung control of viral and bacterial infection, but an overwhelming inflammatory response often leads to the onset of acute respiratory distress syndrome. IL-10 plays a crucial role in regulating the balance between antimicrobial immunity and immunopathology. In the present study, we investigated the role of IL-10 in acute lung injury induced by influenza A virus and methicillin-resistant Staphylococcus aureus coinfection. This unique coinfection model resembles patients with acute pneumonia undergoing appropriate antibiotic therapies. Using global IL-10 and IL-10 receptor gene-deficient mice, as well as in vivo neutralizing antibodies, we show that IL-10 deficiency promotes IFN-γ-dominant cytokine responses and triggers acute animal death. Interestingly, this extreme susceptibility is fully preventable by IFN-γ neutralization during coinfection. Further studies using mice with Il10ra deletion in selective myeloid subsets reveal that IL-10 primarily acts on mononuclear phagocytes to prevent IFN-γ/TNF-α hyperproduction and acute mortality. Importantly, this antiinflammatory IL-10 signaling is independent of its inhibitory effect on antiviral and antibacterial defense. Collectively, our results demonstrate a key mechanism of IL-10 in preventing hypercytokinemia and acute respiratory distress syndrome pathogenesis by counteracting the IFN-γ response.

Background: Gut microbiota (GM) have been implicated as important regulators of gastrointestinal symptom which is commonly occurred along with respiratory influenza A virus (IAV) infection, suggesting the involvement of the gut-to-lung axis in a host's response to IAV. IAV primarily destroys airway epithelium tight junctions (TJs) and consequently causes acute respiratory disease syndrome. It is known that GM and their metabolism produce an anti-influenza effect, but their role in IAV-induced airway epithelial integrity remains unknown., Methods: A mouse model of IAV infection was established. GM were analyzed using 16S rRNA gene sequencing, and short-chain fatty acids (SCFAs) levels were measured. GM depletion and fecal microbiota transplantation (FMT) were conducted to validate the role of GM in IAV infection. A pair-feeding experiment was conducted to reveal whether IAV-induced GM dysbiosis is attributed to impaired food intake. Furthermore, human bronchial epithelial (HBE) cells were cocultured with IAV in the presence or absence of acetate. TJs function was analyzed by paracellular permeability and transepithelial electronic resistance (TEER). The mechanism of how acetate affects TJs integrity was evaluated in HBE cells transfected with G protein-coupled receptor 43 (GPR43) short hairpin RNA (shRNA)., Results: IAV-infected mice exhibited lower relative abundance of acetate-producing bacteria (Bacteroides, Bifidobacterium, and Akkermansia) and decreased acetate levels in gut and serum. These changes were partly caused by a decrease in food consumption (due to anorexia). GM depletion exacerbated and FMT restored IAV-induced lung inflammatory injury. IAV infection suppressed expressions of TJs (occludin, ZO-1) leading to disrupted airway epithelial barrier function as evidenced by decreased TEER and increased permeability. Acetate pretreatment activated GPR43, partially restored IAV-induced airway epithelial barrier function, and reduced inflammatory cytokines levels (TNF-α, IL-6, and IL-1β). Such protective effects of acetate were absent in HBE cells transfected with GPR43 shRNA. Acetate and GPR43 improved TJs in an AMP-activated protein kinase (AMPK)-dependent manner., Conclusion: Collectively, our results demonstrated that GM protected airway TJs by modulating GPR43-AMPK signaling in IAV-induced lung injury. Therefore, improving GM dysbiosis may be a potential therapeutic target for patients with IAV infection., (© 2024. The Author(s).)

Influenza is a significant public health and economic threat around the world. Epidemiological studies have demonstrated a close association between influenza pandemics and cardiovascular mortality. Moreover, it has been shown that there is a decrease in cardiovascular mortality in high-risk patients following vaccination with the influenza vaccine. Here, we have investigated the role of anti-viral STAT1 signaling in influenza-induced myocarditis. Wild-type mice (C57BL/6) were infected with either influenza A/PR/8/34 or control, and cellular response and gene expression analysis from the heart samples were assessed 7 days later. The expression of interferon response genes STAT1, STAT2, Mx1, OASL2, ISG15, chemokines CCL2, CCL3, CXCL9 and CXCL10, and the frequency of neutrophils (CD45 + CD11b + Ly6G + ) and CD4+ T cells (CD45 + CD4 + ) were all significantly increased in influenza-infected mice when compared to vehicle controls. These data suggest that influenza infection induces interferons, inflammatory chemokines, and cellular recruitment during influenza infection. We further investigated the role of STAT1 in influenza-induced myocarditis. The frequency of neutrophils and the levels of lipocalin 2 were significantly increased in STAT1 -/- mice when compared to WT controls. Finally, we investigated the role of Lcn2 in viral-induced myocarditis. We found that in the absence of Lcn2, there was preserved cardiac function in Lcn2 -/- mice when compared to WT controls. These data suggest that the absence of Lcn2 is cardioprotective during viral-induced myocarditis., (© 2024. The Author(s).)

Nontypeable Haemophilus influenzae (NTHi) is a major otitis media (OM) pathogen, with colonization a prerequisite for disease development. Most acute OM is in children <5 years old, with recurrent and chronic OM impacting hearing and learning. Therapies to prevent NTHi colonization and/or disease are needed, especially for young children. Respiratory viruses are implicated in driving the development of bacterial OM in children. We have developed an infant mouse model of influenza-driven NTHi OM, as a preclinical tool for the evaluation of safety and efficacy of clinical therapies to prevent NTHi colonization and the development of OM. In this model, 100% of infant BALB/cARC mice were colonized with NTHi, and all developed NTHi OM. Influenza A virus (IAV) facilitated the establishment of dense (1 × 10 5 CFU/mL) and long-lasting (6 days) NTHi colonization. IAV was essential for the development of NTHi OM, with 100% of mice in the IAV/NTHi group developing NTHi OM compared with 8% of mice in the NTHi only group. Histological analysis and cytokine measurements revealed that the inflammation observed in the middle ear of the infant mice with OM reflected inflammation observed in children with OM. We have developed the first infant mouse model of NTHi colonization and OM. This ascension model uses influenza-driven establishment of OM and reflects the clinical pathology of bacterial OM developing after a respiratory virus infection. This model provides a valuable tool for testing therapies to prevent or treat NTHi colonization and disease in young children., Competing Interests: The authors declare no conflict of interest.

Influenza-associated encephalopathy (IAE) is extremely acute in onset, with high lethality and morbidity within a few days, while the direct pathogenesis by influenza virus in this acute phase in the brain is largely unknown. Here we show that influenza virus enters into the cerebral endothelium and thereby induces IAE. Three-weeks-old young mice were inoculated with influenza A virus (IAV). Physical and neurological scores were recorded and temporal-spatial analyses of histopathology and viral studies were performed up to 72 h post inoculation. Histopathological examinations were also performed using IAE human autopsy brains. Viral infection, proliferation and pathogenesis were analyzed in cell lines of endothelium and astrocyte. The effects of anti-influenza viral drugs were tested in the cell lines and animal models. Upon intravenous inoculation of IAV in mice, the mice developed encephalopathy with brain edema and pathological lesions represented by micro bleeding and injured astrocytic process (clasmatodendrosis) within 72 h. Histologically, massive deposits of viral nucleoprotein were observed as early as 24 h post infection in the brain endothelial cells of mouse models and the IAE patients. IAV inoculated endothelial cell lines showed deposition of viral proteins and provoked cell death, while IAV scarcely amplified. Inhibition of viral transcription and translation suppressed the endothelial cell death and the lethality of mouse models. These data suggest that the onset of encephalopathy should be induced by cerebral endothelial infection with IAV. Thus, IAV entry into the endothelium, and transcription and/or translation of viral RNA, but not viral proliferation, should be the key pathogenesis of IAE., (© 2024. The Author(s).)

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome 1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection 6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Respiratory tract virus infections cause millions of hospitalizations worldwide each year. Severe infections lead to lung damage that coincides with persistent inflammation and a lengthy repair period. Vaccination and antiviral therapy help to mitigate severe infections before or during the acute stage of disease, but there are currently limited specific treatment options available to individuals experiencing the long-term sequelae of respiratory viral infection. Herein, C57BL/6 mice were infected with influenza A/PR/8/34 as a model for severe viral lung infection and allowed to recover for 21 days. Mice were treated with rapamycin, a well-characterized mammalian target of rapamycin complex 1 (mTORC1) inhibitor, on days 12 to 20 after infection, a time period after viral clearance. Persistent inflammation following severe influenza infection in mice was primarily driven by macrophages and T cells. Uniform manifold approximation and projection analysis of flow cytometry data revealed that lung macrophages had high activation of mTORC1, an energy-sensing kinase involved in inflammatory immune cell effector functions. Rapamycin treatment reduced lung inflammation and the frequency of exudate macrophages, T cells, and B cells in the lung, while not impacting epithelial progenitor cells or adaptive immune memory. These data highlight mTORC1's role in sustaining persistent inflammation following clearance of a viral respiratory pathogen and suggest a possible intervention for post-viral chronic lung inflammation., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Streptococcus pneumoniae ( Sp ) frequently causes secondary pneumonia after influenza A virus (IAV) infection, leading to high morbidity and mortality worldwide. Concomitant pneumococcal and influenza vaccination improves protection against coinfection but does not always yield complete protection. Impaired innate and adaptive immune responses have been associated with attenuated bacterial clearance in influenza virus-infected hosts. In this study, we showed that preceding low-dose IAV infection caused persistent Sp infection and suppression of bacteria-specific T-helper type 17 (Th17) responses in mice. Prior Sp infection protected against subsequent IAV/ Sp coinfection by improving bacterial clearance and rescuing bacteria-specific Th17 responses in the lungs. Furthermore, blockade of IL-17A by anti-IL-17A antibodies abrogated the protective effect of Sp preinfection. Importantly, memory Th17 responses induced by Sp preinfection overcame viral-driven Th17 inhibition and provided cross-protection against different Sp serotypes following coinfection with IAV. These results indicate that bacteria-specific Th17 memory cells play a key role in providing protection against IAV/ Sp coinfection in a serotype-independent manner and suggest that a Th17-based vaccine would have excellent potential to mitigate disease caused by coinfection. IMPORTANCE Streptococcus pneumoniae ( Sp ) frequently causes secondary bacterial pneumonia after influenza A virus (IAV) infection, leading to increased morbidity and mortality worldwide. Current pneumococcal vaccines induce highly strain-specific antibody responses and provide limited protection against IAV/ Sp coinfection. Th17 responses are broadly protective against Sp single infection, but whether the Th17 response, which is dramatically impaired by IAV infection in naïve mice, might be effective in immunization-induced protection against pneumonia caused by coinfection is not known. In this study, we have revealed that Sp -specific memory Th17 cells rescue IAV-driven inhibition and provide cross-protection against subsequent lethal coinfection with IAV and different Sp serotypes. These results indicate that a Th17-based vaccine would have excellent potential to mitigate disease caused by IAV/ Sp coinfection., Competing Interests: The authors declare no conflict of interest.

Background: Stability of intestinal flora is not only important for maintaining stable immune functions; it is also a key immune channel communicating the interaction between lung and intestine. In this study, probiotics and fecal microbiota transplantation (FMT) were used to regulate influenza-infected mice with antibiotic-induced intestinal dysbiosis and the effects of intestinal microorganisms on these mice were subsequently observed and evaluated., Methods: Mice are housed in a normal environment with intranasal infection with influenza virus (FM1). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine messenger RNA expression and lung viral replication of toll-like receptor 7 (TLR7), myeloid differentiation primary reaction 88 (MyD88) and nuclear factor κB (ss) p65 in the TLR7 signaling pathway. Western blotting is used to measure the expression levels of TLR7, MyD88, and NF-κB p65 proteins. Flow cytometry was used to detect the proportion of Th17/T regulated cells., Results: Results showed that compared with the simple virus group, both diversity and species of intestinal flora in influenza-infected mice with antibiotic-induced intestinal dysbiosis were lower, in vivo viral replication was significantly increased, lung and intestinal tissues were seriously damaged, degree of inflammation increased, expression of the TLR7 signaling pathway increased, and the Th1/Th2:Th17/Treg ratio decreased. Probiotics and FMT effectively regulated intestinal flora, improved pathological lung changes and inflammation caused by influenza infection, and adjusted the TLR7 signaling pathway and the Th1/Th2:Th17/Treg ratio. This effect was not obvious in TLR7-⁣/- mice.In summary, by affecting the TLR7 signaling pathway, intestinal microorganisms reduced the inflammatory response in the lungs of influenza-infected mice with imbalances in antibiotic flora., Conclusions: By affecting the TLR7 signaling pathway, intestinal microorganisms reduced the inflammatory response in the lungs of influenza-infected mice with imbalances in antibiotic flora. In summary, damage to lung tissue and intestinal mucosa in influenza-infected mice with antibiotic-induced intestinal dysbiosis is more serious compared to simple virus-infected mice. Improving intestinal flora using probiotics or FMT can alleviate intestinal inflammation and improve pulmonary inflammation through the TLR7 signaling pathway., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s). Published by IMR Press.)

Pathogen infection causes a stereotyped state of sickness that involves neuronally orchestrated behavioural and physiological changes 1,2 . On infection, immune cells release a 'storm' of cytokines and other mediators, many of which are detected by neurons 3,4 ; yet, the responding neural circuits and neuro-immune interaction mechanisms that evoke sickness behaviour during naturalistic infections remain unclear. Over-the-counter medications such as aspirin and ibuprofen are widely used to alleviate sickness and act by blocking prostaglandin E2 (PGE2) synthesis 5 . A leading model is that PGE2 crosses the blood-brain barrier and directly engages hypothalamic neurons 2 . Here, using genetic tools that broadly cover a peripheral sensory neuron atlas, we instead identified a small population of PGE2-detecting glossopharyngeal sensory neurons (petrosal GABRA1 neurons) that are essential for influenza-induced sickness behaviour in mice. Ablating petrosal GABRA1 neurons or targeted knockout of PGE2 receptor 3 (EP3) in these neurons eliminates influenza-induced decreases in food intake, water intake and mobility during early-stage infection and improves survival. Genetically guided anatomical mapping revealed that petrosal GABRA1 neurons project to mucosal regions of the nasopharynx with increased expression of cyclooxygenase-2 after infection, and also display a specific axonal targeting pattern in the brainstem. Together, these findings reveal a primary airway-to-brain sensory pathway that detects locally produced prostaglandins and mediates systemic sickness responses to respiratory virus infection., (© 2023. The Author(s).)

Influenza A virus infection causes considerable morbidity and mortality each year globally, and secondary bacterial infection further exacerbates the severity and fatality of the initial viral infection. Mast cells have substantial roles in protecting the respiratory tract mucosa, while their role in viral and bacterial co-infection remains unclear. The present study revealed that secondary Staphylococcus aureus infection significantly aggravated the activation of mast cells during the initial H1N1 infection both in vivo and in vitro, which was closely related to the increased inflammatory lung injury and mortality. Meanwhile, the secondary S. aureus infection suppressed autophagy and promoted inflammatory mediators released by mast cells through activating the PI3K/Akt signaling pathway. Blocking PI3K/Akt pathway by LY294002, an inhibitor of Akt phosphorylation, could rescue autophagy and inhibit the release of inflammatory mediators. Furthermore, based on the influenza A viral and secondary bacterial infected mice model, we showed that the combination of LY294002 and antiviral drug oseltamivir could effectively reduce the inflammatory damage and pro-inflammatory cytokines releasing in lungs, recovering body weight loss and improving the survival rate from the co-infections. In conclusion, secondary bacterial infection can inhibit autophagy and stimulate mast cell activation through the PI3K/Akt pathway, which might explain why secondary bacterial infection would cause severe and fatal consequences following an initial influenza A viral infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Viral pneumonia is a common complication caused by Influenza A virus infection and is characterized by severe pulmonary inflammation. A previous study showed that berberine (BBR) significantly ameliorated the pulmonary inflammation in mice with influenza viral pneumonia but its underlying mechanism is not entirely understood. In this study, we reproduced the mouse model of influenza viral pneumonia through intranasal infection of A/Puerto Rico/8/34 (H1N1), to further investigate the anti-inflammatory mechanism of BBR based on nucleotide-binding oligomerization domain-like receptor protein (NLRP) 3 inflammasome activation and Gasdermin D (GSDMD)-mediated pyroptosis. Consistent with MCC950 (10 mg/kg, a specific NLRP3 inflammasome inhibitor), BBR (10 mg/kg) obviously improved the weight loss and survival rate of infected mice, alleviated their pulmonary inflammation, and suppressed the accumulation of tumor necrosis factor and interleukin (IL)-6 in lungs without obvious inhibition on viral multiplication (hemagglutinin titer and nucleoprotein messenger RNA). Moreover, BBR (10 mg/kg) reduced the expressions of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and cysteinyl aspartate-specific proteinase (Caspase)1 (Caspase1 precursor [Pro-caspase1] + Caspase1p20 subunit) and the ratio of Caspase1p20 subunit to Caspase1, thus inhibiting the NLRP3 inflammasome activation and resulting in the decreased contents of mature IL-1β and IL-18 in lungs. The GSDMD expression (GSDMD precursor [Pro-GSDMD] + GSDMD-N terminal [NT]) and the ratio of GSDMD-NT to GSDMD were also declined by BBR (10 mg/kg). These evidence indicate that BBR may ameliorate pulmonary inflammation in mice with influenza viral pneumonia through inhibiting NLRP3 inflammasome activation, as well as depressing GSDMD-mediated pyroptosis via declining GSDMD expression and restraining NLRP3 inflammasome-mediated GSDMD activation., (© 2022 Wiley Periodicals LLC.)

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

Porcine respiratory disease complex, which is caused by a combination of pathogens, including swine influenza A virus (SIV) and porcine reproductive respiratory syndrome virus (PRRSV), results in significant economic losses in pig production systems. Nasopharynx-associated lymphoid tissue (NALT) plays an important role in the uptake of pathogens and defence of the nasal mucosa in rodents and humans. We characterized NALT M cells in pigs and detected SIV antigen and PRRSV nucleic acid in NALT using histopathological, immunohistochemical and in-situ hybridization analyses. All SIV- and PRRSV-positive cases examined had suppurative nasopharyngitis and pneumonia. M cells were detected by immunohistochemistry and the distribution of M cells showed an increase in the middle section of NALT. SIV antigen was detected in M cells and PRRSV nucleic acid was demonstrated in the cytoplasm of macrophages in NALT. We believe that SIV and PRRSV infection in the upper respiratory tract induces local immunosuppression and these results confirm that swine NALT is a location for virus replication and may be strongly associated with the development of pneumonia in pigs., Competing Interests: Conflict of Interest Statement The authors declared no conflicts of interest with respect of the research, authorship or publication of this article., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The expanding knowledge on the systemic influence of the human microbiome suggests that fecal samples are underexploited sources of new beneficial strains for extra-intestinal health. We have recently shown that acetate, a main circulating microbiota-derived molecule, reduces the deleterious effects of pulmonary Streptococcus pneumoniae and enteric Salmonella enterica serovar Typhimurium bacterial post-influenza superinfections. Considering the beneficial and broad effects of acetate, we intended to isolate a commensal strain, producing acetate and potentially exploitable in the context of respiratory infections. We designed successive steps to select intestinal commensals that are extremely oxygen-sensitive, cultivable after a freezing process, without a proinflammatory effect on IL-8 induction, and producing acetate. We have identified the Blautia faecis DSM33383 strain, which decreased the TNFα-induced production of IL-8 by the intestinal epithelial cell line HT-29. The beneficial effect of this bacterial strain was further studied in two preclinical models of post-influenza Streptococcus pneumoniae ( S.p ) and Salmonella enterica serovar Typhimurium ( S.t ) superinfection. The intragastrical administration of Blautia faecis DSM33383 led to protection in influenza-infected mice suffering from an S.p . and, to a lesser extent, from an S.t secondary infection. Altogether, this study showed that Blautia faecis DSM33383 could be a promising candidate for preventive management of respiratory infectious diseases.

Influenza A virus (IAV) infection triggers an exuberant host response that promotes acute lung injury. However, the host response factors that promote the development of a pathologic inflammatory response to IAV remain incompletely understood. In this study, we identify an interferon-γ (IFN-γ)-regulated subset of monocytes, CCR2 + monocytes, as a driver of lung damage during IAV infection. IFN-γ regulates the recruitment and inflammatory phenotype of CCR2 + monocytes, and mice deficient in CCR2 (CCR2 -/- ) or IFN-γ (IFN-γ -/- ) exhibit reduced lung inflammation, pathology, and disease severity. Adoptive transfer of wild-type (WT) (IFN-γR1 +/+ ) but not IFN-γR1 -/- CCR2 + monocytes restore the WT-like pathological phenotype of lung damage in IAV-infected CCR2 -/- mice. CD8 + T cells are the main source of IFN-γ in IAV-infected lungs. Collectively, our data highlight the requirement of IFN-γ signaling in the regulation of CCR2 + monocyte-mediated lung pathology during IAV infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

It has been established that blood vessels are a target for influenza virus; however, the mechanism by which virus affects the cardiovascular system remains unknown. The aim of the study is the identification of histological changes and changes in the functional activity of the pulmonary and mesenteric blood vessels of Wistar rats. Wistar rats were intranasally infected with the influenza A(H1N1)pdm09 virus. At 24 and 96 h post infection (hpi), histopathological changes were observed in lung tissues with the absence of histological changes in mesenteric tissues. The functional activity of pulmonary and mesenteric arteries was determined using wire myography. In pulmonary arteries, there was a tendency towards an increase in integral response to the vasodilator and a decrease in the integral response to the vasoconstrictor at 24 hpi (compared with control). At 96 hpi, a tendency towards a decrease in the integral response to the vasoconstrictor persisted, while the response to acetylcholine was slightly increased. The functional activity of the mesenteric blood vessels was inverted: a significant decrease in the integral response to the vasodilator and an increase in the response to the vasoconstrictor at 24 hpi were observed; at 96 hpi, the integral response to the vasoconstrictor persisted, while the response to the vasodilator remained significantly reduced. Obtained data indicate the development of endothelial dysfunction in non-lethal and clinically non-severe experimental influenza virus infection.

Obesity is an independent risk factor for morbidity and mortality in response to influenza infection. However, the underlying mechanisms by which obesity impairs immunity are unclear. Herein, we investigated the effects of diet-induced obesity on pulmonary CD8 + T cell metabolism, cytokine production, and transcriptome as a potential mechanism of impairment during influenza virus infection in mice. Male C57BL/6J lean and obese mice were infected with sub-lethal mouse-adapted A/PR/8/34 influenza virus, generating a pulmonary anti-viral and inflammatory response. Extracellular metabolic flux analyses revealed pulmonary CD8 + T cells from obese mice, compared with lean controls, had suppressed oxidative and glycolytic metabolism at day 10 post-infection. Flow cytometry showed the impairment in pulmonary CD8 + T cell metabolism with obesity was independent of changes in glucose or fatty acid uptake, but concomitant with decreased CD8 + GrB + IFNγ + populations. Notably, the percent of pulmonary effector CD8 + GrB + IFNγ + T cells at day 10 post-infection correlated positively with total CD8 + basal extracellular acidification rate and basal oxygen consumption rate. Finally, next-generation RNA sequencing revealed complex and unique transcriptional regulation of sorted effector pulmonary CD8 + CD44 + T cells from obese mice compared to lean mice following influenza infection. Collectively, the data suggest diet-induced obesity increases influenza virus pathogenesis, in part, through CD8 + T cell-mediated metabolic reprogramming and impaired effector CD8 + T cell function., (©2021 Society for Leukocyte Biology.)

The porcine respiratory disease complex (PRDC) is responsible for significant economic losses in the pig industry worldwide. Porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus are major viral contributors to PRDC. Vaccines are cost-effective measures for controlling PRRS, however, their efficacy in the context of co-infections has been poorly investigated. In this study, we aimed to determine the effect of PRRSV-2 and swine influenza H3N2 virus co-infection on the efficacy of PRRSV modified live virus (MLV) vaccination, which is widely used in the field. Following simultaneous challenge with contemporary PRRSV-2 and H3N2 field isolates, we found that the protective effect of PRRS MLV vaccination on clinical disease and pathology was abrogated, although viral load was unaffected and antibody responses were enhanced. In contrast, co-infection in non-immunized animals reduced PRRSV-2 viremia and H3N2 virus load in the upper respiratory tract and potentiated T cell responses against both PRRSV-2 and H3N2 in the lung. Further analysis suggested that an upregulation of inhibitory cytokines gene expression in the lungs of vaccinated pigs may have influenced responses to H3N2 and PRRSV-2. These findings provide important insights into the effect of viral co-infections on PRRS vaccine efficacy that may help identify more effective vaccination strategies against PRDC in the field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chrun, Maze, Vatzia, Martini, Paudyal, Edmans, McNee, Manjegowda, Salguero, Wanasen, Koonpaew, Graham and Tchilian.)

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently a serious public health concern worldwide. Notably, co-infection with other pathogens may worsen the severity of COVID-19 symptoms and increase fatality. Here, we show that co-infection with influenza A virus (IAV) causes more severe body weight loss and more severe and prolonged pneumonia in SARS-CoV-2-infected hamsters. Each virus can efficiently spread in the lungs without interference by the other. However, in immunohistochemical analyses, SARS-CoV-2 and IAV were not detected at the same sites in the respiratory organs of co-infected hamsters, suggesting that either the two viruses may have different cell tropisms in vivo or each virus may inhibit the infection and/or growth of the other within a cell or adjacent areas in the organs. Furthermore, a significant increase in IL-6 was detected in the sera of hamsters co-infected with SARS-CoV-2 and IAV at 7 and 10 days post-infection, suggesting that IL-6 may be involved in the increased severity of pneumonia. Our results strongly suggest that IAV co-infection with SARS-CoV-2 can have serious health risks and increased caution should be applied in such cases., (© 2021. The Author(s).)

People with diabetes mellitus are susceptible to both cardiovascular disease and severe influenza A virus infection. We hypothesized that diabetes also increases risks of influenza-associated cardiac complications. A murine type 1 (streptozotocin-induced) diabetes model was employed to investigate influenza-induced cardiac distress. Lung histopathology and viral titres revealed no difference in respiratory severity between infected control and diabetic mice. However, compared with infected control mice, infected diabetic mice had increased serum cardiac troponin I and creatine-kinase MB, left ventricular structural changes and right ventricular functional alterations, providing the first experimental evidence of type I diabetes increasing risks of influenza-induced cardiovascular complications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sinclair, Bloxham, Chiu, Chew, Russell, Yoshikawa, Bielefeldt-Ohmann, Steele, Hulme, Verzele, Noye, Wu, Reichelt, Thomas, Gallo, Redd and Short.)

Primary influenza virus (IV) infection can predispose hosts to secondary infection with Haemophilus influenzae (H. influenzae), which further increases the severity and mortality of the disease. While adhesion molecules play a key role in the host inflammatory response and H. influenzae colonization, it remains to be clarified which types of adhesion molecules are associated with H. influenzae colonization and invasion following IV infection. In this study, we established a mouse model of co-infection with influenza A virus (A/Puerto Rico/8/34, H1N1) (PR8) and non-typeable H. influenzae (NTHi) and found that sequential infection with PR8 and NTHi induced a lethal synergy in mice. This outcome may be possibly due to increased NTHi loads, greater lung damage and higher levels of cytokines. Furthermore, the protein levels of intracellular adhesion molecules-1 (ICAM-1) and Fibronectin (Fn) were significantly increased in the lungs of coinfected mice, but the levels of carcinoembryonic adhesion molecule (CEACAM)-1, CEACAM-5 and platelet-activating factor receptor (PAFr) were unaffected. Both the protein levels of ICAM-1 and Fn were positively correlated with NTHi growth. These results indicate the correlation between adhesion molecules, including ICAM-1 and Fn, and NTHi growth in secondary NTHi pneumonia following primary IV infection., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Inflammatory cytokine storm is a known cause for acute respiratory distress syndrome. In this study, we have investigated the role of IFN-γ in lethal lung inflammation using a mouse model of postinfluenza methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. To mimic the clinical scenario, animals were treated with antibiotics for effective bacterial control following MRSA superinfection. However, antibiotic therapy alone is not sufficient to improve survival of wild-type animals in this lethal acute respiratory distress syndrome model. In contrast, antibiotics induce effective protection in mice deficient in IFN-γ response. Mechanistically, we show that rather than inhibiting bacterial clearance, IFN-γ promotes proinflammatory cytokine response to cause lethal lung damage. Neutralization of IFN-γ after influenza prevents hyperproduction of TNF-α, and thereby protects against inflammatory lung damage and animal mortality. Taken together, the current study demonstrates that influenza-induced IFN-γ drives a stepwise propagation of inflammatory cytokine response, which ultimately results in fatal lung damage during secondary MRSA pneumonia, despite of antibiotic therapy., (Copyright © 2021 by The American Association of Immunologists, Inc.)

In this study we assessed the effects of antigen exposure in mice pre-sensitized with allergen following viral infection on changes in lung function, cellular responses and tight junction expression. Female BALB/c mice were sensitized to ovalbumin and infected with influenza A before receiving a second ovalbumin sensitization and challenge with saline, ovalbumin (OVA) or house dust mite (HDM). Fifteen days post-infection, bronchoalveolar inflammation, serum antibodies, responsiveness to methacholine and barrier integrity were assessed. There was no effect of infection alone on bronchoalveolar lavage cellular inflammation 15 days post-infection; however, OVA or HDM challenge resulted in increased bronchoalveolar inflammation dominated by eosinophils/neutrophils or neutrophils, respectively. Previously infected mice had higher serum OVA-specific IgE compared with uninfected mice. Mice previously infected, sensitized and challenged with OVA were most responsive to methacholine with respect to airway resistance, while HDM challenge caused significant increases in both tissue damping and tissue elastance regardless of previous infection status. Previous influenza infection was associated with decreased claudin-1 expression in all groups and decreased occludin expression in OVA or HDM-challenged mice. This study demonstrates the importance of the respiratory epithelium in pre-sensitized individuals, where influenza-infection-induced barrier disruption resulted in increased systemic OVA sensitization and downstream effects on lung function.

Although coagulation abnormalities, including microvascular thrombosis, are thought to contribute to tissue injury and single- or multiple-organ dysfunction in severe influenza, the detailed mechanisms have yet been clarified. This study evaluated influenza-associated abnormal blood coagulation utilizing a severe influenza mouse model. After infecting C57BL/6 male mice with intranasal applications of 500 plaque-forming units of influenza virus A/Puerto Rico/8/34 (H1N1; PR8), an elevated serum level of prothrombin fragment 1 + 2, an indicator for activated thrombin generation, was observed. Also, an increased gene expression of oxidized low-density lipoprotein (LDL) receptor-1 (Olr1), a key molecule in endothelial dysfunction in the progression of atherosclerosis, was detected in the aorta of infected mice. Body weight decrease, serum levels of cytokines and chemokines, viral load, and inflammation in the lungs of infected animals were similar between wild-type and Olr1 knockout (KO) mice. In contrast, the elevation of prothrombin fragment 1 + 2 levels in the sera and intravascular thrombosis in the lungs by PR8 virus infection were not induced in KO mice. Collectively, the results indicated that OLR1 is a critical host factor in intravascular thrombosis as a pathogeny of severe influenza. Thus, OLR1 is a promising novel therapeutic target for thrombosis during severe influenza., (© 2021. The Author(s).)

The synergism of the influenza virus and respiratory tract pathogens is known to exacerbate diseases in both humans and animals. The mechanism of the co-infection of associated respiratory tract pathogens is explored in this study. Co-infection has a directional effect when influenza virus or other pathogens occur in a different order. In the present study, we used a mouse animal model to study the synergism of influenza virus and Streptococcus suis co-infection in different orders of administration. We found that the group infected with bacteria alone did not show any clinical symptoms, but the group infected with the virus alone showed 100 % mortality and clinical signs typical in infected mice. In the bacteria infected following virus pre-exposure group, the mice died before the virus-infected group and showed severer clinical signs. When the influenza virus was administered after the bacteria, the infected mice showed reduced mortality compared with mice administered the influenza virus alone. The results indicated that the order of infection significantly affected the outcome of the co-infection of these two pathogens in the mice. However, the underlying mechanism was unclear. Therefore, a transcriptome analysis of mouse lungs was conducted to explore the potential mechanism. The results showed that inflammation and cell damage signaling pathways were upregulated, which may have contributed to the increased mortality in the secondary bacterial infection group. Upregulated innate immunity may have been a major cause of reduced mortality when the bacteria were inoculated before the virus infection., (Copyright © 2021. Published by Elsevier B.V.)

The H9N2 avian influenza viruses infect poultry worldwide, and can potentially cause a human pandemic without adaptation. Vitamin D3 (D3) is increasingly being recognized for its extra-skeletal roles, such as the inflammatory and immune responses to infection. The aim of this study was to analyze the changes in vitamin D metabolizing enzymes and vitamin D receptor (VDR) in the lung tissues of mice infected with H9N2. The mice were intranasally inoculated with the appropriate dose of the virus, and various clinical indices were measured on days 3, 7, 14 and 21 post-infection. H9N2 infection significantly increased the expression levels of 1α-hydroxylase mRNA and protein, which is the activating enzyme of 25-hydroxyvitamin D (25(OH)D 3 ), but had no significant effect on the 25(OH)D 3 inactivating enzyme 24-hydroxylase, indicating that inactive D3 might be converted to its active form in the H9N2-infected lungs. Furthermore, a significant increase was also observed in the VDR mRNA and protein levels, suggesting enhanced responsiveness of the lung tissues to 1, 25(OH) 2 D 3 post H9N2 infection. In addition, daily 25(OH)D 3 injection from day 2-14 post-infection did not affect the clinical signs, virus replication and cytokine (IL-1β and TNF-α) production in the lungs of the infected mice. Given that the biological effects of D3 rely on its activation, and the binding of 1, 25(OH) 2 D 3 to VDR in specific tissues, our findings provide novel insights into the possible role of vitamin D in the development and progression of influenza., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

There is an urgent need for new drugs for patients with acute respiratory distress syndrome (ARDS), including those with coronavirus disease (COVID-19). ARDS in influenza-infected mice is associated with reduced concentrations of liponucleotides (essential precursors for de novo phospholipid synthesis) in alveolar type II (ATII) epithelial cells. Because surfactant phospholipid synthesis is a primary function of ATII cells, we hypothesized that disrupting this process could contribute significantly to the pathogenesis of influenza-induced ARDS. The goal of this study was to determine whether parenteral liponucleotide supplementation can attenuate ARDS. C57BL/6 mice inoculated intranasally with 10,000 plaque-forming units/mouse of H1N1 influenza A/WSN/33 virus were treated with CDP (cytidine 5'-diphospho)-choline (100 μg/mouse i.p.) ± CDP -diacylglycerol 16:0/16:0 (10 μg/mouse i.p.) once daily from 1 to 5 days after inoculation (to model postexposure influenza prophylaxis) or as a single dose on Day 5 (to model treatment of patients with ongoing influenza-induced ARDS). Daily postexposure prophylaxis with CDP-choline attenuated influenza-induced hypoxemia, pulmonary edema, alterations in lung mechanics, impairment of alveolar fluid clearance, and pulmonary inflammation without altering viral replication. These effects were not recapitulated by the daily administration of CTP (cytidine triphosphate) and/or choline. Daily coadministration of CDP-diacylglycerol significantly enhanced the beneficial effects of CDP-choline and also modified the ATII cell lipidome, reversing the infection-induced decrease in phosphatidylcholine and increasing concentrations of most other lipid classes in ATII cells. Single-dose treatment with both liponucleotides at 5 days after inoculation also attenuated hypoxemia, altered lung mechanics, and inflammation. Overall, our data show that liponucleotides act rapidly to reduce disease severity in mice with severe influenza-induced ARDS.

Background: Secondary bacterial coinfections are ranked as a leading cause of hospitalization and morbid conditions associated with influenza. Because vitamin A deficiency (VAD) and insufficiency are frequent in both developed and developing countries, we asked how VAD influences coinfection severity., Methods: VAD and control mice were infected with influenza virus for evaluation of inflammatory cytokines, cellular immune responses, and viral clearance. Influenza-infected mice were coinfected with Streptococcus pneumoniae to study weight loss and survival., Results: Naive VAD mouse lungs exhibited dysregulated immune function. Neutrophils were enhanced in frequency and there was a significant reduction in RANTES (regulated on activation of normal T cells expressed and secreted), a chemokine instrumental in T-cell homing and recruitment. After influenza virus infection, VAD mice experienced failures in CD4+ T-cell recruitment and B-cell organization into lymphoid structures in the lung. VAD mice exhibited higher viral titers than controls and slow viral clearance. There were elevated levels of inflammatory cytokines and innate cell subsets in the lungs. However, arginase, a marker of alternatively activated M2 macrophages, was rare. When influenza-infected VAD animals were exposed to bacteria, they experienced a 100% mortality rate., Conclusion: Data showed that VAD dysregulated the immune response. Consequently, secondary bacterial infections were 100% lethal in influenza-infected VAD mice., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Despite vaccination and antivirals, influenza remains a communicable disease of high burden, with limited therapeutic options available to patients that develop complications. Here, we report the development and preclinical characterization of Adipose Stromal Cell (ASC) concentrated secretome (CS), generated by process adaptable to current Good Manufacturing Practices (cGMP) standards. We demonstrate that ASC-CS limits pulmonary histopathological changes, infiltration of inflammatory cells, protein leak, water accumulation, and arterial oxygen saturation (spO2) reduction in murine model of lung infection with influenza A virus (IAV) when first administered six days post-infection. The ability to limit lung injury is sustained in ASC-CS preparations stored at -80 °C for three years. Priming of the ASC with inflammatory factors TNFα and IFNγ enhances ASC-CS ability to suppress lung injury. IAV infection is associated with dramatic increases in programmed cell death ligand (PDL1) and angiopoietin 2 (Angpt2) levels. ASC-CS application significantly reduces both PDL1 and Angpt2 levels. Neutralization of PDL1 with anti-mouse PDL1 antibody starting Day6 onward effectively ablates lung PDL1, but only non-significantly reduces Angpt2 release. Most importantly, late-phase PDL1 neutralization results in negligible suppression of protein leakage and inflammatory cell infiltration, suggesting that suppression of PDL1 does not play a critical role in ASC-CS therapeutic effects.

Rationale: Patients with and without cardiovascular diseases have been shown to be at risk of influenza-mediated cardiac complications. Recent clinical reports support the notion of a direct link between laboratory-confirmed influenza virus infections and adverse cardiac events., Objective: Define the molecular mechanisms underlying influenza virus-induced cardiac pathogenesis after resolution of pulmonary infection and the role of necroptosis in this process., Methods and Results: Hearts from wild-type and necroptosis-deficient (MLKL [mixed lineage kinase domain-like protein]-KO) mice were dissected 12 days after initial influenza A virus (IAV) infection when viral titers were undetectable in the lungs. Immunofluorescence microscopy and plaque assays showed presence of viable IAV particles in the myocardium without generation of interferon responses. Global proteome and phosphoproteome analyses using high-resolution accurate mass-based LC-MS/MS and label-free quantitation showed that the global proteome as well as the phosphoproteome profiles were significantly altered in IAV-infected mouse hearts in a strain-independent manner. Necroptosis-deficient mice had increased survival and reduced weight loss post-IAV infection, as well as increased antioxidant and mitochondrial function, indicating partial protection to IAV infection. These findings were confirmed in vitro by pretreatment of human and rat myocytes with antioxidants or necroptosis inhibitors, which blunted oxidative stress and mitochondrial damage after IAV infection., Conclusions: This study provides the first evidence that the cardiac proteome and phosphoproteome are significantly altered post-pulmonary influenza infection. Moreover, viral particles can persist in the heart after lung clearance, altering mitochondrial function and promoting cell death without active replication and interferon responses. Finally, our findings show inhibition of necroptosis or prevention of mitochondrial damage as possible therapeutic interventions to reduce cardiac damage during influenza infections. Graphic Abstract: A graphic abstract is available for this article.

Adverse early-life exposures have a lasting negative impact on health. Neonatal hyperoxia that is a risk factor for bronchopulmonary dysplasia confers susceptibility to influenza A virus (IAV) infection later in life. Given our previous findings that the circadian clock protects against IAV, we asked if the long-term impact of neonatal hyperoxia vis-à-vis IAV infection includes circadian disruption. Here, we show that neonatal hyperoxia abolishes the clock-mediated time of day protection from IAV in mice, independent of viral burden through host tolerance pathways. We discovered that the lung intrinsic clock (and not the central or immune clocks) mediated this dysregulation. Loss of circadian protein, Bmal1 , in alveolar type 2 (AT2) cells recapitulates the increased mortality, loss of temporal gating, and other key features of hyperoxia-exposed animals. Our data suggest a novel role for the circadian clock in AT2 cells in mediating long-term effects of early-life exposures to the lungs., Competing Interests: YI, AN, ST, KF, TB, NL, KT, MM, GW, GF, SS No competing interests declared, AS Reviewing editor, eLife, (© 2021, Issah et al.)

Streptococcus pneumoniae is an opportunistic human pathogen that causes invasive diseases, including pneumonia, with greater health risks upon influenza A virus (IAV) co-infection. To facilitate pathogenesis studies in vivo, we developed an inducible CRISPR interference system that enables genome-wide fitness testing in one sequencing step (CRISPRi-seq). We applied CRISPRi-seq to assess bottlenecks and identify pneumococcal genes important in a murine pneumonia model. A critical bottleneck occurs at 48 h with few bacteria causing systemic infection. This bottleneck is not present during IAV superinfection, facilitating identification of pneumococcal pathogenesis-related genes. Top in vivo essential genes included purA, encoding adenylsuccinate synthetase, and the cps operon required for capsule production. Surprisingly, CRISPRi-seq indicated no fitness-related role for pneumolysin during superinfection. Interestingly, although metK (encoding S-adenosylmethionine synthetase) was essential in vitro, it was dispensable in vivo. This highlights advantages of CRISPRi-seq over transposon-based genetic screens, as all genes, including essential genes, can be tested for pathogenesis potential., Competing Interests: Declaration of Interests The authors declare no conflicting interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Influenza A viruses cause severe respiratory illnesses in humans and animals. Overreaction of the innate immune response to influenza virus infection results in hypercytokinemia, which is responsible for mortality and morbidity. However, the mechanism by which influenza induces hypercytokinemia is not fully understood. In this study, we established a mouse-adapted H9N2 virus, MA01, to evaluate the innate immune response to influenza in the lung. MA01 infection caused high levels of cytokine release, enhanced pulmonary injury in mice, and upregulated CD83 protein in dendritic cells and macrophages in the lung. Influenza virus neuraminidase (NA) unmasked CD83 protein and contributed to high cytokine levels. Furthermore, we provide evidence that CD83 is a sialylated glycoprotein. Neuraminidase treatment enhanced lipopolysaccharide (LPS)-stimulated NF-κB activation in RAW264.7 cells. Anti-CD83 treatment alleviated influenza virus-induced lung injury in mice. Our study indicates that influenza virus neuraminidase modulates CD83 status and contributes to the "cytokine storm," which may suggest a new approach to curb this immune injury. IMPORTANCE The massive release of circulating mediators of inflammation is responsible for lung injury during influenza A virus infection. This phenomenon is referred to as the "cytokine storm." However, the mechanism by which influenza induces the cytokine storm is not fully understood. In this study, we have shown that neuraminidase unmasked CD83 protein in the lung and contributed to high cytokine levels. Anti-CD83 treatment could diminish immune damage to lung tissue. The NA-CD83 axis may represent a target for an interruption of influenza-induced lung damage., (Copyright © 2021 American Society for Microbiology.)

Introduction: Epidemiological studies revealed that maternal exposure to influenza A (H1N1) and Toxoplasma gondii (T. gondii) infection during pregnancy may increase the risk for mood disorders of the offspring. However, the impact of maternal infections in different stages of neural development and the nature of antigens remain to be elucidated., Objective: This study investigated behavioral impairments induced by maternal immune activation (MIA) due to H1N1 or T. gondii infection during preborn neurodevelopment., Methods: Maternal infection with influenza or toxoplasma was mimicked by administration of influenza vaccine antigens or suspension of soluble T. gondii antigen (STAg) in pregnant Balb/c mice at E6 or E16. Adult male offspring were evaluated for anxiety-like and depressive-like behavior in elevated plus maze (EPM) and forced swimming test (FST)., Results: In FST, immobility time at E6 and E16 increased when the mothers were treated with both antigen solutions. There was increased immobility in the pups whose mothers were treated with STAg at E16. MIA with influenza antigens reduced the exploration of the open arms of EPM for the pups whose progenitors received treatment at E6 and E16. The animals at E6 exhibited a greater number of stretch-attend postures compared with the saline group. STAg at E6 reduced the time of exploration in the open arms and increased the number of stretch-attend postures compared with the saline group., Conclusion: These results suggest that immunological responses to H1N1 or T. gondii during pregnancy may impact differently the susceptibility of adult offspring to mood disorder., (© 2020 S. Karger AG, Basel.)

Secondary methicillin-resistant Staphylococcus aureus (MRSA) infection is a cause of severe pneumonia with high mortality during influenza A virus (IAV) pandemics. Alveolar macrophages (AMs) mount cellular defenses against IAV and MRSA infection, which occurs via the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome. However, the activity and function of the NLRP3 inflammasome in MRSA pneumonia secondary to IAV infection remain unclear. To clarify this, we studied MRSA infection secondary to IAV both in vitro and in mouse model. The expression of the NLRP3 inflammasome was evaluated by quantitative reverse transcription polymerase chain reaction, immunofluorescence, Western blot, and enzyme-linked immunosorbent assay. The lung pathology and the rate of weight change were observed. We found that IAV infection for 1 week activated NLRP3 inflammasome. The enhanced expression of NLRP3, caspase-1, and cleaved caspase-1 was associated with MRSA infection secondary to IAV, but the expression of interleukin (IL)-1β decreased in superinfection with MRSA both in vitro and in vivo. The aggravated inflammatory pathology in MRSA pneumonia secondary to IAV infection was associated with decreased expression of IL-1β. And increased weight loss in MRSA pneumonia secondary to IAV infection was related to decreased concentration of IL-1β in serum. It infers that superinfection with MRSA reduces expression of IL-1β someway, and decreased expression of IL-1β impairs the host immunity and leads to aggravated pneumonia. These results contributed to our understanding of the detailed activity of the NLRP3 inflammasome, IL-1β, and their relationship with aggravation of MRSA pneumonia secondary to IAV infection. Immunotherapy targeting the IL-1β signaling pathway could be possible therapeutic strategy for secondary MRSA pneumonia., (© 2020 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Background: The highly pathogenic avian influenza A/H5N1 virus is one of the causative agents of acute lung injury (ALI) with high mortality rate. Studies on therapeutic administration of bone marrow-derived mesenchymal stem cells (MSCs) in ALI caused by the viral infection have been limited in number and have shown conflicting results. The aim of the present investigation is to evaluate the therapeutic potential of MSC administration in A/H5N1-caused ALI, using a mouse model., Methods: MSCs were prepared from the bone marrow of 9 to 12 week-old BALB/c mice. An H5N1 virus of A/turkey/East Java/Av154/2013 was intranasally inoculated into BALB/c mice. On days 2, 4, and 6 after virus inoculation, MSCs were intravenously administered into the mice. To evaluate effects of the treatment, we examined for lung alveolar protein as an indicator for lung injury, PaO 2 /FiO 2 ratio for lung functioning, and lung histopathology. Expressions of NF-κB, RAGE (transmembrane receptor for damage associated molecular patterns), TNFα, IL-1β, Sftpc (alveolar cell type II marker), and Aqp5+ (alveolar cell type I marker) were examined by immunohistochemistry. In addition, body weight, virus growth in lung and brain, and duration of survival were measured., Results: The administration of MSCs lowered the level of lung damage in the virus-infected mice, as shown by measuring lung alveolar protein, PaO 2 /FiO 2 ratio, and histopathological score. In the MSC-treated group, the expressions of NF-κB, RAGE, TNFα, and IL-1β were significantly suppressed in comparison with a mock-treated group, while those of Sftpc and Aqp5+ were enhanced. Body weight, virus growth, and survival period were not significantly different between the groups., Conclusion: The administration of MSCs prevented further lung injury and inflammation, and enhanced alveolar cell type II and I regeneration, while it did not significantly affect viral proliferation and mouse morbidity and mortality. The results suggested that MSC administration was a promissing strategy for treatment of acute lung injuries caused by the highly pathogenic avian influenza A/H5N1 virus, although further optimization and combination use of anti-viral drugs will be obviously required to achieve the goal of reducing mortality.

Various respiratory viral infections in general and seasonal influenza in particular may increase the susceptibility to bacterial infections. Plague caused by Yersinia pestis endangers large populations during outbreaks or bioterrorism attacks. Recommended antibiotic countermeasures include well-established protocols based on animal studies and corroborated by effective treatment of human cases. Until now, prior exposure to viral respiratory infections was not taken into consideration when selecting the appropriate treatment for plague. Here, we show that as late as 25 days after exposure to influenza virus, convalescent mice still exhibited an increased susceptibility to sublethal doses of Y. pestis, presented with aberrant cytokine expression, and impaired neutrophil infiltration in the lungs. Increased levels of M2 alveolar macrophages and type II epithelial cells, as well as induction in metalloproteases expression and collagen and laminin degradation, suggested that the previous viral infection was under resolution, correlating with enhanced susceptibility to plague. Surprisingly, postexposure prophylaxis treatment with the recommended drugs revealed that ciprofloxacin was superior to doxycycline in mice recovering from influenza infection. These results suggest that after an influenza infection, the consequences, such as impaired immunity and lung tissue remodeling and damage, should be considered when treating subsequent Y. pestis exposure.

Background: Influenza A virus (IAV) causes a wide range of extrarespiratory complications. However, the role of host factors in these complications of influenza virus infection remains to be defined., Methods: Here, we sought to use transcriptional profiling, virology, histology, and echocardiograms to investigate the role of a high-fat diet in IAV-associated cardiac damage., Results: Transcriptional profiling showed that, compared to their low-fat counterparts (LF mice), mice fed a high-fat diet (HF mice) had impairments in inflammatory signaling in the lung and heart after IAV infection. This was associated with increased viral titers in the heart, increased left ventricular mass, and thickening of the left ventricular wall in IAV-infected HF mice compared to both IAV-infected LF mice and uninfected HF mice. Retrospective analysis of clinical data revealed that cardiac complications were more common in patients with excess weight, an association which was significant in 2 out of 4 studies., Conclusions: Together, these data provide the first evidence that a high-fat diet may be a risk factor for the development of IAV-associated cardiovascular damage and emphasizes the need for further clinical research in this area., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Aspergillus fumigatus is an environmental fungus that can cause invasive pulmonary aspergillosis when spores are inhaled into the respiratory tract and invade airway or lung tissue. Influenza is a common respiratory virus that can cause severe respiratory disease, and postinfluenza invasive pulmonary aspergillosis, which is becoming a well-recognized clinical problem, typically occurs in critically ill patients. Mice challenged with influenza A PR/8/34 H1N1 and subsequently challenged with A. fumigatus had increased fungal burden, viral burden, inflammation, and mortality compared with single infected mice. Neutrophil recruitment in the lung of superinfected mice was decreased; however, mice were not neutropenic, and there was no difference in absolute blood neutrophils between groups. Additionally, CXCL1 and CXCL2 were decreased in lungs of superinfected mice compared with controls. IFN levels were increased in mice that received influenza, and deletion of STAT1 resulted in decreased fungal burden, increased airway and lung neutrophils, and increased CXCL1 compared with wild-type mice, whereas deletion of STAT2 did not change fungal burden or airway neutrophilia compared with wild-type mice. These data demonstrate a mechanism by which influenza A-induced STAT1 signaling inhibits neutrophil recruitment and increases susceptibility to postinfluenza invasive pulmonary aspergillosis., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Thrombocytopenia is a common complication of influenza virus infection, and its severity predicts the clinical outcome of critically ill patients. The underlying cause(s) remain incompletely understood. In this study, in patients with an influenza A/H1N1 virus infection, viral load and platelet count correlated inversely during the acute infection phase. We confirmed this finding in a ferret model of influenza virus infection. In these animals, platelet count decreased with the degree of virus pathogenicity varying from 0% in animals infected with the influenza A/H3N2 virus, to 22% in those with the pandemic influenza A/H1N1 virus, up to 62% in animals with a highly pathogenic A/H5N1 virus infection. This thrombocytopenia is associated with virus-containing platelets that circulate in the blood. Uptake of influenza virus particles by platelets requires binding to sialoglycans and results in the removal of sialic acids by the virus neuraminidase, a trigger for hepatic clearance of platelets. We propose the clearance of influenza virus by platelets as a paradigm. These insights clarify the pathophysiology of influenza virus infection and show how severe respiratory infections, including COVID-19, may propagate thrombocytopenia and/or thromboembolic complications., (© 2020 by The American Society of Hematology.)

Ethnopharmacological Relevance: Moslae Herba, a common traditional Chinese herb with special flavor, has potential for treating respiratory and gastrointestinal diseases., Aim of This Study: Lung endothelial barrier dysfunction (LEBD) accelerates the pathogenesis of influenza A virus (IAV)-induced secondary acute lung injury. New strategies against LEBD provide benefits in prevention and treatment of IAV. Previous studies showed that flavonoids (MHF), main bioactivity fraction derived from M. Herba, exerted anti-inflammatory and antiviral activities, but the underlying protection of MHF against IAV-induced acute lung injury remained obscure. The present study was to investigate the protection of MHF against IAV-induced LEBD in vivo and in vitro., Materials and Methods: Mice were intranasally challenged with IAV and orally administered with MHF for 5 days. The pulmonary hyperpermeability of infected mice was evaluated by Evans Blue staining and in vivo imaging. Serum levels of inflammatory cytokines and mediators were detected by ELISA assay. The transepithelial electrical resistance (TER) of human pulmonary microvascular endothelial cells (HPMVECs) was measured by using TER meter. The expressions of key proteins in NOX4-mediated NF-κB/MLCK pathways were determined by western blotting., Results: MHF treatment reduced lung index, W/D ratios, and serum levels of inflammatory factors (IL-6, TNF-α, IL-1β, PLA 2 , LBT 4 and ICAM-1) in IAV-infected mice. Evans blue staining and in vivo imaging results revealed that MHF alleviated IAV-induced barrier dysfunction and pulmonary hyperpermeability. Moreover, luteolin and kaempferol, the main activity compounds in MHF, significantly inhibited TNF-α-induced HPMVEC apoptosis, and downregulated NF-κB/MLCK pathway by targeting NOX4., Conclusion: MHF attenuated IAV-induced barrier dysfunction by suppressing NOX4/NF-κB/MLCK pathway and may serve as a potential agent for the prevention of LEBD and IAV., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Neurodegenerative diseases feature specific misfolded or misassembled proteins associated with neurotoxicity. The precise mechanisms by which protein aggregates first arise in the majority of sporadic cases have remained unclear. Likely, a first critical mass of misfolded proteins starts a vicious cycle of a prion-like expansion. We hypothesize that viruses, having evolved to hijack the host cellular machinery for catalyzing their replication, lead to profound disturbances of cellular proteostasis, resulting in such a critical mass of protein aggregates. Here, we investigated the effect of influenza virus (H1N1) strains on proteostasis of proteins associated with neurodegenerative diseases in Lund human mesencephalic dopaminergic cells in vitro and infection of Rag knockout mice in vivo. We demonstrate that acute H1N1 infection leads to the formation of α-synuclein and Disrupted-in-Schizophrenia 1 (DISC1) aggregates, but not of tau or TDP-43 aggregates, indicating a selective effect on proteostasis. Oseltamivir phosphate, an antiinfluenza drug, prevented H1N1-induced α-synuclein aggregation. As a cell pathobiological mechanism, we identified H1N1-induced blocking of autophagosome formation and inhibition of autophagic flux. In addition, α-synuclein aggregates appeared in infected cell populations connected to the olfactory bulbs following intranasal instillation of H1N1 in Rag knockout mice. We propose that H1N1 virus replication in neuronal cells can induce seeds of aggregated α-synuclein or DISC1 that may be able to initiate further detrimental downstream events and should thus be considered a risk factor in the pathogenesis of synucleinopathies or a subset of mental disorders. More generally, aberrant proteostasis induced by viruses may be an underappreciated factor in initiating protein misfolding., Competing Interests: Competing interest statement: Co-authors A.R.M., S. Sahu, I.S., S. Selvarajah, and V.R.L. are full-time employees of Prosetta Biosciences Inc., located in San Francisco, CA; however, no intellectual property, products, or other commercial interests were pursued during the studies presented here.

Background: Influenza is an acute viral respiratory illness that causes high morbidity and mortality globally. Therapeutic actions are limited to vaccines and a few anti-viral drugs. Polygala (P.) japonica herba is rich in Polygalasaponin F (PSF, C 53 H 86 O 23 ), used for acute bronchitis, pharyngitis, pneumonia, amygdalitis, and respiratory tract infections treatment in China. Hypercytokinemia is often correlated with severe pneumonia caused by several influenza viruses. PSF was reported to have anti-inflammatory effects and its mechanism is associated with the nuclear factor (NF)-κB signaling pathway. The action of PSF to alleviate pulmonary inflammation caused by influenza A virus (IAV) infection requires careful assessment. In the present study, we evaluated the effect and mechanism of PSF on mice with pneumonia caused by influenza H1N1 (A/FM/1/47)., Methods: Mice were infected intranasally with fifteen 50% mouse lethal challenge doses (MLD 50 ) of influenza virus. BALB/c mice were treated with PSF or oseltamivir (oral administration) for 2 h post-infection and received concomitant treatment for 5 days after infection. On day 6 post-infection, 10 mice per group were killed to collect related samples, measure body weight and lung wet weight, and detect the viral load, cytokine, prostaglandins, pathological changes, and cell pathway protein expression in the lungs. In addition, the survival experiments were carried out to investigate the survival of mice. The expression profile of cell pathway proteins was detected and analyzed using a broad pathway antibody array and confirmed the findings from the array by western blotting., Results: Polygalasaponin F and oseltamivir can protect against influenza viral infection in mice. PSF and oseltamivir significantly relieved the signs and symptoms, reduced body weight loss, and improved the survival rate of H1N1-infected mice. Moreover, PSF efficiently decreased the level of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-4, interferon (IFN)-γ, thromboxane A 2 (TXA 2 ), and prostaglandin E 2 (PGE 2 ) in lung tissues of mice infected with influenza virus (p < 0.05-0.01). Oseltamivir had a similar effect to lung cytokine of PSF, but did not decrease the levels of TXA 2 and PGE 2 . There was a twofold or greater increase in four cell pathway protein, namely NF-κB p65 (2.68-fold), I-kappa-B-alpha (IκBα) (2.56-fold), and MAPK/ERK kinase 1 (MEK1) (7.15-fold) assessed in the array induced by influenza virus. Western blotting showed that the expression of these proteins was significantly decreased in lung after influenza virus challenge in PSF and oseltamivir-treated mice (p < 0.05-0.01)., Conclusion: Polygalasaponin F appears to be able to augment protection against IAV infection in mice via attenuation of pulmonary inflammatory responses. Its effect on IAV-induced pulmonary inflammation was associated with suppression of Raf/MEK/ERK and NF-κB expressions.

We conducted a retrospective study to identify the risk factors for pneumonia in hospitalized pediatric patients with influenza B infection. Receiving oseltamivir within the first 48 hours of onset and frequent cough was respectively considered as a protective factor and a risk factor for the influenza B virus-associated pneumonia in hospitalized pediatric patients. Early administration of oseltamivir can reduce the risk of influenza B virus-associated pneumonia.

Objectives: Secondary bacterial pneumonia is common following influenza infection. However, it remains unclear about the underlying molecular mechanisms., Materials and Methods: We established a mouse model of post-influenza S aureus pneumonia using conditional Shp2 knockout mice (LysM Cre/+ :Shp2 flox/flox ). The survival, bacterial clearance, pulmonary histology, phenotype of macrophages, and expression of type I interferons and chemokines were assessed between SHP2 deletion and control mice (Shp2 flox/flox ). We infused additional KC and MIP-2 to examine the reconstitution of antibacterial immune response in LysM Cre/+ :Shp2 flox/flox mice. The effect of SHP2 on signal molecules including MAPKs (JNK, p38 and Erk1/2), NF-κB p65 and IRF3 was further detected., Results: LysM Cre/+ :Shp2 flox/flox mice displayed impaired antibacterial immunity and high mortality compared with control mice in post-influenza S aureus pneumonia. The attenuated antibacterial ability was associated with the induction of type I interferon and suppression of chemo-attractants KC and MIP-2, which reduced the infiltration of neutrophils into the lung upon secondary bacterial invasion. In additional, Shp2 knockout mice displayed enhanced polarization to alternatively activated macrophages (M2 phenotype). Further in vitro analyses consistently demonstrated that SHP2-deficient macrophages were skewed towards an M2 phenotype and had a decreased antibacterial capacity. Moreover, SHP2 modulated the inflammatory response to secondary bacterial infection via interfering with NF-κB and IRF3 signalling in macrophages., Conclusions: Our findings reveal that the SHP2 expression enhances the host immune response and prompts bacterial clearance in post-influenza S aureus pneumonia., (© 2019 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Asthma is a chronic airways condition that can be exacerbated during respiratory infections. Our previous work, together with epidemiologic findings that asthmatics were less likely to suffer from severe influenza during the 2009 pandemic, suggest that additional complications of influenza such as increased susceptibility to bacterial superinfection, may be mitigated in allergic hosts. To test this hypothesis, we developed a murine model of 'triple-disease' in which mice rendered allergic to Aspergillus fumigatus were co-infected with influenza A virus and Streptococcus pneumoniae seven days apart. Significant alterations to known synergistic effects of co-infection were noted in the allergic mice including reduced morbidity and mortality, bacterial burden, maintenance of alveolar macrophages, and reduced lung inflammation and damage. The lung microbiome of allergic mice differed from that of non-allergic mice during co-infection and antibiotic-induced perturbation to the microbiome rendered allergic animals susceptible to severe morbidity. Our data suggest that responses to co-infection in allergic hosts likely depends on the immune and microbiome states and that antibiotics should be used with caution in individuals with underlying chronic lung disease.