Influenza Virus Infection In Mice Research Articles

Vunakizumab-IL22, a Novel Fusion Protein, Promotes Intestinal Epithelial Repair and Protects against Gut Injury Induced by the Influenza Virus

Secondary immune damage to the intestinal mucosa due to an influenza virus infection has gained the attention of investigators. The protection of the intestinal barrier is an effective means of improving the survival rate in cases of severe pneumonia. We developed a fusion protein, Vunakizumab-IL22(vmab-IL22), by combining an anti-IL17A antibody with IL22. Our previous study showed that Vunakizumab-IL22 repairs the pulmonary epithelial barrier in influenza virus-infected mice. In this study, we investigated the protective effects against enteritis given its anti-inflammatory and tissue repair functions. The number of goblet cells and the expression of zonula occludens protein 1(ZO-1), Mucin-2, Ki67 and IL-22R were determined by immunohistochemistry (IHC) and quantitative RT-PCR in influenza A virus (H1N1)-infected mice. The expression of NOD-like receptor pyrin domain containing 3 (NLRP3) and toll- like-receptor-4 (TLR4) was assayed by IHC in the lungs and intestine in HIN1 virus-induced mice to evaluate the whole efficacy of the protective effects on lungs and intestines. Consequently, Cytochrome C, phosphorylation of nuclear factor NF-kappaB (p-NF-κB), IL-1β, NLRP3 and Caspase 3 were assayed by Western blotting in dextran sulfate sodium salt (DSS)-treated mice. Treatment with Vunakizumab-IL22 improved the shortened colon length, macroscopic and microscopic morphology of the small intestine (p < 0.001) significantly, and strengthened the tight junction proteins, which was accompanied with the upregulated expression of IL22R. Meanwhile, Vunakizumab-mIL22 inhibited the expression of inflammation-related protein in a mouse model of enteritis induced by H1N1 and DSS. These findings provide new evidence for the treatment strategy for severe viral pneumonia involved in gut barrier protection. The results suggest that Vunakizumab-IL22 is a promising biopharmaceutical drug and is a candidate for the treatment of direct and indirect intestinal injuries, including those induced by the influenza virus and DSS.

The protective effect of 999 XiaoErGanMao granules on the lungs and intestines of influenza A virus-infected mice

Context Gastrointestinal symptoms are a common complication of influenza virus infection in children, which the gut-lung axis become involved in its biological progress. The protective effect of 999 XiaoErGanMao granules (XEGMG) on multi-organ injury in viral pneumonia remains unclear. Objective To investigate the therapeutic effect of XEGMG on lungs and intestines injury in A/FM/1/47 (H1N1) influenza virus-infected mice. Materials and methods Male BALB/c mice were infected with the 2LD50 H1N1 influenza virus and then treated with XEGMG (6 or 12 g/kg) intragastrically once a day for 4 days. The lung and colon samples were then collected for pathological observation, and assays for inflammatory cytokines and intestinal barrier. Mouse feces were collected to evaluate the intestinal microbiota. Results Treating with XEGMG (12 g/kg) can mitigate body weight loss caused by 2LD50 H1N1 infection. It can also reduce lung index and pathological damage with the decreased inflammatory cytokines such as IL-6 and IL-1β. Furthermore, XEGMG (12 g/kg) can maintain the goblet cell number in the colons to protect the intestinal barrier and regulate the major flora such as Firmicutes, Bacteroidetes, and Muribaculaceae back to normal. Meanwhile, the expression of IL-17A in the colon tissues was significantly lower in the group of XEGMG (6, 12 g/kg) compared to H1N1 group. Discussion and conclusions XEGMG can protect against H1N1 invasion involved in gut-lung axis regulation. The results provide new evidence for the protective effect of XEGMG, which is beneficial to vulnerable children.

Inhibitory Effects and Related Molecular Mechanisms of Huanglian-Ganjiang Combination Against H1N1 Influenza Virus.

The online version contains supplementary material available at 10.1007/s43450-023-00372-z.

Mitigation of the Oxidative Damage in Liver Caused by Influenza Virus Infection in Mice by an Effective Combination of Oseltamivir and S-adenosyl-L-methionine (SAM)

This study aimed to estimate the protective effect of a combination of S-Adenosyl-L-methionine (SAM) as a precursor of glutathione and oseltamivir as a specific inhibitor of virus replication on oxidative damages caused by influenza infection in the liver of infected mice. Albino mice were inoculated with 10×LD50 of influenza virus A/Aichi/2/68(H3N2). Oseltamivir was applied for five days after infection, twice per day, in a dose of 2.5 mg/kg. SAM was applied for ten days in a 100 mg/kg dose, starting five days before inoculation. Markers of oxidative stress, mortality rate, mean survival time, index, and protection coefficient were followed. Influenza infection causes severe oxidative damage to the liver. All combinations of SAM and oseltamivir restored the levels of the biochemical markers to those in healthy animals and improved the virological parameters. A combination of SAM 100 mg/kg and oseltamivir 2.5 mg/kg, which is 1/4 of the optimal therapeutic mice dose, exhibits protection index and affects most effectively all the tested parameters. This study provides an easy-to-apply approach with a good therapeutic potential for co-medicating influenza infection with a specific antiviral agent and an antioxidant precursor.

Effect of Dietary Supplementation and Intranasal Administration of Soybean Extract on Resistance to Influenza Virus Infection in Mice

Effect of Dietary Supplementation and Intranasal Administration of Soybean Extract on Resistance to Influenza Virus Infection in Mice

Effect of [6]-gingerol on viral neuraminidase and hemagglutinin-specific T cell immunity in severe influenza

BackgroundH1N1 influenza viruses are the leading cause of severe respiratory virus infections worldwide. Resistance to the current selections of anti-influenza agents are still of concern. The Zingiber officinale root is one of the most heavily consumed dietary substances. It is also been traditionally used against common cold in Ayurveda and Chinese herbal medicine. PurposeInvestigation on in vitro and in vivo effect of [6]-gingerol from Zingiber officinale against influenza virus infection. MethodsIn vitro antiviral property of [6]-gingerol was measured by the plaque reduction assay in which MDCK cells were infected with PR8 strain of H1N1 influenza A virus in the presence of gingerol. Gingerol-mediated inhibition of influenza neuraminidase activity was tested by MUNANA assay in vitro. In vivo activity was tested in PR8 strain of H1N1 influenza virus infected mice. Modulation of anti-influenza immunity was tested by the study of hemagglutinin-specific T cells in vivo. Results[6]-Gingerol significantly reduced in vitro infection by PR8 strain of H1N1 influenza virus, with an IC50 value 2.25±0.18 μM. The inhibition was associated with the inhibition of viral neuraminidase activity, over 75% of the NA enzymatic activity of 2.5 × 103 PFUs of PR8 strain influenza virus at a concentration of 2 μM. Oseltamivir (Tamiflu) served as a positive control, which inhibited the NA enzymatic activity with an IC50 value of 1.88±0.05 μM. In H1N1 influenza virus infected mice, gingerol treatment (oral, once daily for 5 days) reduced virus load in the lungs and attenuated body weight loss and mortality. Gingerol inhibited viral neuraminidase-mediated intracellular TGF-β activation of lung-infiltrating CD4+ T cells. With the constrained active-TGF-β and immune suppression, influenza hemagglutinin-specific Th1 and Th17 immunity was increased. Conclusion[6]-Gingerol from Z. officinale is a potent anti-influenza compound that inhibits viral neuraminidase activity and boosts hemagglutinin-specific CD4+ T cell response to the infection. Taken together, gingerol has the potential to be further evaluated for clinical applications in human influenza.

Anti-influenza properties of tiliroside isolated from Hibiscus mutabilis L.

Ethnopharmacological relevanceFu Rong Ye (FRY), the leaf of Hibiscus mutabilis L., is a Chinese medicinal herb used to treat coughs and respiratory diseases. FRY is the major herbal component of the patent medicine Fupo Ganmao Granules for treating common cold. However, its anti-influenza active components and mechanism were not identified. AimHere, we aim to a) isolate the anti-influenza phytochemicals from FRY extract and b) explore its anti-flu mechanism. Material and methodsBioassay guided isolation was performed to get anti-influenza virus components. Influenza virus infected cells and mouse model were employed for efficacy evaluation. ResultsUsing bioassay-guided isolation, the flavonoid tiliroside was obtained, which inhibited four IAV strains in MDCK cells with EC50 ranging from 3.87 to 27.61 μM by suppressing the viral ribonucleoprotein activity. Tiliroside also significantly downregulated the expression of cytokines/chemokines in A549 cells, and protected 50% of PR8-infected BALB/c mice from death and at 800 mg/kg/day, improved lung edema conditions. ConclusionTiliroside is effective for influenza virus infection treatment and promising for further drug development. This study is the first to demonstrate that tiliroside in FRY acts against influenza virus.

Predicting Influenza A Virus Infection in the Lung from Hematological Data with Machine Learning.

The tracking of pathogen burden and host responses with minimally invasive methods during respiratory infections is central for monitoring disease development and guiding treatment decisions. Utilizing a standardized murine model of respiratory influenza A virus (IAV) infection, we developed and tested different supervised machine learning models to predict viral burden and immune response markers, i.e., cytokines and leukocytes in the lung, from hematological data. We performed independently in vivo infection experiments to acquire extensive data for training and testing of the models. We show here that lung viral load, neutrophil counts, cytokines (such as gamma interferon [IFN-γ] and interleukin 6 [IL-6]), and other lung infection markers can be predicted from hematological data. Furthermore, feature analysis of the models showed that blood granulocytes and platelets play a crucial role in prediction and are highly involved in the immune response against IAV. The proposed in silico tools pave the path toward improved tracking and monitoring of influenza virus infections and possibly other respiratory infections based on minimally invasively obtained hematological parameters. IMPORTANCE During the course of respiratory infections such as influenza, we do have a very limited view of immunological indicators to objectively and quantitatively evaluate the outcome of a host. Methods for monitoring immunological markers in a host's lungs are invasive and expensive, and some of them are not feasible to perform. Using machine learning algorithms, we show for the first time that minimally invasively acquired hematological parameters can be used to infer lung viral burden, leukocytes, and cytokines following influenza virus infection in mice. The potential of the framework proposed here consists of a new qualitative vision of the disease processes in the lung compartment as a noninvasive tool.

Targeted delivery of an anti-inflammatory corticosteroid to Ly6C/G-positive cells abates severity of influenza A symptoms

The distribution of Ly6C/G-positive cells in response to an infection of the mouse respiratory tract with influenza A virus was followed noninvasively over time by immuno-positron emission tomography. We converted nanobodies that recognize Ly6C and Ly6G, markers of neutrophils and other myeloid cells, as well as an influenza hemagglutinin-specific nanobody, into 89Zr-labeled PEGylated positron emission tomography (PET) imaging agents. The PET images showed strong accumulation of these imaging agents in the lungs of infected mice. Immunohistochemistry of influenza virus-infected mice and control mice, injected with a biotinylated and PEGylated version of the Ly6C/G-specific nanobody, showed the presence of abundant Ly6C/G-positive myeloid cells and positivity for Ly6C/G on bronchial epithelium in influenza virus-infected mice. This is consistent with focal inflammation in the lungs, a finding that correlated well with the immuno-PET results. No such signals were detected in control mice. Having shown by PET the accumulation of the Ly6C/G-specific nanobody in infected lungs, we synthesized conjugates of Ly6C/G-specific nanobodies with dexamethasone to enable targeted delivery of this immunosuppressive corticosteroid to sites of inflammation. Such conjugates reduced the weight loss that accompanies infection, while the equivalent amount of free dexamethasone was without effect. Nanobody-drug conjugates thus enable delivery of drugs to particular cell types at the appropriate anatomic site(s). By avoiding systemic exposure to free dexamethasone, this strategy minimizes its undesirable side effects because of the much lower effective dose of the nanobody-dexamethasone conjugate. The ability to selectively target inflammatory cells may find application in the treatment of other infections or other immune-mediated diseases.

Common and species-specific molecular signatures, networks, and regulators of influenza virus infection in mice, ferrets, and humans.

Molecular responses to influenza A virus (IAV) infections vary between mammalian species. To identify conserved and species-specific molecular responses, we perform a comparative study of transcriptomic data derived from blood cells, primary epithelial cells, and lung tissues collected from IAV-infected humans, ferrets, and mice. The molecular responses in the human host have unique functions such as antigen processing that are not observed in mice or ferrets. Highly conserved gene coexpression modules across the three species are enriched for IAV infection–induced pathways including cell cycle and interferon (IFN) signaling. TDRD7 is predicted as an IFN-inducible host factor that is up-regulated upon IAV infection in the three species. TDRD7 is required for antiviral IFN response, potentially modulating IFN signaling via the JAK/STAT/IRF9 pathway. Identification of the common and species-specific molecular signatures, networks, and regulators of IAV infection provides insights into host-defense mechanisms and will facilitate the development of novel therapeutic interventions against IAV infection.

Integrated network pharmacology and intestinal flora analysis to determine the protective effect of Xuanbai–Chengqi decoction on lung and gut injuries in influenza virus-infected mice

Ethnopharmacological relevanceXuanbai–Chengqi decoction (XBCQ) is a traditional Chinese medicine (TCM) compound used in the treatment of pulmonary infection in China. Despite the popular usage of XBCQ, its underlying protective roles and the associated molecular mechanisms with the gut-lung axis in influenza remain unclear. Aim of the studyWe aimed to explore the protective effects and the underlying mechanism of XBCQ efficacy on lung and intestine injuries induced by influenza A virus as well as to identify the main active components through integrated network pharmacology, intestinal flora analysis and pathway validation. Materials and methodsThe potential active components and therapeutic targets of XBCQ in the treatment of influenza were hypothesized through a series of network pharmacological strategies, including components screening, targets prediction and bioinformatics analysis. Inflammatory cytokines and pathway proteins were assayed to validate the results of network pharmacology. Then the mechanism of XBCQ alleviating lung and intestine injuries was further explored via intestinal flora analysis. The important role of Rhubarb in the formula was verified by removing Rhubarb. ResultsXBCQ could significantly improve the survival rate in IAV-infected mice. The network pharmacology results demonstrated that JUN, mitogen-activated protein kinase (MAPK), and tumor necrosis factor (TNF) are the key targets of XBCQ that can be useful in influenza treatment as it contains the core components luteolin, emodin, and aloe-emodin, which are related to the pathways of TNF, T-cell receptor (TCR), and NF-κB. Verification experiments demonstrated that XBCQ could significantly alleviate the immune injury of the lungs and the gut of the mice, which is attributable to the inhibition of the release of inflammatory cytokines (such as TNF-α, IL-6, and IL-1β), the downregulation of the protein expression levels of Toll-like receptors-7 (TLR7), MyD88, and p–NF–κB65, and the reduction in the relative abundance of Enterobacteriaceae and Proteus, while an increase in that of Firmicutes and Lachnospiraceae. The overall protective role of XBCQ contributing to the treatment of the lungs and the gut was impaired when Rhubarb was removed from XBCQ. ConclusionsOur results suggest that the efficacy of XBCQ is related to the inhibition of the immune injury and remodeling of the intestinal flora, wherein Rhubarb plays an important role, which cumulatively provide the evidence applicable for the treatment of viral pneumonia induced by a different respiratory virus with XBCQ.

Vaccination with Deglycosylated Modified Hemagglutinin Broadly Protects against Influenza Virus Infection in Mice and Ferrets.

Recent efforts have been directed toward the development of universal influenza vaccines inducing broadly neutralizing antibodies to conserved antigenic supersites of Hemagglutinin (HA). Although several studies raise the importance of glycosylation in HA antigen design, whether this theory can be widely confirmed remains unclear; which influenza HA with an altered glycosylation profile could impact the amplitude and focus of the host immune response. Here, we evaluated the characteristics and efficacy of deglycosylated modified HA proteins, including monoglycosylated HA (HAmg), unglycosylated HA (HAug), and fully glycosylated HA (HAfg), without treatment with H3N2 Wisconsin/67/2005. Our results showed that HAug could induce a cross-strain protective immune response in mice against both H3N2 and H7N9 subtypes with better antibody-dependent cellular cytotoxicity (ADCC) than the HAmg- and HAfg-immunized groups, which suggested that highly conserved epitopes that were masked by surface glycosylation may be exposed and thus promote the induction of broad antibodies that recognize the hidden epitopes. This strategy may also supplement the direction of deglycosylated modified HA for universal influenza vaccines.

Forsythiaside A improves Influenza A virus infection through TLR7 signaling pathway in the lungs of mice

BackgroundInfluenza A virus infection due to drug resistance and side effects of the conventional antiviral drugs yet remains a serious public health threat for humans and animals. Forsythiaside A is an effective ingredient isolated from the Chinese herbal medicine forsythia. It has various pharmacological effects and has a good therapeutic effect against a variety of infectious diseases. This study aimed to further explore the immunological mechanism of Forsythiaside A in the treatment of influenza virus-infected mice and its effect on the Toll-like receptor 7 (TLR7) signaling pathway in the lungs of these mice.MethodsC57/BL6J mice and TLR7−/− mice were infected with the FM1 strains (H1N1 and A/FM/1/4) of the Influenza A virus. Each group of experimental mice were divided into the mock, virus, oseltamivir, and Forsythiaside A groups. Weight change, lung index change, and the mRNA and protein expression levels of key factors in the TLR7 signaling pathway were detected. Flow cytometry was used to detect the changes in the Th1/Th2 and Th17/Treg ratios.ResultsAfter infection with the Influenza A virus, the weight loss of C57/BL6J mice treated with forsythoside A and oseltamivir decreased, and the pathological tissue sections showed that the inflammatory damage was reduced. The expression levels of the key factors, TLR7, myeloid differentiation factor 88(Myd88), and nuclear factor-kappa B (NF-κB) in the TLR7 signaling pathway were significantly reduced. Flow cytometry showed that Th1/Th2 and Th17/Treg ratios decreased after Forsythiaside A treatment. In the TLR7−/− mice, there was no significant change after Forsythiaside A treatment in the virus group.ConclusionsForsythiaside A affects the TLR7 signaling pathway in mouse lung immune cells and reduces the inflammatory response caused by the Influenza A virus FM1 strain in mouse lungs.

Viral PB1-F2 and host IFN-γ guide ILC2 and T cell activity during influenza virus infection

Functional plasticity of innate lymphoid cells (ILCs) and T cells is regulated by host environmental cues, but the influence of pathogen-derived virulence factors has not been described. We now report the interplay between host interferon (IFN)-γ and viral PB1-F2 virulence protein in regulating the functions of ILC2s and T cells that lead to recovery from influenza virus infection of mice. In the absence of IFN-γ, lung ILC2s from mice challenged with the A/California/04/2009 (CA04) H1N1 virus, containing nonfunctional viral PB1-F2, initiated a robust IL-5 response, which also led to improved tissue integrity and increased survival. Conversely, challenge with Puerto Rico/8/1934 (PR8) H1N1 virus expressing fully functional PB1-F2, suppressed IL-5+ ILC2 responses, and induced a dominant IL-13+ CD8 T cell response, regardless of host IFN-γ expression. IFN-γ-deficient mice had increased survival and improved tissue integrity following challenge with lethal doses of CA04, but not PR8 virus, and increased resistance was dependent on the presence of IFN-γR+ ILC2s. Reverse-engineered influenza viruses differing in functional PB1-F2 activity induced ILC2 and T cell phenotypes similar to the PB1-F2 donor strains, demonstrating the potent role of viral PB1-F2 in host resistance. These results show the ability of a pathogen virulence factor together with host IFN-γ to regulate protective pulmonary immunity during influenza infection.

In vitro and in vivo antiviral activity of Maqian (Zanthoxylum myriacanthum var. pubescens) essential oil and its major constituents against strains of influenza virus

Zanthoxylum myriacanthum var. pubescens (Maqian) is an economically important plant that is widely consumed as a flavoring agent or indigenous medicine by local ethnic groups in Southwest China. The pharmacological activities, such as the anti-inflammatory activity of Maqian essential oil, have been highlighted in recent years. In this study, the in vitro and in vivo anti-influenza activities of Maqian essential oil and its major ingredient, d/L-limonene, were assessed. The Maqian essential oil and d/L-limonene were effective against the influenza A and influenza B viruses in vitro. Notably, the Maqian essential oil was highly inhibitive against the drug-resistant influenza virus strain with an EC50 of 1.87 ± 1.04 μg/ml and a selective index of 85.13. Mode-of-action analysis revealed that Maqian essential oil and d/L-limonene inhibited the influenza virus at the stage of intracellular replication. For in vivo therapeutic efficacy, both intragastric and intranasal administration of d/L-limonene protected over 50% of mice from lethal influenza virus challenge. Both infectious viral particles and viral genome RNA were decreased approximately 2-fold in the d/L-limonene-treated mice compared with the virus group. Treatment with d/L-limonene potently improved the pathological features of the influenza virus-infected mice. Analysis of the gene expression level revealed that the major proinflammatory cytokines, including IL-1β, IL-6 and TNF-α, were significantly decreased in the d/L-limonene treated mouse lung tissues. Interestingly, we found that D-limonene was more potent than L-limonene in inhibiting proinflammatory cytokines. Transcriptome analysis revealed 32 host genes that participated in the antiviral process, most of which were related to the immune system and signal transduction. Combined with the antiviral and immunoregulatory effects of limonene, we believe that this multi-functional monoterpene has potential to become a novel candidate against influenza virus infections.

Antiviral Activity and Underlying Action Mechanism of Euglena Extract against Influenza Virus.

It is difficult to match annual vaccines against the exact influenza strain that is spreading in any given flu season. Owing to the emergence of drug-resistant viral strains, new approaches for treating influenza are needed. Euglena gracilis (hereinafter Euglena), microalga, used as functional foods and supplements, have been shown to alleviate symptoms of influenza virus infection in mice. However, the mechanism underlying the inhibitory action of microalgae against the influenza virus is unknown. Here, we aimed to study the antiviral activity of Euglena extract against the influenza virus and the underlying action mechanism using Madin–Darby canine kidney (MDCK) cells. Euglena extract strongly inhibited infection by all influenza virus strains examined, including those resistant to the anti-influenza drugs oseltamivir and amantadine. A time-of-addition assay revealed that Euglena extract did not affect the cycle of virus replication, and cell pretreatment or prolonged treatment of infected cells reduced the virus titer. Thus, Euglena extract may activate the host cell defense mechanisms, rather than directly acting on the influenza virus. Moreover, various minerals, mainly zinc, in Euglena extract were found to be involved in the antiviral activity of the extract. In conclusion, Euglena extract could be a potent agent for preventing and treating influenza.

Early oseltamivir reduces risk for influenza-associated aspergillosis in a double-hit murine model

ABSTRACT Invasive pulmonary aspergillosis (IPA) is a life-threatening fungal infection occurring mainly in immunocompromised patients. We recently identified IPA as an emerging co-infection with high mortality in critically ill, but otherwise immunocompetent influenza patients. The neuraminidase inhibitor oseltamivir is the current standard-of-care treatment in hospitalized influenza patients; however, its efficacy in influenza-associated pulmonary aspergillosis (IAPA) is not known. Therefore, we have established an imaging-supported double-hit mouse model to investigate the therapeutic effect of oseltamivir on the development of IAPA. Immunocompetent mice received intranasal instillation influenza A or PBS followed by orotracheal inoculation with Aspergillus fumigatus 4 days later. Oseltamivir treatment or placebo was started at day 0, day 2, or day 4. Daily monitoring included micro-computed tomography and bioluminescence imaging of pneumonia and fungal burden. Non-invasive biomarkers were complemented with imaging, molecular, immunological, and pathological analysis. Influenza virus-infected immunocompetent mice developed proven airway IPA upon co-infection with Aspergillus fumigatus, whereas non-influenza-infected mice fully cleared Aspergillus, confirming influenza as a risk factor for developing IPA. Longitudinal micro-CT showed pulmonary lesions after influenza infection worsening after Aspergillus co-infection, congruent with bioluminescence imaging and histology confirming Aspergillus pneumonia. Early oseltamivir treatment prevented severe influenza pneumonia and mitigated the development of IPA and associated mortality. A time-dependent treatment effect was consistently observed with imaging, molecular, and pathological analyses. Hence, our findings underscore the importance of initiating oseltamivir as soon as possible, to suppress influenza infection and mitigate the risk of potentially lethal IAPA disease.

ADAM10 partially protects mice against influenza pneumonia by suppressing specific myeloid cell population.

The influenza virus infection poses a serious health threat worldwide. Myeloid cells play pivotal roles in regulating innate and adaptive immune defense. A disintegrin and metalloproteinase (ADAM) family of proteins contributes to various immune responses; however, the role of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) in influenza virus infection remains largely unknown. Herein, we investigated its role, focusing on myeloid cells, during influenza virus infection in mice. ADAM10 gene (Adam10)flox/flox/Lyz2-Cre (Adam10ΔLyz2) and control Adam10flox/flox mice were intranasally infected with 200 plaque-forming units of influenza virus A/H1N1/PR8/34. Adam10ΔLyz2 mice exhibited a significantly higher mortality rate, stronger lung inflammation, and a higher virus titer in the lungs than control mice. Macrophages and inflammatory cytokines, such as TNF-α, IL-1β, and CCL2, were increased in bronchoalveolar lavage fluid from Adam10ΔLyz2 mice following infection. CD11b+Ly6G-F4/80+ myeloid cells, which had an inflammatory monocyte/macrophage-like phenotype, were significantly increased in the lungs of Adam10ΔLyz2 mice. Adoptive transfer experiments suggested that these cells likely contributed to the poorer prognosis in Adam10ΔLyz2 mice. Seven days after infection, CD11b+Ly6G-F4/80+ lung cells exhibited significantly higher arginase-1 expression levels in Adam10ΔLyz2 mice than in control mice, whereas an arginase-1 inhibitor improved the prognosis of Adam10ΔLyz2 mice. Enhanced granulocyte-macrophage colony-stimulating factor (GM-CSF)/GM-CSF receptor signaling likely contributed to this process. Collectively, these results indicate that myeloid ADAM10 protects against influenza virus pneumonia and may be a promising therapeutic target.

Influenza virus infection selectively triggers the accumulation and persistence of more potent Helios-expressing Foxp3+ regulatory T cells in the lungs.

Foxp3+ regulatory T cells (Tregs) represent a special lineage of CD4+ T cells. Analysis of Treg response during primary and secondary influenza virus infection clearly demonstrates a robust accumulation of Tregs into the infected lungs and the existence of a population of long-lived antigen-specific memory Tregs in the same tissues after resolution of the infection. However, it remains unknown whether these Tregs co-express Helios, a member of the Ikaros transcription factor family. In this study, Foxp3+ Helios+ and Foxp3+ Helios- Tregs in the lungs, mLNs and spleens of influenza virus-infected and uninfected control mice were tracked. The data show that while there is a co-existence of Foxp3+ Helios+ and Foxp3+ Helios- Tregs in the tissues, the accumulated Tregs in the lungs and lung-draining mediastinal lymph nodes (mLNs) of the infected mice are highly enriched for Foxp3+ Helios+ cells. It was further demonstrated that, after the clearance of primary infection, Foxp3+ Helios+ cells have the ability to persist in the tissues over their Helios- counterparts. More importantly, Foxp3+ Helios+ Tregs accumulated in an accelerated kinetics during recall response to reinfection. In vitro analysis of Treg suppressive function revealed that Foxp3+ Helios+ Tregs are more capable of suppressing influenza virus-specific CD8+ T cell activation, cytokine production and proliferation. Together, the data provide new insights into Treg responses during primary and secondary influenza virus infection and suggest that Foxp3+ Helios+ Tregs predominantly drive the Treg responses.

Puerarin reduces impairment of intestinal and adipose immune responses to influenza virus infection in mice.

Influenza is an acute viral respiratory disease that can also cause gastroenteritis-like symptoms, such as abdominal pain, nausea, vomiting, and diarrhea. Immune dysfunction of adipose tissue is involved in the occurrence and prognosis of influenza viral pneumonia. In this study, we analyzed intestinal and adipose immune responses in mice infected with influenza virus and found that the impairment of intestinal and adipose immunity to influenza virus infection could be reversed by treatment with puerarin, a medicinal compound isolated from Pueraria lobata (called “gegen” in Chinese). We found that the lungs, small intestines (duodenum, ileum, jejunum) and large intestines (colon and rectum) of infected mice showed obvious inflammatory lesions, with significantly increased levels of virus, inflammatory cytokines (interleukin [IL]-6, IL-17, and tumor necrosis factor-α), Toll-like receptors 3, 4, and 9, and integrin αvβ3 and α4, and a decreased level of secreted IgA compared to the normal control group (NC) (P < 0.05-0.001). Influenza virus infected mesenteric lymph nodes and adipose tissue, and adipokines (leptin, visfatin, “chemerin”, and adiponectin) of lung and mesenteric adipose tissue were dysregulated. Puerarin treatment reversed the impairment of the intestinal and adipose immune responses in mice infected with influenza virus. Our findings suggest that influenza virus can infect adipose tissue and lead to intestinal adipose immune dysfunction in normal-weight mice and that the impairment of the intestinal and adipose immune response to influenza virus infection can be reversed by puerarin treatment.