Search

Regulatory T (T(reg)) cells are an immunosuppressive population that are required to maintain peripheral tolerance and prevent tissue damage from immunopathology, via anti-inflammatory cytokines, inhibitor receptors and metabolic disruption. Here we show that T(reg) cells acquire an effector-like state, yet remain stable and functional, when exposed to interferon gamma (IFNγ) during infection with lymphocytic choriomeningitis and influenza A virus. T(reg) cell-restricted deletion of the IFNγ receptor (encoded by Ifngr1), but not the interleukin 12 (IL12) receptor (encoded by Il12rb2), prevented T(H)1-like polarization (decreased expression of T-bet, CXC motif chemokine receptor 3 and IFNγ) and promoted T(H)2-like polarization (increased expression of GATA-3, CCR4 and IL4). T(H)1-like T(reg) cells limited CD8(+) T cell effector function, proliferation and memory formation during acute and chronic infection. These findings provide fundamental insights into how T(reg) cells sense inflammatory cues from the environment (such as IFNγ) during viral infection to provide guidance to the effector immune response. This regulatory circuit prevents prolonged immunoinflammatory responses and shapes the quality and quantity of the memory T cell response.

Immune responses to influenza vaccination tend to be lower among older, frequently vaccinated adults. Use of egg-free influenza vaccines is increasing, but limited data exist on factors associated with their immunogenicity in older adults.Community-dwelling older adults ≥ 56 years of age were enrolled in a prospective, observational study of immunogenicity of 2018-2019 influenza vaccine. Hemagglutination inhibition (HAI) antibody titers were measured pre-vaccination (Day 0) and four weeks after vaccination (Day 28) to calculate geometric mean titers, seropositivity (HAI titers ≥ 1:40), seroconversion (fourfold rise in HAI titer with post-vaccination titer ≥ 1:40) and geometric mean fold rise (GMFR). Linear regression models assessed the association of predictors of GMFR for each vaccine antigen.Among 91 participants who received egg-free influenza vaccines, 84 (92.3 %) received quadrivalent recombinant influenza vaccine (RIV4, Flublok, Sanofi Pasteur), and 7 (7.7 %) received quadrivalent cell culture-based influenza vaccine (ccIIV4, Flucelvax, Seqirus). Pre-vaccination seropositivity was 52.8 % for A(H1N1), 94.5 % for A(H3N2), 61.5 % for B/Colorado and 48.4 % for B/Phuket. Seroconversion by antigen ranged from 16.5 % for A(H1N1) and B/Colorado to 37.4 % for A(H3N2); 40 participants failed to seroconvert to any antigen. Factors independently associated with higher GMFR in multivariable models included lower pre-vaccination HAI antibody titer for A(H1N1), B/Colorado and B/Phuket, and younger age for A(H1N1).Overall pre-vaccination seropositivity was high and just over half of the cohort seroconverted to ≥ 1 vaccine antigen. Antibody responses were highest among participants with lower pre-vaccination titers. Among older adults with high pre-existing antibody titers, approaches to improve immune responses are needed.

Background Few studies have focused on the immune response to more recent influenza vaccine formulations such as cell-cultured inactivated influenza vaccine (ccIIV4) or live attenuated influenza vaccine (LAIV4) in older children and young adults, or differences in immunoglobulin response using newer antibody landscape technology. Methods Participants ages 4-21 were randomized to receive ccIIV4 (n =112) or LAIV4 (n=118). A novel high-throughput multiplex influenza antibody detection assay (MIADA) was used to provide detailed IgG, IgA, and IgM antibody isotypes, along with hemagglutination inhibition levels (HAI), measured pre- and 28 days post vaccination. Results The HAI and immunoglobulin isotype response to ccIIV4 was greater than LAIV4, with significant increases in IgG but not IgA or IgM. The youngest participants had the highest LAIV4 response. Prior LAIV4 vaccination was associated with a higher response to current season ccIIV4. Cross-reactive A/Delaware/55/2019(H1N1)pdm09 antibodies were present pre-vaccination and increased in response to ccIIV4, but not LAIV4. Immunoglobulin assays strongly correlated with and confirmed the findings of HAI titers to measure immune response. Conclusions Age and prior season vaccination may play a role in the immune response in children and young adults to ccIIV4 and LAIV4. While immunoglobulin isotypes provide high level antigen-specific information, HAI titers alone can provide a meaningful representation of day 28 post vaccination response.

Cytokine storm describes a life-threatening, systemic inflammatory syndrome characterized by elevated levels of proinflammatory cytokines and immune cell hyperactivation associated with multi-organ dysfunction. Matrix-bound nanovesicles (MBV) are a subclass of extracellular vesicle shown to down-regulate proinflammatory immune responses. The objective of this study was to assess the efficacy of MBV in mediating influenza-induced acute respiratory distress syndrome and cytokine storm in a murine model. Intravenous administration of MBV decreased influenza-mediated total lung inflammatory cell density, proinflammatory macrophage frequencies, and proinflammatory cytokines at 7 and 21 days following viral inoculation. MBV decreased long-lasting alveolitis and the proportion of lung undergoing inflammatory tissue repair at day 21. MBV increased the proportion of activated anti-viral CD4 + and CD8 + T cells at day 7 and memory-like CD62L + CD44 + , CD4 + , and CD8 + T cells at day 21. These results show immunomodulatory properties of MBV that may benefit the treatment of viral-mediated pulmonary inflammation with applicability to other viral diseases such as SARS-CoV-2.

BackgroundThe ongoing circulation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a diagnostic challenge because symptoms of coronavirus disease 2019 (COVID-19) are difficult to distinguish from other respiratory diseases. Our goal was to use statistical analyses and machine learning to identify biomarkers that distinguish patients with COVID-19 from patients with influenza.MethodsCytokine levels were analyzed in plasma and serum samples from patients with influenza and COVID-19, which were collected as part of the Centers for Disease Control and Prevention's Hospitalized Adult Influenza Vaccine Effectiveness Network (inpatient network) and the US Flu Vaccine Effectiveness (outpatient network).ResultsWe determined that interleukin (IL)-10 family cytokines are significantly different between COVID-19 and influenza patients. The results suggest that the IL-10 family cytokines are a potential diagnostic biomarker to distinguish COVID-19 and influenza infection, especially for inpatients. We also demonstrate that cytokine combinations, consisting of up to 3 cytokines, can distinguish SARS-CoV-2 and influenza infection with high accuracy in both inpatient (area under the receiver operating characteristics curve [AUC] = 0.84) and outpatient (AUC = 0.81) groups, revealing another potential screening tool for SARS-CoV-2 infection.ConclusionsThis study not only reveals prospective screening tools for COVID-19 infections that are independent of polymerase chain reaction testing or clinical condition, but it also emphasizes potential pathways involved in disease pathogenesis that act as potential targets for future mechanistic studies.

Staphylococcus aureus can complicate preceding viral infections, including influenza virus. A bacterial infection combined with a preceding viral infection, known as superinfection, leads to worse outcomes than a single infection. Most of the pulmonary infection literature focuses on the changes in immune responses to bacteria between homeostatic and virally infected lungs. However, it is unclear how much of an influence bacterial virulence factors have in single or superinfection. Staphylococcal species express a broad range of cell wall-anchored proteins (CWAs) that have roles in host adhesion, nutrient acquisition, and immune evasion. We screened the importance of these CWAs using mutants lacking individual CWAs

BackgroundChronic rhinosinusitis (CRS) is a common, yet underreported and understudied manifestation of upper respiratory disease in people with cystic fibrosis (CF). There are currently no standard of care guidelines for the management of CF CRS, but treatment of upper airway disease may ameliorate lower airway disease. We sought to inform future treatment guidelines by determining whether changes to sinus microbial community diversity and specific taxa known to cause CF lung disease are associated with increased respiratory disease and inflammation.MethodsWe performed 16S rRNA gene sequencing, supplemented with cytokine analyses, microscopy, and bacterial culturing, on samples from the sinuses of 27 adults with CF CRS at the University of Pittsburgh’s CF Sinus Clinic. At each study visit, participants underwent endoscopic paranasal sinus sampling and clinical evaluation. We identified key drivers of microbial community composition and evaluated relationships between diversity and taxa with disease outcomes and inflammation.FindingsSinus community diversity was low and the composition was unstable, with many participants exhibiting alternating dominance between Pseudomonas aeruginosa and Staphylococci over time. Despite a tendency for dominance by these two taxa, communities were highly individualized and shifted composition during exacerbation of sinus disease symptoms. Exacerbations were also associated with communities dominated by Staphylococcus spp. Reduced microbial community diversity was linked to worse sinus disease and the inflammatory status of the sinuses (including increased IL-1β). Increased IL-1β was also linked to worse sinus endoscopic appearance, and other cytokines were linked to microbial community dynamics.InterpretationTo our knowledge, this is the largest longitudinal study of microbial communities and cytokine secretion in CF CRS. Our work revealed previously unknown instability of sinus microbial communities and a link between inflammation, lack of microbial community diversity, and worse sinus disease.FundingCystic Fibrosis Foundation and US National Institutes of Health.Research in ContextEvidence before this studyA search of the PubMed database on October 11, 2021 with the terms [cystic fibrosis sinus microbiome] yielded 16 results, and we have identified seven primary research articles on the CF CRS microbiome (including re-analyses of existing datasets). Most are cross-sectional cohort analyses, along with one prior longitudinal study of four adults at the University of Auckland, New Zealand. Together, these prior studies reveal similarities between CF CRS and CF sputum microbiomes, including low community diversity associated with sinus disease, the presence of common CF-associated microbes in the sinuses, and prevalence of sinus communities dominated by P. aeruginosa or Staphylococcus aureus. High levels of IL-1β are linked to the presence of nasal polyps in CF CRS, and polymorphisms in the IL-1 receptor antagonist gene are associated with risk of CRS outside of the context of CF. Two prior studies of this cohort have been performed by our laboratory. One describes clinical indicators of CF sinus disease and the other links sinus infection biogeography to P. aeruginosa evolutionary genomics.Added value of this studyOur study is the first to examine longitudinal relationships between the host immune response (through cytokine profiling) and microbiota dynamics in CF CRS, including linking elevated IL-1β to worse sinus disease through reduced sinus microbial community diversity. The longitudinal nature of our study also allowed us to uncover striking temporal instability of microbial communities in approximately half of our cohort’s sinus microbial communities over two years, including switching between communities dominated by P. aeruginosa and Staphylococcus spp. This instability could hinder attempts to link the relative abundance of taxa to clinical outcomes of interest in cross-sectional studies (e.g., markers of disease progression). We also identified patterns of synergy and antagonism between specific taxa, and impacts of the host immune response in the sinuses on community composition.Implications of all the available evidenceTogether with prior CF CRS microbiome studies, our study underscores similarities between sinus and lower respiratory tract microbial community structure in CF, and we show how community structure tracks with inflammation and several disease measures. This work strongly suggests that clinical management of CRS could be leveraged to improve overall respiratory health in CF. Our work implicates elevated IL-1β in reduced microbiota diversity and worse sinus disease in CF CRS, suggesting applications for existing therapies targeting IL-1β. Finally, the widespread use of highly effective CFTR modulator therapy has led to less frequent availability of spontaneous expectorated sputum for microbiological surveillance of lung infections. A better understanding of CF sinus microbiology could provide a much-needed alternative site for monitoring respiratory infection status by important CF pathogens.

SARS-CoV-2 vaccines BNT162b2, mRNA-1273, and Ad26.COV2.S received emergency use authorization by the U.S. Food and Drug Administration in 2020/2021. Individuals being vaccinated were invited to participate in a prospective longitudinal comparative study of immune responses elicited by the three vaccines. In this observational cohort study, immune responses were evaluated using a SARS-CoV-2 spike protein receptor-binding domain ELISA, SARS-CoV-2 virus neutralization assays and an IFN- γ ELISPOT assay at various times over six months following initial vaccination. mRNA-based vaccines elicited higher magnitude humoral responses than Ad26.COV2.S; mRNA-1273 elicited the most durable humoral response, and all humoral responses waned over time. Neutralizing antibodies against the Delta variant were of lower magnitude than the wild-type strain for all three vaccines. mRNA-1273 initially elicited the greatest magnitude of T cell response, but this declined by 6 months. Declining immunity over time supports the use of booster dosing, especially in the setting of emerging variants.

Physical frailty’s impact on hemagglutination inhibition antibody titers (HAI) and peripheral blood mononuclear cell (PBMC) transcriptional responses after influenza vaccination is unclear. Physical frailty was assessed using the 5-item Fried frailty phenotype in 168 community- and assisted-living adults ≥55 years of age during an observational study. Blood was drawn before, 3, 7, and 28 days post-vaccination with the 2017-2018 inactivated influenza vaccine. HAI response to the A/H1N1 strain was measured at Days 0 and 28 using seropositivity, seroconversion, log2 HAI titers, and fold-rise in log2 HAI titers. RNA sequencing of PBMCs from Days 0, 3 and 7 was measured in 28 participants and compared using pathway analyses. Frailty was not significantly associated with any HAI outcome in multivariable models. Compared with non-frail participants, frail participants expressed decreased cell proliferation, metabolism, antibody production, and interferon signaling genes. Conversely, frail participants showed elevated gene expression in IL-8 signaling, T-cell exhaustion, and oxidative stress pathways compared with non-frail participants. These results suggest that reduced effectiveness of influenza vaccine among older, frail individuals may be attributed to immunosenescence-related changes in PBMCs that are not reflected in antibody levels.

Development of acute respiratory distress syndrome (ARDS) in influenza A virus (IAV)-infected mice is associated with inhibition of ATII (alveolar type II) epithelial cell de novo phosphatidylcholine synthesis, and administration of the phosphatidylcholine precursor cytidine 5′-diphosphocholine (CDP-choline) attenuates IAV-induced acute respiratory distress syndrome in mice. We hypothesized inhibition of phosphatidylcholine synthesis would also impact the function of ATII cell mitochondria. To test this hypothesis, adult C57BL/6 mice of both sexes were inoculated intranasally with 10,000 pfu/mouse influenza A/WSN/33 (H1N1). Control mice were mock-infected with virus diluent. Mice were treated with saline vehicle or CDP-choline (100 μg/mouse i.p.) once daily from 1 to 5 days postinoculation (dpi). ATII cells were isolated by a standard lung digestion protocol at 6 dpi for analysis of mitochondrial function. IAV infection increased uptake of the glucose analog fludeoxyglucose F 18 by the lungs and caused a switch from oxidative phosphorylation to aerobic glycolysis as a primary means of ATII cell ATP synthesis by 6 dpi. Infection also induced ATII cell mitochondrial depolarization and shrinkage, upregulation of PGC-1α, decreased cardiolipin content, and reduced expression of mitofusin 1, OPA1, DRP1, complexes I and IV of the electron transport chain, and enzymes involved in cardiolipin synthesis. Daily CDP-choline treatment prevented the declines in oxidative phosphorylation, mitochondrial membrane potential, and cardiolipin synthesis resulting from IAV infection but did not fully reverse the glycolytic shift. CDP-choline also did not prevent the alterations in mitochondrial protein expression resulting from infection. Taken together, our data show ATII cell mitochondrial dysfunction after IAV infection results from impaired de novo phospholipid synthesis, but the glycolytic shift does not.

Influenza associated bacterial super-infections have devastating impacts on the lung and can result in increased risk of mortality. New strains of influenza circulate throughout the population yearly promoting the establishment of immune memory. Nearly all individuals have some degree of influenza memory prior to adulthood. Due to this we sought to understand the role of immune memory during bacterial super-infections. An influenza heterotypic immunity model was established using influenza A/PR/8/34 and A/X31. We report here that influenza experienced mice are more resistant to secondary bacterial infection with methicillin-resistant Staphylococcus aureus as determined by wasting, bacterial burden, pulmonary inflammation, and lung leak, despite significant ongoing lung remodeling. Multidimensional flow cytometry and lung transcriptomics revealed significant alterations in the lung environment in influenza-experienced mice compared with naïve animals. These include changes in the lung monocyte and T cell compartments, characterized by increased expansion of influenza tetramer specific CD8+ T cells. The protection that was seen in memory experienced mouse model is associated with the reduction in inflammatory mechanisms making the lung less susceptible to damage and subsequent bacterial colonization. These findings provide insight into how influenza heterotypic immunity re-shapes the lung environment and the immune response to a re-challenge event, which is highly relevant to the context of human infection.

Staphylococcus aureus can complicate preceding viral infections, including influenza virus. A bacterial infection combined with a preceeding viral infection, known as super-infection, leads to worse outcomes compared to single infection. Most of the super-infection literature focuses on the changes in immune responses to bacteria between homeostatic and virally infected lungs. However, it is unclear how much of an influence bacterial virulence factors have in super-infection. Staphylococcal species express a broad range of cell wall-anchored proteins (CWAs) that have roles in host adhesion, nutrient acquisition, and immune evasion. We screened the importance of these CWAs using mutants lacking individual CWAs in vivo in both bacterial pneumonia and influenza super-infection. In bacterial pneumonia, lacking individual CWAs led to varying decreases in bacterial burden, lung damage, and immune infiltration into the lung. However, the presence of a preceding influenza infection partially abrogated the requirement for CWAs. In the screen, we found that the uncharacterized CWA S. aureus surface protein D (SasD) induced changes in both inflammatory and homeostatic lung markers. We further characterized a SasD mutant (sasD A50.1) in the context of pneumonia. Mice infected with sasD A50.1 had decreased bacterial burden, inflammatory responses, and mortalty compared to wildtype S. aureus. Mice also had reduced levels of IL-1β compared with wildtype, likely derived from macrophages. Reductions in IL-1β transcript levels as well as increased macrophage viability implicate altered macrophage cell death pathways. These data identify a novel virulence factor for S. aureus that influences inflammatory signaling within the lung.ImportanceStaphylococcus aureus is a common commensal bacteria that can cause severe infections, such as pneumonia. In the lung, viral infections increase the risk of staphylococcal pneumonia, leading to combined infections known as super-infections. The most common virus associated with S. aureus pneumonia is influenza, and super-infections lead to worse patient outcomes compared to either infection alone. While there is much known about how the immune system differs between healthy and virally infected lungs, the role of bacterial virulence factors in super-infection is less understood. The significance of our research is identifying new bacterial virulence factors that play a role in the initiation of infection and lung injury, which could lead to future therapies to prevent pulmonary single or super-infection with S. aureus.

Background Current influenza vaccine effectiveness (VE) improvement efforts focus on minimizing egg adaptation mutations during manufacture. This study compared immune response of two FDA-approved quadrivalent inactivated influenza vaccines in an unblinded randomized controlled trial. Methods Participants were 144 community dwelling, healthy children/adolescents aged 4–20 years, randomized 1:1 in blocks of 4 to a vaccine grown in cell culture (ccIIV4 [Flucelvax®]; n = 85); or in egg medium (IIV4 [Fluzone ®]; n = 83). Blood was drawn at day 0 prevaccination and at day 28 (19–35 days) post vaccination. Hemagglutination inhibition (HI) assays against A/H1N1 and both B strains and microneutralization (MN) assays against egg-based and cell-based A/H3N2 strains were conducted. The primary outcome measure was seroconversion (day 28/day 0 titer ratio ≥ 4 with day 28 titer ≥ 40). Secondary outcomes were elevated titers (day 28 HI titer ≥ 1:110), geometric mean titers (GMTs) and mean fold rise (MFR) in titers. Outcomes were compared for 74 ccIIV4 recipients and 70 IIV4 recipients, and for those vaccinated and unvaccinated the previous year. Only the HI and MN laboratory analysis team was blinded to group assignment. Results In this racially diverse (81% non-white) group of children with a median age of 14 years, baseline demographics did not differ between vaccine groups. At day 0, half or more in each vaccine group had elevated HI or MN titers. Low seroconversion rates (14%-35%) were found; they did not differ between groups. Among 2018–19 ccIIV4 recipients, those unvaccinated in the previous season showed significantly higher MFR against A/H1N1 and A/H3N2 cell-grown virus than the previously vaccinated. Similar results were found for MFR against B/Victoria among 2018–2019 IIV4 recipients. Conclusion In mostly older children with high baseline titers, no differences in seroconversion or other measures of antibody titers were found between ccIIV4 and IIV4 recipients against egg- and cell-grown influenza vaccine viruses. Clinical Trials No NCT03614975.

Acute influenza virus infections are a global public health concern accounting for millions of illnesses worldwide ranging from mild to severe with, at time, severe complications. Once an individual is infected, the immune system is triggered in response to the pathogen. This immune response can be beneficial ultimately leading to the clearance of the viral infection and establishment of immune memory mechanisms. However, it can be detrimental by increasing susceptibility to secondary bacterial infections and resulting in permanent changes to the lung architecture, in the form of fibrotic sequelae. Here, we review influenza associated bacterial super-infection, the formation of T-cell memory, and persistent lung injury resulting from influenza infection.

The human immune response to inactivated influenza vaccine is dynamic and impacted by age and preexisting immunity. Our goal was to identify postvaccination transcriptomic changes in peripheral blood mononuclear cells from children. Blood samples were obtained before and at 3 or 7 days postvaccination with 2016-2017 quadrivalent inactivated influenza vaccine and RNA sequencing was performed. There were 1,466 differentially expressed genes (DEGs) for the Day 0-Day 3 group and 513 DEGs for the Day 0-Day 7 group. Thirty-three genes were common between the two groups. The majority of the transcriptomic changes at Day 3 represented innate inflammation and apoptosis pathways. Day 7 DEGs were characterized by activation of cellular processes, including the regulation of cytoskeleton, junctions, and metabolism, and increased expression of immunoglobulin genes. DEGs at Day 3 were compared between older and younger children revealing increased inflammatory gene expression in the older group. Vaccine history in the year prior to the study was characterized by robust DEGs at Day 3 with decreased phagosome and dendritic cell maturation in those who had been vaccinated in the previous year. PBMC responses to inactivated influenza vaccination in children differed significantly by the timing of sampling, patient age, and vaccine history. These data provide insight into the expected molecular pathways to be temporally altered by influenza vaccination in children.

Angiotensin-converting enzyme 2 (ACE2) is a potent negative regulator capable of restraining overactivation of the renin–angiotensin system, which contributes to exuberant inflammation after bacterial infection. However, the mechanism through which ACE2 modulates this inflammatory response is not well understood. Accumulating evidence indicates that infectious insults perturb ACE2 activity, allowing for uncontrolled inflammation. In the current study, we demonstrate that pulmonary ACE2 levels are dynamically varied during bacterial lung infection, and the fluctuation is critical in determining the severity of bacterial pneumonia. Specifically, we found that a pre-existing and persistent deficiency of active ACE2 led to excessive neutrophil accumulation in mouse lungs subjected to bacterial infection, resulting in a hyperinflammatory response and lung damage. In contrast, pre-existing and persistent increased ACE2 activity reduces neutrophil infiltration and compromises host defense, leading to overwhelming bacterial infection. Further, we found that the interruption of pulmonary ACE2 restitution in the model of bacterial lung infection delays the recovery process from neutrophilic lung inflammation. We observed the beneficial effects of recombinant ACE2 when administered to bacterially infected mouse lungs following an initial inflammatory response. In seeking to elucidate the mechanisms involved, we discovered that ACE2 inhibits neutrophil infiltration and lung inflammation by limiting IL-17 signaling by reducing the activity of the STAT3 pathway. The results suggest that the alteration of active ACE2 is not only a consequence of bacterial lung infection but also a critical component of host defense through modulation of the innate immune response to bacterial lung infection by regulating neutrophil influx.

BackgroundHemagglutination inhibition (HAI) titers to the live-attenuated influenza vaccine (LAIV4) are typically lower than its counterpart egg-based inactivated influenza vaccines (IIV). Similar comparisons have not been made between LAIV4 and the 4-strain, cell-culture inactivated influenza vaccine (ccIIV4). We compared healthy children and young adult HAI titers against the 2019-2020 LAIV4 and ccIIV4.MethodsParticipants aged 4-21 years were randomized 1:1 to receive ccIIV4 (n =100) or LAIV4 (n=98). Blood was drawn prevaccination and on day 28 (21-35) post vaccination. HAI assays against egg-grown A/H1N1, A/H3N2, both vaccine B strains and cell-grown A/H3N2 antigens were conducted. Outcomes were geometric mean titers (GMT) and geometric mean fold rise (GMFR) in titers.ResultsGMTs to A/H1N1, A/H3N2 and B/Victoria increased following both ccIIV and LAIV and to B/Yamagata following ccIIV (pConclusionThe HAI response to ccIIV4 was greater than LAIV4 in this study of mostly older children, and day 0 HAI titers inversely predicted GMFR for both vaccines. For both vaccines, the A/H3N2 cell-grown antigen levels were lower than egg-grown, but the GMFR for cell-grown and egg-grown did not differ significantly within vaccine type.Clinical Trials NoNCT03982069

Influenza (IAV) neuraminidase (NA) is a glycoprotein required for the viral exit from the cell. NA requires disulfide bonds for proper function. We have recently demonstrated that protein disulfide isomerase (PDI)A3 is required for oxidative folding of IAV hemagglutinin (HA), and viral propagation. However, it not known whether PDIs are required for NA maturation or if these interactions represent a putative target for the treatment of influenza infection. We sought to determine whether PDIA3 is required for disulfide bonds of NA, its activity, and propagation of the virus. Requirement of disulfides for NA oligomerization and activity were determined using biotin switch and redox assays in WT and PDIA3−/− in A549 cells. A PDI specific inhibitor (LOC14) was utilized to determine the requirement of PDIs in NA activity, IAV burden, and inflammatory response in A549 and primary mouse tracheal epithelial cells. Mice were treated with the inhibitor LOC14 and subsequently examined for IAV burden, NA activity, cytokine, and immune response. IAV-NA interacts with PDIA3 and this interaction is required for NA activity. PDIA3 ablation or inhibition decreased NA activity, viral burden, and inflammatory response in lung epithelial cells. LOC14 treatment significantly attenuated the influenza-induced inflammatory response in mice including the overall viral burden. These results provide evidence for PDIA3 inhibition suppressing NA activity, potentially providing a novel platform for host-targeted antiviral therapies.

COVID-19 has had an unprecedented global impact on human health. Understanding the Ab memory responses to infection is one tool needed to effectively control the pandemic. Among 173 outpatients who had virologically confirmed SARS-CoV-2 infection, we evaluated serum Ab concentrations, microneutralization activity, and enumerated SARS-CoV-2–specific B cells in convalescent human blood specimens. Serum Ab concentrations were variable, allowing for stratification of the cohort into high and low responders. Neither participant sex, the timing of blood sampling following the onset of illness, nor the number of SARS-CoV-2 spike protein–specific B cells correlated with serum Ab concentration. Serum Ab concentration was positively associated with microneutralization activity and participant age, with participants under the age of 30 showing the lowest Ab level. These data suggest that young adult outpatients did not generate as robust Ab memory, compared with older adults. Body mass index was also positively correlated with serum Ab levels. Multivariate analyses showed that participant age and body mass index were independently associated with Ab levels. These findings have direct implications for public health policy and current vaccine efforts. Knowledge gained regarding Ab memory following infection will inform the need for vaccination in those previously infected and allow for a better approximation of population-wide protective immunity.

Background Type III interferon, or interferon lambda (IFNλ) is a crucial antiviral cytokine induced by influenza infection. While IFNλ is important for anti-viral host defense, published data demonstrate that IFNλ is pathogenic during influenza/bacterial super-infection. It is known that polymorphisms in specific IFNλ genes affect influenza responses, but the effect of IFNλ subtypes on bacterial super-infection is unknown. Methods Using an established model of influenza, Staphylococcus aureus super-infection, we studied IFNλ3-/- and control mice to model a physiologically relevant reduction in IFNλ and to address its role in super-infection. Results Surprisingly, IFNλ3-/- mice did not have significantly lower total IFNλ than co-housed controls, and displayed no change in viral or bacterial clearance. Importantly, both control and IFNλ3-/- mice displayed a positive correlation between viral burden and total IFNλ in the bronchoalveolar lavage during influenza/bacterial super-infection, suggesting that higher influenza viral burden drives a similar total IFNλ response regardless of IFNλ3 gene integrity. Interestingly, total IFNλ levels positively correlated with bacterial burden, while viral burden and bronchoalveolar lavage cellularity did not. Conclusions These data suggest IFNλ2 can compensate for IFNλ3 to mount an effective antiviral and defense, revealing a functional redundancy in these highly similar IFNλ subtypes. Further, the IFNλ response to influenza, as opposed to changes in cellular inflammation or viral load, significantly correlates with susceptibility to bacterial super-infection. Moreover, the IFNλ response is regulated and involves redundant subtypes, suggesting it is of high importance to pulmonary pathogen defense.

BackgroundThree SARS-CoV-2 vaccines, two based on mRNA, BNT162b2 and mRNA-1273, and one based on an adenovirus platform, Ad26.COV2.S, received emergency use authorization by the U.S. Food and Drug Administration in 2020/2021. These vaccines displayed clinical efficacy in initial studies against confirmed COVID-19 of 95.0%, 94.1%, and 66.9%, respectively.MethodsIndividuals receiving one of these vaccines were invited to participate in a prospective longitudinal comparative study of immune responses elicited by the three vaccines. In this observational cohort study, humoral responses were evaluated using a SARS-CoV-2 receptor-binding domain (RBD) ELISA and a SARS-CoV-2 virus neutralization assay at mean of 21-31 days and 45-63 days following each initial vaccination. IFN-γ ELISPOT assays were conducted with peripheral blood mononuclear cells obtained at a median of 45-63 days after each initial vaccination.ResultsThe two mRNA-based platforms elicited similar RBD ELISA responses and neutralizing antibody responses. The adenovirus-based vaccine elicited significantly lower RBD ELISA and SARS-CoV-2 virus neutralization activity. The mRNA-1273 vaccine elicited significantly higher spike glycoprotein-specific T cell responses than either the BNT162b2 or the Ad26.COV2.S vaccines.ConclusionsBoth mRNA based vaccines elicited higher magnitude humoral responses than Ad26.COV2.S and mRNA1273 elicited the highest magnitude of T cell response. Neutralizing antibody titers correlated with reported estimates of vaccine efficacy.Summary of key pointsWe compared antigen specific humoral and T cell responses following vaccination with BNT162b2, mRNA-1273, or Ad26.COV2.S. Both mRNA based vaccines elicited higher magnitude humoral responses than Ad26.COV2.S and mRNA1273 elicited the highest magnitude of T cell response.

Secondary bacterial pneumonia is a significant complication of severe influenza infection and Staphylococcus aureus and Streptococcus pneumoniae are the primary pathogens of interest. IL-22 promotes S. aureus and S. pneumoniae host defense in the lung through epithelial integrity and induction of antimicrobial peptides and is inhibited by the soluble decoy receptor IL-22 binding protein (IL-22BP). Little is known about the effect of the IL-22/IL-22BP regulatory pathway on lung infection, and it has not been studied in the setting of super-infection. We exposed wild-type and IL-22BP−/− mice to influenza A/PR/8/34 for six days prior to infection with S. aureus (USA300) S. pneumoniae. Super-infected IL-22BP−/− mice had decreased bacterial burden and improved survival compared to controls. IL-22BP−/− mice exhibited decreased inflammation, increased lipocalin 2 expression, and deletion of IL-22BP was associated with preserved epithelial barrier function with evidence of improved tight junction stability. Human bronchial epithelial cells treated with IL-22Fc showed evidence of improved tight junctions compared to untreated cells. This study revealed that IL-22BP−/− mice are protected during influenza, bacterial super-infection, suggesting that IL-22BP has a pro-inflammatory role and impairs epithelial barrier function likely through interaction with IL-22

Background and objective Chronic lower respiratory diseases (CLRD) increase the risk of type 2 diabetes, which in turn may worsen lung function. Metformin, a common antidiabetic with anti-inflammatory and antioxidant properties, may improve respiratory outcomes. Therefore, we examined the association of metformin use with the risk of mortality from CLRD. Methods We analysed data from the National Health and Nutrition Examination Survey during 1988-1994 and 1999-2010 for participants aged 40 years or older who had diabetes and were followed up for mortality through 2011. Information on prescription medicine was collected at baseline and CLRD-related mortality during follow-up was defined using the 10th Revision of the International Classification of Diseases (ICD-10). Cox proportional hazards modelling was used to determine the mortality hazard ratio (HR) associated with metformin use, adjusting for relevant covariates. Results A total of 5266 participants with a median follow-up of 6.1 years were included. The prevalence of metformin use was 31.9% and 1869 participants died during follow-up, including 72 of CLRD. In the adjusted Cox proportional regression analysis, metformin was associated with a decreased risk of CLRD mortality in the overall population (HR: 0.39, 95% CI: 0.15-0.99) and among participants with baseline CLRD (HR: 0.30, 95% CI: 0.10-0.93), after adjusting for age, gender, race/ethnicity, cigarette smoking, body mass index, current asthma and chronic obstructive pulmonary disease (COPD), insulin and other diabetic medications, and glycohaemoglobin level. We found no association between other antidiabetic medications and CLRD mortality. Conclusion In this sample representative of the U.S. population, metformin was associated with lower CLRD mortality in adults with diabetes.

Regulatory T cells (Tregs) are an immunosuppressive cell population that inhibits immune cell function to maintain peripheral tolerance and homeostasis. We have previously shown that Treg-- response to interferon gamma (IFNγ) is required for response to cancer immunotherapy. We aimed to broaden these findings and determine the impact of Treg response to IFNγ and the IFNγ-inducing cytokine IL12, during viral infection. Mice with Treg-restricted deletion of the IFNγ receptor (IFNGR1, Ifngr1L/LFoxp3Cre-YFP) or the IL12 receptor β2 subunit (IL12Rβ2, Il12b2L/LFoxp3Cre-YFP), were infected with the acute (Armstrong), or chronic (Clone 13), strain of lymphocytic choriomeningitis virus (LCMV). Surprisingly, Treg response to IFNγ but not IL12 induced helper T cell 1 (TH1)-like polarization (expression of T-bet, CXCR3 and IFNγ) of Tregs in mice with chronic LCMV infection. Importantly, this effector-like TH1 Treg state was required for adequate suppression of effector T cells during LCMV infection. Additionally, during LCMV infection, Treg-restricted deletion of IFNGR1 not only prevented TH1-like polarization but also reverted these Tregs to a TH2-like state (expression of GATA-3, CCR4 and IL-4). scRNAseq of viral antigen-specific CD8+ T cells from Clone 13 infected mice showed that Ifngr1-deficient Tregs favored CD8+ T cell memory. These findings were confirmed in an Armstrong/Listeria monocytogenes-GP33 memory model. Further, Ifngr1 deletion from Tregs enhanced response to CD8+ T cell-mediated vaccination when challenged with Clone 13. These findings provide fundamental insight on how Tregs sense inflammatory cues from the environment (IFNγ) during viral infection to prevent overt tissue damage and shape the memory response. Supported by grants from NIH (F32 CA247004-01, T32 CA082084, P01 AI108545, R35 CA263850, R01 CA203689, R01 DK130897, P30 DK120531, P30 CA047904, R01 HL107380, R01 CA206517, R01 AI138504)

Influenza virus infection causes 3-5 million cases of severe illness and 250,000-500,000 deaths worldwide annually. Although pneumonia is the most common complication associated with influenza, there are several reports demonstrating increased risk for cardiovascular diseases. Several clinical case reports, as well as both prospective and retrospective studies, have shown that influenza can trigger cardiovascular events including myocardial infarction (MI), myocarditis, ventricular arrhythmia, and heart failure. A recent study has demonstrated that influenza-infected patients are at highest risk of having MI during the first seven days of diagnosis. Influenza virus infection induces a variety of proinflammatory cytokines and chemokines and recruitment of immune cells as part of the host immune response. Understanding the cellular and molecular mechanisms involved in influenza-associated cardiovascular diseases will help to improve treatment plans. This review discusses the direct and indirect effects of influenza virus infection on triggering cardiovascular events. Further, we discussed the similarities and differences in epidemiological and pathogenic mechanisms involved in cardiovascular events associated with coronavirus disease 2019 (COVID-19) compared to influenza infection.

Asthma is a highly prevalent, chronic respiratory disease that impacts millions of people worldwide and causes thousands of deaths every year. Asthmatics display different phenotypes with distinct genetic components, environmental causes, and immunopathologic signatures, and are broadly characterized into type 2-high or type 2-low (non-type 2) endotypes by linking clinical characteristics, steroid responsiveness, and molecular pathways. Regardless of asthma severity and adequate disease management, patients may experience acute exacerbations of symptoms and a loss of disease control, often triggered by respiratory infections. The interferon (IFN) family represents a group of cytokines that play a central role in the protection against and exacerbation of various infections and pathologies, including asthma. Type I and III IFNs in particular play an indispensable role in the host immune system to fight off pathogens, which seems to be altered in both pediatric and adult asthmatics. Impaired IFN production leaves asthmatics susceptible to infection and with uncontrolled type 2 immunity, promotes airway hyperresponsiveness (AHR), and inflammation which can lead to asthma exacerbations. However, IFN deficiency is not observed in all asthmatics, and alterations in IFN expression may be independent of type 2 immunity. In this review, we discuss the link between type I and III IFNs and asthma both in general and in specific contexts, including during viral infection, co-infection, and bacterial/fungal infection. We also highlight several studies which examine the potential role for type I and III IFNs as asthma-related therapies.

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.

Pulmonary infection is a leading cause of hospitalization in world. Lung damage due to infection and host mediated pathology can have life threatening consequences. Factors that limit lung injury and/or promote epithelial barrier function and repair are highly desirable as immunomodulatory therapeutics. Over the last decade, interleukin-22 has been shown to have pulmonary epithelial protective functions at the mucosal immune interface with bacterial and viral pathogens. This article summarizes recent findings in this area and provides perspective regarding the role of IL-22 in mucosal host defense.

Rationale: The role of FSTL-1 (follistatin-like 1) in lung homeostasis is unknown. Objectives: We aimed to define the impact of FSTL-1 attenuation on lung structure and function and to identify FSTL-1–regulated transcriptional pathways in the lung. Further, we aimed to analyze the association of FSTL-1 SNPs with lung disease. Methods: FSTL-1 hypomorphic (FSTL-1 Hypo) mice underwent lung morphometry, pulmonary function testing, and micro–computed tomography. Fstl1 expression was determined in wild-type lung cell populations from three independent research groups. RNA sequencing of wild-type and FSTL-1 Hypo mice identified FSTL-1–regulated gene expression, followed by validation and mechanistic in vitro examination. FSTL1 SNP analysis was performed in the COPDGene (Genetic Epidemiology of Chronic Obstructive Pulmonary Disease) cohort. Measurements and Main Results: FSTL-1 Hypo mice developed spontaneous emphysema, independent of smoke exposure. Fstl1 is highly expressed in the lung by mesenchymal and endothelial cells but not immune cells. RNA sequencing of whole lung identified 33 FSTL-1–regulated genes, including Nr4a1, an orphan nuclear hormone receptor that negatively regulates NF-κB (nuclear factor-κB) signaling. In vitro, recombinant FSTL-1 treatment of macrophages attenuated NF-κB p65 phosphorylation in an Nr4a1-dependent manner. Within the COPDGene cohort, several SNPs in the FSTL1 region corresponded to chronic obstructive pulmonary disease and lung function. Conclusions: This work identifies a novel role for FSTL-1 protecting against emphysema development independent of smoke exposure. This FSTL-1–deficient emphysema implicates regulation of immune tolerance in lung macrophages through Nr4a1. Further study of the mechanisms involving FSTL-1 in lung homeostasis, immune regulation, and NF-κB signaling may provide additional insight into the pathophysiology of emphysema and inflammatory lung diseases.

© 2020 van der Velden et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The stress-induced kinase, c-Jun-N-terminal kinase 1 (JNK1) has previously been implicated in the pathogenesis of lung fibrosis. However, the exact cell type(s) wherein JNK1 exerts its pro-fibrotic role(s) remained enigmatic. Herein we demonstrate prominent activation of JNK in bronchial epithelia using the mouse models of bleomycin- or AdTGFβ1-induced fibrosis. Furthermore, in lung tissues of patients with idiopathic pulmonary fibrosis (IPF), active JNK was observed in various regions including type I and type II pneumocytes and fibroblasts. No JNK activity was observed in adjacent normal tissue or in normal control tissue. To address the role of epithelial JNK1, we ablated Jnk1 form bronchiolar and alveolar type II epithelial cells using CCSP-directed Cre recombinase-mediated ablation of LoxP-flanked Jnk1 alleles. Our results demonstrate that ablation of Jnk1 from airway epithelia resulted in a strong protection from bleomycin- or adenovirus expressing active transforming growth factor beta-1 (AdTGFβ1)-induced fibrosis. Ablation of the Jnk1 allele at a time when collagen increases were already present showed a reversal of existing increases in collagen content. Epithelial Jnk1 ablation resulted in attenuation of mesenchymal genes and proteins in lung tissue and preserved expression of epithelial genes. Collectively, these data suggest that epithelial JNK1 contributes to the pathogenesis of pulmonary fibrosis. Given the presence of active JNK in lungs from patients with IPF, targeting JNK1 in airway epithelia may represent a potential treatment strategy to combat this devastating disease.

We previously showed that the mitochondrial fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD) is expressed in alveolar type II pneumocytes and that LCAD-/- mice have altered breathing mechanics and surfactant defects. Here, we hypothesized that LCAD-/- mice would be susceptible to influenza infection. Indeed, LCAD-/- mice demonstrated increased mortality following infection with 2009 pandemic influenza (A/CA/07/09). However, the mortality was not due to increased lung injury, as inflammatory cell counts, viral titers, and histology scores all showed non-significant trends toward milder injury in LCAD-/- mice. To confirm this, LCAD-/- were infected with a second, mouse-adapted H1N1 virus (A/PR/8/34), to which they responded with significantly less lung injury. While both strains become increasingly hypoglycemic over the first week post-infection, LCAD-/- mice lose body weight more rapidly than wild-type mice. Surprisingly, while acutely fasted LCAD-/- mice develop hepatic steatosis, influenza-infected LCAD-/- mice do not. They do, however, become more hypothermic than wild-type mice and demonstrate increased blood lactate values. We conclude that LCAD-/- mice succumb to influenza from bioenergetic starvation, likely due to increased reliance upon glucose for energy.

Calprotectin is a heterodimer of the proteins S100A8 and S100A9, and it is an abundant innate immune protein associated with inflammation. In humans, calprotectin transcription and protein abundance are associated with asthma and disease severity. However, mechanistic studies in experimental asthma models have been inconclusive, identifying both protective and pathogenic effects of calprotectin. To clarify the role of calprotectin in asthma, calprotectin-deficient S100A9(−/−) and wild-type (WT) C57BL/6 mice were compared in a murine model of allergic airway inflammation. Mice were intranasally challenged with extracts of the clinically relevant allergen, Alternaria alternata (Alt Ext), or PBS every third day over 9 days. On Day 10, BAL fluid and lung tissue homogenates were harvested and allergic airway inflammation was assessed. Alt Ext challenge induced release of S100A8/S100A9 to the alveolar space and increased protein expression in the alveolar epithelium of WT mice. Compared with WT mice, S100A9(−/−) mice displayed significantly enhanced allergic airway inflammation, including production of IL-13, CCL11, CCL24, serum IgE, eosinophil recruitment, and airway resistance and elastance. In response to Alt Ext, S100A9(−/−) mice accumulated significantly more IL-13(+)IL-5(+)CD4(+) T-helper type 2 cells. S100A9(−/−) mice also accumulated a significantly lower proportion of CD4(+) T regulatory (Treg) cells in the lung that had significantly lower expression of CD25. Calprotectin enhanced WT Treg cell suppressive activity in vitro. Therefore, this study identifies a role for the innate immune protein, S100A9, in protection from CD4(+) T-helper type 2 cell hyperinflammation in response to Alt Ext. This protection is mediated, at least in part, by CD4(+) Treg cell function.

Childhood asthma is a complex disease. In this study, we aim to identify genes associated with childhood asthma through a multiomics "vertical" approach that integrates multiple analytical steps using linear and logistic regression models. In a case-control study of childhood asthma in Puerto Ricans (n = 1,127), we used adjusted linear or logistic regression models to evaluate associations between several analytical steps of omics data, including genome-wide (GW) genotype data, GW methylation, GW expression profiling, cytokine levels, asthma-intermediate phenotypes, and asthma status. At each point, only the top genes/single-nucleotide polymorphisms/probes/cytokines were carried forward for subsequent analysis. In step 1, asthma modified the gene expression-protein level association for 1,645 genes; pathway analysis showed an enrichment of these genes in the cytokine signaling system (n = 269 genes). In steps 2-3, expression levels of 40 genes were associated with intermediate phenotypes (asthma onset age, forced expiratory volume in 1 second, exacerbations, eosinophil counts, and skin test reactivity); of those, methylation of seven genes was also associated with asthma. Of these seven candidate genes, IL5RA was also significant in analytical steps 4-8. We then measured plasma IL-5 receptor α levels, which were associated with asthma age of onset and moderate-severe exacerbations. In addition, in silico database analysis showed that several of our identified IL5RA single-nucleotide polymorphisms are associated with transcription factors related to asthma and atopy. This approach integrates several analytical steps and is able to identify biologically relevant asthma-related genes, such as IL5RA. It differs from other methods that rely on complex statistical models with various assumptions.

Influenza is an annual, global healthcare concern. Secondary bacterial pneumonia is a severe complication associated with primary influenza virus infection that often results in critical morbidity and mortality. We have identified influenza-induced suppression of antibacterial type 17 immunity as a mechanism for enhanced susceptibility to bacterial superinfection. We have shown that influenza-induced type I IFN impairs type 17 activation. STAT1 is a transcription factor involved in IFN signaling that is shared by types I, II, and III IFN. In this study, we investigated the role of STAT1 signaling during influenza and methicillin-resistant Staphylococcus aureus superinfection. STAT1−/− mice had increased morbidity and airway inflammation compared with control mice during influenza monoinfection. Despite this worsened antiviral response, STAT1−/− mice were protected from superinfection bacterial burden and mortality compared with controls. Type 17 immune activation was increased in lymphocytes in STAT1−/− mice during superinfection. The elevation in type 17 immunity was not related to increased IL-23 production, because type I IFN could inhibit IL-23 expression in a STAT1-independent manner. STAT1−/− APCs were inherently biased toward type 17 polarization compared with control cells. Further, STAT1−/− dendritic cells produced attenuated IL-6 and TNF-α upon heat-killed S. aureus stimulation compared with control. Overall, these data indicate that STAT1 signaling plays a detrimental role in influenza and methicillin-resistant Staphylococcus aureus superinfection by controlling the magnitude of type 17 immune activation.

Approximately 2 billion people are infected with Mycobacterium tuberculosis (Mtb), resulting in 1.4 million deaths every year. Among Mtb-infected individuals, clinical isolates belonging to the W-Beijing lineage are increasingly prevalent, associated with drug resistance, and cause severe disease immunopathology in animal models. Therefore, it is exceedingly important to identify the immune mechanisms that mediate protection against rapidly emerging Mtb strains, such as W-Beijing lineage. IL-22 is a member of the IL-10 family of cytokines with both protective and pathological functions at mucosal surfaces. Thus far, collective data show that IL-22 deficient mice are not more susceptible to aerosolized infection with less virulent Mtb strains. Thus, in this study we addressed the functional role for the IL-22 pathway in immunity to emerging Mtb isolates, using W-Beijing lineage member, Mtb HN878 as a prototype. We show that Mtb HN878 stimulates IL-22 production in TLR2 dependent manner and IL-22 mediates protective immunity during chronic stages of Mtb HN878 infection in mice. Interestingly, IL-22-dependent pathways in both epithelial cells and macrophages mediate protective mechanisms for Mtb HN878 control. Thus, our results project a new protective role for IL-22 in emerging Mtb infections.

The absent in melanoma 2 (AIM2) inflammasome plays an important role in many viral and bacterial infections, but very little is known about its role in RNA virus infection, including influenza A virus (IAV). In this study, we have designed in vivo and in vitro studies to determine the role of AIM2 in infections with lethal doses of IAVs A/PR8/34 and A/California/07/09. In wild-type mice, IAV infection enhanced AIM2 expression, induced dsDNA release, and stimulated caspase-1 activation and release of cleaved IL-1β in the lung, which was significantly reduced in AIM2-deficient mice. Interestingly, AIM2 deficiency did not affect the transcription of caspase-1 and IL-1β. In addition, AIM2-deficient mice exhibited attenuated lung injury and significantly improved survival against IAV challenges, but did not alter viral burden in the lung. However, AIM2 deficiency did not seem to affect adaptive immune response against IAV infections. Furthermore, experiments with AIM2-specific small interfering RNA–treated and AIM2-deficient human and mouse lung alveolar macrophages and type II cells indicated a macrophage-specific function of AIM2 in regulation of IAV-stimulated proinflammatory response. Collectively, our results demonstrate that influenza infection activates the AIM2 inflammasome, which plays a critical role in IAV-induced lung injury and mortality. AIM2 might serve as a therapeutic target for combating influenza-associated morbidity and mortality without compromising the host antiviral responses.

Influenza A is a highly contagious respiratory virus that causes seasonal epidemics and occasional worldwide pandemics. The primary cause of influenza-related mortality is bacterial superinfection. There are numerous mechanisms by which preceding influenza infection attenuates host defense, allowing for increased susceptibility to bacterial pneumonia. Herein, we demonstrate that influenza inhibits Staphylococcus aureus-induced production of interleukin-33 (IL-33). Restoration of IL-33 during influenza A and methicillin-resistant S. aureus superinfection enhanced bacterial clearance and improved mortality. Innate lymphoid Type 2 cells and alternatively activated macrophages are not required for IL-33-mediated protection during superinfection. We show that IL-33 treatment resulted in neutrophil recruitment to the lung, associated with improved bacterial clearance. These findings identify a novel role for IL-33 in antibacterial host defense at the mucosal barrier.

International audience; no abstract

Background Influenza attacks the epithelium of the lung, causing cell death and disruption of the epithelial barrier leading to fluid buildup in the lung and impairment of gas exchange. Limited treatment options for severe influenza pneumonia prioritize the need for the discovery of effective therapies. IL-22 is a cytokine that promotes tissue integrity and has strong promise as a treatment option. While research has been focused on the cytokine itself, there is limited understanding of the regulation of the IL-22 receptor (IL-22Ra1) at the epithelial surface during infection. Methods IL-22Ra1 levels were measured by qRT-PCR, western blot and immunofluorescence following H1N1 influenza infection (A/PR/8/34 H1N1) or synthetic TLR3 mimetic, Poly (I:C). Regulation of the receptor was determined using STAT inhibitors (STAT1, STAT3 and PanSTAT inhibitors), TLR3 inhibition, and neutralization of interferon alpha receptor 2 (IFNAR2). Significance was determined by a p-value of greater than 0.05. Significance between two groups was measured using unpaired t-test and significance between more than two groups was measured using one-way ANOVA with Tukey Multiple Comparison Test. Results Here we show both in vivo and in vitro that IL-22Ra1 was induced as early as 24 h after influenza (H1N1 PR8) infection. This induction was triggered by toll-like receptor 3 (TLR3) as a TLR3 mimetic [Poly (I:C)] also induced IL-22Ra1 and inhibition of endosomal formation required for TLR3 function inhibited this process. This upregulation was dependent upon IFNβ signaling through STAT1. Importantly, induction of IL-22Ra1 significantly increased IL-22 signaling as evidenced by pSTAT3 levels following IL-22 treatment. Conclusion Collectively, these data suggest epithelial cells may optimize the beneficial effects of IL-22 through the induction of the IL-22 receptor during viral infection in the lung.