Macrophage CD14 impacts immune defenses against influenza virus in allergic hosts.

Asthma and influenza are leading causes of worldwide morbidity and mortality. Although these two conditions can co-exist in the same patient, the immune parameters that impact disease outcomes are not fully elucidated. The importance of macrophages to both conditions suggested a role for CD14, a co-receptor for endotoxin, as a regulatory mechanism for innate immune responses during asthma and influenza co-morbidity. Herein, we hypothesized that parameters of influenza morbidity will be reduced in the absence of CD14. Age and gender matched wild-type (WT) and CD14 knock-out (KO) mice were subjected to our validated model of Aspergillus-induced model of asthma and/or influenza. Characteristics of disease pathogenesis were investigated using standard methods in weight loss, flow cytometry, airway resistance, histology, quantitative real-time PCR, and viral titer quantification. The absence of CD14 did not have an impact on morbidity as these mice were equally susceptible to disease with similar airway resistance. Peribronchovascular inflammation and goblet cell content were equivalent between WT and KO mice in asthma alone and asthma and influenza co-morbidity. Co-morbid KO mice had less lymphocytes and eosinophils in the airways although their lung viral burden was equivalent to WT. Inflammatory gene signatures were altered in co-morbid mice in each genotype. CD14 expression on macrophages is necessary for airway inflammation but not for viral pathogenesis in allergic hosts.

Eosinophils: Nemeses of Pulmonary Pathogens?

PURPOSE OF REVIEW: Eosinophils are short-lived granulocytes that contain a variety of proteins and lipids traditionally associated with host defense against parasites. The primary goal of this review is to examine more recent evidence that challenged this rather outdated role of eosinophils in the context of pulmonary infections with helminths, viruses, and bacteria. RECENT FINDINGS: While eosinophil mechanisms that counter parasites, viruses, and bacteria are similar, the kinetics and impact may differ by pathogen type. Major antiparasitic responses include direct killing and immunoregulation, as well as some mechanisms by which parasite survival/growth is supported. Antiviral defenses may be as unembellished as granule protein-induced direct killing or more urbane as serving as a conduit for better adaptive immune responses to the invading virus. Although sacrificial, eosinophil DNA emitted in response to bacteria helps trap bacteria to limit dissemination. Herein, we discuss the current research redefining eosinophils as multifunctional cells that are active participants in host defense against lung pathogens. Eosinophils recognize and differentially respond to invading pathogens, allowing them to deploy innate defense mechanisms to contain and clear the infection, or modulate the immune response. Modern technology and animal models have unraveled hitherto unknown capabilities of this surreptitious cell that indubitably has more functions awaiting discovery.

Eosinophils Promote Antiviral Immunity in Mice Infected with Influenza A Virus.

Eosinophils are multifunctional cells of the innate immune system linked to allergic inflammation. Asthmatics were more likely to be hospitalized but less likely to suffer severe morbidity and mortality during the 2009 influenza pandemic. These epidemiologic findings were recapitulated in a mouse model of fungal asthma wherein infection during heightened allergic inflammation was protective against influenza A virus (IAV) infection and disease. Our goal was to delineate a mechanism(s) by which allergic asthma may alleviate influenza disease outcome, focused on the hypothesis that pulmonary eosinophilia linked with allergic respiratory disease is able to promote antiviral host defenses against the influenza virus. The transfer of eosinophils from the lungs of allergen-sensitized and challenged mice into influenza virus-infected mice resulted in reduced morbidity and viral burden, improved lung compliance, and increased CD8+ T cell numbers in the airways. In vitro assays with primary or bone marrow-derived eosinophils were used to determine eosinophil responses to the virus using the laboratory strain (A/PR/08/1934) or the pandemic strain (A/CA/04/2009) of IAV. Eosinophils were susceptible to IAV infection and responded by activation, piecemeal degranulation, and upregulation of Ag presentation markers. Virus- or viral peptide-exposed eosinophils induced CD8+ T cell proliferation, activation, and effector functions. Our data suggest that eosinophils promote host cellular immunity to reduce influenza virus replication in lungs, thereby providing a novel mechanism by which hosts with allergic asthma may be protected from influenza morbidity.

Immune responses to fungal aeroallergen in Heligmosomoides polygyrus-infected mice vary by age.

Parasite infections in the developing world have been considered to promote resistance to immune-mediated diseases such as asthma. Mouse studies have shown that helminths and their products reduce the development of allergic asthma. Since epidemiologic studies that show similar protection are in relation to geohelminth infections that occur in early life, we hypothesized that the parasite-mediated protection against asthma may differ by age. Mice infected with Heligmosomoides polygyrus at 3-weeks of age had similar asthma phenotype compared to mice infected at 28-weeks of age wherein airway eosinophilia was unaltered but tissue inflammation and GC metaplasia were reduced. In contrast, mice infected at 18-weeks of age had elevated macrophagic airway inflammation with accompanying tissue pathology. The presence of γδ T cells and Treg cells in the airways was also regulated by age at worm infection. Our findings demonstrate the importance of age in immune responses that may regulate gut and lung diseases.

A novel cytotoxic sequence contributes to influenza A viral protein PB1-F2 pathogenicity and predisposition to secondary bacterial infection.

Enhancement of cell death is a distinguishing feature of H1N1 influenza virus A/Puerto Rico/8/34 protein PB1-F2. Comparing the sequences (amino acids [aa] 61 to 87 using PB1-F2 amino acid numbering) of the PB1-F2-derived C-terminal peptides from influenza A viruses inducing high or low levels of cell death, we identified a unique I68, L69, and V70 motif in A/Puerto Rico/8/34 PB1-F2 responsible for promotion of the peptide's cytotoxicity and permeabilization of the mitochondrial membrane. When administered to mice, a 27-mer PB1-F2-derived C-terminal peptide with this amino acid motif caused significantly greater weight loss and pulmonary inflammation than the peptide without it (due to I68T, L69Q, and V70G mutations). Similar to the wild-type peptide, A/Puerto Rico/8/34 elicited significantly higher levels of macrophages, neutrophils, and cytokines in the bronchoalveolar lavage fluid of mice than its mutant counterpart 7 days after infection. Additionally, infection of mice with A/Puerto Rico/8/34 significantly enhanced the levels of morphologically transformed epithelial and immune mononuclear cells recruited in the airways compared with the mutant virus. In the mouse bacterial superinfection model, both peptide and virus with the I68, L69, and V70 sequence accelerated development of pneumococcal pneumonia, as reflected by increased levels of viral and bacterial lung titers and by greater mortality. Here we provide evidence suggesting that the newly identified cytotoxic sequence I68, L69, and V70 of A/Puerto Rico/8/34 PB1-F2 contributes to the pathogenesis of both primary viral and secondary bacterial infections.

The immune profile associated with acute allergic asthma accelerates clearance of influenza virus.

Asthma was the most common comorbidity in hospitalized patients during the 2009 influenza pandemic. For unknown reasons, hospitalized asthmatics had less severe outcomes and were less likely to die from pandemic influenza. Our data with primary human bronchial cells indicate that changes intrinsic to epithelial cells in asthma may protect against cytopathology induced by influenza virus. To further study influenza virus pathogenesis in allergic hosts, we aimed to develop and characterize murine models of asthma and influenza comorbidity to determine structural, physiological and immunological changes induced by influenza in the context of asthma. Aspergillus fumigatus-sensitized and -challenged C57BL/6 mice were infected with pandemic H1N1 influenza virus, either during peak allergic inflammation or during airway remodeling to gain insight into disease pathogenesis. Mice infected with the influenza virus during peak allergic inflammation did not lose body weight and cleared the virus rapidly. These mice exhibited high eosinophilia, preserved airway epithelial cell integrity, increased mucus, reduced interferon response and increased insulin-like growth factor-1. In contrast, weight loss and viral replication kinetics in the mice that were infected during the late airway remodeling phase were equivalent to flu-only controls. These mice had neutrophils in the airways, damaged airway epithelial cells, less mucus production, increased interferons and decreased insulin-like growth factor-1. The state of the allergic airways at the time of influenza virus infection alters host responses against the virus. These murine models of asthma and influenza comorbidity may improve our understanding of the epidemiology and pathogenesis of viral infections in humans with asthma.

Hyaluronan deposition and co-localization with inflammatory cells and collagen in a murine model of fungal allergic asthma.

OBJECTIVE: Allergic asthma is a chronic inflammatory disease of the airways characterized by excessive inflammation and remodeling of the extracellular matrix (ECM) and associated cells of the airway wall. Under inflammatory conditions, hyaluronan (HA), a major component of the ECM, undergoes dynamic changes, which may in turn affect the recruitment and activation of inflammatory cells leading to acute and chronic immunopathology of allergic asthma. METHODS: In the present study, we measured the changes in HA levels generated at sites of inflammation, and examined its effect on inflammatory responses and collagen deposition in an Aspergillus fumigatus murine inhalational model of allergic asthma. RESULTS: We found that HA levels are elevated in allergic animals and that the increase correlated with the influx of inflammatory cells 5 days after the second allergen challenge. This increase in HA levels appeared largely due to upregulation of hyaluronidase-1 (HYAL1) and hyaluronidase-2 (HYAL2). Furthermore, HA co-localizes with areas of new collagen synthesis and deposition. CONCLUSIONS: Overall, our findings contribute to the growing literature that focuses on the components of ECM as inflammatory mediators rather than mere structural support products. The evidence of HA localization in fungal allergic asthma provides the impetus to study HA more closely with allergic leukocytes in murine models. Further studies examining HA's role in mediating cellular responses may help to develop targets for treatment in patients with severe asthma due to fungal sensitization.

Eosinophils as Modulators of Host Defense during Parasitic, Fungal, Bacterial, and Viral Infections.

Eosinophils, traditionally associated as central innate effector cells with type-2 immunity during allergic and helminth parasitic diseases, have recently been revealed to have important roles in tissue homeostasis as well as host defense in a broader variety of infectious diseases. In a dedicated session at the 2023 biennial conference of the International Eosinophil Society titled "Eosinophils in Host Defense", the multifaceted roles eosinophils play against diverse pathogens ranging from parasites to fungi, bacteria, and viruses was presented. In this review, the session speakers offer a comprehensive summary of recent discoveries across pathogen classes, positioning eosinophils as pivotal leukocytes in both host defense and pathology. By unraveling the intricacies of eosinophil engagement in host resistance, this exploration may provide valuable insights not only to understand specific underpinnings of the eosinophil functions related to each class of pathogens, but also to develop novel therapeutics effective against a broad spectrum of infectious diseases.

Association between diabetic status and risk of all-cause and cause-specific mortality on dialysis following first kidney allograft loss.

Background: Diabetes mellitus (DM) is associated with a greater risk of mortality in kidney transplant patients, primarily driven by a greater risk of cardiovascular disease (CVD)-related mortality. However, the associations between diabetes status at time of first allograft loss and mortality on dialysis remain unknown. Methods: All patients with failed first kidney allografts transplanted in Australia and New Zealand between 2000 and 2020 were included. The associations between diabetes status at first allograft loss, all-cause and cause-specific mortality were examined using competing risk analyses, separating patients with diabetes into those with pre-transplant DM or post-transplant diabetes mellitus (PTDM). Results: Of 3782 patients with a median (IQR) follow-up duration of 2.7 (1.1-5.4) years, 539 (14%) and 390 (10%) patients had pre-transplant DM or developed PTDM, respectively. In the follow-up period, 1336 (35%) patients died, with 424 (32%), 264 (20%) and 199 (15%) deaths attributed to CVD, dialysis withdrawal and infection, respectively. Compared to patients without DM, the adjusted subdistribution HRs (95% CI) for pre-transplant DM and PTDM for all-cause mortality on dialysis were 1.47 (1.17-1.84) and 1.47 (1.23-1.76), respectively; for CVD-related mortality were 0.81 (0.51-1.29) and 1.02 (0.70-1.47), respectively; for infection-related mortality were 1.84 (1.02-3.35) and 2.70 (1.73-4.20), respectively; and for dialysis withdrawal-related mortality were 1.71 (1.05-2.77) and 1.51 (1.02-2.22), respectively. Conclusions: Patients with diabetes at the time of kidney allograft loss have a significant survival disadvantage, with the excess mortality risk attributed to infection and dialysis withdrawal.

Postinfectious Pulmonary Complications: Establishing Research Priorities to Advance the Field: An Official American Thoracic Society Workshop Report.

Continued improvements in the treatment of pulmonary infections have paradoxically resulted in a growing challenge of individuals with postinfectious pulmonary complications (PIPCs). PIPCs have been long recognized after tuberculosis, but recent experiences such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic have underscored the importance of PIPCs following other lower respiratory tract infections. Independent of the causative pathogen, most available studies of pulmonary infections focus on short-term outcomes rather than long-term morbidity among survivors. In this document, we establish a conceptual scope for PIPCs with discussion of globally significant pulmonary pathogens and an examination of how these pathogens can damage different components of the lung, resulting in a spectrum of PIPCs. We also review potential mechanisms for the transition from acute infection to PIPC, including the interplay between pathogen-mediated injury and aberrant host responses, which together result in PIPCs. Finally, we identify cross-cutting research priorities for the field to facilitate future studies to establish the incidence of PIPCs, define common mechanisms, identify therapeutic strategies, and ultimately reduce the burden of morbidity in survivors of pulmonary infections.

Host mitochondria: more than an organelle in SARS-CoV-2 infection.

Since December 2019, the world has been facing viral pandemic called COVID-19 (Coronavirus disease 2019) caused by a new beta-coronavirus named severe acute respiratory syndrome coronavirus-2, or SARS-CoV-2. COVID-19 patients may present with a wide range of symptoms, from asymptomatic to requiring intensive care support. The severe form of COVID-19 is often marked by an altered immune response and cytokine storm. Advanced age, age-related and underlying diseases, including metabolic syndromes, appear to contribute to increased COVID-19 severity and mortality suggesting a role for mitochondria in disease pathogenesis. Furthermore, since the immune system is associated with mitochondria and its damage-related molecular patterns (mtDAMPs), the host mitochondrial system may play an important role during viral infections. Viruses have evolved to modulate the immune system and mitochondrial function for survival and proliferation, which in turn could lead to cellular stress and contribute to disease progression. Recent studies have focused on the possible roles of mitochondria in SARS-CoV-2 infection. It has been suggested that mitochondrial hijacking by SARS-CoV-2 could be a key factor in COVID-19 pathogenesis. In this review, we discuss the roles of mitochondria in viral infections including SARS-CoV-2 infection based on past and present knowledge. Paying attention to the role of mitochondria in SARS-CoV-2 infection will help to better understand the pathophysiology of COVID-19 and to achieve effective methods of prevention, diagnosis, and treatment.

Dissecting Phenotype from Genotype with Clinical Isolates of SARS-CoV-2 First Wave Variants.

The emergence and availability of closely related clinical isolates of SARS-CoV-2 offers a unique opportunity to identify novel nonsynonymous mutations that may impact phenotype. Global sequencing efforts show that SARS-CoV-2 variants have emerged and then been replaced since the beginning of the pandemic, yet we have limited information regarding the breadth of variant-specific host responses. Using primary cell cultures and the K18-hACE2 mouse, we investigated the replication, innate immune response, and pathology of closely related, clinical variants circulating during the first wave of the pandemic. Mathematical modeling of the lung viral replication of four clinical isolates showed a dichotomy between two B.1. isolates with significantly faster and slower infected cell clearance rates, respectively. While isolates induced several common immune host responses to infection, one B.1 isolate was unique in the promotion of eosinophil-associated proteins IL-5 and CCL11. Moreover, its mortality rate was significantly slower. Lung microscopic histopathology suggested further phenotypic divergence among the five isolates showing three distinct sets of phenotypes: (i) consolidation, alveolar hemorrhage, and inflammation, (ii) interstitial inflammation/septal thickening and peribronchiolar/perivascular lymphoid cells, and (iii) consolidation, alveolar involvement, and endothelial hypertrophy/margination. Together these findings show divergence in the phenotypic outcomes of these clinical isolates and reveal the potential importance of nonsynonymous mutations in nsp2 and ORF8.

Impact of Therapeutics on Unified Immunity During Allergic Asthma and Respiratory Infections.

Asthma is a common chronic respiratory disease that affects millions of people worldwide. Patients with allergic asthma, the most prevalent asthma endotype, are widely considered to possess a defective immune response against some respiratory infectious agents, including viruses, bacteria and fungi. Furthermore, respiratory pathogens are associated with asthma development and exacerbations. However, growing data suggest that the immune milieu in allergic asthma may be beneficial during certain respiratory infections. Immunomodulatory asthma treatments, although beneficial, should then be carefully prescribed to avoid misuse and overuse as they can also alter the host microbiome. In this review, we summarize and discuss recent evidence of the correlations between allergic asthma and the most significant respiratory infectious agents that have a role in asthma pathogenesis. We also discuss the implications of current asthma therapeutics beyond symptom prevention.

Human matters in asthma: Considering the microbiome in pulmonary health.

Microbial communities form an important symbiotic ecosystem within humans and have direct effects on health and well-being. Numerous exogenous factors including airborne triggers, diet, and drugs impact these established, but fragile communities across the human lifespan. Crosstalk between the mucosal microbiota and the immune system as well as the gut-lung axis have direct correlations to immune bias that may promote chronic diseases like asthma. Asthma initiation and pathogenesis are multifaceted and complex with input from genetic, epigenetic, and environmental components. In this review, we summarize and discuss the role of the airway microbiome in asthma, and how the environment, diet and therapeutics impact this low biomass community of microorganisms. We also focus this review on the pediatric and Black populations as high-risk groups requiring special attention, emphasizing that the whole patient must be considered during treatment. Although new culture-independent techniques have been developed and are more accessible to researchers, the exact contribution the airway microbiome makes in asthma pathogenesis is not well understood. Understanding how the airway microbiome, as a living entity in the respiratory tract, participates in lung immunity during the development and progression of asthma may lead to critical new treatments for asthma, including population-targeted interventions, or even more effective administration of currently available therapeutics.

Mitochondrial Population in Mouse Eosinophils: Ultrastructural Dynamics in Cell Differentiation and Inflammatory Diseases.

Mitochondria are multifunctional organelles of which ultrastructure is tightly linked to cell physiology. Accumulating evidence shows that mitochondrial remodeling has an impact on immune responses, but our current understanding of the mitochondrial architecture, interactions, and morphological changes in immune cells, mainly in eosinophils, is still poorly known. Here, we applied transmission electron microscopy (TEM), single-cell imaging analysis, and electron tomography, a technique that provides three-dimensional (3D) views at high resolution, to investigate mitochondrial dynamics in mouse eosinophils developing in cultures as well as in the context of inflammatory diseases characterized by recruitment and activation of these cells (mouse models of asthma, H1N1 influenza A virus (IAV) infection, and schistosomiasis mansoni). First, quantitative analyses showed that the mitochondrial area decrease 70% during eosinophil development (from undifferentiated precursor cells to mature eosinophils). Mitophagy, a consistent process revealed by TEM in immature but not in mature eosinophils, is likely operating in mitochondrial clearance during eosinophilopoiesis. Events of mitochondria interaction (inter-organelle membrane contacts) were also detected and quantitated within developing eosinophils and included mitochondria-endoplasmic reticulum, mitochondria-mitochondria, and mitochondria-secretory granules, all of them significantly higher in numbers in immature compared to mature cells. Moreover, single-mitochondrion analyses revealed that as the eosinophil matures, mitochondria cristae significantly increase in number and reshape to lamellar morphology. Eosinophils did not change (asthma) or reduced (IAV and Schistosoma infections) their mitochondrial mass in response to inflammatory diseases. However, asthma and schistosomiasis, but not IAV infection, induced amplification of both cristae numbers and volume in individual mitochondria. Mitochondrial cristae remodeling occurred in all inflammatory conditions with the proportions of mitochondria containing only lamellar or tubular, or mixed cristae (an ultrastructural aspect seen just in tissue eosinophils) depending on the tissue/disease microenvironment. The ability of mitochondria to interact with granules, mainly mobilized ones, was remarkably captured by TEM in eosinophils participating in all inflammatory diseases. Altogether, we demonstrate that the processes of eosinophilopoiesis and inflammation-induced activation interfere with the mitochondrial dynamics within mouse eosinophils leading to cristae remodeling and inter-organelle contacts. The understanding of how mitochondrial dynamics contribute to eosinophil immune functions is an open interesting field to be explored.

Initiation and Pathogenesis of Severe Asthma with Fungal Sensitization.

Fungi represent one of the most diverse and abundant eukaryotes on earth, and their ubiquity and small proteolytically active products make them pervasive allergens that affect humans and other mammals. The immunologic parameters surrounding fungal allergies are still not fully elucidated despite their importance given that a large proportion of severe asthmatics are sensitized to fungal allergens. Herein, we explore fungal allergic asthma with emphasis on mouse models that recapitulate the characteristics of human disease, and the main leukocyte players in the pathogenesis of fungal allergies. The endogenous mycobiome may also contribute to fungal asthma, a phenomenon that we discuss only superficially, as much remains to be discovered.

Murine Models of Eosinophil Function in Fungal and Viral Infections.

Eosinophils are granulocytes that were historically considered to be terminally differentiated at the time of bone marrow egress. However, more recent evidence provides a new outlook on these cells as complex immunomodulators that are involved in host defense and homeostasis. Our work established a role for eosinophils as mediators of antiviral immune responses during influenza in hosts that were sensitized and challenged with fungal allergens. Herein, we describe methods for working with murine eosinophils in the context of influenza A virus.

Eosinophil Responses at the Airway Epithelial Barrier during the Early Phase of Influenza A Virus Infection in C57BL/6 Mice.

Eosinophils, previously considered terminally differentiated effector cells, have multifaceted functions in tissues. We previously found that allergic mice with eosinophil-rich inflammation were protected from severe influenza and discovered specialized antiviral effector functions for eosinophils including promoting cellular immunity during influenza. In this study, we hypothesized that eosinophil responses during the early phase of influenza contribute to host protection. Using in vitro and in vivo models, we found that eosinophils were rapidly and dynamically regulated upon influenza A virus (IAV) exposure to gain migratory capabilities to traffic to lymphoid organs after pulmonary infection. Eosinophils were capable of neutralizing virus upon contact and combinations of eosinophil granule proteins reduced virus infectivity through hemagglutinin inactivation. Bi-directional crosstalk between IAV-exposed epithelial cells and eosinophils occurred after IAV infection and cross-regulation promoted barrier responses to improve antiviral defenses in airway epithelial cells. Direct interactions between eosinophils and airway epithelial cells after IAV infection prevented virus-induced cytopathology in airway epithelial cells in vitro, and eosinophil recipient IAV-infected mice also maintained normal airway epithelial cell morphology. Our data suggest that eosinophils are important in the early phase of IAV infection providing immediate protection to the epithelial barrier until adaptive immune responses are deployed during influenza.

An inhalation model of airway allergic response to inhalation of environmental Aspergillus fumigatus conidia in sensitized BALB/c mice.

Fungal exposure may elicit a number of pulmonary diseases in man, including allergic asthma. Fungal sensitization is linked to asthma severity, although the basis for this increased pathology remains ambiguous. To create conditions simulating environmental fungal allergen exposure in a human, nose-only inhalation delivery of Aspergillus fumigatus conidia was employed in mice previously sensitized to Aspergillus antigen extract. BALB/c mice were immunized with subcutaneous and intraperitoneal injections of soluble A. fumigatus extract in alum, which was followed by three intranasal inoculations of the same fungal antigens dissolved in saline to elicit global sensitization in a manner similar to other published models. The animals were then challenged with a 10-min inhaled dose of live conidia blown directly from the surface of a mature A. fumigatus culture. After a single challenge with inhaled A. fumigatus conidia, allergic pulmonary inflammation and airway hyperresponsiveness were significantly increased above that of either naïve animals or animals that had been sensitized to A. fumigatus antigens but not challenged with conidia. The architecture of the lung was changed by inhalation of conidia when compared to controls in that there were significant increases in epithelial thickness, goblet cell metaplasia, and peribronchial collagen deposition. Additionally, α-smooth muscle actin staining of histological sections showed visual evidence of increased peribronchial smooth muscle mass after fungal challenge. In summary, the delivery of live A. fumigatus conidia to the sensitized airways of BALB/c mice advances the study of the pulmonary response to fungi by providing a more natural route of exposure and, for the first time, demonstrates the consistent development of fibrosis and smooth muscle changes accompanying exposure to inhaled fungal conidia in a mouse model.