Magnetic resonance and computed tomographic imaging characteristics and potential molecular mechanisms of feline meningioma associated calvarial hyperostosis.

Meningiomas are the most common feline primary brain tumours, and calvarial hyperostosis (CH) is frequently documented in association with this neoplastic entity. The clinical significance of and mechanisms driving the formation of CH in cats with meningiomas are poorly understood, although tumour invasion into the skull and tumour production of cytokines and enzymes have been implicated as causes of CH in humans. This retrospective study investigated relationships between signalment, MRI or CT imaging features, histopathologic tumour characteristics, alkaline phosphatase (ALP) isoenzyme concentrations, tumour expression of matrix metalloproteinases (MMP)-2, MMP-9, and interleukin-6 (IL-6), and progression free survival times (PFS) following surgical treatment in 27 cats with meningiomas with (n = 15) or without (n = 12) evidence of CH. No significant differences in breed, age, sex, body weight, tumour grade, tumour volume, peritumoral edema burden, ALP isoenzyme concentrations, tumour Ki-67 labelling indices or MMP-2 or MMP-9 expression and activity, or PFS were noted between cats with or without CH. There was a trend towards higher serum (p = .06) and intratumoral (p = .07) concentrations of IL-6 in cats with CH, but these comparisons were not statistically significant. Histologic evidence of tumour invasion into bone was observed in 5/12 (42%) with CH and in no (0/6) cats without CH, although this was not statistically significant (p = .07). Tumour invasion into bone and tumour production of IL-6 may contribute to the formation of meningioma associated CH in cats, although larger studies are required to further substantiate these findings and determine their clinical relevance.

Rhinovirus-induced epithelial RIG-I inflammasome suppresses antiviral immunity and promotes inflammation in asthma and COVID-19.

Rhinoviruses and allergens, such as house dust mite are major agents responsible for asthma exacerbations. The influence of pre-existing airway inflammation on the infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely unknown. We analyse mechanisms of response to viral infection in experimental in vivo rhinovirus infection in healthy controls and patients with asthma, and in in vitro experiments with house dust mite, rhinovirus and SARS-CoV-2 in human primary airway epithelium. Here, we show that rhinovirus infection in patients with asthma leads to an excessive RIG-I inflammasome activation, which diminishes its accessibility for type I/III interferon responses, leading to their early functional impairment, delayed resolution, prolonged viral clearance and unresolved inflammation in vitro and in vivo. Pre-exposure to house dust mite augments this phenomenon by inflammasome priming and auxiliary inhibition of early type I/III interferon responses. Prior infection with rhinovirus followed by SARS-CoV-2 infection augments RIG-I inflammasome activation and epithelial inflammation. Timely inhibition of the epithelial RIG-I inflammasome may lead to more efficient viral clearance and lower the burden of rhinovirus and SARS-CoV-2 infections.

In vivo bronchial epithelial interferon responses are augmented in asthma on day 4 following experimental rhinovirus infection.

Despite good evidence of impaired innate antiviral responses in asthma, trials of inhaled interferon-ß given during exacerbations showed only modest benefits in moderate/severe asthma. Using human experimental rhinovirus infection, we observe robust in vivo induction of bronchial epithelial interferon response genes 4 days after virus inoculation in 25 subjects with asthma but not 11 control subjects. This signature correlated with virus loads and lower respiratory symptoms. Our data indicate that the in vivo innate antiviral response is dysregulated in asthma and open up the potential that prophylactic rather than therapeutic interferon therapy may have greater clinical benefit.

Histotripsy Ablation in Preclinical Animal Models of Cancer and Spontaneous Tumors in Veterinary Patients: A Review.

New therapeutic strategies are direly needed in the fight against cancer. Over the last decade, several tumor ablation strategies have emerged as stand-alone or combination therapies. Histotripsy is the first completely noninvasive, nonthermal, and nonionizing tumor ablation method. Histotripsy can produce consistent and rapid ablations, even near critical structures. Additional benefits include real-time image guidance, high precision, and the ability to treat tumors of any predetermined size and shape. Unfortunately, the lack of clinically and physiologically relevant preclinical cancer models is often a significant limitation with all focal tumor ablation strategies. The majority of studies testing histotripsy for cancer treatment have focused on small animal models, which have been critical in moving this field forward and will continue to be essential for providing mechanistic insight. While these small animal models have notable translational value, there are significant limitations in terms of scale and anatomical relevance. To address these limitations, a diverse range of large animal models and spontaneous tumor studies in veterinary patients have emerged to complement existing rodent models. These models and veterinary patients are excellent at providing realistic avenues for developing and testing histotripsy devices and techniques designed for future use in human patients. Here, we provide a review of animal models used in preclinical histotripsy studies and compare histotripsy ablation in these models using a series of original case reports across a broad spectrum of preclinical animal models and spontaneous tumors in veterinary patients.

miR-122 promotes virus-induced lung disease by targeting SOCS1.

Virus-induced respiratory tract infections are a major health burden in childhood, and available treatments are supportive rather than disease modifying. Rhinoviruses (RVs), the cause of approximately 80% of common colds, are detected in nearly half of all infants with bronchiolitis and the majority of children with an asthma exacerbation. Bronchiolitis in early life is a strong risk factor for the development of asthma. Here, we found that RV infection induced the expression of miRNA 122 (miR-122) in mouse lungs and in human airway epithelial cells. In vivo inhibition specifically in the lung reduced neutrophilic inflammation and CXCL2 expression, boosted innate IFN responses, and ameliorated airway hyperreactivity in the absence and in the presence of allergic lung inflammation. Inhibition of miR-122 in the lung increased the levels of suppressor of cytokine signaling 1 (SOCS1), which is an in vitro-validated target of miR-122. Importantly, gene silencing of SOCS1 in vivo completely reversed the protective effects of miR-122 inhibition on RV-induced lung disease. Higher miR-122 expression in nasopharyngeal aspirates was associated with a longer time on oxygen therapy and a higher rate of treatment failure in 87 infants hospitalized with moderately severe bronchiolitis. These results suggest that miR-122 promotes RV-induced lung disease via suppression of its target SOCS1 in vivo. Higher miR-122 expression was associated with worse clinical outcomes, highlighting the potential use of anti-miR-122 oligonucleotides, successfully trialed for treatment of hepatitis C, as potential therapeutics for RV-induced bronchiolitis and asthma exacerbations.

Rhinovirus-induced CCL17 and CCL22 in Asthma Exacerbations and Differential Regulation by STAT6.

The interplay of type-2 inflammation and antiviral immunity underpins asthma exacerbation pathogenesis. Virus infection induces type-2 inflammation-promoting chemokines CCL17 and CCL22 in asthma; however, mechanisms regulating induction are poorly understood. By using a human rhinovirus (RV) challenge model in human airway epithelial cells in vitro and mice in vivo, we assessed mechanisms regulating CCL17 and CCL22 expression. Subjects with mild to moderate asthma and healthy volunteers were experimentally infected with RV and airway CCL17 and CCL22 protein quantified. In vitro airway epithelial cell- and mouse-RV infection models were then used to define STAT6- and NF-κB-mediated regulation of CCL17 and CCL22 expression. Following RV infection, CCL17 and CCL22 expression was higher in asthma, which differentially correlated with clinical and immunological parameters. Air-liquid interface-differentiated primary epithelial cells from donors with asthma also expressed higher levels of RV-induced CCL22. RV infection boosted type-2 cytokine-induced STAT6 activation. In epithelial cells, type-2 cytokines and STAT6 activation had differential effects on chemokine expression, increasing CCL17 and suppressing CCL22, whereas NF-κB promoted expression of both chemokines. In mice, RV infection activated pulmonary STAT6, which was required for CCL17 but not CCL22 expression. STAT6-knockout mice infected with RV expressed increased levels of NF-κB-regulated chemokines, which was associated with rapid viral clearance. Therefore, RV-induced upregulation of CCL17 and CCL22 was mediated by NF-κB activation, whereas expression was differentially regulated by STAT6. Together, these findings suggest that therapeutic targeting of type-2 STAT6 activation alone will not block all inflammatory pathways during RV infection in asthma.

Low-dose 17α-ethinyl estradiol (EE) exposure exacerbates lupus renal disease and modulates immune responses to TLR7/9 agonists in genetically autoimmune-prone mice.

Estrogens have been shown to regulate the immune system and modulate multiple autoimmune diseases. 17α-ethinyl estradiol (EE), a synthetic analog of 17ß-estradiol, is prescribed commonly and found in oral contraceptives and hormone replacement therapies. Surprisingly, few studies have investigated the immunoregulatory effects of exposure to EE, especially in autoimmunity. In this study, we exposed autoimmune-prone female MRL/lpr mice to a human-relevant dose of EE through the oral route of exposure. Since lupus patients are prone to infections, groups of mice were injected with viral (Imiquimod, a TLR7 agonist) or bacterial (ODN 2395, a TLR9 agonist) surrogates. We then evaluated autoimmune disease parameters, kidney disease, and response to in vivo TLR7/9 pathogenic signals. EE-exposed mice had increased proteinuria as early as 7 weeks of age. Proteinuria, blood urea nitrogen, and glomerular immune complex deposition were also exacerbated when compared to controls. Production of cytokines by splenic leukocytes were altered in EE-exposed mice. Our study shows that oral exposure to EE, even at a very low dose, can exacerbate azotemia, increase clinical markers of renal disease, enhance glomerular immune complex deposition, and modulate TLR7/9 cytokine production in female MRL/lpr mice. This study may have implications for EE-exposure risk for genetically lupus-prone individuals.

Airway Epithelial Cells Generate Pro-inflammatory Tenascin-C and Small Extracellular Vesicles in Response to TLR3 Stimuli and Rhinovirus Infection.

Viral infections are a common cause of asthma exacerbations, with human rhinoviruses (RV) the most common trigger. RV signals through a number of different receptors, including toll-like receptor (TLR)3. Tenascin-C (TN-C) is an immunomodulatory extracellular matrix protein present in high quantities in the airway of people with asthma, and expression is also upregulated in nasal lavage fluid in response to RV infection. Respiratory viral infection has been demonstrated to induce the release of small extracellular vesicles (sEV) such as exosomes, whilst exosomal cargo can also be modified in the bronchoalveolar lavage fluid of people with asthma. These sEVs may potentiate airway inflammation and regulate the immune response to infection. This study characterizes the relationship between RV infection of bronchial epithelial cells and the release of TN-C, and the release of sEVs following stimulation with the TLR3 agonist and synthetic viral mimic, poly(I:C), as well as the function of the released protein/vesicles. The BEAS-2B airway epithelial cell line and primary human bronchial epithelial cells (PBECs) from asthmatic and non-asthmatic donors were infected with RV or treated with poly(I:C). TN-C expression, release and localization to sEVs was quantified. TN-C expression was also assessed following intra-nasal challenge of C57BL/6 mice with poly(I:C). BEAS-2B cells and macrophages were subsequently challenged with TN-C, or with sEVs generated from BEAS-2B cells pre-treated with siRNA targeted to TN-C or control. The results revealed that poly(I:C) stimulation induced TN-C release in vivo, whilst both poly(I:C) stimulation and RV infection promoted release in vitro, with elevated TN-C release from PBECs obtained from people with asthma. Poly(I:C) also induced the release of TN-C-rich sEVs from BEAS-2B cells. TN-C, and sEVs from poly(I:C) challenged cells, induced cytokine synthesis in macrophages and BEAS-2B cells, whilst sEVs from control cells did not. Moreover, sEVs with ~75% reduced TN-C content did not alter the capacity of sEVs to induce inflammation. This study identifies two novel components of the inflammatory pathway that regulates the immune response following RV infection and TLR3 stimulation, highlighting TN-C release and pro-inflammatory sEVs in the airway as relevant to the biology of virally induced exacerbations of asthma.

Bronchial mucosal IFN-α/ß and pattern recognition receptor expression in patients with experimental rhinovirus-induced asthma exacerbations.

BACKGROUND: The innate immune system senses viral infection through pattern recognition receptors (PRRs), leading to type I interferon production. The role of type I interferon and PPRs in rhinovirus-induced asthma exacerbations in vivo are uncertain. OBJECTIVES: We sought to compare bronchial mucosal type I interferon and PRR expression at baseline and after rhinovirus infection in atopic asthmatic patients and control subjects. METHODS: Immunohistochemistry was used to detect expression of IFN-α, IFN-ß, and the PRRs: Toll-like receptor 3, melanoma differentiation-associated gene 5, and retinoic acid-inducible protein I in bronchial biopsy specimens from 10 atopic asthmatic patients and 15 nonasthmatic nonatopic control subjects at baseline and on day 4 and 6 weeks after rhinovirus infection. RESULTS: We observed IFN-α/ß deficiency in the bronchial epithelium at 3 time points in asthmatic patients in vivo. Lower epithelial IFN-α/ß expression was related to greater viral load, worse airway symptoms, airway hyperresponsiveness, and reductions in lung function during rhinovirus infection. We found lower frequencies of bronchial subepithelial monocytes/macrophages expressing IFN-α/ß in asthmatic patients during infection. Interferon deficiency at baseline was not accompanied by deficient PRR expression in asthmatic patients. Both epithelial and subepithelial PRR expression were induced during rhinovirus infection. Rhinovirus infection-increased numbers of subepithelial interferon/PRR-expressing inflammatory cells were related to greater viral load, airway hyperresponsiveness, and reductions in lung function. CONCLUSIONS: Bronchial epithelial IFN-α/ß expression and numbers of subepithelial IFN-α/ß-expressing monocytes/macrophages during infection were both deficient in asthmatic patients. Lower epithelial IFN-α/ß expression was associated with adverse clinical outcomes after rhinovirus infection in vivo. Increases in numbers of subepithelial cells expressing interferon/PRRs during infection were also related to greater viral load/illness severity.

17ß-Estradiol and 17α-Ethinyl Estradiol Exhibit Immunologic and Epigenetic Regulatory Effects in NZB/WF1 Female Mice.

17α-Ethinyl estradiol (EE), a synthetic analog of natural estrogen 17ß-estradiol (E2), is extensively used in hormonal contraceptives and estrogen replacement therapy, and it has also been found in sewage effluents. Given that E2 is a well-known immunomodulator, surprisingly there has been only limited information on the cellular and molecular immunologic consequences of exposure to EE. To address this fundamental gap, we directly compared the effects of EE with E2 on splenic leukocytes of New Zealand Black × New Zealand White F1 progeny (NZB/WF1) mice during the preautoimmune period. We found that EE and E2 have common, as well as distinctive, immunologic effects, with EE exposure resulting in more profound effects. Both EE and E2 increased numbers of splenic neutrophils, enhanced neutrophil serine proteases and myeloperoxidase expression, promoted the production of nitric oxide and monocyte chemoattractant protein-1, and altered adaptive immune T cell subsets. However, activation of splenic leukocytes through the T cell receptor or Toll-like receptor (TLR)4 revealed not only common (IL-10), but also hormone-specific alterations of cytokines (IFNγ, IL-1ß, ΤΝFα, IL-2). Furthermore, in EE-exposed mice, TLR9 stimulation suppressed IFNα, in contrast to increased IFNα from E2-exposed mice. EE and E2 regulated common and hormone-specific expression of immune-related genes. Furthermore, EE exposure resulted in more marked alterations in miRNA expression levels than for E2. Only EE was able to reduce global DNA methylation significantly in splenic leukocytes. Taken together, our novel data revealed that EE and E2 exposure confers more similar effects in innate immune system-related cell development and responses, but has more differential regulatory effects in adaptive immune-related cell development and responses.

Human Rhinovirus Impairs the Innate Immune Response to Bacteria in Alveolar Macrophages in Chronic Obstructive Pulmonary Disease.

Rationale: Human rhinovirus (HRV) is a common cause of chronic obstructive pulmonary disease (COPD) exacerbations. Secondary bacterial infection is associated with more severe symptoms and delayed recovery. Alveolar macrophages clear bacteria from the lung and maintain lung homeostasis through cytokine secretion. These processes are defective in COPD. The effect of HRV on macrophage function is unknown. Objectives: To investigate the effect of HRV on phagocytosis and cytokine response to bacteria by alveolar macrophages and monocyte-derived macrophages (MDM) in COPD and healthy control subjects. Methods: Alveolar macrophages were obtained by bronchoscopy and MDM by adherence. Macrophages were exposed to HRV16 (multiplicity of infection 5), polyinosinic:polycytidylic acid (poly I:C) 30 µg/ml, IFN-ß 10 µg/ml, IFN-γ 10 µg/ml, or medium control for 24 hours. Phagocytosis of fluorescently labeled Haemophilus influenzae or Streptococcus pneumoniae was assessed by fluorimetry. CXCL8 (IL-8), IL-6, TNF-α (tumor necrosis factor-α), and IL-10 release was measured by ELISA. Measurements and Main Results: HRV significantly impaired phagocytosis of H. influenzae by 23% in MDM (n = 37; P = 0.004) and 18% in alveolar macrophages (n = 20; P < 0.0001) in COPD. HRV also significantly reduced phagocytosis of S. pneumoniae by 33% in COPD MDM (n = 20; P = 0.0192). There was no effect in healthy control subjects. Phagocytosis of H. influenzae was also impaired by poly I:C but not IFN-ß or IFN-γ in COPD MDM. HRV significantly reduced cytokine responses to H. influenzae. The IL-10 response to H. influenzae was significantly impaired by poly I:C, IFN-ß, and IFN-γ in COPD cells. Conclusions: HRV impairs phagocytosis of bacteria in COPD, which may lead to an outgrowth of bacteria. HRV also impairs cytokine responses to bacteria via the TLR3/IFN pathway, which may prevent resolution of inflammation leading to prolonged exacerbations in COPD.

Innate activation of human primary epithelial cells broadens the host response to Mycobacterium tuberculosis in the airways.

Early events in the human airways determining whether exposure to Mycobacterium tuberculosis (Mtb) results in acquisition of infection are poorly understood. Epithelial cells are the dominant cell type in the lungs, but little is known about their role in tuberculosis. We hypothesised that human primary airway epithelial cells are part of the first line of defense against Mtb-infection and contribute to the protective host response in the human respiratory tract. We modelled these early airway-interactions with human primary bronchial epithelial cells (PBECs) and alveolar macrophages. By combining in vitro infection and transwell co-culture models with a global transcriptomic approach, we identified PBECs to be inert to direct Mtb-infection, yet to be potent responders within an Mtb-activated immune network, mediated by IL1ß and type I interferon (IFN). Activation of PBECs by Mtb-infected alveolar macrophages and monocytes increased expression of known and novel antimycobacterial peptides, defensins and S100-family members and epithelial-myeloid interactions further shaped the immunological environment during Mtb-infection by promoting neutrophil influx. This is the first in depth analysis of the primary epithelial response to infection and offers new insights into their emerging role in tuberculosis through complementing and amplifying responses to Mtb.

Control of lupus nephritis by changes of gut microbiota.

BACKGROUND: Systemic lupus erythematosus, characterized by persistent inflammation, is a complex autoimmune disorder with no known cure. Immunosuppressants used in treatment put patients at a higher risk of infections. New knowledge of disease modulators, such as symbiotic bacteria, can enable fine-tuning of parts of the immune system, rather than suppressing it altogether. RESULTS: Dysbiosis of gut microbiota promotes autoimmune disorders that damage extraintestinal organs. Here we report a role of gut microbiota in the pathogenesis of renal dysfunction in lupus. Using a classical model of lupus nephritis, MRL/lpr, we found a marked depletion of Lactobacillales in the gut microbiota. Increasing Lactobacillales in the gut improved renal function of these mice and prolonged their survival. We used a mixture of 5 Lactobacillus strains (Lactobacillus oris, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus johnsonii, and Lactobacillus gasseri), but L. reuteri and an uncultured Lactobacillus sp. accounted for most of the observed effects. Further studies revealed that MRL/lpr mice possessed a "leaky" gut, which was reversed by increased Lactobacillus colonization. Lactobacillus treatment contributed to an anti-inflammatory environment by decreasing IL-6 and increasing IL-10 production in the gut. In the circulation, Lactobacillus treatment increased IL-10 and decreased IgG2a that is considered to be a major immune deposit in the kidney of MRL/lpr mice. Inside the kidney, Lactobacillus treatment also skewed the Treg-Th17 balance towards a Treg phenotype. These beneficial effects were present in female and castrated male mice, but not in intact males, suggesting that the gut microbiota controls lupus nephritis in a sex hormone-dependent manner. CONCLUSIONS: This work demonstrates essential mechanisms on how changes of the gut microbiota regulate lupus-associated immune responses in mice. Future studies are warranted to determine if these results can be replicated in human subjects.

Mucosal Type 2 Innate Lymphoid Cells Are a Key Component of the Allergic Response to Aeroallergens.

RATIONALE: Newly characterized type 2 innate lymphoid cells (ILC2s) display potent type 2 effector functionality; however, their contribution to allergic airways inflammation and asthma is poorly understood. Mucosal biopsy used to characterize the airway mucosa is invasive, poorly tolerated, and does not allow for sequential sampling. OBJECTIVES: To assess the role of ILC2s during nasal allergen challenge in subjects with allergic rhinitis using novel noninvasive methodology. METHODS: We used a human experimental allergen challenge model, with flow cytometric analysis of nasal curettage samples, to assess the recruitment of ILC2s and granulocytes to the upper airways of subjects with atopy and healthy subjects after allergen provocation. Soluble mediators in the nasal lining fluid were measured using nasosorption. MEASUREMENTS AND MAIN RESULTS: After an allergen challenge, subjects with atopy displayed rapid induction of upper airway symptoms, an enrichment of ILC2s, eosinophils, and neutrophils, along with increased production of IL-5, prostaglandin D2, and eosinophil and T-helper type 2 cell chemokines compared with healthy subjects. The most pronounced ILC2 recruitment was observed in subjects with elevated serum IgE and airway eosinophilia. CONCLUSIONS: The rapid recruitment of ILC2s to the upper airways of allergic patients with rhinitis, and their association with key type 2 mediators, highlights their likely important role in the early allergic response to aeroallergens in the airways. The novel methodology described herein enables the analysis of rare cell populations from noninvasive serial tissue sampling.

Vitamin D increases the antiviral activity of bronchial epithelial cells in vitro.

BACKGROUND: By modulating the antiviral immune response via vitamin D receptor, the active form of vitamin D (1,25-dihydroxyvitamin D, calcitriol) could play a central role in protection against respiratory virus infections. This in vitro study tested the hypothesis that respiratory viruses modulate vitamin D receptor expression in human bronchial epithelial cells and this modulation affects the antiviral response to exogenous vitamin D. METHODS: Human primary bronchial epithelial cells were infected with rhinoviruses and respiratory syncytial virus in the presence or absence of vitamin D. Expression of vitamin D receptor, 1α-hydroxylase (1α(OH)ase), 24-hydroxylase (24(OH)ase), innate interferons, interferon stimulated genes and cathelicidin were measured by quantitative polymerase chain reaction. The antiviral effect of vitamin D on rhinovirus replication was determined by measurement of virus load. A direct inactivation assay was used to determine the antiviral activity of cathelicidin. RESULTS: Both RV and RSV decreased vitamin D receptor and 24(OH)ase and, in addition, RSV increased 1α(OH)ase expression in epithelial cells. Vitamin D decreased rhinovirus replication and release, and increased rhinovirus-induced interferon stimulated genes and cathelicidin. Furthermore, cathelicidin had direct anti-rhinovirus activity. CONCLUSIONS: Despite lower vitamin D receptor levels in rhinovirus-infected epithelial cells, exogenous vitamin D increased antiviral defences most likely via cathelicidin and innate interferon pathways.

Identification of novel macrolides with antibacterial, anti-inflammatory and type I and III IFN-augmenting activity in airway epithelium.

BACKGROUND: Exacerbations of asthma and COPD are triggered by rhinoviruses. Uncontrolled inflammatory pathways, pathogenic bacterial burden and impaired antiviral immunity are thought to be important factors in disease severity and duration. Macrolides including azithromycin are often used to treat the above diseases, but exhibit variable levels of efficacy. Inhaled corticosteroids are also readily used in treatment, but may lack specificity. Ideally, new treatment alternatives should suppress unwanted inflammation, but spare beneficial antiviral immunity. METHODS: In the present study, we screened 225 novel macrolides and tested them for enhanced antiviral activity against rhinovirus, as well as anti-inflammatory activity and activity against Gram-positive and Gram-negative bacteria. Primary bronchial epithelial cells were grown from 10 asthmatic individuals and the effects of macrolides on rhinovirus replication were also examined. Another 30 structurally similar macrolides were also examined. RESULTS: The oleandomycin derivative Mac5, compared with azithromycin, showed superior induction (up to 5-fold, EC50 = 5-11 µM) of rhinovirus-induced type I IFNß, type III IFNλ1 and type III IFNλ2/3 mRNA and the IFN-stimulated genes viperin and MxA, yet had no effect on IL-6 and IL-8 mRNA. Mac5 also suppressed rhinovirus replication at 48 h, proving antiviral activity. Mac5 showed antibacterial activity against Gram-positive Streptococcus pneumoniae; however, it did not have any antibacterial properties compared with azithromycin when used against Gram-negative Escherichia coli (as a model organism) and also the respiratory pathogens Pseudomonas aeruginosa and non-typeable Haemophilus influenzae. Further non-toxic Mac5 derivatives were identified with various anti-inflammatory, antiviral and antibacterial activities. CONCLUSIONS: The data support the idea that macrolides have antiviral properties through a mechanism that is yet to be ascertained. We also provide evidence that macrolides can be developed with anti-inflammatory, antibacterial and antiviral activity and show surprising versatility depending on the clinical need.

Interferon response of the cystic fibrosis bronchial epithelium to major and minor group rhinovirus infection.

Rhinoviruses (RVs) are associated with exacerbations of cystic fibrosis (CF), asthma and COPD. There is growing evidence suggesting the involvement of the interferon (IFN) pathway in RV-associated morbidity in asthma and COPD. The mechanisms of RV-triggered exacerbations in CF are poorly understood. In a pilot study, we assessed the antiviral response of CF and healthy bronchial epithelial cells (BECs) to RV infection, we measured the levels of IFNs, pattern recognition receptors (PRRs) and IFN-stimulated genes (ISGs) upon infection with major and minor group RVs and poly(IC) stimulation. Major group RV infection of CF BECs resulted in a trend towards a diminished IFN response at the level of IFNs, PRRs and ISGs in comparison to healthy BECs. Contrary to major group RV, the IFN pathway induction upon minor group RV infection was significantly increased at the level of IFNs and PRRs in CF BECs compared to healthy BECs.

Increased nuclear suppressor of cytokine signaling 1 in asthmatic bronchial epithelium suppresses rhinovirus induction of innate interferons.

BACKGROUND: Rhinovirus infections are the dominant cause of asthma exacerbations, and deficient virus induction of IFN-α/ß/λ in asthmatic patients is important in asthma exacerbation pathogenesis. Mechanisms causing this interferon deficiency in asthmatic patients are unknown. OBJECTIVE: We sought to investigate the expression of suppressor of cytokine signaling (SOCS) 1 in tissues from asthmatic patients and its possible role in impaired virus-induced interferon induction in these patients. METHODS: We assessed SOCS1 mRNA and protein levels in vitro, bronchial biopsy specimens, and mice. The role of SOCS1 was inferred by proof-of-concept studies using overexpression with reporter genes and SOCS1-deficient mice. A nuclear role of SOCS1 was shown by using bronchial biopsy staining, overexpression of mutant SOCS1 constructs, and confocal microscopy. SOCS1 levels were also correlated with asthma-related clinical outcomes. RESULTS: We report induction of SOCS1 in bronchial epithelial cells (BECs) by asthma exacerbation-related cytokines and by rhinovirus infection in vitro. We found that SOCS1 was increased in vivo in bronchial epithelium and related to asthma severity. SOCS1 expression was also increased in primary BECs from asthmatic patients ex vivo and was related to interferon deficiency and increased viral replication. In primary human epithelium, mouse lung macrophages, and SOCS1-deficient mice, SOCS1 suppressed rhinovirus induction of interferons. Suppression of virus-induced interferon levels was dependent on SOCS1 nuclear translocation but independent of proteasomal degradation of transcription factors. Nuclear SOCS1 levels were also increased in BECs from asthmatic patients. CONCLUSION: We describe a novel mechanism explaining interferon deficiency in asthmatic patients through a novel nuclear function of SOCS1 and identify SOCS1 as an important therapeutic target for asthma exacerbations.

Novel antiviral properties of azithromycin in cystic fibrosis airway epithelial cells.

Virus-associated pulmonary exacerbations, often associated with rhinoviruses (RVs), contribute to cystic fibrosis (CF) morbidity. Currently, there are only a few therapeutic options to treat virus-induced CF pulmonary exacerbations. The macrolide antibiotic azithromycin has antiviral properties in human bronchial epithelial cells. We investigated the potential of azithromycin to induce antiviral mechanisms in CF bronchial epithelial cells. Primary bronchial epithelial cells from CF and control children were infected with RV after azithromycin pre-treatment. Viral RNA, interferon (IFN), IFN-stimulated gene and pattern recognition receptor expression were measured by real-time quantitative PCR. Live virus shedding was assessed by assaying the 50% tissue culture infective dose. Pro-inflammatory cytokine and IFN-ß production were evaluated by ELISA. Cell death was investigated by flow cytometry. RV replication was increased in CF compared with control cells. Azithromycin reduced RV replication seven-fold in CF cells without inducing cell death. Furthermore, azithromycin increased RV-induced pattern recognition receptor, IFN and IFN-stimulated gene mRNA levels. While stimulating antiviral responses, azithromycin did not prevent virus-induced pro-inflammatory responses. Azithromycin pre-treatment reduces RV replication in CF bronchial epithelial cells, possibly through the amplification of the antiviral response mediated by the IFN pathway. Clinical studies are needed to elucidate the potential of azithromycin in the management and prevention of RV-induced CF pulmonary exacerbations.