Toward targeted treatments for silicosis.

PURPOSE OF REVIEW: There has been a rapid increase in silicosis cases, particularly related to artificial stone. The key to management is avoidance of silica exposure. Despite this, many develop progressive disease and there are no routinely recommended treatments. This review provides a summary of the literature pertaining to pharmacological therapies for silicosis and examines the plausibility of success of such treatments given the disease pathogenesis. RECENT FINDINGS: In-vitro and in-vivo models demonstrate potential efficacy for drugs, which target inflammasomes, cytokines, effector cells, fibrosis, autophagy, and oxidation. SUMMARY: There is some evidence for potential therapeutic targets in silicosis but limited translation into human studies. Treatment of silicosis likely requires a multimodal approach, and there is considerable cross-talk between pathways; agents that modulate both inflammation, fibrosis, autophagy, and ROS production are likely to be most efficacious.

Harnessing endogenous peptide compounds as potential therapeutics for severe influenza.

BACKGROUND: Excessive pulmonary inflammation and damage are characteristic features of severe influenza virus infections. LAT8881 is a synthetic, 16 amino acid cyclic peptide form of a naturally occurring C-terminal fragment of human growth hormone with therapeutic efficacy against influenza. Shorter, linear peptides are typically easier to manufacture and formulate for delivery than larger cyclic peptides. A 6 amino acid linear peptide fragment of LAT8881, LAT9997, was investigated as a potential influenza therapy. METHODS: LAT9997 was evaluated for its potential to limit disease in a preclinical mouse model of severe influenza infection. RESULTS: Intranasal treatment of mice with either LAT8881 or LAT9997 from day 1 following influenza infection significantly improved survival outcomes. Initiating LAT9997 treatment at the onset of severe disease, also significantly improved disease severity. Greater disease resistance in LAT9997-treated mice correlated with reduced lung immunopathology, damage markers, vascular leak, and epithelial cell death. Treatment reduced viral loads, cytokines, and neutrophil infiltration in the airways, yet maintained protective alveolar macrophages in a dose-dependent manner. Sequential trimming of N- and C-terminal amino acids from LAT9997 revealed a structure-activity relationship. CONCLUSIONS: These findings provide preclinical evidence that therapeutic LAT9997 treatment limits viral burden and characteristic features of severe influenza, including hyperinflammation and lung damage.

Aggregated Hendra virus C-protein activates the NLRP3 inflammasome to induce inflammation.

BACKGROUND: Hendra virus is an emerging virus with a geographically broad host reservoir. In humans, Hendra virus causes excessive inflammatory disease of the lung and nervous system. Our current understanding as to how Hendra virus or what factors induce inflammation is limited and as such, there are currently no therapeutic options available for patients who contract Hendra virus. Recent studies have identified viral aggregating proteins as drivers of inflammation in influenza A virus and SARS-CoV-2 virus. In this study, we sought to identify potential aggregating Hendra virus proteins as proof-of-concept that inflammasome activation may induce inflammation and contribute to disease pathology. RESULTS: Here, we have identified that a peptide analogue of Hendra virus C protein (termed HeVc) forms aggregates and activates the NLRP3 inflammasome through phagocytic uptake into cells in vitro. Treatment of cells with the specific NLRP3 inhibitor MCC950 ameliorated IL-1ß secretion responses in vitro. Critically, in vivo intranasal inoculation of mice with aggregated HeVc peptide induced pulmonary inflammation, suggesting HeVc may drive immunopathology during infection. Importantly, mice treated with MCC950 demonstrated reduced IL-1ß secretion into the bronchoalveolar space, highlighting the role of NLRP3 in host HeV infections and a potential therapeutic strategy to reduce disease pathology. CONCLUSION: Taken together, these results identify Hendra virus C protein as a possible contributor to immunopathology during Hendra virus infections. Importantly, these studies highlight a potential role for NLRP3 in driving disease-associated inflammation, critically identifying a possible therapeutic strategy to alleviate disease-associated inflammation of infected patients through targeting of the NLRP3 inflammasome.

Gasdermin D promotes hyperinflammation and immunopathology during severe influenza A virus infection.

Excessive inflammation and tissue damage during severe influenza A virus (IAV) infection can lead to the development of fatal pulmonary disease. Pyroptosis is a lytic and pro-inflammatory form of cell death executed by the pore-forming protein gasdermin D (GSDMD). In this study, we investigated a potential role for GSDMD in promoting the development of severe IAV disease. IAV infection resulted in cleavage of GSDMD in vivo and in vitro in lung epithelial cells. Mice genetically deficient in GSDMD (Gsdmd-/-) developed less severe IAV disease than wildtype mice and displayed improved survival outcomes. GSDMD deficiency significantly reduced neutrophil infiltration into the airways as well as the levels of pro-inflammatory cytokines TNF, IL-6, MCP-1, and IL-1α and neutrophil-attracting chemokines CXCL1 and CXCL2. In contrast, IL-1ß and IL-18 responses were not largely impacted by GSDMD deficiency. In addition, Gsdmd-/- mice displayed significantly improved influenza disease resistance with reduced viral burden and less severe pulmonary pathology, including decreased epithelial damage and cell death. These findings indicate a major role for GSDMD in promoting damaging inflammation and the development of severe IAV disease.

Flu on the Brain: Identification of Highly Pathogenic Influenza in the Brains of Wild Carnivores in The Netherlands.

Highly pathogenic avian influenza (HPAI) viruses circulate in wild birds and can infect domestic poultry [...].

Interferon-ε is a tumour suppressor and restricts ovarian cancer.

High-grade serous ovarian cancers have low survival rates because of their late presentation with extensive peritoneal metastases and frequent chemoresistance1, and require new treatments guided by novel insights into pathogenesis. Here we describe the intrinsic tumour-suppressive activities of interferon-ε (IFNε). IFNε is constitutively expressed in epithelial cells of the fallopian tube, the cell of origin of high-grade serous ovarian cancers, and is then lost during development of these tumours. We characterize its anti-tumour activity in several preclinical models: ovarian cancer patient-derived xenografts, orthotopic and disseminated syngeneic models, and tumour cell lines with or without mutations in Trp53 and Brca genes. We use manipulation of the IFNε receptor IFNAR1 in different cell compartments, differential exposure status to IFNε and global measures of IFN signalling to show that the mechanism of the anti-tumour activity of IFNε involves direct action on tumour cells and, crucially, activation of anti-tumour immunity. IFNε activated anti-tumour T and natural killer cells and prevented the accumulation and activation of myeloid-derived suppressor cells and regulatory T cells. Thus, we demonstrate that IFNε is an intrinsic tumour suppressor in the female reproductive tract whose activities in models of established and advanced ovarian cancer, distinct from other type I IFNs, are compelling indications of potential new therapeutic approaches for ovarian cancer.

Preclinical Mouse Model of Silicosis.

Silicosis is an untreatable occupational lung disease caused by chronic inhalation of crystalline silica. Cyclical release and reuptake of silica particles by macrophages and airway epithelial cells causes repeated tissue damage, characterized by widespread inflammation and progressive diffuse fibrosis. While inhalation is the main route of entry for silica particles in humans, most preclinical studies administer silica via the intratracheal route. In vivo mouse models of lung disease are valuable tools required to bridge the translational gap between in vitro cell culture and human disease. This chapter describes a mouse model of silicosis which mimics clinical features of human silicosis, as well as methods for intranasal instillation of silica and disease analysis. Lung tissue can be collected for histological assessment of silica particle distribution, inflammation, structural damage, and fibrosis in sections stained with hematoxylin and eosin or Masson's trichrome. This approach can be extended to other chronic fibrotic lung diseases where inhalation of small damaging particles such as pollutants causes irreversible disease.

A novel dual NLRP1 and NLRP3 inflammasome inhibitor for the treatment of inflammatory diseases.

Objectives: Inflammasomes induce maturation of the inflammatory cytokines IL-1ß and IL-18, whose activity is associated with the pathophysiology of a wide range of infectious and inflammatory diseases. As validated therapeutic targets for the treatment of acute and chronic inflammatory diseases, there has been intense interest in developing small-molecule inhibitors to target inflammasome activity and reduce disease-associated inflammatory burden. Methods: We examined the therapeutic potential of a novel small-molecule inhibitor, and associated derivatives, termed ADS032 to target and reduce inflammasome-mediated inflammation in vivo. In vitro, we characterised ADS032 function, target engagement and specificity. Results: We describe ADS032 as the first dual NLRP1 and NLRP3 inhibitor. ADS032 is a rapid, reversible and stable inflammasome inhibitor that directly binds both NLRP1 and NLRP3, reducing secretion and maturation of IL-1ß in human-derived macrophages and bronchial epithelial cells in response to the activation of NLPR1 and NLRP3. ADS032 also reduced NLRP3-induced ASC speck formation, indicative of targeting inflammasome formation. In vivo, ADS032 reduced IL-1ß and TNF-α levels in the serum of mice challenged i.p. with LPS and reduced pulmonary inflammation in an acute model of lung silicosis. Critically, ADS032 protected mice from lethal influenza A virus challenge, displayed increased survival and reduced pulmonary inflammation. Conclusion: ADS032 is the first described dual inflammasome inhibitor and a potential therapeutic to treat both NLRP1- and NLRP3-associated inflammatory diseases and also constitutes a novel tool that allows examination of the role of NLRP1 in human disease.

Naturally derived cytokine peptides limit virus replication and severe disease during influenza A virus infection.

Objectives: Novel host-targeted therapeutics could treat severe influenza A virus (IAV) infections, with reduced risk of drug resistance. LAT8881 is a synthetic form of the naturally occurring C-terminal fragment of human growth hormone. Acting independently of the growth hormone receptor, it can reduce inflammation-induced damage and promote tissue repair in an animal model of osteoarthritis. LAT8881 has been assessed in clinical trials for the treatment of obesity and neuropathy and has an excellent safety profile. We investigated the potential for LAT8881, its metabolite LAT9991F and LAT7771 derived from prolactin, a growth hormone structural homologue, to treat severe IAV infection. Methods: LAT8881, LAT9991F and LAT7771 were evaluated for their effects on cell viability and IAV replication in vitro, as well as their potential to limit disease in a preclinical mouse model of severe IAV infection. Results: In vitro LAT8881 treatment enhanced cell viability, particularly in the presence of cytotoxic stress, which was countered by siRNA inhibition of host lanthionine synthetase C-like proteins. Daily intranasal treatment of mice with LAT8881 or LAT9991F, but not LAT7771, from day 1 postinfection significantly improved influenza disease resistance, which was associated with reduced infectious viral loads, reduced pro-inflammatory cytokines and increased abundance of protective alveolar macrophages. LAT8881 treatment in combination with the antiviral oseltamivir phosphate led to more pronounced reduction in markers of disease severity than treatment with either compound alone. Conclusion: These studies provide the first evidence identifying LAT8881 and LAT9991F as novel host-protective therapies that improve survival, limit viral replication, reduce local inflammation and curtail tissue damage during severe IAV infection. Evaluation of LAT8881 and LAT9991F in other infectious and inflammatory conditions of the airways is warranted.

Constitutive expression and distinct properties of IFN-epsilon protect the female reproductive tract from Zika virus infection.

The immunological surveillance factors controlling vulnerability of the female reproductive tract (FRT) to sexually transmitted viral infections are not well understood. Interferon-epsilon (IFNɛ) is a distinct, immunoregulatory type-I IFN that is constitutively expressed by FRT epithelium and is not induced by pathogens like other antiviral IFNs α, ß and λ. We show the necessity of IFNɛ for Zika Virus (ZIKV) protection by: increased susceptibility of IFNɛ-/- mice; their "rescue" by intravaginal recombinant IFNɛ treatment and blockade of protective endogenous IFNɛ by neutralising antibody. Complementary studies in human FRT cell lines showed IFNɛ had potent anti-ZIKV activity, associated with transcriptome responses similar to IFNλ but lacking the proinflammatory gene signature of IFNα. IFNɛ activated STAT1/2 pathways similar to IFNα and λ that were inhibited by ZIKV-encoded non-structural (NS) proteins, but not if IFNε exposure preceded infection. This scenario is provided by the constitutive expression of endogenous IFNε. However, the IFNɛ expression was not inhibited by ZIKV NS proteins despite their ability to antagonise the expression of IFNß or λ. Thus, the constitutive expression of IFNɛ provides cellular resistance to viral strategies of antagonism and maximises the antiviral activity of the FRT. These results show that the unique spatiotemporal properties of IFNε provides an innate immune surveillance network in the FRT that is a significant barrier to viral infection with important implications for prevention and therapy.

Another One Fights the Dust: Targeting the NLRP3 Inflammasome for the Treatment of Silicosis.

Silicosis is a multifaceted lung disease, characterized by persistent inflammation and structural remodeling. Despite its poor prognosis, there are no treatments currently available for patients with silicosis. Recent preclinical findings in models of lung fibrosis have suggested a major role for the NLRP3 (nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3) inflammasome in silica-driven inflammation and fibrosis. This review outlines the beneficial effects of targeting the NLRP3 inflammasome in in vitro cell experiments and in in vivo animal models, whereby inflammation and fibrosis are abrogated after NLRP3 inflammasome inhibition. Although preclinical evidence is promising, studies that explore NLRP3 inflammasomes in the clinical setting are warranted. In particular, there is still a need to identify biomarkers that may be helpful for the early detection of silicosis and to fully elucidate mechanisms underlying these beneficial effects to further develop or repurpose existing anti-NLRP3 drugs as novel treatments that limit disease progression.

Too young to die? How aging affects cellular innate immune responses to influenza virus and disease severity.

Influenza is a respiratory viral infection that causes significant morbidity and mortality worldwide. The innate immune cell response elicited during influenza A virus (IAV) infection forms the critical first line of defense, which typically is impaired as we age. As such, elderly individuals more commonly succumb to influenza-associated complications, which is reflected in most aged animal models of IAV infection. Here, we review the important roles of several major innate immune cell populations in influenza pathogenesis, some of which being deleterious to the host, and the current knowledge of how age-associated numerical, phenotypic and functional cell changes impact disease development. Further investigation into age-related modulation of innate immune cell responses, using appropriate animal models, will help reveal how immunity to IAV may be compromised by aging and inform the development of novel therapies, tailored for use in this vulnerable group.

Interleukin-1ß exacerbates disease and is a potential therapeutic target to reduce pulmonary inflammation during severe influenza A virus infection.

Hyperinflammatory responses including the production of NLRP3-dependent interleukin (IL)-1ß is a characteristic feature of severe and fatal influenza A virus (IAV) infections. The NLRP3 inflammasome has been shown to play a temporal role during severe IAV immune responses, with early protective and later detrimental responses. However, the specific contribution of IL-1ß in modulating IAV disease in vivo is currently not well defined. Here, we identified that activation of NLRP3-dependent IL-1ß responses occurs rapidly following HKx31 H3N2 infection, prior to the onset of severe IAV disease. Mature IL-1ß was detectable in vivo in both hemopoietic and nonhemopoietic cells. Significantly, therapeutic inhibition of IL-1ß in the airways with intranasal anti-IL-1ß antibody treatment from day 3 postinfection, corresponding to the onset of clinical signs of disease, significantly prolonged survival and reduced inflammation in the airways. Importantly, early targeting of IL-1ß from day 1 postinfection also improved survival. Together, these studies specifically define a role for IL-1ß in contributing to the development of hyperinflammation and disease and indicate that targeting IL-1ß is a potential therapeutic strategy for severe IAV infections.

Die Another Way: Interplay between Influenza A Virus, Inflammation and Cell Death.

: Influenza A virus (IAV) is a major concern to human health due to the ongoing global threat of a pandemic. Inflammatory and cell death signalling pathways play important roles in host defence against IAV infection. However, severe IAV infections in humans are characterised by excessive inflammation and tissue damage, often leading to fatal disease. While the molecular mechanisms involved in the induction of inflammation during IAV infection have been well studied, the pathways involved in IAV-induced cell death and their impact on immunopathology have not been fully elucidated. There is increasing evidence of significant crosstalk between cell death and inflammatory pathways and a greater understanding of their role in host defence and disease may facilitate the design of new treatments for IAV infection.

RIPLET, and not TRIM25, is required for endogenous RIG-I-dependent antiviral responses.

The innate immune system is our first line of defense against viral pathogens. Host cell pattern recognition receptors sense viral components and initiate immune signaling cascades that result in the production of an array of cytokines to combat infection. Retinoic acid-inducible gene-I (RIG-I) is a pattern recognition receptor that recognizes viral RNA and, when activated, results in the production of type I and III interferons (IFNs) and the upregulation of IFN-stimulated genes. Ubiquitination of RIG-I by the E3 ligases tripartite motif-containing 25 (TRIM25) and Riplet is thought to be requisite for RIG-I activation; however, recent studies have questioned the relative importance of these two enzymes for RIG-I signaling. In this study, we show that deletion of Trim25 does not affect the IFN response to either influenza A virus (IAV), influenza B virus, Sendai virus or several RIG-I agonists. This is in contrast to deletion of either Rig-i or Riplet, which completely abrogated RIG-I-dependent IFN responses. This was consistent in both mouse and human cell lines, as well as in normal human bronchial cells. With most of the current TRIM25 literature based on exogenous expression, these findings provide critical evidence that Riplet, and not TRIM25, is required endogenously for the ubiquitination of RIG-I. Despite this, loss of TRIM25 results in greater susceptibility to IAV infection in vivo, suggesting that it may have an alternative role in host antiviral defense. This study refines our understanding of RIG-I signaling in viral infections and will inform future studies in the field.

Repurposing drugs targeting the P2X7 receptor to limit hyperinflammation and disease during influenza virus infection.

BACKGROUND AND PURPOSE: Severe influenza A virus (IAV) infections are associated with damaging hyperinflammation that can be fatal. There is an urgent need to identify new therapeutic agents to treat severe and pathogenic IAV infections. Repurposing of drugs with an existing and studied pharmacokinetic and safety profile is a highly attractive potential strategy. We have previously demonstrated that the NLRP3 inflammasome plays time-dependent roles during severe IAV infection with early protective responses and later dysregulation leading to excessive inflammation, contributing to disease severity. EXPERIMENTAL APPROACH: We tested two existing drugs, probenecid and AZ11645373, to target P2X7 receptor signalling and dampen NLRP3 inflammasome responses during severe IAV infection. In vitro, the drugs were assessed for their ability to limit NLRP3 inflammasome-dependent IL-1ß secretion in macrophage cultures. In vivo, their effects were assessed on hyperinflammation and disease during severe IAV infection in C57BL/6 mice. KEY RESULTS: Treatment of macrophages with probenecid or AZ11645373 in vitro diminished NLRP3 inflammasome-dependent IL-1ß secretion. Intranasal therapeutic treatment of mice displaying severe influenza disease with probenecid or AZ11645373 reduced pro-inflammatory cytokine production, cellular infiltrates in the lung, and provided protection against disease. Importantly, these drugs could be administered at either early or late stage of disease and provide therapeutic efficacy. CONCLUSIONS AND IMPLICATIONS: Our study demonstrates that the anti-inflammatory drugs probenecid and AZ11645373, which have documented pharmacokinetics and safety profiles in humans, are effective at dampening hyperinflammation and severe influenza disease providing potentially new therapeutic strategies for treating severe or pathogenic IAV infections.