Comparative Efficacy of Mayaro Virus-Like Particle Vaccines Produced in Insect or Mammalian Cells.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes often debilitating rheumatic disease in tropical Central and South America. There are currently no licensed vaccines or antiviral drugs available for MAYV disease. Here, we generated Mayaro virus-like particles (VLPs) using the scalable baculovirus-insect cell expression system. High-level secretion of MAYV VLPs in the culture fluid of Sf9 insect cells was achieved, and particles with a diameter of 64 to 70 nm were obtained after purification. We characterize a C57BL/6J adult wild-type mouse model of MAYV infection and disease and used this model to compare the immunogenicity of VLPs from insect cells with that of VLPs produced in mammalian cells. Mice received two intramuscular immunizations with 1 µg of nonadjuvanted MAYV VLPs. Potent neutralizing antibody responses were generated against the vaccine strain, BeH407, with comparable activity seen against a contemporary 2018 isolate from Brazil (BR-18), whereas neutralizing activity against chikungunya virus was marginal. Sequencing of BR-18 illustrated that this virus segregates with genotype D isolates, whereas MAYV BeH407 belongs to genotype L. The mammalian cell-derived VLPs induced higher mean neutralizing antibody titers than those produced in insect cells. Both VLP vaccines completely protected adult wild-type mice against viremia, myositis, tendonitis, and joint inflammation after MAYV challenge. IMPORTANCE Mayaro virus (MAYV) is associated with acute rheumatic disease that can be debilitating and can evolve into months of chronic arthralgia. MAYV is believed to have the potential to emerge as a tropical public health threat, especially if it develops the ability to be efficiently transmitted by urban mosquito vectors, such as Aedes aegypti and/or Aedes albopictus. Here, we describe a scalable virus-like particle vaccine against MAYV that induced neutralizing antibodies against a historical and a contemporary isolate of MAYV and protected mice against infection and disease, providing a potential new intervention for MAYV epidemic preparedness.

Mouse models of COVID-19 recapitulate inflammatory pathways rather than gene expression.

How well mouse models recapitulate the transcriptional profiles seen in humans remains debatable, with both conservation and diversity identified in various settings. Herein we use RNA-Seq data and bioinformatics approaches to analyze the transcriptional responses in SARS-CoV-2 infected lungs, comparing 4 human studies with the widely used K18-hACE2 mouse model, a model where hACE2 is expressed from the mouse ACE2 promoter, and a model that uses a mouse adapted virus and wild-type mice. Overlap of single copy orthologue differentially expressed genes (scoDEGs) between human and mouse studies was generally poor (≈15-35%). Rather than being associated with batch, sample treatment, viral load, lung damage or mouse model, the poor overlaps were primarily due to scoDEG expression differences between species. Importantly, analyses of immune signatures and inflammatory pathways illustrated highly significant concordances between species. As immunity and immunopathology are the focus of most studies, these mouse models can thus be viewed as representative and relevant models of COVID-19.

ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions.

SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intrapulmonary hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lung epithelium. RNA-Seq and histological analyses illustrated that this model involved an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mouse lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.

Injection site vaccinology of a recombinant vaccinia-based vector reveals diverse innate immune signatures.

Poxvirus systems have been extensively used as vaccine vectors. Herein a RNA-Seq analysis of intramuscular injection sites provided detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. The multiple adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFNγ, TNF and IL1ß, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such injection site vaccinology approaches should inform refinements in poxvirus-based vector design.

Neuroinvasiveness of the MR766 strain of Zika virus in IFNAR-/- mice maps to prM residues conserved amongst African genotype viruses.

Zika virus (ZIKV) strains are classified into the African and Asian genotypes. The higher virulence of the African MR766 strain, which has been used extensively in ZIKV research, in adult IFNα/ß receptor knockout (IFNAR-/-) mice is widely viewed as an artifact associated with mouse adaptation due to at least 146 passages in wild-type suckling mouse brains. To gain insights into the molecular determinants of MR766's virulence, a series of genes from MR766 were swapped with those from the Asian genotype PRVABC59 isolate, which is less virulent in IFNAR-/- mice. MR766 causes 100% lethal infection in IFNAR-/- mice, but when the prM gene of MR766 was replaced with that of PRVABC59, the chimera MR/PR(prM) showed 0% lethal infection. The reduced virulence was associated with reduced neuroinvasiveness, with MR766 brain titers ≈3 logs higher than those of MR/PR(prM) after subcutaneous infection, but was not significantly different in brain titers of MR766 and MR/PR(prM) after intracranial inoculation. MR/PR(prM) also showed reduced transcytosis when compared with MR766 in vitro. The high neuroinvasiveness of MR766 in IFNAR-/- mice could be linked to the 10 amino acids that differ between the prM proteins of MR766 and PRVABC59, with 5 of these changes affecting positive charge and hydrophobicity on the exposed surface of the prM protein. These 10 amino acids are highly conserved amongst African ZIKV isolates, irrespective of suckling mouse passage, arguing that the high virulence of MR766 in adult IFNAR-/- mice is not the result of mouse adaptation.

Are the Relevant Risk Factors Being Adequately Captured in Empirical Studies of Smoking Initiation? A Machine Learning Analysis Based on the Population Assessment of Tobacco and Health Study.

INTRODUCTION: Cigarette smoking continues to pose a threat to public health. Identifying individual risk factors for smoking initiation is essential to further mitigate this epidemic. To the best of our knowledge, no study today has used machine learning (ML) techniques to automatically uncover informative predictors of smoking onset among adults using the Population Assessment of Tobacco and Health (PATH) study. AIMS AND METHODS: In this work, we employed random forest paired with Recursive Feature Elimination to identify relevant PATH variables that predict smoking initiation among adults who have never smoked at baseline between two consecutive PATH waves. We included all potentially informative baseline variables in wave 1 (wave 4) to predict past 30-day smoking status in wave 2 (wave 5). Using the first and most recent pairs of PATH waves was found sufficient to identify the key risk factors of smoking initiation and test their robustness over time. The eXtreme Gradient Boosting method was employed to test the quality of these selected variables. RESULTS: As a result, classification models suggested about 60 informative PATH variables among many candidate variables in each baseline wave. With these selected predictors, the resulting models have a high discriminatory power with the area under the specificity-sensitivity curves of around 80%. We examined the chosen variables and discovered important features. Across the considered waves, two factors, (1) BMI, and (2) dental and oral health status, robustly appeared as important predictors of smoking initiation, besides other well-established predictors. CONCLUSIONS: Our work demonstrates that ML methods are useful to predict smoking initiation with high accuracy, identifying novel smoking initiation predictors, and to enhance our understanding of tobacco use behaviors. IMPLICATIONS: Understanding individual risk factors for smoking initiation is essential to prevent smoking initiation. With this methodology, a set of the most informative predictors of smoking onset in the PATH data were identified. Besides reconfirming well-known risk factors, the findings suggested additional predictors of smoking initiation that have been overlooked in previous work. More studies that focus on the newly discovered factors (BMI and dental and oral health status,) are needed to confirm their predictive power against the onset of smoking as well as determine the underlying mechanisms.

The evolution of age-specific smoking cessation rates in the United States from 2009 to 2017: a Kalman filter based approach.

BACKGROUND: Tracking the US smoking cessation rate over time is of great interest to tobacco control researchers and policymakers since smoking cessation behaviors have a major effect on the public's health. Recent studies have employed dynamic models to estimate the US cessation rate through observed smoking prevalence. However, none of those studies has provided annual estimates of the cessation rate by age group. Hence, the primary objective of this study is to estimate annual smoking cessation rates specific to different age groups in the US from 2009 to 2017. METHODS: We employed a Kalman filter approach to investigate the annual evolution of age-group-specific cessation rates, unknown parameters of a mathematical model of smoking prevalence, during the 2009-2017 period using data from the 2009-2018 National Health Interview Surveys. We focused on cessation rates in the 25-44, 45-64 and 65 + age groups. RESULTS: The findings show that cessation rates followed a consistent u-shaped curve over time with respect to age (i.e., higher among the 25-44 and 65 + age groups, and lower among 45-64-year-olds). Over the course of the study, the cessation rates in the 25-44 and 65 + age groups remained nearly unchanged around 4.5% and 5.6%, respectively. However, the rate in the 45-64 age group exhibited a substantial increase of 70%, from 2.5% to 2009 to 4.2% in 2017. The estimated cessation rates in all three age groups tended to converge to the weighted average cessation rate over time. CONCLUSIONS: The Kalman filter approach offers a real-time estimation of cessation rates that can be helpful for monitoring smoking cessation behavior.

Monitoring the Increase in the U.S. Smoking Cessation Rate and Its Implication for Future Smoking Prevalence.

INTRODUCTION: We calculate the U.S. adult smoking cessation rate for 2014-2019, compare it to the historical trend, and estimate the implication for future smoking prevalence. METHODS: We repeated an earlier analysis, which examined the cessation rate from 1990 to 2014, extending the period to 2019. Employing National Health Interview Survey (NHIS) and National Survey on Drug Use and Health (NSDUH) data, we estimated the adult cessation rate in 6-year intervals, using weighted nonlinear least squares. We then employed a meta-regression model to test whether the cessation rate has increased beyond expectation. We used cessation rate estimates and smoking initiation rate estimates to project smoking prevalence in 2030 and eventual steady-state prevalence. RESULTS: The annual cessation rate increased 29% using NHIS data (from 4.2% in 2008-2013 to 5.4% in 2014-2019) and 33% with NSDUH data (4.2%-5.6%). The cessation rate increase accounts for 60% of a smoking prevalence decline in the most recent period exceeding the 1990-2013 predicted trend. The remaining 40% owes to declining smoking initiation. With current initiation and cessation rates, smoking prevalence should fall to 8.3% in 2030 and eventually reach a steady state of 3.53%. CONCLUSIONS: The smoking cessation rate continued to increase during 2014-2019. NHIS and NSDUH results are practically identical. The larger share (60%) of the smoking prevalence decrease, beyond expectation, attributable to the increased cessation rate is encouraging since the positive health effects of cessation occur much sooner than those derived from declining initiation. IMPLICATIONS: The smoking cessation rate in the United States continues to increase, accelerating the decline in smoking prevalence. This increase suggests that the Healthy People 2030 goal of 5% adult smoking prevalence, while ambitious, is attainable. Our findings can be used in simulation and statistical models that aim to predict future prevalence and population health effects due to smoking under various scenarios.

The association between smoking behaviors and prices and taxes per cigarette pack in the United States from 2000 through 2019.

OBJECTIVE: The conclusions on how tax and price increases affect smoking behaviors are mixed. This work is devoted to re-evaluating the relationship between cigarette prices and taxes and smoking behaviors. METHODS: Using 2000-2019 Behavioral Risk Factor Surveillance System data, we employed linear mixed-effect models to re-examine the impact of cigarette prices and taxes on smoking prevalence and the proportion of current smokers having tried to quit smoking in the past 12 months. All the analyses were conducted for the general population, then by age group, gender, race/ethnicity, and income level. RESULTS: The results indicate that higher cigarette prices and taxes were associated with a decrease in smoking prevalence and an increased likelihood of quitting smoking. Cigarette tax and price increases produced the most powerful impact on the smoking prevalence of 18- to 24-year-olds. The estimates also show that males tended to be more price-sensitive than females. Raising cigarette prices and taxes was estimated to be more effective in reducing the smoking prevalence among non-Hispanic Blacks and Hispanics when compared to non-Hispanic whites. Cigarette price and tax changes were likely to have a smaller effect on individuals with annual income under $25,000 relative to individuals with higher income levels. CONCLUSIONS: Increases in cigarette prices and taxes are significantly associated with a reduction in smoking prevalence and an increased likelihood of quitting smoking among adults across different demographic and socioeconomic groups. However, as cigarette price and tax changes disproportionately affect low-income individuals, raising cigarette prices and taxes may deepen income disparities.

Simple rapid in vitro screening method for SARS-CoV-2 anti-virals that identifies potential cytomorbidity-associated false positives.

BACKGROUND: The international SARS-CoV-2 pandemic has resulted in an urgent need to identify new anti-viral drugs for treatment of COVID-19. The initial step to identifying potential candidates usually involves in vitro screening that includes standard cytotoxicity controls. Under-appreciated is that viable, but stressed or otherwise compromised cells, can also have a reduced capacity to replicate virus. A refinement proposed herein for in vitro drug screening thus includes a simple growth assay to identify drug concentrations that cause cellular stress or "cytomorbidity", as distinct from cytotoxicity or loss of viability. METHODS: A simple rapid bioassay is presented for antiviral drug screening using Vero E6 cells and inhibition of SARS-CoV-2 induced cytopathic effects (CPE) measured using crystal violet staining. We use high cell density for cytotoxicity assays, and low cell density for cytomorbidity assays. RESULTS: The assay clearly illustrated the anti-viral activity of remdesivir, a drug known to inhibit SARS-CoV-2 replication. In contrast, nitazoxanide, oleuropein, cyclosporine A and ribavirin all showed no ability to inhibit SARS-CoV-2 CPE. Hydroxychloroquine, cyclohexamide, didemnin B, γ-mangostin and linoleic acid were all able to inhibit viral CPE at concentrations that did not induce cytotoxicity. However, these drugs inhibited CPE at concentrations that induced cytomorbidity, indicating non-specific anti-viral activity. CONCLUSIONS: We describe the methodology for a simple in vitro drug screening assay that identifies potential anti-viral drugs via their ability to inhibit SARS-CoV-2-induced CPE. The additional growth assay illustrated how several drugs display anti-viral activity at concentrations that induce cytomorbidity. For instance, hydroxychloroquine showed anti-viral activity at concentrations that slow cell growth, arguing that its purported in vitro anti-viral activity arises from non-specific impairment of cellular activities. The cytomorbidity assay can therefore rapidly exclude potential false positives.

Is mammography screening beneficial: An individual-based stochastic model for breast cancer incidence and mortality.

The benefits of mammography screening have been controversial, with conflicting findings from various studies. We hypothesize that unmeasured heterogeneity in tumor aggressiveness underlies these conflicting results. Based on published data from the Canadian National Breast Screening Study (CNBSS), we develop and parameterize an individual-based mechanistic model for breast cancer incidence and mortality that tracks five stages of breast cancer progression and incorporates the effects of age on breast cancer incidence and all-cause mortality. The model accurately reproduces the reported outcomes of the CNBSS. By varying parameters, we predict that the benefits of mammography depend on the effectiveness of cancer treatment and tumor aggressiveness. In particular, patients with the most rapidly growing or potentially largest tumors have the highest benefit and least harm from the screening, with only a relatively small effect of age. However, the model predicts that confining mammography to populations with a high risk of acquiring breast cancer increases the screening benefit only slightly compared with the full population.

Consequences of a match made in hell: the harm caused by menthol smoking to the African American population over 1980-2018.

BACKGROUND: For many years, national surveys have shown a consistently disproportionately high prevalence of menthol smokers among African Americans compared with the general population. However, to our knowledge, no prior study has quantified the harm that menthol smoking has caused on that population. In this work, we estimate the public health harm that menthol cigarettes have caused to the African American community over the last four decades. METHODS: Using National Health Interview Survey data, we employed a well-established simulation model to reproduce the observed smoking trajectory over 1980-2018 in the African American population. Then, we repeat the experiment, removing the effects of menthol on the smoking initiation and cessation rates over that period, obtaining a new hypothetical smoking trajectory. Finally, we compared both scenarios to calculate the public health harm attributable to menthol cigarettes over 1980-2018. RESULTS: Our results show that menthol cigarettes were responsible for 1.5 million new smokers, 157 000 smoking-related premature deaths and 1.5 million life-years lost among African Americans over 1980-2018. While African Americans constitute 12% of the total US population, these figures represent, respectively, a staggering 15%, 41% and 50% of the total menthol-related harm. DISCUSSION: Our results show that menthol cigarettes disproportionally harmed African Americans significantly over the last 38 years and are responsible for exacerbating health disparities among that population. Removing menthol cigarettes from the market would benefit the overall US population but, particularly, the African American community.

Lower temperatures reduce type I interferon activity and promote alphaviral arthritis.

Chikungunya virus (CHIKV) belongs to a group of mosquito-borne alphaviruses associated with acute and chronic arthropathy, with peripheral and limb joints most commonly affected. Using a mouse model of CHIKV infection and arthritic disease, we show that CHIKV replication and the ensuing foot arthropathy were dramatically reduced when mice were housed at 30°C, rather than the conventional 22°C. The effect was not associated with a detectable fever, but was dependent on type I interferon responses. Bioinformatics analyses of RNA-Seq data after injection of poly(I:C)/jetPEI suggested the unfolded protein response and certain type I interferon responses are promoted when feet are slightly warmer. The ambient temperature thus appears able profoundly to effect anti-viral activity in the periphery, with clear consequences for alphaviral replication and the ensuing arthropathy. These observations may provide an explanation for why alphaviral arthropathies are largely restricted to joints of the limbs and the extremities.

RNA-Seq analysis of chikungunya virus infection and identification of granzyme A as a major promoter of arthritic inflammation.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus causing epidemics of acute and chronic arthritic disease. Herein we describe a comprehensive RNA-Seq analysis of feet and lymph nodes at peak viraemia (day 2 post infection), acute arthritis (day 7) and chronic disease (day 30) in the CHIKV adult wild-type mouse model. Genes previously shown to be up-regulated in CHIKV patients were also up-regulated in the mouse model. CHIKV sequence information was also obtained with up to ≈8% of the reads mapping to the viral genome; however, no adaptive viral genome changes were apparent. Although day 2, 7 and 30 represent distinct stages of infection and disease, there was a pronounced overlap in up-regulated host genes and pathways. Type I interferon response genes (IRGs) represented up to ≈50% of up-regulated genes, even after loss of type I interferon induction on days 7 and 30. Bioinformatic analyses suggested a number of interferon response factors were primarily responsible for maintaining type I IRG induction. A group of genes prominent in the RNA-Seq analysis and hitherto unexplored in viral arthropathies were granzymes A, B and K. Granzyme A-/- and to a lesser extent granzyme K-/-, but not granzyme B-/-, mice showed a pronounced reduction in foot swelling and arthritis, with analysis of granzyme A-/- mice showing no reductions in viral loads but reduced NK and T cell infiltrates post CHIKV infection. Treatment with Serpinb6b, a granzyme A inhibitor, also reduced arthritic inflammation in wild-type mice. In non-human primates circulating granzyme A levels were elevated after CHIKV infection, with the increase correlating with viral load. Elevated granzyme A levels were also seen in a small cohort of human CHIKV patients. Taken together these results suggest granzyme A is an important driver of arthritic inflammation and a potential target for therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00281294.

SerpinB2 deficiency in mice reduces bleeding times via dysregulated platelet activation.

SerpinB2, also known as plasminogen activation inhibitor type 2 (PAI-2), is classically viewed as an inhibitor of fibrinolysis. However, we show herein a distinct, hitherto unrecognized role for SerpinB2 in hemostasis. Mice deficient in SerpinB2 expression and mice with an active site mutation in SerpinB2, both showed significant reductions in tail bleeding times. This hemostatic phenotype was associated with platelets, with SerpinB2 and SerpinB2-urokinase complexes clearly present in platelet fractions, and immunohistochemistry of blood clots suggesting SerpinB2 is associated with platelet aggregates. Thromboelastography illustrated faster onset of clot formation in blood from SerpinB2 deficient mice, whereas clotting of platelet-free plasma was unaffected. The results appear consistent with the low circulating SerpinB2 levels and hypercoagulation seen during pre-eclampsia; however, SerpinB2 was not detected in human platelets.

Optimized Synthesis of ZSM-11 Catalysts using 1,8-Diaminooctane as a Structure-Directing Agent.

Developing new zeolite catalysts for (petro)chemical applications is nontrivial owing to challenges that include the identification of commercially-viable syntheses. The vast majority of syntheses require the use of an organic structure-directing agent (OSDA), which has economic and environmental shortcomings. In the search for alternative zeolites to common industrial catalysts, such as ZSM-5 (MFI), a promising candidate is ZSM-11 (MEL), a close structural analogue of MFI. ZSM-11 is comprised of 3-dimensional straight channels that have less diffusion limitations than MFI. Side-by-side comparisons of ZSM-11 and ZSM-5 catalysts reveal the former exhibits significantly longer time-on-stream lifetime in many reactions; however, there are several difficulties associated with the preparation of ZSM-11, which include the ability to identify inexpensive OSDAs that produce small crystals in high yield. Here, we examine ZSM-11 synthesis using 1,8-diaminooctane (DAO), which produces ZSM-11 crystals with sizes of ca. 300 nm. We demonstrate that the use of MEL seeds allows for an order of magnitude reduction in the quantity of DAO without sacrificing crystal purity or yield (>80 %). Catalytic tests using methanol to hydrocarbons as a representative reaction show that H-ZSM-11 lifetime and selectivity are similar for samples prepared with and without seeds. Collectively our findings highlight an efficient method to produce ZSM-11 as a potential alternative to ZSM-5 for catalytic applications.

SerpinB2 Deficiency Results in a Stratum Corneum Defect and Increased Sensitivity to Topically Applied Inflammatory Agents.

SerpinB2 (plasminogen activator inhibitor type 2) is constitutively expressed at high levels by differentiating keratinocytes in mice and humans; however, the physiological function of keratinocyte SerpinB2 remains unclear. Herein, we show that SerpinB2(-/-) mice are more susceptible to contact dermatitis after topical application of dinitrofluorobenzene, and show enhanced inflammatory lesions after topical applications of phorbol ester. Untreated SerpinB2(-/-) mice showed no overt changes in epithelial structure, and we were unable to find evidence for a role for keratinocyte SerpinB2 in regulating immunity, apoptosis, IL-1ß production, proteasomal activity, or wound healing. Instead, the phenotype was associated with impaired skin barrier function and a defective stratum corneum, with SerpinB2(-/-) mice showing increased transepidermal water loss, increased overt loss of stratum corneum in inflammatory lesions, and impaired stratum corneum thickening after phorbol ester treatment. Immunoblotting suggested that SerpinB2 (cross-linked into the cornified envelope) is present in the stratum corneum and retains the ability to form covalent inhibitory complexes with urokinase. Data suggest that the function of keratinocyte SerpinB2 is protection of the stratum corneum from proteolysis via inhibition of urokinase, thereby maintaining the integrity and barrier function of the stratum corneum, particularly during times of skin inflammation. Implications for studies involving genetically modified mice treated with topical agents and human dermatological conditions, such as contact dermatitis, are discussed.

STAT-3 contributes to pulmonary fibrosis through epithelial injury and fibroblast-myofibroblast differentiation.

Lung fibrosis is the hallmark of the interstitial lung diseases. Alveolar epithelial cell (AEC) injury is a key step that contributes to a profibrotic microenvironment. Fibroblasts and myofibroblasts subsequently accumulate and deposit excessive extracellular matrix. In addition to TGF-ß, the IL-6 family of cytokines, which signal through STAT-3, may also contribute to lung fibrosis. In the current manuscript, the extent to which STAT-3 inhibition decreases lung fibrosis is investigated. Phosphorylated STAT-3 was elevated in lung biopsies from patients with idiopathic pulmonary fibrosis and bleomycin (BLM)-induced fibrotic murine lungs. C-188-9, a small molecule STAT-3 inhibitor, decreased pulmonary fibrosis in the intraperitoneal BLM model as assessed by arterial oxygen saturation (control, 84.4 ± 1.3%; C-188-9, 94.4 ± 0.8%), histology (Ashcroft score: untreated, 5.4 ± 0.25; C-188-9, 3.3 ± 0.14), and attenuated fibrotic markers such as diminished α-smooth muscle actin, reduced collagen deposition. In addition, C-188-9 decreased the expression of epithelial injury markers, including hypoxia-inducible factor-1α (HIF-1α) and plasminogen activator inhibitor-1 (PAI-1). In vitro studies show that inhibition of STAT-3 decreased IL-6- and TGF-ß-induced expression of multiple genes, including HIF-1α and PAI-1, in AECs. Furthermore, C-188-9 decreased fibroblast-to-myofibroblast differentiation. Finally, TGF-ß stimulation of lung fibroblasts resulted in SMAD2/SMAD3-dependent phosphorylation of STAT-3. These findings demonstrate that STAT-3 contributes to the development of lung fibrosis and suggest that STAT-3 may be a therapeutic target in pulmonary fibrosis.

CCR2 deficiency promotes exacerbated chronic erosive neutrophil-dominated chikungunya virus arthritis.

UNLABELLED: Chikungunya virus (CHIKV) is a member of a globally distributed group of arthritogenic alphaviruses that cause weeks to months of debilitating polyarthritis/arthralgia, which is often poorly managed with current treatments. Arthritic disease is usually characterized by high levels of the chemokine CCL2 and a prodigious monocyte/macrophage infiltrate. Several inhibitors of CCL2 and its receptor CCR2 are in development and may find application for treatment of certain inflammatory conditions, including autoimmune and viral arthritides. Here we used CCR2(-/-) mice to determine the effect of CCR2 deficiency on CHIKV infection and arthritis. Although there were no significant changes in viral load or RNA persistence and only marginal changes in antiviral immunity, arthritic disease was substantially increased and prolonged in CCR2(-/-) mice compared to wild-type mice. The monocyte/macrophage infiltrate was replaced in CCR2(-/-) mice by a severe neutrophil (followed by an eosinophil) infiltrate and was associated with changes in the expression levels of multiple inflammatory mediators (including CXCL1, CXCL2, granulocyte colony-stimulating factor [G-CSF], interleukin-1ß [IL-1ß], and IL-10). The loss of anti-inflammatory macrophages and their activities (e.g., efferocytosis) was also implicated in exacerbated inflammation. Clear evidence of cartilage damage was also seen in CHIKV-infected CCR2(-/-) mice, a feature not normally associated with alphaviral arthritides. Although recruitment of CCR2(+) monocytes/macrophages can contribute to inflammation, it also appears to be critical for preventing excessive pathology and resolving inflammation following alphavirus infection. Caution might thus be warranted when considering therapeutic targeting of CCR2/CCL2 for the treatment of alphaviral arthritides. IMPORTANCE: Here we describe the first analysis of viral arthritis in mice deficient for the chemokine receptor CCR2. CCR2 is thought to be central to the monocyte/macrophage-dominated inflammatory arthritic infiltrates seen after infection with arthritogenic alphaviruses such as chikungunya virus. Surprisingly, the viral arthritis caused by chikungunya virus in CCR2-deficient mice was more severe, prolonged, and erosive and was neutrophil dominated, with viral replication and persistence not being significantly affected. Monocytes/macrophages recruited by CCL2 thus also appear to be important for both preventing even worse pathology mediated by neutrophils and promoting resolution of inflammation. Caution might thus be warranted when considering the use of therapeutic agents that target CCR2/CCL2 or inflammatory monocytes/macrophages for the treatment of alphaviral (and perhaps other viral) arthritides. Individuals with diminished CCR2 responses (due to drug treatment or other reasons) may also be at risk of exacerbated arthritic disease following alphaviral infection.