ABSTRACT
PURPOSE: Polysorbate excipients are commonly used as surfactants to stabilize therapeutic proteins in formulations. Degradation of polysorbates could lead to particle formation and instability of the drug formulation. We investigated how the fatty acid composition of polysorbate 80 impacts the degradation profile, particle formation, and product stability under stress conditions. METHODS: Two polysorbate 80-containing therapeutic protein formulations were reformulated with either Polysorbate 80 NF synthesized from a fatty acid mixture that contains mainly oleic acid (≥58%) or a version of polysorbate 80 synthesized with high oleic acid (>98%). Stress conditions, including high temperature and esterase spiking, were applied and changes to both the polysorbate and the therapeutic protein product were investigated for stability, purity, innate immune response and biological activity. RESULTS: The addition of esterase and storage at 37°C led to significant hydrolysis of the polysorbate and increases in sub-visible particle formation for both polysorbates tested. The fatty acid composition of polysorbate 80 did not directly alter the stability profile of either therapeutic protein as measured by size exclusion chromatography, or significantly impact innate immune response or biological activity. However, formulations with Polysorbate 80 NF showed greater propensity for sub-visible particle formation under stress conditions. CONCLUSIONS: These results suggest that composition of fatty acids in polysorbate 80 may be a promoter for sub-visible particulate formation under the stress conditions tested but may not impact protein aggregation or biological activity.
Subject(s)
Excipients/chemistry , Fatty Acids/chemistry , Polysorbates/chemistry , Rituximab/chemistry , Cell Line, Tumor , Chemistry, Pharmaceutical , Drug Compounding/methods , Humans , Immunity, Innate/drug effects , Leukocytes, Mononuclear , Protein Stability , Rituximab/pharmacology , Rituximab/therapeutic useABSTRACT
The interleukin 4 receptor (IL-4R) is a central mediator of T helper type 2 (T(H)2)-mediated disease and associates with either the common gamma-chain to form the type I IL-4R or with the IL-13R alpha1 chain (IL-13Ralpha1) to form the type II IL-4R. Here we used Il13ra1-/- mice to characterize the distinct functions of type I and type II IL-4 receptors in vivo. In contrast to Il4ra-/- mice, which have weak T(H)2 responses, Il13ra1-/- mice had exacerbated T(H)2 responses. Il13ra1-/- mice showed much less mortality after infection with Schistosoma mansoni and much more susceptibility to Nippostrongylus brasiliensis. IL-13Ralpha1 was essential for allergen-induced airway hyperreactivity and mucus hypersecretion but not for fibroblast or alternative macrophage activation. Thus, type I and II IL-4 receptors exert distinct effects on immune responses.
Subject(s)
Interleukin-13 Receptor alpha1 Subunit/physiology , Receptors, Interleukin-4, Type II/physiology , Th2 Cells/immunology , Allergens/immunology , Animals , Antigens, Helminth/immunology , Bronchial Hyperreactivity/immunology , Cells, Cultured , Disease Susceptibility , Fibroblasts/immunology , Interleukin-13 Receptor alpha1 Subunit/genetics , Macrophages/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Mucus/metabolism , Nippostrongylus/physiology , Schistosoma mansoni/immunology , Schistosoma mansoni/physiology , Schistosomiasis mansoni/immunology , Schistosomiasis mansoni/mortality , Strongylida Infections/immunologyABSTRACT
Respiratory syncytial virus (RSV) infects small foci of respiratory epithelial cells via infected droplets. Infection induces expression of type I and III interferons (IFNs) and proinflammatory cytokines, the balance of which may restrict viral replication and affect disease severity. We explored this balance by infecting two respiratory epithelial cell lines with low doses of recombinant RSV expressing green fluorescent protein (rgRSV). A549 cells were highly permissive, whereas BEAS-2B cells restricted infection to individual cells or small foci. After infection, A549 cells expressed higher levels of IFN-ß-, IFN-λ-, and NF-κB-inducible proinflammatory cytokines. In contrast, BEAS-2B cells expressed higher levels of antiviral interferon-stimulated genes, pattern recognition receptors, and other signaling intermediaries constitutively and after infection. Transcriptome analysis revealed that constitutive expression of antiviral and proinflammatory genes predicted responses by each cell line. These two cell lines provide a model for elucidating critical mediators of local control of viral infection in respiratory epithelial cells.IMPORTANCE Airway epithelium is both the primary target of and the first defense against respiratory syncytial virus (RSV). Whether RSV replicates and spreads to adjacent epithelial cells depends on the quality of their innate immune responses. A549 and BEAS-2B are alveolar and bronchial epithelial cell lines, respectively, that are often used to study RSV infection. We show that A549 cells are permissive to RSV infection and express genes characteristic of a proinflammatory response. In contrast, BEAS-2B cells restrict infection and express genes characteristic of an antiviral response associated with expression of type I and III interferons. Transcriptome analysis of constitutive gene expression revealed patterns that may predict the response of each cell line to infection. This study suggests that restrictive and permissive cell lines may provide a model for identifying critical mediators of local control of infection and stresses the importance of the constitutive antiviral state for the response to viral challenge.
Subject(s)
Cytokines/immunology , Epithelial Cells/immunology , Gene Expression Regulation/immunology , Respiratory Mucosa/immunology , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Viruses/immunology , A549 Cells , Epithelial Cells/virology , Humans , Respiratory Mucosa/virology , Respiratory Syncytial Virus Infections/pathologyABSTRACT
Type III IFNs are important mediators of antiviral immunity. IFN-λ4 is a unique type III IFN because it is produced only in individuals who carry a dG allele of a genetic variant rs368234815-dG/TT. Counterintuitively, those individuals who can produce IFN-λ4, an antiviral cytokine, are also less likely to clear hepatitis C virus infection. In this study, we searched for unique functional properties of IFN-λ4 that might explain its negative effect on hepatitis C virus clearance. We used fresh primary human hepatocytes (PHHs) treated with recombinant type III IFNs or infected with Sendai virus to model acute viral infection and subsequently validated our findings in HepG2 cell line models. Endogenous IFN-λ4 protein was detectable only in Sendai virus-infected PHHs from individuals with the dG allele, where it was poorly secreted but highly functional, even at concentrations < 50 pg/ml. IFN-λ4 acted faster than other type III IFNs in inducing antiviral genes, as well as negative regulators of the IFN response, such as USP18 and SOCS1 Transient treatment of PHHs with IFN-λ4, but not IFN-λ3, caused a strong and sustained induction of SOCS1 and refractoriness to further stimulation with IFN-λ3. Our results suggest unique functional properties of IFN-λ4 that can be important in viral clearance and other clinical conditions.
Subject(s)
Alleles , Hepatocytes/immunology , Interferons/genetics , Interleukins/genetics , Respirovirus Infections/immunology , Sendai virus/immunology , Adolescent , Adult , Aged , Endopeptidases/genetics , Female , Hep G2 Cells , Hepacivirus/immunology , Hepatitis C/genetics , Hepatitis C/immunology , Hepatocytes/virology , Humans , Immunity , Interferons/metabolism , Interleukins/metabolism , Male , Middle Aged , Polymorphism, Single Nucleotide , Suppressor of Cytokine Signaling 1 Protein/genetics , Ubiquitin Thiolesterase , Up-Regulation , Viral Load , Young AdultABSTRACT
Plasmacytoid dendritic cells (pDC) are rare cells found in peripheral blood and lymphoid tissues. pDC are considered to be "professional" type I IFN-producing cells and produce 10- to 100-fold more IFN-α than other cell types in response to enveloped viruses or synthetic TLR7 and TLR9 agonists. In this study, purified pDC were found to express high levels of IFN-λ receptor mRNA, as well as cell-surface IFN-λ receptor. We have developed intracellular flow cytometry assays using Abs to IFN-λ1/3 or -λ2 to assess the expression of IFN-λ proteins by pDC. We observed that a subset of human pDC expresses only intracellular IFN-α, whereas another subset produces both IFN-α and IFN-λ after stimulation with virus or the TLR9 agonist, CpG A; the cells that coexpressed IFN-α and IFN-λ were the cells with the highest levels of IFN-α expression. Ab cross-linking of CD4 or CD303 molecules on pDC inhibited both HSV-induced IFN-λ and IFN-α production. Like the production of IFN-α, the HSV-induced IFN-λ production in pDC was mediated through TLR9 and independent of virus replication. Exogenous IFN-λ treatment of pDC resulted in increased virus-induced expression of both IFN-α and IFN-λ. In addition, both exogenous IFN-λ and -α inhibited dexamethasone-induced apoptosis of pDC. We conclude that pDC are major producers of IFN-λ1 and -λ2 in response to viral stimulation and also express functional receptors for this cytokine. Thus, IFN-λ can serve as an autocrine signal to strengthen the antiviral response of pDC by increasing IFN-α and IFN-λ production, resulting in prolonged pDC survival.
Subject(s)
Dendritic Cells/immunology , Dendritic Cells/metabolism , Interleukins/biosynthesis , Interleukins/physiology , Cells, Cultured , Dendritic Cells/virology , HEK293 Cells , Hep G2 Cells , Herpesvirus 1, Human/immunology , Humans , Influenza A virus/immunology , Interferons , Interleukins/genetics , Receptors, Cytokine/metabolism , Sendai virus/immunologyABSTRACT
Rapid depletion of groundwater and climate change mediated shifting precipitation pattern is forcing farmers to look for alternative irrigation options like wastewater. However, routine irrigation with trace metal contaminated wastewaters could potentially pollute soil as well as cause health risks through the consumption of food products grown in contaminated soil. Thus, the present study aimed to investigate the trace metals build-up status in topsoil and potato (Solanum tuberosum L.) tubers upon continuous irrigation with coalmine effluent contaminated wastewater compared to irrigation with groundwater and surface water over three consecutive years. Soil pollution status and human health risk associated with consumption of potato tubers grown on wastewater-irrigated soil was also assessed in this study. Three separate experimental sites differing in irrigation source (groundwater, surface water, and coalmine wastewater) were selected near Barapukuria Coal Mining Company Limited located at Parbatipur upazilla of Dinajpur district, Bangladesh. Nine trace metals namely arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb), and zinc (Zn) were estimated. Results showed significantly higher trace metal content in both soil and potato tubers due to wastewater irrigation. Wastewater suitability for irrigation regarding Cd, Cr, Cu, Fe, Ni and Pb were off the permissible level although the soil contamination with trace metals and their levels in potato tubers remained within the safety limit. Health risk assessment revealed that, consumption of potato tubers grown in wastewater-irrigated soil remained safe although health risk associated with Cr was almost at the border. The study exclusively highlighted the core massage that, trace metal contamination of both soil and potatoes cultivated in them was increasing alarmingly due to three years of wastewater-irrigation. Although the extent of contamination was below critical limit, it can potentially become hazardous in years to come unless wastewater-irrigation is checked. This study was successful to provide valuable insights regarding the potential environmental and human health threats that might arise due to unmindful irrigation of contaminated coalmine wastewater. Besides, this study should prove useful in strategizing safety measures for cropping under trace metal contaminated soils and for planning industrial effluent disposal to avoid agricultural soil contamination.
ABSTRACT
The human beta-coronavirus strain, OC43, provides a useful model for testing the antiviral activity of various agents. We compared the activity of several antiviral drugs against OC43, including remdesivir, chloroquine, interferon (IFN)-ß, IFN-λ1, and IFN-λ4, in two distinct cell types: human colorectal carcinoma cell line (HCT-8 cells) and normal human bronchial epithelial (NHBE) cells. We also tested whether these agents mediate additive, synergistic, or antagonistic activity against OC43 infection when used in combination. When used as single agents, remdesivir exhibited stronger antiviral activity than chloroquine, and IFN-ß exhibited stronger activity than IFN-λ1 or IFN-λ4 against OC43 in both HCT-8 and NHBE cells. Anakinra (IL-1 inhibitor) and tocilizumab (IL-6 inhibitor) did not mediate any antiviral activity. The combination of IFN-ß plus chloroquine or remdesivir resulted in higher synergy scores and higher expression of IFN-stimulated genes than did IFN-ß alone. In contrast, the combination of remdesivir plus chloroquine resulted in an antagonistic interaction in NHBE cells. Our findings indicate that the combined use of IFN-ß plus remdesivir or chloroquine induces maximal antiviral activity against human coronavirus strain OC43 in primary human respiratory epithelial cells. Furthermore, our experimental OC43 virus infection model provides an excellent method for evaluating the biological activity of antiviral drugs.
Subject(s)
Coronavirus Infections , Coronavirus OC43, Human , Humans , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Interferon-beta/pharmacology , Interferon-beta/therapeutic use , Coronavirus OC43, Human/genetics , Coronavirus OC43, Human/metabolism , Chloroquine/pharmacology , Chloroquine/therapeutic use , Coronavirus Infections/drug therapy , Interferons/metabolismABSTRACT
Drought is a major abiotic stress that severely limits sustainable wheat (Triticum aestivum L.) productivity via morphological and physio-biochemical alterations of cellular processes. The complex nature and polygenic control of drought tolerance traits make breeding tolerant genotypes quite challenging. However, naturally occurring variabilities among wheat germplasm resources could potentially help combating drought. The present study was conducted to assess the drought tolerance of 18 Bangladeshi hexaploid wheat genotypes, focusing on the identification of potent sources of diversity by combining microsatellite markers, also known as single sequence repeat markers, and morpho-physiological characteristics that might help accelerating wheat crop improvement programs. Initially, the genotypes were evaluated using 25 microsatellite markers followed by an on-field evaluation of 7 morphological traits (plant height, spike number, spike length, grains per spike, 1000-grain weight, grain yield, biological yield) and 6 physiological traits (SPAD value, membrane stability index, leaf relative water content, proline content, canopy temperature depression, and leaf K+ ion content). The field-trial was conducted in a factorial fashion of 18 wheat genotypes and two water regimes (control and drought) following a split-plot randomized complete block design. Regardless of genotype, drought was significantly damaging for all the tested traits; however, substantial variability in drought stress tolerance was evident among the genotypes. Spike length, 1000-grain weight, SPAD value, leaf relative water content, canopy temperature depression, proline content, and potassium (K+) ion content were the most representative of drought-induced growth and yield impairments and also correlated well with the contrasting ability of genotypic tolerance. Microsatellite markers amplified 244 alleles exhibiting 79% genetic diversity. Out of 25 markers, 23 was highly polymorphic showing 77% average polymorphism. Morpho-physiological trait-based hierarchical clustering and microsatellite marker-based neighbor-jointing clustering both revealed three genotypic clusters with 71% co-linearity between them. In both cases, the genotypes Kanchan, BAW-1147, BINA Gom 1, BARI Gom 22, BARI Gom 26, and BARI Gom 33 were found to be comparatively more tolerant than the other tested genotypes, showing potential for cultivation in water-deficit environments. The findings of this study would contribute to the present understanding of drought tolerance in wheat and would provide a basis for future genotype selection for drought-tolerant wheat breeding programs.
ABSTRACT
Sugarcane (Saccharum officinarum L.), a globally cultivated carbohydrate producing crop of industrial importance is being challenged by soil salinity due to its glycophytic nature. Water stress coupled with cellular and metabolic alterations resulting from excess sodium (Na+) ion accumulation is irreversibly damaging during early crop developmental stages that often results in complete crop failure. This study therefore aimed to explore the potential of salicylic acid as a sett priming material to mitigate the negative effects of salt stress on sugarcane during germination and early growth stages. Five doses of salicylic acid (0 [hydropriming] [control], 0.5 mM, 1 mM, 1.5 mM and 2 mM) were tested against three levels of salinity (0.5 dS m-1 [control], 4 dS m-1, and 8 dS m-1) within a polyhouse environment. Results revealed 11.2%, 18.5%, 25.4%, and 38.6%, average increase in final germination, germination energy, seedling length and seedling vigor index respectively with a subsequent reduction of 21% mean germination time. Investigations during early seedling growth revealed 21.6%, 17.5%, 27.0%, 39.9%, 10.7%, 11.5%, 17.5%, 47.9%, 35.3% and 20.5% overall increase in plant height, total leaf area, shoot dry matter, root dry matter, leaf greenness, relative water content, membrane stability index, proline content, total antioxidant activity and potassium (K+) ion accumulation respectively with a subsequent reduction of 24.9% Na+ ion accumulation and 35.8% Na+/K+ ratio due to salicylic acid priming. Germination, seedling growth and recovery of physiochemical traits were highly satisfactory in primed setts than non-primed ones even under 8 dS m-1 salinity level. This study should provide useful information for strategizing salinity management approaches for better productivity of sugarcane.
ABSTRACT
Type I interferons (IFN-alpha/beta) induce apoptosis in certain tumor cell lines but not others. Here we describe a mutation in STAT2 that confers an apoptotic effect in tumor cells in response to type I IFNs. This mutation was introduced in a conserved motif, PYTK, located in the STAT SH2 domain, which is shared by STAT1, STAT2, and STAT3. To test whether the tyrosine in this motif might be phosphorylated and affect signaling, Y631 of STAT2 was mutated to phenylalanine (Y631F). Although it was determined that Y631 was not phosphorylated, the Y631F mutation conferred sustained signaling and induction of IFN-stimulated genes. This prolonged IFN response was associated with sustained tyrosine phosphorylation of STAT1 and STAT2 and their mutual association as heterodimers, which resulted from resistance to dephosphorylation by the nuclear tyrosine phosphatase TcPTP. Finally, cells bearing the Y631F mutation in STAT2 underwent apoptosis after IFN-alpha stimulation compared with wild-type STAT2. Therefore, this mutation reveals that a prolonged response to IFN-alpha could account for one difference between tumor cell lines that undergo IFN-alpha-induced apoptosis compared with those that display an antiproliferative response but do not die.
Subject(s)
Apoptosis/drug effects , Interferon Type I/pharmacology , Mutation/genetics , Phosphotyrosine/metabolism , STAT1 Transcription Factor/metabolism , STAT2 Transcription Factor/metabolism , src Homology Domains , Amino Acid Motifs , Amino Acid Sequence , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Conserved Sequence , Humans , Janus Kinases/metabolism , Molecular Sequence Data , Phosphorylation/drug effects , STAT1 Transcription Factor/chemistry , STAT2 Transcription Factor/chemistry , Transcription, Genetic/drug effectsABSTRACT
PEGylated recombinant human granulocyte colony stimulating factor (pegfilgrastim) is used clinically to accelerate immune reconstitution following chemotherapy and is being pursued for biosimilar development. One challenge to overcome in pegfilgrastim biosimilar development is establishing pharmacokinetic (PK) similarity, which is partly due to the degree of PK variability. We herein report that commercially available G-CSF and PEG ELISA detection kits have different capacities to detect pegfilgrastim aggregates that rapidly form in vitro in physiological conditions. These aggregates can be observed using SDS-PAGE, size-exclusion chromatography, dynamic light scattering, and real-time NMR analysis and are associated with decreased bioactivity as reflected by reduced drug-induced cellular proliferation and STAT3 phosphorylation. Furthermore, individual variability in the stability and detectability of pegfilgrastim in human sera is also observed. Pegfilgrastim levels display marked subject variability in sera from healthy donors incubated at 37 °C. The stability patterns of pegfilgrastim closely match the stability patterns of filgrastim, consistent with a key role for pegfilgrastim's G-CSF moiety in driving formation of inactive aggregates. Taken together, our results indicate that individual variability and ELISA specificity for inactive aggregates are key factors to consider when designing and interpreting studies involving the measurement of serum pegfilgrastim concentrations.
Subject(s)
Biological Variation, Individual , Filgrastim/pharmacokinetics , Polyethylene Glycols/pharmacokinetics , Animals , Cell Line, Tumor , Cell Proliferation , Enzyme-Linked Immunosorbent Assay/standards , Humans , Mice , STAT3 Transcription Factor/metabolismABSTRACT
Secretoglobin (SCGB) 3A1 and 3A2 are members of the small molecular weight secretoglobin gene superfamily. SCGB3A1 is a tumor suppressor gene, whereas SCGB3A2 has anti-inflammatory properties. Both genes are mainly expressed in the lung and trachea in mice. Whether the expression and/or function of these two genes are related is not known. Here we show that the expression of SCGB3A1 and SCGB3A2 are bidirectionally regulated by oncostatin M (OSM) when examined in a mouse transformed Clara cell line (mtCC); SCGB3A1 is up-regulated by OSM, while SCGB3A2 is down-regulated in a time- and dose-dependent manner. OSM-activated STAT3/5, through binding to the STAT-binding element located at -201 to -209 bp in the mouse Scgb3a1 gene promoter, and the extracellular signal-regulated kinase (ERK)- and p38-mitogen-activated protein kinase (MAPK) pathways are responsible for the OSM-induced up-regulation of SCGB3A1 expression. On the other hand, the -113 to -273 bp region in the Scgb3a2 promoter appears to be responsible for the OSM induced down-regulation of the gene. No significant differences in the levels or patterns of specific DNA-binding proteins were found in the -113 to -273 bp region as determined by electrophoretic mobility shift assays. Neither the ERK- nor p38-MAPK pathways were involved in the OSM-induced reduction of Scgb3a2 promoter activity. These results suggest that OSM-induced suppression of SCGB3A2 expression is an indirect effect of OSM. Expression of the Clara cell marker, CYP2F2, was markedly decreased upon OSM treatment in parallel with the decrease of SCGB3A2 expression in mtCC cells. The differential regulation of Scgb3a1 and Scgb3a2 gene expression by OSM may explain the unique functions of these genes in the lung.
Subject(s)
Gene Expression Regulation/drug effects , Oncostatin M/pharmacology , Proteins/genetics , Animals , Base Sequence , MAP Kinase Signaling System/drug effects , Mice , Mice, Inbred C57BL , Molecular Sequence Data , NIH 3T3 Cells , Oncostatin M/administration & dosage , Phenotype , Protein Binding/drug effects , Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Respiratory System/metabolism , Respiratory System/pathology , Response Elements/genetics , STAT Transcription Factors/metabolism , SecretoglobinsABSTRACT
Recently discovered type III IFNs (IFN-lambda) exert their antiviral and immunomodulatory activities through a unique receptor complex composed of IFN-lambdaR1 and interleukin-10 receptor 2. To further study type III IFNs, we cloned and characterized mouse IFN-lambda ligand-receptor system. We showed that, similar to their human orthologues, mIFN-lambda2 and mIFN-lambda3 signal through the IFN-lambda receptor complex, activate IFN stimulated gene factor 3, and are capable of inducing antiviral protection and MHC class I antigen expression in several cell types including B16 melanoma cells. We then used the murine B16 melanoma model to investigate the potential antitumor activities of IFN-lambdas. We developed B16 cells constitutively expressing murine IFN-lambda2 (B16.IFN-lambda2 cells) and evaluated their tumorigenicity in syngeneic C57BL/6 mice. Although constitutive expression of mIFN-lambda2 in melanoma cells did not affect their proliferation in vitro, the growth of B16.IFN-lambda2 cells, when injected s.c. into mice, was either retarded or completely prevented. We found that rejection of the modified tumor cells correlated with their level of IFN-lambda2 expression. We then developed IFN-lambda-resistant B16.IFN-lambda2 cells (B16.IFN-lambda2Res cells) and showed that their tumorigenicity was also highly impaired or completely abolished similar to B16.IFN-lambda2 cells, suggesting that IFN-lambdas engage host mechanisms to inhibit melanoma growth. These in vivo experiments show the antitumor activities of IFN-lambdas and suggest their strong therapeutic potential.
Subject(s)
Interferons/immunology , Melanoma, Experimental/immunology , Melanoma, Experimental/therapy , Receptors, Interferon/immunology , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Cloning, Molecular , Cricetinae , Female , Humans , Interferons/genetics , Ligands , Melanoma, Experimental/genetics , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Receptors, Interferon/genetics , Signal Transduction , TransfectionABSTRACT
The influenza virus NS1 protein interacts with a wide range of proteins to suppress the host cell immune response and facilitate virus replication. The amino acid sequence of the 2009 pandemic virus NS1 protein differed from sequences of earlier related viruses. The functional impact of these differences has not been fully defined. Therefore, we made mutations to the NS1 protein based on these sequence differences, and assessed the impact of these changes on host cell interferon (IFN) responses. We found that viruses with mutations at position 171 replicated efficiently but did not induce expression of interferon genes as effectively as wild-type viruses in A459 lung epithelial cells. The decreased ability of these NS1 mutant viruses to induce IFN gene and protein expression correlated with decreased activation of STAT1 and lower levels of IFN-stimulated gene (ISG) expression. These findings demonstrate that mutations at position 171 in the NS1 protein result in decreased expression of IFN and ISGs by A549 cells. Consequently, these viruses may be more virulent than the parental strains that do not contain mutations at position 171 in the NS1 protein.
Subject(s)
Epithelial Cells/immunology , Immunity, Innate , Influenza A Virus, H1N1 Subtype/genetics , Influenza, Human/immunology , Interferons/immunology , Viral Nonstructural Proteins/genetics , Amino Acid Motifs , Epithelial Cells/virology , Host-Pathogen Interactions , Humans , Influenza A Virus, H1N1 Subtype/chemistry , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H1N1 Subtype/physiology , Influenza, Human/genetics , Influenza, Human/virology , Interferons/genetics , Mutation , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/immunology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/immunology , Virus ReplicationABSTRACT
Influenza A viruses pose a constant potential threat to human health. In view of the innate antiviral activity of interferons (IFNs) and their potential use as anti-influenza agents, it is important to know whether viral resistance to these antiviral proteins can arise. To examine the likelihood of emergence of IFN-λ1-resistant H1N1 variants, we serially passaged the A/California/04/09 (H1N1) strain in a human lung epithelial cell line (Calu-3) in the presence of increasing concentrations of recombinant IFN-λ1 protein. To monitor changes associated with adaptation of this virus to growth in Calu-3 cells, we also passaged the wild-type virus in the absence of IFN-λ1. Under IFN-λ1 selective pressure, the parental virus developed two neuraminidase (NA) mutations, S79L and K331N, which significantly reduced NA enzyme activity (↓1.4-fold) and sensitivity to IFN-λ1 (↓Ë20-fold), respectively. These changes were not associated with a reduction in viral replication levels. Mutants carrying either K331N alone or S79L and K331N together induced weaker phosphorylation of IFN regulatory factor 3 (IRF3), and, as a consequence, much lower expression of the IFN genes (IFNB1, IFNL1 and IFNL2/3) and proteins (IFN-λ1 and IFN-λ2/3). The lower levels of IFN expression correlated with weaker induction of tyrosine-phosphorylated STAT1 and reduced RIG-I protein levels. Our findings demonstrate that influenza viruses can develop increased resistance to the antiviral activity of type III interferons.
Subject(s)
Drug Resistance, Viral/drug effects , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/physiology , Interleukins/pharmacology , Amino Acid Substitution/genetics , Animals , Antiviral Agents/pharmacology , Cell Line , DEAD Box Protein 58/metabolism , DNA-Directed RNA Polymerases/metabolism , Dogs , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation/drug effects , Humans , Immunity, Innate/drug effects , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H1N1 Subtype/growth & development , Interferon Regulatory Factor-3/metabolism , Interferons , Mutation/genetics , Neuraminidase/genetics , Phosphorylation/drug effects , Receptors, Immunologic , Receptors, Virus/genetics , Recombination, Genetic/genetics , STAT1 Transcription Factor/metabolism , Sequence Analysis, DNA , Virus Replication/drug effectsABSTRACT
Several novel interleukin (IL)-10-related cytokines have recently been discovered. These include IL-22, IL-26, and the interferon-lambda (IFN-lambda) proteins IFN-lambda1 (IL-29), IFN-lambda2 (IL-28A), and IFN-lambda3 (IL-28B). The ligand-binding chains for IL-22, IL-26, and IFN-lambda are distinct from that used by IL-10; however, all of these cytokines use a common second chain, IL-10 receptor-2 (IL-10R2; CRF2-4), to assemble their active receptor complexes. Thus, IL-10R2 is a shared component in at least four distinct class II cytokine-receptor complexes. IL-10 binds to IL-10R1; IL-22 binds to IL-22R1; IL-26 binds to IL-20R1; and IFN-lambda binds to IFN-lambdaR1 (also known as IL-28R). The binding of these ligands to their respective R1 chains induces a conformational change that enables IL-10R2 to interact with the newly formed ligand-receptor complexes. This in turn activates a signal-transduction cascade that results in rapid activation of several transcription factors, particularly signal transducer and activator of transcription (STAT)3 and to a lesser degree, STAT1. Activation by IL-10, IL-22, IL-26, or IFN-lambda can be blocked with neutralizing antibodies to the IL-10R2 chain. Although IL-10R2 is broadly expressed on a wide variety of tissues, only a subset of these tissues expresses the ligand-binding R1 chains. The receptors for these cytokines are often present on cell lines derived from various tumors, including liver, colorectal, and pancreatic carcinomas. Consequently, the receptors for these cytokines may provide novel targets for inhibiting the growth of certain types of cancer.
Subject(s)
Cytokines/metabolism , Membrane Glycoproteins/metabolism , Receptors, Cytokine/metabolism , Animals , Humans , Interleukin-10 Receptor beta Subunit , Receptors, Interferon/genetics , Receptors, Interleukin/metabolism , Interferon gamma ReceptorABSTRACT
Macrophages coexpress both the interleukin (IL)-2Rγ chain (γ(c)) and IL-13Rα1. These receptor chains can heterodimerize with IL-4Rα to form type I or type II IL-4 receptor complexes, respectively. We used macrophages derived from Il2rg and Il13ra1 knockout (KO) mice to evaluate the requirements for these receptor chains for induction of the alternative macrophage activation (AMA) pathway by IL-4 and IL-13. Absence of γ(c) significantly decreased activation of STAT6 by IL-4 but not IL-13. However, although activation of STAT6 by IL-4 was markedly reduced in γ(c) KO macrophages, it was not abolished, indicating that IL-4 can still signal through type II IL-4 receptors via the IL-13Rα1 chain. IL-13 failed to activate STAT6 in macrophages derived from Il13ra1 KO mice; however, these cells remained fully responsive to IL-4. The inability of IL-13 but not IL-4 to signal in Il13ra1(-/-) macrophages correlated with the inability of IL-13 but not IL-4 to induce expression of genes such as Arg1, Retnla and Ccl11 that are characteristically expressed by alternatively activated macrophages. In addition, IL-13 but not IL-4 failed to induce membrane fusion and giant cell formation by Il13ra1 KO macrophages. These findings demonstrate that the IL-13Rα1 chain is essential for induction of the AMA pathway by IL-13 but not IL-4.
Subject(s)
Complement Pathway, Alternative , Interleukin-13 Receptor alpha1 Subunit/metabolism , Interleukin-13/metabolism , Interleukin-4/metabolism , Macrophages/immunology , Animals , Arginase/genetics , Arginase/metabolism , CD11 Antigens/genetics , CD11 Antigens/metabolism , Cells, Cultured , Complement Pathway, Alternative/genetics , Gene Expression Regulation/genetics , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Interleukin Receptor Common gamma Subunit/genetics , Interleukin-13/genetics , Interleukin-13 Receptor alpha1 Subunit/genetics , Interleukin-4/genetics , Membrane Fusion/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , STAT6 Transcription Factor/metabolismABSTRACT
Environmental factor(s), such as viral infection, has been implicated as one of the triggering events leading to neuroinflammation in multiple sclerosis. This study underlines the importance of double-stranded RNA (dsRNA), the active component of a viral infection, in inducing the expression of inducible nitric oxide synthase (iNOS) in human astroglia. DsRNA in the form of synthetic polyinosinic-polycytidylic acid (poly IC) induced expression of iNOS and iNOS promoter-driven luciferase activity through activation of nuclear factor (NF)-kappaB and CCAAT/enhancer-binding proteinbeta (C/EBPbeta). In addition, we show that inhibitors of protein kinase R attenuated iNOS by suppressing the activation of NF-kappaB but not C/EBPbeta. In contrast, knock down of p38 mitogen-activated protein kinase (MAPK) attenuated iNOS by suppressing the activation of C/EBPbeta but not NF-kappaB. This study delineates a novel role of dsRNA in inducing the expression of iNOS through dsRNA-activated protein kinase (PKR)-mediated activation of NF-kappaB and p38-mediated activation of C/EBPbeta in human astroglia that may participate in virus-induced neurological abnormalities.
Subject(s)
Astrocytes/enzymology , Nitric Oxide Synthase/metabolism , RNA, Double-Stranded/metabolism , eIF-2 Kinase/metabolism , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cell Line , Cells, Cultured , Enzyme Activation , Enzyme Induction/drug effects , Enzyme Inhibitors/pharmacology , Genes, Reporter , Humans , Luciferases/genetics , Luciferases/metabolism , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Nitric Oxide Synthase Type II , Poly I-C/pharmacology , Promoter Regions, Genetic , Time Factors , Transcriptional Activation , Transfection , p38 Mitogen-Activated Protein KinasesABSTRACT
TLR agonists such as LPS and poly(I:C) induce expression of type I IFNs, such as IFN-α and -ß, by macrophages. To examine the role of IFN-ß in the induction of ISGs by LPS, we compared the ability of LPS to induce ISGF3 activity and ISG expression in bone marrow-derived macrophages from WT and Ifnb1(-/-) mice. We found that LPS treatment activated ISGF3 and induced expression of ISGs such as Oas1, Mx1, Ddx58 (RIG-I), and Ifih1 (MDA5) in WT macrophages, but not in macrophages derived from Ifnb1(-/-) mice or Ifnar1(-/-) mice. The inability of LPS to induce activation of ISGF3 and ISG expression in Ifnb1(-/-) macrophages correlated with the failure of LPS to induce activation of STAT1 and -2 in these cells. Consistent with these findings, LPS treatment also failed to induce ISG expression in bone marrow-derived macrophages from Stat2 KO mice. Although activation of ISGF3 and induction of ISG expression by LPS was abrogated in Ifnb1(-/-) and Ifnar1(-/-) macrophages, activation of NF-κB and induction of NF-κB-responsive genes, such as Tnf (TNF-α) and Il1b (IL-1ß), were not affected by deletion of either the IFN-ß or IFN-αR1 genes. These findings demonstrate that induction of ISGF3 activity and ISG expression by LPS is critically dependent on intermediate production of IFN-ß and autocrine signaling through type I IFN receptors.
Subject(s)
Gene Expression Regulation , Interferon-beta/metabolism , Macrophages/immunology , Macrophages/metabolism , Animals , Gene Expression Regulation/drug effects , Interferon-beta/genetics , Lipopolysaccharides/immunology , Mice , Mice, Knockout , Receptor, Interferon alpha-beta/genetics , Receptor, Interferon alpha-beta/metabolism , STAT1 Transcription Factor/metabolism , STAT2 Transcription Factor/metabolism , Toll-Like Receptors/agonists , Toll-Like Receptors/metabolism , Transcription Factors/metabolismABSTRACT
This study compared the ability of IFN-α and IFN-λ to induce signal transduction and gene expression in primary human hepatocytes, PBLs, and monocytes. IFN-α drug products are widely used to treat chronic HCV infection; however, IFN-α therapy often induces hematologic toxicities as a result of the broad expression of IFNARs on many cell types, including most leukocytes. rIFN-λ1 is currently being tested as a potential alternative to IFN-α for treating chronic HCV. Although IFN-λ has been shown to be active on hepatoma cell lines, such as HepG2 and Huh-7, its ability to induce responses in primary human hepatocytes or leukocytes has not been examined. We found that IFN-λ induces activation of Jak/STAT signaling in mouse and human hepatocytes, and the ability of IFN-λ to induce STAT activation correlates with induction of numerous ISGs. Although the magnitude of ISG expression induced by IFN-λ in hepatocytes was generally lower than that induced by IFN-α, the repertoire of regulated genes was quite similar. Our findings demonstrate that although IFN-α and IFN-λ signal through distinct receptors, they induce expression of a common set of ISGs in hepatocytes. However, unlike IFN-α, IFN-λ did not induce STAT activation or ISG expression by purified lymphocytes or monocytes. This important functional difference may provide a clinical advantage for IFN-λ as a treatment for chronic HCV infection, as it is less likely to induce the leukopenias that are often associated with IFN-α therapy.