RESUMEN
Therapeutic proteins can induce immune responses that affect their safety and efficacy. Product aggregates and innate immune response modulating impurities (IIRMI) are risk factors of product immunogenicity. In this study, we use Intravenous Immunoglobulin (IVIG), Avastin, and Human Serum Albumin (HSA) to explore whether increased aggregates activate innate immune cells or modify the response to IIRMI. We show that increased aggregates (shaken or stirred) in IVIG and Avastin, but not HSA, induced activation of MAPKs (pp38, pERK and pJNK) and transcription of immune-related genes including IL8, IL6, IL1ß, CSF1, CCL2, CCL7, CCL3, CCL24, CXCL2, IRAK1, EGR2, CEBPß, PPARg and TNFSF15 in human PBMC. The immunomodulatory effect was primarily mediated by FcγR, but not by TLR. Interestingly, increased aggregates in IVIG or Avastin magnified innate immune responses to TLR2/4 agonists, but diminished responses to TLR3/9 agonists. This study shows that IIRMI and aggregates can modify the activity of immune cells potentially modifying the milieu where the products are delivered highlighting the complex interplay of different impurities on product immunogenicity risk. Further, we show that aggregates could modify the sensitivity of PBMC-based assays designed to detect IIRMI. Understanding and managing immunogenicity risk is a critical component of product development and regulation.
Asunto(s)
Inmunidad Innata/inmunología , Inmunoglobulinas Intravenosas/inmunología , Albúmina Sérica Humana/inmunología , Animales , Formación de Anticuerpos/inmunología , Bevacizumab/inmunología , Células Cultivadas , Células HEK293 , Humanos , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/inmunología , Transcripción Genética/inmunologíaRESUMEN
Immunogenicity can have devastating consequences on the safety and efficacy of therapeutic proteins. Therefore, evaluating and mitigating the risk of product immunogenicity is critical for the development these products. This study, showed that Betaseron and Extavia, which are reported to be more immunogenic among IFNß products in clinical usage, contain residual innate immune response modulating impurities (IIRMIs) capable of activating NF-κB and induced expression of inflammatory mediators. These IIRMIs were undetectable in Rebif or Avonex. The stimulatory effect was attributed solely to IIRMIs because it was evident in murine cells lacking the interferon receptor (IFNAR). The IIRMIs in Betaseron and Extavia triggered NF-κB activation in HEK-293 cells bearing TLR2 and TLR4 in MyD88 dependent manner. Importantly, the IIRMIs in Betaseron induced up-regulation of IL-6, IL-1ß, and ccl5 in the skin of IFNAR knock out mice following subcutaneous administration. This indicates that trace level IIRMIs in Betaseron could contribute to the higher immunogenicity rates seen in clinics. Together these data suggest that cell based assays can reveal subtle but clinically relevant differences in IIRMIs following manufacturing changes or between products with the same active ingredients but different manufacturing processes. Appreciating these differences may inform immunogenicity risk assessments.
Asunto(s)
Contaminación de Medicamentos , Interferon beta-1b/normas , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Células Cultivadas , Células HEK293 , Humanos , Interferon beta-1b/química , Interleucinas/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Endotoxin tolerance is a complex phenomenon characterized primarily by decreased production of proinflammatory cytokines, chemokines, and other inflammatory mediators, whereas the expression of other genes are induced or unchanged. Endotoxin tolerance is induced by prior exposure of murine macrophages/human monocytes, experimental animals, or people to TLR ligands. Although recent studies reported a possible relationship between endotoxin tolerance and differentiation of alternatively activated macrophages (AA-MΦs or M2), we show in this study that LPS pretreatment of IL-4Rα(-/-) and STAT6(-/-) macrophages, which fail to develop into AA-MΦs, resulted in tolerance of proinflammatory cytokines, as well as molecules and chemokines previously associated with AA-MΦs (e.g., arginase-1, mannose receptor, CCL2, CCL17, and CCL22). In contrast to LPS, wild-type (WT) MΦs pretreated with IL-4, the prototype inducer of AA-MΦs, did not induce endotoxin tolerance with respect to proinflammatory cytokines, AA-MΦ-associated chemokines, negative regulators, NF-κB binding and subunit composition, and MAPKs; conversely, IL-13(-/-) macrophages were tolerized equivalently to WT MΦs by LPS pretreatment. Further, IL-4Rα deficiency did not affect the reversal of endotoxin tolerance exerted by the histone deacetylase inhibitor trichostatin A. Like WT mice, 100% of LPS-tolerized IL-4Rα-deficient mice survived LPS + d-galactosamine-induced lethal toxicity and exhibited decreased serum levels of proinflammatory cytokines and AA-MΦ-associated chemokines induced by LPS challenge compared with nontolerized mice. These data indicate that the signaling pathways leading to endotoxin tolerance and differentiation of AA-MΦs are dissociable.
Asunto(s)
Diferenciación Celular/inmunología , Endotoxinas/inmunología , Tolerancia Inmunológica/inmunología , Macrófagos/inmunología , Animales , Diferenciación Celular/genética , Línea Celular , Endotoxinas/metabolismo , Tolerancia Inmunológica/genética , Interleucina-13/genética , Interleucina-13/inmunología , Interleucina-13/metabolismo , Lipopolisacáridos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/inmunología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , FN-kappa B/genética , FN-kappa B/inmunología , FN-kappa B/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/inmunología , Factor de Transcripción STAT6/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Transcriptoma/genética , Transcriptoma/inmunologíaRESUMEN
IL-33, a member of the IL-1 family of cytokines, is produced by many cell types, including macrophages, yet its regulation is largely unknown. Treatment of primary murine macrophages with a panel of TLR (e.g., TLR2, TLR3, TLR4, and TLR9) agonists and non-TLR (e.g., MDA5, RIG-I) agonists revealed a pattern of gene and protein expression consistent with a role for IFN regulatory factor-3 (IRF-3) in the expression of IL-33. Accordingly, induction of IL-33 mRNA was attenuated in IRF-3(-/-) macrophages and TBK-1(-/-) mouse embryonic fibroblasts. Despite the fact that all IL-33 agonists were IRF-3 dependent, LPS-induced IL-33 mRNA was fully inducible in IFN-ß(-/-) macrophages, indicating that IL-33 is not dependent on IFN-ß as an intermediate. Epinephrine and Bordetella pertussis adenylate cyclase toxin (ACT), cAMP-activating agents, activate CREB and greatly synergize with LPS to induce IL-33 mRNA in macrophages. Both LPS-induced and ACT/LPS-enhanced expression of IL-33 mRNA was partially, but significantly, inhibited by the protein kinase A inhibitor H-89 but not by tyrosine kinase or protein kinase C inhibitors. Two IL-33 mRNA species derived from two alternative promoters encode full-length IL-33; however, the shorter "A" species is preferentially induced by all IL-33-inducing agonists except Newcastle disease virus, a RIG-I agonist that induced expression of both "A" and "B" transcripts. Together, these studies greatly extend what is currently known about the regulation of IL-33 induction in macrophages stimulated by bacterial and viral agonists that engage distinct innate immune signaling pathways.
Asunto(s)
Interleucinas/biosíntesis , Receptores Toll-Like/agonistas , Receptores Toll-Like/fisiología , Activación Transcripcional/inmunología , Animales , Células Cultivadas , Fibroblastos/inmunología , Fibroblastos/microbiología , Fibroblastos/virología , Inmunidad Innata/genética , Factor 3 Regulador del Interferón/deficiencia , Factor 3 Regulador del Interferón/genética , Interleucina-33 , Interleucinas/genética , Ligandos , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/biosíntesis , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptores Toll-Like/metabolismo , Activación Transcripcional/genéticaRESUMEN
Ligation of FcgammaR concurrent with LPS stimulation of murine macrophages results in decreased IL-12 and increased IL-10 production. Because PI3K deficiency has been associated with increased IL-12, we hypothesized that PI3K was central to the anti-inflammatory effect of FcgammaR ligation on TLR-induced IL-12. FcgammaR ligation of macrophages increased pAKT, a correlate of PI3K activity, above levels induced by TLR4 or TLR2 agonists. This increase was blocked by PI3K inhibitors, wortmannin or LY294002, as was the effect of FcgammaR ligation on TLR-induced IL-12 and IL-10. LPS-induced binding of NF-kappaB to the IL-12 p40 promoter NF-kappaB-binding site was not affected by FcgammaR ligation at 1 h; however, by 4 h, NF-kappaB binding was markedly inhibited, confirmed in situ by chromatin immunoprecipitation analysis. This effect was wortmannin sensitive. Although TLR-induced IkappaBalpha degradation was not affected by FcgammaR ligation, IkappaBalpha accumulated in the nuclei of cells treated with LPS and FcgammaR ligation for 4 h, and was blocked by PI3K inhibitors. LPS-induced IFN regulatory factor-8/IFN consensus sequence-binding protein mRNA, and an IFN regulatory factor-8-dependent gene, Nos2, were inhibited by concurrent FcgammaR ligation, and this was also reversed by wortmannin. Thus, FcgammaR ligation modulates LPS-induced IL-12 via multiple PI3K-sensitive pathways that affect production, accumulation, and binding of key DNA-binding proteins required for IL-12 induction.