Development and characterization of anti-IL-5 monoclonal antibody Fab fragment for blocking IL-5/IL-5Rα binding.

Interleukin-5 (IL-5) is a homodimeric cytokine that is a crucial regulator of the proliferation, activation, and maturation of eosinophils. Anti-IL-5 monoclonal antibodies, which block the binding of IL-5 to the IL-5 receptor subunit alpha (IL-5Rα), have been successfully used to treat eosinophilic (EOS) asthma. The currently marketed monoclonal antibody drugs require repeated injections for administration, which seriously affect patient compliance and high systemic exposure for injectable drug delivery. Here we successfully screened and developed the Fab (fragment of antigen binding), which is 1/3rd the molecular weight of IgG, favoring inhalation-mediated delivery to the lungs, making it more effective for asthma treatment. The 20A12-Fab-H12L3 can bind to IL-5 with a binding constant of 1.236E-09 M while significantly inhibiting the IL-5/IL-5Rα complex formation. We found that the light chain amino acids (S46 and F71) significantly affected the antibody expression during humanization. The 20A12-Fab-H12L3 significantly inhibited the proliferation of TF-1 cells and blocked the IL-5 binding to the IL-5Rα-overexpressing human embryonic kidney (HEK)-293 cells in vitro. Therefore, based on the mutant IL-5 binding with Fab, we explained why antibodies blocked IL-5 binding to IL-5Rα. Thus, this study provided a candidate pharmaceutical antibody for inhalation drug delivery.

Gut stem cell necroptosis by genome instability triggers bowel inflammation.

The aetiology of inflammatory bowel disease (IBD) is a multifactorial interplay between heredity and environment1,2. Here we report that deficiency in SETDB1, a histone methyltransferase that mediates the trimethylation of histone H3 at lysine 9, participates in the pathogenesis of IBD. We found that levels of SETDB1 are decreased in patients with IBD, and that mice with reduced SETDB1 in intestinal stem cells developed spontaneous terminal ileitis and colitis. SETDB1 safeguards genome stability3, and the loss of SETDB1 in intestinal stem cells released repression of endogenous retroviruses (retrovirus-like elements with long repeats that, in humans, comprise approximately 8% of the genome). Excessive viral mimicry generated by motivated endogenous retroviruses triggered Z-DNA-binding protein 1 (ZBP1)-dependent necroptosis, which irreversibly disrupted homeostasis of the epithelial barrier and promoted bowel inflammation. Genome instability, reactive endogenous retroviruses, upregulation of ZBP1 and necroptosis were all seen in patients with IBD. Pharmaceutical inhibition of RIP3 showed a curative effect in SETDB1-deficient mice, which suggests that targeting necroptosis of intestinal stem cells may represent an approach for the treatment of severe IBD.

ZBP1 mediates interferon-induced necroptosis.

Interferons (IFNs) play an important role in immunomodulatory and antiviral functions. IFN-induced necroptosis has been reported in cells deficient in receptor-interacting protein kinase 1 (RIPK1), Fas-associated protein with death domain (FADD), or caspase-8, but the mechanism is largely unknown. Here, we report that the DNA-dependent activator of IFN regulatory factors (ZBP1, also known as DAI) is required for both type I (ß) and type II (γ) IFN-induced necroptosis. We show that L929 fibroblast cells became susceptible to IFN-induced necroptosis when RIPK1, FADD, or Caspase-8 was genetically deleted, confirming the antinecroptotic role of these proteins in IFN signaling. We found that the pronecroptotic signal from IFN stimulation depends on new protein synthesis and identified ZBP1, an IFN-stimulated gene (ISG) product, as the de novo synthesized protein that triggers necroptosis in IFN-stimulated cells. The N-terminal domain (ND) of ZBP1 is important for ZBP1-ZBP1 homointeraction, and its RHIM domain in the C-terminal region interacts with RIPK3 to initiate RIPK3-dependent necroptosis. The antinecroptotic function of RIPK1, FADD, and caspase-8 in IFN-treated cells is most likely executed by caspase-8-mediated cleavage of RIPK3, since the inhibitory effect on necroptosis was eliminated when the caspase-8 cleavage site in RIPK3 was mutated. ZBP1-mediated necroptosis in IFN-treated cells is likely physiologically relevant, as ZBP1 KO mice were significantly protected against acute systemic inflammatory response syndrome (SIRS) induced by TNF + IFN-γ.

Nucleoporin Seh1 Interacts with Olig2/Brd7 to Promote Oligodendrocyte Differentiation and Myelination.

Nucleoporins (Nups) are involved in neural development, and alterations in Nup genes are linked to human neurological diseases. However, physiological functions of specific Nups and the underlying mechanisms involved in these processes remain elusive. Here, we show that tissue-specific depletion of the nucleoporin Seh1 causes dramatic myelination defects in the CNS. Although proliferation is not altered in Seh1-deficient oligodendrocyte progenitor cells (OPCs), they fail to differentiate into mature oligodendrocytes, which impairs myelin production and remyelination after demyelinating injury. Genome-wide analyses show that Seh1 regulates a core myelinogenic regulatory network and establishes an accessible chromatin landscape. Mechanistically, Seh1 regulates OPCs differentiation by assembling Olig2 and Brd7 into a transcription complex at nuclear periphery. Together, our results reveal that Seh1 is required for oligodendrocyte differentiation and myelination by promoting assembly of an Olig2-dependent transcription complex and define a nucleoporin as a key player in the CNS.

Inactivation of Cyclic AMP Response Element Transcription Caused by Constitutive p38 Activation Is Mediated by Hyperphosphorylation-Dependent CRTC2 Nucleocytoplasmic Transport.

The p38 signal transduction pathway can be activated transiently or constitutively, depending on the contexts in which the activation occurs. However, the biological consequence of constitutive activation of p38 is largely unknown. After screening 300 transcriptional cofactors, we identified CRTC2 as a downstream substrate of constitutively activated p38. Constitutive, rather than transient, activation of p38 led to hyperphosphorylation of CRTC2, resulting in CRTC2 cytosolic relocation and subsequent inactivation of cyclic AMP response element (CRE)-mediated transcription. Interestingly, the cytosolic translocation of CRTC2 depended on phosphorylation accumulation at multiple sites (≥11 phosphoserine/phosphothreonine residues) but not on specific sites. The hyperphosphorylation-driven nucleocytoplasmic transport of CRTC2 may not be a rare case of nuclear export of proteins, as we also observed that constitutively activated p38 promoted FOS nuclear export in a hyperphosphorylation-dependent manner. Collectively, our study uncovered a previously unknown mechanism of inactivation of selected transcription, which results from hyperphosphorylation-driven nucleocytoplasmic transport of cofactors or transcription factors mediated by constitutively active kinase.

RIP3 targets pyruvate dehydrogenase complex to increase aerobic respiration in TNF-induced necroptosis.

Receptor-interacting protein kinase 3 (RIP3)-regulated production of reactive oxygen species (ROS) positively feeds back on tumour necrosis factor (TNF)-induced necroptosis, a type of programmed necrosis. Glutamine catabolism is known to contribute to RIP3-mediated ROS induction, but the major contributor is unknown. Here, we show that RIP3 activates the pyruvate dehydrogenase complex (PDC, also known as PDH), the rate-limiting enzyme linking glycolysis to aerobic respiration, by directly phosphorylating the PDC E3 subunit (PDC-E3) on T135. Upon activation, PDC enhances aerobic respiration and subsequent mitochondrial ROS production. Unexpectedly, mixed-lineage kinase domain-like (MLKL) is also required for the induction of aerobic respiration, and we further show that it is required for RIP3 translocation to meet mitochondria-localized PDC. Our data uncover a regulation mechanism of PDC activity, show that PDC activation by RIP3 is most likely the major mechanism activated by TNF to increase aerobic respiration and its by-product ROS, and suggest that RIP3-dependent induction of aerobic respiration contributes to pathologies related to oxidative stress.

Recognition of cytosolic DNA attenuates glucose metabolism and induces AMPK mediated energy stress response.

Both viral infection and DNA transfection expose single-stranded or double-stranded DNA to the cytoplasm of mammalian cells. Recognition of cytosolic DNA activates a series of cellular responses, including induction of pro-inflammatory genes such as type I interferon through the well-known cGAS-STING pathway. Here we show for the first time that intracellular administration of either single or double stranded interferon stimulating DNA (ISD), but not poly(dA) suppresses cell growth in many different cell types. Suppression of cell growth by cytosolic DNA is cGAS/STING independent and associated with inhibition of glucose metabolism, ATP depletion and subsequent cellular energy stress responses including activation of AMPK and inactivation of mTORC1. Our results suggest that in concert with but independent of innate immune response, recognition of cytosolic DNA induced cellular energy stress potentially functions as a metabolic barrier to viral replication.

Ppm1b negatively regulates necroptosis through dephosphorylating Rip3.

The auto-phosphorylation of murine receptor-interacting protein 3 (Rip3) on Thr 231 and Ser 232 in the necrosome is required to trigger necroptosis. However, how Rip3 phosphorylation is regulated is still largely unknown. Here we identified protein phosphatase 1B (Ppm1b) as a Rip3 phosphatase and found that Ppm1b restricts necroptosis in two settings: spontaneous necroptosis caused by Rip3 auto-phosphorylation in resting cells, and tumour necrosis factor-α (TNF)-induced necroptosis in cultured cells. We revealed that Ppm1b selectively suppresses necroptosis through the dephosphorylation of Rip3, which then prevents the recruitment of mixed lineage kinase domain-like protein (Mlkl) to the necrosome. We further showed that Ppm1b deficiency (Ppm1b(d/d)) in mice enhanced TNF-induced death in a Rip3-dependent manner, and the role of Ppm1b in inhibiting necroptosis was evidenced by elevated Rip3 phosphorylation and tissue damage in the caecum of TNF-treated Ppm1b(d/d) mice. These data indicate that Ppm1b negatively regulates necroptosis through dephosphorylating Rip3 in vitro and in vivo.

RIP1/RIP3 binding to HSV-1 ICP6 initiates necroptosis to restrict virus propagation in mice.

Necroptosis is a form of programmed necrosis that is mediated by signaling complexes containing the receptor-interacting protein 3 (RIP3) and RIP1 kinases. We show that RIP3 and its interaction with the herpes simplex virus type 1 (HSV-1) protein ICP6 triggers necroptosis in infected mouse cells and limits viral propagation in mice. ICP6 interacts with RIP1/RIP3 through its RHIM domain and forms dimers/oliogmers by its C-terminal R1 domain. These binding events result in RIP1-RIP3 hetero- and RIP3-RIP3 homo-interactions and subsequent necroptosis of HSV-1-infected mouse cells. However, ICP6 RHIM cannot trigger necroptosis and even inhibits TNF-induced necroptosis in human cells. As the RHIM domain in murine cytomegalovirus protein vIRA can inhibit necroptosis in both human and mouse cells, these data suggest that both viral and host RHIM sequences determine whether the virus-host RHIM interaction is pro- or anti-necroptotic and that some viruses may evolve to escape this restriction.

The Gßγ-Src signaling pathway regulates TNF-induced necroptosis via control of necrosome translocation.

Formation of multi-component signaling complex necrosomes is essential for tumor necrosis factor α (TNF)-induced programmed necrosis (also called necroptosis). However, the mechanisms of necroptosis are still largely unknown. We isolated a TNF-resistant L929 mutant cell line generated by retrovirus insertion and identified that disruption of the guanine nucleotide-binding protein γ 10 (Gγ10) gene is responsible for this phenotype. We further show that Gγ10 is involved in TNF-induced necroptosis and Gß2 is the partner of Gγ10. Src is the downstream effector of Gß2γ10 in TNF-induced necroptosis because TNF-induced Src activation was impaired upon Gγ10 knockdown. Gγ10 does not affect TNF-induced activation of NF-κB and MAPKs and the formation of necrosomes, but is required for trafficking of necrosomes to their potential functioning site, an unidentified subcellular organelle that can be fractionated into heterotypic membrane fractions. The TNF-induced Gßγ-Src signaling pathway is independent of RIP1/RIP3 kinase activity and necrosome formation, but is required for the necrosome to function.