NFκB1 inhibits memory formation and supports effector function of ILC2s in memory-driven asthma.

Background: ILC2s are capable of generating memory. The mechanism of memory induction and memory-driven effector function (trained immunity) in ILC2s is unknown. Objective: NFκB1 is preferentially expressed at a high level in ILC2s. We examined the role of NFkB1 in memory induction and memory-driven effector function in a mouse model of asthma. Methods: Intranasal administration of Alternaria, flexivent, ELISA, histology, real-time PCR, western blot, flow cytometry and immunofluorescence staining. Results: NFκB1 was essential for the effector phase of memory-driven asthma. NFκB1 was critical for IL33 production, ILC2 generation, and production of type-2 cytokines, which resulted in eosinophilic inflammation and other features of asthma. NFκB1 induction of type-2 cytokines in ILC2s was independent of GATA3. NFκB1 was important for allergen induction of ILC3s and FoxP3+ Tregs. NFκB1 did not affect Th2 cells or their cytokine production. In contrast to its protagonistic role in the effector phase, NFκB1 had an antagonistic role in the memory phase. NFκB1 inhibited allergen-induced upregulation of memory-associated repressor and preparedness genes in ILC2s. NFκB1 upregulated RUNX1. NFκB1 formed a heterodimer with RUNX1 in ILC2s. Conclusions: NFκB1 positively regulated the effector phase but inhibited the induction phase of memory. The foregoing pointed to an interdependent antagonism between the memory induction and the memory effector processes. The NFκB1-RUNX1 heterodimer represented a non-canonical transcriptional activator of type-2 cytokines in ILC2s.

Regulation of transferrin receptor trafficking by optineurin and its disease-associated mutants.

Transferrin receptor (TFRC) is a transmembrane protein that plays a crucial role in mediating homeostasis of iron in the cell. The binding of transferrin (that is bound to iron) to TFRC at the cell membrane generally starts endocytosis of TFRC-transferrin complex, which leads to formation of vesicles that are positive for TFRC. These vesicles travel to the early endosomes and later to the endocytic recycling compartment. Release of iron occurs in the early endosomes because of acidic pH. Major fraction of the transferrin and TFRC is transported back to the cell membrane; however, a minor fraction of it is transported to lysosomes through the process of autophagy. Optineurin (OPTN) is a multi-functional adaptor protein that plays a pivotal role in the control of TFRC trafficking, recycling and autophagy dependent degradation. Optineurin also plays a role in cargo-selective and non-selective autophagy. Here, we review our understanding of the function of OPTN in regulating TFRC trafficking, recycling and autophagy dependent degradation. We also discuss the mechanisms by which certain disease-associated mutations of OPTN alter these processes.

Isolation and Characterization of Conventional and Non-conventional Type 2 Innate Lymphoid Cells (ILC2s) from Human Peripheral Blood Mononuclear Cells (PBMCs).

Innate lymphoid cells (ILCs) are a relatively new family of lymphoid cells that lack lineage cell surface markers but produce various effector cytokines. Based on phenotype and function, the group 2 ILCs (ILC2s) mirror the features of the adaptive CD4+ Th2 cell subset. In humans, they are traditionally characterized as the Lin-IL7Rα+CRΤΗ2+CD161+ cell population that produces type 2 cytokines - IL-5 and IL-13. However, the commonly used surface markers for human ILC2s leave a majority of type 2 cytokine-producing ILC2s unaccounted for. Recently, we characterized a distinct type 2 cytokine-producing Lin- population that lacks surface expression of canonical CRTH2 but expresses CD30 and TNFR2. Herein, we describe a detailed protocol for isolation, staining, and analysis of the conventional Lin-CRTH2+IL7Ra+ and the non-conventional Lin-CD30+TNFR2+ ILC2 populations.

The molecular and epigenetic mechanisms of innate lymphoid cell (ILC) memory and its relevance for asthma.

Repetitive exposure of Rag1-/- mice to the Alternaria allergen extract generated a form of memory that elicited an asthma-like response upon a subthreshold recall challenge 3-15 wk later. This memory was associated with lung ICOS+ST2+ ILC2s. Genetic, pharmacologic, and antibody-mediated inhibition and adoptive transfer established an essential role for ILC2s in memory-driven asthma. ATAC-seq demonstrated a distinct epigenetic landscape of memory ILC2s and identified Bach2 and AP1 (JunD and Fosl2) motifs as major drivers of altered gene accessibility. scRNA-seq, gene knockout, and signaling studies suggest that repetitive allergenic stress induces a gene repression program involving Nr4a2, Zeb1, Bach2, and JunD and a preparedness program involving Fhl2, FosB, Stat6, Srebf2, and MPP7 in memory ILC2s. A mutually regulated balance between these two programs establishes and maintains memory. The preparedness program (e.g., Fhl2) can be activated with a subthreshold cognate stimulation, which down-regulates repressors and activates effector pathways to elicit the memory-driven phenotype.

Sprouty2 positively regulates T cell function and airway inflammation through regulation of CSK and LCK kinases.

The function of Sprouty2 (Spry2) in T cells is unknown. Using 2 different (inducible and T cell-targeted) knockout mouse strains, we found that Spry2 positively regulated extracellular signal-regulated kinase 1/2 (ERK1/2) signaling by modulating the activity of LCK. Spry2-/- CD4+ T cells were unable to activate LCK, proliferate, differentiate into T helper cells, or produce cytokines. Spry2 deficiency abrogated type 2 inflammation and airway hyperreactivity in a murine model of asthma. Spry2 expression was higher in blood and airway CD4+ T cells from patients with asthma, and Spry2 knockdown impaired human T cell proliferation and cytokine production. Spry2 deficiency up-regulated the lipid raft protein caveolin-1, enhanced its interaction with CSK, and increased CSK interaction with LCK, culminating in augmented inhibitory phosphorylation of LCK. Knockdown of CSK or dislodgment of caveolin-1-bound CSK restored ERK1/2 activation in Spry2-/- T cells, suggesting an essential role for Spry2 in LCK activation and T cell function.

Optimal identification of human conventional and nonconventional (CRTH2-IL7Rα-) ILC2s using additional surface markers.

BACKGROUND: Human type 2 innate lymphoid cells (ILC2s) are identified by coupled detection of CRTH2 and IL7Rα on lineage negative (Lin-) cells. Type 2 cytokine production by CRTH2-IL7Rα- innate lymphoid cells (ILCs) is unknown. OBJECTIVE: We sought to identify CRTH2-IL7Rα- type 2 cytokine-producing ILCs and their disease relevance. METHODS: We studied human blood and lung ILCs from asthmatic and control subjects by flow cytometry, ELISA, RNA sequencing, quantitative PCR, adoptive transfer to mice, and measurement of airway hyperreactivity by Flexivent. RESULTS: We found that IL-5 and IL-13 were expressed not only by CRTH2+ but also by CRTH2-IL7Rα+ and CRTH2-IL7Rα- (double-negative [DN]) human blood and lung cells. All 3 ILC populations expressed type 2 genes and induced airway hyperreactivity when adoptively transferred to mice. The frequency of type 2 cytokine-positive IL7Rα and DN ILCs were similar to that of CRTH2 ILCs in the blood and lung. Their frequency was higher in asthmatic patients than in disease controls. Transcriptomic analysis of CRTH2, IL7Rα, and DN ILCs confirmed the expression of mRNA for type 2 transcription factors in all 3 populations. Unexpectedly, the mRNA for GATA3 and IL-5 correlated better with mRNA for CD30, TNFR2, ICOS, CCR4, and CD200R1 than for CRTH2. By using a combination of these surface markers, especially CD30/TNFR2, we identified a previously unrecognized ILC2 population. CONCLUSIONS: The commonly used surface markers for human ILC2s leave a majority of type 2 cytokine-producing ILC2s unaccounted for. We identified top GATA3-correlated cell surface-expressed genes in human ILCs by RNA sequencing. These new surface markers, such as CD30 and TNFR2, identified a previously unrecognized human ILC2 population. This ILC2 population is likely to contribute to asthma.

BAP1 links metabolic regulation of ferroptosis to tumour suppression.

The roles and regulatory mechanisms of ferroptosis (a non-apoptotic form of cell death) in cancer remain unclear. The tumour suppressor BRCA1-associated protein 1 (BAP1) encodes a nuclear deubiquitinating enzyme to reduce histone 2A ubiquitination (H2Aub) on chromatin. Here, integrated transcriptomic, epigenomic and cancer genomic analyses link BAP1 to metabolism-related biological processes, and identify cystine transporter SLC7A11 as a key BAP1 target gene in human cancers. Functional studies reveal that BAP1 decreases H2Aub occupancy on the SLC7A11 promoter and represses SLC7A11 expression in a deubiquitinating-dependent manner, and that BAP1 inhibits cystine uptake by repressing SLC7A11 expression, leading to elevated lipid peroxidation and ferroptosis. Furthermore, we show that BAP1 inhibits tumour development partly through SLC7A11 and ferroptosis, and that cancer-associated BAP1 mutants lose their abilities to repress SLC7A11 and to promote ferroptosis. Together, our results uncover a previously unappreciated epigenetic mechanism coupling ferroptosis to tumour suppression.

Optineurin promotes autophagosome formation by recruiting the autophagy-related Atg12-5-16L1 complex to phagophores containing the Wipi2 protein.

Autophagy is a quality-control mechanism that helps to maintain cellular homeostasis by removing damaged proteins and organelles through lysosomal degradation. During autophagy, signaling events lead to the formation of a cup-shaped structure called the phagophore that matures into the autophagosome. Recruitment of the autophagy-associated Atg12-5-16L1 complex to Wipi2-positive phagophores is crucial for producing microtubule-associated protein 1 light chain 3-II (LC3-II), which is required for autophagosome formation. Here, we explored the role of the autophagy receptor optineurin (Optn) in autophagosome formation. Fibroblasts from Optn knock-out mouse showed reduced LC3-II formation and a lower number of autophagosomes and autolysosomes during both basal and starvation-induced autophagy. However, the number of Wipi2-positive phagophores was not decreased in Optn-deficient cells. We also found that the number of Atg12/16L1-positive puncta and recruitment of the Atg12-5-16L1 complex to Wipi2-positive puncta are reduced in Optn-deficient cells. Of note, Optn was recruited to Atg12-5-16L1-positive puncta, and interacted with Atg5 and also with Atg12-5 conjugate. A disease-associated Optn mutant, E478G, defective in ubiquitin binding, was also defective in autophagosome formation and recruitment to the Atg12-5-16L1-positive puncta. Moreover, we noted that Optn phosphorylation at Ser-177 was required for autophagosome formation but not for Optn recruitment to the phagophore. These results suggest that Optn potentiates LC3-II production and maturation of the phagophore into the autophagosome, by facilitating the recruitment of the Atg12-5-16L1 complex to Wipi2-positive phagophores.

661W is a retinal ganglion precursor-like cell line in which glaucoma-associated optineurin mutants induce cell death selectively.

A photoreceptor cell line, 661W, derived from a mouse retinal tumor that expresses several markers of cone photoreceptor cells has been described earlier. However, these cells can be differentiated into neuronal cells. Here, we report that this cell line expressed certain markers specific to retinal ganglion cells such as Rbpms, Brn3b (Pou4f2), Brn3c (Pou4f3), Thy1 and γ-synuclein (Sncg), and some other markers of neuronal cells (beta-III tubulin, NeuN and MAP2). These cells also expressed Opn1mw, a cone-specific marker and nestin, a marker for neural precursor cells. Two glaucoma-associated mutants of OPTN, E50K and M98K, but not an amyotrophic lateral sclerosis-associated mutant, E478G, induced cell death selectively in 661W cells. However, in a motor neuron cell line, NSC34, E478G mutant of OPTN but not E50K and M98K induced cell death. We conclude that 661W is a retinal ganglion precursor-like cell line, which shows properties of both retinal ganglion and photoreceptor cells. We suggest that these cells could be utilized for exploring the mechanisms of cell death induction and cytoprotection relevant for glaucoma pathogenesis. RGC-5 cell line which probably arose from 661W cells showed expression of essentially the same markers of retinal ganglion cells and neuronal cells as seen in 661W cells.

Defects in autophagy caused by glaucoma-associated mutations in optineurin.

Certain mutations in optineurin (gene OPTN) are associated with primary open angle glaucoma. Optineurin is ubiquitously expressed but it shows high level of expression in certain cells and tissues including retinal ganglion cells. It interacts with many proteins, often acting as an adaptor to link two or more proteins. These interactions play a crucial role in mediating various functions of optineurin such as membrane vesicle trafficking, autophagy, signal transduction etc. Autophagy is basically a quality control mechanism to remove damaged proteins and organelles through lysosomal degradation. Optineurin was identified as an autophagy receptor that directly interacts with autophagosomal protein, LC3, and ubiquitin. These interactions are important for autophagy receptor function. Autophagy receptors recruit their cargo and take it to autophagosomes which fuse with lysosomes to form autolysosomes where degradation of proteins takes place. Optineurin interacts with a motor protein, myosinVI, and this interaction is involved in mediating fusion of autophagosomes with lysosomes. A glaucoma-associated mutant of optineurin, E50K, impairs autophagy as well as vesicle trafficking, leading to death of retinal cells by apoptosis. E50K-OPTN-induced block in autophagy is dependent on a GTPase activating protein, TBC1D17. The E50K mutant also causes other changes in the cells such as altered interaction with TBK1 protein kinase, aggregate formation, generation of reactive oxygen species and inhibition of proteasome, which may contribute to pathogenesis. A polymorphism of optineurin, M98K, associated with glaucoma, causes enhanced autophagy leading to transferrin receptor degradation and apoptotic death of retinal cells. M98K-OPTN-induced autophagic cell death is dependent on Rab12 GTPase. Thus, an optimum level of optineurin-mediated autophagy is crucial for survival of retinal cells, and impaired autophagy is likely to contribute to glaucoma pathogenesis. How impaired autophagy caused by optineurin mutants leads to apoptosis and cell death, is yet to be explored.

A Glaucoma-Associated Variant of Optineurin, M98K, Activates Tbk1 to Enhance Autophagosome Formation and Retinal Cell Death Dependent on Ser177 Phosphorylation of Optineurin.

Certain missense mutations in optineurin/OPTN and amplification of TBK1 are associated with normal tension glaucoma. A glaucoma-associated variant of OPTN, M98K, induces autophagic degradation of transferrin receptor (TFRC) and death in retinal cells. Here, we have explored the role of Tbk1 in M98K-OPTN-induced autophagy and cell death, and the effect of Tbk1 overexpression in retinal cells. Cell death induced by M98K-OPTN was dependent on Tbk1 as seen by the effect of Tbk1 knockdown and blocking of Tbk1 activity by a chemical inhibitor. Inhibition of Tbk1 also restores M98K-OPTN-induced transferrin receptor degradation. M98K-OPTN-induced autophagosome formation, autophagy and cell death were dependent on its phosphorylation at S177 by Tbk1. Knockdown of OPTN reduced starvation-induced autophagosome formation. M98K-OPTN expressing cells showed higher levels of Tbk1 activation and enhanced phosphorylation at Ser177 compared to WT-OPTN expressing cells. M98K-OPTN-induced activation of Tbk1 and its ability to be phosphorylated better by Tbk1 was dependent on ubiquitin binding. Phosphorylated M98K-OPTN localized specifically to autophagosomes and endogenous Tbk1 showed increased localization to autophagosomes in M98K-OPTN expressing cells. Overexpression of Tbk1 induced cell death and caspase-3 activation that were dependent on its catalytic activity. Tbk1-induced cell death possibly involves autophagy, as shown by the effect of Atg5 knockdown, and requirement of autophagic function of OPTN. Our results show that phosphorylation of Ser177 plays a crucial role in M98K-OPTN-induced autophagosome formation, autophagy flux and retinal cell death. In addition, we provide evidence for cross talk between two glaucoma associated proteins and their inter-dependence to mediate autophagy-dependent cell death.

M98K-OPTN induces transferrin receptor degradation and RAB12-mediated autophagic death in retinal ganglion cells.

Mutations in the autophagy receptor OPTN/optineurin are associated with the pathogenesis of glaucoma and amyotrophic lateral sclerosis, but the underlying molecular basis is poorly understood. The OPTN variant, M98K has been described as a risk factor for normal tension glaucoma in some ethnic groups. Here, we examined the consequence of the M98K mutation in affecting cellular functions of OPTN. Overexpression of M98K-OPTN induced death of retinal ganglion cells (RGC-5 cell line), but not of other neuronal and non-neuronal cells. Enhanced levels of the autophagy marker, LC3-II, a post-translationally modified form of LC3, in M98K-OPTN-expressing cells and the inability of an LC3-binding-defective M98K variant of OPTN to induce cell death, suggested that autophagy contributes to cell death. Knockdown of Atg5 reduced M98K-induced death of RGC-5 cells, further supporting the involvement of autophagy. Overexpression of M98K-OPTN enhanced autophagosome formation and potentiated the delivery of transferrin receptor to autophagosomes for degradation resulting in reduced cellular transferrin receptor levels. Coexpression of transferrin receptor or supplementation of media with an iron donor reduced M98K-induced cell death. OPTN complexes with RAB12, a GTPase involved in vesicle trafficking, and M98K variant shows enhanced colocalization with RAB12. Knockdown of Rab12 increased transferrin receptor level and reduced M98K-induced cell death. RAB12 is present in autophagosomes and knockdown of Rab12 resulted in reduced formation of autolysosomes during starvation-induced autophagy, implicating a role for RAB12 in autophagy. These results also show that transferrin receptor degradation and autophagy play a crucial role in RGC-5 cell death induced by M98K variant of OPTN.