BATF sustains homeostasis and functionality of bone marrow Treg cells to preserve homeostatic regulation of hematopoiesis and development of B cells.

Bone marrow Treg cells (BM Tregs) orchestrate stem cell niches crucial for hematopoiesis. Yet little is known about the molecular mechanisms governing BM Treg homeostasis and function. Here we report that the transcription factor BATF maintains homeostasis and functionality of BM Tregs to facilitate homeostatic regulation of hematopoiesis and B cell development. Treg-specific ablation of BATF profoundly compromised proportions of BM Tregs associated with reduced expression of Treg effector molecules, including CD44, ICOS, KLRG1, and TIGIT. Moreover, BATF deficiency in Tregs led to increased numbers of hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), and granulocyte-macrophage progenitors (GMPs), while reducing the functionality of myeloid progenitors and the generation of common lymphoid progenitors. Furthermore, Tregs lacking BATF failed to support the development of B cells in the BM. Mechanistically, BATF mediated IL-7 signaling to promote expression of effector molecules on BM Tregs and their homeostasis. Our studies reveal a previously unappreciated role for BATF in sustaining BM Treg homeostasis and function to ensure hematopoiesis.

PTEN directs developmental and metabolic signaling for innate-like T cell fate and tissue homeostasis.

Phosphatase and tensin homologue (PTEN) is frequently mutated in human cancer, but its roles in lymphopoiesis and tissue homeostasis remain poorly defined. Here we show that PTEN orchestrates a two-step developmental process linking antigen receptor and IL-23-Stat3 signalling to type-17 innate-like T cell generation. Loss of PTEN leads to pronounced accumulation of mature IL-17-producing innate-like T cells in the thymus. IL-23 is essential for their accumulation, and ablation of IL-23 or IL-17 signalling rectifies the reduced survival of female PTEN-haploinsufficient mice that model human patients with PTEN mutations. Single-cell transcriptome and network analyses revealed the dynamic regulation of PTEN, mTOR and metabolic activities that accompanied type-17 cell programming. Furthermore, deletion of mTORC1 or mTORC2 blocks PTEN loss-driven type-17 cell accumulation, and this is further shaped by the Foxo1 and Stat3 pathways. Collectively, our study establishes developmental and metabolic signalling networks underpinning type-17 cell fate decisions and their functional effects at coordinating PTEN-dependent tissue homeostasis.

Ubiquitin-binding domain in ABIN1 is critical for regulating cell death and inflammation during development.

ABIN1 is a polyubiquitin-binding protein known to regulate NF-κB activation and cell death signaling. Mutations in Abin1 can cause severe immune diseases in human, such as psoriasis, systemic lupus erythematosus, and systemic sclerosis. Here, we generated mice that disrupted the ubiquitin-binding domain of ABIN1 (Abin1UBD/UBD) died during later embryogenesis owing to TNFR1-mediated cell death, similar to Abin1-/- mice. Abin1UBD/UBD cells were rendered sensitive to TNF-α-induced apoptosis and necroptosis as the inhibition of ABIN1UBD and A20 recruitment to the TNF-RSC complex leads to attenuated RIPK1 deubiquitination. Accordingly, the embryonic lethality of Abin1UBD/UBD mice was rescued via crossing with RIPK1 kinase-dead mice (Ripk1K45A/K45A) or the co-deletion of Ripk3 and one allele of Fadd, but not by the loss of Ripk3 or Mlkl alone. Unexpectedly, Abin1UBD/UBD mice with the co-deletion of Ripk3 and both Fadd alleles died at E14.5. This death was caused by spontaneous RIPK1 ubiquitination-dependent multiple inflammatory cytokines over production and could be rescued by the co-deletion of Ripk1 or Tnfr1 combined with Ifnar. Collectively, these data demonstrate the importance of the ABIN1 UBD domain, which mediates the ABIN1-A20 axis, at limiting RIPK1 activation-dependent cell death during embryonic development. Furthermore, our findings reveal a previously unappreciated ubiquitin pathway that regulates RIPK1 ubiquitination by FADD/Casp8 to suppress spontaneous IKKε/TBK1 activation.

Heteroplasmic and homoplasmic m.616T>C in mitochondria tRNAPhe promote isolated chronic kidney disease and hyperuricemia.

Inherited kidney diseases are the fifth most common cause of end-stage renal disease (ESRD). Mitochondrial dysfunction plays a vital role in the progression of inherited kidney diseases, while mitochondrial-transfer RNA (mt-tRNA) variants and their pathogenic contributions to kidney disease remain largely unclear. In this study, we identified the pathogenic mt-tRNAPhe 616T>C mutation in 3 families and documented that m.616T>C showed a high pathogenic threshold, with both heteroplasmy and homoplasmy leading to isolated chronic kidney disease and hyperuricemia without hematuria, proteinuria, or renal cyst formation. Moreover, 1 proband with homoplamic m.616T>C presented ESRD as a child. No symptoms of nervous system evolvement were observed in these families. Lymphoblast cells bearing m.616T>C exhibited swollen mitochondria, underwent active mitophagy, and showed respiratory deficiency, leading to reduced mitochondrial ATP production, diminished membrane potential, and overproduction of mitochondrial ROS. Pathogenic m.616T>C abolished a highly conserved base pair (A31-U39) in the anticodon stem-loop which altered the structure of mt-tRNAPhe, as confirmed by a decreased melting temperature and slower electrophoretic mobility of the mutant tRNA. Furthermore, the unstable structure of mt-tRNAPhe contributed to a shortage of steady-state mt-tRNAPhe and enhanced aminoacylation efficiency, which resulted in impaired mitochondrial RNA translation and a significant decrease in mtDNA-encoded polypeptides. Collectively, these findings provide potentially new insights into the pathogenesis underlying inherited kidney disease caused by mitochondrial variants.

The glutathione peroxidase Gpx4 prevents lipid peroxidation and ferroptosis to sustain Treg cell activation and suppression of antitumor immunity.

T regulatory (Treg) cells are crucial to maintain immune tolerance and repress antitumor immunity, but the mechanisms governing their cellular redox homeostasis remain elusive. We report that glutathione peroxidase 4 (Gpx4) prevents Treg cells from lipid peroxidation and ferroptosis in regulating immune homeostasis and antitumor immunity. Treg-specific deletion of Gpx4 impairs immune homeostasis without substantially affecting survival of Treg cells at steady state. Loss of Gpx4 results in excessive accumulation of lipid peroxides and ferroptosis of Treg cells upon T cell receptor (TCR)/CD28 co-stimulation. Neutralization of lipid peroxides and blockade of iron availability rescue ferroptosis of Gpx4-deficient Treg cells. Moreover, Gpx4-deficient Treg cells elevate generation of mitochondrial superoxide and production of interleukin-1ß (IL-1ß) that facilitates T helper 17 (TH17) responses. Furthermore, Treg-specific ablation of Gpx4 represses tumor growth and concomitantly potentiates antitumor immunity. Our studies establish a crucial role for Gpx4 in protecting activated Treg cells from lipid peroxidation and ferroptosis and offer a potential therapeutic strategy to improve cancer treatment.

BATF Regulates T Regulatory Cell Functional Specification and Fitness of Triglyceride Metabolism in Restraining Allergic Responses.

Preserving appropriate function and metabolism in regulatory T (Treg) cells is crucial for controlling immune tolerance and inflammatory responses. Yet how Treg cells coordinate cellular metabolic programs to support their functional specification remains elusive. In this study, we report that BATF couples the TH2-suppressive function and triglyceride (TG) metabolism in Treg cells for controlling allergic airway inflammation and IgE responses. Mice with Treg-specific ablation of BATF developed an inflammatory disorder characterized by TH2-type dominant responses and were predisposed to house dust mite-induced airway inflammation. Loss of BATF enabled Treg cells to acquire TH2 cell-like characteristics. Moreover, BATF-deficient Treg cells displayed elevated levels of cellular TGs, and repressing or elevating TGs, respectively, restored or exacerbated their defects. Mechanistically, TCR/CD28 costimulation enhanced expression and function of BATF, which sustained IRF4 activity to preserve Treg cell functionality. Thus, our studies reveal that BATF links Treg cell functional specification and fitness of cellular TGs to control allergic responses, and suggest that therapeutic targeting of TG metabolism could be used for the treatment of allergic disease.

Podocyte apoptosis in diabetic nephropathy by BASP1 activation of the p53 pathway via WT1.

AIMS: Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. BASP1 (brain acid-soluble protein) is up-regulated in podocyte-specific protein phosphatase 2A knockout mice (Pod-PP2A-KO) that develop kidney dysfunction. Here, we explore the role of BASP1 for podocytes in DN. METHODS: BASP1 was assessed in kidneys from DN patients and DN mouse models, podocyte specific BASP1 knockout mice (Pod-BASP1-KO mice) were generated and studied in vivo. Furthermore, podocyte injury and apoptosis were measured after BASP1 knockdown and overexpression in a mouse podocyte cell line (MPC5). Potential signalling pathways involved in podocyte apoptosis were detected. RESULTS: BASP1 expression was up-regulated in DN patients compared to normal controls. BASP1 specific deletion in podocytes protected against podocyte injury by reducing the loss of expression of slit diaphragm molecules and foot process effacement in the DN model. BASP1 promoted actin cytoskeleton rearrangements and apoptosis in the MPC5 podocyte line. Molecules involved in the p53 pathway were down-regulated in BASP1 knockdown podocytes treated with high glucose compared to controls. BASP1 promoted podocyte apoptosis and P53 pathway activation through co-repression with Wilms' tumour 1 transcription factor (WT1). CONCLUSION: BASP1 activates the p53 pathway through modulation of WT1 to induce podocyte apoptosis in diabetic nephropathy.

Mosaic PKHD1 in Polycystic Kidneys Caused Aberrant Protein Expression in the Mitochondria and Lysosomes.

Autosomal recessive polycystic kidney disease (ARPKD) is a severe renal cystic disease caused mainly by the polycystic kidney and hepatic disease 1 (PKHD1). However, the genetic cause, pathologic features, and mechanism of action of ARPKD are not well known. Here, we identified a family with ARPKD. Two siblings harbored biallelic variants in PKHD1 (c.7205G>A, c.7973T>A). We determined that the "de novo" variant, c.7205G>A, arose from the mosaicism of the father and had a 7.4% level. Pathologic characterization, using biopsy analysis, was evidenced with predominant cystic dilation in proximal tubules, slight ectasia of collecting ducts, defective ciliogenesis, and impaired cell-cell junctions in renal tubules and collecting ducts. Exosome proteomics in the urine from patients with ARPKD were markedly different from those of controls, with the most significant alterations occurring in mitochondrial and lysosomal proteins. Expression of the proteins of OXPHOS was downregulated sharply, in parallel with upregulated expression of the proteins involved in glycolysis in patients with ARPKD. Several lysosomal proteins associated with renal lesions were more abundant in the exosome of the patient than in controls. Moreover, the lysosomal enzyme sulfamidase, which is produced by the SGSH gene, was abrupt uniquely in the exosome of the patient. Consistently, swollen mitochondria and abundant lysosomes were visualized in the mutant tubular epithelial cells of patients with mutant PKHD1. Collectively, these findings provide new insights on the pathophysiology of the polycystic kidney due to PKHD1 deficiency. PKHD1 mosaicism should be considered in genetic testing of ARPKD patients.

STAT5 promotes accessibility and is required for BATF-mediated plasticity at the Il9 locus.

T helper cell differentiation requires lineage-defining transcription factors and factors that have shared expression among multiple subsets. BATF is required for development of multiple Th subsets but functions in a lineage-specific manner. BATF is required for IL-9 production in Th9 cells but in contrast to its function as a pioneer factor in Th17 cells, BATF is neither sufficient nor required for accessibility at the Il9 locus. Here we show that STAT5 is the earliest factor binding and remodeling the Il9 locus to allow BATF binding in both mouse and human Th9 cultures. The ability of STAT5 to mediate accessibility for BATF is observed in other Th lineages and allows acquisition of the IL-9-secreting phenotype. STAT5 and BATF convert Th17 cells into cells that mediate IL-9-dependent effects in allergic airway inflammation and anti-tumor immunity. Thus, BATF requires the STAT5 signal to mediate plasticity at the Il9 locus.

Ubiquitination of RIPK1 suppresses programmed cell death by regulating RIPK1 kinase activation during embryogenesis.

The ubiquitination status of RIPK1 is considered to be critical for cell fate determination. However, the in vivo role for RIPK1 ubiquitination remains undefined. Here we show that mice expressing RIPK1K376R which is defective in RIPK1 ubiquitination die during embryogenesis. This lethality is fully rescued by concomitant deletion of Fadd and Ripk3 or Mlkl. Mechanistically, cells expressing RIPK1K376R are more susceptible to TNF-α induced apoptosis and necroptosis with more complex II formation and increased RIPK1 activation, which is consistent with the observation that Ripk1K376R/K376R lethality is effectively prevented by treatment of RIPK1 kinase inhibitor and is rescued by deletion of Tnfr1. However, Tnfr1-/- Ripk1K376R/K376R mice display systemic inflammation and die within 2 weeks. Significantly, this lethal inflammation is rescued by deletion of Ripk3. Taken together, these findings reveal a critical role of Lys376-mediated ubiquitination of RIPK1 in suppressing RIPK1 kinase activity-dependent lethal pathways during embryogenesis and RIPK3-dependent inflammation postnatally.

Protein phosphatase 2A modulates podocyte maturation and glomerular functional integrity in mice.

BACKGROUND: Protein phosphorylation & dephosphorylation are ubiquitous cellular processes that allow for the nuanced and reversible regulation of protein activity. Protein phosphatase 2A (PP2A) is a multifunction phosphatase that is well expressed in all cell types of kidney during early renal development, though its functions in kidney remains to be elucidated. METHODS: PP2A conditional knock-out mice was generated with PP2A fl/fl mice that were crossed with Podocin-Cre mice. The phenotype of Pod-PP2A-KO mice (homozygous for the floxed PP2A allele with Podocin-Cre) and littermate PP2A fl/fl controls (homozygous for the PP2A allele but lacking Podocin-Cre) were further studied. Primary podocytes isolated from the Pod-PP2A-KO mice were cultured and they were then employed with sing label-free nano-LC - MS/MS technology on a Q-exactive followed by SIEVE processing to identify possible target molecular entities for the dephosphorylation effect of PP2A, in which Western blot and immunofluorescent staining were used to analyze further. RESULTS: Pod-PP2A-KO mice were developed with weight loss, growth retardation, proteinuria, glomerulopathy and foot process effacement, together with reduced expression of some slit diaphragm molecules and cytoskeleton rearrangement of podocytes. Y box protein 1 (YB-1) was identified to be the target molecule for dephosphorylation effect of PP2A. Furthermore, YB-1 phosphorylation was up-regulated in the Pod-PP2A-KO mice in contrast to the wild type controls, while total and un-phosphorylated YB-1 both was moderately down-regulated in podocytes from the Pod-PP2A-KO mice. CONCLUSION: Our study revealed the important role of PP2A in regulating the development of foot processes and fully differentiated podocytes whereas fine-tuning of YB-1 via a post-translational modification by PP2A regulating its activity might be crucial for the functional integrity of podocytes and glomerular filtration barrier.

Embryonic Lethality and Host Immunity of RelA-Deficient Mice Are Mediated by Both Apoptosis and Necroptosis.

In mammalian cells, signaling pathways triggered by TNF can be switched from NF-κB activation to apoptosis and/or necroptosis. The in vivo mechanisms underlying the mutual regulation of these three signaling pathways are poorly understood. In this article, we report that the embryonic lethality of RelA-deficient mice is partially prevented by the deletion of Rip3 or Mlkl, but it is fully rescued by the combined ablation of Fadd and Rip3 or Mlkl or by blocking RIP1 kinase activity (RIP1K45A). RelA-/-Fadd-/-Rip3-/- triple-knockout (TKO) and RelA-/-Rip1K45A/K45A mice displayed bacterial pneumonia leading to death ∼2 wk after birth. Moreover, RelA-/-Rip1K45A/K45A mice, but not TKO mice, developed severe inflammation associated with inflammatory skin lesion. Antibiotic treatment improved bacterial pneumonia, extended the lifespan of TKO and RelA-/-Rip1K45A/K45A mice, and alleviated skin inflammation in RelA-/-Rip1K45A/K45A mice. These results show the mechanisms underlying the in vivo mutual regulation between NF-κB activation and the cell death pathway and provide new insights into this interplay in embryonic development and host immune homeostasis.

MLKL and FADD Are Critical for Suppressing Progressive Lymphoproliferative Disease and Activating the NLRP3 Inflammasome.

MLKL, a key component downstream of RIPK3, is suggested to be a terminal executor of necroptosis. Genetic studies have revealed that Ripk3 ablation rescues embryonic lethality in Fadd- or Caspase-8-deficient mice. Given that RIPK3 has also been implicated in non-necroptotic pathways including apoptosis and inflammatory signaling, it remains unclear whether the lethality in Fadd(-/-) mice is indeed caused by necropotosis. Here, we show that genetic deletion of Mlkl rescues the developmental defect in Fadd-deficient mice and that Fadd(-/-)Mlkl(-/-) mice are viable and fertile. Mlkl(-/-)Fadd(-/-) mice display significantly accelerated lymphoproliferative disease characterized by lymphadenopathy and splenomegaly when compared to Ripk3(-/-)Fadd(-/-) mice. Mlkl(-/-)Fadd(-/-) bone-marrow-derived macrophages and dendritic cells have impaired NLRP3 inflammasome activation associated with defects in ASC speck formation and NF-κB-dependent NLRP3 transcription. Our findings reveal that MLKL and FADD play critical roles in preventing lymphoproliferative disease and activating the NLRP3 inflammasome.

Gambogenic acid inhibits LPS-simulated inflammatory response by suppressing NF-κB and MAPK in macrophages.

Inflammation is a response of body tissues to injury and infection. Compounds that can inhibit inflammation have been shown to have potential therapeutic clinical application. Gambogenic acid (GEA) has potent antitumor and anti-inflammatory activities. Herein, the molecular mechanisms of GEA's anti-inflammatory effect were investigated in lipopolysaccharide (LPS)-stimulated macrophage cells. The results showed that pretreatment with GEA could markedly inhibit interleukin (IL)-1α, IL-1ß, tumor necrosis factor-α, IFN-ß, IL-12b, and IL-23a production in a dose-dependent manner in LPS-induced model. Furthermore, this drug significantly reduced the release of nitric oxide (NO), and impaired the protein level of inducible NO synthase and the cyclooxygenase 2. The finding also showed that the effect of GEA may be related to the suppression of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathway. These results indicate that GEA could suppress LPS-simulated inflammatory response partially by attenuating NO synthesis and NF-κB and MAPK activation, suggesting that it may become a potent therapeutic agent for the treatment of inflammatory diseases.

A novel sgRNA selection system for CRISPR-Cas9 in mammalian cells.

CRISPR-Cas9 mediated genome editing system has been developed as a powerful tool for elucidating the function of genes through genetic engineering in multiple cells and organisms. This system takes advantage of a single guide RNA (sgRNA) to direct the Cas9 endonuclease to a specific DNA site to generate mutant alleles. Since the targeting efficiency of sgRNAs to distinct DNA loci can vary widely, there remains a need for a rapid, simple and efficient sgRNA selection method to overcome this limitation of the CRISPR-Cas9 system. Here we report a novel system to select sgRNA with high efficacy for DNA sequence modification by a luciferase assay. Using this sgRNAs selection system, we further demonstrated successful examples of one sgRNA for generating one gene knockout cell lines where the targeted genes are shown to be functionally defective. This system provides a potential application to optimize the sgRNAs in different species and to generate a powerful CRISPR-Cas9 genome-wide screening system with minimum amounts of sgRNAs.