FoxD1 expression identifies a distinct subset of hepatic stellate cells involved in liver fibrosis.

Hepatic stellate cells (HSCs) are pericytes of the liver responsible for liver fibrosis and cirrhosis, which are the end stages of chronic liver diseases. TGF-ß activates HSCs, leading to the differentiation of myofibroblasts in the process of liver fibrosis. While the heterogeneity of HSCs is appreciated in the fibrotic liver, it remains elusive which HSC subsets mainly contribute to fibrosis. Here, we show that the expression of the pericyte marker FoxD1 specifically marks a subset of HSCs in FoxD1-fate tracer mice. HSCs fate-mapped by FoxD1 were preferentially localized in the portal and peripheral areas of both the homeostatic and fibrotic liver induced by carbon tetrachloride. Furthermore, the deletion of Cbfß, which is necessary for TGF-ß signaling, in FoxD1-expressing cells ameliorated liver fibrosis. Thus, we identified an HSC subset that preferentially responds to liver injuries.

Dysfunction of type 1 and type 2 immune cells: a lesson from exhausted-like ILC2s and their activation-induced cell death.

The concept of immune cell exhaustion/dysfunction has developed mainly to understand impaired type 1 immune responses especially by CD8 T cells against tumors or virus-infected cells and has been applied to other lymphocytes. Natural killer (NK) cells and CD4 T cells support the efficient activation of CD8 T cells but exhibit a dysfunctional phenotype in tumor microenvironments and in chronic virus infections. In contrast, the concept of type 2 immune cell exhaustion/dysfunction is poorly established. Group 2 innate lymphoid cells (ILC2s) and T-helper 2 (Th2) cells are the major lymphocyte subsets that initiate and expand type 2 immune responses for antiparasitic immunity or allergy. In mouse models of chronic parasitic worm infections, Th2 cells display impaired type 2 immune responses. Chronic airway allergy induces exhausted-like ILC2s that quickly fall into activation-induced cell death to suppress exaggerated inflammation. Thus, the modes of exhaustion/dysfunction are quite diverse and rely on the types of inflammation and the cells. In this review, we summarize current knowledge of lymphocyte exhaustion/dysfunction in the context of type 1 and type 2 immune responses and discuss ILC2-specific regulatory mechanisms during chronic allergy.

TIGIT mediates activation-induced cell death of ILC2s during chronic airway allergy.

While group-2 innate lymphoid cells (ILC2s) are highly proliferative in allergic inflammation, the removal of overactivated ILC2s in allergic diseases has not been investigated. We previously showed that chronic airway allergy induces "exhausted-like" dysfunctional ILC2s expressing T cell immunoreceptor with Ig and ITIM domains (TIGIT). However, the physiological relevance of these cells in chronic allergy remains elusive. To precisely identify and monitor TIGIT+ ILC2s, we generated TIGIT lineage tracer mice. Chronic allergy stably induced TIGIT+ ILC2s, which were highly activated, apoptotic, and were quickly removed from sites of chronic allergy. Transcripts from coding genes were globally suppressed in the cells, possibly due to reduced chromatin accessibility. Cell death in TIGIT+ ILC2s was enhanced by interactions with CD155 expressed on macrophages, whereas genetic ablation of Tigit or blockade by anti-TIGIT antagonistic antibodies promoted ILC2 survival, thereby deteriorating chronic allergic inflammation. Our work demonstrates that TIGIT shifts the fate of ILC2s toward activation-induced cell death, which could present a new therapeutic target for chronic allergies.

Trained innate lymphoid cells in allergic diseases.

Group 2 innate lymphoid cells (ILC2s) reside in peripheral tissues such as the lungs, skin, nasal cavity, and gut and provoke innate type 2 immunity against allergen exposure, parasitic worm infection, and respiratory virus infection by producing TH2 cytokines. Recent advances in understanding ILC2 biology revealed that ILC2s can be trained by IL-33 or allergic inflammation, are long-lived, and mount memory-like type 2 immune responses to any other allergens afterwards. In contrast, IL-33, together with retinoic acid, induces IL-10-producing immunosuppressive ILC2s. In this review, we discuss how the allergic cytokine milieu and other immune cells direct the generation of trained ILC2s with immunostimulatory or immunosuppressive recall capability in allergic diseases and infections associated with type 2 immunity. The molecular mechanisms of trained immunity by ILCs and the physiological relevance of trained ILC2s are also discussed.

Increased fatty acyl saturation of phosphatidylinositol phosphates in prostate cancer progression.

Phosphoinositides (PIPs) participate in many cellular processes, including cancer progression; however, the metabolic features of PIPs associated with prostate cancer (PCa) are unknown. We investigated PIPs profiles in PTEN-deficient prostate cancer cell lines, human prostate tissues obtained from patients with PCa and benign prostate hyperplasia (BPH) specimens using mass spectrometry. In immortalized normal human prostate PNT1B cells, PTEN deficiency increased phosphatidylinositol tris-phosphate (PIP3) and decreased phosphatidylinositol mono- and bis-phosphate (PIP1 and PIP2), consistent with PTEN's functional role as a PI(3,4,5)P3 3-phosphatase. In human prostate tissues, levels of total (sum of all acyl variants) phosphatidylinositol (PI) and PIP1 in PCa were significantly higher than in BPH, whereas PIP2 and PIP3 contents were significantly lower than in BPH. PCa patients had significantly higher proportion of PI, PIP1, and PIP2 with 0-2 double bonds in acyl chains than BPH patients. In subgroup analyses based on PCa aggressiveness, mean total levels of PI with 0-2 double bonds in acyl chains were significantly higher in patients with pathological stage T3 than in those with pathological stage T2. These data indicate that alteration of PIPs level and the saturation of acyl chains may be associated with the development and aggressiveness of prostate cancer, although it is unknown whether this alteration is causative.

Phosphorylation of TMEM55B by Erk/MAPK regulates lysosomal positioning.

TMEM55B is first identified as phosphatidylinositol-4,5-P24-phosphatases (PtdIns-4,5-P24-phosphatases) that catalyse dephosphorylation of PtdIns-4,5-P2 to PtdIns-5-P. We demonstrate for the first time that TMEM55B is phosphorylated by Erk/MAPK and that this mechanism participates in regulation of lysosomal clustering. Exposure of RAW264.7 macrophages to various stimuli induces phosphorylation of TMEM55B on Ser76 and Ser169, sites corresponding to consensus sequences (PX(S/T)P) for phosphorylation by MAPK. Of these stimuli, Toll-like receptor ligands most strongly induce TMEM55B phosphorylation, and this is blocked by the MEK1/2 inhibitor U0126. However, phosphorylation does not impact intrinsic phosphatase activity of TMEM55B. TMEM55B has recently been implicated in starvation induced lysosomal translocation. Amino acid starvation induces perinuclear lamp1 clustering in RAW264.7 macrophages, which was attenuated by shRNA-mediated knock-down or CRISPR/Cas9-mediated knock-out of TMEM55B. Cells exposed to U0126 also exhibit attenuated lamp1 clustering. Overexpression of TMEM55B but not TMEM55A notably enhances lamp1 clustering, with TMEM55B mutants (lacking phosphorylation sites or mimicking the phosphorylated state) exhibiting lower and higher efficacies (respectively) than wild-type TMEM55B. Collectively, results suggest that phosphorylation of TMEM55B by Erk/MAPK impacts lysosomal dynamics.

Sac1 Phosphoinositide Phosphatase Regulates Foam Cell Formation by Modulating SR-A Expression in Macrophages.

Macrophages endocytose modified low-density lipoproteins (LDL) vigorously via scavenger receptor A (SR-A) to become foam cells. In the present study, we found that Sac1, a member of the Sac family of phosphoinositide phosphatases, increases the protein level of SR-A and upregulates foam cell formation. Mouse macrophages (RAW264.7) were transfected with short hairpin RNAs (shRNAs) against Sac1. Sac1 knockdown decreased cell surface SR-A levels and impaired acetylated LDL-induced foam cell formation. Transfection of Sac1-knockdown cells with shRNA-resistant flag-Sac1 effectively rescued the expression of SR-A. Glycosylation of SR-A was largely attenuated by Sac1 knockdown, but neither mRNA expression nor protein degradation of SR-A were affected. These results suggest that Sac1 maintains SR-A protein levels by modulating SR-A glycosylation.

Lysophosphatidylinositol-acyltransferase-1 is involved in cytosolic Ca2+ oscillations in macrophages.

Lysophosphatidylinositol-acyltransferase-1 (LPIAT1) specifically catalyzes the transfer of arachidonoyl-CoA to lysophosphoinositides. LPIAT-/- mice have been shown to have severe defects in the brain and liver; however, the exact molecular mechanisms behind these conditions are not well understood. As immune cells have been implicated in liver inflammation based on disfunction of LPIAT1, we generated Raw264.7 macrophages deficient in LPIAT1, using shRNA and CRISPR/Cas9. The amount of C38:4 species in phosphoinositides, especially in PtdInsP2 , was remarkably decreased in these cells. Unlike in wild-type cells, LPIAT1-deficient cells showed prolonged oscillations of intracellular Ca2+ upon UDP stimulation, which is known to activate phospholipase Cß through the Gq-coupled P2Y6 receptor, even in the absence of extracellular Ca2+ . It is speculated that the prolonged Ca2+ response may be relevant to the increased risk of liver inflammation induced by LPIAT1 disfunction.

PIKfyve accelerates phagosome acidification through activation of TRPML1 while arrests aberrant vacuolation independent of the Ca2+ channel.

PIKfyve phosphorylates PtdIns(3)P to PtdIns(3, 5)P2. One of the best characterized effector downstream of PtdIns(3, 5)P2 is a lysosomal Ca2+ channel, TRPML1. Although it has been reported that TRPML1 is involved in phagosome-lysosome fusion, the relevance of the Ca2+ channel in phagosome acidification has been denied. In this article, however, we demonstrated that the phagosome acidification was dependent on TRPML1. Based on the classical idea that Fluorescein isothiocyanate (FITC)-fluorescence is highly sensitive to acidic pH, we could estimate the phagosome acidification by time laps imaging. FITC-zymosan fluorescence that was engulfed by macrophages, decreased immediately after the uptake while the extinction of FITC-zymosan fluorescence was delayed in PIKfyve-deficient cells. The acidification arrest was completely rescued in the presence of Ca2+ ionophore A23187. Cells treated with a PIKfyve inhibitor, apilimod, also showed delayed phagosome acidification but were rescued by the overexpression of TRPML1. Additionally, TRPML1 agonist, ML-SA1 was effective to acidify the phagosome in PIKfyve-deficient cells. Another phenotype observed in PIKfyve-deficient cells is vacuole formation. Unexpectedly, enlarged vacuole formation in PIKfyve-deficient cells was not rescued by Ca2+ or over expression of TRPML1. It is likely that the acidification and vacuolation arrest is bifurcating downstream of PIKfyve.

PIKfyve regulates melanosome biogenesis.

PIKfyve, VAC14, and FIG4 form a complex that catalyzes the production of PI(3,5)P2, a signaling lipid implicated in process ranging from lysosome maturation to neurodegeneration. While previous studies have identified VAC14 and FIG4 mutations that lead to both neurodegeneration and coat color defects, how PIKfyve regulates melanogenesis is unknown. In this study, we sought to better understand the role of PIKfyve in melanosome biogenesis. Melanocyte-specific PIKfyve knockout mice exhibit greying of the mouse coat and the accumulation of single membrane vesicle structures in melanocytes resembling multivesicular endosomes. PIKfyve inhibition blocks melanosome maturation, the processing of the melanosome protein PMEL, and the trafficking of the melanosome protein TYRP1. Taken together, these studies identify a novel role for PIKfyve in controlling the delivery of proteins from the endosomal compartment to the melanosome, a role that is distinct from the role of PIKfyve in the reformation of lysosomes from endolysosomes.

TMEM55a localizes to macrophage phagosomes to downregulate phagocytosis.

TMEM55a (also known as PIP4P2) is an enzyme that dephosphorylates the phosphatidylinositol (PtdIns) PtdIns(4,5)P2 to form PtdIns(5)P in vitro However, the in vivo conversion of the polyphosphoinositide into PtdIns(5)P by the phosphatase has not yet been demonstrated, and the role of TMEM55a remains poorly understood. Here, we found that mouse macrophages (Raw264.7) deficient in TMEM55a showed an increased engulfment of large particles without affecting the phagocytosis of Escherichia coli Transfection of a bacterial phosphatase with similar substrate specificity to TMEM55a, namely IpgD, into Raw264.7 cells inhibited the engulfment of IgG-erythrocytes in a manner dependent on its phosphatase activity. In contrast, cells transfected with PIP4K2a, which catalyzes PtdIns(4,5)P2 production from PtdIns(5)P, increased phagocytosis. Fluorescent TMEM55a transfected into Raw264.7 cells was found to mostly localize to the phagosome. The accumulation of PtdIns(4,5)P2, PtdIns(3,4,5)P3 and F-actin on the phagocytic cup was increased in TMEM55a-deficient cells, as monitored by live-cell imaging. Phagosomal PtdIns(5)P was decreased in the knockdown cells, but the augmentation of phagocytosis in these cells was unaffected by the exogenous addition of PtdIns(5)P. Taken together, these results suggest that TMEM55a negatively regulates the phagocytosis of large particles by reducing phagosomal PtdIns(4,5)P2 accumulation during cup formation.

The effect and possible clinical efficacy of in vivo inhibition of neutrophil extracellular traps by blockade of PI3K-gamma on the pathogenesis of microscopic polyangiitis.

OBJECTIVE: Neutrophil extracellular traps (NETs) are peculiar structures composed of the externalized chromatin with intracellular proteins and formed by activated neutrophils in a reactive oxygen species (ROS)-dependent manner. Aberrant NETs are considered to be autoantigens for anti-neutrophil cytoplasmic antibodies (ANCAs) underling the development of microscopic polyangiitis (MPA). However, little is known regarding the therapeutic efficacy of in vivo inhibition of NET formation (NETosis) on MPA pathogenesis. This study determines whether reducing NETosis prevents ANCA production and improves characteristic involvement. METHODS: A mouse model of MPA induced by administering a novel extract from Candida albicans was devised. By applying this method to mice lacking phosphoinositide 3-kinase gamma (PI3K-gamma), which is indispensable for ROS production in neutrophils, we investigated the levels of in vivo NETs, ANCA titers and histological damage. RESULTS: Our model exhibited accumulation of NETs in vivo, elevation of ANCA titers and characteristic pathologies mimicking human MPA, including small-vessel vasculitis and crescentic glomerulonephritis. Strikingly, these abnormalities were reduced by genetically and/or pharmacologically blocking PI3K-gamma. Moreover, a pharmacological PI3K-gamma blockade decreased the levels of human NETs. CONCLUSION: Our results suggest that in vivo inhibition of NETosis by blocking PI3K-gamma could be a promising therapeutic strategy for the pathogenesis of MPA.

Phosphoinositide phosphatase Sac3 regulates the cell surface expression of scavenger receptor A and formation of lipid droplets in macrophages.

The findings of this study suggest that the phosphoinositide phosphatase Sac3 maintains the protein level of scavenger receptor A (SR-A) and regulates foam cell formation. RAW264.7 macrophages were transfected with short hairpin RNAs that target Sac3. The knockdown decreased the level of the cell surface SR-A and suppressed the acetylated low density lipoprotein-induced foam cell formation. The associated regulator of PIKfyve (ArPIKfyve) is a scaffold protein that protects Sac3 from proteasome-dependent degradation. The knockdown of ArPIKfyve decreased Sac3, cell surface SR-A, and foam cell formation. The knockdown of PIKfyve had no effect on SR-A protein levels. These results suggest that the ArPIKfyve-Sac3 complex regulates SR-A protein levels independently of its effect on PIKfyve activity.

Vps34 regulates myofibril proteostasis to prevent hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common heart disease with a prevalence of 1 in 500 in the general population. Several mutations in genes encoding cardiac proteins have been found in HCM patients, but these changes do not predict occurrence or prognosis and the molecular mechanisms underlying HCM remain largely elusive. Here we show that cardiac expression of vacuolar protein sorting 34 (Vps34) is reduced in a subset of HCM patients. In a mouse model, muscle-specific loss of Vps34 led to HCM-like manifestations and sudden death. Vps34-deficient hearts exhibited abnormal histopathologies, including myofibrillar disarray and aggregates containing αB-crystallin (CryAB). These features result from a block in the ESCRT-mediated proteolysis that normally degrades K63-polyubiquitinated CryAB. CryAB deposition was also found in myocardial specimens from a subset of HCM patients whose hearts showed decreased Vps34. Our results identify disruption of the previously unknown Vps34-CryAB axis as a potentially novel etiology of HCM.

The ML1Nx2 Phosphatidylinositol 3,5-Bisphosphate Probe Shows Poor Selectivity in Cells.

Phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P2) is a quantitatively minor phospholipid in eukaryotic cells that plays a fundamental role in regulating endocytic membrane traffic. Despite its clear importance for cellular function and organism physiology, mechanistic details of its biology have so far not been fully elucidated. In part, this is due to a lack of experimental tools that specifically probe for PtdIns(3,5)P2 in cells to unambiguously identify its dynamics and site(s) of action. In this study, we have evaluated a recently reported PtdIns(3,5)P2 biosensor, GFP-ML1Nx2, for its veracity as such a probe. We report that, in live cells, the localization of this biosensor to sub-cellular compartments is largely independent of PtdIns(3,5)P2, as assessed after pharmacological, chemical genetic or genomic interventions that block the lipid's synthesis. We therefore conclude that it is unwise to interpret the localization of ML1Nx2 as a true and unbiased biosensor for PtdIns(3,5)P2.

INPP4B Is a PtdIns(3,4,5)P3 Phosphatase That Can Act as a Tumor Suppressor.

UNLABELLED: Inositol polyphosphate 4-phosphatase B (INPP4B) has been identified as a tumor suppressor mutated in human breast, ovary, and prostate cancers. The molecular mechanism underlying INPP4B's tumor-suppressive role is currently unknown. Here, we demonstrate that INPP4B restrains tumor development by dephosphorylating the PtdIns(3,4,5)P3 that accumulates in situations of PTEN deficiency. In vitro, INPP4B directly dephosphorylates PtdIns(3,4,5)P3. In vivo, neither inactivation of Inpp4b (Inpp4b(Δ/Δ)) nor heterozygous deletion of Pten (Pten(+/-)) in mice causes thyroid abnormalities, but a combination of these mutations induces malignant thyroid cancers with lung metastases. At the molecular level, simultaneous deletion of Inpp4b and Pten synergistically increases PtdIns(3,4,5)P3 levels and activates AKT downstream signaling proteins in thyroid cells. We propose that the PtdIns(3,4,5)P3 phosphatase activity of INPP4B can function as a "back-up" mechanism when PTEN is deficient, making INPP4B a potential novel therapeutic target for PTEN-deficient or PIK3CA-activated cancers. SIGNIFICANCE: Although INPP4B expression is reduced in several types of human cancers, our work on Inpp4B-deficient mice provides the first evidence that INPP4B is a bona fide tumor suppressor whose function is particularly important in situations of PTEN deficiency. Our biochemical data demonstrate that INPP4B directly dephosphorylates PtdIns(3,4,5)P3.

Inpp5e increases the Rab5 association and phosphatidylinositol 3-phosphate accumulation at the phagosome through an interaction with Rab20.

Phosphoinositide 5'-phosphatases have been implicated in the regulation of phagocytosis. However, their precise roles in the phagocytic process are poorly understood. We prepared RAW264.7 macrophages deficient in Inpp5e (shInpp5e) to clarify the role of this lipid phosphatase. In the shInpp5e cells, the uptake of solid particles was increased and the rate of phagosome acidification was accelerated. As expected, levels of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 were increased and decreased respectively, on the forming phagocytic cups of these cells. Unexpectedly, the most prominent consequence of the Inpp5e deficiency was the decreased accumulation of PtdIns3P and Rab5 on the phagosome. The expression of a constitutively active form of Rab5b in the shInpp5e cells rescued the PtdIns3P accumulation. Rab20 has been reported to regulate the activity of Rabex5, a guanine nucleotide exchange factor for Rab5. The association of Rab20 with the phagosome was remarkably abrogated in the shInpp5e cells. Over-expression of Rab20 increased phagosomal PtdIns3P accumulation and delayed its elimination. These results suggest that Inpp5e, through functional interactions with Rab20 on the phagosome, activates Rab5, which, in turn, increases PtdIns3P and delays phagosome acidification.

Phosphatidylinositol-3,5-bisphosphate: metabolism and physiological functions.

Phosphatidylinositol (PtdIns) is a membrane phospholipid composed of diacylglycerol and a D-myo-inositol head group. In mammals, the hydroxyl groups at the D3, D4 and D5 positions of the inositol ring can be phosphorylated to yield seven phosphoinositide derivatives. PtdIns-3,5-bisphosphate [PtdIns(3,5)P2] is the most recently discovered species of phosphoinositide that is generated by the phosphorylation of PtdIns(3)P at the D5 position by PtdIns phosphate kinase and catabolized through the dephosphorylation by myotubularin family of phosphatases. Genetic and biochemical analyses of the enzymes metabolizing PtdIns(3,5)P2 have revealed that this phospholipid is involved in the control of endolysosomal systems and plays crucial roles in various mammalian tissues. In this article, we review the current state of knowledge of the metabolic/physiological functions of PtdIns(3,5)P2, and describe how disruption of these functions may contribute to human diseases.

Critical roles of type III phosphatidylinositol phosphate kinase in murine embryonic visceral endoderm and adult intestine.

The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] controls multiple steps of the intracellular membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of PtdIns(3,5)P(2) in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P(2), is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell's apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that PtdIns(3,5)P(2) metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease.

Essential roles of PIKfyve and PTEN on phagosomal phosphatidylinositol 3-phosphate dynamics.

PtdIns(3)P (phosphatidylinositol 3-phosphate) is a signaling molecule important for phagosome maturation. The major role of Vps34 in production of phagosomal PtdIns(3)P has been indicated. However, the fate of the newly generated PtdIns(3)P has not been well described. Here we show that elimination of PtdIns(3)P from phagosomal membrane was significantly delayed in RAW264.7 macrophages lacking PTEN or PIKfyve. In the PTEN-deficient cells treated with a PIKfyve inhibitor, degradation of PtdIns(3)P was almost lost, indicating that PTEN and PIKfyve are two major players in phagosomal PtdIns(3)P metabolism.