ALBERT

Description: Proper development of the CNS axon-glia unit requires bi-directional communication between axons and oligodendrocytes (OLs). We show that the signaling lipid phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is required in neurons and in OLs for normal CNS myelination. In mice, mutations of Fig4, Pikfyve or Vac14, encoding key components of the PI(3,5)P2 biosynthetic complex, each lead to impaired OL maturation, severe CNS hypomyelination and delayed propagation of compound action potentials. Primary OLs deficient in Fig4 accumulate large LAMP1+ and Rab7+ vesicular structures and exhibit reduced membrane sheet expansion. PI(3,5)P2 deficiency leads to accumulation of myelin-associated glycoprotein (MAG) in LAMP1+perinuclear vesicles that fail to migrate to the nascent myelin sheet. Live-cell imaging of OLs after genetic or pharmacological inhibition of PI(3,5)P2 synthesis revealed impaired trafficking of plasma membrane-derived MAG through the endolysosomal system in primary cells and brain tissue. Collectively, our studies identify PI(3,5)P2 as a key regulator of myelin membrane trafficking and myelinogenesis.

Description: Key Points Different isoforms of PIP5KIγ fulfill unique functions in platelets.

Description: Platelets release different types of secretory granules into their local environment, and this allows them to contribute to a variety of physiologic processes. These platelet granules include alpha granules, dense granules, and lysosomes, which all derive from the endosomal-lysosomal system. However, the mechanism of the biogenesis of each type of granule is not completely understood. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] is a membrane phosphoinositide that is essential for the regulation of membrane homeostasis, as well as for vesicle trafficking and cargo transport along the endolysosomal system in mammals. PtdIns(3,5)P2 is synthesized on endosomes by the lipid kinase PIKfyve. Given the role of PIKfyve-mediated PtdIns(3,5)P2 production in the endolysosomal pathway in other mammalian cells, we hypothesized that PtdIns(3,5)P2 was an essential regulator for the biogenesis of granules within platelets. To analyze the contribution of PtdIns(3,5)P2 to platelet granule biogenesis, we generated mice lacking PIKfyve kinase activity specifically in their platelets and in their megakaryocytes (PIKfyveflox/flox Pf4-Cre). We found that when compared with the control mice, PIKfyveflox/flox Pf4-Cre mice contained higher levels of the lysosomal enzyme, β-hexosaminidase, within their platelets and within their plasma. The PIKfyve-ablated platelets also released excessive β-hexosaminidase ex vivo upon stimulation with ADP, collagen or thrombin. However, the percentage of the total cellular β-hexosaminidase that was released from the PIKfyve-ablated platelets was comparable with that of control platelets. This suggests that the increased release of β-hexosaminidase from the PIKfyve-ablated platelets is due to the excessive storage of this enzyme within these platelets and not because of increased efficiency of lysosome secretion. In addition, we observed that PIKfyve-ablated platelets expressed increased amounts of Lysosomal Associated Membrane Protein 1 (LAMP-1), a marker of late endosomes and lysosomes. However, PIKfyve-ablated platelets expressed normal amounts of Early Endosome Antigen 1 (EEA-1), a marker of early endosomes. These results suggest that PIKfyve is critical for a component of platelet lysosome biology that occurs after the maturation of early endosomes. Together, these data demonstrate that PIKfyve is essential for the homeostasis of the endolysosomal system in platelets. Notably, the generation and secretion of the alpha granule components were intact in the PIKFyve-ablated platelets. This was shown by the normal expression of von Willebrand factor, platelet basic protein, and platelet factor 4. Likewise, secretion of ATP stored in the dense granules was similar between the PIKfyveflox/flox Pf4-Cre mice and their control littermates. Together, these results suggest that PIKfyve plays an essential regulatory role along the endolysosomal pathway in platelets, and the loss of PIKfyve in platelets can lead to an abnormality of lysosomal storage. Unexpectedly, we also found that the loss of PIKfyve exclusively within platelets triggers an inappropriate inflammatory response. This is shown by the massive tissue infiltration of aberrant vacuolated macrophages. In turn, this leads to multiple organ defects that impair development, body mass, and survival in mice. Moreover, mice lacking PIKfyve within their platelets developed accelerated arterial thrombosis in vivo, despite having normal platelet aggregation ex vivo. It is also remarkable that mice lacking PIKfyve in their platelets attenuated their organ defects when the secretion of their platelet lysosomes were inhibited in vivo. Collectively, our study demonstrates that PIKfyve is an essential regulator of platelet lysosome biogenesis. This study also highlights the previously unrecognized and important contributions of platelet lysosomal storage to inflammation, arterial thrombosis, and macrophage biology. Disclosures: No relevant conflicts of interest to declare.

Description: Phosphatidylinositol (PtdIns) is a relatively rare phospholipid in cell membranes. However, because of its unique ability to be transiently phosphorylated, it is critical for platelet signaling and vascular plug formation. Phosphatidylinositol transfer proteins (PITPs) facilitate the transfer of aqueous insoluble phosphatidylinositol in vitro from one cell membrane to another. Although murine platelets contain multiple PITP variants, PITPα is by far the most abundant isoform. Platelets are best known for their contribution to hemostasis, however, several lines of evidence indicate that they also contribute to tumor metastasis formation. To test the hypothesis that platelet phosphoinositide signaling contributes to tumor dissemination, we asked whether deletion of platelet PITPα impacts tumor metastasis formation by engineering PITPαfl/fl PF4Cre+ mice that lack PITPα only within their platelets and megakaryocytes. We found that loss of PITPα in platelets decreased PtdIns(4)P and PtdIns(4,5)P2 production by 30-40%. Even more striking was the 80% reduction in IP3 formation following thrombin-stimulation. However, we found no significant defect in the platelet ex vivo aggregation in response to typical doses of most of the platelet agonists, and only a small defect in response to low doses of thrombin. We investigated whether platelets lacking PITPα normally formed thrombi in vivo by using three well-established murine models. First, we observed an extremely minor (but statistically real) prolongation of the tail bleeding time in the PITPαfl/fl PF4Cre+ mice. Second, we found that mice lacking PITPα in their platelets had no defect in forming intravascular clots in response to a chemical induced carotid injury. Finally, we observed that thrombosis and platelet a-granule secretion in response to a laser-induced injury were completely normal in the knockout mice. Therefore, despite the biochemical defect in phosphoinositide signaling induced by the loss of PITPα, there was essentially no hemostastic defect. To determine whether PITPα-mediated phosphoinositide metabolism in platelets is required for tumor dissemination, we utilized a well-characterized B16F10 melanoma model of tumor metastasis. We observed that lung metastasis formation was reduced by 47%±18% in mice lacking PITPα in their platelets. We also found that during the first 3 hours after tumor injection, the control mice, but not the PITPαfl/fl PF4Cre+ mice, developed a rapid and transient thrombocytopenia. Histology analysis of the lung tissue at this time point revealed the presence of 30% more clots in the lung tissue of control mice. Further analysis showed that these thrombi were actually heterogenous complexes composed of tumor cells surrounded by platelets and fibrin. To understand why platelet PITPα influences tumor-induced fibrin formation, we investigated the ability of tumor cells to cause thrombin generation in platelet rich plasma derived from PITPαfl/fl PF4Cre+ and PITPαfl/fl PF4Cre- mice. We observed that the loss of PITPα in platelets resulted in an 88% reduction of thrombin generation compared to the controls. Furthermore, we found that PITPαfl/fl PF4Cre+ platelets also have impaired Annexin V binding suggesting that the defective fibrin formation seen in PITPαfl/fl PF4Cre+ mice is likely due to a role of PITPα in the exposure of phosphatidylserine on the platelet surface that is required for thrombin generation. Finally, we observed a mucosal immune response composed of NK-cells, T-cells, and neutrophils that was strikingly hyperplastic in mice lacking platelet PITPα at 48 hours after tumor injection, but was essentially absent in the control mice. Together, these findings demonstrate that PITPα-mediated phosphoinositide metabolism within platelets is not essential for platelet plug formation in vivo, but is required for the dissemination of tumors in vivo. Our work further demonstrates that platelet PITPα is required for tumor-induced phosphatidylserine exposure and thrombin generation. This process induces a shroud of platelets and fibrin that surround the surface of tumor cells, and thereby protects the tumors from elimination by the mucosal immune system. These results demonstrate that it is possible to clearly distinguish the platelet signaling processes required for platelet plug formation from those processes that augment metastasis formation. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 262 Platelets play key roles in hemostasis and thrombosis, as well as in non-hemostatic processes, including atherosclerosis and inflammation. Although platelet secretory products are thought to be essential mediators in these processes, the mechanism of cross-talk between platelets and cells of both the vascular and immune systems is poorly understood. Here, we report that the inactivation of a phosphoinositide kinase, PIKFyve, specifically in platelets, leads to the secretion of aberrant platelet granules and tissue infiltration of vacuolated macrophages, thus promoting both inflammation and thrombosis in mice. We recently generated a mouse model with the intent to analyze the role of phosphoinositide synthesis in platelet granule biogenesis. These mice were genetically engineered to ablate platelet-specific PIKFyve kinase activity (PF4Cre+ PIKFyvefl/fl) necessary to synthesize phosphatidylinositol(3,5)P2. The PF4Cre+ PIKFyvefl/fl mice formed aberrant platelet granules, but unexpectedly developed hair loss, body-swelling, osteopenia, and multiorgan failure, leading to death by 5–6 months of age. Necropsy revealed extensive tissue infiltration of large vacuolated macrophages. These findings were associated with accelerated arterial thrombosis in vivo. Because the targeting strategy was not anticipated to directly affect macrophages, we analyzed tissue expression of the PF4-Cre transgene in the PF4Cre+ PIKFyvefl/fl mouse by crossing it with a Cre-dependent LacZ reporter mouse. The PF4-Cre expression was confirmed to be limited to platelets and megakaryocytes, and not in any other cells, including the vacuolated macrophages. This suggests that the macrophage-driven phenotype is mediated by macrophage-extrinsic factors, likely by PIKFyve-null platelets. To examine whether the macrophage phenotype is mediated by hematopoietic cells, PF4 Cre+ PIKFyvefl/fl bone marrow cells were transplanted into lethally irradiated WT mice. The recipient mice engrafted with PF4 Cre+ PIKFyvefl/fl donor cells developed tissue infiltration of vacuolated macrophages in multiple organs, recapitulating the phenotype of PF4 Cre+ PIKFyvefl/fl mice. Conversely, the lethally irradiated PF4 Cre+ PIKFyvefl/fl recipient mice repopulated with WT donor cells completely reverted their hair loss and body swelling, and resolved their tissue accumulation of vacuolated macrophages. Moreover, while PF4 Cre+ PIKFyvefl/fl mice survive for only 5–6 months, the PF4 Cre+ PIKFyvefl/fl mice that were recipients of normal hematopoietic progenitor cells appear to have a normal lifespan. Together, our data demonstrate that the macrophage-driven phenotype is exclusively mediated by hematopoietic cells. To determine whether the released granules from PIKFyve-null platelets mediate the macrophage phenotype, PF4 Cre+ PIKFyvefl/fl mice were crossed with Bloc1−/− mice. The Bloc1 complex is required for vesicle docking and fusion during the biogenesis and secretion of platelet granules. The PF4 Cre+ PIKFyvefl/fl mice that were also homozygous for the Bloc1 mutation did not develop the macrophage-driven pleomorphic phenotype typically seen in PF4 Cre+ PIKFyvefl/fl mice. This suggests that the secretion of granules from PIKFyve-null platelets is required to drive the macrophage response in PF4 Cre+ PIKFyvefl/fl mice. Notably, PIKFyve-null megakaryocytes displayed numerous enlarged vacuoles in the cytoplasm by electron microscopy. In addition, despite normal secretion of the alpha and dense granule cargos, PIKFyve-null platelets contained and released elevated levels of lysosomal enzymes. Although ex vivo study of PIKFyve-null platelet aggregation was normal, in vivo analysis of platelet thrombus formation with a FeCl3-induced carotid artery injury model showed an accelerated formation of arterial thrombus indicating that PF4 Cre+ PIKFyvefl/fl mice were prothrombotic. Collectively, our data show that the loss of PIKFyve kinase activity in platelets results in the formation of aberrant platelet granules with increased release of lysosomal enzymes, and promotes the development of macrophage-driven inflammation and thrombosis. This study provides further evidence that secretion of platelet granules modulates primary immunity, and contributes to a wide range of biological processes that extend beyond hemostasis. Disclosures: No relevant conflicts of interest to declare.

Description: Phosphatidylinositol and its phosphorylated derivatives, phosphoinositides, are minor constituents of phospholipids at the cellular membrane level. Nevertheless, phosphatidylinositol and phosphoinositides represent essential components of intracellular signaling that regulate diverse cellular processes, including platelet plug formation. Accumulating evidence indicates that the metabolism of phosphoinositides is temporally and spatially modulated by the opposing effects of specific phosphoinositide-metabolizing enzymes, including lipid kinases, lipid phosphatases, and phospholipases. Each of these enzymes generates a selective phosphoinositide or second messenger within precise cellular compartments. Intriguingly, phosphoinositide-metabolizing enzymes exist in different isoforms, which all produce the same phosphoinositide products. Recent studies using isoform-specific mouse models and chemical inhibitors have elucidated that the different isoforms of phosphoinositide-metabolizing enzymes have nonredundant functions and provide an additional layer of complexity to the temporo-spatial organization of intracellular signaling events. In this review, we will discuss recent advances in our understanding of phosphoinositide organization during platelet activation.

Description: The biogenesis and degradative functions of the lysosome in macrophages and other cells is critically dependent on the membrane trafficking along the endolysosomal system. This trafficking is tightly regulated by several signaling molecules including the endosomal phosphoinositide PtdIns(3,5)P2, which is synthesized from PtdIns(3)P in mammals by the lipid kinase, PIKfyve. Although PtdIns(3,5)P2 deficiency is associated with neurodegeneration in mice and humans, the critical importance of PIKfyve to normal macrophage function is poorly understood. To address this, we genetically engineered mice to lack PIKfyve in their macrophages by pairing PIKfyvefl/fl mice with mice that are transgenic for the myeloid-specific LysM-Cre (PIKfyvefl/fl LysM-Cre). PIKfyvefl/fl LysM-Cre mice develop weight gain and massive hepatosplenomegaly. Immunohistochemical analyses of the liver and spleen from PIKfyvefl/fl LysM-Cre mice demonstrate tissue infiltration of mononuclear cells with numerous cytoplasmic vacuoles that stain with the macrophage-specific marker, anti-F4/80 antibody. To verify that these vacuolated cells result from PIKfyve ablation, we utilized genetic lineage tracing by crossing PIKfyvefl/fl LysM-Cre mice with mice transgenic for a Cre-dependent β-galactosidase reporter. Histochemical analyses using a chromogenic substrate, x-gal, reveal β-galactosidase activity in cells with small cytoplasmic vacuoles, demonstrating Cre expression and therefore PIKfyve ablation in these cells. However, β-galactosidase activity is absent in cells with large vacuoles, indicating that these cells do not express Cre and still contain PIKfyve. These findings suggest that the cytoplasmic vacuolation resulted from both cell-autonomous and non-cell-autonomous effects of PIKfyve deletion. To investigate the macrophage-specific effects of PIKfyve ablation in the lysosomal biogenesis and functions, we isolated macrophages from the bone marrow and spleen of PIKfyvefl/fl LysM-Cre mice using magnetic beads bound to anti-F4/80 antibody. Immunoblot analysis of F4/80+ PIKfyve-null macrophages reveal increased expression of lysosomal proteins including Cathepsin D, Cathepsin S, and LAMP-1, demonstrating that PIKfyve is essential for lysosome homeostasis in macrophages. Recent studies showed that the transcription factor TFEB is a master regulator of lysosomal biogenesis and function. When TFEB is phosphorylated by mTORC1 on the lysosomal membrane, TFEB is retained in the cytosol, and therefore unable to promote gene transcription. However, when TFEB is de-phosphorylated, TFEB translocates to the nucleus where it is transcriptionally active. Since PtdIns(3,5)P2 was previously shown to regulate the recruitment of mTORC1 to the lysosome, we hypothesized that the synthesis of PtdIns(3,5)P2 by PIKfyve regulates TFEB phosphorylation and indirectly controls TFEB transcriptional activity. To test this hypothesis, we studied the expression of TFEB in the F4/80+ macrophages isolated from the control and PIKfyvefl/fl LysM-Cre mice. While wild-type macrophages express TFEB in both phosphorylated and dephosphorylated forms, PIKfyve-null macrophages primarily express the dephosphorylated form of TFEB. This suggests that PIKfyve is required for the phosphorylation of TFEB and consequently PIKfyve prevents TFEB’s ability to transcribe lysosomal genes. Thus, PIKfyve ablation within macrophages upregulates the transcription of lysosomal genes via TFEB activation. Our study suggests that PIKfyve is a critical regulator of lysosomal homeostasis and demonstrates a previously unrecognized link between PIKfyve, PtdIns(3,5)P2 synthesis, and the TFEB pathway in the regulation of lysosomal biogenesis and function within macrophages. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 549 In response to agonist stimulation, platelets undergo a rapid reorganization of their actin cytoskeleton. This process involves simultaneous disassembly and assembly of filamentous actin, and is one of the earliest phenomena seen in platelet activation. Ex vivo flow models suggest that the platelet cytoskeleton is required for platelet adhesion that can withstand the shear conditions found within the arterial vascular system. The signaling pathways that link external stimuli with actin assembly are believed to include polyphosphoinositides, small GTP-binding proteins, and actin binding proteins. Extrapolations of data, mostly derived from tissue culture cell lines, suggest that a central component of this signaling cascade is the small GTP binding protein, RhoA. A few studies using a RhoA-specific pharmacologic inhibitor, C3 exotoxin, suggest that RhoA is essential for platelet spreading and focal adhesion formation. These findings support the hypothesis that RhoA within platelets is critical for the cytoskeletal dependent processes that contribute to platelet plug formation. To determine the true in vivo role of RhoA within platelets, we utilized a murine genetic approach. Mice were genetically modified to contain conditional RhoA null mutation by inserting LoxP sites flanking exon 3. This exon encodes the P-loop and Switch 1 domains within this protein. RhoA fl/fl mice were crossed with Platelet factor 4 (PF4) expressing Cre mice. The PF4 promotor leads to Cre expression exclusively in platelets and megakaryocytes, thereby producing homologous recombination at the LoxP sites, and deletion the critical exon only within these cells. This end result of this breeding strategy produced RhoA fl/fl PF4 Cre+ mice that specifically lacked RhoA only in their platelets and megakaryocytes. RhoA fl/fl PF4 Cre+ mice were compared with their RhoA fl/fl PF4 Cre- littermates. RhoA fl/fl PF4 Cre+ mice appeared normal, but had platelet counts that were 30% +/− 3% lower than normal. The mean platelet volume was also increased by 25 % +/− 7 % in the RhoA-null platelets. Review of the peripheral blood smears confirmed that the mice had macrothrombocytopenia, but did not reveal any abnormalities in the erythrocytes or leukocytes of the mice. Examination of the bone marrows from RhoA fl/fl PF4 Cre+ mice demonstrated that they had at least as many megakaryocytes as RhoA fl/fl PF4 Cre- mice. But compared to the control cells, the RhoA-null megakaryocytes were larger, more lobulated, and had more cytoplasm. Furthermore, the thromobocytopenia is probably not due to splenic sequestration because the spleens of RhoA fl/fl PF4 Cre- mice were only minimally larger (less than 10%) than those of the control mice. These results suggest that the mechanism for thrombocytopenia is due to peripheral destruction. Platelets derived from RhoA fl/fl PF4 Cre+ mice were studied ex vivo, and were found to undergo shape change and aggregate normally in response to thrombin, collagen, and the thromboxane A2 analog, U46619. Surprisingly, platelet adhesion and cell spreading was also unaffected by the loss of RhoA. It is also remarkable that total F-actin (as assessed by phalloidin staining) was identical in the platelets derived from RhoA fl/fl PF4 Cre+ and RhoA fl/fl PF4 Cre- mice. Our results definitively refute the model that RhoA is an essential component of platelet actin dynamics and platelet adhesion. Instead our findings surprisingly indicate that loss of the platelet RhoA causes macrothrombocytopenia. Our data suggests that the development of macrothrombocytopenia is due to an intrinsic platelet abnormality that leads to a shortened platelet lifespan. Disclosures: No relevant conflicts of interest to declare.

Description: Phosphatidylinositol (PI) comprises less than one percent of the total phospholipid content in platelets. However, PI serves important roles in second messenger signaling and membrane trafficking. The inositol head groups of PI can be phosphorylated by PI kinases to generate seven different phosphoinositides (PtdIns), each capable of producing unique signaling events. The biogenesis of these PtdIns is restricted to various organelle compartments, and PtdIns cannot diffuse freely within the aqueous cytosol. Therefore, the synthesis of phosphoinositides is tightly regulated in space as well as in time. Class I phosphatidylinositol transfer proteins (PITPs) facilitate the exchange of PtdIns between different membrane compartments by shuttling these phospholipids across the aqueous cytoplasm. Two PITP isoforms (α and β) are expressed in platelets. We have generated a mouse model in which these isoforms are deleted in megakaryocytes and in platelet lineages by using a CRE/Lox strategy. Mice lacking either of the PITP isoforms (PITPαfl/fl PF4 Cre+ or PITPβfl/fl PF4 Cre +) or mice lacking both of the isoforms of PITP (PITPαfl/fl PITPβfl/fl PF4 Cre+) in their platelets and in their megakaryocytes appeared normal, and exhibited no evidence of spontaneous hemorrhage. Mice lacking individual isoforms of PITP exhibited mild thrombocytopenia, while mice lacking both PITP isoforms in their platelets and in their megakaryocytes had platelet counts that were 45% ± 4% less than the platelet counts of their matched littermate controls. These knockout mice had no splenomegaly, and the double knockout platelets had a normal lifespan when infused into wild type recipient mice. Together, these findings suggest that the loss of PITPα and PITPβ in platelets and in megakaryocytes causes thrombocytopenia due to decreased platelet production. Analysis of the megakaryocytes in PITPαfl/fl PITPβfl/fl PF4 Cre+ mice demonstrates that the relative number of megakaryocytes within their bone marrow was unaffected by the loss of both PITP isoforms (control 1.04 ± 0.12% versus knockout 0.98 ± 0.07%). These knockout megakaryocytes also expressed proplatelets in tissue culture as efficiently (44 ± 7% of megakaryocytes had a least one proplatelet) as wild type megakaryocytes (40.9 ± 2.9%). These data suggest that the thrombocytopenia in PITPαfl/fl PITPβfl/fl PF4 Cre+ mice is due to a failure of their megakaryocyte proplatelet extensions to release platelets into the circulation. It is notable that we have also found that the deletion of either PITPα or PITPβ leads to decreased in vitro synthesis of phosphoinositides, such as PI(4)P and PI(4,5)P2, and second messengers, such as IP3. Together, these data indicate that PITPα and PITPβ in megakaryocytes are critical for normal proplatelet release. We speculate that this is due to the loss of specific phosphoinositides required during thrombopoiesis. Disclosures: No relevant conflicts of interest to declare.

Description: Macrophages are professional phagocytes essential for host defense and tissue homeostasis. These functions are critically dependent on the proper biogenesis and degradative activity of lysosomes. Through the biosynthetic pathway, lysosomes receive newly synthesized lysosomal enzymes, which are necessary for the degradation of endogenous and exogenous materials obtained via endocytosis or autophagy pathways. Recent studies identified the endosomal phosphoinositide PI(3,5)P2 as a critical regulator of the trafficking pathways along the endolysosomal system. PI(3,5)P2 is synthesized by the lipid kinase PIKfyve in mammals. In our previous work, we demonstrated that PIKfyve is vital in the lysosomal biogenesis and homeostasis in platelets and PIKfyve deficiency could lead to arterial thrombosis and inflammation in vivo. However, despite the critical importance of lysosomal biogenesis and functions in macrophages, the role of PIKfyve in these cells is unknown. To address the role of PIKfyve in macrophages, we generated mice lacking PIKfyve in their macrophages by pairing PIKfyvefl/fl mice with mice that are transgenic for the myeloid-specific LysM-Cre. As expected, macrophage expression of PIKfyve was normal in wild-type mice, partially reduced in the PIKfyvefl/+ LysM-Cre mice, and undetectable in the PIKfyvefl/fl LysM-Cre mice. To validate the tissue specificity of LysM-Cre, PIKfyvefl/fl LysM-Cre mouse was crossed with the Cre-dependent YFP reporter mouse. LysM-Cre induced YFP expression was present predominantly in the monocytes and neutrophils, and minimally in lymphocytes. As they matured, PIKfyvefl/fl LysM-Cre mice developed abdominal distention due to severe hepatosplenomegaly. Histological analysis of their liver and spleen demonstrated tissue infiltration of cells with numerous cytoplasmic vacuoles. These vacuolated cells stained with markers of neutrophils and macrophages by immunostaining. Immunophenotyping analysis of white blood cells from the peripheral blood demonstrated elevated counts of monocytes and neutrophils but decreased number of lymphocytes in the PIKfyvefl/fl LysM-Cre mice. In addition, PIKfyvefl/fl LysM-Cre mice had increased levels of inflammatory cytokines compared to control mice. Together, these data indicate that PIKfyve deficiency in myeloid cells induce inflammatory responses and suggest that PIKfyve in myeloid cells is essential to prevent pathological inflammatory responses. To investigate the effects of PIKfyve ablation in lysosome biogenesis and functions in macrophages, we examined the macrophages isolated from the bone marrow and spleen of control and PIKfyvefl/fl LysM-Cre mice. Immunofluorescence analysis showed enlarged vacuoles staining with LAMP-1 (marker of late endosomes and lysosomes) in PIKfyve-null macrophages.Immunoblotting analyses of total lysates from PIKfyve-null macrophages revealed excessive levels of lysosomal proteins including LAMP-1 and Cathepsin D. In spite of the increased levels of lysosomal proteins, intralysosomal proteolysis was defective. This demonstrates that PIKfyve is essential not only for macrophage lysosomal biogenesis but also for the degradative function in lysosomes. Intriguingly, PIKfyve deficiency was associated with reduced phosphorylation of the transcription factor TFEB, which is key regulator of the lysosomal gene expression. We found that compared to wild-type macrophages, PIKfyve-null macrophages had significantly reduced levels of the phosphorylated forms of TFEB and primarily expressed the dephosphorylated form of TFEB. These findings provide a clear link between PIKfyve, lysosome biogenesis, and TFEB. Taken together, our study demonstrates that PIKfyve is essential in both the biogenesis and function of macrophage lysosomes. Furthermore, our work demonstrates a previously unrecognized connection between phosphoinositide signaling, the transcription factor TFEB, and lysosomes in professional phagocytic cells. Whether this link is perturbed in human diseases would be an interesting direction of investigation. Disclosures No relevant conflicts of interest to declare.