Escherichia coli infection. Investigation of PHLPP1 function in macrophages reveals that it controls the magnitude and duration of inflammatory signaling by dephosphorylating the transcription factor STAT1 on Ser727 to inhibit its activity, reduce its promoter residency, and reduce the expression of target genes involved in innate immunity and cytokine signaling. This previously undescribed function of PHLPP1 depends on a bipartite nuclear localization signal in its unique N-terminal extension. Our data support a model in which nuclear PHLPP1 dephosphorylates STAT1 to control the magnitude and duration of inflammatory signaling in macrophages.Inflammation is an essential aspect of innate immunity but also contributes to diverse human diseases. Although much is known about the kinases that control inflammatory signaling, less is known about the opposing phosphatases. Here we report that deletion of the gene encoding PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) protects mice from lethal lipopolysaccharide (LPS) challenge and live  Gene expression is an exquisitely regulated process that maintains cellular homeostasis and orchestrates appropriate responses to environmental stimuli such as hormones, cytokines, and pathogenic microbes . HomeostMacrophages are among the first responders to infection, engaging foreign pathogens via pattern recognition receptors, including the Toll-like receptors (TLRs). TLRs are a conserved family of cell surface or phagosome-associated receptors that discriminate distinct features of microbial and viral pathogens, including lipoproteins (TLR1/2/6), lipopolysaccharide (LPS) (TLR4), flagellin (TLR5), single-stranded RNA (TLR7/8), double-stranded RNA (TLR3), and double-stranded DNA (TLR9) . Upon paSTAT1 is the founding member of the STAT transcription factor family and serves as a paradigm for how phosphorylation regulates transcription factor structure, function, and localization . In the Fgf18 expression, suggesting PHLPP1 inhibition could be a strategy to promote cartilage regeneration and repair (PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) is one of the newest members of the phosphatome . Originad repair . PHLPP1 d repair .PHLPP1 is unusual among protein phosphatases in that its regulatory modules and catalytic domain are on the same polypeptide. Most notably, it has a PH domain essential for dephosphorylation of protein kinase C (PKC) , a PDZ l-/-Phlpp1 mice have improved survival following infection with Escherichia coli (E. coli), indicating a role of the phosphatase in innate immunity. Since macrophages are key in the initial response to lipopolysaccharide (LPS) from Gram-negative bacteria such as E. coli, we further explored the role of PHLPP1 in controlling LPS-dependent signaling in this cell type. The STAT1 binding motif was identified from the most common promoter sequences of 199 genes that remained elevated following LPS treatment of bone marrow-derived macrophages (BMDMs) from -/-Phlpp1 mice compared to those from wild-type (WT) mice. We validated common transcriptional targets of PHLPP1 and STAT1, showing that loss of PHLPP1 upregulates the transcription of several genes including guanylate binding protein 5 (Gbp5), whereas loss of STAT1 downregulates them. Cellular studies revealed that dephosphorylation of STAT1 on Ser727 suppresses its transcriptional activity by a mechanism that depends both on the catalytic activity of PHLPP1 and a previously undescribed nuclear localization signal (NLS) in the NTE of PHLPP1. Taken together, our results identify PHLPP1 as a major player in the resolution of inflammatory signaling.Here we report that nuclear-localized PHLPP1 opposes STAT1 Ser727 phosphorylation to inhibit its transcriptional activity and promote normal resolution of inflammatory signaling. We find that E. coli bacteria in WT and -/-Phlpp1 mice. Surprisingly, absence of PHLPP1 provided a strong protective effect; at a dose where more than 50% of WT mice died within 12 hr of E. coli challenge, 50% of the -/-Phlpp1 mice remained alive after 10 days  injection of Gram-negative  10 days . Similar WT mice . To undenjection . Serum lnjection . In cont WT mice . By cont-/- mice . Note thE. coli infection and LPS challenge, we analyzed the transcriptome of BMDMs isolated from WT or -/-Phlpp1 mice before and after stimulation by the major LPS component, Kdo2-Lipid A (KLA), for 1, 6, or 24 hr  24 hr later. Another set of genes exhibited reduced expression 24 hr following KLA treatment  signaling-dependent pathways , a suite of tools for motif discovery and Next Generation Sequencing (NGS) analysis (−18) and IRF (p<10−9) motifs  motifs  in the p and IRF . We selerophages . Thus, lCd69, Ifit2, and Gbp5 at 6 hr compared to a control siRNA transfection, with transcript levels dropping to near baseline by 24 hr  on the Ifit2 promoter and at a later time (24 hr) on the Cd69 promoter. Thus, PHLPP1 suppresses KLA-stimulated binding of STAT1 to its promoters and thereby reduces transcription of its target genes.If PHLPP1 suppresses STAT-regulated gene transcription, we reasoned that 1] knockdown of a STAT family member should reduce transcription of the same genes affected by loss of PHLPP1 and 2] knockdown of PHLPP1 should enhance STAT binding to its promoters. STAT1 is required for LPS-induced gene expression in macrophages  and implby 24 hr . The effer 24 hr . In cont-/-Phlpp1  mice and the kinetics and magnitude of KLA-triggered phosphorylation at each of the two STAT1 sites were compared. Loss of PHLPP1 in BMDMs led to a robust increase in STAT1 phosphorylation on the regulatory site Ser727 but did not affect Tyr701 phosphorylation  were co-transfected with a firefly luciferase reporter construct containing GAS promoter elements, as well as a renilla luciferase controlled by a constitutive CMV promoter as an internal control. STAT1 promoter activity was assessed by monitoring luminescence following IFNγ stimulation. -/-Phlpp1 MEFs compared to WT MEFs at both 6 hr and 24 hr. Pre-treatment of cells with okadaic acid, under conditions that increased the phosphorylation of PP2A-sensitive substrates  in the N-terminal extension unique to the PHLPP1 isozyme  and exit from (NES) the nucleus. First, we identify a bipartite NLS in the NTE of PHLPP1 whose integrity is necessary for the phosphatase to regulate the transcriptional activity of STAT1. Second, we identify an NES in the segment between the LRR and PP2C domain that drives export out of the nucleus. Under the ‘unstimulated’ conditions of our immunofluorescence, PHLPP1 localized primarily to the cytosol, suggesting masking of the NLS and exposure of the NES. Inputs that regulate the exposure of the NLS and NES are likely important regulators of PHLPP1 function.-/-Phlpp1 mice contain a consensus STAT-binding motif in their proximal promoters. Our studies have demonstrated a direct interaction between PHLPP1 and STAT1, thus it is highly likely that PHLPP1 is recruited to gene promoters through its association with STAT1. Elevated STAT1 occupancy and delayed dismissal kinetics of STAT1 from its target promoters in -/-Phlpp1 macrophages indicate a major function of PHLPP1-dependent dephosphorylation in termination of STAT1 signaling and its dismissal from chromatin.Our transcriptomic data support a key role for PHLPP1 in the resolution of the inflammatory response specific to genes downstream of type II IFN signaling pathways. This suggests the possibility that PHLPP1 can selectively discriminate between inflammatory promoters that are differentially regulated by distinct transcription factor families. Surprisingly, over 50% of the inflammatory genes that fail to properly resolve in the macrophages from Stat1 on the background of a mouse that has enhanced TLR4 signaling  provides protection against LPS-induced toxemic death compared to mice with normal STAT1 levels  or DNA binding (Q463H and E320Q), increase the susceptibility of otherwise healthy patients to mycobacterial and viral infection . This in1 levels . AlthougPHLPP1NES-NTE.OA (459616) was purchased from Millipore. Gö6983 (365251) and staurosporine (569397) were purchased from Calbiochem. Antibody against HA (11867425001) was purchased from Roche; antibodies against GFP (2555), STAT1 (9172), phosphorylated Ser727 on STAT1 (9177), phosphorylated Tyr701 on STAT1 (7649), phosphorylated Thr202/Tyr204 on Erk1/2 (9101), total Erk1/2 (9102), and phosphorylated Thr308 on Akt (9275) were purchased from Cell Signaling. Antibody against total Akt (126811) was obtained from AbCam. Antibodies against PHLPP1 were purchased from Cosmo (KIAA0606) and Proteintech (22789–1-AP); antibodies against FLAG (F3165), β-Actin (A2228), and α-tubulin (T6074) were purchased from Sigma-Aldrich. The pcDNA3 HA-tagged PHLPP1 and PHLPP2 constructs for mammalian cell expression were described previously . Full-le2 atmosphere for 7 days, then treated with 100 ng/ml KLA  for noted times. Peritoneal macrophages were collected by flushing mouse peritoneal cavities with PBS following 48 hr post peritoneal injection with 3 ml of thioglycolate . BMDMs were obtained by PBS flush of femurs and tibias , red blolycolate .-/-Phlpp1 mice stably expressing shp53 were a kind gift from Lloyd Trotman (CSHL) and have been described previously  from triplicate experiments and quantified using a NanoDrop Spectrophotometer (ThermoFisher Scientific). RNA was either reverse transcribed into cDNA for quantitative real-time PCR using gene-specific primers or used for next-generation library preparation. For cDNA generation, one μg of total mRNA was reverse transcribed using the SuperScript III Reverse Transcriptase (ThermoFisher Scientific). The resulting cDNA (25 ng) was used to perform real-time PCR using SYBR Green Master Mix (ThermoFisher Scientific) and 50 nM mix of forward and reverse primers. The real-time PCR values for individual genes were normalized to the house keeping gene, T method . The pri36B4_qPCR_F AATCTCCAGAGGCACCATTG36B4_qPCR_R CCGATCTGCAGACACACACTCd69_qPCR_F CTATCCCTTGGGCTGTGTTAATCd69_qPCR_R ACATGGTGGTCAGATGATTCCIfit2_qPCR_F GAGTTTGAGGACAGGGTGTTTAIfit2_qPCR_R AGACCTCTGCAGTGCTTTACGbp5_qPCR_F GGAAGTGCTGCAGACCTATTGbp5_qPCR_R GCTCTTTCTTGTTCCGCTTTAChttp://homer.ucsd.edu/homer/. Data visualization was performed using Microsoft Excel, JavaTreeGraph and software packages available in R.Libraries were prepared from two biological replicates per condition. RNA-Seq libraries were prepared as previously described . SequencStat1: L-058881). Thioglycollate-elicited peritoneal macrophages were transfected with 30 nM siRNA for 48 hr using Deliver X (Affymetrix) according to the manufacturer’s instructions prior to being stimulated with KLA for designated times.SMART siRNA pools for examined genes were purchased from Dharmacon . Elutions were subsequently de-crosslinked overnight at 65°C and DNA was purified using ChIP DNA Clean and Concentrator (Zymo Research) and DNA was used for qPCR. The primer sequences used in this study are:ChIP assays were performed as described before . Cells wCd69_ChIP_F TCCCTGCTGTCTGAAATGTGCd69_ChIP_R GTGGAAGGATGTCTTCGATTCTIfit2_ChIP_F GCATTGTGCAAGGAGAATTCTATGIfit2_ChIP_R TTCCGGAATTGGGAGAGAGAGbp5_ChIP_F TAAACAGCGCTTGAAACAATGAGbp5_ChIP_R AGGCTTGAATGTCACTGAACTACells were plated in a 96-well plate and transfected when approximately 80% confluent. Transfections of pRL-CMV encoding Renilla luciferase , togethe2HPO4 (pH 7.5), 1 mM sodium pyrophosphate, 20 mM NaF, 2 mM EDTA, 2 mM EGTA, 1% SDS, 1 mM DTT, 1 µM microcystin, 20 µM benzamidine, 40 µg/ml leupeptin, and 1 mM PMSF and then were sonicated briefly. For co-immunoprecipitation, cells were lysed and the detergent-solubilized cell lysates were incubated with an anti-HA antibody  at 4°C overnight. Samples were incubated with protein A/G PLUS-Agarose (Santa Cruz Cat sc-2003) for 1 hr at 4°C and washed three times in lysis buffer containing 0.3 M NaCl and 0.1% Triton X 100. Bound proteins and lysates were separated by SDS/PAGE gel and analyzed by western blot.DNA was transfected into HEK-293T cells using FuGene 6. Cells were collected 24 hr post-transfection and then lysed in a buffer containing 50 mM NaHeLa cells were plated on glass coverslips and transfected using FuGene 6. 24 hr after transfection, cells were fixed with 4% paraformaldehyde for 20 min at room temperature, followed by fixation with 100% methanol for 3 min at −20°C. Cells were permeabilized and blocked in 0.3% Triton X 100% and 3% BSA for 30 min at room temperature, followed by three 5 min washes in PBS-T. Primary antibodies were diluted at the following dilutions: mouse anti-HA, 1:500; rabbit anti-α-tubulin , 1:200. Secondary antibodies were diluted at the following dilutions: Alexa647 anti-mouse , 1:500; Alexa488 anti-rabbit , 1:500. Coverslips were mounted onto slides with ProLong Diamond Antifade Mountant with DAPI . Images were acquired on a Zeiss Axiovert 200M microscope (Carl Zeiss Microimaging Inc) using an iXon Ultra 888 EMCCD camera (ANDOR) controlled by MetaFluor software (Molecular Devices) and analyzed on ImageJ (NIH). The Nuclear to Cytoplasmic ratio was calculated as follows: the mean signal intensity was measured for a region of the nucleus and cytoplasm for each cell, and the mean signal intensity of the background was subtracted from these values. Then the Nuclear to Cytoplasmic ratio was calculated by dividing the background subtracted mean signal intensity for the nuclear signal by the background subtracted value for the cytoplasmic signal.6 cells per plate, 80% transfection efficiency) using Fugene 6. Cells were collected 48 hr post-transfection and lysed in a buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X 100, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM DTT, 1 mM PMSF, 1 µM microcystin, 20 µM benzamidine, and 40 µg/ml leupeptin. The detergent-solubilized cell lysates were incubated with anti-FLAG M2 affinity gel  for 1 hr at 4°C, washed four times in lysis buffer and the beads were resuspended in 40 µl 200 mM Tris, 4 mM DTT, 20 mM MnCl2 for use in in vitro phosphatase assay. STAT1 (0.3 µM)  was phosphorylated in vitro by incubation with recombinant human cdk1/cyclinB (0.2 µM)  at 30°C for 90 min in the presence of 1 mM ATP, and 1 X PK buffer  containing 50 mM Tris, 10 mM MgCl2, 0.1 mM EDTA, 2 mM DTT, 0.01% Brij, pH 7.5, and the reaction was quenched by addition of 144 µM CDK1 inhibitor RO3306 . Phosphorylated STAT1 substrate was added to 1/4 vol of beads with bound PHLPP1 (or to lysis buffer control) and reactions were allowed to proceed for an additional 120 min at 30°C. For the zero minute time point, beads were added after the 120 min incubation and all reactions were immediately quenched with 4xSB (sample buffer). Samples were analyzed by western blot.pCMV 3xFLAG PHLPP1 was transfected into HEK-293T cells plated in four 10 cm plates (approximately 9 × 10E. coli K1 strain RS218 and LPS endotoxemia was induced by injection of purified E. coli O111:B4 LPS (Sigma-Aldrich). The E. coli culture was grown overnight in Luria broth (LB) medium (Hardy Diagnostics) at 37°C with shaking. The bacterial culture was diluted 1:50 in fresh LB, grown to mid-log phase, washed twice with PBS and reconstituted in PBS to yield the appropriate inoculum. For survival experiments, 10 to 14 week-old female C57BL/6 WT and littermate control -/-Phlpp1 mice were injected i.p. with 5 × 107 colony forming units (cfu) E. coli or 15 mg/kg LPS and mouse survival recorded for 10 days following injection. For measurement of serum IL-6, IL-10 and IL-1β levels, mice were injected with 10 mg/kg LPS, and at 4, 8, 12 and 24 hr after injection, 80 µl of blood was collected by submandibular bleeding using a lancet into a serum separating blood collection tubes (BD) that were spun at 1500 × g for 10 min to separate serum. Serum cytokines were quantified by specific ELISA (R and D) following the manufacturer’s protocol. All protocols for mouse experiments were conducted in accordance with the institutional guidelines and were approved by the Institutional Animal Care and Usage Committee (IACUC) at the University of California, San Diego.Bacterial sepsis in mice was induced by injection of  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, including Kim Orth as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor.Thank you for submitting your work entitled "PHLPP1 counter-regulates STAT1-mediated inflammatory signaling" for consideration by eLife. Although the work is seen as important, it is not a complete story and would require experiments that will take longer than the allowed time. If the author chooses to address the issues brought up by these reviewers, we encourage them to submit a new manuscript to eLife with this data and we will do our best to use the same editors and reviewers for the new submission.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in Reviewer #1:-/-Phlpp1 mice have improved survival during E. coli infection and LPS-induced toxicity, indicating an important role for the PHLPP1 phosphatase in innate immune signaling. The authors then performed RNA-seq BMDMs derived from WT and -/-Phlpp1 mice to determine how loss of Phlpp1 altered gene expression and found a significant enrichment in STAT binding motifs in the Phlpp1-null elevated transcripts. Importantly, the authors validated the Phlpp1's effect on STAT1 promoter occupancy through ChIP experiments. Finally, the authors show that dephosphorylation of Ser727 on STAT1 does indeed suppress its activity as a transcription factor and that PHLPP1 specifically dephosphorylates Ser727 in vitro and that loss of Phlpp1 increases basal STAT1 Ser727 phosphorylation in vivo. Although this manuscript elucidates an important level of control in STAT1 signaling the following major and minor concerns must be addressed before this manuscript can be accepted for publication.The authors report that the PHLPP1 phosphatase specifically targets phosphorylated Ser727 on STAT1 and thereby inhibits its activity as a transcription factor. This is an important finding because STAT1 is a transcriptional activator that mediates the cellular response to interferons, cytokines and growth factors. The authors begin by showing that Essential revisions:Figure 2E-G:The text states that these are RT-PCR data (subsection “Loss of PHLPP1 results in a prolonged STAT1-dependent transcription in macrophages”), however the figure legend states that they are RNA-seq values. Which are they? If they are qRT-PCR then are they relative expression values or absolute expression? There is no detailed information in the Materials and methods section as to how the qPCR was performed and no sequences for primers to normalization genes if they are indeed relative values. The reference given is behind a paywall so the common reader would not be able to access any detailed information about the qRT-PCR methods used. The methods used to obtain these data must be further elucidated to be able to determine if the proper controls and normalization were performed.Figure 5:The analysis of the subcellular localization of PHLPP1 would be greatly improved by the addition of an experiment in which nuclear export was inhibited by Leptomcyin B. The authors reported that full-length PHLPP1 is cytosolic, but they failed to take into account that nuclear export/import is an equilibrium process and the only way to tell if a protein is indeed primarily localized in the cytosol is to block nuclear export.It would also strengthen this line of inquiry if the authors tried (even if just with bioinformatics) to identify the native NLS of PHLPP1. The authors add a foreign NLS (from what protein? what type of NLS?) to show that driving the protein to the nucleus inhibits STAT1 promoter activity suggesting nuclear localization of PHLPP1 is important for its activity. They do not elucidate PHLPP1's native NLS, which must exist in order for the effect of nuclear localization of PHLPP1 on STAT1 promoter activity to actually be significant in vivo.in vitro and is out of place in Figure 5 which is all about nuclear localization. Figure 5D should either be incorporated into Figure 6 or moved to the supplement. This would leave room in Figure 5 for the suggested Leptomycin B experiment and NLS bioinformatics in this figure.Figure 5D shows that PHLPP1 dephosphorylates STAT1 Ser727 Figure 7:Can you comment on the fact that pAkt T308 is elevated in all conditions in the PHLPP1 KO in 7A? PHLPP1 dephosphorylation of Ser473 was mentioned in the Discussion section, but not T308.As it is presented, Figure 7C is confusing. It seems like the luciferase assay graph and the pAkt blot should be separated into Figure 7C and 7D or at least the blot needs to be mentioned in the figure legend which currently only explains the luciferase assay for Figure 7C. I understand that the blot is only for validation purposes, but it still needs to be mentioned in the figure legend if it is be included.Subsection “PHLPP1 binds to STAT1 and dephosphorylates Ser727”: Please revise to "…interacts with STAT1 and reduces its promoter activity". You have shown that the PHLPP1 NTE can pull down STAT1 and that the PHLPP1 NTE reduces STAT1 promoter activity, but not exactly that one causes the other.And as these were IPed out of lysate and not shown to directly interact with purified recombinant proteins, a direct interaction cannot be claimed.Reviewer #2:E. coli challenge. Molecular analysis in macrophages revealed that PHLPP1 dephosphorylates Ser727 in STAT1 and thus suppresses inflammatory signaling.The presented paper identifies a potential phosphatase for STAT1. Deletion of the phosphatase-encoding gene Phlpp1 increases survival of mice upon LPS or Strong points of the paper are its detail in the characterization of PHLPP1 and its effect on STAT1. Weak points of the paper are the apparent presence of PHLPP1 even in knockouts, minimal in vivo analysis and exclusive focus on macrophages.1) The manuscript is lacking controls and/or appropriate quantification for their knockdown and knockouts. The authors should show the efficiency of Phlpp1 knockdown by WB or PCR and that siCtl does not affect STAT1. Most importantly, neither the CRISPR knockout  in BMDM nor BMDM from PHLPP1 KO  are convincingly lacking PHLPP1. The band intensity of PHLPP1 is almost the same between knockout and WT. Why is there a band at all for PHLPP1 in PHLPP1 KO? In Figure 6 and Figure S1, there seems to be only one band, whereas in Figure 7C the higher molecular band is marked with the arrow for PHLPP1. The authors have to show convincingly that PHLPP1 is absent in their KO cells.2) The focus of the manuscript is the characterization of molecular interactions of PHLPP1. Nevertheless, the in vivo experiments should be analyzed in more detail. Is there increased dissemination in peripheral organs and blood in WT mice compared to KO mice? The increase in inflammatory cytokines could be either due to a higher inflammatory profile of immune cells or an increased recruitment/number. Thus, ideally the authors would measure recruitment of neutrophils, macrophages and DCs to the peritoneum and lymph nodes.-/-Phlpp1 mice had a diminished initial pro-inflammatory cytokine burst. Only IL-1β levels were consistently higher in KO mice. The authors should analyze more cytokines to draw this conclusion. For in vitro experiments, e.g. in vivo levels of IFNγ would be highly relevant and should be measured.3) The authors show in vivo levels of only 3 cytokines and conclude from 2 cytokines that 4) Figure 5C. Is the transfection efficiency the same? From the images, it seems that different constructs have different transfection efficacy. Confocal images lack scale bars. Bar graphs lack statistical analysis.5) The rationale for choosing GBP5, IFIT2 and CD69 as representative genes should be provided.Reviewer #3:This is an interesting study that seeks to identify roles of the PHLPP1 phosphatase. The authors conclude that PHLPP1 dephosphorylates STAT1 on Ser727 and serves to promote resolution of inflammation by suppressing STAT1 activity. This is an important finding because while the kinases that cause STAT1 Ser727 phosphorylation have been characterized, little is known concerning the mechanism of dephosphorylation.The authors' conclusions are supported by the data presented, although several aspects of the analysis appear incomplete.1) The biological focus of the study is the protection against endotoxin and bacterial sepsis while the mechanistic studies focus on the resolution of inflammation. There is a logical problem here. Indeed, this is acknowledged by the authors who briefly state in subsection “PHLPP1 regulates the innate immune response” that the reduced mortality is most likely associated with a diminished initial cytokine response. The mechanism of the reduced initial cytokine response is not defined. Instead, the manuscript focusses on a second phenotype – reduced resolution – that may have little to do with the reduced mortality associated with endotoxin and bacterial sepsis. This lack of coherence between biology and mechanism needs to be fixed.2) The conclusion that PHLPP1 deficiency promotes sustained STAT1 signaling is not strongly supported by the gene expression data  that show increased expression at 6 hours and a similar level of decline in expression at 24 hours. Since the heat map is presented as the ratio of KO:WT, it is not clear that this expression pattern is not shared by the other genes in this group. A similar argument could be made for some of the genes in the ChIP analysis that show increased binding at all time points . Please clarify. Moreover, this increase in gene expression at all time points correlates poorly with the decrease in the initial cytokine response that may contribute to the protection against sepsis and LPS (see #1 above).in vitro , this is not demonstrated in vivo. For example, knockout macrophages complemented with WT and catalytically inactive PHLPP1 should be compared to investigate the regulation of STAT1 Ser727 phosphorylation.3) The authors conclude (Abstract) that PHLPP1 catalytic activity is required for suppression of STAT1. The data to support this conclusion is incomplete. While the authors do show – by mutational analysis – that PHLPP1 catalytic activity is required for PHLPP1 Ser727 dephosphorylation -/-Phlpp1 BMDM were not used for these studies – these cells prepared from -/-Phlpp1 mice would not express PHLPP1 protein.4) The CRISPR studies are unclear. First, the KO cells still express PHLPP1 . Second, no controls for the CRISPR KO are presented. Third, the conclusion that KLA-induced phosphorylation is sustained at 24 hours in the PHLPP1 KO cells is not convincing . Fourth, it is unclear why 5) No information is provided concerning methods and statistical analysis of the promoter motif analysis .6) No controls for the siRNA SMARTpool study  are presented. Minimally, this requires the use of two independent single oligonucleotides. Preferably also complementation analysis.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your article has been reviewed by three peer reviewers, including Kim Orth as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and Tadatsugu Taniguchi as the Senior Editor.Thank you for submitting your article "PHLPP1 Counter-regulates STAT1-mediated Inflammatory Signaling" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The authors added significant amount of data for their molecular analysis of the role of PHLPP1 in suppressing STAT1 signaling.Essential revisions:Reviewer #1:The authors have satisfied this reviewer with their revisions to the first submission.Reviewer #2:-/-Phlpp1 mice as suggested by reviewer 3 significantly improved the quality of the manuscript and support the claims made.The authors added significant amount of data for their molecular analysis of the role of PHLPP1 in suppressing STAT1 signaling. Comments addressing the concerns of reviewer 1 and the use of BMDMs from In their rebuttal, the authors wrote that they replaced all knockdown experiments with BMDMs. However, siRNA knockdowns of STAT1 are still used in Figure 3. Is there a reason for why the authors did not show that STAT1 is actually knocked down, that siCrtl does not affect STAT1, and the efficiency of the knockdown?The in vivo role of PHLPP1 remains still largely unexplored. The authors acknowledge this and state that this is not in their area of expertise. The cytokine profiles are only measured until 24 hours post injection of LPS though, at which time the mortality rate between the two groups is identical. Difference are observed 48 hours post infection. As authors are unable to repeat the experiment and/or measure more cytokines, this should at least be clearly mentioned. The molecular analysis that the authors focus on seems sound, but the evidence that the observed in vivo phenotype is due to the lack of dephosphorylation of STAT1 is minimal. As the authors suggest exploration of PHLPP1 inhibitors for treatment of sepsis in the Discussion section, this lack of direct evidence should be mentioned.Reviewer #3:The manuscript has been improved. While the authors have not addressed all the points raised in my critique, I believe that the revised manuscript does address the primary criticisms and that the paper is now acceptable for publication. [Editors’ note: the author responses to the first round of peer review follow.]Reviewer #1:The authors report that the PHLPP1 phosphatase specifically targets phosphorylated Ser727 on STAT1 and thereby inhibits its activity as a transcription factor. This is an important finding because STAT1 is a transcriptional activator that mediates the cellular response to interferons, cytokines and growth factors. The authors begin by showing that Phlpp1-/- mice have improved survival during E. coli infection and LPS-induced toxicity, indicating an important role for the PHLPP1 phosphatase in innate immune signaling. The authors then performed RNA-seq BMDMs derived from WT and Phlpp1-/- mice to determine how loss of Phlpp1 altered gene expression and found a significant enrichment in STAT binding motifs in the Phlpp1-null elevated transcripts. Importantly, the authors validated the Phlpp1's effect on STAT1 promoter occupancy through ChIP experiments. Finally, the authors show that dephosphorylation of Ser727 on STAT1 does indeed suppress its activity as a transcription factor and that PHLPP1 specifically dephosphorylates Ser727 in vitro and that loss of Phlpp1 increases basal STAT1 Ser727 phosphorylation in vivo. Although this manuscript elucidates an important level of control in STAT1 signaling the following major and minor concerns must be addressed before this manuscript can be accepted for publication. Essential revisions: Figure 2E-G: The text states that these are RT-PCR data (subsection “Loss of PHLPP1 results in a prolonged STAT1-dependent transcription in macrophages”), however the figure legend states that they are RNA-seq values. Which are they? If they are qRT-PCR then are they relative expression values or absolute expression? There is no detailed information in the Materials and methods section as to how the qPCR was performed and no sequences for primers to normalization genes if they are indeed relative values. The reference given is behind a paywall so the common reader would not be able to access any detailed information about the qRT-PCR methods used. The methods used to obtain these data must be further elucidated to be able to determine if the proper controls and normalization were performed.The data in Figure 2E-G are RNA-Seq, whereas those in Figure 3A-C are qPCR. We have updated the text to indicate “normalized mRNA-Seq data” where appropriate. In addition, we have updated the Materials and methods section to include the primer sequences for 36B4 that were used for normalization. We have also included additional references for the Materials and methods section.Figure 5:The analysis of the subcellular localization of PHLPP1 would be greatly improved by the addition of an experiment in which nuclear export was inhibited by Leptomcyin B. The authors reported that full-length PHLPP1 is cytosolic, but they failed to take into account that nuclear export/import is an equilibrium process and the only way to tell if a protein is indeed primarily localized in the cytosol is to block nuclear export.It would also strengthen this line of inquiry if the authors tried (even if just with bioinformatics) to identify the native NLS of PHLPP1.We have now identified a bipartite NLS in the unstructured N-Terminal Extension (NTE) of PHLPP1 . We show that a construct encoding the NTE localizes to the nucleus, that mutation of either half of the bipartite mutation significantly increases localization in the cytosol, and that mutation of both halves of the bipartite motif results in cytosolic localization that is almost as complete as the addition of a strong NES . Additionally, we show that the decrease in STAT1 promoter activity observed upon overexpression of PHLPP1 in HEK cells depends on a functional NLS: mutation of either or both halves of the NLS reduces or abolishes, respectively, the PHLPP1-dependent effects on STAT1 promoter activity . Additionally, we identify a nuclear exclusion signal (NES) preceding the phosphatase domain . We appreciate the reviewer’s suggestion we address the mechanism for nuclear localization as identification of the NLS provides the first functional role of the NTE, which is unique to PHLPP1. We have also revised the model in Figure 8 to indicate the NLS and NES.The authors add a foreign NLS (from what protein? what type of NLS?) to show that driving the protein to the nucleus inhibits STAT1 promoter activity suggesting nuclear localization of PHLPP1 is important for its activity.We have added the information on the NLS, which is from PKI.They do not elucidate PHLPP1's native NLS, which must exist in order for the effect of nuclear localization of PHLPP1 on STAT1 promoter activity to actually be significant in vivo.See above.Figure 5D shows that PHLPP1 dephosphorylates STAT1 Ser727 in vitro and is out of place in Figure 5 which is all about nuclear localization. Figure 5D should either be incorporated into Figure 6 or moved to the supplement. This would leave room in Figure 5 for the suggested Leptomycin B experiment and NLS bioinformatics in this figure.in vitro dephosphorylation assay  is now presented in the new Figure 4, which focusses exclusively on phosphorylation of Ser727 of STAT1 by PHLPP1. Specifically, the figure shows that (1) STAT1 phosphorylation on Ser727 is enhanced in primary bone marrow derived macrophages from -/-Phlpp1 mice compared to WT mice , (2) pSer727 is dephosphorylated in vitro by PHLPP1 , and (3) STAT1 has reduced phosphorylation on Ser727 in cells overexpressing PHLPP1 .We appreciate these helpful recommendations and have reorganized all the biochemical figures as follows: the Figure 7:Can you comment on the fact that pAkt T308 is elevated in all conditions in the PHLPP1 KO in 7A? PHLPP1 dephosphorylation of Ser473 was mentioned in the Discussion section, but not T308.Phosphate at the hydrophobic motif (Ser473) decreases the phosphatase sensitivity at the activation loop (Thr308), that is why the PHLPP1 KO cells have elevated phosphorylation at both sites, even though only Ser473 is a direct PHLPP. In the revision, we replaced the original Figure 7A,B with a better analysis, which now is the new Figure 5A,B. Here, we make the point that the Ser727 phosphorylation is okadaic-acid insensitive in the primary bone marrow-derived macrophages, under conditions where known PPA sites are okadaic-acid sensitive (the phosphorylation of T308 on Akt and T202/Y204 on Erk increases with okadaic acid yet there is no detectable change on Ser727 phosphorylation).As it is presented, Figure 7C is confusing. It seems like the luciferase assay graph and the pAkt blot should be separated into Figure 7C and 7D or at least the blot needs to be mentioned in the figure legend which currently only explains the luciferase assay for Figure 7C. I understand that the blot is only for validation purposes, but it still needs to be mentioned in the figure legend if it is be included.The luciferase assay is now Figure 6A; we removed the blot and replaced it with the new data in the new Figure 5A,B.Subsection “PHLPP1 binds to STAT1 and dephosphorylates Ser727”: Please revise to "…interacts with STAT1 and reduces its promoter activity". You have shown that the PHLPP1 NTE can pull down STAT1 and that the PHLPP1 NTE reduces STAT1 promoter activity, but not exactly that one causes the other.And as these were IPed out of lysate and not shown to directly interact with purified recombinant proteins, a direct interaction cannot be claimed.Point well taken; text modified as suggested.Reviewer #2:The presented paper identifies a potential phosphatase for STAT1. Deletion of the phosphatase-encoding gene Phlpp1 increases survival of mice upon LPS or E. coli challenge. Molecular analysis in macrophages revealed that PHLPP1 dephosphorylates Ser727 in STAT1 and thus suppresses inflammatory signaling.Strong points of the paper are its detail in the characterization of PHLPP1 and its effect on STAT1. Weak points of the paper are the apparent presence of PHLPP1 even in knockouts, minimal in vivo analysis and exclusive focus on macrophages.1) The manuscript is lacking controls and/or appropriate quantification for their knockdown and knockouts. The authors should show the efficiency of Phlpp1 knockdown by WB or PCR and that siCtl does not affect STAT1. Most importantly, neither the CRISPR knockout  in BMDM nor BMDM from PHLPP1 KO  are convincingly lacking PHLPP1. The band intensity of PHLPP1 is almost the same between knockout and WT. Why is there a band at all for PHLPP1 in PHLPP1 KO?-/-Phlpp1 mice. The new Figure 4A compares the KLA-induced phosphorylation of STAT1 at Ser727 and Tyr701 in these macrophages, with the data from 5 independent experiments quantified in the new Figure 5B.We have replaced all the experiments using CRISPR knockdown with ones using primary BMDM isolated directly from the WT vs. In Figure 6 and Figure S1, there seems to be only one band, whereas in Figure 7C the higher molecular band is marked with the arrow for PHLPP1. The authors have to show convincingly that PHLPP1 is absent in their KO cells.-/-Phlpp1 mice. The immunoreactive band in the PHLPP1 blot in original Figure 7C  that is present in all lanes is β-catenin; all commercially available PHLPP1 antibodies raised to the C-term of PHLPP1 seem to label it .As noted above, Figure 6 (with the CRIPSR knockdown) has been replaced with experiments using BMDM from the 2) The focus of the manuscript is the characterization of molecular interactions of PHLPP1. Nevertheless, the in vivo experiments should be analyzed in more detail. Is there increased dissemination in peripheral organs and blood in WT mice compared to KO mice? The increase in inflammatory cytokines could be either due to a higher inflammatory profile of immune cells or an increased recruitment/number. Thus, ideally the authors would measure recruitment of neutrophils, macrophages and DCs to the peritoneum and lymph nodes.These are very interesting suggestions but beyond the scope of our expertise. Additionally, the two co-first authors are no longer in our labs, and the third author is on maternity leave. While we recruited another postdoc to address all the biochemical revisions, we are not equipped to do whole animal studies. Thus, although very interesting experiments, our current contribution focuses on the molecular mechanisms of how PHLPP1 suppresses STAT1 signaling.-/- mice had a diminished initial pro-inflammatory cytokine burst. Only IL-1β levels were consistently higher in KO mice. The authors should analyze more cytokines to draw this conclusion. For in vitro experiments, e.g. in vivo levels of IFNγ would be highly relevant and should be measured.3) The authors show in vivo levels of only 3 cytokines and conclude from 2 cytokines that Phlpp1We have focused on the mechanistic aspects given our lack of personnel (please see previous comment).4) Figure 5C. Is the transfection efficiency the same? From the images, it seems that different constructs have different transfection efficacy. Confocal images lack scale bars. Bar graphs lack statistical analysis.The transfection efficiency was similar for all constructs and ranged from 50-75%. We have now added a scale bar (15 µm) and statistical analysis.5) The rationale for choosing GBP5, IFIT2 and CD69 as representative genes should be provided.-/-Phlpp1 comparted to WT MEFs. Additionally, they have proximal STAT1 binding motifs in their promoter regions. We have clarified this in the text:We chose these genes because they were significantly elevated in the -/-Phlpp1 compared to WT macrophages and which had proximal STAT1 binding motifs in their promoters to further analyze: Cd69, Ifit2, and Gbp5.”“We next selected three genes whose expression was elevated in the Reviewer #3:This is an interesting study that seeks to identify roles of the PHLPP1 phosphatase. The authors conclude that PHLPP1 dephosphorylates STAT1 on Ser727 and serves to promote resolution of inflammation by suppressing STAT1 activity. This is an important finding because while the kinases that cause STAT1 Ser727 phosphorylation have been characterized, little is known concerning the mechanism of dephosphorylation.The authors' conclusions are supported by the data presented, although several aspects of the analysis appear incomplete.1) The biological focus of the study is the protection against endotoxin and bacterial sepsis while the mechanistic studies focus on the resolution of inflammation. There is a logical problem here. Indeed, this is acknowledged by the authors who briefly state in subsection “PHLPP1 regulates the innate immune response” that the reduced mortality is most likely associated with a diminished initial cytokine response. The mechanism of the reduced initial cytokine response is not defined. Instead, the manuscript focusses on a second phenotype – reduced resolution – that may have little to do with the reduced mortality associated with endotoxin and bacterial sepsis. This lack of coherence between biology and mechanism needs to be fixed.-/-Phlpp1 mice likely arises from an abundance of perturbations to the normal physiology of the mouse. We begin with the mouse experiment because it clearly points to a defect in innate immunity, however the primary focus of our manuscript is to identify a previously undescribed mechanism for the suppression of STAT1 signaling by PHLPP1. This mechanism is likely one of many that contribute to the whole animal phenotype, and we have tried to express this in the manuscript.The biological phenotype of the 2) The conclusion that PHLPP1 deficiency promotes sustained STAT1 signaling is not strongly supported by the gene expression data  that show increased expression at 6 hours and a similar level of decline in expression at 24 hours. Since the heat map is presented as the ratio of KO:WT, it is not clear that this expression pattern is not shared by the other genes in this group. A similar argument could be made for some of the genes in the ChIP analysis that show increased binding at all time points . Please clarify. Moreover, this increase in gene expression at all time points correlates poorly with the decrease in the initial cytokine response that may contribute to the protection against sepsis and LPS (see #1 above).-/-Phlpp1 macrophages compared with WT. We conclude that STAT1 signaling is sustained (or elevated) in the -/-Phlpp1 macrophages relative to the WT macrophages based on the following observations:We acknowledge that the genes evaluated show a decline in expression at 24 hours, however they are expressed at significantly higher levels in the -/-Phlpp1 relative to WT macrophages.1) There are elevated genes in the 2) The promoter regions of these genes have an enrichment of STAT1 binding motifs relative to genome background.-/-Phlpp1 macrophages relative to WT cells.3) The ChIP signal for STAT1 is elevated on representative promoters in 4) STAT1 phosphorylation is enhanced and sustained in Phlpp1 KO macrophages relative to WT.3) The authors conclude (Abstract) that PHLPP1 catalytic activity is required for suppression of STAT1. The data to support this conclusion is incomplete. While the authors do show – by mutational analysis – that PHLPP1 catalytic activity is required for PHLPP1 Ser727 dephosphorylation in vitro , this is not demonstrated in vivo. For example, knockout macrophages complemented with WT and catalytically inactive PHLPP1 should be compared to investigate the regulation of STAT1 Ser727 phosphorylation.in vitro.Our new experiments focus on primary BMDM which are have exceptionally low transfection efficiency and not amenable to the rescue experiment noted. When we overexpress a catalytically-dead PHLPP1 PP2C domain, it no longer regulates STAT1 transcriptional activity whereas an active PP2C does. These cell-based assays are consistent with PHLPP1 regulating STAT1 via a catalytic mechanism. Additionally, PHLPP1 dephosphorylates Ser727 -/- BMDM were not used for these studies – these cells prepared from Phlpp1-/- mice would not express PHLPP1 protein.4) The CRISPR studies are unclear. First, the KO cells still express PHLPP1 . Second, no controls for the CRISPR KO are presented. Third, the conclusion that KLA-induced phosphorylation is sustained at 24 hours in the PHLPP1 KO cells is not convincing . Fourth, it is unclear why Phlpp1-/-Phlpp1 BMDM. We have replaced all the CRISPR KO with experiments using the BMDM; see new Figure 4A,B and new Figure 5A,B.The reviewer brings up an excellent point, to use the 5) No information is provided concerning methods and statistical analysis of the promoter motif analysis .We have updated the text to include more details on the promoter analysis; we have also updated the figure legends.6) No controls for the siRNA SMARTpool study  are presented. Minimally, this requires the use of two independent single oligonucleotides. Preferably also complementation analysis.These studies were internally controlled for with a control siRNA and a SMARTpool of four oligos synthesized by Dharmacon. The purpose of this experiment was to demonstrate that these are targets of STAT1, and the presented data support this conclusion.[Editors' note: the author responses to the re-review follow.]Essential revisions:Reviewer #2:-/- mice as suggested by reviewer 3 significantly improved the quality of the manuscript and support the claims made.The authors added significant amount of data for their molecular analysis of the role of PHLPP1 in suppressing STAT1 signaling. Comments addressing the concerns of reviewer 1 and the use of BMDMs from Phlpp1In their rebuttal, the authors wrote that they replaced all knockdown experiments with BMDMs. However, siRNA knockdowns of STAT1 are still used in Figure 3. Is there a reason for why the authors did not show that STAT1 is actually knocked down, that siCrtl does not affect STAT1, and the efficiency of the knockdown?Apologies for the misleading statement – we replaced all the biochemical/mechanistic experiments addressing PHLPP1 loss with ones using BMDMS, but the reviewer is correct in noting that the validation experiments in Figure 3 were with siRNA knockdown of STAT1. The knockdown of STAT1 was confirmed by the RNA-Seq. Also, given the clear effect of the STAT1 knockdown, but not siCtrl, on genes with STAT1 promoters, we did not feel that addressing efficiency for these experiments was necessary.The in vivo role of PHLPP1 remains still largely unexplored, which you acknowledge, stating that this is not in their area of expertise. The cytokine profiles are only measured until 24 hours post injection of LPS though, at which time the mortality rate between the two groups is identical. Differences are observed at 48 hours post infection. As you cannot repeat the experiment and/or measure more cytokines, this should at least be clearly mentioned. The molecular analysis that you focus on appears sound, but the evidence that the observed in vivo phenotype is due to the lack of dephosphorylation of STAT1 is minimal. As you suggest exploration of PHLPP1 inhibitors for treatment of sepsis in the Discussion section, this lack of direct evidence must be mentioned.These are all good points and we have adjusted the text as in the Results section:“Note that cytokine levels were measured up to 24 h post LPS injection, when the protective effect of PHLPP1 loss was not yet apparent.”and in the Discussion section:E. coli-induced sepsis and LPS-induced endotoxemia in mice, our data indicate that PHLPP1 inhibitors could be explored as adjunctive therapies to antibiotics and supportive care of patients with Gram-negative sepsis, a leading cause of mortality in intensive care units.”"Although the current study does not provide direct evidence that enhanced phosphorylation of STAT1 causes the protective effect of PHLPP1 loss on both


Perhaps the most important feature of neutrophils is their ability to rapidly change shape. In the bloodstream, the neutrophils circulate as almost spherical cells, with the ability to deform in order to pass along narrower capillaries. Upon receiving the signal to extravasate, they are able to transform their morphology and flatten onto the endothelium surface. This transition, from a spherical to a flattened morphology, is the first key step which neutrophils undergo before moving out of the blood and into the extravascular tissue space. Once they have migrated through tissues towards sites of infection, neutrophils carry out their primary role—killing infecting microbes by performing phagocytosis and producing toxic reactive oxygen species within the microbe-containing phagosome. Phagocytosis involves the second key morphology change that neutrophils undergo, with the formation of pseudopodia which capture the microbe within an internal vesicle. Both the spherical to flattened stage and the phagocytic capture stage are rapid, each being completed within 100 s. Knowing how these rapid cell shape changes occur in neutrophils is thus fundamental to understanding neutrophil behaviour. This article will discuss advances in our current knowledge of this process, and also identify an important regulated molecular event which may represent an important target for anti-inflammatory therapy. When neutrophils undergo spreading on the endothelium  or phagocytosis, there is an apparent expansion of the plasma membrane by nearly 200% ,2. HowevA number of mechanisms which could provide additional plasma membrane for this process are all unlikely to operate within neutrophils ,2. FusioA number of elegant studies which characterise the biophysical properties of the neutrophil plasma membrane have been made. Essentially, these studies used micropipettes attached to a vacuum pump to draw the plasma membrane up into the micropipette tip. This enables both the tension in the cell membrane and the amount of membrane available to be measured. Moderate suction can draw some of the neutrophil membrane up into the micropipette, equivalent to 5% expansion of the neutrophil membrane ,11,12. HIn non-muscle cells, such as neutrophils, actin exerts a pushing force on the plasma membrane, rather than the contractile force of actin-myosin in muscle cells. This force is achieved by polymerisation of monomeric actin pushing against the plasma membrane. In neutrophils, there is a significant amount of polymerised actin beneath the cell cortex ,15, whicScanning electron microscopy reveals that the plasma membrane which forms the phagocytic cup is devoid of microridges ,18, in c2+ within the neutrophil or macrophage [2+ signal is probably induced by immobilisation of cell surface adhesion molecules, such a β2-integrin (CD11b/CD18). Experimentally, immobilising these molecules alone causes a Ca2+ signal [2+ signal is not a consequence of spreading, but rather the cause, because (i) it occurs before the cell spreads and (ii) a cytosolic Ca2+ signal alone can induce cell spreading. For example, without integrin engagement, experimentally-induced Ca2+ signals are able to cause neutrophil spreading in the absence of other stimuli. Neutrophil spreading occurs in response to experimentally-induced elevations of cytosolic Ca2+ achieved by either photolytic uncaging of cytosolic caged Ca2+ [3 [2+ as the trigger for neutrophil spreading.It has long been known that immediately before spreading onto a surface, there is a transient rise in global cytosolic free Cacrophage ,22. This+ signal ,24,25. Tged Ca2+  or cytos Ca2+ [3 ,28. This2+-activated enzyme responsible for this effect. The use of pharmacological inhibitors has eliminated a number of potential Ca2+ targets including calmodulin, protein kinase C (PKC) and calcineurin. However, a number of chemically unrelated inhibitors of the Ca2+ activated protease, µ-calpain, inhibit neutrophil spreading [2+ and is thus considered to be a regulatory protease rather than having a simple degradatory role. Its name reflects this, being a portmanteau word combining the molecular element for Ca2+ regulation  and cysteine protease activity (from papain). Like many other proteases, µ-calpain is promiscuous, with no identified amino acid sequence required for recognition of the cleavage site [2+ activation site on µ-calpain [2-integrin adhesion has also been shown to depend on µ-calpain activity [2+ signal is seen to proceed immediately before the acceleration phase of engulfment, [2+ and acceleration is broken by inhibition of µ-calpain activity [2+ activation of µ-calpain as the signalling axis which leads to cell surface “unwrinkling”. However, it has been more difficult identifying the Capreading ,28. µ-Caage site . Howeverage site . There a-calpain ,31,32 whactivity ,34. By culfment, ,36 and tactivity . TogetheIn order to understand the mechanism by which activated µ-calpain mediates the release of cell surface microridges, and so permit cell spreading, we must first consider the molecules involved in maintaining the cell surface microridges.Cell surface wrinkles and microridges are maintained by proteins, especially of the ezrin/radixin/moesin (ERM) family . These cEzrin, previously known as p81, the epidermal growth factor (EGF) receptor tyrosine kinase substrate, villin and cytovillin is a member of the ERM family of membrane-cytoskeletal linker proteins. Ezrin was named in tribute to Ezra Cornell, a co-founder of Cornell University where it was first purified from the microvilli of epithelial cells . Ezrin a2) binding [The phosphorylation status of ERM proteins correlates with their functional activity as crosslinkers between the plasma membrane and the actin cytoskeleton. Ezrin has two main functional domains, located at the N- and C- termini . When ph binding  and phos binding ,49,50 wh binding . Ezrin was first suggested to play a role in crosslinking the plasma membrane to the F-actin cytoskeleton by Gould and colleagues  on accou2 in the plasma membrane [2+ and the activation of protease μ-calpain is important. ERM proteins bind to phosphatidylinositol 4-phosphate (PIP) and PIPmembrane ,60, whicmembrane . Thus, Tmembrane ,62. Howemembrane ,62. In c2+ [2+-dependent manner. This allows µ-calpain to associate with the plasma membrane in regions of elevated Ca2+ concentration [2+ binding protein calmodulin, and contains four EF motifs, similar to domain VI of the smaller subunit.µ-Calpain is a member of the calpain family of 15 known isoforms. The two most studied members are µ-calpain  and m-calpain , which share 62% sequence similarity . µ-Calpa2+ ,65 Domai2+ . Domain ntration . Domain 2+ has therefore been proposed as the mechanism for neutrophil membrane expansion and cell spreading.µ-Calpain is a modulator protease rather than a degradative protease. Calpain modifies the activity or the behaviour of its substrate protein, rather than totally destroying it. However, experimentally, more than 100 substrates of µ-calpain have been reported. Although these can be broadly categorised into three main groups, including (i) transcription factors, (ii) kinases/phosphatases, and (iii) cytoskeletal and membrane-associated proteins . The rol2+ concentration required for in vitro activation, 20–60 µM Ca2+ for µ-calpain and 0.3–1.3 mM Ca2+ for m-calpain respectively [2+ concentrations exceed the global cytosolic free Ca2+ concentrations reported in neutrophils, which peaks at 1 µM . Mathematical modelling Ca2+ concentration has predicted that, if the Ca2+ influx channels were distributed uniformly over the neutrophil surface when the bulk cytosolic reached 1 µM, the concentration of Ca2+ that could be reached within the microridges would be very high [2+ are predicted to be sufficient to activate µ-calpain locally at these Ca2+ hotspots, specifically, at locations were ezrin would also be expected . ,72. 2+ c+ influx ,74. It iembrane,  the Ca2+an 30 µM . This isan 30 µM , suggestery high . Dependiery high . Thus, iexpected . 2+ concentrations reached in the bulk cytosol and so activation per se must occur only at the cell periphery.Although calpain activation has been demonstrated by immunoelectron microscopy in fixed cell , the mea2+ to (ii) µ-calpain activation, leading to (iii) the cleavage of ezrin and (iv) the release of cell surface wrinkles and microridges which (iv) permit actin polymerisation at the cell cortex to push the unfolding plasma membrane and (v) result in a rapid transition into the spread morphology or the localised formation of pseudopodia required for phagocytosis  the signalling from an elevation of cytosolic Caocytosis . The resocytosis . This sereported ,30,31,89


Here, we investigate signaling factors and sources of Ca2+ rise in OLs in the mouse brainstem. Glutamate puff or axon fiber stimulation induces a Ca2+ rise in pre‐myelinating OLs, which is primarily mediated by Ca2+‐permeable AMPA receptors. During glutamate application, inward currents via AMPA receptors and elevated extracellular K+ caused by increased neuronal activity collectively lead to OL depolarization, triggering Ca2+ influx via P/Q‐ and L‐type voltage‐gated Ca2+ (Cav) channels. Thus, glutamate is a key signaling factor in dynamic communication between neurons and OLs that triggers Ca2+ transients via AMPARs and Cav channels in developing OLs. The results provide a mechanism for OL Ca2+ dynamics in response to neuronal input, which has implications for OL development and myelination.Communication between neurons and developing oligodendrocytes (OLs) leading to OL Ca Dynamic interactions between neurons and OLs modulate development of OL lineage cells and myelination to control conduction speed in the central nervous system  and NMDA receptors, and thus can directly sense the glutamate release from neurons  channels are potential indirect effectors of glutamatergic input, because activation of AMPA receptors is able to depolarize and activate voltage‐gated ion channels in OL lineage cells  of the auditory brainstem. The heavily myelinated axon fibers in the MNTB, which are important for ensuring the reliability and temporal fidelity of conduction, undergo activity‐dependent myelination during development  Institutional Animal Care and Use Committee . Heterozygous CNPase‐Cre mice, generated by Dr. Klaus Nave  and obtained from Dr. Manzoor Bhat (UTHSCSA), were bred with homozygote Cre‐dependent GCAMP6f mice  obtained from Dr. Martin Paukert (UTHSCSA). Heterozygote CNPase‐Cre;GCaMP6f offspring of either sex between the ages of postnatal Day 8 (P8) and P12 were used for experiments. Genotyping was accomplished using PCR.2.22, 0.1 CaCl2, 25 glucose, 25 NaHCO3, 1.25 NaH2PO4, 0.4 ascorbic acid, 3 myo‐inositol, and 2 Na‐pyruvate, pH 7.3–7.4, when bubbled with carbogen . After cutting, slices were incubated in recovery aCSF bubbled with carbogen at 35°C for 30 min, and thereafter at room temperature.Transverse brainstem slices (200 μm thick) were prepared from P8–P12 mice. After rapid decapitation, the brainstem was removed from the skull and immersed in ice‐cold artificial cerebrospinal fluid (aCSF) containing (in mM): 125 NaCl, 2.5 KCl, 3 MgCl2.3Brainstem slices (100 μm thick) were cut and subsequently fixed with 4% (wt/vol) paraformaldehyde in phosphate buffer solution (PBS) for 20 min. Free‐floating sections were blocked in 4% goat serum and 0.3% Triton X‐100 in PBS for 1 hr. Slices were incubated with antibodies for chicken anti‐green fluorescent protein , rabbit anti‐oligodendrocyte transcription factor 2 , mouse anti‐neuronal nuclei  overnight at 4°C and subsequently incubated with different Alexa dye–conjugated secondary antibodies  for 2 hr at room temperature. Slices were mounted onto Superfrost slides in photobleaching‐protective medium . Stained slices were viewed with laser lines at 488, 555, and 633 nm using a 40× oil‐immersion objective on a confocal laser‐scanning microscope . Stack images were acquired at a digital size of 1,024 × 1,024 pixels with optical section separation  of 0.5 μm and were later cropped to the relevant part of the field without changing the resolution.2.42+ imaging was performed in normal aCSF at room temperature (22–24°C). Normal aCSF was the same as slicing aCSF, but with 1 mM MgCl2 and 2 mM CaCl2. Fluorescence images were acquired at 20 Hz sampling frequency using an ORCA Flash4.0 CMOS camera  and HCImage software , using wide‐field fluorescence microscope illumination (X‐Cite 120Q excitation light source with 120‐watt lamp). 1 mM glutamate  or 40 mM KCl  was puff‐applied through a glass pipette (5–7 psi for ~1 s), the tip of which was placed 50–100 μm away from the cell body. GCaMP6f‐GFP or Fluo‐4  fluorescence was measured when excited at 488 nm. Background subtractions were done for cells filled with Fluo‐4. Fluorescent regions of interest (ROIs) were defined manually. All values are expressed as a change in the fluorescence calculated as ΔF/F, where F is the basal fluorescence of the ROI and ΔF is the change in fluorescence of the ROI at the peak response relative to the F value. 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione , 1‐naphthyl acetyl spermine , D‐AP5 , dantrolene , CdCl2 , 4‐aminopyridine , tertraethylammonium , BaCl2 , tetrodotoxin , nifedipine , and ω‐agatoxin‐IVA  were bath‐applied.Ca2.52+ current recordings, CsCl‐based pipette solution included (in mM): 150 CsCl, 10 TEA‐Cl, 1 MgCl2, 10 HEPES, 2 ATP, 0.3 GTP, 10 phosphocreatine, and 50 μM Fluo‐4, pH = 7.3. Ca2+ currents were analyzed after leak subtraction using a “traditional” p/4 stimulus train in EPC10‐Patchmaster. For current clamp and AMPAR current recordings, K‐gluconate‐based pipette solution contained (in mM): 130 K‐gluconate, 20 KCl, 5 Na2‐phosphocreatine, 10 HEPES, 4 Mg‐ATP, 0.3 GTP, and 50 μM Fluo‐4, pH = 7.3. Occasionally, pipette solution additionally contained Alexa 568 (50 μM) to visualize the cell through dye‐filling during whole‐cell recordings. Patch electrodes had resistances of 4–6 MΩ, and the series resistance (Rs) was <25 MΩ before compensation.Whole‐cell patch‐clamp recordings were performed in normal aCSF at room temperature (22–24°C) using the voltage‐ or current‐clamp mode of the EPC‐10 . For Ca2.62+ imaging and electrophysiology data were analyzed and presented using Igor Pro . Statistical significance was determined using paired Student's t test . p Values < .05 were considered significant. Data values are reported as means ± standard error of the mean (SEM).Ca33.1In the MNTB of the mouse auditory brainstem, GCaMP6f was expressed specifically in OLs, which were Olig2+ and NeuN‐, Figure a and wer2+ rise, as recorded by a transient increase in fluorescence  and the decay time from the peak of the Ca2+ response to the resting Ca2+ level was 13.92 ± 0.52 s in 113 individual cells from 19 animals. Glutamate‐induced Ca2+ rise in OLs was replicable and consistent in amplitude after a two‐minute recovery period. Morphologically, the processes of GCaMP6f + cells did not align with axons to generate myelin sheaths  application 50–100 μm from the OL cell body resulted in a Ca mM applis Figure .2+ transients in OLs using their antagonists. Before drug application, we confirmed the reliability of a Ca2+ response by verifying consistent Ca2+ rises in the OL in response to two consecutive glutamate puffs. The individual Ca2+ responses in the presence of antagonists were normalized to this control response. In the presence of Naspm, a Ca2+‐permeable AMPAR antagonist, the amplitude of glutamate‐induced Ca2+ transient was significantly decreased to 40.8 ± 5.46% of the control response from the same cell . We tested whether NMDA receptor activation contributes to glutamate‐induced Ca2+ rise in OLs. D‐AP5, an NMDA receptor antagonist, had no significant effect on glutamate‐induced Ca2+ rise in OLs . Other studies have shown the presence of Ca2+‐induced Ca2+ release (CICR) in OLs .OL lineage cells express ionotropic glutamate receptors  and receive glutamatergic input from neurons , the amplitude of AMPAR currents was −36.3 ± 6.04 pA in response to glutamate puff, which simultaneously induced a Ca2+ rise , indicating that glutamate induced AMPAR currents through both Ca2+‐permeable and ‐impermeable AMPARs in OLs. Interestingly, the Ca2+ rise in response to glutamate puff was completely inhibited in the presence of Naspm . Thus, Ca2+ influx through Ca2+‐permeable AMPARs is mainly responsible for the Ca2+ rise when the membrane voltage was clamped at −70 mV. Notably, during GCaMP6f imaging when the membrane potential was not clamped, there was a significant portion of the Ca2+ rise remaining in the presence of Naspm, which was completely inhibited by CNQX . This result indicates that glutamate triggers Ca2+ entry through Cav channels in pre‐myelinating OLs.How do Cav channels. To study the correlation between glutamate‐induced depolarization and Ca2+ influx via Cav channel in pre‐myelinating OLs, we recorded membrane potential changes and simultaneously monitored a Ca2+ transient from a single OL . This remaining Ca2+ response was significantly reduced by the application of CdCl2 reduced to 24.3 ± 6.30% of responses in the presence of CNQX and Naspm , although TTX application resulted in a decrease in the Ca2+ rise in the presence of TTX to 74.7 ± 6.04% of control responses  and depolarization . These results suggest that elevated extracellular K+ due to increased firing rate of neurons partially contributes to depolarization and Ca2+ rise in OLs, and that the larger remaining response is due to AMPAR‐mediated currents.We next determined whether increased neuronal activity and elevated extracellular K3.4v channels involved in Ca2+ influx in OLs in response to glutamate puff. We specifically examined L‐type and P/Q‐type channels, which are expressed in OL lineage cells , an L‐type Cav channel blocker, reduced the glutamate‐induced Ca2+ response in GCaMP6f + OLs to 38.0 ± 5.09% , while agatoxin (200 nM), a P/Q‐type Cav channel blocker, reduced the amplitude to 20.6 ± 2.61% of the control response from the same cell . The combination of agatoxin and CdCl2 reduced the Ca2+‐impermeable AMPAR‐mediated Ca2+ response to 6.4 ± 2.26% of control responses in the same cell . Thus, P/Q‐type and L‐type Cav channels play a significant role in OL Ca2+ rise in response to glutamate puff.Furthermore, we identified the types of Cav channel currents in OLs, voltage‐activated Ca2+ currents (ICa) were elicited from OLs in whole‐cell voltage‐clamp recordings. ICa was shown in response to depolarization steps from −80 mV to 60 mV and was completely inhibited by CdCl2  relationship curve. The peak of ICa at 0 mV was −79.86 ± 10.47 pA . The presence of ICa and effect of Cav channel antagonists on glutamate‐induced Ca2+ responses demonstrates that Ca2+ influx via Cav channels importantly contributes to Ca2+ rise in pre‐myelinating OLs during glutamate‐induced depolarization.To confirm the presence of functional P/Q‐type Ca2 Figure c. Partian Figure d. The vo2+ response mediated by P/Q‐type and L‐type Cav channels using whole‐cell voltage‐clamp. A depolarization pulse, mimicking the time course of the depolarization recorded in response to glutamate puff, resulted in a Ca2+ rise measured with GCaMP6f fluorescence. The Ca2+ rise was detectable in response to ~25 mV depolarization from −65 mV to −40 mV and was partially blocked by agatoxin application , and the combination of Naspm and CNQX reduced the response to 28.4 ± 3.16% of control responses in the same cell . During axon fiber stimulation, increased neuronal activity leads to the accumulation of extracellular K+ and consequently OL depolarization (Battefeld et al., + concentration using KCl puff depolarized and induced Ca2+ influx through Cav channels in OLs without AMPAR activation (Figure 2+ rise in OLs is mediated by AMPARs and Cav channels as sources of Ca2+ influx.We next examined whether neuronal activity causes glutamate‐mediated Ca42+ transients and initiate downstream signaling processes. Here, we studied what factors mediate the dynamic communication between neurons and OLs leading to OL Ca2+ rise and the primary source of OL Ca2+ transients in response to neuronal input. The results reveal that increased neuronal activity leads to Ca2+ influx in OLs through glutamatergic signaling, mediated by AMPARs, depolarization, and L‐type and P/Q‐type Cav channels. These findings support that glutamatergic signaling is a key mechanism for neuron‐OL interaction that could lead to various downstream Ca2+ signaling pathways important for myelination and OL function in the mammalian nervous system.During development, neuronal activity drives physiological responses in OL lineage cells that influence OL maturation and myelination. It is proposed that neuronal activity triggers release of signaling factors to elicit OL Ca2+ responses using pharmacological approaches. For example, bath application of CdCl2 can block axonal or presynaptic Ca2+ channels, inhibiting vesicular glutamate release, as well as oligodendroglial Ca2+ channels, and thus a mixed response could be observed. Glutamate puff could be activating AMPARs on neighboring cells, resulting in increased firing and release of other molecules or ions to trigger OL Ca2+ transients. During periods of increased action potential firing, the concentration of K+ in the extracellular area increases, which in turn depolarizes OLs (Battefeld et al., + release from neighboring cells partially contributes to OL Ca2+ transients. This depolarization could then activate Cav channels in OLs, resulting in a Ca2+ rise. In addition, ATP and adenosine are other possible molecules, which are released in response to axon stimulation and have been shown to induce a Ca2+ rise in OL lineage cells (Hamilton et al., 2+ transients and explain the slow and prolonged kinetics of the Ca2+ rise in OLs in response to electrical stimulation of axons seen in Figure 2+ dynamics in OLs are a result of a summation of various inputs over time when neuronal activity increases.Due to the overlap in expression of channels and receptors in both neurons and glia in the brain slice, it was difficult to isolate effects on single OLs and to quantify the contribution of each cell type to OL Ca2+ rise to delineate sources of Ca2+ influx in OLs in response to glutamatergic input. An alternative mediator of Ca2+ transient duration may be a secondary Ca2+ source, such as release from internal stores in OLs. Amplitude and duration of Ca2+ transients in OLs induced by AMPA application were reduced after blocking ryanodine receptors (Ruiz et al., 2+ transients. We tested the effect of dantrolene, a blocker of CICR, on glutamate‐induced Ca2+ rise in OLs, but there was no significant effect (Figure 2+ rise mediated by Cav channels and Ca2+‐permeable AMPARs, similarly observed in the present study. In contrast to our results, Ca2+ influx triggered CICR and additionally amplified Ca2+ rise in hippocampal NG2+ cells (Haberlandt et al., 2+ source, a recent study demonstrates that mitochondrial Ca2+ release mediates microdomain Ca2+ transients and large Ca2+ waves in myelin sheaths (Battefeld, Popovic, de Vries, & Kole, 2+ regulation is important for myelin remodeling.In this study, we examined OL Cat Figure  demonstr2+ rise in OLs in response to glutamatergic input? Glutamate has been repeatedly shown to induce pathological damage to cultured OLs (Alberdi, Sánchez‐Gómez, Marino, & Matute, 2+ rise and downstream cascades leading to mitochondrial damage and apoptosis (Leuchtmann, Ratner, Vijitruth, Quab, & McDonald, 2+ through either AMPAR or Cav channels could induce necrosis of OLs in pathological conditions. However, dynamic Ca2+ transients are beneficial for signaling cascades of physiological processes of OLs, such as increased OL lineage cell survival and migration, as well as myelination (Gautier et al., 2+ response in OLs in zebrafish, which was associated with myelin elongation or retraction, depending on the dynamics of the Ca2+ response (Baraban et al., v channels as an important Ca2+ influx pathway for Ca2+ dynamics in developing OLs. Recent studies suggest the potential role of AMPARs in OL development and myelination (Chen et al., v channels or Nav channels throughout the OL lineage impaired OL development and myelination (Berret et al., v channels, as well as other signaling mechanisms, can play a role in OL Ca2+ dynamics as important mediators of glutamate‐mediated signaling to impact OL development.What is the functional relevance of Ca2+ rise in OLs has been observed in brain areas other than the MNTB. Neuronal activity induced myelination‐associated Ca2+ rises in OLs in the zebrafish spinal cord (Baraban et al., 2+ rise in response to glutamate or depolarization has been observed in the rodent optic nerve and hippocampus (Haberlandt et al., 2+ rise in pre‐myelinating OLs in the MNTB, but could be relevant for Ca2+ transients in developing OLs in other brain areas. Understanding the physiological properties of OLs and mechanisms of neuron‐OL interaction throughout the OL lineage and throughout the brain is critical for further comprehension of their roles in brain function. The mechanism of Ca2+ rise in pre‐myelinating OLs resulting from depolarization and neuronal input demonstrated in this study provides important insight into the dynamic physiological properties of OLs.Dynamic interaction between active axons and OLs in the auditory brainstem is particularly relevant to auditory function, because this brain region is heavily myelinated and operates at the upper limits of action potential frequency and speed required for sound localization (Kim, Renden, & von Gersdorff, The authors declare no competing interests.T.B. performed all experiments, analyses, and drafted the manuscript. J.H.K. supervised the project and revised the manuscript. All authors reviewed and approved the final manuscript.


Intensive multi-agent chemotherapy is an important cornerstone of curative-intent treatment for acute myeloid leukemia (AML). This is unlikely to change soon despite recent approval of several new targeted drugs  which may be used best as add-on to, rather than replacement of, conventional chemotherapeutics. Numerous regimens have been developed for AML therapy, including standard-dose cytarabine with an anthracycline (e.g. “7+3”), the combination of mitoxantrone, etoposide and cytarabine (“MEC”), or the combination of fludarabine, cytarabine, granulocyte colony stimulating factor (G-CSF) and idarubicin (“FLAG-Ida”), among others. Since established regimens are ineffective in many patients, however, there is ongoing interest in new or refined chemotherapy backbones for people with newly-diagnosed as well as relapsed/refractory AML.2 on days 1-5), cytarabine , G-CSF, and mitoxantrone  in adults with primary refractory AML or early relapsed disease had acceptable tolerability and encouraging activity – among 118 treated people, a complete remission (CR) rate of 58% was obtained  without MRD). 4- and 8-week mortality was 5% at both timepoints. Comparisons between patients with primary refractory vs. relapsed disease revealed no difference in tolerability or rates of subsequent hematopoietic cell transplantation. After adjustment, CLAG-M with mitoxantrone at 16 mg/m2 was associated with statistically significantly longer overall survival and non-significantly longer relapse-free survival compared to 30 similar patients treated at our institution with CLAG-M with regular-dose mitoxantrone, while remission rates were not different. After controlling for prognostic factors, CR/CRi rates were higher, and survival longer, in people treated with CLAG-M at 16 mg/m2 compared to 36 matched patients treated with decitabine-primed MEC  years, with primary refractory (C (d/MEC ).2 (newly-diagnosed disease) or 16 mg/m2 (relapsed/refractory disease) are safe and relatively well-tolerated in fit younger and older adults with AML and other high-grade myeloid neoplasms; whether this RP2D difference reflects a greater vulnerability of patients with relapsed/refractory disease to higher doses of mitoxantrone or represents a chance finding due to the small patient numbers is unknown. Escalating the mitoxantrone dose may provide additional anti-leukemia efficacy over the dose conventionally used with CLAG-M, although non-randomized comparisons and small cohort sizes limit the conclusions we can draw from our analyses.Overall, the results from our institutional phase 1/2 trial indicate CLAG-M with mitoxantrone up to 18 mg/mneg remissions than with 7+3 – most achieved with the first cycle of therapy – makes CLAG-M an attractive regimen for initial therapy of AML although randomized comparisons are needed to validate this finding. Because of the general tolerability of CLAG-M with escalated doses of mitoxantrone, institutional efforts are ongoing to test add-on of sorafenib (NCT02728050) or gemtuzumab ozogamicin (NCT03531918) in fit younger and older adults with newly-diagnosed AML. In parallel, this regimen is currently in medically less-than-fit adults with newly-diagnosed AML.Our study adds to the evidence supporting the use of CLAG-M for intensive AML re-induction therapy, perhaps particularly with higher mitoxantrone doses. Perhaps more importantly, our study points to a role of this regimen in the upfront setting where it is currently not commonly used. Demonstration of a higher rate of MRD