Cellular and transcriptional impacts of Janus kinase and/or IFN-gamma inhibition in a mouse model of primary hemophagocytic lymphohistiocytosis

BackgroundPrimary hemophagocytic lymphohistiocytosis (pHLH) is an inherited inflammatory syndrome driven by the exuberant activation of interferon-gamma (IFNg)-producing CD8 T cells. Towards this end, ruxolitinib treatment or IFNg neutralization (aIFNg) lessens immunopathology in a model of pHLH in which perforin-deficient mice (Prf1–/–) are infected with Lymphocytic Choriomeningitis virus (LCMV). However, neither agent completely eradicates inflammation. Two studies combining ruxolitinib with aIFNg report conflicting results with one demonstrating improvement and the other worsening of disease manifestations. As these studies used differing doses of drugs and varying LCMV strains, it remained unclear whether combination therapy is safe and effective.MethodsWe previously showed that a ruxolitinib dose of 90 mg/kg lessens inflammation in Prf1–/– mice infected with LCMV-Armstrong. To determine whether this dose controls inflammation induced by a different LCMV strain, we administered ruxolitinib at 90mg/kg to Prf1–/– mice infected with LCMV-WE. To elucidate the impacts of single agent versus combination therapy, Prf1–/– animals were infected with LCMV, treated or not with ruxolitinib, aIFNg or both agents, and analyzed for disease features and the transcriptional impacts of therapy within purified CD8 T cells.ResultsRuxolitinib is well-tolerated and controls disease regardless of the viral strain used. aIFNg, administered alone or with ruxolitinib, is most effective at reversing anemia and reducing serum IFNg levels. In contrast, ruxolitinib appears better than aIFNg, and equally or more effective than combination therapy, at lessening immune cell expansion and cytokine production. Each treatment targets distinct gene expression pathways with aIFNg downregulating IFNg, IFNa, and IL-6-STAT3 pathways, and ruxolitinib downregulating IL-6-STAT3, glycolysis, and reactive oxygen species pathways. Unexpectedly, combination therapy is associated with upregulation of genes driving cell survival and proliferation.ConclusionsRuxolitinib is tolerated and curtails inflammation regardless of the inciting viral strain and whether it is given alone or in combination with aIFNg. When administered at the doses used in this study, the combination of ruxolitinb and aIFNg appears no better than treatment with either drug alone in lessening inflammation. Further studies are warranted to elucidate the optimal doses, schedules, and combinations of these agents for the treatment of patients with pHLH

The National Cancer Institute’s Community Networks Program Initiative to Reduce Cancer Health Disparities: Outcomes and Lessons Learned

We describe reach, partnerships, products, benefits, and lessons learned of the 25 Community Network Programs (CNPs) that applied community-based participatory research (CBPR) to reduce cancer health disparities

Structural brain correlates of childhood inhibited temperament: an ENIGMA-Anxiety Mega-analysis

NWORubicon 019.201SG.022Pathways through Adolescenc

Control of Anther Cell Differentiation by the Small Protein Ligand TPD1 and Its Receptor EMS1 in <i>Arabidopsis</i>

<div><p>A fundamental feature of sexual reproduction in plants and animals is the specification of reproductive cells that conduct meiosis to form gametes, and the associated somatic cells that provide nutrition and developmental cues to ensure successful gamete production. The anther, which is the male reproductive organ in seed plants, produces reproductive microsporocytes (pollen mother cells) and surrounding somatic cells. The microsporocytes yield pollen via meiosis, and the somatic cells, particularly the tapetum, are required for the normal development of pollen. It is not known how the reproductive cells affect the differentiation of these somatic cells, and vice versa. Here, we use molecular genetics, cell biological, and biochemical approaches to demonstrate that TPD1 (TAPETUM DETERMINANT1) is a small secreted cysteine-rich protein ligand that interacts with the LRR (Leucine-Rich Repeat) domain of the EMS1 (EXCESS MICROSPOROCYTES1) receptor kinase at two sites. Analyses of the expressions and localizations of TPD1 and EMS1, ectopic expression of TPD1, experimental missorting of TPD1, and ablation of microsporocytes yielded results suggesting that the precursors of microsporocyte/microsporocyte-derived TPD1 and pre-tapetal-cell-localized EMS1 initially promote the periclinal division of secondary parietal cells and then determine one of the two daughter cells as a functional tapetal cell. Our results also indicate that tapetal cells suppress microsporocyte proliferation. Collectively, our findings show that tapetal cell differentiation requires reproductive-cell-secreted TPD1, illuminating a novel mechanism whereby signals from reproductive cells determine somatic cell fate in plant sexual reproduction.</p></div

Genetic ablation of microsporocytes shows the interdependence of tapetal cell and microsporocyte differentiation.

<p>(<b>A</b>) A wild-type anther lobe at stage 5 showing four layers of anther wall cells (indicated by red dots, the same hereinafter) and microsporocytes. (<b>B-E</b>) <i>SDS</i>:<i>SDS-BANASE</i> anther lobes at stage 5, which we divided into three classes. Class I: four somatic cell layers, including one organized single-cell layer that surrounds the microsporocytes and is made of cells that are morphologically similar to tapetal cells (<b>B</b>). Class II: four somatic cell layers, including a monolayer of vacuolated tapetal-like cells (<b>C</b>) and three somatic cell layers that contains delaminated vacuolated tapetal-like cells (<b>D</b>). Degenerating microsporocytes are observed in Class-II anthers. Class III: three somatic cell layers and excess microsporocytes (<b>E</b>). Among 60 T1 plants analyzed by semi-thin section, 16.7% (10/60) were Class I, 70.0% (42/60) were Class II, and 13.3% (8/60) were Class III. (<b>F</b>-<b>J</b>) <i>In situ</i> hybridization results showing that the expression of the tapetal cell marker gene, <i>A9</i> at stage 5 was strong in the tapetum of the wild-type anther (<b>F</b>), but was progressively decreased in tapetal-like cells from Class-I (<b>G</b>) and Class-II (<b>H</b>, <b>I</b>) <i>SDS</i>:<i>SDS-BANASE</i> anthers. No <i>A9</i> expression was detected in the Class-III <i>SDS</i>:<i>SDS-BANASE</i> anther (<b>J</b>). (<b>K</b>-<b>O</b>) <i>In situ</i> hybridization results showing the expression of the microsporocyte marker gene, <i>SDS</i>, in anthers. In wild-type anthers, <i>SDS</i> expression was weak at stage 4 in precursors of microsporocytes (<b>K</b>), but strong at stage 5 in microsporocytes (<b>L</b>). <i>SDS</i> was weakly expressed in the microsporocytes of <i>SDS</i>:<i>SDS-BANASE</i> anthers (<b>M-O</b>). The <i>SDS</i> expression domain was relatively expanded in <i>SDS</i>:<i>SDS-BANASE</i> Class-II and -III anthers (<b>N, O</b>). E, epidermis; En, endothecium; M, microsporocyte; ML, middle layer; PM, precursor of microsporocyte; T, tapetal cell; and TL, tapetal-like cell. Scale bars, 10 μm. (<b>P</b>, <b>Q</b>) We used qRT-PCR to examine expression levels of <i>A9</i> and <i>BARNASE</i> in anthers from three representative transgenic plants of each class. From Class I to Class III anthers, the expression of <i>A9</i> progressively decreased (<b>P</b>), while that of <i>BARNASE</i> increased (<b>Q</b>).</p

The putative signal peptide of TPD1 is required for its normal function.

<p>To test if the putative signal peptide of TPD1 and the N-terminal signal peptide-directed secretion of TPD1 are required for its function in anther cell fate determination, vectors encoding TPD1s in which the putative signal peptide had been replaced with known secreted signal peptides were introduced into the <i>tpd1</i> mutant and assessed for their ability to rescue the <i>tpd1</i> phenotype. (<b>A</b>) Schematic diagrams showing the structures of the <i>TPD1</i>:<i>TPD1sp-</i>Δ<i>TPD1</i>, <i>TPD1</i>:Δ<i>TPD1</i>, <i>TPD1</i>:<i>CLV3sp-</i>Δ<i>TPD1</i>, and <i>TPD1</i>:<i>PAP1sp-</i>Δ<i>TPD1</i> constructs. Red bar: the TPD1 putative signal peptide, Orange bar: the CLV3 signal peptide, Purple bar: the PAP1 signal peptide, Cyan bar: the non-conserved N-terminal region, Blue bar: the conserved C-terminal domain, and ΔTPD1: TPD1 without the putative signal peptide. The 2.7-kb <i>TPD1</i> promoter was used for all constructs. (<b>B</b>-<b>G</b>) Compared with wild-type plants (<b>B</b>), primary inflorescences from 80% (96/120) of <i>TPD1</i>:<i>TPD1sp-</i>Δ<i>TPD1</i>/<i>tpd1</i> (<b>D</b>), 72% (36/50) of <i>TPD1</i>:<i>CLV3sp-</i>Δ<i>TPD1</i>/<i>tpd1</i> (<b>F</b>), and 70% (28/40) of <i>TPD1</i>:<i>PAP1sp-</i>Δ<i>TPD1</i>/<i>tpd1</i> (<b>G</b>) plants showed normal siliques, whereas 100% (88/88) of <i>TPD1</i>:Δ<i>TPD1/tpd1</i> plants (<b>E</b>) exhibited short siliques comparable to those of <i>tpd1</i> plants (<b>C</b>). Scale bars, 5 mm. (<b>H</b>-<b>M</b>) Pollen viability tests performed using Alexander pollen staining show functional pollen grains (red-stained) in wild-type (<b>H</b>), <i>TPD1</i>:<i>TPD1sp-</i>Δ<i>TPD1</i>/<i>tpd1</i> (<b>I</b>), <i>TPD1</i>:<i>CLV3sp-</i>Δ<i>TPD1/tpd1</i> (<b>L</b>), and <i>TPD1</i>:<i>PAP1sp-</i>Δ<i>TPD1</i>/<i>tpd1</i> (<b>M</b>) anthers, but not in <i>tpd1</i> (<b>J</b>) or <i>TPD1</i>:Δ<i>TPD1/tpd1</i> (<b>K</b>) anthers. Scale bars, 50 μm. (<b>N</b>-<b>S</b>) Semi-thin sections of stage-5 anthers showing normal anther cell differentiation in wild-type (<b>N</b>), <i>TPD1</i>:<i>TPD1sp-</i>Δ<i>TPD1</i>/<i>tpd1</i> (<b>O</b>), <i>TPD1</i>:<i>CLV3sp-</i>Δ<i>TPD1/tpd1</i> (<b>R</b>), and <i>TPD1</i>:<i>PAP1sp-</i>Δ<i>TPD1</i>/<i>tpd1</i> (<b>S</b>) anthers, but not in <i>TPD1</i>:<i>△TPD1</i>/<i>tpd1</i> (<b>Q</b>) or <i>tpd1</i> (<b>P</b>) anthers, which lacked tapetal cells and exhibited excess microsporocytes. E, epidermis; En, endothecium; ML, middle layer; T, tapetal cell; and M, microsporocyte. Scale bars, 10 μm.</p

TPD1 is a secreted protein whose localization at the plasma membrane depends on EMS1.

<p><i>35S</i>:<i>TPD1sp-GFP-ΔTPD1</i> and <i>35S</i>:<i>TPD1sp-GFP-ΔTPD1 35S</i>:<i>EMS1</i> transgenic plants were used to analyze the localization and secretion of TPD1 in root cells. (<b>A</b>-<b>C</b>) Confocal images of <i>35S</i>:<i>TPD1sp-GFP-ΔTPD1</i> root cells showing TPD1 proteins [arrows, (<b>A</b>)], FM4-64-stained trafficking vesicles [arrows, (<b>B</b>)], and GFP signals overlapping with trafficking vesicles [arrows, (<b>C</b>)]. (<b>D</b>, <b>E</b>) Confocal images of <i>35S</i>:<i>TPD1sp-GFP-ΔTPD1</i> root cells treated without (<b>D</b>) or with (<b>E</b>) BFA, which blocks the formation of Golgi-derived vesicles. Arrows in (<b>E</b>) indicate aggregated TPD proteins. (<b>F</b>-<b>H</b>) Confocal images of <i>35S</i>:<i>TPD1sp-GFP-ΔTPD1 35S</i>:<i>EMS1</i> root cells showing TPD1sp-GFP-ΔTPD1 proteins at the plasma membrane (<b>F</b>: GFP alone, <b>G</b>: FM4-64-stained, and <b>H</b>: merged). Scale bars, 10 μm.</p

TPD1 is processed into a 13-kD small protein in planta.

<p>(<b>A</b>) The TPD1 protein sequence. Red color: the putative signal peptide, Cyan: the non-conserved N-terminal region, Blue: the conserved C-terminal domain, Orange: cysteine residues (numbers indicate their positions), Pink “KR”: the potential dibasic cleavage site, and Underlined: the sequence of identified mature TPD1. (<b>B</b>) To examine the processing of TPD1, constructs encoding GFP-TPD1 fusion proteins were generated and introduced into the <i>tpd1</i> mutant. Schematic diagrams showing the constructs used for the complementation experiments. The 2.7-kb <i>TPD1</i> promoter was used in all constructs. Red bar: the putative signal peptide (Sp) of TPD1, Green bar: GFP, Cyan bar: the non-conserved N-terminal region, Blue bar: the conserved C-terminal domain, ΔTPD1: TPD1 without the putative signal peptide, and Pink line: K135GR135G mutations. All constructs were transformed into <i>tpd1</i> heterozygous plants. PCR genotyping was carried out to determine the <i>tpd1</i> mutant background of T2 transgenic plants. Transgenic plants showing siliques comparable in size to those of wild-type plants were counted as complemented plants. (<b>C</b>-<b>G</b>) Alexander pollen staining of mature anthers reveals viable pollen grains in wild-type (<b>C</b>) and <i>TPD1</i>:<i>TPD1sp-GFP-ΔTPD1/tpd1</i> (<b>D</b>) plants, whereas no pollen is observed in <i>TPD1</i>:<i>TPD1sp-ΔTPD -GFP/tpd1</i> (<b>E</b>), <i>TPD1</i>:<i>GFP-ΔTPD1/tpd1</i> (<b>F</b>), and <i>TPD1</i>:<i>TPD1sp-GFP-ΔTPD1</i> ^{<i>K135G R136G</i>}/<i>tpd1</i> (<b>G</b>) anthers. Scale bars, 50 μm. (<b>H</b>-<b>L</b>) Semi-thin sections of stage-6 anthers show normal tapetal cells and microsporocytes in wild-type (<b>H</b>) and <i>TPD1</i>:<i>TPD1sp-GFP-ΔTPD1/tpd1</i> (<b>I</b>) plants, whereas a complete lack of tapetal cells and the presence of excess microsporocytes are seen in <i>TPD1</i>:<i>TPD1sp-ΔTPD -GFP/tpd1</i> (<b>J</b>), <i>TPD1</i>:<i>GFP-ΔTPD1/tpd1</i> (<b>K</b>), and <i>TPD1</i>:<i>TPD1sp-GFP-ΔTPD1</i> ^{<i>K135G R136G</i>}/<i>tpd1</i> (<b>L</b>) anthers. E, epidermis; En, endothecium; M, microsporocyte; ML, middle layer; and T, tapetal cell. Scale bars, 10 μm. (<b>M</b> and <b>N</b>) Western blot analysis of the processing of GFP-fused TPD1 proteins. (<b>M</b>) No band was seen for wild-type (WT) or <i>TPD1</i>:<i>TPD1sp-ΔTPD -GFP/tpd1</i> plants, whereas <i>TPD1</i>:<i>TPD1sp-GFP-ΔTPD1/tpd1</i> plants exhibited a 41-kD band. By subtracting 28 kD for GFP, we calculate that the mature TPD1 protein is about 13 kD in size. (<b>N</b>) Wild-type (WT) plants showed no band; <i>TPD1</i>:<i>TPD1sp-GFP-ΔTPD1/tpd1</i> plants exhibited a 41-kD band; <i>TPD1</i>:<i>GFP-ΔTPD1/tpd1</i> plants exhibited a non-cleaved 45-kD band; and <i>TPD1</i>:<i>TPD1sp-GFP-ΔTPD1</i>^{<i>K135G R136G</i>}<i>/tpd1</i> plants exhibited a non-cleaved 48-kD band. Arrowheads indicate the 41-kD bands. Bottom: Coomassie blue staining of RuBisCO.</p

The mature TPD1 interacts with the EMS1 LRR domain at two sites.

<p>Bimolecular Fluorescence Complementation (BiFC) experiments were performed using <i>Arabidopsis</i> mesophyll protoplasts to test the interaction between the mature TPD1 and EMS1 and determine the TPD1-interacting site(s) in EMS1. <i>35S</i>:<i>EYFP</i> was used as a control to monitor the transfection efficiency. At least three independent experiments were performed for each assay. (<b>A</b>) Schematic diagrams showing the truncated and mutated versions of TPD1. Red bar: the putative signal peptide of TPD1, Cyan bar: the non-conserved N-terminal region, Blue bar: the conserved C-terminal domain, with cysteine positions shown as vertical orange lines, Yellow oval: nEYFP (N-terminal EYFP) inserted right after the signal peptide of TPD1, and Pink line in the <i>nEYFP- TPD1</i>^{<i>K135G R136G</i>} construct: K135GR136G mutations. Size bar indicates TPD1 alone, and does not include nEYFP. (<b>B</b>-<b>D</b>) Confocal images showing that cEYFP-LRR (the full-length EMS1 LRR domain) interacts with nEYFP-TPD1 (<b>B</b>) and nEYFP-ΔC2 (<b>C</b>), but not nEYFP- TPD1^{K135G R136G} (<b>D</b>). Scale bars, 10 μm. (<b>E</b>-<b>O</b>) Identification of TPD1-binding domains in EMS1. (<b>E</b>) Schematic diagrams showing the primary structures of EMS1 and the truncated/mutated versions used for the BiFC assays. SP: signal peptide, LRRNT: leucine-rich repeat containing N-terminus, LRR: leucine-rich repeat, GAP: non-leucine-rich repeat gap region between the two LRRs, OJM: outer juxtamembrane domain, IJM: inner juxtamembrane domain, TM: transmembrane domain, KD: kinase domain, Red bar in the cEYFP-EMS1^K104N construct: K104N mutation. All EMS1 constructs contained SP, OJM, TM, and IJM. Orange oval represents the C-terminal EYFP (cEYFP). Size bar indicates EMS1 alone, and does not include cEYFP. (<b>F</b>-<b>O</b>) Confocal images showing that the mature TPD1 (nEYFP-ΔC2) interacts with cEYFP-EMS1 [the full-length EMS1, (<b>F</b>)], cEYFP-LRR [the full-length EMS1 LRR domain, (<b>G</b>)], cEYFP-ΔLRR-I (<b>J</b>), cEYFP-ΔLRR-III (<b>L</b>), and cEYFP-ΔLRR-IV (<b>N</b>), but not with cEYFP-KD (<b>H</b>), cEYFP-BRI1 [(<b>I)</b>, negative control], cEYFP-ΔLRR-II (<b>K</b>), cEYFP-EMS1^K104N (<b>M</b>), or cEYFP-ΔLRR-V (<b>O</b>). Scale bars, 10 μm.</p

Localization of TPD1 and EMS1 in anthers.

<p>(<b>A</b>-<b>T</b>) Confocal images showing merges of red chlorophyll autofluorescence and green GFP signals, with the exceptions of (<b>G</b>) and (<b>H</b>). (<b>A</b>, <b>B</b>) In <i>TPD1</i>:<i>mGFP5er</i> stage-4 (<b>A</b>) and stage-5 (<b>B</b>) anthers, the <i>TPD1</i> promoter was active only in precursors of microsporocytes and microsporocytes, respectively. (<b>C</b>, <b>D</b>) In the <i>TPD1</i>:<i>TPD1sp-GFP-ΔTPD1/tpd1</i> stage-4 anther, TPD1 proteins were detected in precursors of microsporocytes (<b>D</b>, high magnification of <b>C</b>). (<b>E</b>, <b>F</b>) In the <i>TPD1</i>:<i>TPD1sp-GFP-ΔTPD1/tpd1</i> stage-5 anther, TPD proteins were mainly localized in microsporocytes, but were also detected at the surface of tapetal cells (<b>F</b>, high magnification of <b>E</b>). (<b>G</b>, <b>H</b>) TPD1 proteins were localized in vesicle-like compartments of microsporocytes isolated from <i>TPD1</i>:<i>TPD1sp-GFP-ΔTPD1/tpd1</i> stage-5 anthers (<b>H,</b> confocal image merged with DIC-viewed microsporocytes). (<b>I, J</b>) In the <i>TPD1</i>:<i>TPD1sp-GFP-ΔTPD1/ems1</i> stage-5 anther, the TPD1 localization domain was expanded and TPD1 proteins were evenly distributed in microsporocytes as these anthers lacked tapetal cells (<b>J</b>, high magnification of <b>I</b>). (<b>K, L</b>) In <i>TPD1</i>:<i>GFP-ΔTPD1</i> (<b>K</b>) and <i>TPD1</i>:<i>TPD1sp-GFP-ΔTPD1</i>^{<i>K135G R136G</i>} (<b>L</b>) stage-5 anthers, TPD1 proteins were restricted to microsporocytes, regardless of the presence of EMS1. (<b>M</b>, <b>N</b>) In the <i>EMS1</i>:<i>mGFP5er</i> stage-4 anther, the <i>EMS1</i> promoter was active in outer secondary parietal cells (OSPC) and inner secondary parietal cells (ISPC) (<b>N</b>, high magnification of <b>M</b>). (<b>O</b>, <b>P</b>) In the early <i>EMS1</i>:<i>mGFP5er</i> stage-5 anther, the <i>EMS1</i> promoter was active in the middle layer (ML) and precursors of tapetal cells (PT) (<b>P</b>, high magnification of <b>O</b>). (<b>Q</b>, <b>R</b>) In the <i>EMS1</i>:<i>mGFP5er</i> stage-5 anther, <i>EMS1</i> promoter activity was only detected in tapetal cells (T) (<b>R</b>, high magnification of <b>Q</b>). (<b>S</b>, <b>T</b>) In the <i>EMS1</i>:<i>EMS1-3xGFP/ems1</i> stage-5 anther, EMS1 proteins were only observed at surfaces of tapetal cells (<b>T</b> shows a higher magnification of <b>S</b>). (<b>U, V</b>) EM-immunolabeling results showing TPD1 (<b>U</b>) and EMS1 (<b>V</b>) proteins at the plasma membrane of precursors of tapetal cells from <i>TPD1</i>:<i>TPD1sp-GFP-</i>Δ<i>TPD1/tpd1</i> and <i>EMS1</i>:<i>EMS1-3xGFP/ems1</i> early stage-5 anthers, respectively. For each <i>GFP</i> fusion gene, at least 15 independent T2 plants were observed. Similar GFP signals were observed from >90% tested plants. The anther stage was determined by FM4-64 staining (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006147#pgen.1006147.s007" target="_blank">S5 Fig</a>) after GFP images were acquired. ISPC, inner secondary parietal cell; M, microsporocyte; ML, middle layer; OSPC, outer secondary parietal cell; PM, precursor of microsporocyte; PT, precursor of tapetal cell; S, stage; and T, tapetal cell. (<b>A</b>-<b>C</b>, <b>E</b>, <b>I</b>, K, <b>L</b>, <b>M</b>, <b>O</b>, <b>Q, S</b>) Scale bars, 50 μm. (<b>D</b>, <b>F</b>, <b>J</b>, <b>N</b>, <b>P</b>, <b>R, T</b>) Scale bars, 20 μm. (<b>G</b>, <b>H</b>) Scale bars, 10 μm. (<b>U</b>, <b>V</b>) Scale bars, 0.2 μm.</p