The balance between their levels is critical for tissue patterning, with Pk-Sple being the major functional isoform in the eye. Here we uncover a post-translational role for Nemo kinase in limiting the amount of the minor isoform Pk. We identified Pk as a Nemo substrate in a genome-wide in vitro band-shift screen. In vivo, nemo genetically interacts with pkpk but not pksple and enhances PCP defects in the eye and leg. Nemo phosphorylation limits Pk levels and is required specifically in the R4 photoreceptor like the major isoform, Pk-Sple. Genetic interaction and biochemical data suggest that Nemo phosphorylation of Pk leads to its proteasomal degradation via the Cullin1/SkpA/Slmb complex. dTAK and Homeodomain interacting protein kinase (Hipk) may also act together with Nemo to target Pk for degradation, consistent with similar observations in mammalian studies. Our results therefore demonstrate a mechanism to maintain low levels of the minor Pk isoform, allowing PCP complexes to form correctly and specify cell fate.Planar cell polarity (PCP) instructs tissue patterning in a wide range of organisms from fruit flies to humans. PCP signaling coordinates cell behavior across tissues and is integrated by cells to couple cell fate identity with position in a developing tissue. In the fly eye, PCP signaling is required for the specification of R3 and R4 photoreceptors based upon their positioning relative to the dorso-ventral axis. The ‘core’ PCP pathway involves the asymmetric localization of two distinct membrane-bound complexes, one containing Frizzled  and the other Van Gogh . Inhibitory interactions between the cytosolic components of each complex reinforce asymmetric localization. Prickle (Pk) and Spiny-legs (Pk-Sple) are two antagonistic isoforms of the  For functional tissues to form, individual cells must correctly orient themselves and function appropriately for their particular location in the body. The Planar Cell Polarity (PCP) complexes transmit one set of spatial cues by acting as signposts to mark direction across an epithelial layer. PCP signals can direct and coordinate cell differentiation, the behavior of groups of cells, or the orientation of individual cellular protrusions, depending on the tissue. PCP signals act as a polarization relay with two different complexes being positioned on opposite sides of each cell. This pattern of polarity is transmitted to neighboring cells and so extends across the tissue. In the fly eye, PCP signals control the differentiation of a pair of photoreceptors, R3 and R4, where the cell that is positioned closer to the dorso-ventral midline becomes R3. An excess of the PCP protein Prickle prevents the proper assembly of PCP complexes in the eye and so alters R3/R4 fate. Here we show that Nemo kinase is required in the R4 cell to phosphorylate Prickle and promote its degradation by the proteasome. Maintenance of low Prickle levels allows proper formation of PCP complexes, cell fate specification, and eye development. Drosophila to humans, through input into cellular orientation across tissues, individual cell fate decisions, and the coordinated movement of groups of cells S-Methionine, and then incubated with purified Nmo in the presence of unlabeled ATP. Reduced mobility on Anderson gels was used as the criterion to select candidate substrates. Surprisingly, one of the candidates identified in this screen was Prickle, an ‘upstream’ core component of PCP complexes. Nmo, but not dRok or Hpo kinases, was able to induce a band shift of Prickle  or over-expressed individual Pk and Pk-Sple isoforms . We first examined the interaction between nmo and pksple1 in the eye. Wild-type and nmoP hypomorphs show wild-type ommatidial chirality, in addition to the previously-described underrotation in nmo mutant clusters  is sufficient to unsettle the balance between Pk and Pk-Sple and cause defects: act-EGFP-Pk animals  B, H) w-;; nmoPC, I) w-; pksple1D, J) w-; pksple1; nmoPE) mδ-LacZF) w-; pksple1; nmoP, mδ-LacZK) w-;; act-EGFP-Pk/+L) w-;; act-EGFP-Pk/nmoDBS2 B) mδ-LacZC) y-,w-, eyFLP; pkpk1; nmoDB, FRT80B/p[w+], FRT80BD)pkpk1E) y-,w-, eyFLP; pkpk-sple13; nmoDB, FRT80B/p[w+], FRT80BF) w; pkpk-sple13G) w-;; act-EGFP-Pk/+H) w-;; act-EGFP-Pk/nmoDBS3 A-B) w-C) w-; pksple1D) w-;; nmoPE) w-; pksple1; nmoPF) w-;; act-EGFP-Pk/+G) w-;; act-EGFP-Pk/nmoDBS4 A,C-D) w; UbxFLP/+; act-FRT-STOP-FRT-EGFP-Sple/+B, E) w; UbxFLP/+; act-FRT-STOP-FRT-EGFP-Sple/nmoDBF) w;; sevGal4, UAS-Sple/+G) w;; sevGal4, UAS-Sple/UAS-nmoIRH) w;; sevGal4, UAS-Sple/nmoDBB) w-/Y;; sevGal4/UAS-mycPk-WTC) w-/Y;; sevGal4/UAS-mycPk-mut1&2E) w-/Y; sevGal4, UAS-Pk/+F) w-/Y; sevGal4, UAS-Pk/+; nmoP/+G) w-/Y; sevGal4, UAS-Pk/+; nmoDB/+H) w-/Y; sevGal4, UAS-Pk/+; UAS-NmoGFP/+S5 C) w-/Y; pksple1; sevGal4/ UAS-mycPk-WTD) top) w-/Y; nubGal4/+ middle) w-/Y;; nubGal4/UAS-mycPk-WT bottom) w-/Y;; nubGal4/UAS-mycPk-mut1&2E) sev-Sple-WT/+F) sev-Sple-MUT1&2/+G) w; pkpk-sple13; sev-Sple-Mut1&2/+A) y-, w-, eyFLP; pksple1; nmoDB,FRT80B/p[w+], FRT80BA) For blot w-: w-;; actGal4/UAS-Pk-WT: w-;; actGal4/UAS-Pk-Mut1&2B) For blot w-: w-;; act-EGFP-Pk/+: w-;; act-EGFP-Pk/nmoDBC) w-; sevGal4,UAS-Pk/+D) w-; sevGal4,UAS-Pk/+; UAS-DNProsβ2/+E) w-;; act-EGFP-Pk/+F) w-;GMRGal4, UAS-DNProsβ6/+; act-EGFP-Pk/+H) For blot w-;; act-EGFP-Pk/+ and w-;GMRGal4, UAS-DNProsβ6/+; act-EGFP-Pk/+S6 A) w; Ubx-flp/+; act-FRT-STOP-FRT-EGFP-Sple/+B) w; Ubx-flp/+; act-FRT-STOP-FRT-EGFP-Sple/nmoDBF) w; sevGal4/+; UAS-DNProsβ2/+G) w-; GMRGal4, UAS-DNProsβ6/+A) w-; sevGal4, UAS-Pk/+B) w-/Y; sevGal4, UAS-Pk/+; UAS-Cul1IR/+C) w-/Y; sevGal4, UAS-Pk/+; UAS-SkpAIR/+D) w-/Y; sevGal4, UAS-Pk/+; slmb00295/+F) For blot w-;; act-EGFP-Pk/+: w-;; act-EGFP-Pk/ slmb00295: w-;GMRGal4, UAS-DNProsβ2/+; act-EGFP-Pk/+S7 A) w-/Y; sevGal4/+; UAS-Cul1IR/+B) w-/Y; sevGal4/+; UAS-SkpAIR/+C) w-/Y; sevGal4/+; UAS-slmbIR/+D) w-/Y; sevGal4, UAS-Pk/+; UAS-SlmbIR/+w-/w-, dTAK179; sevGal4, UAS-Pk/+B) w-/Y; sevGal4, UAS-Pk/+; nmoIR/+C) w-/Y; sevGal4, UAS-Pk/+; HipkIR/+D) w-/Y; sevGal4, UAS-Pk/+; UAS-wIR/+2 or Fisher’s exact test were performed on adult eye data based on the number in each category, depending on which criteria were met regarding sample size and values of each category. Data in graphs are shown as a percentage for clarity.Tangential eye sections were prepared as described . Three teyFLP; pksple1; nmoDBFRT80B/pw+FRT80B were analyzed. The white pigment marks all heterozygous cells and those homozygous for pw+FRT80B following eyFLP-mediated recombination. Cells lacking white pigment were therefore homozygous for the nmoDB allele. Tangential eye sections were carefully studied to identify R3/R4 pairs in which one cell lacked pigment (nmo- mutant) and the other produced pigment and therefore had at least one functioning copy of nmo (nmo+/+ or nmo+/-). Pairs were scored for the number of times the R3/R4 fate decision was correctly resolved (WT) or where PCP defects occurred , when either R3 had Nmo function or when Nmo function was only present in R4. Over 140 R3/R4 pairs were analyzed from 12 individual adults. Mosaic eye data were analyzed by Fisher’s exact test. For comparison to the pksple1 and pksple1; nmoP mutants, the data from Animals of the genotype Adult legs were dissected and incubated overnight in 70% ethanol. Legs were then washed three times in PBS with 0.1% Triton-X100 and once in PBS. Legs were mounted in 80% glycerol in PBS. The Mann-Whitney U test was used to compare number of tarsal joints. Over 54 legs were analyzed per genotype. Wings were dissected and incubated in PBS with 0.1% Triton-X100 for at least an hour before being mounted in 80% glycerol in PBS.Immunofluorescence was performed on third instar larval eye discs and imaged as described , using tDrosophila β-catenin, whose levels are unaffected by Nmo P kinase assays were done using 1μg of the indicated Gst protein in 20μl reactions using 0.5μl Nmo (about 350 ng) as described [[35]S labeled in vitro translated proteins , pβTH_common [Gst and His tag fusion proteins were prepared as in , 25, 28.pUAS attB_myc-Pk-Wt and pUAS attB_myc-Pk-mut were cloned as follows:Pk EcoRINterm Myc ATG F AACGCACCATGGAACAAAAACTTATTTCTGAAGAAGATCTGATGGATACCCCAAATCAAATGCC,PKRA 1232 R XhoI CCGCTCGAGAAAGCCGGCGATAGCTGGTG). The 3′ region was digested using SalI and NotI from either pBS2SKP_PkBE (WT) or pBS2SKP_PkBE_mut to generate myc-Pk-WT or myc-Pk-mut, respectively. These fragments were ligated into pUAS attB.Pk EcoRINtermMycATGF and PKRA 1232R XhoI primers were used to amplify the 5′ region and to add a myc tag  was cloned into the corresponding BssHII / EcoRI sites of pBS2SKP_Sple_cloneable [Sple.The generation of ribed in . The conribed in  cloned aribed in ). For seloneable . From thS1 FigA) Purification of baculovirus-expressed Nmo kinase. Nmo kinase is marked by *. (B-C) Band-shift assays of S2 cell culture lysates. Cells were transfected with HA-Pk and myc-Nmo expression constructs. WT, but not a kinase dead (KD) Nmo isoform mutant, induced a band shift of HA-Pk (B); the band shift is sensitive to phosphatase-treatment (PPase) (C).Nmo phosphorylates Pk. ((JPG)Click here for additional data file.S2 Fignmo interacts with the Pk isoform in the eye. (A) Schematic of photoreceptor arrangement and corresponding arrows used in all adult eye sections. The arrangement of R3/R4 (in grey) gives a chirality to each ommatidium, denoted by black arrow  and red arrow . The line of mirror symmetry between the dorsal and ventral halves of the eye is called the equator. When R3 and R4 are not specified correctly, symmetrical clusters can form, denoted by straight green arrow. (B) Image of whole wt eye disc shown in mδ-lacZ marking R4) is shown in green and Elav  is in blue. (C) nmoDB clone in a pkpk1 background marked by lack of pigment. Note that despite the rotation defects within the nmoDB clone (shaded in grey below) there is no effect on chirality. (D) A pkpk1 mutant eye, which looks like wild-type (equator marked by organe line in upper panel), is shown for comparison. (E) nmoDB clone in a pk13 (null) background marked by lack of pigment. Note that despite the enhancement of rotation defects in the clone (shaded in grey below) there is no enhancement of pk13 chirality defects. Loss of photoreceptors is marked by an open circle. A pk13 mutant eye is shown for comparison (F). See (I) for quantification of symmetrical clusters in (C-F). (G-H) Loss of nmo function enhances an overexpression of Pk: act-EGFP-Pk, gain-of-function eye phenotype. Chirality defects occur in act-EGFP-Pk/+ eyes (G) and the proportion of defects increases in act-EGFP-Pk/nmoDB animals  Quantification of symmetrical clusters within nmoDB clones and the surrounding control tissue in pkpk1 and pk13 backgrounds. The equivalent experiment in a pksple1 background is included . There is only an increase in symmetrical clusters in the pksple1; nmoDB clone.(JPG)Click here for additional data file.S3 Fignmo does not interact with the Pk isoform in the wing. (A) Overview of a wild-type adult wing, rectangle outlining the region shown in (B-G). There are no wing PCP defects in any of the following genotypes: wt (B), pksple1 (C), nmoP (D), pksple1; nmoP (E), act-EGFP-Pk (o/e Pk)(F) or act-EGFP-Pk (o/e Pk)/nmoDB (G).(JPG)Click here for additional data file.S4 Fignmo does not interact with the Pk-Sple isoform. (A-B) nmo+/- loss-of-function (LOF) does not affect Pk-Sple overexpression (o/e). act-EGFP-Sple eyes look wild-type (A), and are not affected by nmo+/- LOF heterozygosity. (B). (C-E) act-EGFP-Sple wings show wing hair polarity reversals (overview for box position in (C), magnified view in (D) and this phenotype is not modified by nmo LOF (E). (F-I) sevenless(sev)Gal4-driven Sple overexpression phenotype in the eye (F) is not affected by reduction of nmo function, via RNAi (G) or nmo mutation (H); quantified in panel I .(JPG)Click here for additional data file.S5 FigA) Full size radiograph and coomassie-stained gel as shown in B-C) Increasing the amount of Pk in pksple1 mutants enhances chirality defects, particularly the proportion of symmetrical clusters (C), quantified in (B **** P<0.0001). This effect is similar to nmo LOF (data from D) nubbin(nub)-Gal4-driven Pk and PkMut1&2 expression in the wing. Compared to control , overexpression of Pk and PkMut1&2 display similar PCP phenotypes. (E-G) Direct sevenless (sev)-driven Pk-Sple overexpression (sev-Sple) phenotype in the eye (E) is not affected by mutation of all eight Nmo phosphorylation sites  and does not interfere with the function of Pk-Sple, as SpleMut1&2 fully rescues the pk13 (null) phenotype (G).Nmo limits Pk but not Pk-Sple activity. ((JPG)Click here for additional data file.S6 FigA-C) Loss of nmo function increases Pk but not Pk-Sple protein level in eye discs. The relative amount of EGFP-Sple protein in a wt or nmoDB/+ background was calculated and normalized to γ-tubulin levels. A representative blot is shown in (A), the fold change in a nmoDB/+ background is shown for each independent experiment in (C). Quantification of fold change increase from each independent experiment for EGFP-Pk is shown in (B). (D-E) Mutation of Nmo phosphorylation sites or co-expression of dominant negative proteasome components (DNProsβ6) increases Pk protein level in eye discs. Quantification of the fold change in PkMut1&2 to PkWT (D) or EGFP-Pk in w1118 or GMR>DNProsbeta6 backgrounds (E) from each independent experiment. (F-G) Expression of dominant negative proteasome components does not cause chirality phenotypes in the eye: sevenless>DNProsβ2 (F) and GMR>DNProsβ6 (G.) (H-J) Full length blots from H and J. Three different exposures are shown in (I) and the red color in the Armadillo blot indicates saturation.Nmo phosphorylation promotes proteasomal degradation of Pk but not Pk-Sple. ((JPG)Click here for additional data file.S7 FigA-C) Knockdown of SCF components on its own does not cause chirality defects in the eye: sev>Cul1IR (A), sev>SkpAIR (B), sev>slmbIR (C). (D) Knockdown of slmb using RNAi (D) enhances the sev>Pk gain-of-function phenotype compared to sev>Pk, and wIR control samples . For quantification and related genotypes see E-F) Full-length blot (E) and quantification of the fold change of EGFP-Pk in w1118 or slmb+/- backgrounds from independent experiments (F) of The Cul1/SkpA/Slmb SCF complex promotes Pk degradation. Click here for additional data file.


Apc1 inactivation in the intestine: the hyperactivation of Wnt target gene expression, excess number of ISCs, and hyperplasia of the epithelium. In contrast, only Ebd, but not Ewg, mediates the Wnt-dependent regulation of ISC proliferation during homeostasis. Therefore, in the adult intestine, Ebd acts independently of Ewg in physiological Wnt signaling, but cooperates with Ewg to induce the hyperactivation of Wnt target gene expression following Apc1 loss. These findings have relevance for human tumorigenesis, as Jerky (JRK/JH8), the human Ebd homolog, promotes Wnt pathway hyperactivation and is overexpressed in colorectal, breast, and ovarian cancers. Together, our findings reveal distinct requirements for Ebd and Ewg in physiological Wnt pathway activation versus oncogenic Wnt pathway hyperactivation following Apc1 loss. Such differentially utilized transcription cofactors may offer new opportunities for the selective targeting of Wnt-driven cancers.Wnt/β-catenin signal transduction directs intestinal stem cell (ISC) proliferation during homeostasis. Hyperactivation of Wnt signaling initiates colorectal cancer, which most frequently results from truncation of the tumor suppressor Adenomatous polyposis coli (APC). The β-catenin-TCF transcription complex activates both the physiological expression of Wnt target genes in the normal intestinal epithelium and their aberrantly increased expression in colorectal tumors. Whether mechanistic differences in the Wnt transcription machinery drive these distinct levels of target gene activation in physiological versus pathological states remains uncertain, but is relevant for the design of new therapeutic strategies. Here, using a Drosophila model, we demonstrate that two evolutionarily conserved transcription cofactors, Earthbound (Ebd) and Erect wing (Ewg), are essential for all major consequences of  APC. However, as Wnt signaling is also required for normal intestinal homeostasis, the selective therapeutic targeting of oncogenic Wnt signaling remains a major challenge. Through a forward genetic screen, we previously identified two suppressors of Drosophila Apc1, Earthbound (Ebd) and Erect wing (Ewg), as transcription cofactors of the Wnt pathway. Here, we analyze the roles of these two factors in the Wnt-dependent control of intestinal stem cell proliferation. We find that both Ebd and Ewg are essential for the hyperactivation of Wnt signaling and the consequent epithelial hyperplasia resulting from Apc1 inactivation. Moreover, Ebd, but not Ewg, is also required for the Wnt-dependent maintenance of normal intestinal homeostasis. Together, our findings reveal differential requirements for two highly conserved transcriptional cofactors in Wnt pathway activation versus hyperactivation. The identification of such factors may provide potential selectivity for the targeting of Wnt-driven cancers.The identification of effective therapy for colorectal cancer, which is a leading cause of cancer-related death, is imperative. Wnt pathway components have promise as therapeutic targets, since more than 90% of colon cancers are triggered by mutations that overactivate this pathway, particularly in the tumor suppressor  The evolutionarily conserved Wnt/β-catenin signal transduction pathway directs fundamental cellular processes across metazoans, whereas deregulation of this pathway is associated with numerous human congenital disorders and cancers ,2. In thAPC, initiates the development of the vast majority of colorectal cancers , mouse anti-Discs large , mouse anti-Prospero , mouse anti-Delta , rabbit anti-dsRed , mouse anti-β-gal , rabbit anti-β-gal , rabbit anti-phospho-histone H3 (Ser10) , rabbit anti-phospho-histone H3  and Cat. no. 9713 (Ser28), 1:100), guinea pig anti-Ebd1 Adult fly intestines were dissected in PBS and fixed in 4% paraformaldehyde in PBS for 45 minutes at room temperature. For Delta staining, intestines were fixed in 8% paraformaldehyde, 200mM Na cacodylate, 100mM sucrose, 40 mM KOAc, 10 mM NaOAc, and 10mM EGTA for 20 minutes at room temperature . TissuesMitotic clones were generated using the MARCM system . Developesgts  driver. For knock down during development, crosses were set up at 18°C and shifted to 29°C 6 days later . Progeny of desired genotypes were dissected 2–3 days after eclosion. For knock down during adulthood, crosses were maintained at 18°C until eclosion, and progeny of desired genotypes were then shifted to 29°C for 14 days before analysis.To induce temporal knockdown in ISCs, control or specific RNAi lines were crossed to the Myo1A-Gal4 with the temperature-sensitive Gal4 repressor, Gal80ts. Crosses were maintained at 22°C and on the day of eclosion, progeny of desired genotypes were shifted to the restrictive temperature (29°C) for 7 days.To induce temporal knockdown in ECs, RNAi experiments were performed using esgtsF/O: esg-Gal4 tub-Gal80tsUAS-GFP; UAS-flp Act>CD2>Gal4), crosses were maintained at 18°C and 3-5-day-old progeny of the desired genotypes were shifted to 29°C. The marked esg+ cell lineages were analyzed 14 days later.To induce temporal knockdown using the “escargot flip out” system -lacZ positive cells. For quantification of pH3-positive cells, the total number of pH3-positive cells in the posterior midgut of the indicated genotypes was counted. For quantification of pH3-positive cells near MARCM clones, the number of pH3-positive cells in a field of 4000 µm2 around the clone was counted.For quantification of ISCs, flies were stained with anti-Delta (Dl) and anti-Prospero antibodies. Images of the midgut R5a region  were obtFor quantification of intestinal structure, 40x Z-stack confocal images of posterior midguts of desired genotypes were acquired. The maximum number of epithelial layers and maximum epithelial height were measured using NIS-elements software (Nikon).esgts flip out” guts was performed by measuring overall GFP intensity within two areas per posterior midgut and normalizing that value by the total number of cells in the field.Quantification of GFP intensity in “fz3-RFP intensity was performed with ImageJ (NIH). For each clone examined , intensities of three separate areas within the clone and areas of identical size outside the clone were measured. The average intensities of the three areas were compared to the average intensities of their control counterparts.Quantification of stat-GFP intensity was performed using Imaris software (Bitplane). Stat-GFP-positive cells within a field (40μm × 40μm) surrounding an ebd1 mutant clone, or in an equal field at least 50μm away from the ebd1 mutant clone, were identified and their intensity was measured.Quantification of All statistical tests were performed using Prism .Apc1Q8 7-day-old females were dissected in nuclease-free PBS and processed for transcriptomic analysis. Total RNA from 30 adult midguts per sample was extracted using Trizol following manufacturer’s instructions. Triplicate samples of each of the genotypes were prepared. RNA was sent to the Microarray facility at the University of Manchester where it was used to hybridize Drosophila Affymetrix 2.0 chips. The CEL files were subject to RMA normalization and log2 transformation followed by differential gene expression analysis by the Beatson Institute Bioinformatics department. Microarray data were deposited in the GEO database (GSE99071). GO term analysis was performed via “PANTHER GO-slim”  upon loss of Apc1 is suppressed by further inactivation of ewg (C and F). Nuclei are marked with DAPI. Anterior to the left.Expansion of Scale bars: 100 μm.control:nkd(UpE2)-lacZ/+; Apc1Q8/+Genotypes: Apc1:nkd(UpE2)-lacZ/+; Apc1Q8ewg Apc1:ewgP1; nkd(UpE2)-lacZ/+; Apc1Q8.(TIF)Click here for additional data file.S9 FigApc1 mutant guts when compared to the wild type controls and the top enriched GO terms  are visualized.A GO term analysis was performed on biological processes deregulated in (TIF)Click here for additional data file.S10 FigApc1. Misexpression of both sets of genes is rescued in ebd1 Apc1 and ewg Apc1 double mutants. This is an independent biological replicate of the data shown in Quantitative RT-PCR of genes up- (A) or down-regulated (B) by loss of control:Apc1Q8/+Genotypes: Apc1:Apc1Q8ebd Apc1:ebd1240Apc1Q8ewg Apc1:ewgP1; Apc1Q8.(TIF)Click here for additional data file.S11 Fig(A) Sequence of TCF core (HMG: High Mobility Group) consensus DNA binding sites, TCF Helper site, as well as Ewg consensus DNA binding sites.fz3-RFP (D) has an Ewg consensus binding site.(B-D) Each of the Wingless reporters contains at least one TCF HMG consensus binding site and TCF Helper site, but only (TIF)Click here for additional data file.S12 Figfz3-RFP reporter is 100% conserved within the melanogaster group (except that it is absent in the Drosophila yakuba species), and with only one substitution in the evolutionarily distant Drosophila ananassae and Drosophila willistoni species.Using an Evoprinter analysis to identify site conservation, we found that the Ewg consensus binding site within the (TIF)Click here for additional data file.S13 Figof fz3-RFP (A-B”) or nkd-lacZ (C-D”), reporters for Wingless signaling (in magenta), is retained in ewg null mutant clones, suggesting that Ewg is not required for Wingless-dependent expression of fz3 or nkd. Clones are marked with GFP (green). Magnified views of the boxed regions in (A or C) are shown in (B-B” and D-D”), respectively.Expression Scale bars: (A and C) 50 μm and (B-B” and D-D”) 10 μm.hs-flp tub-Gal80 FRT19A/ewg2FRT19A; fz3-RFP/+; tub-Gal4 UAS-mCD8::GFP/+Genotypes: (A-B”) hs-flp tub-Gal80 FRT19A/ewg2FRT19A; tub-Gal4 UAS-mCD8::GFP/nkd-lacZ.(C-D”) (TIF)Click here for additional data file.S14 Figesg>GFP  and observed as small cells with high levels of membrane-associated Arm and the absence of Prospero (Pros) staining , are either present as single cells or doublets distributed evenly in ebd1/+ control guts.(A-C) Progenitor cells, marked with esg>GFP positive progenitor cells (asterisk in F), with strong Arm staining, are observed in ebd1240 mutants.(D-F) Chains of small ebd1 and heterozygous mutant for ebd2 (ebd15 ebd2136/ ebd1240) contain multi-cell clusters of esg>GFP positive cells (arrowhead in I), as well as chains of progenitor cells (asterisk).(G-I) Flies homozygous mutant for esg>GFP positive cells is increased in flies homozygous mutant for both ebd1 and ebd2 (ebd15ebd2136/ ebd1240ebd2136).(J-L) The prominence of clusters of ebd1 and ebd1 ebd2/ebd1 mutants have higher numbers of esg>GFP positive progenitor cells compared to controls. **** P<0.0001 (Mann-Whitney test).(M) esg>GFP positive cells.(N) Proportion of posterior midguts containing chains and clusters of Scale bars: (A-L) 10 μm.ebd1/+:esg-Gal4 UAS-GFP; ebd1240/+Genotypes: ebd1:esg-Gal4 UAS-GFP; ebd1240ebd1ebd2/ebd1:esg-Gal4 UAS-GFP; ebd15 ebd2136/ ebd1240ebd1ebd2:esg-Gal4 UAS-GFP; ebd15ebd2136/ ebd1240ebd2136.(TIF)Click here for additional data file.S15 Figebd1 clones are larger than control clones, indicating that Ebd1 is required for homeostasis of intestinal tissue during adulthood. Transient clones were excluded by restricting the analysis to clones of two or more cells.Adult control:y w hs-flp/+; tub-Gal4 UAS-GFP+; FRT2A tub-Gal80/FRT2AGenotypes: ebd1:y w hs-flp/+; tub-Gal4 UAS-GFP/+; FRT2A tub-Gal80/ebd1240FRT2A.(TIF)Click here for additional data file.S16 Fig(A-A”) Fixed intestines were immunostained with the Ebd1 antibody and signals are detected in all intestinal epithelial cell types, including ECs (yellow arrow), progenitors (white arrow) and EEs (orange arrow).ebd1 null mutant clones (marked with GFP).(B-C”‘) Ebd1 staining is specifically diminished inside enterocytes in Scale bars: (A-A”) 10 μm, (B and B’) 50 μm and (C-C”‘) 10 μm.Genotypes: (A-A”) Canton Sebd1:y w hs-flp/+; tub-Gal4 UAS-GFP/+; FRT2A tub-Gal80/ebd1240FRT2A.(B-C”’) (TIF)Click here for additional data file.S17 FigUAS-GFP-lacZ driven by ebd1-Gal4  exhibits expression in ECs (yellow arrow), progenitors (white arrow) and EEs (orange arrow).(A-A”) UAS-GFP-lacZ alone.(B-B”) No signal is detected with Scale bars: 10 μm.ebd1(104336)>U-GFP-lacZGenotypes: (A-A”) U-GFP-lacZ/+.(B-B”) (TIF)Click here for additional data file.S18 Figebd1 expression leads to increased numbers of progenitor cells (compare D-F to A-C). Progenitor cells are marked with esg-lacZ (green) or are identified as small cells with strong Arm staining and lack of Prospero (Pros) staining (magenta). Flies were analyzed seven days post-eclosion.(A-F) RNAi-mediated disruption of (G-I) The number of progenitor cells in flies analyzed one day post-eclosion is not increased, indicating that Ebd1 regulates stem cell proliferation non-autonomously in adults, but not during development.Scale bars: 10 μm.control:Myo1A-Gal4 UAS-GFP tub-Gal80ts/esg-lacZGenotypes: Myo>ebd1i3: Myo1A-Gal4 UAS-GFP tub-Gal80ts/esg-lacZ; UAS-ebd1 RNAi#3.(TIF)Click here for additional data file.S19 FigGBE-Su(H)-lacZ, magenta) are readily detected in midguts of newly eclosed Apc1 mutants (C). In contrast, this defect is not observed in age-matched ebd1 mutants (B) and is suppressed in ebd1 Apc1 double mutants (D). Nuclei are labeled by DAPI (turquoise).(A-D) Compared with controls (A), excess EBs (marked by GBE-Su(H)-lacZ positive cells in newly eclosed control, ebd1, Apc1 and ebd1 Apc1 mutants. **** P<0.0001, *** P<0.001, ns: not significant (t-test).(E) Quantification of Scale bars: (A-D) 10 μm.control:GBE-Su(H)-lacZ/+Genotypes: ebd1:GBE-Su(H)-lacZ/+; ebd1240Apc1:GBE-Su(H)-lacZ/+; Apc1Q8ebd1 Apc1:GBE-Su(H)-lacZ; ebd1240Apc1Q8.(TIF)Click here for additional data file.


Drosophila Zasp PDZ domain protein, and is required for myofibril assembly and maintenance. We show by in vitro biochemistry that the PDZ domain plus a C-terminal extension is the only area of Zasp52 involved in the interaction with α-actinin. In addition, site-directed mutagenesis of 5 amino acid residues in the N-terminal part of the PDZ domain, within the PWGFRL motif, abolish binding to α-actinin, demonstrating the importance of this motif for α-actinin binding. Rescue assays of a novel Zasp52 allele demonstrate the crucial importance of the PDZ domain for Zasp52 function. Flight assays also show that a Zasp52 mutant suppresses the α-actinin mutant phenotype, indicating that both proteins are core structural Z-disc proteins required for optimal Z-disc function.Z-discs are organizing centers that establish and maintain myofibril structure and function. Important Z-disc proteins are α-actinin, which cross-links actin thin filaments at the Z-disc and Zasp PDZ domain proteins, which directly interact with α-actinin. Here we investigate the biochemical and genetic nature of this interaction in more detail. Zasp52 is the major  α-actinin/+ heterozygous animals show irregularities in wing beat frequency, which can be suppressed by removing one copy of Zasp52. This suggests that both proteins are required at fixed levels at the Z-disc to support optimal functionality.Although Zasp PDZ domain proteins are known to bind α-actinin and play a role in muscle assembly and maintenance, the details and importance of this interaction have not been assessed. Here we demonstrate that a conserved motif in the N-terminal part of the Zasp52 PDZ domain is responsible for α-actinin binding and that a C-terminal extension of the PDZ domain is required for optimal α-actinin binding. We show using transgenic animals that in the absence of the PDZ domain no aspect of myofibril assembly can be rescued. Intriguingly,  Drosophila, Zasp52 has a PDZ, ZM (Zasp-like motif) and four LIM domains; while Zasp66 and Zasp67 only feature the N-terminal PDZ domain and a weakly conserved ZM domain. Zasp52 colocalizes with α-actinin at Z-discs and plays a role in myofibril assembly and maintenance [Drosophila, a ZASP/Cypher and ENH double knock-out in mice demonstrates partial redundancy in myofibril assembly [C. elegans ortholog ALP-1 displays defects in actin filament organization, but motility defects are much milder than in vertebrates or Drosophila [Like most animals, invertebrates have three main types of muscles, body wall, heart, and visceral muscle, but in contrast to vertebrates, all of them are striated. A particularly highly organized muscle is the indirect flight muscle (IFM) in insects, a stretch-activated fibrillar muscle . Slidingntenance , 17, 18.ntenance , 20. Furntenance . Mutationtenance , 7, 21. assembly . The sinosophila –25. Mutaosophila , 27.α-actinin heterozygous flight defect is suppressed by removal of one copy of Zasp52. Our data indicate that Zasp PDZ domain family proteins are core scaffold proteins of muscles.In this study, we explore the relationship of Zasp52 and α-actinin. We show that even though different Zasp52 deletion transgenes co-immunoprecipitate α-actinin and localize to Z-discs, only an extended PDZ domain mediates direct interaction of Zasp52 with α-actinin. Through site-directed mutagenesis we also demonstrate the importance of the PWGFRL motif in α-actinin binding. A rescue assay confirms the importance of the PDZ domain of Zasp52 for myofibril assembly. Finally, we show genetically that the Zasp52 α-actinin interaction is required for IFM function, because the We have previously shown that Zasp52 binds directly to α-actinin via an N-terminal construct containing the PDZ, ZM, and LIM1 domain . Both PDWe noticed that Mef2-Gal4-mediated expression of transgenes results in various muscle defects such as wavy myofibrils . To asseOur data show that multiple domains of Zasp52 are able to mediate Z-disc localization irrespective of expression level, and therefore raise the question if different domains of Zasp52 can bind directly to α-actinin.We have previously shown a direct interaction of Zasp52-PK containing a PDZ, ZM, and LIM1 domain with α-actinin . To deteWe previously identified 16 highly conserved amino acids in the N-terminal half of the Zasp52 PDZ domain as the defining feature of Zasp PDZ domain proteins , and proZasp52 alleles. Recently, a large number of MiMIC insertions based on the Minos transposon were created in Drosophila inserting splice acceptor sites followed by stop codons at various positions in the genome [Zasp52 locus: MI02988 after exon 2, MI07547 after exon 8, and MI00979 after exon 15 BSC427 than homozygously BSC427 adults myofibril assembly in IFM is severely disrupted. In many myofibrils, Z-discs and M-lines are no longer distinguishable (Zasp52MI00979/Df(2R)BSC427 looks similar to the previously described RNAi lines iZasp52ex20 and iZasp52ex16 (Zasp52MI07547/Df(2R)BSC427 shows no obvious defects BSC427 IFM defects by expressing our transgenes with UH3-Gal4. We started with the most common isoform, Zasp52-PR, which can indeed fully rescue all IFM defects and localizes normally to Z-discs (Zasp52MI00979/Df(2R)BSC427 mutants, which resulted in no rescue BSC427, M{3xP3-RFP.attP}ZH-86Fb, y w, and Actn8 from the Bloomington Drosophila Stock Center; UH3-Gal4 has been described previously [ΔPDZ, UAS-FLAG-Zasp52-PRΔZM, UAS-FSH-Zasp52-PK and UAS-FSH-Zasp52-LIM234 (this study). Mef2-Gal4; UAS constructs and UH3-Gal4; UAS stocks were generated by standard genetic crosses.The following fly stocks were used: Mef2-Gal4, eviously ; UAS-FLAZasp52 and α-actinin, Actn8/FM7 was crossed to y w, and Zasp52MI02988/CTG and Zasp52MI00979/CTG were crossed to Actn8/FM7 or y w and incubated at 20°C.To test for genetic interaction between y w (for RK and LIM234) and M{3xP3-RFP.attP}ZH-86Fb  embryos.Transgenic flies were generated by injecting sequence-verified plasmids into ΔPDZ (deleting amino acids 11–84 of Zasp52-PR) and pUAST-attb-Zasp52-RRΔZM (deleting amino acids 144–169 of Zasp52-PR). GST-Zasp52-RK and GST-Zasp52-LIM234 were cloned from EST LP01550 (for RK) and RH03424 (for LIM234) into pGEX-5X-1 . Zasp52-PKP18DW19F, Zasp52-PPR22T Zasp52-PPG25W, Zasp52-PPG26D, Zasp52-PDZ, Zasp52-ZM, Zasp52-LIM1, Zasp52-STOP155, Zasp52-STOP143, and Zasp52-STOP112 were synthesized or generated by site-directed mutagenesis by GenScript and cloned into pGEX-5X-1 to generate GST constructs.Flag-Zasp52-RR (FlyBase ID: FBtr0329912), FSH-Zasp52-RK (FlyBase ID: FBtr0302163) and FSH-Zasp52-LIM234 were synthesized by GenScript and cloned into pUAST-attb  and pUASt . PCR-me2 fly thoraces were homogenized in 2x SDS sample buffer. Protein samples were resolved by 8% SDS-PAGE and then detected by immunoblotting. Antibodies employed were anti-Zasp52 , anti-F2, 1 mM DTT, 5% glycerol, 0.5% Triton X-100 and complete EDTA-free protease inhibitor; Roche). Protein extracts were then incubated with prewashed anti-FLAG M2 affinity resin (Sigma-Aldrich) for 3 hours at 4°C. After incubation, the beads were washed three times with wash buffer . Bound proteins were eluted by boiling in 2x SDS sample buffer. Eluates were analyzed by SDS-PAGE and by immunoblotting. Antibodies were used at the following dilution: rat anti-α-actinin MAC276 antibody at 1:2000 (Babraham Bioscience Technologies); mouse anti-FLAG antibody at 1:5000 (Sigma-Aldrich). The immunoreaction was visualized by ECL (Millipore).70 adult fly thoraces were cut in half and were homogenized in lysis buffer  for 3 hours at 4°C. The beads retaining the GST-tagged proteins were washed three times with above buffer with 250 mM NaCl and 0.5% Triton X-100. Subsequently, rabbit skeletal muscle α-actinin (Cytoskeleton) was added and incubated for another 3 hours at 4°C. Final washes were in above buffers with 150 mM NaCl and 0.2% Triton X-100. Beads were resuspended in SDS sample buffer and analyzed by SDS-PAGE and immunoblotting.For GST pulldown assays, 2, 2 mM EGTA, 5 mM DTT, 0.5% Triton X-100, 50% glycerol) overnight at -20°C. IFMs were dissected, washed and then fixed with 4% paraformaldehyde in relaxing solution  with protease inhibitors. Incubations of primary antibodies and Alexa 594-phalloidin (ThermoFisher Scientific) were carried out overnight at 4°C, followed by secondary antibody incubation for 3 hours at room temperature. Primary antibodies used were rat anti-α-actinin MAC276 , rat anti-Kettin KIg16 MAC155 , and anti-FLAG . Fluorescently labeled secondary antibodies of the Alexa series (ThermoFisher Scientific) were used at a 1:400 dilution. Samples were mounted in ProLong Gold antifade solution (ThermoFisher Scientific).Half thoraces were glycerinated .The wing beat frequency of a fly was determined using an optical tachometer as previously described . In thisS1 FigConfocal microscopy of IFM stained with anti-Flag antibody visualizing the transgene in green, anti-α-actinin antibody to label Z-discs in purple, as well as phalloidin to visualize actin thin filaments in red. Weak UH3-Gal4-mediated expression of UAS-FSH-Zasp52-LIM234 still results in Z-disc localization and reduces overexpression defects. Scale bar, 10 μm.(TIF)Click here for additional data file.S2 FigA) Zasp52-PK, Zasp52-LIM234, Zasp52-PP, Zasp52-PDZ, Zasp52-ZM, and Zasp52-LIM1 run on a SDS-PAGE gel after purification. Asterisks indicate the fusion protein. (B) Zasp52-PP, Zasp52-STOP155, Zasp52-STOP143, Zasp52-STOP112, and Zasp52-PDZ run on a SDS-PAGE gel after purification. (C) Zasp52-PK and Zasp52-PKP18DW19F mutant variant run on a SDS-PAGE gel after purification. (D) Zasp52-PP and Zasp52-PPR22T, Zasp52-PPG25W, and Zasp52-PPG26D point mutant variants run on a SDS-PAGE gel after purification. Molecular weight marker is indicated in kD.GST and Zasp52 domain GST fusions run on a SDS-PAGE gel after purification. ((TIF)Click here for additional data file.S3 FigA) Confocal microscopy of Zasp52MI02988/Df(2R)BSC427 versus homozygous Zasp52MI02988 IFM stained with phalloidin to visualize actin thin filaments in green and anti-Kettin antibody to visualize Z-discs in red. Homozygous Zasp52MI02988 IFM show no obvious defects. (B) Quantification of IFM defects in C) Confocal microscopy of Zasp52MI00979/Df(2R)BSC427 IFM rescued with UH3-Gal4-expressed UAS-Zasp52-PR and stained with anti-Flag antibody to visualize the transgene in blue, phalloidin to visualize actin thin filaments in green and anti-Kettin antibody to visualize Z-discs in red. Zasp52-PR cannot rescue Zasp52MI00979.((TIF)Click here for additional data file.S4 FigActn8/+, Zasp52MI02988/+, and Actn8/+; Zasp52MI02988/+ exhibit no obvious defects.Confocal microscopy of IFM of heterozygotes stained with phalloidin to visualize actin thin filaments in red and anti-Kettin antibody to visualize Z-discs in green. (TIF)Click here for additional data file.


TH9 cells are less well-characterized than other T cell subsets, and factors that control their development and function have only recently been identified. It is now clear that TH9 cells play critical roles in immune-mediated diseases, including allergic airway, autoimmune and inflammatory bowel diseases, and cancer. Thus, the promotion or suppression of TH9 cell differentiation, transcriptional control and function may provide novel treatments for clinical inflammation, autoimmune diseases and tumors.Naïve CD4 Naïve disease –8 , and RBP-Jk  [H1-specific transcriptional factor, thereby promoting TH9 differentiation [H2 cells with low IL-4 expression. PU.1-deficient T cells produce less IL-9, and ectopic expression of PU.1 increases IL-9 production. Reduced PU.1 expression in human IL-9-secreting T cell cultures also reduced IL-9 production. Mechanistic studies have shown that PU.1 likely influences TH9 differentiation by interfering with GATA3 activation or by recruiting the histone acetyltransferase (HAT) proteins Gcn5 and PCAF to the Il9 locus [TGFβ is also required for T region) , 43. In ntiation , 39. PU.l9 locus , 38.H9 differentiation [H9 differentiation. SIRT1 deficiencies induced by either conditional deletion in mouse CD4+T cells or small interfering RNA (siRNA) in mouse or human T cells increased, while ectopic SIRT1 expression inhibited, IL-9 production. Additionally, glycolytic activation through the mTOR-hypoxia-inducible factor-1α (HIF1α) pathway was required for TH9 cell differentiation. SIRT1 may therefore function as a gatekeeper of the downstream mTOR-HIF1α axis  expressions and TH17 cell generation, thereby promoting TH9 cell differentiation [Transcriptional factors downstream of IL-2 are critical for Tntiation , and IL-ntiation , 42.H9 cell differentiation. Ligation of OX40 triggers sustained activation of the non-canonical NF-kB pathway in CD4+T cells during TH9 cell differentiation [Il9 promoter region and triggers Il9 transcription under TH9-inducing conditions. The non-canonical alternative NF-kB pathway probably also acts together with other factors to promote TH9 differentiation, suggesting that it restricts the capacity of NF-kB to interact with other transcription factors at the Il9 locus. NFAT1 (nuclear factor of activated T cells) is also required together with NF-kB for IL-9 production in CD4+T cells [Il9 promoter region.The transcription factors NF-kB and NFAT also modulate Tntiation , 36. The+T cells . NFAT1 aH9 cell differentiation, during which cells integrate the Treg-inducing TGF-β signal and the TH2-inducing IL-4 signal to develop a specific TH9 cell phenotype. TGF-β signaling induces Foxp3, which is a negative regulator of TH9 cell differentiation; ectopic Foxp3 expression reduces IL-9 production in TH9 cells [H2 and TH17, but not TH9, cell differentiation. GATA3 and STAT6 are also expressed in TH2 cells. Thus, each of these transcriptional factors likely plays integral roles in Il9 gene expression and production in CD4+T cells. Therefore, these transcription factors may not directly involved in the transcriptional regulation of the Il9 gene, but act rather as molecules downregulating negative factors during TH9 differentiation, such as Foxp3, or upregulating positive factors, such as IRF4, GATA3 and STAT6, during TH9 cell differentiation.Transcription factors control the secretion of specific cytokines that direct T cell differentiation and differentiating T cells integrate multiple, and sometimes conflicting, signals as they differentiate into particular subsets. This is especially true for TH9 cells , 46. TheH9 cells have protective or pathological roles in several clinical diseases, including allergic diseases, autoimmune diseases, ulcerative colitis, infection with various pathogens, and anti-tumor immunity  and airways strongly correlate with allergic airway diseases. TH9 cell-derived IL-9 can exacerbate disease conditions by increasing inflammatory cell infiltration and activation within the respiratory tract. Numbers of circulating CD4+T cells and IL-9 secretion are increased in serum from allergic lung disease patients compared to controls [H9 cell transfers increased the development of allergic airway diseases following OVA challenge, while anti-IL-9 treatment reduced disease severity [H9 cells are critical for the induction of allergic airway disease. Similarly, our work showed that severe pathogenic lung inflammation and inflammatory cell recruitment, including eosinophil infiltration in bronchoalveolar lavage fluid (BALF), were increased in SIRT1-deficient mice compared to WT mice. Additionally, more of the CD4+T cells isolated from lung DLNs of SIRT1-deficient mice were IL-9+. Importantly, administration of an anti-IL-9 antibody reversed pathological lung tissue damage and infiltration of inflammatory cells, including eosinophils and IL-9+CD4+T cells in SIRT1-deficient mice. Thus, SIRT1 is required for the suppression of TH9 cell differentiation and alleviates TH9-associated allergic airway inflammation [Allergic airway diseases are characterized by Tammation . In addio asthma –55. Airwo asthma . Such stcontrols , 58. Impseverity , 58–60. ammation .H2 and TH9 cells may play different roles in allergic airway diseases. We propose that, during the acute inflammation phase, activated TH2 cells secrete multiple cytokines . Subsequently, various cytokines promote mast cell recruitment , eosinophil maturation (IL-5 and GM-CSF), basophil infiltration (IL-4), and the initiation of mucus metaplasia (IL-13). During the chronic phase of inflammation, repeated exposure to allergenic antigens and damaged epithelium promotes the secretion of various cytokine growth factors, including TGFβ, from epithelial cells, eosinophils, macrophages, and even mast cells. In the presence of TGFβ and IL-4, TH2 cells differentiate into TH9 cells, which promote the progression of allergic airway diseases [Tdiseases , 61, 62.H17 and iTreg cells have been implicated in the pathological mechanisms of IBD. However, studies have also shown that TH9 cells are involved in IBD [H9 cells into lymphopenic recipient mice  aggravated colitis, but the underlying mechanisms remain unknown. TH9 cells contribute to the pathogenesis of IBD, particularly UC, by regulating intestinal barrier integrity and immunological function [+PU.1+T cells than control patients or patients with CD, indicating that TH9 cells are at least partly responsible for IBD pathogenesis. In acute and chronic IBD mouse models, numbers of IL-9-producing cells and IL-9R levels increased during colitis. In a novel IL-9 reporter mouse, CD4+T cells, but not other cell types such as innate lymphoid cells, are the principal source of IL-9 in colitis. Importantly, treatment with an IL-9 antibody or IL-9 knockout in mice considerably reduces colitis, as indicated by weight loss, generation of reactive oxygen species, and clinical scores, indicating that IL-9 promotes the progression of colitis. Thus, targeting IL-9 activity may improve diagnosis and treatment in UC patients.Inflammatory bowel diseases (IBD), which include Crohn's diseases (CD) and ulcerative colitis (UC), are characterized by sustained inflammation, mucosal barrier defects, and frequent intestinal infections. Td in IBD –65. For function , 66, 67.H9 cells have been implicated in conditions that involve central nervous system inflammation, such as systemic lupus erythematosus (SLE), experimental autoimmune encephalitis (EAE), and systemic sclerosis (SSc) [H9 cells into Rag1−/− mice result in more severe EAE than transfer of TH1 or regular TH9 cells, indicating that TH9 cells play a critical role in the development of EAE [−/− mice develop less severe EAE than their WT counterparts following either immunization with myelin proteolipid protein  peptide in the presence of Complete Freund's Adjuvant (CFA) or adoptive transfer of PLP (180-199) peptide-specific effector T cells from WT littermates. EAE-resistant IL-9−/− mice exhibited considerably fewer infiltrating immune cells in the CNS, as well as reduced IL-17 and IFNγ expression. IL-9 deficiency also reduced PLP peptide-specific IL-17 and IFNγ levels [H17 cells and IL-6-producing macrophages in the central nervous system, and with decreased numbers of mast cells in regional lymph nodes [−/− EAE mice had more TH1 cells and TH17 cells than WT mice. Additionally, the inhibitory activity of Treg cells was reduced in IL-9R−/− mice compared to WT mice [Tis (SSc) . Adoptivt of EAE , 68–70. t of EAE , 43, 71.γ levels , 73. In ph nodes . However+IL-9+ T cells was elevated in SLE patients. Moreover, IL-9 expression in T cells and serum IL-9 levels in 8 untreated active SLE patients decreased 1, 2, and 3 weeks after treatment with methylprednisolone [H9 plays an important role in the pathogenesis of SLE.To further investigate these possibilities, mRNA and serum IL-9 levels were assessed in the peripheral blood of SLE patients and healthy controls . The pernisolone . Thus, TH9 cells also results in anti-helminth activity. Experiments in IL-9−/− mice and IL-9-fluorescent reporter mice indicate that IL-9 is crucial in the initiation of host-protective responses in the early type 2 immune response against Nippostrongylusbrasiliensis [H9 cells, but not TH2 cells, caused rapid worm expulsion and marked basophilia and increased mast cell numbers in Rag2−/− mice [H9 cells and IL-9 are also involved in human Echinococcusgranulosus infection [H9 cells with the effector memory cell phenotype was found in tuberculous pleural effusion [H9 cells and IL-9 a play critical and non-redundant roles in host-protective immunity against pathogenic infection.IL-9 production in Tiliensis . Adoptiv−/− mice . TH9 celnfection . Compareeffusion . Taken tH9 cells are present in metastatic pleural effusion [H9 also has anti-tumor properties in mice [−/− mice, and treatment with rIL-9 inhibited this growth. Interestingly, adoptive transfer of anti-tumor TH9 cells inhibited tumor growth, and administration of an anti-IL-9 mAb reversed this effect. Moreover, treatment with exogenous IL-9 suppressed the growth of B16F10 melanoma and LLC-1, but not EL-4, tumors. Mechanistic investigation revealed that IL-9R expression is negligible in B16F10 and LLC-1 cells, and IL-9 had minimal effects on growth in these cells [H9 cells exerts anti-tumor effects by inhibiting the subcutaneous lung metastasis of B16F10 and TH9 cells. In addition, Treg, TH17, and TH2 cells also secrete low levels of IL-9. A considerable portion of TH9 cells acquire the TH1 phenotype and produce IFN-γ in vivo [H9 cell plasticity therefore plays a role in various pathological processes, including cancer.Teffusion  and humaeffusion . TH9 als in mice , 79  acts as an antigen and induces TH2 responses in Cca-bearing mice, thus inhibiting tumor growth primarily by converting intra-Cca Tregs cells into TH9 cells [H9 cells have stronger anti-tumor effects than TH17 or TH1 cells [Several studies have reported direct regulatory mechanisms of Tnd TRAIL . Most blory role . Two recory role , 87 alsoH9 cells . FinallyH1 cells , but theH9 cells are likely also critical for anti-tumor immunity. IL-9 produced by CD4+T cells is responsible for increased survival and function in myeloid DCs, which contribute to anti-tumor immunity [H9 cells also induce host anti-tumor CD8+CTL responses by upregulating the CCL20/CCR6-dependent recruitment of dendritic cells (DCs) to local tumor tissues [H9-mediated immune regulation is crucial for anti-tumor immunity. Moreover, tumor-specific IL-9-producing CD8+Tc9 cells also exert potent anti-tumor effects [H9-inducing conditions secreted different cytokines and were less cytolytic in vitro, but exhibited much stronger anti-tumor effects in OT-I/B16-OVA and Pmel-1/B16 melanoma mouse models. Adoptive transfer of Tc9 cells results in differentiation into IFNγ- and granzyme-B (GrzB)-producing cytolytic Tc1-like effector cells. This suggests that the anti-tumor activities of TH9 cells are partially due to their effects on Tc9 cells.Indirect regulatory mechanisms of Timmunity . TH9 cel tissues . These f effects . Tc9 celH9 cells also exerts critical anti-tumor effects. IL-1β promotes the secretion of cytokines IL-9, IL-10, and IL-21 from TH9 cells through STAT1-IRF1-dependent mechanisms [H9 differentiation increased IL-21, but not IL-9, secretion, and increased IL-21-dependent anti-tumor effects [+CTL activity, all of which promote anti-tumor immunity [In addition to IL-9, IL-21 secreted by Tchanisms , 90, 91. effects –94. IL-2immunity , 94.+T cells differentiate into one of several functional effector cell classes. In addition to the classical TH1 and TH2 lineages, TH17 cells have been extensively characterized. Recently, new subsets of IL-9-producing CD4+T cells have been induced in vitro by IL-4 and TGFβ and identified in vivo. IL-9 is a pleiotropic cytokine produced by TH2 cells, mast cells, and eosinophils. Due to the critical role of IL-9-producing CD4+T cells in immune-mediated diseases, TH9 cells have been extensively investigated in mouse and human studies during the past 20 years. These studies have demonstrated that TH9 cells contribute to both immune responses and immunopathological diseases. Moreover, they suggest that promoting or suppressing TH9 cell differentiation, transcriptional control, and function may provide novel treatments for TH9-associated inflammation, autoimmune diseases, and tumors. Specifically, administration of IL-9 antibodies effectively eliminated TH9 cells and ameliorated or aggravatedTH9-associated diseases.Following antigen stimulation, naïve CD4


To prospectively assess the diagnostic performance of high resolution image-navigated 3-dimensional late gadolinium enhancement (iNAV-3D LGE) magnetic resonance imaging (CMR) for the detection of myocardial necrosis in a routine clinical setting.iNAV-3D LGE is a novel CMR technique which allows for direct respiratory motion correction of the heart. However, its performance in real-life clinical scenarios has not yet been established.23 consecutive patients referred for CMR examination including scar imaging were prospectively enrolled. Gadolinium enhanced (0.20 mmol/kg Gd-DTPA) navigated high resolution (2 mm3 isotropic) 3D T1-weighted gradient-echo inversion recovery sequence using image-based navigation in comparison with a conventional two-dimensional (2D LGE) sequence were performed in random order by using a 1.5-T clinical MR imaging system. Images were assessed qualitatively with regard to the detection of global and segmental LGE and transmurality. Additional subjective image quality assessement including image quality, mean LGE signal intensity and LGE-myocardial/ blood pool sharpness on a 4-point scale was performed.Interpretable images were obtained in all 2D-LGE and in 22/ 23 iNAV-3D LGE exams, resulting in a total of 22 complete sequence datasets and 352 segments. LGE was detected in 5 patients with ischemic pattern, in 8 with non-ischaemic pattern, while it was absent in 9 cases. Figure  There weIn this study, imaging performance and quality scores of iNAV-3D LGE images were comparable to those of 2D LGE in a prospective clinical setting. iNAV-3D LGE may potentially offer a reliable alternative for high quality scar imaging.


The JAVELIN Solid Tumor clinical trials group recently reported the results of a multi-center open-label phase 1b study NCT01772004) evaluating avelumab in patients with progressive or platinum-resistant metastatic or recurrent non-small cell lung cancer (NSCLC) [004 evaluin vitro [in vitro.Among the five currently FDA-approved anti-PD-1/PD-L1 agents, avelumab is unique due to its unmodified fragment crystallizable (Fc) domain which may potentiate antibody-dependent cell-mediated cytotoxicity (ADCC) via interaction with CD16 (FcɣRIIIa) on natural killer (NK) cells . The impin vitro . Howeverin vitro . This strd line of therapy) and with other driver mutations . Multiple studies across multiple treatment modalities including immunotherapy have shown lower response rates with more advanced disease / later lines of therapy.With a median follow up of 8.8 months in the NSCLC dose-expansion cohort, the JAVELIN investigators enrolled 184 patients with stage IIIB or IV NSCLC with disease progression after prior platinum-based doublet therapy for metastatic disease across 58 academic centers . This stn = 22), and 1-year progression-free and overall survival rates were 18% and 36%, respectively. Ongoing follow-up has confirmed an additional 4 responses, raising the ORR to 14.1% (n = 26). Accounting for these factors, ORR was similar (12%-14.1% vs. 14%-20%) in this study compared with randomized prospective studies evaluating other PD-1 or PD-L1 inhibitors in the second-line advanced NSCLC setting [vs. 11.4%) [Objective response rate (ORR) was 12% . These findings are exploratory and hypothesis-generating at present but add to a growing compendium of literature attempting to clarify the appropriate use of PD-L1 expression as a predictive biomarker for patient selection. A preliminary exposure-response analysis of this cohort of NSCLC patients explored the relationship between PD-L1 expression and avelumab exposure (trough concentration after first dose) [To explore potential biomarkers of response to avelumab, clinical responses were stratified by PD-L1 expression. A proprietary assay for PD-L1 expression (anti-PD-L1 rabbit mAb clone 73-10) using a cutoff value of ≥1% on tumor cells showed a progression-free survival benefit for PD-L1 positive patients relative to PD-L1 negative counterparts (12.0 In consideration of this heterogenous, heavily pre-treated, PD-L1 non-selected advanced NSCLC patient population, avelumab demonstrated a toxicity profile and anti-tumor activity similar to other anti-PD-1 and PD-L1 mAbs .


DrNOD2) and RIG-I (DrRIG-I) in innate immune responses. Luciferase assays were conducted to determine the activation of NF-κB and interferon signaling. Morpholino-mediated knockdown and mRNA-mediated rescue were performed to further confirm the regulatory roles between DrNOD2 and DrRIG-I. Results showed that DrNOD2 and DrRIG-I shared conserved structural hallmarks with their mammalian counterparts, and activated DrRIG-I signaling can induce DrNOD2 production. Surprisingly, DrNOD2-initiated signaling can also induce DrRIG-I expression, indicating that a mutual regulatory mechanism may exist between them. Studies conducted using HEK293T cells and zebrafish embryos showed that DrRIG-I could negatively regulate DrNOD2-activated NF-κB signaling, and DrNOD2 could inhibit DrRIG-I-induced IFN signaling. Moreover, knocking down DrRIG-I expression by morpholino could enhance DrNOD2-initiated NF-κB activation, and vice versa, which could be rescued by their corresponding mRNAs. Results revealed a mutual feedback regulatory mechanism underlying NOD2 and RIG-I signaling pathways in teleosts. This mechanism reflects the coordination between cytosolic antibacterial and antiviral PRRs in the complex network of innate immunity.Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and retinoic acid-inducible gene I (RIG-I) are two important cytosolic pattern recognition receptors (PRRs) in the recognition of pathogen-associated molecular patterns (PAMPs), initiating innate antibacterial and antiviral signaling pathways. However, the relationship between these PRRs, especially in teleost fish models, is rarely reported. In this article, we describe the mutual regulation of zebrafish NOD2 ( Host defense against pathogen infection is initiated by the recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs). Well-recognized PRRs include toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors (CLRs), and the family of DNA sensors ,3,4,5,6.NOD2 is a member of the NLR family and a general cytoplasmic sensor for most bacteria ,10,11. SRIG-I is an essential cytoplasmic receptor for recognizing various RNA viruses . This PRAlthough NOD2 and RIG-I are important in innate antibacterial/viral processes in the hosts, studies on the correlation between the NOD2 and RIG-I signaling pathways are limited. Previous studies showed that NOD2 could be significantly induced by IFN-β produced through the activation of RIG-I signaling pathway upon viral stimulation ,20. HoweDrNOD2 and DrRIG-I were highly conserved and similar to their mammalian counterparts [DrRIG-I and DrNOD2 could induce each other, and a close negative mutual regulation between these molecules was observed. Our results provide insights into the coordination of cytosolic antibacterial and antiviral PRRs. This information can improve the understanding on the functional modulation among innate signaling pathways in the complex innate immunity network from an evolutionary perspective.Teleosts were used in several studies to investigate the structures, signaling pathways, and antipathogen functions of NOD2 and RIG-I ,33,34,35terparts ,37,38. TDrRIG-I and DrNOD2 with their mammalian counterparts to investigate the mutual regulation of NOD2 and RIG-I signaling pathways using zebrafish. We used SMART and Pfam databases to refer the protein domains and found that DrRIG-I and DrNOD2 possessed all the functional domains shared by their Homo sapiens counterparts  was used, instead of wild-type DrRIG-I, because the latter remains in an auto-inhibited state without ligand stimulation [DrNOD2, a mutant without the LRR domain (DrNOD2 (ΔLRR)) was also used to activate the NOD2 signaling. Our results showed that DrNOD2 was induced at 6 and 12 h post injection (hpi) of DrRIG-I (CARD) and reached its highest level at 6 h. Surprisingly, we also observed the induction of DrRIG-I when DrNOD2 signaling was activated at 24 hpi (DrRIG-I could be observed as early as 12 hpi in the DrNOD2 (ΔLRR)-injected group  alone led to a robust NF-κB activation in the HEK293T cells (DrRIG-I or DrRIG-I-CARD was co-injected (DrRIG-I (ΔCARD) was also used as negative control, and this mutant could not inhibit the DrNOD2- and DrNOD2 (ΔLRR)-activated NF-κB signaling -mediated NF-κB activation and TNF-α expression. Good rescue was achieved with the simultaneous injection of DrRIG-I mRNA .To prove our hypothesis, we determined the function of 3T cells A and zeb3T cells B. Activainjected A,B. The ignaling B. The Drobserved C. Co-admG-I mRNA D,E. MDP- embryos F. These DrNOD2 in DrRIG-I-induced IFN signaling were also examined. DrRIG-I (CARD) overexpression induced the ISG15 expression in the HEK293T cells (DrNOD2 or DrNOD2 (ΔLRR) was co-injected (DrNOD2 (ΔCARD) could not inhibit the DrRIG-I (CARD)-initiated Mx activation (DrNOD2 by MO (DrNOD2 mRNA decreased the level of activation (DrNOD2 or DrNOD2 (ΔLRR) was co-injected in zebrafish embryos .The roles of 3T cells A and Mx 3T cells B. ISG15 injected A,B. The tivation B. HighlyD2 by MO C resultetivation D,E. Furt embryos F. These RIG-I and NOD2 are two of the most important cytosolic PRRs participating in the recognition of viral and bacterial invasion in mammals. In the present study, we used zebrafish as an attractive model organism to investigate the NOD2- and RIG-I-mediated immunology. The zebrafish was selected because of its conserved structural and functional characteristics compared with its mammalian counterparts.DrNOD2 expression and that NOD2 signaling activation could induce DrRIG-I expression. These observations suggest a feedback regulatory mechanism between the two signaling pathways. Further evaluation showed that the overexpression of DrRIG-I or DrRIG-I-CARD in the HEK293T cells and zebrafish embryos could significantly inhibit DrNOD2 and DrNOD2 (ΔLRR)-activated NF-κB signaling. Similarly, administration of DrNOD2 or DrNOD2 (ΔLRR) in cells and embryos could significantly suppress DrRIG-I (CARD)-induced IFN signaling. Further knockdown and rescue experiments also confirmed this proposition. Therefore, negative feedback and mutual regulation exist between DrNOD2 and DrRIG-I signaling pathways. Moreover, the results showed the physiological significance of this mutual feedback regulation. DrRIG-I could negatively regulate MDP-initiated NOD2 signaling, and DrNOD2 could negatively regulate LMW poly I:C initiated RIG-I signaling. NOD2 was recently confirmed to interact with RIG-I in a zebrafish cell line (ZF4) by co-immunoprecipitation analysis [DrNOD2 and DrRIG-I signaling pathways may have resulted from the sequestering of the CARDs of DrRIG-I and DrNOD2 away from their adaptors, MAVS and RIPK2, respectively.We used the zebrafish model to demonstrate that RIG-I signaling activation could induce analysis , and we analysis . These dDrNOD2 and DrRIG-I could be induced by their corresponding signaling pathway and negative regulate the signaling. We believe that this feedback might be of great biological significance in innate antibacterial or antiviral immunity. For example, when the host NOD2/RIG-I signaling is activated via bacterial/viral infection, a feedback regulation might contribute to maintain the homeostasis of immune responses. This characteristic prevents excessive immune reactions. Meanwhile, the induced NOD2 or RIG-I would alert the host, leading to a much faster and stronger immune response against a secondary bacterial or viral invasion. Thus, dysfunction in this feedback regulation may result in various pathological disorders. Secondary bacterial infection commonly develops after viral infections, and this process is accompanied by bursts of inflammatory responses [Therefore, esponses . In addiesponses . We infeDanio rerio) weighing 0.5–1 g and measuring 1–2 cm in length were purchased from the National Zebrafish Resources of China. The fish were kept in tanks with recirculating water at 28 °C and fed daily with commercial pellets at 0.7% of their body weight. The fish were acclimatized and evaluated for overall fish health at least two weeks before the experiments.Wild-type AB zebrafish  between the EcoRI and XhoI sites to construct the eukaryotic expression vector (pCMV-DrNOD2). The LRR motif-deleted mutant (ΔLRR) construct (pCMV-DrNOD2 (ΔLRR)) and the CARD motif-deleted mutant (ΔCARD) construct (pCMV-DrNOD2 (ΔCARD)) were cloned and inserted into the pCMV-HA between the sites of EcoRI and XhoI. The CARD motif-deleted mutant (ΔCARD) construct of DrRIG-I was cloned and inserted into the pcDNA6 between the sites of BamHI and EcoRI. The NF-κB luciferase vector was purchased from Clontech , while the pRL-TK vector was obtained from Promega . Human ISG15 and zebrafish Mx promoters (hISG15-pro-luc and DrMx-pro-luc), as well as the RIG-I expression plasmids pcDNA6-DrRIG-I and pcDNA6-DrRIG-I (CARD), were constructed in our previous study [The open-reading frame (ORF) of us study ,45. All DrRIG-I and splice junction MO of DrNOD2 were designed, synthesized by Gene Tools , and dissolved in water (2 mM). The MO sequences used were as follows: DrRIG-I MO, 5′-GATTCTCCTTCTCCAGCTCGTACAT-3′; and DrNOD2 MO, 5′-ACCTGCCAAAAATCCAACATGGTTA-3′. The 5′UTR sequence (complement to the MO sequence) of DrRIG was amplified with DrRIG-I F1 and R1 primers (DrRIG-I MO. Along with DrRIG-I MO or standard control MO (4 ng/embryo), the constructed vector was injected into one-cell stage embryos (100 pg/embryo). The embryos were collected at 24 hpi, and the GFP fluorescence was visualized through an Olympus MVX10 MacroView. The splice inhibition efficiency of DrNOD2 MO was examined through RT-PCR. The one-cell stage embryos of zebrafish were injected with DrNOD2 MO (4 or 6 ng/embryo) or standard control MO (6 ng/embryo). The embryos were collected at 24 hpi for RNA isolation and cDNA reverse transcription. A forward primer in exon 1 and reverse primer in exon 2 were used to detect the deletion of exon 2  according to the manual and then solubilized in DEPC water.The translation-blocking MOs of  primers , and thef exon 2 . Capped v/v) FBS , penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 °C in 5% CO2. Cells were seeded into six-well plates to allow growth until 70–90% confluence on the day of transfection. Transient transfection and cell lysate preparation were performed as previously described [HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% (escribed .HEK293T cells or zebrafish embryos were transfected (1 µg/mL) or injected (100 pg/embryo) with relative stimulant plasmids and NF-κB/ISG15/Mx luciferase reporter vectors. The pRL-TK renilla luciferase reporter plasmid was used as internal control. An empty control plasmid was then added to ensure same amounts of the total DNA. Subsequently, the cells and embryos were lysed at 24 hpi, and dual-luciferase reporter assay was performed as described previously . LuciferDrRIG-I (CARD) or pCMV-DrNOD2 or pCMV-DrNOD2 (ΔLRR) (100 pg/embryo) was injected into one-cell stage embryos. The mRNA levels of NOD2 or RIG-I relative to β-actin were examined at 6, 12, and 24 hpi using RT-PCR to determine whether RIG-I-initiated signaling induced NOD2 production and vice versa. The empty plasmid injection group was set as control. RT-PCR was conducted with the following parameters: (1) 40 cycles of amplification at 95 °C for 30 s and 60 °C for 20 s; (2) melting curve analysis at 95 °C for 5 s, 65 °C for 15 s, and 95 °C for 15 s; and (3) cooling at 40 °C for 30 s. Relative gene expression was calculated using the 2Ct−∆∆ method with NOD2/RIG-I, which were initially normalized against β-actin. The primers used are shown in pcDNA6-DrRIG-I (CARD) (Myc tag) and pCMV-DrNOD2 (HA tag) were transfected into HEK293T cells. At 48h post transfection, cells were lysed with cold lysis buffer ) containing protease inhibitor mixture  for 30 min at 4 °C. lysates were centrifuged for 15 min at 14,000 rpm and the supernatants were incubated with mouse anti-HA Ab  at 4 °C overnight and then incubated with protein A-agarose beads (Roche) for 4 h. The obtained samples were subjected to Western blot assays using rabbit anti-c-Myc tag and HRP-conjugated goat anti-rabbit IgG Ab (Abcam) antibodies, and visualized with ECL reagents as described before.pcDNA6-DrNOD2 and DrRIG-I was examined in the HEK293T cells and zebrafish embryos. The role of DrRIG-I in the DrNOD2-activated NF-κB signaling was analyzed by administering pCMV-DrNOD2 or pCMV-DrNOD2 (ΔLRR), alone or together, with pcDNA6-DrRIG-I, pcDNA6-DrRIG-I (CARD), or pcDNA6-DrRIG-I (ΔCARD) and the NF-κB luciferase reporter plasmid and internal control plasmid pRL-TK to the HEK293T cells or one-cell stage zebrafish embryos. The CARD deletion mutant was used as the negative control. The role of DrNOD2 in DrRIG-I-induced IFN signaling was analyzed by administering pcDNA6-DrRIG-I (CARD), alone or together, with pCMV-DrNOD2, pCMV-DrNOD2 (ΔLRR), or pCMV-DrNOD2 (ΔCARD). The IFN luciferase reporter plasmid (hISG15-pro-luc in HEK293T and DrMx-pro-luc in embryo) and control plasmid pRL-TK were also administered. The CARD deletion mutant was used as a negative control. The cells/embryos were harvested and lysed at 24 h post transfection/injection for dual-luciferase reporter assay.The mutual regulation between DrRIG-I in DrNOD2-initiated pathways and DrNOD2 in DrRIG-I-initiated pathways. For the former, one-cell stage embryos were injected with pCMV-DrNOD2/pCMV-DrNOD2 (ΔLRR) (100 pg/embryo) and DrRIG-I MO (4 ng/embryo), or together with capped DrRIG-I mRNA (100 pg/embryo). For the latter, one-cell stage embryos were injected with pcDNA6-DrRIG-I (CARD) (100 pg/embryo) and DrNOD2 MO (4 ng/embryo) or together with capped DrNOD2 (100 pg/embryo) mRNA. The embryos in each group were harvested at 24 hpi and lysed for dual-luciferase reporter assay. Furthermore, qRT-PCR was conducted to examine the relative expression levels of TNFα  and Mx .Knockdown and rescue experiments were conducted to further confirm the roles of DrRIG-I and DrNOD2 by evaluating the role of DrRIG-I in MDP-initiated NOD2 signaling and DrNOD2 in LMW poly I:C-initiated RIG-I signaling. For DrRIG-I, one-cell stage embryos were administered with 2 nL (1 µg/μL) of MDP, alone or together, with pcDNA6-DrRIG-I or pcDNA6-DrRIG-I (CARD) (100 pg/embryo), as well as NF-κB luciferase reporter plasmids. The embryos were collected at 24 hpi, and luciferase assays were conducted to examine the NF-κB activation level. For DrNOD2, one-cell stage embryos were administered with 4 nL (1 µg/μL) of LMW poly I:C alone or together with pCMV-DrNOD2/pCMV-DrNOD2 (ΔLRR) (100 pg/embryo), as well as Mx luciferase reporter plasmids. The embryos were collected at 24 hpi, and luciferase assays were conducted to examine Mx activation level.We further confirmed the physiological significance of the mutual negative regulatory roles between t-test for paired samples. Statistical significance were considered at * p < 0.05 and ** p < 0.01.Data from three independent experiments were expressed as mean ± SD. Groups were compared statistically using Student’s


NK cell activation following high-dose viral challenge was likely mediated by activated dendritic cells (DCs) and macrophages and the NK cell-stimulating cytokines interleukin 15 (IL-15) and IL-18. Neutralization of these cytokines decreased NK cell functions and increased viral loads, whereas IL-15 and IL-18 therapy improved NK cell activity. Here we demonstrate that virus dose positively correlates with antiviral NK cell activity and function, which are at least partly driven by IL-15 and IL-18. Our results suggest that NK cell activity may be therapeutically enhanced by administering IL-15 and IL-18 in virus infections that inadequately activate NK cells.Natural killer (NK) cells are part of the innate immune system and recognize virus-infected cells as well as tumor cells. Conflicting data about the beneficial or even detrimental role of NK cells in different infectious diseases have been described previously. While the type of pathogen strongly influences NK cell functionality, less is known about how the infection dose influences the quality of a NK cell response against retroviruses. In this study, we used the well-established Friend retrovirus (FV) mouse model to investigate the impact of virus dose on the induction of antiviral NK cell functions. High-dose virus inoculation increased initial virus replication compared to that with medium- or low-dose viral challenge and significantly improved NK cell activation. Antiviral NK cell activity, including IMPORTANCE In infections with retroviruses, like HIV and FV infection of mice, NK cells clearly mediate antiviral activities, but they are usually not sufficient to prevent severe pathology. Here we show that the initial infection dose impacts the induction of an antiviral NK cell response during an acute retroviral infection, which had not investigated before. High-dose infection resulted in a strong NK cell functionality, whereas no antiviral activities were detected after low- or medium-dose infection. Interestingly, DCs and macrophages were highly activated after high-dose FV challenge, which corresponded with increased levels of NK cell-stimulating cytokines IL-15 and IL-18. IL-15 and IL-18 neutralization decreased NK cell functions, whereas IL-15 and IL-18 therapy improved NK cell activity. Here we show the importance of cytokines for NK cell activation in retroviral infections; our findings suggest that immunotherapy combining the well-tolerated cytokines IL-15 and IL-18 might be an interesting approach for antiretroviral treatment. NK cells belong to the innate arm of the immune system and provide an early defense against viruses and transformed cells. NK cells use a complex array of germ line-encoded receptors and do not need clonal expansion or receptor rearrangement to initiate a response , 2. They11–22–25–in vivo modulation of several immune cell populations  was determined by using the early activation marker CD69  and effector functions  of NK cells in medium-dose-infected mice at 5 dpi compared to those in uninfected mice or infected mice at 3 dpi (data not shown). Taken together, these results demonstrate that a medium-dose infection did not induce NK cell activation, maturation, or effector function at day 3 postinfection, whereas high virus inoculation resulted in a detectable NK cell response.In a previous study it was demonstrated that a low- or medium-dose FV infection did not result in a potent activation of NK cells with antiviral activity . To addrker CD69 . We deteNK cells . FurtherNK cells . Remarkaculation . In viraculation . We therNK cells . Medium-in vitro by using the MHC-I-negative target cell line YAC-1 or the FV-transformed murine leukemia cell line FBL-3  (+ CD317+ DCs (pDCs) (−] F4/80+ CD11b+)  , CD11c+ s (pDCs) , and mac CD11b+)  in the s CD11b+) , pDCs (F CD11b+) , and mac CD11b+) , reveale+ macrophages and CD11c+ DCs from spleens of naive and high-dose-infected mice and measured the mRNA levels of IL-15 , proliferating (KI67+), and mature (KLRG1+). We also detected higher percentages of effector NK cells, measured by FasL and granzyme B (GzmB) expression. After cytokine stimulation, percentages of IFN-γ+ NK cells were also increased in comparison to those in untreated mice. To analyze the therapeutic effect of the exogenous IL-15 and IL-18 therapy, we measured the viral loads in treated and untreated animals that were infected with a medium dose of FV  and the European Health Law of the Federation of Laboratory Animal Science Associations (FELASA). Experiments were done using female inbred C57BL/6 mice  that were between 7 and 10 weeks old at day 0 of the experiment. For the experiments, an FV complex containing B-tropic Friend murine leukemia helper virus and polycythemia-inducing spleen focus-forming virus was used. The FV stock was prepared as a 15% spleen cell homogenate from BALB/c mice infected 14 days previously with 3,000 SFFU of FV. Mice were injected intravenously with 0.15 ml of phosphate-buffered saline (PBS) containing 5,000 SFFU (low dose), 20,000 SFFU (medium dose), or 40,000 SFFU (high dose) of FV. By injection of 20,000 or 40,000 SFFU of FV, mice received 20,000 or 40,000 infectious virus particles that can induce the proliferation of spleen cells Mus dunnis cells were prepared. Cultures were incubated for 3 days, then fixed with ethanol, stained with the F-MuLV envelope-specific monoclonal antibody 720, and developed with a peroxidase-conjugated goat anti-mouse antibody and aminoethylcarbazol for the detection of foci.For the detection of infectious centers, 10-fold dilutions of single-cell suspensions onto −) population was performed by exclusion of singlets, dead cells, T cells, and NK cells. After surface staining, granzyme B (GB11) was stained intracellularly after fixation with BD Cytofix/Cytoperm. Cells were fixed with the Foxp3/transcription factor staining buffer set (eBioscience) to analyze the expression of KI67 (B56). For IFN-γ (XMG1.2) and FasL (MFL3) restimulation, cells were restimulated with ionomycin (500 ng/ml), phorbol myristate acetate , monensin (1×), and brefeldin A (2 μg/ml) diluted in Iscove's modified Dulbecco's medium (IMDM) buffer and incubated for 3 h at 37°C. Splenocytes were fixed with a BD Cytofix/Cytoperm fixation/permeabilization kit. For the detection of caspase 1 activity, splenocytes were stained for surface markers and fixed with the BD Cytofix/Cytoperm kit. After fixation, monoclonal IgG1 caspase 1 antibody (Santa Cruz Biotechnology) was added for 30 min. After a washing, cells were incubated for 30 min with an allophycocyanin-coupled anti-IgG1 antibody (BD Pharmingen).Surface stainings were done using antibodies against the following: CD3 (17A2), CD4 (RM4-5), CD8 (53-6.7), CD11b (M1/70), CD11c (N418), CD49b (DX5), CD80 (16-10A1), CD107a (ID4B), CD317 (927), F4/80 (BM8), KLRG1 (2F1), and NK1.1 (PK136). For the exclusion of dead cells, splenocytes were stained with a Zombie UV fixable viability kit (BioLegend). Gating of the lineage-negative (linin vitro using 1 × 104 carboxyfluorescein succinimidyl ester (CFSE)-stained YAC-1 or FBL-3 tumor cells and 25 × 104 isolated NK cells from the spleens of naive or FV-infected mice. NK cells were isolated using a MagniSort mouse NK cell enrichment kit (eBioscience). The cytotoxic assay was performed in 96-well U-bottom plates. The cells were coincubated for 24 h in a humidified 5% CO2 atmosphere at 37°C. Cells were washed once and stained with fixable viability dye (eBioscience) to exclude dead cells. After a washing, cells were directly analyzed by flow cytometry.NK cell cytotoxicity was tested in vivo NK cell cytotoxicity assay was executed using 5 × 105 RMA/S cells labeled with 10 μM CFSE  and 5 × 105 RMA cells labeled with a 2.5 μM concentration of the cell proliferation dye eFluor 670 (eBioscience) per mouse. Cells were mixed in a ratio of 1:1 and injected intraperitoneally (i.p.) at 3 dpi. NK cell-depleted mice were used as negative controls. Mice were sacrificed at 5 dpi, and intraperitoneal lavage was performed with 10 ml of PBS to obtain cells. Cells were washed once, resuspended in buffer containing fixable viability dye (eBioscience) for the exclusion of dead cells, and analyzed with a flow cytometer. The ratio of CFSE-labeled target cells (RMA/S) versus eFluor 670-labeled nontarget cells (RMA) was calculated normalized to the ratio in NK cell-depleted animals.An + cells were depleted.NK cells were ablated by intraperitoneal injection of 0.2 ml of supernatant fluid containing NK1.1-specific monoclonal antibody PK136. For depletion of NK cells, depletion antibody was injected 1 day prior to and 2 days after FV infection. More than 90% of NK1.1Murine spleens were harvested and grinded using 70-μm strainers. Cells were collected in 1 ml of PBS. After centrifugation, supernatants of splenocytes were used for enzyme-linked immunosorbent assays (ELISAs). ELISAs were performed according to the manufacturer's instructions with a mouse IFN-α (multiple subtypes) bioluminescent ELISA kit (InvivoGen), IL-12 p70 ELISA MAX Deluxe (BioLegend), mouse IL-15 ELISA Ready-SET-Go! (eBioscience), and mouse IL-18 Platinum ELISA (eBioscience).+  or CD11c+  cells with anti-PE microbeads (Miltenyi Biotec), utilizing DNA/RNA Shield (Zymoresearch) and an innuPREP RNA minikit . cDNA was synthesized using innuScript reverse transcriptase . cDNA was stored at −20°C.Total RNA was isolated from splenocytes and F4/80Real time-PCR analysis for the quantification of IFN-α, IL-12, IL-15, and IL-18 mRNA was performed using innuMIX quantitative PCR (qPCR) MasterMix SyGreen . Oligonucleotide sequences (Biomers) for tested genes were as follows: for β-actin, 5′-AAATCGTGCGTGACATCAAA-3′ and 5′-CAAGAAGGAAGGCTGGAAAA-3′; for IFN-α, 5′-ATGGCTAGGCTCTGTGCTTTCC-3′ and 5′-AGGGCTCTCCAGACTTCTGCTCTG-3′; for IL-12 p40, 5′-ACAGCACCAGCTTCTTCATCAG-3′ and 5′-TCTTCAAAGGCTTCATCTGCAA-3′; and for IL-15, 5′-CATTTTGGGCTGTGTCAGTG-3′ and 5′-TCTTCAAAGGCTTCATCTGCAA-3′. The Mm-Il18-1-SG QuantiTect primer assay (Qiagen) was used. The quantitative mRNA levels were determined by using Rotor-Gene Q series software (Qiagen) and were normalized to β-actin mRNA expression levels.Mice were injected every day i.p. with IL-15-neutralizing  and IL-18-neutralizing  antibodies starting 1 day before high-dose FV infection.Mice were infected with a medium dose of FV. Starting from day 0, mice were injected i.p. twice with IL-15  and three times with IL-18 .Statistical analyses and graphical presentations were done with GraphPad Prism version 6 and Excel 2010. Statistical differences between two different groups were determined by the Mann-Whitney test. Analyses including several groups were tested using the nonparametric Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks and Dunn's multiple-comparison test or ordinary one-way ANOVA and Tukey multiple-comparison test.


Conversely, the induction of protective tumour-specific CTLeff and their recruitment into the tumour remain challenging tasks. Here we show that lymphocytic choriomeningitis virus (LCMV) can be engineered to serve as a replication competent, stably-attenuated immunotherapy vector (artLCMV). artLCMV delivers tumour-associated antigens to dendritic cells for efficient CTL priming. Unlike replication-deficient vectors, artLCMV targets also lymphoid tissue stroma cells expressing the alarmin interleukin-33. By triggering interleukin-33 signals, artLCMV elicits CTLeff responses of higher magnitude and functionality than those induced by replication-deficient vectors. Superior anti-tumour efficacy of artLCMV immunotherapy depends on interleukin-33 signalling, and a massive CTLeff influx triggers aninflammatory conversion of the tumour microenvironment. Our observations suggest that replicating viral delivery systems can release alarmins for improved anti-tumour efficacy. These mechanistic insights may outweigh safety concerns around replicating viral vectors in cancer immunotherapy.Viral infections lead to alarmin release and elicit potent cytotoxic effector T lymphocyte (CTL Viruses trigger potent cytotoxic T cell responses, whereas anti-tumour immunity has been difficult to establish. Here the authors engineer a replicating viral delivery system for tumour-associated antigens, which induces alarmin release, innate activation and protective anti-tumour immunity in mice. Experimeff responses to several replicating RNA and DNA viruses+ T cells, thereby enhancing clonal expansion, effector differentiation and ultimately CTL efficacyWe and others have previously reported that interleukin-33 (IL-33) is a key driver of potent and protective CTLeff responses. The activation of critical innate immune pathways including alarmins represents a discriminating feature of replicating microbial delivery systems, which might be decisive for the success of active cancer immunotherapy.Hence we engineer a TAA delivery platform based on live-attenuated LCMV (artLCMV). Unlike replication-deficient rLCMV vectors, artLCMV targets not only dendritic but also lymphoid stromal cells, thereby triggering the IL-33–ST2 alarmin pathway and inducing superior TAA-specific CTLGPC) and nucleoprotein (NP) genes onto artificially duplicated S segments . Genetic and phenotypic stability represent key criteria for manufacturing and clinical translation of live-attenuated viral vector systems29GPC under 3′UTR control, we generated viruses with an artificial genome organization  as well as of the type I and type II interferon genes, AGR mice readily reveal an attenuated virus' reversion to virulencein vivo attenuation of r3LCMVrec, NP and GPC sequences in a single RNA molecule were detected inten out of ten r3LCMV-GFP-infected AGR mice from two independent series of experiments. In contrast, the blood of 11 artLCMV-infected mice was free of detectable Srec RNA. Sequence analysis of Srec segments revealed that individual non-homologous recombination events had occurred in each animal, with breakpoints in the GFP–IGR border region  grew to LCMVwt titres in cell culture  expressing vector (artLCMV-OVA). It induced OVA epitope-specific CTL numbers, which were substantially higher than those elicited by an OVA-recombinant adenovirus 5-based vector (rAd-OVA) or by a replication-deficient LCMV-based vector  responses in blood and spleen exceeded those of the other delivery systems by ∼tenfold  immunization , artLCMV infected a significantly higher number of conventional dendritic cells and macrophages than its replication-deficient counterpart rLCMV (eff responses in particularIl1rl1−/−), both in spleen and blood  gp38+ fibroblastic reticular cells (FRC) reported IL-33 in naïve animals (IL-33-GFP+), whereas CD31+ blood endothelial cells (BEC) were virtually IL-33-GFP-negative (+IL-33-GFP+) FRCs, confirming that replicating artLCMV but not replication-deficient rLCMV infected IL-33-expressing FRCs in spleen (+) IL-33-GFP+ BEC and FRC frequencies declined sharply between day 3 and 7 after immunization . Unlike rLCMV-OVA and rAd-OVA, which showed partial or no clinical benefit, respectively, when administered to mice with an established solid tumour, treatment with artLCMV-OVA afforded substantial tumour control and prolonged the animals' survival in a CD8t manner . Importat manner . Moreove−/− mice . These d−/− mice , altoget−/− mice . Of note−/− mice . Anti-PD−/− mice .eff in blood of EG7-OVA tumour-bearing mice than did artLCMV-GFP, rAd-OVA or rLCMV-OVA , MC57 cells (CRL-2295), EL-4 cells (TIB-39), EG7 thymoma cells  and P815 mastocytoma cells (TIB-64) were obtained from ATCC. Stably transfected BHK-21 cells expressing the LCMV-NP and -GP proteins, respectively , and GP-expressing 293T cells have previously been described26GPC-encoding S segment cDNA (pol-I-S-GPCtag) was generated by codon-optimizing the 255 C-terminal nucleotides of the viral GPC ORF, creating a novel ORF named ‘GPCtag'. To create a Srec#1 expression plasmid, we substituted the IGR in pol-I-S-GPCtag for a synthetic cDNA fragment (Genscript) as described in The origin, passage and titration of the LCMV strains Armstrong and Clone 13 strains have been described2660Il33/−− micehttp://www.cdb.riken.jp/arg/mutant%20mice%20list.html). AGRAG mice Il1rl1/−− (ref. Il33gfp/wt) were generated by intercrossing IL-33−/− mice with the ZP3-Cre germ line deleter strain to remove the neomycin cassette. C57BL/6, BALB/c and DBA/2 wild-type mice were eitherpurchased from Charles River and Janvier Labs or were bred at the Institut für Labortierkunde of the University of Zurich, Switzerland under specific pathogen-free conditions. Unless specified otherwise, rAd-OVA vectors and rVACC-OVA were administered in their respective optimal dose range of 108 viral particles i.v. and 106 PFU i.v., respectively665 PFU i.v., except for virus tracing experiments in C57BL/6 mice (106 PFU) and experiments in AGRAG mice (104 PFU). rLCMV vectors were used at 105 PFU for standard immunization experiments or at 106 PFU for immunization withtumour self antigens, for tumour immunotherapy and for Cr51 release experiments. Except for prime-boost immunization experiments in 2 inhalation in accordance with the Swiss law. PD-1 blocking antibody  was administered at a dose of 12.5 mg kg−1 intraperitoneally. CD8+ T cells were depleted by injecting 200 μg anti-CD8 antibody  intraperitoneally. Animal experiments were performed at the Universities of Geneva and Basel in accordance with the Swiss law for animal protection. Permission was granted by the Directiongénérale de la santé, Domaine de l′expérimentation animale of the Canton of Geneva and by the Veterinäramt Basel-Stadt, respectively. Experimental groups were sex- and age-matched. Animals in tumour therapy experiments were assigned to groups in a manner to assure even distribution of tumour volumes between groups at the time of tumour therapy. The groups were neither randomized, nor were experiments conducted in a blinded manner. The Swiss law for animal protection requires that mice with wounds on the tumour or exhibiting signs of distress  be euthanized by CO2 inhalation irrespective of tumour size and diameter. Accordingly, animals not reaching humane endpoints of tumour volume or diameter were excluded from survival curves.−/− ref.  and ZP3-NP-specific primer (5′-GGCTCCCAGATCTGAAAACTGTT-3′). PCR amplification used the same primer together with a GPC-specific primer (5′-GCTGGCTTGTCACTAATGGCTC-3′). Amplified products were purified for Sanger sequencing (Microsynth). Whole-cell RNA was extracted from tumour tissue using QIAzol (QIAGEN). nCounter Nanostring Mouse Inflammation v2 assay and Applied Biosystems TaqMan RT-PCR assays were used for quantification of gene expression. TaqMan results were normalized to GAPDH.Viral RNA was extracted from cell culture supernatant or serum of infected mice using the QIAamp Viral RNA Mini Kit . Reverse-transcription was performed with ThermoScript RT-PCR System (Invitrogen) and an LCMV-+B220− lymphocytes. BD Trucount Absolute Counting Tubes were used to determine absolute cell counts in blood. Spleen lymphocyte countswere determined in a Neubauer chamber. Peptide-MHC tetramer-binding CTLs were back calculated. Cytokine profiles after restimulation with peptide  were determined in intracellular cytokine assaysAntibodies against CD4 (RM4-5 or GK1.5), CD8 (53-6.7), CD45R/B220 (RA3-6B2), CD45.2 (104), Ter-119 (TER-119), CD31 (390), gp38 , CD3 (17A2), Klrg1 (2F1), CD127 (A7R34), CD11c (N418), CD11b (M1/70), CD19 (6D5), NK1.1 (PK136), CD90.2 (30-H12), GR-1 (RB6-8C5), IFN-γ (XMG1.2), TNF (MP6-XT22) and IL-2 (JES6-5H4) were from Biolegend, Pharmingen and eBioscience. All fluorescently labelled monoclonal antibodies were used at a 1:100 dilution, except for gp38 (Podoplanin) and Ter-119, which were diluted 1:1,000, and 1:10, respectively. Dead cells were excluded with Zombie UV Fixable Viability Kit . OVA- (SIINFEKL) and P1A epitope- (LPYLGWLVF) specific CTLs were identified as peptide-MHC class I tetramers (TCMetrix) binding cells amongst CD8−1 Collagenase IV (Worthington), 40 μg ml−1 DNAseI (Roche) and 2% (vol/vol) FCS), were cut into pieces and digested. Erythrocytes were lysed in 0.15 M NH4Cl/10 mM KHCO3/0.1 mM EDTA and haematopoietic CD45+ cells were depleted using anti-CD45 beads (Miltenyi Biotec). For TIL analysis, tumours were cut and digested with accutase (PAA), Collagenase IV (Worthington), Hyaluronidase (Sigma) and DNAseI (Roche) and red blood cells were lysed. Mononuclear cells were isolated using a Histopaque-1119 gradient.Splenic single-cell suspensions were prepared by mechanical disruption. The isolation of stromal cells from spleen has been described6 tumour cells were implanted subcutaneously in the right flank. Tumour growth was monitored three times per week and the longest (‘length') and the shortest diameter (‘width') were measured using a caliper. Tumour volumes (mm3) were calculated as 1/2 (length*widtĥ2). In accordance with the Swiss law for animal protection, mice were euthanized when tumour volumes exceeded 1,500 mm3 or when the longest median tumour diameter exceeded 20 mm.Suspensions containing 10For immunohistochemical bright-field staining, tissues were prepared in HOPE fixative (DCS Innovative) and paraffin embedded. Upon inactivation of endogenous peroxidases, tissue sections were incubated with rat-anti-mouse CD8a  or rat-anti-LCMV NP . Bound primary antibodies were visualized with biotin-labelled anti-rat antibody (diluted 1:100) and streptavidin-peroxidase staining method using polymerized 3,3′-diaminobenzidine .For detection of GFP in virus-infected organs, cryosections (10 μm) were prepared from PFA-fixed tissues and nuclei were stained with 4′,6-diamidino-2-phenylindole .Immunostained bright-field sections and fluorescence sections of virus-infected tissues were scanned using a Panoramic Digital Slide Scanner 250 FLASH II (3DHISTECH). White balance was adjusted and contrast was linearly enhanced using the tools ‘levels', ‘curves', ‘brightness' and ‘contrast' in Photoshop CS6 (Adobe).IL-33gfp/gfp and IL-33gfp/wt, cryosections (8–10 μm) were prepared from PFA-fixed spleens. For detection of nuclear IL-33, rat anti-gp38 , rabbit anti-GFP  and goat anti-IL33  were used as primary antibodies. As secondary antibodies donkey-anti-rat IgG HRP , donkey-anti-rabbit Alexa488 (diluted 1:500) and donkey-anti-goat Alexa647 (diluted 1:300) were used (Jackson Immunoresearch). HRP was visualized using a Tyramide Cy3 (Invitrogen) reaction. Nuclei were stained with DAPI. Immunostainings were visualized using an AxioImager Z.1 microscope (Zeiss), followed by image processing in Adobe Photoshop.For GFP detection in naïve ex vivo cytotoxicity of splenocytes was determined in a 5-h assay on Cr51-loaded EL-4 target cells pulsed with SIINFEKL peptide according to established proceduresPrimary Serum interferon-alpha levels were determined by ELISA using the Verikine Mouse Interferon Alpha ELISA Kit .t-tests. Single values of multiple groups were compared by one-way analysis of variance (ANOVA), followed by Bonferroni post hoc test or Dunnett's post hoc test when comparing against a reference group. A Bonferroni correction was made when comparing multiple parameters. Two-way ANOVA with Bonferroni post hoc test was used to compare multiple groups with multiple measurements. Survival curves were analysed by log-rank tests with Bonferroni correction. For the comparison of tumour growth curves, the area under the curve was comparedFor statistical analysis, GraphPad Prism software  was used. Differences between two groups were assessed using unpaired two-tailed Student's Viral load data were log-converted to obtain a near-normal distribution prior to statistical analysis. Variances within different groups in a given experiment were similar. In accordance with current standard practice in the field of viral immunology, only very substantial differences were reported, obviating the need for variance testing.P values of P<0.05 were considered significant (*), P<0.01 (**) and P<0.001 (***) as highly significant.Nanostring data that support the findings of this study have been deposited in National Center for Biotechnology Information Gene Expression Omnibus (GEO) with the accession code GSE84039. The nucleotide sequences of recombinant and recombined LCMV segments have been deposited in GenBank with the accession codes KX462116-KX462128. All relevant data are available from the authors upon request.How to cite this article: Kallert, S. M. et al. Replicating viral vector platform exploits alarmin signals for potent CD8+ T cell-mediated tumour immunotherapy. Nat. Commun.8, 15327 doi: 10.1038/ncomms15327 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary References