Summary During immune responses, pro-and anti-inflammatory mechanisms must be balanced to ensure pathogen clearance while limiting tissue damage. Monocyte-derived cells contribute to both processes, yet the underlying regulatory circuits remain incompletely defined. Here, we show that a subset of PDPN + IL-7R + monocyte-derived cells that impair effector CD4 ⁺ T cell–mediated control of intracellular pathogens, and thus perpetuate the infection. Fibroblast-derived IL-7 drives this immunosuppressive program, which is up-regulated in response to IFNγ. We thus uncover a cytokine-dependent feedback circuit in which elevated IFNγ induces IL-7 production by fibroblasts, licensing immunosuppressive monocyte-derived cells that restrain CD4⁺ T cell responses. This mechanism links excessive inflammation to immune suppression at the expense of pathogen control. Targeting this feedback loop may enable therapeutic strategies that enhance antimicrobial immunity while preserving tissue integrity.

BACKGROUND:Macrophages exhibit high heterogeneity and plasticity, which is essential for their multifaceted roles in host defense and tissue regeneration. Mast cells (MCs) respond rapidly to injury or infection by releasing intact secretory granules, thereby initiating and potentiating innate and adaptive immunity. OBJECTIVE:Because MCs reside in close proximity to macrophages in the skin, we decoded the impact of exocytosed MC granules (MCGs) on macrophage phenotype and function. METHODS:We tracked the fate of MCGs and studied their specific effects on macrophage plasticity and functional characteristics in vivo and in vitro in mouse and human samples using advanced imaging, functional assays, and transcriptomic profiling. RESULTS:We found that intact MCGs are engulfed by macrophages in vivo in murine models and in vitro. MCG ingestion boosted macrophage functional capacities and resulted in an atypical plasticity that contains both alternatively activated and classically activated macrophage features, suggesting increased efficiency in their multifaceted roles. In addition, the engulfment of intact MCGs by macrophages led to a specific transcriptome reprogramming. Importantly, both the process of MCG ingestion and its functional impact was confirmed with human MCs and macrophages in vitro and in situ in healthy human skin explants and in psoriatic patient lesional skin. CONCLUSION:MCs enhance the function of macrophages and drive them to atypical polarization through granule-mediated intercellular communication. Our results suggest that beyond acute inflammation, MCs act as important controllers of host defense and integrity, supporting their emerging relevance as therapeutic targets.

Gut-draining mesenteric and celiac lymph nodes (mLNs and celLNs) critically contribute to peripheral tolerance toward food and microbial antigens by supporting the de novo induction of regulatory T cells (Tregs). These tolerogenic properties of mLNs and celLNs are stably imprinted within stromal cells (SCs) by microbial signals and vitamin A (VA), respectively. Here, we report that a single, transient gastrointestinal infection in the neonatal, but not adult, period durably abrogates the efficient Treg-inducing capacity of celLNs by altering the subset composition and gene expression profile of celLNSCs. These cells carry information about the early-life pathogen encounter until adulthood and durably instruct migratory dendritic cells entering the celLN with reduced tolerogenic properties. Mechanistically, transiently reduced VA levels cause long-lasting celLN functional impairment, which can be rescued by early-life treatment with VA. Together, our data highlight the therapeutic potential of VA to prevent sequelae post gastrointestinal infections in infants.

Abstract Background Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils. Methods Ly6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1β. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied. Results Strikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals. Conclusions Taken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.

Introduction Arterial and venous thrombosis represent significant causes of mortality and morbidity in patients with Myeloproliferative Neoplasms (MPNs). While aberrant platelet and leukocyte activity has been identified as important cellular switchboards, the precise molecular chronology of pathologic blood coagulation in MPNs remains elusive. Our previous research (Edelmann et al., JCI, 2018) has demonstrated that JAK2-V617F induced over-activation of β1/β2 integrins and aberrant neutrophil adhesion to VCAM-1 and ICAM-1 are critical in pathological thrombus formation in MPNs. However, there is limited molecular data on the role of neutrophil adhesion in venous thrombosis associated with CALR mutations. This study aims to elucidate the molecular mechanisms underlying thrombosis formation in CALR-del52 and JAK2-V617F mutated MPNs, with a particular focus on the role of neutrophils. Methods We employed the inferior vena cava (IVC) stenosis mouse model, using Vav1-Cre x JAK2+/+ and Vav1-Cre x JAK2VF/+ mice (Mullally et al., 2010), as well as Vav1-Cre x CALR+/+ and Vav1-Cre x CALR+/del52 mice (Li et al., 2018). Thrombus size and weight and plasma cytokines levels were assessed. The role of integrins was investigated by treatment with and without anti-VLA-4/β2 integrin and anti-isotype antibodies. Additionally, MPN patients harboring JAK2-V617F and CALR mutations, along with healthy volunteers, were enrolled in the study. Citrated plasma samples were collected from these participants to profile cytokine levels using a multiplex assay for human cytokines. Furthermore, neutrophil extracellular trap (NET) formation was investigated using immunofluorescence microscopy. Results Using the IVC stenosis model, we observed a significant increase in thrombus size (p<0.0001) and weight (p<0.0022) in CALR-mutated mice (n=5) compared to CALR-WT mice (n=6). The fold change in thrombus size compared to the respective WT control post-ligation was found to be 4-fold in JAK2-V617F mice (Edelmann et al., JCI 2018) and only 2-fold in CALR-del52 mice. Additionally, thrombus induction time was 4 hours in JAK2-V617F mice and 12 hours in CALR-del52 mice. Profiling inflammatory cytokines revealed a significant elevation of G-CSF (25,898 ± 2,665 pg/ml; p=0.0001), IL-6 (466.4 ± 123.3 pg/ml; p=0.0036), IL-1α (73.33 ± 19.86 pg/ml; p=0.0042), IL-10 (20.33 ± 7.3 pg/ml; p=0.0203), and CXCL-1 (509 ± 131.7 pg/ml; p=0.0031) in both CALR-WT and CALR-del52 mouse models post ligation of IVC. Anti-VLA-4/β2 integrin antibody treatment in CALR-del52 mice (n=5) resulted in marked suppression of thrombus size (p<0.0001) and weight (p<0.0001), alongside with a reduction in elevated cytokine levels (G-CSF, p=0.0631; IL-6, p=0.0724; IL-1α, p=0.0374; IL-10, p=0.0840; CXCL-1, p=0.1637) compared to the IgG control (n=5) treatment group. Similarly, increases in G-CSF, IL-6, CXCL-1, and CCL-2 were observed in JAK2-V617F mutated mice (n=5) following IVC ligation. Furthermore, plasma profiling of JAK2-V617F patients (n=6) with a history of thrombosis showed upregulation of IL-6 (p=0.0078), IL-8 (p=0.0176), IL-10 (p=0.0175), sVCAM-1 (p=0.0001), CXCL-12 (p=0.0027), and MPO (p=0.0431) compared with heathy donors. Additionally, we selected one of the elevated cytokines post-ligation, CXCL-1, and used it to stimulate JAK2-V617F-mutated murine neutrophils. This stimulation of isolated murine neutrophils induced NETosis, as evidenced by CitH3 and DAPI staining. Conclusion Our study highlights the crucial role of neutrophil adhesion and cytokine modulation in venous thrombosis associated with CALR-del52 and JAK2-V617F mutations in MPNs. The significant suppression of thrombus size and inflammatory cytokine levels following anti-VLA-4/β2 integrin antibody treatment in CALR-del52 and JAK2-V617F mice suggests a potential therapeutic target. The similar cytokine profiles in JAK2-V617F mouse models and MPN patients underscore the relevance of our findings to human disease. These insights advance our understanding of the molecular mechanisms of thrombosis in MPNs, potentially guiding future therapeutic strategies.

Asthma is one of the most common chronic respiratory diseases and is characterized by airway inflammation, increased mucus production, and structural changes in the airways. Recently, there is increasing evidence that the disease is much more heterogeneous than expected, with several distinct asthma endotypes. Based on the specificity of T cells as the best-known driving force in airway inflammation, bronchial asthma is categorized into T helper cell 2 (Th2) and non-Th2 asthma. The most studied effector cells in Th2 asthma include T cells and eosinophils. In contrast to Th2 asthma, much less is known about the pathophysiology of non-Th2 asthma, which is often associated with treatment resistance. Besides T cells, the interaction of myeloid cells such as monocytes/macrophages and mast cells with the airway epithelium significantly contributes to the pathogenesis of asthma. However, the underlying molecular regulation and particularly the specific relevance of this cellular network in certain asthma endotypes remain to be understood. In this review, we summarize recent findings on the regulation of and complex interplay between epithelial cells and the “nonclassical” innate effector cells mast cells and monocytes/macrophages in Th2 and non-Th2 asthma with the ultimate goal of providing the rationale for future research into targeted therapy regimens.

CD4+ T cells play a central role in orchestrating the immune response in asthma, with dysregulated ion channel profiles and altered metabolic signatures contributing to disease progression and severity. An important classification of asthma is based on the presence of T-helper cell type 2 (Th2) inflammation, dividing patients into Th2-high and Th2-low endotypes. These distinct endotypes have implications for disease severity, treatment response, and prognosis. By elucidating how ion channels and energy metabolism control Th cells in asthma, this review contributes to the pathophysiological understanding and the prospective development of personalized therapeutic treatment strategies for patients suffering from distinct asthma endotypes.

BACKGROUND:Infection by beet cyst nematodes (BCN, Heterodera schachtii) causes a serious disease of sugar beet, and climatic change is expected to improve the conditions for BCN infection. Yield and yield stability under adverse conditions are among the main breeding objectives. Breeding of BCN tolerant sugar beet cultivars offering high yield in the presence of the pathogen is therefore of high relevance.RESULTS:To identify causal genes providing tolerance against BCN infection, we combined several experimental and bioinformatic approaches. Relevant genomic regions were detected through mapping-by-sequencing using a segregating F2 population. DNA sequencing of contrasting F2 pools and analyses of allele frequencies for variant positions identified a single genomic region which confers nematode tolerance. The genomic interval was confirmed and narrowed down by genotyping with newly developed molecular markers. To pinpoint the causal genes within the potential nematode tolerance locus, we generated long read-based genome sequence assemblies of the tolerant parental breeding line Strube U2Bv and the susceptible reference line 2320Bv. We analyzed continuous sequences of the potential locus with regard to functional gene annotation and differential gene expression upon BCN infection. A cluster of genes with similarity to the Arabidopsis thaliana gene encoding nodule inception protein-like protein 7 (NLP7) was identified. Gene expression analyses confirmed transcriptional activity and revealed clear differences between susceptible and tolerant genotypes.CONCLUSIONS:Our findings provide new insights into the genomic basis of plant-nematode interactions that can be used to design and accelerate novel management strategies against BCN.

Most clinically applied cancer immunotherapies rely on the ability of CD8 + cytolytic T cells to directly recognize and kill tumour cells 1–3 . These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment 4–6 . The ability of CD4 + effector cells to contribute to antitumour immunity independently of CD8 + T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified 7–10 . Here, we describe a mechanism whereby a small number of CD4 + T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8 + T cell targeting. The CD4 + effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II + CD11c + antigen-presenting cells. We show that T helper type 1 cell-directed CD4 + T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4 + T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4 + T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8 + T cells and natural killer cells and advance cancer immunotherapies.

T helper (Th) cells provide immunity to pathogens but also contribute to detrimental immune responses during allergy and autoimmunity. Th2 cells mediate asthmatic airway inflammation and Th1 cells are involved in the pathogenesis of multiple sclerosis. T cell activation involves complex transcriptional networks and metabolic reprogramming, which enable proliferation and differentiation into Th1 and Th2 cells. The essential trace element zinc has reported immunomodulatory capacity and high zinc concentrations interfere with T cell function. However, how high doses of zinc affect T cell gene networks and metabolism remained so far elusive. Herein, we demonstrate by means of transcriptomic analysis that zinc aspartate (UNIZINK), a registered pharmaceutical infusion solution with high bioavailability, negatively regulates gene networks controlling DNA replication and the energy metabolism of murine CD3/CD28-activated CD4 + T cells. Specifically, in the presence of zinc, CD4 + T cells show impaired expression of cell cycle, glycolytic and tricarboxylic acid cycle genes, which functionally cumulates in reduced glycolysis, oxidative phosphorylation, metabolic fitness and viability. Moreover, high zinc concentrations impaired nuclear expression of the metabolic transcription factor MYC, prevented Th1 and Th2 differentiation in vitro and reduced Th1 autoimmune central nervous system (CNS) inflammation and Th2 asthmatic airway inflammation induced by house dust mites in vivo. Together, we find that higher zinc doses impair the metabolic fitness of CD4 + T cells and prevent Th1 CNS autoimmunity and Th2 allergy.

Abstract Current clinically applied cancer immunotherapies largely focus on the ability of CD8+ cytolytic T-cells to directly recognise and kill tumour cells1–3. These strategies are limited by the emergence of MHC-I-deficient or IFN-unresponsive tumour cells and the development of an immunosuppressive tumour microenvironment4–6. CD4+ effector T-cells can contribute to tumour immune defence independent of CD8+ T-cells. However, the potential and the mechanisms of CD4+ T-cell-mediated anti-tumour immunity are incompletely understood7–12. Here, we show how an indirect CD4+ T-cell-mediated mode of action, that is fundamentally different from CD8+ T-cells, enables the eradication of tumours that would otherwise escape direct T-cell targeting. CD4+ effector T-cells preferentially cluster at tumour invasive margins where they engage in antigen-specific interactions with MHC-II+CD11c+ cells, while CD8+ T-cells briskly infiltrate tumour tissues. CD4+ T-cells and innate immune stimulation reprogram the tumour-associated inflammatory monocyte network towards IFN-activated antigen-presenting and tumouricidal effector phenotypes. This results in an amplification loop driving the release of T-cell-derived IFNγ and myeloid cell-derived nitric oxide which cooperatively induce apoptotic death of MHC-deficient and IFN-unresponsive tumour cells that escape cytolytic CD8+ T-cell therapy. Exploiting the ability of CD4+ T-cells to orchestrate indirect inflammatory killing of tumour cells complements the direct cytolytic activity of T-cells to advance cancer immunotherapies.

Background: Severe traumatic injury has been associated with high susceptibility for the development of secondary complications caused by dysbalanced immune response. As the first line of the cellular immune response, neutrophils and monocytes recruited to the site of tissue damage and/or infection, are divided into three different subsets according to their CD16/CD62L and CD16/CD14 expression, respectively. Their differential functions have not yet been clearly understood. Thus, we evaluated the phenotypic changes of neutrophil and monocyte subsets among their functionality regarding oxidative burst and the phagocytic capacity in severely traumatized patients. Methods: Peripheral blood was withdrawn from severely injured trauma patients (TP; n = 15, ISS ≥ 16) within the first 12 h post-trauma and from healthy volunteers (HV; n = 15) and stimulated with fMLP and PMA. CD16dimCD62Lbright (immature), CD16brightCD62Lbright (mature) and CD16brightCD62Ldim (CD62Llow) neutrophil subsets and CD14brightCD16− (classical), CD14brightCD16+ (intermediate) and CD14dimCD16+ (non-classical) monocyte subsets of HV and TP were either directly analyzed by flow cytometry or the examined subsets of HV were sorted first by fluorescence-activated cell sorting and subsequently analyzed. Subset-specific generation of reactive oxygen species (ROS) and of E. coli bioparticle phagocytosis were evaluated. Results: In TP, the counts of immature neutrophils were significantly increased vs. HV. The numbers of mature and CD62Ldim neutrophils remained unchanged but the production of ROS was significantly enhanced in TP vs. HV and the stimulation with fMLP significantly increased the generation of ROS in the mature and CD62Ldim neutrophils of HV. The counts of phagocyting neutrophils did not change but the mean phagocytic capacity showed an increasing trend in TP. In TP, the monocytes shifted toward the intermediate phenotype, whereas the classical and non-classical monocytes became less abundant. ROS generation was significantly increased in all monocyte subsets in TP vs. HV and PMA stimulation significantly increased those level in both, HV and TP. However, the PMA-induced mean ROS generation was significantly lower in intermediate monocytes of TP vs. HV. Sorting of monocyte and neutrophil subsets revealed a significant increase of ROS and decrease of phagocytic capacity vs. whole blood analysis. Conclusions: Neutrophils and monocytes display a phenotypic shift following severe injury. The increased functional abnormalities of certain subsets may contribute to the dysbalanced immune response and attenuate the antimicrobial function and thus, may represent a potential therapeutic target. Further studies on isolated subsets are necessary for evaluation of their physiological role after severe traumatic injury.

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrated that MC-derived tumor necrosis factor (TNF) was crucial for neutrophil extravasation to sites of contact hypersensitivity-induced skin inflammation by promoting intraluminal crawling. MC-derived TNF directly primed circulating neutrophils via TNF receptor-1 (TNFR1) while being dispensable for endothelial cell activation. The MC-derived TNF was infused into the bloodstream by directional degranulation of perivascular MCs that were part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules boosted neutrophil extravasation. Pronounced and rapid intravascular MC degranulation was also observed upon IgE crosslinking or LPs challenge indicating a universal MC potential. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.

Neutrophils represent one of the first immune cell types recruited to sites of infection, where they can control pathogens by phagocytosis and cytotoxic mechanisms. Intracellular pathogens such as Leishmania major can hijack neutrophils to establish an efficient infection. However the dynamic interactions of neutrophils with the pathogen and other cells at the site of the infection are incompletely understood. Here, we have investigated the role of Ly6G, a homolog of the human CD177 protein, which has been shown to interact with cell adhesion molecules, and serves as a bona fide marker for neutrophils in mice. We show that Ly6G deficiency decreases the initial infection rate of neutrophils recruited to the site of infection. Although the uptake of L. major by subsequently recruited monocytes was tightly linked with the concomitant uptake of neutrophil material, this process was not altered by Ly6G deficiency of the neutrophils. Instead, we observed by intravital 2-photon microscopy that Ly6G-deficient neutrophils entered the site of infection with delayed initial recruitment kinetics. Thus, we conclude that by promoting neutrophils' ability to efficiently enter the site of infection, Ly6G contributes to the early engagement of intracellular pathogens by the immune system.

Site-directed mutagenesis facilitates the experimental validation of gene function and can speed up plant breeding by producing new genetic variability or by reproducing previously known gene variants in other than their original genetic backgrounds. However, its application is challenging in wheat owing to high genomic redundancy and highly genotype-dependent DNA transfer methods (Koeppel et al., 2019). In wheat, large chromosomal regions are hardly amenable to meiotic recombination, which limits the potential for trait improvements. The era of transgenesis facilitated the generation of desired traits through the transfer of recombinant DNA into elite backgrounds. This technology, however, is limited by long and costly regulatory evaluation processes owing to publicly overrated method-specific risks. As another option, the use of meiotically recombinant and genetically fixed doubled haploids proved very useful for accelerating crop improvement (Kalinowska et al., 2019). Viable methods of in planta haploid induction via uniparental genome elimination are available in species such as Arabidopsis through modification of CENTROMERIC HISTONE 3 (CENH3) (Ravi and Chan, 2010), in maize and rice via knockout of a sperm-specific phospholipase gene (Kelliher et al., 2017; Yao et al., 2018), and in wheat through intergeneric crossing with maize (Laurie and Bennett, 1988). Haploid induction coupled with site-directed mutagenesis has previously been reported in Arabidopsis, maize and wheat (Kelliher et al., 2019). However, in wheat, no unambiguous evidence has been provided yet, considering that a mutated target sequence was shown for just a single event. Furthermore, proof of heritability of site-directed mutations is still lacking. The present study involves intergeneric pollination of wheat with cas9/guide RNA (gRNA)-transgenic maize to facilitate site-directed mutagenesis in any wheat germplasm of choice. For exemplification of this principle, new allelic variants were generated for the wheat genes BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and SEMI-DWARF 1 (SD1) which are involved in the regulation of plant height. The present approach relies on the expression of cas9 and wheat gene-specific gRNA in maize sperm cells. Therefore, transgenic maize carrying a ubiquitously expressed GFP was analysed, and conspicuous fluorescence was found in sperm cells (Figure 1a). Two Cas9/gRNA target motifs for TaBRI1 and one for TaSD1 were selected. These proved to be conserved across all two (AABB) or three (AABBDD) homeologues of the target genes in durum and bread wheat, respectively. Corresponding gRNAs were cloned into generic vectors used to transform maize (Budhagatapalli et al., 2016). Two hundred maize T0 plants carrying wheat target-specific cas9/gRNA-encoding T-DNAs were prescreened by qRT-PCR analysis. Per target motif, five maize transgenics with high cas9 and gRNA expression were selected for pollination of wheat (Figure 1b). Upon these intergeneric crosses, embryos were rescued in vitro. Regenerated wheat plants were then subjected to PCR-based mutation analysis by Sanger sequencing. For BRI1 target motif 1, three, two and one mutants were obtained out of 83, 44 and 10 plants in genotypes BW, W5 and D6, respectively. Two plants out of 4 and 3 carried mutations for BRI1 target motif 2 in genotypes W5 and D7, respectively. In addition, seven mutants for the SD1 target motif 1 were obtained from 17, 5 and 8 plants in genotypes BW, K15 and S96, respectively (Figure 1c). Subcloning and Sanger sequencing of target motif-derived amplicons of M1 plants indicated that all bread wheat mutants for BRI1 and SD1 were invariably homozygous, whereas those in durum genotypes D6 and D7 were chimeric (Figure 1c). This may be due to differences in cas9 and gRNA expression, the time point of male genome activation and the activity of DNA repair. In the present approach, mutations can be induced at various phases before and after the zygote undergoes mitosis (Figure 1d). Mutations induced during G1 and early S phase are more likely to occur owing to Cas9 and gRNA molecules pre-produced in the sperm rather than to zygotic de novo transgene expression. Resultant embryos are expectedly non-chimeric with regard to the induced mutations (Figure 1d-i). Alternatively, after chromatid duplication, Cas9 may trigger mutations in one chromatid or independently in either of the sister chromatids (Figure 1d-ii). In this scenario, the daughter cell that has received a mutated wheat chromatid during the first embryonic mitosis itself undergoes S phase, by which the mutated allele becomes genetically fixed across the two sister chromatids, while the other daughter cell has received a non-mutated or differently mutated chromatid and thus gives rise to a genetically distinct sector. Consequently, embryos formed via mutagenesis during G2 phase are expectedly chimeric (Figure 1d-ii). In the course of initial embryonic cell divisions upon wheat x maize crosses, maize chromosomes are eliminated due to asynchronous processing in terms of DNA replication, condensation and centromere formation (Laurie and Bennett, 1988). In total, 15 independent target gene-specific mutants were identified out of 174 wheat plants from which good-quality Sanger sequences of target motifs had been retrieved. Mutants were obtained in six wheat backgrounds, including the three spring-type bread wheats BW, W5 and K15, the winter-type bread wheat S96, and the two durum wheats D6 and D7 (Figure 1c). Mutations were found in all three target motifs addressed (Figure 1c). None of the 15 mutants carried any transgene. Across the genotypes, the efficiency in mutant plant formation ranged from 3.6% to 50% (Figure 1c). The BRI1 and SD1 genes are known to play an important role in plant height. Therefore, loss-of-function mutants may entirely fail to develop. In addition, knockouts of BRI1 and SD1 (GA20ox) in Arabidopsis lead to male sterility, as they regulate key genes of anther and pollen development (Plackett et al., 2012; Ye et al., 2010). The haploid plants obtained in the present work were subjected to colchicine treatment. As a result, 7 out of 15 mutants were fertile (Figure 1c). In M2, progenies of doubled-haploid mutants SD1-TM1-DH04-AABBdd of genotype K15 and SD1-TM1-DH07-AABBdd of genotype BW proved to have invariably inherited the very same mutations detected in their M1 progenitors (2-bp deletion and 48-bp insertion in the D subgenome, respectively) (Figure 1c). These primary mutants were thereby confirmed to be non-chimeric and true breeding. The M2 plants displayed a reduced plant height phenotype. At the anthesis stage, the height of tiller 1 exhibited an average reduction of 6 and 5 cm compared with the wild-type in genotypes K15 and BW, respectively (Figure 1e). The weak phenotype of these mutants is likely due to the still functional SD1 homeologues of the A and B genomes which may largely compensate the loss of function of the sd1 alleles of the D genome. In conclusion, the principle of haploid induction coupled with site-directed mutagenesis was exemplified in wheat using the two target genes BRI1 and SD1 which control the agronomically important trait plant height. Major advances achieved in this work include reduced genotype dependence of site-directed mutagenesis in wheat, the opportunity of creating a whole variety of mutations using just one cas9/gRNA-transgenic (pollinator) plant as well as the production of T-DNA-free and frequently homozygous M1 plants. There is still scope for increasing the efficiency of this approach, for example by stronger transgene expression at the relevant time point or by the development of improved protocols for in planta production of doubled haploids. We thank Andrea Müller, Petra Hoffmeister, Josef Bergstein, Margit Lang, Anke Halbach and her team, Janett Paper, Jenny Osterburg and Dr. Katja Kempe for their excellent support. We also thank Dr. Andreas Jacobi and Dr. Edgar Müller for selecting and providing appropriate wheat genotypes. The study was financially supported by the Federal Ministry of Food and Agriculture (FKZ 2814603113). The authors declare no conflicts of interest. J.K. conceived the project concept, and N.B., T.H., H.B. and A.E.M. designed and performed the experiments. N.B. and J.K. wrote the manuscript, and T.H., S.H. and A.E.M. reviewed and edited the manuscript. All authors read and approved the manuscript.

Upon the onset of inflammatory responses, bacterial pathogens are confronted with altered tissue microenvironments which can critically impact on their metabolic activity and growth. Changes in these parameters have however remained difficult to analyze over time, which would be critical to dissect the interplay between the host immune response and pathogen physiology. Here, we established an in vivo biosensor for measuring the growth rates of Staphylococcus aureus ( S . aureus ) on a single cell-level over days in an ongoing cutaneous infection. Using intravital 2-photon imaging and quantitative fluorescence microscopy, we show that upon neutrophil recruitment to the infection site and bacterial uptake, non-lethal dampening of S . aureus proliferation occurred. This inhibition was supported by NADPH oxidase activity. Therefore, reactive oxygen production contributes to pathogen containment within neutrophils not only by killing S . aureus , but also by restricting the growth rate of the bacterium.

The integrin LFA-1 (CD11a/CD18) plays a critical role in the interaction of T cells with antigen presenting cells (APCs) to promote lymphocyte differentiation and proliferation. This integrin can be present either in a closed or in an open active conformation and its activation upon T-cell receptor (TCR) stimulation is a critical step to allow interaction with APCs. In this study we demonstrate that the serine/threonine kinase Ndr2 is critically involved in the initiation of TCR-mediated LFA-1 activation (open conformation) in T cells. Ndr2 itself becomes activated upon TCR stimulation and phosphorylates the intracellular integrin binding partner Filamin A (FLNa) at serine 2152. This phosphorylation promotes the dissociation of FLNa from LFA-1, allowing for a subsequent association of Talin and Kindlin-3 which both stabilize the open conformation of LFA-1. Our data suggest that Ndr2 activation is a crucial step to initiate TCR-mediated LFA-1 activation in T cells.

JAK2-V617F–positive chronic myeloproliferative neoplasia (CMN) commonly displays dysfunction of integrins and adhesion molecules expressed on platelets, erythrocytes, and leukocytes. However, the mechanism by which the 2 major leukocyte integrin chains, b1 and b2, may contribute to CMN pathophysiology remained unclear. b1 (a4b1; VLA-4) and b2 (aLb2; LFA-1) integrins are essential regulators for attachment of leukocytes to endothelial cells. We here showed enhanced adhesion of granulocytes from mice with JAK2-V617F knockin (JAK2+/VF mice) to vascular cell adhesion molecule 1– (VCAM1-) and intercellular adhesion molecule 1–coated (ICAM1-coated) surfaces. Soluble VCAM1 and ICAM1 ligand binding assays revealed increased affinity of b1 and b2 integrins for their respective ligands. For b1 integrins, this correlated with a structural change from the lowto the high-affinity conformation induced by JAK2-V617F. JAK2-V617F triggered constitutive activation of the integrin inside-out signaling molecule Rap1, resulting in translocation toward the cell membrane. Employing a venous thrombosis model, we demonstrated that neutralizing anti– VLA-4 and anti–b2 integrin antibodies suppress pathologic thrombosis as observed in JAK2+/VF mice. In addition, aberrant homing of JAK2+/VF leukocytes to the spleen was inhibited by neutralizing anti-b2 antibodies and by pharmacologic inhibition of Rap1. Thus, our findings identified cross-talk between JAK2-V617F and integrin activation promoting pathologic thrombosis and abnormal trafficking of leukocytes to the spleen. Research Article Hematology

Growing sugar beet (Beta vulgaris L. ssp. vulgaris) as a winter crop in cool temperate climates is expected to increase yield potential. However, this requires bolting resistance after winter. One strategy to achieve complete bolting resistance is to accumulate genes for bolting delay from various genetic resources within the B.vulgaris gene pool. To identify such genes, a QTL mapping was performed in a segregating population derived from a biennial leaf beet with delayed bolting after winter. The population was tested for bolting delay after winter in two different experiments with natural or artificial vernalization. Three QTL for bolting delay were mapped on linkage groups 3, 5 and 9 affecting bolting time by up to 19 days. These QTL could be combined with recently reported bolting QTL to develop a winter sugar beet with complete bolting resistance.

Hepatotropic viruses such as hepatitis C virus cause life-threatening chronic liver infections in millions of people worldwide. Targeted in vivo antigen-delivery to cross-presenting dendritic cells (DCs) has proven to be extraordinarily efficient in stimulating antigen-specific T cell responses. To determine whether this approach would as well be suitable to induce local antiviral effector T cells in the liver we compared different vaccine formulations based on either the targeting of DEC-205 or TLR2/6 on cross-presenting DCs or formulations not involving in vivo DC targeting. As read-outs we used in vivo hepatotropic adenovirus challenge, histology and automated multidimensional fluorescence microscopy (MELC). We show that targeted in vivo antigen delivery to cross-presenting DCs is highly effective in inducing antiviral CTLs capable of eliminating virus-infected hepatocytes, while control vaccine formulation not involving DC targeting failed to induce immunity against hepatotropic virus. Moreover, we observed distinct patterns of CD8+ T cell interaction with virus-infected and apoptotic hepatocytes in the two DC-targeting groups suggesting that the different vaccine formulations may stimulate distinct types of effector functions. Our findings represent an important step toward the future development of vaccines against hepatotropic viruses and the treatment of patients with hepatic virus infection after liver transplantation to avoid reinfection.