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.

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.

The virulence of intracellular pathogens relies largely on the ability to survive and replicate within phagocytes but also on release and transfer into new host cells. Such cell-to-cell transfer could represent a target for counteracting microbial pathogenesis. However, our understanding of the underlying cellular and molecular processes remains woefully insufficient. Using intravital 2-photon microscopy of caspase-3 activation in the Leishmania major-infected (L. major-infected) live skin, we showed increased apoptosis in cells infected by the parasite. Also, transfer of the parasite to new host cells occurred directly without a detectable extracellular state and was associated with concomitant uptake of cellular material from the original host cell. These in vivo findings were fully recapitulated in infections of isolated human phagocytes. Furthermore, we observed that high pathogen proliferation increased cell death in infected cells, and long-term residency within an infected host cell was only possible for slowly proliferating parasites. Our results therefore suggest that L. major drives its own dissemination to new phagocytes by inducing host cell death in a proliferation-dependent manner.

Cell fate determinants and polarity regulators influence the fine balance between self-renewal and differentiation in HSC and leukemic stem cells (LSC). Previously our group provided first evidence that inactivation of the cell fate regulator Llgl1 in murine HSCs leads to alterations in self-renewal, proliferative capacity and differentiation (Heidel et al., 2013). The impact of Llgl1 on malignant transformation and especially acute myeloid leukemia (AML) development had not been investigated in detail. Inactivation of LLGL1 in several human AML cell lines (MOLM13, HEL, MV4;11, HL-60) by CRISPR/Cas9 resulted in decreased proliferation of leukemic cells. Consistently, xenograft studies using LLGL1-depleted human AML cells resulted in delayed disease development compared to non-targeting control (median survival NT-control: 37.5 days vs. LLGL1-KD: 92 days; p=0.0002). Genetic inactivation of LLGL1 in primary AML patient cells resulted in reduced colony formation in vitro and improved survival of recipient mice in a patient-derived xenograft model of AML. To recapitulate these findings in defined model systems of murine AML, we used a conditional Llgl1 knockout mouse model (Klezovitch et al, 2004) and intercrossed it with AML-models harboring different driver mutations: MllAF9 (Cerral et al., 1995) or Flt3ITD/ITD (Lee et al., 2006). Conditional deletion of Llgl1 in MllAF9 knockin mice using an Mx1-Cre-recombinase (Cre+) resulted in significant delay of leukemia development (median survival +/+: 174 days; -/-: 203 days; p=0.0361). To confirm these findings, we investigated a second oncogenic model and induced deletion of Llgl1 in Flt3ITD/ITD knockin mice. Leukemic Flt3ITD/ITD; Llgl1+/+; Cre+ animals (n=12) showed rapid onset of leukemia development, whereas the Flt3ITD/ITD; Llgl1-/-; Cre+ animals (n=25) showed significant delay in leukemia progression (median survival +/+: 38 days; -/-: 51 days; p=0.0005). Llgl1-/- animals show reduced peripheral blast counts (WBC +/+: 332.558 Gpt/l; -/-: 141.083 Gpt/l; p=0.0012) and decreased organ infiltration. Importantly, immunophenotypic analysis of bone marrow (BM) compartments indicated a shift of immature cells towards a more differentiated state. The overall abundance of leukemic Lin-Kit+Sca-1+ (LSK) cells was reduced in Flt3ITD/ITD; Llgl1-/- animals compared to Flt3ITD/ITD; Llgl1+/+ controls (LSK +/+: 13099±3144/106 BMC; -/-: 8117±2810 /106 BMC; p=0.0227). In contrast, we found increased numbers of Lin-Kit+Sca-1+FcgR+CD34+ of leukemic granulocyte-macrophage progenitors (LGMPs: +/+: 8306±1963/106 BMC; -/-: 14755±4783/106 BMC; p=0.0141) upon deletion of Llgl1 in Flt3ITD/ITD animals. These findings indicate that genetic inactivation of Llgl1 results in loss of leukemia stemness and shifts the leukemia stem cell phenotype from HSC-like to GMP-like LSCs. To test for this hypothesis, we performed global transcriptome analysis of sorted LSK cells of Flt3ITD/ITD mice early after genetic deletion of Llgl1. Among the significantly downregulated genes, we found several members of the HoxA-cluster, consistent with the immunophenotypic loss of stemness. Gene-set-enrichment analysis revealed downregulation of leukemia stem cell signatures (Gal et al., 2006) and enrichment of a gene set associated with LGMPs (compared to leukemic-HSC) signatures (Krivtsov et al., 2006). To validate whether loss of Hox-gene expression is relevant for impaired leukemia development, we performed rescue experiments in vivo. LSK cells derived from Flt3ITD/ITD; Llgl1+/+ or Flt3ITD/ITD; Llgl1-/- animals were retrovirally transduced with HoxA9 or empty vector control (eV). When transplanted into sublethally irradiated recipient mice, overexpression of HoxA9 (OE) in Llgl1-/-; Flt3ITD/ITD cells rescued the disease phenotype comparable to Flt3ITD/ITD; Llgl1+/+ cells (median survival +/+;eV: 62.50%; -/-; OE: 55.56%). In contrast, empty vector control Flt3ITD/ITD; Llgl1-/- recipients showed no disease development (median survival -/-; eV: 100%). Taken together our results may provide first evidence for a functional role of Llgl1 in models of AML. In contrast to its function in normal HSCs, deletion of Llgl1 appears to reduce stemness of leukemic stem cells and impair leukemia development in vitro and in vivo. Experiments to assess for the effects of Llgl1 on LSC polarity and cell fate decisions are currently under way.

Background One of the most common complications of hip arthroplasty is excessive blood loss that could necessitate allogenic blood transfusion, which is further associated with other complications, such as infections, transfusion reactions or immunomodulation. In gynecology, 4DryField®PH, an absorbable polysaccharide-based formulation, is used for hemostasis and adhesion prophylaxis. In this study, we evaluated its hemostatic effect in patients undergoing hip bipolar hemiarthroplasty following intracapsular femoral neck fracture. Methods We studied 40 patients with intracapsular femoral neck fractures (Garden III or IV) admitted at our institution between July 2016 and November 2017. We included patients above 60 years with simple fracture and without pathologic fractures. Patients were randomized into intervention and control groups. The intervention group received 5 g of 4DryField® PH (subfascially and subcutaneously) during wound closure. Three drainages were inserted in a standardized manner (submuscular, subfascial, and subcutaneous) and drainage volume was measured immediately before extraction. Total blood loss was calculated using Mercuriali’s formula and standard hemograms upon admission and five days after surgery. Volume of postoperative hematoma was measured using point-of-care ultrasound seven days after surgery. Results Volume of the postoperative hematoma was reduced by 43.0 mL. However, significant reduction of total blood loss and drainage volume was not observed. Conclusions We observed that 4DryField® PH had a local hemostatic effect, thereby reducing volume of the postoperative hematoma. However, this reduction was small and had no effect on the total blood loss. Further studies are warranted to improve the application algorithm. Trial registration DRKS, DRKS00017452 , Registered 11 June 2019 – Retrospectively registered.

Nitric oxide (NO) is an important antimicrobial effector but also prevents unnecessary tissue damage by shutting down the recruitment of monocyte-derived phagocytes. Intracellular pathogens such as Leishmania major can hijack these cells as a niche for replication. Thus, NO might exert containment by restricting the availability of the cellular niche required for efficient pathogen proliferation. However, such indirect modes of action remain to be established. By combining mathematical modeling with intravital 2-photon biosensors of pathogen viability and proliferation, we show that low L. major proliferation results not from direct NO impact on the pathogen but from reduced availability of proliferation-permissive host cells. Although inhibiting NO production increases recruitment of these cells, and thus pathogen proliferation, blocking cell recruitment uncouples the NO effect from pathogen proliferation. Therefore, NO fulfills two distinct functions for L. major containment: permitting direct killing and restricting the supply of proliferation-permissive host cells.

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.

Janus kinases (JAKs) mediate responses to cytokines, hormones and growth factors in haematopoietic cells1,2. The JAK gene JAK2 is frequently mutated in the ageing haematopoietic system3,4 and in haematopoietic cancers5. JAK2 mutations constitutively activate downstream signalling and are drivers of myeloproliferative neoplasm (MPN). In clinical use, JAK inhibitors have mixed effects on the overall disease burden of JAK2-mutated clones6,7, prompting us to investigate the mechanism underlying disease persistence. Here, by in-depth phosphoproteome profiling, we identify proteins involved in mRNA processing as targets of mutant JAK2. We found that inactivation of YBX1, a post-translationally modified target of JAK2, sensitizes cells that persist despite treatment with JAK inhibitors to apoptosis and results in RNA mis-splicing, enrichment for retained introns and disruption of the transcriptional control of extracellular signal-regulated kinase (ERK) signalling. In combination with pharmacological JAK inhibition, YBX1 inactivation induces apoptosis in JAK2-dependent mouse and primary human cells, causing regression of the malignant clones in vivo, and inducing molecular remission. This identifies and validates a cell-intrinsic mechanism whereby differential protein phosphorylation causes splicing-dependent alterations of JAK2–ERK signalling and the maintenance of JAK2V617F malignant clones. Therapeutic targeting of YBX1-dependent ERK signalling in combination with JAK2 inhibition could thus eradicate cells harbouring mutations in JAK2. Inhibition of YBX1, a downstream target of the Janus kinase JAK2, sensitizes myeloproliferative neoplasm cells to JAK and could provide a means to eradicate such cells in human haematopoietic cancers.

The survival of peripheral T cells is dependent on their access to peripheral LNs (pLNs) and stimulation by IL‐7. In pLNs fibroblastic reticular cells (FRCs) and lymphatic endothelial cells (LECs) produce IL‐7 suggesting their contribution to the IL‐7‐dependent survival of T cells. However, IL‐7 production is detectable in multiple organs and is not restricted to pLNs. This raises the question whether pLN‐derived IL‐7 is required for the maintenance of peripheral T cell homeostasis. Here, we show that numbers of naive T cells (TN) remain unaffected in pLNs and spleen of mice lacking Il7 gene activity in pLN FRCs, LECs, or both. In contrast, frequencies of central memory T cells (TCM) are reduced in FRC‐specific IL‐7 KO mice. Thus, steady state IL‐7 production by pLN FRCs is critical for the maintenance of TCM, but not TN, indicating that both T cell subsets colonize different ecological niches in vivo.

Purpose Cell fate determinants Scrib and Llgl1 influence self-renewal capacity of hematopoietic stem cells (HSCs). Scrib-deficient HSCs are functionally impaired and lack sufficient repopulation capacity during serial transplantation and stress. In contrast, loss of Llgl1 leads to increased HSC fitness, gain of self-renewal capacity and expansion of the stem cell pool. Here, we sought to assess for shared and unique molecular functions of Llgl1 and Scrib by analyzing their interactome in hematopoietic cells. Methods Interactome analysis was performed by affinity purification followed by mass spectrometry. Motility, migration and adhesion were assessed on primary murine HSCs, which were isolated by FACS sorting following conditional deletion of Scrib or Llgl1, respectively. Imaging of Scrib-deficient HSCs was performed by intravital 2-photon microscopy. Results Comparison of Scrib and Llgl1 interactome analyses revealed involvement in common and unique cellular functions. Migration and adhesion were among the cellular functions connected to Scrib but not to Llgl1. Functional validation of these findings confirmed alterations in cell adhesion and migration of Scrib-deficient HSCs in vitro and in vivo. In contrast, genetic inactivation of Llgl1 did not affect adhesion or migratory capacity of hematopoietic stem cells. Conclusion Our data provide first evidence for an evolutionarily conserved role of the cell fate determinant Scrib in HSC adhesion and migration in vitro and in vivo, a unique function that is not shared with its putative complex partner Llgl1.

Cell fate determinants influence self-renewal potential of hematopoietic stem cells. Scribble and Llgl1 belong to the Scribble polarity complex and reveal tumor-suppressor function in drosophila. In hematopoietic cells, genetic inactivation of Llgl1 leads to expansion of the stem cell pool and increases self-renewal capacity without conferring malignant transformation. Here we show that genetic inactivation of its putative complex partner Scribble results in functional impairment of hematopoietic stem cells (HSC) over serial transplantation and during stress. Although loss of Scribble deregulates transcriptional downstream effectors involved in stem cell proliferation, cell signaling, and cell motility, these effectors do not overlap with transcriptional targets of Llgl1. Binding partner analysis of Scribble in hematopoietic cells using affinity purification followed by mass spectometry confirms its role in cell signaling and motility but not for binding to polarity modules described in drosophila. Finally, requirement of Scribble for self-renewal capacity also affects leukemia stem cell function. Thus, Scribble is a regulator of adult HSCs, essential for maintenance of HSCs during phases of cell stress.

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, β1 and β2, may contribute to CMN pathophysiology remained unclear. β1 (α4β1; VLA-4) and β2 (αLβ2; 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 β1 and β2 integrins for their respective ligands. For β1 integrins, this correlated with a structural change from the low- to 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-β2 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-β2 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.

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

The family of cold shock proteins (CSPs) is highly conserved and consists of 8 members, including Ybx1-3, Csde1 and Lin28. Ybx1 is a multifunctional DNA/RNA binding protein that modulates gene transcription and translation during inflammation and malignant transformation. Recently, our group identified Ybx1 as a mediator of Jak2 signaling in MPN that protects Jak2-mutated cells from Jak-inhibitor induced apoptosis.

Campylobacter jejuni is one of the leading infectious causes of food-borne illness around the world. Its ability to persistently colonize the intestinal tract of a broad range of hosts, including food-producing animals, is central to its epidemiology since most infections are due to the consumption of contaminated food products. Using a highly saturated transposon insertion library combined with next-generation sequencing and a mouse model of infection, we have carried out a comprehensive genome-wide analysis of the fitness determinants for growth in vitro and in vivo of a highly pathogenic strain of C. jejuni. A comparison of the C. jejuni requirements to colonize the mouse intestine with those necessary to grow in different culture media in vitro, combined with isotopologue profiling and metabolic flow analysis, allowed us to identify its metabolic requirements to establish infection, including the ability to acquire certain nutrients, metabolize specific substrates, or maintain intracellular ion homeostasis. This comprehensive analysis has identified metabolic pathways that could provide the basis for the development of novel strategies to prevent C. jejuni colonization of food-producing animals or to treat human infections.

Several genes and signaling pathways control the fine balance between self-renewal and differentiation in hematopoietic stem cells and potentially also in leukemic stem cells (LSC). Phospholipase C family members are key mediators of calcium signaling which play an important role in differentiation and proliferation of immune cells but also contribute to malignant transformation and tumorigenesis. Plcg1 is highly expressed in hematopoietic stem- and progenitor cells and also in myeloid leukemia. Plcg1 gets activated by cell extrinsic receptor stimulation and integrates signals from the cell surface. Its influence on proliferation and differentiation of hematopoietic cells may be largely independent of other bone fide mediators of self-renewal and stem cell viability such as STAT-, MEK-ERK or AKT-signaling. To which extent Plcg1-dependent signal integration is required for function and maintenance of leukemic stem cells remained so far elusive.

SummaryThermophilic Campylobacter species colonize the intestine of agricultural and domestic animals commensally but cause severe gastroenteritis in humans. In contrast to other enteropathogenic bacteria, Campylobacter has been considered to be non‐glycolytic, a metabolic property originally used for their taxonomic classification. Contrary to this dogma, we demonstrate that several Campylobacter coli strains are able to utilize glucose as a growth substrate. Isotopologue profiling experiments with 13C‐labeled glucose suggested that these strains catabolize glucose via the pentose phosphate and Entner‐Doudoroff (ED) pathways and use glucose efficiently for de novo synthesis of amino acids and cell surface carbohydrates. Whole genome sequencing of glycolytic C. coli isolates identified a genomic island located within a ribosomal RNA gene cluster that encodes for all ED pathway enzymes and a glucose permease. We could show in vitro that a non‐glycolytic C. coli strain could acquire glycolytic activity through natural transformation with chromosomal DNA of C. coli and C. jejuni subsp. doylei strains possessing the ED pathway encoding plasticity region. These results reveal for the first time the ability of a Campylobacter species to catabolize glucose and provide new insights into how genetic macrodiversity through intra‐ and interspecies gene transfer expand the metabolic capacity of this food‐borne pathogen.

The optimal treatment for proximal humeral fractures remains under debate. In this article, we report the mid-term results of patients who underwent the less-invasive implantation of a polyaxial locking plate for displaced proximal humeral fractures.

Proximal humeral fractures remain a surgical challenge, and scientific discussions are commonly focused on their ideal treatment. One possible treatment involves the use of an angle stable plate osteosynthesis. However, which material can most feasibly be used to attach the greater tuberosity to the implant remains unknown. In two prospective, non-randomized trials, we compared the results of a FiberWire® and a wire cerclage. A total of 104 patients with 3- and 4-part fractures were included in this examination. In 25 cases, the greater tuberosity was fixated with a FiberWire®, size 2, while 79 cases received a wire cerclage. Plate osteosynthesis was constantly performed via the anterolateral delta-split approach using the NCB®-PH-plate by Zimmer®. The patients were followed clinically at discharge, 6 weeks and 6 months postoperatively and were examined for relevant complications. Age and gender were equally distributed in both groups. Concerning the follow-up after 6 weeks, a significant benefit concerning shoulder function was observed in the FiberWire®-group (wire cerclage: 39.20±11.85, 95% CI 32.37-44.56, FiberWire®: 45.84±16.80, 95% CI 28.34-61.56: p=0.049). After 6 months, the difference between the groups was not significant (wire cerclage: 58.13±18.73, 95% CI 50.25-72.40, FiberWire®: 68.85±23.10, 95% CI 46.83-99.53: p=0.06).

Scribble-complex is a polarity module that has been described to regulate cell polarity as well as migration, adhesion and cellular architecture in several cellular models. Recently, we provided first evidence that loss of the putative Scribble complex member Llgl1 enhances fitness and self-renewal potential of hematopoietic stem cells (HSCs) in vivo. However, deletion by itself did not cause leukemia. Here, we further investigate the role of putative complex partners Llgl1 and Scribble in murine HSC function. We used a previously published conditional knockout mouse models for Llgl1 and Scribble crossed with an inducible Mx1-Cre-recombinase. Administration of pIpC led to complete deletion of Scribble (Scrib-/-) and Llgl1 (Llgl1-/-) as determined by PCR. Following deletion of Scribble steady state hematopoiesis was not impaired, however, (in contrast to the phenotype observed for Llgl1-/- HSCs) genetic inactivation of Scribble led rather to a functional decrease of HSC repopulation capacity in serial transplantations. Tumor suppressor activity appeared to be conserved for Llgl1 but not for Scribble depending on the oncogenic background inducing acute myeloid leukemia in vivo. Global gene expression profiling of the respective Llgl1-/- and Scrib-/- HSC subsets and proteomic analysis of Scrib complex binding partners are currently under way to further dissect the opposing phenotypes of Scribble complex partners observed in hematopoietic and leukemic stem cell function.