Hematopoietic stem and progenitor cells (HSPCs) are localized within specialized niches of the bone marrow (BM). However, during hematological disorders or infections, the functionality of these cells in the BM is compromised, leading to extramedullary hematopoiesis (EMH). Chronic inflammation drives EMH, yet its impact on HSPCs outside the BM is poorly understood. Using a mouse model of chronic autoinflammatory disease, we demonstrated the presence of extramedullary HSPCs in blood, spleen, and inflamed tails and paws. Single-cell transcriptomics revealed a unique expression profile in extramedullary HSCs, with significant up-regulation of Cd53, MHCII-associated, and immunosuppressive genes. We further demonstrated that extramedullary CD53+ HSPCs act as antigen-presenting cells, promoting the development of regulatory T cells (Treg cells) to control chronic inflammation at extramedullary sites. Conversely, Treg cells exert a protective role on extramedullary HSPCs. Together, our findings revealed a mutually supportive relationship between a unique subset of HSPCs and T cells in inflamed tissues during chronic inflammation.

WBP1L is a broadly expressed transmembrane adaptor protein involved in regulating hematopoietic stem cell function and T cell development. It interacts with NEDD4-family E3 ubiquitin ligases and regulates important chemokine receptor CXCR4. Using tandem affinity purification coupled with mass spectrometry, we identified novel WBP1L interactions with the IFNγ receptor and the Cullin-RING ubiquitin ligases CRL1β-TrCP1/2. We found that WBP1L interaction with the IFNγ receptor serves to downregulate proximal IFNγ receptor signaling in female macrophages, while the interaction with CRL1β-TrCP1/2 ubiquitin ligases regulates WBP1L protein levels. Disrupting this interaction, as well as inhibiting proteasome activity or neddylation, increased WBP1L protein levels, demonstrating that CRL1β-TrCP1/2 ubiquitin ligases regulate WBP1L protein abundance. These data provide important insights into the mechanisms controlling WBP1L function.

Chronic recurrent multifocal osteomyelitis (CRMO) is a childhood autoinflammatory disorder characterized by spontaneous sterile inflammation and destruction of bone tissue with unclear pathophysiology and no specific diagnostic markers. Although in murine models the disease is mediated by neutrophils harboring a mutation of a negative regulator of immune response—adaptor protein Pstpip2, this cell type was never comprehensively analyzed in patients with CRMO and no mutations or SNPs in human PSTPIP2 associated with human disease were found.In this study, we analyzed neutrophils from 34 patients with CRMO and 7 age-matched controls. In agreement with murine models, we observed hyperactivation of neutrophils represented by increased CD62L shedding and upregulation of proinflammatory pathways (such as TLRs, NFκB, or interferon response) in patients with CRMO. Expression of IL-1 was not elevated in CRMO neutrophils, suggesting IL-1 is not a crucial mediator of the human disease. This finding is supported by the inefficiency of anti-IL-1 therapy in patients with CRMO. Importantly, we found reduced PSTPIP2 protein level in CRMO neutrophils, providing the evidence for a putative mechanistic link between the molecular causes of CRMO and its murine model – reduced levels of PSTPIP2. In search for a potential diagnostic markers, we identified CRMO-specific neutrophil gene signature that strongly correlates with disease severity and the age of disease onset.Together, our data suggest that, similar to murine models, pathophysiology of CRMO involves hyperactivation of neutrophils accompanied by reduced expression of PSTPIP2 but without clinically relevant contribution of IL1. We described the transcriptome of neutrophils in CRMO, identified deregulated pathways, and defined CRMO-specific neutrophil gene signature, providing a solid base for the future development of CRMO diagnostic tools.

Hematopoietic stem cells (HSCs) are localized within specialized niches of the bone marrow (BM). However, during hematological disorders or infections, the functionality of HSCs in the BM is compromised, leading to extramedullary hematopoiesis (EMH). Chronic inflammation drives EMH, yet its impact on HSCs outside the BM is poorly understood. Using a mouse model of chronic autoinflammatory disease, we demonstrated the presence of extramedullary HSCs in blood, spleen, and inflamed tails and paws. Single-cell transcriptomics revealed a unique expression profile in extramedullary HSCs, with significant upregulation of Cd53, MHCII-associated, and immunosuppressive genes. We further demonstrated that extramedullary CD53+HSCs act as antigen-presenting cells, promoting the development of regulatory T cells (Tregs) to control chronic inflammation at extramedullary sites. Conversely, Tregs exert a protective role on extramedullary HSCs. Altogether, our findings revealed a mutually supportive relationship between a unique subset of HSCs and T cells in inflamed tissues during chronic inflammation. ### Competing Interest Statement The authors have declared no competing interest.

Styrene-maleic acid (SMA) and similar amphiphilic copolymers are known to cut biological membranes into lipid nanoparticles/nanodiscs containing membrane proteins apparently in their relatively native membrane lipid environment. Our previous work demonstrated that membrane raft microdomains resist such disintegration by SMA. The use of SMA in studying membrane proteins is limited by its heterogeneity and the inability to prepare defined derivatives. In the present paper, we demonstrate that some amphiphilic peptides structurally mimicking SMA also similarly disintegrate cell membranes. In contrast to the previously used copolymers, the simple peptides are structurally homogeneous. We found that their membrane-disintegrating activity increases with their length (reaching optimum at 24 amino acids) and requires a basic primary structure, that is, (XXD)n, where X represents a hydrophobic amino acid (optimally phenylalanine), D aspartic acid, and n is the number of repeats of these triplets. These peptides may provide opportunities for various well-defined potentially useful modifications in the study of membrane protein biochemistry. Our present results confirm a specific character of membrane raft microdomains.

The knowledge about the contribution of the innate immune system to health and disease is expanding. However, to obtain reliable results, it is critical to select appropriate mouse models for in vivo studies. Data on genetic and phenotypic changes associated with different mouse strains can assist in this task. Such data can also facilitate our understanding of how specific polymorphisms and genetic alterations affect gene function, phenotypes, and disease outcomes. Extensive information is available on genetic changes in all major mouse strains. However, comparatively little is known about their impact on immune response and in particular on innate immunity. Here, we analyzed a mouse model of chronic multifocal osteomyelitis (CMO), an autoinflammatory disease driven exclusively by the innate immune system, which is caused by an inactivating mutation in the Pstpip2 gene. We investigated how the genetic background of BALB/c, C57BL/6J, and C57BL/6NCrl strains alters the molecular mechanisms controlling disease progression. While all mice developed the disease, symptoms were significantly milder in BALB/c and partially also in C57BL/6J when compared to C57BL/6NCrl. Disease severity correlated with the number of infiltrating neutrophils and monocytes and with the production of chemokines attracting these cells to the site of inflammation. It also correlated with increased expression of genes associated with autoinflammation, rheumatoid arthritis, neutrophil activation, and degranulation, resulting in altered neutrophil activation in vivo. Together, our data demonstrate striking effects of genetic background on multiple parameters of neutrophil function and activity influencing the onset and course of the CMO disease.

In steady-state hematopoiesis, hematopoietic stem cells (HSCs) reside in specialized niches of the bone marrow (BM). Nevertheless, during hematological disorders or infections, HSC function in the BM is diminished, and there is a need to create new sites of hematopoiesis. This compensatory mechanism is known as extramedullary hematopoiesis (EMH). Remarkably, chronic inflammation promotes EMH, but the impact of chronic inflammation on HSCs outside the BM is poorly understood. Here, using mice suffering from a progressive chronic autoinflammatory disease described as chronic multifocal osteomyelitis (CMO), we observed increased numbers of functional HSCs in blood, spleen, and inflamed paws of CMO mice compared to WT controls. Single cell transcriptomics revealed that HSCs in CMO EMH sites have a unique expression profile, characterized by upregulated Cd53 that distinguishes HSCs from BM HSCs in WT or CMO mice. This upregulation correlates positively with MHCII-associated and immunosuppressive genes such as Cd274 (PD-L1) and Icosl. We observed that CD53+ HSCs isolated from CMO EMH sites displayed lower proliferation but enhanced myeloid colony-forming capacity in comparison to CD53- HSCs. Further, CD53+Lin-c-kit+ cells isolated from CMO EMH sites strongly induced proliferation and survival of T cells in comparison to CD53-Lin-c-kit+ cells. Additionally, CD53+Lin-c-kit+ also promoted the development of Tregs in vitro. Altogether, our findings revealed a unique type of EMH HSCs characterized by enhanced expression of CD53, MHCII, and immunosuppressive genes. Notably, under chronic inflammation, EMH CD53+ HSCs might act as antigen presenting cells inducing naive T cell proliferation and the development of Tregs, suggesting an immunoregulatory role at the periphery. This work was supported by GACR24-10938S and IMG institutional funding RVO68378050.

WW domain binding protein 1-like (WBP1L), also known as outcome predictor of acute leukemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which was shown to correlate with ETV6-RUNX1 translocation and favorable prognosis in childhood leukemia. It has a broad expression pattern in hematopoietic and non-hematopoietic cells. Our previous work described WBP1L as a regulator of CXCR4 signaling and hematopoiesis. Here, we show that hematopoiesis in the mice with Wbp1l germline deletion is dysregulated, already at the level of hematopoietic stem cells and early progenitors. We further demonstrate that thymi of WBP1L-deficient mice are significantly enlarged and contain increased numbers of thymocytes of all subsets. This can potentially be explained by increased generation of multipotent progenitors 4 (MPP4) in the bone marrow, from which the thymus-seeding progenitors are derived. We also observed increases in multiple cell types in the blood. In addition, we show that WBP1L regulates hematopoietic stem cell functionality and leukocyte progenitor proliferation and gene expression during hematopoietic stem and progenitor cell transplantation, which contribute to more efficient engraftment of WBP1L-deficient cells. WBP1L thus emerges as a regulator of hematopoietic stem and progenitor cell function, which controls leukocyte numbers at the steady state and after bone marrow transplantation.

Neutrophilic inflammation is a hallmark of many monogenic autoinflammatory diseases; pathomechanisms that regulate extravasation of damaging immune cells into surrounding tissues are poorly understood. Here we identified three unrelated boys with perinatal-onset of neutrophilic cutaneous small vessel vasculitis and systemic inflammation. Two patients developed liver fibrosis in their first year of life. Next-generation sequencing identified two de novo truncating variants in the Src-family tyrosine kinase, LYN , p.Y508*, p.Q507* and a de novo missense variant, p.Y508F, that result in constitutive activation of Lyn kinase. Functional studies revealed increased expression of ICAM-1 on induced patient-derived endothelial cells (iECs) and of β2-integrins on patient neutrophils that increase neutrophil adhesion and vascular transendothelial migration (TEM). Treatment with TNF inhibition improved systemic inflammation; and liver fibrosis resolved on treatment with the Src kinase inhibitor dasatinib. Our findings reveal a critical role for Lyn kinase in modulating inflammatory signals, regulating microvascular permeability and neutrophil recruitment, and in promoting hepatic fibrosis.

Background: Chronic inflammation (CI) is a hallmark of autoinflammatory disorders and is characterized by excessive production of cytokines. CI has been proposed as a factor that promotes solid tumor progression and metastasis. Accordingly, population-based studies identified a history of CI diseases as a risk factor for leukemia development. In order to investigate how CI affects hematopoietic stem cells (HSCs), as well as how it promotes leukemia development, we employed a mouse model suffering from chronic multifocal osteomyelitis (CMO). CMO mice exhibit a progressive autoinflammatory disorder that resembles human chronic recurrent multifocal osteomyelitis. CMO mice are asymptomatic until 7 weeks old, and after develop swollen paws, tail kinks, increased bone marrow (BM) cellularity, number of granulocytes, and production of cytokines. Aims: To investigate (1) how sterile CI affects HSCs, (2) identify the mechanisms that are mediating these effects, and (3) determine whether the inflammatory environment can accelerate AML development. Methods: Flow cytometry, generation of murine chimeras, limiting dilution transplantation assays, competitive BM transplantations, RNA sequencing, intraperitoneal injection of IL-6-, IL-6R-blocking antibody or Stat3 inhibitor, phospho-flow analysis, survival assays, MLL-AF9 retroviral infection, TP53 deficient mouse model Results: Our data showed that CMO HSCs are expanded in asymptomatic mice, and that this expansion continued as the mice grew older. Limiting dilution transplantation assays indicated that CMO HSCs are functionally impaired. Interestingly, MyD88-deficient CMO mice did not develop the inflammatory phenotype, however their HSC population was still expanded and functionally impaired. Next, we generated chimeras to investigate the effect of the CMO immune compartment and the CMO BM niche on HSCs. Our results showed that both CMO compartments affect HSC functionality. Furthermore, RNAseq analysis suggested that the loss of HSC function is in part mediated by the IL-6/Jak/Stat3 signaling pathway. Indeed, we detected increased levels of IL-6 in CMO serum, BM, as well as increased pStat3 levels in CMO HSCs. Treatment of CMO mice with IL-6- or IL-6R-blocking antibody significantly prevented the HSC expansion, while targeting pStat3 prevented their expansion and functional impairment. Remarkably, MLL-AF9 leukemic cells demonstrated a faster leukemic onset in CMO mice than in WT mice, and they exhibited enhanced growth in vitro when IL-6 was added to the cultures. Next, we investigated whether CI promotes cancer progression in a TP53 deficient mouse model. We crossed CMO mice to TP53-deficient mice, and monitored their survival. Remarkably, CMO/Tp53+/- double mutants showed significantly accelerated tumor development and succumbed to disease faster than Tp53+/- non-inflammatory mice. TP53 deficient mice do not exhibit myeloid transformation, however, we observed AML development in one CMO/Tp53+/- mouse, suggesting that CI might induce AML transformation in some genetically predisposed murine models. Summary/Conclusion: Altogether, our data indicate that CI has a detrimental effect on HSCs and highlight the possibility of adding clinically available Stat3 inhibitors to the current treatment in order to preserve stem cell functions. Further, our results suggest that CI acts as an additional factor in the development of AML, providing additional understanding of the mechanisms of transformation. The work was partially funded by the National Institute for Cancer Research (Program EXCELES, ID Project No. LX22NPO5102)-Funded by the European Union-Next Generation EU. Keywords: Inflammation, MLL, STAT3, Bone marrow niche

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: Chronic inflammation is a condition characterized by a long-term increased in cytokines and chemokines. It has been reported that chronic inflammatory conditions favor the establishment of new hematopoietic sites outside of the bone marrow (BM), a condition known by the term of extramedullary hematopoiesis (EMH). EMH has been described in pathological conditions in which the BM is inefficient and unable to fulfill the hematopoietic demands. Nevertheless, little is known about how chronic inflammation induces EMH, how it affects hematopoietic stem cells (HSCs), and what are the mechanisms leading to it. Here, we employed a mouse model of Chronic Multifocal Osteomyelitis (CMO) which develops sterile chronic inflammation due to a mutation in the Pstpip2 gene. CMO mice exhibit expansion of the myeloid compartment, swollen paws, and kinks in the tail. Recently, we reported that HSC in the BM of CMO mice present reduced functionality due to hyperactivation of the IL-6/Jak/Stat3 signaling pathway. Aims: The aims of this study are (1) to determine whether CMO mice develop EMH, (2) to functionally characterized the HSCs in EMH sites in CMO mice, and (3) to decipher the mechanisms that promote EMH under chronic inflammatory conditions. Methods: Flow cytometry assays, transplantation assays, generation of chimeras, extreme limiting dilution transplantation assays, in vitro cultivation, RT-PCR, ELISA, gelatinase assays, MMP9 administration in vivo, genome-wide gene expression profile, mass spectrometry. Results: Since EMH is commonly accompanied by the presence of HSCs in circulation, we assessed the presence of Lin-ckit+Sca-1+CD48-CD150+ cells in peripheral blood of CMO mice. We observed increased number of HSCs in circulation of CMO mice, and colony culture assays and murine transplantation experiments demonstrated that these HSCs were functional. Next, we investigated whether CMO mice had HSCs in spleen, a common site of EMH. We observed that CMO mice exhibited higher number of HSCs in spleen, however their functionality was reduced in comparison to wt splenic HSCs. These observations prompted us to search for additional sites of EMH which could further support hematopoiesis under chronic inflammatory conditions. Interestingly, we detected the presence of HSCs in the inflamed paws of CMO mice, and remarkably paw HSCs were able to engraft and reconstitute hematopoiesis in lethally irradiated mice. Further, we investigated which compartment was mediating the occurrence of EMH in CMO mice. Using murine chimeras, we observed that while the BM niche from CMO mice was not mediating the presence of HSCs in the sites of EMH, the CMO immune cells were responsible for the presence of HSCs in peripheral blood, spleen, and paws. Mechanistically, we showed that despite the increased presence of HSC anchoring tools to the BM (CXCR4, VLA-4, and SDF-1), HSCs in CMO conditions were mobilizing to EMH sites. Transcriptomic analysis determined elevated levels of metalloproteinases in CMO mice, and ELISA and gelatinase assays verified the elevated levels and activity of MMP9. Next, we showed that blocking MMP9 activity with SB-3CT prevented mobilization of HSCs in CMO mice. Summary/Conclusion: Altogether, our data indicate that chronic inflammation promotes HSCs mobilization from the BM and leads to the development of EMH. We determined that the immune cells in CMO mice, but not the non-hematopoietic BM niche, mediate HSC mobilization by producing high levels of activated MMP9. The work was partially funded by the National Institute for Cancer Research (Program EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Keywords: Hematopoietic stem and progenitor cells, Hematopoietic stem cell, Inflammation, Hematopoietic cell transplantation

Toll-like receptor (TLR) signaling relies on Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor proteins that recruit downstream signaling molecules to generate tailored immune responses. In addition, the palmitoylated transmembrane adaptor protein family member Scimp acts as a non-TIR-containing adaptor protein in macrophages, scaffolding the Src family kinase Lyn to enable TLR phosphorylation and proinflammatory signaling responses. Here we report the existence of a smaller, naturally occurring translational variant of Scimp (Scimp TV1), which is generated through leaky scanning and translation at a downstream methionine. Scimp TV1 also scaffolds Lyn, but in contrast to full-length Scimp, it is basally rather than lipopolysaccharide (LPS)-inducibly phosphorylated. Macrophages from mice that selectively express Scimp TV1, but not full-length Scimp, have impaired sustained LPS-inducible cytokine responses. Furthermore, in granulocyte macrophage colony-stimulating factor-derived myeloid cells that express high levels of Scimp, selective overexpression of Scimp TV1 enhances CpG DNA-inducible cytokine production. Unlike full-length Scimp that localizes to the cell surface and filopodia, Scimp TV1 accumulates in intracellular compartments, particularly the Golgi. Moreover, this variant of Scimp is not inducibly phosphorylated in response to CpG DNA, suggesting that it may act via an indirect mechanism to enhance TLR9 responses. Our findings thus reveal the use of alternative translation start sites as a previously unrecognized mechanism for diversifying TLR responses in the innate immune system.

IntroductionAutoinflammatory diseases are characterized by dysregulation of innate immune system leading to spontaneous sterile inflammation. One of the well-established animal models of this group of disorders is the mouse strain Pstpip2cmo. In this strain, the loss of adaptor protein PSTPIP2 leads to the autoinflammatory disease chronic multifocal osteomyelitis. It is manifested by sterile inflammation of the bones and surrounding soft tissues of the hind limbs and tail. The disease development is propelled by elevated production of IL-1β and reactive oxygen species by neutrophil granulocytes. However, the molecular mechanisms linking PSTPIP2 and these pathways have not been established. Candidate proteins potentially involved in these mechanisms include PSTPIP2 binding partners, PEST family phosphatases (PEST-PTPs) and phosphoinositide phosphatase SHIP1.MethodsTo address the role of these proteins in PSTPIP2-mediated control of inflammation, we have generated mouse strains in which PEST-PTP or SHIP1 binding sites in PSTPIP2 have been disrupted. In these mouse strains, we followed disease symptoms and various inflammation markers.ResultsOur data show that mutation of the PEST-PTP binding site causes symptomatic disease, whereas mice lacking the SHIP1 interaction site remain asymptomatic. Importantly, both binding partners of PSTPIP2 contribute equally to the control of IL-1β production, while PEST-PTPs have a dominant role in the regulation of reactive oxygen species. In addition, the interaction of PEST-PTPs with PSTPIP2 regulates the production of the chemokine CXCL2 by neutrophils. Its secretion likely creates a positive feedback loop that drives neutrophil recruitment to the affected tissues.ConclusionsWe demonstrate that PSTPIP2-bound PEST-PTPs and SHIP1 together control the IL-1β pathway. In addition, PEST-PTPs have unique roles in the control of reactive oxygen species and chemokine production, which in the absence of PEST-PTP binding to PSTPIP2 shift the balance towards symptomatic disease.

Cells communicate with their environment via surface receptors, but nanoscopic receptor organization with respect to complex cell surface morphology remains unclear. This is mainly due to a lack of accessible, robust and high-resolution methods. Here, we present an approach for mapping the topography of receptors at the cell surface with nanometer precision. The method involves coating glass coverslips with glycine, which preserves the fine membrane morphology while allowing immobilized cells to be positioned close to the optical surface. We developed an advanced and simplified algorithm for the analysis of single-molecule localization data acquired in a biplane detection scheme. These advancements enable direct and quantitative mapping of protein distribution on ruffled plasma membranes with near isotropic 3D nanometer resolution. As demonstrated successfully for CD4 and CD45 receptors, the described workflow is a straightforward quantitative technique to study molecules and their interactions at the complex surface nanomorphology of differentiated metazoan cells.

Chronic inflammation represents a major threat to human health since long‐term systemic inflammation is known to affect distinct tissues and organs. Recently, solid evidence demonstrated that chronic inflammation affects hematopoiesis; however, how chronic inflammation affects hematopoietic stem cells (HSCs) on the mechanistic level is poorly understood. Here, we employ a mouse model of chronic multifocal osteomyelitis (CMO) to assess the effects of a spontaneously developed inflammatory condition on HSCs. We demonstrate that hematopoietic and nonhematopoietic compartments in CMO BM contribute to HSC expansion and impair their function. Remarkably, our results suggest that the typical features of murine multifocal osteomyelitis and the HSC phenotype are mechanistically decoupled. We show that the CMO environment imprints a myeloid gene signature and imposes a pro‐inflammatory profile on HSCs. We identify IL‐6 and the Jak/Stat3 signaling pathway as critical mediators. However, while IL‐6 and Stat3 blockage reduce HSC numbers in CMO mice, only inhibition of Stat3 activity significantly rescues their fitness. Our data emphasize the detrimental effects of chronic inflammation on stem cell function, opening new venues for treatment.