CHIP and hips: clonal hematopoiesis is common in patients undergoing hip arthroplasty and is associated with autoimmune disease.

Clonal hematopoiesis (CH) is an age-related condition predisposing to blood cancer and cardiovascular disease (CVD). Murine models demonstrate CH-mediated altered immune function and proinflammation. Low-grade inflammation has been implicated in the pathogenesis of osteoarthritis (OA), the main indication for total hip arthroplasty (THA). THA-derived hip bones serve as a major source of healthy hematopoietic cells in experimental hematology. We prospectively investigated frequency and clinical associations of CH in 200 patients without known hematologic disease who were undergoing THA. Prevalence of CH was 50%, including 77 patients with CH of indeterminate potential (CHIP, defined as somatic variant allele frequencies [VAFs] ≥2%), and 23 patients harboring CH with lower mutation burden (VAF, 1% to 2%). Most commonly mutated genes were DNMT3A (29.5%), TET2 (15.0%), and ASXL1 (3.5%). CHIP is significantly associated with lower hemoglobin, higher mean corpuscular volume, previous or present malignant disease, and CVD. Strikingly, we observed a previously unreported association of CHIP with autoimmune diseases (AIDs; multivariable adjusted odds ratio, 6.6; 95% confidence interval, 1.7-30; P = .0081). These findings underscore the association between CH and inflammatory diseases. Our results have considerable relevance for managing patients with OA and AIDs or mild anemia and question the use of hip bone-derived cells as healthy experimental controls.

Clonal hematopoiesis and its impact on the aging osteo-hematopoietic niche.

Clonal hematopoiesis (CH) defines a premalignant state predominantly found in older persons that increases the risk of developing hematologic malignancies and age-related inflammatory diseases. However, the risk for malignant transformation or non-malignant disorders is variable and difficult to predict, and defining the clinical relevance of specific candidate driver mutations in individual carriers has proved to be challenging. In addition to the cell-intrinsic mechanisms, mutant cells rely on and alter cell-extrinsic factors from the bone marrow (BM) niche, which complicates the prediction of a mutant cell's fate in a shifting pre-malignant microenvironment. Therefore, identifying the insidious and potentially broad impact of driver mutations on supportive niches and immune function in CH aims to understand the subtle differences that enable driver mutations to yield different clinical outcomes. Here, we review the changes in the aging BM niche and the emerging evidence supporting the concept that CH can progressively alter components of the local BM microenvironment. These alterations may have profound implications for the functionality of the osteo-hematopoietic niche and overall bone health, consequently fostering a conducive environment for the continued development and progression of CH. We also provide an overview of the latest technology developments to study the spatiotemporal dependencies in the CH BM niche, ideally in the context of longitudinal studies following CH over time. Finally, we discuss aspects of CH carrier management in clinical practice, based on work from our group and others.

Immune-monitoring of myelodysplastic neoplasms: Recommendations from the i4MDS consortium.

Advancements in comprehending myelodysplastic neoplasms (MDS) have unfolded significantly in recent years, elucidating a myriad of cellular and molecular underpinnings integral to disease progression. While molecular inclusions into prognostic models have substantively advanced risk stratification, recent revelations have emphasized the pivotal role of immune dysregulation within the bone marrow milieu during MDS evolution. Nonetheless, immunotherapy for MDS has not experienced breakthroughs seen in other malignancies, partly attributable to the absence of an immune classification that could stratify patients toward optimally targeted immunotherapeutic approaches. A pivotal obstacle to establishing "immune classes" among MDS patients is the absence of validated accepted immune panels suitable for routine application in clinical laboratories. In response, we formed International Integrative Innovative Immunology for MDS (i4MDS), a consortium of multidisciplinary experts, and created the following recommendations for standardized methodologies to monitor immune responses in MDS. A central goal of i4MDS is the development of an immune score that could be incorporated into current clinical risk stratification models. This position paper first consolidates current knowledge on MDS immunology. Subsequently, in collaboration with clinical and laboratory specialists, we introduce flow cytometry panels and cytokine assays, meticulously devised for clinical laboratories, aiming to monitor the immune status of MDS patients, evaluating both immune fitness and identifying potential immune "risk factors." By amalgamating this immunological characterization data and molecular data, we aim to enhance patient stratification, identify predictive markers for treatment responsiveness, and accelerate the development of systems immunology tools and innovative immunotherapies.

Transition from an autoimmune-prone state to fatal autoimmune disease in CCR7 and RORγt double-deficient mice is dependent on gut microbiota.

Autoimmunity is associated with a strong genetic component, but onset and persistence of clinically apparent autoimmune diseases often require an additional environmental trigger. The balance between immunity and tolerance is regulated by numerous molecular factors including nuclear hormone and homeostatic chemokine receptors. The nuclear hormone receptor RORγt and the chemokine receptor CCR7 are both essentially involved in functional lymphoid organogenesis and maintenance of lymphocyte homeostasis. Lack of one or the other impairs thymic T cell development and alters T cell homeostasis. Mice deficient for both, Ccr7(-/-)Rorγt(-/-), succumbed early to acute destructive inflammation, characterized by massive recruitment of inflammatory leukocytes, pro-inflammatory cytokine and autoantibody production, and wasting disease. Antibiotic-treatment of mice before disease onset reduced the overall gut microflora and abrogated the development of fatal mucosal inflammation. Hence, commensal bacteria and a confined tissue-specific inflammatory milieu serve as complementary trigger to initiate the lethal pathophysiologic process in Ccr7(-/-)Rorγt(-/-) mice.

Cooperative function of CCR7 and lymphotoxin in the formation of a lymphoma-permissive niche within murine secondary lymphoid organs.

Lymphoma cell survival and progression are putatively dependent on a specific microanatomic localization within secondary lymphoid organs. Despite compelling data correlating homeostatic chemokine receptor expression and human lymphoma pathogenesis, genetic models that either mimic lymphoma dissemination or dissect a crosstalk of lymphoma and stromal cells are missing. Applying the genetically tractable Eµ-Myc transgenic mouse model, we show that the chemokine receptor CCR7 regulates Eµ-Myc lymphoma homing to lymph nodes and distinctive microanatomic sites of the spleen. CCR7-controlled access of lymphoma cells to the splenic T-cell zone led to a significant survival advantage compared with CCR7-deficient lymphoma cells, which were excluded from this zone. Within the niche, lymphoma cells stimulated a reciprocal cross-talk with gp38(+) fibroblastic reticular cells. This reciprocal cooperation program was mediated by lymphoma B cell-presented lymphotoxin, which acted on lymphotoxin-ß-receptor-bearing stromal cells followed by alteration of stromal cellular composition. Cross-talk inhibition by lymphotoxin-α deletion and using a lymphotoxin-ß receptor-immunoglobulin fusion protein impaired lymphoma growth. Thus, abrogation of CCR7-governed migration and of sustained lymphotoxin signaling could provide new targets in lymphoma therapy.

Activation of distinct inflammatory pathways in subgroups of LR-MDS.

Aberrant innate immune signaling has been identified as a potential key driver of the complex pathophysiology of myelodysplastic neoplasms (MDS). This study of a large, clinically and genetically well-characterized cohort of treatment-naïve MDS patients confirms intrinsic activation of inflammatory pathways in general mediated by caspase-1, interleukin (IL)-1ß and IL-18 in low-risk (LR)-MDS bone marrow and reveals a previously unrecognized heterogeneity of inflammation between genetically defined LR-MDS subgroups. Principal component analysis resolved two LR-MDS phenotypes with low (cluster 1) and high (cluster 2) levels of IL1B gene expression, respectively. Cluster 1 contained 14/17 SF3B1-mutated cases, while cluster 2 contained 8/8 del(5q) cases. Targeted gene expression analysis of sorted cell populations showed that the majority of the inflammasome-related genes, including IL1B, were primarily expressed in the monocyte compartment, consistent with a dominant role in determining the inflammatory bone marrow environment. However, the highest levels of IL18 expression were found in hematopoietic stem and progenitor cells (HSPCs). The colony forming activity of healthy donor HSPCs exposed to monocytes from LR-MDS was increased by the IL-1ß-neutralizing antibody canakinumab. This work reveals distinct inflammatory profiles in LR-MDS that are of likely relevance to the personalization of emerging anti-inflammatory therapies.

Manifestation of spontaneous and early autoimmune gastritis in CCR7-deficient mice.

Autoimmune gastritis is a common autoimmune disorder characterized by chronic inflammatory cell infiltrates, atrophy of the corpus and fundus, and the occurrence of autoantibodies to parietal cell antigen. In CCR7-deficient mice, autoimmune gastritis developed spontaneously and was accompanied by metaplasia of the gastric mucosa and by the formation of tertiary lymphoid organs at gastric mucosal sites. T cells of CCR7-deficient mice showed an activated phenotype in the gastric mucosa, mesenteric lymph nodes, and peripheral blood. In addition, elevated serum IgG levels specific to gastric parietal cell antigen were detected. Because the role of organized lymphocytic aggregates at this inflammatory site is not completely understood, we first analyzed the cellular requirements for the formation of these structures. Autoreactive CD4(+) T cells were pivotal for tertiary lymphoid follicle formation, most likely in cooperation with dendritic cells, macrophages, and B cells. Second, we analyzed the necessity of secondary lymph nodes and tertiary lymphoid organs for the development of autoimmune gastritis using CCR7 single- and CCR7/lymphotoxin α double-deficient mice. Strikingly, manifestation of autoimmune gastritis was observed in the absence of secondary lymph nodes and preceded the development of tertiary lymphoid organs. Taken together, these findings identify an inflammatory process where gastric autoreactive T cells independent of organized tertiary lymphoid organs and classic lymph nodes can induce and maintain autoimmune gastritis.

Compartment-specific mutational landscape of clonal hematopoiesis.

Clonal hematopoiesis (CH) is characterized by somatic mutations in blood cells of individuals without hematologic disease. While the mutational landscape of CH in peripheral blood (PB) has been well characterized, detailed analyses addressing its spatial and cellular distribution in the bone marrow (BM) compartment are sparse. We studied CH driver mutations in healthy individuals (n = 261) across different anatomical and cellular compartments. Variant allele frequencies were higher in BM than PB and positively correlated with the number of driver variants, yet remained stable during a median of 12 months of follow-up. In CH carriers undergoing simultaneous bilateral hip replacement, we detected ASXL1-mutant clones in one anatomical location but not the contralateral side, indicating intra-patient spatial heterogeneity. Analyses of lineage involvement in ASXL1-mutated CH showed enriched clonality in BM stem and myeloid progenitor cells, while lymphocytes were particularly involved in individuals carrying the c.1934dupG variant, indicating different ASXL1 mutations may have distinct lineage distribution patterns. Patients with overt myeloid malignancies showed higher mutation numbers and allele frequencies and a shifting mutation landscape, notably characterized by increasing prevalence of DNMT3A codon R882 variants. Collectively, our data provide novel insights into the genetics, evolution, and spatial and lineage-specific BM involvement of CH.

Luspatercept restores SDF-1-mediated hematopoietic support by MDS-derived mesenchymal stromal cells.

The bone marrow microenvironment (BMME) plays a key role in the pathophysiology of myelodysplastic syndromes (MDS), clonal blood disorders affecting the differentiation, and maturation of hematopoietic stem and progenitor cells (HSPCs). In lower-risk MDS patients, ineffective late-stage erythropoiesis can be restored by luspatercept, an activin receptor type IIB ligand trap. Here, we investigated whether luspatercept can modulate the functional properties of mesenchymal stromal cells (MSCs) as key components of the BMME. Luspatercept treatment inhibited Smad2/3 phosphorylation in both healthy and MDS MSCs and reversed disease-associated alterations in SDF-1 secretion. Pre-treatment of MDS MSCs with luspatercept restored the subsequent clonogenic potential of co-cultured HSPCs and increased both their stromal-adherence and their expression of both CXCR4 and ß3 integrin. Luspatercept pre-treatment of MSCs also increased the subsequent homing of co-cultured HSPCs in zebrafish embryos. MSCs derived from patients who had received luspatercept treatment had an increased capacity to maintain the colony forming potential of normal but not MDS HSPCs. These data provide the first evidence that luspatercept impacts the BMME directly, leading to a selective restoration of the ineffective hematopoiesis that is a hallmark of MDS.

Integrating the "Immunome" in the Stratification of Myelodysplastic Syndromes and Future Clinical Trial Design.

Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis and often include a dysregulation and dysfunction of the immune system. In the context of population aging, MDS incidence is set to increase substantially, with exponential increases in health care costs, given the limited and expensive treatment options for these patients. Treatment selection is mainly based on calculated risk categories according to a Revised International Prognostic Scoring System (IPSS-R). However, although IPSS-R is an excellent predictor of disease progression, it is an ineffective predictor of response to disease-modifying therapies. Redressing these unmet needs, the "immunome" is a key, multifaceted component in the initiation and overall response against malignant cells in MDS, and the current omission of immune status monitoring may in part explain the insufficiencies of current prognostic stratification methods. Nevertheless, integrating these and other recent molecular advances into clinical practice proves difficult. This review highlights the complexity of immune dysregulation in MDS pathophysiology and the fine balance between smoldering inflammation, adaptive immunity, and somatic mutations in promoting or suppressing malignant clones. We review the existing knowledge and discuss how state-of-the-art immune monitoring strategies could potentially permit novel patient substratification, thereby empowering practical predictions of response to treatment in MDS. We propose novel multicenter studies, which are needed to achieve this goal.

A variant erythroferrone disrupts iron homeostasis in SF3B1-mutated myelodysplastic syndrome.

Myelodysplastic syndromes (MDS) with ring sideroblasts are hematopoietic stem cell disorders with erythroid dysplasia and mutations in the SF3B1 splicing factor gene. Patients with MDS with SF3B1 mutations often accumulate excessive tissue iron, even in the absence of transfusions, but the mechanisms that are responsible for their parenchymal iron overload are unknown. Body iron content, tissue distribution, and the supply of iron for erythropoiesis are controlled by the hormone hepcidin, which is regulated by erythroblasts through secretion of the erythroid hormone erythroferrone (ERFE). Here, we identified an alternative ERFE transcript in patients with MDS with the SF3B1 mutation. Induction of this ERFE transcript in primary SF3B1-mutated bone marrow erythroblasts generated a variant protein that maintained the capacity to suppress hepcidin transcription. Plasma concentrations of ERFE were higher in patients with MDS with an SF3B1 gene mutation than in patients with SF3B1 wild-type MDS. Thus, hepcidin suppression by a variant ERFE is likely responsible for the increased iron loading in patients with SF3B1-mutated MDS, suggesting that ERFE could be targeted to prevent iron-mediated toxicity. The expression of the variant ERFE transcript that was restricted to SF3B1-mutated erythroblasts decreased in lenalidomide-responsive anemic patients, identifying variant ERFE as a specific biomarker of clonal erythropoiesis.

A Reduction in Intracellular Reactive Oxygen Species Due to a Mutation in NCF4 Promotes Autoimmune Arthritis in Mice.

AIMS: The mechanisms linking deficits in the phagocytic NADPH oxidase 2 (NOX2) complex to autoimmunity are so far incompletely understood. Deficiency in neutrophil cytosolic factor 1 (NCF1) inactivates the NOX2 complex, leading to a dramatic reduction of intra- and extracellular reactive oxygen species (ROS) and enhanced susceptibility to autoimmune disease. The contribution of intracellular NOX2 activity to autoimmune regulation is, however, unknown. Another component of the NOX2 complex, NCF4, directs the NOX2 complex to phagosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns3P) and has been proposed to regulate intracellular ROS levels. To address the impact of NCF4 and selective changes in intracellular ROS production on autoimmune inflammation, we studied collagen-induced arthritis (CIA) and mannan-induced psoriatic arthritis-like disease (MIP) in mice lacking NCF4 and mice with a mutation in the PtdIns3P-binding site of NCF4. RESULTS: Targeted deletion of Ncf4 (Ncf4-/-) led to severe defects in overall ROS production due to concomitant reduction of NCF2 and NCF1. These mice displayed delayed neutrophil apoptosis and enhanced innate immune responses, and they developed aggravated CIA and MIP. Disruption of the PtdIns3P-binding site by targeted mutation (Ncf4*/*) resulted in selective defects in intracellular NOX2 activity, which entailed milder effects on innate immunity and MIP but clearly promoted susceptibility to CIA. Innovation and Conclusion: This is, to our knowledge, the first study addressing the development of autoimmunity in an organism with selectively compromised NOX2-dependent intracellular ROS levels. Our data reveal a specific role for NCF4-mediated intracellular ROS production in regulating autoimmunity and chronic inflammation. Antioxid. Redox Signal. 25, 983-996.

Hydrogen peroxide as an immunological transmitter regulating autoreactive T cells.

SIGNIFICANCE: An unexpected finding, revealed by positional cloning of genetic polymorphisms controlling models for rheumatoid arthritis, exposed a new function of Ncf1 and NADPH oxidase (NOX) 2 controlled oxidative burst. RECENT ADVANCES: A decreased capacity to produce ROS due to a natural polymorphism was found to be the major factor leading to more severe arthritis and increased T cell-dependent autoimmunity. CRITICAL ISSUES: In the vein of this finding, we here review a possible new role of ROS in regulating inflammatory cell and autoreactive T cell activity. It is postulated that peroxide is an immunologic transmitter secreted by antigen-presenting cells that downregulate the responses by autoreactive T cells. FUTURE DIRECTIONS: This may operate at different levels of T cell selection and activation: during negative selection in the thymus, priming of T cells in draining lymph nodes, and while interacting with macrophages in peripheral target tissues.

CCR7 regulates lymphocyte egress and recirculation through body cavities.

T and B lymphocytes recirculate among blood, lymph, and extralymphoid tissues to ensure immune surveillance and the establishment of self-tolerance. The underlying mechanisms regulating homeostatic lymphocyte recirculation through body cavities are not fully understood. Here, we demonstrate that the homeostatic chemokine receptor CCR7 regulates homeostatic recirculation of lymphocytes through body cavities. CCR7 deficiency results in massive accumulation of CD4(+) and CD8(+) T cells and B-2 B cells in the peritoneal and pleural cavities. The increase in B-2 B and T lymphocytes is not associated with an altered maturation and/or activation status of these cells. Mechanistically, an increase in peritoneal lymphocyte numbers is caused by impaired egress of CCR7-deficient lymphocytes from body cavities. These results establish that CCR7 plays a crucial role in lymphocyte exit from the PerC.

The chemokine receptor CXCR5 is pivotal for ectopic mucosa-associated lymphoid tissue neogenesis in chronic Helicobacter pylori-induced inflammation.

Ectopic lymphoid follicles are a key feature of chronic inflammatory autoimmune and infectious diseases, such as rheumatoid arthritis, Sjögren's syndrome, and Helicobacter pylori-induced gastritis. Homeostatic chemokines are considered to be involved in the formation of such tertiary lymphoid tissue. High expression of CXCL13 and its receptor, CXCR5, has been associated with the formation of ectopic lymphoid follicles in chronic infectious diseases. Here, we defined the role of CXCR5 in the development of mucosal tertiary lymphoid tissue and gastric inflammation in a mouse model of chronic H. pylori infection. CXCR5-deficient mice failed to develop organized gastric lymphoid follicles despite similar bacterial colonization density as infected wild-type mice. CXCR5 deficiency altered Th17 responses but not Th1-type cellular immune responses to H. pylori infection. Furthermore, CXCR5-deficient mice exhibited lower H. pylori-specific serum IgG and IgA levels and an overall decrease in chronic gastric immune responses. In conclusion, the development of mucosal tertiary ectopic follicles during chronic H. pylori infection is strongly dependent on the CXCL13/CXCR5 signaling axis, and lack of de novo lymphoid tissue formation attenuates chronic immune responses.