Mathis Richter

Neutrophils are the most abundant circulating leukocytes in humans and act as key effectors of innate immunity. Severe congenital neutropenia (SCN) encompasses a heterogeneous group of disorders characterized by early onset of recurrent infections. Mutations in Jagunal homolog 1 (Jagn1) cause SCN, yet the mechanisms linking Jagn1 deficiency to defective neutrophil function and neutropenia remain unclear. Here, we identify Jagn1 as a critical regulator that connects intracellular protein trafficking and surface glycosylation during neutrophil development. Using neutrophils derived from Hoxb8-ER–immortalized progenitors, we show that Jagn1 expression is restricted to early progenitor stages and transcriptionally controlled by the transcription factor CCAAT/enhancer-binding protein α. Loss of Jagn1 delays neutrophil differentiation, resulting in progenitor accumulation. Functionally, Jagn1-deficient murine neutrophils exhibit defective E-selectin–dependent rolling and chemokine-induced adhesion in ex vivo and in vitro flow chambers, mirrored by impaired rolling, adhesion, and transmigration in vivo during tumor necrosis factor–induced inflammation. Mechanistically, lectin-binding and static adhesion assays revealed reduced sialylation of adhesion-relevant receptors on Jagn1-deficient neutrophils, leading to impaired E-selectin and CXCL8 binding. In addition, NLRP3-dependent S100A8/A9 release from Jagn1-deficient neutrophils was reduced after E-selectin stimulation. These findings define Jagn1 as a key determinant of neutrophil protein sialylation by regulating endoplasmic reticulum–Golgi posttranslational glycosylation, thereby ensuring proper sialylation of adhesion-relevant molecules, including E-selectin ligands and CXCR2. This mechanism provides a unifying explanation for the clinical and cellular phenotypes in Jagn1-associated SCN and may offer new therapeutic approaches in the treatment of patients with loss-of-function mutation in Jagn1.

Systemic inflammatory responses shape clinical outcomes in acute cardiovascular disease. Because of their functional plasticity and rapid turnover, neutrophils have emerged as dynamic indicators of inflammatory stress. Here we profile the appearance of distinct neutrophil maturation stages in patients with ST-elevation myocardial infarction, heart failure and stroke. Our data reveal the mobilization of immature neutrophils in all groups; however, patients with ST-elevation myocardial infarction exhibited the most pronounced engagement of this response, including the appearance of CD16 ^low CD10 ^neg preneutrophils (preNeus), the final mitotic neutrophil progenitor, which was associated with disease outcome. Plasma cytokine profiling identified a coordinated inflammatory signature associated with preNeu mobilization, consistent with emergency granulopoiesis. PreNeus were identifiable as immature granulocytes in routine blood count analysis, where they predicted 30-day mortality better than established biomarkers in two clinical cohorts. Immature granulocytes also remained independently associated with survival in multivariate models incorporating risk factors, enabling the immediate identification of vulnerable patients with ST-elevation myocardial infarction upon hospital admission.

Extramedullary organs such as the spleen can assume granulopoiesis as a supportive mechanism to cope with increased demands during persistent inflammation. However, the quantitative output of extramedullary granulopoiesis is limited, and whether the spleen provides neutrophils of a qualitative difference remains unclear. Here, we found that splenic stress granulopoiesis is associated with distinct neutrophil production and differentiation trajectories. Myeloid progenitors in the spleen engaged in accelerated production of neutrophils with an immature phenotype. Yet, neutrophils generated during persistent stress granulopoiesis were fully competent to exert antimicrobial functions and were necessary to contain bacterial invasion in the bladder. Activation of type I interferon signaling in the spleen was required for splenic neutrophil priming, and its loss impaired antibacterial host defense. Thus, the spleen provides an immunological environment for stress-induced rapid production and priming of highly active neutrophils to meet demands during infection.

Background: Neutrophils guarantee a prompt and robust host response to pathogens. Yet, overshooting neutrophil activation leads to tremendous collateral damage in tissues. In advanced chronic liver disease (ACLD), neutrophils are exposed to the highly immunogenic milieu of the portal circulation prior to entering the liver sinusoids. In alcohol-related liver disease (ALD), neutrophils occupy a central role and therefore mechanisms regulating neutrophil activity pose a possible therapeutic target. Objective: Evaluate the impact of the portal milieu on neutrophils in ACLD with portal hypertension. Design: We conducted a prospective study of patients undergoing transjugular intrahepatic portosystemic shunt placement. Paired blood samples were obtained from the portal vein (PV) and superior vena cava (SVC); the neutrophil phenotype was assessed by spectral flow cytometry; plasma was analysed by cytokine quantification, reporter assays and metabolomics. Effects of tryptophan supplementation on neutrophil function and phenotype were tested in isolated neutrophils. Results: Patients with ALD but not non-ALD showed highly activated CD10 ^high CD11b ^high neutrophils in the portal circulation. PV plasma induced this phenotype in SVC neutrophils. Analysis of portal plasma revealed no differences in cytokines or toll-like receptor (TLR)/nucleotide-binding oligomerisation domain-containing protein (NOD) ligands but decreased local tryptophan concentrations in patients with ALD. In vitro supplementation of tryptophan ameliorated activation of isolated neutrophils. Conclusion: Our findings identify portal tryptophan as a local regulator of neutrophil activation in ACLD. The link between low tryptophan levels and heightened neutrophil reactivity, especially in ALD, underscores the role of the gut–liver metabolic crosstalk in immune modulation and highlights a potential therapeutic target for mitigating neutrophil-driven liver injury.

Neutrophils, the first cells to arrive at the site of inflammation, are rather short-lived cells and thus have to be constantly replenished. During neutrophil development, vesicle dynamics need to be fine-tuned and impaired vesicle trafficking has been linked to failure in neutrophil maturation. Here, we characterized the role of VPS18 as a central core component of CORVET & HOPS tethering complexes for neutrophil development. Using CRISPR/Cas9-engineered Hoxb8 cells with heterozygous mutations in Vps18 , we found that VPS18 deficiency interfered with neutrophil development due to tethering complex instability. As a result, vesicle dynamics were impaired with a strong increase in LC3B-II and p62 levels, indicating autophagosome accumulation and reduced autophagic flux. With transmission electron microscopy, we verified the increase in autophagosomes and also found irregularly shaped vesicular structures in Vps18 mutants. Subsequently, Vps18 mutant neutrophil progenitors underwent premature apoptosis. We described a novel patient with a heterozygous stop-gain mutation in VPS18 suffering from neutropenia and recurrent infections. To verify our findings in the human system, we used human induced pluripotent stem cells (iPSCs). Upon differentiation into neutrophils, loss of VPS18 resulted in an almost complete absence of iPSC-derived developing neutrophils. Heterozygous VPS18 mutant and patient mutation-harboring iPSCs were characterized by strongly reduced numbers of developing neutrophils. Zebrafish larvae with heterozygous mutations in vps18 were also characterized by significantly reduced neutrophil numbers. This study shows the pivotal impact of VPS18 for adequate vesicle dynamics during neutrophil development which might be relevant in the context of vesicle trafficking during granulopoiesis and congenital neutropenia.

Neutrophils exhibit remarkable phenotypic and functional diversity across tissues and diseases^1,2, yet the lack of understanding of how this immune compartment is globally organized challenges translation to the clinic. Here we performed single-cell transcriptional profiling of neutrophils spanning 47 anatomical, physiological and pathological scenarios to generate an integrated map of the global neutrophil compartment in mice, which we refer to as NeuMap. NeuMap integrates and expands existing models^3,4 to generate fundamental new insights; it reveals that neutrophils organize in a finite number of functional hubs that distribute sequentially during maturation to then branch out into interferon-responsive and immunosuppressive states, as well as a functionally silent state that dominates in the healthy circulation. Computational modelling and timestamp analyses identify prototypical trajectories that connect these hubs, and reveal that the dynamics and preferred paths vary during health, inflammation and cancer. We show that TGFβ, IFNβ and GM-CSF push neutrophils along the different trajectories, and projection of chromatin accessibility sites onto NeuMap reveals that the transcription factor JUNB controls angiogenic and immunosuppressive states and promotes tissue revascularization. The architecture of NeuMap appears to be conserved across sex, environmental and genetic backgrounds, as well as in humans. Finally, we show that NeuMap enables inference of the pathophysiological state of the host by profiling blood neutrophils. Our study delineates the global architecture of the neutrophil compartment and establishes a framework for exploration and exploitation of neutrophil biology.

Neonatal encephalopathy caused by hypoxia-ischemia (HI) leads to a strong neutrophil infiltration. The long-held assumption that neutrophils act exclusively as tissue-damaging cells, is challenged by increasing evidence of a profound neutrophil heterogeneity. Here, we uncovered a pronounced phenotypical and functional diversification of neutrophils in neonatal mice depending on the disease stage. Neutrophil infiltration was biphasic, peaking 1 and 7 days after HI. Early brain-infiltrating neutrophils displayed a hyperactivated phenotype, whereas neutrophils at day 7 exhibited an angiogenic phenotype with high Siglec-F expression. Acute neutrophil depletion protected against neural cell death, associated with decreased hyperactivity in adolescent animals. Delayed neutrophil depletion impaired vascular and oligodendrocyte regeneration, resulting in exacerbated alterations of anxiety-related behavior and myelination deficits. These findings suggest a divergent function of neutrophils, with early neutrophils aggravating tissue damage and late neutrophils contributing to neurological recovery. The disease stage-dependent neutrophil diversification offers new possibilities to identify disease-stage-specific therapeutic targets.

An acute inflammatory response to infection or sterile injury involves an adequate activation and recruitment of leukocytes. Activation of β2-integrins is required for neutrophil recruitment and is also mandatory for various neutrophil cell-intrinsic functions. Guanosine triphosphatases (GTPases) are key regulators of the actin cytoskeleton and are required for β2-integrin activation. Myosin-IXb (Myo9b), a Rho GTPase-activating protein, is essential for regulating Rho activity in neutrophils. Yet, the exact molecular mechanism through which Myo9b regulates β2-integrin activity and neutrophil recruitment into inflamed tissue is unknown. We demonstrate that Myo9b deficiency causes RhoA overactivation, increases actin cytoskeleton rearrangement in neutrophils, decreases neutrophil recruitment into the kidney, and improves kidney function in murine models of acute kidney injury. Loss of Myo9b also affects neutrophil effector functions and causes increased rolling velocity, decreased adhesion, impaired crawling, and strongly reduced transmigration of neutrophils in vivo. Mechanistically, Myo9b regulates RhoA activity, which is required for chemokine- and selectin-induced talin-1 recruitment to β2-integrins. Thus, Myo9b is a crucial regulator of important signaling pathways in neutrophils and is required for an adequate immune response triggered by chemokines and selectins.

Inflammatory cardiovascular diseases are the leading cause of death worldwide. Neutrophils, key drivers during inflammation, have consistently been associated with adverse outcomes and mortality in cardiovascular pathologies. Given the uniquely high plasticity and turnover of neutrophils, we profiled the appearance of distinct neutrophil maturation stages as sensitive and specific biomarkers in patients with ST-elevation myocardial infarction (STEMI), heart failure and stroke. Our data reveal a neutrophilia driven by mobilization of immature neutrophils in all groups; however, STEMI patients exhibited selective appearance of CD16 ^neg CD10 ^neg preNeus, the final mitotic neutrophil progenitor. PreNeus predicted 30-day mortality better than established biomarkers and were identifiable as immature granulocytes in blood counter analysis, hence enabling identification of high-risk STEMI patients upon hospital admission to guide tailored intervention.