Journal of Leukocyte Biology

Tracking mature neutrophils remains challenging due to the lack of reliable cell surface markers. Although CD101 is a promising candidate for mature neutrophils, its stability under pathological conditions is unclear. Using a CD101-tdTomato reporter mouse model, we confirmed that the reporting system does not alter CD101 expression, and tdTomato fluorescence is predominantly expressed in mature neutrophils across peripheral tissues. Further analysis revealed that CD101+ and tdTomato+ neutrophils display identical characteristics of mature neutrophil, including poly-segmented nuclei, cell size, and key functions under homeostasis. By comparing tdTomato fluorescence with CD101 protein levels, we demonstrate that reduced CD101 expression under pathological states was not attributed to shedding or degradation. Our finding enhances CD101 as a robust and reliable marker of neutrophil maturity, providing a foundation for future applications in spatial transcriptomics and lineage tracing studies to dissect neutrophil heterogeneity and function. Highlights of the studyO_LIIn CD101-tdTomato homozygous mice, tdTomato is predominantly expressed in neutrophils and labels nearly 100% of mature neutrophils, aligning with the phenotype of CD101+ mature neutrophils; C_LIO_LIThe CD101-tdTomato reporting system does not interrupt CD101 expression or neutrophil functions; C_LIO_LICD101 remains a stable and reliable cell surface marker for labeling mature neutrophils, even under pathological conditions. C_LI

Expression of the Csf1r gene in cells of the mononuclear phagocyte lineage is regulated by a conserved enhancer, the fms-intronic regulatory element (FIRE). In mice with a germ-line deletion of FIRE (Fireko) CSF1R expression is undetectable in bone marrow progenitors and classical monocytes. Fireko mice lack subpopulations of macrophages in the brain and periphery but develop normally. Here we show that loss of CSF1R expression in Fireko mice is partly overcome by CSF2 in vitro and inflammatory recruitment in vitro. Analysis of heterozygous mutant mice and deletion of the conserved AP1 motif in FIRE provide evidence that continuous receptor synthesis determines CSF1 responsiveness. The absence of macrophages in kidney and heart of Fireko mice was not associated with detectable loss of physiological function. In a model of renal injury macrophage recruitment and histopathology were similar in WT and Fireko mice. Tissue resident macrophages that were depleted in Fireko mice, including microglia, were replaced by donor-derived cells following intraperitoneal adoptive transfer of wild-type bone marrow at weaning. The Fireko mouse provides a novel platform to dissect the functions of tissue resident macrophages in development, homeostasis and pathology. Summary StatementThis study describes a unique model of selective tissue resident macrophage deficiency arising from dysregulated expression of the mouse Csf1r gene.

BackgroundTrained immunity is a durable functional reprogramming of innate immune cells characterized by enhanced responsiveness upon secondary challenge. While metabolic rewiring and epigenetic remodeling are well-established features of this process, the contribution of ubiquitin-mediated post-translational regulation remains poorly defined. MethodsWe performed an integrative analysis of publicly available human transcriptomic datasets derived from monocytes, macrophages, and PBMCs exposed to established training stimuli ({beta}-glucan, Bacillus Calmette-Guerin [BCG], and hemin-{beta}-glucan) followed by secondary stimulation. A curated panel of deubiquitinating enzymes (DUBs) and E3 ubiquitin ligases with established immune functions was analyzed for differential expression. Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted to evaluate higher-order convergence across independent datasets. ResultsAcross multiple trained immunity models, we identified reproducible transcriptional remodeling of ubiquitin-modifying enzymes. USP25, OTUB1, and TRIM25 were consistently upregulated following restimulation, whereas several chromatin- and cytokine-regulatory DUBs--including USP3, USP4, USP7, USP16, MYSM1, and USP38--were downregulated. Normalization to RPMI-restimulated controls reduced many activation-associated signals; however, USP25 remained persistently elevated, suggesting a stable training-associated signature. Pathway enrichment analysis independently demonstrated significant engagement of ubiquitin-related functional categories across datasets, supporting coordinated reorganization of ubiquitin regulatory networks. ConclusionThese findings identify selective transcriptional remodeling of the ubiquitin- proteasome system as a recurring feature of trained immunity. Integrating ubiquitin signaling into the established metabolic-epigenetic framework expands the conceptual model of innate immune memory and suggests that ubiquitin-modifying enzymes function as modulatory rheostats shaping immune amplitude and stability. Future functional and proteomic studies are required to determine whether these transcriptional signatures directly mediate trained immunity phenotypes.

We show that clonal hematopoiesis is associated with an increased incidence of autoimmune hemolytic anemia. The hazard ratios of autoimmune hemolytic anemia and immune thrombocytopenia associated with clonal hematopoiesis were similar.

BackgroundHumans and mice display elevated levels of IL-27, an immunosuppressive cytokine shown to increase during neonatal bacterial sepsis and compromise survival. This study explores two hypotheses for regulation of IL-27 expression: 1) decreased DNA methylation in newborns that contributes to increased expression of IL-27 genes; 2) neonatal hormones regulate IL-27 expression through upstream hormone response elements (HREs). MethodsWhole genome methyl-seq analysis of neonatal and adult blood-derived macrophages identified differentially methylated regions (DMRs) at steady-state. Quantitative PCR (qPCR) measured expression of IL-27 genes (IL27p28 and EBI3) in human and murine neonatal macrophages stimulated in vitro with synthetic glucocorticoid or progesterone. Confocal microscopy and chromatin immunoprecipitation (ChIP) of glucocorticoid receptor (GR) assessed translocation into the nucleus and binding to the EBI3 promoter. ResultsThe IL-27p28 promoter contained DMRs that were increased in the neonatal cohort. The analysis did not identify DMRs within the EBI3 promoter. Dexamethasone stimulation increased EBI3 gene expression in human and murine neonatal macrophages. GR localized to the nucleus in response to dexamethasone and was enriched at the EBI3 upstream regulatory region. ConclusionThese data suggest glucocorticoid (GC) signaling increases EBI3 expression. This has importance in the context of antenatal GC administration that may increase IL-27 levels. Impact Statement{blacksquare} Elevated expression of IL-27 in early life impairs the host response to invasive bacterial infection in neonates. {blacksquare}Understanding the regulatory mechanisms contributing to increased IL-27 during the neonatal period is necessary to reduce susceptibility to infection in this vulnerable population. {blacksquare}The methylation status of the IL-27 genes in macrophages from neonatal and adult blood donors does not suggest regulation of differential expression with age. {blacksquare}Glucocorticoids are a signal that can induce EBI3 gene expression in a GR-dependent manner. {blacksquare}Glucocorticoid therapy for premature infants may increase IL-27 expression and promote enhanced susceptibility to infection.

Alveolar macrophages (AMs) are tissue-resident and the primary immune cells in the airspace. Following perturbations in the lungs, these AMs that are derived from the fetal liver, become depleted and are transiently replaced by myeloid cells that use lung-specific cues to differentiate into myeloid-derived AMs. While these myeloid-derived AMs are critically important in a range of pulmonary diseases, including post-influenza bacterial pneumonia, it remains challenging to fully understand their function due to a lack of ex vivo models that recapitulate key differences observed in vivo between AMs and myeloid-derived AMs. Here, we overcome this limitation by expanding our recently developed model of fetal liver-derived alveolar macrophages (FLAMs) to differentiate myeloid progenitors in the presence of GM-CSF and TGF{beta}, key cytokines that drive tissue resident AM functions. These myeloid-derived alveolar-like macrophages (MAMs) express AM surface markers and look similar morphologically to FLAMs, however, they remain more inflammatory than FLAMs. Mechanistic studies found that differential CpG methylation at inflammatory loci, basal transcriptional expression, and metabolic flux all contribute to the hyperinflammatory state of MAMs. Importantly, we find that while FLAMs are highly dependent of lipid metabolism, MAMs are more glycolytic and this hardwired metabolism is not easily overcome to mute their inflammatory state. Finally, we found that MAMs and FLAMs both function within the lung environment following transfer into mice lacking AMs. While both MAMs and FLAMs stably seed the lungs and reverse pulmonary proteinosis, MAMs remain highly inflammatory in the lungs following an LPS model of acute lung injury. Taken together our results find that MAMs are a reproducible model of myeloid-derived AMs and lays the groundwork to better understand how these important immune cells contribute to pulmonary homeostasis and responses to lung perturbations. These future studies will help to identify new targets that can be modulated to prevent severe pulmonary disease outcomes.

Immune memory responses are rapid and qualitatively distinct from primary responses. They typically develop in the presence of antigen-experienced memory T and B cells and pre-existing antibodies. Although the contribution of T and B cells to recall responses is well defined, the contribution of antibody "memory" and the mechanisms by which pre-existing antibodies modulate the development of germinal center and plasma cell responses is not precisely understood. Here we report on mechanisms that mediate antibody enhancement of germinal center (GC) and plasmablast (PB) compartments, and the parallel process by which they change the affinity threshold for B cell recruitment into immune responses. The data indicate that antibody-mediated enhancement of GC and PB responses is Fc gamma receptor (Fc{gamma}R) dependent and largely complement receptor 1 and 2 (CR1/2) independent. In contrast, the reduction in the affinity threshold for GC entry is independent of both Fc{gamma}Rs and CR1/2. SummaryCipolla et al. show that antibody can modulate immune responses via both Fc gamma receptor dependent and independent mechanisms. These mechanisms influence both the magnitude and composition of the germinal center response.

Objective: To characterize genome-wide DNA methylation patterns associated with sepsis using the Infinium Methylation EPIC v2.0 platform and to evaluate the feasibility of pooled methylation profiling in a pilot critical care cohort. Design: Single-center pilot epigenome-wide association study using pooled whole-blood genomic DNA and pool-level bioinformatic analysis. Setting: Academic medical center. Patients: Fifty critically ill adults enrolled within 48 hours of illness onset and 20 healthy controls. Interventions: None. Measurements and Main Results: Critically ill patients required mechanical ventilation and/or vasopressor support. Sepsis was defined according to Sepsis-3 criteria. Seventy individual samples were organized into 14 intended pools of 5 individuals each: 7 sepsis pools, 3 critically ill non-septic pools, and 4 healthy-control pools. One critically ill non-septic pool was excluded because of poor DNA quality, yielding 13 analyzable pools. For the primary pooled comparison, 7 sepsis pools were compared with 6 non-sepsis comparator pools comprising 2 critically ill non-septic and 4 healthy-control pools. After quality control and preprocessing with SeSAMe, 876,094 CpG sites were retained. The initial pool-level screen identified 170,897 candidate differentially methylated regions. Application of stringent secondary filters (false discovery rate <= 1%, absolute delta-beta >= 7.5%, and >= 5 CpGs per region) yielded a high-confidence subset with marked directional skewing, including 155 hypomethylated and 32 hypermethylated regions in sepsis. Differentially methylated region-associated genes were enriched in myeloid leukocyte activation, myeloid leukocyte-mediated immunity, defense response to bacterium, neutrophil granule biology, and hematopoietic cell lineage pathways. Additional signals involved microRNA-associated targets, ribosome biogenesis, RNA processing, long noncoding RNAs, and previously uncharacterized loci. Conclusions: In this pilot pooled EPIC v2.0 study, sepsis was associated with a biologically coherent, predominantly hypomethylated methylation signature enriched in myeloid and host-defense pathways. These findings support the feasibility of pooled methylation profiling for discovery-oriented sepsis biobank studies but should be interpreted as hypothesis-generating given the pool-level design, limited effective sample size, heterogeneous comparator group, and lack of direct validation against individual-level methylation profiles.

RationaleSepsis is a life-threatening syndrome causing significant morbidity and mortality especially in the aging population. Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition of clonal expansion of hematopoietic stem cells harboring somatic mutations associated with increased incidence of chronic illness and all-cause mortality. ObjectiveEvaluate the association of pre-illness CHIP with mortality and morbidity in patients admitted to the ICU with sepsis. MethodsWe performed a retrospective study using a de-identified electronic health record linked with a DNA biorepository. We identified adult patients with sepsis who had DNA collected prior to ICU admission. We tested the association between CHIP status, determined from whole-genome sequencing, and ICU mortality, organ support-free days, and long-term survival adjusting for age, sex, race and Sequential Organ Failure Assessment (SOFA) score on ICU admission. Measurements and Main ResultsPre-illness CHIP was associated with increased sepsis mortality (OR = 1.54, 95% CI 1.13 to 2.07, P = 0.005) and fewer days alive and free of organ support (-1.7 days, 95% CI -3.2 to -0.2, P = 0.028) after adjusting for age, sex, race, and SOFA score. In sepsis survivors, CHIP was also associated with increased long-term mortality after discharge (HR 1.40, 95% CI 1.01 to 1.93, P = 0.041). ConclusionsPre-illness CHIP was independently associated with increased mortality and morbidity in critically-ill adults with sepsis. These findings suggest that CHIP is a risk factor for sepsis severity. Elucidating the mechanism underlying this association could uncover new therapeutic interventions for sepsis.

BackgroundImmune dysfunction in sepsis and critical illness is biologically heterogeneous, yet available stratification frameworks leave many patients unclassified. We hypothesized that ex vivo cytokine-induction responses would define a continuous axis of functional immune responsiveness and identify a low-response state enriched in sepsis. MethodsIn this prospective observational study, 39 critically ill adults enrolled within 48 hours of ICU admission and 6 healthy controls underwent standardized whole-blood stimulation with lipopolysaccharide, anti-CD3/anti-CD28 antibodies, and PMA/ionomycin, with selected wells additionally supplemented with interleukin-7 or granulocyte-macrophage colony-stimulating factor. Interleukin-6, tumor necrosis factor, and interferon-gamma responses were quantified and referenced to subject-specific unstimulated baselines. A patient-anchored primary feature matrix was used to derive a continuous immune axis by principal component analysis, and a cross-validated 5-feature MiniResponder score was developed as a portable summary measure. ResultsAmong critically ill patients, induced cytokine responses organized along a dominant continuous axis of functional immune responsiveness; the first principal component explained 53.3% of between-patient variance. MiniResponder captured this axis and showed a lower-shifted distribution in sepsis. Using a control-referenced threshold defined by the 10th percentile of the healthy-control distribution, 19 of 39 patients (48.7%) were classified as low-response, including 15 of 21 patients with sepsis (71.4%) and 4 of 18 critically ill patients without sepsis (22.2%) (odds ratio 8.75, Fisher exact P=0.004). In exploratory analyses, lower MiniResponder scores were associated with greater unadjusted improvement in Sequential Organ Failure Assessment score from day 1 to days 3-9 (rho=-0.33; P=0.046), but this association attenuated after adjustment for baseline SOFA score (beta=-0.10; 95% CI-0.36 to 0.27). ConclusionsEx vivo immune profiling identified a continuous patient-anchored axis of functional immune responsiveness in critical illness that can be summarized by a compact 5-feature score. A control-referenced low-response state was enriched in sepsis. This framework may complement existing biomarker-based stratification approaches and support future enrichment strategies in sepsis trials.

Haematopoiesis and differentiation of immune cells from haematopoietic stem and progenitor cells (HSPCs) are essential to core aspects of health and disease. A key player in haematopoiesis and HSPC differentiation is the cytoskeleton, which governs cell division and lineage bias. Despite insights using mouse models, regulation of haematopoiesis by the septin cytoskeleton is mostly unknown. Septins are unconventional filament forming proteins best known for roles in cell division and host defence. To investigate septin-mediated host defence in vivo, we generated septin-deficient zebrafish models for infection with Mycobacterium marinum. Unexpectedly, septin-deficient larvae were protected from mycobacterial infection due to significantly increased macrophage numbers, reduced cell death, and enhanced inflammatory responses. Underlying this, we found that septin-deficient larvae produce significantly more HSPCs and show myeloid lineage bias, establishing a requirement for septins in haematopoiesis. In agreement with classical HSPC hierarchy, increased myeloid production in septin-deficient larvae is at the expense of erythroid lineage production. Our findings that septins play a role in haematopoiesis is consistent with hallmarks of haematological disorders in which septin dysfunction has been implicated, including acute myeloid leukaemia, myelodysplastic syndrome, and platelet disorder Bernard-Soulier syndrome. These results highlight zebrafish as a new model to investigate septin-mediated haematopoiesis and application of septin-based medicines to treat blood disorders.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterised by sustained type I interferon signalling and widespread immune dysregulation. Low-density neutrophils (LDNs) are expanded in SLE and display pro-inflammatory and tissue-damaging properties. However, their metabolic phenotype remains poorly defined. Here, we performed a comprehensive metabolic characterisation of circulating LDNs and normal-density neutrophils (NDNs) from patients with SLE and matched healthy individuals (HC). Neutrophil subsets were isolated from peripheral blood of SLE patients and HC donors using a two-step protocol of negative selection and Percoll density centrifugation. Immunophenotyping phenotype was carried out by flow cytometry to assess phenotypic expression of common neutrophil markers CD15, CD16, CD10, CD66b, CD62L, MPO, and IL-1{beta}. Bioenergetic profiling of LDNs and NDNs was performed in situ using the Seahorse MitoStress test to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Metabolic flexibility and phenotypic alterations were assessed in LDNs and NDNs following inhibiting mitochondrial metabolism with oligomycin and glycolysis with 2DG. We found that SLE LDNs exhibit an immature phenotype compared with autologous and healthy NDNs, as determined transcriptionally by C/EBP{varepsilon} and by surface protein expression levels of CD10. Both LDNs and NDNs from SLEDAI[&ge;]4 patients demonstrated significantly elevated ECAR relative to HC neutrophils. Further, SLE LDNs displayed enhanced metabolic flexibility, with the capacity to switch towards a glycolytic phenotype under metabolic stress conditions. Inhibition of glycolysis altered the inflammatory and maturation-associated phenotype of both SLE neutrophil subsets, indicating a direct link between cellular metabolism and pathogenic neutrophil function. Collectively, these findings identify fundamental metabolic alterations in SLE neutrophil subsets and support neutrophil immunometabolism as a potential therapeutic target in SLE.

Inflammation is a key driver of hematopoietic dysfunction in myeloid malignancies, but its role in the context of hypomethylating therapy remains incompletely understood. Although 5-Azacytidine is used posttransplant in high-risk myelodysplastic syndrome (MDS), only 50% of patients show a clinical response. We provide evidence that inherent inflammatory properties of healthy donor CD34+ stem cells exist that are likely to contribute to the "response" seen in MDS patients. These are linked to epigenetic priming of the myeloid niche, resulting in S100A8/A9-driven inflammatory program that promotes functionality of immature NK cells. Using in vitro differentiation systems, multi-omic profiling, and a S100A9-/- mouse model, we find that 5-AzaC modulates inflammatory transcriptional programs through epigenetic rewiring of upstream regulatory elements. Loss of S100A9 disrupts myeloid differentiation, impairs NK cell maturation, and alters key developmental regulators including CEBPB, JUN, and NFIL3. In vivo, 5-AzaC restores these defects and primes NK cells in a time- and context-dependent manner. Re-analysis of the published Australian MDS/CMML cohort shows that "responders" display increased S100A8/A9 expression together with enhanced IFN-{gamma}, IL6-JAK-STAT3, and TNF signaling. These findings suggest that inflammatory myeloid programs may serve as predictive biomarkers and therapeutic targets to enhance NK cell-mediated graft-versus-leukemia activity posttransplant. SummaryO_LIWe provide compelling evidence that inherent properties of healthy donor CD34+ hematopoietic stem cells (SCs) exist that are likely to contribute to the "response" seen upon pre-emptive posttransplant 5-AzaC therapy of patients with high-risk myelodysplastic syndrome (MDS). C_LIO_LIThese properties are linked to a distinct form of epigenetic plasticity at upstream-located transcription factor (TF) binding sites. This may indirectly contribute to acute S100A8/A9-driven inflammation, which is demonstrable in distinct monocyte subsets and, importantly, also in NK cells thereby determining the characteristics of inflammatory monocyte-NK cell crosstalk. C_LIO_LIMice with a targeted deletion of S100A9 fail to upregulate CEBPB / JUN and NFIL3 which results in impaired myeloid priming and dysfunctional NK cell maturation, respectively. C_LIO_LIRe-analysis of the Australian MDS/CMML cohort confirms that MDS patients that "respond" to 5-AzaC exhibit activated IFN-{gamma}, IL6-JAK-STAT3, and TNF-signaling pathways in the context of upregulated S100A8/A9 after six months of treatment. C_LIO_LIOur study indicates that screening of healthy donors SCs for specific inflammatory markers in early developing monocytes could be used as a marker to predict which donor will have the potential of generating a S100A8/A9-driven inflammatory response. This may help identify patients with MDS as well as AML who are likely to benefit from low-dose, short-term 5-AzaC therapy as early as day 7 after transplantation, potentially resulting in increased graft-versus-leukemia (GvL) activity. C_LI

Patients with a dominant mutation in the Rho GTPase RAC2, RAC2E62K, which hyperactivates the protein, suffer from a combined immunodeficiency characterized by recurrent bacterial and fungal infections and severe T cell lymphopenia. Patient neutrophils have elevated F-actin and superoxide production yet fail to control growth of S. aureus, and the mechanism underlying this killing defect is unknown. Here we report that hyperactive Rac2 primes neutrophils for primary granule degranulation, potentially depleting myeloperoxidase (MPO) needed for intraphagosomal microbial killing. Using a Rac2+/E62K mouse model, we show that mature bone marrow neutrophils have decreased side scatter, elevated surface CD63, and reduced intracellular MPO. Interestingly, bone marrow architecture and neutrophil development in the mice are normal. Rac2+/E62K neutrophils are hyperactivated, with increased CD11b expression, cell spreading, and bioparticle phagocytosis. In the spleen, Rac2+/E62K mice display extramedullary granulopoiesis and an accumulation of degranulating neutrophils. Splenic T cells, but not B cells, show elevated surface phosphatidylserine, an "eat me" signal that sensitizes them to phagocytic clearance and provides a candidate mechanism for the selective T cell lymphopenia. Together these findings suggest that hyperactive Rac2 compromises antimicrobial neutrophil function and drives selective T cell clearance in the spleen.

Legionnaires disease (LD), a pneumonia caused by Legionella pneumophila intracellular bacterium, leads to intensive care unit (ICU) admission in 20-40% of cases. While these ICU-LD patients display severe lung injury or septic shock, their functional immune response remains poorly understood. The present study aimed, through a large immune gene expression assessment, to improve the understanding of immune cell functionality after whole blood LPS ex vivo stimulation in ICU-LD patients compared with non-ICU. Both ICU and non-ICU-LD displayed altered gene expression indicating that both patients immune cells are less able to respond to the LPS ex vivo stimulus than a healthy population. ICU-LD patients had 1.6-fold greater number of less-expressed genes (35/93 vs 22/93, p=0.039), and lower Log2(FC) of these genes (median [IQR]: -1.9 [-2.6;-1.5] vs -1.2 [-1.7;-0.9], p=0.0011) than non-ICU-LD. Seven genes were significantly less expressed by ICU-LD patients (IRF7, MX1, NFKBI2, NFKBIA, RELB, SRC, TIM3; p-value range: 0.029-0.0080). Top five gene ontology biological processes, subcellular localisations, and reactome pathways (STRING database) uniquely enriched in ICU-LD-patients and related less-expressed genes were cellular response to LPS (CCL2, NFKBIA, IRAK2, TIM3, SRC, NFKB1), regulation of IFN-{beta} production (IRF7, RIG1, OAS2, RELB), I-{kappa}B/NF-{kappa}B complex (NFKBIA, NFKB1, NFKB2), IFN regulatory factor complex (RIG1, IRF7), and TRAF6-mediated NF-{kappa}B activation pathway (NFKBIA, NFKB1, NFKB2, RIG1). Immune gene expression alterations in LD after LPS stimulation were found herein, with more pronounced alterations in ICU-LD patients. A reduced expression of key genes and pathways involved in controlling Legionella proliferation in ICU-LD patients may contribute to increased disease severity.

In mammals, most of the iron is found in the heme of red blood cells (RBCs), which must be recycled to support erythropoiesis in the bone marrow. Splenic red pulp macrophages (RPMs) play a crucial role in this process by phagocytosing senescent RBCs, metabolizing the heme and releasing iron back into the blood. Free cytoplasmic iron generates toxic reactive oxygen species, yet iron-specific adaptations of RPMs are not well documented. We previously reported that autophagy prevents ferroptosis in Langerhans cells, a cutaneous phagocyte subset. Thus, we hypothesized that autophagy may be important for the regulation of RPM metabolism and their maintenance of systemic iron homeostasis. To study this, we used Atg5flox/flox and Cd169cre mouse models to delete ATG5 in CD169+ macrophages, including RPMs. Atg5-deficient RPMs were decreased in number, and the remaining ones showed increased generation of toxic lipid peroxides. Spleens of Atg5{Delta}Cd169 mice were enlarged and contained more RBCs. Finally, autophagy impairment in RPMs exacerbated RBC loss in a model of phenylhydrazine-induced anemia. Our findings exemplify how dysregulation of macrophage metabolism alters their function and can disrupt tissue homeostasis upon challenge.

Neutrophils recruited to the airways are important for innate lung defense and can release neutrophil extracellular traps (NETs) to capture and eliminate microbes. While NETs are not abundant in healthy airways, uncontrolled NETosis is a known pathological feature and contributor to both chronic and acute respiratory diseases. Prior studies have shown that mucin glycoproteins secreted in the oral cavity and cervicovaginal tract can modulate NETosis, but it remains unknown whether mucins secreted in the respiratory tract influence NET formation. In these studies, we discovered that human airway mucus strongly inhibits NETosis in primary human neutrophils in a sialic acid dependent manner. In comparison, mucus produced by human airway epithelial cells genetically engineered to lack either MUC5B or MUC5AC secreted airway mucins showed a reduced ability to suppress NETosis. To assess how the lung microenvironment in obstructive lung diseases may influence mucus-dependent NET formation, we engineered a synthetic, mucin-laden hydrogel model with physical properties resembling that of mucus in a healthy lung and a disease-affected lung. When neutrophils were cultured on these gel substrates, we found that increasing gel stiffness led to a significantly greater extent of NETosis. Together these data demonstrate a new functional role of airway mucus in modulating neutrophil homeostasis in the respiratory tract and provide evidence that mucus dysfunction in disease can impair its ability to regulate NETosis.

Klebsiella pneumoniae is a leading cause of pneumonia and bacteremia and is especially dangerous in healthcare settings. Despite massive clinical significance, the mechanisms used by macrophages to kill K. pneumoniae are not well defined. Macrophages are critical for controlling K. pneumoniae as mice lacking monocyte-derived or alveolar macrophages have higher bacterial tissue burdens and mortality. Two prominent mechanisms used by macrophages to kill bacteria are the production of reactive oxygen species (ROS) via the NADPH oxidase NOX2 and reactive nitrogen species (RNS) via the inducible nitric oxide synthase iNOS. Previously, we found that K. pneumoniae uses similar genetic factors to survive during bacteremia and within macrophages. The ability of these factors to enhance intracellular fitness was significantly correlated with resistance against RNS, not ROS. Here, we aimed to define whether macrophage ROS and RNS contribute to intracellular K. pneumoniae clearance. Using wild-type, Cybb-/-, and Nos2-/- cells, we measured K. pneumoniae survival within macrophages lacking such defenses. NOX2 was dispensable for K. pneumoniae clearance, and ROS was undetectable in K. pneumoniae-infected macrophages. We confirmed that ROS was undetectable within alveolar-like macrophages, indicating a conserved ROS evasion phenotype across macrophage subsets. Instead, iNOS significantly contributed to macrophage clearance of K. pneumoniae and enhanced cytokine production. iNOS likely enhances K. pneumoniae clearance through coordination of immunity and RNS. Activation of pathways upstream of iNOS may be the most relevant to supporting effective macrophage control of K. pneumoniae. This study defines unexpected differential roles for ROS and RNS in macrophage clearance of K. pneumoniae.

The formation of the premetastatic niche prepares distant tissues for tumor cell engraftment. Endothelial cells are critical mediators of premetastatic niche formation, orchestrating extravasation of circulating tumor cells and critical pro-tumor immune cells, such as neutrophils. In mouse models of breast cancer, we show that primary tumors upregulate the non-signaling chemokine receptor ACKR1 in the endothelium of the lung premetastatic niche. ACKR1-expressing venules were found to be preferential sites of neutrophil and tumor cell localization within lung tissue. A newly generated conditional ACKR1 allele was used to show that endothelial-specific removal of ACKR1 expression significantly reduces metastatic engraftment in the lung. When ACKR1 is activated by tumor-secreted factors, endothelial ACKR1 functions to promote neutrophil recruitment within the lung parenchyma. We conclude that ACKR1 is a critical component of the endothelial response to tumors at the metastatic site of the lung, leading to neutrophil recruitment and promotion of tumor cell metastasis. SUMMARYEndothelial cells play critical roles in breast cancer metastasis. ACKR1 is upregulated in the endothelium of the lung metastatic niche in response to primary mammary tumors. Endothelial ACKR1 expression was found to promote neutrophil infiltration into the metastatic niche and support breast tumor cell metastasis to the lung.

Mounting evidence indicates that T cells can operate in an innate-like mode challenging the classical description of T cells as strictly adaptive immune effectors. T cells can engage innate pattern recognition receptors to mount rapid but antigen-nonspecific responses to infection or cellular stress. This study observed that CD8+ T cells, and to a lesser extent also CD4+ T cells, responded to viral proteins in the mouse lung quickly in an innate-like fashion. We employed intravital lung microscopy to visualize infiltration of CD8+ T cells into the lung following intratracheal instillation of the SARS-CoV-2 envelope (E)-protein. Here, we demonstrate acute recruitment of CD8+ from the pulmonary microcirculation into the lung as early as 4 and 24 hours after (E)-protein instillation. The acute infiltration of CD8+ T cells was not observed in Tlr2-/- mice. Immunohistochemistry analysis of mouse lungs revealed T cell accumulation in nodular inflammatory foci (NIF) of the lung at perivascular regions and around large airways. Stimulating spleen-derived CD8+ T cells from wild-type mice with (E)-protein ex vivo in combination with cytokines or TCR agonists significantly upregulated CD69 and activated secretion of interferon (IFN){gamma} which was not observed with CD8+ T cells isolated from Tlr2-/- mice. These findings indicate rapid bystander activation of CD8+ T cells by the SARS-CoV-2 envelope (E)-protein that depends on (E)-protein sensing by TLR2. This innate-like CD8+ T cell response to SARS-CoV-2 (E)-protein may offer novel opportunities for diagnostic and therapeutic development, warranting further investigation.