Abstract Introduction IL-2 is central to CD8 T cell responses and signals via JAK-STAT5, RAF-ERK-MAPK, and PI3K-AKT pathways, but how STAT5-dominant output is enforced from this shared receptor is unclear. We hypothesized that uncharacterized IL-2-induced, STAT5-regulated E3 ubiquitin ligases sculpt this signaling hierarchy to optimize antiviral CD8 T cell function. Methods RNA-seq of human CD8 T cells stimulated with IL-2 ± JAK inhibition plus STAT5 ChIP-seq identified IL-2/STAT5-regulated ubiquitin genes. Rnf144a-/- mice and mixed bone marrow chimeras were used to define CD8-intrinsic roles during influenza infection. RNF144A localization and substrates were mapped by biochemical and imaging assays, and whole-blood transcriptomes from patients with moderate or severe influenza were analyzed to relate RNF144A expression and gene signatures to clinical severity. Results IL-2 broadly remodeled the T cell ubiquitin program, with RNF144A emerging as the most strongly induced STAT5-bound E3 ligase. RNF144A localized to the plasma membrane, associated with IL-2Rβ and STAT5, and enhanced STAT5 recruitment and phosphorylation, sustaining STAT5-dependent transcription. In parallel, RNF144A acted as a bona fide E3 ligase that directly polyubiquitinated RAF1 for degradation, reducing ERK activation and thereby preserving JAK-STAT5 dominance over RAF-ERK-MAPK output. CD8 T cells from Rnf144a-/- mice showed impaired IL-2-induced effector gene expression, degranulation, and cytokine production, and CD8-intrinsic deficiency in mixed chimeras reduced antigen-specific responses and worsened weight loss and lung inflammation after influenza infection. In human influenza, RNF144A expression was reduced in severe disease, inversely correlated with an ERK-MAPK target gene signature, and discriminated severe from moderate cases with performance comparable to established severity markers. Conclusion RNF144A is an IL-2-STAT5-induced E3 ligase that enforces STAT5-dominant signaling and limits viral immunopathology. Funding Source N/A Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Rationale: Pulmonary arterial hypertension (PAH) is a rare, female-predominant disease characterized by an inflammatory, proliferative arteriopathy that results in progressive narrowing of pre-capillary pulmonary arterioles and eventually death from right heart failure. Pre-clinical PAH animal models are critical to the development of novel therapeutics and the most common primary endpoint of these studies is improvement in histopathological lung vessel remodeling. However, current methods for evaluating pulmonary vascular histopathology are subjective, time-consuming and inconsistently applied across studies. To address these limitations, our group sought to develop a deep learning algorithm with the ability to perform rigorous and non-biased histopathological assessments in the rat SU-5416/Hypoxia (SuHx) model of PAH. Methods: In order to develop an algorithm that recognizes pulmonary arterioles and determines the extent of vessel occlusion, DeepLabV3+ (Visiopharm®)—a multi-layered feature-detecting neural network—was trained to detect background, arteriole wall, and airway wall using manually annotated Masson Trichrome stained lung sections from SuHx (n=15) and healthy control (n=11) rats. DeepLabV3+ maintains denser feature maps and is thus better able to utilize context in decision making compared to similar neural networks. The model was trained to detect airways in addition to arterioles and background in order to prevent inaccurate labeling of airways as arterioles due to their similar morphology. The model was allowed to train to 200,000 iterations to maximize learning and minimize overfitting. A validation set was generated using lung sections which had not been displayed to the model during training (SuHx: n=3, control: n=2). Because the data set used to create the model included more SuHx animals than control animals, a greater image area was used from control animals for both the training and validation sets. Results: Precision, sensitivity, and dice scores (dice score >0.5 is average, >0.7 is good) were calculated per pixel and per object (see figure below). Based on these metrics, the deep learning algorithm performed better in lung sections from SuHx animals. Notably, many of the “false positives” identified by the deep learning algorithm were indeed arterioles that were either inadvertently or intentionally excluded from the validation set due to pre-specified criteria such as size and wall clarity. Conclusion: Lung vessel segmentation using a deep learning algorithm is feasible, however, based on the current performance metrics, further refinements are required to improve model validity. In addition, a more exhaustive validation set with less pre-specified criteria for vessel inclusion may be more appropriate for assessing model performance.

Thymic stromal lymphopoietin (TSLP) is a type I cytokine that promotes allergic responses and mediates type 2 immunity. A balance between effector T cells (Teffs), which drive the immune response, and regulatory T cells (Tregs), which suppress the response, is required for proper immune homeostasis. Here, we report that TSLP differentially acts on Teffs versus Tregs to balance type 2 immunity. As expected, deletion of TSLP receptor (TSLPR) on all T cells (Cd4CreCrlf2fl/fl mice) resulted in lower numbers of T helper 2 (TH2) cells and diminished ovalbumin-induced airway inflammation, but selective deletion of TSLPR on Tregs (Foxp3YFP-Cre/YCrlf2fl/fl mice) resulted in increased interleukin-5 (IL-5)- and IL-13-secreting TH2 cells and lung eosinophilia. Moreover, TSLP augmented the expression of factors that stabilize Tregs. During type 2 immune responses, TSLPR-deficient Tregs acquired TH2-like properties, with augmented GATA3 expression and secretion of IL-13. TSLP not only is a driver of TH2 effector cells but also acts in a negative feedback loop, thus promoting the ability of Tregs to limit allergic inflammation.

Introduction/Purpose:During the septic cardiomyopathy, the mechanism and relationship to outcome of changes in left ventricular (LV) end diastolic volume (EDV) and ejection fraction (EF) remains obscure. We compared serial changes in LVEF and LVEDV to successive alterations in LV wall ultrastructure, water content, and total mass to investigate whether these measures can explain their basis. Methods:We performed cardiac magnetic resonance imaging at 0,6,18,30,42,54, and 92h post-bacterial challenge in a large-animal model (n=57) that mimics human septic cardiomyopathy. LV tissue was obtained for electron microscopy (EM) upon death and 66h in sacrificed survivors. Results:Between 0-6h post-challenge, LV compliance and EDV reached its greatest decline. Non-survivors (n=18) exhibited significantly greater reductions in LVEDV, along with more myocyte edema, mitochondrial swelling and myofilament fragmentation on EM. This increased tissue damage may explain why non-survivors developed worse LV compliance and a greater decline in LVEDV, which persisted until death. From 6-30h, LVEDV significantly improved to baseline in non-survivors, while survivors experienced ∼20% increases (n=39). Concurrently, there was significant LV mass loss and increases in percent water content that were significantly associated with increases in LVEDV. This is consistent with a passive mechanism for rapidly improving LV compliance and EDV. Full recovery of EF required additional days. We hypothesize the prolonged significant mass loss over 5d reflects an active process for remodeling fragmented myofilaments, eliminating myocyte edema, and mitochondrial swelling, ultimately restoring contractile function. Conclusion:The septic cardiomyopathy constitutes a diffuse ultrastructural injury to myocytes with three phases. Initially, there is a decrease in LVEDV, and EF due to myocyte damage within 6h of bacterial challenge; next, the patient sees a passive LVEDV recovery from 6-30h, where LV mass loss increases relative wall percent water content, which facilitates wall compliance and LVEDV; and lastly, the patient sees mass loss beyond 30h consistent with an active repair mechanism of myocytes, returning systolic function to normal. Therefore, EDV changes are a pathophysiological biomarker for sepsis outcomes. A lower LVEDV indicates persistent unrepairable ultrastructure damage with worsening wall compliance and poorer outcomes. LVEDV dilation is a sign of near-full recovery of ultrastructure injury, augmenting wall compliance and improving outcomes. Clinical Implications:We explain herein why septic cardiomyopathy findings don't have clinical implications like heart failure. Septic patients who exhibit signs of heart failure, low LVEF with high EDVs, are doing well - reflecting mild myocyte injury, effective damaged tissues clearance, increased relative LV wall water content, and compliance. This augments the LVEDV, lowering the LVEF. Septic patients who deteriorate rapidly, contrary to heart failure patients, show high/normal LVEF and low/normal LVEDV. Here, the myocyte damage is severe, leading to insufficient wall repair, and this decreased wall compliance persists, preventing the LV from dilating and making LVEDV low which ultimately raises the LVEF.

In USA, six million individuals with Sub-Saharan ancestry carry two APOL1 high-risk variants, which increase the risk for kidney diseases. Whether APOL1 high-risk variants increase other diseases under dyslipidemia remains unclear and requires further investigation.We characterized a mouse model to investigate the role of APOL1 in dyslipidemia and cardiovascular diseases. Transgenic mice carrying APOL1 (G0 and G1 variants)on bacterial artificial chromosomes (BAC/APOL1 mice) were crossed with the ApoE knock-out (ApoE-KO) dyslipidemia and atherosclerosis mouse model. The compound transgenic mice were evaluated for the impact of APOL1 on systemic phenotypes. ApoE-KO mice carrying APOL1-G0 and APOL1-G1 did not show differences in the extent of atherosclerotic lesions or aortic calcification, as evaluated by Sudan IV staining and radiographic examination, respectively. However, ~20% of ApoE-KO; BAC/APOL1-G1 mice developed hydrocephalus and required euthanasia. The hydrocephalus was communicating and likely was due to excess cerebrospinal fluid produced by the choroid plexus, where epithelial cells expressed APOL1. Single-nuclear RNA-seq of choroid plexus identified solute transporter upregulation and mTORC2 pathway activation in APOL1-G1-expressing epithelial cells. Further, in the All of Us cohort, we found higher hydrocephalus prevalence among individuals with the APOL1-G1 variant in both recessive and dominant models, supporting the mouse findings. While APOL1-G1 expression in ApoE-KO mice did not worsen cardiovascular disease phenotypes, we uncovered hydrocephalus as a novel APOL1 risk allele-mediated phenotype. These findings extend the spectrum of APOL1-associated pathologies.

Apolipoprotein A-I (APOA1), the major protein of high-density lipoproteins, has anti-inflammatory functions. APOA1 is primarily produced in the liver; however, it is not known whether neutrophils are a cellular source. Here, we assessed whether human neutrophils express APOA1. Peripheral blood and bronchoalveolar lavage fluid (BALF) were obtained from healthy volunteers (HVs) and asthmatics. Peripheral blood neutrophils from HVs expressed APOA1 at both the mRNA and protein levels, while confocal microscopy demonstrated that APOA1 was localized to a unique population of intracytoplasmic granules. In HVs and asthmatics, APOA1 was preferentially expressed by neutrophils with high side-scatter (SSChigh) in blood and BALF. Furthermore, APOA1+ SSChigh neutrophils were characterized as a population with high levels of caspase-3/caspase-7 activation and CCR5 expression. Since APOA1 has anti-inflammatory functions, this suggests that APOA1 expression by neutrophils may represent a mechanism to attenuate excessive inflammatory responses in health and disease.

Individuals with sickle cell disease (SCD) experience red blood cell sickling, hemolysis, endothelial dysfunction, and vaso-occlusive crises that can lead to substantial organ damage and a significant decrease in adult life expectancy. Allogeneic hematopoietic cell transplantation (HCT) is the most widely available curative option for SCD; however, due to overt organ damage, adults with SCD cannot tolerate myeloablative conditioning. Thus, we adopted a non-myeloablative (NM) regimen for patients with severe organ damage. However, little is known about the impact of NM HCT on organ function in adults with SCD. Here, we utilized the humanized Townes SCD murine model, which closely mimics the SCD phenotype, to assess the impact of NM HCT on the heart, lungs, kidneys, liver, brain, and neurocognitive function. We hypothesized that organ damage would reverse or cease to progress compared to mice that did not undergo NM HCT. We established a congenic model by transplanting female donor C57BL/6 (CD45.1) bone marrow cells into female Townes HbSS (CD45.2) recipients. The transplant conditioning regimen included low-dose total body irradiation and sirolimus. Follow-up analysis was performed serially over 6 months (endpoint) to assess the engraftment status and organ function. Our results demonstrate that NM HCT achieved an average donor myeloid chimerism of 17.76 ±1.89% by the endpoint, with a significant reduction in the sickle hemoglobin fraction post-HCT (from 99.63 ±0.22% to 29.11 ±3.52%, p < 0.0001). Hemoglobin concentration was significantly higher in transplanted, 11.97 ±0.27 g/dL, compared to HbSS mice, 9.59 ±0.22 g/dL (p < 0.0001). In addition, the total bilirubin level normalized in HbSS transplanted compared to HbSS control mice (0.29 ±0.15 mg/dL, compared to 0.89 ±0.30 mg/dL, p < 0.0001). Cardiopulmonary function was evaluated by testing the maximal treadmill running capacity of mice. The average running time until exhaustion at the endpoint was significantly higher in HbSS transplanted (23.89 ±1.25 min) compared to HbSS control mice (17.90 ±1.32 min, p = 0.0005). Echocardiogram analysis showed that HbSS transplanted mice had a similar ejection fraction (EF), 61.1 ±1.59%, to the HbAA mice, 59.74 ±0.55%, at the endpoint (p > 0.05). In contrast, control HbSS mice had a significantly lower EF at 54.84 ±0.99% compared to HbSS transplanted and HbAA mice, p < 0.0001, p = 0.0017, respectively. In addition, HbSS control mice had a significantly higher LV systolic (LVSV, 48.4 ±4.63 uL) and diastolic volume (LVDV, 106.61 ± 9.06 uL) compared to HbSS transplanted mice (29.34 ± 4.35 uL and 73.1 ± 8.06 uL, respectively, p < 0.0001). LVSV and LVDV values for HbSS transplant were similar to HbAA mice (24.14 ±0.89 uL and 59.9 ± 2.17 uL, respectively, p > 0.05) (Panel A). The average cardiac mass in HbSS transplanted mice at the endpoint was significantly lower at 138.65 ±9.12 mL/min compared to the HbSS control mice at 189.95 ±13.18 mL/min (p < 0.0001). Left renal Doppler scans at the endpoint showed a significantly lower resistivity index in HbSS transplanted (0.64 ±0.03) compared to HbSS control mice (0.78 ± 0.03, p = 0.0028). Compared to the resistivity index for HbAA controls (0.48 ±0.01), HbSS control and transplanted mice were significantly higher (p < 0.0001) (Panel B). This significant improvement in the renal blood flow was reflected in the Doppler images of the same HbSS mouse pre- and post-HCT (Panel C). In sum, NM HCT successfully achieved mixed donor/recipient chimerism with improved hemoglobin and hemolysis markers. Reversal of the SCD phenotype in transplanted HbSS mice led to improvement in the cardiopulmonary and renal morphology and function post-HCT. LV End Systolic Volume: A. Left Ventricle (LV) systolic volume. Noninvasive echocardiography performed on Townes mice. Separated by genotype, AA, SS and SS Transplanted. Comparing mice at baseline, 10 weeks old, to the endpoint, 38 weeks old. Each bar represents cumulative data with a n = 12-19 per group. (Mean ± SEM) *P < 0.0001Left Renal Doppler: Resistivity Index (RI). Noninvasive ultrasound performed on Townes mice. Separated by genotype, AA, SS and SS Transplanted. Comparing mice at baseline, 10 weeks old, to the endpoint, 38 weeks old. Each data point represents individual data with a n = 12-19 per group. (Mean ± SEM) *P < 0.0001Left Renal Doppler Image. Pre-transplant (baseline) and Post-transplant (endpoint) Renal doppler scan of Townes mice at 2 months old pre-transplant (baseline) and 9 months old post-transplant (endpoint)

Cytokine-mediated STAT5 protein activation is vital for lymphocyte development and function. In vitro tyrosine phosphorylation of a C-terminal tyrosine is critical for activation of STAT5A and STAT5B; however, the importance of STAT5 tyrosine phosphorylation in vivo has not been assessed. Here we generate Stat5a and Stat5b tyrosine-to-phenylalanine mutant knockin mice and find they have greatly reduced CD8+ T-cell numbers and profoundly diminished IL-2-induced proliferation of these cells, and this correlates with reduced induction of Myc, pRB, a range of cyclins and CDKs, and a partial G1→S phase-transition block. These mutant CD8+ T cells also exhibit decreased IL-2-mediated activation of pERK and pAKT, which we attribute in part to diminished expression of IL-2Rβ and IL-2Rγ. Our findings thus demonstrate that tyrosine phosphorylation of both STAT5A and STAT5B is essential for maximal IL-2 signaling. Moreover, our transcriptomic and proteomic analyses elucidate the molecular basis of the IL-2-induced proliferation of CD8+ T cells. The importance of STAT5 tyrosine phosphorylation on T cells has not been investigated in vivo. Here the authors generate STAT5A and STAT5B tyrosine mutant knockin mice and show that Stat5a/b tyrosine is required for maximal IL-2 signaling and CD8 + T cell proliferation.

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, activates NOTCH1 signaling and plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in PAECs activated AKT and decreased DLL4 expression. DLL4 loss was also seen in lungs of patients with IPAH and HPAH. Over-expression of DLL4 in PAECs induced BMPR2 promoter activity and exogenous DLL4 increased BMPR2 mRNA through NOTCH1 activation. Furthermore, DLL4/NOTCH1 signaling blocked AKT activation, decreased proliferation and reversed EndoMT in BMPR2-silenced PAECs and ECs from IPAH patients. PPARγ, suppressed by BMPR2 loss, was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH PAECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Finally, leniolisib, a well-tolerated oral PI3Kδ/AKT inhibitor, decreased cell proliferation, induced apoptosis and reversed markers of EndoMT in BMPR2-silenced PAECs. Restoring DLL4/NOTCH1/PPARγ signaling and/or suppressing AKT activation may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

ABSTRACT BACKGROUND In hypoxic and pseudohypoxic rodent models of pulmonary arterial hypertension (PAH), hypoxia-inducible factor (HIF) inhibition reduces disease severity. However, HIF activation alone, due to genetic alterations or use of inhibitors of prolyl hydroxylase domain (PHD) enzymes, has not been definitively shown to cause PAH in humans, indicating the involvement of other mechanisms. METHODS Pseudohypoxia was investigated in primary human lung endothelial cells by silencing PHD2, and in Tie2-Cre / Phd2 knockout mice, a rodent model of PAH. Lung vascular endothelial cells from PAH patients, and lung tissue from both SU5416/hypoxia PAH rats and PAH patients, were examined for validation. RESULTS PHD2 silencing or inhibition, while activating HIF2α, induces apoptosis-resistance, hypo-proliferation, and IFN/STAT activation in endothelial cells, independent of HIF signaling. Mechanistically, PHD2 deficiency activates AKT and ERK, inhibits JNK, and reduces AIP1 (ASK1-interacting protein 1), all independent of HIF2α. Like PHD2, AIP1 silencing affects these same kinase pathways and produces a similar dysfunctional endothelial cell phenotype, which can be partially reversed by AKT inhibition. These findings are corroborated in lung tissues of rodent PAH models and pulmonary vascular endothelial cells and tissues from PAH patients. CONCLUSIONS PHD2 deficiency in lung vascular endothelial cells induces an apoptosis-resistant, inflammatory, and hypo-proliferative phenotype. AKT activation and AIP1 loss, but not HIF signaling, drive these aberrant phenotypic changes. Our study suggests that HIF blockade alone may not suffice for PAH therapy; targeting PHD2, AKT, and AIP1 has the potential for developing more effective treatment. GRAPHIC ABSTRACT Highlights PHD2 silencing in human lung vascular endothelial cells suppresses apoptosis, inhibits proliferation, and activates STAT signaling, effects that persist despite HIF2α inhibition or knockdown. PHD2 silencing activates AKT and ERK, inhibits JNK, and decreases AIP1, all independently of HIF2α Like PHD2, AIP1 silencing led to similar alterations in kinase signaling and endothelial cell phenotypes, which are partially reversed by ATK inhibition. These in vitro findings align with observations in lung vascular endothelial cells and tissues from rodent models of PAH as well as PAH patients.

Background:Septic shock, in humans and in our well-established animal model, is associated with increases in biventricular end diastolic volume (EDV) and decreases in ejection fraction (EF). These abnormalities occur over 2 days and reverse within 10 days. Septic non-survivors do not develop an increase in EDV. The mechanism for this cardiac dysfunction and EDV differences is unknown. Methods:Purpose-bred beagles randomized to receive intrabronchial Staphylococcus aureus (n=27) or saline (n=6) were provided standard ICU care including sedation, mechanical ventilation, and fluid resuscitation to a pulmonary arterial occlusion pressure of over 10mmHg. No catecholamines were administered. Over 96h, cardiac magnetic resonance imaging, echocardiograms, and invasive hemodynamics were serially performed, and laboratory data was collected. Tissue was obtained at 66h from six septic animals. Results:From 0-96h after bacterial challenge, septic animals vs. controls had significantly increased left ventricular wall edema (6%) and wall thinning with loss of mass (15%) which was more pronounced at 48h in non-survivors than survivors. On histology, edema was located predominantly in myocytes, the interstitium, and endothelial cells. Edema was associated with significantly worse biventricular function (lower EFs), ventricular-arterial coupling, and circumferential strain. In septic animals, from 0-24h, the EDV decreased from baseline and, despite cardiac filling pressures being similar, decreased significantly more in non-survivors. From 24-48h, all septic animals had increases in biventricular chamber sizes. Survivors biventricular EDVs were significantly greater than baseline and in non-survivors, where biventricular EDVs were not different from baseline. Preload, afterload, or HR differences did not explain these differential serial changes in chamber size. Conclusion:Systolic and diastolic cardiac dysfunction during sepsis is associated with ventricular wall edema. Rather than differences in preload, afterload, or heart rate, structural alterations to the ventricular wall best account for the volume changes associated with outcome during sepsis. In non-survivors, from 0-24h, sepsis induces a more severe diastolic dysfunction, further decreasing chamber size. The loss of left ventricular mass with wall thinning in septic survivors may, in part explain, the EDV increases from 24-48h. However, these changes continued and even accelerated into the recovery phase consistent with a reparative process rather than ongoing injury.

Background Septic shock is associated with increases in end‐diastolic volume (EDV) and decreases in ejection fraction that reverse within 10 days. Nonsurvivors do not develop EDV increases. The mechanism is unknown. Methods and Results Purpose‐bred beagles (n=33) were randomized to receive intrabronchial Staphylococcus aureus or saline. Over 96 hours, cardiac magnetic resonance imaging and echocardiograms were performed. Tissue was obtained at 66 hours. From 0 to 96 hours after bacterial challenge, septic animals versus controls had significantly increased left ventricular wall edema (6%) and wall thinning with loss of mass (15%). On histology, the major finding was nonocclusive microvascular injury with edema in myocytes, the interstitium, and endothelial cells. Edema was associated with significant worsening of biventricular ejection fractions, ventricular‐arterial coupling, and circumferential strain. Early during sepsis, (0–24 hours), the EDV decreased; significantly more in nonsurvivors (ie, greater diastolic dysfunction). From 24 to 48 hours, septic animals' biventricular chamber sizes increased; in survivors significantly greater than baseline and nonsurvivors, whose EDVs were not different from baseline. Preload, afterload, or heart rate differences did not explain these differential changes. Conclusions The cardiac dysfunction of sepsis is associated with wall edema. In nonsurvivors, at 0 to 24 hours, sepsis induces a more severe diastolic dysfunction, further decreasing chamber size. The loss of left ventricular mass with wall thinning in septic survivors may, in part, explain the EDV increases from 24 to 48 hours because of a potentially reparative process removing damaged wall tissue. Septic cardiomyopathy is most consistent with a nonocclusive microvascular injury resulting in edema causing reversible systolic and diastolic dysfunction with more severe diastolic dysfunction being associated with a decreased EDV and death.

The Krebs cycle enzyme aconitate decarboxylase 1 (ACOD1) mediates itaconate synthesis in monocytes and macrophages. Previously, we reported that administration of 4-octyl itaconate to lupus-prone mice abrogated immune dysregulation and clinical features. In this study, we explore the role of the endogenous ACOD1/itaconate pathway in the development of TLR7-induced lupus (imiquimod [IMQ] model). We found that, in vitro, ACOD1 was induced in mouse bone marrow-derived macrophages and human monocyte-derived macrophages following TLR7 stimulation. This induction was partially dependent on type I IFN receptor signaling and on specific intracellular pathways. In the IMQ-induced mouse model of lupus, ACOD1 knockout (Acod1-/-) displayed disruptions of the splenic architecture, increased serum levels of anti-dsDNA and proinflammatory cytokines, and enhanced kidney immune complex deposition and proteinuria, when compared with the IMQ-treated wild-type mice. Consistent with these results, Acod1-/- bone marrow-derived macrophages treated in vitro with IMQ showed higher proinflammatory features. Furthermore, itaconate serum levels in systemic lupus erythematosus patients were decreased compared with healthy individuals, in association with disease activity and specific perturbed cardiometabolic parameters. These findings suggest that the ACOD1/itaconate pathway plays important immunomodulatory and vasculoprotective roles in systemic lupus erythematosus, supporting the potential therapeutic role of itaconate analogs in autoimmune diseases. The Journal of Immunology, 2024, 213: 419-434.

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by pathologic vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in pulmonary artery endothelial cells (PAECs) activated AKT and suppressed the expression of DLL4. Consistent with these in vitro findings, increased AKT activation and reduced DLL4 expression was found in the small pulmonary arteries of patients with PAH. Increased NOTCH1 activation through exogenous DLL4 blocked AKT activation, decreased proliferation and reversed EndoMT. Exogenous and overexpression of DLL4 induced BMPR2 and PPRE promoter activity, and BMPR2 and PPARG mRNA in idiopathic PAH (IPAH) ECs. PPARγ, a nuclear receptor associated with EC homeostasis, suppressed by BMPR2 loss was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH ECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Directly blocking AKT or restoring DLL4/NOTCH1/PPARγ signaling may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.