Citations

Abstract Microglia, the major resident immune cells in the central nervous system, serve as the frontline soldiers against cerebral ischemic injuries, possibly along with metabolic alterations. However, signaling pathways involved in the regulation of microglial immunometabolism in ischemic stroke remain to be further elucidated. In this study, using single‐nuclei RNA sequencing, a microglial subcluster up‐regulated in ischemic brain tissues is identified, with high expression of Igf1 and Trem2, neuroprotective transcriptional signature and enhanced oxidative phosphorylation. Microglial depletion by PLX3397 exacerbates ischemic brain damage, which is reversed by repopulating the microglia with high Igf1 and Trem2 phenotype. Mechanistically, Igf1 serves as one of the major down‐stream molecules of Trem2, and Trem2‐Igf1 signaling axis regulates microglial functional and metabolic profiles, exerting neuroprotective effects on ischemic stroke. Overexpression of Igf1 and supplementation of cyclocreatine restore microglial glucometabolic levels and cellular functions even in the absence of Trem2. These findings suggest that Trem2‐Igf1 signaling axis reprograms microglial immunometabolic profiles and shifts microglia toward a neuroprotective phenotype, which has promising therapeutic potential in treating ischemic stroke.

Primary Myelofibrosis (PMF) is an aggressive myeloproliferative neoplasm (MPN) characterized by constitutional symptoms, cytopenias, splenomegaly, extramedullary hematopoiesis, bone marrow (BM) fibrosis, and a propensity to transform to acute myeloid leukemia (AML). Despite advances in understanding the underlying genetic abnormalities in MPN and the development of JAK2 inhibitors to treat MF, there is an urgent need to devise new treatment strategies, particularly for triple negative myelofibrosis (TN-MF) cases that lack mutations in the JAK2 kinase pathway and who have inferior outcomes. To identify potential oncogenic drivers in TN-MF, we performed cytogenetic analyses and targeted exome sequencing of 98 genes commonly mutated in myeloid malignancies in 584 MF patients identified from the Total Cancer Care database at Moffitt Cancer Center. Notably, there were no significant differences in the somatic mutation profiles (other than JAK2 activating mutations) between TN vs. JAK2/CALR/MPL mutant MF patients. However, trisomy of chromosome 8 occurs more frequently in TN-MF vs. other subtypes (26.7% vs. 6.2%, p=0.0001). To identify oncogenic drivers in trisomy 8+ TN-MF, we performed scRNA-seq analysis from 18,651 cells ( n=15,850 from 3 normal donors and n=2,801 from a trisomy 8+ TN-MF patient) and identified a total of 1,260 genes that are differentially regulated in hematopoietic stem cells (HSCs) of trisomy 8+ TN-MF compared to normal donors. Among these, 131 genes were located on chromosome 8 and MYC was one of the top 5 genes that are significantly upregulated in trisomy 8+ HSCs. Additional immunohistochemistry staining demonstrated that MYC protein levels in BM cells were significantly higher in trisomy 8+ TN-MF patients vs. trisomy 8- patients. To assess whether MYC might be a driver of MPN, we established an Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC transgenic mouse model that inducibly overexpresses MYC in HSCs following treatment of polyinosinic-polycytidylic acid (pIpC). Forced MYC expression induced profound anemia, monocytosis, megakaryocytic atypia, splenomegaly, increased BM collagen deposition/fibrosis, extramedullary hematopoiesis in spleen and liver, and significantly reduced OS vs. Mx1-Cre +/- wild type controls (median OS 258 days vs. not reached (NR), p<0.0001). MYC also promoted expansion of HSCs, myeloid progenitors, and Gr-1 +/CD11b + mature myeloid cells in BM and spleen; thus, MYC was sufficient to provoke MF-like disease independent of JAK2 pathway mutations. Subsequent scRNA-seq analysis of 25,232 cells ( n=13,552 from control and n=11,680 from a MYC transgenic mouse) revealed that S100a9 mRNA levels were increased by MYC in most major hematopoietic cell types. Supporting oncogenic roles of S100A9 in MYC-driven MF, deletion of S100a9 in Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC;S100a9 -/- mice abolished MYC-induced MF phenotypes and significantly improved OS (median OS NR vs. 225 days, p=0.0345). To test whether inhibition of MYC can suppress MF disease progression in vivo, lethally irradiated CD45.1 +/CD45.2 + WT mice were transplanted with BM cells harvested from CD45.2 + Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC mice that were treated with pIpC 20 weeks prior to transplant. MYCi975, a small molecule that inhibits MYC, reduced MYC and S100a9 protein levels in BM and spleen cells. Further, MYCi975 effectively suppressed MYC-driven MF phenotypes and disease progression, and significantly improved OS (median OS NR vs. 252 days, p=0.0109). In summary, our studies are the first to describe an oncogenic role of MYC in MF pathogenesis, where MYC provokes an alarmin-driven inflammatory circuit, and where the MYC-S100A9 axis represents a therapeutic vulnerability for TN-MF patients. Accordingly, our results provide a strong rationale for testing agents targeting MYC or S100A9 in early phase clinical trials in MPNs having increased MYC levels or activity.

Our group has reported that transient CD19 antigen masking of leukemic cells with the CD19 monoclonal antibody (mAb), tafasitamab, resulted in reduction of CD19 chimeric antigen receptor T cell (CART19) apoptosis, enhanced CAR T proliferation, improved anti-tumor activity, diminished tumor pyroptosis, and reduced severity of cytokine release syndrome (CRS) in preclinical models. In this report, we aimed to study the mAb characteristics responsible for these observations. Specifically, we hypothesized that binding affinity of CD19 mAbs might affect the subsequent activation and apoptosis of CART19 as well as tumor cell pyroptosis in preclinical models. To test our hypothesis, we first determined the affinities of the CD19-targeting antibodies tafasitamab, HD37 and FMC63 to recombinant human CD19 protein. Tafasitamab demonstrated the strongest binding affinity among the three tested antibodies [tafasitamab ( K D: 1.0±0.2 nM), HD37 ( K D: 4.1±0.3 nM), FMC63 ( K D: 3.4±1.2 nM)]. Next, we studied CART19 apoptosis in the presence of these CD19 mAbs with different binding affinities. Here, we co-cultured CART19, CD19 + mantle cell lymphoma JeKo, and 400 μg/mL of CD19 mAb for 1 hour and measured CD3 + 7-AAD - Annexin V + T cells with flow cytometry. The number of apoptotic CART19 cells was significantly reduced by tafasitamab co-treatment compared to HD37, FMC63, or control IgG [control IgG (mean = 19.1, standard deviation (SD) = 0.8), tafasitamab (mean = 13.1, SD = 2.9), HD37 (mean = 17.8, SD = 0.8), FMC63 (mean = 18.4, SD = 1.0)]. We also performed an in vitro high mobility group box 1 (HMGB1) assay to assess if different affinities of the CD19 mAbs affect tumor cell pyroptosis triggered by CART19. Here, we co-cultured CART19, CD19 + JeKo, and 400 μg/mL of CD19 mAb for 24 hours and assessed HMGB1 on JeKo. In analogy to the apoptosis data, tafasitamab was the only mAb that prevented tumor cell pyroptosis, indicated by reduced HMGB1 expression (Fig 1A). Given these results, we aimed to study the impact of CD19 occupancy with different CD19 mAbs prior to CART19 infusion on tumor pyroptosis in vivo. We utilized a mouse model that mimics CAR T toxicities. Immunocompromised NSG mice were inoculated via tail vein with 5x10 6 leukemic cells derived from patients with relapsed/refractory acute lymphoblastic leukemia. Two weeks later (day -7), tumor burden was assessed by peripheral blood sampling. Leukemic cells were defined as murine CD45 -, human CD45 + CD19 + CD20 + via flow cytometry, and volumetric absolute number was calculated. Mice were then randomized according to tumor burden to receive IgG control [Group 1 (G1)], tafasitamab (G2), HD37 (G3), or FMC63 (G4) (5 mg/kg, ip) at day -7. CRS was determined by weight loss, development of motor weakness, poor body posture, and elevated serum cytokine levels. At day 0, all mice received 3x10 6 luciferase + CART19 via tail vein. Mice were monitored with daily bioluminescent imaging (BLI) to track and quantify CART19 expansion and physical assessment to detect toxicity. BLI on day 4 revealed that CART19 expansion in the tafasitamab-treated group (G2, mean = 1x10 7, SD = 2.3x10 7) was significantly lower than in the control group (mean = 3.5x10 7, SD = 9.3x10 7), the HD37-treated group (G3, mean = 4x10 7, SD = 6.6x10 7), and the FMC63-treated group (G4, mean = 2x10 7, SD = 3.59x10 7), [G1 vs G2: 95% confidence interval (CI) 3.5x10 7 to 1.1x10 8, p <0.0001, G1 vs G3: 95% CI -7.5x10 7 to -1.1x10 8, p=0.005, G1 vs G4: 95% CI -4.5x10 7 to 1.9x10 7, p=0.71] indicating that tafasitamab pretreatment significantly reduced early activation of CART19 compared to IgG control and HD37 in this model. Mice were bled on day 4 to analyze plasma cytokine. Tafasitamab-treated mice (G2) secreted significantly less IFNγ (Fig 1B), IL10 (Mean G1: 78, G2: 2.52, G3: 12.2, G4: 13.9 pg/mL), MDC (Mean G1: 4072, G2: 445, G3: 921, G4: 1364 pg/mL), and MIP1β (Mean G1: 283, G2: 59.1, G3: 148, G4: 123 pg/mL) compared to G1, G3, and G4. Satellite mice were euthanized on day 1 and spleens were harvested. Western blot analysis of splenocytes revealed that G1, 3, and 4 showed significantly lower expression of apoptosis-inducing protein gasdermin E on leukemic cells compared to G2, indicating reduced tumor pyroptosis in G2. In summary, application of the high affinity mAb tafasitamab prior to CART19 resulted in reduced CAR T apoptosis, diminished tumor cell pyroptosis, and reduction of CRS compared to lower affinity CD19 mAbs in preclinical models.

Nonalcoholic fatty liver disease (NAFLD) is considered an independent risk factor for the development of cardiovascular disease. However, the association between changes in NAFLD status and the risk of cardiovascular disease (CVD) remains uncertain. Starting January 1, 2013, participants were followed until the occurrence of CVD event, death, or December 31, 2020. This was a population-based cohort study that included data from adults aged ≥ 20, who underwent 2 consecutive health screenings from 2009 to 2012. NAFLD was defined as a Fatty Liver Index ≥ 60 at each screening. The primary endpoint was a CVD event, which encompassed ischemic heart disease and cerebrovascular disease. The association between changes in NAFLD status and the risk of CVD was determined using multivariable Cox proportional hazards regression. This cohort comprised 4656,305 adults with a median age of 53 years. During 36,396,968 person-years of follow-up, 238,933 (5.1%) CVD events were observed. Compared to patients with no NAFLD at both screenings, patients who developed NAFLD at the second screening exhibited an increased risk of CVD (adjusted hazard ratio, 1.15; 95% confidence interval, 1.13–1.17). In contrast, individuals who recovered from NAFLD at the second screening demonstrated a reduced CVD risk compared to those with persistent NAFLD (adjusted hazard ratio, 0.91; 95% confidence interval, 0.90–0.92). The reversal of NAFLD is associated with a reduced risk of CVD. Therefore, focusing on NAFLD treatment could serve as a clinical target for lowering CVD risk.

Abstract The long-term physiological consequences of SARS-CoV-2, termed Post-Acute Sequelae of COVID-19 (PASC), are rapidly evolving into a major public health concern. The underlying cellular and molecular etiology remain poorly defined but growing evidence links PASC to abnormal immune responses and/or poor organ recovery post-infection. Yet, the precise mechanisms driving non-resolving inflammation and impaired tissue repair in the context of PASC remain unclear. With insights from three independent clinical cohorts of PASC patients with abnormal lung function and/or viral infection-mediated pulmonary fibrosis, we established a clinically relevant mouse model of post-viral lung sequelae to investigate the pathophysiology of respiratory PASC. By employing a combination of spatial transcriptomics and imaging, we identified dysregulated proximal interactions between immune cells and epithelial progenitors unique to the fibroproliferation in respiratory PASC but not acute COVID-19 or idiopathic pulmonary fibrosis (IPF). Specifically, we found a central role for lung-resident CD8+ T cell macrophage interactions in maintaining Krt8hi transitional and ectopic Krt5+ basal cell progenitors, thus impairing alveolar regeneration and driving fibrotic sequelae after acute viral pneumonia. Mechanistically, CD8+ T cell derived IFN-γ and TNF stimulated lung macrophages to chronically release IL-1β, resulting in the abnormal accumulation of dysplastic epithelial progenitors and fibrosis. Notably, therapeutic neutralization of IFN-γ and TNF, or IL-1β after the resolution of acute infection resulted in markedly improved alveolar regeneration and restoration of pulmonary function. Together, our findings implicate a dysregulated immune-epithelial progenitor niche in driving respiratory PASC. Moreover, in contrast to other approaches requiring early intervention, we highlight therapeutic strategies to rescue fibrotic disease in the aftermath of respiratory viral infections, addressing the current unmet need in the clinical management of PASC and post-viral disease.

Local production of growth factors within the airway with autocrine/paracrine effects can promote features of asthma. Here, we show that glial-derived neurotrophic factor (GDNF) is a procontractile and proremodeling factor that contributes to allergen-induced airway hyperreactivity and tissue remodeling in a mouse model of asthma. Blocking GDNF signaling attenuates allergen-induced airway hyperreactivity and remodeling, suggesting a novel approach to alleviating structural and functional changes in the asthmatic airway.