Levels Of CD11b Research Articles

Preclinical efficacy of CDK7 inhibitor–based combinations against myeloproliferative neoplasms transformed to AML

AbstractRising blast percentage or secondary acute myeloid leukemia (sAML) transformation in myeloproliferative neoplasms (MPNs) leads to JAK inhibitor (JAKi) therapy resistance and poor survival. Here, we demonstrate that treatment with the CDK7 inhibitor (CDK7i) SY-5609 depletes phenotypically characterized post-MPN sAML stem/progenitor cells. In cultured post-MPN sAML SET2 and HEL as well as patient-derived (PD) post-MPN sAML cells, SY-5609 treatment inhibited growth and induced lethality while sparing normal cells. RNA-sequencing analysis after SY-5609 treatment demonstrated reduced messenger RNA (mRNA) expression of MYC, MYB, CDK4/6, PIM1, and CCND1 but increased mRNA levels of CDKN1A and BCL2L1. Mass spectrometry of SY-5609–treated MPN-sAML cells also demonstrated reduced c-Myc, c-Myb, PIM1, and CDK4/6 but increased p21, caspase 9, and BAD protein levels. CRISPR-mediated CDK7 depletion also reduced the viability of HEL cells. CyTOF analysis of SY-5609–treated PD post-MPN sAML stem/progenitor cells showed reduced c-Myc, CDK6, and PU.1 but increased protein levels of CD11b, p21, and cleaved caspase 3. Cotreatment with SY-5609 and ruxolitinib was synergistically lethal in HEL, SET2, and PD post-MPN sAML cells. A CRISPR screen in SET2 and HEL cells revealed BRD4, CBP, and p300 as codependencies with SY-5609 treatment. Accordingly, cotreatment with SY-5609 and the BETi OTX015 or pelabresib or with the CBP/p300 inhibitor GNE-049 was synergistically lethal in MPN-sAML cells (including those exhibiting TP53 loss). Finally, in the HEL-Luc/GFP xenograft model, compared with each agent alone, cotreatment with SY-5609 and OTX015 reduced post-MPN sAML burden and improved survival without inducing host toxicity. These findings demonstrate promising preclinical activity of the CDK7i-based combinations with BETi or HATi against advanced MPNs, including post-MPN sAML.

The role of IL15 in angiotensin II-mediated cardiac remodelling

Abstract Hypertension (HTN) mediated target organ damage, such as cardiac remodelling, is a major cause of death and disability. Cytokines are important regulators of cardiac function, inflammation, and remodelling in cardiovascular disease. Increased levels of interleukin 15 (IL15) have been noted in HTN, but its role in HTN heart disease remains unknown. This study investigates the role of the IL15 axis in the pathogenesis of cardiac remodelling in HTN. Male 12-week-old IL15 receptor α knock-out (IL15RAKO), IL15 knock-out (IL15KO) and matching wild type (WT) mice were administered vehicle or angiotensin II (AngII) (490 ng/min/kg) for 2 weeks by osmotic minipump. Blood pressure was measured by tail cuff (n=5-8). Cardiac function was analysed by echocardiography (n=3-4). Cardiac infiltrating immune cells were analysed by flow cytometry (n=2-3 for IL15RAKO, n=6-10 for IL15KO). Cardiac remodelling was assessed by picrosirius red, WGA staining (n=5-8) and expression of markers of remodelling using RT-qPCR and western blots (n=5-8). Data are presented as mean±SEM and analysed using two-way ANOVA. AngII infusion caused a significant increase in systolic blood pressure in WT and IL15KO animals. In contrast, in IL15RAKO, the response to AngII was blunted. This was in line with a decrease in ejection fraction, however only WT mice had an increase in global longitudinal strain (-20.3±2.0 vs -10.9±1.2, p=0.0039 in WT and 19.9±0.8 vs -16.0±2.1, p=0.1878 in IL15RAKO). HTN was associated with increased cardiac IL15 protein levels in mice. The heart-to-body ratio, along with cardiomyocyte cross-sectional, were significantly increased in WT, but not in IL15RAKO HTN mice. Furthermore, histological analysis showed significantly higher cardiac collagen accumulation in WT but not IL15RAKO upon AngII infusion (5.8±1.0% in WTvvs. 2.1±0.5% in IL15RAKO, p=0.0021). This was in line with a higher expression of col1a1 in WT but not IL15RAKO HTN hearts (20.0±6.0 vs 4.5±1.7, p=0.0173). Similarly, the FN1 protein level was significantly increased in WT but not IL15RAKO AngII-treated mice. These changes were associated with a higher number of Cd11b+ macrophages accumulated in WT hypertensive hearts only. In contrast to IL15RAKO, mice lacking IL15 showed similar increases in global longitudinal strain, heart hypertrophy, and cardiomyocyte cross-sectional area as WT mice upon AngII infusion. Similarly, IL15KO hypertensive hearts showed a significant increase of col1a1 and FN1 in comparison to vehicle-treated mice. A similar level of Cd11b+ macrophages was found in hypertensive IL15KO and WT mice's hearts. In conclusion, IL15RA, but not IL15, plays a critical role in cardiac remodelling mediated by AngII infusion. This suggests that the IL15-IL15RA axis may play an important yet complex role in hypertensive heart disease.

Combined administration of atorvastatin and mesenchymal stem cells synergizes to attenuate inflammation and promote efferocytosis in atherosclerotic mice

Abstract Background Atherosclerosis (AS), characterized by chronic systemic and lesional inflammation, is by far the most frequent underlying cause of cardio-cerebrovascular diseases. Recently, impaired efferocytosis, referring to defective removal of dead cells, was identified as another essential factor contributing to the development of AS, which is partly attributed to upregulation of CD47 and explain the benefits of atorvastatin (ATV). Mesenchymal stem cells (MSCs) were reported to protect against AS by inhibiting inflammation; while whether ATV and MSCs could synergize to attenuate inflammation and promote efferocytosis and consequently, reduce AS burden, remains unclear. Methods To establish the AS model, 6-week-old male ApoE-/- mice fed with Western diet for 12 weeks were used and grouped randomly (n=15 per group). The scheme of administration of ATV and human umbilical cord MSCs was shown in Figure 1A. After 6-week treatment, mice were sacrificed, and hearts and aortas were harvested. Oil Red O staining was used to evaluate the lesional burden. Immunofluorescence staining was used to assess the inflammation and efferocytosis in the plaque. A high level of CD11b+ cells (macrophages) infiltration was considered an indicator of inflammation; a high level of cleaved caspase 3 puncta unmerged with macrophages (Mac3+ area) was considered an indicator of impaired efferocytosis. Results Human umbilical cord MSCs were identified as CD73+CD90+CD105+CD45-CD14- cells. Evoked inflammation and defective clearance of apoptotic cells in the plaque were observed (Figure 1B, C). ATV and MSC treatment inhibited macrophage infiltration and activated efferocytosis. Given the mechanistic idea that upregulated CD47 blocks the Signal regulatory protein α (SIRPα) / src homology-2 (SH2)-domain-containing protein tyrosine phosphatases (SHP) signal and leads to efferocytosis impairment, we evaluated the expression levels of CD47 in plaque and phosphorylated SHP (p-SHP) in lesional macrophages (Figure 2A, B). The elevation of CD47 expression and consequent downregulation of p-SHP was reversed by both ATV and MSCs, with a lower lesion burden observed in NS+MSC, ATV+PBS and ATV+MSC groups. All benefits were more pronounced in the group received combined treatment (ATV+MSC). Conclusions The results indicated ATV and MSC administration could synergizes to attenuate inflammation and promote efferocytosis in AS plaque, and the combined treatment led to the lowest lesion burden. CD47/p-SHP signal plays an essential role in the activation of efferocytosis. The mechanisms underlying the inhibition of CD47 are currently under investigation.

Prognostic Significance of CD11b-, CD8-, and CD163-Positive Tumor-Infiltrating Immune Cells in Distal Bile Duct Cancer.

Background: Distal bile duct cancer is an aggressive malignancy. Tumor-infiltrating immune cells (TIICs) in the tumor microenvironment are crucial for predicting prognosis in various cancers. In this study, we analyzed TIICs based on CD11b, CD163, and CD8 expression, and evaluated their association with clinicopathologic factors and prognosis in distal bile duct cancer. Methods: A total of 90 patients who underwent curative resection for distal bile duct cancer were enrolled. We analyzed CD11b+ tumor-infiltrating myeloid cells (TIMs), CD163+ tumor-infiltrating macrophages (TAMs), and CD8+ tumor-infiltrating lymphocytes (TILs) using immunohistochemistry and tissue microarrays. The correlation between TIICs and clinicopathologic characteristics was assessed. Results: Low levels of CD11b+ TIMs (p < 0.001) and high levels of CD8+ TILs (p = 0.003) were significantly associated with improved overall survival (OS). A combined low level of CD11b+ TIMs and high level of CD8+ TILs was identified as an independent favorable prognostic factor (hazard ratio, 0.159; confidence interval, 0.061-0.410; p < 0.001). Conclusions: CD11b+ TIMs play a crucial role in the tumor microenvironment and the prognosis of distal bile duct cancer. The combined analysis of CD11b+ TIMs and CD8+ TILs can predict survival in patients with distal bile duct cancer.

The Effect of Photobiomodulation and Akkermansia muciniphila on THP-1 Derived Macrophage Polarization Treated with Gliadin Peptide.

Introduction: Photobiomodulation (PBM) and Akkermansia muciniphila have been shown to be effective in improving inflammatory conditions with positive effects on increasing the population of anti-inflammatory M2 macrophages (MQs). In this study, gliadin-stimulated THP-1 derived MQs were treated with A. muciniphila and PBM to evaluate their effects on promoting the polarization of M2 MQs. Methods: The human monocyte cell line (THP-1) was differentiated to MQs. MQs were stimulated with 200 μg/mL gliadin for 24 hours and then treated with PBM 810 nm alone and in combination with A. muciniphila for the following 24 hours to evaluate their effects on MQs polarization. THP-1 derived MQs were also treated with PBM and A. muciniphila to evaluate their effects on non-stimulated MQs. CD11b, CD80, and CD206 levels were evaluated by using the flow cytometry technique. Moreover, the expression of some M1 and M2-related cytokines was determined. Results: PBM therapy of gliadin-stimulated MQs decreased IL-6 and increased TGF-β, IL-10 and TNF-α expression compared with gliadin exposed MQs. PBM along with A. muciniphila treatment induced IL-6, TNF-α, and IL-10 expression in MQs in comparison to the untreated group. It also elevated TGF-β, IL-10 and TNF-α levels in gliadin-triggered MQs in comparison to gliadin-stimulated MQ cells. Conclusion: The result of this study showed the potential of PBMT and A. muciniphila for modulating inflammatory responses and MQs polarization. This may open new perspectives to find possible therapeutic targets for celiac diseases.

Immunomodulator Effect of Polyherbal Extract Turmeric-Meniran on Macrophages Profile of Mice Cancer Model by DMBA Injection

The immune system has responsibility for various infections, including cancer. Macrophage cells are divided into Macrophage 1 (M1) and Macrophage 2 (M2) based on polarization against cancer cells. Alternative medicine, such as herbal medicine, have potential to modulate the immune system. This study was conducted to evaluate the potential of the combination of Meniran and Turmeric extracts for regulating immune system cells, especially macrophage cells profile against cancer. This experiment was used Mus musculus cancer model that were injected subcutaneously with 7,12-dimethylbenz[a]anthracene (DMBA) 45 mg/kg body weight. Animal models were treated with Meniran and Turmeric extract combination for 2 weeks. The immunocompetent cell parameters were analyzed using flow cytometry, with markers CD11b cells and cytokines secreted in macrophage [i.e., interleukin (IL)-6, IL-10, also tumor necrosis factor α (TNF-α)]. The study showed that DMBA injection on mice increased the cell level of CD11b+ and CD11b+IL10+ (M2), at the same time decreased the cell level of M1, i.e., CD11b+IL6+ and CD11b+TNF-α+ compared with the Normal group. The Meniran and Turmeric extract combination treatment on DMBA-injected mice decreased the level of the cells of CD11b+ and CD11b+IL10+, meanwhile it increased the level of the cells of CD11b+IL6+ and CD11b+TNF-α+. The result indicated that the Meniran and Turmeric combination had the potential to modulate the immune system, especially macrophage cells profile against cancer.

Emergence of novel CD3+ Macrophages in Brown Adipose Tissue Following Adrenergic Stimulation

Abstract Macrophages play a vital role in maintaining tissue homeostasis and defending against infections and cancer. Brown adipose tissue (BAT) is essential for expending surplus energy during cold stress, while white adipose tissue functions as the primary energy reservoir. The sympathetic nervous system responds to cold stress by releasing norepinephrine (NE), activating BAT through β3-adrenergic receptor. BAT-infiltrated macrophages exert opposing effects on thermogenesis, regulating the interplay between BAT cells and sympathetic nerves to either inhibit thermogenesis by clearing NE or enhance it by supporting sympathetic innervation of BAT. In mouse interscapular BAT, we identified a novel subset of CD3+ macrophages (CD45+ CD3+ CD4+ CD8+ CD11b+ F4/80+), whose frequency increased with chronic adrenergic stimulation. Despite the potential for T cell-monocyte doublets in flow cytometry, CD3+ macrophages demonstrated a unique surface expression pattern, lacking T cell markers CD62L and TCRβ. Furthermore, they showed elevated levels of Ly6C, MHCII, F4/80, and CD11b compared to T cells and CD3- macrophages. CD3+ macrophages exhibit heterogeneity in MHCII expression, with subsets expressing low and high levels. Since MHCII-expressing macrophages play a crucial role in regulating sympathetic nerve-BAT interaction, further characterization of CD3+ macrophages is pivotal for understanding the intricate mechanisms that govern adaptive thermogenesis regulation.

Apolipoprotein E controls Dectin-1-dependent development of monocyte-derived alveolar macrophages upon pulmonary β-glucan-induced inflammatory adaptation

The lung is constantly exposed to the outside world and optimal adaptation of immune responses is crucial for efficient pathogen clearance. However, mechanisms that lead to lung-associated macrophages’ functional and developmental adaptation remain elusive. To reveal such mechanisms, we developed a reductionist model of environmental intranasal β-glucan exposure, allowing for the detailed interrogation of molecular mechanisms of pulmonary macrophage adaptation. Employing single-cell transcriptomics, high-dimensional imaging and flow cytometric characterization paired with in vivo and ex vivo challenge models, we reveal that pulmonary low-grade inflammation results in the development of apolipoprotein E (ApoE)-dependent monocyte-derived alveolar macrophages (ApoE+CD11b+ AMs). ApoE+CD11b+ AMs expressed high levels of CD11b, ApoE, Gpnmb and Ccl6, were glycolytic, highly phagocytic and produced large amounts of interleukin-6 upon restimulation. Functional differences were cell intrinsic, and myeloid cell-specific ApoE ablation inhibited Ly6c+ monocyte to ApoE+CD11b+ AM differentiation dependent on macrophage colony-stimulating factor secretion, promoting ApoE+CD11b+ AM cell death and thus impeding ApoE+CD11b+ AM maintenance. In vivo, β-glucan-elicited ApoE+CD11b+ AMs limited the bacterial burden of Legionella pneumophilia after infection and improved the disease outcome in vivo and ex vivo in a murine lung fibrosis model. Collectively these data identify ApoE+CD11b+ AMs generated upon environmental cues, under the control of ApoE signaling, as an essential determinant for lung adaptation enhancing tissue resilience.

Intracellular Accumulation and Secretion of YKL-40 (CHI3L1) in the Course of DMSO-Induced HL-60 Cell Differentiation.

YKL-40 (CHI3L1) is a matrix glycoprotein stored in human neutrophil-specific granules and released upon activation. While it is implicated in inflammation, cancer progression, and cell differentiation, its exact physiological role remains unclear. This study investigated the intracellular expression and secretion of YKL-40 by untreated and DMSO-treated HL-60 cells in association with surface expression of CD11b and CD66b throughout the differentiation process (up to 120 h). Secreted YKL-40 protein and mRNA levels of YKL-40, CD66b, and CD11b were measured by ELISA and quantitative RT-PCR, respectively. The intracellular YKL-40 and surface CD11b and CD66b expression were assessed by flow cytometry. A significant increase in CD11b expression confirmed DMSO-induced differentiation of HL-60 cells. Upon DMSO stimulation, YKL-40 mRNA expression increased in a time-dependent manner, unlike CD66b. The lack of CD66b (a granulocyte maturation and activation marker) on the surface of HL-60 cells might suggest that DMSO treatment did not induce full maturation or activation. The intracellular YKL-40 protein expression was increasing up to 96 h of DMSO treatment and then declined. YKL-40 secretion into the culture medium was detectable only at later time points (96 and 120 h), which was correlated with a decreased proliferation of DMSO-treated HL-60 cells. These findings suggest sequential changes in YKL-40 production and secretion during DMSO-induced differentiation of HL-60 cells and might contribute to a better understanding of YKL-40's involvement in both physiological processes and disease development, including multiple sclerosis.

FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.

Abstract 5344: Myeloid populations upregulated in prostate cancer are associated with colitis induced colorectal cancer

Abstract Chronic systemic inflammation caused by diseases like ulcerative colitis (UC) and Crohn’s disease (CD) increases the risk of developing colorectal cancer (CRC). Research indicates that patients with UC are more susceptible to prostate cancer (PCa) and individuals with PCa may also be at a higher risk for developing CRC. However, these relationships are not well defined. Here, we explored these relationships using preclinical mouse models of dextran sulfate sodium (DSS)-induced colitis (DSS-UC) and in young and aged healthy wild-type mice and conditional transgenic mice harboring Pten-deficient (KO) or Pten/Trp53-deficient (DKO) PCa. After a 25-week follow-up, DSS-induced UC caused more severe colitis in prostate tumor-bearing DKO compared to healthy WT mice. None of the DSS WT mice developed tumors, whereas 75% (3/4 mice) of DSS DKO mice did, including two out of four mice that developed multiple tumors. Overall, DKO mice with prostate tumors had increased levels of CD11b+/Gr1+ in peripheral blood, prostate, and colon compared with WT mice. Quantitative immunohistochemical analyses revealed that DKO mice with prostate tumors had a significantly higher 2.8-fold increase in the levels of Gr1+ cells in the colon compared to heathy WT mice. Overall, DSS-induced colitis further increased Gr1+ cells in the colon of WT and DKO mice by 2.5- and 2-fold, respectively. Gr1+ cells comprised 3.0%, 4.7%, and 10.1% of cells in normal colon from control WT mice and inactive and active regions of the colon in DSS-exposed WT mice, respectively. In DKO mice, Gr1+ cells comprised 7.0%, 19.2%, and 23.6% of cells in normal colon from control mice and inactive and active regions of the colon in DSS-exposed mice, respectively. CD68+, which corresponds to M1 polarized macrophages comprised 0.9%, 1.8%, and 20.6% of cells in normal colon from control WT mice and inactive and active regions of the colon in DSS-exposed WT mice, respectively, However, in DKO mice CD68+ cells comprised 3.0%, 2.8% and 1.6% of cells in normal colon from control mice and inactive and active regions of the colon in DSS-exposed mice, respectively. Our study suggests that Gr1 cells that were abundant in PCa-bearing mice were also present in colons and could have contributed to CRC. Moreover, CD68+ macrophages in the colons of healthy mice may have decreased tumor promoting effects of DSS. This study provides preclinical evidence showing that systemic inflammation in the presence of an existing malignancy increases the risk of a second malignancy, in this case linking PCa to colitis-induced-CRC. Citation Format: Yurie Kura, Marco A. De Velasco, Kazuko Sakai, Mamoru Hashimoto, Syogo Adomi, Takafumi Minami, Kazuhiro Yoshimura, Kazutoshi Fujita, Kazuto Nishio, Hirotsugu Uemura. Myeloid populations upregulated in prostate cancer are associated with colitis induced colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5344.

Systemic neutrophil degranulation and emergency granulopoiesis in patients with Clostridioides difficile infection

ObjectivesClostridioides difficile infection (CDI) is characterized by neutrophilia in blood, with a high leukocyte count accompanying severe infection. In this study, we characterized peripheral blood neutrophil activation and maturity in CDI by (i) developing a method to phenotype stored neutrophils for disease-related developmental alterations and (ii) assessing neutrophil-associated biomarkers. MethodsWe stored fixed leukocytes from blood collected within 24 h of diagnosis from a cohort of hospitalized patients with acute CDI. Additional study cohorts included recurrent CDI patients at time of and two months after FMT therapy and a control healthy cohort. We assessed levels of neutrophil surface markers CD66b, CD11b, CD16 and CD10 by flow cytometry. Plasma neutrophil elastase and lipocalin-2 were measured using ELISA, while G-CSF, GM-CSF and cytokines were measured using O-link Proteomic technology. ResultsCD66b+ neutrophil abundance assessed by flow cytometry correlated well with complete blood counts, establishing that neutrophils in stored blood are sufficiently well-preserved for phenotyping by flow cytometry. Neutrophil abundance was significantly increased in CDI patients compared to healthy controls. Emergency granulopoiesis in acute CDI patients was evidenced by lower neutrophil surface expression of CD10, CD11b and CD16. CD10+ staining of neutrophils started to recover within 3–7 days of CDI treatment. Neutrophil activation and degranulation were higher in acute CDI as assessed by plasma neutrophil elastase and lipocalin-2. Biomarker levels in immunocompetent subjects were associated with recurrence and fatal outcomes. ConclusionsNeutrophil activation and emergency granulopoiesis characterize the early immune response in acute CDI, with plasma degranulation biomarkers predictive of disease severity.

Network pharmacology analysis and experimental validation of Xiao-Qing-Long-Tang’s therapeutic effects against neutrophilic asthma

BackgroundXiao-Qing-Long-Tang (XQLT), a classical Chinese herbal medicine formula, has been extensively used for allergic asthma treatment. However, there is limited research on its anti-inflammatory effects and mechanisms specifically in neutrophilic asthma (NA). PurposeThis study aims to investigate the potential therapeutic effects of XQLT against NA using a combination of network pharmacology and experimental validation. Study designBy utilizing traditional Chinese medicine and disease databases, we constructed an XQLT-asthma network to identify potential targets of XQLT for NA. In the experimental phase, we utilized an ovalbumin (OVA)/lipopolysaccharide (LPS)-induced model for neutrophilic asthma and examined the therapeutic effects of XQLT. ResultsOur research identified 174 bioactive components within XQLT and obtained 140 target genes of XQLT against asthma. Functional enrichment analysis revealed that these target genes were primarily associated with inflammation and cytokines. In the experimental validation, mice induced with OVA-LPS showcased eosinophilic and neutrophilic cell infiltration in peri-bronchial areas, elevated levels of IL-4 and IL-17 in both serum and lung, increased percentages of Th2 and Th17 cells in the spleen, as well as elevated levels of CD11b+ and CD103+ dendritic cells (DCs) within the lung. Treatment with XQLT effectively reduced IL-4 and IL-17 levels, decreased the percentages of Th2, Th17, CD11b+, and CD103+ DCs, and improved inflammatory cell infiltrations in lung tissues. These findings serve as a foundation for the potential clinical application of XQLT in neutrophilic asthma.

Abstract P14: Efficacy of FHD-286 monotherapy and combinations against Menin inhibitor sensitive and resistant AML cells

Abstract BRG1 (SMARCA4) and BRM (SMARCA2) are the core ATPase within the BAF chromatin remodeler complexes. FHD-286 (Foghorn Therapeutics) is a potent, selective, oral inhibitor of BRG1/BRM in clinical development in AML. Present studies show that FHD-286 induces marked in vitro differentiation and loss of viability in AML cell lines and patient-derived (PD) AML cells with MLLr or mutant (mt) NPM1. FHD-286 treatment concordantly reduced genome-wide ATAC-Seq and RNA-Seq peaks, with negative enrichment at the gene-sets of MYC and E2F targets, mTORC1 signaling, translation initiation/elongation and DNA replication. FHD-286 reduced the occupancy of BRG1 and H3K27Ac on the chromatin. Mass spectrometry revealed FHD-286 reduced protein levels of c-Myc (and its targets), PLK1, BCL2, PU.1 and CDK4, but increased HEXIM1, CDKN1A/1B, CEBPα and CD11b levels. CyTOF analysis in the phenotypically characterized, PD, AML stem/progenitor cells with MLL1r or mtNPM1 showed decline in the protein levels of c-Myc, PU.1, BRG1, MEF2C, PBX3, BCL2 and MCL1, but increase in cleaved PARP and caspase-3 levels. In a PD xenograft (PDX) model of MLL1r AML, monotherapy with FHD-286 (oral gavage) for 4 to 6-weeks reduced AML burden and improved survival. FHD-286 treatment also significantly inhibited the AML-initiating potential, by reducing AML burden and improving survival of the re-engrafted mice with the FHD-286-treated PDX cells. Co-treatment with FHD-286 and BET inhibitor (OTX015), Menin inhibitor (MI, SNDX-50469), decitabine or venetoclax, exerted synergistic lethality in AML cell lines and PD AML cells with MLL1r or mtNPM1. Importantly, co-treatment with FHD-286 and OTX015, SNDX-5613 (oral gavage), venetoclax or decitabine vs each drug alone significantly reduced AML burden and improved survival in the PDX models of AML cells with MLLr or with mtNPM1, without significant toxicity. Notably, FHD-286 also induced significant lethality in MI-resistant AML cell lines and PD AML cells. Through repeated shocks with LD90 dosing of MI, followed by drug washout and recovery, we have generated MLL1r (MV4-11-MITR) and mtNPM1 (OCI-AML3-MITR) AML cell lines that are tolerant/resistant (TR) to MI (without exhibiting Menin mutations) with LD50 values greater than 1 µM. A domain-specific CRISPR screen in MV4-11-MITR cells identified several druggable co-dependencies (e.g., EP300 and MOZ) and co-enrichments (SMARCA4, CREBBP and BRD4) with MI. Accordingly, co-treatment with FHD-286 and OTX015, SNDX-50469 or CBP/p300 inhibitor GNE781 exerted synergistic lethality in MV4-11-MITR and OCI-AML3-MITR cells as well as in PD MITR AML cells with MLL1r or mtNPM1. FHD-286 and OTX015 or SNDX-5613 treatment also reduced AML burden in mice bearing OCI-AML3-MITR xenografts. These findings demonstrate pre-clinical efficacy of FHD-286-based rational combinations and underscore their promise against MI-sensitive or resistant AML with MLL1r or mtNPM1. Citation Format: Warren C Fiskus, Jessica Piel, Murphy Hentemann, Mike Collins, Christopher P Mill, Branko Cuglievan, Courtney D DiNardo, Kapil N Bhalla. Efficacy of FHD-286 monotherapy and combinations against Menin inhibitor sensitive and resistant AML cells [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P14.

A potential mechanism clue to the periodic storm from microglia activation and progressive neuron damage induced by paraquat exposure.

Paraquat (PQ), is characterized by neurotoxicity, which increases the potential risk of Parkinson's disease (PD) exposure in the long-term and low doses. Triggering microglia activation and neuroinflammation is deemed an early event resulting in PD. However, the underlying pathogenesis of PD by PQ is not clear yet. In this article, C57BL/6J mice treated with PQ could successfully act out Parkinson-like. In addition, we observed the fluorescence intensity enhancement of Iba-1 activated microglia with released pro-inflammatory, all ahead of both the damage of dopaminergic neurons in the substantia nigra and corpus striatum of the brain. Surprisingly, the injection of minocycline before PQ for many hours not only can effectively improve the neurobehavioral symptoms of mice but inhibit the activation of microglia and the release of pro-inflammatory substances, even controlling the gradual damage and loss of neurons. A further mechanism of minocycline hampered the expression levels of key signaling proteins PI3K, PDK1, p-AKT, and CD11b (the receptor of microglia membrane recognition), while a large number of inflammatory factors. Our results suggested that the CD11b/PI3K/NOX2 pathway may be a clue that microglia-mediated inflammatory responses and neuronal damage in a PQ-induced abnormal behavior Parkinson-like mouse.

Distinguishing the impact of distinct obstructive sleep apnea syndrome (OSAS) and obesity related factors on human monocyte subsets

Obstructive sleep apnea syndrome (OSAS) and obesity go hand in hand in the majority of patients and both are associated with a systemic inflammation, immune disturbance and comorbidities such as cardiovascular disease. However, the unambiguous impact of OSAS and obesity on the individual inflammatory microenvironment and the immunological consequences of human monocytes has not been distinguished yet. Therefore, aim of this study was to investigate the impact of OSAS and obesity related factors on the inflammatory microenvironment by performing flow cytometric whole blood measurements of CD14/CD16 monocyte subsets in normal weight OSAS patients, patients with obesity but without OSAS, and patients with OSAS and obesity, compared to healthy donors. Moreover, explicitly OSAS and obesity related plasma levels of inflammatory mediators adiponectin, leptin, lipocalin and metalloproteinase-9 were determined and the influence of different OSAS and obesity related factors on cytokine secretion and expression of different adhesion molecules by THP-1 monocytes was analysed. Our data revealed a significant redistribution of circulating classical and intermediate monocytes in all three patient cohorts, but differential effects in terms of monocytic adhesion molecules CD11a, CD11b, CD11c, CX3CR1, CD29, CD49d, and plasma cytokine levels. These data were reflected by differential effects of OSAS and obesity related factors leptin, TNFα and hypoxia on THP-1 cytokine secretion patterns and expression of adhesion molecules CD11b and CD49d. In summary, our data revealed differential effects of OSAS and obesity, which underlines the need for a customized therapeutic regimen with respect to the individual weighting of these overlapping diseases.

Notable Efficacy of Co-Treatment with FHD-286, a Dual BRG1/BRM ATP-Ase Inhibitor, and Menin or BET Inhibitor, Decitabine or Venetoclax Against AML with MLL-r or Mutant NPM1

BRG1 (SMARCA4) and BRM (SMARCA2) are the core ATPase within the multi-protein, ATP-dependent, chromatin remodeling BAF complexes that regulate gene transcription. FHD-286 is a potent, selective, orally administered, inhibitor of BRG1/BRM in early clinical development as a therapy for AML. In present studies, we determined that exposure to FHD-286 (10 to 100 nM) for 4 to 7 days induced CD11b and morphologic features of differentiation and loss of viability in AML cell lines (MOLM13, MV4-11 and OCI-AML3) and patient-derived (PD) AML cells with MLL1r or mtNPM1. Notably, FHD-286 also induced significant lethality in Menin inhibitor (MI) resistant AML cell lines and PD AML cells lacking Menin mutations. ChIP-Seq analysis showed that exposure to FHD-286 significantly reduced the occupancy of BRG1 and H3K27Ac on the chromatin and reduced the core transcriptional regulatory circuitry in MOLM13 cells. FHD-286 treatment concordantly reduced genome-wide ATAC-Seq and RNA-Seq peaks, with negative enrichment at the gene-sets of MYC and E2F targets, mTORC1 signaling, translation initiation/elongation and DNA replication. scRNA-Seq analysis in PD AML bone marrow cells with MLL1r or mtNPM1 showed significant decline in the myeloid/erythroid progenitor (MEP) cells. Mass spectrometry conducted on FHD-286-treated vs control cells revealed a significant decline in the protein levels of c-Myc and its targets, as well as of PLK1, BCL2, PU.1 and CDK4, but exhibited increased levels of HEXIM1, CDKN1A/1B, CEBPα and CD11b. Moreover, CyTOF analysis of PD AML cells with MLL1r or mtNPM1 showed significant decline in the protein levels of c-Myc, PU.1, BRG1, MEF2C, PBX3, BCL2 and MCL1, but exhibited increased levels of cleaved PARP and Caspase-3 in the AML stem/progenitor cells defined by high expression of CLEC12A, CD123, CD99 and CD33 but low expression of CD11b. A domain-specific CRISPR screen targeting epigenetic regulators revealed significant co-enrichments with FHD-286 treatment of the gRNAs against BRD4 and LSD1, suggesting them as the epigenetic mechanisms of FHD-286 resistance. Consistent with findings noted above, co-treatment with FHD-286 and venetoclax, BET inhibitor (OTX015), decitabine or MI (SNDX-50469) exerted synergistic lethality in AML cell lines (MOLM13, MV4-11 and OCI-AML3) and PD AML cells with MLL1r or mtNPM1 (delta synergy scores between 10 to 33 by the ZIP method). In a PD xenograft (PDX) model of MLL1r AML with FLT3 mutation, monotherapy with FHD-286 (oral gavage) for 4 to 6-weeks was significantly effective in reducing AML burden and improving overall survival. Separately, in vivo FHD-286 treatment vs vehicle control, also significantly inhibited the AML-initiating potential, by reducing the AML burden and improving survival of the re-engrafted mice with the FHD-286-treated PDX cells. Moreover, co-treatment with FHD-286 and venetoclax or decitabine or OTX015, vs each drug alone, or vehicle control, significantly reduced the AML burden and improved survival, without significant toxicity in the PDX model. Finally, co-treatment with FHD-286 and OTX015 or SNDX-5613 (oral gavage) was significantly more effective than each drug alone in reducing the AML burden and overall survival of mice engrafted with a separate PDX model of AML cells with mtNPM1 and FLT3-ITD, without significant toxicity. These findings demonstrate the pre-clinical efficacy of FHD-286-based rational combinations and underscore their promise against AML with MLL1r or mtNPM1.

Decreased toll-like receptor 4 and CD11b/CD18 expression on peripheral monocytes of hypertensive patients correlates with a lesser extent of endothelial damage: a preliminary study.

Low-grade chronic inflammation is recognized to contribute to the physiopathology of arterial hypertension. Therefore, this study aimed to assess the pro-inflammatory phenotype of peripheral monocytes of hypertensive patients by analyzing Toll-like receptor 4 (TLR4) and CD11b/CD18 surface expression. In the second part, the influence of phenotypic alterations of monocytes on the endothelial status reflected by circulating endothelial cells (CECs) was evaluated. The study included 60 patients with arterial hypertension, who were divided into two subgroups based on the disease severity according to the applicable criteria. The mild hypertension and resistant hypertension groups included 30 patients each. The control group consisted of 33 normotensive volunteers matched for age and sex. Both in the entire group of patients and individual subgroups, reduced surface expression of TLR4 and CD11b/CD18 was found compared to normotensive volunteers. A reduced percentage of monocytes with the CD14 + TLR4 + immunophenotype was correlated with a lower MFI level of CD18 and CD11b in the entire group of patients and after division only in the mild hypertension group. Reduced surface expression of TLR4 in hypertensive patients correlated with a lower number of CECs. This relationship was not observed in the resistant hypertension group; instead, an independent effect of reduced CD11b/CD18 expression on the reduction of CEC number was demonstrated. Our preliminary study showed for the first time that hypertension of varying severity is accompanied by phenotypic changes in monocytes, manifested by reduced surface expression of both TLR4 and CD11b/CD18. These phenotypic changes were associated with a reduced degree of endothelial injury. Our study opens a new, unexplored area of research on the protective features of peripheral monocytes in hypertension.

Candida-induced granulocytic myeloid-derived suppressor cells are protective against polymicrobial sepsis.

Polymicrobial intra-abdominal infections are serious clinical infections that can lead to life-threatening sepsis, which is difficult to treat in part due to the complex and dynamic inflammatory responses involved. Our prior studies demonstrated that immunization with low-virulence Candida species can provide strong protection against lethal polymicrobial sepsis challenge in mice. This long-lived protection was found to be mediated by trained Gr-1+ polymorphonuclear leukocytes with features resembling myeloid-derived suppressor cells (MDSCs). Here we definitively characterize these cells as MDSCs and demonstrate that their mechanism of protection involves the abrogation of lethal inflammation, in part through the action of the anti-inflammatory cytokine interleukin (IL)-10. These studies highlight the role of MDSCs and IL-10 in controlling acute lethal inflammation and give support for the utility of trained tolerogenic immune responses in the clinical treatment of sepsis.

Dexmedetomidine attenuates neuroinflammation and microglia activation in LPS-stimulated BV2 microglia cells through targeting circ-Shank3/miR-140-3p/TLR4 axis.

It has been shown that dexmedetomidine (Dex) could attenuate postoperative cognitive dysfunction (POCD) via targeting circular RNAs (circRNAs). Circ-Shank3 has been found to be involved in the neuroprotective effects of Dex against POCD. However, the role of circ-Shank3 in POCD remains largely unknown. Reverse transcription quantitative PCR (RT-qPCR) was performed to detect circ-Shank3 and miR-140-3p levels in lipopolysaccharide (LPS)-treated microglia BV-2 cells in the absence or presence of Dex. The relationship among circ-Shank3, miR-140-3p and TLR4 was confirmed by dual-luciferase reporter assay. Additionally, Western blot and immunofluorescence (IF) assays were conducted to evaluate TLR4, p65 and Iba-1 or CD11b levels in cells. In this study, we found that Dex notably decreased circ-Shank3 and TLR4 levels and elevated miR-140-3p level in LPS-treated BV2 cells. Mechanistically, circ-Shank3 harbor miR-140-3p, functioning as a miRNA sponge, and then miR-140-3p targeted the 3'-UTR of TLR4. Additionally, Dex treatment significantly reduced TLR4 level and phosphorylation of p65, and decreased the expressions of microglia markers Iba-1 and CD11b in LPS-treated BV2 cells. As expected, silenced circ-Shank3 further reduced TLR4, p65 and Iba-1 and CD11b levels in LPS-treated BV2 cells in the presence of Dex, whereas these phenomena were reversed by miR-140-3p inhibitor. Collectively, our results found that Dex could attenuate the neuroinflammation and microglia activation in BV2 cells exposed to LPS via targeting circ-Shank3/miR-140-3p/TLR4 axis. Our results might shed a new light on the mechanism of Dex for the treatment of POCD.