Myeloid Differentiation Research Articles

Abstract 103: Prostate cancer bone metastasis: Therapeutic targeting of tumor associated macrophages

Abstract Introduction: Castration-resistant bone metastatic prostate cancer is a morbid and deadly disease with an exceedingly low response rates to available immunotherapies. Understanding the mechanisms behind immune evasion and tumor progression is critical to improving therapy. Historically, bone metastases have been challenging to model in the laboratory. Our foundational work was built upon fresh patient samples taken directly from the operating room for analysis across multiple platforms: IHC, single cell expression profiling, and spatial transcriptomics. We compared tumors from patients with bone metastatic disease to tumors from patients with disease localized to the prostate. Our work (Cancer Cell, 2021 and Nature Communications, 2023) has consistently pointed towards dysregulated myeloid populations within the tumor microenvironment, particularly TREM2+ tumor associated macrophages (TAMs), as key drivers of disease progression. Methods: Our patient samples and murine pre-clinical models of prostate cancer highlighted several specifically upregulated molecules and pathways in TAMs. Here we focus on TAMs in vitro and in vivo using a new and genetically tractable myeloid model to interrogate the mechanistic role of key pathways, and how they may contribute to T-cell immune suppression. The ER-Hoxb8 system of conditional myeloid differentiation permitted the generation of isogenic cell lines to interrogate the roles of key molecules TREM2, MSR1, APOE, and MERTK through overexpression or CRISPR/Cas9 knockout. Results: Using a system of co-culture in vitro, we quantified the role of these proteins in suppressing T-cell proliferation and cytokine production, as well as their role in TAM differentiation and function. In vivo, these lines were co-injected subcutaneously with syngeneic RM1-BM3 tumor cells to quantify their effect on tumor growth and T-cell suppression in vivo. In addition to localized subcutaneous tumors, bone metastatic disease was established by intracardiac injection. Furthermore, established tumors were treated with TAM-directed small molecule therapy (e.g., MERTK inhibition) as well as immune checkpoint inhibition (anti-PD1) alone, and in combination. Conclusions: TAMs are immune suppressive myeloid cells that are not found in normal tissues. They are specifically recruited to tumors and highly enriched in bone metastatic disease. Transcriptomic analyses highlighted signaling pathways potentially amenable to productive therapeutic targeting. The ER-Hoxb8 system was highly effective in generating a genetically tractable model of TAM for study in vitro and in vivo. Our work supports the hypothesis that the specific targeting of TAMs, by focusing on critical signaling pathways, can be effective in the treatment of pre-clinical models of prostate cancer. This new approach merits further study as a potential strategy for the treatment of patients with bone metastases. Citation Format: Hanyu Zhang, Shenglin Mei, Nathan E. Jeffries, Douglas M. Dahl, Chin-Lee Wu, Philip J. Saylor, David B. Sykes. Prostate cancer bone metastasis: Therapeutic targeting of tumor associated macrophages [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 103.

Abstract 741: Distinct functional effects of ILT family members in mediating the activity of an ILT2/ILT4/ILT5 cross-reactive monoclonal antibody in releasing the suppression on innate and adaptive anti-tumor immunity

Abstract The expression of ILT (LILRB) family members and their major ligand HLA-G within solid tumors are associated with immune suppression and poor patient survival. Blocking multiple ILT family members might be more potent than targeting individual members by overcoming compensatory resistances within tumor microenvironment. Based on this hypothesis, we developed a monoclonal antibody (mAb) LITCHI 7 which reacts to three major ILT family members, ILT2, ILT4 and ILT5, and blocks ligand binding to these receptors. LITCHI 7 exhibits a broad binding capability to a wide range of human myeloid and T cell subsets that express ILT2, ILT4, and/or ILT5. Binding to monocyte-derived dendritic cells (moDC), LITCHI 7 released the inhibition on activating Fcγ receptor. On LPS-treated macrophages, LITCHI 7 enhanced inflammatory macrophage differentiation. The effect is primarily mediated by blocking ILT4 since ILT4 mono-specific mAb is as potent as cross-reactive LITHI 7 in modulating myeloid differentiation although both ILT2 and ILT5 are highly expressed by different myeloid subsets. The result was further confirmed by CRISPR Cas9 knock down of ILT2 or ILT5. In the absence of either ILT2 or ILT5, the accumulation of CD14+ monocytes and inflammatory macrophages were not changed during differentiation. While different ILT family members are generally enriched on myeloid cells, ILT2 is also expressed on NK cells and effector memory CD45RA+ T cells (Temra). Blocking ILT2 by LITCHI 7 enhanced TCR signaling when co-culturing the MART-1 specific T cell reporter line with MART-1/HLA-G positive melanoma cells. The effect of LITCHI 7 is mediated by ILT2 blockade since knocking out ILT2, but not ILT4 and ILT5, reduced accumulation of Temra in activated PBMC. To assess whether immune modulation of LITCHI 7 can be translated into anti-tumor activity by blocking different ILT family members, tumor killing assays were performed using innate and adaptive immune effector cells. LITCHI7 enhanced NK cell-mediated cytotoxicity on HLA-G+ tumor cells by blocking ILT2. Both ILT2 and ILT4 blockade play a role in macrophage phagocytosis on lymphoma cells since LITCHI 7 as well as individual anti-ILT2 or ILT-4 mAbs increased macrophage phagocytosis to a similar level. Finally, LITCHI 7 could boost in vitro T cell-mediated killing of CD19+ lymphoma by CD19 specific CAR-T cells in the presence of CD33+ myeloid cells. In summary, LITCHI 7 can enhance both innate and adaptive anti-tumor immunity through ILT2 or ILT4 blockade directly on effector cells or indirectly through myeloid cell reprogramming. As clinical trials are ongoing by targeting ILT2 and ILT4, blocking multiple ILT family members through a single cross-reactive mAb may provide broad anti-tumor effects and advantages when combining with additional oncology treatments. Citation Format: Shiming Ye, Dong Zhang, Brenal Singh, Jaishree Bankoti, Diane Cohen, Siusze Tan, Donghee Choi, Jonathan Hickson, Min-Zu Wu, Urvi Kolhatkar, Mikhail Binnewies, Wei Jin, Jason Hall, John Engelhardt, Alexander Shoemaker. Distinct functional effects of ILT family members in mediating the activity of an ILT2/ILT4/ILT5 cross-reactive monoclonal antibody in releasing the suppression on innate and adaptive anti-tumor immunity [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 741.

Abstract 1703: SMARCA2/4 degraders relieve the differentiation block in AML via changes in chromatin looping and accessibility

Abstract Acute Myeloid Leukemia (AML) is characterized by the unfettered proliferation and poor differentiation of myeloid cells causing reduced survival. The mammalian BAF complexes (also known as SWI/SNF) are important regulators of MYC and the cell cycle. There are three BAF complexes (cBAF, ncBAF and pBAF) that share several subunits. SMARCA2 and 4 are mutually exclusive ATPase subunits within the BAF complexes that use ATP hydrolysis and helicase activity to remodel chromatin via nucleosome sliding and removal. PBRM1 is a SMARCA4 associated factor present in pBAF. The association of SMARCA4 with regulatory elements of transcription factors makes it an intriguing target for AML. To evaluate the role of SMARC2/4 in AML, we analyzed RNA transcripts from highly purified primary AML initiating populations and control samples. Sorted leukemia stem cells (Lin-ve, CD34+, CD38-) overexpressed SMARCA2/4 when compared to healthy cells. A novel and highly selective SMARCA2/4 and PBRM1 PROTAC degrading agent AU-15330 was used to investigate the therapeutic effectiveness of targeting BAF complexes as well as the role of SMARCA2/4 in leukemic pathogenesis. Our data demonstrate that AU-15330 effectively degrades SMARCA2/4 and PBRM1 in human leukemia cell lines with varying mutational profiles; MV4-11 was the most sensitive cell line. When evaluating healthy CD34+ HSPCs as well as patient derived AML cells in colony assays, AU-15330 treatment increased differentiation in patient AML samples with minimal effects on healthy CD34+ cells. A xenograft of patient derived AML cells in irradiated mice was performed to determine the requirement of SMARCA2/4 in leukemia stem cell maintenance. Mice treated with AU-15330 had greater myeloid differentiation, fewer human cells in their bone marrows and reduced leukemia stem cell activity, seen in secondary transplants. To understand the underlying mechanism of how SMARCA2/4 and PBRM1 promote leukemic pathogenesis, we used a multi-omic analysis of transcription and chromatin dynamics in MV4-11 cells: ATAC-seq and RNA-seq identified downregulated genes with decreased accessibility, PRO-seq determined early transcriptional changes and HiChIP showed changes in enhancer promoter loops with SMARC2/4 degradation. Several leukemia related transcription factors as well as inflammatory genes were identified from these analyses as possible targets of the BAF complexes in the context of AML. Finally, to determine the effect of SMARCA2/4 degradation on pBAF association with chromatin, single cell imaging was performed of U2OS cells with HALO-tagged PBRM1 treated with the degrader or DMSO. pBAF in the degrader treated cells improved engagement with target chromatin however, a decrease in the stability of the complex binding to the nucleosome when compared to DMSO treated cells was shown. Taken together, these studies will determine the role of SMARCA2/4 in leukemia disease. Citation Format: Sarah Aminov, Kith Pradhan, Srabani Sahu, Shanisha Gordon-Mitchell, Emma Rabinovich, Ariel Fromowitz, Joyeeta Chakraborty, Srinivas Aluri, Rongbao Zhao, Nayem Haque, Milagros Carbajal, Aditi Shastri, Ulrich Steidl, Susanta Samajdarr, Akhil Kumar, Murali Ramachandra, Robert A. Coleman, Kristy Stengel, Amit K. Verma. SMARCA2/4 degraders relieve the differentiation block in AML via changes in chromatin looping and accessibility [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 1703.

Abstract 3229: IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets

Abstract Block in differentiation and accumulation of undifferentiated blasts are hallmarks of Acute Myeloid Leukemia (AML) and epigenetic processes have been shown to play a critical role in hematological malignancies. Paralogs CREBBP and EP300, in association with co-factors, are hijacked during leukemogenesis by aberrant transcriptional factors, thus driving blast proliferation and maintaining the undifferentiated phenotype. Here, we introduce IACS-16559, a bromodomain inhibitor targeting CREBBP/EP300. IACS-16559 is highly selective for the bromodomains of CBP and EP300 versus BRD4, showing a dose-dependent inhibition of H3K27 acetylation residue in vivo and in vitro. In addition, IACS-16559 pharmacokinetic properties show low clearances, high oral exposures and predicted QD dosing in human. We screened AML cell lines in vitro for their response to IACS-16559 and identified specific subtypes of AML that exhibit heightened sensitivity to CBP/EP300 inhibition, specifically AML with AML1-ETO and MLL-AF9 rearrangements, and AML featuring mutations in the nucleophosmin (NPM1c) gene. IACS-16559 inhibited proliferation of AML responder cell lines in vitro, halted cell cycle progression, with an accumulation of cells in G1 and a decrease of cells in S phase. IACS-16559 did not induce apoptosis across a panel of AML cell lines, indicating that induction of apoptosis is not a major mechanism of action of CBP/EP300 inhibition. To augment the anti-tumor effects of IACS-16559, we prioritized the pharmacological combination of CBP/EP300 and MLL-Menin inhibitors. This combination synergistically impeded cell proliferation and potently triggered apoptosis in vitro and in vivo, whereas none of the individual agents alone had this effect. The changes in gene and surface marker expression produced by these treatments, as single agents and in combination with synergistic effects, were in line with an induced myeloid differentiation phenotype. Collectively, our findings provide a compelling basis for further exploring the use of a CBP/EP300 bromodomain inhibitor in conjunction with an inhibitor of the MLL-Menin interaction in a subset of AML patients with MLL rearrangements or NPM1 mutations. Citation Format: Ciro Zanca, Michael Soth, Guang Gao, Jason Gay, Ningping Feng, Christopher Bristow, Kang Le, Quanyun Xu, Phuong Nguyen, Yongying Jiang, Teresa Perry, Faika Mseeh, Paul Leonard, Jason Cross, Virginia Giuliani, Joe Marszalek, Philip Jones, Tim Heffernan. IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets [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 3229.

Autophagy Inhibition-induced Cytosolic DNA Sensing Combined with Differentiation Therapy Induces Irreversible Myeloid Differentiation in Leukemia Cells.

Clinical effects on AML therapy are closely associated with reactivating the normal myeloid differentiation potential in leukemia cells. This study shows that autophagosome formation inhibitors activate the cytosolic DNA-sensor signaling, thereby augmenting conventional differentiation therapy to induce irreversible differentiation and cell growth arrest in several types of AML cell lines.

The CDK4/6 Inhibitor Palbociclib Synergizes with ATRA to Induce Differentiation in AML.

Differentiation therapy based on ATRA almost cured acute promyelocytic leukemia (APL). However, it is disappointing that ATRA is not effective against other acute myeloid leukemia (AML) subtypes. Developing new and effective anti-AML therapies that promote leukemia differentiation is necessary. The CDK4/6-cyclin D pathway is a key initiator of the G1-S phase transition, which determines cell fate. Herein, we investigated whether the CDK4/6 inhibitor palbociclib would synergize with ATRA to promote leukemia differentiation in vitro and in vivo. Our findings revealed that CDK4/6-cyclin D pathway genes were aberrantly expressed in AML, and we observed that palbociclib sensitized AML cells to ATRA-induced morphologic, biochemical, and functional changes indicative of myeloid differentiation. The combination of palbociclib and ATRA attenuated AML cell expansion in vivo. These enhanced differentiation effects may be associated with the regulation of transcription factors, including RARα, E2F1, and STAT1. Overall, our findings demonstrate that CDK4/6 inhibition sensitizes AML cells to ATRA and could guide the development of novel therapeutic strategies for patients with AML.

Development of recombinant protein-based nanoparticle systems for inducing tumor cell apoptosis: In vitro evaluation of their cytotoxic and apoptotic effects on cancer cells

Myeloid leukemia cell differentiation protein (Mcl-1) is a B-cell lymphoma 2 (Bcl-2) family antiapoptotic protein that regulates cellular apoptosis. Mcl-1 expression is important for cancer cell survival and a prospective target in cancer treatment. Bcl-2 homology 3 (BH3) mimetic peptides, such as the Mcl-1-specific MS1 peptide that targets conserved BH3 domains, are highly specific Mcl-1 inhibitors. However, the application of these peptides is limited due to their metabolic instability, degradation by cellular proteases, and low cell permeability. In this study, it was aimed to form nanostructures to increase the cellular uptake and stability of the Mcl-1 inhibitor MS1 peptide. The Bim BH3 mimetic MS1 peptide was produced recombinantly in fusion with mNeonGreen fluorescent protein and polyhistidine (6xHisTag). Self-assembled protein nanoparticles were fabricated from the recombinant MS1-mNeonGreen protein. Furthermore, the MS1-mNeonGreen protein was encapsulated into polymeric nanoparticles. The cytotoxic effects of these nanoparticles on cancer cells [cervical cancer cell line (HeLa), lung cancer cell line (A549) and breast cancer cell line (MDA-MB-231)] and healthy cell line [Human umbilical vein endothelial cells (HUVEC)] was analyzed by MTT test. The apoptotic effect of nanoparticles on these cells was investigated by determining the antiapoptotic and proapoptotic protein levels. As a result, it was revealed that the nanoparticles have a specific cytotoxic effect against the cells and induce apoptosis.

MiR-515-5p targeting Toll-like receptor 4 regulates myeloid differentiation primary response gene 88/nuclear factor-kappa B pathway to inhibit apoptosis and inflammatory response of osteoarthritis chondrocytes

To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis (OA). Human cartilage cell line C28/I2 was cultured in vitro and treated with 10 ng/mL interleukin 1β (IL-1β) for 24 hours to construct an in vitro OA model. C28/I2 cells were transfected with miR mimics, mimics negative control (NC), over expression (oe)-NC, and oe-Toll-like receptor 4 (TLR4), respectively, and then treated with 10 ng/mL IL-1β for 24 hours to establish OA model. Cell proliferation capacity was detected by cell counting kit 8 and 5-Ethynyl-2'-deoxyuridine, cell apoptosis and cell cycle were detected by flow cytometry, and B-cell lymphoma 2 protion (Bcl-2), Bcl-2-associated X protein (Bax), cleaved-Caspase-3, TLR4, myeloid differentiation primary response gene 88 (MyD88), p65 and phosphorylated p65 (p-p65) protein expression levels were detected by Western blot. Real-time fluorescence quantitative PCR was used to detect mRNA expression levels of miR-515-5p and TLR4, and ELISA was used to detect pro-inflammatory factor prostaglandin E2 (PGE2), tumor necrosis factor α (TNF -α), and IL-6 levels in cell supernatant. The potential binding sites between miR-515-5p and TLR4 were predicted by BiBiServ2 database, and the targeting relationship between miR-515-5p and TLR4 was verified by dual luciferase reporting assay. After the treatment of C28/I2 cells with IL-1β, the expressions of miR-515-5p and Bcl-2 protein and the proliferation ability of C28/I2 cells significantly reduced. The expression levels of Bax and cleaved-Caspase-3 protein, the levels of pro-inflammatory factors (PGE2, TNF-α, IL-6) in the supernatant of C28/I2 cells, and the apoptosis of C28/I2 cells significantly increased. In addition, the proportion of the cells at S phase and G 2 phase decreased significantly, and the proportion of cells at G 1 phase increased significantly, suggesting that the cell cycle was blocked after IL-1β treatment. After transfection with miR mimics, the expression level of miR-515-5p in the cells significantly up-regulated, partially reversing the apoptosis of OA chondrocytes induced by IL-1β, and alleviating the cycle arrest and inflammatory response of OA chondrocytes. After treating C28/I2 cells with IL-1β, the mRNA and protein levels of TLR4 significantly increased. Overexpression of miR-515-5p targeted inhibition of TLR4 expression and blocked activation of MyD88/nuclear factor κB (NF-κB) pathway. Overexpression of TLR4 could partially reverse the effect of miR mimics on IL-1β-induced apoptosis and inflammation of OA chondrocytes. miR-515-5p negatively regulates the expression of TLR4, inhibits the activation of MyD88/NF-κB pathway and apoptosis of OA chondrocytes, and effectively alleviates the inflammatory response of the cells.

Impact of Toll-Like Receptors (TLRs) and TLR Signaling Proteins in Trigeminal Ganglia Impairing Herpes Simplex Virus 1 (HSV-1) Progression to Encephalitis: Insights from Mouse Models.

Herpes simplex virus 1 (HSV-1) or simplexvirus humanalpha 1 is a neurotropic virus that is responsible for orofacial infections in humans. More than 70% of the world's population may have seropositivity for HSV-1, and this virus is a leading cause of sporadic lethal encephalitis in humans. The role of toll-like receptors (TLRs) in defending against HSV-1 infection has been explored, including the consequences of lacking these receptors or other proteins in the TLR pathway. Cell and mouse models have been used to study the importance of these receptors in combating HSV-1, how they relate to the innate immune response, and how they participate in the orchestration of the adaptive immune response. Myeloid differentiation factor 88 (MyD88) is a protein involved in the downstream activation of TLRs and plays a crucial role in this signaling. Mice with functional MyD88 or TLR2 and TLR9 can survive HSV-1 infection. However, they can develop encephalitis and face a 100% mortality rate in a dose-dependent manner when MyD88 or TLR2 plus TLR9 proteins are non-functional. In TLR2/9 knockout mice, an increase in chemokines and decreases in nitric oxide (NO), interferon (IFN) gamma, and interleukin 1 (IL-1) levels in the trigeminal ganglia (TG) have been correlated with mortality.

Investigation of the acute pathogenesis of spondyloarthritis/HLA-B27-associated anterior uveitis based on genome-wide association analysis and single-cell transcriptomics

BackgroundPatients with spondyloarthritis (SpA)/HLA-B27-associated acute anterior uveitis (AAU) experience recurring acute flares, which pose significant visual and financial challenges. Despite established links between SpA and HLA-B27-associated AAU, the exact mechanism involved remains unclear, and further understanding is needed for effective prevention and treatment.MethodsTo investigate the acute pathogenesis of SpA/HLA-B27-associated AAU, Mendelian randomization (MR) and single-cell transcriptomic analyses were employed. The MR incorporated publicly available protein quantitative trait locus data from previous studies, along with genome-wide association study data from public databases. Causal relationships between plasma proteins and anterior uveitis were assessed using two-sample MR. Additionally, colocalization analysis was performed using Bayesian colocalization. Single-cell transcriptome analysis utilized the anterior uveitis dataset from the Gene Expression Omnibus (GEO) database. Dimensionality reduction, clustering, transcription factor analysis, pseudotime analysis, and cell communication analysis were subsequently conducted to explore the underlying mechanisms involved.ResultsMendelian randomization analysis revealed that circulating levels of AIF1 and VARS were significantly associated with a reduced risk of developing SpA/HLA-B27-associated AAU, with AIF1 showing a robust correlation with anterior uveitis onset. Colocalization analysis supported these findings. Single-cell transcriptome analysis showed predominant AIF1 expression in myeloid cells, which was notably lower in the HLA-B27-positive group. Pseudotime analysis revealed dendritic cell terminal positions in differentiation branches, accompanied by gradual decreases in AIF1 expression. Based on cell communication analysis, CD141+CLEC9A+ classic dendritic cells (cDCs) and the APP pathway play crucial roles in cellular communication in the Spa/HLA-B27 group.ConclusionsAIF1 is essential for the pathogenesis of SpA/HLA-B27-associated AAU. Myeloid cell differentiation into DCs and decreased AIF1 levels are also pivotal in this process.

MyD88-mediated signaling is critical for the generation of seizure responses and cognitive impairment in a model of anti-N-methyl-D-aspartate receptor encephalitis.

We previously demonstrated that interleukin-1 receptor-mediated immune activation contributes to seizure severity and memory loss in anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. In the present study, we assessed the role of the myeloid differentiation primary response gene 88 (MyD88), an adaptor protein in Toll-like receptor signaling, in the key phenotypic characteristics of anti-NMDAR encephalitis. Monoclonal anti-NMDAR antibodies or control antibodies were infused into the lateral ventricle of MyD88 knockout mice (MyD88-/-) and control C56BL/6J mice (wild type [WT]) via osmotic minipumps for 2 weeks. Seizure responses were measured by electroencephalography. Upon completion of the infusion, the motor, anxiety, and memory functions of the mice were assessed. Astrocytic (glial fibrillary acidic protein [GFAP]) and microglial (ionized calcium-binding adaptor molecule 1 [Iba-1]) activation and transcriptional activation for the principal inflammatory mediators involved in seizures were determined using immunohistochemistry and quantitative real-time polymerase chain reaction, respectively. As shown before, 80% of WT mice infused with anti-NMDAR antibodies (n = 10) developed seizures (median = 11, interquartile range [IQR] = 3-25 in 2 weeks). In contrast, only three of 14 MyD88-/- mice (21.4%) had seizures (0, IQR = 0-.25, p = .01). The WT mice treated with antibodies also developed memory loss in the novel object recognition test, whereas such memory deficits were not apparent in MyD88-/- mice treated with anti-NMDAR antibodies (p = .03) or control antibodies (p = .04). Furthermore, in contrast to the WT mice exposed to anti-NMDAR antibodies, the MyD88-/- mice had a significantly lower induction of chemokine (C-C motif) ligand 2 (CCL2) in the hippocampus (p = .0001, Sidak tests). There were no significant changes in the expression of GFAP and Iba-1 in the MyD88-/- mice treated with anti-NMDAR or control antibodies. These findings suggest that MyD88-mediated signaling contributes to the seizure and memory phenotype in anti-NMDAR encephalitis and that CCL2 activation may participate in the expression of these features. The removal of MyD88 inflammation may be protective and therapeutically relevant.

Functional mechanism of baicalein in alleviating severe acute pancreatitis-acute lung injury by blocking the TLR4/MyD88/TRIF signaling pathway.

Severe acute pancreatitis-acute lung injury (SAP-ALI) is a disease with high mortality. This study aims to explore the mechanism of baicalein on SAP-ALI in rats by blocking toll-like receptor-4 (TLR4)/myeloid differentiation primary response gene 88 (MyD88)/TIR-domain-containing adapter-inducing interferon-β (TRIF) signal pathway. The SAP-ALI rat model was established by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg), with pancreas and intestines turned over, injected with 3.5% sodium taurocholate backward into the bile-pancreatic duct at 0.1 mL/100 g for 12h, and treated with baicalein, lipopolysaccharide (LPS), miR-182 agomir, or miR-182 antagomir. The TLR4/MyD88/TRIF pathway was activated using LPS in SAP-ALI rats after baicalein treatment. Baicalein attenuated inflammatory cell infiltration, alveolar wall edema, decreased W/D ratio and levels of TLR4, MyD88, and TRIF in the lung tissues, reduced levels of inflammatory factors in pancreatic and lung tissues and BALF, diminished ROS, and elevated GSH, SOD and CAT in pancreatic and lung tissues of SAP-ALI rats. Activation of the TLR4/MyD88/TRIF pathway partly abrogated baicalein-mediated improvements in inflammation and oxidative stress in SAP-ALI rats. miR-182 targeted TLR4. miR-182 suppressed inflammation and oxidative stress in SAP-ALI rats by targeting TLR4. Inhibition of miR-182 partly nullified baicalein-mediated attenuation on inflammation and oxidative stress in SAP-ALI rats. In conclusion, baicalein can inhibit the TLR4/MyD88/TRIF pathway and alleviate inflammatory response and oxidative stress in SAP-ALI rats by upregulating miR-182 and suppressing TLR4, thus ameliorating SAP-ALI.

Acquired immune resistance is associated with interferon signature and modulation of KLF6/c-MYB transcription factors in myeloid leukemia.

Resistance to immunity is associated with the selection of cancer cells with superior capacities to survive inflammatory reactions. Here, we tailored an ex vivo immune selection model for acute myeloid leukemia (AML) and isolated the residual subpopulations as "immune-experienced" AML (ieAML) cells. We confirmed that upon surviving the immune reactions, the malignant blasts frequently decelerated proliferation, displayed features of myeloid differentiation and activation, and lost immunogenicity. Transcriptomic analyses revealed a limited number of commonly altered pathways and differentially expressed genes in all ieAML cells derived from distinct parental cell lines. Molecular signatures predominantly associated with interferon and inflammatory cytokine signaling were enriched in the AML cells resisting the T-cell-mediated immune reactions. Moreover, the expression and nuclear localization of the transcription factors c-MYB and KLF6 were noted as the putative markers for immune resistance and identified in subpopulations of AML blasts in the patients' bone marrow aspirates. The immune modulatory capacities of ieAML cells lasted for a restricted period when the immune selection pressure was omitted. In conclusion, myeloid leukemia cells harbor subpopulations that can adapt to the harsh conditions established by immune reactions, and a previous "immune experience" is marked with IFN signature and may pave the way for susceptibility to immune intervention therapies.

TRIF-IFN-I pathway in Helicobacter-induced gastric cancer in an accelerated murine disease model and patient biopsies

Helicobacter pylori (H.pylori) infection is a known cause of many digestive diseases, including gastritis, peptic ulcers, and gastric cancer. However, the underlying mechanisms by which H.pylori infection triggers these disorders are still not clearly understood. Gastric cancer is a slow progressing disease, which makes it difficult to study. We have developed an accelerated disease progression mouse model, which leverages mice deficient in the myeloid differentiation primary response 88 gene (Myd88-/-) infected with Helicobacter felis (H.felis). Using this model and gastric biopsy samples from patients, we report that activation of the Toll/interleukin-1 receptor (TIR)-domain-containing adaptor inducing interferon-β (TRIF)-type I interferon (IFN-I) signaling pathway promotes Helicobacter-induced disease progression toward severe gastric pathology and gastric cancer development. Further, results implicated downstream targets of this pathway in disease pathogenesis. These findings may facilitate stratification of Helicobacter-infected patients and thus enable treatment prioritization of patients.

ST2825, independent of MyD88, induces reactive oxygen species-dependent apoptosis in multiple myeloma cells

Myeloid differentiation factor 88 (MyD88), which is a key regulator of nuclear factor kappa B (NF-κB), plays an important role in tumorigenesis in lymphoid malignancies such as Waldenstrom's macroglobulinemia (WM). However, its biological function in multiple myeloma (MM), which is a malignant plasma cell disorder like WM, remains unexplored. In this article, we first demonstrated that higher expression MyD88 was significantly correlated with poor survival in patients with MM using multiple publicly available datasets. Interestingly, bioinformatic analysis also revealed that MyD88 gene alteration, which is recognized in nearly 80% of patients with WM, was extremely rare in MM. In addition, ST2825 (a specific inhibitor of MyD88) suppressed cell growth followed by apoptosis. Furthermore, ST2825 induced intracellular reactive oxygen species (ROS) in MM cells, and N-acetyl-l-cysteine, which is known as a ROS scavenger, significantly decreased the number of apoptotic MM cells evoked by ST2825 treatment. Taken together, our results indicated that ST2825 leads to ROS-dependent apoptosis in MM cells and could be an attractive therapeutic candidate for patients with MM. By highlighting the pathological mechanism of MyD88 in MM, this study also provides novel treatment strategies to conquer MM.

Deletion of MyD88 in T Cells Improves Antitumor Activity in Melanoma

Cytotoxic CD8+ T cells are central to the antitumor immune response by releasing cytotoxic granules that kill tumor cells. They are activated by antigen-presenting cells, which become activated by DAMPs (damage associated molecular patterns) through MyD88. However, the suppressive tumor microenvironment promotes T-cell tolerance to tumor antigens, in part by enhancing the activity of immune checkpoint molecules that prevent CD8+ T-cell activation and cytotoxicity. MyD88 limits CD4+ T-cell activation during cardiac adaptation to stress. A similar mechanism is hypothesized to exist in CD8+ T cells that could be modulated to improve antitumor immunity. Herein, adoptive transfer of MyD88-/- CD8+ T cells in melanoma-bearing T-cell-deficient mice resulted in slower tumor growth, greater intratumoral T-cell accumulation, and higher melanoma cell death compared with transfer of wild-type CD8+ T cells. These findings were also observed in T-cell-specific MyD88-/- mice compared with wild-type littermates implanted with melanoma. Mechanistically, deletion of MyD88 enhanced CD8+ T-cell activation and survival, and T-cell receptor induced degranulation of cytotoxic molecules, overall improving the killing of melanoma cells. This enhanced cytotoxicity was retained in mice bearing tumors expressing the specific antigen for which cytotoxic T-cells were restricted. This study's results demonstrate a conserved mechanism for MyD88 in modulating CD8+ T-cell activation and represent a novel target in improving cancer immunotherapy.

KCTD5 regulates Ikaros degradation induced by chemotherapeutic drug etoposide in hematological cells.

Therapy-related leukemia carries a poor prognosis, and leukemia after chemotherapy is a growing risk in clinic, whose mechanism is still not well understood. Ikaros transcription factor is an important regulator in hematopoietic cells development and differentiation. In the absence of Ikaros, lymphoid cell differentiation is blocked at an extremely early stage, and myeloid cell differentiation is also significantly affected. In this work, we showed that chemotherapeutic drug etoposide reduced the protein levels of several isoforms of Ikaros including IK1, IK2 and IK4, but not IK6 or IK7, by accelerating protein degradation, in leukemic cells. To investigate the molecular mechanism of Ikaros degradation induced by etoposide, immunoprecipitation coupled with LC-MS/MS analysis was conducted to identify changes in protein interaction with Ikaros before and after etoposide treatment, which uncovered KCTD5 protein. Our further study demonstrates that KCTD5 is the key stabilizing factor of Ikaros and chemotherapeutic drug etoposide induces Ikaros protein degradation through decreasing the interaction of Ikaros with KCTD5. These results suggest that etoposide may induce leukemic transformation by downregulating Ikaros via KCTD5, and our work may provide insights to attenuate the negative impact of chemotherapy on hematopoiesis.

Mechanism of Shenling Baizhu Powder on treatment of alcoholic liver disease by regulating TLR4/NLRP3 pathway

This study aims to investigate the regulatory effects of Shenling Baizhu Powder(SBP) on cellular autophagy in alcoholic liver disease(ALD) and its intervention effect through the TLR4/NLRP3 pathway. A rat model of chronic ALD was established by gavage of spirits. An ALD cell model was established by stimulating BRL3A cells with alcohol. High-performance liquid chromatography(HPLC) was utilized for the compositional analysis of SBP. Liver tissue from ALD rats underwent hematoxylin-eosin(HE) and oil red O staining for pathological evaluation. Enzyme-linked immunosorbent assay(ELISA) was applied to quantify lipopolysaccharides(LPS), tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1β), and interleukin-18(IL-18) levels. Quantitative reverse transcription polymerase chain reaction(qRT-PCR) was conducted to evaluate the mRNA expression of myeloid differentiation factor 88(MyD88) and Toll-like receptor 4(TLR4). The effect of different drugs on BRL3A cell proliferation activity was assessed through CCK-8 analysis. Western blot analysis was performed to examine the protein expression of NOD-like receptor pyrin domain-containing 3(NLRP3), nuclear factor-kappa B P65(NF-κB P65), phosphorylated nuclear factor-kappa B P65(p-P65), caspase-1, P62, Beclin1, and microtubule-associated protein 1 light chain 3(LC3Ⅱ). The results showed that SBP effectively ameliorated hepatic lipid accumulation, reduced liver function, mitigated hepatic tissue inflammation, and reduced levels of LPS, TNF-α, IL-1β, and IL-18. Moreover, SBP exhibited the capacity to modulate hepatic autophagy induced by prolonged alcohol intake through the TLR4/NLRP3 signaling pathway. This modulation resulted in decreased expression of LC3Ⅱ and Beclin1, an elevation in P62 expression, and the promotion of autolysosome formation. These research findings imply that SBP can substantially enhance liver function and mitigate lipid irregularities in the context of chronic ALD. It achieves this by regulating excessive autophagic responses caused by prolonged spirit consumption, primarily through the inhibition of the TLR4/NLRP3 pathway.

Activated mesenchymal stem/stromal cells promote myeloid cell differentiation via CCL2/CCR2 signaling

Myeloid cells, which originate from hematopoietic stem/progenitor cells (HSPCs), play a crucial role in mitigating infections. This study aimed to explore the impact of mesenchymal stem/stromal cells (MSCs) on the differentiation of HSPCs and progenitors through the C-C motif chemokine CCL2/CCR2 signaling pathway. Murine MSCs, identified as PDGFRα+Sca-1+ cells (PαS cells), were found to secrete CCL2, particularly in response to lipopolysaccharide stimulation. MSC-secreted CCL2 promoted the differentiation of granulocyte/macrophage progenitors into the myeloid lineage. MSC-derived CCL2 plays an important role in the early phase of myeloid cell differentiation invivo. Single-cell RNA sequencing analysis confirmed that CCL2-mediated cell fate determination was also observed in human bone marrow cells. These findings provide valuable insights for investigating the invivo effects of MSC transplantation.

Periodontitis and lipopolysaccharides: How far have we understood?

Periodontitis is a ubiquitous chronic inflammatory worldwide disease. The multiplicity of gram-negative microbiomes and their endotoxins, such as lipopolysaccharides (LPS), play a crucial role in its pathogenesis. The detection and consequent effects of LPS occur either via membrane-based cluster of differentiation 14 (CD14)/myeloid differentiation factor 2 (MD2)/Toll-like receptor (TLR)-4 complex activation or through intracellular cytosolic LPS detection that further cascades its effects, resulting in a variety of cell death processes, including apoptosis, pyroptosis, necroptosis, NETosis, and their crosstalk. Irrespective of the detection of LPS, the cellular response is for protecting and resolving the inflammation. However, chronic and exaggerated responses in periodontitis result in the destruction of periodontal structures. This review summarizes the extracellular and cytosolic detection of LPS and its further consequences. Then, it sheds light on methods reported to mitigate the adverse effects of LPS.