Myeloid Lineage Research Articles

Clonal hematopoiesis driven by mutated DNMT3A promotes inflammatory bone loss

Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3aR878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory Tcell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss.

Myeloid Targeted Human MLL-ENL and MLL-AF9 Induces cdk9 and bcl2 Expression in Zebrafish Embryos.

Acute myeloid leukemia (AML) accounts for greater than twenty thousand new cases of leukemia annually in the United States. The average five-year survival rate is approximately 30%, pointing to the need for developing novel model systems for drug discovery. In particular, patients with chromosomal rearrangements in the mixed lineage leukemia (MLL) gene have higher relapse rates with poor outcomes. In this study we investigated the expression of human MLL-ENL and MLL-AF9 in the myeloid lineage of zebrafish embryos. We observed an expansion of MLL positive cells and determined these cells colocalized with the myeloid markers spi1b, mpx, and mpeg. In addition, expression of MLL-ENL and MLL-AF9 induced the expression of endogenous bcl2 and cdk9, genes that are often dysregulated in MLL-r-AML. Co-treatment of lyz: MLL-ENL or lyz:MLL-AF9 expressing embryos with the BCL2 inhibitor, Venetoclax, and the CDK9 inhibitor, Flavopiridol, significantly reduced the number of MLL positive cells compared to embryos treated with vehicle or either drug alone. In addition, cotreatment with Venetoclax and Flavopiridol significantly reduced the expression of endogenous mcl1a compared to vehicle, consistent with AML. This new model of MLL-r-AML provides a novel tool to understand the molecular mechanisms underlying disease progression and a platform for drug discovery.

Dose-dependent effects of Dnmt3a in an inducible murine model of KrasG12D-driven leukemia

DNMT3A mutations are frequently found in clonal hematopoiesis and a variety of hematologic malignancies, including acute myeloid leukemia. An assortment of mouse models have been engineered to explore the tumorigenic potential and malignant lineage bias due to loss of function of DNMT3A in consort with commonly comutated genes in myeloid malignancies, such as Flt3, Nras, Kras, and c-Kit. We employed several tamoxifen-inducible Cre-ERT2 murine model systems to study the effects of constitutively active KrasG12D-driven myeloid leukemia (Kras) development together with heterozygous (3aHet) or homozygous Dnmt3a deletion (3aKO). Due to the rapid generation of diverse nonhematologic tumors appearing after tamoxifen induction, we employed a transplantation model. With pretransplant tamoxifen induction, most Kras mice died quickly of T-cell malignancies regardless of Dnmt3a status. Using posttransplant induction, we observed a dose-dependent effect of DNMT3A depletion that skewed the leukemic phenotype toward a myeloid lineage. Specifically, 64% of 3aKO/Kras mice had exclusively myeloid disease compared with 36% of 3aHet/Kras and only 13% of Kras mice. Here, 3aKO combined with Kras led to increased disease burden, multiorgan infiltration, and faster disease progression. DOT1L inhibition exerted profound antileukemic effects in malignant 3aKO/Kras cells, but not malignant cells with Kras mutation alone, consistent with the known sensitivity of DNMT3A-mutant leukemia to DOT1L inhibition. RNAseq from malignant myeloid cells revealed that biallelic Dnmt3a deletion was associated with loss of cell-cycle regulation, MYC activation, and TNF⍺ signaling. Overall, we developed a robust model system for mechanistic and preclinical investigations of acute myeloid leukemia with DNMT3A and Ras-pathway lesions.

Hematopoietic stem cell transplant for adults with mixed phenotypic acute leukemia.

6517 Background: Mixed phenotype acute leukemia (MPAL) is a rare acute leukemia subtype characterized by expression of lymphoid and myeloid lineage markers on blasts. Consequently, the role of hematopoietic stem cell transplant (HCT) consolidation is unclear. We conducted a retrospective study of MPAL patients treated at our institution integrating the impact of transplant status, chemotherapy, and initial response on survival. Methods: We evaluated patients with MPAL at Fred Hutchinson Cancer Center from 1/2005 – 9/2022, some of whom were previously reported (Huang et al, ASH 2023). Independent pathology review using 2022 WHO criteria verified MPAL diagnosis. Potentially transplant eligible patients were defined as those <75 years of age, diagnosed at least 6 months prior to data retrieval date, and alive 100 days after diagnosis with an evaluable induction response. Log-rank and Cox proportional hazard models were used to compare survival and adjust for baseline covariates. Survival was landmarked at 100 days. Results: Of 55 total patients, 42 were potentially HCT eligible and 30 received HCT (71.4%; 20/30 myeloablative). Patients who did not complete HCT were similar in age (median 46.2, range 18-70 vs median 50.3, range 24 – 74, p = ns) and had similar ECOG (ECOG 0-1 86.6% vs 75.0%, p = ns) at diagnosis. Nine of 12 non-transplanted patients had age-adjusted HCT-CI score ≤ 3. Patients receiving a HCT had better progression free survival (PFS) (p = 0.025) and were more likely to be alive at 48 months (61.6% vs. 32.4%, p = 0.011) with a median overall survival (mOS) of not reached (NR) (95% CI, 43 months – NR) compared to a mOS of 13 months (95% CI, 9 months – NR) for those not transplanted. Given that our prior work showed that the type of induction chemotherapy did not influence OS (p = 0.40) or remission status (p = 0.16) (Huang et al. ASH 2023), we investigated impact of remission status at the time of transplant. Differences in PFS and OS persisted when stratifying patients by induction response status with HCT status (PFS p = 0.0048; OS p < 0.0001). Patients who had a complete response (CR) followed by HCT had the longest mOS = NR (17 of 24 patients alive after 48 mo). Patients who had resistant disease (RD) and completed HCT had mOS = 43 months (95% CI, 43 months– NR). Patients who achieved CR but did not undergo HCT had mOS = 19 months (95% CI, 11 months – NR). Patients who had RD and did not undergo HCT had median OS of 6 months (95% CI, 6 – NR). The hazard ratio for death among patients who underwent HCT was 0.28 (95% CI 0.077 – 0.99, p = 0.049). Conclusions: HCT consolidation was significantly correlated with improved survival (p = 0.011). While potentially biased by selection and limited by small sample size, patients benefitted from undergoing a HCT regardless of response to prior therapy (p < 0.0001). This study suggests that patients with MPAL in CR after induction chemotherapy as well as those with less than complete responses should be considered for HCT consolidation.

Vascular restoration through local delivery of angiogenic factors stimulates bone regeneration in critical size defects

Critical size bone defects represent a significant challenge worldwide, often leading to persistent pain and physical disability that profoundly impact patients’ quality of life and mental well-being. To address the intricate and complex repair processes involved in these defects, we performed single-cell RNA sequencing and revealed notable shifts in cellular populations within regenerative tissue. Specifically, we observed a decrease in progenitor lineage cells and endothelial cells, coupled with an increase in fibrotic lineage cells and pro-inflammatory cells within regenerative tissue. Furthermore, our analysis of differentially expressed genes and associated signaling pathway at the single-cell level highlighted impaired angiogenesis as a central pathway in critical size bone defects, notably influenced by reduction of Spp1 and Cxcl12 expression. This deficiency was particularly pronounced in progenitor lineage cells and myeloid lineage cells, underscoring its significance in the regeneration process. In response to these findings, we developed an innovative approach to enhance bone regeneration in critical size bone defects. Our fabrication process involves the integration of electrospun PCL fibers with electrosprayed PLGA microspheres carrying Spp1 and Cxcl12. This design allows for the gradual release of Spp1 and Cxcl12 in vitro and in vivo. To evaluate the efficacy of our approach, we locally applied PCL scaffolds loaded with Spp1 and Cxcl12 in a murine model of critical size bone defects. Our results demonstrated restored angiogenesis, accelerated bone regeneration, alleviated pain responses and improved mobility in treated mice.

TGF-β Signalling Regulates Cytokine Production in Inflammatory Cardiac Macrophages during Experimental Autoimmune Myocarditis.

Myocarditis is characterized by an influx of inflammatory cells, predominantly of myeloid lineage. The progression of myocarditis to a dilated cardiomyopathy is markedly influenced by TGF-β signalling. Here, we investigate the role of TGF-β signalling in inflammatory cardiac macrophages in the development of myocarditis and post-inflammatory fibrosis. Experimental autoimmune myocarditis (EAM) was induced in the LysM-Cre × R26-stop-EYFP × Tgfbr2-fl/fl transgenic mice showing impaired TGF-β signalling in the myeloid lineage and the LysM-Cre × R26-stop-EYFP control mice. In EAM, immunization led to acute myocarditis on day 21, followed by cardiac fibrosis on day 40. Both strains showed a similar severity of myocarditis and the extent of cardiac fibrosis. On day 21 of EAM, an increase in cardiac inflammatory macrophages was observed in both strains. These cells were sorted and analysed for differential gene expression using whole-genome transcriptomics. The analysis revealed activation and regulation of the inflammatory response, particularly the production of both pro-inflammatory and anti-inflammatory cytokines and cytokine receptors as TGF-β-dependent processes. The analysis of selected cytokines produced by bone marrow-derived macrophages confirmed their suppressed secretion. In conclusion, our findings highlight the regulatory role of TGF-β signalling in cytokine production within inflammatory cardiac macrophages during myocarditis.

The miR-29-3p family suppresses inflammatory osteolysis.

Osteoclasts are the cells primarily responsible for inflammation-induced bone loss, as is particularly seen in rheumatoid arthritis. Increasing evidence suggests that osteoclasts formed under homeostatic versus inflammatory conditions may differ in phenotype. While microRNA-29-3p family members (miR-29a-3p, miR-29b-3p, miR-29c-3p) promote the function of RANKL-induced osteoclasts, the role of miR-29-3p during inflammatory TNF-α-induced osteoclastogenesis is unknown. We used bulk RNA-seq, histology, qRT-PCR, reporter assays, and western blot analysis to examine bone marrow monocytic cell cultures and tissue from male mice in which the function of miR-29-3p family members was decreased by expression of a miR-29-3p tough decoy (TuD) competitive inhibitor in the myeloid lineage (LysM-cre). We found that RANKL-treated monocytic cells expressing the miR-29-3p TuD developed a hypercytokinemia/proinflammatory gene expression profile in vitro, which is associated with macrophages. These data support the concept that miR-29-3p suppresses macrophage lineage commitment and may have anti-inflammatory effects. In correlation, when miR-29-3p activity was decreased, TNF-α-induced osteoclast formation was accentuated in an in vivo model of localized osteolysis and in a cell-autonomous manner in vitro. Further, miR-29-3p targets mouse TNF receptor 1 (TNFR1/Tnfrsf1a), an evolutionarily conserved regulatory mechanism, which likely contributes to the increased TNF-α signaling sensitivity observed in the miR-29-3p decoy cells. Whereas our previous studies demonstrated that the miR-29-3p family promotes RANKL-induced bone resorption, the present work shows that miR-29-3p dampens TNF-α-induced osteoclastogenesis, indicating that miR-29-3p has pleiotropic effects in bone homeostasis and inflammatory osteolysis. Our data supports the concept that the knockdown of miR-29-3p activity could prime myeloid cells to respond to an inflammatory challenge and potentially shift lineage commitment toward macrophage, making the miR-29-3p family a potential therapeutic target for modulating inflammatory response.

Lactococcus lactis subsp. cremoris C60 Upregulates Macrophage Function by Modifying Metabolic Preference in Enhanced Anti-Tumor Immunity

Lactococcus lactis subsp. cremoris C60 is a probiotic strain of lactic acid bacteria (LAB) which induces various immune modifications in myeloid lineage cells. These modifications subsequently regulate T cell function, resulting in enhanced immunity both locally and systemically. Here, we report that C60 suppresses tumor growth by enhancing macrophage function via metabolic alterations, thereby increasing adenosine triphosphate (ATP) production in a murine melanoma model. Intragastric (i.g.) administration of C60 significantly reduced tumor volume compared to saline administration in mice. The anti-tumor function of intratumor (IT) macrophage was upregulated in mice administered with C60, as evidenced by an increased inflammatory phenotype (M1) rather than an anti-inflammatory/reparative (M2) phenotype, along with enhanced antigen-presenting ability, resulting in increased tumor antigen-specific CD8+ T cells. Through this functional modification, we identified that C60 establishes a glycolysis-dominant metabolism, rather than fatty acid oxidation (FAO), in IT macrophages, leading to increased intracellular ATP levels. To address the question of why orally supplemented C60 exhibits functions in distal places, we found a possibility that bacterial cell wall components, which could be distributed throughout the body from the gut, may induce stimulatory signals in peripheral macrophages via Toll-like receptors (TLRs) signaling activation. Thus, C60 strengthens macrophage anti-tumor immunity by promoting a predominant metabolic shift towards glycolysis upon TLR-mediated stimulation, thereby increasing substantial energy production.

Efficient enzyme-free method to assess the development and maturation of the innate and adaptive immune systems in the mouse colon

Researchers who aim to globally analyze the gastrointestinal immune system via flow cytometry have many protocol options to choose from, with specifics generally tied to gut wall layers of interest. To get a clearer idea of the approach we should use on full-thickness colon samples from mice, we first undertook a systematic comparison of three tissue dissociation techniques: two based on enzymatic cocktails and the other one based on manual crushing. Using flow cytometry panels of general markers of lymphoid and myeloid cells, we found that the presence of cell-surface markers and relative cell population frequencies were more stable with the mechanical method. Both enzymatic approaches were associated with a marked decrease of several cell-surface markers. Using mechanical dissociation, we then developed two minimally overlapping panels, consisting of a total of 26 antibodies, for serial profiling of lymphoid and myeloid lineages from the mouse colon in greater detail. Here, we highlight how we accurately delineate these populations by manual gating, as well as the reproducibility of our panels on mouse spleen and whole blood. As a proof-of-principle of the usefulness of our general approach, we also report segment- and life stage-specific patterns of immune cell profiles in the colon. Overall, our data indicate that mechanical dissociation is more suitable and efficient than enzymatic methods for recovering immune cells from all colon layers at once. Additionally, our panels will provide researchers with a relatively simple tool for detailed immune cell profiling in the murine gastrointestinal tract, regardless of life stage or experimental conditions.

In vivo imaging system (IVIS) therapeutic assessment of tyrosine kinase inhibitor-loaded gold nanocarriers for acute myeloid leukemia: a pilot study.

Acute myeloid leukemia (AML) is a malignancy in the myeloid lineage that is characterized by symptoms like fatigue, bleeding, infections, or anemia, and it can be fatal if untreated. In AML, mutations in tyrosine kinases (TKs) lead to enhanced tumor cell survival. The most frequent mutations in TKs are reported in Fms-like tyrosine kinase 3 (FLT3), Janus kinase 2 (JAK2), and KIT (tyrosine-protein kinase KIT), making these TKs potential targets for TK inhibitor (TKI) therapies in AML. With 30% of the mutations in TKs, mutated FLT3 is associated with poor overall survival and an increased chance of resistance to therapy. FLT3 inhibitors are used in FLT3-mutant AML, and the combination with hypomethylating agents displayed promising results. Midostaurin (MDS) is the first targeted therapy in FLT3-mutant AML, and its combination with chemotherapy showed good results. However, chemotherapies induce several side effects, and an alternative to chemotherapy might be the use of nanoparticles for better drug delivery, improved bioavailability, reduced drug resistance and induced toxicity. The herein study presents MDS-loaded gold nanoparticles and compares its efficacy with MDS alone, on both in vitro and in vivo models, using the FLT3-ITD-mutated AML cell line MV-4-11 Luc2 transfected to express luciferin. Our preclinical study suggests that MDS-loaded nanoparticles have a better tumor inhibitory effect than free drugs on in vivo models by controlling tumor growth in the first half of the treatment, while in the second part of the therapy, the tumor size was comparable to the cohort that was treatment-free.

Age, sex and breed effect on laboratory parameters in natural Babesia canis infection

We tested the hypothesis that age, breed, and sex are related to hematology, biochemistry, acute phase proteins (APPs), seroreactivity and level of parasitemia in dogs with an acute phase response (APR) due to Babesia canis infection. The study enrolled 61 privately owned dogs that naturally acquired B. canis infection. Groups were formed according to the age: young dogs less than one year, and adult dogs more than one year old. Moreover, the group of males was compared to females and purebred to mixed breed dogs. Seroreactivity was tested with immunofluorescence antibody test, level of parasitemia with real-time polymerase chain reaction (real-time PCR), hematology, and biochemistry with automatic analyzers, serum amyloid A with enzyme-linked immunosorbent assay, fibrinogen with heat precipitation and ceruloplasmin and paraoxonase-1 with manual spectrophotometric methods. For protein separation agarose gel electrophoresis was used. The main changes in the whole population of B. canis-infected dogs were fever, pancytopenia, and change in APPs level. One-third of young, and 96% of adult dogs were seropositive (P < 0.001). The level of parasitemia was higher in the young dogs (P < 0.001). Erythroid lineage parameters (P < 0.01), and leukocytes (P < 0.05) were lower in the young, when compared to the adult dogs. Young dogs had lower total globulins (P < 0.001), β- and γ-globulins (P < 0.001), and higher α-globulins (P = 0.022) than adult dogs. Young dogs had higher concentrations of phosphate (P = 0.003) and cholesterol (P < 0.001) and lower amylase (P = 0.014) and lipase activity (P = 0.020) than adult ones. Male dogs had lower neutrophil count than females (P = 0.035), and purebred dogs had more band neutrophils than mixed breed dogs (P = 0.004). In conclusion, dogs with natural Babesia canis infection at a young age have more severe anemia and APR including leukopenia than adults. Male and purebred dogs might also have more severe APR than females and mix-breeds, as they have more pronounced changes related to the myeloid lineage.

Targetable leukaemia dependency on noncanonical PI3Kγ signalling.

Phosphoinositide-3-kinase-γ (PI3Kγ) is implicated as a target to repolarize tumour-associated macrophages and promote antitumour immune responses in solid cancers1-4. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukaemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid and dendritic lineages. This dependency is characterized by innate inflammatory signalling and activation of phosphoinositide 3-kinase regulatory subunit 5 (PIK3R5), which encodes a regulatory subunit of PI3Kγ5 and stabilizes the active enzymatic complex. We identify p21 (RAC1)-activated kinase 1 (PAK1) as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency and find that dephosphorylation of PAK1 by PI3Kγ inhibition impairs mitochondrial oxidative phosphorylation. Treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukaemias with activated PIK3R5. In addition, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukaemia xenografts with low baseline PIK3R5 expression, as residual leukaemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Together, our study reveals a targetable dependency on PI3Kγ-PAK1 signalling that is amenable to near-term evaluation in patients with acute leukaemia.

Increased levels of GM-CSF and CXCL10 and low CD8+ memory stem T Cell count are markers of immunosenescence and severe COVID-19 in older people

BackgroundAgeing leads to altered immune responses, resulting in higher susceptibility to certain infections in the elderly. Immune ageing is a heterogeneous process also associated with inflammaging, a low-grade chronic inflammation. Altered cytotoxic T cell responses and cytokine storm have previously been described in severe COVID-19 cases, however the parameters responsible for such immune response failures are not well known. The aim of our study was to characterize CD8+ T cells and cytokines associated with ageing, in a cohort of patients aged over 70 years stratified by COVID-19 severity.ResultsOne hundred and four patients were included in the study. We found that, in older people, COVID-19 severity was associated with (i) higher level of GM-CSF, CXCL10 (IP-10), VEGF, IL-1β, CCL2 (MCP-1) and the neutrophil to lymphocyte ratio (NLR), (ii) increased terminally differentiated CD8+T cells, and (ii) decreased early precursors CD8+ T stem cell-like memory cells (TSCM) and CD27+CD28+. The cytokines mentioned above were found at higher concentrations in the COVID-19+ older cohort compared to a younger cohort in which they were not associated with disease severity.ConclusionsOur results highlight the particular importance of the myeloid lineage in COVID-19 severity among older people. As GM-CSF and CXCL10 were not associated with COVID-19 severity in younger patients, they may represent disease severity specific markers of ageing and should be considered in older people care.

The Transcription Factor Mohawk Facilitates Skeletal Muscle Repair via Modulation of the Inflammatory Environment.

Efficient repair of skeletal muscle relies upon the precise coordination of cells between the satellite cell niche and innate immune cells that are recruited to the site of injury. The expression of pro-inflammatory cytokines and chemokines such as TNFα, IFNγ, CXCL1, and CCL2, by muscle and tissue resident immune cells recruits neutrophils and M1 macrophages to the injury and activates satellite cells. These signal cascades lead to highly integrated temporal and spatial control of muscle repair. Despite the therapeutic potential of these factors for improving tissue regeneration after traumatic and chronic injuries, their transcriptional regulation is not well understood. The transcription factor Mohawk (Mkx) functions as a repressor of myogenic differentiation and regulates fiber type specification. Embryonically, Mkx is expressed in all progenitor cells of the musculoskeletal system and is expressed in human and mouse myeloid lineage cells. An analysis of mice deficient for Mkx revealed a delay in postnatal muscle repair characterized by impaired clearance of necrotic fibers and smaller newly regenerated fibers. Further, there was a delay in the expression of inflammatory signals such as Ccl2, Ifnγ, and Tgfß. This was coupled with impaired recruitment of pro-inflammatory macrophages to the site of muscle damage. These studies demonstrate that Mkx plays a critical role in adult skeletal muscle repair that is mediated through the initial activation of the inflammatory response.

Monocyte Production of C1q Potentiates CD8+ T-Cell Function Following Respiratory Viral Infection.

Respiratory viral infections remain a leading cause of morbidity and mortality. Using a murine model of human metapneumovirus, we identified recruitment of a C1q-expressing inflammatory monocyte population concomitant with viral clearance by adaptive immune cells. Genetic ablation of C1q led to reduced CD8+ T-cell function. Production of C1q by a myeloid lineage was necessary to enhance CD8+ T-cell function. Activated and dividing CD8+ T cells expressed a C1q receptor, gC1qR. Perturbation of gC1qR signaling led to altered CD8+ T-cell IFN-γ production, metabolic capacity, and cell proliferation. Autopsy specimens from fatal respiratory viral infections in children exhibited diffuse production of C1q by an interstitial population. Humans with severe coronavirus disease (COVID-19) infection also exhibited upregulation of gC1qR on activated and rapidly dividing CD8+ T cells. Collectively, these studies implicate C1q production from monocytes as a critical regulator of CD8+ T-cell function following respiratory viral infection.

MiRNA-7145-cuedc2 axis controls hematopoiesis through JAK1/STAT3 signaling pathway

Hematopoiesis ensures tissue oxygenation, and remodeling as well as immune protection in vertebrates. During embryogenesis, hemangioblasts are the source of all blood cells. Gata1a and pu.1 are co-expressed in hemangioblasts before hemangioblasts are differentiated into blood cells. However, the genes that determine the differentiation of hemangioblasts into myeloid or erythroid cell lineages have not been fully uncovered. Here we showed that miRNA-7145, a miRNA with previously unknown function, was enriched in erythrocytes at the definitive wave, but not expressed in myeloid cells. Overexpression and loss-of-function analysis of miRNA-7145 revealed that miRNA-7145 functions as a strong inhibitor for myeloid progenitor cell differentiation while driving erythropoiesis during the primitive wave. Furthermore, we confirmed that cuedc2 is one of miRNA-7145 targeted-genes. Overexpression or knock-down of cuedc2 partially rescues the phenotype caused by miRNA-7145 overexpression or loss-of-function. As well, overexpression and loss-of-function analysis of cuedc2 showed that cuedc2 is required for myelopoiesis at the expense of erythropoiesis. Finally, we found that overexpression of zebrafish cuedc2 in 293 T cell inhibits the JAK1/STAT3 signaling pathway. Collectively, our results uncover a previously unknown miRNA-7145-cuedc2 axis, which regulate hematopoiesis through inhibiting the JAK1/STAT3 signaling pathway.

Altered Histone Deacetylase Versus Histone Acetyltransferase Activity in Peripheral Blood Mononuclear Cells from Apparently Healthy Young Adults with Prior Exposure to Adverse Childhood Experiences

Introduction: Adverse childhood experiences (ACEs) are severe psychosocial stressors which occur in early developmental periods and promote later life cardiometabolic disease in a dose-dependent manner. ACE-related risk may be mediated in part by epigenetic alterations, particularly in myeloid lineage immune cells. Histone acetylation is a dynamic epigenetic process that regulates gene expression via modification of chromatin structure and is dictated by the opposed actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). An appropriate balance between HDAC and HAT activity is critical for maintaining cellular function, and an imbalance has been implicated in the premature development of human diseases including cardiometabolic disease. Glucocorticoids (e.g., cortisol) are well-described regulators of immune function and can influence histone acetylation through glucocorticoid receptors. Thus, we hypothesized that HDAC:HAT activity would be altered in young adults with prior ACE exposure, and that HDAC:HAT activity would be associated with salivary cortisol concentrations. Purpose: We examined HDAC:HAT activity in peripheral blood mononuclear cells (PBMCs) in apparently healthy young adults with versus without prior exposure to ACEs and explored whether circulating cortisol was associated with HDAC:HAT activity. Methods: PBMCs were isolated from whole blood collected within 1 hour of wakening from 27 young adult women who reported high (≥ 4 ACEs) ACE exposure (ACE4+; mean ± SD; age = 21 ± 3 y, BMI = 25.7 ± 4.5 kg/m2) and from 14 young adult women who reported no ACE exposure (CON; age = 21 ± 3 y, BMI = 26.1 ± 6 kg/m2). Salivary cortisol was also collected within 1 hour of wakening and 8 hours later. Nuclear proteins were isolated from PBMCs and HDAC and HAT activities were quantified by commercially available assay kits. Salivary cortisol was quantified via ELISA. Independent samples t-tests were used to examine differences in HDAC:HAT, HDAC, and HAT activity. Pearson correlation coeffcients were used to examine the relation of HDAC:HAT activity with morning and afternoon cortisol concentrations, and the diurnal cortisol slope in the complete sample. Results: HDAC:HAT activity was reduced in ACE4+ versus CON (140.4 ± 52 vs. 189.6 ± 42 arbitrary units, p = 0.004). This difference was primarily driven by reduced HDAC activity (13,706 ± 4,696 vs. 18,060 ± 3,641 ng/h/mg, p = 0.004) in ACE4+ versus CON, whereas HAT activity was not different (99.7 ± 11 vs. 96.1 ± 9 ng/h/mg, p = 0.30). HDAC:HAT activity was not associated with morning (r=0.27, p = 0.07) or afternoon (r=-0.23, p = 0.16) cortisol, but was associated with the diurnal cortisol slope (r=-0.37, p = 0.02). Discussion: Young adults with prior ACE exposure have an altered balance between HDAC and HAT activity in PBMCs that appears to be driven by lower HDAC activity, and which is negatively related to diurnal cortisol slope. These findings warrant further exploration, particularly regarding the specific HDACs implicated, their role in reduced cellular function as it relates to disease risk, and whether these can be modulated to improve function among those with ACE exposure. Further, our data support future examination of the role of glucocorticoid signaling in regulating histone acetylation in this population. This study was supported by the Center for Integrative Research on Childhood Adversity (Grant #P20GM109097), through the National Institute of General Medical Sciences (to NDMJ and TKT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

T-bet Regulates Ion Channels and Transporters and Induces Apoptosis in Intestinal Epithelial Cells.

T-bet, encoded by TBX21, is extensively expressed across various immune cell types, and orchestrates critical functions in their development, survival, and physiological activities. However, the role of T-bet in non-immune compartments, notably the epithelial cells, remains obscure. Herein, a Tet-O-T-bet transgenic mouse strain is generated for doxycycline-inducible T-bet expression in adult animals. Unexpectedly, ubiquitous T-bet overexpression causes acute diarrhea, intestinal damage, and rapid mortality. Cell-type-specific analyses reveal that T-bet-driven pathology is not attributable to its overexpression in CD4+ T cells or myeloid lineages. Instead, inducible T-bet overexpression in the intestinal epithelial cells is the critical determinant of the observed lethal phenotype. Mechanistically, T-bet overexpression modulates ion channel and transporter profiles in gut epithelial cells, triggering profound fluid secretion and subsequent lethal dehydration. Furthermore, ectopic T-bet expression enhances gut epithelial cell apoptosis and markedly suppresses colon cancer development in xenograft models. Collectively, the findings unveil a previously unrecognized role of T-bet in intestinal epithelial cells for inducing apoptosis, diarrhea, and local inflammation, thus implicating its potential as a therapeutic target for the treatment of cancer and inflammatory diseases.

A Comprehensive Flow Cytometry Panel for Analysis of Idiopathic Subglottic Stenosis.

To present a comprehensive flow cytometry panel for idiopathic subglottic stenosis (iSGS). Controlled ex vivo cohort study. Tertiary care academic hospital in a metropolitan area. Flow cytometry and single-cell RNA sequencing were performed on 9 paired normal and scar tissue samples from iSGS patients. Flow cytometry was used to assess the presence of myeloid (CD11b, CD14, CD15, Siglec8), lymphoid (CD3, CD4, CD8, gamma delta [γδ], FOXP3), endothelial (CD31), fibroblast (CD90, SMA), and epithelial (CD326, CK5) markers. On flow cytometry, iSGS scar is characterized by an increased presence of myeloid, lymphoid, endothelial, and fibroblast cell types, but a decreased presence of epithelial cells. In the myeloid lineage, iSGS scar samples demonstrated increased CD11b+ monocytes (P < .001), Siglec8+ eosinophils (P = .03), and CD14+ monocytes (P = .02). In the lymphoid lineage, iSGS scar demonstrated increased CD3+ T-cells (P < .001), CD4+ helper T-cells (P < .001), γδ+ T-cells (P < .001), and FOXP3+ regulatory T-cells (P = .002). iSGS scar exhibited specific increases in CD90+ (P = .04) and SMA+ (P < .001) fibroblasts but decreased CD326+ (E-cadherin) epithelial cells (P = .01) relative to normal samples. We present a comprehensive flow cytometry panel for iSGS. This flow panel may serve as a common platform among airway scientists to elucidate the cellular mechanisms underpinning iSGS and other upper airway pathologies. Scar iSGS samples demonstrate a distinct cellular profile relative to normal iSGS specimens, exhibiting increased fibroblast, endothelial, and inflammatory cell types but decreased epithelium.

Study of Patterns of Leukaemia in a Tertiary Care Hospital

Introduction: Leukaemia arises from abnormal proliferation of hematopoietic or lymphoid cells, categorized as acute or chronic and originating from lymphoid, myeloid, or mixed lineages. Clinical manifestations result from leukemic cell proliferation and tissue infiltration. Objectives: This study aimed to determine the frequency and clinicopathological profile of leukaemia cases. Material and Methods: A three-year cross-sectional observational study was carried out in the Department of Pathology, Indira Gandhi Medical College, Shimla. Diagnosis of Leukaemia was based on complete blood count, peripheral smear examination, bone marrow analysis and cytochemistry wherever required. Data comprising of patient details, clinical examination and haematological parameters were analysed. Results: A total of 163 leukaemia cases were identified, including 102 (63%) acute and 61(37%) cases of chronic leukaemia. Acute leukaemia consisted of 58 cases of Acute Myeloid Leukaemia (AML), 25 Acute Lymphoblastic Leukaemia (ALL), and 19 undifferentiated leukaemia (UL), while chronic cases comprised 47 Chronic Myeloid Leukaemia (CML) and 14 Chronic Lymphocytic Leukaemia (CLL). Most patients were in the age group of 31-60 years, with a male predominance (M:F 1.7:1). Conclusions: Early diagnosis and typing are vital for prompt leukaemia treatment. Despite advanced technologies, cytomorphological examination with cytochemistry remains crucial, especially in resource-limited settings like India. Keywords: Leukaemia, acute, chronic, cytomorphology, observational study