NKAP Must Associate with HDAC3 to Regulate Hematopoietic Stem Cell Maintenance and Survival.

NKAP is a multifunctional nuclear protein that associates with the histone deacetylase HDAC3. Although both NKAP and HDAC3 are critical for hematopoietic stem cell (HSC) maintenance and survival, it was not known whether these two proteins work together. To assess the importance of their association in vivo, serial truncation and alanine scanning was performed on NKAP to identify the minimal binding site for HDAC3. Mutation of either Y352 or F347 to alanine abrogated the association of NKAP with HDAC3, but did not alter NKAP localization or expression. Using a linked conditional deletion/re-expression system in vivo, we demonstrated that re-expression of the Y352A NKAP mutant failed to restore HSC maintenance and survival in mice when endogenous NKAP expression was eliminated using Mx1-cre and poly-IC, whereas re-expression of wild type NKAP maintained the HSC pool. However, Y352A NKAP did restore proliferation in murine embryonic fibroblasts when endogenous NKAP expression was eliminated using ER-cre and tamoxifen. Therefore, Y352 in NKAP is critical for association with HDAC3 and for HSC maintenance and survival but is not important for proliferation of murine embryonic fibroblasts, demonstrating that NKAP functions in different complexes in different cell types.

Murine T Cell Maturation Entails Protection from MBL2, but Complement Proteins Do Not Drive Clearance of Cells That Fail Maturation in the Absence of NKAP.

Recent thymic emigrants that fail postpositive selection maturation are targeted by complement proteins. T cells likely acquire complement resistance during maturation in the thymus, a complement-privileged organ. To test this, thymocytes and fresh serum were separately obtained and incubated together in vitro to assess complement deposition. Complement binding decreased with development and maturation. Complement binding decreased from the double-positive thymocyte to the single-positive stage, and within single-positive thymocytes, complement binding gradually decreased with increasing intrathymic maturation. Binding of the central complement protein C3 to wild-type immature thymocytes required the lectin but not the classical pathway. Specifically, MBL2 but not MBL1 was required, demonstrating a unique function for MBL2. Previous studies demonstrated that the loss of NKAP, a transcriptional regulator of T cell maturation, caused peripheral T cell lymphopenia and enhanced complement susceptibility. To determine whether complement causes NKAP-deficient T cell disappearance, both the lectin and classical pathways were genetically ablated. This blocked C3 deposition on NKAP-deficient T cells but failed to restore normal cellularity, indicating that complement contributes to clearance but is not the primary cause of peripheral T cell lymphopenia. Rather, the accumulation of lipid peroxides in NKAP-deficient T cells was observed. Lipid peroxidation is a salient feature of ferroptosis, an iron-dependent nonapoptotic cell death. Thus, wild-type thymocytes naturally acquire the ability to protect themselves from complement targeting by MBL2 with maturation. However, NKAP-deficient immature peripheral T cells remain scarce in complement-deficient mice likely due to ferroptosis.

Treg-specific deletion of NKAP results in severe, systemic autoimmunity due to peripheral loss of Tregs.

Regulatory T cells are critical for the generation and maintenance of peripheral tolerance. Conditional deletion of the transcriptional repressor NKAP in Tregs using Foxp3-YFP-cre NKAP conditional knockout mice causes aggressive autoimmunity characterized by thymic atrophy, lymphadenopathy, peripheral T cell activation, generation of autoantibodies, immune infiltration into several organs, and crusty skin at 3 weeks of age, similar to that of "scurfy" Foxp3-mutant mice. While Treg development in the thymus proceeds normally in the absence of NKAP, there is a severe loss of thymically-derived Tregs in the periphery. NKAP-deficient Tregs have a recent thymic emigrant phenotype, and are attacked by complement in a cell-intrinsic manner in the periphery. Previously, we demonstrated that NKAP is required for conventional T cell maturation as it prevents complement-mediated attack in the periphery. We now show that Tregs undergo a similar maturation process as conventional T cells, requiring NKAP to acquire complement resistance after thymic egress.

Three-Dimensional Echocardiographic Evaluation of Mitral Apparatus during Preload Manipulation in Patients with Hypertrophic Cardiomyopathy.

BACKGROUND: The three-dimensional (3D) dynamic change of mitral geometry during preload manipulation has not been fully investigated. We investigated how preload manipulation affected the mitral apparatus geometry in hypertrophic cardiomyopathy (HCM) patients using 3D echocardiography. METHODS AND RESULTS: Twenty five HCM patients, thirteen with obstructive HCM (HOCM) and twelve with nonobstructive HCM (HNCM), and six healthy controls were studied. Subjects underwent 3D echocardiography during rest, leg raising, the Valsalva maneuver, and the Valsalva maneuver after nitroglycerin intake (NTG-Valsalva). Left ventricular outflow tract (LVOT) pressure gradients, mitral annular area, annular circumference, and the tenting volume of the mitral leaflets were measured. Standardized annular area significantly decreased during the NTG-Valsalva maneuver in all 3 groups (▵2.23 mm(2) /m(2) in control, P = 0.031; ▵0.46 mm(2) /m(2) in HNCM, P = 0.012; ▵1.3 mm(2) /m(2) in HOCM, P = 0.013). Standardized annular area decrease during the Valsalva maneuver alone was more prominent in HNCM patients (▵0.57 mm(2) /m(2) , P = 0.009) than HOCM patients (▵0.3 mm(2) /m(2) , P = 0.094). Standardized mitral tenting volume during the NTG-Valsalva maneuver significantly decreased only in HOCM patients (▵1.18 mm(3) /m(2) , P = 0.046). CONCLUSION: Decreased mitral annular area and changes in leaflets tenting volume during preload reduction might affect the development of LVOT obstruction. Our data suggest the importance of preserving the saddle-shaped of the mitral annulus in management of HCM with LVOT obstruction.

Prophylactic Intrawound Antibiotics Significantly Reduce the Risk of Deep Infections in Fracture Fixation: Subgroup Meta-analyses of the Type of Fracture, Antibiotics, and Organism.

OBJECTIVES: To analyze the efficacy of subgroups of various intrawound local antibiotics in reducing the rate of fracture-related infections. DATA SOURCES AND STUDY SELECTION: PubMed, MEDLINE via Ovid, Web of Science, Cochrane database, and Science Direct were searched for articles in English on July 5, 2022, and December 15, 2022. STUDY SELECTION: All clinical studies comparing the incidence of fracture-related infection between the administration of prophylactic systemic and topical antibiotics in fracture repair were analyzed. DATA EXTRACTION: Cochrane collaboration's assessment tool and the methodological bias and the methodological index for nonrandomized studies were used to detect bias and evaluate the quality of included studies, respectively. DATA SYNTHESIS: RevMan 5.3 software (Nordic Cochrane Centre, Denmark) was used to conduct the meta-analyses and generate forest plots. CONCLUSIONS: From 1990 to 2021, 13 studies included 5309 patients. Nonstratified meta-analysis showed that intrawound administration of antibiotics significantly decreased the overall incidence of infection in both open and closed fractures, regardless of the severity of open fracture and antibiotics class [OR = 0.58, ( P = 0.007)] [OR = 0.33, ( P < 0.00001)], respectively. The stratified analysis revealed that prophylactic intrawound antibiotics significantly lowered infection rate in open fracture patients with Gustilo-Anderson type I (OR = 0.13, P = 0.004), type II (OR = 0.29, P = 0.0002), type III (OR = 0.21, P < 0.00001), when either tobramycin PMMA beads (OR = 0.29, P < 0.00001) or vancomycin powder (OR = 0.51, P = 0.03) was applied. This study demonstrates prophylactic administration of intrawound antibiotics significantly decreases the overall incidence of infection in all subgroups of surgically fixated fractures but does not affect the patient's length of hospital stay. LEVEL OF EVIDENCE: Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.

HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment.

CD4 and CD8 T cells are vital components of the immune system. We found that histone deacetylase 3 (HDAC3) is critical for the development of CD4 T cells, as HDAC3-deficient DP thymocytes generate only CD8SP thymocytes in mice. In the absence of HDAC3, MHC Class II-restricted OT-II thymocytes are redirected to the CD8 cytotoxic lineage, which occurs with accelerated kinetics. Analysis of histone acetylation and RNA-seq reveals that HDAC3-deficient DP thymocytes are biased towards the CD8 lineage prior to positive selection. Commitment to the CD4 or CD8 lineage is determined by whether persistent TCR signaling or cytokine signaling predominates, respectively. Despite elevated IL-21R/γc/STAT5 signaling in HDAC3-deficient DP thymocytes, blocking IL-21R does not restore CD4 lineage commitment. Instead, HDAC3 binds directly to CD8-lineage promoting genes. Thus, HDAC3 is required to restrain CD8-lineage genes in DP thymocytes for the generation of CD4 T cells.

The Interaction between NKAP and HDAC3 Is Critical for T Cell Maturation.

NKAP and HDAC3 are critical for T cell maturation. NKAP and HDAC3 physically associate, and a point mutation in NKAP, NKAP(Y352A), abrogates this interaction. To evaluate the significance of NKAP and HDAC3 association in T cell maturation, transgenic mice were engineered for cre-mediated endogenous NKAP gene deletion coupled to induction of NKAP(Y352A) or a wild type (WT) control transgene, NKAP(WT), in double positive thymocytes or regulatory T cells (Tregs). T cell maturation was normal in mice with endogenous NKAP deletion coupled to NKAP(WT) induction. However, severe defects occurred in T cell and Treg maturation and in iNKT cell development when NKAP(Y352A) was induced, recapitulating NKAP deficiency. Conventional T cells expressing NKAP(Y352A) failed to enter the long-term T cell pool, did not produce cytokines, and remained complement susceptible, whereas Tregs expressing NKAP(Y352A) were eliminated as recent thymic emigrants leading to lethal autoimmunity. Overall, these results demonstrate the significance of NKAP-HDAC3 association in T cells.

Histone deacetylase 3 is required for iNKT cell development.

NKT cells are a distinct subset that have developmental requirements that often differ from conventional T cells. Here, we show that NKT-specific deletion of Hdac3 results in a severe reduction in the number of iNKT cells, particularly of NKT1 cells. In addition, there is decreased cytokine production by Hdac3-deficient NKT2 and NKT17 cells. Hdac3-deficient iNKT cells have increased cell death that is not rescued by transgenic expression of Bcl-2 or Bcl-xL. Hdac3-deficient iNKT cells have less Cyto-ID staining and lower LC3A/B expression, indicative of reduced autophagy. Interestingly, Hdac3-deficient iNKT cells also have lower expression of the nutrient receptors GLUT1, CD71 and CD98, which would increase the need for autophagy when nutrients are limiting. Therefore, Hdac3 is required for iNKT cell development and differentiation.

The differentiation of ROR-γt expressing iNKT17 cells is orchestrated by Runx1.

iNKT cells are a unique lineage of T cells that recognize glycolipid presented by CD1d. In the thymus, they differentiate into iNKT1, iNKT2 and iNKT17 effector subsets, characterized by preferential expression of Tbet, Gata3 and ROR-γt and production of IFN-γ, IL-4 and IL-17, respectively. We demonstrate that the transcriptional regulator Runx1 is essential for the generation of ROR-γt expressing iNKT17 cells. PLZF-cre Runx1 cKO mice lack iNKT17 cells in the thymus, spleen and liver. Runx1-deficient iNKT cells have altered expression of several genes important for iNKT17 differentiation, including decreased expression of IL-7Rα, BATF and c-Maf and increased expression of Bcl11b and Lef1. However, reduction of Lef1 expression or introduction of an IL-7Rα transgene is not sufficient to correct the defect in iNKT17 differentiation, demonstrating that Runx1 is a key regulator of several genes required for iNKT17 differentiation. Loss of Runx1 leads to a severe decrease in iNKT cell numbers in the thymus, spleen and liver. The decrease in cell number is due to a combined decrease in proliferation at Stage 1 during thymic development and increased apoptosis. Thus, we describe a novel role of Runx1 in iNKT cell development and differentiation, particularly in orchestrating iNKT17 differentiation.

An Essential Role for the Transcription Factor Runx1 in T Cell Maturation.

The transcription factor Runx1 has essential roles throughout hematopoiesis. Here, we demonstrate that Runx1 is critical for T cell maturation. Peripheral naïve CD4(+) T cells from CD4-cre Runx1 cKO mice are phenotypically and functionally immature as shown by decreased production of TNF-α upon TCR stimulation. The loss of peripheral CD4(+) T cells in CD4-cre Runx1 cKO mice is not due to defects in homeostasis or decreased expression of IL-7Rα, as transgenic expression of IL-7Rα does not rescue the loss of CD4(+) T cells. Rather, immature Runx1-deficient CD4(+) T cells are eliminated in the periphery by the activation and fixation of the classical complement pathway. In the thymus, there is a severe block in all aspects of intrathymic T cell maturation, although both positive and negative selection are unaltered. Thus, loss of Runx1 leads to the earliest characterized block in post-positive selection intrathymic maturation of CD4 T cells.

Comparison of 1-deoxynojirimycin and aqueous mulberry leaf extract with emphasis on postprandial hypoglycemic effects: in vivo and in vitro studies.

Carbohydrate digestion by α-glucosidase and subsequent glucose uptake at the brush border are critical for postprandial blood glucose control. Any specific inhibitors are useful as hyperglycemia modulating agents. In this study, it was postulated that an array of active components in mulberry leaf extract (MLE) may provide higher potency in inhibiting intestinal glucose absorption compared to the single component 1-deoxynojirimycin (DNJ), which is recognized as a promising inhibitor of intestinal glucose absorption. Both MLE and DNJ were active in inhibiting α-glucosidase. However, in Caco-2 cells, only MLE showed significant inhibition of 2-deoxyglucose uptake, whereas DNJ was ineffective. For glucose loading, co-administration of MLE resulted in potent inhibitions of glucose responses compared to those by DNJ in Sprague Dawley (SD) rats, but this was not found for maltose loading. These novel findings add evidence that the unabsorbed phytochemicals in MLE compete with glucose for intestinal glucose transporters, but DNJ itself does not. We also evaluated the timing of MLE consumption. By administering MLE for 30 min before glucose loading, the incremental area under the curve (IAUC) was significantly lowered in the rats, as compared to a simultaneously administered group. Similarly, cellular glucose uptake was significantly reduced in Caco-2 cells following pretreatment.