Essential role for histone deacetylase 11 (HDAC11) in neutrophil biology

Abstract

Epigenetic changes in chromatin structure have been recently associated with the deregulated expression of critical genes in normal and malignant processes. HDAC11, the newest member of the HDAC family of enzymes, functions as a negative regulator of IL-10 expression in APCs, as previously described by our lab. However, at the present time, its role in other hematopoietic cells, specifically in neutrophils, has not been fully explored. In this report, for the first time, we present a novel physiologic role for HDAC11 as a multifaceted regulator of neutrophils. Thus far, we have been able to demonstrate a lineage-restricted overexpression of HDAC11 in neutrophils and committed neutrophil precursors (promyelocytes). Additionally, we show that HDAC11 appears to associate with the transcription machinery, possibly regulating the expression of inflammatory and migratory genes in neutrophils. Given the prevalence of neutrophils in the peripheral circulation and their central role in the first line of defense, our results highlight a unique and novel role for HDAC11. With the consideration of the emergence of new, selective HDAC11 inhibitors, we believe that our findings will have significant implications in a wide range of diseases spanning malignancies, autoimmunity, and inflammation.

Keywords: epigenetics; gene regulation; innate immunity.

Figure 1

The HDAC11 message is differentially expressed in various stages of myelopoiesis. (A) Dynamic visualization of the HDAC11 message using a Tg‐HDAC11‐eGFP mouse in myeloid compartments. BM was extracted from Tg‐HDAC11‐eGFP (as well as C57BL/6 mice as control, non‐eGFP‐expressing cells; gray), and cells were labeled (as indicated above) with specific cell surface markers for identification of each population. HDAC11 expression was determined by flow cytometry analysis of eGFP reporter gene expression, where the expression of the eGFP protein corresponds to the activation of HDAC11 transcriptional machinery (representative presentation from 2 individual experiments). SSC‐H, Side‐scatter‐height. (B) Monocytes, promyelocytes, and neutrophils from Tg‐HDAC11‐eGFP mouse BM cells were sorted using the FACSAria (BD Biosciences) device with 99% purity. Cells were lysed using TRIzol reagent (Thermo Fisher Scientific), as well as radioimmunoprecipitation assay buffer (Cell Signaling Technology, Danvers, MA, USA), and RNA as well as protein was extracted, respectively. Immunoblotting [Western blot (WB)] was performed using 10% SDS gel, the image was resolved using the Dura ECL reagent (Pierce; Thermo Fisher Scientific; inset), and qRT‐PCR analysis was performed using HDAC11 primers (graph). Sorted cell populations were also morphologically confirmed (lower). (C) Coexpression of the HDAC11 message and eGFP message was confirmed by qRT‐PCR using eGFP primers (error bars = sem; data representative of 3 individual experiments; n = 3). (D) Monocytes, promyelocytes, neutrophils, B cells, and T cells were sorted from C57BL/6 WT mouse BM cells using the FACSAria (BD Biosciences) device with 98% purity. Cells were lysed using TRIzol reagent (Thermo Fisher Scientific), and RNA was extracted. qRT‐PCR analysis was performed using HDAC11 primer. This was done to confirm the HDAC11 expression profile in a non‐Tg setting (error bars = sem; data representative of 3 individual experiments; n = 3). (E) Four normal human donor peripheral blood source leukocyte samples (purchased from OneBlood, Florida Blood Bank) were sorted for monocytic and neutrophylic populations using the FACSAria (BD Biosciences) device with a 96% purity and then examined for the expression of the HDAC11 message using qRT‐PCR analysis, and the fold change between neutrophils and monocytes was normalized to the lowest expressing monocytic population (error bars = sem; n = 4). (B–E) Note: subpopulations were compared and normalized to monocytes (as they have the lowest expression of HDAC11).

Figure 2

Expression of HDAC11 in APL samples and functional consequences of its manipulation in this model. (A) HL60 cells were treated with ATRA for 72 h, the expression of the HDAC11 message was measured by qRT‐PCR, and the differentiation of HL60 cells to granulocytic‐like cells was examined by morphologic analysis (Below bar graph; error bars = sem; data representative of 3 individual experiments). NT, Nontreated control. (B) The differentiation of HL60 cells to granulocytic‐like cells was examined using MPO (a marker for cell's gain of granularity; upper), as well as CD11b expression (lower; representative figure from 3 individual experiments).

Figure 3

Phenotypic consequences of HDAC11‐deficient PNs. (A) PNs from C57BL/6 WT and HDAC11KO mice were collected post‐thyoglycolate injection (5% at 18 h). The cells were treated with 2.5 μg/ml LPS for 2 and 4 h. Expression of inflammatory genes TNF‐α and IL‐6 were measured by qRT‐PCR. NT, nontreated control. (B) Protein concentrations for TNF‐α and IL‐6 were measured by CBA analysis. (C) CBC from C57BL/6 WT and HDAC11KO (n = 6/group). LYM, lymphocyte; MONO, monocyte; GRAN, granulocyte. (D) The recruitment of HDAC11 protein to chromatin fragments of TNF‐α and IL‐6 promoters was analyzed using the Pfaffl method [46] and is presented relative to input before immunoprecipitation, and the enrichment ratio was normalized to the IgG control (PNs isolated from C57BL/6 WT). (E) PNs isolated from HDAC11KO mice were used in another ChIP assay as negative control (error bars = sem; data presented for each graph are representative of 2 individual experiments).

Figure 4

Enhanced migratory and phagocytic capacity of HDAC11KO neutrophils. (A) Migration assay analysis between C57BL/6 WT and HDAC11KO PNs using the Transwell system and 2 × 10⁶/sample. Expression of migratory genes was analyzed in PNs isolated from WT and HDAC11KO mice using qRT‐PCR analysis (data generated from 4 individual experiments); ∗∗P < 0.01. (B) mRNA expression of CXCL2 and CXCR2 was analyzed in C57BL/6 WT and HDAC11KO mice at steady state (data generated from 2 individual experiments). (C) Expression of surface CXCR2 protein on neutrophils from HDAC11KO versus C47BL/6 WT mice. gMFI, geometric mean fluorescence intensity; ∗P < 0.05. (D) Phagocytic ability of PNs isolated from C57BL/6 WT and HDAC11KO mice was analyzed in the presence of pHrodo Red E. coli BioParticles loaded with a pH‐sensitive fluorescent dye and analyzed as a measure of cell engulfment and lysis in real time (RFP measurement by microscopy; error bars = sem; statistical analysis was done using 2‐way ANOVA; data presented are representative of 2 individual experiments). (E) The recruitment of HDAC11 protein‐to‐chromatin fragments of CXCL2 and CXCR2 promoters was analyzed in the presence and absence of LPS stimulation. (F) PNs from HDAC11KO mice were used in another ChIP assay as a negative control. The values obtained for these ChIP experiments were analyzed using the Pfaffl method [46] and are presented relative to input before immunoprecipitation; the enrichment ratio was normalized to the IgG control (error bars = sem; representative figure from 2 individual experiments).

Figure 5

Septic shock experiment comparing HDAC11KO with C56BL/6 mice. A cohort (n = 8) of HDAC11KO and C57BL/6 mice was injected with 15 mg/kg LPS via TV. Survival graph representing both groups in timeline up to 72 h (data presented are representative of 2 individual experiments).

Figure 6

Observation of splenomegaly as well as hypercellularity with granulocytic expansion in BM of HDAC11KO mice. (A) Representative sections of spleens (H&E stain) harvested from aged C57BL/6 WT (upper) and HDAC11 KO (lower) mice showing a marked expansion of the red pulp (left; original magnification, ×40), as a result of replacement of the mostly lymphocytic red pulp cellularity observed on C57BL/6 WT mice with mostly trilineage extramedullary hematopoiesis on HDAC11KO mice (right; original magnification, ×400). (B) Representative sections of femurs (H&E stain) harvested from aged C57BL/6 WT (upper) and HDAC11 KO (lower) mice showing a marked increase in the BM cellularity on HDAC11KO mice compared with WT mice (left; original magnification, ×20), mostly as a result of an expansion on maturing neutrophils (right; original magnification, ×200; n = 5/group).