HDAC6 Deletion Decreases Pristane-induced Inflammation Open Access

Systemic lupus erythematosus (SLE) is an autoimmune disease in which body tissues and organs are attacked by their own immune system (1). In SLE, autoantibodies are generated, resulting in the formation of circulating Ag–Ab complexes that become lodged in tiny capillaries and different organ systems, including the kidney and skin (2). Histone acetylation has been shown to play a critical role in controlling gene expression (3, 4). The process of acetylation of histone proteins frequently leads to enhanced transcription, whereas deacetylation is linked to gene repression (5). We and others have previously reported that HDAC inhibitors showed efficacy in the treatment of allergy, cancer, and autoimmune diseases, in particular SLE (6). However, the use of pan-HDAC inhibitors has shown significant adverse effects limiting the use for chronic conditions (7). HDAC6 modulates protein degradation by deacetylating nonhistone proteins, such as heat shock protein 90, α-tubulin, and NF-κB, and has been shown to play a role in the modulation of immune response (8). It has been demonstrated that overexpression of HDAC6 in macrophages significantly increased the expression of proinflammatory cytokines, including IL-6, IL-1β, and TNF-α, via the upregulation of AP-1 and NF-κB signaling pathways (9). HDAC6 inhibition has been shown to interfere with the NF-κB signaling pathway and decrease inflammation by increasing acetylation of NF-κB, which results in decreased nuclear translocation (10, 11).

One commonly used animal model to study lupus is the pristane-induced disease model. Pristane (2, 6, 10, and 14 tetramethylpentadecane) is a naturally occurring branched alkane found in mineral oil and in low concentration in vegetables (12). When injected into the peritoneum in mice, pristane induces lupus-like symptoms over time, including the activation of immune cells and the production of autoantibodies against the host’s own DNA and nuclear Ags (13). This resembles the autoimmune response observed in human SLE. Interestingly, after pristane administration, in the first 2 wk, severe alveolar hemorrhage occurs in C57BL/6 (B6) mice; symptoms include endothelial damage, hemorrhage, and alveolar and perivascular inflammation (small vessel vasculitis and capillaritis) (14). This leads to a mortality rate as high as 20% in the pristane-treated B6 mice (15). In our current studies, we sought to determine whether HADC6 deletion would decrease the inflammation seen with pristane administration. Furthermore, we sought to determine the mechanism by which HADC6 inhibition alters macrophage activation.

Female wild-type (WT) B6 mice and HDAC6 knockout (KO) mice on B6 background were purchased from The Jackson Lab (Bar Harbor, ME). The animals were kept in accordance with the Institutional Animal Care and Use Committee at Virginia Tech College of Veterinary Medicine, in a pathogen-free environment with a regular 12-h light/dark cycle. All animal procedures were performed in compliance with the Institutional Animal Care and Use Committee–approved guidelines. WT and HDAC6 KO mice on the B6 background were administered a single dose of 0.5 ml of pristane (Sigma Aldrich, St. Louis, MO) or PBS i.p. at 8–12 wk of age. Mice were sacrificed at 10 d after pristane injection. At sacrifice, body weight and spleen weight were measured, sera were collected, and splenocytes and peritoneal cells were harvested for flow cytometry.

At sacrifice, spleens were collected and minced into small pieces, then transferred on the 70-μm cell strainer over a 50-ml conical tube. The spleen pieces were minced and pressed through the strainer with the plunger end of a syringe. The cells were washed through the strainer with PBS and then collected after centrifuge. The cell pellet was resuspended in 2–5 ml of cold 1× RBC Lysis buffer (eBioscience, San Diego, CA) for 5 min on ice to lyse the RBCs. The cell suspension was washed with cold PBS and centrifuged. Splenocytes were collected for flow cytometry analysis. Mouse peritoneal cells were extracted as previously published (16). In brief, each mouse’s abdominal skin was covered in 70% alcohol after euthanasia. A tiny incision was created so that the intact peritoneal wall could be seen. In the peritoneal cavity, 5 ml of ice-cold PBS was injected. Peritoneal fluid was collected after a little massage and centrifuged at 500 × g for 10 min at 4°C. The cells were then resuspended for flow cytometry.

Attune NxT (Thermo Fisher Scientific, Waltham, MA) or BD FACSAria II (BD Biosciences, San Jose, CA) flow cytometer was used for flow cytometry; peritoneal cells and splenocytes were extracted, blocked with anti-mouse CD16/32 (eBioscience), and stained with fluorochrome-conjugated Abs. Anti-mouse Abs used in this study included PerCP/Cy5.5 CD19 (115534; BioLegend), FITC CD3 (100204; BioLegend), allophycocyanin CD69 (104514; BioLegend), PE CD4 (BD 553730), PE/Cy7 CD138 (142513; BioLegend), PerCp/Cy5.5 CD11b (101227; BioLegend), allophycocyanin/Cy7 Ly6c (128025; BioLegend), and BV421 Ly6G (127627; BioLegend). The gating strategy for splenocytes is in Supplemental Fig. 1.

Using the RNeasy mini kit (Qiagen, Hilden, Germany) as directed by the manufacturer, total RNA was extracted, purified, and then reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Real-time PCR was performed using SYBR Green master mix on an ABI 7500 fast system (Applied Biosystems, Foster City, CA). The endogenous reference genes were GAPDH. The comparative ΔCt method was used to calculate the relative mRNA expression levels. Every experiment’s samples were conducted in technical triplicates, with each sample consisting of three reaction wells that produced an average threshold cycle value. Primer sequences are listed as follows: Isg15, forward 5′-GGT GTC CGT GAC TAA CTC CAT-3′, reverse 5′-CTG TAC CAC TAG CAT CAC TGT G-3′; Mx1, forward 5′-GAT CCG ACT TCA CTT CCA GAT GG-3′, reverse 5′-CAT CTC AGT GGT AGT CAA CCC-3′; Irf9, forward 5′-TGT CTG GAA GAC TCG CCT AC-3′, reverse 5′-GCA ACA TCC ATA CGA CCT CTC T-3′; Ifnb, forward 5′-CAG CTC CAA GAA AGG ACG AAC-3′, reverse 5′-GGC AGT GTA ACT CTT CTG CAT-3′; Irf7, forward 5′-TGC TGT TTG GAG ACT GGC TAT-3′, reverse 5′-TCC AAG CTC CCG GCT AAG T-3′; Oas1a, forward 5′-GCC TGA TCC CAG AAT CTA TGC-3′, reverse 5′-GAG CAA CTC TAG GGC GTA CTG-3′; Cxcl10, forward 5′-CCA AGT GCT GCC GTC ATT TTC-3′, reverse 5′-GGC TCG CAG GGA TGA TTT CAA-3′; Gapdh, forward 5′-AGG TCG GTG TGA ACG GAT TTG-3′, reverse 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′.

J774A.1 cells were purchased from ATCC (TIB-67) and cultured in DMEM supplemented with 10% heat-inactivated FBS, penicillin, and streptomycin (100 μg/ml) at 37°C in a 5% CO₂ humidified incubator. When cells were 80% confluent, cells were treated with the selective HDAC6 inhibitor ACY-738 (Adooq Bioscience, Irvine, CA) and stimulated with LPS (Sigma Aldrich) and IFN-γ (R&D Systems, Minneapolis, MN) for 24 h.

BCA protein assay kit (Thermo Fisher Scientific) was used to measure protein concentrations after total protein was extracted and lysed in radioimmunoprecipitation buffer containing 1% protease and phosphatase inhibitor mixture (Thermo Fisher Scientific). Equal amounts of protein in the lysates were mixed with SDS loading buffer and boiled for 5 min. The protein samples were separated by electrophoresis on 4–12% SDS-PAGE gel (Bio-Rad) and transferred to polyvinylidenedifluoride membranes. The membrane was then blocked with 0.1% Tween 20 in TBS containing 5% BSA. The membranes were then probed with primary and secondary Abs. The blot was scanned on an Odyssey Clx Imager (LI-COR Biosciences, Lincoln, NE). All Abs were purchased from Cell Signaling Technology (Danvers, MA). The primary Abs dilution ratio is 1:1000. The Western blot Abs and catalog number are as follows: NF-κB p65 (8242S), acetyl-NF-κB p65 (Lys310) (12629S), inducible NO synthase (iNOS; 13120S), Histone H3 (4499T), acetyl-Histone H3 (Lys9) (9649T), acetyl-α-Tubulin (Lys40) (5335T), α-Tubulin (3873T), phospho-Stat1 (Tyr701) (9167T), Stat1 (14994T), phospho-NF-κB p65 (Ser536) (3033T), anti-mouse IgG (H + L) (5257P), and anti-rabbit IgG (H + L) (5151P). Using ImageJ software and densitometric analysis, we quantified and normalized the amounts of proteins to housekeeping protein.

The one-way ANOVA for multiple comparisons or the Student t test for single comparisons were used to evaluate statistical differences between sample groups. Data are shown as mean ± SEM. Statistical significance was determined by p value (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

The splenomegaly is one of the indicators of inflammation. To determine the role of HDAC6 in splenomegaly, we compared HDAC6^−/− mice with WT mice 10 d after pristane injection. WT and HDAC6^−/− mice on the B6 background were administered 0.5 ml of pristane or PBS at 12 wk of age. After 10 d of pristane injection, the mice were euthanized, and we measured body weight (Fig. 1A), spleen weight (Fig. 1B), and spleen-to-body weight ratio (Fig. 1C). Although body weights were not significantly altered in any group, spleen weights were significantly increased in the pristane-treated HDAC6^−/− mice compared with the PBS-treated HDAC6^−/− mice. In addition, the spleen-to-body weight ratio was significantly increased in the pristane-treated HDAC6^−/− mice compared with the PBS-treated HDAC6^−/− mice. The results show that HDAC6 deletion does not affect splenomegaly induced by pristane.

The inflammation caused by pristane is defined by the ongoing recruitment of leukocytes, such as lymphocytes, macrophages, and neutrophils, to the peritoneal cavity (17, 18). To assess the impact of HDAC6 deletion on early inflammation in pristane-induced lupus, we compared peritoneal cells in WT and HDAC6^−/− mice 10 d after pristane administration. At sacrifice, cells were collected from the peritoneum and assessed by flow cytometry. Inflammatory monocytes were identified as CD11b⁺Ly6C⁺⁺ (Fig. 2A), and neutrophils were identified as CD11b⁺Ly6G⁺ (Fig. 2B). In both WT and HDAC6^−/− mice, pristane administration increased the number of inflammatory monocytes and neutrophils compared with PBS controls. In the pristane-treated HDAC6^−/− mice, there was significantly less recruitment of inflammatory monocytes (Fig. 2C) and neutrophils (Fig. 2D) compared with the pristane-treated WT mice. Taken together, these data demonstrated that HDAC6 deletion decreased the early inflammatory response to pristane.

The administration of pristane via peritoneal injection can elicit a localized inflammatory reaction and induce lipogranuloma (19). Pristane-induced inflammation is correlated with the continuous recruitment of leukocytes in the spleen, which are subsequently directed toward the site of inflammation (17). To examine the responses of splenic leukocytes to pristane, we compared immune cells in WT and HDAC6^−/− mice 10 d after pristane administration. Ten days after pristane administration, the spleens were removed and splenocytes were isolated. Flow cytometry was used to assess whole splenic cells. The percent of CD4⁺ T cells (Fig. 3B), CD8⁺ T cells (Fig. 3C), and CD19⁺ B cells (Fig. 3G) in the spleen was unchanged after pristane treatment. The percent of CD3⁺ T cells was decreased in pristane-treated HDAC6^−/− mice compared with PBS-treated HDAC6^−/− mice (Fig. 3A). The number of CD3⁺ T cells was reduced in PBS-treated HDAC6^−/− mice compared with PBS-treated WT mice (Fig. 3D). CD69 is an early activation marker in immune cells and is implicated in T cell differentiation. In T cells, the number of CD69⁺ cells was significantly increased in the pristane-treated HDAC6^−/− mice compared with the pristane-treated WT mice (Fig. 3E, 3F). In B cells, the number of CD69⁺ cells was increased by pristane treatment, and the number of CD69⁺ cells was significantly increased in the pristane-treated HDAC6^−/− mice compared with the pristane-treated WT mice (Fig. 3H). Furthermore, the percentages of CD19⁺CD138⁺ plasmablasts (Fig. 3I), as well as CD19⁻CD138⁺ plasma cells (Fig. 3J), were significantly increased in the pristane-treated HDAC6^−/− mice compared with PBS-treated HDAC6^−/− mice. CD69 can regulate regulatory T (Treg)/Th17 differentiation and is crucial for immunosuppression activity (20). In summary, these data indicate that HDAC6 KO mice are shielded from early inflammation induced by pristane injection.

Type I IFNs have been identified as crucial pathogenic cytokines in SLE and are closely associated with the severity of the disease (21). To determine the potential involvement of type I IFN in the initial inflammatory response in mice treated with pristane, we evaluated the expression of IFN-stimulated genes in the peritoneum of these mice. After 10 d of pristane treatment, a portion of the peritoneal cells was collected, and mRNA was isolated. We used real-time quantitative PCR to assess several IFN signature genes. We examined the expression of the IFN signature genes Ifnb (Fig. 4A), Mx1 (Fig. 4B), Oas1a (Fig. 4C), Irf7 (Fig. 4D), Irf9 (Fig. 4E), Cxcl10 (Fig. 4F), and Isg15 (Fig. 4G) and normalized the expression to the housekeeping gene GAPDH. Pristane treatment increased type I IFN signature genes except for IFN-β, which was decreased. Furthermore, there was no significant difference between pristane-treated HDAC6^−/− mice and pristane-treated WT mice. Taken together, these results indicate that HDAC6 deletion has little effect on pristane-induced IFN signature genes expression.

We have demonstrated that HDAC6 deletion inhibited the peritoneal recruitment of inflammatory monocytes after pristane treatment. Inflammatory monocytes possess a high degree of infiltration capability and can transform into inflammatory macrophages. To investigate further the role of HDAC6 inhibition on macrophage function, we used the J774A.1 cell line. Initially, experiments were conducted to determine the proper concertation of the selective HDAC6 inhibitor ACY-738 that would increase α-tubulin acetylation without increasing histone acetylation. We found that with 1 μM ACY-738 treatment, α-tubulin was acetylated, but not histone H3 (Fig. 5A). When J774A.1 cells were stimulated with a mixture of LPS/IFN-γ, we found that 1 μM concertation of ACY-738 increased NF-κB acetylation and decreased total NF-κB protein level without affecting NF-κB phosphorylation (Fig. 5B). Surprisingly, when macrophages were treated with ACY-738 and stimulated with LPS/IFN-γ, we measured an increase in iNOS expression (Fig. 5B). Furthermore, STAT1 phosphorylation was significantly increased by ACY-738 with LPS/IFN-γ stimulation (Fig. 5C). These results suggest that HDAC6 inhibitor ACY-738 downregulates total NF-κB protein level while upregulating iNOS expression via the STAT1 pathway.

HDAC6 dysregulation plays a significant role in the development and progression of various human diseases (22, 23). HDAC6 is an enzyme responsible for removing acetyl groups from various cytosolic proteins and transcription factors that influence cell signaling and gene expression (24, 25). Dysregulation of HDAC6 has been linked to several pathological conditions, including cancer, neurodegenerative disorders, autoimmune diseases, and cardiovascular ailments. In cancer, overexpression of HDAC6 can promote uncontrolled cell growth and inhibit cell death, contributing to tumorigenesis and tumor progression (26–28). In neurodegenerative diseases such as Alzheimer’s and Parkinson’s, altered HDAC6 activity has been associated with abnormal protein aggregates and impaired cellular clearance mechanisms, contributing to neuronal damage and degeneration (29–34). In inflammation, and in particular SLE, we have previously reported that selective HDAC6 inhibition can decrease disease in lupus mice (6, 35–37). The pristane-induced lupus model is one frequently used animal model for lupus research. The B6 pristane-induced lupus mouse model does not develop lupus-like disease until several months after the initial injection (13). However, these mice often develop severe alveolar hemorrhage that may result in up to 20% mortality by 2 wk after injection (15). In our present studies, we sought to determine whether HDAC6 deletion would decrease early inflammation in this lupus mouse model.

We found that gene deletion of HDAC6 significantly decreased inflammatory monocytes and neutrophils accumulated in the peritoneum 10 d after pristane injection compared with WT mice. This was particularly interesting because HDAC6 inhibition has been shown to decrease neutrophil activation in Pseudomonas aeruginosa–induced inflammation and infection in cystic fibrosis (38). Moreover, studies by Yan et al. (39) found differing effects of HADC6 gene deletion in the LPS-induced model of acute peritonitis. They found that neutrophil activation was largely unaffected and macrophage activation was enhanced in the WT animals, whereas it was blunted in the HDAC6^−/− animals. This apparent dichotomy could be because of the different mechanisms by which the immune system is activated.

In contrast, the intracellular mechanisms of pristane-induced inflammation are complex and involve the activation of immune cells and various signaling pathways. Although the exact mechanisms may vary depending on the specific context and cell types involved, pristane is believed to act through both pattern recognition receptors, such as TLRs, and NOD-like receptors (NLRs) on the surface or within the cytoplasm of immune cells (40). TLR activation, in particular, can trigger intracellular signaling cascades, including NF-κB and MAPK pathways, which are central to the regulation of inflammatory gene expression (41–46).

In addition, pristane-induced inflammation has been associated with the activation of inflammasomes and multiprotein complexes that regulate the processing and release of proinflammatory cytokines, especially IL-1β (47). Furthermore, the NLRP3 (NOD-like receptor family, pyrin domain-containing inflammasome) has been implicated in pristane-induced inflammation (48). Through these mechanisms, pristane has been linked to the development of autoimmune diseases and can lead to the production of autoantibodies (49).

In regard to T and B cell activation, pristane has been shown to activate B cells and promote the production of Abs. It induces the formation of long-lived Ab-secreting cells in the peritoneal cavity of mice. We found that there was an increase in B cell activation with pristane treatment, and the increase in B cell activation was greater in the HDAC6^−/− animals compared with the WT mice. Furthermore, there was an increase in the plasma cells in the pristane-treated HDAC6^−/− mice compared with the pristane-treated WT mice. Similarly, pristane has been reported to activate certain subsets of T cells (50). It can influence the differentiation and activation of CD4⁺ Th cells (51). We found increased activation markers of T cells, along with increased B cell differentiation to plasma cells in HDAC6^−/− mice treated with pristane. It has been reported that pristane increases autoantibody production and nephritis through TLR7 or IFNR-mediated type I IFN production (52).

CD69 is a classic early marker of lymphocyte activation because of its rapid expression on the plasma membrane after stimulation (53). Apart from its inherent significance as an activation marker, contemporary data indicate that CD69 plays a crucial role in regulating immunological responses. In particular, CD69 controls the secretion of IL-10, IL-17, IL-22, and IFN-γ, as well as the differentiation of Treg cells (53). CD69 KO mice show enhanced Th17 proinflammatory responses (54). Yu et al. (55) found that CD69 stimulates the production of IL-10, which is crucial for the suppressive role of Tregs. Specifically, they found the proliferation of CD4⁺ T cells is considerably inhibited by CD69⁺ Tregs from IL10^+/+ mice as opposed to IL10^−/− mice. They also found the development of inflammatory bowel disease in mice was significantly prevented by the adoptive transfer of CD69⁺ Tregs, but not CD69⁻ Tregs or CD69⁺ Tregs deficient in IL-10. In the mouse model of collagen-induced arthritis, the lack of CD69 intensifies the inflammatory and destructive process in the afflicted joints (56). CD69 functions as a suppressor of autoimmune reactivity and inflammation by controlling the production of TGF-β, a cytokine that subsequently reduces the production of other proinflammatory substances (56). In a cardiomyopathy model, the absence of CD69 amplifies the inflammatory process and exacerbates Th17-mediated cardiac tissue damage, which promotes heart failure (57). According to their research, CD69 negatively controls cardiac inflammation, heart-specific Th17 responses, and the progression of heart failure in experimental autoimmune myocarditis (57). Another group studying myocardial infarction found that Cd69^−/− mice exhibited robust IL-17⁺ γδ T cell responses after ischemia, which exacerbated myocardial inflammation and, as a result, impaired cardiac function (58). They also found the recruitment of IL-17⁺ γδ T cells was reduced by the adoptive transfer of CD69⁺ Tregs into Cd69^−/− mice after left anterior descending ligation, thereby increasing survival (58). The transplantation of CD4⁺ T cells from CD69 KO mice into Rag-1^−/− immunodeficient animals results in an accelerated form of colitis, characterized by an increased production of IL-17, IFN-γ, and TNF-α (59). The absence of CD69 resulted in heightened synthesis of proinflammatory cytokines, diminished induction of Foxp3⁺ Treg cells, compromised development of oral tolerance, and exacerbated colitis (59). CD69-deficient animals have been seen to exhibit heightened tissue inflammation in the initial phase of Listeria monocytogenes infection (60). In (NZB × NZW)F1 mice, a model of SLE, the number of CD4⁺CD69⁺ T cells increases, which leads to a suppressive effect in vitro by inhibiting the synthesis of IL-2 (61). In summary, in addition to serving as an activation marker, CD69 also participates in regulating Treg/Th17 differentiation and is crucial for immunosuppression (20, 62). Our result shows that the number of CD69⁺ T and B cells was increased in HDAC6^−/− mice after pristane treatment, which indicates that HDAC6 deletion protects mice from early inflammation.

Inflammatory monocytes exhibit a specific pattern of movement toward areas of inflammation, where they generate inflammatory cytokines and contribute to both local and systemic inflammation (63). Inflammatory monocytes can transform into inflammatory macrophages. Based on the result that HDAC6 deletion inhibited the peritoneal recruitment of inflammatory monocytes, we conducted in vitro studies to further characterize the mechanism of activation of the monocytes. J774A.1 is a mouse monocyte/macrophage cell line and can be used in immunology research. We found that HDAC6 inhibitor ACY-738 inhibited the NF-κB signaling in stimulated J774A.1 cells. NF-κB triggers the expression of many genes that promote inflammation. Our results of in vitro experiments provide more evidence that HDAC6 promotes inflammation. Surprisingly, we found HDAC6 inhibition did not suppress iNOS expression in LPS/IFN-γ–stimulated J774A.1 cells. IFN-γ interacted with LPS synergistically to induce iNOS expression; we also found that neither LPS nor IFN-γ alone was sufficient to induce iNOS expression in J774A.1 cells (Supplemental Fig. 2). Wang et al. (64) have previously reported that macrophages from HDAC6^−/− mice have decreased iNOS production with LPS stimulation. Because LPS acts predominantly through NF-κB to induce iNOS, the IFN-γ pathway acting through STAT1 to induce iNOS expression can act independently from NF-κB. IFN-γ can potentiate proinflammatory signaling by priming macrophages for antimicrobial actions because it induces NO production and inhibits NLRP3 inflammasome activation. Furthermore, the contribution by p38 to the induction of iNOS and apoptosis is independent of NF-κB nuclear translocation (65). Discrepancies in our results from Wang and coworkers (64) could be because of many factors, including using a different inhibitor, different cell type, different concentration, and not pretreating the cells before stimulation. In our studies, we added ACY-738 concurrent with LPS/IFN-γ for 24 h. In their studies, they pretreated the cells for 3 h before 12 h of LPS stimulation.

In summary, we discovered that in the early inflammatory response to pristane, HDAC6 deletion inhibited the recruitment of inflammatory monocytes and neutrophils in the peritoneum. HDAC6 deletion promoted the number of CD69⁺ T and B cells, and CD69 was reported to function as a suppressor of inflammation. The HDAC6 inhibitor was found to increase NF-κB acetylation and decrease total NF-κB protein level in in vitro investigations using J774A.1 cells.

The authors have no financial conflicts of interest.

We appreciate Melissa Makris for allowing use of Virginia Tech’s flow cytometry core laboratory.