Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, diminishes lymphoproliferation in the Fas -deficient MRL/lpr(-/-) murine model of autoimmune lymphoproliferative syndrome (ALPS)

Abstract

Objective: Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of apoptosis, often presenting in childhood. Similarly, MRL/lpr(-/-) mice homozygous for Fas mutations develop an ALPS-like disease with autoimmunity, lymphadenopathy, splenomegaly, and expansion of double-negative T cells. Currently, there are no proven therapies with adequate safety margins for sustained abolition of the lymphoproliferation associated with ALPS. We sought to test the ability of valproic acid (VPA), a histone deacetylase inhibitor, to induce apoptosis and inhibit lymphoproliferation.

Materials and methods: Human peripheral blood mononuclear cells from patients with ALPS and normal controls were tested in vitro to determine the efficacy of VPA at inducing cell death. VPA was used in vivo to control lymphoproliferation in MRL/lpr(-/-) mice, a model for ALPS.

Results: VPA induced cell death in vitro, and was partially inhibited by the pan caspase inhibitor, Z-VAD-FMK. MRL/lpr(-/-) mice treated with VPA for 8 weeks showed significant reductions in spleen and lymph node weights and cellularity compared to controls. A concomitant decrease in double-negative T cells was observed in the spleen, lymph nodes, and peripheral blood. Serum levels of VPA peaked 1 hour after injection, and a 2.5-fold increase in histone acetylation was observed in the spleen at 4 hours after injection.

Conclusion: Based on our data, VPA is effective at reducing lymphoproliferation in mice, and is currently being studied in a clinical trial as a lympholytic agent in patients with ALPS.

Figure 1. Valproic acid induces cell death in vitro

PBMCs were incubated with valproic acid (VPA) for 48 hours in the presence or absence of the caspase inhibitor, Z-VAD-FMK (50uM). A. Normal control (N=2). B. Patient with type 1a ALPS (N=2).

Figure 2. Lymphoid organ weights and cellularity are reduced with valproic acid or romidepsin

(A). Thirty mice in each group received 500 mg/kg valproic acid (VPA) or vehicle only (control) intraperitoneally 5 times per week for 8 weeks. Lymph node (LN) weight was the sum of inguinal, axillary, and brachial lymph nodes; lymph node cellularity was based on inguinal nodes only. (B) Eight mice in each group received three doses of 2 mg/kg romidepsin or vehicle i.v. Spleen weight, and spleen, lymph node, and bone marrow cellularity were determined.

Figure 3. Double-negative T cells are reduced with valproic acid treatment

Absolute cell numbers in the (A) spleen and (A) inguinal lymph nodes were determined based on cell number per organ and percentages based on flow cytometry. The absolute lymphocyte types in the (C) blood was based on flow cytometry percentage and peripheral blood lymphocyte absolute numbers. Flow cytometry analysis of cells stained with antibodies to CD3, CD4, CD8 and CD45R (B220). N=30/group for spleen and lymph node values and 17–19/group for blood.

Figure 4. Differential blood counts of mice treated with valproic acid

Mice received 500 mg/kg valproic acid (VPA) or vehicle only (control) intraperitoneally 5 times per week for 8 weeks. N=17–19/group.

Figure 5. Serum levels peak at one hour after valproic acid treatment

Eight to 10 mice in each group received a single injection of 500 mg/kg valproic acid (VPA) intraperitoneally and were sacrificed at given time points post injection. PBS shows mice that received phosphate buffered saline only. Serum valproic acid levels were determined.

Figure 6. Histone acetylation peaks at four hours after valproic acid treatment

Two mice for each time point received a single injection of 500 mg/kg valproic acid (VPA) and were sacrificed at given times after injection. Control shows mice that received vehicle only (PBS). Splenocytes were used to determine acetylation of histone 3 by dot blot assay. Acetylated histone 3 (ACh3)/GAPDH ratios are plotted against time.