Melanocortin Receptor-Mediated Anti-Inflammatory Effect of Acthar® Gel in Human Myeloid Cells

Abstract

Introduction

Acthar® Gel, a complex mixture of porcine adrenocorticotropic hormone analogs that activates all 5 melanocortin receptor (MCR) subtypes, is an approved noncorticosteroid treatment for multiple sclerosis (MS) exacerbations.

Methods

MCR expression and anti-inflammatory effects of Acthar Gel in human monocyte-derived macrophages (MDMs) and human brain-derived microglia were investigated following lipopolysaccharide stimulation in vitro.

Results

MC1R was expressed at substantially higher levels than the other MCR subtypes in human MDMs and was the only MCR gene detected in human adult microglia. As shown by microarray gene expression analysis, polarization of MDMs to a pro-inflammatory phenotype increased the expression and secretion of interleukin-6, tumor necrosis factor α, and CXC motif chemokine ligand 10, which were inhibited in a dose-dependent manner with Acthar Gel treatment.

Conclusions

These results are consistent with previous insights that Acthar Gel has an immunomodulatory mechanism distinct from glucocorticoids alone and suggest that Acthar Gel can improve clinical outcomes in MS and other inflammation-mediated central nervous system disorders by inhibiting multiple pro-inflammatory cytokine signaling pathways.

Introduction

High-dose corticosteroids are standard-of-care treatment for many autoimmune disorders, including acute exacerbations in multiple sclerosis (MS), which are caused by inflammation in the central nervous system (CNS). However, corticosteroids are also associated with substantial short- and long-term adverse effects (e.g., weight gain, hypertension, depression) [1, 2]. Acthar^® Gel, a complex mixture of adrenocorticotropic hormone analogs and other components derived from porcine pituitary extract, is 1 of only 2 approved noncorticosteroid therapies for acute exacerbations of MS in adults in the USA and has been shown to reduce the time to resolution of MS exacerbations compared with placebo [3–7].

The immunomodulatory effects of Acthar Gel are distinct from those of glucocorticoids [8–11], which are natural immunomodulators [12]. Acthar Gel activates all 5 melanocortin receptors (MCRs) (MC1R to MC5R), of which MC1R, MC3R, and MC5R are expressed in inflammatory cells such as lymphocytes and monocytes/macrophages [6, 13]. Signaling through these receptors leads to a range of different downstream immunomodulatory effects, including inhibition of B-cell proliferation and immunoglobulin G production in vitro and reduction of active helper and cytotoxic T cells in mice [14, 15].

MCRs also mediate anti-inflammatory effects and have been implicated in the modulation of pro-inflammatory cytokines [16]. In inflammatory cells within the CNS, MCRs play a key role in MS by protecting oligodendroglia (myelin-producing cells) from cytotoxins that promote the pathogenesis of MS lesions [13]. In MS, infiltrating macrophages (monocyte-derived macrophages [MDMs]) and resident macrophages (microglia) both play important roles in neuroinflammation and the production of pro-inflammatory cytokines and chemokines, such as interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and CXC motif chemokine ligand 10 (CXCL10), which promote demyelination and development of new CNS lesions [17]. In this brief report, we investigate the anti-inflammatory effects of Acthar Gel on MCRs and pro-inflammatory cytokine production in human myeloid cells following lipopolysaccharide (LPS) stimulation in vitro.

Methods

Human Myeloid Cell Isolation and Culture

Monocyte-Derived Macrophages

Peripheral blood mononuclear cells were kindly provided by Dr. Jack Antel (McGill University, Montreal, Canada). These cells were isolated from whole blood samples using Ficoll-Paque PLUS medium (GE Healthcare) and density gradient centrifugation as described previously [18]. CD14+ cells were isolated and purified from 95% to 99% purity using human CD14 MicroBeads (Miltenyi Biotec) according to the manufacturer’s instructions. Monocytes were cultured (5 × 10⁵ cells/mL, 37°C, 5% CO₂) in RPMI media supplemented with 10% fetal bovine serum, 0.1% penicillin/streptomycin, and 0.1% glutamine. MDMs were differentiated in media supplemented with 25 ng/mL MDM colony-stimulating factor for 7 days. Methods for cell treatments and assessments were previously reported [19].

Human Adult Microglia

Human adult microglia, also kindly provided by Dr. Jack Antel (McGill University, Montreal, Canada), were isolated from brain tissue that was surgically resected from patients with pharmacologically intractable, nonmalignant temporal lobe epilepsy as described previously [20, 21]. Briefly, brain tissue was mechanically dissociated and digested with trypsin and DNase I prior to filtration through nylon mesh. Subsequently, tissues underwent density gradient ultracentrifugation to remove myelin. Dissociated cells were plated at 2 × 10⁶ cells/mL in MEM media supplemented with 5% fetal bovine serum, 0.1% penicillin/streptomycin, and 0.1% glutamine. This protocol has been reported to yield ≥90% microglia purity via CD11c staining by flow cytometry [21]. Microglia were cultured (37°C, 5% CO₂) for 3 days, and then collected and plated at 1 × 10⁵ cells/mL and maintained in culture for 7 days, during which cells were polarized.

Polarization

MDMs and microglia were polarized to an M1 (pro-inflammatory) phenotype using 100 ng/mL LPS (serotype 0127:B8) and 20 ng/mL interferon γ as described previously [20]. This stimulates cell signaling cascades to increase the transcription and secretion of pro-inflammatory cytokines and chemokines such as IL-6, TNFα, and CXCL10 [17, 22].

Treatments

Cells (5 × 10⁵/well) were incubated with Acthar Gel at 4 dilutions (1:11 [1.82 IU/mL], 1:22 [0.91 IU/mL], 1:55 [0.36 IU/mL], or 1:110 [0.18 IU/mL]) for 3 h, followed by treatment with 100 ng/mL LPS for at least 24 h. The control gel (Mallinckrodt) dilutions were identical to Acthar Gel but did not contain any active pharmaceutical ingredient. For MC1R expression experiments, cells were also treated with 1 µm dexamethasone. Unless otherwise specified, data were gathered from 6 independent experiments, from 6 separate donors.

Gene Expression

Gene expression of polarized cells was measured using GeneChip Human Gene 2.0 ST microarray (Affymetrix). Raw intensity data were normalized, averaged, transformed to a log₂ scale, and analyzed using Affymetrix Expression Console software.

RNA Extraction and Quantitative Polymerase Chain Reaction

Cells were lysed in TRIzol, total RNA was extracted using Qiagen RNeasy kits with MinElute spin columns, and complementary DNA was generated with reverse transcriptase (Thermo Fisher Canada). Subsequently, quantitative polymerase chain reaction of IL-6, TNFα, and CXCL10 was performed using TaqMan reagents and a Thermo Fisher thermocycler. GADPH was used as the reference gene for normalizing expression data. All methods were performed according to the respective manufacturer’s protocols.

Enzyme-Linked Immunosorbent Assay

Cell culture supernatants were assayed for IL-6, TNFα, and CXCL10 using enzyme-linked immunosorbent assay kits (BD Biosciences Canada) according to the manufacturer’s protocol.

Statistical Analyses

One-way ANOVA, with Dunnett’s post hoc correction for multiple comparisons when appropriate, was performed using GraphPad Prism (v6.07). p < 0.05 were considered statistically significant.

Results

Expression of MCR Genes

In human MDMs, the gene expression of MC1R was significantly higher than the expression of MC2R, MC3R, MC4R, or MC5R (n = 6; p < 0.0001; Fig. 1a). MC1R mRNA expression was not affected by the polarization conditions (data not shown). In human adult microglia, MC1R was the only MCR expressed (Fig. 1b). LPS with or without dexamethasone led to modest reductions in MC1R gene expression in MDMs (Fig. 1c) and adult microglia (Fig. 1d) relative to untreated cells, but the reductions were not statistically significant. Furthermore, treatment of LPS-polarized cells with Acthar Gel did not have any significant effects on MC1R mRNA.

Fig. 1.

Expression of MCR genes in human MDMs and microglia. The expression of the 5 MCR genes was determined in MDMs (a) and microglia (b) by microarray analysis. In a, **** indicates p < 0.0001 for MC1R vs. all other receptor genes (MC2R, MC3R, MC4R, and MC5R) using one-way ANOVA (n = 6). In b, the expression of MC2R, MC3R, MC4R, and MC5R was below the limit of detection. In addition, the mean (SE) fold change in expression of MC1R was measured by quantitative PCR in MDMs (c) and microglia (d) after treatment with LPS, LPS + Dex, control gel, or Acthar Gel, relative to untreated cells. ANOVA, analysis of variance; Dex, dexamethasone; FPKM, fragments per kilobase of transcript per million fragments mapped; LPS, lipopolysaccharide; MCR, melanocortin receptor; MDM, monocyte-derived macrophage; PCR, polymerase chain reaction; SE, standard error.

Expression and Secretion of Pro-Inflammatory Cytokines in MDMs and Microglia

LPS activation of human MDMs increased the mRNA expression of M1 pro-inflammatory cytokines and chemokines IL-6 (Fig. 2a), TNFα (Fig. 2b), and CXCL10 (Fig. 2c). Subsequent treatment with Acthar Gel significantly reduced the mRNA expression (Fig. 2a–c) and secretion of these cytokines relative to LPS alone in a dose-dependent manner (Fig. 2d–f, respectively; n = 6) in human MDMs. However, in human adult microglia, Acthar Gel did not reduce mRNA or protein expression of these LPS-stimulated pro-inflammatory molecules (data not shown).

Fig. 2.

Effect of LPS and Acthar Gel on the production of pro-inflammatory cytokines in human MDMs. Mean (SD) fold change in mRNA expression and protein secretion of IL-6 (a, d), TNFα (b, e), and CXCL10 (c, f) in MDM cells treated with LPS and Acthar Gel (black) or control gel (dark gray), relative to unstimulated cells. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs. treatment with LPS alone using one-way ANOVA (n = 6). ANOVA, analysis of variance; CXCL10, CXC motif chemokine ligand 10; IL-6, interleukin-6; LPS, lipopolysaccharide; MDM, monocyte-derived macrophage; SD, standard deviation; TNFα, tumor necrosis factor α.

Discussion

Our results in MDMs are in agreement with previous studies showing that MC1R is expressed in monocytes, macrophages, and microglia, and that endogenous melanocortins, as well as adrenocorticotropic hormone (1–24), inhibit the production of pro-inflammatory cytokines in murine microglial cell lines [6, 13, 23]. It is not clear why the control gel significantly inhibited the mRNA expression of IL-6, TNFα, and CXCL10 at low dilutions (1:11 to 1:22) in MDMs since the control gel does not contain any biologically active peptides, but it is possible that the gel reduces cell viability. Nevertheless, these effects were not observed at high dilutions (1:55 to 1:110) in contrast with Acthar Gel, which contained active components and showed significant inhibitory effects at all dilutions (1:11 to 1:110) in MDMs. Although Acthar Gel significantly reduced LPS-stimulated pro-inflammatory cytokines in MDMs (infiltrating macrophages), we did not observe this immunomodulatory effect in human adult microglia (resident macrophages), where MC1R was the only MCR detected. Reasons for these differences are unclear, but this could suggest that MCRs other than MC1R may play a more prominent role in modulating the inflammatory response of Acthar Gel in these cell types.

On the basis of our observations and the plasticity of MDMs [17], we hypothesize that Acthar Gel acts primarily as an immunomodulator of IL-6, TNFα, and CXCL10 signaling pathways and ameliorates inflammatory CNS disorders, such as MS and infantile spasms (a form of epilepsy that occurs in infancy), via a mechanism that is distinct from that of corticosteroids. In MS, Acthar Gel may facilitate resolution of exacerbations, particularly in patients who have intolerance or inadequate response to high-dose corticosteroids, by repolarizing MDMs away from the inflammatory M1 phenotype to reduce immune activity in lesions [3–5, 24]. This mechanism of action is consistent with the established role of MCRs in modulating macrophage function in inflammatory processes [25]. In addition, blockade of IL-6, TNF receptor 1, and CXCL10 signaling has shown therapeutic potential in murine models of MS and other demyelinating diseases [26–28]. Similarly, pro-inflammatory cytokines including IL-6 and TNFα are implicated in the generation and exacerbation of seizures such as infantile spasms [29]. It remains unclear whether the ability of Acthar Gel to suppress production of these cytokines may explain the differences in treatment response to Acthar Gel vs. corticosteroids in randomized controlled trials for treatment of infantile spasms [30, 31]. These hypotheses, while intriguing, are limited to the in vitro conditions of our experimental paradigm. Further in vitro, in vivo, and clinical studies are needed to elucidate the immunomodulatory mechanisms of Acthar Gel in each of these therapeutic areas.

This brief report has several limitations and future directions that should be considered. First, the human brain-derived microglia culture was established using a protocol that has been reported to yield ≥90% microglia purity [21]; however, the purity of the microglia used herein was not directly confirmed. Therefore, we cannot rule out the possibility of contamination of other cell types (e.g., fibroblasts) affecting MCRs in the microglia culture. Additional studies are needed to clarify differences in Acthar Gel immunomodulation in MDMs (infiltrating macrophages) and microglia (resident macrophages). Second, the use of parametric statistical tests (e.g., one-way ANOVA) that assume normal distribution may not be appropriate for all datasets, especially fold-change values or percentages. Further testing with more robust statistical analyses is needed to confirm these findings. Finally, the precise role of MC1R or other MCR subtypes in the anti-inflammatory effects of Acthar Gel must be further clarified via additional experiments utilizing blockage of 1 or more MCRs.

Acknowledgments

Research support was provided by Dr. Jack Antel of McGill University (Montreal, Canada). Medical writing support was provided by Alexander Simon from Citrus Health Group, Inc. (Chicago, IL, USA), in accordance with Good Publication Practice 2022 guidelines.

Statement of Ethics

For isolation of peripheral blood mononuclear cells, Institutional Review Board (IRB) approval for peripheral blood collection from healthy human donors was obtained from the Montreal Neurological Institute and Hospital ethics review board (IRB No. ANTJ2001/1). Anonymized resected brain tissue, from which human adult microglia were isolated, was obtained from the Montreal Neurological Institute and Hospital Department of Pathology, with ethics approval granted under the same IRB No. ANTJ2001/1, which did not require individual patient consent.

Conflict of Interest Statement

Kyle Hayes is an employee of Mallinckrodt Pharmaceuticals. Dale Wright is a former employee of Mallinckrodt Pharmaceuticals.

Funding Sources

This study, including research and medical writing support, was funded by Mallinckrodt Pharmaceuticals (Bridgewater, NJ, USA).

Author Contributions

Kyle Hayes and Dale Wright were involved in the design of the study; collection, analysis, and interpretation of the data; and writing and reviewing the manuscript.

Funding Statement

This study, including research and medical writing support, was funded by Mallinckrodt Pharmaceuticals (Bridgewater, NJ, USA).

Data Availability Statement

The data that support the findings of this study are not available to the general public due to privacy reasons but are available to qualified researchers upon reasonable request to the corresponding author.