The antioxidant MnTBAP does not effectively downregulate CD4 expression in T cells in vivo

Abstract

The antioxidant MnTBAP was previously shown to down-regulate the surface expression of CD4 molecule in T cells. This observation obviously holds great potential impact in a number of pathological human conditions, including autoimmunity. Three different single doses of MnTBAP reduced the frequency of CD4^high cells. However, the median florescent intensity (MFI) was not different. Initiation of in vivo pharmacotherapy or vehicle control was performed inC57BL/6 mice that were actively immunized for experimental autoimmune encephalomyelitis (EAE). In contrast to published reports, the mean frequency of CD4^high cells, and the median fluorescent intensity (MFI) of CD4 was similar in both treatment groups. 25-day survival following active immunization among the MnTBAP treated animals compared to vehicle controls was16.6 ± 6.9 days vs 23.6 ± 2.7 days; (P value <0.05). We conclude that MnTBAP (Sack and Herzog, 2009 (Sack and Herzog, 2009)) does not effectively downregulate CD4 expression in T cells in vivo, probably due to extensive mechanism that distinguishes it from an in vitro model (Harding, 1993 (Harding, 1993)) possesses toxic properties that may limit its clinic use in possible doses that could deliver the immunomodulation through down regulation of CD4 expression, and (Saizawa et al., 1987 (Saizawa et al., 1987)) has limited availability in specific tissues, including the CNS.

Vector-assisted gene transplant holds great potential for targeted gene therapy. The ensuing adaptive immune response to viral vectors or the transgene products by the recipient has remained a major obstacle to their widespread use. Even with optimized dosing and tissue specificity, the incomplete transfection efficacy necessitates repeated dosing. Consequently, immune mediated neutralization and clearing of vector particles increases with each successive dose (Sack and Herzog, 2009). Intuitively, immunomodulation to diminish these adaptive immune responses presents a plausible biological and pharmacological solution to this problem.

CD4⁺ T helper cells are central mediators of adaptive immune responses against viral epitopes and transgene-encoded determinants. They recognize immunogenic peptide sequences in the context of major histocompatibility complex class II (MHC II), become activated, and clonally expand (Harding, 1993). In addition, they mediate antibody class switching and affinity maturation in antigen-specific B cells, culminating in a robust immunological memory. Expression of CD4 molecule, a co-receptor of the T cell receptor (TCR), on these cells is required for recognition of cognate antigens presented by B cells and myeloid antigen presenting cells (Saizawa et al., 1987).

A recent article by Da Rocha and colleagues demonstrated the down-regulatory effects of the cell-permeable synthetic antioxidant with superoxide dismutase-like activity Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) on the surface expression of CD4 molecule in T cells (Da Rocha et al., 2019). The authors’ observation obviously holds great potential impact in a number of pathological human conditions, including vector-assisted gene therapy, transplant medicine, and autoimmunity.

Confirming some of the observations made by Da Rocha et al., we found that in vitro exposure of CD4⁺ T lymphocytes to three different single doses (100 μM, 200 μM, and 400 μM) of MnTBAP reduced the frequency of CD4^high cells (Fig. 1). This reduction was statistically significant compared to untreated samples (P value <0.0001) (Fig. 2). However, the median florescent intensity (MFI) levels were not different between treatment groups (Fig. 2). To explore potential effects of MnTBAP on CD4 cell surface expression in vivo, we employed a CD4⁺ T cell-mediated model of central nervous system (CNS) autoimmunity, experimental autoimmune encephalomyelitis (EAE) (Zamvil and Steinman, 1990). We hypothesized that MnTBAP treatment would result in attenuated CD4⁺ T cell recognition of, and proliferation to their cognate myelin antigen. C57BL/6 mice (H-2b) (4 to 6 animals per treatment group in 3 study cohorts) were started on daily intraperitoneal MnTBAP (80 mg/kg from a 20 mM stock solution of MnTBAP in 75 mM NaOH diluted with PBS as described by Da Rocha et al), or vehicle control (equal volume NaOH solution diluted with phosphate buffered saline (PBS)). Other controls included equal volume of intraperitoneal PBS injections or “no-intervention”. The experimental animals were treated for 5 days following active immunization of all experimental animals with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG_p35–55) in Complete Freund’s Adjuvant (CFA), as previously described (Hussain et al., 2018). In contrast to the published findings by Da Rocha et al., the mean frequency of CD4^high cells (2.4 ± 1.9 vs. 1.6 ± 0.8), and the median fluorescent intensity (MFI) of CD4 was similar in MnTBAP and vehicle treatment groups (15.4 ± 3.2 vs 15.9 ± 4.3 in MnTBAP vs controls, respectively; P value = 0.8) (Fig. 2). The mean ± SE of EAE severity scores was higher among the “no intervention” controls, but the results were not significantly different from vehicle-treated or PBS-treated animals (Fig. 3A). Furthermore, MnTBAP treatment with the above-mentioned doses proved to be toxic, observable as rapid weight loss and significantly lower 25-day survival following active immunization among the MnTBAP treated animals compared to vehicle controls (16.6 ± 6.9 days vs 23.6 ± 2.7 days; P value <0.05). Fig. 3B shows the probability of survival among the animals in each experimental group. The high mortality rate among the MnTBAP-treated animals did not allow for a meaningful analysis of disease severity in comparison to controls. We also assessed extended low dose MnTBAP injections (50% of daily dose for 10 days) to circumvent this issue. However, animal mortality prior to onset of clinical EAE (day 12–14 post immunization) remained the same (data not shown). Of note was the lack of clinical EAE among the MnTBAP-treated mice that survived in all study cohorts. This observation would have substantiated the results reported by Da Rocha et al. However, compared to “no intervention” controls, the overall EAE severity was reduced in vehicle-treated or PBS-injected controls as well. This prompted the conclusion that the observed alleviation of symptoms or lack of disease incidence in study animals that received multiple injections could be induced by the stressful setting of the study. Specifically, intraperitoneal injection of MnTBAP solution or the vehicle (pH level > 10 in both injection solutions with the preparation method described by Da Rocha et al) was noticeably irritating for the animals, and elicited prolonged agitation following each injection.

Fig. 1.

Multiparameter flow cytometry of samples following in vitro and in vivo exposure to MnTBAP. (A-D) Singlet events were gated for viable CD45⁺ CD3⁺ CD4⁺ cells. (D) A higher frequency of untreated cells expressed high levels of surface CD4 (CD4^high) in vitro compared to (E-G) cells treated with three different single doses (100 μM, 200 μM, and 400 μM) of MnTBAP. (H and I) There was no significant difference between treated and untreated cells after in vivo exposure to MnTBAP regarding the expression of CD4. A representative experiment is shown.

Fig. 2.

(A) The frequency of CD4^high T cells in vitro was significantly lower with three different single doses (100 μM, 200 μM, and 400 μM) of MnTBAP than in the no treatment control group (P value<0.0001, Student t-test). (B) However, there was no significant difference regarding the MFI of CD4 (P value>0.05, Mann Whitney U test). (C & D) There were no differences between the treatment groups regarding the frequency of CD4⁺ T cells and the mean fluorescent intensity (MFI) of CD4 following in vivo experiments.

Fig. 3.

(A) MnTBAP ameliorates active experimental autoimmune encephalomyelitis (EAE), compared to vehicle control, PBS control, and mice that were not subjected to any intervention. The mean ± SE of EAE scores were significantly different between controls however; the groups that received multiple injections had a less severe disease course and lower incidence of clinical EAE. MnTBAP-treated mice that survived the treatment never developed clinical EAE. (N = 5 experimental animals per group). (B) MnTBAB increases mortality in experimental animals. The probability of 25-day survival among animals in each study group is presented. MnTBAP-treated mice had significantly lower survival rates (P value <0.005); (N = 15; data show pooled analysis of three separate cohorts).

In solution, MnTBAP has a dark blue-green appearance. Post-mortem tissue assessment revealed extensive infiltration of MnTBAP throughout internal organs evident by discoloration of oral mucosa, skin and subcutaneous tissues, intestines, liver, kidneys, lymph nodes, and spleen. In contrast, cardiac muscle, lungs, and the CNS were not discolored (Fig. 4A–C). This finding was indicative of a persistent presence of MnTBAP inside select tissues without complete clearance of the agent, which may have triggered the high mortality rates in recipient animals. Furthermore, this observation suggests limited CNS bioavailability of MnTBAP.

Fig. 4.

Gross anatomy of post-mortem tissues showed blue-green discoloration (A) of serous membranes in the peritoneum and subcutaneous tissue, spleen, lymph nodes and lymphatic vessels (red arrows) (B), the gall bladder and biliary vessels (red arrows), and (C) retroperitoneal serous membranes in MnTBAP-treated animals. Representative photographs of 15 experimental animals that underwent post-mortem assessments are shown.

Given the above results, we conclude that MnTBAP (Sack and Herzog, 2009) does not effectively downregulate CD4 expression in T cells in vivo, (Harding, 1993) possesses toxic properties that may limit its clinic use in possible doses that could deliver the immunomodulation through down regulation of CD4 expression, and (Saizawa et al., 1987) has limited availability in specific tissues, including the CNS. While the in vitro effects of MnTBAP appear promising, its clinical usefulness in other pre-clinical models of human vector-assisted gene therapy, transplant medicine, and autoimmunity needs to be demonstrated.