Mitochondrial Autoantibodies and the Role of Apoptosis in Pemphigus Vulgaris

Abstract

Pemphigus vulgaris (PV) is an IgG autoantibody-mediated, potentially fatal mucocutaneous disease manifested by progressive non-healing erosions and blisters. Beyond acting to inhibit adhesion molecules, PVIgGs elicit a unique process of programmed cell death and detachment of epidermal keratinocytes termed apoptolysis. Mitochondrial damage by antimitochondrial antibodies (AMA) has proven to be a critical link in this process. AMA act synergistically with other autoantibodies in the pathogenesis of PV. Importantly, absorption of AMA inhibits the ability of PVIgGs to induce blisters. Pharmacologic agents that protect mitochondrial function offer a new targeted approach to treating this severe immunoblistering disease.

Full text

Citation: Hutchison, D.M.; Hosking,
A.-M.; Hong, E.M.; Grando, S.A.
Mitochondrial Autoantibodies and
the Role of Apoptosis in Pemphigus
Vulgaris. Antibodies 2022,11, 55.
https://doi.org/10.3390/
antib11030055
Academic Editor: Kyle T. Amber
Received: 3 August 2022
Accepted: 23 August 2022
Published: 25 August 2022
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antibodies
Review
Mitochondrial Autoantibodies and the Role of Apoptosis in
Pemphigus Vulgaris
Dana M. Hutchison 1,2,3 , Anna-Marie Hosking 1, Ellen M. Hong 2,4 and Sergei A. Grando 1,5,6,*
1Department of Dermatology, University of California Irvine, Irvine, CA 92697, USA
2Beckman Laser Institute, University of California Irvine, Irvine, CA 92612, USA
3Department of Internal Medicine, Riverside Community Hospital, Riverside, CA 92501, USA
4Hackensack Meridian School of Medicine, Nutley, NJ 07110, USA
5Department of Biochemistry, University of California Irvine, Irvine, CA 92697, USA
6Institute for Immunology, University of California Irvine, Irvine, CA 92697, USA
*Correspondence: sgrando@hs.uci.edu; Tel.: +1-949-824-2713; Fax: +1-949-824-2993
Abstract:
Pemphigus vulgaris (PV) is an IgG autoantibody-mediated, potentially fatal mucocuta-
neous disease manifested by progressive non-healing erosions and blisters. Beyond acting to inhibit
adhesion molecules, PVIgGs elicit a unique process of programmed cell death and detachment of
epidermal keratinocytes termed apoptolysis. Mitochondrial damage by antimitochondrial antibodies
(AMA) has proven to be a critical link in this process. AMA act synergistically with other autoan-
tibodies in the pathogenesis of PV. Importantly, absorption of AMA inhibits the ability of PVIgGs
to induce blisters. Pharmacologic agents that protect mitochondrial function offer a new targeted
approach to treating this severe immunoblistering disease.
Keywords: pemphigus vulgaris; apoptosis; apoptosis; antimitochondrial autoantibodies
1. Introduction
Pemphigus encompasses a family of rare, potentially lethal autoimmune blistering
dermatoses involving the skin and mucosal surfaces. The word ‘pemphigus’ derives from
the Greek “pemphix”, which means blister. Its earliest use dates back to Hippocrates in
460–370 B.C. [
1
]. In modern history, the disease was first described by an Irish physician
in 1788 [
2
], however, our current understanding of pemphigus pathophysiology began
in 1964 with the discovery of autoantibodies in the sera of pemphigus vulgaris (PV) pa-
tients directed against the cell surface of keratinocytes [
3
]. The disease is associated with
both circulating and tissue-bound IgG autoantibodies, and manifested by the loss of cell–
cell adhesion of keratinocytes (acantholysis), and formation of non-healing suprabasal
intraepidermal blisters.
IgG antibodies against desmoglein-1 (Dsg1) and desmoglein-3 (Dsg3), calcium-dependent
cell adhesion molecules of the cadherin family, have been considered to play a primary role in
the development of PV. However, explanation of the pathogenesis remains controversial [
4
].
Clinically, the detection of anti-Dsg3 reactivity, with or without anti-Dsg1 reactivity, is helpful
in diagnosing PV. However, there have been a number of reports of patients in whom no
reactivity was found, challenging the notion of an exclusive role these proteins have in the
biologic mechanism of keratinocyte cohesion and their autoantibodies in blister formation in
patients (reviewed in Ref. [5]).
Proteomic studies have led to the discovery of additional major defined types of non-
Dsg proteins targeted by pemphigus autoantibodies, including: mitochondrial proteins,
desmocollin 1 and 3 (Dsc1 and Dsc3), various nicotinic and muscarinic acetylcholine recep-
tor subtypes, thyroid peroxidase, human leukocyte antigen (HLA) molecules, and secretory
pathway Ca
2+
/Mn
2+
-ATPase isoform 1 (SPCA1) encoded by the ATP2C1 gene, which is
mutated in Hailey-Hailey disease [
6
]. A “multiple hit” hypothesis has been proposed [
7
],
Antibodies 2022,11, 55. https://doi.org/10.3390/antib11030055 https://www.mdpi.com/journal/antibodies

Antibodies 2022,11, 55 2 of 8
wherein various non-Dsg autoantibodies against keratinocytes act synergistically with
anti-Dsg autoantibodies to cause blistering. These non-Dsg autoantibodies can induce
changes seen in PV, including keratinocyte shrinkage, cell–cell detachment, and triggering
of apoptotic signaling events (reviewed in Ref. [
4
]). For example, non-Dsg autoantibodies
against Dsc3, M3 muscarinic acetylcholine receptor (M3AR), and SPCA1 isolated from the
sera of patients with anti-Dsg1/3 autoantibody-negative PV were found to be pathogenic,
working synergistically with each other to cause acantholysis [
8
]. Thus, recent discoveries
of numerous non-Dsg autoantibody species further develop our understanding of PV and
implicate additional cell metabolism and signaling pathways involved in acantholysis [
6
,
9
].
In this article, we focus on targets of PV autoimmunity within the mitochondrion, as an-
timitochondrial antibodies (AMA) have proven to be a critical link in the
pathogenesis of PV.
2. Apoptolysis
Beyond acting at the keratinocyte cell membrane to block the function of adhesion
molecules, PVIgGs elicit the signaling events that trigger the keratinocyte cell death pro-
gram. The term “apoptolysis” has been coined to describe the distinct autoantibody-
induced process of keratinocyte structural damage and detachment (acantholysis) followed
by death (apoptosis), which is unique to PV. Acantholysis and apoptosis are inseparable in
PV and are mediated by the same cell death enzymes [
10
]. Apoptosis refers to programmed
cell death—a pathway not activated by inflammation, but rather by cysteine aspartate
proteases, or caspases. This cell death pathway can be activated by cellular damage (intrin-
sic pathway) or by signaling molecules (extrinsic, death receptor-initiated pathway), and
ultimately results in the formation of apoptotic bodies which are then cleared by phago-
cytic cells [
11
]. The best-known cell death pathways are apoptosis, oncosis, and necrosis,
however others have recently been described by the Nomenclature Committee on Cell
Death (NCCD) [
12
]. Additional, newly described apoptolytic pathways that play a role in
the pathophysiology of skin blistering characteristic of PV should be further investigated.
Mitochondria play a critical role in programmed cell death (reviewed in Ref. [
13
]).
Initiation of apoptotic pathways ultimately disrupt the inner mitochondrial membrane,
resulting in loss of the mitochondrial transmembrane potential and leakage of pro-apoptotic
proteins into the cell cytosol, including the release of cytochrome c (CytC), a marker
for mitochondrial outer membrane permealization and early apoptosis, and subsequent
activation of caspases [
14
]. The disruption of mitochondrial energy production, combined
with cleavage of adhesion and structural molecules, causes cytoskeleton collapse and the
keratinocyte to shrink [
10
]. The fundamental feature of apoptolysis is that anti-keratinocyte
antibodies in PV cause basal keratinocytes only to shrink, but not to die, giving rise to
their “tombstone” appearance on histopathology. This is distinct from the classic apoptotic
processes in the epidermis of patients with Stevens-Johnson Syndrome/Toxic Epidermal
Necrolysis, in which apoptosis leads to sloughing of the entire epidermis, including its
basal layer [15].
In PV, the apoptotic pathway is activated long before morphological evidence of
acantholysis [
16
,
17
]. The hypothetical sequence of apoptolysis development in PV has
five consecutive steps: (i) Pathogenic autoantibodies bind PV antigens on the surface of
keratinocytes and pro-apoptolytic signals are transduced. (ii) Activation of EGFR, mTOR,
Src, p38 MAPK and other signaling pathways increase intracellular calcium and initiate
programmed cell death enzymatic cascades predominately in basal keratinocytes. (iii)
Executioner caspases cleave tonofilaments, leading to their collapse and retraction, while
inter-desmosomal adhesion complexes are phosphorylated and dissociate. This results in
basal cell contraction, a crossing step of both the apoptotic and early acantholytic pathways.
The majority of desmosomes remain intact and bridge collapsing keratinocytes. (iv) The
continued degradation of structural proteins by the programmed cell death enzymes lead
to cytoskeleton collapse and complete separation of shrinking keratinocytes (visible acan-
tholysis). The sloughed cell membrane pieces trigger production of scavenging (secondary)
autoantibodies to Dsg, Dsc, E-cadherin and other adhesion molecules attached to the cell

Antibodies 2022,11, 55 3 of 8
membrane. (v) The end result is rounding up and apoptotic death of acantholytic cells
resulting from irreversible damage to mitochondrial and nuclear proteins by the same
cell death enzymes giving rise to a “tombstone” appearance of the surviving basal ker-
atinocytes [
10
]. The more recent observations of the pathogenic role of AMA, however,
indicate that damage of mitochondria occurs at an early stage of apoptolysis.
3. Mitochondrial Damage by AMA in PV
Patients with PV produce PVIgG antibodies targeting a variety of proteins, including
those at the inner and outer mitochondrial membrane, as well as the mitochondrial ma-
trix [
18
–
20
]. Utilizing protein microarray, the most common antigen targets recognized by
AMA in PV have been identified from a large cohort of patients (Table 1) [
18
]. Based on
the known functions of these proteins, AMA likely lead to mitochondrial dysfunction by
altering cellular ability to produce or inactivate reactive oxygen species, perform oxidative
phosphorylation, and participate in oxygen respiration. The exact mechanism of mitochon-
drial damage likely varies greatly among PV patients, consistent with the strikingly wide
spectrum of disease severity and treatment response [
20
]. Importantly, absorption of AMA
inhibits the ability of PVIgGs to induce keratinocyte detachment and blistering [19].
Table 1. Mitochondrial autoantibodies in patients with PV (adapted from Kalantari et al. [18]) *.
Symbol Antigen Localization on
Mitochondria
Frequency
PV (%) Control (%)
ABAT-V1 4-Aminobutyrate aminotransferase, mitochondrial; 50 kDa Matrix 19 4
ALDH4A1 Aldehyde dehydrogenase 4 family, member A1 Matrix 23 5
CPT1B Carnitine O-palmitoyltransferase 1B Outer membrane 18 5
CRAT Carnitine O-acetyltransferase Inner membrane 28 7
CYB5B Cytochrome b5type B; 21 kDa Outer membrane 19 1
ETFA Electron transfer flavoprotein, αprotein Matrix 19 4
ETFB Electron transfer flavoprotein, βprotein Matrix 21 3
FDXR-V2 NADPH:adrenodoxin oxidoreductase Matrix 25 6
FH Fumarate hydratase (fumarase) Mitochondrion 29 3
MAOB Amine oxidase (flavin-containing) B Outer membrane 27 5
ME2 NAD-dependent malic enzyme Matrix 18 6
ME3 NADP-dependent malic enzyme, mitochondrial Matrix 23 8
MLYCD Malonyl-CoA decarboxylase Mitochondrion 29 4
NDUFA9 NADH dehydrogenase [ubiquinone] 1αsubcomplex subunit 9; 39 kDa Matrix 20 3
NDUFA13
NADH dehydrogenase [ubiquinone] 1
α
subcomplex subunit 13; 16 kDa
Inner membrane 24 6
NDUFB10 NADH dehydrogenase [ubiquinone] 1βsubcomplex subunit 10 Matrix 17 2
NDUFV3 NADH dehydrogenase [ubiquinone] flavoprotein 3; 9 kDa Inner membrane 19 4
NDUFS6 NADH dehydrogenase [ubiquinone] iron-sulfur protein 6; 13 kDa Inner membrane 24 6
PC Pyruvate carboxylase Matrix 32 5
PDK4 Pyruvate dehydrogenase kinase, isozyme 4 Matrix 24 4
PDHA1 Pyruvate dehydrogenase E1 component αsubunit, somatic form Glycolysis 30 3
PMPCB Mitochondrial processing peptidase βsubunit Mitochondrial
organization 31 4
PRODH Proline oxidase Matrix 25 6
SOD2 Superoxide dismutase [Mn] Matrix 23 2
TIMM44 Mitochondrial import inner membrane translocase subunit Inner membrane 20 4
* Every PV serum analyzed in the referenced study contained an autoantibody to at least one mitochondrial
protein (data not shown).
Antibodies to mitochondrial proteins, among anti-keratinocyte antibodies and sev-
eral other soluble pathogenic factors, act synergistically to activate keratinocyte cell death
pathways in PV (Figure 1). In an organ culture of neonatal mouse skin—an
in vitro
model
of PV—AMA and anti-Dsg1/3 autoantibodies acted synergistically to induce acantholy-
sis [
21
]. In that study, treatment with AMA alone did not result in acantholysis, however
the combination of AMA and a mixture of anti-Dsg antibodies induced acantholysis. The
AMA/anti-Dsg3 combination induced epidermal splitting suprabasally and the AMA/anti-
Dsg1 combination induced epidermal splitting subcorneally, which is characteristic of
pemphigus foliaceus. Moreover, while acantholysis was observed following treatment with
high concentrations of the human anti-Dsg single-chain variable fragment (scFv), at low,

Antibodies 2022,11, 55 4 of 8
physiologic doses the scFv was not able to induce keratinocyte detachment. Acantholysis
was only observed when it was combined with AMA [
21
]. The synergy of AMA with other
autoantibodies in PV implies that a simultaneous hit is required to alter the keratinocyte
ability to maintain epidermal integrity. It is theorized that the binding of a single type of au-
toantibody only induces reversible changes in the keratinocyte, such that the cell maintains
its ability to recover via self-repair mechanisms. Irreversible keratinocyte damage only
occurs following presumed synchronized inactivation of salvage pathways by partnering
autoantibodies, leading to loss of epidermal integrity [21].
Antibodies 2022, 11, x FOR PEER REVIEW 4 of 9
Table 1. Mitochondrial autoantibodies in patients with PV (adapted from Kalantari et al. [18]) *.
Symbol Antigen Localization on Mitochondria
Frequency
PV (%) Control (%)
ABAT-V1 4-Aminobutyrate aminotransferase, mitochondrial; 50 kDa Matrix 19 4
ALDH4A1 Aldehyde dehydrogenase 4 family, member A1 Matrix 23 5
CPT1B Carnitine O-palmitoyltransferase 1B Outer membrane 18 5
CRAT Carnitine O-acetyltransferase Inner membrane 28 7
CYB5B Cytochrome b5 type B; 21 kDa Outer membrane 19 1
ETFA Electron transfer flavoprotein, α protein Matrix 19 4
ETFB Electron transfer flavoprotein, β protein Matrix 21 3
FDXR-V2 NADPH:adrenodoxin oxidoreductase Matrix 25 6
FH Fumarate hydratase (fumarase) Mitochondrion 29 3
MAOB Amine oxidase (flavin-containing) B Outer membrane 27 5
ME2 NAD-dependent malic enzyme Matrix 18 6
ME3 NADP-dependent malic enzyme, mitochondrial Matrix 23 8
MLYCD Malonyl-CoA decarboxylase Mitochondrion 29 4
NDUFA9 NADH dehydrogenase [ubiquinone] 1α subcomplex subunit 9; 39 kDa Matrix 20 3
NDUFA13 NADH dehydrogenase [ubiquinone] 1α subcomplex subunit 13; 16 kDa Inner membrane 24 6
NDUFB10 NADH dehydrogenase [ubiquinone] 1β subcomplex subunit 10 Matrix 17 2
NDUFV3 NADH dehydrogenase [ubiquinone] flavoprotein 3; 9 kDa Inner membrane 19 4
NDUFS6 NADH dehydrogenase [ubiquinone] iron-sulfur protein 6; 13 kDa Inner membrane 24 6
PC Pyruvate carboxylase Matrix 32 5
PDK4 Pyruvate dehydrogenase kinase, isozyme 4 Matrix 24 4
PDHA1 Pyruvate dehydrogenase E1 component α subunit, somatic form Glycolysis 30 3
PMPCB Mitochondrial processing peptidase β subunit Mitochondrial organization 31 4
PRODH Proline oxidase Matrix 25 6
SOD2 Superoxide dismutase [Mn] Matrix 23 2
TIMM44 Mitochondrial import inner membrane translocase subunit Inner membrane 20 4
* Every PV serum analyzed in the referenced study contained an autoantibody to at least one mito-
chondrial protein (data not shown).
Figure 1. Hypothetical multipathogenic mechanism of interconnected signaling cascades leading to
keratinocyte apoptolysis in pemphigus (modified from Marchenko et al. [19]). AMA: antimitochon-
drial antibodies; Anti-Dsg3 PVAb: anti-desmoglein 3 PV antibody; Cs: caspase; Dsg3: desmoglein-
3; EGFR: epidermal growth factor receptor; FasL: Fas ligand; FasR: Fas receptor; JNK: c-Jun N-ter-
minal kinase; mTOR: mammalian target of rapamycin; NO: nitric oxide; nDPVAb: non-Dsg PV an-
tibodies; OPVAg: other PV antigens; PKC: protein kinase C; PVAb: PV antibody; Src: SRC proto-
oncogene, nonreceptor tyrosine kinase; TNF-α: tumor necrosis factor-α.
Figure 1.
Hypothetical multipathogenic mechanism of interconnected signaling cascades leading to
keratinocyte apoptolysis in pemphigus (modified from Marchenko et al. [
19
]). AMA: antimitochon-
drial antibodies; Anti-Dsg3 PVAb: anti-desmoglein 3 PV antibody; Cs: caspase; Dsg3: desmoglein-3;
EGFR: epidermal growth factor receptor; FasL: Fas ligand; FasR: Fas receptor; JNK: c-Jun N-terminal
kinase; mTOR: mammalian target of rapamycin; NO: nitric oxide; nDPVAb: non-Dsg PV antibodies;
OPVAg: other PV antigens; PKC: protein kinase C; PVAb: PV antibody; Src: SRC proto-oncogene,
nonreceptor tyrosine kinase; TNF-α: tumor necrosis factor-α.
Human skin contains a complex non-neuronal cholinergic network composed of
the cytotransmitter acetylcholine and its nicotinic and muscarinic receptors [
22
]. These
receptors are involved in keratinocyte cell–cell and cell-matrix adhesion and some are
targeted by PV autoantibodies (reviewed in Ref. [
6
]). In a neonatal mouse model of PV,
preabsorption of PVIgGs with recombinant pemphaxin, a low-affinity dual muscarinic and
nicotinic receptor for acetylcholine, eliminated the acantholytic activity of PVIgGs. In turn,
the acantholytic activity could be restored by the addition of anti-pemphaxin antibody
back to the preabsorbed PVIgG fraction [
23
]. In addition to being present on the surface
of keratinocytes, nicotinic acetylcholine receptors have been found on the mitochondrial
outer membrane (mt-nAChRs) and are one of the targets of AMA. Stimulation of mt-
nAChR prevents apoptosis by inhibiting mitochondrial permeability transition pore (mPTP)
opening, thus preventing CytC release from the organelle [
20
]. Interestingly, nicotinergic
stimulation has been shown to protect keratinocytes from apoptolysis [20].
Among non-Dsg autoantibodies in PV patient sera, that increase activity of pathways
involved in apoptolysis, a combination of anti-M3AR, anti-SPCA1 and Dsc3 has been
identified [
8
]. When each of the above autoantibodies was tested alone in a neonatal mouse
skin explant model, none were able to solely induce acantholysis. However, a mixture of
all three produced an acantholytic effect similar to that of PVIgGs. When the combination

Antibodies 2022,11, 55 5 of 8
was further tested in a model of PV in BABL/c mice, it was also found to be sufficient to
disrupt epidermal integrity
in vivo
[
8
]. Thus, antibodies altering vital cell functions (i.e.,
anti-M3AR), cell adhesion (i.e., anti-Dsc3) and Ca
2+
metabolism (i.e., anti-SPCA1) appear
to work synergistically to produce an acantholytic effect similar to that of total PVIgGs in a
clinically relevant manner.
It has been shown that the binding of anti-M3AR or anti-SPCA1 autoantibodies to
keratinocytes leads to mitochondrial damage and the release of CytC and activation of the
caspase 9 pathway [
24
]. Anti-SPCA1 produced a 10-fold, and anti-M3AR autoantibody a
4–5 fold, increase in levels of CytC and Cs-9, respectively [
24
]. Further, anti-M3AR and
anti-SPCA1 autoantibodies worked synergistically in a 3D culture of human epidermis.
A mixture of anti-SPCA1 and anti-M3AR autoantibodies resulted in changes in the mor-
phology of human epidermis consistent with acantholysis, including “bubbling” at the
epidermis while basal cells remained intact at the dermal-epidermal junction [
24
]. However,
when given alone, anti-M3AR did not cause morphological changes in that
in vitro
model,
while anti-SPCA1 given alone resulted in shrinkage of epidermal keratinocytes [24].
SPCA1 is located on the Golgi apparatus, and it is thought that a defect in SPCA1 Ca
2+
sequestration contributes to Golgi stress leading to apoptosis [
25
]. The Golgi complex is
capable of transducing pro-apoptotic signals which are partially mediated through caspase
2 (Cs-2) that localizes to the Golgi apparatus [
26
,
27
]. A recent study demonstrated that the
effects of PV anti-SPCA1 autoantibody on mitochondrial CytC release were abolished in
the presence of a Cs-2 inhibitor [
24
]. These findings suggest that anti-SPCA1 autoantibodies
alter mitochondrial function through Cs-2, triggering early pro-apoptotic events. Notably,
SPCA1 is encoded by the ATP2C1 gene, which is mutated in benign chronic pemphigus
(also known as Hailey-Hailey disease) [28].
The neonatal Fc receptor (FcRn) may, in part, mediate the pathogenic effects of PV
autoantibodies to intracellular self-antigens, including SPCA1 and those present in mito-
chondria. Following binding of PVIgG to FcRn on the cell membrane of keratinocytes,
complexes of PVIgG-FcRn are internalized and trafficked to the mitochondria, where they
are released from endosomes [
21
]. The complexes dissociate and AMA reach mitochon-
dria, triggering early apoptotic events and cell shrinkage. However, this AMA-induced
damage is reversible. Interestingly, cells lacking FcRn do not internalize PVIgGs and AMA
is therefore unable to reach the mitochondria [
21
]. Further, pretreatment of mouse ker-
atinocytes with anti-FcRn antibody, which functionally inactivates FcRn, prevents shrinkage
of keratinocytes as well as other AMA-dependent changes in mitochondrial integrity and
metabolism [
21
]. Since FcRn is predominantly expressed in the basal epidermal layer [
29
],
basal and suprabasal keratinocytes should respond differently to the PVIgGs entering
keratinocytes via FcRn-mediated mechanism. This may explain the suprabasal location of
epidermal split in PV, as only basal keratinocytes shrink, thereby separating themselves
from suprabasal keratinocytes. However, the exact mechanism by which PVIgGs enter ker-
atinocytes to reach the mitochondrial target antigens, and why other cell types in the body
that contain the same mitochondrial antigens are not affected by PVIgGs,
is still unknown.
New research has implicated that the thioredoxin-2 (Trx2)/apoptosis signal-regulating
kinase 1 (ASK1) pathway may play a key role in mediating mitochondrial injury in PV [
30
].
ASK1, a serine/threonine kinase, is activated by oxidative stress and triggers apoptosis.
One of the functions of Trx2 is to inactivate ASK1 by forming a complex with the molecule,
thereby preventing its phosphorylation and activation. Elevated levels of reactive oxygen
species, which are found following mitochondrial injury by AMA, oxidize the cysteine
residues of Trx2, promoting the dissociation of the Trx2-ASK1 complex and allowing
activated ASK1 to trigger apoptotic events. In an
in vitro
study of keratinocytes cultured
with PV sera, the Trx2/ASK1 cascade was abnormally activated, with decreased local
expression of Trx2, an increased amount of phosphorylated ASK1, and an increased rate
of apoptosis compared to control cells [
29
]. In a mouse model, the overexpression of
Trx2 decreased ASK1 phosphorylation, the apoptotic rate, and relieved acantholysis and
blister formation. Thus, Trx2 appears to have a protective role in mitochondrial injury

Antibodies 2022,11, 55 6 of 8
and compounds targeting the Trx2/ASK1 pathway may help prevent progression of PV
in the future.
4. Efficacy of Mitochondrion Protective Agents in Pemphigus Patients
While the antigen specificities of AMA produced by individual PV patients is highly
variable (Table 1), uniform protection from mitochondrial damage can be achieved with non-
steroidal mitochondrion-protective pharmacologic agents [
18
]. Growing evidence suggests
that the use of mitochondrion-protecting drugs, such as cyclosporine A (CsA), tetracyclines
and nicotinamide (also called niacinamide), are justified in the treatment of PV [
18
]. In
addition to inhibiting the production of cytokines involved in T-cell activation, CsA can
protect mitochondria by binding cyclophilin D and inhibiting opening of mPTP, allowing
the mitochondrion to retain a high transmembrane potential (
∆ψ
m) [
31
–
33
]. Tetracyclines,
such as minocycline and doxycyline, inhibit mPTP opening by reducing mitochondrial
uptake of Ca
2+
, inhibiting loss of
∆ψ
m and preventing CytC release [
34
]. Niacinamide, one
of the two principle forms of vitamin B
3
, is a precursor of the coenzyme NAD
+
consumed
during ATP generation in the mitochondrial electron transport chain. Vitamin B
3
is thought
to help cells retain high-quality mitochondria by activating autophagy of mitochondria
with low
∆ψ
m, which indicates a damaged (depolarized) cell [
33
]. Animal studies pro-
vided evidence that pharmacologic protection of mitochondria with CsA, minocycline,
and nicotinamide prevents PVIgGs-mediated induction of skin blisters in mouse skin [
18
],
which is in keeping with clinical reports that PV lesions can be partially controlled by these
agents in the absence of systemic steroids [35–37].
One interesting observation is that nicotine competes with PVIgGs for binding to
mt-nAChRs, thereby inhibiting mitochondrial CytC release in a dose-dependent fashion,
and prevents intrinsic apoptosis in keratinocytes [
20
]. The potential therapeutic effect
of nicotinergic stimulation in PV has been reported in a case study [
38
] as well as in
epidemiological data showing a beneficial effect of smoking on PV [
39
–
41
]. This may be in
part due to nicotinic agonism at mt-nAChRs protecting mitochondria.
Additionally, sirolimus (also known as rapamycin) has been proposed to protect
keratinocytes from PVIgG aggression through a poorly understood mechanism. In an
experimental setting, pretreatment with sirolimus prevented acantholysis in a mouse
model of PV [
42
]. In a clinical setting, within two weeks of initiating therapy with sirolimus,
PV lesions on a man with severe side effects to prednisone completely healed, allowing him
to rapidly taper off prednisone and remain lesion free on a maintenance dose of 2 mg/day
of sirolimus. Studies in other mitochondrial disorders have suggested that sirolimus
improves cellular function by reducing the number of dysfunctional mitochondria within
an organelle, thereby preserving mitochondrial integrity [43].
At the UC Irvine Immunobullous Clinic, PV patients are successfully treated with
a multidrug therapeutic approach including mitochondrion-protecting agents (minocy-
cline or doxycycline 200 mg/day + niacinamide 1.5 g/day), in addition to intravenous
immunoglobulin, or IVIg, systemic corticosteroids, and an immunosuppressive cytotoxic
drug (mycophenolate mofetil, azathioprine or cyclophosphamide) [
44
]. The synergy of the
drugs utilized in this protocol allows for rapid achievement and maintenance of clinical re-
mission in approximately 88% of pemphigus patients with a smaller than usual cumulative
dose of systemic corticosteroids. Indeed, while these mitochondrion protective agents are
already utilized in the treatment of PV, novel pharmacologic prospects which protect and
or compensate for disrupted mitochondrial function may offer an even safer, nonsteroidal
approach to treating PV in the future.
5. Conclusions and Future Directions
The acantholytic process in PV is complex and involves autoantibodies directed
against various keratinocyte proteins that maintain adhesion and other vital cell functions.
While different pathogenic autoantibodies act synergistically in the pathogenesis of PV,
pharmacological protection or the elimination of a single antibody may suffice to protect

Antibodies 2022,11, 55 7 of 8
epidermal integrity and halt development of the disease. Further characterization of the
role of individual AMA causing mitochondrial injury in the pathogenesis of PV may lead
to development of personalized pharmacologic therapies to correct mitochondrial abnor-
malities unique to individual PV patients. Future studies to improve our understanding
of the immunopathogenesis of PV should, therefore, aid in the development of novel and
more efficient therapeutic modalities.
Author Contributions:
Conceptualization, D.M.H., S.A.G.; Writing—Original Draft Preparation,
D.M.H.; Writing—Review and Editing, E.M.H., A.-M.H., S.A.G.; Figures, D.M.H., E.M.H. All authors
have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Conflicts of Interest: The authors declare no conflict of interest.
Disclaimer:
The views expressed in this publication represent those of the author(s) and do not
necessarily represent the official views of HCA Healthcare or any of its affiliated entities.
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