storms (see Glossary). A key to effective interventions is to trigger robust antiviral defenses
but with checked in?ammation. Therefore, molecules that suppress both viral replication and
Aging
Highlights
NAD
+
can exert both antiviral and anti-
in?ammatoryeffectsinmiceandhumans,
which might be bene?cial during co-
ronavirus disease 2019 (COVID-19)
infections.
Compared with healthy individuals,
NAD
+
concentrations in tissues and or-
gansarelowerinolderindividualsandin
patients with diseases associated with
severe COVID-19 symptoms, including
diabetes and cardiovascular disease.
Viral infections, including coronavirus
infections, have been reported to further
deplete cellular NAD
+
stores.
Many NAD
+
-dependent enzymes,
including members of the sirtuin and
poly-ADP-ribose polymerase (PARP)
families, display potent antiviral activities.
Severe acute respiratory syndrome co-
ronavirus 2 (SARS-CoV-2) and a variety
of viruses possess ADP-ribosyl hydrolase
activity, which counteracts the activity of
PARPs.
Viruses such as SARS-CoV-2 can
hyperactivate the immune system
by activating nuclear factor kappa B
(NF-κB) and the NOD-, LRR-, and
pyrin domain-containing protein 3
(NLRP3) in?ammasome, potentially
leading to deadly cytokine storms.
NAD
+
-boostingcompoundsmaycoun-
teract these processes.
Several NAD
+
-boosting compounds
and molecules that target NAD
+
-
producing or -consuming enzymes
are in clinical development as putative
anti-in?ammatory or antiviral drugs.
Institute, Paul F. Glenn Center for
Trends in
more susceptible to developing severe disease could lead to new candidate strategies for
therapeutic intervention.
Biology of Aging Research, Harvard
Medical School, Boston, MA, USA
2
These authors contributed equally.
diseases af?ict boththe very oldand young,makingthisdemographicpatternfor COVID-19 sus-
ceptibility unusual [5]. A better understanding of the mechanisms that render older individuals
AtragicandpoorlyunderstoodaspecttoCOVID-19istheincreasedsusceptibilityoftheelderlyto
severe forms of the disease [3]. Rates of hospitalization, intensive care unit (ICU) admission, and
death increase with age, while the young are often left relatively unscathed [4]. Most infectious
1
Department of Genetics, Blavatnik
in?ammation may be particularly important for ?ghting severe COVID-19 [1]. A growing body
of evidence shows that the metabolite NAD
+
is a mediator of both antiviral and anti-
in?ammatory mechanisms. Based on this evidence, we posit that therapies that boost
NAD
+
concentrations might play a role in preventing and treating severe COVID-19 and
other viral infections.
Firstdescribedasa yeastfermentationfactoroveracentury ago,today, NAD
+
hasrisentoprom-
inence as a regulator of healthy aging. Low levels of NAD
+
in tissues and organs are associated
with aging, metabolic syndrome, and in?ammation, while dietary interventions that slow age-
related diseases increase NAD
+
concentrations [2]. Here, we review the epidemiological and
mechanistic data supporting a role for NAD
+
in modulating the outcomes of viral infections,
with a focus on SARS-CoV and SARS-CoV-2. We also explore ongoing approaches to boost
NAD
+
levels for therapeutic bene?t in the clinic.
NAD
+
and risk factors for severe COVID-19
Opinion
NAD
+
in COVID-19 and viral infections
Minyan Zheng ,
1,2
Michael B. Schultz,
1,2
and David A. Sinclair
1,
NAD
+
, as an emerging regulator of immune responses during viral infections, may
be a promising therapeutic target for coronavirus disease 2019 (COVID-19). In this
Opinion, we suggest that interventions that boost NAD
+
levels might promote anti-
viral defenseand suppress uncontrolled in?ammation. Wediscuss the association
between low NAD
+
concentrations and risk factors for poor COVID-19 outcomes,
including aging and common comorbidities. Mechanistically, we outline how viral
infectionscanfurtherdepleteNAD
+
anditsrolesinantiviraldefenseandin?amma-
tion. We also describe how coronaviruses can subvert NAD
+
-mediated actions via
genes that remove NAD
+
modi?cations and activate the NOD-, LRR-, and pyrin
domain-containing protein 3 (NLRP3) in?ammasome. Finally, we explore ongoing
approaches to boost NAD
+
concentrations in the clinic to putatively increase
antiviral responses while curtailing hyperin?ammation.
NAD
+
as a modulator of viral infection outcomes
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viral infections
render the body a battle?eld, requiring mobilization of vast resources to mount an effective
defense. These defenses can back?re when uncontrolled, resulting in deadly cytokine
Immunology
Trends in Immunology, April 2022, Vol. 43, No. 4 https://doi.org/10.1016/j.it.2022.02.001 283
? 2022 Elsevier Ltd. All rights reserved.
Correspondence:
david_sinclair@hms.harvard.edu
(D.A. Sinclair).
Trends in Immunology
Wepositthatoneculpritmaybeade?ciency in NAD
+
, the concentration of which decreases
during aging in nearly every tissue studied across species [6]. In rodents, this decrease has been
observed in tissues and cells that are relevant to COVID-19 infection, such as the skeletal muscle,
liver endothelial cells, and macrophages [7–10]. A growing body of human data corroborates an
association between age and low NAD
+
concentrations across multiple tissues including the
skin, blood, liver, and muscle [10–13,117].
WhyNAD
+
levelsfallduringagingisanactiveareaofresearch.Studiesinmicehaveimplicatedan
increase in the concentrations and activity of NAD
+
-consuming enzymes such as CD38, poly
(ADP-ribose) polymerase 1 (PARP1)1, and sterile alpha and TIR motif containing 1 (SARM1)
[9,14–17], as well as an insuf?cient ?ux from NAD
+
-producing enzymes such as nicotinamide
phosphoribosyltransferase (NAMPT), indoleamine 2,3-dioxygenase (IDO), and quinolinate
phosphoribosyltransferase (QPRT) [10,18–20]. These enzymes all serve as potential pharmaco-
logical targets to raise NAD
+
concentrations (Figure 1).
Comorbidities
NAD
+
concentrations are also low in certain comorbidities associated with COVID-19
severity. One such comorbidity is insulin resistance (IR) and diabetes mellitus [21]. Speci?cally, in
mouse models, genetic (KK/HIJ), diet-induced (high-fat diet), streptozotocin-induced, and
age-associated IR have been associated with lower NAD
+
concentrations in liver and white adipose
tissue, while restoration of NAD
+
with NAD
+
boosters such as nicotinamide ribose (NR) and
nicotinamide mononucleotide (NMN) (oral, intraperitoneal,or infusion) reverses IR[22–26]. In humans,
metabolic syndrome and obesity have been associated with low NAD
+
concentrations in adipose
tissue [27]. NMN was recently shown to improve insulin sensitivity in prediabetic women, and further
clinical studies are ongoing, discussed later in this review [28](Table 1, Key table). In addition, a
recently developed oral antidiabetic medication, imeglimin, can enhance glucose-stimulated ATP
generation and induce the synthesis of NAD
+
in pancreatic islets derived from diseased rodents
with type 2 diabetes [29].
Another risk factor for severe COVID-19 is cardiovascular disease (CVD) [30]. Several preclinical
studies demonstrate that raising NAD
+
concentrations in endothelial cells reverses vascular and
endothelialdysfunction[8,31].Furtherstudieshaveshownabene?cialroleforNAD
+
incardiovas-
cular function in mouse models, including models of dyslipidemia, ischemia–reperfusion injury,
and diastolic heart failure [32,33]. This link is supported in humans by epidemiological data
demonstrating a correlation between dietary intake of the NAD
+
precursor niacin (nicotinic acid)
and vascular health, including brachial-artery ?ow-mediated dilation and serum low-density lipo-
protein (LDL) concentrations [34]. In fact, niacin is an USA FDA-approved therapy for reducing
LDL, ApoB, and triglycerides, and for raising high-density lipoprotein (HDL). Additionally, multiple
ongoing clinical studies are testing the effects of other NAD
+
boosters such as NMN and NR on
hypertension and heart failure (discussed later) (Table 1).
Mechanisms of NAD
+
in viral infections
Viral infections can deplete NAD
+
concentrations
Not only are low NAD
+
concentrations associated with risk factors for poor COVID-19 outcomes,
butcertainviralinfectionscanfurtherdepleteNAD
+
ininfectedcells.Forexample,lowerNAD
+
con-
centrations have been reported in human peripheral blood leukocytes infected with HIV-1 in vitro
[35],human ?broblastsinfectedwithherpessimplexvirus1(HSV-1)[36],andintheskeletalmuscle
of individuals coinfected with HIV-1 and hepatitis C virus [37]. These effects are presumably due to
the induction of NAD
+
-consuming enzymes such as CD38 and PARPs [17]. For instance, CD8
+
T lymphocytes expressing CD38 have been proposed as a marker for HIV-1-mediated disease
284 Trends in Immunology, April 2022, Vol. 43, No. 4
Trends in Immunology
progression [38]. The decline of NAD
+
concentrations in human ?broblasts induced by HSV-1
infection is associated with increased protein poly(ADP-ribosyl)ation, and can be blocked by phar-
macological inhibition of PARP1/PARP2 [36].
Trendsrends inin ImmunologyImmunology
Figure 1. NAD
+
metabolism and points of pharmacological intervention. Enzymes involved in NAD
+
biosynthesis
and hydrolysis play important roles in in?ammation and immunity. Biosynthetic pathways include the NAD
+
salvage
pathway, which recycles nicotinamide to form NMN, then NAD
+
, and the de novo pathway that begins with tryptophan.
Hydrolysis of NAD
+
is largely carried out by PARPs, which tag target proteins with poly- or mono-(ADP ribose); sirtuins,
which remove acyl groups and create O-acyl-ADP-ribose; and CD38, BST, and SARM1, which create (cyclic)-ADP-ribose
[44]. There are multiple points of potential pharmacological intervention throughout NAD
+
metabolic pathways. Created
with BioRender.com. Abbreviations: i, inhibitor; NAMPT, nicotinamide phosphoribosyltransferase; NMN, nicotinamide
mononucleotide; NR, nicotinamide ribose; NSP3, nonstructural protein 3; PARP, poly(ADP-ribose) polymerase; SARM1,
sterile alpha and TIR motif containing 1; SIRT, sirtuin.
Glossary
Apoptosis-associated speck-like
protein containing a caspase
recruitment domain (ASC):
in?ammasome adaptor that plays a key
role inthe assemblyand activationofthe
in?ammasome. The NLRP3
in?ammasome consists of a sensor
(NLRP3), an adaptor (ASC), and an
effector (caspase-1). Upon
sensing-speci?c triggers, NLRP3
undergoes conformational changes
which catalyze ASC oligomerization to
form a macromolecular signaling complex
known as the ASC speck. ASC then
recruits caspase-1. Active caspase-1
cleaves pro-IL-1β and pro-IL-18, which
are vital cytokines during infection and
in?ammation.
Cytokine storm: life-threatening event
triggered by uncontrolled in?ammatory
responses with the release of a large
amounts of proin?ammatory cytokines,
including IL-6, IL-1, TNFα, and IFN. This
leads to the recruitment and expansion
of various immune cells into injury sites
which can result in tissue and organ
damage.
Histone H3 lysine 9 (H3K9)
deacetylation:epigeneticmodi?cation;
H3K9 is generally acetylated when a
gene is transcriptionally activated and is
deacetylated (and/or methylated) when
ageneissilenced.
Imeglimin: novel oral agent for the
treatment of type 2 diabetes. Its
mechanism of action involves
ampli?cation of glucose-stimulated insulin
secretion and enhanced insulin action.
Approved for use in Japan in 2021.
P2X purinoceptor 7: expressed in an
increasingnumberofcelltypes;primarily
mediates in?ammation and cell death;
ligand-gatedcationchannelactivated by
high concentrations of extracellular ATP,
triggering the assembly and activation of
the NLRP3 in?ammasome and
subsequent release of IL-1β and IL-18.
Poly(ADP-ribose) polymerases
(PARPs): with coenzyme NAD
+
,PARP
enzymes catalyze ADP-ribosylation or the
transferofADP-ribosylgroupsfrom NAD
+
to nucleophilic side chains of proteins.
They can also ADP-ribosylate DNA and
RNA. In humans, there are 17 identi?ed
PARPs. PARPs participate in diverse
cellular functions such as DNA repair,
apoptosis, unfolded protein response,
pathogen response, and in?ammation.
Sirtuins: enzymes that couple NAD
+
degradation to deacylation (e.g.,
deacetylation, desuccinylation, and
Trends in Immunology, April 2022, Vol. 43, No. 4 285
demyristoylation). With NAD
+
, sirtuins
remove acyl groups from lysine residues
on proteins, sense intracellular NAD
+
concentrations, and transduce a signal
via protein deacylation. In humans, there
are seven sirtuins at different cellular
locations that regulate diverse functions,
including transcription, genome stability,
metabolism, and cell signaling.
Stress granules (SGs): dense,
membraneless, organelles in the cytosol
that appear in response to cellular stress
to promotecellsurvival.Ribonucleoprotein
assemblies that store mRNAs stalled at
translation initiation. The introduction of
viral RNA into the cytoplasm triggers the
formation of stress granules.
TNF receptor-associated factor 3
(TRAF3): member of the TRAF family;
contains a RING domain with E3
ubiquitin ligase activity, and a TRAF
domain mediating protein–protein
interactions; functions in in?ammation,
antiviral defense, and apoptosis. There
are seven TRAF proteins identi?ed in
mammals.
Toll/interleukin-1 receptor domain:
intracellular signaling domain in proteins
that mediates protein–protein
interactions between Toll-like receptors
and signal transduction proteins. When
activated, the TIR domain recruits
cellular adaptor proteins and induces
downstream activation of kinases.
Trends in Immunology
Similar depletion of NAD
+
has been reported with coronavirus infections. Speci?cally, blood from
severe COVID-19 patients contains lower amounts of the NAD
+
precursor NMN compared with
blood from healthy individuals [39]. Moreover, expression changes in genes involved in NAD
+
synthesis and utilization such as Nampt, Parp9, Parp12, and Parp14 have been observed in ep-
ithelial cell lines and enterocyte organoids infected withSARS-CoV-2 compared withmock infec-
tion[40].SimilarNAD
+
-relatedgeneexpressionchangeswerealsoreportedinthelungtissueofa
deceased COVID-19 patient and in the bronchoalveolar lavage ?uid of SARS-CoV-2-infected in-
dividuals relative to healthy controls [40]. Furthermore, infection of a coronavirus, mouse hepatitis
virus (MHV), in mouse bone-marrow-derived macrophages (BMDMs) induced NAD
+
depletion
[40] as well as increased gene expression of many PARPs including Parp7, Parp9, Parp10,
Parp11,Parp12,Parp13,andParp14relativetomockinfection[41].Theseobservationssuggest
that NAD
+
metabolic pathways are under increased demand during SARS-CoV-2 infection, and
highlights the potential relevance of NAD
+
in modulating COVID-19 disease outcomes.
NAD
+
-consuming enzymes in antiviral mechanisms
NAD
+
harbors an important role in fueling the activity of enzymes that regulate mammalian
immune responses [42,43]. Classically, NAD
+
participates in redox processes, but it also
participates in non‐redox reactions in which it is hydrolyzed, and it non‐enzymatically regulates
protein–protein interactions [16](Figure 1). In these latter functions, NAD
+
acts as a signaling
molecule, serving as a marker of energy availability and directing a cell to respond to metabolic
changes via the action of NAD
+
-utilizing enzymes [44].
PARPs and sirtuins are two NAD
+
-dependent enzyme families that participate in immune
responses [42,43]. By adding or removing post-translational modi?cations on key proteins
such as nuclear factor kappa B (NF-κB), they can coordinate the intensity of in?ammatory and
immune responses [42]. This places NAD
+
in an important position for both promoting strong
immune responses to pathogens, and for keeping those responses in check.
PARP1, the pre-eminent member of the PARP family, is a potent coactivator of the proin?amma-
tory transcription factor NF-κB, and therefore participates in initiating speci?c immune responses
[42]. However, this is a double-edged sword, as PARP1 may also increase the severity of cyto-
kine storms as it regulates the expression of many NF-κB-dependent cytokines and chemokines
[42]. Indeed, inhibiting or deleting PARP1 has ameliorated the severity of symptoms in several
in?ammatory disease rodent models including asthma and colitis [45–51]. For example, PARP1
inhibition (pharmacological or genetic) has prevented ovalbumin-induced lung in?ammation in
mice [45] and in a guinea pig model of asthma [46]. Treatment with PARP inhibitors has attenu-
ated in?ammation associated with colitis seenininterleukin-10(IL-10)de?cient (Il10
-/-
)mice
[47], as well as in trinitrobenzene sulfonic acid-treated rats [49]. Furthermore, PARP1 knockout
(KO) (Parp1
-/-
) mice are protected from dextran sulfate sodium-induced colitis compared with
wild-type mice [48].
Several PARPs harbor potent antiviral functions [42,52–59]. Indeed, PARP13 is a powerful anti-
viralfactor thatrecognizesvariousviruses fromseveralfamilies,including Retroviridae, Filoviridae,
Alphaviridae, and Hepadnaviridae [53–56]. It binds to speci?c sequences of viral RNAs during in-
fection and mediates their degradation via the cellular mRNA decay machinery; however, these
functions are not dependent on PARP-mediated ADP-ribosylation [54–56]. Expression of
PARP7, PARP10, and the long isoform of PARP12 (PARP12L) ef?ciently inhibits cellular transla-
tion and the replication of Venezuelan equine encephalitis virus and other alphaviruses in verte-
brate cells [52,59]. These effects of PARP12L are dependent on its catalytic activity. Moreover,
PARP9 and PARP14 are also upregulated in macrophages stimulated by interferon (IFN)-γ and
286 Trends in Immunology, April 2022, Vol. 43, No. 4
Key table
Table 1. Selected clinical trials involving NAD
+
boosters
Clinicaltrials.
gov ID
Phase Interventions Duration Type Enrollment
(participants)
Inclusion criteria Primary endpoint Completion
NCT03151239 N/A NMN, 250 mg/d or
placebo
8 wk Randomized,
double-blind,
placebo-controlled
25 Prediabetic
postmenopausal
women age 55–75 yr
Change in muscle
insulin sensitivity
June 2021
NCT05175768 N/A NMN, NMN
+L-leucine, or
placebo
Up to 28
d
Randomized,
double-blind,
placebo-controlled
375 Individuals age >40 yr
hospitalized with
COVID-19 requiring
supplemental oxygen
COVID-19
associated fatigue
December
2022
NCT04903210 IV NMN, 800 mg/d +
lifestyle modi?cation
or lifestyle
modi?cation alone
8 wk Randomized, single
blind
20 Individuals age 18–65
yr with mild essential
hypertension (BP
130/80–159/99)
Hypertension
(?ow-mediated
dilation and
brachial–ankle pulse
wave velocity)
July 2022
NCT04664361 N/A NMN 250 mg/d,
NMN 500 mg/d, or
placebo
38 d Randomized,
double-blind,
placebo-controlled
150 Healthy men age
20–49 yr with regular
moderate physical
activity
Muscle recovery
(post-endurance
Wingate Anaerobic
Test)
September
2022
NCT02950441 II NR 1 g/d or
placebo
21 d,
followed
by
washout
and
crossover
Randomized,
double-blind,
placebo-controlled,
crossover
12 Men age 70–80 yr Mitochondrial
function
(respirometry) and
NAD
+
concentrations
in muscle biopsy
September
2019
NCT02921659 I/II NR 1 g/d or
placebo
6wk,
followed
by
crossover
Randomized,
double-blind,
placebo-controlled,
crossover
30 Individuals age 55–79
yr
Treatment-emergent
adverse events
October
2016
NCT04040959 II NR 1 g/d or
placebo
3 mo Randomized,
double-blind,
placebo-controlled
118 Individuals age 35–80
yr with chronic kidney
disease stage III or IV
Arterial stiffness
(carotid–femoral
pulse wave velocity)
September
2024
NCT03821623 II NR 1 g/d or
placebo
3 mo Randomized,
double-blind,
placebo-controlled
118 Individuals age ≥50 yr
with systolic blood
pressure between
120 and 139 mmHg
Resting systolic
blood pressure
December
2023
NCT04528004 I NR dose escalation
to 1 g/d or placebo
14 d Randomized,
double-blind,
placebo-controlled
40 Adults with end-stage
heart failure NYHA
class IV
Whole blood NAD
+
concentrations
August
2024
NCT04407390 II NR 1 g/d or
placebo
14 d Randomized,
double-blind,
placebo-controlled
100 Individuals age ≥70 yr
with COVID-19
Hypoxic respiratory
failure
May 2022
NCT04818216 II NR 1 g/d or
placebo
10 d Randomized,
double-blind,
placebo-controlled
100 Adults hospitalized
with COVID-19 and
acute kidney injury
Whole blood NAD
+
concentrations,
adverse events,
thrombocytopenia
June 2023
NCT04573153 II/III NR + serine +
L-carnitine tartrate +
N-acetylcysteine +
hydroxychloroquine
vs. placebo +
hydroxychloroquine
14 d Randomized,
placebo-controlled
400 Adults with
COVID-19,
ambulatory and
symptomatic
Hospitalization rate March
2021
(continued on next page)
Trends in Immunology
Trends in Immunology, April 2022, Vol. 43, No. 4 287
Primary endpoint Completion
Cognitive functioning
measured by
executive functioning
and memory
composite scores
December
2022
Trends in Immunology
haveopposing rolesinmacrophageactivation[58].PARP9 activatesIFNγ–STAT1 signaling and
induces proin?ammatory activation while PARP14 ADP-ribosylation reduces STAT1 phos-
phorylationinIFNγ-treatedhumanmacrophages[58].,Additionally,thenucleocapsidproteins
of several coronaviruses, including SARS-CoV, MERS-CoV, and MHV, are ADP-ribosylated in
infected cells, presumably by PARPs, which may indicate a common use of this pathway
among viruses. However, the functional consequences of such ADP‐ribosylation remain to
be investigated [57]. We argue that since PARP enzymatic activity requires NAD
+
[44], main-
taining a suf?cient NAD
+
concentration may be crucial for achieving PARP-related antiviral
mechanisms.
Sirtuins also play a role in antiviral defenses [43,60–64]. Indeed, sirtuin 1 (SIRT1) KO or inhibition
promotes the lifecycle and replication of vesicular stomatitis virus in mouse embryonic ?broblasts
(MEFs) and Kaposi’s sarcoma-associated herpesvirus in human lymphoma cell lines [61,62].
Disruption of SIRT1 also increases HPV16 E1–E2 replication [60]. Moreover, knockdown via
siRNA of each of the seven sirtuins in human ?broblast cells promoted the growth of a di-
verse set of human viruses after infection, including human cytomegalovirus (CMV), HSV1,
adenovirus type 5, and in?uenza virus (H1N1) [63]. Furthermore, SIRT1-activating drugs
such as resveratrol and CAY10602 have inhibited the replication of these viruses [63].
SIRT6 promotes tumor necrosis factor (TNF)α secretion, as evidenced from the suppression
of TNFα release from SIRT6 KO MEFs, whereby TNFα secretion would be expected to pro-
mote the eradication of pathogens [64]. Indeed, pharmacological inhibition and siRNA
knockdown of SIRT6 and NAMPT, an NAD
+
-synthetic enzyme (Figure 1), in mouse ?bro-
Table 1. (continued)
Clinicaltrials.
gov ID
Phase Interventions Duration Type Enrollment
(participants)
Inclusion criteria
NCT04809974 IV NR 2 g/d or
placebo
22 wk Randomized,
double-blind,
placebo-controlled
100 Individuals age 18–65
yr, 2+ mo out from
COVID-19 PCR
diagnosis, currently
PCR negative, with
persistent cognitive
and physical
dif?culties
(long-COVID)
blasts promoted CMV replication [65].
Other NAD
+
-utilizing enzymes that also play roles during immune responses include CD38
[66], BST1 [2], and SARM1 [43]. Indeed, CD38 and BST1 are highly expressed on the sur-
face of macrophages and lymphocytes and produce extracellular cyclic(ADP-ribose), a
calcium-mobilizing second messenger that is important for immune cell activation [2,66,67].
SARM1, another potent NADase, contains a Toll/IL-1 receptor domain, which might elicit
neuroprotective innate immune responses, as suggested from mouse models of neurode-
generation [68].
Viruses ?ghting back
One antiviral mechanism that is associated with PARP activity is the formation of cytosolic,
membraneless organelles called stress granules (SGs) to sequester viral RNAs and arrest pro-
viraltranslation[69,70].Indeed,ADP-ribosylationofSGcomponentsbyPARPspromotesthefor-
mation of antiviral SGs [71–73]. Several antiviral PARPs including PARP5a, 12, 13, 14, and 15
localize to SGs in human cells, presumably to execute this modi?cation [71].
288 Trends in Immunology, April 2022, Vol. 43, No. 4
Trends in Immunology
As a counter-mechanism to the host, many viruses, including coronaviruses and alphaviruses,
deploy ADP-ribosylhydrolases to remove SG-promoting ADP-ribose modi?cations [74–77].
SARS-CoV-2 has such a domain with mono-ADP-ribosylhydrolase activity in its largest
encoded protein, nonstructural protein 3 (NSP3) [79,80]. Ectopic expression of the SARS-
CoV-2NSP3macrodomainreversesPARP9-dependent ADP-ribosylationoftargetproteins
[81]. In live viruses, this domain is important for both viral replication and virulence. Mutations
of this domain in MHV and SARS-CoV led to attenuated viral replication, and thus, the mutant
viruses were unable to cause lung disease in infected mice, while treatment with pan-PARP in-
hibitors enhanced SARS-CoV replication and inhibited IFN production in BMDMs infected
with macrodomain-de?cient mutant coronavirus [41,82]. These ?ndings suggest that the
SARS-CoV-2 NSP3 protein might also interfere with SG formation and thereby allow for
evasion of cellular antiviral responses (Figure 2). We argue that providing more NAD
+
to
fuel the activity of PARPs might shift the antiviral balance back in favor of host cells.
Viruses can drive uncontrolled in?ammation
One of the potential catastrophic effects of SARS-CoV-2 infections is cytokine storms. Indeed,
the concentrations of circulating proin?ammatory factors such as IL-1, IL-6, and TNFα are
strongly associated with ICU admission and mortality in COVID-19 patients [83,84]. Studies of
the ?rst SARS pandemic coronavirus, SARS-CoV, have shown that viral proteins play an active
role in the processing and release of the two speci?cproin?ammatory cytokines, IL-1β and
IL-18, by enhancing NF-κB transcriptional activity and promoting the formation of the NLRP3
in?ammasome (Figure 2)[85–90].
The SARS-CoV proteins that facilitate these processes include open reading frame (ORF)3a,
envelope protein (E), and ORF8b [85–90]. ORF3a mediates NF-κB activation through
TRAF3-dependent ubiquitination of an NF-κB inhibitory subunit, and mediates speck formation
of the in?ammasome subunit ASC, which accompanies assembly of the NLRP3 in?ammasome
in human cells [86]. Additionally, ORF3a has transmembrane domains and ion channel (IC)
activity that drives K
+
ef?ux, which further promotes activation of the NLRP3 in?ammasome.
Indeed, IL-1β secretion was completely blocked when BMDMs, stimulated with lentiviruses
expressing the SARS-CoV ORF3a, were treated with K
+
-rich medium [78,85]. The SARS-CoV E
protein also promotes in?ammasome activation through its intracellular activity; it forms lipid–protein
channels at the endoplasmic reticulum (ER)–Golgi intermediate compartment, and the resulting Ca
2
+
stimulates the activation of the NLRP3 in?ammasome. The E protein also promotes IL-1β release
in mammalian cells expressing the NLRP3 in?ammasome, while E protein mutants lacking ion
conductance cannot boost IL-1β secretion [87,88]. ORF8b causes in?ammasome activation in
human macrophages and interacts directly with NLRP3 in vitro; moreover, it can form protein
aggregates and trigger ER stress and lysosomal damage that activate the in?ammasome in HeLa
cells [89]. These processes not only promote virulence but may also facilitate viral replication
[87,90]. For example, a comparative study of the functional motifs included within the SARS-CoV
viroporins showed that full-length E and ORF3a proteins were required for maximal SARS-CoV
replication and virulence in infected mice [87].
As with SARS-CoV, SARS-CoV-2 can also activate the in?ammasome [91]. Active NLRP3
in?ammasomes have been found in peripheral blood mononuclear cells (PBMCs) and tissues
from deceased COVID-19 patients upon autopsy, along with higher concentrations of serum
IL-18 which correlated with COVID-19 severity. SARS-CoV-2 may activate the in?ammasome
through mechanisms similar to those of SARS-CoV [91]. Overexpression of SARS-CoV-2
ORF3a in human cells can induce K
+
ef?ux, NLRP3 activation, and IL-1β release. Restricting
K
+
ef?ux with K
+
-rich media impairs the ability of ORF3a to trigger NLRP3 in?ammasome
Trends in Immunology, April 2022, Vol. 43, No. 4 289
Trends in Immunology
assembly, as evidenced from coimmunoprecipitation (co-IP) assays [92]. SARS-CoV-2 N
protein can interact directly with NLRP3, supported by results of reciprocal co-IP and confocal
microscopy, thus promoting in?ammasome activation and IL-1β release in cells. In Nlrp3
+/+
mice infected with adeno-associated virus (AAV)-Lung-N, serum IL-1β concentrations were in-
creased (detected via ELISA), whereas IL-1β was not induced by AAV-N in the sera of
Nlrp3
?/?
mice [93]. The higher cytokine concentrations stemming from NLRP3 activation
Trendsrends inin ImmunologyImmunology
Figure 2. Regulation oftheNLRP3in?ammasomebySARS-CoVinfection and NAD
+
. Severalproteinsencodedby
SARS-CoV promote NLRP3 in?ammasome activity and the release of proin?ammatory cytokines. ORF3a activates NF-κB
through TRAF3-dependent ubiquitination, which facilitates ASC speck formation and the assembly of NLRP3
in?ammasome [86]. ORF3a also has transmembrane domains and ion channel activity that drives a K
+
ef?ux [85].
E protein is located at the ER–Golgi compartment and promotes Ca
2+
ef?ux [87,88]. ORF8bdirectly interacts with
NLRP3. These mechanisms all activate the in?ammasome [89]. Host cell SIRT1, SIRT2, and SIRT3 all suppress the
NLRP3 in?ammsome [94–98]. SIRT1 deacetylates NF-κB, suppressing its activity, and reduces oxidative stress,
decreasing in?ammasome activation. SIRT2 deacetylates NLRP3. SIRT3 suppresses mitochondrial ROS production,
decreasing in?ammasome activation. SIRT6 reduces in?ammation via H3K9 deacetylation in the promoters of NF-κB
target genes [99]. PARPs promote antiviral SG formation through ADP-ribosylation of SG components [71–73].
Created with BioRender.com. Abbreviations: Ac, acetylation;ACE-2,angiotensin-convertingenzyme2;ASC,
apoptosis-associated speck-like protein containing a caspase recruitment domain Casp1, caspase-1; ER,
endoplasmic reticulum; NF-κB, nuclear factor κB; NLRP3, NOD-, LRR-, and pyrin domain-containing protein 3; ORF,
open reading frame; SARS-CoV, severe acute respiratory syndrome coronavirus 1; SIRT, sirtuin; TRAF3, TNF
receptor-associated factor 3; Ub, ubiquitination.
290 Trends in Immunology, April 2022, Vol. 43, No. 4
Trends in Immunology
suggest an increased likelihood of uncontrolled in?ammation in response to SARS-CoV-2
infection.
NAD
+
-consuming enzymes in anti-in?ammatory mechanisms
NAD
+
may contribute to the resolution of in?ammation, and to limiting or preventing the effects of
cytokine storms; it might do so by increasing the activity of sirtuins (Figure 2)[44]. SIRT1, SIRT2,
and SIRT3 all suppress the activity of NF-κB and the NLRP3 in?ammasome via multiple mecha-
nisms [94–98]. From a biochemical standpoint, SIRT1 physically interacts with and deacetylates
NF-κB and thereby suppressesitstranscriptionalactivity in humanepithelialcells[95]. SIRT1also
inhibits lipopolysaccharide-induced NLRP3 in?ammasome activation by reducing oxidative
stress, as demonstrated in human trophoblasts with an shRNA knockdown of SIRT1 [94].
SIRT2 directly deacetylates NLRP3 and inactivates the NLRP3 in?ammasome, as shown in
SIRT2 KO (Sirt2
-?
) mouse macrophages [97]. SIRT3 mediates in?ammasome activation by sup-
pressingmitochondrialreactiveoxygenspecies(ROS)production,whichcanactivatetheNLRP3
in?ammasome. SiRNA depletion of SIRT3 in human macrophages results in increased ROS
production and in?ammasome activation compared with controls [98].SIRT6alsopromotes
the resolution of in?ammation via histone H3 lysine 9 (H3K9) deacetylation in the promoters
of NF-κB target genes [99]. However, the effects of sirtuins on the NLRP3 in?ammasome have
yet to be extensively studied in the context of viral infection.
WepositthatNAD
+
boosters that suppress NF-κBandNLRP3in?ammasome activity might
also represent potential treatments to ameliorate the in?ammatory symptoms of COVID-19.
Indeed, treatment with NR, an NAD
+
precursor, for 24 hours has been reported to decrease
thereleaseofIL-1β and blunt in?ammasome activation in PBMCs from healthy and fasted
individuals [98]. NR also promotes SIRT1 expression, suppresses NLRP3 expression, and
reduces secretion of proin?ammatory factors TNFα and IL-6 in mouse hepatocytes [100].
Another NAD
+
precursor, nicotinic acid, also reduced in?ammation in a rodent model of type
2 diabetes (KK/HIJ mice) by modulating NLRP3 activity [26]. However, consideration should
be given to the fact that some studies show that extracellular nucleotides such as NAD
+
can
also promote in?ammation via the activation of P2X7 receptors in mouse macrophages and
T cells [101].
The promise of NAD
+
boosters in the clinic
Adding more fuel
Given that low NAD
+
concentrations might exacerbate COVID-19 severity, boosting the levels of
this metabolite in at-risk populations is one potential therapeutic strategy for mitigating severe
disease. The most straightforward way to raise NAD
+
concentrations is to provide additional pre-
cursors to boost NAD
+
synthesis [2]. Canonically, NAD
+
cannot be taken up directly by the cell,
but its precursors can [102,115]. The NAD
+
precursors nicotinic acid, NR, and NMN, all have pu-
tative transporters, high safety thresholds, and are orally bioavailable [2]. They do, however, vary
intheirpharmacokineticpro?lesandseemtoraiseNAD
+
concentrationstodifferentextentsindif-
ferent tissues [103], although this remains an area of active investigation.
Structurally, the closest molecule to NAD
+
is NMN, requiring only one enzymatic step to be con-
verted to NAD
+
. In a double blind, placebo-controlled trial, NMN was shown to increase insulin
sensitivityinprediabeticwomen [28] (NCT03151239;Table1). It iscurrently being studiedinhos-
pitalized COVID-19 patients with hypertension and metabolic syndrome for its effects on fatigue,
duration of hospitalization, and viral load (NCT05175768; Table 1). Ongoing clinical trials with
NMN are also investigating its effect on hypertension and postexercise muscle recovery
(NCT04903210 and NCT04664361; Table 1).
Trends in Immunology, April 2022, Vol. 43, No. 4 291
Outstanding questions
Why are NAD
+
tissue levels reduced
during aging? Many hypotheses exist
to date, but there is currently no
consensus, especially as multiple
mechanisms may be involved.
ArelowNAD
+
concentrations associated
with severe COVID-19? There is lit-
tle data to date showing a direct
association.
CanraisingNAD
+
tissueconcentrations
reduce COVID-19 symptoms, severity,
and mortality in the elderly and in the
young?
Can NAD
+
boosters reduce COVID-19
severity after the onset of symptoms,
or only prophylactically?
What might be the best approach for
raising NAD
+
concentrations in the
clinic: would this be NMN, NR, activa-
tors of NAD
+
biosynthesis, or inhibitors
of enzymes that consume NAD
+
?
What role does NAD
+
playinpromoting
SG-dependent antiviral mechanisms?
How does SARS-CoV-2 modulate
NAD
+
andNSP3toincreasevirulence?
Trends in Immunology
NR, which is two enzymatic steps removed from NAD
+
, is also being studied clinically. In one
placebo-controlled trial, oral NR reduced circulating concentrations of in?ammatory cytokines
IL-2, IL-5, and IL-6 [104] (NCT02950441; Table 1). In another trial, oral NR reduced blood pres-
sure and possibly aortic stiffness in healthy middle-aged and older adults [105] (NCT02921659;
Table 1). At the time this review was written, over 30 active studies testing NR had been regis-
tered on clinicaltrials.gov, including many for COVID-19 risk factors such as vascular disease,
hypertension, and heart failure (NCT04040959, NCT03821623, and NCT04528004; Table
1). At least four ongoing studies are directly testing NR in COVID-19 patients, some in patients
with particular risk factors such as advanced age or acute kidney injury, with readouts such as
rates of hospitalization, respiratory failure, and long-COVID symptoms (NCT04407390,
NCT04818216, NCT04573153, and NCT04809974; Table 1).
Other approaches to raising NAD
+
concentrations
Another approach to raising NAD
+
concentrations in humans is to inhibit the activity of NAD
+
-
consuming enzymes. For instance, as PARP1 is a major consumer of NAD
+
in cells [42], PARP1
inhibitors such as olaparib have been extensively studied for the treatment of breast and ovarian
cancer, given that PARP1 is synthetically lethal with BRCA mutations [106]. Regarding COVID-19,
several PARP1 inhibitors, CVL218 and stenoparib, were recently shown to limit SARS-CoV-2
replication and proin?ammatory cytokine production in human PBMCs and lung cells. One
hypothesized mechanism of action is via inhibition of PARP1-dependent NAD
+
depletion [107,116].
Several inhibitors of other NAD
+
-consuming enzymes are in preclinical development, including
against CD38 [108,109], SARM1 [110], and ACMSD (which consumes a precursor of NAD
+
in
the de novo pathway) [20]. In humans, treatment with luteolin, a naturally occurring CD38
inhibitor, reduced serum TNF and IL-6 concentrations [111].
The activation of NAD
+
biosynthetic enzymes is another strategy to raise NAD
+
concentrations; a
small molecule NAMPT activator, SBI-797812, is currently in preclinical development for cardio-
vascular and metabolic disease [112]. Finally, a virally‐encoded protein is a potential target that
interfereswithNAD
+
metabolism:themacrodomainofNSP3canantagonizetheantiviralactivities
of host PARPs,although the implications of this activity entail further investigation for SARS-CoV-
2[41,80–82]. A few inhibitors of NSP3 were recently identi?ed in a chemical screen and were
shown to exhibit antiviral properties against Chikungunya virus in human epithelial cells [113].
Concluding remarks
NAD
+
metabolism appears to be linked to infections of SARS-CoV-2 and other viruses via multiple
lines of evidence, epidemiological and mechanistic. Further research is warranted to better under-
stand its mechanistic role and utility as a target of intervention (see Outstanding questions). Clinical
translation of basic scienti?c discoveries is dif?cult, and there are many examples of antiviral and
anti-in?ammatory molecules that have shown early promise but have not materialized. However,
while human studies are still at an early stage, we are excited about the promise of interventions
thatmodulatetheconcentrationsofNAD
+
ortheactivityofNAD
+
-consumingenzymes.Suchinter-
ventions may soon have a place in our arsenal to ?ght current and future viral infections.
Declaration of interests
D.A.S. is a founder, equity owner, advisor to, director of, board member of, consultant to, investor in and/or inventor on
patents licensed to GlaxoSmithKline, Segterra, Animal Biosciences, AFAR, Cohbar, Galilei, Zymo Research, Immetas,
EdenRoc Sciences and af?liates (Arc-Bio, Dovetail Genomics, Claret Bioscience, MetroBiotech, Astrea, Liberty Biosecurity
and Delavie), Life Biosciences, and Levels Health. D.A.S. is an inventor on a patent application licensed to Elysium Health.
More at https://sinclair.hms.harvard.edu/david-sinclairs-af?liations.
292 Trends in Immunology, April 2022, Vol. 43, No. 4
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type 2 diabetes and neuropathy in mice. Sci. Rep. 6, 26933
49. Zingarelli, B. et al. (2003) Inhibitors of poly (ADP-ribose) poly-
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