Hindawi
Journal of Immunology Research
Volume 2022, Article ID 8873536, 13 pages
https://doi.org/10.1155/2022/8873536
Research Article
The Pharmacologically Active Alkaloid Cryptolepine Activates a
Type 1 Interferon Response That Is Independent of MAVS and
STING Pathways
Seth A. Domfeh ,1,2,3 Patrick W. Narkwa ,4 Osbourne Quaye ,1,2 Kwadwo A. Kusi ,1,2,5
Bright S. Addy ,3 Sian Lant,6 Rebecca P. Sumner ,6 Carlos Maluquer de Motes ,6
Gordon A. Awandare ,1,2 Charles Ansah ,7 and Mohamed Mutocheluh 4
1West African Centre for Cell Biology of Infectious Pathogens, University of Ghana, Legon, Ghana
2Department of Biochemistry, Cell and Molecular Biology, School of Biological Sciences University of Ghana, Legon, Ghana
3Department of Biochemistry and Biotechnology, College of Science, Kwame Nkrumah University of Science and Technology,
Kumasi, Ghana
4Department of Clinical Microbiology, School of Medicine and Dentistry, Kwame Nkrumah University of Science and Technology,
Kumasi, Ghana
5Department of Immunology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana,
Legon, Ghana
6Department of Microbial Sciences, University of Surrey, Guildford, UK
7Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah
University of Science and Technology, Kumasi, Ghana
Correspondence should be addressed to Mohamed Mutocheluh; mutocheluh@gmail.com
Received 10 March 2022; Revised 25 May 2022; Accepted 1 July 2022; Published 26 July 2022
Academic Editor: Kai Wang
Copyright © 2022 Seth A. Domfeh et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Type 1 interferons (IFN-1) are pleiotropic cytokines with well-established anticancer and antiviral properties, particularly in
mucosal tissues. Hence, natural IFN-1-inducing treatments are highly sought after in the clinic. Here, we report for the first
time that cryptolepine, a pharmacoactive alkaloid in the medicinal plant Cryptolepis sanguinolenta, is a potent IFN-1 pathway
inducer. Cryptolepine increased the transcript levels of JAK1, TYK2, STAT1, STAT2, IRF9, and OAS3, as well as increased the
accumulation of STAT1 and OAS3 proteins, similar to recombinant human IFN-α. Cryptolepine effects were observed in
multiple cell types including a model of human macrophages. This response was maintained in MAVS and STING-deficient
cell lines, suggesting that cryptolepine effects are not mediated by nucleic acids released upon nuclear or organelle damage. In
agreement, cryptolepine did not affect cell viability in concentrations that triggered potent IFN-1 activation. In addition, we
observed no differences in the presence of a pharmacological inhibitor of TBK1, a pleiotropic kinase that is a converging point
for Toll-like receptors (TLRs) and nucleic acid sensors. Together, our results demonstrate that cryptolepine is a strong inducer
of IFN-1 response and suggest that cryptolepine-based medications such as C. sanguinolenta extract could be potentially tested
in resource-limited regions of the world for the management of chronic viral infections as well as cancers.
1. Introduction of microbial molecular patterns by the cellular pattern rec-
ognition receptors (PRRs). Cytosolic RNA is normally
Type 1 interferons are a family of pleiotropic cytokines and sensed by the melanoma differentiation-associated gene 5
the main products of the innate immune response. IFN-1 (MDA5) and retinoic acid-inducible gene 1 (RIG-1) which
are produced by several cells typically via the recognition leads to the phosphorylation of TANK-bind kinase 1
2 Journal of Immunology Research
(TBK1) through MAVS (mitochondrial antiviral-signalling malaria [21]. Although studies have investigated some of
protein) reviewed in [1]. Cytosolic DNA, on the other hand, the pharmacological properties of cryptolepine [22–24],
is mostly sensed by the cyclic GMP-AMP synthase (cGAS) there is still scanty data on the immunomodulatory effects
that generates the cyclic GMP-AMP (cGAMP) resulting in of cryptolepine specifically its effect on the IFN-1 response
the activation of TBK1 through STING (stimulator of IFN pathway.
genes protein) [2]. The activated TBK1 from these sensing Here, we established the mechanisms via which cryptole-
pathways phosphorylates the IFN-regulatory factor 3 (IRF3) pine modulates the IFN-1 response pathway. We observed
leading to the release of IFN-1 from the infected cells [3]. no differences in the IFN-1 response activation by cryptole-
After their release, the IFN-1 interact with IFN-α recep- pine in the absence of MAVS and STING, as well as
tors (IFNARs) on the infected and nearby cells resulting in impaired TBK1 signalling. Our findings provide evidence
the activation of the Janus kinase-signal transducer and acti- of the immune-boosting properties of C. sanguinolenta. This
vator of transcription (JAK-STAT) pathway. This causes the supports further studies to evaluate and potentially repur-
expression of numerous genes containing IFN-sensitive pose the beneficial effects of this plant for the treatment of
response elements (ISRE) in their promoter regions. These chronic viral infections as well as cancers.
IFN-stimulated genes (ISGs) such as 2′5′-oligoadenylate
synthase (OAS) and IFN-induced protein with tetratrico- 2. Materials and Methods
peptide repeats (IFIT), among others, are reviewed in [4].
These ISGs facilitate the antiviral and anticancer effects of 2.1. Chemicals and Reagents. The cryptolepine used in this
IFN-1 reviewed in [5]. Moreover, the IFN-1 pathway exerts study was prepared and stored as we have previously
an anti-inflammatory response via the reduction in the levels described [24]. It was isolated and purified from the roots
of interleukin 1 (IL-1) and other inflammatory mediators of C. sanguinolenta by Professor Kwesi Boadu Mensah as
[6]. Hence, impaired IFN-1 response has been reported to described [25]. Recombinant Human Interferon Alpha 2b
be allied to the increased susceptibility to viral infections (IFN-α) (cat # 11105-1) was procured from PBL Assay Sci-
and cancers [7]. ence (Piscataway, USA). HSV-60/LyoVec (cat # tlrl-hsv60c),
IFN-1 are well-known immune mediators in the fight 3′3 ′-cGAMP Fluorinated (cat # tlrl-nacgaf), and Poly(I:C)-
against viral infections, cancer, and autoimmune diseases HMW/LyoVec (cat # tlrl-piclv) were purchased from Invi-
and have a key role in immune protection in mucosal tissues vogen (San Diego, USA). GSK8612 (cat # HY-111941) was
[8, 9], the first line of defence against many pathogens. The purchased from MedChemExpress (New Jersey, USA).
integral role of IFN-1 in the mucosal barrier functions and TransIT-2020 Transfection Reagent (cat # MIR 5400) was
immunity regulation has been demonstrated using animal purchased from Mirus (Madison, USA). Dual Luciferase
models lacking IFN signalling, which fails to develop suitable Reporter Assay System (cat # E1960) was procured from
immune responses to pathogenic microbes or their products Promega (Madison, USA). Phorbol 12-myristate 13-acetate
[10]. The unique biological effects of endogenous IFN-1 (cat # P1585), fludarabine (cat # F9813), and thiazolyl blue
have generated considerable interest in the use of recombi- tetrazolium bromide (MTT) powder (cat # M5655-1G) were
nant IFNs in the treatment of cancers, viral infections, and procured from Sigma-Aldrich (St. Louis, USA). Anti-STAT1
immunological disorders [11, 12]. Although recombinant antibody (cat # ab210524), anti-OAS3 antibody (cat #
IFNs are effective, they have been reported with immuno- ab154270), goat anti-rabbit IgG H&L (HRP) (cat #
suppressive effects [13] and are also associated with numer- ab205718), and anti-beta actin antibody (cat # ab8227) were
ous adverse effects including fatigue, hepatotoxicity, retinal purchased from Abcam (Cambridge, USA). Anti-IFN-α/
vascular complication, and biphasic thyroid dysfunction βRα antibody (cat # sc-7391) was procured from Santa Cruz
[14, 15]. Further, in low- and middle-income countries Biotechnology (Texas, USA). Pierce enhanced chemilumi-
(LMICs), IFN-based therapies are hindered by the unavail- nescence (ECL) western blotting substrate (cat # 32109),
ability and high cost [16]. Owing to these limitations, there radioimmunoprecipitation assay (RIPA) buffer (cat #
is a high interest in natural therapies that are less toxic, 89900), protease and phosphatase inhibitor cocktail (cat #
cheaper, and culturally acceptable [17]. 78440), ethylenediaminetetraacetic acid (cat # 78440), Gene-
Most people residing in LMICs use medicinal plants for JET RNA purification kit (cat # K0732), Maxima first-strand
their basic health care needs [18]. Moreover, these medicinal cDNA synthesis kit with dsDNase (cat # K1672), and ABso-
plants are easily accessible in these countries [19]. However, lute qPCR ROX mix (cat # AB1139A) were purchased from
an important limitation in our ability to exploit these ThermoFisher Scientific (Massachusetts, USA). The chemi-
medicinal plants therapeutically is the absence of molecular cals and reagents were prepared in line with the manufactur-
studies revealing their immunomodulatory properties. Cryp- ers’ instructions.
tolepine is the main biologically active alkaloid in the West
African medicinal plant Cryptolepis sanguinolenta (Lindl.) 2.2. Cell Culture. The human monocytic leukaemia (THP1)
Schlechter (Apocynaceae). This plant has been reported to cells stably expressing the interferon-induced protein with
exhibit a plethora of pharmacoeffects including antimalarial, tetratricopeptide repeats 1- (IFIT1-) driven Gaussia lucifer-
antihyperglycaemic, antibacterial, and anti-inflammatory ase gene (THP1-IFIT1-GLuc cells (parent cells), THP1-
effects in diverse animal models reviewed in [20]. Owing to IFIT1-GLuc-MAVS−/− cells (MAVS KO cells), and THP1-
the convincing pharmacological properties of cryptolepine, IFIT1-GLuc-STING−/− cells (STING KO cells)) have been
C. sanguinolenta is used in Ghana for the treatment of previously described [26–30]. How the various THP1 cells
Journal of Immunology Research 3
(parent and KO (knockout) cells) were modified and vali- was further validated by resolving on 1% agarose gel. The
dated have been described previously [27, 29] (Supplemen- complementary DNA (cDNA) was synthesized using the
tary data S3). Also, human hepatoma cells (HepG2, ATCC Maxima first-strand cDNA synthesis kit with dsDNase in
HB‐8065), as well as human embryonic kidney cells line with the manufacturer’s instructions. The target genes
(HEK293, ATCC CRL-1573), were bought from the Ameri- (JAK1, TYK2, STAT1, STAT1, IRF9, and OAS3) were ampli-
can Type Culture Collection (ATCC, Manassas, USA). The fied using the ABsolute qPCR ROX mix with β-actin
HepG2 and HEK293 cells were used to buttress some of (ACTB) as an endogenous control. The primers and probes
the results obtained using the THP1 cells (Supplementary used in this study were synthesized by Biomers, Germany
data S1, S2, S4–S7). The THP1 cells were cultured in the (Supplementary data S8). The amplification was done using
RPMI‐1640 Medium (ATCC) supplemented with 0.05mM the StepOnePlus Real-Time PCR System (Applied Biosys-
2-β‐mercaptoethanol (Bio-Rad, California, USA, cat # tems, USA). After the initial holding for 10 minutes at
1610710), 100μg/mL streptomycin and 100 IU/mL penicillin 95°C, the PCR reaction was followed by 40 cycles for 15 sec-
(Gibco Life Technologies Ltd., Paisley, UK, cat # 15140122), onds at 95°C and 60 seconds at 60°C. The relative quantifica-
and 10% foetal bovine serum (FBS, ATCC 30-2020) to con- tion of the target genes was calculated using the comparative
stitute the complete growth medium. However, the HepG2 CT method. The relative levels of the target genes after the
and HEK293 cells were grown in Eagle’s Minimum Essential normalisation to the endogenous gene (ACTB) were derived
Medium (EMEM, ATCC) containing 1% MEM nonessential from the 2-ΔΔCT values as done previously [31].
amino acids (ScienCell, Carlsbad, USA, cat # 0823), 100μg/
mL streptomycin, 100 IU/mL penicillin, and 10% FBS to 2.6. Western Blotting. The protein levels of STAT1 and
make the complete growth medium. All the cells (THP1, OAS3 were assessed using western blot to corroborate the
HepG2, and HEK293) were cultured under a humidified expression of the mRNA levels. This is because the mRNA
condition at 37°C and in 5% carbon dioxide. levels do not always translate into proteins [32, 33]. The
protein level of β-actin (ACTB) was included as a loading
2.3. Cytotoxicity Assay. All the THP1 cells (parent, MAVS, control. The parent THP1 cells were differentiated into mac-
and STING KO cells) were seeded each at 8 × 104 cells per rophages and treated as in the RT-qPCR. The total proteins
well in a 100μL complete growth medium containing were extracted using an ice-cold RIPA buffer which contains
100nM PMA (phorbol 12-myristate 13-acetate) in 96-well a protease and phosphatase inhibitor cocktail, as well as eth-
plates. At 72-hour postdifferentiation, the derived macro- ylenediaminetetraacetic acid, following the manufacturer’s
phages were treated with 0.5–4μM cryptolepine in a instructions. The extracted proteins were denatured in a
100μL PMA-free complete growth medium. The viability sample buffer containing 2-beta mercaptoethanol. The dena-
of the cells was assessed after 24-hour posttreatment using tured proteins were resolved using 4–20% gradient poly-
an MTT-based assay as we have previously described [24]. acrylamide gel and further transferred onto polyvinylidene
difluoride (PVDF) membrane. The membrane was blocked
2.4. Luciferase Reporter Gene Assay. All the THP1 cells (par- in 5% nonfat dried milk in 1X Tris-buffered saline contain-
ent, MAVS, and STING KO cells) were seeded at 8 × 104 ing Tween-20 (TBST). The proteins of interest were probed
cells per well in a 100μL complete growth medium using the primary specific antibodies: anti-STAT1 antibody
containing 100 nM PMA in 96-well plates. At 72-hour post- (1 : 1000), anti-OAS3 antibody (1 : 1000), and anti-ACTB
differentiation, the derived macrophages were cultured with antibody (1 : 1000) in 3% nonfat dried milk in 1X TBST.
cryptolepine alone or together with fludarabine (STAT1 The PVDF membrane was incubated with the primary
inhibitor), GSK8612 (TBK1 inhibitor), anti-IFNAR1 block- antibodies for 18 hours at 4°C. The PVDF membrane
ing antibody, Poly(I:C) (an agonist of RIG-1/MDA5-MAVS was then probed with the goat anti-rabbit IgG H&L
axis), HSV-60 (an agonist of cGAS-STING axis), and IFN-α (HRP) secondary antibody (1 : 20000) at room temperature
(an agonist of JAK-STAT axis) when necessary in 100μL for 60 minutes. The PVDF membrane was incubated with
PMA-free complete growth medium. After 24 hours of incu- the Pierce enhanced chemiluminescence western blotting
bation, the Gaussia luciferase activity (which is the IFIT1 substrate to visualise the bands. The images were taken
induction and the measure of the IFN-1 pathway response) using the C-DIGIT blot scanner (Li-COR Bioscience,
was assessed in the presence of coelenterazine (2μg/mL) as USA) and further assessed using the ImageJ software
previously done [30]. (National Institute of Health).
2.5. Reverse Transcriptase-Quantitative Polymerase Chain 2.7. Statistical Analysis. The data were analysed using Micro-
Reaction (RT-qPCR). The parent THP1 cells were differenti- soft Excel (version 2019) where applicable. The viability of
ated into macrophages at 2 × 106 cells per well in a 6-well the cells was assessed as previously described [24]. The IFIT1
plate and cocultured in the presence of 60μM fludarabine induction fold was derived from the ratio of IFIT1 induction
and cryptolepine (2 and 4μM) or 400 IU/mL IFN-α (positive (which is the IFN-1 pathway induction) in the cells cultured
control). At 24-hour postculture, the total RNA was with cryptolepine or the ligands to the cells cultured without
extracted with the GeneJET RNA Purification Kit in line cryptolepine or the ligands [17]. Assessments among multi-
with the manufacturer’s protocols. The purity and yield of ple treatments were done using the one-way analysis of var-
the extracted RNA were assessed by spectroscopy (Nano- iance (ANOVA) and Bonferroni’s test for the post hoc
Drop 1000, Thermo Scientific), and the quality of the RNA analysis, whereas assessments between two treatments were
4 Journal of Immunology Research
45.0 120.0
⁎⁎⁎⁎
⁎⁎
⁎⁎
30.0 ⁎ ⁎⁎ 80.0
⁎⁎
⁎⁎⁎
⁎⁎
15.0 ⁎ 40.0
⁎
⁎
ns
0.0 0.0
CRYPT (𝜇M) – 0.5 1 2 4 – 0 1 2 3 4
IFN-𝛼 (IU/mL) – – – – – 400 Cryptolepine (𝜇M)
(a) (b)
Figure 1: Cryptolepine activates IFN-1 response in THP1-derived macrophages. The derived macrophages from the parent THP1 cells were
cultured with 0.5–4 μM cryptolepine (CRYPT). IFN-α (400 IU/mL) was employed as an experimental positive control. The IFIT1 induction
(a), as well as cell viability (b), was evaluated after 24 h. Data are shown as the means, from three varied experiments with each done in
triplicate, and error bars represent the standard deviations. ∗p < 0:05, ∗∗p < 0:01, ∗∗∗p < 0:001, and ∗∗∗∗p < 0:0001; ns: difference not
significant (one-way ANOVA and Bonferroni’s test).
conducted using Student’s t-test. In all comparisons, a p cological inhibitor of STAT1, suggesting that cryptolepine
value less than 0.05 was considered statistically significant. effects were STAT1 dependent.
3. Results 3.3. Cryptolepine Increases the Protein Levels of STAT1 and
OAS3 in THP1-Derived Macrophages. We then assessed
3.1. Cryptolepine Activates the IFN-1 Pathway in a Dose- whether the transcriptional changes resulted in increased
Dependent Manner in Multiple Cells. To study the ability protein levels. Cryptolepine increased the protein levels of
of cryptolepine to induce IFN-1 responses, we initially used STAT1 (Figure 3(a)) and OAS3 (Figure 3(b)) in the derived
differentiated human THP-1 monocytes expressing Gaussia macrophages. In agreement with the previous results
luciferase (GLuc) under the control of the promoter of the (Figures 2(c) and 2(f)), the elevated STAT1 and OAS3 levels
IRF3- and ISRE-dependent IFIT1 gene [26]. Cryptolepine were reversed in the presence of fludarabine similar to the
at 0.5–4μM increased the IFIT1 induction (which is a IFN-α-positive control.
measure of the IFN-1 pathway response) in a dose-
dependent manner and with at least two-fold induction in
the presence of 1μM cryptolepine in the derived macro- 3.4. IFN-1 Pathway Activation in the Presence of
phages (Figure 1(a)). Recombinant human IFN-α 2b (here- Cryptolepine Does Not Require MAVS, STING, and TBK1
after referred to as IFN-α, JAK-STAT agonist) was in THP1-Derived Macrophages. IFN-1 are typically pro-
included in the experiment as a positive control. Moreover, duced by activation of PRRs such as TLRs or cytosolic recep-
the concentrations of cryptolepine used in this experiment tors. Engagement of IFN-1 with their cognate receptor
(0.5–4μM) were not toxic to the differentiated macrophages results in activation of the IFN-1 pathway. To establish
since the viability of the cells remained > 80% after 24 hours whether the effects of cryptolepine on the IFN-1 pathway
(Figure 1(b)). To determine whether the observed effects were mediated by cytosolic nucleic acid sensors, we tested
were unique to the human macrophage model used, we cryptolepine in THP-1 cells unable to respond to cytosolic
tested HepG2 and HEK293 cells. Similar patterns of IFN-1 RNA or DNA due to the absence of the main RNA and
pathway induction by cryptolepine were observed in these DNA sensors MAVS and STING (Supplementary data S3
cell lines (Supplementary data S1), suggesting that the cryp- and [27–29]). IFIT1 induction by cryptolepine was indistin-
tolepine mechanism of action is not cell type specific. guishable in the macrophages derived from the MAVS and
STING-deficient cells as compared to the parent cells
3.2. Cryptolepine Increases the mRNA Levels of JAK1, TYK2, (Figures 4(a) and 4(b)). As expected, MAVS-deficient cells
STAT1, STAT2, IRF9, and OAS3. To confirm the effects of were unable to respond to Poly(I:C) (a synthetic dsRNA
cryptolepine on the IFN-1 pathway, we assessed endogenous analogue) (Figure 4(a)), and STING-deficient cells were
gene expression by RT-qPCR. Cryptolepine increased the unable to respond to HSV-60 (a DNA motif derived from
expressions of key transcripts of the IFN-1 response pathway herpes simplex virus-1 genome) (Figure 4(b)). These results
such as JAK1, TYK2, STAT1, STAT2, and IRF9, as well as indicate that cryptolepine activates the IFN-1 response inde-
OAS3 (a well-established ISG), in a dose-dependent manner pendent of MAVS and STING signalling, suggesting that
(Figure 2). A similar increase in mRNA levels was observed cryptolepine does not trigger intracellular damage resulting
in HepG2 cells (Supplementary data S2). Except for JAK1 in the leakage of nucleic acids from organelles or the nuclear
and TYK2, the elevated mRNA levels induced by cryptole- envelope. This was in line with the absence of noticeable
pine were reduced in the presence of fludarabine, a pharma- toxicity in the deficient cells exposed to cryptolepine
IFIT1 induction fold
Cell viability (%)
Journal of Immunology Research 5
15.0 4.5
⁎⁎⁎ ⁎⁎
⁎⁎
⁎⁎ ⁎⁎
10.0 ⁎⁎ 3.0 ⁎⁎
⁎⁎
⁎
5.0 1.5
⁎
0.0 0.0
FLUD (60 𝜇M) – + – – + – + FLUD (60 𝜇M) – + – – + – +
CRYPT (𝜇M) – – 2 4 4 – – CRYPT (𝜇M) – – 2 4 4 – –
IFN-𝛼 (IU/mL) – – – – – 400 400 IFN-𝛼 (IU/mL) – – – – – 400 400
(a) (b)
15.0 15.0
⁎⁎⁎ ⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
10.0 ⁎⁎⁎ 10.0
⁎⁎⁎
⁎⁎
5.0 ⁎⁎ 5.0 ⁎⁎
0.0 0.0
FLUD (60 𝜇M) – + – – + – + FLUD (60 𝜇M) – + – – – – –
CRYPT (𝜇M) – – 2 4 4 – – CRYPT (𝜇M) – – 2 4 4 – –
IFN-𝛼 (IU/mL) – – – – – 400 400
IFN-𝛼 (IU/mL) – – – – – 400 400
(c) (d)
15.0 15.0
⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎
⁎⁎⁎ ⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
10.0 ⁎⁎⁎ 10.0
⁎⁎
5.0 ⁎⁎5.0
0.0 0.0
FLUD (60 𝜇M) – + – – – – – FLUD (60 𝜇M) – + – – – – –
CRYPT (𝜇M) – – 2 4 4 – – CRYPT (𝜇M) – – 2 4 4 – –
IFN-𝛼 (IU/mL) – – – – – 400 400 IFN-𝛼 (IU/mL) – – – – – 400 400
(e) (f)
Figure 2: Cryptolepine increases JAK1, TYK2, STAT1, STAT2, IRF9, and OAS3 mRNA levels in THP1-derived macrophages. The derived
macrophages from the parent THP1 cells were cultured with cryptolepine (CRYPT), fludarabine (FLUD), or IFN-α as indicated. The target
genes were assessed after 24 h by RT-qPCR with gene-specific primers and probes using β-actin as an endogenous control. Data are shown
as the means, from three varied experiments with each done in triplicate, and error bars represent the standard deviations. ∗p < 0:05, ∗∗p
< 0:01, and ∗∗∗p < 0:001 (one-way ANOVA and Bonferroni’s test).
(Figures 4(c) and 4(d)) similar to the parental cells were dependent on TBK1 activation, we tested cryptole-
(Figure 1(b)). pine in the presence of the TBK1 inhibitor GSK8612.
Upon recognition of RNA and DNA by cytosolic TBK1 inhibition in the macrophages derived from the par-
nucleic acid sensors, adaptors MAVS and STING mediate ent THP1 cells was first established using the HSV-60.
activation of the kinase TBK1. TBK1 is also a converging There was a dose-dependent suppression of the IFIT1
point for IFN-1 activation mediated by TLRs. To establish induction by GSK8612 in the presence of HSV-60
whether the effects of cryptolepine on the IFN-1 pathway (Figure 5(a)). The GSK8612 at 2μM was used to inhibit
STAT1 mRNA
IRF9 mRNA (relative expression) JAK1 mRNA(relative expression) (relative expression)
STAT2 mRNA
OAS3 mRNA (relative expression) TYK2 mRNA(relative expression) (relative expression)
6 Journal of Immunology Research
4.5 4.5
⁎ ⁎⁎ ⁎ ⁎⁎
⁎ ⁎
3.0 ns 3.0 ns
⁎ ⁎
1.5 1.5
0.0 0.0
STAT1 OAS3
ACTB ACTB
FLUD (60 𝜇M) – – – – – + FLUD (60 𝜇M) – – – – – +
CRYPT (𝜇M) – 2 4 4 – – CRYPT (𝜇M) – 2 4 4 – –
IFN-𝛼 (IU/mL) – – – – 400 400 IFN-𝛼 (IU/mL) – – – – 400 400
(a) (b)
Figure 3: Cryptolepine increases STAT1 and OAS3 protein levels in THP1-derived macrophages. The derived macrophages were cotreated
with fludarabine (FLUD) and cryptolepine (CRYPT) or IFN-α. The target proteins were assessed after 24 h by western blotting using the
indicated antibodies. β-Actin (ACTB) was included as a protein loading control. The images are representative of three varied
experiments. ∗p < 0:05 and ∗∗p < 0:01; ns: difference not significant (one-way ANOVA and Bonferroni’s test).
60.0 60.0
ns ⁎⁎⁎
40.0 ns 40.0 ns
⁎⁎⁎
20.0 ns 20.0 ns
ns
0.0 0.0
CRYPT (4 𝜇M) – + – – – CRYPT (4 𝜇M) – + – – –
Poly(I:C) (4 𝜇g/mL) – – + – – Poly(I:C) (4 𝜇g/mL) – – + – –
HSV-60 (4 𝜇g/mL) – – – + – HSV-60 (4 𝜇g/mL) – – – + –
IFN-𝛼 (400 IU/mL) – – – – + IFN-𝛼 (400 IU/mL) – – – – +
Control Control
MAVS KO STING KO
(a) (b)
120.0 120.0
80.0 80.0
40.0 40.0
0.0 0.0
0 1 2 3 4 0 1 2 3 4
Cryptolepine (𝜇M) Cryptolepine (𝜇M)
(c) (d)
Figure 4: Cryptopine activates IFN-1 response in MAVS and STING-deficient THP1-derived macrophages. The derived macrophages from the
MAVS (a) and STING (b) deficient THP1 cells were cultured with cryptolepine (CRYPT), Poly(I:C), HSV-60, or IFN-α. The derived macrophages
from the parent THP1 cells served as the control to which the IFIT1 induction in the deficient cells was compared. Also, the cytotoxic effects of
cryptolepine on the MAVS (c) and STING (d) deficient cells were assessed. Data are shown as the means, from three varied experiments with
each done in triplicate, and error bars represent the standard deviations. ∗∗∗p < 0:001; ns: difference not significant (Student’s t-test).
Cell viability (%)
STAT1
(relative band intensity)
IFIT1induction fold
Cell viability (%)
OAS3
(relative band intensity)
IFIT1 induction fold
Journal of Immunology Research 7
60.0 60.0
⁎⁎
⁎⁎
⁎⁎
40.0
40.0 ⁎⁎
⁎
20.0 ns
20.0
0.0
GSK8612 (2 𝜇M) – + – + – + – +
0.0
HSV-60 (4 𝜇g/mL) – – – – – – + +
GSK8612 (𝜇m) – 2 – 1 2 CRYPT (2 𝜇M) – – + + – – – –
HSV-60 (4 𝜇g/mL) – – + + + IFN-a (4 IU/mL) – – – – + + – –
(a) (b)
Figure 5: Cryptolepine activates IFN-1 response regardless of TBK1 inhibition in THP1-derived macrophages. The derived macrophages
from the parent THP1 cells were cocultured with GSK6812 and HSV-60 (a) or GSK6812 and cryptolepine (CRYPT) (b), and the IFIT1
induction was evaluated after 24 h. Data are shown as the means, from three varied experiments with each done in triplicate, and error
bars represent the standard deviations. ∗p < 0:05, ∗∗p < 0:01, and ∗∗∗p < 0:001; ns: difference not significant (one-way ANOVA and
Bonferroni’s test (a) or Student’s t-test (b)).
the TBK1 in the derived macrophages because this concen- 3.6. Cryptolepine Enhances IFN-1 Pathway Activation by
tration suppressed over 50% of the IFIT1 induction. Further, Poly(I:C), HSV-60, and IFN-α. To determine whether
the IFIT1 induction by cryptolepine was unaffected by cryptolepine effects could negatively or positively impact
GSK8612 (Figure 5(b)). These results imply that cryptolepine responses induced by innate immune agonists, we cultured
activates the IFN-1 response independent of TBK1 signalling. differentiated macrophages in the presence of cryptolepine
Also, similar patterns of results as observed in the derived together with IFN-α, Poly(I:C), or HSV-60 (Figures 7(a)–
macrophages were observed in HepG2 and HEK293 cells 7(c), respectively). Cryptolepine enhanced IFIT1 induction
(Supplementary data S4). in all conditions tested, and this was suppressed when
STAT1 was inhibited (Figure 7(d)). These data together
indicate that cryptolepine-mediated IFN-1 activation syner-
3.5. IFN-1 Pathway Activation in the Presence of gises with activation induced by other agonists and suggest
Cryptolepine Requires IFNAR1 and STAT1. We then gained that, during viral infection, the IFN-1 response will be
more mechanistic insights using IFNAR antibody blockade amplified in the presence of cryptolepine.
and STAT1 inhibitor. We first determined a dose-
dependent suppression of the IFIT1 induction in the pres-
ence of the anti-IFNAR1 blocking antibody and IFN-α 4. Discussion
(Figure 6(a)). The anti-IFNAR1 blocking antibody at
10μg/mL suppressed over 50% of the IFIT1 induction and IFN-1 play a significant function in the innate immune
was used to block IFNAR1 in further experiments. Further, response as the first line of defence against pathogens. Before
when the IFNAR1 was blocked in the derived macrophages infections, the exposure of cells to IFN-1 has been reported
from the parent THP1 cells, the IFIT1induction by cryptole- to induce an antiviral state that subsequently averts produc-
pine was over 50% suppressed (Figure 6(b)). Similar patterns tive viral infection [34, 35]. Also, the IFN-1 have been
of results as observed in the derived macrophages were reported to be hepatoprotective through the induction of
observed in HepG2 and HEK293 cells (Supplementary the anti-inflammatory IL-1 receptor antagonist [36]. Fur-
data S5). Further, the effect of STAT1 inhibition on the ther, the IFN-1 play a key role in immune protection in
IFIT1 induction by cryptolepine was assessed. There was mucosal tissues leading to the restriction of enteric virus
a dose-dependent suppression of the IFIT1 induction by infections [37–39]. Moreover, IFN-1 have anticancer effects
fludarabine in the presence of IFN-α (Figure 6(c)). Fludar- against non-Hodgkin lymphoma, chronic myeloid leukae-
abine at 60μM was used to inhibit STAT1 because this mia, and hairy cell leukaemia [40]. With these beneficial
concentration suppressed over 50% of the IFIT1 induction. properties, recombinant IFN-1 have been used in the
Next, when STAT1 was inhibited in the derived macro- management of viral infections, cancers, and immunological
phages from the parent THP1 cells, the IFIT1 induction disorders [11, 12].
by cryptolepine was over 80% suppressed (Figure 6(d)). Medicinal plants are emerging as sources of compounds
Also, similar patterns of results as observed in the derived with notable pharmacological effects reviewed in [41]. One
macrophages were observed in HepG2 and HEK293 cells such compound that needs to be investigated is cryptolepine,
(Supplementary data S6). Taken together, these results which is the major pharmacoactive alkaloid in C. sanguino-
demonstrate that cryptolepine activates the IFN-1 pathway lenta. The aqueous root extract of C. sanguinolenta is being
via IFNAR1 and STAT1. used for years as a tonic that is often taken daily with no
IFIT1 induction fold
IFIT1 induction fold
8 Journal of Immunology Research
45.0 30.0
⁎⁎
⁎⁎
⁎
30.0 ⁎ 20.0 ⁎⁎
15.0 10.0
0.0 0.0
aIFNAR1 ( g/mL) – 10 – 5 10 aIFNAR1 (10 𝜇g/mL) – + – + – +𝜇
CRYPT (4 𝜇m) – – + + – –
IFN-𝛼 (400 IU/mL) – – + + + IFN-𝛼 (400 IU/mL) – – – – + +
(a) (b)
48.0 33.0
⁎⁎⁎⁎ ⁎⁎⁎
⁎⁎⁎
⁎⁎
32.0 ⁎⁎⁎ 22.0 ⁎⁎⁎
⁎⁎
⁎⁎
16.0 11.0
0.0 0.0
FLUS (60 𝜇M) – + – + – +
Fludarabine (mM) – 80 – 40 60 80
CRYPT (4 𝜇M) – – + + – –
IFN-𝛼 (400 IU/mL) – – + + + + IFN-𝛼 (400 IU/mL) – – – – + +
(c) (d)
Figure 6: Cryptolepine activates IFN-1 response through IFNAR1 and STAT1 in THP1-derived macrophages. The derived macrophages
from the parent THP1 cells were cocultured with the anti-IFNAR1 blocking antibody (aIFNAR1) and IFN-α (a) or cryptolepine
(CRYPT) (b), and fludarabine (FLUD) and IFN-α (c) or cryptolepine (d) and the IFIT1 induction was evaluated after 24 h. Data are
shown as the means, from three varied experiments with each done in triplicate, and error bars represent the standard deviations. ∗p <
0:05, ∗∗p < 0:01, ∗∗∗p < 0:001, and ∗∗∗∗p < 0:0001 (one-way ANOVA and Bonferroni’s test (a and c) or Student’s t-test (b and d)).
reported toxicity and has hence been used in Ghana for clearance [49, 50]. This could explain the mechanism
the management of malaria [21]. Besides its plasmodioci- through which cryptolepine exhibits its antiplasmodial effect
dal activity, this medicinal plant has numerous pharmaco- and the ethnomedical use of C. sanguinolenta for the treat-
logical effects including, but not limited to, anticancer, ment of malaria.
antipyretic, anti-inflammatory, antithrombotic, antidia- The anticancer role of STAT1 is considered to be
betic, antiprotozoal, antifungal, antibacterial, and antihy- important during the onset of tumourigenesis where it
pertensive effects reviewed in [42]. Studies have shown promotes the destruction of transformed cells by the
that these pharmacological effects of C. sanguinolenta are immune system [51]. Moreover, STAT1 signalling has
attributed to mainly the presence of its main alkaloid been reported to impede the proliferation of hepatocellular
cryptolepine [22, 24, 43–45]. carcinoma through the induction of p53 signalling [52].
Cryptolepine activated IFN-1 response similar to recom- Hence, the increase in levels of STAT1 protein in the pres-
binant human IFN-α which supports the beneficial proper- ence of cryptolepine (Figure 8) gives credence to its anti-
ties of cryptolepine similar to IFN-1. The IFN-1 response cancer properties [23, 24, 43].
pathway is known to be hepatoprotective and antiviral [36, During mitochondrial stress, there is the leakage of mito-
46], and therefore, it could be deduced that cryptolepine chondrial (mt) DNA into the cytoplasm [53]. The mtDNA
has hepatoprotective properties, and this supports the eth- has been shown to trigger the cGAS-STING signalling and
nomedical use of C. sanguinolenta for the suggested man- IFN-1 production [54, 55]. Hence, the activation of IFN-1
agement of viral hepatitis in Ghana. Moreover, OAS3 was response by cryptolepine independent of STING implies that
reported to restrict intracellular replication of Mycobacte- cryptolepine is not causing substantial cellular and mito-
rium tuberculosis [47]. Therefore, the increase in levels of chondrial stress. This is supported by the absence of
OAS3 protein in the presence of cryptolepine (Figure 8) sup- noticeable toxicity in the cells exposed to cryptolepine con-
ports the ethnopharmacological use of C. sanguinolenta in centrations used to activate the IFN-1 response pathway.
the management of tuberculosis [48]. Further, the IFN-1 Some cancer cells, including ovarian and liver cancer cells,
response is activated during malaria which leads to parasite exhibit impaired STING signalling [56, 57]. Also, defective
IFIT1 induction fold IFIT1 induction fold
IFIT1 induction fold IFIT1 induction fold
Journal of Immunology Research 9
33.0
30.0
⁎⁎
⁎⁎
20.0 22.0
⁎⁎
⁎⁎
⁎⁎
10.0 11.0 ⁎⁎
⁎ ⁎
0.0 0.0
0 1 2 3 4 0 1 2 3 4
Cryptolepine (𝜇M) Cryptolepine (𝜇M)
IFN + Poly(I:C) +
IFN – Poly(I:C) –
(a) (b)
39.0
⁎⁎⁎
26.0
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
13.0
0.0
0 1 2 3 4
Cryptolepine (𝜇M)
HSV-60 +
HSV-60 –
(c)
39.0
⁎⁎⁎
⁎⁎⁎
⁎⁎⁎
26.0
⁎⁎⁎
13.0 ⁎⁎⁎
0.0
FLUD (60 𝜇M) – + – + – + – + – + – +
CRYPT (4 𝜇M) – – – – + + – – + + + +
Poly(I:C) (0.5 𝜇g/mL) – – + + + + – – – – – –
HSV-60 (0.5 𝜇g/mL) – – – – – – + + + + – –
IFN- (50 𝜇g/mL) – – – – – – – – – – + +
(d)
Figure 7: Cryptolepine enhances IFN-1 response activation by Poly(I:C), HSV-60, and IFN-α in THP1-derived macrophages. The derived
macrophages from the parent THP1 cells were cultured with cryptolepine and 50 IU/mL IFN-α (a), 0.5 μg/mL Poly(I:C) (b), and 0.5μg/mL
HSV-60 (c), and the IFIT1 induction was assessed after 24 h. Moreover, the macrophages were cocultured with fludarabine (FLUD), IFN-α
(Poly(I:C) or HSV-60), and cryptolepine, and the IFIT1 induction was evaluated after 24 h (d). Data are shown as the means, from three
varied experiments with each done in triplicate, and error bars represent the standard deviations. ∗p < 0:05, ∗∗p < 0:01, ∗∗∗p < 0:001, and
∗∗∗∗p < 0:0001 (Student’s t-test).
IFIT1 induction fold
IFIT1induction fold IFIT1 induction fold
IFIT1 induction fold
10 Journal of Immunology Research
CRYPT
ISGs: OAS3, IFIT1,…
Antiviraleffects
Anticancereffects
Otherimmuneeffects
Figure 8: Proposed upregulation of the IFN-1 response pathway by cryptolepine. Cytosolic RNA is detected by the MDA5 or RIG-1 which
leads to the activation of IRF3 via the TBK1. However, cytosolic DNA is recognised by cGAS which then stimulates the STING-dependent
signalling, leading to the phosphorylation of IRF3. The phosphorylated IRF3 homodimerises and moves into the nucleus causing the release
of the IFN-1 (mainly IFN-α/β). The secreted IFNs simulate the later phase of the IFN-1 pathway (JAK-STAT axis) through the IFNARs,
which transmit a cascade of signalling events to activate the promoters of ISGF3 and induce the production of numerous IFN-responsive
proteins which facilitate anticancer and antiviral responses. This study has demonstrated that cryptolepine activates the IFN-1 response
pathway via the JAK-STAT axis (as indicated by arrows) but is independent of the first phase of the pathway (as indicated by a crossed
arrow). CRYPT: cryptolepine.
STING signalling in human osteosarcoma cells has been Abbreviations
reported to contribute to the growth of HSV-1 [58]. Further,
MAVS dimer although crucial in innate immunity is a target AMP: Adenosine monophosphate
for HCV NS3/4A protease [59]. Moreover, a heterozygous cGAMP: Cyclic guanosine monophosphate-adenosine
mutation in TBK1 impairs TLR3 signalling [60]. Therefore, monophosphate
the activation of IFN-1 response by cryptolepine regardless cGAS: Cyclic GMP-AMP synthase
of the STING,MAVS, and TBK1 could be clinically beneficial. GMP: Guanosine monophosphate
HBV: Hepatitis B virus
5. Conclusion HCC: Hepatocellular carcinoma
HCV: Hepatitis C virus
Taken together, we have shown for the first time that cryp- HRP: Horseradish peroxidase
tolepine is a potent IFN-1 response inducer. Additionally, IFIT: Interferon-induced protein with tetratricopeptide
we have revealed some antimicrobial and immune- repeats
boosting mechanistic activities of cryptolepine. Since crypto- IFN: Interferon
lepine has similar beneficial effects as IFN-1, we recommend IFNAR: Interferon alpha receptor
that further studies should be conducted to potentially ISRE: Interferon stimulated response elements
repurpose C. sanguinolenta preparation as a natural and JAK: Janus kinase
cheaper immunotherapeutic drug for the management of LMICs: Low- and middle-income countries
chronic viral infections as well as cancers in resource- Luc: Luciferase
limited settings of the world. MAVS: Mitochondrial antiviral sensing protein
MDA5: Melanoma differentiation-associated gene 5
6. Limitations OAS: Oligoadenylate synthase
PMA: Phorbol 12-myristate 13-acetate
Although the results from this in vitro study are encourag- RIG-1: Retinoic acid-inducible gene 1
ing, there were some limitations. The experiments were not STAT: Signal transducer and activator of transcription
performed in primary cells and in vivo. Also, the effects of STING: Stimulator of interferon genes
cryptolepine on IRF3 phosphorylation, IFN-α, and IFN-β TANK: TRAF family member-associated NFκB activator
levels were not assessed due to financial constraints. Further, TBK: TANK-binding kinase
the actual interaction between cryptolepine and the IFNARs
was not assessed. Hence, we recommend that further studies Data Availability
should be conducted in primary cells and in vivo as well as
ascertain the effects of cryptolepine on type 2 and 3 IFN The datasets used during the current study are available
pathways. from the corresponding author on reasonable request.
Journal of Immunology Research 11
Conflicts of Interest [6] G. Guarda, M. Braun, F. Staehli et al., “Type I interferon
inhibits interleukin-1 production and inflammasome activa-
The authors declare that they have no competing interests. tion,” Immunity, vol. 34, no. 2, pp. 213–223, 2011.
[7] A. J. Lee and A. A. Ashkar, “The dual nature of type I and type
II interferons,” Frontiers in Immunology, vol. 9, 2018.
Authors’ Contributions
[8] N. E. Mangan and K. Y. Fung, “Type I interferons in regulation
MM conceived and supervised the work. SAD and PWN of mucosal immunity,” Immunology & Cell Biology, vol. 90,
planned and carried out the experiments and analysed the no. 5, pp. 510–519, 2012.
data. SL, RPS, and CMM provided and validated reagents. [9] J. Pott and S. Stockinger, “Type I and III interferon in the gut:
SAD and MM wrote the manuscript. MM, PWN, OQ, tight balance between host protection and immunopathology,”
KAK, BSA, SL, RPS, CMM, GAA, and CA reviewed and edi- Frontiers in Immunology, vol. 8, p. 258, 2017.
ted the manuscript. GAA and MM provided the funding for [10] L. Pastorelli, C. De Salvo, J. R. Mercado, M. Vecchi, and T. T.
the work. All authors read and approved the nal Pizarro, “Central role of the gut epithelial barrier in the path-fi
ogenesis of chronic intestinal inflammation: lessons learned
manuscript. from animal models and human genetics,” Frontiers in Immu-
nology, vol. 4, p. 280, 2013.
Acknowledgments [11] H. Chi, B. E. Hansen, S. Guo et al., “Pegylated interferon alfa-
2b add-on treatment in hepatitis B virus envelope antigen-
Professors Siamon Gordon and David J. Blackbourn both positive chronic hepatitis B patients treated with nucleos
supported Professor Mohamed Mutocheluh’s preliminary (t) ide analogue: a randomized, controlled trial (PEGON),”
work at the University of Surrey, UK, which partly led to Journal of Infectious Diseases, vol. 215, no. 7, pp. 1085–
the conception of this manuscript. This study was supported 1093, 2017.
by a Wellcome/African Academy of Sciences Developing [12] J. Bekisz, S. Baron, C. Balinsky, A. Morrow, and K. C. Zoon,
Excellence in Leadership Training and Science (DELTAS) “Antiproliferative properties of type I and type II interferon,”
grant (DEL-15-007 and 107755/Z/15/Z: Awandare). Addi- Pharmaceuticals, vol. 3, no. 4, pp. 994–1015, 2010.
tional support came from the Kwame Nkrumah University [13] F. E. Lotrich, “Major depression during interferon-α treat-
of Science and Technology Research Fund (KReF) grant ment: vulnerability and prevention,” Dialogues in Clinical
Neuroscience, vol. 11, no. 4, pp. 417–425, 2009.
VC/OGR/15 to Prof. Mohamed Mutocheluh.
[14] A. S. Rahman, M. Amir, S. Ahmed, Q. Jamal, M. Riaz, and
M. A. Siddiqui, “Prevalence of depression after treatment with
Supplementary Materials conventional interferons and ribavirin therapy in patients with
hepatitis C using PHQ-9,” American Journal of Research &
The supplementary material file contains the data obtained Medical Science, vol. 5, no. 1, pp. 2238–2248, 2019.
from the HepG2 and HEK293 cells (Supplementary data [15] R. Certo, G. Giuffrida, A. Campennì, F. Trimarchi, and R. M.
S1, S2, S4 – S7) as well as the validation of the MAVS and Ruggeri, “Thyroid dysfunction during interferon alpha ther-
STING THP1-deficient cells (Supplementary data S 3). Also, apy for chronic hepatitis C: challenges in differential diagnosis
the le contains the primers used in the application of the and treatment,” Atti della Accademia Peloritana deifi
gene of interest (Supplementary data S8). (Supplementary Pericolanti-Classe di Scienze Medico-Biologiche, vol. 106,
no. 2, pp. 1–5, 2018.
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