Methamphetamine facilitates HIV infection of primary human monocytes through inhibiting cellular viral restriction factors

Abstract

Background
Methamphetamine (METH), a potent addictive psychostimulant, is highly prevalent in HIV-infected individuals. Clinically, METH use is implicated in alteration of immune system and increase of HIV spread/replication. Therefore, it is of importance to examine whether METH has direct effect on HIV infection of monocytes, the major target and reservoir cells for the virus.

Results
METH-treated monocytes were more susceptible to HIV infection as evidenced by increased levels of viral proteins (p24 and Pr55Gag) and expression of viral GAG gene. In addition, using HIV Bal with luciferase reporter gene (HIV Bal-eLuc), we showed that METH-treated cells expressed higher luciferase activities than untreated monocytes. Mechanistically, METH inhibited the expression of IFN-λ1, IRF7, STAT1, and the antiviral IFN-stimulated genes (ISGs: OAS2, GBP5, ISG56, Viperin and ISG15). In addition, METH down-regulated the expression of the HIV restriction microRNAs (miR-28, miR-29a, miR-125b, miR-146a, miR-155, miR-223, and miR-382).

Conclusions
METH compromises the intracellular anti-HIV immunity and facilitates HIV replication in primary human monocytes.

Full text

Liuetal. Cell Biosci (2021) 11:194
https://doi.org/10.1186/s13578-021-00703-4
RESEARCH
Methamphetamine facilitates HIV
infection ofprimary human monocytes
throughinhibiting cellular viral restriction
factors
Yu Liu1†, Feng‑Zhen Meng1†, Xu Wang1,2, Peng Wang1,2, Jin‑Biao Liu1, Wen‑Hui Hu1, Won‑Bin Young1 and
Wen‑Zhe Ho1,2*
Abstract
Background: Methamphetamine (METH), a potent addictive psychostimulant, is highly prevalent in HIV‑infected
individuals. Clinically, METH use is implicated in alteration of immune system and increase of HIV spread/replication.
Therefore, it is of importance to examine whether METH has direct effect on HIV infection of monocytes, the major
target and reservoir cells for the virus.
Results: METH‑treated monocytes were more susceptible to HIV infection as evidenced by increased levels of viral
proteins (p24 and Pr55Gag) and expression of viral GAG gene. In addition, using HIV Bal with luciferase reporter gene
(HIV Bal‑eLuc), we showed that METH‑treated cells expressed higher luciferase activities than untreated monocytes.
Mechanistically, METH inhibited the expression of IFN‑λ1, IRF7, STAT1, and the antiviral IFN‑stimulated genes (ISGs:
OAS2, GBP5, ISG56, Viperin and ISG15). In addition, METH down‑regulated the expression of the HIV restriction microR‑
NAs (miR‑28, miR‑29a, miR‑125b, miR‑146a, miR‑155, miR‑223, and miR‑382).
Conclusions: METH compromises the intracellular anti‑HIV immunity and facilitates HIV replication in primary
human monocytes.
Keywords: Methamphetamine, Human immunodeficiency virus, Interferon‑stimulated genes, Monocytes
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Introduction
METH is one of the most widely abused illicit drugs
among HIV-infected individuals. METH use and HIV
infection frequently coexist due to the association of
METH use with engagement of high-risk behaviors
[1–3]. ere is a high prevalence of HIV infection in
METH using population [4, 5]. Among men who have
sex with men, those who use METH are more suscepti-
ble to HIV infection than non-users [6–10]. Clinically,
METH use has been implicated in HIV disease progres-
sion [11]. Active METH users with HIV infection display
higher levels of viral load than non-users [12]. In addi-
tion, METH users have delayed viral suppression after
initiation of antiretroviral therapy (ART), higher levels of
blood HIV RNA, increased frequency of drug resistance
mutations and accelerated progression to AIDS [13–17].
METH abuse contributes to CD4+ T cells depletion,
inflammation/immune activation, and the promotion of
HIV entry and disease progression [18].
Open Access
Cell & Bioscience
*Correspondence: wenzheho@temple.edu
†Yu Liu and Feng‑Zhen Meng contributed equally to this work
1 Department of Pathology and Laboratory Medicine, Temple University
Lewis Katz School of Medicine, 3500 N Broad St., Philadelphia, PA 19140,
USA
Full list of author information is available at the end of the article

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Liuetal. Cell Biosci (2021) 11:194
Cells of monocyte/macrophage lineage are crucial
in initial HIV infection and implicated in the immu-
nopathogenesis of HIV disease. Monocytes are among
the first and major cell types infected by HIV and serve
as reservoirs for the virus. However, unlike tissue mac-
rophages and invitro monocyte-derived macrophages
which are highly susceptible to HIV infection, periph-
eral blood monocytes are refractory to HIV infec-
tion invivo and invitro, and only a small percentage
of monocytes harbor the virus [19, 20]. Despite of
their relative resistance to HIV infection, monocytes
are involved in HIV infection of the central nervous
system (CNS) as they can bring the virus to the brain
[21]. A study reported that HIV-infected monocytes
are more likely to cross the blood brain barrier (BBB) as
compared to uninfected monocytes [22]. Among HIV-
infected METH users, HIV-associated neurocognitive
disorders (HAND) are more frequent and severe [23,
24]. A recent study demonstrated that METH could
enhance HIV infection of neural progenitor cells, a
possible mechanism for the impairment or disruption
of neurocognitive functioning in HIV-infected indi-
viduals with NeuroAIDS [25]. Several studies showed
that elevated extracellular CNS dopamine by METH
abuse could facilitate uninfected and HIV-infected
CD14+CD16+ monocytes transmigration across the
BBB, resulting in the propagation of viral reservoirs and
inflammation in the CNS which contribute to the devel-
opment of HAND [26, 27]. us, it is of great interest
to determine the direct impact of METH on suscepti-
bility of peripheral blood monocytes to HIV infection.
In addition, it is critical to understand the pathologi-
cal effects of METH on the specific intracellular innate
immunity against HIV in monocytes.
Results
METH enhances HIV infection
We first examined cytotoxicity effect of METH on
monocytes. As shown in Additional file 1: Fig. S1,
METH at the concentration as high as 1000μM had
little effect on cell viability. We then studied whether
METH could enhance susceptibility of monocytes
to HIV infection. As demonstrated in Fig. 1A and B,
METH treatment of monocytes dose-dependently
increased the expression of both intracellular and
extracellular HIV GAG gene expression. In addition,
METH-treated monocytes showed higher levels of
HIV (p24 and Pr55Gag) proteins than untreated cells
(Fig.1C and D). e enhancing effect of METH on HIV
p24 protein production was dose-dependent (Fig.1E).
As shown in Fig.1F, METH treatment enhanced lucif-
erase activity in HIV Bal-eLuc-infected cells.
METH suppresses theJAK/STAT signaling pathway
To study the mechanisms by which METH enhances
HIV infection of monocytes (Fig.1), we examined the
effect of METH on IFNs. As shown in Fig.2A, while it
had little effect on IFN-α/β expression, METH treat-
ment significantly suppressed IFN-λ1 expression in
monocytes. In addition, METH inhibited the expression
of phosphorylated IRF7 in a time-dependent fashion
(Fig.2B).
IRF7 is a key regulator for both type I and type III
IFNs during viral infections [28, 29]. The phosphoryla-
tion of IRF7 would directly trigger the transcription of
IFNs and the downstream antiviral signaling, includ-
ing the activation of JAK/STAT pathway and the pro-
duction of ISGs. We also examined whether METH
has a negative impact on the expression of STAT fam-
ily members including STAT1, STAT2 and STAT3,
the crucial factors in JAK/STAT signaling pathway
[30, 31]. As shown in Fig.2C, while METH treatment
of monocytes had little effect on STAT2 and STAT3
expression, it significantly inhibited STAT1 expres-
sion at both mRNA and protein levels, and reduced
the phosphorylation of STAT1. We next examined the
effect of METH on the expression of the intracellu-
lar antiviral ISGs. As shown in Fig.2D, METH dose-
dependently inhibited the expression of the antiviral
ISGs (OAS2, GBP5, ISG56, Viperin, ISG15) at 24 h
post-treatment. In addition, the Western blot analy-
sis demonstrated that METH-treated monocytes had
lower protein levels of the antiviral ISGs than the
untreated cells (Fig.2E).
METH inhibits HIV restriction miRNAs
Our earlier study showed that monocytes contain sig-
nificantly higher levels of the HIV restriction miRNAs
than monocyte-derived macrophages, which explains
why monocytes are refractory to HIV infection [32]. We
thus investigated whether METH negatively influences
the expression of the HIV restriction miRNAs in mono-
cytes. As shown in Fig.2F, METH treatment of mono-
cytes suppressed the expression of the intracellular HIV
restriction miRNAs (miR-28, miR-29a, miR-125b, miR-
146a, miR-155, miR-223, and miR-382). In addition, we
observed lower levels of these HIV restriction miRNAs in
the supernatants of monocyte cultures 36h after METH
treatment compared to those in untreated cells (Fig.2G).
Discussion
Although METH use has been linked to HIV transmis-
sion and infection, its pathological effects on the host
cell-mediated specific innate immunity against HIV

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Liuetal. Cell Biosci (2021) 11:194
Fig. 1 METH enhances HIV infection of primary human monocytes. A–E Monocytes isolated from human peripheral blood were treated with
METH for 24 h and then infected with HIV Bal strain overnight. Cells were washed with PBS three times and cultured in the presence of METH for
72 h. RNAs extracted from cells (A) and the cell‑free supernatants (B) were subjected to the real‑time PCR with HIV GAG gene primers. C, D Proteins
of cells and culture supernatants were analyzed by Western blot using the antibodies against HIV proteins (p24 and Pr55Gag) and GAPDH. E The
cell‑free supernatants were subjected to ELISA assay to quantitatively determine p24 protein level. F Monocytes were treated with METH (150 μM)
for 24 h and then infected with HIV Bal‑eLuc overnight. Cells were washed with PBS three times and cultured in the presence of METH for 24 h or
48 h prior to luminescence assay. Data shown were the mean ± SD of three independent experiments with monocytes from three different donors
(*P < 0.05, **P < 0.01)

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Liuetal. Cell Biosci (2021) 11:194
infection remain to be determined. e earlier stud-
ies reported that METH could enhance HIV infec-
tion of several cell types, including dendritic cells [33],
macrophages [34, 35], CD4+ T cells [36, 37], micro-
glia [38], and neural progenitor cells [25]. However, it
is unclear whether METH facilitates HIV infection of
primary human monocytes. In the present study, we
demonstrated that METH treatment of the monocytes
significantly enhanced HIV infection/replication at
both intracellular and extracellular levels (Fig. 1). To
investigate the underlying mechanisms of METH-medi-
ated HIV enhancement in monocytes, we examined
that the impact of METH on the expression of IFNs-
JAK/STAT signaling pathways. We found that although
METH treatment of monocytes had little effect on
IFN-α and IFN-β expression, it significantly suppressed
IFN-λ1 expression (Fig.2A). IFN-λ can induce type I
IFN-like antiviral response and inhibition of HIV [39,
40]. It is likely that IFN-λ inhibition by METH can result
in reduction of ISGs. e following results indicated
Fig. 2 METH inhibits viral restriction factors. A–E Monocytes from human peripheral blood were treated with METH (150 μM) for the indicated
times or at the indicated concentrations for 24 h. A The cellular RNAs were extracted and subjected to the real‑time PCR for IFN‑α, IFN‑β, and IFN‑λ1
mRNA expression, the culture supernatants were collected and subjected to ELISA for IFN‑λ1 protein expression. B–E The cellular RNAs or proteins
were extracted and subjected to the real‑time PCR or Western blot assays. F, G Monocytes were treated with METH (150 μM) for the indicated times.
Cellular miRNAs were quantified by the real‑time PCR. RNU48 was used as a control gene. miRNAs in the culture supernatants were quantified by
the real‑time PCR. Synthetic Caenorhabditis elegans miRNA‑39 (cel‑miR‑39) was used as a spiked‑in control miRNA for normalization. Data shown
were the mean ± SD of three independent experiments with monocytes from three different donors (*P < 0.05, **P < 0.01)

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Liuetal. Cell Biosci (2021) 11:194
that METH treatment of the cells down-regulated the
expression of the antiviral ISGs, including OAS2, GBP5,
ISG56, Viperin, and ISG15 (Fig.2D and E). ese ISGs
are known to have the ability to restrict HIV replication
at different steps of viral replication cycle [41–45]. For
instance, Krapp etal. demonstrated that the expression
of GBP5 could interfere with the processing and virion
incorporation of the HIV envelope glycoprotein, which
remarkably reduce virion incorporation of mature
gp120 and enhance virion-associated immature gp160
precursor, leading to the inhibition of HIV infectivity
[42]. Okumura etal. showed that ISG15 impaired the
interaction between HIV GAG and tumor susceptibility
gene 101 (Tsg101), and suppressed HIV virion release
[46].
Given that the IFN regulatory factors (IRFs) are
responsible for IFN-JAK/STAT signaling pathway and
the production of the antiviral ISGs, we also exam-
ined the impact of METH on IRFs, particularly IRF1,
IRF3, IRF5 and IRF7, the key players in regulating the
expression of antiviral ISGs and producing an antiviral
state [47]. We found that while METH had little effect
on IRF1, IRF3, IRF5, it specifically suppressed IRF7
expression in monocytes at both mRNA and protein
levels (Fig.2B). IRF7 has a key role for the production
of both type I and type III IFNs during viral infections
[28, 29]. e phosphorylation of IRF7 would directly
trigger the transcription of IFNs and the downstream
antiviral signaling, including the activation of JAK/
STAT pathway. erefore, it is likely that IRF7 suppres-
sion by METH is a possible mechanism for STAT1 inhi-
bition in METH-treated monocytes (Fig. 2C). STAT1
is a crucial regulatory factor in IFNs-mediated induc-
tion of antiviral ISGs [48, 49]. While exact mechanism
by which METH inhibits IRF and STAT remain to be
determined, it is possible that down-regulation of IFN-
λ1 has a negative impact on both IRF7 and STAT1
expression.
In addition to the HIV restriction factors of pro-
tein nature, a cluster of HIV restriction miRNAs have
also been shown to be a contributor to HIV latency in
resting CD4+ T cells [50, 51]. These cellular miRNAs
interact with the 3’-termini of HIV RNA, resulting in
the transcriptional inefficiency and post-transcrip-
tional suppression [52, 53]. Our earlier study showed
that primary human monocytes expressed much
higher levels of miRNAs (miRNA-382, -223, -150,
and -28) than monocyte-derived macrophages, and
the suppression of these miRNAs facilitates HIV-1
infectivity, which provide direct evidence that HIV
restriction miRNAs have a key role in protecting
monocytes/macrophages from HIV-1 infection [32].
Several studies reported that METH use altered the
miRNA expression in human serum or animal model
[54, 55]. We thus determined whether METH could
regulate the expression of the miRNAs that are impli-
cated in HIV infection and persistence. Among the
cellular miRNAs, the miR-29 family members (miR-
29a, miR-29b, and miR-29c) suppress HIV replication
by targeting a highly conserved region of HIV [56].
Importantly, our early study documented that the lev-
els of the cellular HIV restriction miRNAs arenega-
tively correlated with susceptibility of monocytes and
macrophages to HIV infection [32]. We also reported
that ART failed to restore the levels of several HIV
restriction miRNAs in PBMCs of HIV-infected men
who have sex with men who used METH [57]. There-
fore, it is clinically relevant to seek the direct evi-
dence of how METH negatively impacts on the HIV
restriction miRNAs. Our observation that METH
could significantly suppress the expression of the HIV
restriction miRNAs (Fig.2F and G) provide not only
direct evidence for our invivo finding [57], but also an
additional mechanism for METH-mediated enhance-
ment of HIV infection.
It is suggested that METH could facilitate HIV entry
into the cells through up-regulation CXCR4 and CCR5,
the key coreceptors for HIV entry into target cells [33,
34]. However, in contrast to these earlier studies, we did
not observe the enhancing effect of METH on CCR5
expression in monocytes (Additional file 2: Fig. S2).
METH also had no significant impact on the expression
of CD4. is conflicting finding could be due to the
difference in use of different cell types: while we used
primary human monocytes, the previous studies used
monocyte-derived dendritic cells [33] and macrophages
[34] which are more susceptible to HIV infection com-
pared to monocytes [32].
In summary, we demonstrate that METH can enhance
HIV infection of primary human in monocytes through
the inhibition of the multiple cellular antiviral factors
(IFN-λ1, ISGs, and miRNAs). While it is possible that
there are additional mechanisms involved in the METH
action on HIV enhancement, compromising the intra-
cellular immunity against HIV should be responsible
for much of METH-mediated HIV enhancement in
monocytes. ese findings suggest that METH use is a
contributing factor for HIV infection and persistence
in monocytes. As HIV-infected individuals are living
longer with ART and many of infected individuals are
METH users, to further identify the pathological role of
METH in HIV-infected reservoir cells is necessary for

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Liuetal. Cell Biosci (2021) 11:194
understanding the mechanisms of HIV persistence and
developing strategies for the viral eradication.
Conclusions
METH compromises the intracellular anti-HIV immu-
nity and facilitates HIV replication in primary human
monocytes.
Materials andmethods
Cells andreagents
Purified human peripheral blood monocytes were
obtained from Human Immunology Core at the Uni-
versity of Pennsylvania (Philadelphia, PA, USA). The
Core has the Institutional Review Board approval for
blood collection from healthy donors. These blood
samples were screened for all normal viral bloodborne
pathogens and certified to be pathogen free. The pro-
tocol of monocyte isolation was described previously
[34]. Briefly, after the initial purification, greater
than 97% of the cells were monocytes, as determined
by nonspecific esterase staining and flow cytometry
analysis using monoclonal antibody against CD14,
the marker specific for monocytes and macrophages.
Freshly isolated monocytes were cultured in 1640
RPMI (Gibco, New York, USA) medium supplemented
with 10% fetal bovine serum (Corning, New York,
USA), 1% MEM NEAA (Gibco, New York, USA), 1%
L-Glutamine (Gibco, New York, USA) and 1% penicil-
lin–streptomycin solution (Lonza, Walkersville, GA,
USA). Rabbit antibodies against OAS2, GBP5, ISG56,
Viperin, ISG15, IRF7, p-IRF7, STAT1, p-STAT1 and
GAPDH were purchased from Cell Signaling Technol-
ogy (Danvers, MA, USA). Mouse anti-HIV p24 anti-
body was purchased from Abcam (Abcam, Cambridge,
UK). METH was purchased from Sigma Aldrich (St
Louis, MO, USA). METH powder was dissolved in
sterile endotoxin-free water (HyPure™ Cell culture
grade water, GE Healthcare Life Science, Logan, UT,
USA) and stored at 4℃. Trichloroacetic acid (TCA)
and acetone were purchased from Sigma-Aldrich. All
antibodies and reagents for flow cytometry assay were
purchased from BD Bioscience (BD Bioscience, San
Jose, CA, USA).
HIV infection andMETH treatment
HIV Bal strain was obtained from AIDS Reagent Pro-
gram (NIH, Bethesda, MD), HIV Bal with luciferase
reporter gene (HIV Bal-eLuc) was generated by Dr. Won-
Bin Young [58]. Freshly isolated and purified monocytes
were treated with METH at clinically relevant concentra-
tions [59–62] (0, 100, 150, and 250μM) for 24h before
being infected with HIV Bal strain (p24 60ng/106 cells)
or HIV Bal-eLuc (p24 60ng/106 cells) overnight. e cells
were then washed three times with plain RPMI to remove
any unabsorbed virus and cultured in the presence of
METH.
MTS assay
e cytotoxic effect of METH on monocytes was
evaluated by MTS (3-(4, 5-dimethylthiazol-2-yl)-
5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-
zolium, innersalt) assay. Freshly isolated human blood
monocytes (2 × 104 cells/well) were placed in 96-well
round bottom plates, and treated with different con-
centrations of METH (0, 100, 150, 250, 400, 600, and
1000μM) for 96h. e cells were then incubated with
CellTiter 96® AQueous One Solution Reagent (Promega
Corporation, Madison, WI) containing MTS and phena-
zine ethosulfate for 4h at 37℃. Absorbance at 490nm
was measured by a plate reader (SpectraMax i3, Molecu-
lar Devices, Sunnyvale, CA, USA).
ELISA
HIV p24 protein levels in monocyte culture supernatants
were determined by ELISA kit from Abnova (Taipei, Tai-
wan) as instructed by the manufacturer. IFN-λ1 protein
levels in monocyte culture supernatants were determined
by human IL-29 (IFN lambda1) ELISA kit from Invitro-
gen (Invitrogen, CA, USA) according to the manufactur-
er’s instruction.
Flow cytometry assay
Monocytes from human peripheral blood were treated
with METH (150μM) for 24h. en the cells were col-
lected and washed with a cell staining buffer prior to
staining with PE mouse anti-human CD4 antibody and
PE mouse anti-human CCR5 antibody, respectively.
PE-isotype IgG antibody-stained cells were used as the
negative control. e stained cells were measured by a
FACSCanto II (BD Bioscience, CA, USA) and analyzed
using FlowJo software (Tree Star Inc., Ashland, OR,
USA).
Western blot assay
Proteins from monocytes and culture supernatants
were determined by Western blot assay for viral pro-
teins (p24 and Pr55Gag) expression. Monocytes were
lysed with RIPA lysis buffer supplemented with pro-
tease/phosphatase inhibitors (Sigma Aldrich, St Louis,
MO). Proteins from the culture supernatants were
extracted by the TCA/acetone precipitation method.
Briefly, 0.5mL of culture supernatants were precipi-
tated with 0.5mL of 20% TCA at − 20°C for 1h and

Page 7 of 9
Liuetal. Cell Biosci (2021) 11:194
then centrifuged at 11,500rpm for 15min at 4°C. After
three washes with 1mL of ice-cold acetone, the pellet
was lysed with Western blot lysis buffer. e protein
concentrations were determined by the bicinchoninic
acid (BCA) assay (ChemCruz, Dallas, TX). e blots
were incubated with primary antibodies in 5% nonfat
milk in PBS overnight at 4℃, then washed with PBS
containing 0.5% Tween. e blots were further incu-
bated with horseradish peroxidase-conjugated sec-
ond antibodies at room temperature for an hour, then
washed with PBST. e blots were developed with
enhanced chemiluminescence (Amersham, Bucks, UK)
and then exposed to an iBright 1500 imaging analyzer
(Invitrogen, CA,USA).
RNA andmicroRNA extraction andquantication
Freshly isolated monocytes in 48-well plates were
treated with or without METH (100, 150, and 250μM)
for different time points (0, 6, 12, and 24 h). Total
RNAs were extracted with Tri-reagent (Molecular
Research Center, OH, USA). RNA (1μg) was subjected
to reverse transcriptase PCR using reagents from Pro-
mega (Promega, WI, USA). e cDNA sample was then
subjected to the real-time PCR using iQ SYBR Green
Supermix (Bio-Rad Laboratories, CA, USA). All values
were normalized to GAPDH mRNA. e sequences of
oligonucleotide primers used in this study are listed
in Table1. Extracellular miRNAs were extracted from
supernatants of monocyte cultures using the miRNeasy
Mini Kit (Qiagen, CA, USA). e miRNAs from cells
or supernatants were reversely transcribed with miS-
cript Reverse Transcription Kit (Qiagen, CA, USA).
e real-time PCR for the miRNAs quantification was
carried out with miScript Primer Assays using miS-
cript SYBR Green PCR Kit from Qiagen as previously
described [32].
HIV GAG gene quantication
HIV GAG gene copy numbers in monocytes or mono-
cytes culture supernatants were determined by the
real-time PCR. RNAs from cells or the cell-free super-
natants were extracted with Tri-reagent (for tissues,
cells cultured in monolayer, or cell pellets) or Tri-rea-
gent (for whole blood, serum/plasma or cell culture
supernatant) according to the manufacturer’s instruc-
tions, respectively. HIV GAG standards with known
copy numbers were used to quantify viral GAG gene
expression in the culture supernatants.
Statistical analysis
Data were expressed as mean ± standard de viation
(mean ± SD) of three experiments using monocytes from
three different donors. Statistical significance was meas-
ured by Student’s t-test using GraphPad Prism Statistical
Software (GraphPad Software, La Jolla, USA). *P < 0.05
and **P < 0.01 indicate statistic difference between com-
pared groups.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13578‑ 021‑ 00703‑4.
Table 1 Primers Pairs
Gene Orientation Sequence (5′-3′)
GAPDH Forward GGT GGT CTC CTC TGA CTT CAACA
Reverse GTT GCT GTA GCC AAA TTC GTTGT
HIV GAG Forward ATA ATC CAC CTA TCC CAG TAG GAG AAA
Reverse TTT GGT CCT TGT CTT ATG TCC AGA ATGC
OAS2 Forward CAG TCC TGG TGA GTT TGC AGT
Reverse ACA GCG AGG GTA AAT CCT TGA
GBP5 Forward CAG GAA CAA CAG ATG CAG GA
Reverse TCA TCG TTA TTA ACA GTC CTC TGG
ISG56 Forward TTC GGA GAA AGG CAT TAG A
Reverse TCC AGG GCT TCA TTC ATA T
Viperin Forward TGG GTG CTT ACA CCT GCT G
Reverse TGA AGT GAT AGT TGA CGC TGGT
ISG15 Forward GGC TGG GAG CTG ACG GTG AAG
Reverse GCT CCG CCC GCC AGG CTC TGT
IRF1 Forward TGA AGC TAC AAC AGA TGA GG
Reverse AGT AGG TAC CCC TTC CCA TC
IRF3 Forward ACC AGC CGT GGA CCA AGA G
Reverse TAC CAA GGC CCT GAG GCA C
IRF5 Forward AAG CCG ATC CGG CCAA
Reverse GGA AGT CCC GGC TCT TGT TAA
IRF7 Forward TGG TCC TGG TGA AGC TGG AA
Reverse GAT GTC GTC ATA GAG GCT GTTGG
STAT1 Forward CCG TGG CAC TGC ATA CAA TC
Reverse ACC ATG CCG AAT TCC CAA AG
STAT2 Forward CCC CAT CGA CCC CTC ATC
Reverse GAG TCT CAC CAG CAG CCT TGT
STAT3 Forward CTG CCC CAT ACC TGA AGA CC
Reverse TCC TCA CAT GGG GGA GGT AG
IFN‑α Forward TTT CTC CTG CCT GAA GAA CAG
Reverse GCT CAT GAT TTC TGC TCT GACA
IFN‑β Forward GCC GCA TTG ACC ATC TAT GAGA
Reverse GAG ATC TTC AGT TTC GGA GGT AAC
IFN‑λ1 Forward CTT CCA AGC CCA CCC CAA CT
Reverse GGC CTC CAG GAC CTT CAG C
CD4 Forward AGT CCC TTT TAG GCA CTT GC
Reverse GAT CAT TCA GCT TGG ATG G
CCR5 Forward CAA GTG TCA AGT CCA ATC TA
Reverse ACC AAA GAT GAA CAC CAG TG

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Liuetal. Cell Biosci (2021) 11:194
Additional le1: Fig. S1. Effect of METH on the cell viability of human
monocytes. Freshly isolated human monocytes were treated with
METH at the indicated concentrations for 96 hours. The cell viability was
assessed by MTS assay. Data are showed as the absorbance (490 nm)
relative to untreated control, which is defined as 1.0. The results shown
were obtained as mean ± SD from three independent experiments with
triplicate wells.
Additional le2: Fig. S2. Effect of METH on CD4 and CCR5. (A, B) Freshly
isolated human monocytes were treated with METH (150 μM) at the indi‑
cated time points. The cellular RNA was subjected to the real‑time PCR for
CD4 and CCR5 expression. (C, D) Freshly isolated monocytes were treated
with METH (150 μM) for 24 h and then collected for the flow cytometry
analysis of CD4 and CCR5 protein expression. Data are shown in A and B
as mean ± SD from three independent experiments with triplicate wells.
Flow cytometry data shown in C and D are the representative pictures of
three independent experiments.
Acknowledgements
Not applicable.
Authors’ contributions
YL, FZM, XW, WZH designed the research; YL, FZM, PW, JBL, WBY, and WHH
performed the experiments and analyzed the data; YL, FZM, XW, and WZH
interpreted the data and wrote the paper; YL, FZM, XW, JBL, WHH, WZH
discussed and edited the paper. All authors read and approved the final
manuscript.
Funding
This work is financially supported by the NIH (DA 51893 and DA 41302 for WZ
Ho).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from
the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
All authors agree to publish this paper.
Competing interests
No potential conflict of interest was reported by the authors.
Author details
1 Department of Pathology and Laboratory Medicine, Temple University
Lewis Katz School of Medicine, 3500 N Broad St., Philadelphia, PA 19140, USA.
2 Center for Substance Abuse Research, Temple University Lewis Katz School
of Medicine, Philadelphia, PA 19140, USA.
Received: 31 May 2021 Accepted: 27 October 2021
References
1. Marshall BD, Wood E, Shoveller JA, Patterson TL, Montaner JS, Kerr T.
Pathways to HIV risk and vulnerability among lesbian, gay, bisexual,
and transgendered methamphetamine users: a multi‑cohort gender‑
based analysis. BMC Public Health. 2011;11:20.
2. Pitpitan EV, Semple SJ, Zians J, Strathdee SA, Patterson TL. Mood, meth,
condom use, and gender: latent growth curve modeling results from a
randomized trial. AIDS Behav. 2018;22(9):2815–29.
3. Semple SJ, Patterson TL, Grant I. Motivations associated with meth‑
amphetamine use among HIV+ men who have sex with men. J Subst
Abuse Treat. 2002;22(3):149–56.
4. Nerlander LMC, Hoots BE, Bradley H, Broz D, Thorson A, Paz‑Bailey G,
NHBS Group. HIV infection among MSM who inject methamphetamine
in 8 US cities. Drug Alcohol Depend. 2018;190:216–23.
5. Vu NT, Maher L, Zablotska I. Amphetamine‑type stimulants and HIV
infection among men who have sex with men: implications on HIV
research and prevention from a systematic review and meta‑analysis. J
Int AIDS Soc. 2015;18:19273.
6. Morin SF, Steward W T, Charlebois ED, Remien RH, Pinkerton SD,
Johnson MO, Rotheram‑Borus MJ, Lightfoot M, Goldstein RB, Kittel L,
et al. Predicting HIV transmission risk among HIV‑infected men who
have sex with men: findings from the healthy living project. J Acquir
Immune Defic Syndr. 2005;40(2):226–35.
7. Plankey MW, Ostrow DG, Stall R, Cox C, Li X, Peck JA, Jacobson LP. The
relationship between methamphetamine and popper use and risk
of HIV seroconversion in the multicenter AIDS cohort study. J Acquir
Immune Defic Syndr. 2007;45(1):85–92.
8. Shoptaw S, Reback CJ. Associations between methamphetamine use
and HIV among men who have sex with men: a model for guiding
public policy. J Urban Health. 2006;83(6):1151–7.
9. Shoptaw S, Reback CJ. Methamphetamine use and infectious disease‑
related behaviors in men who have sex with men: implications for
interventions. Addiction. 2007;102(Suppl 1):130–5.
10. Urbina A, Jones K. Crystal methamphetamine, its analogues, and HIV
infection: medical and psychiatric aspects of a new epidemic. Clin Infect
Dis. 2004;38(6):890–4.
11. Moore RD, Keruly JC, Chaisson RE. Differences in HIV disease progression
by injecting drug use in HIV‑infected persons in care. J Acquir Immune
Defic Syndr. 2004;35(1):46–51.
12. Ellis RJ, Childers ME, Cherner M, Lazzaretto D, Letendre S, Grant I. Group
HIVNRC: increased human immunodeficiency virus loads in active
methamphetamine users are explained by reduced effectiveness of
antiretroviral therapy. J Infect Dis. 2003;188(12):1820–6.
13. Carrico AW, Johnson MO, Colfax GN, Moskowitz JT. Affective correlates
of stimulant use and adherence to anti‑retroviral therapy among HIV‑
positive methamphetamine users. AIDS Behav. 2010;14(4):769–77.
14. Colfax GN, Vittinghoff E, Grant R, Lum P, Spotts G, Hecht FM. Frequent
methamphetamine use is associated with primary non‑nucleoside
reverse transcriptase inhibitor resistance. AIDS. 2007;21(2):239–41.
15. Fairbairn N, Kerr T, Milloy MJ, Zhang R, Montaner J, Wood E. Crystal meth‑
amphetamine injection predicts slower HIV RNA suppression among
injection drug users. Addict Behav. 2011;36(7):762–3.
16. Kapadia F, Cook JA, Cohen MH, Sohler N, Kovacs A, Greenblatt RM,
Choudhary I, Vlahov D. The relationship between non‑injection drug use
behaviors on progression to AIDS and death in a cohort of HIV seroposi‑
tive women in the era of highly active antiretroviral therapy use. Addic‑
tion. 2005;100(7):990–1002.
17. Massanella M, Gianella S, Schrier R, Dan JM, Perez‑Santiago J, Oliveira MF,
Richman DD, Little SJ, Benson CA, Daar ES, et al. Methamphetamine use
in HIV‑infected individuals affects T‑cell function and viral outcome dur‑
ing suppressive antiretroviral therapy. Sci Rep. 2015;5:13179.
18. Jiang J, Wang M, Liang B, Shi Y, Su Q, Chen H, Huang J, Su J, Pan P, Li
Y, et al. In vivo effects of methamphetamine on HIV‑1 replication: a
population‑based study. Drug Alcohol Depend. 2016;159:246–54.
19. Kedzierska K, Crowe SM. The role of monocytes and macrophages in the
pathogenesis of HIV‑1 infection. Curr Med Chem. 2002;9(21):1893–903.
20. Zhu T. HIV‑1 in peripheral blood monocytes: an underrated viral source. J
Antimicrob Chemother. 2002;50(3):309–11.
21. Strazza M, Pirrone V, Wigdahl B, Nonnemacher MR. Breaking down
the barrier: the effects of HIV‑1 on the blood‑brain barrier. Brain Res.
2011;1399:96–115.
22. Williams DW, Eugenin EA, Calderon TM, Berman JW. Monocyte matura‑
tion, HIV susceptibility, and transmigration across the blood brain barrier
are critical in HIV neuropathogenesis. J Leukoc Biol. 2012;91(3):401–15.
23. Sanmarti M, Ibanez L, Huertas S, Badenes D, Dalmau D, Slevin M, Krupin‑
ski J, Popa‑Wagner A, Jaen A. HIV‑associated neurocognitive disorders. J.
Mol Psychiatry. 2014;2(1):2.
24. Zayyad Z, Spudich S. Neuropathogenesis of HIV: from initial neuroinva‑
sion to HIV‑associated neurocognitive disorder (HAND). Curr HIV/AIDS
Rep. 2015;12(1):16–24.

Page 9 of 9
Liuetal. Cell Biosci (2021) 11:194
25. Skowronska M, McDonald M, Velichkovska M, Leda AR, Park M, Toborek M.
Methamphetamine increases HIV infectivity in neural progenitor cells. J
Biol Chem. 2018;293(1):296–311.
26. Calderon TM, Williams DW, Lopez L, Eugenin EA, Cheney L, Gaskill PJ,
Veenstra M, Anastos K, Morgello S, Berman JW. Dopamine increases
CD14(+)CD16(+) monocyte transmigration across the blood brain
barrier: implications for substance abuse and HIV neuropathogenesis. J
Neuroimmune Pharmacol. 2017;12(2):353–70.
27. Gaskill PJ, Calderon TM, Coley JS, Berman JW. Drug induced increases in
CNS dopamine alter monocyte, macrophage and T cell functions: impli‑
cations for HAND. J Neuroimmune Pharmacol. 2013;8(3):621–42.
28. Honda K, Taniguchi T. IRFs: master regulators of signalling by Toll‑like
receptors and cytosolic pattern‑recognition receptors. Nat Rev Immunol.
2006;6(9):644–58.
29. Osterlund PI, Pietilä TE, Veckman V, Kotenko SV, Julkunen I. IFN regulatory
factor family members differentially regulate the expression of type III IFN
(IFN‑lambda) genes. J Immunol. 2007;179(6):3434–42.
30. Chen K, Liu J, Cao X. Regulation of type I interferon signaling in immunity
and inflammation: a comprehensive review. J Autoimmun. 2017;83:1–11.
31. Hillmer EJ, Zhang H, Li HS, Watowich SS. STAT3 signaling in immunity.
Cytokine Growth Factor Rev. 2016;31:1–15.
32. Wang X, Ye L, Hou W, Zhou Y, Wang YJ, Metzger DS, Ho WZ. Cellular
microRNA expression correlates with susceptibility of monocytes/mac‑
rophages to HIV‑1 infection. Blood. 2009;113(3):671–4.
33. Nair MP, Saiyed ZM, Nair N, Gandhi NH, Rodriguez JW, Boukli N,
Provencio‑Vasquez E, Malow RM, Miguez‑Burbano MJ. Methampheta‑
mine enhances HIV‑1 infectivity in monocyte derived dendritic cells.
J Neuroimmune pharmacology : the official journal of the Society on
NeuroImmune Pharmacology. 2009;4(1):129–39.
34. Liang H, Wang X, Chen H, Song L, Ye L, Wang SH, Wang YJ, Zhou L, Ho WZ.
Methamphetamine enhances HIV infection of macrophages. Am J Pathol.
2008;172(6):1617–24.
35. Wang X, Wang Y, Ye L, Li J, Zhou Y, Sakarcan S, Ho W. Modulation of intra‑
cellular restriction factors contributes to methamphetamine‑mediated
enhancement of acquired immune deficiency syndrome virus infection
of macrophages. Curr HIV Res. 2012;10(5):407–14.
36. Lawson KS, Prasad A, Groopman JE. Methamphetamine enhances HIV‑1
replication in CD4(+) T‑cells via a novel IL‑1beta auto‑regulatory loop.
Front Immunol. 2020;11:136.
37. Prasad A, Kulkarni R, Shrivastava A, Jiang S, Lawson K, Groopman JE.
Methamphetamine functions as a novel CD4(+) T‑cell activator via the
sigma‑1 receptor to enhance HIV‑1 infection. Sci Rep. 2019;9(1):958.
38. Wires ES, Alvarez D, Dobrowolski C, Wang Y, Morales M, Karn J, Harvey
BK. Methamphetamine activates nuclear factor kappa‑light‑chain‑
enhancer of activated B cells (NF‑κB) and induces human immunode‑
ficiency virus (HIV) transcription in human microglial cells. J Neurovirol.
2012;18(5):400–10.
39. Hou W, Wang X, Ye L, Zhou L, Yang ZQ, Riedel E, Ho WZ. Lambda
interferon inhibits human immunodeficiency virus type 1 infection of
macrophages. J Virol. 2009;83(8):3834–42.
40. Wang Y, Li J, Wang X, Zhou Y, Zhang T, Ho W. Comparison of antiviral
activity of lambda‑interferons against HIV replication in macrophages. J
Interferon Cytokine Res. 2015;35(3):213–21.
41. Fagone P, Nunnari G, Lazzara F, Longo A, Cambria D, Distefano G,
Palumbo M, Nicoletti F, Malaguarnera L, Di Rosa M. Induction of OAS
gene family in HIV monocyte infected patients with high and low viral
load. Antiviral Res. 2016;131:66–73.
42. Krapp C, Hotter D, Gawanbacht A, McLaren PJ, Kluge SF, Sturzel CM, Mack
K, Reith E, Engelhart S, Ciuffi A, et al. Guanylate binding protein (GBP) 5
is an interferon‑inducible inhibitor of HIV‑1 infectivity. Cell Host Microbe.
2016;19(4):504–14.
43. Morales DJ, Lenschow DJ. The antiviral activities of ISG15. J Mol Biol.
2013;425(24):4995–5008.
44. Nasr N, Alshehri AA, Wright TK, Shahid M, Heiner BM, Harman AN, Bot‑
ting RA, Helbig KJ, Beard MR, Suzuki K, et al. Mechanism of interferon‑
stimulated gene induction in HIV‑1‑infected macrophages. J Virol.
2017;91(20):e00744.
45. Nasr N, Maddocks S, Turville SG, Harman AN, Woolger N, Helbig KJ, Wilkin‑
son J, Bye CR, Wright TK, Rambukwelle D, et al. HIV‑1 infection of human
macrophages directly induces viperin which inhibits viral production.
Blood. 2012;120(4):778–88.
46. Okumura A, Lu G, Pitha‑Rowe I, Pitha PM. Innate antiviral response targets
HIV‑1 release by the induction of ubiquitin‑like protein ISG15. Proc Natl
Acad Sci USA. 2006;103(5):1440–5.
47. Ikushima H, Negishi H, Taniguchi T. The IRF family transcription factors
at the interface of innate and adaptive immune responses. Cold Spring
Harb Symp Quant Biol. 2013;78:105–16.
48. Brierley MM, Fish EN. Stats: multifaceted regulators of transcription. J
Interferon Cytokine Res. 2005;25(12):733–44.
49. Raftery N, Stevenson NJ. Advances in anti‑viral immune defence:
revealing the importance of the IFN JAK/STAT pathway. Cell Mol Life Sci.
2017;74(14):2525–35.
50. Huang J, Wang F, Argyris E, Chen K, Liang Z, Tian H, Huang W, Squires K,
Verlinghieri G, Zhang H. Cellular microRNAs contribute to HIV‑1 latency in
resting primary CD4+ T lymphocytes. Nat Med. 2007;13(10):1241–7.
51. Pilakka‑Kanthikeel S, Nair MP. Interaction of drugs of abuse and microRNA
with HIV: a brief review. Front Microbiol. 2015;6:967.
52. Swaminathan G, Navas‑Martin S, Martin‑Garcia J. MicroRNAs and HIV‑1
infection: antiviral activities and beyond. J Mol Biol. 2014;426(6):1178–97.
53. Whisnant AW, Bogerd HP, Flores O, Ho P, Powers JG, Sharova N, Stevenson
M, Chen CH, Cullen BR. In‑depth analysis of the interaction of HIV‑1
with cellular microRNA biogenesis and effector mechanisms. mBio.
2013;4(2):e000193.
54. Sandau US, Duggan E, Shi X, Smith SJ, Huckans M, Schutzer WE, Loftis JM,
Janowsky A, Nolan JP, Saugstad JA. Methamphetamine use alters human
plasma extracellular vesicles and their microRNA cargo: an exploratory
study. J Extracell Vesicles. 2020;10(1):e12028–e12028.
55. Zhu L, Zhu J, Liu Y, Chen Y, Li Y, Chen S, Li T, Dang Y, Chen T. Chronic
methamphetamine regulates the expression of MicroRNAs and puta‑
tive target genes in the nucleus accumbens of mice. J Neurosci Res.
2015;93(10):1600–10.
56. Nathans R, Chu CY, Serquina AK, Lu CC, Cao H, Rana TM. Cellular
microRNA and P bodies modulate host‑HIV‑1 interactions. Mol Cell.
2009;34(6):696–709.
57. Liu MQ, Zhao M, Kong WH, Peng JS, Wang F, Qiu HY, Zhu ZR, Tang L,
Sang M, Wu JG, et al. Antiretroviral therapy fails to restore levels of
HIV‑1 Restriction miRNAs in PBMCs of HIV‑1‑infected MSM. Medicine.
2015;94(46):e2116.
58. Yin C, Zhang T, Qu X, Zhang Y, Putatunda R, Xiao X, Li F, Xiao W, Zhao H,
Dai S, et al. In vivo excision of HIV‑1 provirus by saCas9 and multiplex
single‑guide RNAs in animal models. Mol Ther. 2017;25(5):1168–86.
59. Kalasinsky KS, Bosy TZ, Schmunk GA, Reiber G, Anthony RM, Furukawa
Y, Guttman M, Kish SJ. Regional distribution of methamphetamine in
autopsied brain of chronic human methamphetamine users. Forensic Sci
Int. 2001;116(2–3):163–9.
60. Klette KL, Kettle AR, Jamerson MH. Prevalence of use study for
amphetamine (AMP), methamphetamine (MAMP), 3,4‑methylenedioxy‑
amphetamine (MDA), 3,4‑methylenedioxy‑methamphetamine (MDMA),
and 3,4‑methylenedioxy‑ethylamphetamine (MDEA) in military entrance
processing stations (MEPS) specimens. J Anal Toxicol. 2006;30(5):319–22.
61. Schepers RJ, Oyler JM, Joseph RE Jr, Cone EJ, Moolchan ET, Huestis MA.
Methamphetamine and amphetamine pharmacokinetics in oral fluid and
plasma after controlled oral methamphetamine administration to human
volunteers. Clin Chem. 2003;49(1):121–32.
62. Takayasu T, Ohshima T, Nishigami J, Kondo T, Nagano T. Screening and
determination of methamphetamine and amphetamine in the blood,
urine and stomach contents in emergency medical care and autopsy
cases. J Clin Forensic Med. 1995;2(1):25–33.
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