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Immune activation and
inflammation in lactating women
on combination antiretroviral
therapy: role of gut dysfunction
and gut microbiota imbalance
Privilege Tendai Munjoma
1
*, Panashe Chandiwana
1
,
Jacqueline Wyss
2,3
, Arthur John Mazhandu
1
,
Sebastian Bruno Ulrich Jordi
2,3
, Rutendo Gutsire
1
,
Leolin Katsidzira
4
, Bahtiyar Yilmaz
2,3
†
, Benjamin Misselwitz
2,3
†
and Kerina Duri
1
†
1
Immunology Unit, Department of Laboratory Diagnostic and Investigative Sciences, University of
Zimbabwe Faculty of Medicine and Health Sciences (UZ-FMHS), Harare, Zimbabwe,
2
Department of
Visceral Surgery and Medicine, Inselspital, Bern University Hospital, University of Bern,
Bern, Switzerland,
3
Department for Biomedical Research, Maurice Müller Laboratories, University of
Bern, Bern, Switzerland,
4
Department of Internal Medicine, University of Zimbabwe Faculty of
Medicine and Health Sciences (UZ-FMHS), Harare, Zimbabwe
Introduction: Combination antiretroviral therapy (cART) effectively controls HIV;
however, chronic low-level viremia and gut microbiota dysbiosis remain significant
drivers of gut and systemic inflammation. In this study, we explored the relationship
between gut microbiota composition, intestinal inflammation, microbial
translocation, and systemic inflammation in women on cART in Sub-Saharan Africa.
Methods: We conducted a study in HIV-infected and HIV-uninfected lactating
women followed up at 6 weeks and 6 months postpartum in Harare, Zimbabwe. We
used 16S ribosomal Ribonucleic Acid (rRNA) sequencing and MesoScale Discovery
V-Plex assays to examine the gut microbiome and to quantify plasma inflammatory
biomarkers, respectively. In addition, we measured fecal calprotectin, plasma
lipopolysaccharide-binding protein (LBP), and soluble cluster of differentiation 14
(sCD14) by enzyme-linked immunosorbent assay to assess gut inflammation,
microbial translocation, and monocyte/macrophage activation.
Results: A group of 77 lactating women were studied, of which 35% were HIV-
infected. Fecal calprotectin levels were similar by HIV status at both follow-up
time points. In the HIV-infected group at 6 weeks postpartum, fecal calprotectin
was elevated: median (interquartile range) [158.1 µg/g (75.3–230.2)] in women
who had CD4+ T-lymphocyte counts <350 cells/µL compared with those with
≥350 cells/µL [21.1 µg/g (0–58.4)], p = 0.032. Plasma sCD14 levels were
significantly higher in the HIV-infected group at both 6 weeks and 6 months
postpartum, p < 0.001. Plasma LBP levels were similar, but higher levels were
observed in HIV-infected women with elevated fecal calprotectin. We found
significant correlations between fecal calprotectin, LBP, and sCD14 with
proinflammatory cytokines. Gut microbial alpha diversity was not affected by
Frontiers in Immunology frontiersin.org01
OPEN ACCESS
EDITED BY
Giulia Carla Marchetti,
University of Milan, Italy
REVIEWED BY
Camilla Tincati,
University of Milan, Italy
Eugenio Nelson Cavallari,
Sapienza University of Rome, Italy
*CORRESPONDENCE
Privilege Tendai Munjoma
privilegemunjoma@gmail.com;
ptmunjoma@medsch.uz.ac.zw
†
These authors have contributed
equally to this work and share
last authorship
RECEIVED 21 August 2023
ACCEPTED 17 October 2023
PUBLISHED 16 November 2023
CITATION
Munjoma PT, Chandiwana P, Wyss J,
Mazhandu AJ, Jordi SBU, Gutsire R,
Katsidzira L, Yilmaz B, Misselwitz B and
Duri K (2023) Immune activation and
inflammation in lactating women on
combination antiretroviral therapy:
role of gut dysfunction and gut
microbiota imbalance.
Front. Immunol. 14:1280262.
doi: 10.3389/fimmu.2023.1280262
COPYRIGHT
© 2023 Munjoma, Chandiwana, Wyss,
Mazhandu,Jordi,Gutsire,Katsidzira,Yilmaz,
Misselwitz and Duri. This is an open-access
article distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that
the original publication in this journal is
cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms.
TYPE Original Research
PUBLISHED 16 November 2023
DOI 10.3389/fimmu.2023.1280262
HIV status and was not affected by use of antibiotic prophylaxis. HIV significantly
affected microbial beta diversity, and significant differences in microbial
composition were noted. The genera Slackia and Collinsella were relatively
more abundant in the HIV-infected group, whereas a lower relative abundance
of Clostriduim sensu_stricto_1 was observed. Our study also found correlations
between gut microbial taxa abundance and systemic inflammatory biomarkers.
Discussion and conclusion: HIV-infected lactating women had increased
immune activation and increased microbial translocation associated with
increased gut inflammation. We identified correlations between the gut
inflammation and microbial composition, microbial translocation, and systemic
inflammation. The interplay of these parameters might affect the health of this
vulnerable population.
KEYWORDS
fecal calprotectin, microbial translocation, systemic inflammation, HIV, gut microbiota,
lactating women, resource limited setting
Introduction
People living with HIV in Sub-Saharan Africa (SSA) constitute
about 54% of the world’s HIV-infected population (1). The
introduction of combination antiretroviral therapy (cART) has
reduced the burden of HIV in SSA with a significant reduction in
morbidity and mortality. However, chronic persistent low-level
viremia due to residual HIV remains a significant contributor to
microbial translocation, chronic monocyte activation, and
inflammation in this population (2–4). Understanding the
relationship between these biological systems contributes to the
global effort of interventions toward the mitigation of HIV-
associated morbidities.
The acute phase of HIV infection involves depletion of the CD4+
T-cell population, causing major damage to gut-associated lymphoid
tissue, which is not fully restored by cART (5,6). Upon gut
inflammation, neutrophils serve as a reliable defense mechanism.
Gut inflammation causes increased gut permeability and release of
calprotectin from neutrophils. The fecal calprotectin levels are thus a
useful marker of gut inflammation and an indirect marker of intestinal
permeability (7). Faecal calprotectin levels have been reported to be
higherintheHIV-infectedpopulationevenifoncARTcomparedwith
that in HIV-uninfected peers (8,9). Despite these studies, the influence
of gut inflammation on microbial translocation and systemic
inflammation is still insufficiently understood.
Inflammation of the gut epithelial lining causes translocation of
microbial antigens into circulation driving HIV disease progression
through monocyte activation and inflammation (10). The monocyte/
macrophage bound cluster of differentiation 14 (CD14) is a co-
receptor for lipopolysaccharide (LPS) and causes the secretion of
soluble CD14 (sCD14) (11) on exposure to bacterial toxins. Thus,
both LPS-binding protein (LBP) and sCD14 are considered
biomarkers of endotoxemia and intestinal permeability, which also
alters the gut microbiota (12). Increased systemic inflammation with
accompanied gut permeability has been observed in cART-treated
women (13). However, long-term exposure to cART has been shown
to decrease biomarkers of gut permeability, microbial translocation,
and vascular injury in adults with chronic HIV infection (6,14,15).
A healthy gut microbiota is usually dominated by commensal
microorganisms that continually face perturbations such as HIV-
induced gut damage and antibiotics. Dysbiosis as a result of HIV
infection is generally characterized by a decrease in alpha diversity
(16,17) with a low abundance of Bacteroides and an increased
abundance of Prevotella (18). However, further evidence is needed
to further understand the impact of HIV-associated gut microbiota
dysbiosis on the production of proinflammatory cytokines and the
consequent systemic inflammation (19). Microbial antigens
translocated into circulation causes immune activation with
higher levels being associated with increased T-cell activation in
cART-treated individuals (15). In early chronic HIV infection,
circulating LPS has been shown to be a predictor of HIV disease
progression independent of HIV viremia and CD4+ T-lymphocyte
count (20). Evidence of the role of HIV-induced gut microbiota
dysbiosis in microbial translocation and inflammation has been
conflicting due to possible confounders in HIV management such
as antibiotic prophylaxis and cART.
To gain an understanding of the intricate relationships between
the processes, we hypothesized that microbial dysbiosis and
inflammation of the gut due to HIV infection cause microbial
translocation and, ultimately, systemic inflammation. Our study
aims to provide insights into the gut microbiota diversity and
abundance in HIV-infected and HIV-uninfected lactating women
at 6 weeks and 6 months postpartum. Biomarkers of systemic
inflammation and their association with gut microbiota
abundance, gut inflammation, and microbial translocation
were investigated.
Munjoma et al. 10.3389/fimmu.2023.1280262
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Materials and methods
Study design
This investigation was performed as a prospective longitudinal
study nested in the University of Zimbabwe Birth Cohort Study
(UZBCS). The UZBCS has been previously described in detail (21).
In brief, lactating women were longitudinally followed up at 6 weeks
and 6 months postpartum as part of a longitudinal follow-up to 2
years after birth.
Study participants
The study followed up women enrolled in the UZBCS who were
HIV-infected and HIV-uninfected and receiving postnatal care
services. They were monitored at 6 weeks and 6 months after
giving birth at four primary healthcare clinics located in areas with
low socio-economic status in Harare, Zimbabwe (Budiriro,
Glenview, Kuwadzana, and Rujeko clinics).
Inclusion and exclusion criteria
We recruited pregnant women beyond the 20th week of pregnancy
seeking antenatal care services. All participants gave written informed
consent to participate and had been tested for HIV. Women who failed
to adhere to the study procedures due to any health disorders such as
mental issues were not included in this sub-study. For this particular
study, only women who enrolled in the UZBCS in 2019 and had stool
samples available were included. These women were then followed up
at 6 weeks and 6 months after giving birth.
Data collection, sample collection,
and storage
Data were collected using paper-based approved questionnaires
and entered into a Research Electronic Data Capture (REDCap)
database—a secure, web-based software platform designed to support
data capture for research studies (22). A physical examination
including anthropometric assessments was carried out by trained
and qualified nurses. A total of 4 mL of whole venous blood samples,
collected using ethylenediamine-tetraacetic acid as an anticoagulant,
were obtained from each participant. The blood collection procedure
was carried out by trained and qualified nurses, ensuring adherence
to proper protocols. The collected samples were promptly processed
within a maximum time frame of 6 h from the time of collection.
Plasma was isolated and stored at −80°C until enzyme-linked
immunosorbent assays (ELISAs) and MesoScale Discovery (MSD)
V-plex assays were performed. About 50 g of feces was collected in
sterile containers, aliquoted, and stored at −80°C until fecal
calprotectin and DNA extraction assays were done.
HIV RNA load and CD4+
T-lymphocyte counts
Results of HIV RNA load and CD4+ T-lymphocyte counts
measured during the third trimester of pregnancy were obtained
from the UZBCS REDCap database. The assaying methods for these
HIV disease progression markers were previously described (21).
All HIV-infected women in this study were taking cART at both
follow-up time points, and used a formulation of efavirenz,
lamivudine, and tenofovir disoproxil fumarate (Tenolam-E),
following the World Health Organization guidelines (23).
Systemic inflammation and
immune biomarkers
The MSD multi-spot V-plex assays (Rockville, Maryland, USA)
were used to quantify proinflammatory and vascular injury immune
markers in 60 µL of plasma. The assays were carried out following the
manufacturer’s instructions and as previously described (24).
Originally, 48 biomarkers were quantifiedinplasma,and,forthis
study, the proinflammatory and vascular injury V-plex panels were of
interest. The proinflammatory V-plex panel included interferon-
gamma (IFN-g), interleukin-1-beta (IL-1b), interkeukin-2 (IL-2),
interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8),
interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), interleukin-13
(IL-13), and tumor necrosis factor (TNF), and the vascular injury V-
plex panel included serum amyloid A (SAA), C-reactive protein (CRP),
vascular cell adhesion molecule 1 (VCAM-1), and intercellular
AdhesionMolecule1(ICAM-1).ThebiomarkerSAAwasexcluded
from our analyses due to calibration failure in one of the assays.
Biomarker of gut inflammation
Fecal calprotectin was quantified from stool using a Buhlmann
fecal calprotectin sandwich ELISA assay (EK-CAL2-WEX,
Schönenbuch, Switzerland) based on the manufacturer’s
instructions. The Buhlmann Calex Cap (B-CALEX-C200,
Schönenbuch, Switzerland) was utilized to prepare stool extracts
following the manufacturer’s instructions. The extracts were then
diluted at a ratio of 1:5 with an appropriate incubation buffer before
proceeding with the assay. All assays were conducted in duplicate,
and ELISA plates were read at 450 nm using Gen 5 software
(BioTek, Winooski, VT, USA). Fecal calprotectin concentrations
were determined using a standard curve and categorized based on
the manufacturer’s clinical cutoffs: normal (<80 µg/g), borderline/
grey zone (80–160 µg/g), and elevated (>160 µg/g).
Munjoma et al. 10.3389/fimmu.2023.1280262
Frontiers in Immunology frontiersin.org03
Biomarkers of microbial translocation and
monocyte activation
Plasma LBP and sCD14 levels were quantified using ELISA
assays (Hycult Biotech, Wayne, USA) according to the
manufacturer’s instructions. Absorbance was read at 450 nm, and
sample concentrations were determined from a standard curve. All
assays were performed in duplicate, and ELISA plates were read
using Gen 5 software (BioTek, Winooski, VT, USA).
Stool DNA Extraction and 16S
rRNA sequencing
Fecal samples were collected into sterile 50-mL sample cups,
aliquoted into 2-mL tubes, and stored at −80°C prior to assays. Fecal
DNA extraction was carried out from about 250 mg of stool sample
using the QIAamp PowerFecal Pro DNA kit (Qiagen, Dusseldorf,
Germany) as previously described (25). Total bacterial DNA was
eluted with 70 mL of elution buffer and then stored at −20°C prior to
PCR amplification. The eluted DNA was amplified using PCR,
targeting the V5 and V6 regions of the 16S rRNA gene. Previously
described bacteria-specific primers (forward: 5′-
CCATCTCATCCCTGCGTGTCTCCGACTCAGC-barcode-
ATTAGATACCCYGGTAGTCC-3′and reverse: 5′-CCTCTCTAT
GGGCAGTCGGTGATA CGAGCTGACGACARCCATG-3′) were
utilized (26). PCR conditions consisted of an initial denaturation at
94°C for 5 min, followed by 35 cycles of denaturation at 94°C for
1 min, annealing at 46°C for 20 s, elongation at 72°C for 30 s, and a
final elongation at 72°C for 7 min.
The PCR amplicons were run on 1% agarose gel electrophoresis
at 100 volts for 1 h, with an expected product length of
approximately 350 base pairs. The amplicons were purified using
the QIAQuick Gel Extraction Kit (Qiagen, Dusseldorf, Germany).
The concentration of amplicons was determined using a Qubit
dsDNA HS Assay Kit on the Qubit 3.0 Fluorometer (ThermoFisher
Scientific) and then set to 26 pM for sequencing library preparation.
Sequencing was carried out on the Ion PGM™System
(ThermoFisher Scientific) using an Ion PGM™Sequencing kit
and chip, following a previously described method (27).
Data analysis
Sociodemographic and participant
characteristics
Data analysis was conducted using R software version 4.2.2
(http://www.r-project.org/). Continous variables were tested for
normality using the Shapiro–Wilk test, and data were
summarized using median and interquartile range (IQR) or using
mean ± standard deviation (SD) where appropriate. Continous data
between groups were compared using the Mann–Whitney U-test,
Kruskal–Wallis test, or Student T-test depending on the
distribution of the data. Categorical data were reported as
proportions and associations determined by Fisher’s exact test or
Chi-squared test where appropriate.
Microbial translocation, monocyte
activation, gut inflammation, and systemic
inflammatory bıomarkers
Continous data were tested for normalcy using the Shapiro–
Wilk test and data reported as median (IQR) or mean ± SD where
appropriate, depending on data distribution. Concentrations of
proinflammatory and vascular injury immune markers below the
assay detection limit were assigned the concentration of the lowest
calibrator as previously described (24). A q-value was calculated to
correct for multiple testing using the Bonferroni test, and q < 0.05
was considered significant. Correction was done for the 48
biomarkers originally tested during the assays. Spearman rho (r)
correlation coefficient was used to determine associations between
biomarkers of HIV disease progression, gut inflammation,
microbial translocation, and systemic inflammation.
Computational analysis of 16S rRNA
microbial data
The Fastq sequencing files generated from Ion Torrent PGM™
System were processed using the Quantitative Insights into Microbial
Ecology 2 (QIIME2) version 2021.11.0 pipeline (https://qiime2.org/), as
previously described (26,28,29). Amplicon sequence variants were
assigned with a 97% sequence identity threshold, using the default
optionsinQIIME2aswellastheq2-feature-classifier plugin and a
Naïve Bayes classifier. Taxonomic weights were assembled using the
SILVA database (https://www.arb-silva.de/).
The feature table and mapping file were used to generate a
phyloseq object in R (version 4.2.2) package phyloseq (30). Only
samples with more than 2,000 high-quality reads were further
analyzed. Diversity within communities was determined using
alpha diversity indices (Simpson and Shannon index), and inter-
community diversity was determined using beta diversity [Bray–
Curtis dissimilarity using principal coordinate analysis (PCoA)] (31).
Mann–Whitney U-tests and Adonis (PERMANOVA) tests for alpha
diversity and beta diversity were performed to test for significance of
any group differences, respectively. Taxonomy profiling was
performed using microbiome Multivariable Association with Linear
Models (MaAsLin2) package (https://huttenhower.sph.harvard.edu)
to determine associations of the gut microbiota with categorical and
continuous variables (32). A q-value was calculated to correct for
multiple testing using the Benjamini–Hochberg (BH) false discovery
rate (FDR) correction as a default step in MaAsLin2. A q-value <0.05
was considered significant, and microbiota plots were generated using
the package phyloseq and GraphPad prism version 9.0.0 (GraphPad,
San Diego, CA).
Munjoma et al. 10.3389/fimmu.2023.1280262
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Results
Ofthe97womenenrolledsince2019,77lactatingwomenwere
successfully followed up at both 6 weeks and 6 months postpartum.
Maternal socio-demographics, concurrent medications, and clinical
characteristics at 6 weeks postpartum are shown in Table 1.TheHIV-
infected women [median age, 32 years (IQR, 29–35)] were older than
the HIV-uninfected counterparts [median age, 26 years (IQR, 21–30)].
At 6 weeks postpartum, all HIV-infected women were on cART,
and 15 (55.6%) were taking cotrimoxazole prophylaxis. In all these
women, cART was started pre-conception or during pregnancy. HIV-
infected women were more likely to be on antibiotics during the study
period, p < 0.0001. In addition, HIV-infected women had a
significantly higher mid-upper–arm circumference (MUAC)
(27.0 cm; IQR, 25.7–28.0) compared with their HIV-uninfected peers
(25.4 cm; IQR, 24.0–27.3), p = 0.031. Interestingly, HIV-infected
women had a higher body mass index (BMI) compared with their
uninfected counterparts (p =0.020),asindicatedinTable 1.
Socio-demographics and clinical characteristics at 6 months
postpartum are shown in Table 2. HIV-infected women were more
likely to be in employed and to have a higher median monthly family
income (Table 2). There were no associations between HIV infection
status and toilet facilities, drinking water sources, and water
treatment. Similar to the 6-week postpartum findings, HIV-infected
women were more likely to be on antibiotics (see Table 1). In the
HIV-infected group 55.6% and 57.1% reported taking cotrimoxazole
antibiotics at 6 weeks and 6 months postpartum, respectively.
Biomarkers of gut inflammation and gut
microbial translocation
Biomarkers of intestinal inflammation (fecal calprotectin),
microbial translocation, and immune activation (plasma LBP and
sCD14) were quantified to assess the association between HIV
infection with gut inflammation and microbial gut translocation.
Overall, fecal calprotectin levels did not significantly differ by HIV
infection status and antibiotic use at 6 weeks and 6 months
postpartum. However, at 6 weeks, higher fecal calprotectin levels
(158.1 µg/g; IQR, 75.3–230.2) were observed in HIV-infected
women with third-trimester CD4+ T-lymphocyte counts <350
cells/µL compared with those with ≥350 cells/µL (21.1 µg/g; IQR,
0–58.4), p = 0.032. In a sub-analysis of the HIV infected at 6 weeks
postpartum, we investigated the effects of cART and HIV viremia.
We stratified cART duration into early (<2 years) and long-term
(≥2 years) (33) and HIV RNA load into low level viremia (<200
copies/mL) and viremic (≥200 copies/mL) groups (4). Fecal
calprotectin levels were non-significantly higher in unsuppressed
(HIV RNA > 1,000 copies/mL) versus suppressed (≤1,000 copies/
mL) in viremic versus low level viremia groups (p = 0.610 and p =
0.614, respectively). Levels were non-significantly higher (116.7 µg/
g; IQR, 43.3–230) in the early ART group compared with that in the
long-term ART group (61.3 µg/g; IQR, 16.3–165.3).
HIV-infected participants had significantly higher plasma sCD14
levels (29.7 ng/mL; IQR, 22.4–35.5) at 6 weeks postpartum when
TABLE 1 Socio-demographic and clinical data of the study participants
at 6 weeks postpartum (n =77), stratified by HIV status.
HIV-
infected
(n = 27)
HIV-
uninfected
(n = 50)
p-
value
Social demographics
Age (years)
[Median (IQR)] 32 (29–35) 26 (21–30) 3.95e-05
Breastfeeding type
Exclusive
Mixed
25 (92.6%)
2 (7.4%)
42 (84%)
8 (16%)
0.479
Postpartum alcohol use
Yes
No
1 (3.7%)
26 (96.3%)
1 (2%)
49 (98%)
1.000
Household meals
per day
[Median (IQR)] 3 (2-3) 3 (2-3) 0.569
Average stool frequency
Once daily
Greater or equal to
twice daily
Once every 2 days
21 (77.8%)
4 (14.8%)
2 (7.4%)
37 (75.5%)
9 (18.4%)
3 (6.1%)
Missing=1
0.883
Concurrent medications
Antibiotics use
Yes
No
15 (55.6%)
12 (44.4%)
2 (4%)
48 (96%)
4.48e-07
Anti-acid use
Yes
No
0 (0%)
27 (100%)
3 (6%)
47 (94%)
0.547
cART use
Yes
No
27 (100%)
0 (0%)
NA _
Clinical data
MUAC (cm)
[Median (IQR)] 27.0 (25.7–28.0) 25.4 (24.0–27.3) 0.031
BMI (kg/m
2
)
[Median (IQR)] 24.2 (22.1–25.6) 22.1 (19.6–23.9) 0.020
Mode of delivery
Spontaneous
Caesarean section
24 (88.9)
3 (11.1%)
50 (100%)
0 (0%)
0.039
cART duration
(months)
[Median (IQR); min-
max]
40.8 (13.5–86.1);
1.8–1455.4
NA _
cART duration group
Early ART (<2 years)
Long-term ART
(≥2 years)
8 (29.6%)
19 (70.4%)
NA _
Third trimester CD4
count (cells/µL)
[Median (IQR);
min-max]
355 (253–449);
176–635
NA _
Third trimester HIV
RNA suppression
Suppressed (≤1,000 23 (85.2%) NA _
(Continued)
Munjoma et al. 10.3389/fimmu.2023.1280262
Frontiers in Immunology frontiersin.org05
compared with their uninfected counterparts (18.9 ng/mL; IQR,
15.9–21.2), p < 0.0001 (Figure 1A). Similar results were found at 6
months postpartum where increased sCD14 levels were found in
HIV-infected (33.2 ng/mL; IQR, 25.0–36.2) compared with that in
HIV-uninfected women (22.7 ng/mL; IQR, 19.0–27.0), p = 0.0006.
IntheHIV-infectedgroupat6weekspostpartum,sCD14levels
were similar when compared by cART duration and HIV viremia
groups (p = 0.449 and p = 0.921 respectively). In all women, at 6 weeks
postpartum, those taking antibiotics had significantly higher plasma
sCD14 levels (24.4 ng/mL; IQR, 22.1–35.3) (Figure 1B)comparedwith
those not taking antibiotics (20.1 ng/mL; IQR, 16.5–24.7). Plasma
sCD14 levels did not differ by antibiotic use in the subgroups of HIV-
infected and HIV-uninfected women (Figure 1C). At 6 months
postpartum, only HIV-infected women were on regular antibiotics,
and no significant difference in median sCD14 levels was noted on
comparison by antibiotic usage. Furthermore, plasma sCD14 levels at 6
weeksand6monthspostpartumdidnotdifferbetweenwomenwith
elevated and normal fecal calprotectin levels.
Plasma LBP levels were similar between the HIV subgroups at 6
weeks or 6 months postpartum. In the HIV-infected group at 6 weeks
postpartum, LBP levels were similar when compared by cART duration
and HIV viremia groups (p = 0.632 and p = 0.453, respectively). At the
same time point, most HIV-infected women (88.2%) were on
cotrimoxazole prophylaxis, and there was no significant difference in
plasma LBP levels by antibiotic use. At 6 weeks postpartum,
significantly lower LBP levels were noted in women with normal
fecal calprotectin levels compared with those with elevated levels [18.2
ng/mL (IQR, 14.7–26.5) versus 26.4 ng/ml (IQR, 19.9–31.8), p = 0.022]
(Figure 2A). A similar trend was observed in the HIV-infected
subgroupatthesametimepoint(Figure 2B).
Correlation between biomarkers of gut
inflammation, microbial translocation, and
systemic inflammation
To determine whether biomarkers of gut inflammation (fecal
calprotectin) and microbial translocation (sCD14 and LBP) are
associated with the systemic immune environment, a correlation
matrix of these biomarkers and 13 proinflammatory cytokines and
chemokines was computed (Figure 3). These correlations were
calculated after stratification by HIV status to minimize confounding
by known effects of HIV on the cytokine and chemokine environment.
TABLE 1 Continued
HIV-
infected
(n = 27)
HIV-
uninfected
(n = 50)
p-
value
copies/mL)
Unsuppressed (>1,000
copies/mL)
4 (14.8%)
Third trimester HIV
Viremia
Low-level (<200 copies/
mL)
Viremic (≥200 copies/
mL)
23 (85.2%)
4 (14.8%)
NA _
BMI, body mass index; MUAC, mid-upper–arm circumference; IQR, interquartile range;
HIV, human immunodeficiency virus; CD4, cluster of differentiation 4; cART, combination
anti-retroviral therapy; RNA, ribonucleic acid. Statistical analysis: Group comparisons were
done using Mann–Whitney U-test or Fisher’s exact test where appropriate. P-values in bold
font are statistically significant at p < 0.05.
TABLE 2 Socio-demographic, water, hygiene and sanitation and
concurrent medications at 6 months postpartum (n = 77), stratified by
HIV status.
HIV-
infected
(n = 28)
HIV-
uninfected
(n = 49)
p-
value
Social demographics
Employment status
Employed
Unemployed
14 (50%)
14 (50%)
11 (22.4%)
38 (77.6%)
0.044
Household size
[Median (IQR)] 5 (4–5) 4 (3–5) 0.074
Family monthly income
(USD)
[Median (IQR)] 1,150 (800–
1,450)
650 (480–1,285) 0.017
Water, hygiene and sanitation
Toilet facility in household
Flush (outside/inside)
Blair
27 (96.4%)
1 (3.6%)
47 (95.9%)
2 (4.1%)
0.155
Households sharing toilet
[Median (IQR)] 1 (1–4) 3 (2–4) 0.106
Main drinking water source
Borehole
Piped water into dwelling
Protected well
18 (64.3%)
1 (3.6%)
9 (32.1%)
22 (44.9%)
5 (10.2%)
22 (44.9%)
0.293
Drinking water treatment
No
Yes
21 (75%)
7 (25%)
42 (85.7%)
7 (14.3%)
0.357
Current sewer burst/overspill
No
Yes
25 (89.3%)
3 (10.7%)
41 (85.4%)
7 (14.6%)
Missing = 1
0.736
Current diarrhea
(participant/household
member)
No
Yes
26 (92.9%)
2 (7.1%)
42 (89.4%)
5 (10.6%)
Missing = 2
0.705
Average stool frequency
Once daily
Greater or equal to twice daily
Once every 2 days
23 (82.1%)
4 (14.3%)
1 (3.6%)
34 (69.4%)
14 (28.6%)
1 (2%)
0.357
Concurrent medications
Antibiotics use
Yes
No
16 (57.1%)
12 (42.9%)
0 (0%)
49 (100%)
2.23e-09
Anti-acid use
Yes
No
0 (0%)
27 (100%)
Missing = 1
0 (0%)
49 (100%)
_
IQR, interquartile range; HIV, human immunodeficiency virus. Statistical analysis: Group
comparisons were done using the Mann–Whi tney U-test or Fi sher’s exact test where
appropriate. P-values in bold font are statistically significant at p < 0.05.
Munjoma et al. 10.3389/fimmu.2023.1280262
Frontiers in Immunology frontiersin.org06
In the HIV-uninfected women, at 6 weeks postpartum, fecal
calprotectin and LBP correlated positively with IL-10 (r= 0.38,
p = 0.0376) and IL-13 (r= 0.47, p = 0.0082), respectively (Figure 3A).
In the same group at 6 months postpartum, LBP correlated positively
with IL-6 (r= 0.40, p = 0.024) and fecal calprotectin (r= 0.31,
p = 0.032), whereas fecal calprotectin correlated positively with IL-2
(r= 0.43, p = 0.018) and IFN-g(r= 0.42, p = 0.022). The biomarker
sCD14 correlated positively with IL-2 (r= 0.36, p = 0.046) in this
subgroup at 6 months postpartum (Figure 3C).
In the HIV-infected group, pregnancy third-trimester CD4+ T-
lymphocyte count (En CD4) and pregnancy third-trimester HIV
RNA load (En VL) were included in the correlation analysis at 6
weeks postpartum (Figure 3B). We assumed that the time since the
third trimester of pregnancy may have caused insignificant changes
in CD4+ T-lymphocyte counts and HIV RNA load. Interestingly,
third-trimester CD4+ T-lymphocyte counts negatively correlated
with LBP (r=−0.47, p = 0.019), IL-6 (r=−0.43, p = 0.033) and
CRP (r=−0.42, p = 0.035). Third-trimester HIV RNA levels
(En VL) positively correlated with eight proinflammatory
cytokines and chemokines, p < 0.05 (Figure 3B). In the HIV-
infected group at 6 weeks postpartum, fecal calprotectin
correlated positively with LBP (r= 0.44, p = 0.027), IL-2
(r= 0.42, p = 0.037), and IFN-g(r= 0.49, p = 0.012), whereas
LBP correlated positively with IL-2 (r= 0.56, p = 0.002), IFN-g
(r= 0.48, p = 0.010), and IL-6 (r= 0.43, p = 0.026).
At 6 weeks postpartum, sCD14 levels positively correlated
with IL-8 (r= 0.42, p = 0.029), TNF (r= 0.39, p = 0.044), and
IL-6 (r= 0.42, p = 0.029) in the HIV-infected group (Figure 3B). At
6 months postpartum, fecal calprotectin positively correlated
with IL-12p70 (r= 0.41, p= 0.044) in the HIV-infected
group (Figure 3D), whereas LBP levels positively correlated with
IL-2 (r= 0.50, p = 0.010), IL-1b(r= 0.42, p = 0.037), IL-6 (r= 0.68,
p = 0.0002), TNF (r= 0.50, p = 0.011), and IFN-g(r= 0.55,
p = 0.004). In the same group, plasma sCD14 levels positively
correlated with CRP (r= 0.52, p = 0.006) (Figure 3D).
Comparison of biomarkers of
systemic inflammation and vascular
injury according to HIV infection
status and follow-up time point
To explore the impact of HIV infection on the cytokine/
chemokine environment, we conducted a comparative analysis of
median biomarker levels between HIV-infected and HIV-
uninfected women at 6 weeks and 6 months postpartum
(Supplementary Figure 1). We did not observe any statistically
significant differences in the biomarker levels between the HIV-
infected and HIV-uninfected women even after applying multiple
testing correction to account for potential false positives.
BCA
FIGURE 1
Plasma sCD14 levels. Plasma sCD14 levels in all participants at 6 weeks postpartum, according to HIV infection status (A), antibiotic use (B), and
stratified for HIV status (C). Statistics: Mann–Whitney U-test.
BA
FIGURE 2
Plasma LBP levels by fecal calprotectin group. Plasma LBP levels in all participants (A) and HIV-infected women (B) at 6 weeks postpartum stratified
for fecal calprotectin group. Statistics: Mann–Whitney U-test.
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Gut microbiota assessment at 6 weeks and
6 months postpartum
For gut microbiota analysis, samples from 73 of the 77
participants at 6 weeks and 65 of the 77 participants at 6 months
postpartum could be used. There were four participants with
missing stool samples at 6 weeks and five participants with
missing stool samples at 6 months. Seven samples were dropped
from analysis due to low sequence read numbers. Overall, alpha
diversity measures (Shannon and Simpson index) did not differ
significantly between the two follow-up time points or by HIV
status (Figure 4A). However, there was a significant difference in
beta diversity in all participants between the two time points
(p = 0.001). Beta diversity also differed significantly by HIV status
at 6 weeks (Figure 4B) and 6 months (Figure 4C) postpartum.
We conducted a comparative analysis of the gut microbiota in the
HIV-infected group at 6 weeks postpartum, considering third-
trimester HIV RNA suppression (cutoff of ≤1,000 copies/mL) and
third-trimester CD4+ T-lymphocyte status (cutoff of ≤350 cells/µL).
There were no significant differences in the alpha diversity (assessed
by Shannon and Simpson indices) and beta diversity according to
HIV RNA suppression and CD4+ T-lymphocyte status. In addition,
there were no significant differences in either alpha or beta diversity
stratified by antibiotic use in the HIV-infected group at 6 weeks and 6
months postpartum (data not shown). All HIV-infected participants
in our study were receiving cART; therefore, the effects of HIV and
cART on the gut microbiota could not be separated.
Our study examined the mean relative abundance of the top 10
phyla of the gut microbiota in all women (Figure 5). The
predominant phyla identified were Firmicutes (86.4%),
Actinobacteriota (7.6%), Bacteroidota (4.9%), Proteobacteria (0.8%),
Spirochaetota (0.08%), Verrucomicrobiota (0.06%), and
Desulfobacterota (0.04%). In addition to that, the mean relative
abundance of the top 10 genera was also determined. The
predominant genera were Clostriduim_sensu_stricto_1 (16.6%),
Romboutsia (16.1%), Agathobacter (5.3%), Faecalibacterium (4.9%),
Prevotella (4.4%), Sarcina (4.4%), and Blautia (3.8%). Notably, in the
HIV-infected group, a lower relative abundance of the genera
B
CD
A
FIGURE 3
Correlation matrix analysis of plasma biomarkers in HIV-infected and HIV-uninfected women at 6 weeks and 6 months postpartum. HIV-uninfected
participants at 6 weeks postpartum (A), HIV-infected participants at 6 weeks postpartum (B), HIV-uninfected participants at 6 months postpartum
(C), and HIV-infected participants at 6 months postpartum (D). Correlations marked by X were either not present or non-significant at p < 0.05.
Munjoma et al. 10.3389/fimmu.2023.1280262
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FIGURE 5
Overall relative abundance for the top 10 phyla of the gut microbiota. The top 10 phyla by order of decreasing relative abundance at both 6 weeks
and 6 months postpartum are indicated.
BC
A
FIGURE 4
Microbiota characteristics at 6 weeks and 6 months postpartum according to HIV status. Alpha diversity (Shannon and Simpson index) comparison
by HIV status at both 6 weeks and 6 months postpartum (A), PCoA for beta diversity (Bray–Curtis) comparison by HIV infection status at 6 weeks
postpartum (B), and PCoA for beta diversity (Bray-Curtis) comparison by HIV status at 6 months postpartum (C).
Munjoma et al. 10.3389/fimmu.2023.1280262
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Akkermansia and Collinsella was noted at 6 months postpartum
compared with that at 6 weeks postpartum. However, in the same
group, a higher relative abundance of the genera Lachnospira,
Prevotella,Bacteroides, and UCG.005 from the Oscillospiraceae
family was noted at 6 months compared with that at 6 weeks
postpartum. These findings highlight dynamic shifts in the relative
abundance of specific taxa in the postpartum period.
The gut microbiota was compared to determine taxa
significantly associated with HIV infection status at both 6 weeks
and 6 months postpartum. At 6 weeks postpartum, HIV infection
was significantly associated with lower abundance of genus
Clostriduim_sensu_stricto_1 and family Clostridiaceae from the
Firmicutes phylum (Figure 6A). At the same time point, HIV
infection was significantly associated with a higher abundance of
taxa from the Actinobacteriota phylum including the genera Slackia
and Collinsella (Figure 6A). At 6 months postpartum, only taxa
from the Firmicutes phylum significantly differed by HIV infection
status (Figure 6B). The genus Clostriduim_sensu_stricto_1 was
significantly abundant in the HIV-uninfected group at both 6
weeks and 6 months postpartum.
We investigated longitudinal changes in the gut microbiota from
pregnancy to 6 months postpartum. The pregnancy gut microbiota
from our study population has been previously described in detail (34).
In brief, species richness was lower (Shannon, p = 0.0092, and Simpson,
p = 0.012) in the HIV-infected women compared with that in the
uninfected peers. Beta diversity assessed using Bray–Curtis dissimilarity
index showed significant differences in diversity between HIV-infected
and HIV-uninfected pregnant women. Alpha diversity did not differ by
CD4+ T-lymphocyte group and viral load suppression using similar
cutoffs used in our study. Infection with HIV was associated with
reduced abundance of Clostridium,Bacteroides,Bifidobacterium,and
Faecalibacterium with an observed increase in Actinomyces.
In HIV-uninfected women, there were no differences in
microbial evenness between pregnancy and the two follow-up
time points (Supplementary Figure 2A). However, a significance
was noted in microbial richness (p = 0.017) when comparing the
three time points. In the HIV-infected group, both evenness and
richness indices showed an increase from pregnancy to 6 months
postpartum although the difference was non-significant for
evenness (Supplementary Figure 2B). Beta diversity comparison
showed a significant difference (p = 0.001) when compared by time
point in HIV-uninfected (Supplementary Figure 2C) and HIV-
infected groups, p = 0.001 (Supplementary Figure 2D).
Furthermore, the effects of cART duration and HIV viremia on
the gut microbiota in the HIV-infected group were investigated at 6
weeks postpartum. No differences were observed for both Alpha
and Beta diversity when compared between early versus long-term
cART groups and low-level viremia versus viremic groups.
Association of inflammatory and
vascular injury biomarkers with
the gut microbiota abundance
We determined the association of the gut microbiota with
inflammatory and vascular injury biomarkers after stratification by
HIV infection status using the MaAsLin2 package in R. Only
associations remaining significant (q < 0.05) after BH FDR
correction were reported (Table 3). To test for microbial taxa
associations with proinflammatory and vascular injury biomarkers,
microbial features that appeared in ≥25% of the total number of
samples assayed. In the HIV-infected group at 6 months postpartum,
the genera Catenibacterium and Haemophilus positively associated
with levels of IL-2 and IL-6 respectively (q = 0.021 and q =
0.017, respectively).
In the HIV-uninfected group at 6 months, the Actinobacteriota
phylum positively associated with LBP levels (q = 0.018), whereas the
Firmicutes phylum negatively associated with IL-2 levels (q = 0.041).
The family Coriobacteriaceae positively associated with IL-2 levels
(q = 0.029), and the genus Clostridia_UCG.014 negatively associated
with IFN-glevels (Table 3). In the same group, the genus Actinomyces
positively associated with fecal calprotectin levels (q = 0.032).
B
A
FIGURE 6
Association of bacterial taxa with HIV infection status. (A) Comparisons at 6 weeks postpartum and (B) 6 months postpartum are shown. *p < 0.05
and ***p < 0.001.
Munjoma et al. 10.3389/fimmu.2023.1280262
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Discussion
Our study addresses associations of gut inflammation, microbial
composition, and systemic inflammation in HIV-infected lactating
women on cART in SSA where data have been scarce. None of the
women in our study suffered from AIDS or HIV-related symptoms,
and the levels of systemic inflammation were low. Therefore, no
strong HIV-related alterations were observed; however, we would like
to emphasize the following key results.
1. In our study population, similar levels of gut inflammation
(fecal calprotectin levels), microbial translocation (LBP
levels), and important descriptors of systemic
inflammation (TNF, IL-6, IL-8, and CRP) were found in
HIV-infected and HIV-uninfected individuals.
2. HIV infection can affect intestinal inflammation in more
immune-compromised individuals, because fecal
calprotectin levels correlated with third trimester CD4+
T-lymphocyte counts.
3. HIV-infected participants showed increased levels monocyte
activation (increased sCD14 levels), despite taking cART
and prophylactic antibiotics.
4. There is a relationship between intestinal inflammation,
microbial translocation, and systemic inflammation
because fecal calprotectin, LBP, and sCD14 correlated
with biomarkers of systemic inflammation.
5. Stratified by HIV status, the gut microbiota differed in beta
diversity but not alpha diversity.
6. Several gut microbial taxa were significantly associated with
HIV status and systemic inflammation.
TABLE 3 Gut microbial taxa and systemic inflammation.
Feature Metadata Coefficient N N not 0% q-value
HIV-infected (6 weeks postpartum)
p:Proteobacteria.c:Gammaproteobacteria.o:Burkholderiales.f:Oxalobacteraceae CRP −0.6080931 25 4 0.023359
p:Firmicutes.c:Clostridia.o:Lachnospirales.f:Lachnospiraceae.g:Eubacterium_eligens_group ICAM-1 0.43383237 25 3 0.000235
HIV-uninfected (6 weeks postpartum)
p:Firmicutes.c:Bacilli.o:Lactobacillales.f:Leuconostocaceae.g:Weissella IL-4 0.9326621 48 9 6.04e-08
HIV-infected (6 months postpartum)
p:Firmicutes.c:Bacilli.o:Erysipelotrichales.f:Erysipelatoclostridiaceae.g:Catenibacterium IL-2 1.32753279 23 9 0.021138
p:Proteobacteria.c:Gammaproteobacteria.o:Pasteurellales.f:Pasteurellaceae.g:Haemophilus IL-6 1.58409509 23 6 0.017096
p:Firmicutes.c:Clostridia.o:Lachnospirales.f:Lachnospiraceae.g:Eubacterium_eligens_group IL-2 0.55998378 23 4 0.001936
p:Firmicutes.c:Clostridia.o:Lachnospirales.f:Lachnospiraceae.g:Eubacterium_eligens_group IL-10 0.60010197 23 4 0.000557
p:Firmicutes.c:Clostridia.o:Lachnospirales.f:Lachnospiraceae.g:Anaerostignum IL-4 0.45052327 23 3 0.017532
p:Firmicutes.c:Clostridia.o:Lachnospirales.f:Lachnospiraceae.g:Butyrivibrio IL-6 0.76872521 23 3 4.03e-05
p:Firmicutes.c:Clostridia.o:Lachnospirales.f:Lachnospiraceae.g:Butyrivibrio TNF 0.70911458 23 3 0.001431
HIV-uninfected (6 months postpartum)
p:Actinobacteriota LBP 0.83076488 42 42 0.018545
p:Firmicutes IL-2 −0.1308627 42 42 0.041327
p:Actinobacteriota.c:Coriobacteriia.o:Coriobacteriales.f:Coriobacteriaceae IL-2 0.91371356 42 41 0.029222
p:Firmicutes.c:Clostridia.o:Clostridia_UCG.014.f:Clostridia_UCG.014.g:Clostridia_UCG.014 IFN-g−1.5113457 42 36 0.033319
p:Actinobacteriota.c:Actinobacteria.o:Actinomycetales.f:Actinomycetaceae.g:Actinomyces FC 0.97433139 42 32 0.03262
p:Firmicutes.c:Bacilli.o:Erysipelotrichales.f:Erysipelatoclostridiaceae ICAM-1 2.02529785 42 27 0.033465
p:Firmicutes.c:Bacilli.o:Erysipelotrichales.f:Erysipelotrichaceae.g:Solobacterium ICAM-1 1.74957426 42 12 0.033465
p:Actinobacteriota.c:Coriobacteriia.o:Coriobacteriales.f:Eggerthellaceae.g:Eggerthella FC 1.21725587 42 9 0.03262
p:Actinobacteriota.c:Coriobacteriia.o:Coriobacteriales.f:Eggerthellaceae.g:Eggerthella IL-2 1.3634006 42 9 0.0208
p:Firmicutes.c:Bacilli.o:Lactobacillales.f:Lactobacillaceae.g:Lactobacillus IL-8 1.04516168 42 9 0.041506
p:Fusobacteriota.c:Fusobacteriia.o:Fusobacteriales.f:Fusobacteriaceae FC 1.30098884 42 6 0.001424
Association between gut microbial taxa and microbial translocation, and systemic and gut inflammation biomarkers. N = total number of samples used in model; N not 0% = number of samples
in which microbial feature is not 0%; FC, fecal calprotectin.
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Effect of HIV infection on
biomarkers of gut inflammation
and microbial translocation
In our population of asymptomatic HIV-infected and HIV-
uninfected lactating women, intestinal inflammation (as assessed by
fecal calprotectin levels) was not affected by HIV status. Subgroups
with elevated fecal calprotectin levels included women with third-
trimester CD4+ T-lymphocyte counts <350 cells/µL consistent with
findings from previous studies (35,36). Other studies in HIV-
infected individuals described similar (37,38) or elevated fecal
calprotectin levels (8,9), most likely depending on the degree of gut
mucosal damage and immune dysfunction despite taking cART
(39). In our study, above normal median fecal calprotectin levels
were observed in HIV-infected cART-experienced individuals, in
agreement with studies in cART-naïve Italians (40,41).
In line with an overall well-preserved intestinal barrier with
similar translocation of LPS and microbial antigens into the
systemic circulation, we found no effects of HIV status on plasma
LBP levels in our population. This contradicts previous studies
showing elevated plasma LBP levels in HIV-infected adults from
Europe and Africa (3,10). However, our findings resembled results
from studies in Ugandan, American, and Chinese adults (8,42–44).
Moreover, a study in Swedish HIV-infected adults revealed a
decrease in LBP upon the commencement of cotrimoxazole
prophylaxis (45). However, plasma LBP correlated positively with
fecal calprotectin levels in some of our analyses, confirming that gut
inflammation can impact the translocation of microbes and/or LPS
in HIV-infected individuals on cART.
HIV-infected individuals in our study showed activation of
innate immunity, indicated by persistently high plasma sCD14
levels, a biomarker for innate immune activation including
monocytes and macrophages (46), and an independent predictor
for mortality in HIV-infected individuals on cART (47,48). Elevated
sCD14 levels were also found in HIV-infected European and African
children as well as African and Chinese adults (2,3,8,13,36,43,44,
46,49,50). We did not determine the source of monocyte activation
in our population but our results are consistent with HIV-induced
microbial translocation potentially driving monocyte/macrophage
activation as demonstrated in another study (47).
In our study, plasma sCD14 levels did not differ significantly by
antibiotic use and correlated neither with fecal calprotectin nor with
plasma LBP levels. These findings are inconsistent with another
study in cART-naïve Ugandan adults with recent HIV infection,
which reported significant correlations between LBP and sCD14
levels (51). Differences between both study populations and/or
cotrimoxazole prophylaxis in our study might explain this
discrepancy; however, this warrants further investigations.
Association of systemic inflammation
biomarkers with HIV infection, microbial
translocation, and gut inflammation
In line with an overall preserved immune function in our study
population, we found overall similar plasma levels of important
systemic inflammatory biomarkers (TNF, IL-6, IL-8, and CRP) in
HIV-infected and HIV-uninfected women, consistent with previous
work (2,17,52). Our results thus likely reflect protection from
systemic inflammation due to cART treatment, as observed in other
studies (14,53). The situation was shown to be different in cART-
naïve and more immune-suppressed cART-experienced individuals
who showed increased levels of inflammatory biomarkers (49,
53–57).
However, our detailed analyses revealed some effects of HIV
infection on the intestinal immune system: Fecal calprotectin
positively correlated with proinflammatory cytokines in the HIV-
infected group, consistent with findings from another study (8).
Furthermore, in the HIV-infected group, LBP, a biomarker of
microbial translocation, positively correlated with IL-2, IL-6, 1L-
1b, IFN-g, and CRP. Moreover, as shown in some (58,59) but not
all previous studies (60), CD4+ T-lymphocyte counts inversely
correlated with LBP, CRP, and IL-6. These findings indicate some
effects of gut inflammation and microbial translocation on systemic
inflammation mainly in the more immune-compromised
individuals in our study.
Furthermore, some cytokines seem to be sensitive to low-level
HIV viremia; we observed a strong correlation of third-trimester
HIV RNA levels with IL-4, IL-10, IL-6, IL-13, IL-1b, IL-13, IL-
12p70, and TNF. Similar findings were also observed in suppressed
and unsuppressed HIV-infected French and African participants
(51,61). Some effects of HIV infection on the immune system are
likely mediated by macrophage/monocyte activation and
inflammation (62). In our and a previous Spanish study (4), the
monocyte/macrophage activation marker sCD14 correlated with
IL-2, IL-6, TNF, and CRP.
Microbial translocation might also be relevant in the HIV-
uninfected group, where we found a positive association of taxa
from the Actinobacteriota phylum with LBP and fecal calprotectin,
as well as the genus Actinomyces with fecal calprotectin in line with
existing knowledge (63).
The gut microbiota and its association with
microbial translocation, gut, and systemic
inflammation biomarkers
HIV infection can lead to a decrease in gut microbiota diversity,
potentially resulting in the loss of beneficial bacteria and the
proliferation of harmful ones (64). In contrast, the gut microbiota
richness and evenness in our study were similar between the HIV-
infected and HIV-uninfected groups in line with previous studies in
cART-treated American and Spanish adults as well as South African
and Italian children (37,50,65–68). In contrast, in other studies,
reduced (16,17,64,69) or increased (41) alpha diversity was
observed in HIV-infected cART-naïve individuals. Furthermore,
in our study, microbiota richness or evenness was not influenced by
immune status, in agreement with some (34,44,70,71) but not all
previous studies in HIV-infected individuals (72). Most likely,
cART-mediated viral suppression can restore or preserve immune
status sufficiently that gross effects of HIV on the gut microbiota are
no longer detectable.
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We observed a decrease in microbial richness in the HIV-
uninfected group from pregnancy to 6 weeks postpartum, possibly
related to effects of birth and lactation. We also observed increasing
richness in the HIV-infected group from pregnancy to 6 months
after birth. The increase could have been an effect of better maternal
compliance with cART in pregnancy and lactation to prevent HIV
mother-to-child transmission, which would also drive a partial
restoration of the gut-associated lymphoid tissue. However, in
other studies, cART use caused substantial alterations to the
composition of the gut microbiota (41) and a decrease in alpha
diversity with time (73,74).
We show significant inter-community differences (beta
diversity) of the gut microbiota between HIV-uninfected and
HIV-infected lactating women on cART. Similar findings were
observed in cART-treated American, Spanish, and Chinese adults
as well as South African and Italian cART-treated children (37,44,
50,64,66,67,75). However, no significant variability in gut
microbiota composition due to HIV was found in other studies
(17,68), possibly related to varying degrees of immune dysfunction
or co-infection in the study populations.
At phylum level, we found high abundance of Firmicutes,
Actinobacteriota, and Proteobacteria in all participants as
previously reported in Asian and American cART-treated adults
(44,68,76). Increased relative abundances of taxa from the
Actinobacteriota phylum were observed in the HIV-infected
group at 6 weeks postpartum in line with a Japanese study in
cART-treated adults (76).
We found a higher relative abundance of the order
Coriobacteriales and the genera Collinsella and Slackia in the
HIV-infected group, consistent with previous findings in
ZimbabweanchildrenandcART-experienced Japanese
participants (75,76). The genus Collinsella has been previously
linked to detrimental outcomes such as obesity, non-alcoholic
steatohepatitis, and dyslipidemia (77). The lower relative
abundance of genus Collinsella in the HIV-infected group noted
by Zhao et al. was inconsistent with our results but concurred with
other studies in American and Chinese adults (44,68).
At genus level, we observed lower relative abundance of genus
Clostridium sensu_stricto_1 in the HIV-infected group at 6 weeks
postpartum, consistent with previous studies in African and
Chinese adults (44,59).Clostridiumspecies are important
obligate anaerobes in the human gut with a significant role in
fermentation and metabolism of carbohydrates and amino acids. In
another Chinese study, Clostridium sensu_stricto_1 positively
correlated with CD4+ and CD8+ T-lymphocytes counts,
suggesting this taxon as a potential marker of improved immune
status in HIV-infected participants (70).
In our study, the relative abundance of the genera Romboutsia
and Clostridium sensu_stricto_1 was lower in the HIV-infected
group at 6 months postpartum in line with a study in South
African children (37). The significantly lower relative abundances
of Romboutsia and Clostridium sensu_stricto_1 in the HIV-infected
women may signify compromised metabolism and imbalanced
intestinal homeostasis compared with the HIV-uninfected peers.
In line with the beneficial role of Clostridium species, we found a
negative association of the genus Clostridia_UCG.014 with IFN-gin
the HIV-uninfected at 6 months postpartum, supporting a role in
attenuating inflammation within the human gut.
More than 50% of the HIV-infected participants in our study
were taking cotrimoxazole prophylaxis following the local and
WHO guidelines (78). In our study, cotrimoxazole prophylaxis
had no significant effects on general descriptors of the gut
microbiota consistent with findings from a study in HIV-
uninfected Ugandan adults and children (79,80). These findings
were confirmed with antibiotics other than cotrimoxazole in HIV-
infected and HIV-uninfected individuals (69). However, in a study
in African children, significant differences were noted in seven
species of the gut microbiota with lower abundances in the
cotrimoxazole treated group (80).
Assessing the relationship between HIV-associated microbial
dysbiosis and systemic inflammation, we identified significant
correlations between the relative abundance of certain taxa and
plasma inflammatory biomarkers, warranting further investigations.
Our findings were inconsistent with studies in Australian cART-naïve
adults and Italian cART-experienced children where correlation of
gut microbiota with systemic cytokines and microbial translocation
markers was not found (17,50).
Strengths and limitations of the study
The strengths of our study lie in its comprehensive approach,
encompassing longitudinal assessments of multiple intestinal and
systemic inflammatory biomarkers, as well as the gut microbiota in
HIV-infected and HIV-uninfected controls from the same
community. However, there are limitations that should be
acknowledged. First, because of the inclusion criteria of our study,
we were unable to disentangle the effects of cART and HIV infection,
as all HIV-infected women were receiving cART. Furthermore, the
relatively small sample size hampers the generalizability of our
conclusions, and it is possible that certain associations between
microbial taxa and inflammatory biomarkers may have been
overlooked. We are therefore also underpowered to draw
meaningful conclusions from cART duration and HIV viremia
group comparisons. Moreover, our study exclusively comprised
lactating female participants, with no age-matched non-lactating
controls that may restrict the generalizability of our findings to
other populations. In addition, we did not directly assess microbial
translocation, and measurements of 16S rDNA levels, as previously
done (15) and could be used in follow-up studies. Finally, non-
bacterial infections, such as protozoa, were not tested in our study.
Conclusion
In our study population of HIV-infected lactating women on
cART without AIDS and HIV-related symptoms, many important
parameters of the immune system were not affected by HIV.
However, we identified effects of HIV on gut inflammation,
microbial composition, and translocation as well as some
cytokines, in line with a role of intestinal pathology contributing
to systemic inflammation in HIV.
Munjoma et al. 10.3389/fimmu.2023.1280262
Frontiers in Immunology frontiersin.org13
Data availability statement
The data presented in this manuscript are tabulated in the main
paper and Supplementary Materials. All sequencing data generated
in the preparation of this manuscript, files and entire details of
used samples used has been deposited in https://doi.org/10.6084/
m9.figshare.24455392.v1.
Ethics statement
The studies involving humans were approved by The Joint
Parirenyatwa Hospitals and University of Zimbabwe Research
Ethics Committee (JREC), JREC/114/20 and the Medical Research
Council of Zimbabwe (MRCZ), MRCZ/A/2663. The studies were
conducted in accordance with the local legislation and institutional
requirements. The participants provided their written informed
consent to participate in this study.
Author contributions
PM: Conceptualization, Formal Analysis, Investigation,
Methodology, Writing –original draft. PC: Investigation,
Methodology, Writing –review & editing. JW: Formal Analysis,
Writing –review & editing. AM: Methodology, Writing –review &
editing. SJ: Formal Analysis, Writing –review & editing. RG:
Writing –review & editing. LK: Conceptualization, Writing –
review & editing. BY: Funding acquisition, Methodology,
Supervision, Writing –review & editing. BM: Conceptualization,
Funding acquisition, Investigation, Supervision, Writing –review &
editing. KD: Conceptualization, Investigation, Supervision, Writing
–review & editing.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This work
was supported financially by the Botnar Foundation, Department of
Visceral Surgery and Medicine, Inselspital, Bern University,
Switzerland and the Welcome Trust. BM was supported by the Swiss
National Science Foundation (SNF), Grant Number: 320030_197815
and BY is supported by SNF Ambizione Grant: PZ00P3_185880.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fimmu.2023.1280262/
full#supplementary-material
SUPPLEMENTARY FIGURE 1
Plasma proinflammatory and vascular injury biomarkers in HIV infected and
HIV uninfected women. Comparison of plasma proinflammatory and vascular
injury biomarkers in HIV-infected vs. HIV uninfected women at 6 weeks
postpartum and 6 months postpartum. Significance after Bonferroni
correction is indicated as non-significant (ns), q<0.05 (*), q< 0.01 (**) or q<
0.001 (***). CRP, c-reactive protein; ICAM, intercellular adhesion molecule;
IL, Interleukin; TNF, tumour necrosis factor; INF-g, interferon gamma; VCAM,
vascular cell adhesion molecule.
SUPPLEMENTARY FIGURE 2
Comparison of pregnancy, 6 weeks and 6 months postpartum gut
microbiota. Alpha diversity comparison in HIV-uninfected women (A) and
HIV-infected women (B). Beta diversity comparison in HIV-uninfected
women (C) and HIV-infected women (D).
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