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\begin{document}

\title{Respiratory syncytial virus infection disrupts pulmonary microbiota and
immune barriers to induce microglia phenotype shift}



\author[1]{Yurong Tan}%
\author[2]{Ousman Bajinka}%
\author[1]{Zhongxiang Tang}%
\author[1]{Yu Mao}%
\author[1]{Xiangjie Qiu}%
\author[1]{Alansana Darboe}%
\affil[1]{Central South University Department of Medical Microbiology}%
\affil[2]{Central South University Xiangya School of Medicine}%


\vspace{-1em}



  
  \date{September 19, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Background}: The lung-brain-axis is an emerging biological
pathway that is being investigated in relation to microbiome medicine.
Increasing evidence suggest that pulmonary viral infections can lead to
distinct pathological imprints in the brain, thereby the need to explore
and understand this mechanism and find possible interventions.~
\textbf{Objective:}~ This study used RSV infection in mice as a model to
establish the potential lung-brain axis phenomenon. We hypothesized that
RSV infection could disrupt the lung microbiota, thereby compromising
the immune barriers and thus induces significant shift in microglia
phenotype.~ \textbf{Methods}: Mice were randomized into the Control,
Ampicillin, RSV, and RSV+Ampicillin treated groups (n = 6 each).
Ampicillin was given intratracheal instillation and seven days after the
respective treatments, the mice were anesthetized. Hematoxylin-eosin
(HE) staining of lung tissue to detect histopathology.
Immunofluorescence label of specific target antigens in both the lung
and brain tissues,~namely, Malondialdehyde (MDA) and Superoxide
dismutase (SOD) were used as~markers of cellular damage. RT-qPCR was
used to detect viral RNA in both tissues,~ ELISA to
measure~IL-1\selectlanguage{greek}β,\selectlanguage{english}~iNOS,~IL-10 and Arg1~in the supernatant~and~16s DNA
technology were used to detect the lung microflora.~ \textbf{Results}:
We found out that RSV infection induces~elevated oxidative stress,
reduced anti-oxidant and caused significant dysbacteriosis in the lungs
of the mice. Pulmonary microbes were found affecting~Th1-type
immunoreactivity~induced by RSV~infection and eventually, microbiota in
lung~induced~microglia phenotype~shift in the brain of the mice.~
\textbf{Conclusion.~}This study was able to establish that RSV infection
can disrupt the pulmonary microbiome and~immune barriers~to
induce~microglia phenotype~shift. Thus,~we recommend a large sample size
study with robust data analysis for the long-term effects of antibiotics
and RSV infection on brain physiology.%
\end{abstract}%



\sloppy


\textbf{Title:} Respiratory syncytial virus infection disrupts pulmonary
microbiota and immune barriers to induce microglia phenotype shift

\textbf{Authors} : Ousman Bajinka \textsuperscript{1,2,3}, Zhongxiang
Tang\textsuperscript{1}, Yu Mao\textsuperscript{1}, Xiangjie
Qiu\textsuperscript{1}, Alansana Darboe,\textsuperscript{3} Yurong
Tan\textsuperscript{1}

\textbf{Affliations} :

\textsuperscript{1} Department of Medical Microbiology, Xiangya School
of Medicine, Central South University, Changsha 410078, Hunan, China.

\textsuperscript{2} Medicine and Allied Health Sciences, University of
The Gambia, Banjul, The Gambia.

\textsuperscript{3} Functional Cell Biology International Center for
Genetic and Biotechnology (ICGEB), Trieste, Italy.

* \textbf{Corresponding author} : Dr Yurong Tan, Department of
Microbiology, Xiangya School of Medicine, Central South University,
Changsha 410078, Hunan, China. Tel: 86-18711187944. E-mail:
yurongtan@csu.edu.cn

\textbf{Abstract:}

\textbf{Background} : The lung-brain-axis is an emerging biological
pathway that is being investigated in relation to microbiome medicine.
Increasing evidence suggest that pulmonary viral infections can lead to
distinct pathological imprints in the brain, thereby the need to explore
and understand this mechanism and find possible
interventions.~\textbf{Objective:} ~ This study used RSV infection in
mice as a model to establish the potential lung-brain axis phenomenon.
We hypothesized that RSV infection could disrupt the lung microbiota,
thereby compromising the immune barriers and thus induces significant
shift in microglia phenotype.~\textbf{Methods} : Mice were randomized
into the Control, Ampicillin, RSV, and RSV+Ampicillin treated groups (n
= 6 each). Ampicillin was given intratracheal instillation and seven
days after the respective treatments, the mice were anesthetized.
Hematoxylin-eosin (HE) staining of lung tissue to detect histopathology.
Immunofluorescence label of specific target antigens in both the lung
and brain tissues,~namely, Malondialdehyde (MDA) and Superoxide
dismutase (SOD) were used as~markers of cellular damage. RT-qPCR was
used to detect viral RNA in both tissues,~ ELISA to
measure~IL-1\selectlanguage{greek}β,\selectlanguage{english}~iNOS,~IL-10 and Arg1~in the supernatant~and~16s DNA
technology were used to detect the lung microflora.~\textbf{Results} :
We found out that RSV infection induces~elevated oxidative stress,
reduced anti-oxidant and caused significant dysbacteriosis in the lungs
of the mice. Pulmonary microbes were found affecting~Th1-type
immunoreactivity~induced by RSV~infection and eventually, microbiota in
lung~induced~microglia phenotype~shift in the brain of the
mice.~\textbf{Conclusion.~} This study was able to establish that RSV
infection can disrupt the pulmonary microbiome and~immune barriers~to
induce~microglia phenotype~shift. Thus,~we recommend a large sample size
study with robust data analysis for the long-term effects of antibiotics
and RSV infection on brain physiology.

\textbf{Keywords:} RSV infection, pulmonary microbiome, immune barriers,
microglia shift, lung-brain-axis.

\textbf{Background}

Respiratory viral infections cause asthma, viral pneumonia, and chronic
obstructive pulmonary disease (COPD) {[}1{]}. In general, the leading
cause of hospitalization of infants in the developed world is acute
viral bronchiolitis {[}2{]}. Approximately 80\% of children's lower
respiratory tract infections are caused by RNA-enveloped viruses
{[}3{]}. Using the Respiratory Syncytial Virus (RSV) mouse model, we
were able to conceptualize possible lung-brain axis pathways and found
that uniform metabolomics and pathological changes occurred in the lungs
and brain of mice infected with RSV {[}2{]}. Thus, we hypothesize that
RSV-induced dysbiosis in the lung cavity may induce disruption of immune
barriers and induce a shift in microglial phenotype in the brain.

RSV is a single-stranded, non-segmental RNA envelope virus that is
encoded by a 15.2 kb genome. It belongs to the paramyxovirus family and
the genus of pulmonary viruses. RSV is the most common cause of acute
lower respiratory tract infections in children, leading~to~pneumonia and
bronchiolitis. RSV has been found to cause up to 200,000 under-five
deaths per year and is a highly pathogenic and lethal virus {[}4,5{]}.
RSV has also been shown to be associated with asthma, myocarditis, and
other diseases in the elderly and immunocompromised patients {[}6{]}.
There is considerable evidence that a significant proportion of children
hospitalized with RSV bronchiolitis suffer recurrent wheezing and even
asthma in the months and years following recovery, much more frequently
than non-RSV infected peers {[}7,8{]}.

The human microbiome is composed of many bacteria, archaea, viruses, and
fungi, and is widely distributed in the human oral cavity, respiratory
tract, gastrointestinal tract, urogenital tract, and skin {[}9{]}. The
microbiome is~involved~in the development of a variety of diseases and
is considered the human body's ``Second Genome'' {[}10,11{]}. The
homeostasis of the respiratory microbiome is closely related to the
immune defense function of the respiratory system. It has been found
that microbial signaling is necessary to supplement and activate
anti-inflammatory and regulatory T lymphocytes {[}12{]}. In addition,
there are interactions between respiratory microbiota and respiratory
viruses at many levels.~Moreover, from our conceptual literature
following our previous study, we could establish that post-RSV lung
inflammation induced neuroinflammation {[}13, 14{]}. Furthermore, we
reported that in current microbiome medicine, the focus on connecting
the lung to the brain in the gut-brain axis biological pathway has
associative effects of dysbiotic pulmonary microbes and some
neurological diseases. For example, an increased risk of Parkinson's
disease (PD) and Alzheimer disease (AD) dementia are both associated
with COPD, which is correlated with an altered microbiome {[}13{]}.

Pulmonary~microbes~and their metabolites~may~promote the proliferation
of respiratory viruses and enhance their infectivity {[}15{]}. Symbiotic
flora can also inhibit early replication of influenza virus by
increasing the expression of type I interferon receptor and enhancing
basal interferon signaling by driving pulmonary interstitial cells
{[}16{]}. Respiratory viruses can also cause secondary bacterial
infections by damaging the host's mucosal barrier, affecting immune
function, and altering the abundance and diversity of the
respiratory~microbiota, such as RSV infection, which can alter the nasal
respiratory microbiota {[}17{]}.

Currently, research on pulmonary microbiota is very limited, and the
mechanism of its~interaction with~viral infection is not entirely clear.
Our understanding of the respiratory microbiome is partial. The
mechanism and role of intrapulmonary bacteria in lung function is not
fully understood. This study aims to identify pulmonary bacterial
dysbiosis~after RSV infection~and the influence of dysbiosis in the lung
on the lung-brain axis. In addition, the study sought to identify
the~mechanisms of~lung microbiota~on~RSV~infection by investigating the
differentiation of immune cells in the lung and brain~during RSV
infection.

Material\textbf{s and methods}

\textbf{Animal models}

The study was conducted in accordance with the Helsinki Declaration and
approved by the Medical Ethics Committee of Xiangya School of Medicine.
A week old male neonatal BALB/c mice (8-15 g) were purchased from~Slack
Jinda Laboratory Animal Co. Ltd., Changsha, Hunan. The mice were
acclimatized for one week in a sterile environment with a 12 hrs.
light/dark cycle and a constant temperature of 25\selectlanguage{ngerman}°C and fed adaptively
for 7 days. The healthy mice were then treated randomly into four
groups, namely, the Control (PBS), Ampicillin, RSV, and RSV+Ampicillin
groups (n = 6 each). Ampicillin (Sigma, St. Louis, USA) was given
intratracheal instillation at 100 mg/kg body weight, while other mice
were injected with Phosphate Buffer Saline (PBS) instead. For RSV
infection, the mice were~anesthetized with isoflurane,
5×10\textsuperscript{6}~pfu RSV in 100~\selectlanguage{greek}μ\selectlanguage{english}l was intranasally inoculated
while mice in the RSV+Ampicillin group were treated with 100 mg/kg/d
Ampicillin prior to RSV inoculation and daily after RSV infection. Seven
days after the respective treatments, the mice were anesthetized with
ether and placed in a supine position. An incision was made from the
neck to the bottom of the chest wall, and the left lung was resected,
washed with 0.9\% sodium chloride, and properly analyzed.~~Ampicillin
treatment was added in order to confirm any altered pathological
imprints in the brain for both RSV group alone and the additional
Ampicillin.

\textbf{Hematoxylin-eosin (HE) staining of lung tissue}

The resected lung tissue was fixed with\textbf{~a} 10\% formaldehyde
solution for 48 h, embedded in paraffin, and cut into 4~\selectlanguage{greek}µ\selectlanguage{english}m thick
sections. Tissue sections were heated to 60\selectlanguage{ngerman}°C for 2~h, xylene dewaxed
(twice, 15 min each), and dehydrated using an alcohol gradient (75\%,
95\% and 100\%) for 5 min each. HE stain was performed as per standard
protocols.

\textbf{Immunofluorescence}

Lung~and brain~tissue sections were treated as described above sections
4 \selectlanguage{greek}µ\selectlanguage{english}m thick and boiled with 0.01M citrate buffer for antigen retrieval.
After washing twice with PBS (3 min each), sections were fixed with 4\%
paraformaldehyde for 5 min, washed with PBS, and blocked with normal
goat serum for 20 min (Bioss,~Beijing, China). Subsequently, sections
were incubated overnight with rabbit~anti-RSV major surface glycoprotein
G monoclonal antibody (Bioss, bs-1264R, Beijing, China, 1:500) at
4\selectlanguage{ngerman}°C.~iNOS antibody (18985-1-AP) was purchased from Proteintech Group
(Rosemont, IL, USA).~The following day, TRITC-labeled goat anti-rabbit
antibody (Zen-Bioscience, Chengdu, China, 1:300)~for RSV
determination~was added to the cover glass and incubated in darkness at
room temperature for 1 hr.~Secondary antibodies conjugated with FITC
(a0568) and DAPI staining solution (c1006) were from Beyotime (Shanghai,
China). After the final three washes with PBS, the sections were
counterstained with DAPI (100~ng/ml) for 10 min. After removing the
moisture, the slides were sealed with the anti-fluorescence quenching
agent and observed under the fluorescence microscope (Nikon~Ti-S,
Japan).

\textbf{Malondialdehyde (MDA) and Superoxide dismutase (SOD)}

Fresh lung and brain tissue samples prepared were ground in saline
solution to make 10\% tissue homogenates, followed by centrifugation at
3,500 rpm for 20 min. The resulting supernatant was collected to measure
SOD and MDA content using specific kits according to the manufacturer's
instructions. All commercial test kits were obtained from Jiancheng
Bioengineering Company (Nanjing, China).

\textbf{RT-qPCR}

Total RNA was extracted from lung and brain tissues using Trizol reagent
(Takara, Japan). Primers were synthesized by Sangon Biotech Co., Ltd.
(Shanghai, China), and the sequences are shown in Table 1. Each sample
was reverse transcribed into cDNA using RR036A PrimeScript RT Master Mix
(Perfect Real Time) (Takara, Japan). The~cDNA was then synthesized by
reverse transcription and amplified using 2× SYBR Green qPCR Master Mix
(Bimake, USA) according to the manufacturer's instructions. RT-qPCR was
performed at 95°C for 3 min, and 40 cycles of 95°C for 15 s, 55°C for 30
s, and 72°C for 30 s. GAPDH was the internal reference for the target
genes. Relative expression levels of mRNA were calculated using the
2\selectlanguage{english}\textsuperscript{-ΔΔCT} method.



\textbf{Table 3.1 The primer sequences used in the study.}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{Names} & \textbf{Sequences(5'-3')}\tabularnewline
\midrule
\endhead
\selectlanguage{greek}β-\selectlanguage{english}actin & GTGACGTTGACATCCGTAAAGA\tabularnewline
& GTAACAGTCCGCCTAGAAGCAC\tabularnewline
RORgamma & GCAGGAGCAATGGAAGTCGTC\tabularnewline
& AGGACGGTTGGCATTGATGAG\tabularnewline
GATA-3 & CCGGGTTCGGATGTAAGTCG\tabularnewline
& GCCTTCGCTTGGGCTTGAT\tabularnewline
T-bet & ATCATCACTAAGCAAGGACGGC\tabularnewline
& CCAAGACCACATCCACAAACATC\tabularnewline
Foxp3 & CCTGGTTGTGAGAAGGTCTTCG\tabularnewline
& TGCTCCAGAGACTGCACCACTT\tabularnewline
iNOS & GAGACAGGGAAGTCTGAAGCAC\tabularnewline
& CCAGCAGTAGTTGCTCCTCTTC\tabularnewline
IL-10 & CGGGAAGACAATAACTGCACCC\tabularnewline
& CGGTTAGCAGTATGTTGTCCAGC\tabularnewline
Arg-1 & CATTGGCTTGCGAGACGTAGAC\tabularnewline
& GCTGAAGGTCTCTTCCATCACC\tabularnewline
\bottomrule
\end{longtable}

\textbf{Enzyme Linked Immunosorbent Assay (ELISA)}

The concentrations of IL-1\selectlanguage{greek}β, \selectlanguage{english}iNOS, IL-10 and Arg1 in the supernatant of
each treatment group were measured using ELISA according to the
manufacturer's instructions (Fankew, Shanghai, China). Briefly, the
supernatant collected was diluted 5 times and added to a 96-well plate
with 50 \selectlanguage{greek}μ\selectlanguage{english}L per well. Fifty \selectlanguage{greek}μ\selectlanguage{english}L of biotin-binding antibodies were added to
each well and incubated at 37 for 1 hour. After washing five times with
PBS, 100 \selectlanguage{greek}μ\selectlanguage{english}L of chromogenic agent was added to each well and incubated in
darkness at 37 for 15 min. The optical density (OD) of each well was
detected at a wavelength of 450 nm. Fifty \selectlanguage{greek}μ\selectlanguage{english}L of the stop solution was
added to each well to stop the reactions. The OD value of each well was
measured at 450 nm by a microplate reader. The standard curve was
plotted according to the standard OD value. The concentration of the
target protein in the sample was calculated according to the standard
curve. All ELISA results were expressed as cytokine concentrations
(ng/mL or pg/mL) and performed in triplicates.

\textbf{Construction of 16sRNA library}

16s DNA technology was used to detect lung microflora. Portions of lung
tissue were frozen flash after resection and stored at -80\selectlanguage{ngerman}°C. Total
genomic DNA was extracted using a DNA extraction kit (TIANGEN\_Biotech
Co. Ltd, China) according to the manufacturer's instructions, and its
purity and concentration were determined by the nucleic acid analyzer
(Hangzhou Aosheng Instrument Co. Ltd\selectlanguage{english},China). PCR was performed using
double index fusion primers containing sequencing joints, and the
amplicons were separated by magnetic beads. A qualified library was used
for cluster preparation and sequencing, and after filtering out
low-quality reads, high- quality clean data was analyzed (Illumina,
USA).

\textbf{Cell culture and RSV Infection}

Human lung epithelial~Beas-2b and mouse nerve cells HT-22 cells were
cultured in DMEM or RPMI 1640 plus 10\% fetal bovine serum (FBS) and 1\%
Penicillin-Streptomycin-Amphotericin B solution at 37degC and 5\%
CO\textsubscript{2}. RSV (Long strain/A2 type) was stored at the
Department of Medical Microbiology of the Central South University and
later propagated in Hela cells with RPMI 1640 containing 2\% FBS. Viral
titers were determined using the plaque assay and RSV (1 MOI) was added
to collect cell samples for the next experiment.

Statistical\textbf{~Analysis}

SPSS 21.0 software was used for statistical analysis. Significant fold
changes were analyzed by online features of MetaboAnalyst 2.0.
Intergroup differences were analyzed by\emph{~t} -test and ANOVA as
appropriate, and LSD pairwise comparison was used to analyze intragroup
differences. P \textless{}0.05 was considered statistically significant.

\textbf{Results}

\textbf{RSV infection induced elevated oxidative stress and reduced
antioxidant in the lung}

While Ampicillin alone had no effect on normal lung morphology, RSV
infection promoted epithelial shedding and infiltration of inflammatory
cells (Fig. 1A). In addition,~RSV~immunofluorescence~and titers were
evident in the lung tissue, while there was no significant difference
between RSV and RSV+Amp (Fig. 1B, C).~We then detected plasma MDA and
SOD activity in the lung to determine oxidative stress markers and
antioxidant enzymes, respectively. No significant differences were found
between the RSV and RSV+Amp groups.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. Effects of microbiota on lung injury induced by RSV
infection.} A) HE stained lung sections indicating morphological
features. B) RSV titers in the lung were assayed using real-time PCR. C)
RSV infection was assayed by immunofluorescence (X400). D, MDA and SOD
in the lung were assayed by biochemical kits, (n = 6), ****\emph{p}
\textless{} 0.0001, *** \emph{p} \textless{} 0.001.

\textbf{Pulmonary microbes affect~Th1-type immunoreactivity~induced by
RSV~infection}

With the detection of inflammatory mediators and releases, we found that
IL-1 (pro-inflammatory cytokines), IFN-\selectlanguage{greek}γ \selectlanguage{english}(induce Th1 differentiation),
IL-4 (induce Th2 differentiation) and IL-6 (induce Th17 differentiation
and inhibit Treg differentiation) were significantly elevated. However,
IFN-\selectlanguage{greek}γ \selectlanguage{english}significantly decreased in RSV+Amp treated group when compared
with RSV group (Fig. 2A). We further examined the expression of T cell
differentiation transcription factors. The results showed that
transcription factors RORrt, FoxP3, and T-bet significantly increased,
with T-bet significantly decreasing in the RSV+Amp group when compared
with the RSV group, correlating to the above observation (Fig. 2A).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. Effects of lung microbiota on Th differentiation after
RSV infection (n = 6).} A) lung cytokines were assayed using ELISA. B)
Th differentiation was assayed using real-time RT-qPCR. ****
\emph{p}\textless{} 0.0001, ** \emph{p} \textless{} 0.01 and *
\emph{p}\textless{} 0.05.

\textbf{Microbiota in lung induce microglia phenotype shift}

Microglia influence the immune microenvironment of neurons mainly
through the release of effector factors from different phenotypes after
activation, thus affecting neuronal survival. Therefore, we first
examined the effect of~RSV~and Amp on the microglia M1/M2
phenotype\emph{in~vivo}. Real-time qPCR results~showed that
IL-1\selectlanguage{greek}β\selectlanguage{english}~and~iNOS expression levels~were significantly~higher~after RSV
infection~than those in the control group.~In addition,~IL-1\selectlanguage{greek}β\selectlanguage{english}~and~iNOS
were~further increased after Amp pretreatment prior to RSV infection
(Figure~3A). However, mRNA~levels of~the Arg 1, M2 phenotype marker were
significantly reduced after RSV infection and there was no difference
after Amp pretreatment (Figure~3A). In addition, we examined the
secretion levels of M1 and M2 phenotypic markers~using
ELISA.~The~results showed that the protein expression levels
of~IL-1\selectlanguage{greek}β\selectlanguage{english}~and~iNOS, M1 phenotypic markers were significantly
increased~after RSV infection~(\emph{p}~\textless{} 0.01,~Figure 3B)~and
further increased~after Amp pretreatment prior to RSV infection.
However, IL-10 and Arg1~expression levels were~slightly increased after
RSV infection and~not significantly different~after Amp pretreatment
prior to RSV infection (\emph{p}~\textgreater{} 0.05,~Figure 3B). The
levels of~iNOS in microglia were detected~by confocal
immunofluorescence, Expression~of~iNOS~was significantly increased~after
RSV infection and further elevated after~Amp pretreatment prior to RSV
infection (Figure 3C). These results suggest that~lung microbiota
may~inhibit~microglia activation and polarization towards the M1
phenotype.~We also assessed brain injury by detecting MDA and SOD
activity and found that MDA was significantly increased, and SOD was
reduced in~the brain tissue. And~Amp pretreatment~further promoted the
secretion of MDA and inhibited SOD~in the brain tissues.~

\textbf{Figure 3. Effects of lung microbiota on microglia phenotype
shift after RSV infection.} A) IL-1\selectlanguage{greek}β, \selectlanguage{english}iNOS, IL-10 and Arg1 in the brain
were assayed using real-time RT-qPCR. B) IL-1\selectlanguage{greek}β, \selectlanguage{english}iNOS, IL-10 and Arg1 in
the brain were assayed using real-time ELISA. C) NOS levels in microglia
were detected by confocal immunofluorescence. D) MDA, and SOD in the
brain were assayed by biochemical kits, n=6, **** p \textless{} 0.0001,
** p \textless{} 0.01 and * p \textless{} 0.05.

\textbf{RSV infection induced lung dysbacteriosis}

The effect of RSV infection on the lung microbiota was studied in 6-8
weeks old BALB/c male mice weighing 16-22 g. The mice were divided into
the control group, Ampicillin treated group, RSV infected group and
RSV+Ampicillin treated group. Ampicillin was injected into the tail vein
once a day, while RSV+Ampicillin group was injected into the tail vein
once a day at the same time of infection. Seven days after infection,
16s DNA technology was used to detect lung microflora. The abundance and
species of pulmonary bacteria in mice showed relatively good species
richness and uniformity in the lungs of all four groups. Alpha diversity
across four groups were not significant (\emph{P} \textgreater{} 0.05),
suggesting no significant variation in operational taxonomic unit (OTU)
species richness (data not shown). In the beta diversity analysis, it
was found that the normal control group had the highest diversity, while
the remaining groups showed a significant decrease in pulmonary
bacterial diversity (Fig. 4A). The Venn diagram showed no significant
difference in bacterial abundance between the different groups (Fig.
4B). Moreover, in the RSV infection group, the number of common clinical
pathogens such as \emph{Escherichia, Acinetobacter, Pseudomonas and
Treptococcus} \emph{species} increased significantly, while the number
of probiotics such as \emph{Bifidobacterium, Fibrobacter,
Selenoselomonas and Butyrivibrio} \emph{species} decreased
significantly. Compared to the RSV infected group, dysbacteriosis in the
lung of the RSV+Ampicillin group was more pronounced, and probiotics
decreased further (Fig.4C). By analyzing the metabolic function of
different groups, the results showed that the difference in metabolic
function was mainly manifested in membrane transport function,
carbohydrate metabolism and amino acid metabolism (Fig. 5).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure~4.~Effect of RSV infection on lung microflora.~} A) Beta
diversity analysis among the groups showed that the normal control group
had the highest~bacterial~diversity, while the remaining groups showed a
significant decrease in bacterial diversity. B) Venn diagram show that
there was no significant difference in bacterial abundance between
control and RSV infection groups. C) The results of differential
microflora heat map showed that RSV infection caused significant
dysbacteriosis, and the tendency of dysbacteriosis induced by RSV
infection was more obvious after~Ampicillin~treatment, n= 3.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5. Changes in metabolic function of microflora in lung
after RSV infection.} A) Heat maps showing significant changes in
metabolic function among the different groups. B) Radar maps showed that
the top 10 metabolic differences among groups were mainly the function
of cell membrane transport, carbohydrate metabolism, and amino acid
metabolism.

\textbf{Discussion}

Our findings established the interaction of lung microbiota and RSV
infection. RSV infection can induce bacterial dysbiosis. Although no
significant difference was seen in the bacterial abundance between
groups, in the RSV infection treated group, the number of common
clinical pathogens such as \emph{Escherichia, Acinetobacter,} \emph{and
Pseudomonas} and \emph{Treptococcus} species increased significantly,
while the number of probiotics such as \emph{Bifidobacterium,
Fibrobacter} , \emph{Selenomonas,} and \emph{Butyrivibrio spp} .
decreased significantly. \emph{Fibrobacter} was the most active
cellulolytic bacterium known, producing succinic acid as its main
fermentation product {[}18{]}. Thus, reduction in \emph{Selenomonas} is
a marker of asthma severity {[}19{]}. \emph{Butyrivibrio spp.} was one
of the symbiotic bacteria that can produce short-chain fatty acids,
especially butyrate. Following Ampicillin administration, the number
of\emph{Bifidobacterium} was further reduced compared to the RSV
group.\emph{Bifidobacterium} is the most common probiotic for restoring
beneficial flora {[}20{]}. These indicated that Ampicillin
administration had no benefits in restoring beneficial flora, which may
accelerate bacterial dysbiosis. However, abnormal increases in
opportunistic pathogens in asthma and COPD patients often require
antibiotics to control infections {[}21, 22{]}. Our study indicated that
antibiotics may accelerate bacterial dysbiosis.

An increased MDA is a biomarker of injury, while SOD is a biomarker of
anti-injury. The imbalance between this elevated MDA (oxidative stress)
and SOD (reduced antioxidant) was found to increase the risk of
complications in a study conducted on pregnant women {[}23{]}.~This can
be suggested since increased oxidative stress (the imbalance between
free radicals and stabilizing antioxidant enzymes) can react with DNA,
lipids or protein and lead to degenerative disorders. Moreover, since
SOD as an antioxidant is reduced, it would not lower oxidants with
electron donation and thus non-stabilization of free radicals, which
leads to harmful effects in the cell {[}24{]}.~We found that, while
Ampicillin alone had no effect on MDA and SOD secretions after RSV
infection, MDA was significantly increased and SOD decreased in the
brain tissues after Amp treatment prior to RSV infection, indicating
that lung microbiota can~promote~neuronal survival and inhibit microglia
activation and also polarization towards the M1 phenotype.~While
Ampicillin alone had no effect on normal lung morphology, RSV infection
promoted epithelial shedding and inflammation infiltration.~With the
detection of inflammatory mediator releases, IL-1, IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-4 and IL-6
showed increased levels of~which IFN-\selectlanguage{greek}γ\selectlanguage{english}~significantly~decreased~in
RSV+Amp when compared with RSV.~Reduced IFN-\selectlanguage{greek}γ \selectlanguage{english}was seen with elevated
TGF-\selectlanguage{greek}β \selectlanguage{english}signaling, which is a biomarker for damage in connective tissue
{[}25{]}.~Significant increases in T cell differentiation transcription
factors;~RORrt (for Th17 development), FoxP3 (for Treg cell
development)~and T-bet (as~a regulator of type 1 inflammatory responses)
{[}26{]}~are critical in immune homeostasis {[}27{]}.
However,~the~significant~decreases of IFN-\selectlanguage{greek}γ \selectlanguage{english}and T-bet~in the RSV+Amp
group~when compared with the RSV group~indicates a~decreased Th1-type
immune response in the~lung {[}28{]}.

In correlating RSV-induced dysbiosis in lung tissue with specific CNS
and brain, we detected microglia that affected the neuronal survival.
From our study, Ampicillin alone was seen to increase in IL-1\selectlanguage{greek}β\selectlanguage{english}~and~iNOS
in brain tissues. Inducible nitric oxide synthase (iNOS), one of the
enzymes that produces nitric oxide (NO) from the amino acid l-arginine
confers some anti-inflammatory properties that regulate human asthma
while~IL-1\selectlanguage{greek}β \selectlanguage{english}antibody has some protective effects on the brain from
neuropathology of hypoperfusion {[}29{]}. An increased phenotypic marker
of M1, which are IL-1\selectlanguage{greek}β\selectlanguage{english}~and~iNOS during RSV infection can cause
neurotoxicity and DNA damage respectively {[}30{]}.

Although we were able to explore a lot about short-term intrapulmonary
bacteria alleviating RSV-induced inflammation in the lung,\textbf{~} the
main limitations of the study were the limited sample size, and the lack
of data analysis for the long-term effects of antibiotics and RSV
infection in these BALB/c mice.~ In addition,~metabolomics and
metagenomics will confer a clear revealation as to the further
mechanism.



\textbf{Conclusion}

From our\textbf{~} study, we found that pulmonary microbes were found to
affect~Th1-type immunoreactivity~induced by RSV~infection, and
ultimately, the lung microbiota induced microglia phenotype shift in the
mouse brain tissues. We were able to add scholarly information on the
lung-brain axis through RSV infection. The disruption of the pulmonary
microbiome and~immune barriers~that induce~microglia phenotype~shift in
the brain is related to the mechanism and role of intrapulmonary
bacteria~on RSV infection.~Furthermore, the focus on connecting the lung
to the brain in the gut-brain axis biological pathway has associative
effects of dysbiotic pulmonary microbes and some alterations in the
brain. With these novel microbiome findings and their impact on human
physiology, we recommend a large sample size study with robust data
analysis for the long-term effects of antibiotics and RSV infection on
brain tissue physiology.

\subsubsection*{}

{\label{section}}

\subsubsection*{Declarations}

{\label{declarations}}

\textbf{Funding:} Not applicable.

\textbf{Conflicts of interest:} The authors declare that they have no
conflict of interest.

\textbf{Ethical approval:} The study was approved by the Institutional
Ethic Committee of Xiangya School of Medicine, Central South University.
All applicable international, national, and/or institutional guidelines
for the care and use of animals were followed.

\textbf{Authors' contributions:} OB and ZT designed the study, OB, ZT,
YM and XQ conduct the experiments, YT evaluate the study, OB, LB, RK and
TY wrote the manuscript

\textbf{Availability of data and materials:} The datasets used and/or
analyzed during the current study are available from the corresponding
author on reasonable request.

\textbf{Acknowledgements:} Not applicable.

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