Hedley et al. Journal of Neuroinflammation(2024) 21:158
https://doi.org/10.1186/s12974-024-03150-3

Journal of Neuroinflammation

Open Access

RESEARCH

Neonatal Chlamydia muridarum respiratory
infection causes neuroinflammation within
the brainstem during the early postnatal
periodKateleen E Hedley1,2, Henry M Gomez1,2, Eda Kecelioglu1,2, Olivia R Carroll1,2, Phillip Jobling1,2, Jay C Horvat1,2 andMelissa A Tadros1,2*
Abstract
Respiratory infections are one of the most common causes of illness and morbidity in neonates worldwide. In
the acute phase infections are known to cause wide-spread peripheral inflammation. However, the inflammatory
consequences to the critical neural control centres for respiration have not been explored. Utilising a wellcharacterised model of neonatal respiratory infection, we investigated acute responses within the medulla
oblongata which contains key respiratory regions. Neonatal mice were intranasally inoculated within 24 h ofbirth, with either Chlamydia muridarum or sham-infected, and tissue collected on postnatal day 15, the peak of
peripheral inflammation. A key finding of this study is that, while the periphery appeared to show no sex-specificeffects of a neonatal respiratory infection, sex had a significant impact on the inflammatory response of the
medulla oblongata. There was a distinct sex-specific response in the medulla coincident with peak of peripheral
inflammation, with females demonstrating an upregulation of anti-inflammatory cytokines and males showing
very few changes. Microglia also demonstrated sex-specificity with the morphology of females and males differing
based upon the nuclei. Astrocytes showed limited changes during the acute response to neonatal infection. These
data highlight the strong sex-specific impact of a respiratory infection can have on the medulla in the acuteinflammatory phase.
Keywords Medulla oblongata, Cytokines, Microglia, Astrocytes, Sex-specific*Correspondence:
Melissa A Tadrosmelissa.tadros@newcastle.edu.au
1
School of Biomedical Sciences & Pharmacy, The University of Newcastle
Callaghan, NSW 2308, Australia
2
Hunter Medical Research Institute, New Lambton Heights, NSW, Australia© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Hedley et al. Journal of Neuroinflammation(2024) 21:158

Introduction
There is a critical window of development that ranges
from birth to adolescence where the periphery and central nervous system (CNS) undergo major changes.
During this time, the periphery and CNS are especiallysensitive to insults, and in particular acute inflammatory events [1]. Acute respiratory tract infections are oneof the most common causes of peripheral inflammation,
resulting in significant morbidity and mortality in neonates worldwide, with up to 18% of Australian children
admitted to hospital with respiratory distress within
the first two years of life [2]. One of the major causes of
respiratory tract infections in neonates is Chlamydia
trachomatis [3]. C. trachomatis is passed onto an infant
through vertical transmission during childbirth, resulting in a severe respiratory infection with lung deficits,
wheeze and increased neutrophilic infiltration [4]. Older
infants are more likely to contract Chlamydia pneumonia
via community transmission, which results in a similar
respiratory phenotype of lung dysfunction and significant
inflammation. Neonatal peripheral inflammatory events
have been linked to respiratory dysfunction later in life,
such as asthma [5], however not all aspects of these conditions can be explained by peripheral respiratory deficits alone. The CNS has been proposed as a contributor
to respiratory dysfunction [6] and this dysfunction isdriven by the nuclei that control respiration, housed in
the medulla oblongata of the brainstem [7]. However,the specific neuroinflammatory impacts of a neonatal
respiratory infection on these brainstem centres have notbeen explored.
There are many respiratory regions within the medulla
oblongata forming a circuit that drives respiration, however there are three primary centres that are critical tobasic function of the respiratory system. Inflammationfrom the lungs is thought to affect these nuclei via thevagus nerve, via a number of well described pathways
that ultimately end with the release of inflammatory
mediators [8] within the nuclei that receive and supply
information from the vagus nerve. These are the nucleus
tractus solitarii (NTS), a major integration and dissemination centre for sensory information entering the CNS
through the vagus nerve and the dorsal motor nucleus of
the vagus (DMX), providing parasympathetic pre-ganglionic innervation to the smooth muscle in the bronchi.
The inflammatory mediators stimulated at these nuclei
then go on to interact with microglia and astrocytes
within the CNS, causing a cascade of neuroinflammation[1].
The inflammatory cascade has been shown to be detrimental, not only in the acute phase but also leading tolong-term changes. The neonatal period has been shown
to be particularly suspectable to neuroinflammation, witha pro-inflammatory response that appears to be regulated

Page 2 of 15

by microglia [9] and increased susceptibility to changes inblood brain barrier (BBB) permeability that could allow
infiltration of peripheral immune cells into the CNS [10].
This has linked neonatal neuroinflammation to several
neurological disorders [11], as well as causing disruption
to the local CNS environment, which can alter neuronalsignalling and result in peripheral organ dysfunction, forexample the lungs and heart [12].
In this study, utilising our well-established model ofneonatal respiratory infection using Chlamydia muridarum [13, 14], we aim to provide evidence that a neonatal respiratory infection does acutely affect the
respiratory centres in the medulla oblongata, by altering
the inflammatory profile in a sex-specific manner. We
showed altered inflammatory mediators within the CNS,
including cytokines and chemokines, as well as modulation of microglial activity, the resident immune cell of the
CNS, with limited changes in astrocytes, that maintainthe BBB. Intriguingly, these changes occur in a sex-specific manner, even during this neonatal period.

Methods
Animals

All experiments were performed in accordance with the
National Health and Medical Research Council Australian Code of Practice and as approved by the University of Newcastle Animal Care and Ethics Committee(approval number A-2020-028). Pregnant BALB/c mice
were acquired from the University of Newcastle’s Central
Animal House, with the pups born to these mothers usedas the experimental animals. Animals were housed under
specific pathogen free (SPF), PC2 conditions in individually ventilated cages (Bioresources facility, HMRI Building, New Lambton Heights, NSW, Australia). All animals
were maintained in a temperature (22 °C ± 2) and humidity-controlled environment on a 12 h light/dark cyclewith food (standard chow) and water available ad libitum
for the duration of the study.
Neonatal Chlamydia muridarum respiratory infection
model

The laboratory model of neonatal Chlamydia muridarum(CMU) has been previously published [14]. Briefly, within
24 h of birth, (assigned postnatal day 0 [P0]), neonatal
BALB/c mice were infected intranasally with either Chlamydia muridarum (CMU, 400 inclusion-forming units,
ATCC VR-123, in 5µL sucrose phosphate glutamate buffer [SPG]) or sham-infected with SPG alone. Mice were
weighed and rate of weight gain was calculated (g/d)
throughout the experiments as a measure of health status. Mice were then sacrificed at P15 (peak of peripheral inflammation [13]) and tissues collected as outlined
below. A total of 55 offspring were used for this study
from 13 litters (SPG derived from 6 litters, CMU derivedHedley et al. Journal of Neuroinflammation(2024) 21:158

from 7 litters). Each individual offspring was considered
an individual experimental unit. Mothers were used forone litter only. Molecular and anatomical studies usedboth males and females from SPG and CMU groups,
with each litter being evenly distributed across both techniques. Both males and females were assessed independently, thus experimental groups are as follows; female
SPG (FSPG), female CMU (FCMU), male SPG (MSPG)
and male CMU (MCMU).
RNA extraction and RT-qPCR of lung tissue

For confirmation of infection, lungs at P15 were assessed
for the presence of 16 S, confirming bacteria within thelungs, and a selection of inflammatory markers. Following euthanasia with sodium pentobarbital (150 mg/kg
intraperitoneally [i.p.]), the lungs (multi-lobe, n = 7–8 pergroup) were removed and immediately snap-frozen to-80 OC in liquid nitrogen. The RNA was extracted using
standard protocols [15]. Briefly, the tissue was thawed
and homogenised (1 mL of TRIzol, 4 °C; ThermoFisher
Scientific, Australia) with a tissue tearor homogeniser(Biospec Products, Bartlesville, OK, USA). Chloroform
was added and then centrifuged (12,000× g, 15 min,4 °C) to precipitate out the DNA, and the aqueous phase
containing the RNA was collected. With the addition of
Table 1 Forward and reverse primer sequences
GeneΒ-Actin
18s
HPRT
16 S
IL-1β
TNF⍺
NLRP3
CXCL9
CXCL10
IL-13
KCNN4
Stat1
Stat6
TGFβ
PTGS2
BDNF
IFN𝛾

Forward Primer (5’->3’)​G​C​A​G​G​A​G​T​A​C​G​A​T​G​A​G​T​C​C​G

Reverse Primer (5’->3’)​A​C​G​C​A​G​C ​T​C​A​G​T​A​A​C​A​G​
T​C​C​A​C​C​A​G​A​C ​T ​T​G​C​C​C ​T​C​C​A​A​T​G​A​A​C​G​G​C ​T​A​C​C​A​C​A​T​C​C​A​
A​G​G​A​G​G​C​C​A​G​A​C ​T ​T ​T​G​T ​T​G​G​A​T​​C​A​A​C ​T ​T​G​C​G​C ​T​C​A​T​C ​T ​T​A​G​
T​T​G​A​A
G​C​T​T​T​G​C​G​G​C​A​G​A​A​A​T​G​T​C​G​T ​T ​T ​T​C​G​C ​T​C​G​T ​T​G​C​G​G​G​A​C ​T ​T​A
​G​A​A​G​T ​T​G​A​C​G​G​A​C​C​C​C​A​A​A​A ​G​C​C ​T​G​C​C ​T​G​A​A​G​C ​T​C ​T ​T​G​T ​T​G​C​C ​T​C ​T ​T​C ​T​C​A​T ​T​C​C ​T​G​C ​T ​T​G ​C ​T​G​A​T​G​A​G​A​G​G​G​A​G​G​C​
C​A​T ​T
​G​T​G​G​A​T​G​G​G​T ​T ​T​G​C ​T​G​G​G​A​T​C​C ​T​G​C ​T ​T​C ​T​C​A​C​A​T​G​T​C​G​T​C ​T​C​A​G​A​G​C​C​A​G​A​C​A​G​G​G​T​G​A​A​A ​A​G​C​A​G​T​C​C​C​A​A​A​T​A​T​C​C​G​C​A​A​A​G​C ​T​A​T​G​T​G​G​A​G​G​T​G​C​G​A​C ​T​G​A​G​C ​T​A​G​G​G​A​G​G​A​C​A​A​
G​G​A​A​G​A​C ​T​C​C​C​C ​T​G​T​G​C​A​A​C​G​G​C​A ​G​G​A​G​A​C​C​G​T​A​G​T​G​G​G​G​G​C​C ​T ​T​C​C​A​G​T​C​A​G​G​A​G​G​C​C​A​C​A​T​A​G ​A​G​A​A​A​A​C​A​C​A​G​G​A​G​C​A​G​
G​G​A​T​C​A​G​A​A​A​A​A​C​G​C ​T​G​G​G​A​A​C​​G​G​T​G​G​T​C ​T​C​C​A​G​G​T​C​A​A​T​C​A
A​G​A​T​C​C​A​C​G​A​G​C ​T ​T​C​A​C​A​T ​T​G​G​G​A​G​G​A​T​G​G​T​A​G​C ​T ​T ​T​G​G​C​G​T ​T​C ​T​G​C ​T​G​A​C​C​C​C​C​A​C ​T​G​A​T​A​C​A​G​C​C​C ​T​G​T​A​T ​T​C​C​G​T​C ​T​C​C ​T​A​A​T​A​C ​T​G​G​A​A​G​C​C​G​A​G​C​A​C​C ​A​A​G​A​A​G​T​G​A​A​G​G​G​A​C​A​C​C​C​C​C​C ​T​G​C​A​T​C ​T​G​T ​T​G​G​G​G​A​G​A​C​T​G​G​T​C​A​T​C​A​C ​T​C ​T ​T​C ​T​C​A​C​C ​T​G​C ​T​G​G​A​G​G​A​A​C ​T​G​G​C​A​A​A​A​G​G ​T ​T​G​C ​T​G​A​T​G​G​C​C ​T​G​A​T ​T​G​T​C

Page 3 of 15

isopropyl alcohol, the RNA was precipitated out and pelleted (12,000× g, 10 min, 4 °C), then washed twice with
75% ethanol. The RNA pellets were dissolved in nuclease-free water and RNA concentration and quality were
measured with a NanoDrop 1000 Spectrophotometer(ThermoFisher Scientific, USA).
For cDNA synthesis, any contaminating genomic DNA
remaining in the RNA samples was digested using DNase
I (Sigma-Aldrich, Macquarie Park, Australia; 15 min,room temperature) and then the samples were heated(10 min, 65 °C). Random hexamer primers (50 ng, Meridian Bioscience, Memphis, TN, USA) and dNTPs (10 mM,
Meridian Bioscience, Memphis, TN, USA) were added
and samples were further heated (5 min, 65 °C). Dithiothreitol (10 mM) and Bioscript reverse transcriptase (200units in reaction buffer, Meridian Bioscience, Memphis,
TN, USA) were added, and reverse transcription was performed (10 min, 25 °C; 50 min, 42 °C; 15 min, 70 °C).
All qPCR primers (Table 1) were designed with
Ensembl using the standard primer design criteria. The
primer pairs were then put through NCBI primer BLAST
to ensure primer specificity. A total volume of 12 µl wasused containing: 6 µl iTaq Universal SYBR Green Supermix (Bio-rad, NSW, Australia), 10µM each of forward
and reverse primers, 2000ng cDNA and molecular grade
water to 3 µl. After an initial 2 min 60 °C enzyme activation step, 40 cycles of 95 °C for 15 s (step 1) followedby 60 °C for 30 s (step 2) were completed. Melt curves
were generated to confirm the presence of a single PCR
product. Primers were deemed specific if a single amplified product of appropriate size was detected by melt
curve analysis. Reactions were performed on a Bio-Rad
CFX384 Touch Real-Time PCR System (Bio-Rad, NSW,
Australia) and analysed using the Bio-Rad CFX Manager(version 3.1). For each primer the samples were run ona 384-well plate in triplicate, including a negative water
control on each plate. Delta Ct (ΔCt, threshold cycle) was
determined for each gene relative to the housekeeping
gene, hypoxanthine-guanine phosphoribosyl transferase(HPRT), and the well-established ΔΔCt method [16] wasemployed to enable comparisons between the groups.
RNA extraction and RT-qPCR of medulla oblongata

Brainstems (n = 7–8 per group) were removed following euthanasia (i.p. sodium pentobarbital 150 mg/kg)
and immediately snap-frozen to -80 OC in liquid nitrogen. The medulla oblongata was then separated from thewhole brainstem, using the cerebral peduncles and thedorsal median sulcus as landmarks, and homogenised.
Total cellular RNA was extracted using the RNeasy® Mini
Kit (Qiagen, Hilden, Germany), following manufactures
instructions. RNA concentration and quality were measured with a NanoDrop 1000 Spectrophotometer (ThermoFisher Scientific, USA).

Hedley et al. Journal of Neuroinflammation(2024) 21:158

Any contaminating genomic DNA remaining in the
RNA samples was digested using DNase I (Invitrogen,
Scoresby, Australia). Reverse transcription was then performed using Superscript III (Invitrogen, Scoresby, Australia), according to manufacturer’s instructions. Briefly,
30–200ng of total RNA, 1 µl of oligo(dT)18 (Meridian
Bioscience, Ohio, USA), 1 µl of random hexar (Meridian Bioscience, Ohio, USA), 1 µl of 10µM dNTP (Meridian Bioscience, Ohio, USA), and molecular grade waterto 13 µl, were mixed and heated for 5 min at 65 °C in a
Thermal Cycler (Eppendorf, Germany). Next, 4 µl of 5x
first-strand buffer, 1 µl of 0.1 M DTT, 1 µl RNaseOUT (40U/µl) (Meridian Bioscience, Ohio, USA) and 1 µl SuperScript III RT (200 U/µl) were added and the mixture
was incubated for 60 min at 50 °C, followed by 70 °C for
15 min. Reverse transcription without Superscript III was
also performed to assess genomic DNA contamination.
The same qPCR primers (Table 1) were used and theprotocol was similar to that of the lungs, with the exceptions detailed below. A total volume of 12.5 µl was used
containing: 6.25 µl 2x SensiFAST SYBR (Meridian Bioscience, Ohio, USA), 10µM each of forward and reverse
primers, 1–2.5ng cDNA and molecular grade water to12.5 µl. After an initial 10 min 95 °C enzyme activation
step, 40 cycles of 95 °C for 15 s (step 1) followed by 60 °C
for 30 s (step 2) were completed. Delta Ct (ΔCt, threshold cycle) was determined for each gene relative to thegeometric mean of the housekeeping genes β-Actin and
18 S, and the well-established ΔΔCt method [16] wasemployed to enable comparisons between the groups.
Immunofluorescence labelling of glia

Brainstems (n = 6 per group) were dissected following
euthanasia (i.p. sodium pentobarbital 150 mg/kg), andimmersion-fixed in 4% paraformaldehyde (PFA) in phosphate buffer for 24 h then washed into phosphate buffered saline (PBS). Brainstems were then washed with
3 × 15 min 80% ethanol (EtOH), 3 × 15 min dimethyl
sulfoxide (DMSO) and then 3 × 15 min 100% EtOH.
The brainstems were then placed in a cell culture dishPage 4 of 15

containing melted 1000 MW polyethylene glycol (PEG)
in a 46 OC vacuum oven (Thermoline, Wetherill Park,
NSW) until the tissue had sunk (approximately 1 h). The
tissue was then placed in a cryostat mould and embedded
in 1450 MW PEG. Once hardened, the tissue block was
sectioned at 30 μm on a rotary microtome. Every one infour sections from bregma level − 7.20 mm to -7.76 mm
were taken and placed in PBS. Five sections were selectedusing a brain atlas [17], to ensure the correct region, andwere then blocked in 10% normal donkey serum (NDS,
Jackson ImmunoResearch) for 30 min. For labelling, sections were incubated at room temperature overnightin a solution of antibody diluent (0.3 M NaCl, 7.5mM
Na2HPO4, 2.5mM NaH2PO4, 0.3% TritonX-1000, 0.05%
Na Azide), 10% NDS, ionized calcium binding adaptor
molecule 1 (Iba1; rabbit; 1:250; Wako; 019-19741) andglial fibrillary acidic protein (GFAP; chicken; 1:1000;
Merck Millipore; AB5541). After the primary incubation, sections were rinsed in 3 × 15 min PBS and incubated for 2 h at room temperature with secondary
antibodies Alexa594-donkey-anti-rabbit (1:50; Abcam;
ab150076), Alexa488-donkey-anti-chicken (1:50; Jackson;
JI703545155) and NeuroTrace Blue (1:25; ThermoFisher;
N21479) in antibody diluent. Sections were mounted onslides in buffered glycerol and cover-slipped. Images wereacquired using an Olympus BX50 microscope equippedwith a mercury burner and an Olympus DP72 camera.
Images were processed offline using ImageJ for mean
fluorescent intensity (MFI) and percentage area (% area)
quantification. The NTS and DMX, were selected using
the neuronal marker NeuroTrace Blue and a mouse brain
atlas for identification (Fig. 1) [17] and the same size area
was taken for each image. To calculate MFI and % area
for microglia and astrocytes, an image was converted
into a 16-bit image, then duplicated and one image wasconverted to binary. The binary image was redirectedto the 16-bit image and then the particle analysis was
performed. An average MFI and % area were calculated
for each animal, from the five sections taken. A custom MATLAB script was utilised to analyse microglia

Fig. 1 Brain atlas for identification of nuclei. Nuclei were identified using a brain atlas [17], at bregma level − 7.48 mm, with central canal (CC), area postrema (AP) and hypoglossal nucleus (nXII) for reference, with NTS outlined in red, DMX outlined in green

Hedley et al. Journal of Neuroinflammation(2024) 21:158

number and morphology [18–20]. This peer-reviewed
script identifies the cell body, as determined by density of pixels, and then traces adjoining pixels from thecell body, again determined by their density. All images
from a single nuclei were run at the same time to ensure
consistency.
Statistics

For weight data, statistical differences were determinedusing mixed-effect model, with Sidak post-hoc correction. For RT-qPCR and immunofluorescence, statisticaldifferences were determined using a general linear model
multivariate analysis of variance (ANOVA) or equivalent non-parametric test (Kruskal-Wallis with Dunn’s
post-hoc) using SPSS with Sidak used for post-hoc correction. Significance set at p < 0.05. All graphs presented
use untransformed means and standard errors of means(SEM) and created in GraphPad Prism v9 (GraphPad
Software Inc., San Diego, CA, USA) and Adobe Illustrator 2023.

Results
Neonatal weight gain was significantly reduced due toinfection

Significant decreases in the rate of weight gain (Fig. 2B)of CMU infected mice compared to SPG infected mice
were observed in neonates from days 6 to 15 (endpoint),
with the exception of day 12, as shown in Fig. 2B. A significant decrease in weight gain is consistent with infection in mice.
Confirmation of Chlamydia muridarum infection in the
lungs

Along with the weight data to confirm infection, theexpression of 16 S mRNA was measured at P15 in thelungs. The expression of a number of key inflammatory
mediators were also quantified in the lungs to ensure thatperipheral inflammation was present.
16 S is the central structural component of the bacterial subunit that is conserved in bacteria and allows

Page 5 of 15

identification of bacteria [21]. Given the variability inbacterial titre and clearance, a non-parametric test was
performed, as 16 S expression data failed tests of homogeneity and Levene’s test of equal variances. There was asignificant main effect of treatment, as shown in Fig. 3A,(treatment effect; H (1,21) = 21.06, p < 0.001), this indicatesthat both male and female cohorts were successfully
infected. Chemokine ligand 9 (CXCL9) is thought to aid
in the infiltration and retention of activated T-cells in
response to bacterial infection [22] and will give an indication of the direct response to the bacterial infection.
There was a significant main effect in the expression of
CXCL9 in the lungs at P15, shown in Fig. 3B (treatment
effect; H (1,25) = 21.85, p < 0.001). This indicates that theinitial peripheral response to the bacterial infection is not
sex-specific.
NLR family pyrin domain containing 3 (NLRP3) isa major component of the inflammasome and triggers
the immune response. Once triggered, NLRP3 activates
caspase-1 which then cleaves pro-interleukin 1β into its
mature form, the a highly pro-inflammatory interleukin
1β (IL-1β) [23]. Tumour necrosis factor alpha (TNFα)
is a pro-inflammatory cytokine produced during acuteinflammation [24]. These mediators act together to initiate the inflammatory response in the lungs. At P15,
there was a main effect of treatment on NLRP3, TNFαand IL-1β (treatment effect, NLRP3; F (1,25) = 45.229,
p < 0.001, TNFα; F (1,25) = 13.235, p = 0.001, IL-1β; F(1,25) = 51.409, p < 0.001) with CMU-infected mice having
a significant increase compared to SPG-infected mice, as
shown in Fig. 3C, D, E. There was no other main effects
or interactions. This indicates that in the lungs there isa robust inflammatory response to infection that is not
sex-specific.
Neonatal infection alters inflammatory mediators in the
medulla oblongata

This study examined the expression of a wide range ofinflammatory mediators in the medulla oblongata in theacute phase of peripheral inflammation. Inflammatory

Fig. 2 Acute effects of a neonatal respiratory infection on weight. The A) average gross weight and B) weight gain per day of SPG (solid line) and CMU(dotted line) infected mice, with significance between weight gain on a single day, SPG n = 27, CMU n = 28 (*p < 0.05, **p < 0.01, ****p < 0.0001)

Hedley et al. Journal of Neuroinflammation(2024) 21:158

Page 6 of 15

Fig. 3 Upregulation in the expression of all inflammatory mediators in the lungs following neonatal infection. Graphs show gene expression levels(mean ± SEM) for SPG (solid bars) and CMU (hatched bars) females (grey) and males (black), with total number of samples shown in each bar. Inflammatory
mediator expression; A) 16s (SPG vs. CMU ***p < 0.001), B) CXCL9 (SPG vs. CMU ***p < 0.001), C) NLRP3 (SPG vs. CMU ***p < 0.001), D) TNFα (SPG vs. CMU**p < 0.01) and E) IL-1β (SPG vs. CMU ***p < 0.001)

mediators selected have been shown to be upregulated
peripherally in response to infection, as well as mediatorsthat a play crucial role in regulating the local immune
environment of the CNS.
Upregulation of neuroprotective inflammatory mediators in
infected females

CXCL9 and chemokine ligand 10 (CXCL10) are two
major chemokines that have been associated withimmune cell migration, differentiation and activation[25]. Specifically, CXCL10 is primarily expressed on glia
and recruits lymphocytes and phagocytes to inflamedareas to limit the spread of infection [26, 27] and as is
the case with CXCL9, aids in T-cells responses to bacterial infection. These two chemokines will indicate thestrength of the initial immune response to the bacteria.
At P15 there was a significant treatment x sex effect in theexpression of both CXCL9 and CXCL10 (treatment x sex,
CXCL9; F (1,26) = 6.682, p = 0.016, CXCL10; F (1,27) = 5.472,
p = 0.027). FCMU had a significant increase in the expression of both CXCL9 and CXCL10 compared to FSPG,
as shown in Fig. 4A, B (post-hoc for females, CXCL9; F(1,26) = 15.251, p = 0.001, CXCL10; F (1,27) = 4.843, p = 0.036).
There was no significant difference in MCMU comparedto MSPG (post-hoc for males, CXCL9; F (1,26) = 0.062,

p = 0.805, CXCL10; F (1,27) = 0.024, p = 0.879). These results
suggest that females, unlike their male counterparts, aremounting a stronger response within the CNS to inhibit
the development of infection during the acute phase.
Interleukin-13 (IL-13), functions in the CNS as ananti-inflammatory cytokine and is expressed by reactive
microglia and can produce both beneficial and harmful
effects on the local environment [28]. The IL-13 inflammatory pathway has been shown to modulate potassium calcium-activated channel subfamily N member 4(KCNN4) which codes for KCa3.1 [29]. KCa3.1 is a voltage-independent potassium channel that is activated byintracellular calcium. Activation of this channel helps tosustain calcium entry into cells via voltage independent
pathways and is critical for the inflammatory response in
microglia and astrocytes [30, 31].
At P15, treatment x sex analysis revealed sex-specificeffects of infection on both of these inflammatory mediators (treatment x sex, IL-13; F (1, 26) = 5.820, p = 0.026,
KCNN4; F (1, 27) = 7.661, p = 0.010). For the expression of
IL-13, there was significant difference between FCMU
and FSPG, as shown in Fig. 4C (post-hoc for females;
F (1,26) = 5.281, p = 0.030) while there were no differences between males (post-hoc for males; F (1,26) = 0.245,
p = 0.624). Similarly, KCNN4 expression also showedHedley et al. Journal of Neuroinflammation(2024) 21:158

Page 7 of 15

Fig. 4 Upregulation of inflammatory mediators in the medulla oblongata of infected females. Graphs show A) CXCL9 (FSPG vs. FCMU **p < 0.01), B)
CXCL10 (FSPG vs. FCMU *p < 0.05), C) IL-13 (FSPG vs. FCMU *p < 0.05), D) KCNN4 (FSPG vs. FCMU *p < 0.05), E) BDNF (FSPG vs. FCMU *p < 0.05) and F) TGFβ
relative gene expression levels (mean ± SEM) at P15. Graphs presented as SPG (solid bars), CMU (hatched bars), females (grey) and males (black), with total
number of samples shown in each bar

significant differences between FCMU and FSPG, asshown in Fig. 4D (post-hoc for females; F (1,27) = 6.192,
p = 0.019) while there were no differences between
males (post-hoc for males; F (1,27) = 1.982, p = 0.171). This
indicates that there are significant changes within the
IL-13 inflammatory pathway in females only during theacute inflammatory response to a peripheral respiratory
infection.
Brain derived neurotrophic factor (BDNF) is a crucialcomponent in brain plasticity, neuronal development,
growth and survival, and is implicated in establishing
functional neuronal networks during development [32].Transforming growth factor beta (TGFβ) is a multifactorial cytokine that regulates the behaviour of many
immune cells, such as macrophages and neutrophils,
causing anti-inflammatory effects and forming a network of negative immune regulatory inputs [33]. TGFβ
signalling can regulate BDNF and both are considered
protective when acutely responding to inflammation [34].
At P15, treatment x sex analysis revealed a significant
effect of infection on BDNF expression (Fig. 4E), but nochange to the expression of TGFβ (Fig. 4F) (treatment x
sex, BDNF; F (1, 27) = 5.235, p = 0.031, TGFβ; F (1, 27) = 4.009,
p = 0.055). The expression of BDNF in FCMU wassignificantly increased compared to FSPG, shown in
Fig. 4E (post-hoc for females; F (1,27) = 5.054, p = 0.033)
and there was no significant difference in males (post-hoc
for males; F (1,27) = 0.056, p = 0.815). Together, this suggests that the initial potential protective response in the
medulla oblongata is only seen in females, and it is driven
by BDNF expression, not TGFβ.
Suppression of inflammatory mediators in infected males

Signal transducer and activator of transcription 6 (Stat6)
is required for the promotion of the T-helper type 2(Th2) immune response [35]. There was a significant
treatment x sex interaction for Stat6 (treatment x sex,
Stat6; F (1, 27) = 5.831, p = 0.023). The expression of Stat6
showed a significant decrease in MCMU comparedto MSPG, as shown in Fig. 5A (post-hoc for males; F(1,27) = 5.395, p = 0.028) while there were no differences
between females (post-hoc for females; F (1,27) = 0.017,
p = 0.896). This indicates that there are significant sexspecific changes, with infected males showing a suppression of potential anti-inflammatory pathways in theacute inflammatory response to a peripheral respiratory
infection.

Hedley et al. Journal of Neuroinflammation(2024) 21:158

Page 8 of 15

Fig. 5 Downregulation of inflammatory mediators in the medulla oblongata of infected males. Graphs show A) Stat6 (MSPG vs. MCMU *p < 0.05), B)
PTGS2 (MSPG vs. MCMU *p < 0.05), C) IFNγ, D) Stat1, E) NLRP3, F) TNFα and G) IL-1β relative gene expression levels (mean ± SEM) at P15. Graphs presented
as SPG (solid bars), CMU (hatched bars), females (grey) and males (black), with total number of samples shown in each bar

Prostaglandin-endoperoxide synthase 2 (PTGS2), also
known as cyclooxygenase (COX-2), is a key enzyme
in prostaglandin biosynthesis and during inflammation is associated with oedema and leukocyte trafficking [36]. Treatment x sex analyses revealed a significant
effect on the expression of PTGS2 (Fig. 5B, treatment x
sex, PTGS2; F (1, 26) = 5.439. p = 0.028). MCMU showeda significant decrease compared to MSPG (post-hoc formales; F (1,26) = 4.347, p = 0.047), with no differences seenin females (post-hoc for females; F (1,26) = 1.472, p = 0.236).
This indicates a clear suppression of male PTGS2 in
response to the infection.
Interferon gamma (IFNγ) is a pro-inflammatory cytokine that primarily activates macrophages and stimulates
neutrophils [37]. Signal transducer and activator of transcription 1 (Stat1) increases macrophage inflammatory

responses [38] and is one of the primary transcription
factors activated by IFNγ, thus playing a major role inimmune responses [39]. However, at P15 no significant
differences were seen in either IFNγ or Stat1, shown in
Fig. 5C, D, indicating that, at this timepoint, this pathway
is not heavily involved in the medulla oblongata’s initial
response to a neonatal bacterial infection.
Given the importance of NLRP3, IL-1β, TNFα in theacute phase of the peripheral inflammatory response,
the expression of these inflammatory mediators was also
assessed in the medulla. However, there were no significant differences in NLRP3, TNFα or IL-1β, shown in
Fig. 5E-G, indicating that, at this acute timepoint, thispathway is not heavily involved in the medulla oblongata’s acute response to a neonatal infection.

Hedley et al. Journal of Neuroinflammation(2024) 21:158

Page 9 of 15

Glia in the respiratory centres of the brainstem aresignificantly affected by a neonatal respiratory infection

analysis revealing a significant difference in the mean
fluorescent intensity (MFI) in males, where MCMU
increased compared to MSPG, shown in Fig. 6F (treatment effect; F (1,20) = 5.531, p = 0.029) (post-hoc for males;
F (1,20) = 17.410, p < 0.001). In contrast, females showeda significant difference in % area, with FCMU increasing compared to FSPG, shown in Fig. 6G (treatment
effect; F (1,20) = 15.109, p = 0.001) (post-hoc for females;
F (1,20) = 16.089, p < 0.001). Interestingly, treatment x sex
analysis of microglia in the NTS revealed differences
between the sexes in % area, with FSPG being smaller
than MSPG, shown in Fig. 6G (post-hoc for SPG; F(1,20) = 7.381, p = 0.013). Further morphological analyses
were undertaken to examine the specific characteristicsof the microglia. There was a main effect of treatmentof soma area, Fig. 6H, (treatment effect; F (1,19) = 29.554,
p < 0.001). Again, when treatment x sex analysis was
performed a magnitude difference was observed, with agreater difference observed between MCMU and MSPG
(post-hoc for males; F (1,18) = 21.919, p < 0.001, post-hoc
for females; F (1,18) = 9.039, p = 0.007). There was alsoa treatment x sex effect on the number of secondarybranches (branch points), with FCMU decreasing compared to FSPG (treatment effect; F (1,18) = 3.612, p = 0.073)(post-hoc for females; F (1,18) = 5.076, p = 0.037), withno difference in males. Taken all together, the female’s
response to infection results in more microglia, withlarger somas and retracted branches, which accounts
for the increase in % area. Whereas the male’s response
to infection results in a bigger soma and a greater production of Iba1, as shown by the MFI. This indicates thatwhile both female and male microglia are being affectedin the acute phase of infection, the characteristics of the
response to the insult differs.
In the DMX, treatment x sex analysis (treatment x sex,
F (1, 18) = 17.458, p = 0.001) revealed there was a significant
decrease in the cell number of FCMU compared to FSPG,
shown in Fig. 7E (post-hoc for females; F (1,18) = 5.517,
p = 0.030) while there was a significant increase in cellnumber of MCMU compared to MSPG (post-hoc formales; F (1,18) = 12.674, p = 0.002). This was reflected in theanalysis of MFI (treatment x sex, F (1, 20) = 30.167.
p < 0.001), with a significant increase in the MFI
between MCMU and MSPG, shown in Fig. 7F (post-hoc
for males; F (1,20) = 22.062, p < 0.001), whereas in females,
FCMU saw a significant decrease compared to FSPG,
Fig. 7F (post-hoc for females; F (1,20) = 9.428, p = 0.006).
Unlike the NTS, morphological analyses of the DMX
showed no significant effect of infection on any characteristic, indicating in the acute phase a respiratory infection only impacts the number of microglia present in the
DMX. Interestingly, sex x treatment analysis revealed
differences between the sexes in soma area, with FSPG
having a smaller soma than MSPG, shown in Fig. 6HBoth microglia, labelled with Iba1, and astrocytes,
labelled with GFAP, were assessed in key regions of the
medulla oblongata known to be important to the central
processing of respiratory function. These are the nucleus
tractus solitarii (NTS) and dorsal motor nucleus of thevagus (DMX).
NTS and DMX show significant alterations to microglia
activation and morphology

In the NTS, there was a main effect of treatment on thenumber of cells (Fig. 6E; treatment effect; F (1,18) = 23.946,
p < 0.001). However, when this was further exploredthrough treatment x sex analysis, a significant difference was only present between the number of cells in
FCMU and FSPG (post-hoc for females; F (1,18) = 23.815,
p < 0.001), whereas males were approaching significance(post-hoc for males; F (1,18) = 4.163, p = 0.056). This indicates that the magnitude of change to the number of cells
differs between females and males. This sex-difference
was mirrored in other analyses, with treatment x sex

Fig. 6 Iba1 immunofluorescent images and quantification of microglia in
the NTS. Brainstem sections were labelled with Iba1 to identify microglia
and nuclei were identified using a brain atlas. A-D) representative imagesof all groups showing the area selected for NTS with the area postrema
removed, scale bars represent 100 μm. Graphs show E) number of cells(FSPG vs. MCMU ***p < 0.001), F) MFI (MSPG vs. MCMU ***p < 0.001), G)% area (FSPG vs. FCMU ***p < 0.001, FSPG vs. MSPG *p < 0.05), H-J) units
in pixels, with H) soma area (FSPG vs. FCMU **p < 0.01, MSPG vs. MCMU***p < 0.001), I) number of primary branches and J) number of secondarybranches (branch points) (FSPG vs. FCMU *p < 0.05), (mean ± SEM). Graphspresented as SPG (solid bars), CMU (hatched bars), females (grey) andmales (black), with total number of samples shown in each bar

Hedley et al. Journal of Neuroinflammation(2024) 21:158

Fig. 7 Iba1 immunofluorescent images and quantification of microglia in
the DMX. Brainstem sections were labelled with Iba1 to identify microglia and nuclei were identified using a brain atlas. A-D) representative images of all groups showing the area selected for DMX, scale bars represent
100 μm. Graphs show E) number of cells (FSPG vs. FCMU *p < 0.05, MSPG
vs. MCMU **p < 0.01), F) MFI (FSPG vs. FCMU **p < 0.01, MSPG vs. MCMU***p < 0.001), G) % area, H-J) units in pixels, with H) soma area (FSPG vs.
MSPG *p < 0.05), I) number of primary branches and J) number of secondary branches (branch points) (mean ± SEM). Graphs presented as SPG(solid bars), CMU (hatched bars), females (grey) and males (black), withtotal number of samples shown in each bar(sex effect; F (1,18) = 9.810, p = 0.006) (post-hoc for SPG; F(1,18) = 4.513, p = 0.048). This indicates a different starting
point between male and female microglia regardless ofenvironmental factors.
Astrocyte changes are minimal following neonatal
respiratory infection

There was a main effect of treatment (treatment effect, F(1, 19) = 27.138, p < 0.001) on the % area of NTS GFAP + ve
cells, shown in Fig. 8F. This indicates that astrocytes fromboth sexes show similar responses to infection. There
were no changes in either MFI of the NTS or either
parameter within the DMX.

Discussion
It is well known that the critical period of development
is particularly susceptible to environmental stressors,
however the specific effect within brainstem respiratory
centres remains unknown. In addition, the majority ofdata that is available explores the response to a bacterial mimetic (like lipopolysaccharide [LPS]) and does notinvestigate the more complex response from exposure to
the entire bacterium [40]. This study explores the acuteeffects of a neonatal respiratory infection on the brainstem centres that are essential for respiratory control.

Page 10 of 15

Fig. 8 GFAP immunofluorescent images and quantification of astrocytes
in the NTS and DMX. Brainstem sections were labelled with GFAP to identify astrocytes and nuclei were identified using a brain atlas. A-D) representative images of all groups showing the area selected for NTS with thearea postrema removed, G-J) representative images of all groups showing
the area selected for DMX, scale bars represent 100 μm. Graphs show E)
MFI and F) % area (SPG vs. CMU ***p < 0.001) in the NTS and K) MFI and
L) % area in the DMX (mean ± SEM). Graphs presented as SPG (solid bars),
CMU (hatched bars), females (grey) and males (black), with total numberof samples shown in each bar

There was a significant decrease in the rate of weight gainof the CMU-infected mice as well as a significant increase
in inflammatory mediators in the lungs that were notsex-specific, indicating that there was a robust inflammatory response to a respiratory bacterial infectionin the lungs. In the brainstem, however, inflammatory
mediators showed a strong sex-specific response, with
infected females showing increases in neuroprotective
inflammatory mediators and males showing decreases ina few inflammatory mediators. There were also significant effects on microglia in both males and females. In
the DMX, infected females observed to have a decreasein the MFI and microglia cell number, and a contrasting impact in infected males of a significant increase in
MFI and cell number. Astrocytes demonstrated a limited acute response, with a significant increase in percentage area only occurring in the NTS of both sexes.
Overall, these data indicate that a neonatal respiratory

Hedley et al. Journal of Neuroinflammation(2024) 21:158

bacterial infection not only produces a robust responsein the lungs but also a strong sex-specific response in thebrainstem centres that control respiration. This infection
and subsequent inflammation during a critical period ofdevelopment of the CNS could interrupt key developmental processes, and lead to life-long dysfunction.
The peripheral response to a neonatal respiratory infection

The peripheral response in this model has been well characterised and established in female mice [13]. To identify
any sex-specific peripheral changes and to validate thatthe effects seen in the brainstem are related to infectionof the lungs, infection and the peripheral inflammatory
response had to be confirmed. There was a significant
reduction in weight gain in the CMU-infected animals
from days 6 until endpoint (day 15/2wks). There was also
saw a significant upregulation of 16 S expression in both
FCMU and MCMU at the endpoint (day 15/2wks). This
mirrored previous work from our laboratory showing asignificant reduction in weight gain from days 7 to 23 in
response to infection, with peak of infection at day 12 andpeak of inflammation at day 15/2wks in the lungs [13].
The reduction in weight gain is indicative of infection in
the pups, and coupled with the 16 S expression, successfully confirms infection in this cohort. To ensure there
had been no drift in the model in terms of inflammatory
response, key inflammatory mediators were examined in
the lungs, IL-1β, NLRP3, TNFα and CXCL9. All mediators showed a significant increase in the CMU-infected
mice, regardless of sex, indicating a robust inflammatory
peripheral phenotype that is consistent with our previous
findings.
Sex-specific inflammatory mediator response in the
medulla oblongata

A key finding of this study is that, while a neonatal respiratory infection caused the same peripheral inflammatory response in both males and females, sex did have asignificant impact on the inflammatory response within
the brainstem centres, housed in the medulla oblongata,that control respiration.
With respect to the female response to infectioninduced neuroinflammation, there was significant upregulation in the expression of CXCL9, CXCL10, BDNF,
IL-13 and KCNN4 in the medulla oblongata. In the acuteresponse phase these inflammatory mediators are allconsidered to be neuroprotective, with the exception of
KCNN4. Both CXCL9 and CXCL10 control the spread of
infection in the CNS through the recruitment of effector
cells, such as phagocytes and lymphocytes, to the CNS.
This has been shown in other infections in adult models,such as viral infections; herpes simplex virus [41] and theWest Nile virus [42], where it was linked to reductions inmortality following infection. BDNF is well-established

Page 11 of 15

as a key factor in neuronal development and plasticity
and has been shown to reduce inflammation and neuronal apoptosis in response to bacterial infections [43].
Early-life deficiency of BDNF has been linked to several
mood disorders such as depression [44, 45] and bipolar disorder [46]. Therefore, this increase in females, in
response to the early life infection, could protect the CNS
from inflammation induced damage. Although, IL-13 isa major driver of allergic asthma in the lungs [47], in the
CNS it has anti-inflammatory effects and has been shown
to provide neuroprotection through anti-inflammatory microglial responses in stroke [48] and to promote
recovery after a traumatic brain injury, again throughmicroglia-mediated immune responses [49]. KCNN4
is the only upregulated mediator that would be considered damaging to the CNS. KCNN4 contributes the proinflammatory activation of microglia [50], with blockingof this channel shown to improve CNS outcomes following traumatic brain injury [51, 52] and has been proposed
as a potential therapeutic for microglia neurotoxicity
in Alzheimer’s disease [53]. Given the response in thisstudy of the microglia in females, i.e., decreased numbers and MFI (discussed further below), KCNN4 appears
to be having only a limited effect or the system is plastic
enough to overcome these alterations. Overall, at the 2wk
timepoint in the medulla, there is a strong anti-inflammatory effect in females, which would likely protect the
CNS from damage caused by the peripheral inflammatory event.
Unlike the similar peripheral response observed in thelungs. the response of the medulla oblongata in males toa peripheral inflammatory event is contrasting and significantly muted compared to females. There was a significant decrease in only two inflammatory mediators,
PTGS2 and Stat6, in response to the respiratory infection, with no changes to any other mediator observed.
PTGS2 plays a central role in the inflammatory cascadeof both anti- and pro-inflammatory mediators [54]. Multiple studies have reported that by either inhibiting or
knocking down PTGS2, the CNS response to a systematic inflammatory challenge is exacerbated by increasing
microglia activation and permeability of the BBB to infiltrating peripheral immune cells [55–57]. However, thesestudies have been conducted in adult models of systemic
inflammation and use LPS, a bacterial mimetic, to induce
inflammation. Our recent work exploring the brainstem’s
response to neonatal LPS inflammation begins to fill inthis gap in knowledge [58], demonstrating a reduction in
PTGS2 in males during the acute response and that overall, the response to neonatal LPS followed similar trends
to CMU, with suppression of inflammatory mediators
in males. There is no information surrounding a specificneonatal respiratory infection and the response of PTGS2
in the medulla at the neonatal age. Stat6 helps promoteHedley et al. Journal of Neuroinflammation(2024) 21:158

microglial clearance of dying cells which is key to theresolution of neuroinflammation. Supressing Stat6 has
been shown to exacerbate local inflammation and worsen
long-term outcomes [59, 60]. Again, these studies werecompleted in adult models and the neonatal response
could differ, but there is no literature available for thisearly postnatal age. It is well described in clinical literature that male neonates are more susceptible to infection
due to higher testosterone levels supressing the immune
system [61–63]. Suppression at this age does not allow
the system to correctly respond and clear the infection,
causing increased mortality as well as an increased likelihood of severe long-term health consequences in males.
The differences between the strength of the central andthe peripheral response could relate to timing. The CNS
response to a peripheral infection is known to be slower
than in the periphery itself [64], therefore this would
suggest that at the 2wk timepoint, we are observing the
emerging response within the brainstem to the peripheral respiratory infection. This would explain the strong
upregulation of pro-inflammatory mediators in the lungs
in both sexes, contrasted with the variable shift in the
pro-inflammatory mediators in the medulla. Since only
one timepoint was observed within this acute inflammatory phase, it remains to be seen whether in females,
the significant neuroprotective changes are the initial
response to peripheral infection-induced neuroinflammation or if this a counter measure to reduce a previous
peak in the pro-inflammatory response. The same could
be said for males, with the trends towards suppression of
inflammatory mediators potentially being indicative ofeither the start or the tapering off of the neuroinflammatory response. To determine which of these is true, themedulla’s response to peripheral infection induced neuroinflammation requires a more comprehensive exploration throughout this acute inflammatory phase.
Glial response to neonatal respiratory infection

The acute response of microglia and astrocyte to a
peripheral respiratory infection was nuclei- and sex-specific. Activation appeared to be enhanced in males andsuppressed in females, with further, differential changes
observed between the NTS and DMX, with significant
alterations to microglial morphology and cell numberin the NTS and changes to cell number in the DMX. In
females, the number of microglia in the DMX decreased
in response to a respiratory infection, further indicatinga strong anti-inflammatory neuroprotective phenotype.
Suppression of the female microglia response in the DMX
could be attributed to the increases observed in antiinflammatory immune mediators, mainly IL-13, which
has been shown to reduce neuroinflammation throughinduction of microglia apoptosis [65]. The morphological changes in the NTS suggest a neuroinflammatory

Page 12 of 15

phenotype for both males and females, which could
lead to long-term consequences. At this age, neonatal
microglia have a very similar function to adult microglia
but have a distinct phenotype. Adult microglia are transient in their nature, whereas neonatal microglia exhibit
a more classically defined amoeboid morphology, making the neonatal period a critical window for microglial
development [66]. Activation of neonatal microglia via a
peripheral infection, and subsequent neuroinflammation,
can result in persistent changes in neonatal microglial
function which then remains into adulthood [67]. This
alteration has been linked to neurodevelopment disorders [68] and respiratory deficits [69] in adulthood. The
significant sex-specific responses to infection and thebaseline sex differences observed in the soma area of
the DMX, does indicate that male and female microglia have a distinct phenotype. This is well characterisedin the literature with the sex differences believed to be
derived from large increase in male gonadal hormones
in males and the sex chromosome complement [70]. In
addition, other studies have found that oestrogen receptors on microglia are undetectable in the early life stages[71], which does indicate alternate mechanisms for sexspecific effects. As the NTS modulates and disseminatesafferent sensory information and the DMX sends efferent
information, the different changes to morphology andnumber of microglia would have a distinct impact on signalling both centrally and in the periphery.
Astrocytes showed an increase in % area, in the NTS
only, in both males and females and thus was not sexspecific. The marker used in this study, GFAP, plays arole in supporting BBB function and is elevated withastrocyte activation [72]. Astrocyte activation can leadto thickening and elongation of astrocyte processing thatwould be indicated through altered percentage area [73].
The NTS is the primary integration centre for all incoming peripheral signals from the vagus nerve, including a large component from the lungs, and contributes
to synaptic transmission through gliotransmission andregulation of glutamate [74, 75]. Thus, it is not surprising at this acute stage, astrocytes within the NTS show
the greatest response as this is the first integration centre
to receive the peripheral inflammatory signals and thenfurther distribute these signals around the CNS. This further emphasises the need for a deeper exploration of thisacute inflammatory phase, including ages either side ofthis P15 time point, to more accurately assess the early
response of astrocytes to a neonatal respiratory infection.
Development consequences of a neonatal respiratory
infection

During early postnatal development, neurons associated with respiration undergo maturational changes
which are crucial to ensuring the proper functioning ofHedley et al. Journal of Neuroinflammation(2024) 21:158

the respiratory system. While the fundamental processesof these neurons are established at birth, postnatal finetuning of the system allows many tightly regulated processes to develop to allow rapid and effective responses toenvironmental demands. In rodent studies, the DMX has
been shown to undergo postnatal maturation in neuronal function, with markers of neuronal activation revealing a plateau in activity occurring at days 3–4 and 12 [76].
Further study by McMenamin et al. demonstrated thatduring the first postnatal week DMX neurons received
inhibitory currents from both gamma-aminobutyric acid
(GABA) and glycine sources, which are both fast currents
during this developmental period. By the second postnatal week, there was a switch to only GABAergic inhibitory currents that gradually slowed into adulthood [77].
GABA has known interactions with inflammatory processes [78, 79], therefore, disruption to this developmental switch by neonatal inflammation could potentiallyalter the profile of these currents, leading to hypersensitivity of the neuronal respiratory circuit and adversely
affecting vagal efferent control of the respiratory system.
Similar maturation processes have been described forthe development of NTS neurons, with a downregulation of BDNF at day 12 [80]. This normal developmental reduction leaves the system susceptible to stressors
during this critical window, and given that this Chlamydia model results in a peak of inflammation at day 12,
this could be altering a key developmental trajectory ofneurotransmission within the respiratory circuit. In thisstudy, we do see an increase in BDNF in females, however the long-term changes still need to be assessed.
Other normal developmental processes of NTS neurons
include increases to synapse formation and decreases tomembrane resistance over the first four postnatal weeks[81]. This maturation of NTS neurons renders NTS neurons less sensitive to inputs and represents refinement ofbrainstem circuitry associated with respiration. However,
disruption to this developmental refinement by a neonatal respiratory infection could lead to dysfunction withinthese processes, again resulting in a hypersensitivity ofthe system, and highlights the vulnerability of the system
to a neonatal respiratory infection.
Limitations and future directions

This is one of the first studies to evaluate the acute effects
of a neonatal respiratory infection on the brainstem.
However, there are specific limitations which need to
be investigated in future studies. The medulla oblongata
contains multiple nuclei with a diverse array of functions.
Since the RNA for the expression of inflammatory mediators in this study was extracted from the entire medulla,
conclusions cannot be drawn upon its specificity to individual nuclei, nor the relative inflammatory response
within other regions of the brainstem. In future RT-qPCR

Page 13 of 15

studies, individual nuclei should be isolated to detail eachspecific nuclei’s inflammatory response. Another limitation is while a link is hypothesised between the respiratory infection and the medulla inflammatory response,
this needs to be explored functionally. To validate thishypothesis, knock-out models can be generated to eliminate key neuroinflammatory pathways and functionallyassess the respiratory response under these conditions.

Conclusions
This study clearly demonstrates that a neonatal respiratory infection induces acute sex-specific neuroinflammation in the respiratory centres of the medulla
oblongata, which does not directly mirror the inflammatory response in the periphery. In females, there wasa significant neuroprotective phenotype displayed withincreased anti-inflammatory immune mediators and a
suppression of microglia activation within the DMX only.
Conversely, in males, there was suppression of inflammatory mediators and activation of microglia which could
ultimately lead to worse outcomes. The neonatal period
is a critical window for development and disruption during this time can lead to significant neurodevelopmental challenges and long-term pre-disposition to further
damage. Given these centres control major componentsof respiration, neuroinflammation within these regions
could lead to major respiratory deficits with outcomessuch as sudden infant death syndrome [82], asthma [5]and sleep apnoea [83].
Author contributions
KEH, JCH and MAT conceived and designed the study. KEH performed all
experiments (excluding lung RT-qPCR) and analysis, prepared all figures andwrote the manuscript. HMG and EK performed RT-qPCR on lung tissue. ORC
assisted with the laboratory model and tissue collection. PJ, JCH and MAT
reviewed data and proofread the manuscript. All authors read and approved
the final manuscript.
Funding
We acknowledge the support of funding from the University of Newcastle andCommonwealth Research Training Program.
Data availability
The data sets used and/or analysed during the current study are available
from the corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
All experiments were performed in accordance with the National Health andMedical Research Council Australian Code of Practice and as approved by the
University of Newcastle Animal Care and Ethics Committee (approval number
A-2020-028).
Consent for publication
Not applicable.
Competing interests
All authors declare that they have no known competing financial interests
or personal relationships that could have appeared to influence the work
reported in this paper.

Hedley et al. Journal of Neuroinflammation(2024) 21:158

Received: 29 January 2024 / Accepted: 10 June 2024

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