Journal of Neuroinflammation

Shad et al. Journal of Neuroinflammation(2024) 21:122
https://doi.org/10.1186/s12974-024-03093-9

Open Access

RESEARCH

Modelling lung infection with Klebsiellapneumoniae after murine traumatic brain
injuryAli Shad1,2, Sarah S. J. Rewell1,2, Matthew Macowan3,4, Natasha Gandasasmita1, Jiping Wang5, Ke Chen5,
Ben Marsland3,4, Terence J. O’Brien1,2,4,6, Jian Li5 and Bridgette D. Semple1,2,6*Abstract
Pneumonia is a common comorbidity in patients with severe traumatic brain injury (TBI), and is associated withincreased morbidity and mortality. In this study, we established a model of intratracheal Klebsiella pneumoniae
administration in young adult male and female mice, at 4 days following an experimental TBI, to investigate how
K. pneumoniae infection influences acute post-TBI outcomes. A dose-response curve determined the optimal doseof K. pneumoniae for inoculation (1 x 10^6 colony forming units), and administration at 4 days post-TBI resulted
in transient body weight loss and sickness behaviors (hypoactivity and acute dyspnea). K. pneumoniae infection
led to an increase in pro-inflammatory cytokines in serum and bronchoalveolar lavage fluid at 24 h post-infection,in both TBI and sham (uninjured) mice. By 7 days, when myeloperoxidase + neutrophil numbers had returned tobaseline in all groups, lung histopathology was observed with an increase in airspace size in TBI + K. pneumoniae
mice compared to TBI + vehicle mice. In the brain, increased neuroinflammatory gene expression was observed
acutely in response to TBI, with an exacerbated increase in Ccl2 and Hmox1 in TBI + K. pneumoniae mice comparedto either TBI or K. pneumoniae alone. However, the presence of neuroinflammatory immune cells in the injured
brain, and the extent of damage to cortical and hippocampal brain tissue, was comparable between K. pneumoniae
and vehicle-treated mice by 7 days. Examination of the fecal microbiome across a time course did not reveal any
pronounced effects of either injury or K. pneumoniae on bacterial diversity or abundance. Together, these findings
demonstrate that K. pneumoniae lung infection after TBI induces an acute and transient inflammatory response,
primarily localized to the lungs with some systemic effects. However, this infection had minimal impact on
secondary injury processes in the brain following TBI. Future studies are needed to evaluate the potential longerterm consequences of this dual-hit insult.
Keywords Bacteria, Pneumonia, Hospital-acquired infection, Inflammation, Controlled cortical impact*Correspondence:
Bridgette D. Semple
Bridgette.Semple@monash.edu
1
Department of Neuroscience, The School of Translational Medicine,
Monash University, Level 6 Alfred Centre, 99 Commercial Rd, Melbourne,
VIC 3004 VIC, Australia
2
Alfred Health, Prahran, VIC, Australia

3

Department of Immunology, The School of Translational Medicine,
Monash University, Melbourne, VIC, Australia
4
GIN Discovery Program, The School of Translational Medicine, Monash
University, Melbourne, VIC, Australia
5
Department of Microbiology, Monash Biomedical Discovery Institute,
Monash University, Clayton, VIC, Australia
6
Department of Medicine (Royal Melbourne Hospital), University of
Melbourne, Parkville, VIC, 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.

Shad et al. Journal of Neuroinflammation(2024) 21:122

Background
Traumatic brain injury (TBI) continues to represent
a major global public health burden [1]. Acutely postinjury, the hospitalization of individuals with severe
TBI entails substantial in-hospital healthcare utilization,with higher costs associated with greater TBI severity,
increased length of stay, and surgical interventions [2, 3].
Hospital-acquired infections pose a secondary complication for severe TBI patients, which drive higher healthcare costs by increasing the length of stay in intensive
care, hospital, and in-patient rehabilitation, as well as
increasing the frequency of hospital re-admission andlikelihood of unfavorable long-term outcomes [4–7].
Hospital-acquired or ventilator-acquired pneumonia
is the most commonly-reported infection in this population, affecting approximately 40% of severe TBI patients[8–14]. Susceptibility to pneumonia is heightened after
severe TBI due to the frequent need for tracheal intubation and ventilator support, alongside injury-induced
immunosuppression [15]. However, prophylactic use ofantibiotics is not typically recommended as most studies have failed to demonstrate efficacy at improving outcomes [14, 16].
The most common pathogens that cause nosocomial
infections in TBI patients are opportunistic Gram-negative bacteria such as Klebsiella pneumoniae, and Grampositive Staphylococcus aureus [17–20]. K. pneumoniae iscommonly detected in ventilator-associated pneumonia,
where it causes severe acute lung damage and inflammation leading to respiratory failure in immunocompromised patients, including those with a TBI or spinal
cord injury [21–25]. K. pneumoniae is highly virulent andincreasingly a concern due to multidrug resistance [26],with carbapenem-resistant K. pneumoniae in particularbeing identified by the World Health Organization as a
‘Critical’ priority for the development of new antibiotics[27].
However, despite clinical evidence that bacterial infections lead to worse outcomes for TBI patients, we currently have an incomplete understanding of the biologicalmechanisms that contribute to an additional burden for
the affected patient. An increasing appreciation of TBIinduced immune deficiencies [15, 28], and bi-directional
communication between the acutely-injured brain andlungs [29, 30], provide new directions for targeted therapeutics to improve patient outcomes. Limited preclinical mouse models have examined the consequences ofan infection following TBI [as recently reviewed: [31].
For example, intratracheal infection with Pseudomonas aeruginosa led to increased mortality and reduced
lung bacterial clearance in mice that had sustained asevere TBI 24 h prior (via the controlled cortical impact(CCI) model), compared to similarly-infected uninjured mice [32]. In another study, Doran and colleagues

Page 2 of 18

administered intranasal Streptococcus pneumoniae at 3or 60-days after moderate CCI, and reported higher mortality, poorer functional outcomes, and increased lungand neuroinflammation in mice with the combined insult[33]. By contrast, Vermeij and colleagues found that the
combined insult of an intratracheal infection with S.
aureus and a mild weight-drop injury in male rats did notlead to worse outcomes [34].
The consequences of lung infection with K. pneumoniae after TBI have not previously been examined ina preclinical model. Others have shown that intratracheal
inoculation of naive mice with live K. pneumoniae leads
to the rapid expansion of bacteria in the lungs within
the first 24 h, leading to lung pathology, reduced pulmonary function, and associated morbidity [35–37], similar
to the effects of K. pneumoniae infection in humans. In
the current study, we sought to establish a new modelof K. pneumoniae-mediated lung infection after TBI, toprobe the effects of a secondary immune challenge on
TBI outcomes over a sub-acute period (the first week)
post-infection. We hypothesized that K. pneumoniae
infection would result in poorer outcomes in TBI mice
via increased immune activation and inflammation. We
also examined the gut microbiome in this context, inlight of recent evidence of complex bi-directional interactions between the brain, lungs and gut after brain injury[38–40].

Methods
Animals and ethics

All animal experiments were conducted following
approval from the local Alfred Research Alliance Animal Ethics Committee (#P8032) as well as the Animal
Care and Use Review Office (ACURO) from the Officeof Research Protections, US Department of Defense, andcarried out in accordance with these approved standards
as well as the Australian Code for the Care and Use ofLaboratory Animals as stipulated by the National Healthand Medical Research Council of Australia (NHMRC).
Male and female C57Bl/6J mice were sourced fromthe Walter and Eliza Hall Institute of Medical Researchin Melbourne, Australia, and habituated for one weekprior to the commencement of experiments. Animal
experiments were performed within QC2 facilities at theMonash Research Animal Precinct in Clayton, Australia.
Mice were group housed as same-sex littermates (2–6/
cage; mix of experimental conditions per cage) in Optimice® individually-ventilated cages under a 12-h light/darkcycle with continuous access to food and water.
Controlled cortical impact (CCI) model of TBI

Moderate-severe experimental TBI was induced in
10–12 week old mice using the CCI model, as previously
described [41]. Briefly, anesthesia was induced using 4%

Shad et al. Journal of Neuroinflammation(2024) 21:122

isoflurane in oxygen, and maintained via a nose cone at
1.5%. All animals received buprenorphine (0.05 mg/kg
in saline; s.c. flank) and bupivacaine (1 mg/kg in saline;s.c. scalp) for pain relief before surgery commenced, and
0.5 mL 0.9% saline at the end of the procedure to provide
additional hydration. Animals were stabilized in a stereotaxic frame, and a 3.5 mm diameter craniotomy was
made over the exposed left parietal bone. An electronic
controlled cortical impactor device (eCCE-6.3; Custom
Design and Fabrication Inc., Sandston, VA) delivered an
injury using a 3 mm rounded impactor tip, at 4.5 m/s,
1.7 mm depth and 150 ms duration. Sham animals
underwent an identical surgical procedure, without delivery of the impact. Following CCI (or sham surgery), theskin incision was sutured and antiseptic solution applied.
Animals were allowed to recover in individual cages on aheat mat before being returned to their home cage.
K. pneumoniae administration

K. pneumoniae ATCC 15,380 freeze-dried cultures weresourced from In Vitro Technologies (Noble Park, VIC,
Australia), then cultivated onto nutrient agar plates(Medium Preparation Unit, University of Melbourne,
Victoria, Australia) at 37 °C overnight. For the initial
culture, a maximum number of K. pneumoniae colonies were collected and immersed in cation-adjusted

Page 3 of 18

Mueller-Hinton broth (CAMHB) for an additional 24 hincubation at 37 °C, while shaking at 180 rpm [42]. Midlogarithmic-phase cultures were then prepared with a 3 h
incubation in fresh CAMHB. The bacterial concentration (colony forming units (CFU)/mL) was determinedby measuring the optical density (OD) at 600 nm anddiluted to a working solution.
An initial series of pilot studies were conducted todetermine the optimal dose of K. pneumoniae via intratracheal inoculation to achieve an appropriate lunginfection model, using naïve male and female mice. Inoculation was performed on day 0 followed by tissue collection at 24 h, 48 h and 7 d to evaluate changes in bodyweight, sickness symptoms, survival, and lung bacterial load (Fig. 1a), and testing 4 doses of K. pneumoniae:
1 × 104, 1 × 105, 1 × 106 and 1 × 107 CFU. A separate pilot
cohort (n = 4/group) was used to compare sterile saline
vs. broth (the vehicle for K. pneumoniae inoculation).
For the remaining experiments, mice were randomized
for inoculation with 106 CFU K. pneumoniae or vehicle
on day 4 post-injury, as hospital-acquired infections aremost common during the first week after a TBI [21, 43].
Following anesthesia with 2–4% inhaled isoflurane, anexperienced technician used a MicroSprayer® Aerosolizer(Penn-Century, Philadelphia, PA, USA) to administer 25µL bacterial suspension or vehicle solution directly into

Fig. 1 Dose-dependent responses to K. pneumoniae in Naïve mice. K. pneumoniae (Kp) or vehicle solution was administered intratracheally to naïve
young adult mice (a). Changes in body weight (b), lung bacterial load (c) and % survival (d) were evaluated, with a dose of 106 CFU found to be optimal.
Mean ± SEM; CFU = colony-forming unit. n = 8/group (4 male and 4 female) for % survival (b). In (c), males are indicated by closed circles and females by
open circles

Shad et al. Journal of Neuroinflammation(2024) 21:122

the trachea [44]. Following recovery from the procedure,mice were closely monitored post-treatment for sickness
behavior, general appearance, and weight loss over thetime course.
Additionally, five-fold serial dilutions of both K. pneumoniae and vehicle samples were spiral plated and incubated overnight at 37 °C on nutrient agar plates to verify
the CFU/mL in the administered inoculum, using ProtoCOL 3 software (Synbiosis, USA).
Open field locomotor activity

At 4 h, 24 h, and 7 d after inoculation, all mice underwent an open field test to evaluate locomotor activity andanxiety-like behavior [41]. Mice were individually placedin an empty arena (400 mm W x 400 mm D x 300 mmH) and allowed free exploration for 10 min. Activity wasrecorded using an overhead camera and analyzed using
TopScan Lite v2.0.0 software (CleverSys Inc., Reston, VA,
USA), to quantify the total distance travelled and the proportion of time spent in a predefined center zone (70% of
the total arena size).
Tissue collection for histological analyses

At 7 days post-infection, mice were humanely euthanizedwith 160 mg/kg i.p. sodium pentobarbitone (Lethabarb®,
Virbac, Australia). Cardiac puncture was performed tocollect blood samples for serum cytokine analysis, andthe spleen was removed and weighed. A subset of mice
were transcardially perfused with 0.9% saline then 4%paraformaldehyde (PFA) for fixation. Lungs were inflated
in situ with 4% PFA then removed and post-fixed, andbrains were collected whole. Samples were transferred
to 70% ethanol and sent to the Monash Histology Platform (Monash University, Clayton, Australia) for paraffin processing and embedding. Coronal brain sectionsof 7 μm-thickness spanning approximately − 0.7 mm
to -3.4 mm Bregma were collected. For lung samples,
serial 4 μm-thick sections of the left lung were collected.
Another subset of mice were euthanised by rapid decapitation following sodium pentobarbitone overdose, forcollection of bronchoalveolar lavage fluid (BALF) via insitu lung lavage [45]. Mice were then decapitated, and thebrains extracted from the skull. The cerebral cortices andhippocampi were dissected and rapidly snap-frozen inliquid nitrogen for gene expression analyses.
Histology

To quantify tissue volume atrophy, six equidistant coronalbrain Sect. (280 μm apart, approximately − 0.7 to -3.5 mm
Bregma) were stained with Luxol Fast Blue (LFB) andCresyl Violet (CV) as per a modified protocol [46]. For
analysis, images were captured using a Leica Aperio AT
Turbo Brightfield slide scanner, then FIJI/ImageJ (NIH,
Bethesda, MD; http://imagej.nih.gov/ij) was used toPage 4 of 18

apply the unbiased Cavalieri method to estimate the volume of the intact dorsal cortex and hippocampus in eachhemisphere [47]. Each grid point represented an area of
100 μm², with a sampling frequency of four. Measurements were confined to the dorsal portion of each regionof interest [48]. Group means were expressed as the ratioof ipsilateral to contralateral volumes.
Lung tissue sections were stained with hematoxylin andeosin (H&E) as per a standard protocol by the Monash
Histology Platform, using the Leica ST5010 Autostainer
XL (Leica Biosystems Imaging Inc.). Digital lung slides
were viewed with Aperio ImageScope v.12.4.3 at 10x
magnification. Alveolar airspace size was quantified
using the mean linear intercept (MLI) method [45, 49].
Briefly, 20 equal length lines were drawn over images of
H&E-stained lung cross-sections, then the number ofintercepts with alveolar walls were counted. MLI was calculated by dividing the line length by the average intercepts per line. Analyses were performed on five regionsof interest (ROIs) 500 × 500 μm in size selected randomly
per lung section, and averaged across the top, middle andbottom sections of each lobe.
Immunofluorescence staining

Brain sections were stained for astrocytic marker glial
fibrillary acidic protein (GFAP) and neutrophil marker
myeloperoxidase (MPO) to evaluate cellular immune
responses. Heat-mediated antigen retrieval was performed using DAKO Target Antigen Retrieval Solution(diluted 1:10; Agilent Technologies, USA). After blocking
with 10% normal donkey serum in 0.1% Triton-X100 in
PBS, a rabbit polyclonal GFAP antibody (1:1000; Thermo
Fisher Scientific) and goat polyclonal MPO (1:250; R&D
Systems) were applied. Secondary antibodies used were
donkey anti-rabbit AF 594, donkey anti-goat AF 488,
or donkey anti-goat AF 594 (1:250; Thermo Fisher Scientific) and counterstained using Hoechst dye. Lung
sections were stained with MPO as an indicator of neutrophil infiltration. Sections were dewaxed then rehydrated, followed by heat-mediated antigen retrieval with
Tris-EDTA solution (pH 9.0). After blocking with CASBlock™ Histochemical Reagent (Thermo Fisher Scientific,
Waltham, MA, USA), sections were incubated overnightwith a goat anti-mouse MPO antibody (1:50; R&D Systems, USA) diluted in 1% bovine serum albumin (BSA)in PBS. On day 2, a chicken anti-goat Alexa Fluor (AF)594-conjugated secondary antibody was applied (1:200;
Thermo Fisher Scientific), then counterstained with
4’,6-diamidino-2-phenylindole (DAPI).
Images were captured on a Nikon Eclipse Ti-E inverted
microscope fitted with an Andor Zyla sCMOS camera
and using the Nikon NIS-Elements v.5.30.06 software
at 20x magnification. Stitched images of the entire lungsection or ipsilateral dorsal hemisphere were captured

Shad et al. Journal of Neuroinflammation(2024) 21:122

and exported to FIJI ImageJ for manual cell counts (e.g.,
co-localized MPO/DAPI in the lung; MPO + cells in the
injured brain) or determination of fluorescence percentage area (for GFAP), by a blinded investigator within
specified ROIs (two for the cortex and one for the hippocampus), averaged across 2 sections per brain per marker.
Cytokine analysis of BALF and serum

Cytokine and chemokine concentration was quantified
in BALF and serum samples using a V-PLEX® Cytokine
Panel 1 (mouse) and V-PLEX® Pro-inflammatory Panel 1(mouse) (Meso Scale Diagnostics, LLC, Rockville, MD).
These panels included detection of MCP-1 (CCL2),
MIP-2 (CXCL2), IL-1β, IL-6, KC/GRO, IL-10, and TNFα.
Detection was performed according to the manufacturer’s specifications on a MSD SQ120MM (VB204) instrument, with curves analyzed by Discovery Workbench®
using a 4PL Curve fit for all analytes. Samples wereassayed in duplicate by Crux Biolabs (Bayswater, VIC,
Australia).
Brain quantitative polymerase chain reaction (qPCR)

RNA was isolated from the ipsilateral (injured) cortex
and hippocampus using the RNeasy® Mini Kit (Qiagen,Hilden, Germany). RNA purity was measuring using
the QIAxpert spectrophotometer, then converted to
cDNA using the QuantiTect® Reverse Transcription Kit(Qiagen). High throughput qPCR was performed by theMonash Health Translation Precinct (MHTP) Medical Genomics Facility using the Fluidigm BioMark HD™
(Standard BioTools Inc., San Francisco, CA, USA) in a
Fluidigm 192.24 Dynamic Array™ Integrated Fluidic Circuit for Gene Expression. A panel of 24 genes were analyzed, including the housekeeping genes (HKGs) Ywhaz(Mm01722325_m1), Ppia (Mm02342430_g1) and Hprt
(Mm03024075_m1) (Zamani et al., 2020), as well as thefollowing cytokines, chemokines, immune cell markers,
and immune-regulatory genes: Il-1β (Mm00434228_m1),
Il-1α (Mm00439620_m1), Tnf-α (Mm99999068_m1), Il6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Ccl2
(Mm00441242_m1), Ccr2 (Mm00438270_m1), Cxcl2(Mm00436450_m1) and Cxcr2 (Mm99999117_s1), Cd45
(Mm01293577_m1) and Cd86 (Mm00444543_m1), Gfap
(Mm01253033_m1), Aldh1l1 (Mm03048957_m1), Arg1
(Mm00475988_m1), Tmem119 (Mm00525305_m1),
Trem2 (Mm04209424_g1), Nos2 (Mm00440485_m1),
Tgf1 (Mm01227699_m1), Mapk1 (Mm00442479_m1),
Nfkb1 (Mm00476361_m1), and Hmox1 (Mm00516005_
m1). Relative gene expression was calculated using
the 2− ρρCT method and was normalized to the average
expression of three reference genes.

Page 5 of 18

Fecal microbiome analysis

The fecal microbiome was sampled at five defined acutetime points: (1) immediately prior to the TBI/sham procedure; (2) 24 h after the procedure; (3) on day 4 postTBI/sham (prior to K. pneumoniae/vehicle inoculation);(4) 24 h post-inoculation with K. pneumoniae/vehicle;
and (5) 7 d post-infection. Microbial DNA extraction andsequencing was performed using a modified publishedprotocol [50, 51]. The QIAamp PowerFecal Pro DNA Kit(Qiagen, #51,084) was used to extract fecal DNA according to the manufacturer’s protocol. Three negative controls were included to address potential microbial DNA
contamination at different steps of the procedure. For
validation, positive controls were established by treating
5 µL of ZymoBIOMICS Microbial Community (Zymo
Research) akin to the actual samples, successfully identifying 7 out of 8 bacterial species present in the positive
control samples.
Each sample underwent PCR amplification using
custom barcoded primers targeting the bacterial 16S
rDNA v1-v2 region (F-27/R-338), with the following
sequences: 16S-Forward: 5’-AATGATACGGCGACC A C C G A G ATC TA C A C TATG G TA AT TC C A G MGTTYGATYMTGGCTCAG-3’; and 16S-Reverse:
5’-CAAGCAGAAGACGGCATACGAGATACGAGACTGATTNNNNNNNNNNNNAAGCTGCCTCCCGTAGGAGT-3’. Here, the ‘N’ sequences represented
sample-specific 12-nucleotide Golay barcodes. Accuprime PCR buffer II and Accuprime Taq polymerase(Fisher Scientific) were used for PCR. Subsequent toquantification using a Fragment Analyzer (Agilent Technologies), amplicons were pooled at equimolar ratios,
purified via the AMPure XP bead cleanup system (Beckman Coulter), and sequenced on a MiSeq platform usinga MiSeq Reagent Kit v2.
Microbial taxonomic profiling

Raw sequences were processed using the microbiomedada2 pipeline (https://github.com/respiratory-immunology-lab/microbiome-dada2) using the DADA2(version 1.28.0) R package [52]. Briefly, fastq files were
demultiplexed using the iu-demultiplex function fromillumina-utils tools [53], primers and adapters removed
with cutadapt [54], reads filtered and trimmed, sequencing error models generated, sequences dereplicated,
amplicon sequence variants (ASVs) inferred, paired-ends
merged, and chimeras removed. Bacterial 16 S ASVs
were assigned a taxonomy using the SILVA databasetrain set (version 138) and the SILVA species assignment dataset (version 138) for exact sequence matching. A phylogenetic tree based on ASV sequences wasbuilt by performing a multiple-alignment using DECIPHER R package (version 2.28.0), followed by construction of a neighbor-joining tree as a starting point [55].

Shad et al. Journal of Neuroinflammation(2024) 21:122

Contaminant taxa were identified using the isContaminant function of the decontam (version 1.20.0) R package[56], using DNA extraction and 16 S PCR water control
samples as negative controls. The decontam ‘prevalence’
method was selected to identify contaminants as thosethat were more prevalent in negative controls than true
samples (prevalence threshold = 0.5); contaminants were
removed prior to filtering. Samples containing < 5,000
reads were excluded from the dataset and taxa present
in < 5% of samples or lacking assignment at the phylum
level were filtered out. Phylogeny-weighted Hill-Shannon
diversity was calculated using the hill_phylo function
from the hillR (version 0.5.2) R package [57]. ASV counts
were normalised using Cumulative Sum Scaling (CSS)
using the calcNormFactors function from the MetagenomeSeq (version 1.42.0) package [58], followed by logtransformation.
Statistical analysis

Most statistical analysis was performed using GraphPad Prism v.9.4.1 (GraphPad Software Inc., San Diego,
CA, USA), with significance defined as p < 0.05. Twoand three-way analyses of variance (ANOVA) were performed with Tukey’s post hoc test where appropriate.
Data with 3 independent variables of time, injury, andinfection, were assessed with a 3-way ANOVA. In most
instances, both male and female mice are pooled pergroup, with open circle data points graphically denoting female animals. Potential sex differences were tested
by 3-way ANOVA (factors of sex, injury, and infection),and only reported where significant sex differences weredetected. Data are presented as mean ± SEM.
Microbiome data was analyzed using R (version 4.3.0).
Differences in Hill-Shannon diversity were addressedusing non-parametric Wilcoxon Rank Sum testing.
Principle Coordinate Analysis (PCoA) was performedon the weighted UniFrac distance of normalized ASV
counts using the ordinate function of the phyloseq(version 1.44.0) R package [59]. Permutational Multivariate Analysis of Variance (PERMANOVA) was subsequently performed on the same data with 1,000,000
permutations. Pre-treatment comparisons used themodel ‘distance ∼ injury’, and post-treatment used ‘distance ∼ injury + treatment’. Longitudinal non-parametric
repeated measures Brunner-Langer tests were performed to assess microbial differences due to injury andtreatment using the f2.ld.f1 function from the nparLD(version 2.2) R package [60]. Linear modelling to assess
timepoint-specific group-wise differences was performed
using a custom wrapper around the limma R package[61](https://github.com/mucosal-immunology-lab/microbiome-analysis/wiki/Limma-DA).

Page 6 of 18

Results
Dose-dependent responses to K. pneumoniae in Naïve
mice

We first conducted a series of pilot studies to determine
the optimal dose of K. pneumoniae via intratracheal
administration in naïve male and female C56Bl/6J mice
(Fig. 1a) and evaluated changes in body weight (Fig. 1b),lung bacterial load (Fig. 1c) and mortality (Fig. 1d), alongside observable symptoms of illness. A low dose of 1 × 104
CFU failed to induce any observable symptoms in inoculated mice, and a negligible bacterial load in the lungs at
24 h (< 3 CFU/lung; n = 3; not shown). Mice that received
a dose of 1 × 105 CFU had a low bacterial load, and exhibited only minimal symptoms (inactive). The intermediate dose of 1 × 106 CFU induced overt clinical symptoms
of illness acutely (e.g., inactive, fur erect, hunched posture, acute dyspnea), and an intermediate lung bacterialload. Mice that received the highest dose of 1 × 107 CFU
were found to have a high lung bacterial load, exhibited
pronounced weight loss within 48 h, and presented withsignificant clinical symptoms of acute respiratory illness.
This dose also resulted in considerable mortality (63%;n = 5/8) within the first week post-infection (2/4 males
and 3/4 females), between 2 and 3 days post-inoculation,as animals reached the weight loss threshold requiring
humane euthanasia. No mortality was observed for micethat received a dose of 1 × 106 CFU K. pneumoniae or
vehicle (Fig. 1b).
In a separate pilot experiment, we compared intratracheal treatments of sterile saline only versus broth (thevehicle for K. pneumoniae inoculation), and found no
mortality, comparable body weights, no signs of acuteillness, and no differences in lung histological measures(n = 4/group; not shown). Subsequent experiments therefore employed a broth solution vehicle or a dose of 1 × 106
CFU K. pneumoniae.
K. pneumoniae administration after TBI does not worsen
acute symptoms of illness

To assess whether K. pneumoniae infection after TBI
altered functional outcomes, mice were subjected to TBI
or sham surgery, followed by K. pneumoniae or vehicle
inoculation at 4 days post-injury, and examined over thesubsequent 7 d period (Fig. 2a). The lung bacterial load
at 24 h post-infection was comparable between Sham-Kp
and TBI-Kp mice (t8 = 0.62, p = 0.5529; Fig. 2b), and undetectable in vehicle-treated mice. Spleens were collectedfresh and weighed at either 24 h or 7 d post-infection, as
a surrogate indication of mobilization of the peripheral
immune response. However, neither TBI nor K. pneumoniae altered spleen weight (either raw or relative tobody weight) (not shown; p > 0.05).
Body weights were assessed daily as an indicator ofgeneral health. A main effect of time was observed (F11,

Shad et al. Journal of Neuroinflammation(2024) 21:122

Page 7 of 18

Fig. 2 K. pneumoniae infection causes transient hypoactivity and weight loss in both TBI and Sham mice. The experimental timeline (a), whereby TBI or
sham mice were inoculated with Kp or vehicle after 4 days, then were assessed repeatedly at 4 h, 24 h and 7 d post-infection (OF = Open Field behavior
test). At 24 h post-infection, the lung bacterial load was equivalent in sham and TBI mice (b). Changes in body weight across the 7 d time course (c; n = 10/group) revealed a main effect of time, and a time-x-Kp interaction (*p < 0.05 from post-hoc analyses, 3-way RM ANOVA). In the OF test at 4 h post-infection,a non-significant trend towards reduced time in the center was observed in Kp-treated mice (d), which was resolved by 24 h and 7 d (not shown). Also in
the OF test, TBI induced an increase in activity at 4 h, 24 h and 7 d (e-g; 2-way ANOVAs, main effect of TBI *p < 0.05, ***p < 0.001), while Kp independently reduced activity just at 4 h post-infection (e; ****p < 0.0001). n’s in (d–g) are depicted by individual data points; males indicated by closed circles/diamonds
and females indicated by open circles/diamonds
396=21.30, p < 0.0001), as well as a time-x-Kp interaction(F11, 396=3.75, p < 0.0001). All groups appeared to exhibit
comparable mild weight loss (approximately 2–4%) in thefirst 24 h post-TBI/sham surgery then quickly regained
it, although this change was not statistically significant.
Then, at 24 h (1 day) post-inoculation, both sham-Kp
and TBI-Kp groups showed a similar reduction in bodyweight compared to vehicle-treated groups (post-hoc*p < 0.01 compared to Sham-Vehicle); although again
this was only a transient drop. When cohorts were separated by sex (n = 3/group; Supplementary Fig. 1), it wasnoted that males showed more consistent body weights
across the time course; whereas females showed considerable within-group variability. The effect of Kp on bodyweight was more pronounced in female mice, as a timex-Kp interaction was detected in females (F11, 176=2.39,
p = 0.0087) but not males (F11, 176=1.53, p = 0.1244).
The open field test was conducted to evaluate locomotor activity and anxiety-like behavior. At 4 h postinfection, a non-significant trend was seen with K.
pneumoniae-treated mice appearing to spend less time inthe center of the arena (F1, 35=3.72, p = 0.0619), suggestiveof increased anxiety-like behavior (Fig. 2d). At the sametime, independent main effects of both TBI (F1, 35=61.62,
p = 0.0255) and K. pneumoniae (F1, 35=5.45, p < 0.0001)

were observed for distance traveled in the open field,with TBI mice moving more but K. pneumoniae-treatedmice moving less (Fig. 2e). This hypoactive effect of K.
pneumoniae was transient, as it had resolved by the 24 h(Fig. 2f ) and 7 d (Fig. 2g) time points. However, a main
effect of TBI was observed across the time course (F1,59=6.38, p = 0.0143 at 24 h, and F1, 36=13.67, p = 0.0007 at
7 d).
Elevated serum inflammatory cytokines in response to K.
pneumoniae

For prototypical pro-inflammatory cytokine IL-1β(Fig. 3a), a significant TBI-x-Kp interaction was detected(F1, 16=5.95, p = 0.0267), whereby IL-1β was elevated in
the serum of TBI-Kp mice compared to TBI-vehicle(Tukey’s post-hoc, p = 0.0397). Independent of TBI, K.
pneumoniae induced a robust increase in cytokines IL-6
(Fig. 3b; F1, 15=10.01, p = 0.0064) and TNFα (Fig. 3c; F1,
15=15.86, p = 0.0011), as well as the chemokines CCL2
(Fig. 3e; F1, 16=7.15, p = 0.0167), CXCL1 (Fig. 3f; F1,
15=7.21, p = 0.0163) and CXCL2 (Fig. 3g; F1, 16=17.29,
p = 0.0010). By contrast, levels of anti-inflammatory cytokine IL-10 were not altered by K. pneumoniae or TBI(Fig. 3d).

Shad et al. Journal of Neuroinflammation(2024) 21:122

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Fig. 3 Elevated Serum Cytokines at 24 h Post-Infection. Cytokine levels in serum at 24 h post-infection (a–g), reveal a pro-inflammatory effect of K. pneumoniae, but no influence of a prior TBI on this response (2-way ANOVAs, main effects of Kp *p < 0.05, **p < 0.01). In (a), *p < 0.05 indicates Tukey’s post-hoc
comparison (TBI-vehicle vs. TBI-Kp) following a significant TBI-x-Kp interaction. Males denoted by closed circles/diamonds and females by open circles/
diamonds. n = 4–5/group

Lung inflammation and pathology after K. pneumoniae
infection

Pulmonary inflammation and lung pathology was evaluated in a subset of animals at 24 h and 7 d post-K. pneumoniae infection, to evaluate whether the combined
insult of TBI plus K. pneumoniae worsened pulmonary
outcomes. At 24 h post-infection, when the presence ofbacteria was confirmed in lung homogenates (Fig. 2b),
the analysis of inflammatory cytokines in BALF revealeda robust pro-inflammatory response in K. pneumoniaetreated mice compared to vehicle. Specifically, K. pneumoniae induced a robust increase in cytokines IL-1β(Fig. 4a; F1, 15=4.66, p = 0.0476), IL-6 (Fig. 4b; F1, 15=27.99,
p = 0.0001), TNFα (Fig. 4d; F1, 15=27.82, p < 0.0001) and
IL-10 (Fig. 4e), as well as the chemokines CCL2 (Fig. 4c;
F1, 15=13.43, p = 0.0023), CXCL2 (Fig. 4f; F1, 15=13.66,
p = 0.0022) and CXCL1 (Fig. 4h; F1, 16=12.53, p = 0.0027).

However, a TBI sustained 4 days prior to K. pneumoniae
infection did not alter BALF cytokine levels.
Significant correlations between serum and BALF levels of several cytokines were noted, for IL-1β (r2 = 0.50,
p = 0.0007), TNFα (r2 = 0.32, p = 0.0121), IL-6 (r2 = 0.43,
p = 0.0024), CXCL1 (r2 = 0.63, p < 0.0001), CXCL2(r2 = 0.25, p = 0.0256) and IL-10 (r2 = 0.38, p = 0.0047), anda trend observed for CCL2 (r2 = 0.20, p = 0.0559), suggesting that the local pulmonary response was the sourceof inflammatory mediators in the serum. Considerable
neutrophil infiltration was observed by MPO immunofluorescence staining of lung tissue at 24 h, particularly
associated with vascular thrombosis (Fig. 4g). Qualitatively this infiltration appeared comparable between
Sham-Kp and TBI-Kp groups, although quantification
was not readily possible due to the dense clustering ofthese cells at this time.

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Fig. 4 K. pneumoniae Infection Alters Lung Histology and BALF Cytokine Levels. Cytokine levels in bronchoalveolar fluid (BALF) at 24 h post-infection(a–f, h) reveal a predominantly pro-inflammatory effect of K. pneumoniae but no effect of prior TBI (2-way ANOVAs, main effect of Kp; *p < 0.05; **p < 0.01;****p < 0.0001). Immunostaining for MPO + neutrophils (colocalized with nuclear marker DAPI; g) in lung tissue revealed a robust neutrophilic infiltration
at 24 h post-infection, particularly associated with vascular thrombi (*). By 7 d post-infection, this immune response had resolved, with no differences in
MPO + cell numbers between the groups (i). Representative H&E staining illustrates lung histology at 7 d post-infection (j). Quantification of the mean
linear intercept (MLI) (k), where an increase reflects pathology, revealed an increase in lungs from TBI-Kp mice (2-way ANOVA, TBI-x-Kp interaction; Tukey’s
post-hoc *p < 0.05). Males denoted by closed circles/diamonds and females by open circles/diamonds. n = 4–6/group. Scale bar = 50 μm (g) and 100 μm(j)

By 7 d post-infection, fewer MPO + cells were detected
in lung tissue (Fig. 4i) and quantification revealed similar numbers across all groups (n.s.). Also at 7 d postinfection, calculation of the mean linear intercept (MLI)
to evaluate lung pathology from H&E-stained sections(Fig. 4j) revealed a significant TBI-x-Kp interaction (F1,20=5.56, p = 0.0287) and a trend towards a main effectof K. pneumoniae (F1, 20=3.71, p = 0.0685). From Tukey’s
post-hoc comparisons, K. pneumoniae increased the MLI
in TBI mice specifically (TBI-vehicle vs. TBI-Kp; p < 0.05),
indicating the presence of pathology (reduced structural
integrity of the alveolar septum/increased alveolar size).
The TBI-Kp group trended towards a higher MLI compared to Sham-Kp mice (p = 0.0593; Fig. 4k).
Immune-related gene expression changes in the injured
brain after k. pneumoniae infection

We next considered the potential effect of K. pneumoniae
lung infection on the brain’s immune response to TBI, by
examining the expression profiles of key genes involved
in regulating neuroinflammation–including cytokines,
chemokines, immune cell markers, and downstream signaling mediators. Broadly, at 24 h post- K. pneumoniae
infection (= 5 d post-TBI/sham), most genes exhibited
upregulation in response to TBI in the ipsilateral cortex(Fig. 5a) and ipsilateral hippocampus (Fig. 5h). In thecortex, TBI induced increased expression of cytokines
Il1β (Fig. 5b; F1, 19=6.91, p = 0.0165) and Tnfα (Fig. 5c;
F1, 19=23.03, p = 0.0001), chemokine Ccl2 (Fig. 5d; F1,
19=47.93, p < 0.0001), leukocyte marker Cd45 (Fig. 5e;
F1, 19=17.54, p = 0.0005), as well as immune mediators Arg1 (Fig. 5f; F1, 19=27.83, p < 0.0001) and Hmox1(Fig. 5g; F1, 19=26.20, p < 0.0001). Independently, K. pneumoniae induced an increase in Tnfα only (F1, 19=5.96,
p = 0.0246). However, a significant TBI-x-Kp interaction
was observed for Ccl2 (F1, 19=7.06, p = 0.0156) and Hmox1(F1, 19=9.99, p = 0.0051), as well as a non-significant trend
towards an interaction for Arg1 expression (F1, 19=3.73,
p = 0.0685). From Tukey’s post-hoc analyses, TBI-Kp mice
were found to have higher levels of Ccl2 expression compared to either TBI-vehicle or Sham-Kp mice (Fig. 5d;
p < 0.05). Arg1 expression was only elevated in TBI-Kp
compared to Sham-Kp mice (Fig. 5f ); while for Hmox1,
expression in TBI-Kp mice was higher compared to both
TBI-Vehicle and Sham-Kp mice (Fig. 5g). Altogether,
TBI induced a robust pro-inflammatory immune state in
the injured cortex, and post-injury K. pneumoniae lunginfection appeared to potentiate this response for selectgenes.
In the hippocampus (Fig. 5i-n), examination of thesame key genes revealed main effects of TBI (Il1β: F1,
17=4.91, p = 0.0406; Tnfα: F1, 17=5.86, p = 0.0270; Ccl2: F1,

Shad et al. Journal of Neuroinflammation(2024) 21:122

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Fig. 5 K. pneumoniae (Kp) infection had a minimal impact on immune-related gene expression in the injured brain. Quantatitive PCR evaluated therelative expression of multiple genes of interest, with group means depicted in heatmaps of the ipsilateral cortex (a) and ipsilateral hippocampus (h) at
24 h post-Kp/vehicle (= 5 d post-TBI/sham). Heatmap key illustrates that increasing color saturation reflects higher gene expression relative to the Shamvehicle control group (bolded gene names are those that are also depicted graphically; scale = fold change gene expression vs. Sham-Vehicle). Group
comparisions for select genes of interest are shown for the cortex (b–g) and hippocampus (i–n), with analysis by 2-way ANOVAs followed by Tukey’s posthoc comparisons when a significant TBI-x-Kp interaction was observed (i.e., for d, g and f). Males are indicated by closed circles/diamonds and femalesby open circles/diamonds. n = 4–6/group
17=15.63, p = 0.0010; Cd45: F1, 17=5.00, p = 0.0391; Arg1:

F1, 17=5.76, p = 0.0281; Hmox1: F1, 17=6.21, p = 0.0233).
However, no independent effects of K. pneumoniae wereobserved, nor were there any TBI-x-Kp interactions.
The chemokine Cxcl2 was minimally detected in thecortex, but below the threshold for detection in the hippocampus, while the anti-inflammatory cytokine Il-10
was below detection in both brain regions regardless ofexperimental group.
Cellular neuroinflammation after TBI was not affected by K.
pneumoniae infection

At a sub-acute time point of 7 d post-infection (= 11 d
post-injury), markers of inflammatory immune cells
were detected by immunofluorescence staining oncoronal sections to evaluate whether K. pneumoniae
altered their infiltration or activation (Fig. 6a). Quantification of MPO + cells, presumably neutrophils, revealed
a TBI-induced increase (Figs. 2 and 6a-way ANOVA F1,
20 = 6.04, p = 0.0232). This response was highly variable,
ranging from 0 to 310 in TBI brains. Quantification of
GFAP + astrogliosis was performed by determining the
proportion of GFAP + coverage of a consistent regionof-interest positioned in the core of the cortical lesion(Fig. 6c) or dorsal hippocampus (Fig. 6d). TBI induced
a robust increase in GFAP reactivity at this time in bothregions of ∼ 20% compared to ∼ 5% in sham-operated
mice (F1, 20 =45.63, p < 0.0001; F1, 20 = 37.22, p < 0.0001),
to a similar extent in both vehicle- and K. pneumoniaetreated mice.

Shad et al. Journal of Neuroinflammation(2024) 21:122

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Fig. 6 Immunofluorescence staining of cellular neuroinflammation after TBI and K. pneumoniae infection. Representative immunofluorescence images from the ipsilateral (injured) cortex of a TBI-Vehicle mouse at 11 days post-injury (7 d post-vehicle), illustrating detection of GFAP + astrocytes and
MPO + neutrophils (a). Scale bar = 100 μm. Quantification of the total number of MPO + cells in the injured hemisphere (b), as well as GFAP % area coverage in the lesioned cortex (c) and hippocampus (d), revealed a main effect of TBI but no effects of K. pneumoniae (2-way ANOVA, *p < 0.05, ****p < 0.0001)

Shad et al. Journal of Neuroinflammation(2024) 21:122

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The extent of brain tissue damage after TBI was notaffected by K. pneumoniae

consequences of K. pneumoniae infection in the context
of TBI. In this study, we sought to establish and characterize a new mouse model of experimental TBI combinedwith a pulmonary K. pneumoniae infection, and evaluate
the consequences of this combined insult on acute andsub-acute outcomes.
In pilot studies, we first sought to optimize the doseof K. pneumoniae required to yield a detectable bacterial burden in the lungs alongside relevant symptomology, while keeping mortality of experimental animals low.
Although intratracheal inoculation requires anesthesia,
an additional burden on the animals, we chose this delivery method as it is typically more reproducible than what
can be achieved via inhalation [31]. A dose of 1 × 104 or
1 × 105 CFU had minimal effect on the mice, with few
symptoms, negligible body weight loss, and no mortality. Conversely, a dose of 1 × 107 CFU led to considerable mortality (> 50%) and significant weight loss (≥ 15%
in survivors), at a time course consistent with what has
been reported previously [64]. An intermediate dose of
1 × 106 CFU was therefore chosen, as it led to moderate
body weight loss at 1–2 days post-inoculation, but nomortality. The presence of K. pneumoniae in the lungs
was confirmed at this time, but largely undetectable by
the 7 day time point. This was consistent with previous
reports that live K. pneumoniae rapidly expands in thelungs within the first 24 h before rapid clearance over the
first week [35–37], a process that is largely neutrophilmediated [65].
Next, we performed experimental TBI and infected
mice with 1 × 106 CFU K. pneumoniae at 4 days postinjury, attempting to recapitulate the time frame when
TBI patients are most vulnerable and exposed to hospital-acquired infections. K. pneumoniae induced significant transient weight loss to a similar extent in bothSham-Kp and TBI-Kp groups, with a comparable trajectory of recovery. Infected mice showed sickness
behaviors including a reduction in general activity at
4 h post-injury, but this was resolved by 24 h and 7 days
post-infection; while TBI independently induced a modest increase in activity across the time course, consistent
with previous findings in the CCI mouse model [41, 66].
Peripherally, spleen weights were unaffected by either
TBI or K. pneumoniae, in contrast to a previous study
that reported splenic thrombi and spleen enlargement
due to expansion of myeloid precursors in mice exposure
to lung K. pneumoniae infection [67].
However, serum cytokine levels measured at 24 h after
K. pneumoniae inoculation reflected activation of the
innate immune response, with an increase in pro-inflammatory cytokines IL-6 and TNFα, as well as prototypical chemokines CCL2, CXCL1 and CXCL2. IL-1β waselevated specifically in the TBI-Kp group, suggestive ofan additive effect of TBI and infection for this mediator.

At 7 days post-infection (= 11 days post-TBI), brains
were collected, sectioned, and stained with CV and LFB
to allow for histopathological assessment (Fig. 7a). Volumetric analysis of the remaining ipsilateral cortex tissue revealed a significant effect of TBI on cortex volume(2-way ANOVA F1, 20=97.85, p < 0.0001; Fig. 7b), as well
as loss of hippocampal tissue compared to sham controls(F1, 20=10.55, p = 0.004; Fig. 7c). However, K. pneumoniae
and vehicle-treated mice did not differ. Consideration ofsex as a variable by 3-way ANOVA revealed a main effectof sex on cortical volume only (F1, 16=6.70, p = 0.0198),
but no interactions, indicating that females had a slightly
larger ipsilateral : contralateral cortex volume overall.
Fecal bacterial diversity and composition were not altered
by TBI or lung infection

The fecal microbiome was examined, across a timecourse post-injury and K. pneumoniae infection, as
recent evidence supports a complex bi-directional relationship between the brain and the gut in the context
of neurotrauma as well as in infection-related immune
responses [38–40].
Alpha-diversity, as measured using the phylogenetic
tree-weighted Hill-Shannon metric, was comparable
between injury and treatment groups at all time points(Fig. 8B). Likewise, beta-diversity was similar between
groups, and PERMANOVA tests to assess the impact
of injury and K. pneumoniae on community structure
revealed no significant differences at any time point(Fig. 8C). Fecal samples at all time points showed highabundance of Bacteroides, Roseburia, and Turicibacter(Fig. 8D).
At both the ASV and genus level, non-parametric
repeated-measures Brunner-Langer tests did not detect
any effect of injury, K. pneumoniae, or time on bacterial
abundance. Subsequent linear modelling approaches atindividual time points to investigate potential group-wise
effects also showed no significant differences between
groups.

Discussion
The risk of contracting ventilator-associated pneumonia
is particularly high for hospitalized patients with severe
TBI [17]. As an opportunistic bacterium of the Enterobacteriaceae family, K. pneumoniae is a leading cause ofventilator-acquired pneumonia, urinary tract infections,and sepsis in immunocompromised and critically-ill individuals, including patients with brain injuries [19, 21,62]. Indeed, recent studies of TBI patients in Taiwan andSaudi Arabia reported K. pneumoniae to be among themost prevalent pathogen in pulmonary infections [20,63]. There is thus a clear need to better understand theShad et al. Journal of Neuroinflammation(2024) 21:122

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Fig. 7 K. pneumoniae Infection Did Not Alter Cortical and Hippocampal Neuropathology After TBI. Representative brain sections from each experimental group (a) illustrate the extent of tissue damage from CV/LFB staining. Quantification of remaining intact tissue in the cortex (b) and hippocampus(c), expressed as ipsilateral relative to contralateral regions, demonstrates the considerable loss of tissue as a result of TBI (2-way ANOVA, **p < 0.01;****p < 0.0001), but no effect of K. pneumoniae nor a TBI-x-Kp interaction. Males are indicated by closed circles and females by open circles. Scale bar= 1000 μm

Shad et al. Journal of Neuroinflammation(2024) 21:122

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Fig. 8 The Fecal Microbiome was Not Altered by TBI or Lung Infection. (a) Time line of sample collection. (b) Violin plots representing phylogenetic treeweighted Hill-Shannon alpha-diversity. (c) Principle coordinate analysis (PCoA) plots of the weighted UniFrac distances shown in the first two principal
coordinates for bacterial taxonomic composition. Ellipses represent the 90% confidence interval around the group centroid. Results for PERMANOVA
testing are shown for injury and treatment differences (SS [sum of squares] shows effect size and R2 shows variance explained). (d) Bacterial genus-level
relative abundance data grouped by injury + treatment group and timepoint

Of note, TBI alone did not appear to increase most of themeasured cytokines, although this was likely due to thetime point chosen (past the peak of TBI-induced cytokine release). One limitation here is that the bacterialload in blood was not quantified, although the transient
time course of symptoms in infected animals suggeststhat septicemia was unlikely to be occurring.
In the infected lungs, analysis of BALF revealed arobust pro-inflammatory response associated with K.
pneumoniae infection, of a much higher magnitude thanwhat was observed in the serum. This finding is consistent with evidence that K. pneumoniae induces extensive
inflammation of the lungs [65, 68, 69], and our immunostaining to detect marked neutrophilic pulmonary
infiltration acutely (although quantification of this at anacute time point was not possible in this study due to atechnical issue). With an increasing awareness of brainlung interactions and the potential for an altered immune
response as a consequence of brain injury [as reviewed
elsewhere; e.g., 70], we had hypothesized that TBI alone
may influence lung immune responses. For example,
in models of lung infections with Pseudomonas aeruginosa, some studies have observed reduced bacterial
clearance in animals that had previously sustained a TBI[32], while others have seen a reduced bacterial burden(presumably increased bacterial clearance) in TBI micepost-infection [71–73]. However, this was not the case in
our model. No additive effects were associated with theprior TBI in TBI-Kp mice, nor was there any differencein the bacterial load in the lungs of Sham-Kp versus TBIKp mice. Our findings align with those of Vermeij andcolleagues, who evaluated TBI in adult rats followed byintratracheal administration of heat-killed Staphylococcus aureus and reported that the severity of pneumonia
was not different in TBI compared to sham rats, despite
evidence of TBI alone resulting in significant pulmonary
edema and systemic immunosuppression [34]. Taken
together, evidence suggests that the bacterial load, species, strain and its virulence are all likely to be important
determinants of the effects of experimental infection on
the lungs’ immune response, in addition to other insults(e.g., a prior TBI) and its relative timing and severity.
By 7 days, when published evidence [64] and our pilot
data indicated that most of the bacteria had been cleared(e.g., few if any bacteria detectable, and MPO + cells atbaseline levels), a modest level of lung pathology wasevident from the analysis of H&E staining in the TBI-Kp
group specifically, compared to either TBI or K. pneumoniae alone. One caveat is that we did not measure
the bacterial load at the 7 d time point, so cannot conclusively state that the time course to resolution of K.
pneuomiae infection was comparable in Sham-Kp versus
TBI-Kp mice. However, together, these findings indicatethat the moderate dose of K. pneumoniae used in thisstudy induced a transient pulmonary infection that iswell-countered by the inflammatory immune response.
By contrast, severe K. pneumoniae associated with ahigher bacterial load or more virulent strain can induceShad et al. Journal of Neuroinflammation(2024) 21:122

severe bacterial pneumonia, resulting in hemorrhage andthe formation of necrotic lesions, and progression to bacteremia and sepsis [69]. However, such pathology is typically associated with significant mortality, which would
be incompatible with an ethically-acceptable animalmodel – particularly when combined with the additional
insult of a TBI.
In the injured brain, we hypothesized that a post-injury
K. pneumoniae infection would exacerbate neuroinflammation and neuropathology. Focusing on the injured
cortex and hippocampus at 24 h after K. pneumoniae
infection, we found that many genes involved in regulating neuroinflammation—including cytokines, chemokines, immune cell markers, and downstream signaling
mediators—were upregulated in response to TBI. However, K. pneumoniae had only minimal effects on gene
expression. For example, in the injured cortex, K. pneumoniae independently increased Tnfα expression; and aninteraction between TBI and K. pneumoniae was noted
for Ccl2, Hmox1 and potentially Arg1, whereby the combination of TBI with K. pneumoniae resulted in higher
expression levels. These limited interactions between TBI
and K. pneumoniae warrant further exploration in futurestudies. For example, CCL2 is one of the key chemokines
induced by severe brain injury in both experimental
models and TBI patients [74]. It is also remarkably upregulated in response to K. pneumoniae infection, at leastin lung tissue [75], where it plays a distinct role in the
recruitment of CCR2 + monocytes necessary for bacterial
clearance [76]. Whether a bacterial infection superimposed upon a injured brain alters CCL2/CCR2-mediatedmonocyte activity remains unclear.
These TBI-induced gene expression changes were likely
associated with the robust cellular immune response
detected by IF staining at 7 d post-infection, including
infiltration of MPO + cells (presumably neutrophils) andreactivity of GFAP (presumably astrocytes) in the injured
cortex and hippocampus. The extent of this staining was
similar in both K. pneumoniae and vehicle-treated TBI
groups, suggesting that the detected effects of K. pneumoniae on Tnfα, Ccl2, Hmox1 and Arg1 gene expression
were insufficient to drive a differential cellular immune
response. Similarly, while pronounced tissue loss wasevident in the ipsilateral cortex and hippocampus of TBI
mice, there was no additive effect of K. pneumoniae on
neuropathology. Together, these findings are contrary to
our initial hypothesis, and indicate that the pathophysiological responses to a sequential brain injury and lunginfection are not necessarily interactive.
Finally, we investigated the fecal microbiome across thetime course—from pre-injury up to 7 days post-infection,
considering increasing evidence of complex brain-gut
connections as well as aberrant microbiome profiles asa consequence of TBI [38, 39]. K. pneumoniae-induced

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pneumonia, as well as several other respiratory diseases,
have also been reported to alter the gut microbiome, forexample, by reducing the relative abundance of Bifidobacterium and Clostridium [40]. However, somewhat
surprisingly, we did not detect a robust effect of either
TBI or K. pneumoniae on fecal bacterial diversity or composition. This lack of an effect perhaps suggests that themoderate dose of K. pneumoniae, and transient nature of
the subsequent lung infection, may have been insufficient
to induce significant consequences for the gut. It is also
worth noting that mice were group-housed as same-sex
littermates, with each cage containing a mix of experimental groups to control for potential litter effects. However, as mice are coprophagous, cross-contamination of
fecal material between mice from different experimental groups may have masked possible injury or infection
effects.
Altogether, this study highlights some of the challenges
associated with modeling complex conditions such asconcurrent bacterial infections in brain-injured individuals. It is particularly difficult to achieve an optimalbalance between a robust, reproducible infection which
induces sufficient pathology in an experimental setting,while taking into consideration ethical research principles of refinement and reduction [77]. The rapid clearance of K. pneumoniae bacteria by immunocompetentmice limits the time window for the assessment of potential interactions between infection and neurotrauma,
while also impacting the potential clinical relevance ofthe model. A future possibility would be to employ themodel in neutropenic or immunocompromised mice[e.g., 44], to more closely mimic the altered immune status of a severely-injured patient in the ICU. Alternatively,a higher bacterial load could be trialed incorporating theuse of antibiotics or other clinically-relevant treatment
paradigms [e.g., 64], to better model a patient’s complex
medical management.
Our experimental groups contained both male andfemale mice, and we typically failed to detect any sexuallydimorphic effects; although this may be influenced by alack of statistical power for some experiments when thegroups were sub-divided by sex. Sex differences in acuteneuroinflammation after experimental TBI have beenreported; for example, Doran and colleagues found anincrease in peripheral myeloid cells infiltrating the brain
in males, alongside increased resident microglial proliferation [78]. Sex differences exist in terms of baseline respiratory physiology, and males typically fairing worse thanfemales in models of respiratory disease [79]. The survival of mice after K. pneumoniae infection is reportedly
sex-dependent, mediated by gonadal hormones and/or
oxidative stress responses [67, 80, 81]. In our model, we
anecdotally observed that males exhibited exacerbatedsickness symptoms (e.g., scruffy fur, hunched posture,

Shad et al. Journal of Neuroinflammation(2024) 21:122

immobility) within hours after K. pneumoniae inoculation. However, this did not manifest as statisticallysignificant differences between the sexes in terms of percentage body weight loss or reduced locomotor activity.
Finally, further investigation of a more chronic timecourse may reveal novel insights into the potential
long-term consequences of TBI and infection, even if
the infection is transient and contained. For example,
patients who survive acute infections may be vulnerable
to long-term complications including neurocognitive
dysfunction [82, 83]. In a mouse model of intranasal K.
pneumoniae-induced pneumosepsis treated with ceftriaxone, even after the resolution of infection, surviving
mice showed deficits in exploratory locomotor behavior associated with persistent brain inflammatory gene
expression [64]. Thus, ongoing work in our laboratory is
now exploring the potential long-term consequences ofcombined TBI and K. pneumoniae infections on neuropathology and neurobehavioral outcomes.

Conclusion
Overall, this study found that a moderate-severe TBI
combined with a moderate inoculation of K. pneumoniae
into the lungs had largely independent effects on brain
and lung pathophysiology, respectively. The consequences of K. pneumoniae infection were predominantly
restricted to a robust, acute, and transient pro-inflammatory response in the lungs, alongside a lower magnitude
increase in serum cytokines. K. pneumoniae appeared toinfluence the expression of a subset of immune-related
genes in the injured cortex and hippocampus, but thesechanges did not translate into any effects on the extent
of neuropathology. While experimental paradigms ofcombined TBI and live infection models are rare, studies
incorporating post-TBI infection with Citrobacter rodentium, Pseudomonas aeruginosa, or Streptococcus pneumoniae have all reported that the dual insult worsens
neuroinflammation and mortality [32, 33, 38]. Of note,these studies all incorporated a moderate or severe TBI
model, whereas other studies have suggested that mild
TBI may conversely be protective against P. aeruginosapneumonia [71, 72]. A more complete understanding of
combined neurotrauma and infection insults is necessary
to ensure that model systems can generate data of clinicalrelevance [31]. Despite our findings in this study, mostclinical evidence indicates that pulmonary infections
after severe TBI represent a key determinant of prognosis, and both the prevention and treatment of infections are high priorities in hospital settings [17, 63, 84].
Further, the growing global prevalence of antimicrobial
resistance and hypervirulence in Enterobacteriaceae suchas Kp is a serious public health concern, associated withpoorer outcomes, higher mortality, and limited treatment

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options [85–87]. Further research in this space is therefore warranted.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03093-9.
Supplementary Material 1
Acknowledgements
The authors would like to thank the Monash Histology Platform, Monash
Micro Imaging Platform, Monash Health Translation Precinct (MHTP) Medical
Genomics Facility, Monash Animal Research Platform, and Crux Biolabs fortheir assistance with the project. They also thank A/Prof. Connie Wong and
Dr. Shu Wen Wen from the Centre of Inflammatory Disease at Monash Health,and A/Prof. Margaret Hibbs from the Department of Immunology at Monash’s
School of Translational Medicine, for valuable guidance regarding lunghistology and staining.
Author contributions
Project conceptualization: BDS, TOB, JL. Data access and analysis: BDS, AS,
SSR, MM, NG, JW, KC. Manuscript draft: BDS, AS, SSR, MM. Manuscript edits: All
authors. Manuscript revisions: BDS, AS, TOB. We confirm that this manuscript
is consistent with the Journal’s and publisher’s position on issues involved in
ethical publication.
Funding
This project was supported by an Epilepsy Research Program Idea
Development Award (#W81XWH-19-ERP-IDA) from the US Department of
Defense, awarded to BDS, JL and TOB. BDS was also supported by a Veski
Near-Miss Grant and the Central Clinical School at Monash University. TOB is
supported by an NHMRC Investigator Grant (APP1176426). JL is supported by
an NHMRC Principal Research Fellowship (APP1157909).
Data availability
The datasets supporting the conclusions of this article are available
in the Monash Bridges repository (www.bridges.monash.edu), doi:
10.26180/25058048. (Note: accessible upon publication).

Declarations
Ethics approval
All animal experiments were conducted following approval from the local
Alfred Research Alliance Animal Ethics Committee (#P8032) as well as theAnimal Care and Use Review Office (ACURO) from the Office of ResearchProtections, US Department of Defense, and carried out in accordance withthese approved standards as well as the Australian Code for the Care andUse of Laboratory Animals as stipulated by the National Health and Medical
Research Council of Australia (NHMRC).
Conflict of interest
The authors do not have any conflicts of interest to declare. The funders had
no role in the design of the study; in the collection, analyses, or interpretationof data; in the writing of the manuscript; or in the decision to publish theresults.
Competing interests
The authors declare no competing interests.
Received: 25 January 2024 / Accepted: 5 April 2024

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