Modelling lung infection with Klebsiella pneumoniae after murine traumatic brain injury

Abstract

Pneumonia is a common comorbidity in patients with severe traumatic brain injury (TBI), and is associated with increased 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 dose of 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 to baseline 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 compared to 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 longer-term consequences of this dual-hit insult.

Keywords: Bacteria; Controlled cortical impact; Hospital-acquired infection; Inflammation; Pneumonia.

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

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

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

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)

Fig. 5

K. pneumoniae (Kp) infection had a minimal impact on immune-related gene expression in the injured brain. Quantatitive PCR evaluated the relative 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 Sham-vehicle 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 post-hoc 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 females by open circles/diamonds. n = 4–6/group

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)

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

Fig. 8

The Fecal Microbiome was Not Altered by TBI or Lung Infection. (a) Time line of sample collection. (b) Violin plots representing phylogenetic tree-weighted 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