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. 2016 Apr 18;13(1):80.
doi: 10.1186/s12974-016-0547-1.

Age exacerbates the CCR2/5-mediated neuroinflammatory response to traumatic brain injury

Josh M Morganti  1   2 , Lara-Kirstie Riparip  1 , Austin Chou  1   3 , Sharon Liu  4 , Nalin Gupta  4   5 , Susanna Rosi  6   7   8   9
Affiliations

Age exacerbates the CCR2/5-mediated neuroinflammatory response to traumatic brain injury

Josh M Morganti et al. J Neuroinflammation. .

Abstract

Background: Traumatic brain injury (TBI) is a major risk factor for the development of multiple neurodegenerative diseases, including Alzheimer's disease (AD) and numerous recent reports document the development of dementia after TBI. Age is a significant factor in both the risk of and the incidence of acquired brain injury. TBI-induced inflammatory response is associated with activation of brain resident microglia and accumulation of infiltrating monocytes, which plays a pivotal role in chronic neurodegeneration and loss of neurological function after TBI. Despite the extensive clinical evidence implicating neuroinflammation with the TBI-related sequelae, the specific role of these different myeloid cells and the influence of age on TBI-initiated innate immune response remain unknown and poorly studied.

Methods: We used gene profiling and pathway analysis to define the effect of age on inflammatory response at the time of injury. The recruitment of peripheral CCR2(+) macrophages was delineated using the CX3CR1 (GFP/+) CCR2 (RFP/+) reporter mouse. These responses were examined in the context of CCR2/5 antagonism using cenicriviroc.

Results: Unsupervised gene clustering and pathway analysis revealed that age predisposes exacerbated inflammatory response related to the recruitment and activation of peripheral monocytes to the injured brain. Using a unique reporter animal model able to discriminate resident versus peripherally derived myeloid cells, we demonstrate that in the aged brain, there is an increased accumulation of peripherally derived CCR2(+) macrophages after TBI compared to young animals. Exaggerated recruitment of this population of cells was associated with an augmented inflammatory response in the aged TBI animals. Targeting this cellular response with cenicriviroc, a dual CCR2/5 antagonist, significantly ameliorated injury-induced sequelae in the aged TBI animals.

Conclusions: Importantly, these findings demonstrate that peripheral monocytes play a non-redundant and contributing role to the etiology of trauma-induced inflammatory sequelae in the aged brain.

Keywords: Aging; Antagonist; CCR2; Chemokine; Macrophage; Microglia; Neurotrauma.

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Figures

Fig. 1
Fig. 1
Inflammatory profiling of the TBI brain. Ipsilateral hippocampi pooled from sham and injured animals (n = 8/group) of 3-month (young; Y) and 23-month (aged; A) 24 h after surgery for gene array analysis. Inflammatory profiling array revealed clusters of enrichment and downregulation of genes across three groups; young TBI, aged sham, and aged TBI, relative to young sham expression levels, all data were Log2 transformed. Three specific clusters were examined, wherein the genes within the green cluster were downregulated in the aged TBI group, the red gene cluster showed marked enrichment for aged TBI, and lastly the aquamarine cluster showed similar enrichment of genes as a response to TBI, regardless of age. In heatmap; teal downregulated, red upregulated
Fig. 2
Fig. 2
Age exacerbates inflammatory gene signatures after TBI. Overall, the aged TBI group had exacerbated responses, whether up- or downregulated, to TBI when compared to their young TBI counterparts. Select genes from each of the three clusters were validated using n = 8/group. a Genes from the “green” hierarchical cluster aligned with pooled responses observed in the array. Specifically, detailing that age significantly affected, via downregulation, gene expression of CD163, CX3CL1, and JAK2, but not JMJD3, which had only a visual trend. b Genes selected from the “red” cluster for analysis showed marked upregulation of CD86, CCL5, CCL8, and CCL7 in the aged TBI group. c Similarly, genes from the “aquamarine” hierarchical cluster all displayed significant upregulation in expression response as a function of age, compared to young TBI. YT young TBI; magenta, AS aged sham; aquamarine, AT aged TBI; orange. Young sham expression values are equivalent to zero. Data were analyzed using two-way ANOVA with Tukey’s PSD for multiple comparisons and presented as mean + SEM. *p < 0.05, **p < 0.01 comparing young TBI to young sham. # p < 0.05, ## p < 0.01 comparing aged TBI to young TBI
Fig. 3
Fig. 3
IPA upstream analysis displays heterogeneous regulatory components associated with inflammatory response. Gene array data were loaded into IPA software; genes with a fold change (relative to young sham) ≥1.5 or ≤−1.5 were included for upstream regulator analysis. a Upregulated and downregulated molecules were sorted via their respective activation z-score and the top ten regulators (up and down) are presented for each condition. b, c Representative regulatory networks for putative upstream mediators associated with young TBI and aged TBI, with CCL2 representing an upregulated response and GPX1 representing a downregulated response. Both CCL2 and GPX1 show dissimilar expression responses for young TBI versus aged TBI
Fig. 4
Fig. 4
Gene profiling predicts exaggerated activation and recruitment of monocytes in aged TBI. Gene array data were uploaded into IPA software, genes with a fold change (relative to young sham) ≥1.5 or ≤−1.5 were included for disease and biological function analysis. a Upregulated functions across all three groups were sorted by activation z-score of the aged TBI group, only the top ten functions are presented with the black arrow emphasizing the activation of monocytes. b Functional network diagram of predicted and measured regulators is presented for both young TBI and aged TBI, which highlight an overrepresented activation for this function for aged TBI group. c Using Dbl-Het reporter mice (n = 6/group), macrophages (CD11b+F4/80+) were delineated based upon their relative expression of GFP (CX3CR1) from RFP (CCR2) by flow cytometry. There were relatively very few CCR2+ macrophages (blue box) in the sham animals; however, there was a significant increase in this subpopulation due to age. However, 24 h following TBI, there was a significant increase in the mean number of CCR2+ macrophages (blue box) in the aged animals compared to young. Data were analyzed using Student’s t test and are represented by the mean + SEM. **p < 0.01
Fig. 5
Fig. 5
Dual targeting of CCR2/5 with CVC mitigates TBI-induced macrophage recruitment. a In WT mice, CVC or vehicle was administered BID via oral gavage at 100 mg/kg at 2 and 10 h post surgery before animals were euthanized for various endpoints at 24 h following surgery. b A cohort of WT aged TBI animals (n = 8/group) was used for flow cytometry analysis of macrophage infiltration into the injured brain. Twenty-four hours after injury, there was a significant decrease in the number of peripheral macrophages (CD11b+F4/80+CD45hiLy6C+) in the CVC-treated animals compared to their vehicle-treated counterparts
Fig. 6
Fig. 6
Treatment with CVC attenuates age-related inflammatory and oxidative stress responses. a-c In a separate cohort (n = 8/group), inflammatory clusters were examined for their response to CVC treatment. a–c Correspond to the previously examined green, red, and aquamarine expression clusters, respectively. d Subunits of the NOX2 complex were measured as a response to CVC treatment (n = 8/group). Gene expression data are relative to vehicle-treated aged TBI. Vehicle expression values are equivalent to zero. Data were analyzed using Student’s t test are represented by mean + SEM. *p < 0.05, **p < 0.01, and ***p < 0.001
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