Systemic immunological hypo-responsiveness following polytrauma involving traumatic brain injury and hemorrhagic shock.

Background: The composition and function of the circulating immune pool following traumatic brain injuries (TBI) in both human and animal-based studies are well documented. Mechanisms regulating trauma-related systemic inflammatory responses have been shown to be complex and dynamic.
Moreover, a state of hypo-responsiveness or immunosuppression of the innate and adaptive arms has been reported following TBI, which may render patients susceptible to infections and complications with increased lethality.
Methods: In this study, we investigated the gene expression profile of 45 markers of inflammatory activation and immune responses in circulating peripheral blood leukocytes after in vitro lipopolysaccharide (LPS) endotoxin challenge (Escherichia coli) and following TBI in a gyrencephalic model of TBI polytrauma involving hemorrhagic shock (HS). To validate our custom low-density microarray, we incubated adult ferret whole blood with LPS or vehicle for 4-hours and then assessed mRNA transcripts using RT-qPCR. In our gyrencephalic model of TBI, anesthetized adult male ferrets underwent controlled cortical impact (CCI) and 15% controlled hemorrhage (TBI + HS) or craniotomy (instrumented sham) injuries. Blood was collected at 6 hours post injury (hpi) for hematology labs, clinical chemistries, and mRNA transcript levels using RT-qPCR. For both studies, gene expression Ct values were normalized to a reference gene (HPRT) and a pre-injury blood sample collected immediately prior to craniotomy using the 2-ΔΔCt method.

Results: LPS treatment (1 ug/ml) for 4 hours increased the expression of a number of cytokine and chemokine genes from circulating peripheral blood leukocytes obtained from healthy normal adult male ferrets (n=6). In particular, a total of 23 ferret-specific gene transcripts were elevated relative to controls. Of those, 12 target genes (IL-10, IL-17, TNFɑ, IFNɑ, IFNɣ, TGFβ2, HSP90AB1, TLR2, NFκβ1, FOXp3, MIF, and CSF1) were upregulated 2-10-fold, 6 target genes (IL-1β, IL-8, IFNβ, CCL5, CXCL11, and PTGS2) were upregulated 10-100-fold, and 4 target genes (IL-1ɑ, IL-6, IL-12p40, and CSF3) were upregulated >100-fold, whereas the expression of housekeeping genes (HPRT, GAPDH, and L32) was comparable. Gene expression profiles at 6 hpi differed between instrumented sham controls (n=3) and TBI+HS injured (n =4), with global decreased expression of most genes related to immune activation/function in the TBI+HS injured cohort. This measured hypo-inflammatory phase is indicated

by decreased levels of pro-inflammatory (IL-1α, IL-1β, IL-6, IL-8, TNFα, MCP-1, CCL5, CXCL11, MIF) and anti-inflammatory cytokines/chemokines (IL-10, TGFβ1, TGFβ2), early transcriptional activators (NFκβ1, STAT6, Foxp3, T-bet), heat-shock proteins (HSPa4, HSP90AB1,HSPB1), prostaglandins (PTGS2), apoptosis regulators (caspases 1,3,8), TLR expression (TLR2, TLR3), co-stimulation molecules (CD80, CD86), serine proteases (granzymes) and those mediators that play a role in innate immune cell function including cell activation, proliferation, adhesion, migration, and phagocytosis (CSF1, CSF2, CSF3, ITGAM, CCR3).

Conclusion: As expected, we demonstrated that LPS stimulation of ferret peripheral blood leukocytes triggered a rapid increase and dynamic shift in gene signature transcription patterns of known inflammatory and innate immune cell signaling pathway genes. To our knowledge, this is the first transcriptome custom-low density microarray developed for assessing leukocyte immunological activation and innate immune responses in the ferret model. We used this custom ferret-specific array to elucidate systemic inflammatory responses and other trauma-induced early cell signaling pathways in our TBI+HS model compared to instrumented sham controls. Initial findings from this study have added to our understanding of acute peripheral changes following CCI brain injury and HS, within 6 hpi, indicative of systemic immunological hypo- responsiveness. Furthermore, additional critical investigations are in progress and warranted to delineate if TBI induces a transient state of cellular hypo- responsiveness to subsequent stimulation with LPS. Findings from this study could potentially help identify systemic biomarkers and chemical mediators of injury that correlate with the individual response to combat-relevant trauma and en route care.