Advanced age is associated with worsened outcomes and a unique genomic response in severely injured patients with hemorrhagic shock

Erin L Vanzant, Rachael E Hilton, Cecilia M Lopez, Jianyi Zhang, Ricardo F Ungaro, Lori F Gentile, Benjamin E Szpila, Ronald V Maier, Joseph Cuschieri, Azra Bihorac, Christiaan Leeuwenburgh, Frederick A Moore, Henry V Baker, Lyle L Moldawer, Scott C Brakenridge, Philip A Efron, Inflammation and Host Response to Injury Investigators, Erin L Vanzant, Rachael E Hilton, Cecilia M Lopez, Jianyi Zhang, Ricardo F Ungaro, Lori F Gentile, Benjamin E Szpila, Ronald V Maier, Joseph Cuschieri, Azra Bihorac, Christiaan Leeuwenburgh, Frederick A Moore, Henry V Baker, Lyle L Moldawer, Scott C Brakenridge, Philip A Efron, Inflammation and Host Response to Injury Investigators

Abstract

Introduction: We wished to characterize the relationship of advanced age to clinical outcomes and to transcriptomic responses after severe blunt traumatic injury with hemorrhagic shock.

Methods: We performed epidemiological, cytokine, and transcriptomic analyses on a prospective, multi-center cohort of 1,928 severely injured patients.

Results: We found that there was no difference in injury severity between the aged (age ≥55, n = 533) and young (age <55, n = 1395) cohorts. However, aged patients had more comorbidities. Advanced age was associated with more severe organ failure, infectious complications, ventilator days, and intensive care unit length of stay, as well as, an increased likelihood of being discharged to skilled nursing or long-term care facilities. Additionally, advanced age was an independent predictor of a complicated recovery and 28-day mortality. Acutely after trauma, blood neutrophil genome-wide expression analysis revealed an attenuated transcriptomic response as compared to the young; this attenuated response was supported by the patients' plasma cytokine and chemokine concentrations. Later, these patients demonstrated gene expression changes consistent with simultaneous, persistent pro-inflammatory and immunosuppressive states.

Conclusions: We concluded that advanced age is one of the strongest non-injury related risk factors for poor outcomes after severe trauma with hemorrhagic shock and is associated with an altered and unique peripheral leukocyte genomic response. As the general population's age increases, it will be important to individualize prediction models and therapeutic targets to this high risk cohort.

Figures

Figure 1
Figure 1
Heat map and calculated difference from distance from reference (DFR) for polymorphonuclear neutrophil (PMN) genome-wide expression. Using a false discovery adjusted probability of <0.001 and a two-fold difference in expression, the temporal pattern of the expression of the trauma responsive genes that differed between the matched aged (≥55 years) and young (<55 years old) trauma cohorts with complicated outcomes, as well as healthy controls, is presented. (A) Cluster analysis of the cohorts 0.5 days after injury showed that there were 3,121 probe sets (2,095 genes) with expression that was significant expressed among groups (F-test, P <0.001). In addition, the overall pattern of gene expression was significantly different in each cohort, as determined by leave one out cross-validation. (B) Summary of the DFR score calculated for each patient in the complicated aged and young cohorts at days 0.5, one and four days after injury. Analysis revealed significant differences in the DFRs at all the post trauma time points between the two cohorts when compared to controls. In addition, the advanced age cohort had significantly more aberrant gene expression as compared to the young patients on day 4 (Newman-Keuls multiple comparison test, *P <0.05).
Figure 2
Figure 2
Calculated difference from reference (DFR) for 51 of the 63 known genes that distinguish clinical trajectory. Using a false discovery adjusted probability <0.001 and a twofold difference in expression, the temporal pattern of expression of the 51 genes that differed between the matched aged (≥55 years old) and young (<55 years old) trauma patients with complicated outcomes, as well as healthy controls, was analyzed and used to calculate a DFR score. The summary of the DFR scores for the patients in each cohort at days 0.5, 1.0 and 4.0 after traumatic injury is presented. Statistical analysis at 0.5, one and four days revealed significant differences in the DFRs between the young and aged. On days 0.5 and 1.0, the expression patterns in the young complicated trauma patients were significantly more aberrant from control to those seen in the advanced age cohort. By day 4, the expression patterns in the aged were found to be significantly more aberrant from controls than those seen in the young (Newman-Keuls multiple comparison test, *P <0.05).
Figure 3
Figure 3
Selected gene ontology pathway heat maps in complicated aged and young patients on days 0.5, 1.0 and 4.0 after severe traumatic injury. Dark blue represents upregulation, whereas light blue represents down regulation. In complicated young patients, gene ontology pathway analysis demonstrated that several pathways involved in innate immunity and neutrophil function (that is, antigen processing and presentation and neutrophil chemotaxis pathways) were significantly more aberrant from controls in the acute periods (days 0.5 and 1) than was seen in the aged. In the sub-acute period (day 4) after injury, these patterns switched. The young trended back toward baseline expression values while the aged continued to demonstrate significantly more aberrant expression patterns in pathways involved in innate immunity and neutrophil function (that is, neutrophil activation pathway) (Holm-Sidak, *P <0.05).
Figure 4
Figure 4
Selected pathways from functional pathway analysis between complicated aged and young patients after severe traumatic injury. Functional pathway analysis on day 1 after injury showed that complicated aged patients had significantly downregulated pathways involved in cell survival, function, chemotaxis, immune cell trafficking and hematological system development categories. Graphs display the category broken down into the various subcategories and their corresponding significance level (*Z-score <-2; downregulated). (A) Functional analysis on day 1 after severe traumatic injury showed that aged patients had either an upregulation or down regulation of genes leading to a significant overall downregulation of pathways in the cell death and survival category (that is, cell survival, cell viability, apoptosis of myeloid cell pathways) compared to controls. Young complicated traumatic injury patients did not reach a similar significance. (B) Similarly, functional analysis on day one showed that aged patients had either an upregulation or downregulation of genes leading to a significant overall downregulation of pathways involved in the cellular function and maintenance category (that is, autophagy of cells, leukocyte and blood cell function and cellular homeostasis pathways) as compared to controls. Again, complicated young trauma patients did not reach a similar significance.
Figure 5
Figure 5
Plasma cytokine and chemokine levels in complicated aged and young patients at 12, 24, 96, 185, 336, 504 and 672 hours after severe injury and hemorrhage. The elderly had significantly less cytokine and chemokine concentrations in their plasma after severe blunt trauma. General linear model analysis was performed to examine the significance in relationship of age and time after injury, to the differences seen in the concentrations cytokines and chemokines between the cohorts. Analysis of the plasma demonstrated that both age and time had significant effects on the differences observed for the levels of IL-6, IL-8, IL-10 and MCP-1 (*model P <0.05). Model analysis of IL-1β and TNF-α found that only age had a significant effect on the differences observed (҂P <0.05). Neither age, nor time after traumatic injury, were found to have significant effect on the levels of IP-10 (data not shown).
Figure 6
Figure 6
Our depiction of the summary of the differences in immune response to severe traumatic injury between the young (<55 years old) and the aged (≥55 years old) who experience complicated clinical outcomes. In the acute period (days 0.5 and 1) after trauma, the aged demonstrate a diminished immune response consistent with immuno-senescence as compared to their younger counterparts. This is followed by continued dysregulation in the advanced age patients as the young trend toward controls by day 4 after injury. In the acute and sub-acute periods after injury, the complicated young and aged trauma patients demonstrate unique genomic expression patterns that are temporal in nature, illustrating that their biologic response to severe injury is different, although they had similar outcomes.

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