A Detailed Characterization of the Dysfunctional Immunity and Abnormal Myelopoiesis Induced by Severe Shock and Trauma in the Aged

Abstract

The elderly are particularly susceptible to trauma, and their outcomes are frequently dismal. Such patients often have complicated clinical courses and ultimately die of infection and sepsis. Recent research has revealed that although elderly subjects have increased baseline inflammation as compared with their younger counterparts, the elderly do not respond to severe infection or injury with an exaggerated inflammatory response. Initial retrospective analysis of clinical data from the Glue Grant trauma database demonstrated that despite a similar frequency, elderly trauma patients have worse outcomes to pneumonia than younger subjects do. Subsequent analysis with a murine trauma model also demonstrated that elderly mice had increased mortality after posttrauma Pseudomonas pneumonia. Blood, bone marrow, and bronchoalveolar lavage sample analyses from juvenile and 20-24-mo-old mice showed that increased mortality to trauma combined with secondary infection in the aged are not due to an exaggerated inflammatory response. Rather, they are due to a failure of bone marrow progenitors, blood neutrophils, and bronchoalveolar lavage cells to initiate and complete an emergency myelopoietic response, engendering myeloid cells that fail to clear secondary infection. In addition, elderly people appeared unable to resolve their inflammatory response to severe injury effectively.

Figure 1. Murine survival rates after trauma or trauma and Pseudomonas pneumonia

Aged mice have a significantly lower survival rate when exposed to Pseudomonas pneumonia (Pp) one day after trauma (PT). Young (6–10 weeks old) and aged (20–24 months old) C57Bl/6 mice underwent trauma and/ or exposed to Pseudomonas aeruginosa (PAK, 10⁷ CFU) and survival was monitored. (PT young□;PT aged○; Pp young△; Pp aged +; PT+Pp young◇; PT+Pp aged●). This figure is the combination of five separate experiments; n=9–10; *p<0.05, Log-rank (Mantel-Cox) test.

Figure 2. Plasma cytokine concentrations after PT

There was no significant statistical difference in the plasma cytokine concentrations between young and aged mice one day after PT. Plasma from young and aged mice were collected one day after PT and cytokine/chemokine production was evaluated by Luminex (n=3).

Figure 3. Lung histology and bacterial clearance in young and aged mice after trauma

(A) Histologic evaluation was performed on H&E sections from lung tissue to assess the degree of acute lung injury. (B) Histologic score ranged from 0–3 with 0=no inflammation, 1=mild, 2=moderate, and 3=severe. Representative sections are shown (n=3–5/group). (C) Bronchoalveolar lavage (BAL) fluid was collected and bacterial colony forming units (CFUs) were determined by plating on sheep blood agar. The experiment was performed at least twice (n=6); ** p<0.01, Mann Whitney t-test. (D) Examples of bacterial CFUs on sheep blood agar plates from BAL samples one day after trauma.

Figure 4. Total BAL leukocytes and functional capacity and in young and aged mice after trauma

(A) Young and aged mice underwent trauma (PT) or trauma and Pseudomonas pneumonia (PT+Pp) and sacrificed one day later. BAL fluid was collected and cells were counted using a hematocytometer. Average of two experiments is shown (n=6); * p<0.05, Mann Whitney t-test. (B) BAL cells from young (solid bars) and aged (empty bars) were incubated with FITC latex beads and stained for PMNs (Ly6G⁺CD11b⁺). FITC⁺ cells were considered phagocytic. This figure contains at least three separate experiments (n=6–10/group); *p<0.05, **p<0.01, unpaired t-test.

Figure 5. Microarray analysis of bronchoalveolar lavage (BAL) cells

The genomic response of BAL leukocytes of young and aged mice that were sacrificed one day after trauma. (A) Heat maps of the hierarchical clustering of gene expression patterns and variation between naïve and aged and young trauma (PT) mouse BAL leukocytes. (B) Conditional principal component analysis of naïve and aged and young trauma mouse BAL leukocytes gene expression patterns. (C) Heat maps of the hierarchical clustering of gene expression patterns and variation between aged and young PT mouse BAL leukocytes. (D) Heat maps show the fold change (from naïve) gene expression of the functional category “phagocytosis pathways” (IPA^®) in young and aged mice one day after trauma (fold change expression versus naïve, p<0.001; orange = upregulation, blue = downregulation, white = neither significantly up nor down regulated).

Figure 6. Microarray analysis of circulating leukocytes after trauma in young and aged mice

(A) Heat map of genomic response of aged and young mice after trauma (PT) and compared to naïve control. Most of the genomic changes are upregulated (red), as compared to control in both aged and young mice 2 hours after trauma. After one day, the expression pattern of young mice is more similar to control as compared to aged mice. Three days after the trauma, the genomic differences between the aged and young mice become similar to each other, most of which represents down regulation (blue) of specific gene expression patterns as compared to control. (B) Distance from reference (DFR) calculations confirm that young, but not aged, mice are genomically more similar to control/naïve mice one day after trauma.

Figure 7. Murine hematopoietic cell numbers, function, and transcriptomic expression from young and aged mice one day after trauma

(A) One day after trauma, BM from young and aged mice were analyzed for LSK (lin⁻sca-1⁺ckit⁺) and ST-HSCs (CD150⁻CD135⁺LSK); ***p<0.001 by two-way ANOVA. (B) BM LSKs from young and aged mice were sorted and cultured in methylcellulose media with indicated cytokines. Colonies were counted 10–14 days later; *p<0.05 by paired t-test. (C) Most of the genes that have significant fold expression changes are dissimilar between aged and young HSCs (p<0.0001). (D) Supervised analysis reveals that 593 probe sets (426 genes; p<0.001) can differentiate between aged and young HSCs with 100% mean % correct classification (leave-one-out validation). (E) IPA^® of the Hematopoiesis Pathway reveals down regulation of the cell-mediated immune responses pathways in young mouse HSC as compared to murine HSCs after trauma.

Figure 8. Summary of differences in young and aged emergency myelopoietic responses after severe hemorrhagic shock and injury

(A) Bone marrow cells from young animals have overall increased numbers of ST-HSCs as well as an increased functional capacity of their HSCs as compared to the elderly. After injury, young mice can rapidly upregulate the expression of genes involved in innate immunity. Their HSCs, especially ST-HSCs, are capable of acutely diverting all their genomic resources to rapid myelopoiesis and creating well functional myeloid cells (specifically PMNs). Subsequently, the immune system in the young is more able to return closer to homeostasis than their elderly counterparts, allowing them to appropriately handle secondary infections. (B) ‘Inflammaging’ in the aged engenders genomic and epigenomic HSC changes, as well as local and systemic alterations, inducing the following in HSCs: lower functional frequency, delayed proliferative response, reduced efficiency for BM homing, myeloid-skewed cell production and relatively less ST-HSCs. Granulocytes created through myelopoiesis in this ‘inflammaging’ environment are released into the circulation have inferior bacterial homing and killing functions. However, these granulocyte functions are adequate enough to overcome typical infections and are overcompensated by increased overall myelopoiesis. After severe trauma, aged mice are unable to acutely upregulate inflammation in a similar manner to young mice. In addition, aged HSCs are unable to undergo adequate acute emergency myelopoiesis due to multiple causes, including relatively decreased overall ST-HSC proliferation and numbers. Also, after injury and shock, aged HSCs engender more immature granulocytes with sub-optimal function - we hypothesize these include myeloid-derived suppressor cells. Regardless, these cells are able to overcome some previous dysfunction, such as creation of reactive oxygen species. Unlike young mice that can rapidly return to baseline transcriptomic expression levels in their HSCs and leukocytes after severe injury, aged murine HSCs now maintain a chronic low grade inflammation. This further worsens their HSC and granulocyte function. In addition, these immature granulocytes contribute to this ‘viscous cycle’ of continued low grade inflammation and further creation of dysfunctional myeloid cells. While young mice are able to again undergo appropriate emergency myelopoiesis and combat secondary infections with functional granulocytes, aged mice eventually succumb to sources of sepsis, such as bacterial pneumonia infections.