Protective immunity and defects in the neonatal and elderly immune response to sepsis

Abstract

Populations encompassing extremes of age, including neonates and elderly, have greater mortality from sepsis. We propose that the increased mortality observed in the neonatal and elderly populations after sepsis is due to fundamental differences in host-protective immunity and is manifested at the level of the leukocyte transcriptome. Neonatal (5-7 d), young adult (6-12 wk), or elderly (20-24 mo) mice underwent a cecal slurry model of intra-abdominal sepsis. Both neonatal and elderly mice exhibited significantly greater mortality to sepsis (p < 0.05). Neonates in particular exhibited significant attenuation of their inflammatory response (p < 0.05), as well as reductions in cell recruitment and reactive oxygen species production (both p < 0.05), all of which could be confirmed at the level of the leukocyte transcriptome. In contrast, elderly mice were also more susceptible to abdominal peritonitis, but this was associated with no significant differences in the magnitude of the inflammatory response, reduced bacterial killing (p < 0.05), reduced early myeloid cell activation (p < 0.05), and a persistent inflammatory response that failed to resolve. Interestingly, elderly mice expressed a persistent inflammatory and immunosuppressive response at the level of the leukocyte transcriptome, with failure to return to baseline by 3 d. This study reveals that neonatal and elderly mice have profoundly different responses to sepsis that are manifested at the level of their circulating leukocyte transcriptome, although the net result of increased mortality is similar. Considering these differences are fundamental aspects of the genomic response to sepsis, interventional therapies will require individualization based on the age of the population.

Conflict of interest statement

Conflict of Interest Disclosures None of the authors have disclosed any conflict of interest.

Figures

Figure 1. Neonatal and elderly mice have increased mortality compared to young adult mice with impaired control of bacteria at the site of infection and decreased activation of leukocytes

A. Neonatal and elderly mice had increased mortality after CS sepsis compared to young adult mice with the same insult (P<0.05) over 7 days. Neonate (n=22), Young Adult (n=20), Elderly (n=21). B. One day after sepsis both neonatal and elderly mice had significantly increased bacterial counts in the blood (* p<0.0001, One-way ANOVA), and in the peritoneum †p=0.019, One-way ANOVA); with elderly mice having the most severe impairments in clearing bacteria (Blood-p<0.001, Peritoneal wash-p<0.05; Tukey’s Multiple Comparisons post-hoc test) (n=3–4, results shown are from two or more independent experiments). C. Functional pathway analysis showed that elderly mice had down-regulation of genes involved in the the activation of luekocytes, whereas young adult mice had significant upregulation of these same genes (Supplemental Table 1). Neonatal mice niether up or down regulated a large enough number of genes involved in leukocyte activation at a significance level of p<0.001. Heatmaps show the of gene expression of the functional category “Immune Cell Trafficking-Activation” one day after sepsis in neonatal, young adult, and elderly mice. Orange represents pathways with an over-expression of genes leading to the activation of the pathway, whereas blue represents pathways with an over-expression of genes whose activation will lead to down-regulation of the pathway. White represents genes that are neither up or down regulated to significance level of p<0.001. Graph shows the various subcategories and their corresponding significance level (Z-score) of up or down regulation. From left to right, categories include “activation of leukocytes,” “activation of mononuclear leukocytes,” “activation of granulocytes,” and “activation of neutrophils.” (* Z-score >2). See Supplemental Table 2 for a complete list of genes in presented pathways.

Figure 2. Inflammatory and anti-inflammatory chemokine and cytokine responses

A. Neonatal, young adult, and elderly mice have significant differences in the production of inflammatory and anti-inflammatory cytokine and chemokine responses across all time points after CS sepsis. Significance determined using Two-way ANOVA with Bonferroni multiple comparisons post-hoc test. Symbols placed adjacent to graph titles indicate that age was a significant interaction accounting for a source of variation. Symbols placed within the graph show all significant differences between the age groups for specific cytokine. † = p<0.05. ** = P<0.01. *** = p<0.001. **** = p<0.0001. B. Neonatal cord blood had an attenuated cytokine response compared to adult blood following ex vivo stimulation with LPS across all cytokines studied. † = p<0.05. ** = P<0.01. *** = p<0.001. **** = p<0.0001.

Figure 3. Elderly mice have increased ability to recruit innate immune effector cells to the peritoneum, whereas neonatal mice have a significantly decreased ability to recruit those same cells

A. Elderly mice have increased absolute numbers of neutrophils (CD11b+, Ly6G+)(* p<0.0001, One-way ANOVA) and macrophages († P<0.05, One-way ANOVA)(CD11b+, Ly6G−, F480+) in the peritoneum via flow cytometry one day after sepsis, whereas neonates had significantly decreased numbers. (n=7–8)(p=0.001, Tukey’s Multiple Comparisons). Data shown are from two or more independent experiments. B. IPA functional pathway analysis revealed that elderly mice have significantly more upregulation of pathways involved in the recruitment of neutrophils, myeloid cells, and phagocytes, while neonates have minimal upregulation of these same pathways (* Z-score >2). Heat maps show the of gene expression of the functional category “Immune Cell Trafficking-Recruitment” 24 hours following sepsis. Orange represents pathways with an over-expression of genes leading to the activation of the pathway, whereas white represents genes that are neither up or down regulated to significance level of p<0.001.

Figure 4. Neontal neutrophils and macrophages have decreased ROS production compared to adult mice

A. Neonatal mouse neutrophils (CD11b+, Ly6G+) and macrophages (CD11b+, Ly6G−, F480+) one day after sepsis had decreased ability to produce reactive oxygen species (ROS) in response to PMA stimulation compared to adult mice one day after sepsis (p<0.0001, 2-way ANOVA). B. Functional pathway analysis showed that elderly mice had increased up regulation of genes involved in the synthesis, production, and metabolism of ROS compared to neonatal and/or young adult mice, which is consistent with in vivo results. Heatmaps show the of gene expression of the functional category “Free Radical Scavenging” 2 hours after sepsis in neonatal, young adult, and elderly mice. Orange represents pathways with an over-expression of genes leading to the activation of the pathway and white represents genes that are neither up or down regulated to significance level of p<0.001. Graph shows the various subcategories and their corresponding significance level (Z-score) of up or down regulation.

Figure 5. The neonatal transcriptome is fundamentally different at baseline compared to the murine adult

A. Heat map showing the gene expression patterns of naïve neonatal (N), young adult (A), and elderly (E) control mice. Gene expression patterns from young adult and elderly mice could not be differentiated genomically at baseline by an unsupervised analysis. There were 5,798 probe sets (representing 3987 genes) differentially expressed between neonatal and adult mice (including young adult and elderly) that were significant at p<0.001. The overall pattern of gene expression was significantly different as determined by leave-one-out cross-validation. B. Fold changes of selected immune related genes from neonatal control mice compared to adult control mice. Neonatal mice have increased suppression of genes involved in adaptive immunity including MHC II, and decreased expression of genes involving innate immunity and inflammation (p<0.001). C. Categories of functional pathways containing genes that are either up or down regulated in naïve neonatal control mice as compared to adult control mice from Ingenuity Pathway Analysis (IPA).

Figure 6. Neonatal and elderly mice do not upregulate important immune related pathways 24 hours following sepsis

Heatmaps show the of gene expression of the functional category “Hematological Systems Development and Function” 24 hours following sepsis. Orange represents pathways with an over-expression of genes leading to the activation of the pathway, whereas blue represents pathways with an over-expression of genes whose activation will lead to down-regulation of the pathway. Neonatal mice have a large proportion of their pathways which are predominantly down regulated 24 hours after sepsis, which young the majority of young adult pathways are upregulated. Elderly mice fall in between. The corresponding tables show the top 10 pathways and their corresponding Z-score within the functional category 24 hours after sepsis. Red indicates that the pathway is significantly upregulated, whereas blue indicates that the pathway is significantly down regulated.