This study represents the first developmental-age group comparison of the transcriptomic response of children with septic shock. We show that developmental age strongly influences the early whole blood transcriptomic response. This assertion is supported by direct comparisons of patients with septic shock across four developmental-age groups and by comparisons between the respective developmental-age groups and age-matched controls. The direct comparisons demonstrated minimal differences between the infant, toddler and school-age groups with septic shock. In contrast, age-specific alterations in host response were most profound in the neonate group, as demonstrated by reduced expression of genes representative of several key pathways of the innate and adaptive immune systems.
In the comparisons to age-matched controls, the neonate and school-age groups had the largest number of uniquely regulated genes. The upregulated genes corresponded to several key inflammatory/immunity pathways. Importantly, the number of upregulated genes corresponding to these pathways increased in proportion to developmental age. In contrast, the downregulated genes derived from these comparisons corresponded to adaptive immunity-related pathways, and the number of downregulated genes in each pathway was greatest in the neonate group.
The innate immune system plays a critical role in a successful host response to sepsis, particularly in the neonate (18
). Multiple developmental alterations of innate host response capabilities are present in neonates compared with older age groups, including pathogen recognition receptors, inflammatory signaling pathways and overall innate immune cellular function (19
). Consistent with these observations, our current data demonstrate reduced expression of genes corresponding to the pathogen recognition receptor and TREM-1
pathways, as well as their relevant downstream signaling molecules (for example, Janus kinase 2
], signal transducer and activator of transcription 5
] and extracellular signal regulated kinase 1/2
]; Supplementary ) in the neonate group.
signaling is critical for amplification of the inflammatory responses to microbial products in adults. Inhibition of TREM-1
signaling through antibody-mediated blockade reduced mortality in septic adult animals and has been proposed as a potential therapeutic target for septic shock (24
). Our current data indicate that TREM-1
pathway-related genes are not substantially expressed in neonates with septic shock. Thus, blockade of TREM
signaling may not be biologically warranted in neonates. The notion of a TREM-1
–limited reduced neonatal capacity to produce an intense innate response to a septic challenge is also supported by the attenuated inflammatory response seen in septic murine neonates compared with septic young adult mice (25
). Taken together, these data suggest that the neonate has a relatively reduced capacity to generate as robust an innate immune response to septic shock as seen in older age groups, which may be in part related to alterations in TREM-1
In stark contrast to the largely upregulated transcriptomic responses from all three other developmental-age groups, neonates exhibited predominantly down-regulated responses when compared with age-matched controls. These alterations represented downregulated pathways related to adaptive immunity. It is well known that baseline neonatal adaptive immune responses are distinct from those seen in more mature populations. These alterations in cellular response have been suggested to permit avoidance of perpetual hyperinflammation through regulation of T-cell responses and increased T-cell apoptosis (26
This is the first report describing downregulation of adaptive immunity-related genes during septic shock in neonates compared with age-matched controls. The predominance of downregulated adaptive immune pathways in neonates could be interpreted to support why adaptive immune responses were not critical for survival in an animal model of neonatal polymicrobial sepsis (27
). In distinct contrast, the absence or dysfunction of the adaptive immune system has a profound negative impact on adult survival in preclinical models (28
) and in humans (29
). As these data illustrate, the contribution of adaptive immunity for protection and response against septic shock, and in particular which components may be protective, is unclear in neonates and requires further investigation.
Many attempts have been made to improve immune function in neonates and reduce the incidence and burden of infection (31
). The failure of these interventions in large randomized trials likely reflects underappreciated differences in the functional capacity of the neonatal host response (32
). To successfully modify immune function and improve infection outcomes in human neonates, as has been done in neonatal animal models (27
), consideration of the unique immuno-developmental stage of the neonate must be taken into account.
We acknowledge there are several potential confounding factors in this study. First, there were fewer neonatal patients compared with the other age groups. To address this potential confounder and the associated risk of over-fitting the data, we set the primary filter to require a =two-fold increase in gene expression. We then used a stringent statistical test by setting a false discovery rate at 1% (equivalent to a P value of 0.01).
Second, the neonatal group had a higher mortality rate and a higher PRISM score, thus raising the possibility that the differences in gene expression reflect a poorer physiologic state, rather than differences reflecting developmental age. To address this potential confounder, we calculated the median time (d) to death for all nonsurvivors. There was no significant difference in median days to death (interquartile range) across the four developmental-age groups: neonate = 2 (1.5–5); infant = 7 (5
); toddler = 2 (1
); and school-age = 2 (1
). We also extracted the 1,823 gene probes differentially regulated between the neonate group and controls () and compared these genes between the neonate survivors and nonsurvivors. None of the 1,823 gene probes were differentially regulated between the survivors and nonsurvivors (ANOVA with a false discovery rate of 1%).
Third, neonates had a significantly higher proportion of infections due to gram-positive bacteria compared with the school-age group. This observation raises the possibility that the differences in gene expression described above reflect a pathogen class effect rather than an effect of developmental age. An analysis (same sequential expression and statistical filters as described for the previous analyses) was performed to compare expression data from all patients with gram-negative infection (n = 46) to all patients with gram-positive infections (n = 47) and revealed only 11 differentially regulated probes (Supplementary ).
Fourth, there were a variety of significant differences between the four developmental-age groups with respect to peripheral differential white blood cell counts. Because we used whole blood–derived RNA, it is possible that the differential gene expression patterns described above reflect differences in peripheral white blood cell counts rather than an effect of developmental age. To address this, we analyzed our data for the presence of previously published “signature probe sets” for neutrophils (38 probes), lymphocytes (50 probes) and monocytes (28 probes), respectively (35
). We used the following criteria to assess the presence of the signature probe sets: =300 raw expression values in a least one-half of the subjects in each developmental age category. demonstrates that the signature probe sets were present to a similar degree across the four developmental-age groups. These data indicate that the relative contributions of the three major leukocyte subsets to the whole blood transcriptome expression patterns were not substantially different across the four developmental-age groups.
Expression of leukocyte subset signature probes across the four developmental-age groups of patients with septic shock.
Although we cannot fully correct for all potential confounders, the above analyses indicate that the differences in gene expression reported in this study reflect, at least in part, a direct influence of developmental age. We recognize that whole-blood transcriptome alterations corresponding to specific immune pathways do not yield specific pathophysiology. However, these data do offer insight into the complex, multifactorial heterogeneous host response to sepsis and allow identification of critical differences between age groups.
Children are not small adults, and, in the host response to sepsis, we show that neonates are not small children. Age-specific neonatal and pediatric studies of the host response to sepsis are critically necessary to permit identification of novel, developmentally appropriate translational opportunities that might lead to improved sepsis outcomes.