EARLY DIFFERENTIATION BETWEEN SEPSIS AND STERILE INFLAMMATION VIA URINARY GENE SIGNATURES OF METABOLIC DYSREGULATION.

ABSTRACT: Objective: The aim of this study was to characterize early urinary gene expression differences between patients with sepsis and patients with sterile inflammation and summarize in terms of a reproducible sepsis probability score. Design: This was a prospective observational cohort study. Setting: The study was conducted in a quaternary care academic hospital. Patients: One hundred eighty-six sepsis patients and 78 systemic inflammatory response syndrome (SIRS) patients enrolled between January 2015 and February 2018. Interventions: Whole-genome transcriptomic analysis of RNA was extracted from urine obtained from sepsis patients within 12 hours of sepsis onset and from patients with surgery-acquired SIRS within 4 hours after major inpatient surgery. Measurements and Main Results: We identified 422 of 23,956 genes (1.7%) that were differentially expressed between sepsis and SIRS patients. Differentially expressed probes were provided to a collection of machine learning feature selection models to identify focused probe sets that differentiate between sepsis and SIRS. These probe sets were combined to find an optimal probe set (UrSepsisModel) and calculate a urinary sepsis score (UrSepsisScore), which is the geometric mean of downregulated genes subtracted from the geometric mean of upregulated genes. This approach summarizes the expression values of all decisive genes as a single sepsis score. The UrSepsisModel and UrSepsisScore achieved area under the receiver operating characteristic curves 0.91 (95% confidence interval, 0.86-0.96) and 0.80 (95% confidence interval, 0.70-0.88) on the validation cohort, respectively. Functional analyses of probes associated with sepsis demonstrated metabolic dysregulation manifest as reduced oxidative phosphorylation, decreased amino acid metabolism, and decreased oxidation of lipids and fatty acids. Conclusions: Whole-genome transcriptomic profiling of urinary cells revealed focused probe panels that can function as an early diagnostic tool for differentiating sepsis from sterile SIRS. Functional analysis of differentially expressed genes demonstrated a distinct metabolic dysregulation signature in sepsis.

Chronic Critical Illness Elicits a Unique Circulating Leukocyte Transcriptome in Sepsis Survivors.

Surgical sepsis has evolved into two major subpopulations: patients who rapidly recover, and those who develop chronic critical illness (CCI). Our primary aim was to determine whether CCI sepsis survivors manifest unique blood leukocyte transcriptomes in late sepsis that differ from transcriptomes among sepsis survivors with rapid recovery. In a prospective cohort study of surgical ICU patients, genome-wide expression analysis was conducted on total leukocytes in human whole blood collected on days 1 and 14 from sepsis survivors who rapidly recovered or developed CCI, defined as ICU length of stay ≥ 14 days with persistent organ dysfunction. Both sepsis patients who developed CCI and those who rapidly recovered exhibited marked changes in genome-wide expression at day 1 which remained abnormal through day 14. Although summary changes in gene expression were similar between CCI patients and subjects who rapidly recovered, CCI patients exhibited differential expression of 185 unique genes compared with rapid recovery patients at day 14 (p < 0.001). The transcriptomic patterns in sepsis survivors reveal an ongoing immune dyscrasia at the level of the blood leukocyte transcriptome, consistent with persistent inflammation and immune suppression. Furthermore, the findings highlight important genes that could compose a prognostic transcriptomic metric or serve as therapeutic targets among sepsis patients that develop CCI.

Identification of unique microRNA expression patterns in bone marrow hematopoietic stem and progenitor cells after hemorrhagic shock and multiple injuries in young and old adult mice.

BACKGROUND: After severe trauma, the older host experiences more dysfunctional hematopoiesis of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs), and dysfunctional differentiation of circulating myeloid cells into effective innate immune cells. Our main objective was to compare BM HSPC microRNA (miR) responses of old and young mice in a clinically relevant model of severe trauma and shock. METHODS: C57BL/6 adult male mice aged 8 to 12 weeks (young) and 18 to 24 months (old) underwent multiple injuries and hemorrhagic shock (polytrauma [PT]) that engenders the equivalent of major trauma (Injury Severity Score, >15). Pseudomonas pneumonia (PNA) was induced in some young and old adult mice 24 hours after PT. MicroRNA expression patterns were determined from lineage-negative enriched BM HSPCs isolated from PT and PT-PNA mice at 24 and 48 hours postinjury, respectively. Genome-wide expression and pathway analyses were also performed on bronchoalveolar lavage (BAL) leukocytes from both mouse cohorts. RESULTS: MicroRNA expression significantly differed among all experimental conditions (p < 0.05), except for old-naive versus old-injured (PT or PT-PNA) mice, suggesting an inability of old mice to mount a robust early miR response to severe shock and injury. In addition, young adult mice had significantly more leukocytes obtained from their BAL, and there were greater numbers of polymorphonuclear cells compared with old mice (59.8% vs. 2.2%, p = 0.0069). Despite increased gene expression changes, BAL leukocytes from old mice demonstrated a more dysfunctional transcriptomic response to PT-PNA than young adult murine BAL leukocytes, as reflected in predicted upstream functional pathway analysis. CONCLUSION: The miR expression pattern in BM HSPCs after PT (+/-PNA) is dissimilar in old versus young adult mice. In the acute postinjury phase, old adult mice are unable to mount a robust miR HSPC response. Hematopoietic stem and progenitor cell miR expression in old PT mice reflects a diminished functional status and a blunted capacity for terminal differentiation of myeloid cells.

The role of bone marrow microRNA (miR) in erythropoietic dysfunction after severe trauma.

BACKGROUND: Previous data has shown that severe traumatic injury is associated with bone marrow dysfunction, which manifests as persistent injury-associated anemia. This study sought to identify whether the expression of erythropoiesis-related microRNAs were altered in the bone marrow of trauma patients to determine if these microRNAs play a role in persistent injury-associated anemia. METHODS: Bone marrow was collected from severely injured trauma patients who underwent fracture fixation as well as patients who underwent elective hip replacement. There were 27 trauma patients and 10 controls analyzed. Total RNA and microRNA were isolated from CD34-positive cells using the RNeasy Plus Mini kit, and genome-wide microRNA expression patterns were assayed. Genes with significant expression differences were found using BRB-ArrayTools with a significance of P < .01. RESULTS: There were marked differences in expression of 108 microRNAs in the trauma group when compared with hip replacement patients. Four of these microRNAs play a role in regulating erythropoiesis: microRNA-150, microRNA-223, microRNA15a, and microRNA-24. These microRNAs were all upregulated significantly, with trauma/hip replacement fold changes of 1.7, 1.8, 1.2, and 1.2 respectively, and all act to suppress or regulate erythropoiesis. CONCLUSION: Assessment of the bone marrow microRNA profile in trauma patients compared to those undergoing elective hip replacement revealed the differential expression of microRNA-150, microRNA-223, microRNA-15a, and microRNA-24. These microRNAs all play a role in decreased erythroid progenitor cell growth and provide important insight to the erythropoietic dysfunction seen after trauma.

Transcriptomic responses from improved murine sepsis models can better mimic human surgical sepsis.

Historically, murine models of inflammation in biomedical research have been shown to minimally correlate with genomic expression patterns from blood leukocytes in humans. In 2019, our laboratory reported an improved surgical sepsis model of cecal ligation and puncture (CLP) that provides additional daily chronic stress (DCS), as well as adhering to the Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS) guidelines. This model phenotypically recapitulates the persistent inflammation, immunosuppression, and catabolism syndrome observed in adult human surgical sepsis survivors. Whether these phenotypic similarities between septic humans and mice are replicated at the circulating blood leukocyte transcriptome has not been demonstrated. Our analysis, in contrast with previous findings, demonstrated that genome-wide expression in our new murine model more closely approximated human surgical sepsis patients, particularly in the more chronic phases of sepsis. Importantly, our new model of murine surgical sepsis with chronic stress did not reflect well gene expression patterns from humans with community-acquired sepsis. Our work indicates that improved preclinical murine sepsis modeling can better replicate both the phenotypic and transcriptomic responses to surgical sepsis, but cannot be extrapolated to other sepsis etiologies. Importantly, these improved models can be a useful adjunct to human-focused and artificial intelligence-based forms of research in order to improve septic patients' morbidity and mortality.

Discovery and Validation of Urinary Molecular Signature of Early Sepsis.

Identify alterations in gene expression unique to systemic and kidney-specific pathophysiologic processes using whole-genome analyses of RNA isolated from the urinary cells of sepsis patients. DESIGN: Prospective cohort study. SETTING: Quaternary care academic hospital. PATIENTS: A total of 266 sepsis and 82 control patients enrolled between January 2015 and February 2018. INTERVENTIONS: Whole-genome transcriptomic analysis of messenger RNA isolated from the urinary cells of sepsis patients within 12 hours of sepsis onset and from control subjects. MEASUREMENTS AND MAIN RESULTS: The differentially expressed probes that map to known genes were subjected to feature selection using multiple machine learning techniques to find the best subset of probes that differentiates sepsis from control subjects. Using differential expression augmented with machine learning ensembles, we identified a set of 239 genes in urine, which show excellent effectiveness in classifying septic patients from those with chronic systemic disease in both internal and independent external validation cohorts. Functional analysis indexes disrupted biological pathways in early sepsis and reveal key molecular networks driving its pathogenesis. CONCLUSIONS: We identified unique urinary gene expression profile in early sepsis. Future studies need to confirm whether this approach can complement blood transcriptomic approaches for sepsis diagnosis and prognostication.

Identification of Unique mRNA and miRNA Expression Patterns in Bone Marrow Hematopoietic Stem and Progenitor Cells After Trauma in Older Adults.

Older adults have significantly worse morbidity and mortality after severe trauma than younger cohorts. The competency of the innate immune response decreases with advancing age, especially after an inflammatory insult. Subsequent poor outcomes after trauma are caused in part by dysfunctional leukocytes derived from the host's hematopoietic stem and progenitor cells (HSPCs). Our objective was to analyze the bone marrow (BM) HSPC transcriptomic [mRNA and microRNA (miR)] responses to trauma in older and younger adults. BM was collected intraoperatively <9 days after initial injury from trauma patients with non-mild injury [ISS ≥ 9] or with shock (lactate ≥ 2, base deficit ≥ 5, MAP ≤ 65) who underwent operative fixation of a pelvic or long bone fracture. Samples were also analyzed based on age (<55 years and ≥55 years), ISS score and transfusion in the first 24 h, and compared to age/sex-matched controls from non-cancer elective hip replacement or purchased healthy younger adult human BM aspirates. mRNA and miR expression patterns were calculated from lineage-negative enriched HSPCs. 924 genes were differentially expressed in older trauma subjects vs. age/sex-matched controls, while 654 genes were differentially expressed in younger subjects vs. age/sex-matched control. Only 68 transcriptomic changes were shared between the two groups. Subsequent analysis revealed upregulation of transcriptomic pathways related to quantity, function, differentiation, and proliferation of HSPCs in only the younger cohort. miR expression differences were also identified, many of which were associated with cell cycle regulation. In summary, differences in the BM HSPC mRNA and miR expression were identified between older and younger adult trauma subjects. These differences in gene and miR expression were related to pathways involved in HSPC production and differentiation. These differences could potentially explain why older adult patients have a suboptimal hematopoietic response to trauma. Although immunomodulation of HSPCs may be a necessary consideration to promote host protective immunity after host injury, the age related differences further highlight that patients may require an age-defined medical approach with interventions that are specific to their transcriptomic and biologic response. Also, targeting the older adult miRs may be possible for interventions in this patient population.

Human Regulatory T Cells From Umbilical Cord Blood Display Increased Repertoire Diversity and Lineage Stability Relative to Adult Peripheral Blood.

The human T lymphocyte compartment is highly dynamic over the course of a lifetime. Of the many changes, perhaps most notable is the transition from a predominantly naïve T cell state at birth to the acquisition of antigen-experienced memory and effector subsets following environmental exposures. These phenotypic changes, including the induction of T cell exhaustion and senescence, have the potential to negatively impact efficacy of adoptive T cell therapies (ACT). When considering ACT with CD4+CD25+CD127-/lo regulatory T cells (Tregs) for the induction of immune tolerance, we previously reported ex vivo expanded umbilical cord blood (CB) Tregs remained more naïve, suppressed responder T cells equivalently, and exhibited a more diverse T cell receptor (TCR) repertoire compared to expanded adult peripheral blood (APB) Tregs. Herein, we hypothesized that upon further characterization, we would observe increased lineage heterogeneity and phenotypic diversity in APB Tregs that might negatively impact lineage stability, engraftment capacity, and the potential for Tregs to home to sites of tissue inflammation following ACT. We compared the phenotypic profiles of human Tregs isolated from CB versus the more traditional source, APB. We conducted analysis of fresh and ex vivo expanded Treg subsets at both the single cell (scRNA-seq and flow cytometry) and bulk (microarray and cytokine profiling) levels. Single cell transcriptional profiles of pre-expansion APB Tregs highlighted a cluster of cells that showed increased expression of genes associated with effector and pro-inflammatory phenotypes (CCL5, GZMK, CXCR3, LYAR, and NKG7) with low expression of Treg markers (FOXP3 and IKZF2). CB Tregs were more diverse in TCR repertoire and homogenous in phenotype, and contained fewer effector-like cells in contrast with APB Tregs. Interestingly, expression of canonical Treg markers, such as FOXP3, TIGIT, and IKZF2, were increased in CB CD4+CD127+ conventional T cells (Tconv) compared to APB Tconv, post-expansion, implying perinatal T cells may adopt a default regulatory program. Collectively, these data identify surface markers (namely CXCR3) that could be depleted to improve purity and stability of APB Tregs, and support the use of expanded CB Tregs as a potentially optimal ACT modality for the treatment of autoimmune and inflammatory diseases.

Effect of Beta-Blockade on the Expression of Regulatory MicroRNA after Severe Trauma and Chronic Stress.

BACKGROUND: Beta-blockade administration after lung contusion, hemorrhagic shock, and chronic stress has been shown to improve bone marrow function, decrease hypercatecholaminemia, and reduce inflammation. MicroRNAs (miR) are critical biologic regulators that can downregulate gene expression by causing messenger RNA degradation or inhibition of translation. This study sought to expand our understanding of the molecular mechanisms underlying the reduced inflammatory response after the administration of beta-blockade (BB) in our rodent trauma model. STUDY DESIGN: Male Sprague-Dawley rats aged 8 to 9 weeks were randomized to lung contusion, hemorrhagic shock with daily restraint stress (LCHS/CS) or LCHS/CS plus propranolol (LCHS/CS+BB). Restraint stress occurred 2 hours daily after LCHS. Propranolol (10 mg/kg) was given daily until day 7. Total RNA and miR were isolated from bone marrow and genome-wide miR expression patterns were assayed. Bone marrow cytokine expression was determined with quantitative polymerase chain reaction. RESULTS: LCHS/CS led to significantly increased bone marrow expression of interleukin (IL) 1ß, tumor necrosis factor-α, IL-6, nitric oxide, and plasma C-reactive protein. There were marked differences in expression of 45 miRs in the LCHS/CS+BB group compared with the LCHS/CS group when using a p value <0.001. Rno-miR-27a and miR-25 were upregulated 7- to 8-fold in the rodents who underwent LCHS/CS+BB compared with LCHS/CS alone, and this correlated with reduced bone marrow expression of IL-1ß, tumor necrosis factor-α, IL-6, nitric oxide, and reduced plasma C-reactive protein in the LCHS/CS+BB group. CONCLUSIONS: The genomic and miR expression patterns in bone marrow after LCHS/CS differed significantly compared with rodents that received propranolol after LCHS/CS. The use of BB after severe trauma can help mitigate persistent inflammation by upregulating Rno-miR-27a and miR-25 and reducing inflammatory cytokines in those who remain critically ill.