Nathan J. Klingensmith

The Gut as the Motor of Multiple Organ Dysfunction in Critical Illness

All elements of the gut - the epithelium, the immune system, and the microbiome - are impacted by critical illness and can, in turn, propagate a pathologic host response leading to multiple organ dysfunction syndrome. Preclinical studies have demonstrated that this can occur by release of toxic gut-derived substances into the mesenteric lymph where they can cause distant damage. Further, intestinal integrity is compromised in critical illness with increases in apoptosis and permeability. There is also increasing recognition that microbes alter their behavior and can become virulent based upon host environmental cues. Gut failure is common in critically ill patients; however, therapeutics targeting the gut have proven to be challenging to implement at the bedside. Numerous strategies to manipulate the microbiome have recently been used with varying success in the ICU.

Mechanisms of Intestinal Barrier Dysfunction in Sepsis.

ABSTRACT Intestinal barrier dysfunction is thought to contribute to the development of multiple organ dysfunction syndrome in sepsis. Although there are similarities in clinical course following sepsis, there are significant differences in the host response depending on the initiating organism and time course of the disease, and pathways of gut injury vary widely in different preclinical models of sepsis. The purpose of this study was to determine whether the timecourse and mechanisms of intestinal barrier dysfunction are similar in disparate mouse models of sepsis with similar mortalities. FVB/N mice were randomized to receive cecal ligation and puncture (CLP) or sham laparotomy, and permeability was measured to fluoresceinisothiocyanate conjugated-dextran (FD-4) six to 48 h later. Intestinal permeability was elevated following CLP at all timepoints measured, peaking at 6 to 12 h. Tight junction proteins claudin 1, 2, 3, 4, 5, 7, 8, 13, and 15, Junctional Adhesion Molecule-A (JAM-A), occludin, and ZO-1 were than assayed by Western blot, real-time polymerase chain reaction, and immunohistochemistry 12 h after CLP to determine potential mechanisms underlying increases in intestinal permeability. Claudin 2 and JAM-A were increased by sepsis, whereas claudin-5 and occludin were decreased by sepsis. All other tight junction proteins were unchanged. A further timecourse experiment demonstrated that alterations in claudin-2 and occludin were detectable as early as 1 h after the onset of sepsis. Similar experiments were then performed in a different group of mice subjected to Pseudomonas aeruginosa pneumonia. Mice with pneumonia had an increase in intestinal permeability similar in timecourse and magnitude to that seen in CLP. Similar changes in tight junction proteins were seen in both models of sepsis although mice subjected to pneumonia also had a marked decrease in ZO-1 not seen in CLP. These results indicate that two disparate, clinically relevant models of sepsis induce a significant increase in intestinal permeability mediated through a common pathway involving alterations in claudin 2, claudin 5, JAM-A, and occludin although model-specific differences in ZO-1 were also identified.

New insights into the gut as the driver of critical illness and organ failure

Purpose of review The gut has long been hypothesized to be the ‘motor’ of multiple organ dysfunction syndrome. This review serves as an update on new data elucidating the role of the gut as the propagator of organ failure in critical illness. Recent findings Under basal conditions, the gut absorbs nutrients and serves as a barrier that prevents approximately 40 trillion intraluminal microbes and their products from causing host injury. However, in critical illness, gut integrity is disrupted with hyperpermeability and increased epithelial apoptosis, allowing contamination of extraluminal sites that are ordinarily sterile. These alterations in gut integrity are further exacerbated in the setting of preexisting comorbidities. The normally commensal microflora is also altered in critical illness, with increases in microbial virulence and decreases in diversity, which leads to further pathologic responses within the host. Summary All components of the gut are adversely impacted by critical illness. Gut injury can not only propagate local damage, but can also cause distant injury and organ failure. Understanding how the multifaceted components of the gut interact and how these are perturbed in critical illness may play an important role in turning off the ‘motor’ of multiple organ dysfunction syndrome in the future.

Epidermal Growth Factor Improves Intestinal Integrity and Survival in Murine Sepsis Following Chronic Alcohol Ingestion.

Abstract Epidermal growth factor (EGF) is a cytoprotective protein that improves survival in preclinical models of sepsis through its beneficial effects on intestinal integrity. Alcohol use disorder worsens intestinal integrity and is associated with increased morbidity and mortality in critical illness. We sought to determine whether chronic alcohol ingestion alters the host response to systemic administration of EGF in sepsis. Six-week-old FVB/N mice were randomized to receive 20% alcohol or water for 12 weeks. All mice then underwent cecal ligation and puncture to induce polymicrobial sepsis. Mice were then randomized to receive either intraperitoneal injection of EGF (150 &mgr;g/kg/day) or normal saline. Water-fed mice given EGF had decreased 7-day mortality compared with water-fed mice (18% vs. 55%). Alcohol-fed mice given EGF also had decreased 7-day mortality compared with alcohol-fed mice (48% vs. 79%). Notably, while systemic EGF improved absolute survival to a similar degree in both water-fed and alcohol-fed mice, mortality was significantly higher in alcohol+EGF mice compared with water+EGF mice. Compared with water-fed septic mice, alcohol-fed septic mice had worsened intestinal integrity with intestinal hyperpermeability, increased intestinal epithelial apoptosis, decreased proliferation and shorter villus length. Systemic administration of EGF to septic alcohol-fed mice decreased intestinal permeability compared with septic alcohol-fed mice given vehicle, with increased levels of the tight junction mediators claudin-5 and JAM-A. Systemic administration of EGF to septic alcohol-fed mice also decreased intestinal apoptosis with an improvement in the Bax/Bcl-2 ratio. EGF also improved both crypt proliferation and villus length in septic alcohol-fed mice. EGF administration resulted in lower levels of both pro- and anti-inflammatory cytokines monocyte chemoattractant protein-1, tumor necrosis factor, and interleukin 10 in alcohol-fed mice. EGF is therefore effective at improving both intestinal integrity and mortality following sepsis in mice with chronic alcohol ingestion. However, the efficacy of EGF in sepsis is blunted in the setting of chronic alcohol ingestion, as intestinal integrity and mortality in alcohol-fed mice given EGF improves animals to levels seen in water-fed mice given vehicle but does not approach levels seen in water-fed mice given EGF.

Fecal microbiota transplantation for multiple organ dysfunction syndrome

Approximately 40 trillion bacteria reside inside the human intestine, meaning there are at least as many cells of microbial origin as human origin [1]. While it was once believed that bacteria and humans simply co-existed in the same space, a wide body of evidence now suggests that host–microbial communication is more complex than ever imagined and the microbiome plays a critical role in maintaining host homeostasis. The microbiome is also altered in multiple disease states, including heart disease [2], cancer [3], and Clostridium difficile infection [4], with changes detectable in microbial composition, number, diversity, and virulence compared to healthy controls. While the majority of studies linking the microbiome to disease are associative, there is increasing evidence that the microbiome plays a crucial role in mediating the pathophysiology of multiple acute and chronic illnesses.

Chronic Alcohol Ingestion Delays T Cell Activation and Effector Function in Sepsis

Sepsis is the leading cause of death in intensive care units in the US, and it is known that chronic alcohol use is associated with higher incidence of sepsis, longer ICU stays, and higher mortality from sepsis. Both sepsis and chronic alcohol use are associated with immune deficits such as decreased lymphocyte numbers, impaired innate immunity, delayed-type hypersensitivity reactions, and susceptibility to infections; however, understanding of specific pathways of interaction or synergy between these two states of immune dysregulation is lacking. This study therefore sought to elucidate mechanisms underlying the immune dysregulation observed during sepsis in the setting of chronic alcohol exposure. Using a murine model of chronic ethanol ingestion followed by sepsis induction via cecal ligation and puncture, we determined that while CD4+ and CD8+ T cells isolated from alcohol fed mice eventually expressed the same cellular activation markers (CD44, CD69, and CD43) and effector molecules (IFN-γ, TNF) as their water fed counterparts, there was an overall delay in the acquisition of these phenotypes. This early lag in T cell activation was associated with significantly reduced IL-2 production at a later timepoint in both the CD4+ and CD8+ T cell compartments in alcohol sepsis, as well as with a reduced accumulation of CD8dim activated effectors. Taken together, these data suggest that delayed T cell activation may result in qualitative differences in the immune response to sepsis in the setting of chronic alcohol ingestion.

Sepsis reveals compartment-specific responses in intestinal proliferation and apoptosis in transgenic mice whose enterocytes re-enter the cell cycle

Cell production and death are tightly regulated in the rapidly renewing gut epithelium, with proliferation confined to crypts and apoptosis occurring in villi and crypts. This study sought to determine how stress alters these compartmentalized processes. Wild‐type mice made septic via cecal ligation and puncture had decreased crypt proliferation and increased crypt and villus apoptosis. Fabpi‐TAg mice expressing large T‐antigen solely in villi had ectopic enterocyte proliferation with increased villus apoptosis in unmanipulated animals. Septic Fabpi‐TAg mice had an unexpected increase in villus proliferation compared with unmanipulated littermates, whereas crypt proliferation was decreased. Cell cycle regulators cyclin D1 and cyclin D2 were decreased in jejunal tissue in septic transgenic mice. In contrast, villus and crypt apoptosis were increased in septic Fabpi‐TAg mice. To examine the relationship between apoptosis and proliferation in a compartment‐specific manner, Fabpi‐TAg mice were crossed with fabpl‐Bcl‐2 mice, resulting in expression of both genes in the villus but Bcl‐2 alone in the crypt. Septic bitransgenic animals had decreased crypt apoptosis but had a paradoxical increase in villus apoptosis compared with septic fabpi‐TAg mice, associated with decreased proliferation in both compartments. Thus, sepsis unmasks compartment‐specific proliferative and apoptotic regulation that is not present under homeostatic conditions.— Lyons, J. D., Klingensmith, N. J., Otani, S., Mittal, R., Liang, Z., Ford, M. L., Coopersmith, C. M. Sepsis reveals compartment‐specific responses in intestinal proliferation and apoptosis in transgenic mice whose enterocytes reenter the cell cycle. FASEB J. 31, 5507–5519 (2017). www.fasebj.org

CXCR4 blockade decreases CD4+ T cell exhaustion and improves survival in a murine model of polymicrobial sepsis

Sepsis is a dysregulated systemic response to infection involving many inflammatory pathways and the induction of counter-regulatory anti-inflammatory processes that results in a state of immune incompetence and can lead to multi-organ failure. CXCR4 is a chemokine receptor that, following ligation by CXCL12, directs cells to bone marrow niches and also plays an important role in T cell cosignaling and formation of the immunological synapse. Here, we investigated the expression and function of CXCR4 in a murine model of polymicrobial sepsis. Results indicate that CXCR4 is selectively upregulated on naïve CD4+ and CD8+ T cells and CD4+ central memory T cells following the induction of sepsis, and that CXCR4 antagonism resulted in a significant decrease in sepsis-induced mortality. We probed the mechanistic basis for these findings and found that CXCR4 antagonism significantly increased the number of peripheral CD4+ and CD8+ T cells following sepsis. Moreover, mice treated with the CXCR4 antagonist contained fewer PD-1+ LAG-3+ 2B4+ cells, suggesting that blockade of CXCR4 mitigates CD4+ T cell exhaustion during sepsis. Taken together, these results characterize CXCR4 as an important pathway that modulates immune dysfunction and mortality following sepsis, which may hold promise as a target for future therapeutic intervention in septic patients.

Increased mortality in CD43-deficient mice during sepsis

CD43 is a large transmembrane protein involved in T cell activation. Previous studies of CD43-/- mice in viral models have demonstrated a role for CD43 in Th1/Th2 skewing, activation of Foxp3+ Treg, and T cell apoptosis. However, the role of CD43 during sepsis has never been tested. Thus, we interrogated the role of CD43 during sepsis using a murine cecal ligation and puncture (CLP) model, and found that CD43-/- mice demonstrated significantly worsened mortality compared to B6 mice following CLP. Phenotypic analysis of splenocytes isolated 24 h after septic insult revealed significantly increased apoptosis of central memory cells in both CD4+ and CD8+ T cell compartments in CD43-/- septic mice compared to WT septic mice. Furthermore, CD43-/-septic mice exhibited a prominent Th2 skewing following sepsis relative to WT septic mice, as evidenced by a significant decrease in the frequency of IL-2+ CXCR3+ TH1 cells as a significant increase in the frequency of IL-4+ CCR4+ TH2 cells. Finally, septic CD43-/- animals contained significantly fewer CD25+ Foxp3+ TReg cells as compared to WT septic animals. Importantly, depleting CD25+ Treg eliminated the increased mortality observed in CD43-/- mice. Taken together, these data demonstrate an important role of CD43 in modulating immune dysregulation and mortality following sepsis.

1329: CD43 SIGNALING CONTROLS SURVIVAL, TH2 SKEWING, AND MEMORY T CELL APOPTOSIS DURING SEPSIS.

Crit Care Med 2016 • Volume 44 • Number 12 (Suppl.) investigation quantified the incidence of and risk factors for deterioration of health-related quality of life (HRQL, per PedsQL[TM]) following pediatric sepsis, hypothesizing that sepsis severity would be associated with a decline in HRQL. Methods: We conducted a retrospective cohort study of children admitted 1/20128/2014 to Seattle Children’s Hospital with sepsis using electronic health records to identify patients meeting the 2005 consensus sepsis criteria within 4 hours of hospital presentation. We assessed HRQL for pre-admission baseline and 0.5-4 month post-discharge status, and identified patients who failed to recover (FR) within 4.5 PedsQL[TM] points of their baseline (the minimally clinically significant difference). We determined associations between patient/illness characteristics and FR from baseline to follow-up in univariate analyses and a multivariable generalized linear model. Results: Failure to recover occurred in 22.5% of the 516 patients meeting inclusion criteria and persisted for up to 100 days post-discharge. Factors associated with FR included sepsis category (septic shock 53%, severe sepsis 27%, sepsis 21%, p=0.03), site of infection (CNS 53%, blood 38%, p<0.01), ICU admission (36%, p=0.01), and length of stay (LOS) (p<0.01). In multivariable regression controlling for sex, age, and race/ethnicity, sepsis category was non-significantly associated with FR (vs. sepsis: septic shock RR 1.64 [95% CI 0.97-2.76], severe sepsis RR 1.21 [0.61-2.41]). However, FR was significantly associated with immune compromise (RR 1.58 [1.07-2.35]), private insurance (RR 1.85 [1.202.78], LOS (RR 1.04/day [1.01-1.07]), and time to follow-up (RR 0.92/week [0.86-0.98]). Conclusions: Nearly one-quarter of children surviving sepsis experience a clinically significant decline in HRQL persisting up to 3 months following hospital discharge. Identification of the factors associated with HRQL deterioration is essential to guide interventions to improve long term outcomes.