Redefining the gut as the motor of critical illness

TRMOME-900; No. of Pages 10

Redefining the gut as the motor ofcritical illnessRohit Mittal and Craig M. Coopersmith

Department of Surgery and Emory Center for Critical Care, Woodruff Health Sciences Center, Emory University School of Medicine,

Atlanta, GA 30322, USA

Review

The gut is hypothesized to play a central role in theprogression of sepsis and multiple organ dysfunctionsyndrome. Critical illness alters gut integrity by increas-ing epithelial apoptosis and permeability and by de-creasing epithelial proliferation and mucus integrity.Additionally, toxic gut-derived lymph induces distantorgan injury. Although the endogenous microflora ordi-narily exist in a symbiotic relationship with the gutepithelium, severe physiological insults alter this rela-tionship, leading to induction of virulence factors in themicrobiome, which, in turn, can perpetuate or worsencritical illness. This review highlights newly discoveredways in which the gut acts as the motor that perpetu-ates the systemic inflammatory response in criticalillness.

The gut as the driving force of critical illnessThe gut has long been characterized as the motor ofmultiple organ dysfunction syndrome (MODS) [1]. Thishas been hypothesized to be due to dysregulated crosstalkamong the epithelium, immune system, and endogenousmicroflora of the gut [2] in which loss of balance betweenthese highly interrelated systems leads to the developmentof systemic manifestations of disease, whose reach extendsfar beyond the intestine. Recent dramatic advances in ourunderstanding of how the gut initiates and propagatescritical illness have led to novel investigations into howit may be targeted for therapeutic gain in the intensive careunit (ICU). This is highly significant considering thatcritical illness accounts for up to 39% of total hospital costs[3] and sepsis kills 229 000–360 00 patients a year in theUnited States alone [4], yet a paucity of effective therapiesexist for the sickest ICU patients. The purpose of thisreview is to highlight new insights into the complexbalance that exists between the gut epithelium and theintestinal microbiome, and how perturbations in this rela-tionship can lead to significant morbidity or even death(Figure 1).

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Corresponding author: Coopersmith, C.M. ([email protected]).Keywords: gut; sepsis; critical illness intestine; epithelium; microbiome.

Evolution of the concept of the gut as the motor ofMODSIn the 1980s, the predominant theory connecting the gut toMODS was bacterial translocation, a process wherebyintestinal hyperpermeability allowed intact bacteria toleave the gut lumen, entering previously sterile environ-ments and causing systemic illness [1,5,6]. This theory wascertainly plausible given the fact that multiple investiga-tors over the past 30 years have demonstrated that criticalillness – whether caused by sepsis, trauma, burn, or ische-mia/reperfusion (I/R) – causes overgrowth of pathogenicbacteria within the gut and increased intestinal perme-ability [7–9]. One theoretical mechanism through whichintestinal contents were assumed to gain access to the restof the body was through hematogenous spread via themesenteric circulation to the portal circulation [2,6,10].However, although bacterial translocation was easy todetect in animal models of critical illness, this was notthe case in human trials. A classic study sought to provethe concept of hematogenous spread of bacteria by placingportal vein catheters in patients following abdominal trau-ma. Contrary to prevailing thought, bacteria were detectedin portal or systemic blood in only 2% of patients (andendotoxin in no patients) despite a 30% incidence of multi-ple organ failure [11]. Multiple other studies over the pasttwo decades have also not been able to confirm a directrelationship between intestinal hyperpermeability andbacterial translocation during critical illness. More recent-ly, numerous studies have provided alternative routesthrough which the gut can both drive and perpetuatecritical illness.

The gut–lymph hypothesis

An alternate route through which intestinal contents couldinitiate systemic injury is via the lymph [6,10]. The gut–lymph hypothesis states that following injury, toxic med-iators are released from the intestine, are transportedthrough the mesenteric lymph nodes, and go on to causedamage remote from the intestine.

Several lines of evidence support this theory. First, thereis direct evidence that interrupting the flow of mesentericlymph prevents distant organ damage. Anatomically, gut-derived lymph flows from the mesenteric lymphatic duct tothe pulmonary circulation, and mesenteric lymph duct liga-tion attenuates both lung injury and neutrophil activationfollowing endotoxemia in rats [12]. Further, mesentericlymph collected from mice following trauma–hemorrhagic

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MODS

Microbiome↑ Pathological bacteria

↑ Virulence↑ An�bio�c resistance

Δ Bacterial gene expression

Epithelium↑ Permeability

↑ Apoptosis↓ Barrier func�on

Altered mucus

TRENDS in Molecular Medicine

Figure 1. Relationship between the intestinal epithelium and microbiome. A

complex but mutually beneficial relationship exists within the gut under

homeostatic conditions. However, critical illness disrupts this balance, leading to

pathological changes in both the intestinal epithelium and microbiome, which can

induce and perpetuate multiple organ dysfunction syndrome (MODS).

Review Trends in Molecular Medicine xxx xxxx, Vol. xxx, No. x


shock (T/HS) and injected into trauma–sham shock (T/SS)mice induces lung injury and demonstrates the same toxicproperties of intestinal lymph seen in T/HS [13]. Addition-ally, ligation of the mesenteric lymph duct in either T/HS orin burn injury prevents injury-induced decreases in cardiaccontractility and cardiac output [14–16]. Perhaps most im-portantly, ligation of the mesenteric lymphatic ductimproves survival in multiple animal models of criticalillness [5,17].

Although the precise factors in gut-derived lymph thatinduce injury remain to be determined, several recent stud-ies have shed light on this. Several different proteins andlipid factors are responsible for the damage caused bymesenteric lymph, whereas endotoxin and cytokines havebeen examined as candidate mediators and found not to beassociated with its toxicity [18–21]. Pancreatic proteasesappear to be important in lymph toxicity, because ligation ofthe pancreatic duct in T/HS prevents production of bioactivemesenteric lymph [22]. Further, T/HS-derived lymph hasincreased lipoprotein lipase activity, which induces endo-thelial toxicity [23,24]. Lipase, in turn, generates free fattyacids (mainly palmitic, stearic, oleic, and linoleic acids),which have been demonstrated to have the same cytotoxicityas T/HS-derived lymph and have been speculated to be thekey components in lymph-induced damage [25]. Of note, T/HS-derived lymph leads to activation of Toll-like receptor(TLR) 4, which is necessary for mediation of the inflamma-tory cascade and priming of neutrophils required for lunginjury [26,27]. Lung injury is fully abrogated in TLRdomain-containing adapter-inducing interferon-b (TRIF)deficient mice and partially abrogated in Myd88 deficientmice (Myd88, like TRIF, is a TLR adapter protein), althoughno effect is seen in TLR2 deficient animals.

Pathways involved in lipid absorption and processingmay also play a role in transporting toxic lymph. Intestinalmicrosomal triglyceride transfer protein (Mttp) is crucialfor chylomicron assembly within the lumen and absorptionof lipids via the lymphatic system. Mttp deficient mice havea block in chylomicron assembly and lipid malabsorption[28]. When these animals are subjected to sepsis fromPseudomonas aeruginosa pneumonia, mortality is pre-vented [29]. Although lung injury is unaffected in theseanimals, gut integrity is improved as evidenced by protec-tion against sepsis-induced changes in apoptosis, prolifer-ation, and villus length. Serum levels of interleukin (IL)-6 –which are elevated 8-fold in septic wild type mice – are

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unaltered in knockout animals. This pathway may also beimplicated in human sepsis. In a recent Phase II clinicaltrial, patients on long-term statin therapy (which is startedto lower cholesterol) had lower baseline IL-6 levels, andcontinuation of atorvastatin in patients with severe sepsiswas associated with a survival advantage [30].

Gut-derived lymph is sterile following experimentalT/HS [6]. However, there are multiple reports of bacterialtranslocation to the mesenteric lymph in both humanstudies and animal models of critical illness. In a burnmodel with acute alcohol ingestion, there is culture evi-dence of bacterial translocation within the mesentericlymph nodes [31]. Similarly, translocation of anaerobicbacteria to the mesenteric lymph nodes is noted followinghepatobiliary surgical procedures when assayed by bacte-rium-specific ribosomal RNA-targeted reverse transcrip-tase PCR [32], and translocation of Gram-negativebacteria to mesenteric lymph nodes is also implicated inthe development of bacteremia and/or spontaneous bacte-rial peritonitis in cirrhotic patients [33]. The literaturethus supports a role for both gut-derived sterile toxiclymph as well as bacterial translocation to the lymph ina complex and possibly disease-specific manner.

Intestinal integrityThe gut epithelium plays a number of important roles inmaintaining homeostasis in the healthy host including butnot limited to (i) absorbing food, (ii) acting as a barrieragainst invading pathogens, (iii) producing hormones andcytokines, (iv) producing antimicrobial peptides, and (v)continuously communicating with both the gut-associatedlymphoid tissue (the largest lymphatic organ in the body)and the intraluminal bacteria. Despite these myriad roles,the gut is made up of only a single layer of columnar cellsthat are continuously renewed from multipotent stem cellsoriginating in the crypts of Lieberku hn (Figure 2). Intesti-nal stem cells express TLR4, which regulates whether theyproliferate or die by apoptosis [34,35]. These give rise to thefour major cell lineages, each of which plays a unique rolein the host [2]. As cells migrate up the villus, they differ-entiate into absorptive enterocytes, mucus-producing gob-let cells, and enteroendocrine cells where they die ofapoptosis or are exfoliated whole into the lumen. The finallineage is the Paneth cells, which migrate towards thecrypt base where they produce defensins. Remarkably, theintestine renews itself on a continual basis, replacingnearly all of its cells (except stem cells and longer livedPaneth cells) every 3–5 days.

Critically ill hosts have numerous alterations in gutintegrity that are thought to be important in the progres-sion of sepsis and MODS. Although no single definition ofgut integrity exists, we believe it is appropriate to considersubcellular, cellular, and organ-wide components whenconsidering gut integrity, as alterations at any of theselevels may play a crucial role in the progression of MODS.On a subcellular level, redox is altered with septic perito-nitis oxidizing glutathione/glutathione disulfide redox [36].On a cellular level, crypt proliferation is markedly de-creased [37] and both crypt and villus apoptosis are simul-taneously increased [38] following sepsis. Althoughepithelial migration is slowed in critical illness in a

Commensal bacteria

Enterocyte

Intraepithelial lymphocytes

Stem cells

Paneth cells

Goblet cells

Villus

CryptGALT

Mucus layer

Key:


Go

ucus layer

Key:

mme

teroc

raep

em ce

neth

Co

Ent

Int

Ste

Pan

GALT

Figure 2. Anatomy of the intestine. Epithelial proliferation is initiated in stem cells and is restricted to the crypt. Epithelial cells migrate upwards to the villus (enterocytes,

goblet cells, enteroendocrine cells) or downwards to the crypt base (Paneth cells) where they differentiate. The epithelium is lined by a mucus layer acting as the first line of

defense against the greater than 100 trillion commensal bacteria that reside within the gut lumen. The gut has a complex immune system including intraepithelial

lymphocytes and elements of the gut-associated lymphatic tissue (GALT) present in Peyer’s patches.



TLR4-dependent manner [39], changes in proliferation andapoptosis overwhelm this slowed progression of cells, result-ing in a marked diminution of villus length [40]. Simulta-neously, critical illness induces global alterations in themucus layer (reduced thickness, diminished luminal cover-age, and poor adherence) [41] and gut barrier function [42].

Dysregulated apoptosisThe gut and spleen are the only solid organs in whichapoptosis is reliably found on autopsy in human studiesand animal models of critical illness, and intestinal epi-thelial cells are the only epithelial cells consistently foundto undergo apoptosis in this setting [43,44]. Notably, mul-tiple comorbidities including cancer, chronic alcohol abuse,and aging are all associated with increased gut epithelialapoptosis in sepsis [9,45,46].

The relationship between gut apoptosis and survival iscomplex in animal models of critical illness with varyingdegrees of cell death as well as timing of apoptosis depen-dent on the model analyzed [47]. Overexpression of theantiapoptotic protein Bcl-2 improves survival in murinesepsis models of either peritonitis or P. aeruginosa pneu-monia models, but does not confer a survival advantage inacute lung injury [48–50].

The circuitry controlling apoptosis is complex [51], andseveral mechanisms have been shown to be involved inincreased gut epithelial apoptosis (Figure 3). Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia-in-duced sepsis induces increases in proapoptotic proteinsBax and Bid and the antiapoptotic protein Bcl-xL in themitochondrial pathway [38]. This is consistent with resultsdemonstrating these three mediators are elevated in cecal

ligation and puncture (CLP), a model of septic peritonitis[52]. MRSA pneumonia also simultaneously induces anincrease in Fas ligand but a decrease in Fas, fas-associatedprotein with death domain (FADD), phospho-FADD(pFADD), tumor necrosis factor receptor-1 (TNF-R1),and tumor necrosis factor receptor type 1-associated deathdomain protein (TRADD) in the receptor-mediated path-way. These changes appear to be at least partially organ-ism-specific because P. aeruginosa pneumonia-inducedincreases in gut apoptosis are associated with increasedBcl-2 in the mitochondrial pathway and increased TNF-R1and decreased Fas in the receptor-mediated pathway.Additionally, aged (but not young) mice deficient in heatshock protein 70 have increased mortality compared withage-matched wild type mice, and this is associated withincreased gut apoptosis in knockout mice [53].

Sepsis-induced gut epithelial apoptosis is mediated byCD4+ lymphocytes [52]. Although unmanipulated Rag�/�

mice (that lack lymphocytes) and wild type mice havesimilar amounts of gut apoptosis, CLP induces a 5-foldaugmentation of gut apoptosis in Rag�/� mice, above andbeyond the increase in gut apoptosis induced by sepsis.Subset analysis indicates that CD4+, but not CD8+, gd or Bcells are responsible for the antiapoptotic effect of theadaptive immune system. Notably, the survival benefitconferred by gut overexpression of Bcl-2 is also dependenton lymphocytes because this intestine-specific transgenefails to alter mortality when overexpressed in Rag�/�mice,and adoptively transferring lymphocytes to these animalsrestores their survival benefit.

No current treatment exists which specifically targetsgut epithelial apoptosis. However, targeting global gut

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Fas TNF-R1

Caspase-8

Apoptosome

Bax/Bak

Cytochrome c +

Apaf-1

Death Caspase-9

Procaspase-3Caspase-3

Bcl-2Bcl-xLIntracellular

death signal

Caspas e-2

TNF

FasL

Recep tor-mediatedpathway

Transloca�on

Mitochondrialpathway

Mitochondria

FADD

Procaspase-8

TRADD


Procaspase-9

Bid

tBid

Bax/Bak

Figure 3. Pathways of apoptosis. Apoptosis occurs via the receptor-mediated pathway (green arrows) or mitochondrial pathway (gray arrows). Proapoptotic genes are

shown in blue, and antiapoptotic genes are shown in white. Inactive procaspases (orange) become activated caspases (yellow). Intracellular signals (black arrows)

activating caspase-2 may also initiate the mitochondrial pathway. Sepsis induces apoptosis in an organism- and model-specific manner, in which multiple components of

each pathway are affected. Both pathways converge at active casapase-3, which initiates cell death.



integrity represents an attractive strategy in the treat-ment of sepsis [54]. Epidermal growth factor (EGF) is apotent cytoprotective peptide that has healing effects onthe intestinal mucosa. EGF reverses sepsis-inducedchanges in apoptosis, proliferation, villus length, and per-meability in both CLP and P. aeruginosa pneumonia[40,55]. Notably, giving systemic EGF improves survivalin both of these diseases, even when started 24 h afterpneumonia. In addition, overexpression of EGF in anintestine-specific manner improves both survival and gutintegrity, suggesting that EGF may be a novel therapeuticfor sepsis by targeting gut integrity [40,56]. A similarsurvival advantage is seen after giving IL-15 to mice madeseptic via CLP or P. aeruginosa pneumonia [57]. IL-15 is apluripotent antiapoptotic cytokine that decreases sepsis-induced gut apoptosis, although the relationship betweengut apoptosis and survival in sepsis is associative in light ofthe fact that IL-15 also blocks apoptosis in natural killer(NK) cells, dendritic cells, and CD8+ cells, as well asaltering systemic cytokines.

The mucus layerThe first line of defense against invading microorganismsis the intestinal mucus, and alterations in either thecharacter or quantity of mucus can have a deleteriousimpact on the critically ill host. The unstirred mucus layer

4

is hydrophobic and serves as an important barrier againstthe microflora in the gut lumen.

Mucus is the main barrier preventing digestive enzymesfrom entering the epithelium and causing epithelial dis-ruption, a process that occurs, at least in part, via degra-dation of E-cadherin and TLR4 [58]. This process can beinhibited in shock or peritonitis using tranexamic acid,aprotinin, or 6-amidino-2-naphtyl p-guanidinobenzoatedimethanesulfate to inhibit proteases, and this approachleads to improved survival in preclinical models of criticalillness [59,60]. Because the presence of digestive enzymesin the wall of the intestine can cause autodigestion – aprocess that has been hypothesized to cause MODS – therole of mucus in preventing this cascade may be critical forthe maintenance of homeostasis [61].

I/R induces an acute loss of the mucus layer with adecrease in its hydrophobicity, which, in turn, is closelycorrelated to increased gut permeability [62]. Similarly, T/HS induces breakdown of the mucus layer, and there is adirect correlation between loss of mucus layer and severityof villus injury [63]. Interestingly, this is gender-depen-dent, as mucus is better preserved in female rats, associ-ated to some degree with the estrus cycle [64]. Although themechanisms underlying mucus degradation are not fullyelucidated, it is known that I/R breaks down mucus, atleast in part, by degrading mucin 2 and fragmenting mucin



13 [58]. Additionally, T/HS induces increased reactiveoxygen species (ROS)-mediated mucus damage and lossof total antioxidant capacity immediately after shock,which is ameliorated by dimethyl sulfoxide (a free radicalscavenger). T/HS also induces delayed reactive nitrogenintermediate-mediated mucus damage 3 h later [65].

Digestive enzymes do not disturb mucin integrity inisolation. However, pancreatic proteases have been shownto play a synergistic role in mucus injury and, whencombined with a mucolytic, they worsen mucus injury overa mucolytic alone [66,67]. However, although mucus injuryis associated with villus injury, it is not sufficient in normalrats to cause the production of toxic gut-derived lymph orcause secondary lung injury, suggesting that mucus injuryis additive to other insults but not sufficient to causesystemic injury from the gut.

Intestinal epithelium – a selective barrierAlthough the intestinal tract is lined with only a singlelayer of epithelial cells, it acts as a selective barrier, thatwhen functioning properly, allows physiological move-ment of important elements but prevents pathophysiolog-ical movement of potentially dangerous ones. The gut

ZO

JAMTightjunc�on

Adherensjunc�on

MLCK

Myo

sin

β-catenin

α-catenin 1

ZO

Figure 4. The gut barrier. The single layer of columnar cells in the intestinal epithelium

the paracellular movement of intraluminal contents. Extracellular proteins, JAM, occludi

myosin ring to regulate permeability. MLCK modulates contraction of the actin–myosin r

myosin ring to form the adherens junction. Abbreviations: JAM, junctional adherens m

barrier allows paracellular movement of water, solutes,and immune modulating factors while preventing move-ment of large luminal molecules and microorganisms [68].This is done in large part via the apical tight junction andjunctional adherens molecules (JAMs), which preventluminal contents from escaping into the local extralum-inal environment and potentially from systemic spread(Figure 4).

Changes in intestinal permeability are seen in bothsepsis and noninfectious (T/HS, I/R, burn) models of criticalillness [7,41,69]. Intestinal hyperpermeability occurs whenany of these disease processes alters the expression ofzonula occludens 1 (ZO-1), any of multiple claudin iso-forms, or occludin in the tight junction or alternativelyby altering expression of components of JAMs [70]. Theparacellular route of transport is modulated by multipleintracellular and extracellular factors. Cytokines can acton junctional complexes to modulate permeability. Activa-tion of myosin light chain kinase (MLCK) can also worsenparacellular permeability. MLCK phosphorylates the my-osin light chain, which results in contraction or opening ofthe apical tight junction [70]. MLCK activation is alsoassociated with an increase in IL-6, TNFa, and IL-1b that

ZO

Occludin

Claudin

JAM

Perijunc�onalac�n–myosincomplex

E-cadherin

ZO

β-cateninnin

α-catenin 1

MLCK

Myosin


are held together by tight junctions and adherens junctions (inset), which regulate

ns, and claudins, interact with intracellular ZO proteins and the perijunctional actin–

ing. Extracellular cadherins along with intracellular catenins interact with the actin–

olecule; ZO, zonula occludens; MLCK, myosin light chain kinase.

5



further activates MLCK, which in turn increases intestinalpermeability, at least in part, via altered ZO-1 proteindynamics and occludin removal, ultimately leading to anamplification of the systemic inflammatory response in afeed-forward response [31,71].

Numerous efforts at improving intestinal hyperperme-ability – either in isolation or in conjunction with attemptsto improve other components of gut integrity – have re-cently been described. One strategy involves targetingMLCK. Intraperitoneal injection of the MLCK inhibitorML-9 attenuates burn-induced intestinal hyperpermeabil-ity, and this is associated with normalization of alterationsin claudin-1, ZO-1, and occludin [69]. Similarly, animalssubjected to binge ethanol exposure and burn injury havemorphological damage in their intestines, elevated intes-tinal IL-6 and IL-1b, increased bacterial translocation andreduced ZO-1 and occludin localization in the tight junctioncompared with sham mice [31]. However, mice that receivePIK, an inhibitor of MLCK, do not have any of thesechanges.

Growth factors represent potential therapeutic candi-dates that mediate their effect, at least in part, via modu-lation of the gut barrier. As outlined above, EGF improvessurvival in sepsis, whether given systemically followingCLP or pneumonia or delivered in an intestine-specificmanner via overexpression in transgenic mice [40,55,56].EGF treatment normalizes gut permeability to shamlevels, and this is associated with decreases in claudin-2as well as changes in subcellular localization of this tightjunction mediator. Exogenous EGF also improves gutpermeability in a rat model of I/R when given in a pre-treatment or post-treatment manner, associated withalterations in ZO-1 and occludin expression as well asreduced systemic and intestinal IL-6 and TNFa [72].Exogenous heparin binding epidermal growth factor-likegrowth factor (HB-EGF) also induces beneficial effects.Animals subjected to CLP given exogenous HB-EGF haveimproved survival, associated with prevention of sepsis-induced intestinal hyperpermeability, increased villuslength, gut apoptosis, and bacterial counts [73]. Of note,exogenous HB-EGF is effective in reversing these deficitsin septic HB-EGF deficient animals, which have worsenedgut integrity compared with septic wild type animals. HB-EGF probably promotes its beneficial effects in multiplemanners, including promoting integrin–extracellular ma-trix interactions as well as intercellular adhesions [74]and preventing intestinal stem cells from injury underpathological conditions [75]. Additionally, milk fat glob-ule-EGF factor 8 also improves survival in both CLP andhemorrhagic shock and decreases both inflammation andtissue injury, although its impact on intestinal permeabil-ity is unknown [76,77].

MicrobiomeThe intestinal microbiome is made up of more than 100trillion microorganisms and is continuously changing overthe life of the host, based on diet, age, drug intake, andpresence or absence of disease [78–80]. Increasingly, it isrecognized that the microbiome plays a crucial role in themaintenance of health and that alterations in both thenumber and function of microorganisms in the microbiome

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can have a critical role on survival – both for good and for ill– in critical illness.

The most reductionist method of assessing the role ofthe microbiome in critical illness is using germ-free mice,which entirely lack an endogenous microflora. Consistentwith the complexity of the microbiome, germ-free micehave widely varying responses in preclinical models ofcritical illness. Germ-free mice have markedly higher mor-tality following P. aeruginosa pneumonia and have alteredintestinal epithelial apoptosis [81]. Further, sepsis-in-duced lymphocyte-dependent increases in gut epithelialapoptosis appear to be mediated by the microbiome in lightof the fact that septic germ-free Rag�/� mice have similarlevels of apoptosis as septic germ-free wild type mice(unlike in conventional animals where gut apoptosis ismarkedly higher following sepsis in Rag�/� mice). Altera-tions in mortality in septic germ-free mice are associatedwith lower TNF and IL-1b levels without alterations insystemic cytokines or intestinal proliferation or permeabil-ity. Similar results are found in germ-free mice subjected toKlebsiella pneumoniae pneumonia, which have significant-ly higher mortality than conventional mice, mediated in anIL-10-dependent manner, modulatable by TLR activation[82]. By contrast, germ-free mice have improved survivalcompared with animals with an intact microflora whensubjected to hemorrhagic shock [83]. Additionally, germ-free animals have 100% survival in a model of I/R that is100% lethal in conventional animals, associated with anabrogation of the systemic inflammatory response [84].These disparate responses highlight the importance ofdisease model (and accompanying timing and pathophysi-ology) in mediating mortality in germ-free mice. They alsosuggest that although germ-free mice represent an impor-tant tool in understanding the microbiome, there may alsobe limitations to conclusions that can be drawn because thevery strength of these animals (total absence of endoge-nous flora) is very distinct from ICU patients that have alifetime of host–microbial interactions.

Microbes alter their own virulenceAn important aspect of the microbiome is its interactionwith the host and the role this plays in the progression ofdisease [85]. Although host and bacteria live under com-mensal or symbiotic conditions under homeostasis, altera-tions in this relationship drive bacteria to a morepathogenic phenotype (Figure 5). The concept that bacteriaare able to sense their environmental state, includingdensity of surrounding bacteria, and adjust behavior/viru-lence accordingly is a new direction in critical illnessresearch and represents an evolution in our understandingof how the gut can function as the motor of critical illness.This has profound clinical implications because pathogenidentification alone without attention to its virulence maynot be sufficient for treating critically ill patients.

The prototypical example of inducible bacterial viru-lence is P. aeruginosa, as during injury, compounds arereleased by the host that either bind to or are taken up bythis microorganism, leading to activation of its quorumsensing system, which in turn leads to the development of alethal phenotype [86,87]. Importantly, not only do activat-ed microorganisms employ a variety of virulence tactics

Commensal bacteria

Mucus layer

Tig htjunc�on


Figure 5. Microbiome virulence induction. Commensal bacteria are able to sense the host microenvironment and other bacterial populations around them. Under basal

conditions, the microbiome has a colonizing and/or symbiotic relationship with the host. During critical illness, microorganisms receive signals from both the host and

surrounding bacteria that drive them to a virulent phenotype. Newly pathogenic bacteria can then further injure the already compromised host in a maladaptive positive

feedback loop.



that subvert normal clearance mechanisms but they arealso able to cause sepsis and remote organ failure withoutsystemic dissemination. This is relevant both in isolationand in combination with other injury as evidenced by thefact that mortality is significantly higher in a murinemodel of intestinal I/R with superimposed inoculation ofP. aeruginosa with in vivo virulence expression of thebarrier disrupting adhesin, P. aeruginosa LecA (PA-IL),but not in mutant bacterial strains lacking this virulenceprotein [88]. Another direct example of the acquired viru-lence of P. aeruginosa is seen following surgical insult.When this microorganism is harvested after inoculationinto the cecum of mice subjected to sham surgery, it causesno lethality when injected into the peritoneum of nonin-jured mice. However, when P. aeruginosa is harvestedafter inoculation into the cecum of mice subjected to30% hepatectomy, it causes 100% lethality when injectedinto the peritoneum of noninjured mice, demonstratingthat surgical injury alters the virulence of the organism[89]. In addition, bacteria isolated from the gut of mice thatundergo hepatectomy demonstrate a distinct morphologi-cal appearance (wrinkled) compared with bacteria isolatedfrom sham animals (smooth phenotype). Notably, Candidaalbicans has the ability to alter its own virulence in re-sponse to surgical injury in the same model [90].

Targeting the microbiome for therapeutic gainRecent work has demonstrated that phosphate depletionmay be a key modulatable determinant of altered microbial

virulence. In times of extreme physiological stress, phos-phate is depleted in the host. Local phosphate concentra-tion functions as an important cue by which endogenousflora evaluate the resources and health status of a host,and determine whether they should colonize or invade ahost [91,92]. For example, phosphate limitation induces C.albicans isolated from the gut of critically ill patients toexpress filaments and a lethal phenotype [90]. Further, inthe presence of opioids, virulence is induced in P. aerugi-nosa via quinolone signal production; however, this geneticshift towards virulence is prevented in the presence ofabundant phosphate [93]. These findings suggest the pos-sibility that maintaining local phosphate abundance mayprevent virulence within multiple components of themicrobiome and represent a novel therapeutic in the fightagainst gut-derived sepsis [94].

In light of the observation that altered gut flora mea-sured in stool are associated with an increased risk of deathin patients with systemic inflammation [95], another strat-egy to target the microbiome is the use of probiotics,prebiotics, and synbiotics. Probiotics are exogenous liveorganisms, whereas prebiotics are nondigestible nutrientsthat stimulate commensal bacterial growth. Synbiotics area combination of both probiotics and prebiotics. Althoughtheir clinical use is rising, data on efficacy and mechanismsinduced by stimulating the microbiome are still emerging[96]. Probiotics improve mortality, bacteremia, and pre-vent sepsis-induced changes in gut epithelial apoptosis andproliferation following CLP [97]. Additionally, probiotics

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can attenuate growth of intestinal bacteria, potentiallylimiting endotoxin production, and preventing bacteremia[96]. As a corollary to probiotic/synbiotic administration,there is also increasing evidence that fecal microbiotatransplant is significantly more effective in the treatmentof recurrent Clostridium difficile infection than standardantibiotic therapy by increasing fecal bacterial diversity inrecipients [98].

Concluding remarksThe concept of the gut as the motor of critical illness is now30 years old. Although the original iteration of this theory isnot strongly supported by experimental and observationaldata, the concept that the gut might drive critical illness is asrelevant now as it ever has been. During critical illness, thecombined effect of erosion of the mucus barrier, a shift in thecomposition and virulence of intestinal microbes, and theinability of the host epithelium to regulate its proliferativeand apoptotic response may lead to a tipping point in gutfunction where cascading inflammation drives distant organfailure. Additionally, the formation of toxic gut-derivedlymph and alterations in the microbiome resulting in de-structive host–pathogen crosstalk further propagate thecascade of distal organ injury. Although each of these ab-normalities is likely to be detrimental in isolation, a feed-forward loop probably exists in which damage to one com-ponent of the gut leads to local and systemic injury, which, inturn, damages other components of the gut. Unless broken, acontinuous cycle of injury/amplification/repeat can lead todevastating consequences. In theory, rationale therapiesaimed at restoring gut integrity, the microbiome, and thehomeostatic balance between the two systems represents anexciting avenue in the battle against critical illness, repre-senting a proximal target in the road to multiple organdysfunction that can be impacted early in the course ofcritical illness, prior to global dysregulation. Further studiesneed to be performed in the context of the gut as a multi-component organ, with examination not only of the elementtargeted but also other local and systemic processes. Whichapproach in targeting the gut will be most effective inrevving down its role as a motor of critical illness representsa prime research challenge for the next decade.

AcknowledgmentsThis work was supported by funding from the National Institutes ofHealth (GM072808, GM104323, GM095442).

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Redefining the gut as the motor of critical illness

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