Gut-Lymph-Lung Pathway Mediates Sepsis-Induced Acute Lung Injury

Gut-Lymph-Lung Pathway Mediates Sepsis-Induced Acute Lung Injury

The gut-lymph (GL) theory, first proposed by Deitch et al. in 2006, has its roots in the pathophysiology of critical illness. This theory suggests that intestinal ischemia-reperfusion injury (IRI), which often occurs with restored reperfusion of microcirculation in critically ill patients with injury-induced shock, leads to the absorption of intestinal toxic substances into the mesenteric lymphatic (ML) system. These substances are then transported into the bloodstream, activating the monocyte-macrophage system (MMS) and triggering a systemic inflammatory response that further aggravates intestinal injury. This process of successive iteration results in a cascade amplification of inflammation.

Early studies indicated that bacterial translocation resulting from the destruction of intestinal mucosal integrity poses a threat of multiple organ dysfunction syndrome (MODS). However, recent experimental evidence suggests that GL acts as a carrier of danger-associated molecular patterns to systemic blood circulation and different organs, leading to MODS. Acute respiratory distress syndrome (ARDS) can be induced in healthy rats by injecting GL fluid (GLF) from rat models of traumatic/hemorrhagic shock (T/HS), which involves severe abdominal infection. Simultaneous ligation of GL vessels or drainage of GLF in this model alleviated the conditions of ARDS. Therefore, the gut is considered the origin of systemic inflammatory responses and the engine of MODS, playing an essential role in the initiation of ARDS.

Bioactive Substances: From Mediator to Molecule

In response to injuries such as shock, extensive burns, severe infection, and severe trauma, the gut releases a large number of inflammatory mediators, cytokines, and bioactive substances such as exosomes, high mobility group protein B1, tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), arachidonic acid metabolite leukotriene (LTB4), and hypoxia-inducible factor-1α (HIF-1α), all of which play a pivotal role in ARDS.

Exosomes, extracellular vesicles secreted by immune cells approximately 40 to 100 nm in size, have inflammatory and immunoregulatory effects on target cells. They can transfer lipid mediators such as fatty acids, prostaglandins, and leukotrienes from host cells to target cells, playing an important role in the activation of target cells. The number of exosomes in body fluids is altered by various injuries such as inflammation, trauma, and infection.

Kojima et al. first identified ML exosomes in rats using vesicle flow cytometry. These exosomes primarily originate from intestinal epithelial cells and are unaffected by T/HS. The occurrence of intestinal IRI leads to neutrophil aggregation and the release of granules containing exosomes through endoplasmic reticulum stress mediated by complement 5a receptor, promoting the development of ARDS.

IL-8 homologues are the major cytokines for neutrophil aggregation. Kojima’s results showed that exosomes obtained from ML after T/HS treatment had significantly increased IL-8 expression. Furthermore, injection of ML exosomes into neonatal mice after T/HS increased secretion of MIP-2 (a rat IL-8 homologue) and numbers of infiltrating neutrophils in bronchoalveolar lavage fluid (BALF), leading to acute lung injury (ALI). Moreover, after intestinal IRI, ML exosomes triggered activation of nuclear factor kappa B (NF-κB) in monocytes and macrophages and induced the production of pro-inflammatory cytokines by monocytes and macrophages via a Toll-like receptor (TLR)-dependent signaling pathway.

Sakamoto et al. reported that the lung captured most exosomes injected into ML catheters, suggesting that the lung is the principal target for ML to carry exosomes. Kojima et al. injected ML exosomes into immature mice before and after shock, finding that ML exosomes had moved to the alveolar cavity and pulmonary parenchyma 6 hours after injection and were absorbed by macrophages and CD68+ pulmonary macrophages, indicating that exosomes could reach the lung and be absorbed by macrophages in vivo.

As the central link of the immune response, dendritic cells (DCs) are powerful antigen-presenting cells that can absorb, process, and present antigens. Another study by Kojima et al. showed that after T/HS, expression of immunoregulatory molecules such as major histocompatibility complex II was significantly increased in ML exosomes. Compared with ML exosomes before shock, co-incubation of DCs with ML exosomes after T/HS increased the apoptotic rate of DCs two-fold. Additionally, ML exosomes after T/HS inhibited the antigen-presenting ability, maturation, and antigen-expression function of DCs and promoted their apoptosis. Thus, ML exosomes are a novel mediator of post-injury immunosuppression.

When intestinal IRI occurs, lipopolysaccharide (LPS) in circulating blood activates TLR4. With the assistance of extracellular LPS-binding protein and CD14 myeloid differentiation protein 2, the LPS-stimulating signal is transduced intracellularly, inducing a signal cascade reaction through two myeloid differentiation factor 88-dependent factors to increase expression of inflammatory factors, ultimately triggering an inflammatory response. The inflammatory mediator high mobility group protein B1, as well as cytokines (TNF-α, IL-6, etc.) and LTB4, plays an important role in the GL theory of MODS. However, the key component of this mechanism is still activation of MMS by endotoxin. LPS-activated MMS mainly depends on TLR4, which can transmit the LPS extracellular signal into intracellular signaling with the help of LPS-binding protein, further activating transcription factors such as NF-κB and activator protein 1. Activation of NF-κB and activator protein 1 also induces the release of interferon, intercellular adhesion molecules, and cytokines such as TNF-α and IL-6. The presence of TNF-α, IL-6, and other inflammatory factors in circulating blood can further increase the permeability of intestinal mucosa, thereby increasing intestinal bacteria and endotoxin entering the interstitial space of intestinal mucosa by GL absorption into the lymphatic circulation. This process starts a new round of MMS activation, forming a vicious cascade of inflammatory amplification that ultimately results in distant organ injury.

Zhang et al. showed that levels of TLR4, NF-κB, and human IL-1 receptor-related kinase 4 were increased in the ileum of rats with abdominal infection compared with sham-operated rats. Indeed, levels of TNF-α, IL-6, IL-4, monocyte chemotactic proteins, and intracellular adhesion molecule 1 in plasma, lymph, and BALF, as well as intestinal and lung tissues, were increased by abdominal infection. Furthermore, GL drainage decreased levels of these factors in BALF and lung tissue. These results are consistent with the reduction of lung injury observed in rats infected by GL drainage.

Abnormal oxygen metabolism and cascade amplification of the inflammatory response are important pathophysiologic features of sepsis-induced ALI. HIF-1α, a transcription factor, is considered the principal regulator of ischemia/hypoxia. Studies have shown that HIF-1α could be activated by an inflammatory state, suggesting that HIF-1α is closely related to the inflammatory process. Sun et al. injected septic lymph (SL) of rats and saline into the jugular vein of healthy rats. Injection of SL could obviously induce HIF-1α production, and levels of lung injury scale and myeloperoxidase were considerably increased compared with saline-injected rats. These results indicate that HIF-1α has a potential stimulatory effect on neutrophils in lymph node ALI of sepsis. Activation of neutrophils could lead to endothelial and erythrocyte dysfunction, which may contribute to ALI. Furthermore, preoperative intra-tracheal drip of 3-(5′-hydroxymethyl-2′-furan)-1-benzylamphetazole (YC-1) in SL-infused rats showed a tendency to reduce the signs of pulmonary inflammation injury, especially the level of myeloperoxidase. These results suggest that YC-1 can inhibit HIF-1α expression and the progress of inflammation induced by HIF-1α. Thus, HIF-1α plays an important role in hypoxia and inflammation.

Proteins and Lipids

GL contains a large number of proteins and lipids, which have an essential impact on biologic activity. Research from Fang et al. indicated an increased abundance of serum albumin precursors, creatine kinase B, and actin in ML nodes after shock; most notably, the abundance of serum albumin precursors increased by 8.5-fold. Previous studies have shown that in some inflammatory conditions, such as mycotic keratitis, serum albumin precursors in body fluids are increased, and increased creatine kinase B, a component of the cytoskeleton network, in the circulation after muscle injury is considered to result from passive or active release. Actin is a central component of cytoskeletal structures. Previous studies demonstrated that HS could result in selective destruction of the intestinal actin cytoskeleton. Collectively, these results indicate that serum albumin precursor, creatine kinase B, and actin are associated with the process of inflammation and injury.

Trypsin, a proteolytic enzyme released from damaged cells, can lead to excessive protein catabolism and aggravation of cellular damage. Zhao et al. found that injecting post-HS mesenteric lymph (PHSML) from rats into the veins of normal rats could significantly increase trypsin activity in plasma, suggesting that toxic components in PHSML cause damage to cells and increase the release of trypsin.

Previously, Mittal et al. showed that ALI could be significantly reduced by blocking or neutralizing pancreatic enzymes entering the intestinal cavity in rats with acute pancreatitis. Upon analyzing the histology of GLF after T/HS, Morishita et al. observed a variety of lipids in PHSML, including phosphatidylcholine, lysophosphatidylcholine (LPC), phosphatidylethanolamine, lysophosphatidylethanolamine (LPE), and sphingomyelin. Compared with the sham shock group (T/SS), levels of linoleoyl, arachidonoyls, docosahexaenol LPCs, and LPEs were significantly increased in PHSML of the T/HS group. Significantly increased LPCs and LPEs containing polyunsaturated fatty acids in PHSML can induce cytotoxic reactions, which in turn can induce persistent superoxide anion (O2−) production. Increased O2− production and elastase release have been shown to lead to ARDS/MODS.

Gut phospholipase A (2) (PLA 2), a neutral lipid, was extracted from PHSML. It is responsible for the initiation of pro-inflammatory lipids in PHSML, which is why the cytotoxicity is increased after polymorphonuclear neutrophils (PMNs) oxidative burst. The physiologic concentration of PHSML lipids will trigger PMN respiratory bursts, and the production of superoxide is twice that of lymphatic cells before shock. The systemic use of gut PLA 2 inhibition before HS eliminated the neutrophil initiation effect of PHSML, suggesting that T/HS may be at the core of the pathogenesis of ARDS/MODS, providing a potentially therapeutic mechanism for acute lung injury after shock.

Bacteria and Endotoxins

Intestinal IRI results in impaired intestinal mucosal barriers, intestinal bacterial translocation, and release of inflammatory mediators and cytokines. Early studies suggested that when intestinal IRI occurred, intestinal barrier function was impaired and intestinal permeability increased, leading to bacterial and endotoxin translocation. Bacteria and endotoxin primarily enter systemic blood circulation through the portal vein system and GL system, leading to distant organ injury. Adams et al. found that the GL of rats with HS and burn was sterile, and no endotoxin was detected. Our unpublished results indicate increased endotoxin levels in GL of rats with IRI compared with sham-operated rats. Reasons underlying these conflicting experimental results may be related to differences in bacterial detection techniques and/or use in different laboratories. Deitch et al. suggested that translocation of bacteria and endotoxins may not exist in GL, but instead be a property of intestinal walls and ML nodes, which induce local inflammation and stimulate the production of a large number of cytokines, thus aggravating systemic inflammatory response syndrome and leading to MODS.

Bio-Mechanism: From Phenomenon to Mechanism

In addition to identifying endogenous risk types and alarm factors, TLR4 has been shown to identify and respond to endogenous ligands, suggesting that it can be used as a sensor for tissue damage and microbial invasion. Cells carrying TLR4 in vivo include innate immune system cells such as macrophages, neutrophils, and DCs. Kojima et al. showed that TLR4 was the subject receptor of ML exosome and could induce macrophage inflammation; moreover, the TLR4 signaling pathway was necessary for ML exosomes to induce macrophage activation. Reino et al. showed that the lung permeability of wild-type mice transfused with T/HS lymph was 2.5-times higher than that of mice transfused with T/SS lymph; however, the lung permeability of TLR4-deficient mice injected with T/HS lymph did not increase significantly. There was no difference in myeloperoxidase levels in TLR4-deficient mice injected with T/HS or T/SS lymph nodes. Compared with wild-type mice, inducible nitric oxide synthase (iNOS) expression in the lungs of TLR4-deficient mice injected with T/HS lymphocytes decreased significantly, suggesting that T/HS lymphokines mediate lung injury through TLR4 activation. Moreover, lack of TLR4 attenuates the lung iNOS response to T/HS lymph-induced lung injury.

PHSML contains large amounts of arachidonic acid when they return to the systemic circulation. LTs are products of arachidonic acid metabolized by the 5-lipoxygenase (5-LO) pathway. 5-LO is a rate-limiting enzyme for LT synthesis and is an important factor causing lung dysfunction after secondary T/HS. 5-LO activation is considered to be an important regulator of the pathogenesis of multi-cellular organisms. Monteiro et al.’s study has shown that IRI and even sepsis patients treated with 5-LO inhibitors are significantly protected from lung inflammation and injury. It can be seen that 5-LO products play an important role in lung injury caused by sepsis, and also suggest that the application of 5-LO inhibitors may have clinical therapeutic value. LTs mainly regulate PMN transport in IRI by regulating the expression of adhesion molecules. In vitro studies show that PHSML activate neutrophils, increase endothelial cell monolayer permeability, and may even cause endothelial cell death. Mesenteric lymph enters the subclavian vein through the thoracic duct, which directly flows into the heart and lungs. The lung is the first organ that contacts the mesenteric lymph. Adams et al. demonstrated that T/HS-induced PMN activation is caused by factors carried in intestinal lymph fluids by enhancing PMN’s response to inflammatory mediators. These results support PMN in mediating the conversion of physiologic shock to immune inflammation. Play a leading role in startup. Moreover, the activation of macrophages in the lung not only produces oxygen-free radicals, lysine, and LTs, but also directly affects pulmonary vascular endothelial cells. In addition, TNF, IL-1, IL-6, and IL-8 can increase the adhesion of PMN to vascular endothelial cells, produce and release TNF, oxygen-free radicals, and nitric oxide near the alveolar epithelium, and cause damage to the alveolar epithelium.

Potential Interventional Targets

Gut-Lymph Pathway Blockage

A large number of studies have confirmed that GL ligation or drainage could effectively prevent intestinal toxic substances from entering systemic circulation in rats with IRI, thereby effectively reducing damage to distant organs, such as ALI. However, both interventions are anti-physiologic and require abdominal surgery, which makes them difficult to be performed when intra-abdominal hypertension occurs in acute pancreatitis. The GL pathway is the most direct damage-associated molecular patterns pathway. We will use the GL pathway block as our key research direction in the future to overcome ARDS induced by GL after IRI.

5-Aminoimidazole-4-Carboxyimide Nucleoside

Idrovo et al. showed that 5-aminoimidazole-4-carboxyimide nucleoside (AICAR), an anti-inflammatory cell-penetrating compound, could alleviate organ injury and inflammation after IRI. AICAR treatment significantly reduced intestinal injury scores and water content, improved histologic integrity, alleviated tissue damage and release of pro-inflammatory cytokines, and decreased intestinal bacterial translocation. Attenuated neutrophil chemotaxis and infiltration into the lungs resulted in decreased levels of TNF-α and IL-6 in the lungs. Pulmonary iNOS and COX-2 protein levels decreased, and apoptosis of lung cells was markedly decreased after AICAR treatment.

3-(5′-Hydroxymethyl-2′-Furan)-1-Benzylamphetazole

As a specific inhibitor of HIF-1α, YC-1 inhibits HIF-1α expression in an inflammatory state. Intra-tracheal administration of YC-1 could substantially reduce neutrophil activation, pulmonary interstitial edema, and alveolar exudation in lung tissue, thereby reducing lung injury.

Vagus Nerve Stimulation

CPSI-121 is a derivative of ornithine hydrazide, which can activate the parasympathetic nervous system through an unknown mechanism. Krzyzaniak et al. confirmed that CPSI-121 could prevent intestinal barrier dysfunction after skin burns through a vagus nerve-dependent mechanism. After injecting CPSI-121 intravenously into T/HS rats, Morishita et al. found that the discharge rate of the vagus nerve increased, and both intestinal and lung injuries were prevented. Therefore, CPSI-121 could be used as a vagal-stimulating drug to limit end organ dysfunction after injury as a potential therapeutic strategy for ALI or ARDS.

Low-Level Laser Therapy

Low-level laser therapy (LLLT) generally involves the application of low-power laser or light-emitting diode light in the range of 1 to 500 mW to promote tissue regeneration and reduce inflammation and pain. In animal experiments, lasers have an inhibitory effect on pneumonia. Carvalho et al. showed that pulmonary vascular permeability, peribranchial edema, neutrophil accumulation, pro-inflammatory factor IL-8, and anti-inflammatory factor IL-10 increased dramatically after IRI in mice. After LLLT intervention, pulmonary vascular permeability and IL-8 were decreased, the level of anti-inflammatory factor IL-10 was increased, and peribranchial edema, neutrophil accumulation, and pulmonary inflammation were inhibited. LLLT has exhibited advantages for immune function in various studies.

In summary, GL plays a major role in the pathologic process of IRI-induced ARDS after intestinal IRI. Inflammatory mediators, cytokines, proteins, and lipids in GL and the TLR4 signaling pathway play a universal role in initiation of ARDS. Although recent studies have indicated that the treatment of GL has some therapeutic effect, there is still a lack of definite treatment measures to block lung injury after intestinal IRI. Thus, more research is required to identify prevention and treatment methods for IRI-induced ARDS. Gut is considered to be the origin of systemic inflammation, and the engine of MODS, while GL plays a pivotal role in initiation of extrapulmonary ARDS. Gut lymph purification, as a new technology deriving from continuous blood purification therapy, will become the next potential interventional target.

doi.org/10.1097/CM9.0000000000000928

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