LDK378 improves micro- and macro-circulation via alleviating STING-mediated inflammatory injury in a Sepsis rat model induced by Cecal ligation and puncture

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection [1, 2]. It is an acute systemic reaction to microbes that invade the body, accompanied by a strong innate immune response [3]. With the stimulation of pathogens, proinflammatory cytokines are released, generating systemic inflammatory responses that impair the micro- and macro-circulation function [4, 5]. Septic shock is a subset of sepsis with hemodynamic alterations and metabolic dysfunction associated with organ dysfunction. Alterations in microcirculatory blood flow have been identified in severe sepsis [6] and the severity of theses alteration is associated with a poor outcome [7].

Sepsis remains the primary cause of death in critical care units despite the use of modern antibiotics, ventilator management, and resuscitative therapies, affecting millions of people all over the world each year. There are currently no existing FDA-approved treatment options for sepsis, though many therapeutic clinical trials have been conducted. Mortality rates from sepsis are 41% in Europe and 28.3% in the United States [8]. The cost of sepsis has become the most expensive health-care problem in the United States, at more than 20 billion dollars annually. [9]

New insights into immune dysregulation illustrate an enduring inflammatory state driven by a dysfunctional innate and suppressed adaptive immunity that culminates in persistent organ injury and death. This initial inflammatory process, if not abated, also contributes to organ failure and early mortality [10, 11]. Inflammatory and anti-inflammatory responses, as well as innate and adaptive immune systems, represent potential targets for immune therapy to improve sepsis outcomes. Inflammatory response is mediated by the production of proinflammatory cytokines such as tumor necrosis factor TNF = a, IL-1 and IL-6. However, this early response is followed by a transition to sustained production of anti-inflammatory mediators such as IL-10. Animal studies of sepsis suggested a largely unopposed proinflammatory response to severe sepsis resulted in increased organ injury and mortality, whereas a greater anti-inflammatory response resulted in less severe sepsis.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER)-resident membrane protein which mediates cytosolic pathogen DNA-induced innate immunity and inflammatory responses in host defenses [12]. STING is activated by cyclic dinucleotide, then translocates to the Golgi apparatus, an event that triggers STING assembly with the downstream enzyme TANK-binding kinase 1 (TBK1). This assembly leads to the phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3), Phosphorylated IRF3 forms a homo-dimer to enter the nucleus and functions together with NF-kB (p65) to induce type-I interferon (IFN) and other pro-inflammatory cytokines [13]. STING was identified by investigators screening cDNA libraries for genes that, when overexpressed, were sufficient to activate the production of IFN [14]. Further studies have revealed STING-knockout mice are susceptible to lethal infection, demonstrating the critical role of STING in facilitating an immune response to pathogens [15].

The 3′3’-cGAMP is a type of cyclic dinucleotide (CDNs) and serve as a canonical STING ligand to induce the production of type I IFNs. LDK378, a novel ALK inhibitor approved for advanced-stage non-small cell lung cancer (NSCLC) with ALK gene rearrangement, was recently found to have the effect of blocks 3′3’-cGAMP-induced IFNβrelease in immortalized bone marrow-derived macrophages (iBMDMs). Similar to pharmacological ALK inhibition, genetic inhibition of ALK also attenuated STING ligand-induced expression and release in IBMDMs. ALK seem to play an important role in the regulation of STING signaling pathway [16].

LDK378, a novel ALK inhibitor approved for advanced-stage non-small cell lung cancer (NSCLC) with ALK gene rearrangement, exhibited promising anti-inflammatory activity in animal models of lethal sepsis. Zeng, L. et al. [16] investigated macrophage activation in an in-vitro study and found that ALK inhibition increased septic animal survival in STING-deficient mice in vivo. Activation of the STING pathway by ALK may be a crucial step for its immunological activity and may contribute to the pathogenesis of sepsis.

The cecal ligation and puncture (CLP) model [17, 18] is considered the gold standard in sepsis research. In a CLP model, sepsis develops due to peritoneal contamination with mixed flora in the presence of devitalized or ischemic tissue, thus the resemblance to clinical reality. Furthermore, metabolic, immunological, and apoptotic responses are similar in the CLP model and in human disease.

Microcirculatory dysfunction is a pivotal element of the pathogenesis of sepsis. Because the microcirculation is the primary site of oxygen and nutrient exchange, therapeutic interventions aimed at increasing organ perfusion should be accompanied by improved microvascular perfusion.

This study aimed to demonstrate two hypotheses: first, LDK378 can alleviate microcirculation and hemodynamics of CLP-induced septic rats, thereby mitigating organic injuries and improving the survival rate. Second, LDK378 blocks the STING pathway activation through interfering with TBK-1-mediated signaling transduction.

The balance of inflammatory mediators is closely related to the severity and outcome of sepsis [19, 20]. Inflammatory cytokines play an important role in sepsis, as interplay between the initial inflammatory, and later, the anti-inflammatory responses lead to sepsis-induced organ dysfunction and lethality. Release of pro-inflammatory cytokines, particularly TNF-a, IL-6 is an important component of the host immune response and the role of these molecules in the pathogenesis of sepsis has been well studied. Overproduction of pro-inflammatory cytokines has been demonstrated and concentrations shown to correlate with severity and outcome of sepsis. In sepsis, inflammatory stimuli also activate counter-inflammatory cytokines such as IL-10 that can downregulate the host inflammatory response, and may have a key role to play in controlling the pro-inflammatory cytokine response. In the present study, CLP-induced sepsis caused an inflammatory response and damage in the heart, lung and kidney. The treatment with LDK378 injection ameliorated the pathological changes in main organic tissues caused by sepsis. Furthermore, we demonstrated the inhibitory effects of LDK378 on the sepsis-induced up-expression of TNF-a and IL-6 and the enhanced effects of LDK378 on IL-10 expression in septic rats. These data suggested that the protective effect of an LDK378 injection on organic injury might occur via the regulation of the secretion of inflammatory cytokines.

Because a common event in STING activation by different ligands is the phosphorylation of TBK1, the carboxy1 terminus of STING containing just 39 amino acids is necessary and sufficient to activate TBK1. Furthermore, STING not only activates TBK1 but also recruits IRF3 to TBK1 to activate the IRF3 pathway. Therefore, we examined STING by the expression and phosphorylation of TBK1. STING promotes the activation of TANK binding kinase 1 (TBK1), leading to IFN regulatory factor 3 (IRF-3) translocating into the nucleus for type I IFN transcription. In addition, the activation of STING induces its dimerization and ubiquitination, which are proposed to play important roles in the activation of IRF-3 signaling [21]. The activation of STING facilitates the recruitment of IRF-3 and TBK-1 into a complex where IRF-3 is phosphorylated. Phosphorylated IRF-3 forms dimers and is transported to the nucleus to activate transcription of type I IFN genes [21].

Anaplastic lymphoma kinase (ALK), a tumor-associated receptor tyrosine kinase, is an enzyme which in humans is encoded by the ALK gene. ALK is a member of the insulin receptor superfamily and shows the greatest sequence similarity to leukocyte tyrosine kinase (LTK), which is a receptor protein-tyrosine kinase. Twenty different ALK-fusion proteins that result from various chromosomal rearrangements have been identified, and they have been implicated in the pathogenesis of several diseases including anaplastic large-cell lymphoma, diffuse large B-cell lymphoma, and inflammatory myofibroblastic tumors [22]. A previous study found that both STING-KO mice and TRIF-KO mice were protected from sepsis in a severe CLP model [15], and the ALK-STING pathway was upregulated more in a human sepsis group than a control group. Both pharmacological and genetic disruption of ALK expression can diminish the stimulator of interferon gene-mediated host immune responses to CDNs in monocytes and macrophages [16]. These findings uncover that ALK plays a key role in modulating the inflammatory signaling pathway. But there is no direct interaction between ALK and phosphorylation of core components of the STING pathway. Zeng [16] found that the phosphorylation of epidermal growth factor receptor (EGFR) was up-regulated by 3′3’-cGAMP and c-di-AMP and ALK-EGFR-AKT pathway is a critical driver of STING activation in innate immune cells, the interplay between ALK and EGFR contributes to the AKT-dependent STING activation in macrophages and monocytes. In the present study, LDK378 treatment reduced p-TBK1 and its downstream transcription factors including phosphorylation of IRF3 and NF-kB in CLP-induced septic rats [23, 24]. Transcription factors induce the production of pro-inflammatory cytokines and type I interferon, indicating that LDK378 can regulate the secretion of inflammatory cytokines through its upstream transcription factors.

Microcirculation is comprised of arterioles, capillaries, and draining venules, and it performs vital functions including oxygen delivery and solute exchange. The inflammatory responses to an infection of microcirculation alter regional blood flow, vascular hyperpermeability, leukocyte recruitment, and coagulation [25]. Microcirculation stasis in low-flow states is enhanced by cytokine-stimulated monocytes and endothelial cells, and diminished deformability of red and white blood cells. Inflammation, ischemia, and reperfusion activate endothelial cells, leukocytes, and platelets, which produce oxygen radicals and inflammatory mediators, causing increased vascular permeability. Thus, a self-enhancing reduction in microcirculatory blood flow occurs, which leads to organ failure. The severity of impaired microcirculation is closely related to the severity of sepsis. Hua, Tianfeng [4] found a close relationship between proinflammatory cytokines and microcirculatory parameters and proved that proinflammatory cytokines generated an intense response that impairs microcirculation. In the present study, LDK378 treatment was shown to improve microcirculation, in which a possible mechanism might be by reducing the cytokine release of CLP-induced sepsis, hence, LDK378 may play a vital role in attenuating the organic injury or the fatality rate.

This study has some limitations. First, we only used moderate septic models in the study; severe models characterized by sepsis onset and rapid progression to multi-organ failure were not examined. Second, the study did not investigate the effects of different concentrations of LDK378 on sepsis. The optimal dose and time point of LDK378 administration were not well established. Therefore, further studies are needed to fully develop LDK378 as a therapeutic strategy for the treatment of sepsis. Third, the study did not discuss in-depth the mechanism of the ALK-STING pathway.

LDK378 alleviates dysfunctions in micro- and macro-circulation of septic rats, thereby mitigating organic injuries and improving the survival rate. The protective effect of LDK378 reduces inflammatory injuries by regulating the ALK-STING pathway. This study provides valuable insights into the role of LDK378 in controlling inflammatory responses and reveals its therapeutic potential for inflammatory disorders.