AMPK directly phosphorylates TBK1 to integrate glucose sensing into innate immunity.

Nutrient sensing and damage sensing are two fundamental processes in living organisms. While hyperglycemia is frequently linked to diabetes-related vulnerability to microbial infection, how body glucose levels affect innate immune responses to microbial invasion is not fully understood. Here, we surprisingly found that viral infection led to a rapid and dramatic decrease in blood glucose levels in rodents, leading to robust AMPK activation. AMPK, once activated, directly phosphorylates TBK1 at S511, which triggers IRF3 recruitment and the assembly of MAVS or STING signalosomes. Consistently, ablation or inhibition of AMPK, knockin of TBK1-S511A, or increased glucose levels compromised nucleic acid sensing, while boosting AMPK-TBK1 cascade by AICAR or TBK1-S511E knockin improves antiviral immunity substantially in various animal models. Thus, we identify TBK1 as an AMPK substrate, reveal the molecular mechanism coupling a dual sensing of glucose and nuclei acids, and report its physiological necessity in antiviral defense.

Induced phase separation of mutant NF2 imprisons the cGAS-STING machinery to abrogate antitumor immunity.

Missense mutations of the tumor suppressor Neurofibromin 2 (NF2/Merlin/schwannomin) result in sporadic to frequent occurrences of tumorigenesis in multiple organs. However, the underlying pathogenicity of NF2-related tumorigenesis remains mostly unknown. Here we found that NF2 facilitated innate immunity by regulating YAP/TAZ-mediated TBK1 inhibition. Unexpectedly, patient-derived individual mutations in the FERM domain of NF2 (NF2m) converted NF2 into a potent suppressor of cGAS-STING signaling. Mechanistically, NF2m gained extreme associations with IRF3 and TBK1 and, upon innate nucleic acid sensing, was directly induced by the activated IRF3 to form cellular condensates, which contained the PP2A complex, to eliminate TBK1 activation. Accordingly, NF2m robustly suppressed STING-initiated antitumor immunity in cancer cell-autonomous and -nonautonomous murine models, and NF2m-IRF3 condensates were evident in human vestibular schwannomas. Our study reports phase separation-mediated quiescence of cGAS-STING signaling by a mutant tumor suppressor and reveals gain-of-function pathogenesis for NF2-related tumors by regulating antitumor immunity.

The protein phosphatase PPM1A dephosphorylates and activates YAP to govern mammalian intestinal and liver regeneration.

The Hippo-YAP pathway responds to diverse environmental cues to manage tissue homeostasis, organ regeneration, tumorigenesis, and immunity. However, how phosphatase(s) directly target Yes-associated protein (YAP) and determine its physiological activity are still inconclusive. Here, we utilized an unbiased phosphatome screening and identified protein phosphatase magnesium-dependent 1A (PPM1A/PP2Cα) as the bona fide and physiological YAP phosphatase. We found that PPM1A was associated with YAP/TAZ in both the cytoplasm and the nucleus to directly eliminate phospho-S127 on YAP, which conferring YAP the nuclear distribution and transcription potency. Accordingly, genetic ablation or depletion of PPM1A in cells, organoids, and mice elicited an enhanced YAP/TAZ cytoplasmic retention and resulted in the diminished cell proliferation, severe gut regeneration defects in colitis, and impeded liver regeneration upon injury. These regeneration defects in murine model were largely rescued via a genetic large tumor suppressor kinase 1 (LATS1) deficiency or the pharmacological inhibition of Hippo-YAP signaling. Therefore, we identify a physiological phosphatase of YAP/TAZ, describe its critical effects in YAP/TAZ cellular distribution, and demonstrate its physiological roles in mammalian organ regeneration.

Mst1 shuts off cytosolic antiviral defense through IRF3 phosphorylation.

Cytosolic RNA/DNA sensing elicits primary defense against viral pathogens. Interferon regulatory factor 3 (IRF3), a key signal mediator/transcriptional factor of the antiviral-sensing pathway, is indispensible for interferon production and antiviral defense. However, how the status of IRF3 activation is controlled remains elusive. Through a functional screen of the human kinome, we found that mammalian sterile 20-like kinase 1 (Mst1), but not Mst2, profoundly inhibited cytosolic nucleic acid sensing. Mst1 associated with IRF3 and directly phosphorylated IRF3 at Thr75 and Thr253. This Mst1-mediated phosphorylation abolished activated IRF3 homodimerization, its occupancy on chromatin, and subsequent IRF3-mediated transcriptional responses. In addition, Mst1 also impeded virus-induced activation of TANK-binding kinase 1 (TBK1), further attenuating IRF3 activation. As a result, Mst1 depletion or ablation enabled an enhanced antiviral response and defense in cells and mice. Therefore, the identification of Mst1 as a novel physiological negative regulator of IRF3 activation provides mechanistic insights into innate antiviral defense and potential antiviral prevention strategies.

LncRNA AFAP1-AS1 promotes osteoblast differentiation of human aortic valve interstitial cells through regulating miR-155/SMAD5 axis.

AIM: Degenerative calcific aortic valve disease (DCAVD) is a common valve disease characterized by massive calcium deposits in the aortic valve. Osteoblast differentiation of valve interstitial cells (VICs) is responsible for the formation of calcific nodules. This study aims to explore the function and underlying mechanism of long non-coding RNA (lncRNA) AFAP1-AS1 (actin filament-associated protein 1 antisense RNA 1) in the pathogenesis of DCAVD. METHODS: AFAP1-AS1, miR-155 and mRNA levels were detected by qRT-PCR. Protein levels were measured by Western blot. Calcification deposition was examined by Alizarin Red staining. The interaction between AFAP1-AS1 and miR-155, as well as miR-155 and SMAD5 was evaluated using luciferase reporter assay. RESULTS: AFAP1-AS1 expression was increased both in calcified aortic valves from DCAVD patients and after osteogenic induction in human VICs. Furthermore, AFAP1-AS1 overexpression promoted osteogenic differentiation of VICs, whereas AFAP1-AS1 knockdown inhibited osteogenic differentiation. Mechanistically, AFAP1-AS1 acted as a sponge for miR-155 to elevate SMAD5 expression. Further functional assays revealed that miR-155 mimic and SMAD5 silencing effectively reversed AFAP1-AS1-promoted osteogenic differentiation of VICs. CONCLUSION: Collectively, AFAP1-AS1 promotes osteogenic differentiation of VICs, at least in part, by sponging miR-155 to upregulate SMAD5. This study sheds new light on lncRNA-directed therapeutics in DCAVD.

TBK1, a central kinase in innate immune sensing of nucleic acids and beyond.

Sensing of intracellular and extracellular environments is one of the fundamental processes of cell. Surveillance of aberrant nucleic acids, derived either from invading pathogens or damaged organelle, is conducted by pattern recognition receptors (PRRs) including RIG-I-like receptors, cyclic GMP-AMP synthase, absent in melanoma 2, and a few members of toll-like receptors. TANK-binding kinase 1 (TBK1), along with its close analogue I-kappa-B kinase epsilon, is a central kinase in innate adaptor complexes linking activation of PRRs to mobilization of transcriptional factors that transcribe proinflammatory cytokines, type I interferon (IFN-α/ß), and myriads interferon stimulated genes. However, it still remains elusive for the precise mechanisms of activation and execution of TBK1 in signaling platforms formed by innate adaptors mitochondrial antiviral signaling protein (MAVS), stimulator of interferon genes protein (STING), and TIR-domain-containing adapter-inducing interferon-ß (TRIF), as well as its complex regulations. An atlas of TBK1 substrates is in constant expanding, setting TBK1 as a key node of signaling network and a dominant player in contexts of cell biology, animal models, and human diseases. Here, we review recent advancements of activation, regulations, and functions of TBK1 under these physiological and pathological contexts.

IRF3 activates RB to authorize cGAS-STING-induced senescence and mitigate liver fibrosis.

Cytosolic double-stranded DNA surveillance by cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon Genes (STING) signaling triggers cellular senescence, autophagy, biased mRNA translation, and interferon-mediated immune responses. However, detailed mechanisms and physiological relevance of STING-induced senescence are not fully understood. Here, we unexpectedly found that interferon regulatory factor 3 (IRF3), activated during innate DNA sensing, forms substantial endogenous complexes in the nucleus with retinoblastoma (RB), a key cell cycle regulator. The IRF3-RB interaction attenuates cyclin-dependent kinase 4/6 (CDK4/6)-mediated RB hyperphosphorylation that mobilizes RB to deactivate E2 family (E2F) transcription factors, thereby driving cells into senescence. STING-IRF3-RB signaling plays a notable role in hepatic stellate cells (HSCs) within various murine models, pushing activated HSCs toward senescence. Accordingly, IRF3 global knockout or conditional deletion in HSCs aggravated liver fibrosis, a process mitigated by the CDK4/6 inhibitor. These findings underscore a straightforward yet vital mechanism of cGAS-STING signaling in inducing cellular senescence and unveil its unexpected biology in limiting liver fibrosis.

LncRNA AFAP1-AS1 promotes M1 polarization of macrophages and osteogenic differentiation of valve interstitial cells.

Little is known about the biological functions and underlying mechanisms of long non-coding RNA AFAP1-AS1 in degenerative calcified aortic valve disease (DCAVD). This study aims to explore whether AFAP1-AS1 regulates macrophage polarization in aortic valve calcification. Macrophage polarization and AFAP1-AS1 expression were detected in normal and calcified aortic valves of DCAVD patients. To explore the effect of AFAP1-AS1 on macrophage polarization, gain and loss of function were performed in THP-1 cells, and the percentage of M1 and M2 and the expressions of M1 and M2 markers were analyzed. Meanwhile, osteogenic differentiation was examined in valve interstitial cells (VICs). Compared with normal valves, there were more M1, less M2, and high AFAP1-AS1 expressions in calcified aortic valves, which may indicate a relationship between AFAP1-AS1 and macrophage polarization. AFAP1-AS1 overexpression promoted M1 polarization in lipopolysaccharide (LPS) and interferon gamma (IFN-γ)-treated THP-1 cells but inhibited M2 polarization, as well as augmented VIC osteogenic differentiation. On the contrary, the silence of AFAP1-AS1 could induce macrophage to M2-type and inhibit VIC osteogenic differentiation. These results elucidate that AFAP1-AS1 can promote M1 macrophages polarization to aggravate VIC osteogenic differentiation, playing a role in aortic valve calcification.

The Hippo Pathway in Innate Anti-microbial Immunity and Anti-tumor Immunity.

The Hippo pathway responds to diverse environmental cues and plays key roles in cell fate determination, tissue homeostasis, and organ regeneration. Aberrant Hippo signaling, on the other hand, has frequently been implicated in diversified pathologies such as cancer and immune dysfunction. Here, we summarize the recent but rapid progress in understanding the involvement of the Hippo pathway in innate immunity, with a special focus on the intrinsic mechanisms and mutual interactions between Hippo-YAP signaling and the innate immune response and its physiological impacts on anti-microbial immunity and anti-tumor immunity. Moving forward, we believe that systematic investigations under the physiological setting are needed to draw a clearer picture of the actions of Hippo in innate immunity.

HER2 recruits AKT1 to disrupt STING signalling and suppress antiviral defence and antitumour immunity.

Sensing cytosolic DNA through the cGAS-STING pathway constitutes a widespread innate immune mechanism to monitor cellular damage and microbial invasion. Evading this surveillance is crucial in tumorigenesis, but the process remains largely unexplored. Here, we show that the receptor tyrosine kinase HER2 (also known as ErbB-2 or Neu) potently inhibits cGAS-STING signalling and prevents cancer cells from producing cytokines, entering senescence and undergoing apoptosis. HER2, but not EGFR, associates strongly with STING and recruits AKT1 (also known as PKB) to directly phosphorylate TBK1, which prevents the TBK1-STING association and TBK1 K63-linked ubiquitination, thus attenuating STING signalling. Unexpectedly, we observed that DNA sensing robustly activates the HER2-AKT1 axis, resulting in negative feedback. Accordingly, genetic or pharmacological targeting of the HER2-AKT1 cascade augments damage-induced cellular senescence and apoptosis, and enhances STING-mediated antiviral and antitumour immunity. Thus, our findings reveal a critical function of the oncogenic pathway in innate immune regulation and unexpectedly connect HER2-AKT1 signalling to the surveillance of cellular damage and antitumour immunity.

Hippo signalling governs cytosolic nucleic acid sensing through YAP/TAZ-mediated TBK1 blockade.

The Hippo pathway senses cellular conditions and regulates YAP/TAZ to control cellular and tissue homeostasis, while TBK1 is central for cytosolic nucleic acid sensing and antiviral defence. The correlation between cellular nutrient/physical status and host antiviral defence is interesting but not well understood. Here we find that YAP/TAZ act as natural inhibitors of TBK1 and are vital for antiviral physiology. Independent of transcriptional regulation and through the transactivation domain, YAP/TAZ associate directly with TBK1 and abolish virus-induced TBK1 activation, by preventing TBK1 Lys63-linked ubiquitylation and the binding of adaptors/substrates. Accordingly, YAP/TAZ deletion/depletion or cellular conditions inactivating YAP/TAZ through Lats1/2 kinases relieve TBK1 suppression and boost antiviral responses, whereas expression of the transcriptionally inactive YAP dampens cytosolic RNA/DNA sensing and weakens the antiviral defence in cells and zebrafish. Thus, we describe a function of YAP/TAZ and the Hippo pathway in innate immunity, by linking cellular nutrient/physical status to antiviral host defence.

Lck/Hck/Fgr-Mediated Tyrosine Phosphorylation Negatively Regulates TBK1 to Restrain Innate Antiviral Responses.

Cytosolic nucleic acid sensing elicits interferon production for primary antiviral defense through cascades controlled by protein ubiquitination and Ser/Thr phosphorylation. Here we show that TBK1, a core kinase of antiviral pathways, is inhibited by tyrosine phosphorylation. The Src family kinases (SFKs) Lck, Hck, and Fgr directly phosphorylate TBK1 at Tyr354/394, to prevent TBK1 dimerization and activation. Accordingly, antiviral sensing and resistance were substantially enhanced in Lck/Hck/Fgr triple knockout cells and ectopic expression of Lck/Hck/Fgr dampened the antiviral defense in cells and zebrafish. Small-molecule inhibitors of SFKs, which are conventional anti-tumor therapeutics, enhanced antiviral responses and protected zebrafish and mice from viral attack. Viral infection induced the expression of Lck/Hck/Fgr through TBK1-mediated mobilization of IRF3, thus constituting a negative feedback loop. These findings unveil the negative regulation of TBK1 via tyrosine phosphorylation and the functional integration of SFKs into innate antiviral immunity.

[Clinical evaluation of the heart function early after repair of tetralogy of Fallot: follow-up of 43 patients].

OBJECTIVE: To evaluate the heart function of the patients early after the repair of tetralogy of Fallot (TOF). METHODS: Forty-three patients with TOF, 25 males and 18 females, underwent operation at the age of 2.5 - 52 years (16.7 years on average) and were followed up for 1 - 3.5 years. Twenty-one age-matched healthy persons were used as controls. Tissue Doppler imaging (TDI) was used to measure the values of the peak tricuspid ring velocity during early diastole (Ea), late diastole (Aa), systole, and isovolumic contraction, and isovolumetric contraction acceleration (IVA); and isovolumetric contraction time (ICT), isovolumetric relaxation time (IRT), and isovolumetric contraction velocity (IVV) of the right ventricle. Tei index was calculated using the formula: (ICT + IRT)/ET. Treadmill test was used on the patients aged > 17 to measure the maximal heart rate maximal blood pressure, maximal exercise tolerance (MET), and movement time. RESULTS: The peak tricuspid ring velocity during Ea of the repaired TOF group (rTOF group) was 11.5 +/- 2.6 cm/s, significantly lower than that of the control group (17.1 +/- 2.4 cm/s, P < 0.0001), the peak tricuspid ring velocity during Aa of the rTOF group was 9.6 +/- 1.7 cm/s, significantly lower than that of the control group (12.9 +/- 2.9 cm/s, P < 0.001), the E/A of the rTOF group was 1.16 +/- 0.36, significantly lower than that of the control group (1.36 +/- 0.26, P < 0.05). The IVV of the rTOF group was 7.7 +/- 1.8 cm/s, significantly lower than that of the control group (9.9 +/- 1.4 cm/s, P = 0.0030, and the IVA of the rTOF group was 131.7 +/- 37.6 cm/s(2), significantly lower than that of the control group (222.5 +/- 39.2 cm/s(2), P < 0.001). The Tei index of the rTOF group was 0.58 +/- 0.11, significantly higher than that of the control group (0.52 +/- 0.04, P = 0.029). The maximal heart rate maximal blood pressure, MET, and movement time of the rTOF group were all significantly lower than those of the control group (P < 0.05 or P < 0.01). CONCLUSION: The heart function of the patients undergoing repair of TOF fails to recover to the normal level during a short time after the surgery.

Differences in sensitivity to rocuronium among orbicularis oris muscles innervated by normal or damaged facial nerves and gastrocnemius muscle innervated by somatic nerve in rats: combined morphological and functional analyses.

OBJECTIVES/HYPOTHESIS: To evaluate mechanisms of discrepant responses to the nondepolarizing muscle relaxant rocuronium among normal and injured facial nerve-innervated orbicularis oris and tibial nerve-innervated gastrocnemius, and to provide information for the proper use of muscle relaxants to balance evoked electromyography (EEMG) monitoring and immobility in general anesthesia. STUDY DESIGN: Randomized controlled study. METHODS: Right-sided facial nerve injury was induced by crush axotomy in 18 Sprague-Dawley rats. At different rocuronium concentrations, muscular tension amplitude (MTA) was determined in vitro for normal and injured facial nerve-innervated orbicularis oris and gastrocnemius; the number of unsaturated acetylcholine receptors (AChRs) at end plates was determined by (125) I-α-bungarotoxin staining followed with gamma spectroscopy. The morphological composition of muscle fibers was determined by histological examination. RESULTS: Following rocuronium incubation, the percentage of MTA inhibition (MTAI%) of gastrocnemius was significantly higher than the corresponding values of orbicularis oris (P < .05), and the degree of saturation of AChR in gastrocnemius was significantly greater than that in orbicularis oris (P < .05). The baseline MTA and AChR density of injured-side orbicularis oris was significantly smaller than those of the normal side, whereas no significant difference was found regarding MTAI% and the degree of AChR saturation between the normal and injured side. CONCLUSIONS: The affinity of AChR at end plates and different number of AChR per unit fiber cross-sectional area may be the mechanisms for differential sensitivities to neuromuscular blockers between facial nerve-innervated muscles and somatic nerve-innervated muscles. The lower EEMG responses in the impaired facial nerve-innervated muscles may result from the lower AChR density at end plates compared with the normal facial nerve-innervated muscles.

Right ventricular dysfunction due to right ventricular outflow tract patch.

Doppler tissue imaging analysis was used to examine the relationship between right ventricular function and right ventricular outflow tract damage in 54 patients with repaired tetralogy of Fallot. The patients were divided into three groups: 16 in whom the right ventricular outflow tract was directly sutured (group DS), 23 who had transventricular patch repair (group TVP), and 15 who had transannular patch repair (group TAP). The control group consisted of 16 age-matched patients who underwent patch closure of a ventricular septal defect (group C). The Tei index was obtained from tricuspid and pulmonary Doppler flow velocities. The right ventricular Tei index was significantly greater in groups TVP and TAP than in group DS. Doppler tissue imaging analysis in groups TVP and TAP showed shorter myocardial systolic velocity, diastolic peak velocity, and atrial diastolic peak velocity, lower peak myocardial velocity and acceleration during isovolumic contraction, and prolonged isovolumic relaxation and contraction times compared to groups DS and C. Right ventricular dysfunction is due to the right ventricular outflow tract patch. Thus, the right ventricular outflow tract may be essential for right ventricular ejection and maintenance of right ventricular function.