Interleukin 21 Drives a Hypermetabolic State and CD4+ T-Cell-Associated Pathogenicity in Chronic Intestinal Inflammation.

BACKGROUND & AIMS: Incapacitated regulatory T cells (Tregs) contribute to immune-mediated diseases. Inflammatory Tregs are evident during human inflammatory bowel disease; however, mechanisms driving the development of these cells and their function are not well understood. Therefore, we investigated the role of cellular metabolism in Tregs relevant to gut homeostasis. METHODS: Using human Tregs, we performed mitochondrial ultrastructural studies via electron microscopy and confocal imaging, biochemical and protein analyses using proximity ligation assay, immunoblotting, mass cytometry and fluorescence-activated cell sorting, metabolomics, gene expression analysis, and real-time metabolic profiling utilizing the Seahorse XF analyzer. We used a Crohn's disease single-cell RNA sequencing dataset to infer the therapeutic relevance of targeting metabolic pathways in inflammatory Tregs. We examined the superior functionality of genetically modified Tregs in CD4+ T-cell-induced murine colitis models. RESULTS: Mitochondria-endoplasmic reticulum appositions, known to mediate pyruvate entry into mitochondria via voltage-dependent anion channel 1 (VDAC1), are abundant in Tregs. VDAC1 inhibition perturbed pyruvate metabolism, eliciting sensitization to other inflammatory signals reversible by membrane-permeable methyl pyruvate supplementation. Notably, interleukin (IL) 21 diminished mitochondria-endoplasmic reticulum appositions, resulting in enhanced enzymatic function of glycogen synthase kinase 3 ß, a putative negative regulator of VDAC1, and a hypermetabolic state that amplified Treg inflammatory response. Methyl pyruvate and glycogen synthase kinase 3 ß pharmacologic inhibitor (LY2090314) reversed IL21-induced metabolic rewiring and inflammatory state. Moreover, IL21-induced metabolic genes in Tregs in vitro were enriched in human Crohn's disease intestinal Tregs. Adoptively transferred Il21r-/- Tregs efficiently rescued murine colitis in contrast to wild-type Tregs. CONCLUSIONS: IL21 triggers metabolic dysfunction associated with Treg inflammatory response. Inhibiting IL21-induced metabolism in Tregs may mitigate CD4+ T-cell-driven chronic intestinal inflammation.

Rho-associated protein kinase 1 inhibition in hepatocytes attenuates nonalcoholic steatohepatitis.

BACKGROUND: NASH is the progressive form of NAFLD characterized by lipotoxicity, hepatocyte injury, tissue inflammation, and fibrosis. Previously, Rho-associated protein kinase (ROCK) 1 has been implicated in lipotoxic signaling in hepatocytes in vitro and high-fat diet-induced lipogenesis in vivo. However, whether ROCK1 plays a role in liver inflammation and fibrosis during NASH is unclear. Here, we hypothesized that pathogenic activation of ROCK1 promotes murine NASH pathogenesis. METHODS AND RESULTS: Patients with NASH had increased hepatic ROCK1 expression compared with patients with fatty liver. Similarly, hepatic ROCK1 levels and activity were increased in mice with NASH induced by a western-like diet that is high in fat, fructose, and cholesterol (FFC). Hepatocyte-specific ROCK1 knockout mice on the FFC diet displayed a decrease in liver steatosis, hepatic cell death, liver inflammation, and fibrosis compared with littermate FFC-fed controls. Mechanistically, these effects were associated with a significant attenuation of myeloid cell recruitment. Interestingly, myeloid cell-specific ROCK1 deletion did not affect NASH development in FFC-fed mice. To explore the therapeutic opportunities, mice with established NASH received ROCKi, a novel small molecule kinase inhibitor of ROCK1/2, which preferentially accumulates in liver tissue. ROCK inhibitor treatment ameliorated insulin resistance and decreased liver injury, inflammation, and fibrosis. CONCLUSIONS: Genetic or pharmacologic inhibition of ROCK1 activity attenuates murine NASH, suggesting that ROCK1 may be a therapeutic target for treating human NASH.

Interleukin-21 Drives a Hypermetabolic State and CD4+ T Cell-associated Pathogenicity in Chronic Intestinal Inflammation.

BACKGROUND & AIMS: Incapacitated regulatory T cells (Tregs) contribute to immune-mediated diseases. Inflammatory Tregs are evident during human inflammatory bowel disease (IBD); however, mechanisms driving the development of these cells and their function are not well understood. Therefore, we investigated the role of cellular metabolism in Tregs relevant to gut homeostasis. METHODS: Using human Tregs, we performed mitochondrial ultrastructural studies via electron microscopy and confocal imaging, biochemical and protein analyses using proximity ligation assay, immunoblotting, mass cytometry and fluorescence-activated cell sorting, metabolomics, gene expression analysis, and real-time metabolic profiling utilizing Seahorse XF analyzer. We utilized Crohn's disease single-cell RNA sequencing dataset to infer therapeutic relevance of targeting metabolic pathways in inflammatory Tregs. We examined the superior functionality of genetically-modified Tregs in CD4+ T cell-induced murine colitis models. RESULTS: Mitochondria-endoplasmic reticulum (ER) appositions, known to mediate pyruvate entry into mitochondria via VDAC1, are abundant in Tregs. VDAC1 inhibition perturbed pyruvate metabolism, eliciting sensitization to other inflammatory signals reversible by membrane-permeable methyl pyruvate (MePyr) supplementation. Notably, IL-21 diminished mitochondria-ER appositions, resulting in enhanced enzymatic function of glycogen synthase kinase 3 ß (GSK3ß), a putative negative regulator of VDAC1, and a hypermetabolic state that amplified Treg inflammatory response. MePyr and GSK3ß pharmacologic inhibitor (LY2090314) reversed IL-21-induced metabolic rewiring and inflammatory state. Moreover, IL-21-induced metabolic genes in Tregs in vitro were enriched in human Crohn's disease intestinal Tregs. Adoptively transferred Il21r-/- Tregs efficiently rescued murine colitis in contrast to wild-type Tregs. CONCLUSIONS: IL-21 triggers metabolic dysfunction associated with Treg inflammatory response. Inhibiting IL-21-induced metabolism in Tregs may mitigate CD4+ T cell-driven chronic intestinal inflammation.

Trypanosoma cruzi DNA Polymerase ß Is Phosphorylated In Vivo and In Vitro by Protein Kinase C (PKC) and Casein Kinase 2 (CK2).

DNA polymerase ß plays a fundamental role in the life cycle of Trypanosoma cruzi since it participates in the kinetoplast DNA repair and replication. This enzyme can be found in two forms in cell extracts of T. cruzi epimastigotes form. The H form is a phosphorylated form of DNA polymerase ß, while the L form is not phosphorylated. The protein kinases which are able to in vivo phosphorylate DNA polymerase ß have not been identified yet. In this work, we purified the H form of this DNA polymerase and identified the phosphorylation sites. DNA polymerase ß is in vivo phosphorylated at several amino acid residues including Tyr35, Thr123, Thr137 and Ser286. Thr123 is phosphorylated by casein kinase 2 and Thr137 and Ser286 are phosphorylated by protein kinase C-like enzymes. Protein kinase C encoding genes were identified in T. cruzi, and those genes were cloned, expressed in bacteria and the recombinant protein was purified. It was found that T. cruzi possesses three different protein kinase C-like enzymes named TcPKC1, TcPKC2, and TcPKC3. Both TcPKC1 and TcPKC2 were able to in vitro phosphorylate recombinant DNA polymerase ß, and in addition, TcPKC1 gets auto phosphorylated. Those proteins contain several regulatory domains at the N-terminus, which are predicted to bind phosphoinositols, and TcPKC1 contains a lipocalin domain at the C-terminus that might be able to bind free fatty acids. Tyr35 is phosphorylated by an unidentified protein kinase and considering that the T. cruzi genome does not contain Tyr kinase encoding genes, it is probable that Tyr35 could be phosphorylated by a dual protein kinase. Wee1 is a eukaryotic dual protein kinase involved in cell cycle regulation. We identified a Wee1 homolog in T. cruzi and the recombinant kinase was assayed using DNA polymerase ß as a substrate. T. cruzi Wee1 was able to in vitro phosphorylate recombinant DNA polymerase ß, although we were not able to demonstrate specific phosphorylation on Tyr35. Those results indicate that there exists a cell signaling pathway involving PKC-like kinases in T. cruzi.

The immune response as a therapeutic target in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a complex and heterogeneous disorder considered a liver-damaging manifestation of metabolic syndrome. Its prevalence has increased in the last decades due to modern-day lifestyle factors associated with overweight and obesity, making it a relevant public health problem worldwide. The clinical progression of NAFLD is associated with advanced forms of liver injury such as fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). As such, diverse pharmacological strategies have been implemented over the last few years, principally focused on metabolic pathways involved in NAFLD progression. However, a variable response rate has been observed in NAFLD patients, which is explained by the interindividual heterogeneity of susceptibility to liver damage. In this scenario, it is necessary to search for different therapeutic approaches. It is worth noting that chronic low-grade inflammation constitutes a central mechanism in the pathogenesis and progression of NAFLD, associated with abnormal composition of the intestinal microbiota, increased lymphocyte activation in the intestine and immune effector mechanisms in liver. This review aims to discuss the current knowledge about the role of the immune response in NAFLD development. We have focused mainly on the impact of altered gut-liver-microbiota axis communication on immune cell activation in the intestinal mucosa and the role of subsequent lymphocyte homing to the liver in NAFLD development. We further discuss novel clinical trials that addressed the control of the liver and intestinal immune response to complement current NAFLD therapies.

Initiator-Directed Transcription: Fission Yeast Nmtl Initiator Directs Preinitiation Complex Formation and Transcriptional Initiation.

The initiator element is a core promoter element encompassing the transcription start site, which is found in yeast, Drosophila, and human promoters. This element is observed in TATA-less promoters. Several studies have defined transcription factor requirements and additional cofactors that are needed for transcription initiation of initiator-containing promoters. However, those studies have been performed with additional core promoters in addition to the initiator. In this work, we have defined the pathway of preinitiation complex formation on the fission yeast nmt1 gene promoter, which contains a functional initiator with striking similarity to the initiator of the human dihydrofolate reductase (hDHFR) gene and to the factor requirement for transcription initiation of the nmt1 gene promoter. The results show that the nmt1 gene promoter possesses an initiator encompassing the transcription start site, and several conserved base positions are required for initiator function. A preinitiation complex formation on the nmt1 initiator can be started by TBP/TFIIA or TBP/TFIIB, but not TBP alone, and afterwards follows the same pathway as preinitiation complex formation on TATA-containing promoters. Transcription initiation is dependent on the general transcription factors TBP, TFIIB, TFIIE, TFIIF, TFIIH, RNA polymerase II, Mediator, and a cofactor identified as transcription cofactor for initiator function (TCIF), which is a high-molecular-weight protein complex of around 500 kDa. However, the TAF subunits of TFIID were not required for the nmt1 initiator transcription, as far as we tested. We also demonstrate that other initiators of the nmt1/hDHFR family can be transcribed in fission yeast whole-cell extracts.

Efecto perjudicial de la pandemia por SARS-CoV-2 en la sintomatología digestiva en pacientes con síndrome de intestino irritable / Detrimental impact of SARS-CoV-2 pandemic in the symptomatology of patients with irritable bowel syndrome

Irritable Bowel Syndrome (IBS) is a more frequent disorder in the brain-gut axis interaction in the world. COVID-19 has affected the population's mental health, and its impact on clinical severity in patients with IBS is unknown. Objective: To evaluate the effect of psychosocial stress produced by the pandemic on the severity of gastrointestinal symptoms. Methodology: 54 women and three men with IBS were interviewed by telephone. Factors associated with quality of life, comorbidities, IBS subtype, and COVID-19 diagnosis were asked. Calls were developed between June 2020 to January 2021. Results: 75% had Diarrheal IBS (IBS-D), 67% had comorbidities, 47% with busy work, and 70% in person, five patients (9%) were diagnosed with COVID-19. Of the total, 88% referred to change in gastrointestinal symptoms, 56% increased abdominal pain, and 95% bloating. Abdominal pain was negatively associated with quality of life (p < 0.036), and the incomplete evacuation's sensation positively with difficulty sleeping (p < 0.034). Conclusion: In this study, IBS patients interviewed by telephone reported higher abdominal pain and subjective bloating associated with the pandemic by SARS-CoV-2. Keywords: Irritable

El Síndrome de Intestino Irritable (SII) es uno de los trastornos en la interacción cerebrointestino más frecuentes en el mundo. La pandemia COVID-19 ha afectado la salud mental de la población, siendo desconocido su impacto en la severidad clínica en pacientes con SII. Objetivo: Evaluar el efecto del estrés psicosocial producido por la pandemia en la severidad de síntomas gastrointestinales de pacientes con SII. Metodología: 54 mujeres y 3 hombres con SII fueron entrevistados vía telefónica. Se preguntó por factores asociados a calidad de vida, comorbilidades, subtipo de SII y diagnóstico de COVID-19. Las llamadas se realizaron entre junio de 2020 hasta enero de 2021. Resultados: Un 75% presentó SII Diarreico (SII-D), el 67% comorbilidades, el 47% con trabajo activo y 70% presencial, 5 pacientes (9%) diagnosticados COVID-19. Del total, 88% refirió cambio en síntomas gastrointestinales, 56% aumentó el dolor abdominal y 95% la distensión abdominal. El dolor abdominal se asoció negativamente con la calidad de vida (p < 0,036), y la sensación de evacuación incompleta positivamente con la dificultad para dormir (p < 0,034). Conclusión: En este estudio, los pacientes con SII entrevistados vía telefónica reportaron mayor dolor y distensión abdominal subjetiva asociado a la pandemia por SARS-CoV-2.

Probiotics-Based Treatment as an Integral Approach for Alcohol Use Disorder in Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is one of the leading causes of morbidity among adults with alcohol use disorder (AUD) worldwide. Its clinical course ranges from steatosis to alcoholic hepatitis, progressing to more severe forms of liver damage, such as cirrhosis and hepatocellular carcinoma. The pathogenesis of ALD is complex and diverse elements are involved in its development, including environmental factors, genetic predisposition, the immune response, and the gut-liver axis interaction. Chronic alcohol consumption induces changes in gut microbiota that are associated with a loss of intestinal barrier function and inflammatory responses which reinforce a liver damage progression triggered by alcohol. Alcohol metabolites such as acetaldehyde, lipid peroxidation-derived aldehyde malondialdehyde (MDA), and protein-adducts act as liver-damaging hepatotoxins and potentiate systemic inflammation. Additionally, ethanol causes direct damage to the central nervous system (CNS) by crossing the blood-brain barrier (BBB), provoking oxidative stress contributing to neuroinflammation. Overall, these processes have been associated with susceptibility to depression, anxiety, and alcohol craving in ALD. Recent evidence has shown that probiotics can reverse alcohol-induced changes of the microbiota and prevent ALD progression by restoring gut microbial composition. However, the impact of probiotics on alcohol consumption behavior has been less explored. Probiotics have been used to treat various conditions by restoring microbiota and decreasing systemic and CNS inflammation. The results of some studies suggest that probiotics might improve mental function in Alzheimer's, autism spectrum disorder, and attenuated morphine analgesic tolerance. In this sense, it has been observed that gut microbiota composition alterations, as well as its modulation using probiotics, elicit changes in neurotransmitter signals in the brain, especially in the dopamine reward circuit. Consequently, it is not difficult to imagine that a probiotics-based complementary treatment to ALD might reduce disease progression mediated by lower alcohol consumption. This review aims to present an update of the pathophysiologic mechanism underlying the microbiota-gut-liver-brain axis in ALD, as well as to provide evidence supporting probiotic use as a complementary therapy to address alcohol consumption disorder and its consequences on liver damage.