Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing β cells and involves an interplay between β cells and cells of the innate and adaptive immune systems. We investigated the therapeutic potential of targeting 12-lipoxygenase (12-LOX), an enzyme implicated in inflammatory pathways in β cells and macrophages, using a mouse model in which the endogenous mouse Alox15 gene is replaced by the human ALOX12 gene. Our finding demonstrated that VLX-1005, a potent 12-LOX inhibitor, effectively delayed the onset of autoimmune diabetes in human gene replacement non-obese diabetic mice. By spatial proteomics analysis, VLX-1005 treatment resulted in marked reductions in infiltrating T and B cells and macrophages, with accompanying increases in immune checkpoint molecule PD-L1, suggesting a shift toward an immunosuppressive microenvironment. RNA sequencing analysis of isolated islets and polarized proinflammatory macrophages revealed significant alteration of cytokine-responsive pathways and a reduction in IFN response after VLX-1005 treatment. Our studies demonstrated that the ALOX12 human replacement gene mouse provides a platform for the preclinical evaluation of LOX inhibitors and supports VLX-1005 as an inhibitor of human 12-LOX that engages the enzymatic target and alters the inflammatory phenotypes of islets and macrophages to promote the delay of autoimmune diabetes.

ABSTRACT Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing β cells and involves an interplay between β cells and cells of the innate and adaptive immune systems. We investigated the therapeutic potential of targeting 12-lipoxygenase (12-LOX), an enzyme implicated in inflammatory pathways in β cells and macrophages, using a mouse model in which the endogenous mouse Alox15 gene is replaced by the human ALOX12 gene. Our findings demonstrate that VLX-1005, a potent 12-LOX inhibitor, effectively delays the onset of autoimmune diabetes in human gene replacement non-obese diabetic (NOD) mice. By spatial proteomics analysis, VLX-1005 treatment resulted in marked reductions in infiltrating T and B cells and macrophages with accompanying increases in immune checkpoint molecules PD-L1 and PD-1, suggesting a shift towards an immune-suppressive microenvironment. RNA sequencing analysis of isolated islets from inhibitor-treated mice revealed significant alteration of cytokine-responsive pathways. RNA sequencing of polarized proinflammatory macrophages showed that VLX-1005 significantly reduced the interferon response. Our studies demonstrate that the ALOX12 human replacement gene mouse provides a platform for the preclinical evaluation of LOX inhibitors and supports VLX-1005 as an inhibitor of human 12-LOX that engages the enzymatic target and alters the inflammatory phenotypes of islets and macrophages to promote the delay of autoimmune diabetes.

The prevalence of autism spectrum disorder (ASD) has significantly risen in the past three decades, prompting researchers to explore the potential contributions of environmental factors during pregnancy to ASD development. One such factor of interest is gestational hypothyroxinemia (HTX), a frequent condition in pregnancy associated with cognitive impairments in the offspring. While retrospective human studies have linked gestational HTX to autistic traits, the cellular and molecular mechanisms underlying the development of ASD-like phenotypes remain poorly understood. This study used a mouse model of gestational HTX to evaluate ASD-like phenotypes in the offspring.
To induce gestational HTX, pregnant mice were treated with 2-mercapto-1-methylimidazole (MMI), a thyroid hormones synthesis inhibitor, in the tap-drinking water from embryonic days (E) 10 to E14. A separate group received MMI along with a daily subcutaneous injection of T4, while the control group received regular tap water during the entire pregnancy. Female and male offspring underwent assessments for repetitive, anxious, and social behaviors from postnatal day (P) 55 to P64. On P65, mice were euthanized for the evaluation of ASD-related inflammatory markers in blood, spleen, and specific brain regions. Additionally, the expression of glutamatergic proteins (NLGN3 and HOMER1) was analyzed in the prefrontal cortex and hippocampus.
The HTX-offspring exhibited anxious-like behavior, a subordinate state, and impaired social interactions. Subsequently, both female and male HTX-offspring displayed elevated proinflammatory cytokines in blood, including IL-1β, IL-6, IL-17A, and TNF-α, while only males showed reduced levels of IL-10. The spleen of HTX-offspring of both sexes showed increased Th17/Treg ratio and M1-like macrophages. In the prefrontal cortex and hippocampus of male HTX-offspring, elevated levels of IL-17A and reduced IL-10 were observed, accompanied by increased expression of hippocampal NLGN3 and HOMER1. All these observations were compared to those observed in the Control-offspring. Notably, the supplementation with T4 during the MMI treatment prevents the development of the observed phenotypes. Correlation analysis revealed an association between maternal T4 levels and specific ASD-like outcomes.
This study validates human observations, demonstrating for the first time that gestational HTX induces ASD-like phenotypes in the offspring, highlighting the need of monitoring thyroid function during pregnancy.
Copyright © 2024 González-Madrid, Rangel-Ramírez, Opazo, Méndez, Bohmwald, Bueno, González, Kalergis and Riedel.

Prevalence of autism spectrum disorder (ASD) has increased over the past three decades. In this regard, there is growing evidence suggesting that environmental factors during pregnancy may contribute as risk factors for ASD, potentially playing a role in this rising trend. Gestational hypothyroxinemia (HTX) is an asymptomatic and frequent thyroid condition characterized by low levels of thyroxine (T4) in blood, with normal range of T3 and TSH. When this condition occurs in early pregnancy it impairs fetal neurodevelopment and diminishes cognitive capacities in the offspring. Additionally, retrospective human studies have shown that gestational HTX is associated with autistic outcomes in the offspring, however, there is an absence of knowledge about the cellular and molecular autism-like traits in the offspring gestated under gestational HTX (HTX-offspring). In this work, autistic-like phenotypes were evaluated in HTX-offspring, using a mouse model of gestational HTX. Consistent with autism-like behaviors of mice models, HTX-offspring exhibited anxiety-related behavior and impaired social interaction skills. Similar to humans, these features were more pronounced in male HTXoffspring. At the molecular level, the expression of NLGN3 and HOMER1 was increased in the prefrontal cortex and hippocampus of male HTX-offspring, compared to the offspring gestated under euthyroidism. Moreover, HTX-offspring showed increased proinflammatory cytokines in serum and an increased ratio of Th17 lymphocytes relative to T regulatory lymphocytes (Th17/Treg), as well as high neuroinflammatory-like traits in the prefrontal cortex and hippocampus, which were also more pronounced in male HTX-offspring. Notably, by administering T4 to pregnant mice during the induction of HTX, the offspring was protected from developing the observed autism-like traits. These findings support that gestational HTX constitutes a significant risk factor for the development of autism-like phenotypes in the offspring and strongly emphasizes the importance of monitoring thyroid function in early pregnancy.

Natural killer T (NKT) cells operate distinctly different metabolic programming from CD4 T cells, including a strict requirement for glutamine to regulate cell homeostasis. However, the underlying mechanisms remain unknown. Here, we report that at a steady state, NKT cells have higher glutamine levels than CD4 T cells and that NKT cells increase glutaminolysis on activation. Activated NKT cells use glutamine to fuel the tricarboxylic acid cycle and glutathione synthesis. In addition, glutamine-derived nitrogen enables protein glycosylation via the hexosamine biosynthesis pathway (HBP). Each of these branches of glutamine metabolism seems to be critical for NKT cell homeostasis and mitochondrial functions. Glutaminolysis and HBP differentially regulate interleukin-4 (IL-4) and interferon γ (IFNγ) production. Glutamine metabolism appears to be controlled by AMP-activated protein kinase (AMPK)-mammalian target of rapamycin complex 1 (mTORC1) signaling. These findings highlight a distinct metabolic requirement of NKT cells compared with CD4 T cells, which may have therapeutic implications in the treatment of certain nutrient-restricted diseases.
Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.