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Blocking interaction of the immune checkpoint receptor PD-1 with its ligand PD-L1 is associated with good clinical outcomes in a broad variety of malignancies. High levels of PD-L1 promote tumor growth by restraining CD8 T-cell responses against tumors. Limiting PD-L1 expression and function is therefore critical for allowing the development of antitumor immune responses and effective tumor clearance. Pyruvate kinase isoform M2 (PKM2) is also a key player in regulating cancer as well as immune responses. PKM2 catalyzes the final rate-limiting step of glycolysis. Furthermore, PKM2 as a dimer translocates to the nucleus, where it stimulates hypoxia-inducible factor 1α (Hif-1α) transactivation domain function and recruitment of p300 to the hypoxia response elements (HRE) of Hif-1α target genes. Here, we provide the first evidence of a role for PKM2 in regulating the expression of PD-L1 on macrophages, dendritic cells (DCs), T cells, and tumor cells. LPS-induced expression of PD-L1 in primary macrophages was inhibited by the PKM2 targeting compound TEPP-46. Furthermore, RNA silencing of PKM2 inhibited LPS-induced PD-L1 expression. This regulation occurs through direct binding of PKM2 and Hif-1α to HRE sites on the PD-L1 promoter. Moreover, TEPP-46 inhibited expression of PD-L1 on macrophages, DCs, and T cells as well as tumor cells in a mouse CT26 cancer model. These findings broaden our understanding of how PKM2 may contribute to tumor progression and may explain the upregulation of PD-L1 in the tumor microenvironment.

Glutathione transferase Omega 1 (GSTO1-1) is an atypical GST reported to play a pro-inflammatory role in response to LPS. Here we show that genetic knockout of Gsto1 alters the response of mice to three distinct inflammatory disease models. GSTO1-1 deficiency ameliorates the inflammatory response stimulated by LPS and attenuates the inflammatory impact of a high fat diet on glucose tolerance and insulin resistance. In contrast, GSTO1-1 deficient mice show a more severe inflammatory response and increased escape of bacteria from the colon into the lymphatic system in a dextran sodium sulfate mediated model of inflammatory bowel disease. These responses are similar to those of TLR4 and MyD88 deficient mice in these models and confirm that GSTO1-1 is critical for a TLR4-like pro-inflammatory response in vivo . In wild-type mice, we show that a small molecule inhibitor that covalently binds in the active site of GSTO1-1 can be used to ameliorate the inflammatory response to LPS. Our findings demonstrate the potential therapeutic utility of GSTO1-1 inhibitors in the modulation of inflammation and suggest their possible application in the treatment of a range of inflammatory conditions.

Microalgae when grown under certain conditions can be rich sources of protein and can complement conventional protein sources like fishmeal and soymeal, in the aquaculture and animal feed industry. In this study, evaluation of four marine microalgae strains (Picochlorum sp., Nannochloris sp., Nannochloropsis sp., and Chlorella sp.) revealed that in vitro protein solubility and digestibility may serve as key indicators in determining the suitability of microalgae as a protein ingredient in feed. The greenhouse areal biomass productivities, protein and lipid concentrations of these strains ranged between 9–17 g m−2 day−1, 30–38% and 22–24%, respectively. Preliminary in vitro assays using undisrupted biomass of Picochlorum sp. revealed that its protein solubility was 47% and 67% less and digestibility was 28% and 22% less compared with fishmeal and de-oiled soy flour (DOSF), respectively. However, disruption of Picochlorum sp. biomass resulted in 2.5- and 1.5-fold increase in protein solubility and digestibility, respectively, as compared with undisrupted biomass. Further in vitro studies indicated that the soluble protein fractions differed significantly among the four experimental microalgae. The highest in vitro protein solubility (%) and soluble protein fractions (g kg−1 biomass) recorded in the four strains were the following: Picochlorum sp. (53%; 176 g kg−1), Nannochloris sp. (57%; 217 g kg−1), Nannochloropsis sp. (71%; 214 g kg−1), and Chlorella sp. (53%; 197 g kg−1). In addition, extracts from all these four strains were tested for the presence of trypsin inhibitors and found that all these strains have significantly lower trypsin inhibiting activity (TIA) compared with DOSF. The methodology presented in this study combines growth, biochemical composition, protein solubility, in vitro protein digestibility, and TIA and thus provides a reliable strategy in selection of microalgae as protein feed ingredient.

A variety of innate immune responses and functions are dependent on time of day, and many inflammatory conditions are associated with dysfunctional molecular clocks within immune cells. However, the functional importance of these innate immune clocks has yet to be fully characterized. NRF2 plays a critical role in the innate immune system, limiting inflammation via reactive oxygen species (ROS) suppression and direct repression of the proinflammatory cytokines, IL-1β and IL-6. Here we reveal that the core molecular clock protein, BMAL1, controls the mRNA expression of Nrf2 via direct E-box binding to its promoter to regulate its activity. Deletion of Bmal1 decreased the response of NRF2 to LPS challenge, resulting in a blunted antioxidant response and reduced synthesis of glutathione. ROS accumulation was increased in Bmal1 –/– macrophages, facilitating accumulation of the hypoxic response protein, HIF-1α. Increased ROS and HIF-1α levels, as well as decreased activity of NRF2 in cells lacking BMAL1, resulted in increased production of the proinflammatory cytokine, IL-1β. The excessive prooxidant and proinflammatory phenotype of Bmal1 –/– macrophages was rescued by genetic and pharmacological activation of NRF2, or through addition of antioxidants. Our findings uncover a clear role for the molecular clock in regulating NRF2 in innate immune cells to control the inflammatory response. These findings provide insights into the pathology of inflammatory conditions, in which the molecular clock, oxidative stress, and IL-1β are known to play a role.

WebTreatment of lipopolysaccharide-activated macrophages with the cell-permeable itaconate derivative 4-octyl itaconate activates the anti-inflammatory transcription factor Nrf2 by alkylating key cysteine residues on the KEAP1 protein. Anti-inflammatory effects of itaconate Macrophages are white blood cells that recognize and destroy invading bacterial pathogens, and later tone down inflammation to enable tissue repair. The endogenous metabolite itaconate inhibits a number of inflammatory cytokines during macrophage activation. Luke O'Neill and colleagues investigate the mechanism underlying this process. Treatment of lipopolysaccharide (LPS)-activated macrophages with the cell-permeable itaconate derivative 4-octyl itaconate activates the anti-oxidant and anti-inflammatory transcription factor Nrf2. This activation occurs via alkylation of key cysteine residues on the KEAP1 protein, which blocks KEAP1-dependent proteolysis of Nrf2. Pre-treating mouse models of LPS with the itaconate derivative activates Nrf2 and prolongs the survival of the animals after a lethal dose of LPS. The authors suggest that itaconate derivatives may prove useful in the treatment of inflammatory diseases. The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood 1 , 2 , 3 . Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1 ) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.

MyD88 adaptor-like (MAL) is a critical protein in innate immunity, involved in signaling by several Toll-like receptors (TLRs), key pattern recognition receptors (PRRs). Crystal structures of MAL revealed a nontypical Toll/interleukin-1 receptor (TIR)-domain fold stabilized by two disulfide bridges. We therefore undertook a structural and functional analysis of the role of reactive cysteine residues in the protein. Under reducing conditions, the cysteines do not form disulfides, but under oxidizing conditions they are highly amenable to modification. The solution structure of the reduced form of the MAL TIR domain, determined by NMR spectroscopy, reveals a remarkable structural rearrangement compared with the disulfide-bonded structure, which includes the relocation of a β-strand and repositioning of the functionally important “BB-loop” region to a location more typical for TIR domains. Redox measurements by NMR further reveal that C91 has the highest redox potential of all cysteines in MAL. Indeed, mass spectrometry revealed that C91 undergoes glutathionylation in macrophages activated with the TLR4 ligand lipopolysaccharide (LPS). The C91A mutation limits MAL glutathionylation and acts as a dominant negative, blocking the interaction of MAL with its downstream target MyD88. The H92P mutation mimics the dominant-negative effects of the C91A mutation, presumably by preventing C91 glutathionylation. The MAL C91A and H92P mutants also display diminished degradation and interaction with interleukin-1 receptor-associated kinase 4 (IRAK4). We conclude that in the cell, MAL is not disulfide-bonded and requires glutathionylation of C91 for signaling.

A variety of innate immune responses and functions are dependent on time of day, and many inflammatory conditions are associated with dysfunctional molecular clocks within immune cells. However, the functional importance of these innate immune clocks has yet to be fully characterized. NRF2 plays a critical role in the innate immune system, limiting inflammation via reactive oxygen species (ROS) suppression and direct repression of the proinflammatory cytokines, IL-1β and IL-6. Here we reveal that the core molecular clock protein, BMAL1, controls the mRNA expression of Nrf2 via direct E-box binding to its promoter to regulate its activity. Deletion of Bmal1 decreased the response of NRF2 to LPS challenge, resulting in a blunted antioxidant response and reduced synthesis of glutathione. ROS accumulation was increased in Bmal1−/− macrophages, facilitating accumulation of the hypoxic response protein, HIF-1α. Increased ROS and HIF-1α levels, as well as decreased activity of NRF2 in cells lacking BMAL1, resulted in increased production of the proinflammatory cytokine, IL-1β. The excessive prooxidant and proinflammatory phenotype of Bmal1−/− macrophages was rescued by genetic and pharmacological activation of NRF2, or through addition of antioxidants. Our findings uncover a clear role for the molecular clock in regulating NRF2 in innate immune cells to control the inflammatory response. These findings provide insights into the pathology of inflammatory conditions, in which the molecular clock, oxidative stress, and IL-1β are known to play a role.

The Omega-class cytosolic glutathione transferases (GSTs) have distinct structural and functional attributes that allow them to perform novel roles unrelated to the functions of other GSTs. Mammalian GSTO1-1 has been found to play a previously unappreciated role in the glutathionylation cycle that is emerging as significant mechanism regulating protein function. GSTO1-1-catalyzed glutathionylation or deglutathionylation of a key signaling protein may explain the requirement for catalytically active GSTO1-1 in LPS-stimulated pro-inflammatory signaling through the TLR4 receptor. The observation that ML175 a specific GSTO1-1 inhibitor can block LPS-stimulated inflammatory signaling has opened a new avenue for the development of novel anti-inflammatory drugs that could be useful in the treatment of toxic shock and other inflammatory disorders. The role of GSTO2-2 remains unclear. As a dehydroascorbate reductase, it could contribute to the maintenance of cellular redox balance and it is interesting to note that the GSTO2 N142D polymorphism has been associated with multiple diseases including Alzheimer’s disease, Parkinson’s disease, familial amyotrophic lateral sclerosis, chronic obstructive pulmonary disease, age-related cataract and breast cancer.

The translation of India's extensive traditional knowledge on indigenous medicinal plants into modern therapeutic solutions is contingent upon a systematic framework. While traditional Indian medicine offers a rich source of therapeutic leads, this knowledge is often not structured for modern computational analysis, creating a barrier to systematic drug discovery. To this end, we present GRAYU, a curated and comprehensive online database that integrates data across multiple categories, connecting 1,039 traditional formulations to 12,743 indigenous plants, 129,542 phytochemicals, and 13,480 indicated diseases. GRAYU provides insights into 1,370,257 plant-phytochemical, 116,531 plant-disease, 2,389 plant-formulation, and 4,087 formulation-disease associations. We show potential applications on phytochemical analogs, sustainable plant substitution, and disease-network analysis, highlighting the potential of integrative graphs to decode shared molecular signatures and therapeutic networks across traditional Ayurvedic formulations. GRAYU represents a user-friendly resource for researchers to investigate complex bio-associations and formulate novel therapeutic hypotheses, with insights from traditional Indian medicine. GRAYU organises reported associations and computational relationships and can hint at mechanistic causality or biological activity; however, all outputs require contextual interpretation and further experimental validation. The database is available at https://caps.ncbs.res.in/GRAYU/.

ABSTRACT Background: Aging is a global phenomenon. Depression and loneliness among the elderly are significant mental health concerns, often overlooked and underestimated in society. Globally, depression amounts to a loss of 2% disability-adjusted life years. India is 2nd most populous in the world by elderly population, depression and loneliness are often overlooked due to various reasons. Therefore, this study aims to explore the proportion of depression and loneliness and to determine their association with loneliness and other sociodemographic factors. Methodology: A community-based cross-sectional study was conducted among 132 elders (age ≥60 years) in three wards of the urban area of Bengaluru. Geriatric Depression Scale 15 and Three-Item Loneliness Scale were used to assess depression and loneliness, respectively. The association between depression and loneliness was analyzed with other demographic variables using Chi-square and Bivariate logistic regression. Results: The magnitude of depression and loneliness among the elderly population was found to be 75.7% and 91%, respectively. Depression was significantly associated with elderly 60-69 years, females, with minimal education (illiterate), homemakers, number of family members >4, with no income, and dependent on others. Loneliness is significantly associated with the female gender, with illiterates, those who are not working and homemakers, below poverty line card holders, those with chronic diseases, and those dependent on others compared to their counterparts. There is a significant positive correlation between depression and loneliness scores among the elderly. Conclusion: The higher proportion of depression and loneliness among the elderly in our study emphasizes the necessary supportive measures. It needs to be addressed adequately through a coordinated primary care approach with community and social support.