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Project description:Cytokines interact with their receptors in the extracellular space to control immune responses. How the physicochemical properties of the extracellular space influence cytokine signaling is incompletely elucidated. Here, we show that the activity of interleukin (IL)-2, a critical cytokine in T cell immunity, is profoundly affected by pH, limiting IL-2 signaling within the acidic environment of tumors. Generation of lactic acid by tumors limits STAT5 activation, effector differentiation and anti-tumor immunity by CD8+ T cells and renders high-dose IL-2 therapy poorly effective. Directed evolution enabled selection of a pH-selective IL-2 mutein (Switch-2). Switch-2 binds the IL-2 receptor subunit IL-2Ra with higher affinity, triggers STAT5 activation and drives CD8+ T cell effector function more potently at acidic pH than at neutral pH. Consequently, high-dose Switch-2 therapy induces potent immune activation and tumor rejection with reduced on-target toxicity in normal tissues. Finally, we find that sensitivity to pH is a generalizable property of a diverse range of cytokines with broad relevance to immunity and immunotherapy in healthy and diseased tissues.
2022-12-02 | GSE213440 | GEO
Project description:Cytokines interact with their receptors in the extracellular space to control immune responses. How the physicochemical properties of the extracellular space influence cytokine signaling is incompletely elucidated. Here, we show that the activity of interleukin (IL)-2, a critical cytokine in T cell immunity, is profoundly affected by pH, limiting IL-2 signaling within the acidic environment of tumors. Generation of lactic acid by tumors limits STAT5 activation, effector differentiation and anti-tumor immunity by CD8+ T cells and renders high-dose IL-2 therapy poorly effective. Directed evolution enabled selection of a pH-selective IL-2 mutein (Switch-2). Switch-2 binds the IL-2 receptor subunit IL-2Ra with higher affinity, triggers STAT5 activation and drives CD8+ T cell effector function more potently at acidic pH than at neutral pH. Consequently, high-dose Switch-2 therapy induces potent immune activation and tumor rejection with reduced on-target toxicity in normal tissues. Finally, we find that sensitivity to pH is a generalizable property of a diverse range of cytokines with broad relevance to immunity and immunotherapy in healthy and diseased tissues.
2022-12-02 | GSE213439 | GEO
Project description:BACKGROUND:Ligninolytic peroxidases are divided into three families: manganese peroxidases (MnPs), lignin peroxidases (LiPs), and versatile peroxidases (VPs). The latter two are able to degrade intact lignins, as shown using nonphenolic lignin model compounds, with VP oxidizing the widest range of recalcitrant substrates. One of the main limiting issues for the use of these two enzymes in lignocellulose biorefineries (for delignification and production of cellulose-based products or modification of industrial lignins to added-value products) is their progressive inactivation under acidic pH conditions, where they exhibit the highest oxidative activities. RESULTS:In the screening of peroxidases from basidiomycete genomes, one MnP from Ceriporiopsis subvermispora was found to have a remarkable acidic stability. The crystal structure of this enzyme recently became available and, after comparison with Pleurotus ostreatus VP and Phanerochaete chrysosporium LiP structures, it was used as a robust scaffold to engineer a stable VP by introducing an exposed catalytic tryptophan, with different protein environments. The variants obtained largely maintain the acidic stability and strong Mn(2+)-oxidizing activity of the parent enzyme, and the ability to oxidize veratryl alcohol and Reactive Black 5 (two simple VP substrates) was introduced. The engineered peroxidases present more acidic optimal pH than the best VP from P. ostreatus, enabling higher catalytic efficiency oxidizing lignins, by lowering the reaction pH, as shown using a nonphenolic model dimer. CONCLUSIONS:A peroxidase that degrades lignin at very acidic pH could be obtained by engineering an exposed catalytic site, able to oxidize the bulky and recalcitrant lignin polymers, in a different peroxidase type selected because of its high stability at acidic pH. The potential of this type of engineered peroxidases as industrial biocatalysts in lignocellulose biorefineries is strongly enhanced by the possibility to perform the delignification (or lignin modification) reactions under extremely acidic pH conditions (below pH 2), resulting in enhanced oxidative power of the enzymes.
| S-EPMC4364632 | biostudies-literature
Project description:Enhanced glycolysis and poor perfusion in most solid malignant tumors create an acidic extracellular environment, which enhances tumor growth, invasion, and metastasis. Complex molecular systems have been explored for imaging and treating these tumors. Here, we report the development of a small molecule, LS662, that emits near-infrared (NIR) fluorescence upon protonation by the extracellular acidic pH environment of diverse solid tumors. Protonation of LS662 induces selective internalization into tumor cells and retention in the tumor microenvironment. Noninvasive NIR imaging demonstrates selective retention of the pH sensor in diverse tumors, and two-photon microscopy of ex vivo tumors reveals significant retention of LS662 in tumor cells and the acid tumor microenvironment. Passive and active internalization processes combine to enhance NIR fluorescence in tumors over time. The low background fluorescence allows tumors to be detected with high sensitivity, as well as dead or dying cells to be delineated from healthy cells. In addition to demonstrating the feasibility of using small molecule pH sensors to image multiple aggressive solid tumor types via a protonation-induced internalization and retention pathway, the study reveals the potential of using LS662 to monitor treatment response and tumor-targeted drug delivery.
| S-EPMC4673398 | biostudies-literature
Project description:Interleukin-2 (IL-2) is critical for regulatory T cell (Treg) function and homeostasis. At low doses, IL-2 can suppress immune pathologies by expanding Tregs that constitutively express the high affinity IL-2Rα subunit. However, even low dose IL-2, signaling through the IL2-Rβ/γ complex, may lead to the activation of proinflammatory, non-Treg T cells, so improving specificity toward Tregs may be desirable. Here we use messenger RNAs (mRNA) to encode a half-life-extended human IL-2 mutein (HSA-IL2m) with mutations promoting reliance on IL-2Rα. Our data show that IL-2 mutein subcutaneous delivery as lipid-encapsulated mRNA nanoparticles selectively activates and expands Tregs in mice and non-human primates, and also reduces disease severity in mouse models of acute graft versus host disease and experimental autoimmune encephalomyelitis. Single cell RNA-sequencing of mouse splenic CD4+ T cells identifies multiple Treg states with distinct response dynamics following IL-2 mutein treatment. Our results thus demonstrate the potential of mRNA-encoded HSA-IL2m immunotherapy to treat autoimmune diseases.
| S-EPMC9256694 | biostudies-literature
Project description:SCOPE:The use of human milk products is increasing for high-risk infants. Human milk contains endogenous enzymes that comprise a dynamic proteolytic system, yet biological properties of these enzymes and their activities in response to variations including pH within infants are unclear. Human milk has a neutral pH around 7, while infant gastric pH varies from 2 to 6 depending on individual conditions. This study is designed to determine the specificity of enzyme-substrate interactions in human milk as a function of pH. METHODS AND RESULTS:Endogenous proteolysis is characterized by incubating freshly expressed human milk at physiologically relevant pH ranging from 2 to 7 without the addition of exogenous enzymes. Results show that the effects of pH on endogenous proteolysis in human milk are protein-specific. Further, specific interactions between cathepsin D and ?-lactalbumin are confirmed. The endogenous enzyme cathepsin D in human milk cleaves ?-lactalbumin as the milk pH shifts from 7 to 3. CONCLUSIONS:This study documents that selective proteolysis activated by pH shift is a mechanism for dynamic interactions between human milk and the infant. Controlled proteolysis can guide the use of human milk products based on individual circumstance.
| S-EPMC7231428 | biostudies-literature
Project description:Hemophilia A is a genetic disorder that results in the deficiency of functional factor VIII protein, which plays a key role in blood coagulation. Currently, the majority of hemophilia A patients are treated with repeated infusions of factor VIII protein. Approximately 30% of severe hemophilia A patients develop neutralizing antibodies to factor VIII (known as factor VIII inhibitors) due to treatment, rendering factor VIII protein infusions ineffective. Previously, mice receiving murine IL-2 complexed with α-murine IL-2 mAbs (JES6-1A12) showed a lack of factor VIII inhibitor formation after factor VIII treatment, which was associated with the proliferation and the activation of factor VIII-specific regulatory T cells (Tregs). In this paper, we evaluated if an Fc-fused mutated protein analog of mouse IL-2, named Fc.Mut24, engineered to selectively promote the expansion of Tregs in vivo can modulate factor VIII-specific immune responses. The mice received one intraperitoneal injection of Fc.Mut24. When the regulatory T cell population reached its highest frequency and peak activation, the mice received a hydrodynamic injection of factor VIII plasmid (day 4) followed by a second Fc.Mut24 dose (day 7). Peripheral blood was collected weekly. Flow cytometry was used to characterize the peripheral blood cell populations, while ELISA and Bethesda assays were used to assess the inhibitor concentrations and the functional titers in plasma. The activated partial thromboplastin time assay was used to assess the functional activities of factor VIII in blood. The mice receiving Fc.Mut24 showed a dramatic and transient increase in the population of activated Tregs after Fc.Mut24 injection. Factor VIII gene therapy via hydrodynamic injection resulted in high anti-factor VIII inhibitor concentrations in control PBS-injected mice, whereas the mice treated with Fc.Mut24 produced no inhibitors. Most significantly, there were no inhibitors generated after a second hydrodynamic injection of factor VIII plasmid administered at 19 weeks after the first injection in Fc.Mut24-treated mice. The mice receiving Fc.Mut24 maintained high levels of factor VIII activity throughout the experiment, while the control mice had the factor VIII activity dropped to undetectable levels a few weeks after the first factor VIII plasmid injection. Our data show that human therapies analogous to Fc.Mut24 could potentially provide a method to prevent inhibitor formation and induce long-term immune tolerance to factor VIII in hemophilia patients.
| S-EPMC7198749 | biostudies-literature
Project description:To investigate the genes that are modulated by acidic extracellular pH, we treated the mouse B16-BL6 cells with acidic medium and then analyzed them comprehensively by cDNA microarray.
2024-09-09 | GSE276124 | GEO
Project description:Areas of locally decreased pH are characteristic for many chronic inflammatory diseases such as atherosclerosis and rheumatoid arthritis, acute pathologies such as ischemia reperfusion, and tumor microenvironment. The data on the effects of extracellular acidic pH on inflammation are conflicting with respect to interleukin 1 beta (IL-1β) as one of the most potent proinflammatory cytokines. In this study, we used various mouse- and human-derived cells in order to identify potential species-specific differences in IL-1β secretion pattern in response to extracellular acidification. We found that a short incubation in mild acidic medium caused significant IL-1β release from human macrophages, however, the same effect was not observed in mouse macrophages. Rather, a marked IL-1β suppression was observed when mouse cells were stimulated with a combination of various inflammasome instigators and low pH. Upon activation of cells under acidic conditions, the cytosolic pH was reduced while metabolic activity and the expression of the main inflammasome proteins were not affected by low pH. We show that IL-1β secretion in mouse macrophages is reversible upon restoration of physiological pH. pH sensitivity of NLRP3, NLRC4 and AIM2 inflammasomes appeared to be conferred by the processes upstream of the apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization and most likely contributed by the cell background rather than species-specific amino acid sequences of the sensor proteins.
| S-EPMC7582253 | biostudies-literature
Project description:This study evaluated the influence of three inactivated yeast derivatives (IYDs) used in wine production, namely OptiRed®, OptiWhite® and Noblesse®, on the viability of the probiotic strain Lactobacillus rhamnosus HN001 in an acidic environment. Addition of the IYDs at 3 g/L significantly enhanced the survival of the probiotic bacteria by 2.75-4.05 log cycles after 10-h exposure in a pH 3.0 buffer. Acid stress assay with IYD components obtained after centrifugation and filtration revealed that water-soluble compounds were responsible for improving the acid tolerance of L. rhamnosus HN001 for all three preparations. Differences in protective effect amongst the IYDs on L. rhamnosus HN001 were observed when permeates and retentates of the water-soluble extracts, obtained through ultrafiltration with a 2 kDa membrane, were assayed against the lactic acid bacterium. Chemical analysis of the water-soluble components suggests that low molecular weight polysaccharides, specific free amino acids and/or antioxidants in the 2 kDa permeates could have contributed to the enhanced survival of L. rhamnosus HN001 during acid stress. The contrast amongst the 2 kDa retentates' viability enhancing property may have been attributed to the differences in size and structure of the higher molecular weight carbohydrates and proteins, as the survival of the probiotic did not relate to the concentration of these compounds. These results suggests that oenological IYDs could potentially be applied to probiotic foods for enhancing the acid tolerance of the beneficial microorganisms, and consequently prolonging the shelf life of these products.
| S-EPMC5524659 | biostudies-other