Structural Perspective of Gliadin Peptides Active in Celiac Disease

Gluten fragments released in gut of celiac individuals activate the innate or adaptive immune systems. The molecular mechanisms associated with the adaptive response involve a series of immunodominant gluten peptides which are mainly recognized by human leucocyte antigen (HLA)-DQ2.5 and HLA-DQ8. Other peptides, such as A-gliadin P31–43, are not recognized by HLA and trigger innate responses by several routes not yet well detailed. Among the gluten fragments known to be active in Celiac disease, here we focus on the properties of all gluten peptides with known tri-dimensional structure either those locked into HLA-DQ complexes whose crystals were X-ray analyzed or characterized in solution as free forms. The aim of this work was to find the structural reasons why some gluten peptides prompt the adaptive immune systems while others do not, by apparently involving just the innate immune routes. We propose that P31–43 is a non-adaptive prompter because it is not a good ligand for HLA-DQ. Even sharing a similar ability to adopt polyproline II structure with the adaptive ones, the way in which the proline residues are located along the sequence disfavors a productive P31–43-HLA-DQ binding.     

Gliadin Peptide P31-43 Induces mTOR/NFkβ Activation and Reduces Autophagy: The Role of Lactobacillus paracasei CBA L74 Postbiotc

Celiac disease (CD) is an autoimmune disease characterized by an altered immune response stimulated by gliadin peptides that are not digested and cause damage to the intestinal mucosa. The aim of this study was to investigate whether the postbiotic Lactobacillus paracasei (LP) could prevent the action of gliadin peptides on mTOR, autophagy, and the inflammatory response. Most of the experiments performed were conducted on intestinal epithelial cells Caco-2 treated with a peptic-tryptic digest of gliadin (PTG) and P31-43. Furthermore, we pretreated the Caco-2 with the postbiotic LP before treatment with the previously described stimuli. In both cases, we evaluated the levels of pmTOR, p70S6k, and p4EBP-1 for the mTOR pathway, pNFkβ, and pERK for inflammation and LC 3 and p62 for autophagy. For autophagy, we also used immunofluorescence analysis. Using intestinal organoids derivate from celiac (CD) patients, we analyzed the effect of gliadin after postbiotic pretreatment with LP on inflammation marker NFkβ. Through these experiments, we showed that gliadin peptides are able to induce the increase of the inflammatory response in a more complex model of intestinal epithelial cells. LP postbiotic was able to induce autophagy in Caco-2 cells and prevent gliadin effects. In conclusion, postbiotic pretreatment with LP could be considered for in vivo clinical trials.     

TCR Recognition of Peptide–MHC-I: Rule Makers and Breakers

T cells are a critical part of the adaptive immune system that are able to distinguish between healthy and unhealthy cells. Upon recognition of protein fragments (peptides), activated T cells will contribute to the immune response and help clear infection. The major histocompatibility complex (MHC) molecules, or human leukocyte antigens (HLA) in humans, bind these peptides to present them to T cells that recognise them with their surface T cell receptors (TCR). This recognition event is the first step that leads to T cell activation, and in turn can dictate disease outcomes. The visualisation of TCR interaction with pMHC using structural biology has been crucial in understanding this key event, unravelling the parameters that drive this interaction and their impact on the immune response. The last five years has been the most productive within the field, wherein half of current unique TCR–pMHC-I structures to date were determined within this time. Here, we review the new insights learned from these recent TCR–pMHC-I structures and their impact on T cell activation.     

Molecular and Structural Parallels between Gluten Pathogenic Peptides and Bacterial-Derived Proteins by Bioinformatics Analysis

Gluten-related disorders (GRDs) are a group of diseases that involve the activation of the immune system triggered by the ingestion of gluten, with a worldwide prevalence of 5%. Among them, Celiac disease (CeD) is a T-cell-mediated autoimmune disease causing a plethora of symptoms from diarrhea and malabsorption to lymphoma. Even though GRDs have been intensively studied, the environmental triggers promoting the diverse reactions to gluten proteins in susceptible individuals remain elusive. It has been proposed that pathogens could act as disease-causing environmental triggers of CeD by molecular mimicry mechanisms. Additionally, it could also be possible that unrecognized molecular, structural, and physical parallels between gluten and pathogens have a relevant role. Herein, we report sequence, structural and physical similarities of the two most relevant gluten peptides, the 33-mer and p31-43 gliadin peptides, with bacterial pathogens using bioinformatics going beyond the molecular mimicry hypothesis. First, a stringent BLASTp search using the two gliadin peptides identified high sequence similarity regions within pathogen-derived proteins, e.g., extracellular proteins from Streptococcus pneumoniae and Granulicatella sp. Second, molecular dynamics calculations of an updated α-2-gliadin model revealed close spatial localization and solvent-exposure of the 33-mer and p31-43 peptide, which was compared with the pathogen-related proteins by homology models and localization predictors. We found putative functions of the identified pathogen-derived sequence by identifying T-cell epitopes and SH3/WW-binding domains. Finally, shape and size parallels between the pathogens and the superstructures of gliadin peptides gave rise to novel hypotheses about activation of innate immunity and dysbiosis. Based on our structural findings and the similarities with the bacterial pathogens, evidence emerges that these pathologically relevant gluten-derived peptides could behave as non-replicating pathogens opening new research questions in the interface of innate immunity, microbiome, and food research.     

Molecular Mechanisms Underlying β-Adrenergic Receptor-Mediated Cross-Talk between Sympathetic Neurons and Immune Cells

Cross-talk between the sympathetic nervous system (SNS) and immune system is vital for health and well-being. Infection, tissue injury and inflammation raise firing rates of sympathetic nerves, increasing their release of norepinephrine (NE) in lymphoid organs and tissues. NE stimulation of β₂-adrenergic receptors (ARs) in immune cells activates the cAMP-protein kinase A (PKA) intracellular signaling pathway, a pathway that interfaces with other signaling pathways that regulate proliferation, differentiation, maturation and effector functions in immune cells. Immune–SNS cross-talk is required to maintain homeostasis under normal conditions, to develop an immune response of appropriate magnitude after injury or immune challenge, and subsequently restore homeostasis. Typically, β₂-AR-induced cAMP is immunosuppressive. However, many studies report actions of β₂-AR stimulation in immune cells that are inconsistent with typical cAMP–PKA signal transduction. Research during the last decade in non-immune organs, has unveiled novel alternative signaling mechanisms induced by β₂-AR activation, such as a signaling switch from cAMP–PKA to mitogen-activated protein kinase (MAPK) pathways. If alternative signaling occurs in immune cells, it may explain inconsistent findings of sympathetic regulation of immune function. Here, we review β₂-AR signaling, assess the available evidence for alternative signaling in immune cells, and provide insight into the circumstances necessary for “signal switching” in immune cells.     

Connexin43 Region 266–283, via Src Inhibition,Reduces Neural Progenitor Cell Proliferation Promoted by EGF and FGF-2 and Increases Astrocytic Differentiation

Neural progenitor cells (NPCs) are self-renewing cells that give rise to the major cells in the nervous system and are considered to be the possible cell of origin of glioblastoma. The gap junction protein connexin43 (Cx43) is expressed by NPCs, exerting channel-dependent and -independent roles. We focused on one property of Cx43—its ability to inhibit Src, a key protein in brain development and oncogenesis. Because Src inhibition is carried out by the sequence 266–283 of the intracellular C terminus in Cx43, we used a cell-penetrating peptide containing this sequence, TAT-Cx43_266–283, to explore its effects on postnatal subventricular zone NPCs. Our results show that TAT-Cx43_266–283 inhibited Src activity and reduced NPC proliferation and survival promoted by epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). In differentiation conditions, TAT-Cx43_266–283 increased astrocyte differentiation at the expense of neuronal differentiation, which coincided with a reduction in Src activity and β-catenin expression. We propose that Cx43, through the region 266–283, reduces Src activity, leading to disruption of EGF and FGF-2 signaling and to down-regulation of β-catenin with effects on proliferation and differentiation. Our data indicate that the inhibition of Src might contribute to the complex role of Cx43 in NPCs and open new opportunities for further research in gliomagenesis.     

Expression of IL-8, IL-6 and IL-1β in Tears as a Main Characteristic of the Immune Response in Human Microbial Keratitis

Corneal infections are frequent and potentially vision-threatening diseases, and despite the significance of the immunological response in animal models of microbial keratitis (MK), it remains unclear in humans. The aim of this study was to describe the cytokine profile of tears in patients with MK. Characteristics of ocular lesions such as size of the epithelial defect, stromal infiltration, and hypopyon were analyzed. Immunological evaluation included determination of interleukine (IL)-1β, IL-6, IL-8, IL-10, IL-12 and tumor necrosis factor (TNF)-α in tear samples obtained from infected eyes of 28 patients with MK and compared with their contralateral non-infected eyes. Additionally, frequency of CD4⁺, CD8⁺, CD19⁺ and CD3⁻CD56⁺ cells was also determined in peripheral blood mononuclear cells in patients with MK, and compared with 48 healthy controls. Non-significant differences were observed in the size of the epithelial defect, stromal infiltration, and hypopyon. Nevertheless, we found an immunological profile apparently related to MK etiology. IL-8 > IL-6 in patients with bacterial keratitis; IL-8 > IL-6 > IL-1β and increased frequency of circulating CD3⁻CD56⁺ NK cells in patients with gram-negative keratitis; and IL-8 = IL-6 > IL-1β in patients with fungal keratitis. Characterization of tear cytokines from patients with MK could aid our understanding of the immune pathophysiological mechanisms underlying corneal damage in humans.     

Binding of Amyloid β(1–42)-Calmodulin Complexes to Plasma Membrane Lipid Rafts in Cerebellar Granule Neurons Alters Resting Cytosolic Calcium Homeostasis

Lipid rafts are a primary target in studies of amyloid β (Aβ) cytotoxicity in neurons. Exogenous Aβ peptides bind to lipid rafts, which in turn play a key role in Aβ uptake, leading to the formation of neurotoxic intracellular Aβ aggregates. On the other hand, dysregulation of intracellular calcium homeostasis in neurons has been observed in Alzheimer’s disease (AD). In a previous work, we showed that Aβ(1–42), the prevalent Aβ peptide found in the amyloid plaques of AD patients, binds with high affinity to purified calmodulin (CaM), with a dissociation constant ≈1 nM. In this work, to experimentally assess the Aβ(1–42) binding capacity to intracellular CaM, we used primary cultures of mature cerebellar granule neurons (CGN) as a neuronal model. Our results showed a large complexation of submicromolar concentrations of Aβ(1–42) dimers by CaM in CGN, up to 120 ± 13 picomoles of Aβ(1–42) /2.5 × 10⁶ cells. Using fluorescence microscopy imaging, we showed an extensive co-localization of CaM and Aβ(1–42) in lipid rafts in CGN stained with up to 100 picomoles of Aβ(1–42)-HiLyteTM-Fluor555 monomers. Intracellular Aβ(1–42) concentration in this range was achieved by 2 h incubation of CGN with 2 μM Aβ(1–42), and this treatment lowered the resting cytosolic calcium of mature CGN in partially depolarizing 25 mM potassium medium. We conclude that the primary cause of the resting cytosolic calcium decrease is the inhibition of L-type calcium channels of CGN by Aβ(1–42) dimers, whose activity is inhibited by CaM:Aβ(1–42) complexes bound to lipid rafts.     

Korean Red Ginseng Improves Astrocytic Mitochondrial Function by Upregulating HO-1-Mediated AMPKα–PGC-1α–ERRα Circuit after Traumatic Brain Injury

Heme oxygenase-1 (HO-1) exerts beneficial effects, including angiogenesis and energy metabolism via the peroxisome proliferator-activating receptor-γ coactivator-1α (PGC-1α)–estrogen-related receptor α (ERRα) pathway in astrocytes. However, the role of Korean red ginseng extract (KRGE) in HO-1-mediated mitochondrial function in traumatic brain injury (TBI) is not well-elucidated. We found that HO-1 was upregulated in astrocytes located in peri-injured brain regions after a TBI, following exposure to KRGE. Experiments with pharmacological inhibitors and target-specific siRNAs revealed that HO-1 levels highly correlated with increased AMP-activated protein kinase α (AMPKα) activation, which led to the PGC-1α-ERRα axis-induced increases in mitochondrial functions (detected based on expression of cytochrome c oxidase subunit 2 (MTCO2) and cytochrome c as well as O₂ consumption and ATP production). Knockdown of ERRα significantly reduced the p-AMPKα/AMPKα ratio and PGC-1α expression, leading to AMPKα–PGC-1α–ERRα circuit formation. Inactivation of HO by injecting the HO inhibitor Sn(IV) protoporphyrin IX dichloride diminished the expression of p-AMPKα, PGC-1α, ERRα, MTCO2, and cytochrome c in the KRGE-administered peri-injured region of a brain subjected to TBI. These data suggest that KRGE enhanced astrocytic mitochondrial function via a HO-1-mediated AMPKα–PGC-1α–ERRα circuit and consequent oxidative phosphorylation, O₂ consumption, and ATP production. This circuit may play an important role in repairing neurovascular function after TBI in the peri-injured region by stimulating astrocytic mitochondrial biogenesis.     

Wnt16 Signaling Is Required for IL-1β-Induced Matrix Metalloproteinase-13-Regulated Proliferation of Human Stem Cell-Derived Osteoblastic Cells

We established a differentiation method for homogeneous α7 integrin-positive human skeletal muscle stem cell (α7⁺hSMSC)-derived osteoblast-like (α7⁺hSMSC-OB) cells, and found that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-13-regulated proliferation of these cells. These data suggest that MMP-13 plays a potentially unique physiological role in the regeneration of osteoblast-like cells. Here, we examined whether up-regulation of MMP-13 activity by IL-1β was mediated by Wingless/int1 (Wnt) signaling and increased the proliferation of osteoblast-like cells. IL-1β increased the mRNA and protein levels of Wnt16 and the Wnt receptor Lrp5/Fzd2. Exogenous Wnt16 was found to increase MMP-13 mRNA, protein and activity, and interestingly, the proliferation rate of these cells. Treatment with small interfering RNAs against Wnt16 and Lrp5 suppressed the IL-1β-induced increase in cell proliferation. We revealed that a unique signaling cascade IL-1β→Wnt16→Lrp5→MMP-13, was intimately involved in the proliferation of osteoblast-like cells, and suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation are regulated by Wnt16.     

Effect of δ-Opioid Receptor Activation on BDNF-TrkB vs. TNF-α in the Mouse Cortex Exposed to Prolonged Hypoxia

We investigated whether δ-opioid receptor (DOR)-induced neuroprotection involves the brain-derived neurotrophic factor (BDNF) pathway. We studied the effect of DOR activation on the expression of BDNF and other proteins in the cortex of C57BL/6 mice exposed to hypoxia (10% of oxygen) for 1–10 days. The results showed that: (1) 1-day hypoxia had no appreciable effect on BDNF expression, while 3- and 10-day hypoxia progressively decreased BDNF expression, resulting in 37.3% reduction (p < 0.05) after 10-day exposure; (2) DOR activation with UFP-512 (1 mg/kg, i.p., daily) partially reversed the hypoxia-induced reduction of BDNF expression in the 3- or 10-day exposed cortex; (3) DOR activation partially reversed the hypoxia-induced reduction in functional TrkB (140-kDa) and attenuated hypoxia-induced increase in truncated TrkB (90-kDa) in the 3- or 10-day hypoxic cortex; and (4) prolonged hypoxia (10 days) significantly increased TNF-α level and decreased CD11b expression in the cortex, which was completely reversed following DOR activation; and (5) there was no significant change in pCREB and pATF-1 levels in the hypoxic cortex. We conclude that prolonged hypoxia down-regulates BDNF-TrkB signaling leading to an increase in TNF-α in the cortex, while DOR activation up-regulates BDNF-TrkB signaling thereby decreasing TNF-α levels in the hypoxic cortex.     

T Cells in Multisystem Inflammatory Syndrome in Children (MIS-C) Have a Predominant CD4+ T Helper Response to SARS-CoV-2 Peptides and Numerous Virus-Specific CD4− CD8− Double-Negative T Cells

We studied SARS-CoV-2-specific T cell responses in 22 subacute MIS-C children enrolled in 2021 and 2022 using peptide pools derived from SARS-CoV-2 spike or nonspike proteins. CD4+ and CD8+ SARS-CoV-2-specific T cells were detected in 5 subjects, CD4+ T helper (Th) responses alone were detected in 12 subjects, and CD8+ cytotoxic T cell (CTL) responses alone were documented in 1 subject. Notably, a sizeable subpopulation of CD4− CD8− double-negative (DN) T cells out of total CD3+ T cells was observed in MIS-C (median: 14.5%; IQR 8.65–25.3) and recognized SARS-CoV-2 peptides. T cells bearing the Vβ21.3 T cell receptor (TcRs), previously reported as pathogenic in the context of MIS-C, were detected in high frequencies, namely, in 2.8% and 3.9% of the CD4+ and CD8+ T cells, respectively. However, Vβ21.3 CD8+ T cells that responded to SARS-CoV-2 peptides were detected in only a single subject, suggesting recognition of nonviral antigens in the majority of subjects. Subjects studied 6–14 months after MIS-C showed T cell epitope spreading, meaning the activation of T cells that recognize more SARS-CoV-2 peptides following the initial expansion of T cells that see immunodominant epitopes. For example, subjects that did not recognize nonspike proteins in the subacute phase of MIS-C showed good Th response to nonspike peptides, and/or CD8+ T cell responses not appreciable before arose over time and could be detected in the 6–14 months’ follow-up. The magnitude of the Th and CTL responses also increased over time. In summary, patients with MIS-C associated with acute lymphopenia, a classical feature of MIS-C, showed a physiological response to the virus with a prominent role for virus-specific DN T cells.     

Low Ozone Concentrations Differentially Affect the Structural and Functional Features of Non-Activated and Activated Fibroblasts In Vitro

Oxygen–ozone (O₂–O₃) therapy is increasingly applied as a complementary/adjuvant treatment for several diseases; however, the biological mechanisms accounting for the efficacy of low O₃ concentrations need further investigations to understand the possibly multiple effects on the different cell types. In this work, we focused our attention on fibroblasts as ubiquitous connective cells playing roles in the body architecture, in the homeostasis of tissue-resident cells, and in many physiological and pathological processes. Using an established human fibroblast cell line as an in vitro model, we adopted a multimodal approach to explore a panel of cell structural and functional features, combining light and electron microscopy, Western blot analysis, real-time quantitative polymerase chain reaction, and multiplex assays for cytokines. The administration of O₂–O₃ gas mixtures induced multiple effects on fibroblasts, depending on their activation state: in non-activated fibroblasts, O₃ stimulated proliferation, formation of cell surface protrusions, antioxidant response, and IL-6 and TGF-β1 secretion, while in LPS-activated fibroblasts, O₃ stimulated only antioxidant response and cytokines secretion. Therefore, the low O₃ concentrations used in this study induced activation-like responses in non-activated fibroblasts, whereas in already activated fibroblasts, the cell protective capability was potentiated.     

Microbe-Derived Antioxidants Reduce Lipopolysaccharide-Induced Inflammatory Responses by Activating the Nrf2 Pathway to Inhibit the ROS/NLRP3/IL-1β Signaling Pathway

Inflammation plays an important role in the innate immune response, yet overproduction of inflammation can lead to a variety of chronic diseases associated with the innate immune system; therefore, modulation of the excessive inflammatory response has been considered a major strategy in the treatment of inflammatory diseases. Activation of the ROS/NLRP3/IL-1β signaling axis has been suggested to be a key initiating phase of inflammation. Our previous study found that microbe-derived antioxidants (MA) are shown to have excellent antioxidant and anti-inflammatory properties; however, the mechanism of action of MA remains unclear. The current study aims to investigate whether MA could protect cells from LPS-induced oxidative stress and inflammatory responses by modulating the Nrf2-ROS-NLRP3-IL-1β signaling pathway. In this study, we find that MA treatment significantly alleviates LPS-induced oxidative stress and inflammatory responses in RAW264.7 cells. MA significantly reduce the accumulation of ROS in RAW264.7 cells, down-regulate the levels of pro-inflammatory factors (TNF-α and IL-6), inhibit NLRP3, ASC, caspase-1 mRNA, and protein levels, and reduce the mRNA, protein levels, and content of inflammatory factors (IL-1β and IL-18). The protective effect of MA is significantly reduced after the siRNA knockdown of the NLRP3 gene, presumably related to the ability of MA to inhibit the ROS-NLRP3-IL-1β signaling pathway. MA is able to reduce the accumulation of ROS and alleviate oxidative stress by increasing the content of antioxidant enzymes, such as SOD, GSH-Px, and CAT. The protective effect of MA may be due to its ability of MA to induce Nrf2 to enter the nucleus and initiate the expression of antioxidant enzymes. The antioxidant properties of MA are further enhanced in the presence of the Nrf2 activator SFN. After the siRNA knockdown of the Nrf2 gene, the antioxidant and anti-inflammatory properties of MA are significantly affected. These findings suggest that MA may inhibit the LPS-stimulated ROS/NLRP3/IL-1β signaling axis by activating Nrf2-antioxidant signaling in RAW264.7 cells. As a result of this study, MA has been found to alleviate inflammatory responses and holds promise as a therapeutic agent for inflammation-related diseases.     

Immunorthodontics: Role of HIF-1α in the Regulation of (Peptidoglycan-Induced) PD-L1 Expression in Cementoblasts under Compressive Force

Patients with periodontitis undergoing orthodontic therapy may suffer from undesired dental root resorption. The purpose of this in vitro study was to investigate the molecular mechanisms resulting in PD-L1 expression of cementoblasts in response to infection with Porphyromonas gingivalis (P. gingivalis) peptidoglycan (PGN) and compressive force (CF), and its interaction with hypoxia-inducible factor (HIF)-1α molecule: The cementoblast (OCCM-30) cells were kinetically infected with various concentrations of P. gingivalis PGN in the presence and absence of CF. Western blotting and RT-qPCR were performed to examine the protein expression of PD-L1 and HIF-1α as well as their gene expression. Immunofluorescence was applied to visualize the localization of these proteins within cells. An HIF-1α inhibitor was added for further investigation of necroptosis by flow cytometry analysis. Releases of soluble GAS-6 were measured by ELISA. P. gingivalis PGN dose dependently stimulated PD-L1 upregulation in cementoblasts at protein and mRNA levels. CF combined with P. gingivalis PGN had synergistic effects on the induction of PD-L1. Blockade of HIF-1α inhibited the P. gingivalis PGN-inducible PD-L1 protein expression under compression, indicating an HIF-1α dependent regulation of PD-L1 induction. Concomitantly, an HIF-1α inhibitor decreased the GAS-6 release in the presence of CF and P. gingivalis PGN co-stimulation. The data suggest that PGN of P. gingivalis participates in PD-L1 up-regulation in cementoblasts. Additionally, the influence of compressive force on P. gingivalis PGN-induced PD-L1 expression occurs in HIF-1α dependently. In this regard, HIF-1α may play roles in the immune response of cementoblasts via immune-inhibitory PD-L1. Our results underline the importance of molecular mechanisms involved in bacteria-induced periodontics and root resorption.