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Systemic sclerosis (SSc) is an autoimmune disease characterized by specific autoantibodies, vasculopathy and fibrosis of the skin and internal organs. In SSc, chronic activation of the immune system is largely sustained by endogenous inflammatory mediators that act as damage-associated molecular patterns (DAMPs), which activate Toll-like receptors (TLRs). Major autoantigens are nucleic acids or molecules that are able to bind nucleic acids. It is important to identify solid and predictive biomarkers of both disease activity and disease subtype. CXCL4 has been regarded as a new biomarker for early SSc in recent years, and here, we discuss its modulation over the course of a disease and after pharmacological interventions. Moreover, we provide evidence that CXCL4, in addition to being a biomarker of SSc subtypes and a prognostic marker of disease severity, has a dual pathogenic role in SSc: on the one hand, in complex with self-nucleic acids, CXCL4 acts as a DAMP for IFN-I and pro-inflammatory cytokines’ release by innate immune cells (such as dendritic cells); on the other hand, CXCL4 is a target of both antibodies and T cells, functioning as an autoantigen. CXCL4 is certainly an interesting molecule in inflammation and autoimmunity, not only in SSc, and it may also be considered as a therapy target. Full article

(This article belongs to the Special Issue Antimicrobial Peptides, Cytokines, Chemokines and Danger Molecules in Immunity and Autoimmunity)

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Figure 1
Pleiotropic functions of CXCL4. CXCL4 is a member of kinocidins, with direct capacity for bacterial killing. The image illustrates the most important functions of CXCL4, which are also relevant in autoimmunity and in SSc. Full article ">Figure 2
CXCL4 organizes dsDNA into immunogenic liquid–crystalline complexes suitable for TLR9-mediated IFN-α production. Inter-DNA spacing close to the steric size of TLR9 (d = 3–4 nm) allows the optimal binding of columnar DNA lattices to TLR9-clustered arrays. CXCL4 organizes DNA into liquid–crystalline columnar lattices at an inter-DNA spacing compatible with TLR9 amplification. DNA fragmentation increases the total number of discrete DNA fragments for the same mass of DNA, with optimal close packing of DNA ligands [<a href="#B38-ijms-26-02421" class="html-bibr">38</a>]. Full article ">Figure 3
CXCL4–DNA and CXCL4–RNA complexes activate immune cells via TLRs. As with other well-known DAMPs like Tenascin-C, acting via TLR4 [<a href="#B49-ijms-26-02421" class="html-bibr">49</a>], fibronectin, S100A8 and S100A9 (alarmins), when CXCL4 binds to DNA/RNA, it acts as a DAMP and participates in the inflammation process. Full article ">Figure 4
Model for B-cell activation via CXCL4–DNA/RNA complexes. After stimulation through TLR7/8/9 by CXCL4–DNA/RNA complexes, pDCs become activated and start to produce IFN-I, which, together with BAFF, IL-15, IL-2, help plasma cell differentiation (pDCs can also produce BAFF, [<a href="#B58-ijms-26-02421" class="html-bibr">58</a>], whereas IL-15 and IL-2 can be produced by T cells but also by stroll cells, monocytes, etc. [<a href="#B59-ijms-26-02421" class="html-bibr">59</a>]). Afterward, B-cell transition and differentiation in antibody-secreting plasma cells occurs. In SSc, activated B cells start to produce antibodies, among which there could be anti-CXCL4 antibodies. Full article ">Figure 5
CXCL4 as an autoantigen in SSc. When complexed with nucleic acids, CXCL4 stimulates pDCs and mDCs via TLRs, which activates both cell types and induces the production of IFN-I, IL-12, IL-23 and TNF-α. IFN-I released by pDCs implements the antigen-presenting cell capacity of mDCs, whereas pro-inflammatory cytokines can activate CD4 T cells (including Th17 cells and perhaps CD8 T cells [<a href="#B64-ijms-26-02421" class="html-bibr">64</a>]). T cells specific to CXCL4 are likely to provide B-cell help for autoantibody production [<a href="#B41-ijms-26-02421" class="html-bibr">41</a>] or directly induce an inflammation that favor fibrosis. An algorithm that predicts the binding capacity of CXCL4-derived epitopes to HLA molecules showed that the sequence of CXCL4 possesses “binding motifs” for several HLA-DR alleles represented in Caucasians (DR4, DR1 and DR11), which may explain the recognition by CD4 T cells [<a href="#B41-ijms-26-02421" class="html-bibr">41</a>]. Full article ">

21 pages, 8256 KiB

Open AccessPerspective

Zα and Zβ Localize ADAR1 to Flipons That Modulate Innate Immunity, Alternative Splicing, and Nonsynonymous RNA Editing

by Alan Herbert, Oleksandr Cherednichenko, Terry P. Lybrand, Martin Egli and Maria Poptsova

Int. J. Mol. Sci. 2025, 26(6), 2422; https://doi.org/10.3390/ijms26062422 - 7 Mar 2025

Abstract

The double-stranded RNA editing enzyme ADAR1 connects two forms of genetic programming, one based on codons and the other on flipons. ADAR1 recodes codons in pre-mRNA by deaminating adenosine to form inosine, which is translated as guanosine. ADAR1 also plays essential roles in the immune defense against viruses and cancers by recognizing left-handed Z-DNA and Z-RNA (collectively called ZNA). Here, we review various aspects of ADAR1 biology, starting with codons and progressing to flipons. ADAR1 has two major isoforms, with the p110 protein lacking the p150 Zα domain that binds ZNAs with high affinity. The p150 isoform is induced by interferon and targets ALU inverted repeats, a class of endogenous retroelement that promotes their transcription and retrotransposition by incorporating Z-flipons that encode ZNAs and G-flipons that form G-quadruplexes (GQ). Both p150 and p110 include the Zβ domain that is related to Zα but does not bind ZNAs. Here we report strong evidence that Zβ binds the GQ that are formed co-transcriptionally by ALU repeats and within R-loops. By binding GQ, ADAR1 suppresses ALU-mediated alternative splicing, generates most of the reported nonsynonymous edits and promotes R-loop resolution. The recognition of the various alternative nucleic acid conformations by ADAR1 connects genetic programming by flipons with the encoding of information by codons. The findings suggest that incorporating G-flipons into editmers might improve the therapeutic editing efficacy of ADAR1. Full article

(This article belongs to the Special Issue New Applications in the Diagnosis and Therapy of Diseases, 2nd Edition)

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Zα and RNA editing in innate immunity. (A) Zα binds in a structure-specific fashion to Z-DNA with high affinity. The space-filling representations of P193 and N173 highlight their interaction with ZNA. Loss of function P193A and N175S variants are causal for the Aicardi–Goutières type 6 interferonopathy. (B) The only other protein in the human genome with a Zα domain is ZBP1. It activates inflammatory cell death in response to Z-RNA produced by viruses and endogenous retroelements (EREs) that amplify themselves by RdRp. Z-RNA also forms in stress granules. ADAR1 suppresses the Z-RNA-dependent activation of ZBP1. ADAR1 also prevents the dsRNA activation of PKR (protein kinase R encoded by EIF2AKA). PKR inhibits EIF2α (Eukaryotic Initiation Factor 2 alpha) dependent translation, leading to stress granule formation. ZBP1 interfaces with effector pathways through RHIMs (receptor-interacting protein homotypic interaction motif). (C) Most ALU elements are dimeric with a left and right arm. A- and B-boxes enable transcription by RNA Polymerase 3. The Z-Box present in ALU, as indicated by the red outline in (D), has one guanosine to cytosine substitution disrupting the expected pyrimidine/purine alternation (as indicated by the dot above the residue), The sequence is colored coded by base. The flip of the ALU Z-Box to Z-RNA helps identify transcripts as of host origin as pathogens lack this class of Z-forming element. Full article ">Figure 2
Zβ and RNA Editing. (A) ADAR1 p110 isoform domains. (B) Schematic of the Zβ winged helix-turn-helix domain, with the α-helices highlighted in blue and the β-sheet strands in yellow. The Y→I substitution that greatly diminishes binding to Z-DNA relative to Zα and the location of R328 in the α3 helix are shown with bold type face. (C) The docking of Zβ to a parallel strand GQ with U4 loops, modeled by Molecular Dynamics Simulation. R328 makes key contacts between the protein and DNA/RNA. (D) The sets of G-tetrad pairs that can form RNA-GQ are underlined. The right and left arms correspond to those in <a href="#ijms-26-02422-f001" class="html-fig">Figure 1</a>C. (E) A model of how Zβ enables the loading of p110 first onto a GQ formed during transcription, then onto a folded dsRNA (F) to enable engagement of the deaminase domain (G). (H) ADAR1 p110 and p150 are localized to ALU inverted repeats by different flipon conformations. (I) The sites of NSE are not associated with ALU elements but rather with GQs that are in close proximity. (J) GQ motifs on the antisense strand of some ALU families are close to a cluster of 3′ “AG” splice acceptor sites. Two of these “AG” sites change the reading frame and the third introduces a UAG stop codon into a reading frame. The coding potential of isoforms is further varied by Zβ-dependent editing of the “AG” splice sites and exonic NSE. (K) Through selection, ALU exonization, alternative splicing, and NSE can increase phenotypic diversity. The GQ and ALU elements may be incorporated into an exon or downstream of a splice junction. In the latter case, the GQ promotes editing of the exon when the downstream exon contains an exon complementary site (ECS), while the remnants of an ALU inverted repeat promote folding of the dsRNA editing substrate. In other cases, an ALU-derived GQ may be associated with the 3′ splice junction. Editing of the “AG” acceptor site would result in exon skipping. (L) Molecular Dynamics Simulation of the ADAR1 Zβ domain complexed with rGQ (pink surface) stacked on dGQ (green surface). (M) Close-up view of the interface between Zβ and rGQ. The Arginine 33 side chain penetrates deeply into the rGQ molecule, forming multiple hydrogen bonds and favorable van der Waals contacts with three guanine residues and the RNA backbone. Lysine 31 (corresponding to K326 in human ADAR1 p150) forms hydrogen bonds and a favorable ion-pair interaction with the RNA backbone. Eight water molecules within the interface form hydrogen bonding networks that bridge the protein backbone and amino acid sidechains with the RNA backbones and bases. Full article ">Figure 3
Examples of the GQ forming sequences associated with RNA editing. (A) ALU-associated editing of the RHOA transcript. The red boxes highlight the GQ. (B) The long dsRNA editing substrate formed by the fold back of the two ALU inverted repeats displayed in (C). The red box shows the position of the RHOA exon relative to the ALU elements. (D) The well-characterized K242R edit of NEIL1 (endonuclease VIII-like DNA glycosylase) is associated with a GQ. (E) The NEIL1 editing substrate with the edits indicated by red arrows. (F) The edits and various RNA isoforms of TNFRSF14 pre-mRNA are associated with a GQ motif. (G) Location of the edits in a dsRNA substrate formed by the TNFRSF14 transcript. (H) The FLG (filaggrin) RNA is associated with both a GQ and the antisense CCDST (cervical cancer associated DHX9 suppressive transcript) that could form a dsRNA substrate. (I) The FLG pre-mRNA by itself also folds into a dsRNA substrate. Full article ">

19 pages, 2176 KiB

Open AccessArticle

Evaluating the Immunogenicity of an Intranasal Microparticle Combination Vaccine for COVID-19 and Influenza

by Sharon Vijayanand, Smital Patil, Priyal Bagwe, Revanth Singh, Emmanuel Adediran and Martin J. D’Souza

Vaccines 2025, 13(3), 282; https://doi.org/10.3390/vaccines13030282 - 7 Mar 2025

Abstract

Background: Infectious respiratory pathogens like SARS-CoV-2 and influenza frequently mutate, leading to the emergence of variants. This necessitates continuous updates to FDA-approved vaccines with booster shots targeting the circulating variants. Vaccine hesitancy and needle injections create inconvenience and contribute to reduced global vaccination rates. To address the burden of frequent painful injections, this manuscript explores the potential of non-invasive intranasal (IN) vaccine administration as an effective alternative to intramuscular (IM) shots. Further, as a proof-of-concept, an inactivated combination vaccine for COVID-19 and influenza was tested to eliminate the need for separate vaccinations. Methods: The methods involved encapsulating antigens and adjuvants in poly(lactic-co-glycolic acid) (PLGA) polymer matrices, achieving over 85% entrapment. The vaccine was evaluated in vitro for cytotoxicity and immunogenicity before being administered to 6–8-week-old Swiss Webster mice at weeks 0, 3, and 6. The mice were then assessed for antibody levels and cellular responses. Results: The intranasal microparticle (IN-MP) vaccine induced an innate immune response, autophagy, and were non-cytotoxic in vitro. In vivo, the vaccine led to high levels of virus-specific serum IgM, IgG, and IgA binding antibodies, as well as elevated IgG and IgA levels in the lung wash samples. The antibodies generated demonstrated neutralizing activity against the SARS-CoV-2 pseudovirus. Furthermore, the IN-MP vaccine prompted increased antigen-specific CD4⁺ and CD8⁺ T-cell responses in the vaccinated mice. Conclusions: The IN-MP combination vaccine produced immune responses comparable to or higher than the IM route, indicating its potential as an alternative to IM injections. Full article

(This article belongs to the Special Issue Innovating Vaccine Research in Mucosal Vaccines)

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Preclinical vaccine study timeline and dosing regimen. 6–8-week-old mice were immunized with three doses at weeks 0, 3, and 6. The mice were bled 2 weeks after each dose at weeks 3, 5, 8, and 10 to evaluate serum antibody levels. The mice were challenged intranasally with 50 μL of an 0.5 × LD50 dose of Influenza A Virus, A/Puerto Rico/8-WG/1934 (H1N1) at week 10 and sacrificed 14 days after challenge (week 12). Terminal blood, lymph nodes, spleen, and lungs were isolated and processed for evaluation of immune responses. Full article ">Figure 2
In vitro nitrite release and cytotoxicity assessment. (A). Nitrite released by DCs upon stimulation with various treatment groups. Cell density was adjusted to 3 × 104 cells/well. The nitrite release in the supernatant was assessed using the Griess’s assay. NO plays a role in fighting invading pathogens and activation of signaling molecules in innate immunity. The MP groups produced increased levels of nitrite compared to the suspension vaccine. Addition of adjuvants potentiated the response of the MP vaccine. The (iSCoV-2 + IIV + adjuvants) MP group received only half the dose of unadjuvanted (iSCoV-2 + IIV) MP group (listed in <a href="#vaccines-13-00282-t001" class="html-table">Table 1</a>) and produced response comparable to the unadjuvanted MP combination vaccine. Data are expressed as mean ± SEM (n = 3), One-way ANOVA test, Post hoc Tukey’s multiple comparisons test, ns (non-significant), ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.001. (B,C). Percent cell viability of DCs pulsed with varying concentrations of IIV MP (B) and CPG 7909 MP (C). Cell density was adjusted to 1 × 104 cells/well. The cells were treated with two-fold serial dilutions of the corresponding MP groups at three concentrations, 31.25 µg/mL, 62.5 µg/mL, 125 µg/mL in cDMEM (100 μL/well) for 24 h. 25% v/v DMSO was used as a -ve control, and cells only were used as a +ve control. The % cell viability was >95% for all concentrations tested. Data are expressed as mean ± SEM (n = 3). One-way ANOVA test, Post hoc Dunnett’s multiple comparison test, **** p ≤ 0.0001, ns, non-significant. Full article ">Figure 3
Assessing autophagy in DCs treated with different groups A-D. Fluorescent microscope imaging. (A). Cells only (−ve control). (B). Antigen suspension. (C). Vaccine MP. (D). Vaccine + Adjuvants in MP. (E). Flow cytometry analysis of autophagy. The vaccine MP induces autophagy in DCs which is significantly higher than the antigen suspension group (E). The anitgen suspension induces autophagy to a lesser extent (B). Data expressed as mean ± SEM, n = 4, one-way ANOVA, post hoc Tukey’s multiple comparisons test. *** p ≤ 0.001. Full article ">Figure 4
Virus-specific antibody levels in the serum and lung wash of vaccinated mice. The serum antibodies were assessed for their ability to bind to inactivated SARS-CoV-2 or inactivated influenza A H1N1 using ELISA. The functional activity of the serum antibodies was assessed for SARS-CoV-2 using a pseudovirus neutralization assay. IgG and IgA levels in the lung wash of the vaccination mice were assessed at week 12 using ELISA. (A). SARS-CoV-2-specific IgM. (B). Influenza A H1N1-specific IgM. Serum IgM levels in vaccinated mice peaked at week 2 after the prime dose and subsequently decreased in the following weeks. (C). % neutralization of SARS-CoV-2 pseudovirus by serum antibodies was assessed at weeks 5 and 12. The neutralizing capacity of the antibodies varied for each animal and was found to range between 47 and 99% at week 5, and between 50% and 90% at week 12 for the IN-MP vaccine group. (D). SARS-CoV-2-specific IgG. (E). Influenza A H1N1-specific IgG. The serum IgG levels increased after the prime dose and remained high until week 12. The IN-MP vaccine group exhibited higher antibody levels compared to the IM suspension group. SARS-CoV-2-specific antibody levels dominated over the influenza-specific antibody levels. (F). Virus-specific IgG in lung wash samples. The IN-MP vaccine induced significant IgG levels in lungs. (G). SARS-CoV-2-specific IgA. (H). Influenza A H1N1-specific IgA. The serum IgA levels of the IN-MP vaccine were higher than the IM suspension vaccine for both viruses. (I). Virus-specific IgA in lung wash samples. The IN-MP vaccine induced significant IgA responses in the lung wash samples of vaccinated mice. Responses obtained are compared to no treatment (control) and IM suspension (control) group. Data are expressed as individual values, n = 6. Two-way ANOVA, post hoc Tukey’s multiple comparisons test. ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Full article ">Figure 5
Virus-specific IgG1 and IgG2a levels and IgG1/IgG2a ratio in the serum of vaccinated mice. (A). SARS-CoV-2-specific IgG1. (B). Influenza A H1N1-specific IgG1. (C). SARS-CoV-2-specific IgG2a. (D). Influenza A H1N1-specific IgG2a. (E). SARS-CoV-2-specific IgG1/IgG2a. The SARS-CoV-2-specific IgG1/IgG2a ratio was evaluated which indicated that the mean value was 3.6 for the IN-MP vaccine and 6.2 for IM suspension vaccine. (F). Influenza A H1N1-specific IgG1/IgG2a. For influenza, the mean IgG1/IgG2a was 5.4 for the IN-MP vaccine and 7.9 for the IM suspension vaccine. The Responses obtained are compared to no treatment (control) and IM suspension (control) group. Data are expressed as individual values, n = 6. Two-way ANOVA, post hoc Tukey’s multiple comparisons test. ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Full article ">Figure 6
SARS-CoV-2-specific and Influenza A H1N1-specific CD4+ and CD8+ T-cells in the splenocytes and lymphocytes of vaccinated mice. The antigen-specific responses were evaluated by indirectly stimulating the cells with the respective antigen (5 μg/mL) and IL-2 (100 IU/mL) to activate antigen-primed T-cells in vaccinated mice. The % cells expressing CD4 and CD8 on the T-cell surface was quantified using flow cytometry. The IN-MP vaccine induced high percentages of antigen-specific T-cells expressing CD4 and CD8 levels in the lymphocytes and only CD4+ T cells in the spleen. (A). CD4+ T-cells in the lymphocytes. (B). CD8+ T-cells in the lymphocytes. (C). CD4+ T-cells in the splenocytes (D). CD8+ T-cells in the splenocytes. Responses obtained are compared to no treatment (control) and the IM suspension (control) group. Data are expressed as individual values, n = 6, Two-way ANOVA, post hoc Tukey’s multiple comparisons test. ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Full article ">

24 pages, 4533 KiB

Open AccessArticle

Anti-Tumor Effects of Cecropin A and Drosocin Incorporated into Macrophage-like Cells Against Hematopoietic Tumors in Drosophila mxc Mutants

by Marina Hirata, Tadashi Nomura and Yoshihiro H. Inoue

Cells 2025, 14(6), 389; https://doi.org/10.3390/cells14060389 - 7 Mar 2025

Viewed by 158

Abstract

Five major antimicrobial peptides (AMPs) in Drosophila are induced in multiple sex combs (mxc) mutant larvae harboring lymph gland (LG) tumors, and they exhibit anti-tumor effects. The effects of other well-known AMPs, Cecropin A and Drosocin, remain unexplored. We investigated the tumor-elimination mechanism of these AMPs. A half-dose reduction in either the Toll or Imd gene reduced the induction of these AMPs and enhanced tumor growth in mxc^mbn1 mutant larvae, indicating that their anti-tumor effects depend on the innate immune pathway. Overexpression of these AMPs in the fat body suppressed tumor growth without affecting cell proliferation. Apoptosis was promoted in the mutant but not in normal LGs. Conversely, knockdown of them inhibited apoptosis and enhanced tumor growth; therefore, they inhibit LG tumor growth by inducing apoptosis. The AMPs from the fat body were incorporated into the hemocytes of mutant but not normal larvae. Another AMP, Drosomycin, was taken up via phagocytosis factors. Enhanced phosphatidylserine signals were observed on the tumor surface. Inhibition of the signals exposed on the cell surface enhanced tumor growth. AMPs may target phosphatidylserine in tumors to induce apoptosis and execute their tumor-specific effects. AMPs could be beneficial anti-cancer drugs with minimal side effects for clinical development. Full article

(This article belongs to the Special Issue Drosophila as a Model for Understanding Human Disease)

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36 pages, 1252 KiB

Open AccessReview

The Role of Inflammation in the Pathogenesis of Comorbidity of Chronic Obstructive Pulmonary Disease and Pulmonary Tuberculosis

by Stanislav Kotlyarov and Dmitry Oskin

Int. J. Mol. Sci. 2025, 26(6), 2378; https://doi.org/10.3390/ijms26062378 - 7 Mar 2025

Viewed by 47

Abstract

The comorbid course of chronic obstructive pulmonary disease (COPD) and pulmonary tuberculosis is an important medical and social problem. Both diseases, although having different etiologies, have many overlapping relationships that mutually influence their course and prognosis. The aim of the current review is to discuss the role of different immune mechanisms underlying inflammation in COPD and pulmonary tuberculosis. These mechanisms are known to involve both the innate and adaptive immune system, including various cellular and intercellular interactions. There is growing evidence that immune mechanisms involved in the pathogenesis of both COPD and tuberculosis may jointly contribute to the tuberculosis-associated obstructive pulmonary disease (TOPD) phenotype. Several studies have reported prior tuberculosis as a risk factor for COPD. Therefore, the study of the mechanisms that link COPD and tuberculosis is of considerable clinical interest. Full article

(This article belongs to the Special Issue Molecular Mechanisms, Pathophysiology and Therapeutic Strategies of Chronic Obstructive Pulmonary Disease (COPD))

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Lung tuberculosis (A) and COPD (B), having some common risk factors and mechanisms of development associated with inflammation, form a separate clinical phenotype in which the polymorphism of morphologic, radiologic, and clinical manifestations of tuberculosis plays an important role due to variability in immune response, structural changes in lung tissue, and individual features of disease pathogenesis. Note: A. Respiratory tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. The main morphological manifestation of tuberculosis is the formation of specific granulomas with a characteristic histological structure: the presence of central caseous necrosis surrounded by a shaft of epithelioid cells, macrophages, lymphocytes, and giant Langhans cells, which reflects a cell-mediated immune response to the penetration of the pathogen. At first exposure to mycobacteria, more often in childhood, primary tuberculosis develops, which manifests itself by the formation of a primary affect (Ghon complex) in the lungs. The primary affect is characterized by an area of caseous tissue degeneration in the area of initial pathogen inoculation (predominantly subpleurally in the lower/middle lobes), tuberculous lymphangitis with caseous lymphadenitis of regional nodes, and often reactive pleurisy. The course of primary tuberculosis varies from asymptomatic with subsequent self-healing and calcification of the focus to manifest progressive tuberculosis with the risk of dissemination, development of serious complications and tuberculous meningitis. Secondary tuberculosis develops due to reactivation of latent tuberculosis infection or exogenous MBT superinfection and is characterized by polymorphism of morphological forms, localized mainly in the apical parts of the lungs. Focal tuberculosis is usually represented by single dense fibrous foci (up to 10 mm) within 1–2 segments with minimal perifocal inflammation and a clinically asymptomatic course. In contrast, infiltrative tuberculosis is characterized by predominantly exudative-necrotic changes with extensive areas of caseification surrounded by inflammatory infiltration, with a tendency to destruction of lung tissue and formation of decay cavities. In disseminated tuberculosis, there is hematogenous or lymphogenous spread of the pathogen with the formation of multiple foci and foci of infiltrative character. Tuberculoma is a form of secondary tuberculosis, which is an encapsulated focus of caseosis ranging in size from 1–2 to 5 cm or more, usually clinically silent and often detected during prophylactic radiologic examinations. Fibrotic cavernous tuberculosis is the final stage of development of all forms of respiratory tuberculosis with the formation of thick-walled cavities (caverns) with a three-layer wall (pyogenic, granulation, fibrous zones), which are surrounded by massive pericavitary sclerosis, bronchial deformation and destruction of parenchyma. In fibrotic cavernous tuberculosis, there is a high risk of multidrug-resistant and extensively drug-resistant MBT. B. Chronic obstructive pulmonary disease (COPD) is associated with chronic exposure to inhaled particles and gases, leading to the development of inflammation in the bronchi involving various cells. Prolonged inflammation leads to symptoms, development of bronchial obstruction, and emphysema. Neutrophils and macrophages are involved in bronchial remodeling through the production of elastases, matrix metalloproteinases. Disturbances in pro- and anti-inflammatory polarization of macrophages leads to chronicization of inflammation and development of systemic inflammation. Full article ">Figure 2
Macrophages play an important role in the pathogenesis of both COPD and tuberculosis. Full article ">

11 pages, 1353 KiB

Open AccessArticle

Inflammatory Bowel Disease from the Perspective of Newer Innate Immune System Biomarkers

by Martin Tobi, Fadi Antaki, MaryAnn Rambus, Jason Hellman, James Hatfield, Suzanne Fligiel and Benita McVicker

Gastrointest. Disord. 2025, 7(1), 22; https://doi.org/10.3390/gidisord7010022 - 6 Mar 2025

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Abstract

Background: The perspective of inflammatory bowel disease (IBD) has changed radically since the first decade of the 21st century, and the formerly monolithic components of IBD, ulcerative colitis (UC), and Crohn’s disease (CD) have undergone a fundamental convergence, with realization that there is likely an element of shared pathogenesis. The ground shift began with genomic revelation but with the current emergence of the innate immune system (InImS) as a key player, allowing for improved understanding of the associations between the immune underpinnings of IBD. Methods: Using unique ferritin/fecal p87 (FERAD) or using colonoscopic effluent as denominator (FEREFF) and other ratios to test this hypothesis, we prospectively enrolled 2185 patients with increased risk of colorectal cancer, of whom 31 had UC and 18 CD, with 2136 controls and brought to bear in a convenient measure for the InImS, the FERAD ratio. The FERAD, FEREFF, and NLR ratios have been shown to be effective measures of the InImS in COVID-19 and various cancers. p87 is expressed in gut Paneth cells known to modulate the microbiome by secretion of alpha-defensins, a natural antibiotic. Other related parameters were also evaluated. Results: There was no significant difference between the FERAD ratio in UC and CD. However, differences between IBD entities and controls were substantial. Conclusions: InImS settings in IBD are similar between CD and UC. p87 tissue immunohistochemistry (IHC) is also shared. Other InImS markers, such as the absolute neutrophil/lymphocyte ratio, are also confluent between the two IBD forms. Full article

(This article belongs to the Special Issue Factors Affecting Disease Activity in Children and Adults with Inflammatory Bowel Disease: An Exploration of Pro-Inflammatory and Anti-Inflammatory Elements)

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A flow diagram showing the total number and those of the IBD patients and controls. Full article ">Figure 2
Bar diagram depicting the comparison of FERAD levels in IBD and controls. Full article ">Figure 3
Bar diagram showing mean levels of colonic p87 expression in disease versus controls. Full article ">Figure 4
The crypts are well-oriented and likely represent inactive UC. The Adnab-9 antibody was used in a concentration of 1:50 dilution and developed using the ABC kit (see <a href="#sec3-gastrointestdisord-07-00022" class="html-sec">Section 3</a> below). The magnification in <a href="#gastrointestdisord-07-00022-f004" class="html-fig">Figure 4</a> is 20×. Full article ">Figure 5
A direct correlation exists in IBD patients between initial and final ferritin determinations. Full article ">

17 pages, 3522 KiB

Open AccessArticle

Differential Responses of Pediatric and Adult Primary Epithelial Cells to Human Metapneumovirus and Respiratory Syncytial Virus Infection

by Pius I. Babawale and Antonieta Guerrero-Plata

Viruses 2025, 17(3), 380; https://doi.org/10.3390/v17030380 - 6 Mar 2025

Viewed by 164

Abstract

Human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are pneumoviruses causing lower respiratory tract infections, primarily in infants and children rather than in healthy adults. Human bronchial epithelial cells serve as a viral replication target and source of the innate immune response to these viruses. To better understand the immune responses induced by RSV and HMPV in the pediatric airway epithelium, we comparatively studied pediatric and adult epithelial responses. We used normal human bronchial epithelial (NHBE) cells cultured in an air–liquid interface culture system (ALI), which helps to mimic the architecture of the human lower respiratory tract epithelium. Our results demonstrate differential viral replication patterns and reduced interferons; and inflammatory cytokines’ expression in pediatric cells compared to adult cells. However, pediatric epithelial cells expressed an increased mucus response and induced a stronger pro-inflammatory response in monocyte-derived dendritic cells. These findings reveal age-dependent immune epithelial responses that may contribute to more severe infections by HMPV and RSV. Full article

(This article belongs to the Section Human Virology and Viral Diseases)

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Age-related differences in NHBE cell susceptibility to HMPV and RSV infection. NHBE cells from pediatric and adult donors cultured at the air–liquid interface (ALI) and infected with RSV or HMPV. (A) After 7 days of HMPV and RSV infection, cells were stained with H&E staining to assess cell morphology; Scale bar = 50 μm. (B) Kinetics of viral copy numbers. Data represent mean ± SEM from three donors for each age group. Statistical significance was determined using two-way ANOVA with Šídák’s multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001). Full article ">Figure 2
IFN responses of pediatric and adult NHBE cells to RSV and HMPV infection. NHBE cells were infected with RSV or HMPV. Gene expression of (A) type I IFNs (IFN-α2, IFN-β, IFN-ε, and IFN-ω) and (B) type III IFNs (IFN-λ1 and IFN-λ2/3) was assessed by RT-qPCR at different time points. Statistical significance was determined using two-way ANOVA with Šídák’s multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Non-significant (ns). Full article ">Figure 3
Differential expression of ISGs by pediatric and adult NHBE cells. Adult and pediatric NHBE cells were differentiated in ALI culture and infected with RSV and HMPV. RNA samples were collected at different time points and analyzed for expression of key ISGs (IFIT1, IFIT2, IFIT3, OAS1, MX1, and ISG15) by RT-qPCR. Statistical significance was determined using two-way ANOVA with Šídák’s multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Full article ">Figure 4
Cytokine responses in NHBE cells infected with HMPV or RSV. Adult and pediatric NHBE cells were differentiated in ALI culture and infected with RSV or HMPV. RNA samples were collected at different time points and analyzed by RT-qPCR for the expression of (A) pro-inflammatory cytokines and (B) epithelial alarmins. Statistical significance was determined using two-way ANOVA with Šídák’s multiple comparisons test (* p < 0.05, ** p < 0.01, **** p < 0.0001). Full article ">Figure 5
Cytokine release from human epithelial cells infected with HMPV or RSV. Pediatric and adult NHBE cells were grown in ALI culture and infected with HMPV or RSV. Apical washes were collected at different time points after infection, and concentration of cytokines was determined by LEGENDplex multiplex immunoassay. Statistical significance was determined using two-way ANOVA with Dunnett’s multiple comparisons test (* p < 0.05). Full article ">Figure 6
Mucin expression induced by RSV and HMPV in pediatric and adult NHBE cells. NHBE cells were differentiated in ALI culture and infected with RSV and HMPV. (A) Cells were stained with PAS histological staining on day 7 after infection. Scale bar = 50 μm. (B) Further analysis by RT-qPCR assessed the expression of MUC5AC and MUC5B levels. Statistical significance was determined using two-way ANOVA with Šídák’s multiple comparisons test (** p < 0.01, *** p < 0.001. Non-significant (ns). Full article ">Figure 7
Induction of inflammatory cytokines in mo-DCs co-cultured with either pediatric or adult NHBE cells. mo-DCs were co-cultured with NHBE cells from either pediatric or adult donors infected with HMPV or RSV. (A) Schematic representation of the co-culture setup: Fully differentiated NHBE cells were infected with HMPV or RSV and cultured with mo-DCs for 3 days. mo-DCs were analyzed for the expression of (B) IL-6, (C) TNF-α, and (D) IL-1β by RT-qPCR. Data represent mean ± SEM, n = 4–6. Statistical significance was determined using the Kruskal–Wallis test (* p < 0.05, ** p < 0.01); non-significant (ns). Full article ">

15 pages, 2229 KiB

Open AccessArticle

Resveratrol Upregulates Antioxidant Factors Expression and Downmodulates Interferon-Inducible Antiviral Factors in Aging

by Iara Grigoletto Fernandes, Luana de M. Oliveira, Milena M. de Souza Andrade, Ricardo W. Alberca, Júlia Cataldo Lima, Emanuella Sarmento Alho de Sousa, Anna Julia Pietrobon, Nátalli Zanete Pereira, Anna Cláudia Calvielli Castelo Branco, Alberto José da Silva Duarte and Maria Notomi Sato

Int. J. Mol. Sci. 2025, 26(5), 2345; https://doi.org/10.3390/ijms26052345 - 6 Mar 2025

Viewed by 82

Abstract

Immunosenescence, a process with a dysfunctional immune response that may favor infection is associated with an increase in inflammatory responses mediated by proinflammatory cytokines, characteristic of inflammaging. Aging and immunosenescence have a relationship relating to oxidative stress and inflammaging. Therefore, natural antioxidant compounds could be candidates for the control of the oxidative process. Our purpose was to evaluate the effect of resveratrol (Resv) on the antioxidant, antiviral, and anti-inflammatory responses induced by toll-like receptors (TLRs) 3, 4, and 7/8 agonists stimulation on peripheral blood mononuclear cells (PBMCs) of elderly and healthy female individuals (63–82 years old) and young and healthy female individuals (21–31 years old). Our data show that Resv may upregulate antioxidant factor expression, such as catalase (CAT) and SIRT1, in response to TLR4 and TLR7/8 agonists, similarly in both young and aged groups. Moreover, the Resv anti-inflammatory effect was detected by inhibiting IL-1β, TNF-α, and IL-10 secretion levels, as well as by the chemokines CCL2 and CCL5, induced by TLR4 and TLR7/8 stimulation. Curiously, Resv decreased antiviral genes, such as MxA, STING, and IRF7 expression, possibly by reducing the inflammatory effects of interferon-induced genes. Taken together, our results demonstrate the ability of Resv to stimulate antioxidant factors, leading to a downmodulation of the inflammatory response induced by innate immune stimulation. These findings point out Resv as a strategy to control the upregulation of inflammatory response, even in elderly individuals. Full article

(This article belongs to the Special Issue Potential Health Benefits of Resveratrol: Laboratory-Confirmed Mechanisms of Action)

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Figure 1
Resv downmodulates antiviral factors upon TLR3 activation. (A) Heatmap of antioxidant and antiviral factors expressed by PBMC stimulated with TLR agonist 3 (POLY(I:C)) and addition of Resv. The young group is colored in green, and the elderly group is colored in yellow. Red shade gene expression means above the row average, and blue shade expression means below the average. Z-score represents subtract mean, divided by standard deviation. (B) Comparison of the constitutive gene expression of young healthy volunteers and elderly healthy volunteers by qPCR. The relative expression of the targets was calculated in comparison to the amplification of the constitutive gene, GAPDH, and in comparison, to the non-stimulated situation. N = 9–10 individuals per group. Data are expressed as median and interquartile range. Paired Wilcoxon test: * p < 0.05, ** p < 0.001 (stimulated vs. unstimulated). Full article ">Figure 2
Resv upregulates CAT/SIRT1 expressions while decreasing antiviral factors upon TLR4 activation. (A) Heatmap of antioxidant and antiviral factors expressed by PBMCs stimulated with TLR agonist 4 (LPS) and addition of Resv. The young group is colored in green, and the elderly group is colored in yellow. Red shade gene expression means above the row average, and blue shade expression means below the average. Z-score represents subtract mean, divided by standard deviation. (B) Comparison of the constitutive gene expression of young healthy volunteers and elderly healthy volunteers by qPCR. The relative expression of the targets was calculated in comparison to the amplification of the constitutive gene, GAPDH, and in comparison to the non-stimulated situation. N = 9–10 individuals per group. Data are expressed as median and interquartile range. Paired Wilcoxon test: * p < 0.05, ** p < 0.001 (stimulated vs. unstimulated). Full article ">Figure 3
Resv upregulates CAT expressions while decreasing antiviral factors upon TLR7/8 activation. (A) Heatmap of antioxidant and antiviral factors expressed by PBMC stimulated with TLR agonists 7 and 8 (CL097) and addition of Resv (N = 20). The young group is colored in green, and the elderly group is colored in yellow. Red shade gene expression means above the row average, and blue shade expression means below the average. Z-score represents subtract mean, divided by standard deviation. (B) Comparison of the constitutive gene expression of young healthy volunteers and elderly healthy volunteers by qPCR. The relative expression of the targets was calculated in comparison to the amplification of the constitutive gene, GAPDH, and in comparison to the non-stimulated situation. N = 9–10 individuals per group. Data are expressed as median and interquartile range. Paired Wilcoxon test: * p < 0.05, ** p < 0.001 (stimulated vs. unstimulated). Full article ">Figure 4
Proinflammatory cytokines induced by TLR4 and TLR7/8 stimulation were inhibited by Resv. PBMCs were incubated for 24 h with CL097 (2.5 µg/mL), LPS (1 µg/mL), POLY(I:C) (10 µg/mL), and Resv (100 µM). The production of cytokines IL-1β, TNF-α, IFN-γ, and IL-10 was assessed by flow cytometry. N = 9–10 individuals per group. Data expressed in the median and interquartile range. Unpaired Mann–Whitney test: # p < 0.05 (young vs. elderly). Wilcoxon paired test: * p < 0.05; ** p < 0.01 (stimulated vs. unstimulated). Full article ">Figure 5
CCL2 and CCL5 induced by TLR4 and TLR3 stimulation were inhibited by Resv. PBMCs were incubated for 24 h with CL097 (2.5 µg/mL), LPS (1 µg/mL), and POLY(I:C) (10 µg/mL) in the presence of Resv (100 µM). CCL2 and CCL5 chemokines were assessed by flow cytometry. N = 9–10 individuals per group. Data expressed as median and interquartile range. One-way ANOVA test: * p < 0.05; ** p < 0.01. Unpaired Mann Whitney test: # p < 0.05 (stimulated vs. unstimulated). Full article ">

19 pages, 1133 KiB

Open AccessReview

Polysaccharides with Arabinose: Key Players in Reducing Chronic Inflammation and Enhancing Immune Health in Aging

by Patricia Pantoja Newman, Brenda Landvoigt Schmitt, Rafael Moura Maurmann and Brandt D. Pence

Molecules 2025, 30(5), 1178; https://doi.org/10.3390/molecules30051178 - 6 Mar 2025

Viewed by 190

Abstract

Aging is associated with a decline in physiological performance leading to increased inflammation and impaired immune function. Polysaccharides (PLs) found in plants, fruits, and fungi are emerging as potential targets for therapeutic intervention, but little is known about their effects on chronic inflammation and aging. This review aims to highlight the current advances related to the use of PLs, with the presence of arabinose, to attenuate oxidative stress and chronic and acute inflammation, and their immunomodulatory effects associated with antioxidant status in monocytes, macrophages, and neutrophil infiltration, and leukocyte rolling adhesion in neutrophils. In addition, recent studies have shown the importance of investigating the ‘major’ monosaccharide, such as arabinose, present in several of these polysaccharides, and with described effects on gut microbiome, glucose, inflammation, allergy, cancer cell proliferation, neuromodulation, and metabolic stress. Perspectives and opportunities for further investigation are provided. By promoting a balanced immune response and reducing inflammation, PLs with arabinose or even arabinose per se may alleviate the immune dysregulation and inflammation seen in the elderly, therefore providing a promising strategy to mitigate a variety of diseases. Full article

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Effect of arabinose in different models. Full article ">

20 pages, 1300 KiB

Open AccessArticle

Venomous Cargo: Diverse Toxin-Related Proteins Are Associated with Extracellular Vesicles in Parasitoid Wasp Venom

by Jennifer Chou, Michael Z. Li, Brian Wey, Mubasshir Mumtaz, Johnny R. Ramroop, Shaneen Singh and Shubha Govind

Pathogens 2025, 14(3), 255; https://doi.org/10.3390/pathogens14030255 - 5 Mar 2025

Viewed by 188

Abstract

Unusual membrane-bound particles are present in the venom of the parasitoid wasps that parasitize Drosophila melanogaster. These venom particles harbor about 400 proteins and suppress the encapsulation of a wasp egg. Whereas the proteins in the particles of Leptopilina boulardi venom modify host hemocyte properties, those in L. heterotoma kill host hemocytes. The mechanisms underlying this differential effect are not well understood. The proteome of the L. heterotoma venom particles has been described before, but that of L. boulardi has not been similarly examined. Using sequence-based programs, we report the presence of conserved proteins in both proteomes with strong enrichment in the endomembrane and exosomal cell components. Extracellular vesicle markers are present in both proteomes, as are numerous toxins. Both proteomes also contain proteins lacking any annotation. Among these, we identified the proteins with structural similarity to the ADP-ribosyltransferase enzymes involved in bacterial virulence. We propose that invertebrate fluids like parasitoid venom contain functional extracellular vesicles that deliver toxins and virulence factors from a parasite to a host. Furthermore, the presence of such vesicles may not be uncommon in the venom of other animals. An experimental verification of the predicted toxin functions will clarify the cellular mechanisms underlying successful parasitism. Full article

(This article belongs to the Special Issue Computational Approaches in Mechanisms of Pathogenesis)

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Figure 1
Venom gland morphology. (A) The entire venom gland complex is composed of the long gland (LG, anterior-most); the connecting duct (CD); the reservoir (R); and the ovipositor (Ovi). The ovipositor enters the host larva during egg laying, and the venom contents are deposited into the host at the same time. (B–E) The long glands from Lb17 (B,C) and Lh14 wasps (D,E), stained with Hoechst 33258 and rhodamine phalloidin. For all samples, the anterior end of the long gland is placed in the top left. N = nose; L = long gland lumen; * = secretory cells; triangle = canals. (C,E) Only the red channel is shown to highlight the F-actin-rich canals. Select Z-stack images were assembled from either 9 (for Lb), or 10 (for Lh) optical sections to visualize the 3D views of canal organization. Full article ">Figure 2
Characterization of Lb and Lh EV proteins. (A,B) Enrichment analysis of Lb (A) and Lh (B) EV proteins. The specific cellular compartments found in Vesiclepedia are shown on the X-axis. The primary Y-axis indicates the percentage of genes, calculated by FunRich, as the number of genes within the provided dataset (for Lb or Lh) that are associated with the listed cellular compartment (i.e., plasma membrane, nucleus, etc.) divided by the total number of genes within the provided dataset found within the FunRich/Vesiclepedia database (see Methods). The secondary Y-axis shows the −log10 (p-value). In both species, there is significant enrichment in the exosomes, lysosomes, mitochondria, ER–Golgi compartment, and ribosomes (p < 0.01). The Bonferroni correction was used by the FunRich program to calculate the p-values. Full article ">Figure 3
Pie charts showing the proportions of various classes of toxin-related protein domains. (A,B) The data presented are from 23 Lb (A) and 30 Lh (B) proteins. Seven toxin categories common to both species are shown in the same color. Species-specific toxin categories are italicized. The number of proteins in each category is indicated. See <a href="#app1-pathogens-14-00255" class="html-app">Tables S2 and S3</a> for more details. Full article ">Figure 4
A conserved ART fold and R-S-E motif in wasp sequences. (A) Structural superposition of Lb_284 (XP_051165226.1; tv_blue; RMSD: 1.84 Å), Lb_316 (XP_051176700.1; slate blue; RMSD: 1.61Å), and Lb_340 (XP_051161330.1; marine blue; RMSD: 1.46 Å) with Bacillus cereus C3 exoenzyme (PDB: 4XSH; red). (B) Structural superposition of Lh_005 (XP_043484983.1; slate blue; RMSD: 2.40 Å) with Bacillus cereus C3 exoenzyme (PDB: 4XSH; red). (C) Multiple sequence alignment of three Lb and one Lh putative ARTs visualized in ESPript. Residues shaded red denote identity matches while residues colored red and boxed in blue show similarity matches. Residues colored blue represent NAD+ -interacting residues and those that are shaded in magenta show NAD+-interacting residues that form a putative R-S-E motif. The secondary structure descriptions for the Lb sequences are shown above and those for the Lh sequence are shown below the alignment. See <a href="#app1-pathogens-14-00255" class="html-app">Table S4 and Figures S2–S5</a>. Full article ">

32 pages, 10662 KiB

Open AccessArticle

Characterization of Exhausted T Cell Signatures in Pan-Cancer Settings

by Rifat Tasnim Juthi, Saiful Arefeen Sazed, Manvita Mareboina, Apostolos Zaravinos and Ilias Georgakopoulos-Soares

Int. J. Mol. Sci. 2025, 26(5), 2311; https://doi.org/10.3390/ijms26052311 - 5 Mar 2025

Viewed by 181

Abstract

T cells play diverse roles in cancer immunology, acting as tumor suppressors, cytotoxic effectors, enhancers of cytotoxic T lymphocyte responses and immune suppressors; providing memory and surveillance; modulating the tumor microenvironment (TME); or activating innate immune cells. However, cancer cells can disrupt T cell function, leading to T cell exhaustion and a weakened immune response against the tumor. The expression of exhausted T cell (Tex) markers plays a pivotal role in shaping the immune landscape of multiple cancers. Our aim was to systematically investigate the role of known T cell exhaustion (Tex) markers across multiple cancers while exploring their molecular interactions, mutation profiles, and potential implications for immunotherapy. The mRNA expression profile of six Tex markers, LAG-3, PDCD1, TIGIT, HAVCR2, CXCL13, and LAYN was investigated in pan-cancer. Utilizing data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), The Cancer Proteome Atlas (TCPA), and other repositories, we characterized the differential expression of the Tex markers, their association with the patients’ survival outcome, and their mutation profile in multiple cancers. Additionally, we analyzed the effects on cancer-related pathways and immune infiltration within the TME, offering valuable insights into mechanisms of cancer immune evasion and progression. Finally, the correlation between their expression and sensitivity to multiple anti-cancer drugs was investigated extensively. Differential expression of all six markers was significantly associated with KIRC and poor prognosis in several cancers. They also played a potential activating role in apoptosis, EMT, and hormone ER pathways, as well as a potential inhibitory role in the DNA damage response and RTK oncogenic pathways. Infiltration of different immune cells was also found to be associated with the expression of the Tex-related genes in most cancer types. These findings underline that the reviving of exhausted T cells can be used to enhance the efficacy of immunotherapy in cancer patients. Full article

(This article belongs to the Special Issue Big Data in Multi-Omics)

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16 pages, 3455 KiB

Open AccessArticle

Genome-Wide Identification and Expression Analysis of CrRLK1-like Gene Family in Potatoes (Solanum tuberosum L.) and Its Role in PAMP-Triggered Immunity

by Yazhou Bao, Ru Zhao, Sixian Hu, Xiaoli Li, Like Wang, Ji Wang, Junbin Ji, Weiduo Wang, Changqing Zhu, Jiajia Chen, Ailing Ben, Jinfeng Peng and Tingli Liu

Genes 2025, 16(3), 308; https://doi.org/10.3390/genes16030308 - 4 Mar 2025

Viewed by 186

Abstract

Background: The Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) subfamily, a specialized group within receptor-like kinases (RLKs), was initially identified in C. roseus cell cultures. CrRLK1L plays an important role in the growth, development and stress response of plants. Although CrRLK1L genes have been characterized across multiple plant species, their biological and genetic functions in potatoes (Solanum tuberosum) remains poorly elucidated. Methods: a genome-wide investigation, phylogenetic analysis, chromosome localization, exon–intron structure, conserved motifs, stress-responsive cis-elements, tissue-specific expression patterns, and their effects on pathogen associated molecular patterns (PAMPs) induced reactive oxygen species (ROS) production were analyzed. Results: A total of 29 CrRLK1L genes were identified in the S. tuberosum genome, unevenly distributed across 10 chromosomes and divided into three groups. Tissue-specific expression analysis revealed seven genes highly expressed in all tissues, while CrRLK1L13 was specific to stamens and flowers. Under stress conditions (mannitol, salt, hormone, and heat), StCrRLK1L genes exhibited diverse expression patterns. Functional characterization in Nicotiana benthamiana identified seven ROS suppressors and four ROS enhancers, implicating their roles in PAMP-triggered immunity. Conclusions: This study provides valuable insights into the StCrRLK1L gene family, enhancing our understanding of its functions, particularly in plant innate immunity. Full article

(This article belongs to the Section Plant Genetics and Genomics)

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Chromosomal locations of potato StCrRLK1L gene family members. Twenty-nine genes are mapped to 10 chromosomes based on Phytozome annotations. Full article ">Figure 2
A phylogenetic tree of CrRLK1L-related proteins from A. thaliana (blue circles) and S. tuberosum (red circles). The tree was constructed using ClustalW alignment and MEGA X with 1000 bootstrap replicates. Full article ">Figure 3
Structural and evolutionary analysis of StCrRLK1Ls. (A) Maximum-likelihood phylogenetic tree of 29 StCrRLK1Ls grouped into three subclasses. (B) Exon–intron architecture: green (UTRs), yellow (exons), gray line (introns). (C) Conserved motif distribution, colored by position in protein sequence. Full article ">Figure 4
Cis-element distribution in the StCrRLK1L promoters. Element positions are scaled relative to the translation start site (ATG). Full article ">Figure 5
Heat map of StCrRLK1L expression across tissues in DM potatoes. Color intensity reflects log2-transformed FPKM values. Full article ">Figure 6
Expression dynamics of the StCrRLK1L genes under stress conditions. The color scale was plotted using the log2 mean of FPKM of each gene. Full article ">Figure 7
The Modulation of PAMP-triggered ROS by StCrRLK1Ls. The indicated constructs were transiently expressed by agrobacterium-mediated transient expression for 2 days and subjected to flg22-induced ROS examination (mean ± SD, n ≥ 8, and one-way ANOVA followed by Tukey’s post hoc test; different letters indicate significant difference at p < 0.01). The protein expression is shown in <a href="#app1-genes-16-00308" class="html-app">Figure S1</a>. Full article ">

20 pages, 2766 KiB

Open AccessReview

Biological Effects of Micro-/Nano-Plastics in Macrophages

by Massimiliano G. Bianchi, Lavinia Casati, Giulia Sauro, Giuseppe Taurino, Erika Griffini, Christian Milani, Marco Ventura, Ovidio Bussolati and Martina Chiu

Nanomaterials 2025, 15(5), 394; https://doi.org/10.3390/nano15050394 - 4 Mar 2025

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Abstract

The environmental impact of plastics is worsened by their inadequate end-of-life disposal, leading to the ubiquitous presence of micro- (MPs) and nanosized (NPs) plastic particles. MPs and NPs are thus widely present in water and air and inevitably enter the food chain, with inhalation and ingestion as the main exposure routes for humans. Many recent studies have demonstrated that MPs and NPs gain access to several body compartments, where they are taken up by cells, increase the production of reactive oxygen species, and lead to inflammatory changes. In most tissues, resident macrophages engage in the first approach to foreign materials, and this interaction largely affects the subsequent fate of the material and the possible pathological outcomes. On the other hand, macrophages are the main organizers and controllers of both inflammatory responses and tissue repair. Here, we aim to summarize the available information on the interaction of macrophages with MPs and NPs. Particular attention will be devoted to the consequences of this interaction on macrophage viability and functions, as well as to possible implications in pathology. Full article

(This article belongs to the Section Biology and Medicines)

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The main steps of the interaction of MPs/NPs with macrophages. The figure represents a unitary vision of the interaction of MPs/NPs with macrophages, from changes occurring before the exposure (environmental weathering) to organ disease, as the result of alterations of macrophages at the cell level. For polarization, the solid line represents the favored drive towards M1 polarization, while the dotted line represents the less common drive towards M2 polarization. See text for a more detailed description. Created in BioRender. Bianchi, M. n80u669. Full article ">

12 pages, 1504 KiB

Open AccessArticle

The Role of NFAT5 in Immune Response and Antioxidant Defense in the Thick-Shelled Mussel (Mytilus coruscus)

by Yijiang Bei, Xirui Si, Wenjun Ma, Pengzhi Qi and Yingying Ye

Animals 2025, 15(5), 726; https://doi.org/10.3390/ani15050726 - 4 Mar 2025

Viewed by 188

Abstract

Nuclear Factor of Activated T Cells 5 (NFAT5) is a transcription factor that plays a pivotal role in immune regulation. While its functions have been extensively studied in mammalian immune systems, its role in marine invertebrates, particularly in bivalves, remains largely unexplored. This study provides the first characterization of the NFAT5 gene in the thick-shelled mussel (Mytilus coruscus), investigating its evolutionary characteristics and immunological functions. Using direct RNA sequencing, McNFAT5 was comprehensively analyzed, revealing its critical involvement in the innate immune response of M. coruscus to Vibrio alginolyticus challenge. Differential expression patterns of McNFAT5 were observed across various tissues with the highest expression detected in hemolymphs. The knockdown of McNFAT5 using small interfering RNA (siRNA) led to a significant reduction in the activities of superoxide dismutase (SOD), Na⁺/K⁺-ATPase, and antioxidant enzymes compared to levels observed post-infection. These findings highlight the central role of McNFAT5 in modulating antioxidant defense mechanisms. In conclusion, McNFAT5 is a key regulatory factor in the innate immune system of M. coruscus, providing valuable insights into the immune adaptive mechanisms and evolutionary mechanisms of bivalve immunity. This study contributes to a deeper understanding of the immune regulatory networks in marine invertebrates. Full article

(This article belongs to the Section Animal Physiology)

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Figure 1
Molecular characterization of McNFAT5 (A) SMART-predicted schematic of McNFAT5 functional domains. (B) SWISS-MODEL-generated three-dimensional structure of McNFAT5. (C) Phylogenetic analysis of McNFAT5 with selected species using the neighbor-joining method in MEGA 7.0 with 5000 bootstrap replications. Different colors indicate distinct taxa. The species in red font, Mytilus coruscus, represents the focus of our study. Full article ">Figure 2
McNFAT5 responds to pathogen stimulation. (A) Agarose gel electrophoresis validation of McNFAT5 expression in various tissues of M. coruscus, including foot, gill, mantle, hemolymph, gonad and digestive gland. The presence of clear bands in all tested tissues confirms the expression of McNFAT5 across different tissue types. (B) Immunofluorescence showing translocation of NFAT5 to the nucleus after V. alginolyticus infection. Red: McNFAT5 protein, Blue: DAPI-stained nuclei. Scale bars = 20. Each sample was analyzed in triplicate. Vertical error bars represent the mean ± standard error (SE, n = 3). (C) The β-actin gene of M. coruscus was used as an internal reference to normalize the cDNA templates across all samples. Differential expression across the six tissues was analyzed using one-way ANOVA. Distinct letters indicate statistically significant differences (p < 0.05) in the relative expression levels of McNFAT5 mRNA. Full article ">Figure 3
Temporal expression patterns of McNFAT5 after infection. McNFAT5 mRNA expression was assessed via qRT-PCR at 6 h, 12 h, 24 h, and 48 h post-V. alginolyticus infection. Data are shown as mean ± SD (n = 3) with different letters indicating significant differences (p < 0.05). Full article ">Figure 4
The impact of NFAT5 knockdown on the activity of three key enzymes and antioxidant capacity. (A) The expression of McNFAT5 was downregulated by Si-McNFAT5. (B) Na+, K+-ATPase. (C) SOD. (D) T-AOC. The vertical bars represent the mean ± SD (n = 3). Different letters indicate significant differences (p < 0.05). Full article ">

26 pages, 1442 KiB

Open AccessArticle

The Association of Toll-like Receptor-9 Gene Single-Nucleotide Polymorphism and AK155(IL-26) Serum Levels with Chronic Obstructive Pulmonary Disease Exacerbation Risk: A Case-Controlled Study with Bioinformatics Analysis

by Entsar R. Mokhtar, Salwa I. Elshennawy, Heba Elhakeem, Rayyh A. M. Saleh, Sawsan Bakr Elsawy, Khadiga S. M. Salama, Maha Fathy Mohamed, Rania Hamid Bahi, Hayam H. Mansour, Sammar Ahmed Kasim Mahmoud, Marwa M. Hassan, Sara M. Elhadad, Hanaa Mohammed Eid El Sayed, Aliaa N. Mohamed and Nadia M. Hamdy

Biomedicines 2025, 13(3), 613; https://doi.org/10.3390/biomedicines13030613 - 3 Mar 2025

Viewed by 267

Abstract

Background: A crucial challenge is the determination of chronic obstructive pulmonary disease (COPD) immune-related mechanisms, where one of the important components of the inflammatory axes in COPD is Toll-like receptor-9 (TLR9) and interleukin-26 AK155(IL-26). Aim: To examine the relation between TLR9 (T1237C) SNP rs5743836 and serum levels of AK155(IL-26) with the exacerbation of COPD. Subjects: A total of 96 COPD patients sub-classified into two groups. Materials: DNA was purified from blood samples of stable COPD patients (n = 48) vs. exacerbated COPD patients (n = 48) as well as 42 age- and sex-matched healthy smokers and passive smokers as a control group. Methods: Genotyping for TLR9 rs5743836 (T1237C) polymorphism was performed using real time polymerase chain reaction (RT-PCR). AK155(IL-26) serum levels were determined using ELISA. Results: There is a significantly higher frequency of the mutant homozygous genotype (C/C) and the mutated C allele of TLR9 rs5743836 (T1237C) in COPD patients and in the exacerbated group when compared with the control group and stable COPD patients, respectively, with OR 31.98, 1.8 to 57.7, and OR 3.64, 0.98 to 13.36, respectively. For the mutated C allele, the OR was 3.57, 1.94 to 6.56, p = 0.001, OR 1.83, 1.02 to 3.27, p = 0.041, respectively. In the exacerbated COPD group, there was a significant association between TLR9 rs5743836 SNP and BMI and the lung vital function measures, CRP, and AK155(IL-26). The exacerbated COPD group has higher serum levels of AK155(IL-26) compared with the stable group or when compared with the control group (p = 0.001) for both. AK155(IL-26) serum levels have a positive significant correlation with CRP and BMI and a significant negative correlation with FEV1% and FEV1/FVC in exacerbated COPD patients. Conclusions: Our results demonstrated a relation linking TLR-9 rs5743836 (T1237C) expression and the risk of COPD development and its exacerbation, indicating that dysfunctional polymorphisms of the innate immune genes can affect COPD development and its exacerbation. AK155(IL-26) upregulation was related to decreased lung functionality, systematic inflammatory disease, and COPD exacerbation. Full article

(This article belongs to the Special Issue Advances in Liquid Biopsy from Technological Innovation to Therapeutic Intervention)

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ROC curve analysis of AK155(IL-26) (pg/mL) between COPD patients and control group (A) and exacerbated and stable COPD patients (B). [Sens.: sensitivity; Spec.: specificity; PPV: positive predictive value; NPV: negative predictive value; AUD: Area Under the Curve; p < 0.001 is highly significant]. Full article ">Figure 2
Genes’ relative expression heatmap: (A) AK155(IL-26) and (B) TLR9. Legend is 0.0., with yellowish (low expression) through red to dark red color indicating highly expressed gene. <a href="http://husch.comp-genomics.org/#/info_tissue/Lung" target="_blank">http://husch.comp-genomics.org/#/info_tissue/Lung</a> (accessed on 5 November 2024). Full article ">Figure 3
(A) The receptor combination of IL10RB and IL20RA is unique and specific for AK155(IL26), presented as pathway members <a href="https://omim.org/entry/605679?search=il26&highlight=il26" target="_blank">https://omim.org/entry/605679?search=il26&highlight=il26</a> and <a href="http://signalink.org/node/Q9NPH9" target="_blank">http://signalink.org/node/Q9NPH9</a>, as well as <a href="https://signor.uniroma2.it/relation_result.php?id=Q9NPH9" target="_blank">https://signor.uniroma2.it/relation_result.php?id=Q9NPH9</a>. (B) <a href="https://www.proteinatlas.org/ENSG00000239732-TLR9/structure+interaction#interaction" target="_blank">https://www.proteinatlas.org/ENSG00000239732-TLR9/structure+interaction#interaction</a> and according to <a href="https://signor.uniroma2.it/relation_result.php?id=Q9NR96" target="_blank">https://signor.uniroma2.it/relation_result.php?id=Q9NR96</a> TLR9 activates myeloid differentiation primary protein 88 (MyD88) and a Toll/Interleukin receptor protein domain. (C) The direct link between AK155(IL-26) and TLR-9 evidenced via the STRING version 12.0 database <a href="https://string-db.org/cgi/network?taskId=b6gWhpi8e53F&sessionId=b4XNW7ObLS7n" target="_blank">https://string-db.org/cgi/network?taskId=b6gWhpi8e53F&sessionId=b4XNW7ObLS7n</a>. (D) List 1, with violet color to the left, is AK155(IL-26), and list 2 indicates TLR9, yellow-colored to the right, showing 5 common regulators between AK155(IL-26) as “List 1” and TLR-9 as “List 2,” which are ETS1, SMARCA4, CEBPA, CTCF, and EGR1. This result is evident when interacting via the STRING, as well as protein interaction via STITCH database. (E,F) <a href="https://genome.ucsc.edu/cgi-bin/hgGeneGraph?gene=IL26&1=OK&supportLevel=text&hideIndirect=on&geneCount=20&geneAnnot=drugbank&1=OK&geneCount=20" target="_blank">https://genome.ucsc.edu/cgi-bin/hgGeneGraph?gene=IL26&1=OK&supportLevel=text&hideIndirect=on&geneCount=20&geneAnnot=drugbank&1=OK&geneCount=20</a>. (G) <a href="https://genome.ucsc.edu/cgi-bin/hgGeneGraph?gene=TLR9&1=OK&supportLevel=text&hideIndirect=on&geneCount=20&geneCount=15&geneAnnot=drugbank&1=OK" target="_blank">https://genome.ucsc.edu/cgi-bin/hgGeneGraph?gene=TLR9&1=OK&supportLevel=text&hideIndirect=on&geneCount=20&geneCount=15&geneAnnot=drugbank&1=OK</a> (all accessed on 5 November 2024) [black-colored genes denote treatment hits by Drug Bank; continuous gray line for results indicates dataset interaction curated from source document, and no text-mining line indicates no curated information where text mining is evident; blue continuous line indicates interaction from several datasets with text mining]. Accessed on 5 November 2024. Full article ">

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