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Introductionβ-thalassemia is a genetic hemoglobinopathy characterized by defective β-globin synthesis and ineffective erythropoiesis. Pharmacological induction of fetal hemoglobin (HbF) via γ-globin gene activation represents a promising therapeutic strategy. Total ginsenosides (TG), the principal active constituents of Panax ginseng, have shown epigenetic and transcriptional modulatory properties, yet their role in HbF induction remains unexplored.MethodsWe evaluated the HbF-inducing potential of TG using human erythroleukemia cell line (K562), primary erythroid precursor cells (ErPCs) derived from CD34+ umbilical cord blood, and Townes transgenic mice. TG was administered at varying concentrations in vitro (25–400 μg/mL) and in vivo (50–800 mg/kg/day for 14 days). HbF and γ-globin expression were quantified by flow cytometry, immunofluorescence, and RT-qPCR. Hemoglobin content, cell viability, and hepatic histology were also assessed.ResultsTG significantly induced HbF production and γ-globin gene expression in both cellular models in a dose-dependent manner. In K562 cells, 200 μg/mL TG elevated γ-globin mRNA by 4.29-fold; in ErPCs, the increase was 1.46-fold. HbF-positive cell populations rose markedly without impairing cell viability or morphology. In vivo, TG treatment at 200 and 400 mg/kg led to 2.8- and 3.1-fold increases in F-cell proportions, respectively, surpassing hydroxyurea controls. No hepatotoxicity was observed upon histopathological examination.DiscussionThese findings establish TG as a potent, well-tolerated inducer of HbF through transcriptional activation of the γ-globin gene. Its efficacy across erythroid cell lines, primary progenitor cells, and transgenic mouse models underscores its translational potential as a natural therapeutic agent for β-thalassemia.

Toxocariasis, a neglected zoonotic disease caused by parasites of the Toxocara genus, represents a significant public health concern, with an estimated global seroprevalence of 19%. Despite the well-known respiratory symptoms associated with toxocariasis, the immune response in the lungs during toxocariasis is still poorly understood. This study analyzes both local lung and systemic immune response to T. canis infection and T. canis excretory-secretory antigens (TES) intranasal application in C57BL/6J mice. Lungs, blood, and spleens were collected at specific time points for histopathological analyses, flow cytometry, cytokine profiling, and gene expression studies. The systemic immune response was further assessed by cytokine measurements in splenocyte cultures and the detection of TES-specific antibodies. T. canis infection triggered severe pulmonary inflammation characterized by eosinophilia and mucus accumulation, with persistent inflammation lasting up to 28 days post-infection. Interestingly, this response was not solely driven by Th2-type interleukin production. Cytokine analysis of splenocyte cultures revealed elevated levels of IL-5 and IL-6, along with increased TES-specific IgE and IgG1 antibody concentrations. In contrast, TES application alone induced local eosinophil infiltration and upregulated genes associated with lung repair, though this response was less intense and shorter-lived compared to the infection. Our study is the first to present a comprehensive cytokine proteome analysis in mouse lungs during T. canis infection and stimulation by larval antigens, highlighting the key role of cytokines such as IL-5, IL-6, and IL-33. These findings provide new insights into the pathogenesis of toxocariasis and underscore the need for further research into potential therapeutic targets.

Innate-like T cells (ILT), including γδ T cells (Vδ2s), Natural Killer T cells (NKTs) and Mucosal-associated Invariant T cells (MAITs), integrate innate and adaptive immunity, playing important roles in homeostatic conditions as well as during infection or inflammation. ILT are present on both sides of the fetal-maternal interface, but our knowledge of their phenotypical and functional features in neonates is limited. Using spectral flow cytometry we characterized cord blood ILT in neonates born to healthy women and women living with HIV. We describe extensive phenotypic and functional heterogeneity within the cord Vδ2 cells at baseline and following activation. In neonates born to women with HIV, we observed modest differences in ILT frequencies ex-vivo and altered proportions of Vδ2 cells producing IFNγ+ or TNFα+, both ex-vivo and after expansion, compared to HIV unexposed infants. Consistent with prior studies, infants born to mothers who initiated ART before pregnancy exhibited less immune perturbation overall. Herein we expand our knowledge of ILT at the maternal-fetal interface by a comprehensive phenotypic analysis of these rare subsets.

Atopic dermatitis (AD) is a chronic dermatological disorder characterized by intense pruritus and eczematous lesions. Repeated topical application of 2,4-dinitrofluorobenzene (DNFB) in NC/Nga mice produces AD-like clinical symptoms that closely resemble human AD. N-acetyl-L-alanine (L-NAA), a derivative of L-Alanine, has unknown biological and physiological effects on cutaneous tissue. In this study, we investigated whether L-NAA modifies AD-like symptoms elicited by ongoing DNFB exposure in NC/Nga mice. Topical administration of L-NAA markedly attenuated the development of AD-like cutaneous lesions triggered by DNFB. L-NAA treatment further suppressed DNFB-induced infiltration of eosinophils and mast cells and prevented the increase of serum IgE resulting from DNFB application. L-NAA treatment decreased DNFB-stimulated expression of IL-4, a Th2-associated cytokine, but increased IFN-γ expression, indicative of Th1 activity, within the skin lesions. In addition, L-NAA prevented the DNFB-driven upregulation of GATA3, a central regulator of Th2 lineage differentiation, in CD4＋ cells, with no effect on T-bet, the principal regulator of Th1 cells. These findings indicate that L-NAA can limit Th2 differentiation in the AD mouse model. Therefore, L-NAA may serve as a promising therapeutic and immunomodulatory compound against AD.

BackgroundHalo nevi are clinically common and are characterized by a circle of leukoderma around the central melanocytic nevus. Studies have shown that the pathogenesis of halo nevi is similar to that of vitiligo and is associated with the role of CD8⁺ T lymphocytes in melanocyte destruction. Histopathological findings have revealed neutrophil infiltration in halo nevi; however, the specific immune mechanisms involving neutrophils have not been thoroughly investigated. In the present study, we investigated the role of neutrophils in halo nevi using histopathological and immunological analyses.MethodsTo this end, we examined the infiltration patterns of immune cells in halo nevi, with a particular focus on IFN-γ-induced PD-L1 expression in neutrophils and its potential immunoregulatory effects.ResultsThe results demonstrated that IFN-γ expression in the lesional skin of halo nevi contributed to the induction of PD-L1 expression in neutrophils. PD-L1⁺ neutrophils promoted apoptosis and suppressed the function of CD8⁺ T lymphocytes. Notably, some halo nevi showed a tendency to spontaneous regression, but the underlying mechanisms remain unclear, and this regulatory mechanism influences the local immune response and may facilitate the repigmentation of the surrounding leukoderma in halo nevi.ConclusionsThis study is the first to explore the involvement of neutrophils in halo nevi and reveal the potential immunoregulatory role of PD-L1 in this process. The elucidation of this mechanism not only provides a more comprehensive understanding of autoimmune skin diseases but may also offer new strategies for targeted therapy in other related disorders, such as vitiligo.

IntroductionCCR2, a chemokine receptor critical for immune cell migration, inflammation, and HIV infection, is regulated by poorly understood mechanisms.MethodsThis study investigated the unusually long CCR2 3’-UTR’s role in post-transcriptional regulation.ResultsThe full-length 3′-UTR significantly inhibited reporter gene expression in primary CD4+ T cells and macrophages, likely mediated by RNA binding proteins (RBPs). HnRNPA0, was shown to bind directly to this region and influence CCR2 levels. When the RBP binding sites were mutagenized or the 3′- UTR removed using CRISPR-Cas9 and gRNAs, CCR2 mRNA and protein levels significantly increased. Cell fractionation experiments confirmed that these changes occurred in both the nucleus and cytoplasm. To directly test mRNA stability, we used a-amanitin and found that removing the 3′-UTR nearly doubled the half-life of CCR2 mRNA. Finally, pseudotyping studies revealed CCR2 functions as an HIV co-receptor at ~10% efficiency compared to CCR5.DiscussionThese results show that the CCR2 3′-UTR plays an important role in post-transcriptional regulation and may provide a novel approach to regulating CCR2 activity in inflammatory or infectious diseases.

ABSTRACTActivation of the cGAS‐STING pathway plays an important role in antitumor immunity through maturation of tumor‐infiltrating DCs. DCs engulf extracellular DNA released by dying cancer cells, supporting activation of the cGAS‐STING pathway and concomitant DC maturation. Extracellular DNA in the tumor microenvironment is primarily derived from cells undergoing uncontrolled necrosis or programmed inflammatory death, such as necroptosis, which can be induced when apoptosis pathways are inhibited. Here, we report that caspase inhibition primes activation of a RIPK1/3, MLKL, and STING signaling axis in DCs, resulting in maturation without the need for any further maturation stimuli such as LPS or TNF‐α. Notably, these signaling events do not induce DC death, indicating a nonnecroptotic role of the RIPK1‐RIPK3‐MLKL pathway and novel crosstalk with the STING pathway. Caspase inhibition in DC/cancer cell co‐cultures results in DC maturation, inducing TNF‐α secretion, which delivers the co‐signal to induce cancer cell necroptosis. In summary, we find a collaborative mechanism of the STING and necroptosis pathway in DC maturation, and that activation of the necroptosis pathway has opposite effects on cancer cells and DCs, proposing a possibility for new targets in cancer immunotherapy. In this study, we show that caspase‐8 inhibition induce maturation in DCs through a synergistic crosstalk between the STING signaling pathway and RIPK1‐RIPK3‐MLKL pathway, without induction of cell death in the DCs. The mature DCs secrete TNF‐α that induces cancer cell death in DC/cancer cell co‐cultures upon concomitant caspase‐8 inhibition.

Optineurin (OPTN) is an autophagy adaptor protein involved in selective autophagy, including aggrephagy and mitophagy. Pathogenic mutations in OPTN have also been linked to amyotrophic lateral sclerosis, frontotemporal dementia, and glaucoma, supporting its role in the etiology of neurodegenerative diseases. Despite its established biological roles, knowledge about its potential contribution to Alzheimer’s disease (AD) pathology and neuronal functioning is lacking. AD is characterized by the accumulation of extracellular amyloid-β plaques and intracellular phosphorylated tau (pTau) tangles, with dysfunction in the autophagy-lysosomal pathway exacerbating tau pathology and impairing proteostasis. To investigate the role of OPTN in neuronal proteostasis and AD, we utilized induced pluripotent stem cell-derived neuron (iN) and astrocyte (iA) models. Analyses revealed a significant negative correlation between OPTN and specific pTau epitopes in neurons, as well as a decrease in OPTN protein abundance in brain tissues of individuals with AD. Given these findings, we generated OPTN knockout (KO), heterozygous, and wildtype iNs and iAs using CRISPR/Cas9 editing of iPSCs in two genetic backgrounds. Loss of OPTN in iNs increased specific pTau proteoforms without substantially affecting autophagy processes or mitochondrial respiration. Despite no clear effect on mitochondrial function, several mitochondrial proteins, including OXCT1, were enriched in an unbiased analysis of the OPTN interactome in iNs, as well as proteins involved in intracellular trafficking. Proteomic analyses further identified intracellular clusterin, an AD risk gene, as significantly upregulated in OPTN KO iNs, suggesting OPTN may influence its intracellular processing. Our model system demonstrates modest roles for OPTN in certain neuronal biological processes and potential implications for AD pathogenesis. These findings also suggest that OPTN may exhibit functional redundancy with other autophagy adaptor proteins in human neurons, leading to relatively mild phenotypic changes with complete loss of OPTN.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40478-025-02103-y.

BackgroundThe imbalance of immune checkpoint molecules leads to immune escape of tumor cells. It has been established that KRAS mutation plays a key role in regulating PD-L1 expression of lung adenocarcinoma. However, the specific mechanism by which KRAS mutation regulates PD-L1 expression still needs further been clarified.MethodsThe relationship of KRAS mutation and ZNF24, SLC7A5 and PD-L1 expression in human lung adenocarcinoma tissues and cell lines were analyzed using relative assays. The effects of KRAS mutation on CD8+ T cell-dependent anti-tumor immunity via the ZNF24/SLC7A5/PD-L1 axis were analyzed through in vitro and in vivo experiments. Additionally, we examined whether and how targeting ZNF24 inhibits KRAS mutation-induced PD-L1 expression and evaluated the effect of ZNF24 inhibition and PD-L1 blocking on CD8+ T cell-dependent anti-tumor immunity.ResultsOur results found that KRAS mutation increases the expression of PD-L1 through the ZNF24/SLC7A5 axis and simultaneously inhibits the activation of CD8+ T cells in lung adenocarcinoma. Importantly, we discovered that Daptomycin (DAPT) binds to ZNF24 and inactivates it, representing the first reported inhibitor of ZNF24. DAPT combined with Anti PD-L1 monoclonal antibody may enhance CD8+ T cell-dependent anti-tumor immunity in KRAS mutated lung adenocarcinoma.ConclusionOur study provides the first evidence that KRAS mutation promotes immune escape in lung adenocarcinoma through the ZNF24/SLC7A5/PD-L1 axis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12885-025-14336-0.

Organ transplantation is a lifesaving procedure, with 50,000 transplants happening every year in the United States. However, many patients harbor antibodies and B cells directed against allogeneic human leukocyte antigen (HLA) molecules, notably HLA-A2, greatly decreasing their likelihood of receiving a compatible organ. Moreover, antibody-mediated rejection is a significant contributor to chronic transplant rejection. Current strategies to desensitize patients non-specifically target circulating antibodies and B cells, resulting in poor efficacy and complications. Regulatory T cells (Tregs) are immune cells dedicated to suppressing specific immune responses by interacting with both innate and adaptive immune cells. Here, we genetically modified human Tregs with a chimeric anti-HLA antibody receptor (CHAR) consisting of an extracellular HLA-A2 protein fused to a CD28-CD3zeta intracellular signaling domain, driving Treg activation upon recognition of anti-HLA-A2 antibodies on the surface of alloreactive B cells. We find that HLA-A2 CHAR Tregs get activated specifically by anti-HLA-A2 antibody-producing cells. Of note, HLA-A2 CHAR activation does not negatively affect Treg stability, as measured by expression of the Treg lineage transcription factors FOXP3 and HELIOS. Interestingly, HLA-A2 CHAR Tregs are not cytotoxic towards anti-HLA-A2 antibody-producing cells, unlike HLA-A2 CHAR modified conventional CD4+ T cells. Importantly, HLA-A2 CHAR Tregs recognize and significantly suppress high affinity IgG antibody production by B cells from HLA-A2 sensitized patients. Altogether, our results provide proof-of-concept of a new strategy to specifically inhibit alloreactive B cells to desensitize transplant recipients.

NLRP3 is a target for anti-inflammatory therapies and can be inhibited by the tool compound MCC950. We describe the characterization of new small-molecule inhibitors of NLRP3, BAL-0028 and BAL-0598, that have a distinct mechanism of action and binding site. The NLRP3 inflammasome is an intracellular protein complex that causes inflammation via the release of IL-1β and pyroptosis. NLRP3 activation is associated with many age-related inflammatory diseases, and NLRP3 inhibition is a promising therapeutic strategy. We previously performed a DNA-encoded library screen to identify novel NLRP3-binding molecules. Herein we describe the characterization of BAL-0028 as a potent and specific inhibitor of NLRP3 signaling. Notably, BAL-0028 is a poor inhibitor of mouse NLRP3 but inhibits human and primate NLRP3 with nanomolar potency. Using cellular and biochemical analyses, we demonstrate that BAL-0028 binds to the NLRP3 NACHT domain at a site that is distinct from the MCC950-binding pocket. Using humanized NLRP3 mice, we show that a derivative of BAL-0028, BAL-0598, inhibits NLRP3 activation in vivo in a peritonitis model. Finally, we demonstrate that both BAL-0028 and BAL-0598 inhibit select hyperactive NLRP3 mutations associated with autoinflammatory diseases more potently than MCC950. BAL-0028 and BAL-0598 thus represent a new modality for NLRP3 inhibition in inflammatory diseases.

The kidney maintains fluid homeostasis by reabsorbing essential compounds and excreting waste. Proximal tubule cells, crucial for reabsorbing sugars, ions, and amino acids, are highly susceptible to injury, often leading to pathologies necessitating dialysis or transplants. Human pluripotent stem cell-derived kidney organoids offer a platform to model renal development, function, and disease, but proximal nephron differentiation and maturation in these structures is incomplete. Here, we drive proximal tubule development in pluripotent stem cell-derived kidney organoids by mimicking in vivo proximal differentiation. Transient PI3K inhibition during early nephrogenesis activates Notch signaling, shifting nephron axial differentiation towards epithelial and proximal precursor states that mature to proximal convoluted tubule cells broadly expressing physiology-imparting solute carriers including organic cation and organic anion family members. The “proximal-biased” organoids thus acquire function, and on exposure to nephrotoxic injury, display tubular collapse and DNA damage, and upregulate injury response markers HAVCR1/KIM1 and SOX9 while downregulating proximal transcription factor HNF4A. Here, we show that proximally biased human-derived kidney organoids provide a robust model to study nephron development, injury responses, and a platform for therapeutic discovery. Here, the authors develop pluripotent stem cell-derived kidney organoids by mimicking in vivo proximal differentiation, providing a model to study nephron development, injury responses, and a platform for therapeutic discovery.