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IntroductionHeme oxygenase-1 (HO-1) is a stress-inducible heat shock protein (HSP32) that exerts cytoprotective effects against oxidative stress and inflammation,and is involved in the maintenance of cellular homeostasis. This study aimed to evaluate the expression of HO-1 in natural killer (NK) cells from individuals of different age groups after stimulation with various factors,and to analyze the relationships between the concentration of this cytoprotective protein and parameters corresponding to oxidative stress and inflammation,that is,NOD-like receptor protein 3 (NLRP3),glutathione (GSH),GSH disulfide (GSSG),and interleukin 6 (IL-6).MethodsThe study population comprised three age groups: young adults (age range,19–23 years),older adults aged under 85 years (age range,73–84 years),and older adults aged over 85 years (age range,85–92 years). NLRP3,GSH,and GSSG concentrations were measured in serum,whereas the HO-1 concentration and IL-6 expression were studied in NK cells cultivated for 48 h and stimulated with IL-2,lipopolysaccharide (LPS),or phorbol 12-myristate 13-acetate (PMA) with ionomycin.ResultsThe analysis of serum NLRP3,GSH,and GSSG concentrations revealed no statistically significant differences among the studied age groups. However,some typical trends of aging were observed,such as a decrease in GSH concentration and an increase in both GSSG level,and GSSG/GSH ratio. The highest basal expression of IL-6 and lowest basal content of HO-1 were found in NK cells of adults over 85 years of age. The NK cells in this age group also showed the highest sensitivity to stimulation with the applied factors. Moreover,statistically significant negative correlations were observed between HO-1 and IL-6 expression levels in the studied NK cells.ConclusionsThese results showed that NK cells can express HO-1 at a basal level,which was significantly increased in activated cells,even in the oldest group of adults. The reciprocal relationship between HO-1 and IL-6 expression suggests a negative feedback loop between these parameters. View Publication

Background & AimsType 2 innate lymphoid cells (ILC2s) and interleukin-13 (IL-13) promote the onset of spasmolytic polypeptide-expressing metaplasia (SPEM) cells. However,little is known about molecular effects of IL-13 in SPEM cells. We now sought to establish a reliable organoid model,Meta1 gastroids,to model SPEM cells in vitro. We evaluated cellular and molecular effects of ILC2s and IL-13 on maturation and proliferation of SPEM cells.MethodsWe performed single-cell RNA sequencing to characterize Meta1 gastroids,which were derived from stomachs of Mist1-Kras transgenic mice that displayed pyloric metaplasia. Cell sorting was used to isolate activated ILC2s from stomachs of IL-13-tdTomato reporter mice treated with L635. Three-dimensional co-culture was used to determine the effects of ILC2s on Meta1 gastroids. Mouse normal or metaplastic (Meta1) and human metaplastic gastroids were cultured with IL-13 to evaluate cell responses. Air-Liquid Interface culture was performed to test long-term culture effects of IL-13. In silico analysis determined possible STAT6-binding sites in gene promoter regions. STAT6 inhibition was performed to corroborate STAT6 role in SPEM cells maturation.ResultsMeta1 gastroids showed the characteristics of SPEM cell lineages in vitro even after several passages. We demonstrated that co-culture with ILC2s or IL-13 treatment can induce phosphorylation of STAT6 in Meta1 and normal gastroids and promote the maturation and proliferation of SPEM cell lineages. IL-13 up-regulated expression of mucin-related proteins in human metaplastic gastroids. Inhibition of STAT6 blocked SPEM-related gene expression in Meta1 gastroids and maturation of SPEM in both normal and Meta1 gastroids.ConclusionsIL-13 promotes the maturation and proliferation of SPEM cells consistent with gastric mucosal regeneration. Graphical Abstract View Publication

This cohort study explores whether the rate of biological aging estimated by an epigenetic clock is associated with social determinants of health in a racially and ethnically diverse population. Key PointsQuestionIs the rate of biological aging associated with racial and ethnic differences in social determinants of health?FindingsIn a cohort study among 376 Japanese American,Native Hawaiian,and White adults,low neighborhood socioeconomic status (NSES) was associated with a higher rate of biological aging measured by the DunedinPACE epigenetic clock. Accounting for NSES,race and ethnicity was a significant factor in correlations between diet,educational level,and moderate to vigorous physical activity and DunedinPACE scores.MeaningThe findings suggest that individual sociobehavioral factors might mitigate negative associations between NSES and biological aging,with differences by race and ethnicity. ImportanceVariation in DNA methylation at specific loci estimates biological age,which is associated with morbidity,mortality,and social experiences. Aging estimates known as epigenetic clocks,including the Dunedin Pace of Aging Calculated From the Epigenome (DunedinPACE),were trained on data predominately from individuals of European ancestry; however,limited research has explored DunedinPACE in underrepresented populations experiencing health disparities.ObjectiveTo investigate associations of neighborhood and individual sociobehavioral factors with biological aging in a racially and ethnically diverse population.Design,Setting,and ParticipantsThis cohort study,part of the Multiethnic Cohort study conducted from May 1993 to September 1996 to examine racial and ethnic disparities in chronic diseases,integrated biospecimen and self-reported data collected between April 2004 and November 2005 from healthy Hawaii residents aged 45 to 76 years. These participants self-identified as of Japanese American,Native Hawaiian,or White racial and ethnic background. Data were analyzed from January 2022 to May 2024.Main Outcomes and MeasuresDNA methylation data were generated from monocytes enriched from cryopreserved lymphocytes and used to derive DunedinPACE scores from November 2017 to June 2021. Neighborhood social economic status (NSES) was estimated from 1990 US Census Bureau data to include factors such as educational level,occupation,and income. Individual-level factors analyzed included educational level,body mass index (BMI),physical activity (PA),and diet quality measured by the Healthy Eating Index (HEI). Linear regression analysis of DunedinPACE scores was used to examine their associations with NSES and sociobehavioral variables.ResultsA total of 376 participants were included (113 [30.1%] Japanese American,144 [38.3%] Native Hawaiian,and 119 [31.6%] White; 189 [50.3%] were female). Mean (SE) age was 57.81 (0.38) years. Overall,mean (SE) DunedinPACE scores were significantly higher among females than among males (1.28 [0.01] vs 1.25 [0.01]; P = .005); correlated negatively with NSES (R = −0.09; P = .08),HEI (R = −0.11; P = .03),and educational attainment (R = −0.15; P = .003) and positively with BMI (R = 0.31; P < .001); and varied by race and ethnicity. Native Hawaiian participants exhibited a higher mean (SE) DunedinPACE score (1.31 [0.01]) compared with Japanese American (1.25 [0.01]; P < .001) or White (1.22 [0.01]; P < .001) participants. Controlling for age,sex,HEI,BMI,and NSES,linear regression analyses revealed a negative association between educational level and DunedinPACE score among Japanese American (β,−0.005 [95% CI,−0.013 to 0.002]; P = .03) and Native Hawaiian (β,−0.003 [95% CI,−0.011 to 0.005]; P = .08) participants,yet this association was positive among White participants (β,0.007; 95% CI,−0.001 to 0.015; P = .09). Moderate to vigorous PA was associated with lower DunedinPACE scores only among Native Hawaiian participants (β,−0.006; 95% CI,−0.011 to −0.001; P = .005),independent of NSES.Conclusions and RelevanceIn this study of a racially and ethnically diverse sample of 376 adults,low NSES was associated with a higher rate of biological aging measured by DunedinPACE score,yet individual-level factors such as educational level and physical activity affected this association,which varied by race and ethnicity. These findings support sociobehavioral interventions in addressing health inequities. View Publication

IntroductionHumanization is typically adopted to reduce the immunogenicity of murine antibodies generated by hybridoma technology when used in humans.MethodsTwo different strategies of antibody humanization are popularly employed,including “complementarity determining region (CDR) grafting” and “framework (FR) shuffling” to humanize a murine antibody against human programmed death-1 (PD-1),XM PD1. In CDR-grafting humanization,the CDRs of XM PD-1,were grafted into the human FR regions with high homology to the murine FR counterparts,and back mutations of key residues were performed to retain the antigen-binding affinities. While in FR-shuffling humanization,a combinatorial library of the six murine CDRs in-frame of XM PD-1 was constructed to a pool of human germline FRs for high-throughput screening for the most favorable variants. We evaluated many aspects which were important during antibody development of the molecules obtained by the two methods,including antibody purity,thermal stability,binding efficacy,predicted humanness,and immunogenicity,along with T cell epitope prediction for the humanized antibodies.ResultsWhile the ideal molecule was not achieved through CDR grafting in this particular instance,FR-shuffling proved successful in identifying a suitable candidate. The study highlights FR-shuffling as an effective complementary approach that potentially increases the success rate of antibody humanization. It is particularly noted for its accessibility to those with a biological rather than a computational background. DiscussionThe insights from this comparison are intended to assist other researchers in selecting appropriate humanization strategies for drug development,contributing to broader application and understanding in the field. View Publication

Activation-induced cytidine deaminase (AID) is a B cell-specific mutator required for antibody diversification. However,it is also implicated in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain blood cancers is critical in assessing disease severity and treatment options. We have developed a digital PCR (dPCR) assay that allows us to quantify mutations resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this assay shows that increased AID levels in immature B cells increase genome instability at loci linked to chromosomal translocation formation. This includes the CRLF2 locus that is often involved in translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Hispanics,particularly those with Latin American ancestry. Using dPCR,we characterize the CRLF2 locus in B cell-derived genomic DNA from both Hispanic ALL patients and healthy Hispanic donors and found increased mutations in both,suggesting that vulnerability to DNA damage at CRLF2 may be driving this health disparity. Our ability to detect and quantify these mutations will potentiate future risk identification,early detection of cancers,and reduction of associated cancer health disparities. Rates of Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) continue to rise in Hispanics and Latinos. Authors developed a digital PCR assay to quantify activation-induced cytidine deaminase activity at risk loci involved in cancer etiology that may contribute to this health disparity. View Publication

MIF is a ubiquitous protein involved in proinflammatory processes,which undergoes an oxidation-driven conformational change to oxidized (ox)MIF. We demonstrate that hypochlorous acid,produced by neutrophil-released myeloperoxidase (MPO) under inflammatory conditions,effectively oxidizes MIF into the oxMIF isoform,which is specifically recognized by the anti-oxMIF therapeutic antibody,ON104. NMR investigation of MIF oxidized by the MPO system revealed increased flexibility throughout the MIF structure,including at several catalytic and allosteric sites. Mass spectrometry of MPO-oxMIF revealed methionines as the primary site of oxidation,whereas Pro2 and Tyr99/100 remained almost unmodified. ELISA,SPR and cell-based assays demonstrated that structural changes caused by MPO-driven oxidation promoted binding of oxMIF to its receptor,CD74,which does not occur with native MIF. These data reveal the environment and modifications that facilitate interactions between MIF and its pro-inflammatory receptor,and a route for therapeutic intervention targeting the oxMIF isoform. View Publication

SummarySensing of extracellular ATP (eATP) controls CD8+ T cell function. Their accumulation can occur through export by specialized molecules,such as the release channel Pannexin 1 (Panx1). Whether Panx1 controls CD8+ T cell immune responses in vivo,however,has not been previously addressed. Here,we report that T-cell-specific Panx1 is needed for CD8+ T cell responses to viral infections and cancer. We found that CD8-specific Panx1 promotes both effector and memory CD8+ T cell responses. Panx1 favors initial effector CD8+ T cell activation through extracellular ATP (eATP) export and subsequent P2RX4 activation,which helps promote full effector differentiation through extracellular lactate accumulation and its subsequent recycling. In contrast,Panx1 promotes memory CD8+ T cell survival primarily through ATP export and subsequent P2RX7 engagement,leading to improved mitochondrial metabolism. In summary,Panx1-mediated eATP export regulates effector and memory CD8+ T cells through distinct purinergic receptors and different metabolic and signaling pathways. Graphical abstract Highlights•The hemichannel Panx1 promotes in vivo effector and memory CD8+ T cell responses•Panx1 promotes effector CD8+ T cells via eATP and lactate extracellular accumulation•Panx1 promotes memory CD8+ T cell survival by eATP export and activation of AMPK Natural sciences; Biological sciences; Biochemistry; Immunology; Immune response; Immune system View Publication

Current clinical strategies for the delivery of pulmonary therapeutics to the lung are primarily targeted to the upper portions of the airways. However,targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease. Here,we have developed an mRNA therapeutic for the lower lung using one-component Ionizable Amphiphilic Janus Dendrimers (IAJDs) as a delivery vehicle. We deliver an anti-inflammatory cytokine mRNA,transforming growth factor-beta (TGF-β),to produce transient protein expression in the lower regions of the lung. This study highlights IAJD’s potential for precise,effective,and safe delivery of TGF-β mRNA to the lung. This delivery system offers a promising approach for targeting therapeutics to the specific tissues,a strategy necessary to fill the current clinical gap in treating parenchymal lung injury and disease. View Publication

Trained immunity may play a role in vaccine-induced protection against infections. We showed that the highly efficacious recombinant VZV-gE zoster vaccine (RZV) generated trained immunity in monocytes,natural killer (NK) cells,and dendritic cells (DCs) and that the less efficacious live zoster vaccine did not. RZV stimulated ex vivo gE-specific monocyte,DC and NK cell responses that did not correlate with CD4 + T-cell responses. These responses were also elicited in purified monocyte and NK cell cocultures stimulated with VZV-gE and persisted above prevaccination levels for ≥ 4 years post-RZV administration. RZV administration also increased ex vivo heterologous monocyte and NK cell responses to herpes simplex and cytomegalovirus antigens. ATAC-seq analysis and ex vivo TGFβ1 supplementation and inhibition experiments demonstrated that decreased tgfβ1 transcription resulting from RZV-induced chromatin modifications may explain the development of monocyte trained immunity. The role of RZV-trained immunity in protection against herpes zoster and other infections should be further studied. View Publication

Background & AimsThe immune tolerance induced by hepatitis B virus (HBV) is a major challenge for achieving effective viral clearance,and the mechanisms involved are not well-understood. One potential factor involved in modulating immune responses is mesencephalic astrocyte-derived neurotrophic factor (MANF),which has been reported to be increased in patients with chronic hepatitis B. In this study,our objective is to examine the role of MANF in regulating immune responses to HBV.MethodsWe utilized a commonly used HBV-harboring mouse model,where mice were hydrodynamically injected with the pAAV/HBV1.2 plasmid. We assessed the HBV load by measuring the levels of various markers including hepatitis B surface antigen,hepatitis B envelope antigen,hepatitis B core antigen,HBV DNA,and HBV RNA.ResultsOur study revealed that following HBV infection,both myeloid cells and hepatocytes exhibited increased expression of MANF. Moreover,we observed that mice with myeloid-specific MANF knockout (ManfMye-/-) displayed reduced HBV load and improved HBV-specific T cell responses. The decreased HBV-induced tolerance in ManfMye-/- mice was associated with reduced accumulation of myeloid-derived suppressor cells (MDSCs) in the liver. Restoring MDSC levels in ManfMye-/- mice through MDSC adoptive transfer reinstated HBV-induced tolerance. Mechanistically,we found that MANF promoted MDSC expansion by activating the IL-6/STAT3 pathway. Importantly,our study demonstrated the effectiveness of a combination therapy involving an hepatitis B surface antigen vaccine and nanoparticle-encapsulated MANF siRNA in effectively clearing HBV in HBV-carrier mice.ConclusionThe current study reveals that MANF plays a previously unrecognized regulatory role in liver tolerance by expanding MDSCs in the liver through IL-6/STAT3 signaling,leading to MDSC-mediated CD8+ T cell exhaustion. Graphical abstract View Publication

BackgroundDiffuse large B-cell lymphoma (DLBCL) represents a prevalent malignant tumor,with approximately 40% of patients encountering treatment challenges or relapse attributed to rituximab resistance,primarily due to diminished or absent CD20 expression. Our prior research identified PDK4 as a key driver of rituximab resistance through its negative regulation of CD20 expression. Further investigation into PDK4’s resistance mechanism and the development of advanced exosome nanoparticle complexes may unveil novel resistance targets and pave the way for innovative,effective treatment modalities for DLBCL.MethodsWe utilized a DLBCL-resistant cell line with high PDK4 expression (SU-DHL-2/R). We infected it with short hairpin RNA (shRNA) lentivirus for RNA sequencing,aiming to identify significantly downregulated mRNA in resistant cells. Techniques including immunofluorescence,immunohistochemistry,and Western blotting were employed to determine PDK4’s localization and expression in resistant cells and its regulatory role in phosphorylation of Histone deacetylase 8 (HDAC8). Furthermore,we engineered advanced exosome nanoparticle complexes,aCD20@ExoCTX/siPDK4,through cellular,genetic,and chemical engineering methods. These nanoparticles underwent characterization via Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM),and their cellular uptake was assessed through flow cytometry. We evaluated the nanoparticles’ effects on apoptosis in DLBCL-resistant cells and immune cells using CCK-8 assays and flow cytometry. Additionally,their capacity to counteract resistance and exert anti-tumor effects was tested in a resistant DLBCL mouse model.ResultsWe found that PDK4 initiates HDAC8 activation by phosphorylating the Ser-39 site,suppressing CD20 protein expression through deacetylation. The aCD20@ExoCTX/siPDK4 nanoparticles served as effective intracellular delivery mechanisms for gene therapy and monoclonal antibodies,simultaneously inducing apoptosis in resistant DLBCL cells and triggering immunogenic cell death in tumor cells. This dual action effectively reversed the immunosuppressive tumor microenvironment,showcasing a synergistic therapeutic effect in a subcutaneous mouse tumor resistance model.ConclusionsThis study demonstrates that PDK4 contributes to rituximab resistance in DLBCL by modulating CD20 expression via HDAC8 phosphorylation. The designed exosome nanoparticles effectively overcome this resistance by targeting the PDK4/HDAC8/CD20 pathway,representing a promising approach for drug delivery and treating patients with Rituximab-resistant DLBCL.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-024-02057-0. View Publication

Synthetic Notch (synNotch) receptors are genetically encoded,modular synthetic receptors that enable mammalian cells to detect environmental signals and respond by activating user-prescribed transcriptional programs. Although some materials have been modified to present synNotch ligands with coarse spatial control,applications in tissue engineering generally require extracellular matrix (ECM)-derived scaffolds and/or finer spatial positioning of multiple ligands. Thus,we develop here a suite of materials that activate synNotch receptors for generalizable engineering of material-to-cell signaling. We genetically and chemically fuse functional synNotch ligands to ECM proteins and ECM-derived materials. We also generate tissues with microscale precision over four distinct reporter phenotypes by culturing cells with two orthogonal synNotch programs on surfaces microcontact-printed with two synNotch ligands. Finally,we showcase applications in tissue engineering by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined micropatterns. These technologies provide avenues for spatially controlling cellular phenotypes in mammalian tissues. Synthetic Notch (synNotch) receptors are genetically encoded,modular synthetic receptors that enable mammalian cells to detect environmental signals and respond by activating user-prescribed transcriptional programs. Here the authors apply synNotch receptors to spatially control differentiation of endothelial and skeletal muscle cells in a multicellular construct on assorted biomaterials. View Publication