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Dendritic cells (DCs) are pivotal in regulating allergic asthma. Our research has shown that the absence of Sema3E worsens asthma symptoms in acute and chronic asthma models. However,the specific role of PlexinD1 in these processes,particularly in DCs,remains unclear. This study investigates the role of PlexinD1 in CD11c+ DCs using a house dust mite (HDM) model of asthma. We generated CD11c+ DC-specific PlexinD1 knockout (CD11cPLXND1 KO) mice and subjected them,alongside wild-type controls (PLXND1fl/fl),to an HDM allergen protocol. Airway hyperresponsiveness (AHR) was measured using FlexiVent,and immune cell populations were analyzed via flow cytometry. Cytokine levels and immunoglobulin concentrations were assessed using mesoscale and ELISA,while collagen deposition and mucus production were examined through Sirius-red and periodic acid Schiff (PAS) staining respectively. Our results indicate that CD11cPLXND1 KO mice exhibit significantly exacerbated AHR,characterized by increased airway resistance and tissue elastance. Enhanced mucus production and collagen gene expression were observed in these mice compared to wild-type counterparts. Flow cytometry revealed higher CD11c+ MHCIIhigh CD11b+ cell recruitment into the lungs,and elevated total and HDM-specific serum IgE levels in CD11cPLXND1 KO mice. Mechanistically,co-cultures of B cells with DCs from CD11cPLXND1 KO mice showed significantly increased IgE production compared to wild-type mice.These findings highlight the critical regulatory role of the plexinD1 signaling pathway in CD11c+ DCs in modulating asthma features. View Publication

A portion of the genetic basis for many common autoimmune disorders has been uncovered by genome-wide association studies (GWAS),but GWAS do not reveal causal variants,effector genes,or the cell types impacted by disease-associated variation. We have generated 3D genomic datasets consisting of promoter-focused Capture-C,Hi-C,ATAC-seq,and RNA-seq and integrated these data with GWAS of 16 autoimmune traits to physically map disease-associated variants to the effector genes they likely regulate in 57 human cell types. These 3D maps of gene cis-regulatory architecture are highly powered to identify the cell types most likely impacted by disease-associated genetic variation compared to 1D genomic features,and tend to implicate different effector genes than eQTL approaches in the same cell types. Most of the variants implicated by these cis-regulatory architectures are highly trait-specific,but nearly half of the target genes connected to these variants are shared across multiple autoimmune disorders in multiple cell types,suggesting a high level of genetic diversity and complexity among autoimmune diseases that nonetheless converge at the level of target gene and cell type. Substantial effector gene sharing led to the common enrichment of similar biological networks across disease and cell types. However,trait-specific pathways representing potential areas for disease-specific intervention were identified. To test this,we pharmacologically validated squalene synthase,a cholesterol biosynthetic enzyme encoded by the FDFT1 gene implicated by our approach in MS and SLE,as a novel immunomodulatory drug target controlling inflammatory cytokine production by human T cells. These data represent a comprehensive resource for basic discovery of gene cis-regulatory mechanisms,and the analyses reported reveal mechanisms by which autoimmune-associated variants act to regulate gene expression,function,and pathology across multiple,distinct tissues and cell types. View Publication

Clinical studies have shown that Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is associated with aggressive periodontitis and can potentially trigger or exacerbate rheumatoid arthritis (RA). However,the mechanism is poorly understood. Here,we show that systemic infection with A. actinomycetemcomitans triggers the progression of arthritis in mice anti-collagen antibody-induced arthritis (CAIA) model following IL-1β secretion and cell infiltration in paws in a manner that is dependent on caspase-11-mediated inflammasome activation in macrophages. The administration of polymyxin B (PMB),chloroquine,and anti-CD11b antibody suppressed inflammasome activation in macrophages and arthritis in mice,suggesting that the recognition of lipopolysaccharide (LPS) in the cytosol after bacterial degradation by lysosomes and invasion via CD11b are needed to trigger arthritis following inflammasome activation in macrophages. These data reveal that the inhibition of caspase-11-mediated inflammasome activation potentiates aggravation of RA induced by infection with A. actinomycetemcomitans. This work highlights how RA can be progressed by inflammasome activation as a result of periodontitis-associated bacterial infection and discusses the mechanism of inflammasome activation in response to infection with A. actinomycetemcomitans. View Publication

IntroductionThe early transcription unit 3 (E3) of human adenoviruses (HAdVs) encodes several immunoevasins,including the E3/49K protein,which is unique for species D of HAdVs. It is expressed as surface transmembrane protein and shed. E3/49K of HAdV-D64 binds to the protein tyrosine phosphatase surface receptor CD45,thereby modulating activation of T and NK cells.MethodsConsidering that E3/49K represents the most polymorphic viral protein among species D HAdVs,we demonstrate here that all tested E3/49K orthologs bind to the immunologically important regulator CD45. Thus,this feature is conserved regardless of the pathological associations of the respective HAdV types.ResultsIt appeared that modulation of CD45 is a unique property restricted to HAdVs of species D. Moreover,E3/49K treatment inhibited B cell receptor (BCR) signaling and impaired BCR signal phenotypes. The latter were highly comparable to B cells having defects in the expression of CD45,suggesting E3/49K as a potential tool to investigate CD45 specific functions.ConclusionWe identified B cells as new direct target of E3/49K-mediated immune modulation,representing a novel viral immunosubversive mechanism. View Publication

AbstractChromobox 2 (CBX2),an epigenetic reader and component of polycomb repressor complex 1,is highly expressed in >75% of high-grade serous carcinoma. Increased CBX2 expression is associated with poorer survival,whereas CBX2 knockdown leads to improved chemotherapy sensitivity. In a high-grade serous carcinoma immune-competent murine model,knockdown of CBX2 decreased tumor progression. We sought to explore the impact of modulation of CBX2 on the tumor immune microenvironment (TIME),understanding that the TIME plays a critical role in disease progression and development of therapy resistance. Exploration of existing datasets demonstrated that elevated CBX2 expression significantly correlated with specific immune cell types in the TIME. RNA sequencing and pathway analysis of differentially expressed genes demonstrated immune signature enrichment. Confocal microscopy and co-culture experiments found that modulation of CBX2 leads to increased recruitment and infiltration of macrophages. Flow cytometry of macrophages cultured with CBX2-overexpressing cells showed increased M2-like macrophages and decreased phagocytosis activity. Cbx2 knockdown in the Trp53-null,Brca2-null ID8 syngeneic murine model (ID8 Trp53−/−Brca2−/−) led to decreased tumor progression compared with the control. NanoString immuno-oncology panel analysis suggested that knockdown in Cbx2 shifts immune cell composition,with an increase in macrophages. Multispectral immunohistochemistry (mIHC) further confirmed an increase in macrophage infiltration. Increased CBX2 expression leads to recruitment and polarization of protumor macrophages,and targeting CBX2 may serve to modulate the TIME to enhance the efficacy of immune therapies.Significance:CBX2 expression correlates with the TIME. CBX2 modulation shifts the macrophage population,potentially leading to an immunosuppressive microenvironment,highlighting CBX2 as a target to improve efficacy of immunotherapy. View Publication

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

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

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