bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved level and longevity of protective immune responses in measles virus with efficient replication in blood and lymphoid tissue
Authors: Lin Wen-Hsuan Wendy , Adams Robert J , Griffin Diane E
Published: January 2023

Infection with wild type measles virus (wt-MV) is an important cause of childhood mortality and is also known to induce remarkable life-long protective immunity. Despite the antigenic similarities between wt-MV and live-attenuated measles virus vaccine (LAMV), the protective immunity after measles vaccination is not as robust or long-lived. To identify mechanisms that contribute to the longevity of immune responses induced by wt-MeV infection, we compared parameters of virologic and immunologic responses after respiratory infection with wt-MeV and LAMV in rhesus macaques. Analysis of viral RNA in various tissues revealed distinct in vivo tropisms between the two strains of virus. Wt-MeV caused efficient hematogenous spread and prolonged presence of viral RNA, while LAMV caused transient localized infection in the respiratory tract. Plasma levels of cytokines IL-1β, IL-12 and interferon (IFN)-γ and chemokines CCL2, CCL11, CXCL9 and CXCL11 were increased after wt-MeV, but not LAMV infection. Infection with wt-MeV or LAMV induced comparable levels of viral-specific T cells, but significantly higher levels of protective antibody and more long-lived plasma cells were observed after wt-MeV infection. Therefore, distinct patterns of in vivo virus replication, efficiency of hematogenous spread and persistence of viral RNA are associated with the altered virulence and the level and longevity of the humoral response. Our results establish a link between life-long protective immunity and the ability of wt-MeV to spread efficiently in lymphocytes and to be cleared slowly.
The utility of Benzathine Penicillin G for Rheumatic Fever prevention in the Solomon Islands
Authors: Sika-Paotonu Dianne , Liligeto Urijah
Published: January 2023

Acute Rheumatic Fever (ARF) frequently affects children and young adults and is an autoimmune condition caused by untreated Group A Streptococcal (GAS) infection of the upper respiratory tract (and skin). Repeated or very severe ARF attacks that are left untreated, can lead to Rheumatic Heart Disease (RHD) - permanent cardiac damage. RHD is rare in developed countries and yet remains a significant cause of morbidity and mortality worldwide. Unfortunately the disease burden of ARF and RHD amongst Indigenous and Pacific communities in New Zealand, Australia and the Pacific Region are amongst the highest in the world. In the absence of a GAS vaccine, the recommended preventative measure for ARF/RHD consists of painful monthly injections of a type of Penicillin known as Benzathine Penicillin G (BPG). Otherwise known as secondary prophylaxis (SP), the purpose of these injections is to prevent GAS infections that can trigger recurrence of ARF and either lead to or worsen RHD. The adherence to SP remains challenging across many settings and it is recognised that a new Penicillin that is more appropriate is needed. To support the drug design efforts and development of a new BPG formulation, this work sought to explore BPG SP for Rheumatic Fever in the Solomon Islands by engaging with health professionals, researchers and community leaders and sourcing local information. Access and other issues connected with BPG SP for those having received a previous diagnosis of ARF were identified. Information concerning these challenges are relevant for drug design efforts and SP schedules for ARF/RHD prevention.
Role of acetylcholine receptors in cholinergic pathway-mediated protection against autoimmune diabetes
Authors: Al-Ramadi Basel K , George Junu A , Bashir Ghada , Qureshi Mohamed M. , Mohamed Yassir A , Elsayed Nourhan , Fernandez-Cabezudo Maria J
Published: January 2023

Type I diabetes (T1D) is a T cell-driven autoimmune disease that results in the killing of pancreatic β-cells and, consequently, loss of insulin production. Using the multiple low-dose streptozotocin (MLD-STZ) model of experimental autoimmune diabetes, we previously reported that pretreatment with a specific acetylcholinesterase inhibitor (AChEI), paraoxon, prevented the development of hyperglycemia in C57BL/6 mice. This correlated with an inhibition of T cell infiltration into the pancreatic islets and a reduction in proinflammatory cytokines. The cholinergic anti-inflammatory pathway utilizes nicotinic and muscarinic acetycholine receptors (AChR) expressed on a variety of cell types. In this study, we carried out a comparative analysis on the effect of specific antagonists of the nicotinic- or muscarinic-AChR on the development of autoimmune diabetes. Co-administration of nicotinic-AChR antagonist (mecamylamine) maintained the protective effect of AChEI on the development of hyperglycemia. In contrast, co-administration with muscarinic-AChR antagonist (atropine) mitigated AChEI-mediated protection. Mice pretreated with mecamylamine had an improved response in glucose tolerance test than mice pretreated with atropine. These findings correlated with the extent of islet cell infiltration and with the structure and functionality of the β-cells. Taken together, our data suggest that muscarinic receptors are essential for the protective effect of cholinergic stimulation on autoimmune diabetes.
p38 MAP kinase signaling in microglia plays a sex-specific protective role in CNS autoimmunity
Authors: Krementsov Dimitry N , McGill Mahalia
Published: January 2023

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system, representing the leading cause of non-traumatic neurologic disease in young adults. This disease is 3 times more common in women, but the underlying mechanisms remain unknown. MS is initiated by autoreactive T helper cells, but CNS-infiltrating inflammatory myeloid cells are the main effector cells driving disease pathology. The role of CNS-resident macrophage-like microglial cells is unclear. We have previously shown that genetic ablation of p38α MAP kinase (p38) in the myeloid lineage is protective in the autoimmune model of MS, experimental autoimmune encephalomyelitis (EAE). Strikingly, protection was seen in female mice, and not males. Here, we sought to precisely define the mechanisms responsible. To this end, we used multiple genetic approaches and bone marrow chimeras to ablate p38 in microglial cells, peripheral myeloid cells, or both. Deletion of p38 in both cell types recapitulated the previous sex difference, with reduced EAE severity in females. Unexpectedly, deletion of p38 in the periphery was protective in both sexes, while deletion of p38 in microglia exacerbated EAE only in males. Transcriptional profiling of microglia isolated from the CNS at peak of EAE revealed male-specific p38-dependent transcriptional modules with anti-inflammatory/tissue reparatory functionality. Taken together, these results reveal the presence of a p38-dependent male-specific molecular pathway in microglia that is protective in CNS autoimmunity, suggesting that autoimmunity in males and females may be driven by distinct cellular and molecular pathways, thus informing the design of novel future sex-specific therapeutic approaches.
Developmental exposure to environmental toxicant trichloroethylene alters DNA methylation in polycomb protein binding regions in effector/memory CD4 + T cells from autoimmune-prone mice
Authors: Blossom Sarah J , Byrum Stephanie , Washam Charity L , Patterson John D. , Vyas Kanan K , Gilbert Kathleen M.
Published: January 2023

Trichloroethylene (TCE) is an industrial solvent and drinking water pollutant associated with autoimmunity in human populations. Previous studies in autoimmune-prone MRL+/+ mice revealed autoimmune pathology and CD4 + T cell autoreactivity were more robust when exposure occurred during development rather than adulthood. To determine whether these effects were linked to epigenetic changes, mice were exposed to vehicle control or TCE in the drinking water beginning at gestation until 37 weeks of age. In some mice, TCE was removed from drinking water at 22 weeks of age. Whole genome reduced representation bisulfite sequencing (RRBS) was used to compare methylation of CpG sites in autosomal chromosomes in effector/memory CD4+ T cells. Few significant changes occurred in mice when TCE was removed from drinking water. In contrast, continuous developmental TCE exposure revealed differential methylation of 252 CpG sites relative to controls. When CpG sites were overlapped with the Open Regulatory Annotation database (ORegAnno), continuous TCE treatment resulted in 130 differentially methylated regions (DMRs) that included 12 unique transcription factors. Interestingly, 80% of DMRs occurred in areas known to bind Polycomb group (PcG) proteins that form the polycomb-repressive complex 2 (PRC2) in mammalian cells, namely, SUZ12, EZH2, JARID2, and MTF2. Pathway analysis of the DMRs indicated that TCE primarily altered the methylation of genes asociated with regulation of cellular metabolism and cell signaling pathways. In summary, TCE differentially methylated binding sites of PcG proteins in effector/memory CD4+ cells, and point toward a novel mechanism by which TCE could modulate CD4 + T cell function and promote autoimmunity.
PDLIM4: Target of JMJD3 regulates CD4 T cell Trafficking by connecting S1P1 and cytoskeleton
Authors: Fu Chuntang , Li Qingtian , Zou Jia , Xing Changsheng , Yu Jiaming , Chu Junjun , Yin Bingnan , Luo Mei , Wang Rongfu
Published: January 2023

In 2018 AAI annual meeting, we presented our discover that JMJD3 was bound to the promoter and gene body regions of Pdlim4 gene and it regulated PDLIM4 expression by interacting with zinc finger transcription factor KLF2. PDLIM4 as an filamentous actin (F-actin) binding protein, was involved in the T cell trafficking. However, the role and mechanisms of PDLIM4 in T cell trafficking remain unclear. In this abstract, we showed that PDLIM4 as an adaptor protein connecting S1P1 and filamentous actin (F-actin) was a key regulator of T cell trafficking. Our studies indicated that PDLIM4 interacted with S1P1 by N terminal PDZ domain. The PLDIM4 C terminal LIM domain anchored the complex to filamentous actin (F-actin). Our findings identified Pdlim4 as a novel Jmjd3 target gene that affects T-cell trafficking by cooperating with S1P1 and provided insights into the molecular mechanisms of which Jmjd3 regulates genes participating in thymic emigration.
Role of CXCR3 in natural killer cell migration to melanoma cells
Authors: Park Eun Jae , Kang Yu Jeong , Lee Hong Kyung , Kim Hyung Sook , Hong Jin Tae , Kim Youngsoo , Han Sang-Bae
Published: January 2023

Natural killer (NK) cell recruitment to solid tumors is essential for direct contact between NK and cancer cells and is regulated by chemokine networks. Tumor-derived CXCL10 increases the infiltration of NK cells into solid tumors. In a xenograft mouse model, adoptively transferred human NK cells efficiently infiltrate CXCL10-transduced tumors and enhance regression of tumor growth compared with mock-transduced tumors. Although this study showed that NK cells homed to solid tumor beds in a CXCL10-depedent manner, several questions remain. How do NK cells find cancer cells after extravasation? Which chemokines do B16F10 cells produce? Which chemokine receptors do NK cells use? How do cancer cell-derived chemokines affect NK cell motility? Do these chemokines directly affect NK cell cytotoxicity? To address these issues, we examined the migration dynamics of NK and cancer cells using time-lapse imaging. Although naïve B16F10 cancer cells produce low levels of chemokines, IFN-g-treated B16F10 cells secreted high levels of CXCL10, low levels of CCL5, but did not secrete CCL2, CCL7, or CXCL12. Wild-type NK cells migrated well toward cancer cells and killed them, whereas NK cells deficient in CXCR3 did not. CXCR3-deficient NK cells also showed slower migration speed than did wild-type NK cells. Taken together, our data show that NK cells find cancer cells, at least in part, by sensing CXCL10 produced by cancer cells and suggest that a strategy to increase CXCL10 secretion by cancer cells may improve the efficacy of NK cell–based immunotherapy.
PIEZO1 forms an adhesive-mechanosensitive complex with activated LFA-1 on T lymphocytes
Authors: Aguilar Alicia Virginia , Benson Bryan , Rathkey Joseph , Correa Luis , Li Lucy , Myers Jay , Tomalka Jeffrey , Kennelley Hannah , Abbott Derek , Ransohoff Richard , Huang Alex
Published: January 2023

In order to respond to infection, injury and stress, T lymphocytes must successfully migrate from the bloodstream into inflamed tissue in a process called the leukocyte adhesion cascade. Of these steps, blockade of adhesion has shown clear clinical benefit in autoimmunity, and the step of intraluminal crawling is of great potential clinical importance. In order to coordinate adhesion and crawling while in circulation, leukocytes must respond appropriately to complex hemodynamic forces, including shear stresses and erythrocyte driven margination against the venule walls. Therefore, we hypothesized that the mechanosensitive calcium channel PIEZO1, which is preferentially expressed in T lymphocytes, mediates T lymphocyte responses to force during adhesion and crawling. To test this hypothesis, we genetically ablated PIEZO1 in primary T lymphocytes from healthy human donors using CRISPR/Cas9, and observed crawling ability in vitro. In this assay, PIEZO1 knockout cells exhibited decreased crawling and disrupted morphology. Moreover, we performed immunofluorescence of PIEZO1 on chemokine-activated crawling T lymphocytes, which demonstrated that PIEZO1 redistributes to the contact zone of crawling cells in a pattern reminiscent of a high-affinity LFA-1 focal zone. Additionally, PIEZO1 colocalizes specifically with high affinity LFA-1. Using co-immunoprecipitation indicated that PIEZO1 preferentially associates with the alpha integrin subunit of LFA-1 in its active form, through a conserved amphipathic eight amino acid motif. Taken together, our data suggest that PIEZO1 contributes to coordination of T lymphocyte crawling via interaction with integrins, modulating the affinity state and turnover of LFA-1.
Dendritic Cell-Induced Dissemination of Mycobacterium tuberculosis into the Central Nervous System
Authors: Gilpin Trey E , Walter Fruzsina R , Herbath Melinda , Sandor Matyas , Fabry Zsuzsanna
Published: January 2023

Central nervous system tuberculosis (CNSTB) is the most serious manifestation of extrapulmonary tuberculosis infections representing 5–10% of all cases. Knowledge of CNSTB stems from observations made in 1933 by pathologists Rich and McCordock, who found caseating foci in the brain parenchyma or meninges that rupture into the subarachnoid space leading to diffuse meningeal infection. However, the mechanism of how immobile Mycobacterium tuberculosis, the causative agent of TB crosses the highly regulated blood-brain barrier (BBB) and enters the CNS forming these foci and infection is unknown. We have developed a novel in vitro BBB model that combines Mycobacteria-infected DCs, PBMCs, and primary mouse brain endothelial cells to observe early cellular interaction and aggregate formation at the BBB similar to previous observations by Rich and McCordock. With this model we observed Mycobacteria impedes migration of DCs but increases DC initiation of aggregation on a primary mouse endothelial BBB with a cellular composition similar to pulmonary granulomas. Aggregates induce an inflammatory response by increasing ICAM-1 and VCAM-1 expression and dysregulation of tight junction proteins and mitochondria suggesting barrier damage possibly due to increased iNOS expression observed during within aggregates. Mycobacteria-infected DC transmigration is increased in the presence of aggregation. Previous research from our lab and others have shown DC transmigration across an in vitro BBB rely on MMP-9 secretion while TNF-alpha is necessary for pulmonary granuloma formation and Mtb infection control. Here we will show the role iNOS, MMP-9 and TNF-alpha play in DC-mediated dissemination of Mycobacterium across the BBB.
Mass-cytometry reveals global immune remodeling with multi-lineage hypersensitivity to Type I Interferon in Down syndrome
Authors: Waugh Katherine A. , Pandey Ahwan , Jordan Kimberly R , Smith Keith P , Granrath Ross E , Khanal Santosh , Butcher Eric T , Rachubinski Angela L , McWilliams Jennifer A , Araya Paula , Minter Ross , Dimasi Tiana , Colvin Kelley L. , Baturin Dmitry , Pham Andrew T , Galbraith Matthew D , Yeager Michael E. , Sullivan Kelly D , Porter Christopher C , Hsieh Elena W , Espinosa Joaquin M
Published: January 2023

People with Down syndrome (DS), the genetic condition caused by trisomy of chromosome 21 (chr21), have an altered disease spectrum. For example, adults with DS are largely protected from solid tumors while predisposed to various autoimmune disorders, early-onset Alzheimer’s disease, and severe respiratory infection. While the immune system is known to play a key role in these comorbidities among the disomic population, there is a dearth of immune studies in adults with DS. Here, we applied the single-cell, systems-level approach of mass-cytometry, or Cytometry by Time-Of-Flight (CyTOF), to broadly define phenotypic and functional alterations in immune homeostasis in adults with DS that are relevant to the potentiation of comorbidities associated with T21. We developed an innovative strategy to analyze CyTOF data, by incorporating techniques commonly used in genomics and even topography to eliminate batch bias and efficiently resolve ~100 immune cell types. This approach revealed that adults with DS have global immune dysregulation reminiscent of various inflammatory and autoimmune states enriched among older individuals of the disomic population. Upon stimulation directly ex vivo with Type I Interferon (IFN) and simultaneous detection of diverse phosphorylation events across the lymphoid and myeloid lineages, all immune subsets from individuals with T21 were hyper-responsive, but the degree of response and signaling pathways used varied among specific cell types. Notably, 4 of the 6 IFN receptor subunits for Types I, II, and III IFNs are encoded on chr21. Altogether, these results establish a critical foundation to pursue immune dysregulation and IFN hyperactivity as a driver of the altered disease spectrum in adults with DS.
Interleukin-37 Promotes Colitis-associated Carcinogenesis (CAC) via SIGIRR-mediated Cytotoxic T Cells Exhaustion
Authors: Li Jiong , Teng Xiu , Wei Xiaoqiong , Wang Zhen , Zeng FanLian , Zhou Hong , Zheng Huaping
Published: January 2023

Interleukin-37 (IL-37), a ligand of the IL-1 cytokine family, was identified as a fundamental inhibitor of innate immunity, having emerged as a dual-function cytokine with extracellular (ligation with receptor SIGIRR) and intra-(interaction with smad3) properties for regulating inflammatory response. However, the role and molecular mechanism of IL-37 in CAC has been rarely reported. IL-37 expression was highly expressed in human colorectal cancer tissues and predicted poor prognosis. Indeed, mice with transgenic expression of IL-37 (IL-37tg mice) were highly susceptible to colorectal cancer induced by AOM/DSS and suffered from dramatically increased tumor burdens in the colon. Nevertheless, aggravated epithelial disruption in IL-37tg mice was observed during DSS-induced injury, which was dependent on the presence of commensal bacteria through MAPK and mTOR pathways. Plus, IL-37 did not affect growth or enhance mutagenesis of transformed colonic epithelia cells. These results demonstrated that decreased proliferation of epithelia cells in response to DSS injury in CAC model is unlikely to be responsible for susceptibility in IL-37tg mice. Significant deficiency and dysfunction of cytotoxic T cells was consistently found in colonic lamina propria in IL-37 tg mice. This exhaustion led by IL-37 was dependent on checkpoint SIGIRR-mediated TIR signaling blocking, independent of TGF-β/smad3 signaling agonizing or antagonizing, and not caused by common immune-checkpoints. Our findings highlight a role for IL-37 in harnessing antitumor immunity by inactivation of cytotoxic T cells and establish new inhibitory checkpoint IL-37/SIGIRR in the cancer-immunity cycle as therapeutic targets in colorectal cancer.
Human IL10 promoter haplotypes confer differential outcomes to experimental colitis in humanized BAC mice
Authors: Vacharathit Vimvara , Ma Jinxia , Gunasekera Dilini C. , Shah Palak , Sheh Alexander , Brayton Cory F. , Whary Mark T. , Fox James G. , Bream Jay H.
Published: January 2023

Most genetic variants associated with complex diseases map to non-coding regions of the genome, but determining their phenotypic consequences remains challenging. We devised a bacterial artificial chromosome (BAC) transgenic approach to model the effects of human non-coding single nucleotide polymorphisms (ncSNPs) on complex disease outcomes. As proof-of-concept, we focus on human IL10, a potent anti-inflammatory cytokine and key mediator of gut homeostasis. ncSNPs in the IL10 locus have been strongly associated with a variety of disease risks, including colitis susceptibility, but their biological functions are unclear. We created two genetically humanized IL10BAC transgenic mouse lines based on two common and well-defined −1082/−819/−592 IL10 promoter haplotypes (“ATA” and “GCC”) on a mouse Il10-deficient background. Although both IL10BAC strains were protected from spontaneous colitis, only ATA-bearing mice were resistant to dextran sulfate sodium (DSS)-induced acute colitis, while GCC and WT mice had high rates of morbidity and mortality. We found that ATAs had different kinetics of cell-type-specific IL-10 production pre- and post-DSS treatment compared to GCCs and WTs, which influenced the expression profiles of other cytokines and led to distinct immunophenotypes. The skewed responses culminated in the inability of GCCs and WTs to control DSS-induced bacteremia, resulting in death. Our data suggest that human IL10 ncSNPs impart allele-specific differences in spatiotemporal regulation of IL-10 expression, leading to differential outcomes to experimental colitis. Humanized BAC mice may serve as an important tool in modeling the potential impact of human non-coding variants on complex biological outcomes.
Detecting IL-1β release with a rapid, homogeneous, bioluminescent immunoassay
Authors: O’Brien Martha A. , Lindstedt Anders , Lazar Dan , Cali James
Published: January 2023

IL-1β is an important inflammatory cytokine of the innate immune system, released from monocytes, macrophages, and other cell types in response to a variety of pathogens and danger signals consequent to inflammasome activation. To enable direct measurement of released IL-1β from cells (no transfers or wash steps), two anti-human IL-1β antibodies were respectively labeled with complementary subunits, SmBiT and LgBiT, of the NanoLuc® Binary Technology. When the labeled antibodies bind to released IL-1β, the complementary subunits are brought into proximity, thereby reconstituting a bright luciferase that generates light when the furimazine substrate is added. Luminescence is monitored with a standard plate-reading luminometer. This novel assay effectively detected released IL-1β from differentiated human monocytic THP-1 cells and human peripheral blood mononuclear cells (PBMCs) stimulated with several TLR agonists, including endotoxin and non-endotoxin pyrogens, and could enable a simplified method for a Monocyte Activation Test for pyrogen detection. Released IL-1β can be detected directly in cell culture assay wells or in a secondary plate with transferred culture medium. Benchmarking assay performance to a standard IL-1β ELISA verified the accuracy of this more rapid, homogeneous IL-1β assay method. Of note, this IL-1β assay can be multiplexed with a homogeneous caspase-1 activity assay, providing complementary high throughput methods to identify modulators of inflammasome activation. MCC950, an NLRP3 inflammasome inhibitor, was shown to inhibit IL-1β release from treated THP-1 cells and PBMCs. Application of this homogeneous immunoassay approach to other inflammatory cytokines is currently being explored.
A circulating NAD biosynthetic enzyme is a novel modulator of inflammation
Authors: Manago’ Antonella , Audrito Valentina , Mazzola Francesca , Sorci Leonardo , Gaudino Federica , Gizzi Katiuscia , Incarnato Danny , Minazzato Gabriele , Ianniello Alice , Varriale Antonio , D’Auria Sabato , Mengozzi Giulio , Politano Gianfranco , Oliviero Salvatore , Raffaelli Nadia , Deaglio Silvia
Published: January 2023

Damage-associated molecular patterns (DAMPs) are molecules that can be actively or passively released by injured tissues and that activate the immune system. Here we show for the first time that the NAD biosynthetic enzyme nicotinate phosphoribosyltransferase (NAPRT), the rate-limiting enzymes in the intracellular synthesis of NAD from nicotinic acid, is physiologically present in human sera, where it acts as a novel DAMP. We detected NAPRT in plasma/sera from human donors by antibody-mediated luminex assays and mass spectrometry. Exposure of human and mouse macrophages to NAPRT triggers activation of ERK1/2, phosphorylation of IKKα/β and nuclear translocation of the p65 subunit of the NF-kB complex, with synthesis and secretion of inflammatory cytokines, including IL-1β, IL-8, TNFα and CCL3. Furthermore, NAPRT enhances monocyte differentiation into macrophages, by inducing macrophage colony-stimulating factor. These effects are independent of the NAPRT catalytic activity, but rely on the protein’s binding to TLR4, as demonstrated by showing direct in vitro interaction and by the in vivo lack of NAPRT effects in TLR4−/− macrophages. In line with the finding that NAPRT mediates endotoxin tolerance, sera from patients with sepsis or septic shock contain the highest levels of circulating NAPRT, compared to other chronic inflammatory conditions, including cancer. Importantly, patients with serum NAPRT >15 ng/ml are characterized by a worse clinical outcome, compared to the counterparts.

Together, these data identify NAPRT as a novel endogenous ligand for TLR4 and a critical mediator of inflammation.
Peripheral blood CD115 (CSF-1R) expression is anticoagulant- and time-dependent
Authors: Acuff Nicole V , Divekar Anagha A , Yang Xifeng
Published: January 2023

Human CD115, also known as CSF-1R, is highly expressed by monocytes, macrophages, and dendritic cells. Here we show that detection of human CD115 on peripheral blood monocytes is dependent on both the anticoagulant and the storage time before sample processing. Commercially available CD115 antibody, clone 9-4D2-1E4, shows bright staining on blood samples collected in EDTA and citrate based anticoagulants and a dimmer staining intensity in blood collected in heparin. Clone 9-4D2-1E4 is not compatible with Cyto-Chex™ tubes. Consistent with published reports in mice, human CD115 signal is reduced within hours of collection. CD115 expression is further reduced after overnight storage at ambient temperature and is completely lost two days post collection. Reduced CD115 expression over time is most noticeable in whole blood collected in heparin. These results demonstrate that anti-coagulant and storage time are important considerations for current research projects and clinical trials targeting CD115 (CSF-1R).
CD301b+ SIRPα+ dendritic cells play a non-redundant role in inducing clonal deletion in the thymus medulla
Authors: Breed Elise R , Hogquist Kristin A
Published: January 2023

The thymus contains an assortment of antigen presenting cells (APC) that have distinct locations and antigen processing capabilities. This suggests that they play non-redundant roles in mediating thymocyte selection. Many APC subsets were shown to be capable of driving clonal deletion, however these studies relied primarily on T cell receptor transgenic models. We therefore still do not understand the extent to which distinct APC contribute to clonal deletion in the polyclonal repertoire. Here, we assessed the contribution of different APC subsets to clonal deletion using a cleaved caspase 3-based assay paired with cell type ablation or deficiency. Total deletion frequencies were not altered in the absence of B cells, pDC, or XCR1+ cDC1. In an effort to eliminate SIRPα+ cDC2, we discovered that a substantial proportion of thymic SIRPα+ DC express the lectin CD301b. CD301b+ DCs were localized exclusively within the thymus medulla and depended on IL-4R□. Deficiency of these IL-4/IL-13 signaled DC resulted in a measurable reduction in clonal deletion. These data suggest that CD301b+ SIRPα+ DC represent a distinct population of antigen presenting cells within the thymus and that they are essential for non-redundant deletional tolerance in the polyclonal repertoire.
Hoxa9 silencing of p16
ink4a is essential for murine lymphopoiesis
Authors: Medina Kay L , Gwin Kimberly A.
Published: January 2023

The homeo-domain transcription factor Hoxa9 is required for normal B and T lymphocyte development in bone marrow (BM) and thymus, respectively. Here we show that Hoxa9-deficiency is accompanied by increased expression of the cdkn2a tumor suppressor locus that encodes the cell cycle inhibitor p16Ink4a. Furthermore, we found that somatic deletion of p16Ink4a is sufficient to rescue the B and T cell progenitor deficiencies, as well as the BM lymphoid progenitor pools, in hoxa9−/− mice. In contrast, the hematopoietic stem cell (HSC) and multipotential progenitor (MPP) abnormalities characteristic of Hoxa9-deficient mice were not corrected by loss of p16Ink4a. These new data reveal that Hoxa9 repression of p16ink4a is essential for lymphoid lineage development in BM and thymus. Importantly, they highlight distinct functions for Hoxa9 in regulation of primitive hematopoietic progenitors versus those committed to the B or T cell fates.
A MYC-inducible tumor-suppressor program protects developing B cells from transformation
Authors: Tonc Elena , Chou Chun , Fujii Chika , Hsiung Sunnie , Egawa Takeshi
Published: January 2023

Rapid expansion of developing and activated lymphocytes is essential for establishment of a diverse antigen receptor repertoire and effective immune responses. c-MYC is a multifunctional transcription factor required for regulated proliferation of lymphocytes and their progenitors. However, c-MYC is also strongly oncogenic, both in humans and mice. Nonetheless, transformation of lymphocytes is rare, despite their extensive proliferative activity and endogenous DNA damage caused by RAG and AID activities. Thus, we hypothesized that lymphocytes are protected from transformation by unique tumor suppressor programs, potentially linked with c-MYC activity. Using the Eμ-Myc mouse model, we identified a c-MYC inducible tumor suppressive program mediated by its direct target, transcription factor AP4/Tfap4. AP4 haploinsufficiency significantly accelerated tumorigenesis of developing B cells in a cell-intrinsic manner. These tumors frequently underwent loss-of-heterozygosity of the remaining Tfap4 allele, a hallmark characteristic of other tumor suppressors. Functionally defective somatic variants of AP4 were also identified in primary human lymphomas. Mechanistically, AP4 suppressed expression of the oncogene Erg in developing B cells, which is normally suppressed in c-MYC+ pro/pre-B cells. Reducing Erg expression significantly prolonged the survival of AP4 heterozygous Eμ-Myc mice. AP4 thus protects developing B cells from transformation, at least in part, by restricting co-expression of MYC and additional oncogenes. Our findings indicate that c-MYC engages an active tumor suppressive program in B cells by inducing its direct target AP4, which is the first example of c-MYC-inducible tumor suppressors.
Modulation of RAG2 Interactions With Histone H3K4me3 Through Residue Thr490
Authors: Rodgers William A , Byrum Jennifer , Simpson Destiny , Hoolehan Walker , Rodgers Karla
Published: January 2023

RAG2 is essential for V(D)J recombination in developing lymphocytes, yet its localization and dynamics in cell nuclei is poorly understood. Here, we used single-cell imaging to interrogate RAG2 interactions with transcriptionally active chromatin containing H3K4me3. We measured GFP-labeled full length RAG2 (FL), the RAG2 core region alone (Core), and a T490A mutant of RAG2. Each RAG2 construct localized to nuclear regions that were adjacent to DAPI-rich heterochromatin, where the T490A RAG2 exhibited greater colocalization with H3K4me3 than either FL or Core. Furthermore, mobility measurements showed that T490A had a significantly lower rate of diffusion within the nucleus than either FL or Core proteins. Finally, mutating W453 in the T490A mutant (W453A, T490A), which blocks interactions between the RAG2 PHD region and H3K4me3, decreased the RAG2 colocalization with H3K4me3 and increased its mobility to values similar to that of FL. Altogether, these data show that Thr490 modulates RAG2 localization and dynamics in the pre-B cell nucleus, such as by affecting PHD-dependent interactions between RAG2 and H3K4me3.
NOD2 regulates distinct pathways of endogenous thymic regeneration after injury
Authors: Kinsella Sinéad , Cooper Kirsten , deRoos Paul , Iovino Lorenzo , Jain Reema , Dudakov Jarrod A
Published: January 2023

Although the thymus has a remarkable capacity for repair following damage, the mechanisms underlying this endogenous regeneration remain poorly understood. Our recent studies have identified two key pathways for thymic regeneration; centered on secretion of BMP4 by endothelial cells (ECs) and IL-22 from innate lymphoid cells (ILCs), which can be utilized individually as therapeutic strategies of immune regeneration. The specific regulatory mechanisms that trigger these regeneration-associated factors after damage remain unclear. Here we detail an unexpected role for the pattern recognition receptor Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in orchestrating these distinct pathways of thymic regeneration. After acute injury, Nod2−/− mice exhibited superior overall thymic regeneration, consistent with increased levels of BMP4, IL-22, and Il-23, a key regulator of IL-22 secreted by dendritic cells (DCs). Furthermore, expression of miR29, a downstream effector of NOD2, was significantly reduced in ECs and DCs after injury, and miR29 mimics suppressed Bmp4 and Il23 expression in ECs or DCs, respectively. Consistent with a non-canonical role for NOD2 in sensing RhoGTPases, we found several RhoGTPase family members were downregulated after TBI; and modulation with small molecule inhibitors targeting Rho GTPases led to robust production of BMP4 and IL-23, with enhanced thymic regeneration after acute injury. These findings provide evidence not only of a novel master regulator of endogenous thymic regeneration, but also offer a superior therapeutic strategy for boosting thymic regeneration and T cell reconstitution after damage such as that caused by infection or cytoreductive therapy.
Early Life Adversity Programs Mast Cells Toward a Hyperactive Phenotype into Adulthood
Authors: Maradiaga Nidia C , Pohl Calvin , Mackey Emily , Moeser Adam J.
Published: January 2023

Early life adversity (ELA) is a risk factor in the onset and exacerbation of inflammatory disorders including allergy, asthma and chronic pain disorders such as irritable bowel syndrome (IBS). Our previous studies using murine and porcine models showed that mast cells (MCs), a key immune cell driving allergies and chronic pain disorders, display a hyperactive tissue phenotype in adult animals exposed to ELA. The mechanism by which ELA induces MC hyperactivity in adulthood remains unknown. The aim of this study was to determine if ELA programs bone marrow derived MC progenitors toward a hyperactive phenotype into adulthood by using an ELA model of neonatal maternal separation plus early weaning (NMSEW). Female pups (C57BL/6) were either raised normal handled (NH) or NMSEW. At 10 weeks, serum and mesenteric windows from jejunum were collected to evaluate histamine and tissue MC activation. Bone-marrow derived MCs (BMMCs) from mice were harvested and stimulated with IgE-DNP and IL33 to assess histamine and IL6 release, respectively. NMSEW mice exhibited increased serum histamine levels (~1.5 fold higher, P<0.05), mesenteric MC numbers (~1.5 fold higher, P=0.05) and MC activation (~2 fold higher, P<0.0001). BMMCs from NMSEW mice exhibited greater histamine content, IgE-mediated histamine release and IL33-stimulated IL6 release (P<0.001), compared with NH BMMCs. Our data demonstrates that ELA induces lasting changes in MC progenitors programming them toward a hyperactive phenotype and increased activation in tissue. Future studies will investigate the mechanisms by which ELA drives lasting hyperactivity of MC progenitors and tissue MC hyperactivity, and its role in MC-related disorders in adulthood.
miR-155 has positive and negative roles on cytokine production in mast cells
Authors: Mohammed Zahraa abdulmohsin , Gomez Gregorio
Published: January 2023

Mast cells play a critical role in allergic inflammation by releasing preformed mediators such as histamine and serine neutral proteases, biosynthesizing lipid mediators like prostaglandins and leukotrienes, and synthesizing new cytokines. MicroRNA-155 (miR-155) plays a key regulatory role in the pathogenesis of allergy. The aim of this study was to investigate the biological functions of miR-155 on mast cell mediator release. MiR-155 was increased in human skin mast cells following FcɛRI crosslinking. To explore the role of miR-155 on mast cells function, we used bone marrow-derived mast cells (BMMCs) from wild type, miR-155 KO, and miR-155 Tg mice. MiR-155 had no effect on FcɛRI-induced mast cell degranulation. On the other hand, FcɛRI-induced COX-2 was significantly diminished in the absence of miR-155 suggesting that miR-155 plays a critical role in Prostaglandin D2 biosynthesis. FcɛRI-induced cytokine production was diminished in miR-155 KO BMMCs compared to WT. Interestingly, cytokine production from miR-155 KO BMMCs was increased compared to WT following LPS treatment. These findings indicate that miR-155 acts as a positive regulator in the FcɛRI pathway, and a negative regulator of the TLR4 pathway in mast cells. Current studies are aimed at determining how miR-155 functions in these pathways.
Analysis of allergen-specific T cell and IgE reactivity in milk and egg allergic children in response to progressive forms of baked milk and egg food extracts
Authors: Schulten Véronique MJ , Chen Meng , Laubach Susan , Leonard Stephanie , Peters Bjoern
Published: January 2023

Egg and milk allergy are among the most common food allergies in young children and are commonly outgrown. Developing tolerance is often clinically monitored by food challenge with cooked forms of egg and milk. This study characterizes IgE and T cell reactivity to four progressively cooked food extracts from egg and milk.

Egg and milk-containing food extracts were prepared from most to least cooked foods: egg muffin, meat balls frittata and raw egg; milk muffin, pizza cheese, custard and raw milk. IgE reactivity against extracts was determined by western blot and protein-specific IgE reactivity was quantified. T cell reactivity against the extracts was determined by FluoroSpot (IL-5, IFNγ and IL-10) after 24h restimulation following a two week in vitro expansion culture.

IgE reactivity varied between extracts, in a protein-specific manner. For milk, a protein band at 14 kDa corresponding in size to Bos d 4 is observed most strongly in raw milk and cheese extract, while being less reactive in custard and milk muffin. In contrast, a band at 67 kDa, corresponding to Bos d 6 in size, is most intense in milk muffin compared to raw milk, cheese and custard. For egg, trends are more consistent across the different proteins, with egg muffin showing most IgE reactivity throughout. Analysis of T cell responses showed highest IL-5 production for raw milk extract compared to custard (p=0.058), cheese (p=0.039) and muffin (p=0.097). No differences were observed for IFNγ and IL-10. For egg, no differences between extract-induced T cell reactivity was observed.

Both IgE and T cell reactivity is readily detectable against food extracts from baked forms of egg and milk. Immunological reactivity is not necessarily associated with the degree of cooking.
A proposed role for Interleukin-6 in the modulation of Th2 asthmatic responses
Authors: McLaughlin Erin M , Leon-Ruiz Beatriz
Published: January 2023

The incidence of allergic asthma in the United States and other industrialized countries has been increasing over the past few decades. One proposed explanation for this increase is the hygiene hypothesis, which states that a lack of early childhood exposure to high levels of environmental lipopolysaccharide (LPS) endotoxins from gram-negative bacteria increases the susceptibility to developing allergic diseases. Our lab has recently shown that LPS can indeed reduce Th2-driven, allergen-induced airway inflammation by inducing the up-regulation of the transcription factor T-bet in responding allergen-specific T cells, which we found was required to prevent the Th2 cell differentiation program. Studies have shown that like LPS, interleukin-6 (IL-6) reduces Th2 cell responses in asthma models. Due to these findings, our lab is now investigating the role of IL-6 in LPS-mediated Th2 cell suppression. In addition, some evidence suggests that IL-12 and IL-18 produced by dendritic cells are involved in the induction of T-bet by upregulating IFN-γ in T cells and other lymphoid cells. Here, we investigate the interplay of these cytokines in a house dust mite-induced asthma model in C57BL/6 (B6) mice. Through studies with IL-6 knock out and IL-6 receptor knock out B6 mice, we have shown that IL-6 modulates the expression of the IL-18 receptor on T cells, thus affecting the synergy of IL-18 and IL-12 to induce T-bet and prevent Th2 responses. We hypothesize that IL-6 affects T cell ability to respond to these cytokines by influencing the thresholds of IL-18 and IL-12 needed for T cell responses.
C-X3-C Chemokine Receptor 1 (CX3CR1) Regulates Immunopathogenesis During Fungal Asthma
Authors: Godwin Matthew S , Jones MaryJane , Yu Zhihong , Matalon Sadis , Steele Chad
Published: January 2023

Severe asthma with fungal sensitization (SAFS) defines a subset of severe asthmatics with allergy to multiple fungal species, including A. fumigatus. The C-X3-C chemokine receptor 1 (CX3CR1) is primarily expressed on monocytes, macrophages, and dendridic cells while expression of its ligand, CX3CL1 is increased in the presence of pro-inflammatory cytokines. Here, we show that bronchoalveolar lavage fluid (BALF) and induced sputum from human asthmatics sensitized to fungi demonstrate increased levels of CX3CL1 compared to fungal negative asthmatics, suggesting that CX3CL1 may contribute to fungal asthma severity. To this end, we examined fungal asthma severity in mice deficient in the CX3CL1 receptor (CX3CR1 -/-). The absence of CX3CR1 yielded no significant impact on type 2 biomarkers of asthma severity (e.g., levels of pro-allergic CCL17, CCL22, and IL-33) or lung eosinophil numbers. Rather, CX3CR1 deficiency was associated with heightened type 17 responses (characterized by increased production of IL-1α, IL-1β, IL-6, IL-17A, IL-22, G-CSF and CXCL2) and type 1 responses (characterized by increased IFNγ and CXCL9 production). Moreover, this inflammatory profile correlated with worse lung physiologic responses (e.g., increased dynamic and airway resistance). Taken together, these preliminary results suggest a critical role for CX3CL1 in regulating the immune response (immunopathogenesis) in fungal asthma independent of its role in cell recruitment.
Suppression of allergic airway inflammation by MCPIP1 through suppression of IL-5-producing Th2 cells
Authors: Liu Jianguo , Wang Qinghong , Ning Huan , Hou Rong
Published: January 2023

Allergic inflammation is mediated mainly through production of Th2 cytokines (IL-4, IL-5 and IL-13). Though we have learned much about how Th2 cells are differentiated, our understanding of the Th2 checkpoint mechanisms remains elusive. We recently found that a new RNA-binding protein, monocyte chemotactic protein induced protein-1 (MCPIP1, also named Regnase-1, encoded by Zc3h12a), suppresses Th2 differentiation and Th2 inflammation. Zc3h12a−/− mice spontaneously develop severe airway inflammation similar to asthma patients, with an increase mainly in IL-5- and IL-13-producing Th2 cells in the airway. Zc3h12a−/−CD4 T cells are mostly effector T cells and produce significant amounts of IL-5 and IL-13 but not IL-4. We further found that the differentiation of IL-5/IL-13-producing CD4 T cells is independent of IL-4 in Zc3h12a−/−naïve CD4 T cells. IL-5 expression correlates with the levels of GATA3 in Zc3h12a−/− CD4 T cells. Overexpression of MCPIP1 inhibits GATA3 and IL-5 expression through promoting GATA3 mRNA decay via RNase and CCCH domains. Furthermore, deletion of MCPIP1 in mice results in a significant increase in OVA-specific Th2 cells and severe Th2 inflammation in the airway of receipt mice after T cell transfer. Our study reveals that MCPIP1 is a potent regulator for Th2 development and function, especially the IL-5/IL-13-producing Th2 cells. MCPIP1 is a potentially new target for treatment of asthma and other Th2-mediated allergic diseases.
PAR1 signaling is involved in TCR-mediated CD8 T cell granule exocytosis
Authors: Smith Mindy Marie , Chen Hui , Hasley Rebecca B. , Karpova Tatiana , Catalfamo Marta
Published: January 2023

The Protease Activated Receptor (PAR1) is the receptor of thrombin, the main serine protease of the coagulation cascade. PAR1 is expressed by platelets, endothelial cells and cells of the innate immune system and mediates platelet aggregation and secretion of pro-inflammatory cytokines. PAR1 is expressed by T lymphocytes, however its role in T cell function is not well defined. We have previously shown that human CD4 and CD8 T cells express PAR1. PAR1 expression was higher in CD8 than CD4 T cells and is associated with T cell differentiation. CD8 T cell Effector Memory and Terminal Effector Memory phenotypes have higher PAR1 expression than Naïve T cells. PAR1 activation by thrombin enhanced CD8 T cell function including chemokinesis and cytokine secretion. In the present study, we address the role of PAR1 signaling in CD8 T cell mediated cytotoxicity by the granule exocytosis pathway. We found that expression of PAR1 on CD8 T cells is associated with expression of the transcription factors T-bet and Eomes and effector molecules, granzyme A, granzyme B and perforin. PAR1 activation by thrombin enhanced TcR-induced degranulation in CD8 T cells. In addition, in a redirected killing assay, we show that granule exocytosis and cytotoxicity of target cells was efficiently blocked by PAR1 antagonist (SCH79797). Confocal microscopy of the CD8 T-APC conjugates revealed that PAR1 blockade inhibit actin polymerization and decreased the docking of the Microtubule Organizing Center (MTOC) at the immunological synapse. These findings suggest that PAR1 signaling is involved in several effector functions of CD8 T cells including the cytotoxicity by the granule exocytosis pathway. These data have implications in CD8 T cell function and disease.