bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of B cell development and malignant transformation by microRNA-142
Authors: Wang Wei-Le , Magilnick Nathaniel , Reyes Estefany , Boldin Mark P
Published: January 2023

MicroRNA-142 (miR-142) is a versatile posttranscriptional regulator that plays a crucial role in the regulation of both innate and adaptive immune responses. Abrogation of miR-142 expression in mice results in a profound immunodeficiency that is characterized by hypoimmunoglobulinemia and failure to mount robust humoral immune responses to soluble antigen challenges. Paradoxically, miR-142−/− mice display a significant expansion of the B cell compartment due to the accumulation of immature and mature B lymphocytes. To better understand the role of miR-142 in B cell ontogenesis, we examined the early B cell development in miR-142−/− mice using the Hardy fraction analysis. We found that miR-142 deletion results in a significant increase in the frequency of the pro-B and large pre-B cells (Fractions B and C), while the pre-pro-B cell population (Fraction A) decreased. In contrast, miR-142 ablation had little effect on the frequency of small pre-B cell and immature B cell populations. Furthermore, our data suggest that the accumulation of pro-B and large pre-B cells in miR-142−/− bone marrow is linked to an increase in their survival capacity.

Because genetic lesions that disrupt control of B cell maturation frequently contribute to the malignant transformation of B cells, we decided to examine the role of miR-142 dysregulation in B cell lymphomagenesis. To address this question, we bred miR-142−/− mice with Eμ-myc transgenic mice that spontaneously develop a mix of pre-B cell and mature B cell lymphomas. We found that miR-142 haploinsufficiency dramatically accelerated lymphoma development in this mouse model. Collectively, our results establish miR-142 as an essential negative regulator of B cell maturation and lymphomagenesis.
E protein activity in DC precursors dictates the differentiation outcome of DC subsets.
Authors: Bajana Sandra Indiana , Thomas Kevin , Mengarelli Joni , Sun Xiao-Hong
Published: January 2023

Dendritic cells (DC) comprise plasmacytoid DC (pDC) and conventional DC (cDC) subsets; the latter is further separated into type 1 (cDC1) and type 2 (cDC2). These cells play a role in initiating and modulating immune responses. Their development is controlled by a network of transcription factors including E proteins consisting of E12, E47, HEB and E2-2, which share a basic helix-loop-helix domain mediating dimerization and DNA binding. Their activity is regulated by their antagonists, Id1-4. The balance between E and Id proteins regulates the transcription of E protein target genes. While E2-2 is essential for pDC differentiation and Id2 is indispensable for cDC1 formation, little is known about how the balance between E and Id proteins controls the early steps of the commitment of common DC precursors (CDP) to the diverse DC subsets. To address this, we used a mouse model that inducibly expresses a chimeric protein, ET2, which contains the DNA binding domain of Tal1 and the transactivation domains of E47. It can compete with Id proteins to bind endogenous E proteins and restore their DNA binding activity, thus increasing net E protein activity. Our data indicated that ET2 expression inhibited the differentiation of pDC and cDC2 subsets in vivo and in vitro. In contrast, cDC1 production was slightly elevated in steady state but dramatically increased in allergic reactions. Furthermore, we detected an augmented proportion of the newly identified cDC1 precursors and decreased representation of cDC2 precursors, suggesting E proteins skew CDPs toward the cDC1 lineage. These results are consistent with reports that E proteins activate IRF8 gene transcription. Further investigation is in progress to elucidate the molecular mechanisms.
Parainfluenza virus spread and clearance in a hematopoietic cell transplant model
Authors: Mostafa Heba , Vogel Peter , Baviskar Pradyumna , Russier Marion , Srinivasan Ashok , Russell Charles J
Published: January 2023

Parainfluenza viruses (PIV) are significant causes of morbidity and mortality in patients receiving hematopoietic cell transplant (HCT), particularly in the first 100 days post an allogeneic transplant. In the absence of effective therapeutic or preventive measures, it is critical to identify the immunological determinants that facilitate viral clearance that could be exploited for intervention, especially with respect to lower respiratory involvement. To study the dynamics of PIV infection in HCT host, we developed an allogeneic mouse model that we infect with a murine PIV. Specifically, we use a reporter Sendai virus that allows a daily, non-invasive bioluminescence imaging in living mice. HCT mice were infected shortly before transplant to study viral progression and clearance in the most critical period after transplant. Our data showed a marked prolongation of infection with a significant delay in clearance (21 days). The initiation of clearance correlated with a slight rebound in lymphocytes, which were mostly of the B type with more than 90% chimerism. Changing the initial site of infection by changing the infecting volume from 5 to 30 μL dramatically increased the lung signal, morbidity, and mortality. Treating the mice with Sendai-specific monoclonal antibodies significantly reduced the lung bioluminescence, morbidity, and prevented mortality. Overall, we developed a system that allows studying the correlation between different aspects of the immune system and PIV progression and clearance. Ongoing studies will determine the effect of manipulating the graft, including T cell enrichment versus depletion and the role of natural killer cells on Sendai virus infection.
TCF-1 Regulates Peripheral Treg Homeostasis
Authors: Yang Bi-Huei , Yuan Xiaomei , Dong Yi , Fu Wenxian
Published: January 2023

CD4+Foxp3+ regulatory T (Treg) cells are key players in preventing lethal autoimmunity and excessive inflammation. Based on their functional status, Treg cells can be distinguished into central (resting) and effector (activated) subsets. The homeostatic regulation between these two subsets is essential for Treg biology. However, what controls the dynamics between central and effector Treg cells remains poorly understood. We find that Wnt signaling transcription factor, TCF-1 plays a central role in regulating the differentiation from central to effector Treg cells. Although TCF-1 is crucial for T cell lineage specification, its expression is declined in a precisely-quantitative manner over the course of Treg development. Based on the expression of TCF-1, effector Treg cells can be further divided into two subsets. TCF-1neg subset is characterized as bona fide effector Treg cells. Transgenic modulation of TCF-1 alters the abundance of effector Treg cells. Our data suggest a two-step regulation of peripheral Treg homeostasis and TCF-1 play a critical role in this process.
Mucosal-associated invariant T cells respond to cutaneous microbiota
Authors: Constantinides Michael G , Linehan Jonathan L , Sen Shurjo , Shaik Jahangheer , Roy Sobhan , LeGrand Jess L , Bouladoux Nicolas , Adams Erin J , Belkaid Yasmine
Published: January 2023

The microbiome consists of a diverse array of commensal microorganisms that reside at barrier sites of the body and promote immune homeostasis through the release of microbial products, including derivatives of vitamin synthesis. Mucosal-associated invariant T (MAIT) cells are unconventional T cells that recognize vitamin B2 (riboflavin) derivatives presented by the major histocompatibility class I-like molecule, MR1. MAIT cells are predominantly located in barrier tissues, where they represent a substantial population of non-classical T cells and provide an initial defense to pathogens through their rapid production of either IL-17A or IFNg. However, it remains to be determined whether commensals regulate the development and function of MAIT cells. Here we show that MAIT cells are present in murine skin at a high frequency and their homeostasis requires the microbiota, as these lymphocytes are nearly absent in germ-free animals. Furthermore, application of distinct human commensal bacteria to the skin of mice induces the proliferation of IL-17A-producing MAIT cells that exhibit a unique transcriptional profile. The induction of these cells occurs in a manner that is partially dependent on both antigen presentation and IL-23 signaling. Additionally, MAIT cells stimulated by the cutaneous microbiota provide heterologous protection against subsequent pathogenic challenges. This work identifies MAIT cells as the only cell population that is entirely dependent on the microbiota and reveals the mechanism by which these cells respond to the commensal microbial community. Due to the high frequency of MAIT cells in human skin, these observations suggest that modulation of the skin-resident bacteria may have clinical applications.
Deficiency in vasoactive intestinal peptide signaling causes compositional and functional changes in mouse fecal microbiota
Authors: Bains Manpreet , Laney Caleb , Schuh Jane M , Dorsam Glenn P
Published: January 2023

By 2030, half the human population will be obese contributing to diabetes, cancer and cardiovascular disease. The gut peptide, vasoactive intestinal peptide (VIP) that acts as a regulator of innate and adaptive immunity, is elevated in obese humans. VIP mediates immunity changes through binding two receptors, called VPAC1 and VPAC2. Unfortunately, the pathways linking VIP signaling to obesity are not known. We hypothesized that defective VIP signaling would have an anti-obesity effect by lowering the gut firmicutes/bacteroidetes (f:b) ratio. To this end, knockout mice deficient for VIP, VPAC1 or VPAC2, were used to collect 16S rRNA reads from fecal samples. Operational taxonomic units for each strain were compared against each other to determine changes in bacterial taxa. Moreover, differences in gene functions were analyzed using PICRUSt. These data revealed a significant decrease in the f:b ratio for mice deficient in VIP and VPAC2 compared to wild type controls. Heterozygous and homozygous littermates for the VIP mutation showed the greatest reduction in fecal bacterial richness and clustered differently when compared phylogenetically to wild type or VIP receptor knockout strains. Metabolome predictions indicated that genes regulating the circulatory system and cardiovascular disease were consistently altered in mice deficient for VIP or its two receptors. In summary, deficiency in the VIP signaling axis significantly alters the gut microbiota and microbiome.
The role of gut microbiota in shaping the immune response in relapse-remitting and chronic-progressive mouse models of multiple sclerosis
Authors: Gandy K. Alexa Orr , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

In this study, we used a mouse model of multiple sclerosis (MS), experimental autoimmune encephalitis (EAE), to evaluate the role of gut microbiota in modulating T helper cell function in chronic-progressive (CP) versus relapse-remitting (RR) forms of the disease. We hypothesized that encephalitogenic T cell function in RR-EAE and CP-EAE is differentially shaped by gut microbiota. Metagenomic sequencing of the variable V4 region of the prokaryotic 16S ribosomal RNA gene present in feces derived from naïve mice and mice exhibiting CP-EAE or RR-EAE revealed significantly diverse microbial populations, which may be responsible for differing disease courses. Specifically, the order Bacteroidales was dominantly represented by RR-EAE mice, with very little or no detection in naive or CP-EAE mice, suggesting a potential role for bacteria in this order in shaping tolerant or remittance-favoring conditions. Additionally, Akkermansia and rc4-4, of phyla Verrucomicrobia and Firmicutes, respectively, were identified specifically in CP-EAE, suggesting a potential pathogenic or pro-inflammatory role for bacteria belonging to these taxa, at least in the context of EAE. We performed qRT-PCR in order to test the validity of the sequencing findings, as well as used a gas chromatograph configured with flame-ionization detectors to evaluate short chain fatty acids from cecal flushes. The current study implies a role for gut microbiota in differentially shaping the immune response in models of RRMS and CPMS. Information regarding tolerance–promoting or –inhibiting conditions shaped by gut bacteria can be harnessed for the potential development of therapies for diseases possessing important inflammatory components.
Dose dependent effect of BAFF receptor specific mAb-siRNA conjugate in experimental myasthenia
Authors: Huda Ruksana
Published: January 2023

We investigated potential therapeutic effect of a conjugate of BAFF receptor specific- monoclonal antibody and short interference RNA in a mouse model of myasthenia gravis (EAMG). We found that at high dose, conjugate treatment caused significant accumulation of Fas expressing CD19+/ B220+ cells and increased type 1 interferon in lymph nodes. Low dose did not induce FAS expression but caused marked BAFF receptor deficiency in lymph nodes that was further associated with improved MG symptoms. Conjugate treatment, however, did not reduce the levels of autoantibody. Our findings, thus, reveal a previously undocumented, dose dependent, immunomodulatory distant effect resulting from BAFF receptor specific mAb-siRNA conjugate treatment in an in vivo model of autoimmune disease.
RNA binding protein HuR is required for efficient function but not generation of Foxp3+ Treg
Authors: Ellis Jason S , Ridenhour Suzanne , Glascock Jacqueline , Atasoy Ulus
Published: January 2023

The RNA-binding protein (RBP) HuR (Elavl1) controls differentiation of CD4+ T cells into Th2 and Th17 helper subsets through regulating transcript stability of multiple subset specific targets. We recently generated dLck-Cre HuRfl/fl mice, in which HuR is conditionally knocked out in T cells late during thymic development. There was a 5:1 skewing toward HuR sufficient Treg in these mice, though there was only a small reduction in the total number of peripheral Treg compared to controls. More interestingly, HuR deficient naïve CD4+ T cells from these mice are less susceptible to Treg polarization than control HuR competent T cells and fail to upregulate Foxp3 following activation. We conditionally ablated HuR in Tregs (Foxp3YFP-Cre HuRfl/fl mouse) (HuR KO). These mice have significant failure to thrive in both hemizygous Foxp3YFP-CreHuRfl/fl males (39% weight reduction) and homozygous Foxp3YFP-Cre/YFP-Cre HuRfl/fl females (30% reduction). However, heterozygous Foxp3YFP-Cre/WT HuRfl/fl females have normal weight gain over time. Heterozygous HuR KO females also have a significant shift in the ratio of HuR− Treg to HuR+ Treg indicating a possible survival bias toward Treg expressing WT levels of HuR. Both male and female HuR KO mice have features of scurfy mice, including alopecia and stippled tails. This coincides with multiorgan inflammation which includes lung, skin, stomach and liver. Additionally, naïve HuR KO mice have significantly reduced peripheral Treg and increases in Foxp3− effector T cells with an activated phenotype. Together, these data suggest HuR is critical for effective Treg function and generation of iTreg.
Therapeutic NKG2D Ligation Facilitates Improved Wound Healing
Authors: Schenkel Jason M , Kubasiak John C , Dolubzino Hubert , Kohlhapp Frederick J , Tarabichi Saeed , Lee Michael , Guevara-Patino Jose A , Zloza Andrew
Published: January 2023

NKG2D is a C-type lectin-like receptor in the CD94/NKG2 family and is expressed by a variety of leukocyte populations including natural killer (NK) cells, γδ T cells, CD8+ T cells and certain innate lymphoid cell (ILC) subsets. NKG2D can bind proteins in two protein families – MIC and RAET1/ULBP. Expression of NKG2D ligands is low/absent under homeostatic conditions, but these proteins are upregulated in several contexts, including infection, cancer, physiologic stress, and senescence. NKG2D-driven activation of NK and CD8+ T cells has previously been shown to result in augmented control of infections and tumor growth. However, the role of NKG2D in the context of tissue injury and repair has not been established. Here we tested the effects of augmented NKG2D ligation in the context of a skin injury model. Biopsy punches were used to generate six 3- mm wounds on the dorsal surface of C57BL/6 mice. On days 0 and 1, NKG2D stimulating antibodies or isotype controls were injected into the peritoneal cavity and wound healing was assessed daily. Mice treated with NKG2D activating antibodies had more rapid wound repair than controls – wounds were healed by day 5 in NKG2D-treated mice as compared to day 7 in controls. Analogous results were observed in an ex vivo human skin model. Using a biolistic particle delivery system to induce upregulation of RAE (an NKG2D ligand), we observed more rapid wound repair compared to controls. Genetic ablation of NKG2D resulted in a 40% delay in wound healing. Moreover, depletion of NK1.1+ or CD8+ leukocytes blunted the benefit of NKG2D ligation, suggesting that T cells, NK cells, and/or ILCs may play a role in driving tissue repair. Thus, we demonstrate a potential therapeutic function of NKG2D engagement in tissue regeneration.
Bacterial amyloids from biofilms break tolerance in lupus
Authors: Lee Michael Hweemoon , Tursi Sarah A , Green Benjamin L , Caricchio Roberto , Tükel Çagla , Gallucci Stefania
Published: January 2023

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease, in which infections are considered to play a pathogenic role, but the cellular and molecular culprits remain unknown. Bacterial biofilms are multicellular bacterial communities important in the establishment of chronic infection by pathogens. Bacteria produce amyloids, complex proteins with a conserved beta sheet structure that strengthen the extracellular matrix of their biofilms. We have reported that bacterial and eukaryotic DNA is incorporated into curli fibers, functional bacterial amyloids present in Salmonella and E. coli biofilms. I.P. infections with curli-expressing S. Typhimurium or E. coli Nissle, and also systemic administration of curli/DNA complexes purified from Salmonella biofilms accelerated onset in lupus-prone NZBxW/F1 mice and triggered autoantibodies production in non-predisposed mice, suggesting curli/DNA complexes as novel players in SLE pathogenesis. We present here evidence that curli/DNA complexes can activate multiple B cell subsets in vivo. In addition, curli/DNA complexes are capable of directly inducing in vitro polyclonal activation and proliferation of B cells from wildtype mice and from lupus-prone mice and also from 3H9 mice, which are knocked-in for a rearranged anti-DNA Ig heavy chain and whose B cells are normally tolerized for the self-Antigen. Our results suggest that curli/DNA complexes may induce autoantibody production by directly breaking tolerance in autoreactive B cell.
Enhanced survival of BENTA patient B cells is dependent on MALT1 protease activity
Authors: Arjunaraja Swadhinya , Malinverni Claire , Sukumar Gauthaman , Quanchard Jean , Lott Nathaniel , Dalgard Clifton , Bornancin Frederic , Snow Andrew L
Published: January 2023

BENTA (B cell Expansion with NF-κB and T cell Anergy) disease is a rare, selective B cell lymphoproliferative disorder caused by gain-of-function (GOF) mutations in the lymphocyte scaffolding molecule CARD11. CARD11 is required for downstream activation of NF-κB following antigen receptor engagement, which normally triggers recruitment of BCL10 and MALT1. MALT1 serves both scaffolding and proteolytic functions that initiate and amplify canonical NF-κB signaling. It still remains unclear how continuous CARD11 signaling ultimately drives massive B cell accumulation in BENTA patients. Here we show that in contrast to normal human B cells, BENTA patient B cells exhibited a striking resistance to apoptosis in cell culture, particularly after stimulation. Enhanced survival of patient cells was dependent on MALT1 protease, which is activated constitutively by CARD11 GOF mutants. Treatment with a novel, specific low molecular weight inhibitor of MALT1 protease completely restored apoptosis sensitivity in BENTA patient B cells. RNA-Seq analyses revealed that MALT1 protease inhibition induced a specific subset of pro-apoptotic genes in activated patient B cells, and downregulated multiple genes associated with cell cycle progression and metabolism, in addition to several NF-κB target genes. Although not strictly required for CARD11-dependent NF-κB activation, our results imply that MALT1 protease function governs a pro-survival signaling program in BENTA B cells that likely contributes to the profound B cell lymphocytosis that distinguishes this disease. MALT1 protease thus represents an attractive therapeutic target for reducing B cell burden in these patients, thereby lowering the risk of B cell malignancy later in life.
Development and Characterization of V-PLEX® TH17 Cytokine. Assays
Authors: Yatsula Victoria , Kobayashi Lori , Sharma Deepak , Qualters Elisa , Janaki Lalitha , Weckerle Celine , Harkins Seth B. , Stewart David , Wohlstadter Jacob N.
Published: January 2023

Th17 cytokines are important mediators of the host defense against infection and are increasingly investigated for their role in autoimmune disorders and immune regulation at mucosal surfaces. Here we describe the development, characterization, and analytical validation of a multiplexed immunoassay panel for seven Th17 cytokines, IL-17A, IL-21, IL-22, IL-23, IL-27, IL-31, and MIP-3alpha, on MSD’s V-PLEX platform.

To efficiently and rapidly identify potential antibody pairs, biotinylated capture antibodies and detection antibodies conjugated with SULFO-TAG™ label were screened on MSD’s U-PLEX® platform, which enables the solution phase assembly of capture antibody arrays. Subsequent development used printed arrays of capture antibodies. Antibody concentrations, calibrator curve linearity, dynamic range, specificity, matrix tolerance, and assay robustness were analyzed for each assay during development. Calibration curves demonstrated a three-log dynamic range while achieving a lower limit of quantitation (LLOQ) of less than 1 pg/mL for most assays. Control samples for the assays had CVs of less than 10%. Dilution linearity and spike recovery studies in serum, plasma, urine, and cell culture media were conducted to demonstrate matrix compatibility. Spiked matrix samples were typically found to recover between 80%–120% of the expected value. Cross-reactivity was shown to be less than 0.3% between assays within the panel and less than 0.5% when panned against more than 30 other blood-related biomarkers.

These validated, multiplexed assays provide sensitive measurement of Th17 cytokines in a variety of matrices and can be used as part of a researcher’s pre-clinical and clinical studies.
MicroRNA-142 guards against autoimmunity by controlling Treg cell development and function
Authors: Wang Wei-Le , Ouyang Ching , Cassady Kaniel , Xiong Min , Reyes Estefany , Davis Alicia , Tang Kathie , Zeng Defu , Boldin Mark
Published: January 2023

Regulatory T (Treg) cells are critical in preventing aberrant immune responses. Post-transcriptional gene control by microRNA (miRNA), a large class of small regulatory RNA, has recently emerged as a key genetic element required for Treg cell function. Nevertheless, specific miRNA gene(s) that play a vital role in the regulation of Treg cell activity are still unknown. Here we report that mice with Treg cell-specific ablation of miR-142 (hereafter Foxp3CremiR-142fl/fl mice) developed a fatal systemic autoimmune disorder due to a breakdown in peripheral T cell tolerance. Foxp3CremiR-142fl/fl mice displayed a sharp decrease in abundance and suppressive capacity of Treg cells. Analysis of miR-142-deficient Treg cells revealed excessive IFNγ production and dysregulated IFNγ signaling. We have identified several of the derepressed IFNγ-related genes, including Ifngr2 receptor and Hif1a transcription factor, as direct miR-142 targets. Furthermore, lowering the Hif1a gene dose in Treg cells significantly diminished the hyperactivation of peripheral effector T cells in Foxp3CremiR-142fl/flmice. Thus, miR-142 is an indispensable regulator of Treg cell development and function that mediates its control by attenuating IFNg response.
Downregulation of allograft inflammatory factor-1 prevents B cell infiltration to cardiac tissue during the development of diabetic cardiomyopathy (DCM)
Authors: Sarkar Amrita , Shukla Sanket Kumar , Rafiq Khadija
Published: January 2023

An emerging concept is that inflammation might be linked to alteration of immune system, thus leading to adverse cardiac function, remodeling in DCM. However, the role of inflammatory pathways during development of DCM is poorly understood and controversial, as both positive/negative effects of inflammation have been observed. 8 weeks wild type male mice were injected with streptozotocin (50 mg/kg, 5 days intraperitoneally) and studied 8 weeks after induction of diabetes mellitus having elevated blood glucose levels (>300 mg/dL) and DCM was confirmed through ECG. We found a significant decrease in B cell infiltration into left ventricular heart, but not in other organs, during DCM. Interestingly, downregulation of B cell infiltration correlates with downregulation of a B cell inflammatory molecule expression, Allograft Inflammatory Factor-1 (AIF-1), which has been reported to enhance lymphocyte activation. Our in vitro study showed that B cell migration is regulated by AIF-1 expression. We observed significant migration of B cells to hyperglycemic AIF-1 transfected H9C2cells compared to control cells transfected with an empty vector. In vivo studies showed strong co-localization of AIF-1 and CD19 B-cell marker in nondiabetic heart tissue compared with diabetic heart tissue. Interestingly, AdAIF-1 overexpression promoted B cell migration into diabetic hearts, reduced inflammation and pathological remodeling. These data suggest that AIF-1 downregulation in cardiac myocytes is caused by diabetes that prevents B cell infiltration into hearts, and increase cardiac inflammation that leads to cardiomyocyte death and that overexpression of AIF-1 could be a novel treatment for this condition.
Novel in vitro model of Rich focus formation and mycobacterium infected cell migration at the blood brain barrier
Authors: Walter Fruzsina R. , Gilpin Trey E. , Mergaert Aisha , Sandor Matyas , Fabry Zsuzsanna
Published: January 2023

Tuberculous meningitis is the most lethal form of extrapulmonary tuberculosis with more than 100,000 new cases per year. Mycobacterium tuberculosis (Mtb) invades the central nervous system (CNS) in 1–5 % of all cases with a mechanism not well understood. Today’s knowledge is based on the findings of Rich and McCordock proposing that bacteria disseminate via the blood either within a carrier cell or extracellularly, enter the CNS through the blood-brain barrier (BBB) and form a tuberculoma (Rich focus) which is able to rupture causing meningitis. Previously our laboratory showed that the dissemination of Mycobacterium bovis (BCG) and Mtb from pulmonary granulomas is executed by dendritic cells. Currently no good in vitro model exists which is able to mimic the focus formation and the dissemination of Mtb to the brain. We are the first to assemble a mouse primary cell based in vitro Rich focus model. We show that cellular aggregates consisting of BCG or Mtb infected DCs derived from bone marrow of CD11c eYFP animals and peripheral blood mononuclear cells isolated from P25 T-cell receptor transgenic mice form within 60 minutes in cultures. These in vitro granulomas attach to the brain microvessel endothelial cells and facilitate migration of infected cells across the barrier. Aggregate formation and transmigration interfere with endothelial zonula occludens-1 junctional integrity and induce mitochondrial damage shown by Mitotracker staining. These data shed light on the mechanism how mycobacterium infected cells migrate across the BBB. Understanding the mechanism of Mtb dissemination into the CNS is crucial for developing novel therapeutic target inhibiting TB meningitis.
FMNL1 promotes T cell extravasation and trafficking to sites of inflammation
Authors: Thompson Scott Beckett , Long Robert A. , Chung Jeffrey W. , Estin Miriam L. , Jacobelli Jordan
Published: January 2023

To execute their effector function, activated T cells traffic through the blood stream to sites of inflammation. To enter tissues, T cells extravasate through the vascular endothelial cell wall by a process known as transendothelial migration (TEM). While many of the adhesion molecules and chemokine signaling pathways required for TEM have been previously characterized, little is known about how downstream cytoskeletal effectors mediate the mechanical forces and shape changes needed for T cell TEM. Formin-like 1 (FMNL1) is a terminal cytoskeletal effector directly involved in actin network remodeling. It has been associated with the formation of membrane protrusions that likely function in migration and cell-cell interactions, and is highly expressed by activated T cells. The goal of our study was to examine the role of FMNL1 in activated T cell extravasation and trafficking to sites of inflammation. While activated FMNL1 deficient T cells had normal migration in response to chemokine across 5μm pore transwell membranes, they displayed an impaired ability to migrate through more restrictive 3μm pores. In vitro time-lapse microscopy data suggest that FMNL1 deficient T cells have a reduced ability to complete TEM across brain endothelium. FMNL1 deficient T cells were also impaired in their ability to traffic to the inflamed central nervous system in vivo. Finally, adoptively transferred autoreactive FMNL1 deficient T cells were impaired in their ability to induce Experimental Autoimmune Encephalitis. Together these data suggest that FMNL1 is required for the efficient migration of activated T cells though restrictive endothelial barriers and may be a promising therapeutic target for regulating T cell trafficking in autoimmunity.
Role of Rho kinases in B cell development
Authors: Sambandam Arivazhagan , Caplazi Patrick , Wu xiumin , Jeet Surinder , Liu John , Zhang Juan , Lee Wyne , Pappu Rajita
Published: January 2023

Cytoskeletal rearrangement is a critical component to hematopoietic stem cell development and differentiation into mature leukocyte populations. A number of chemokines and biolipids have been shown to regulate these events via binding to G protein coupled receptors. Many of these receptors couple to the Ga12/13 family of G proteins to regulate actin reorganization via the RhoA small GTPase. The Rho-kinases, Rock1 and Rock2, are activated downstream of RhoA to induce cytoskeletal changes that regulate numerous cellular processes, including contraction, polarity, proliferation, motility, adhesion and viability. A role for RhoA in regulating hematopoiesis and subsequent leukocyte development has been established using genetic studies. However, a similar role for the Rock1 and Rock2 has not been demonstrated. As global deletion of either kinase causes embryonic lethality, we used conditional gene targeting to ablate either the Rock1 or Rock2 allele in adult mice. Rock1flox/flox or Rock2flox/flox mice were crossed to the Rosa26-Cre ERT2 line to globally delete the floxed alleles with tamoxifen. In contrast to deletion of RhoA, conditional deletion of either Rock1 or Rock2 was well-tolerated, with no aberrant effects on hematopoiesis. These results suggest functional redundancy between Rock1 and Rock2 in regulating RhoA activity in hematopoiesis. However, we observed a significant decrease in marginal zone B cell number and frequency in the spleen of mice lacking Rock1. We also observed a follicular B cell defect in the spleen of mice lacking Rock2. These results are consistent with the known roles of GPCRs in B cell migration/positioning, and suggest there are isoform specific functions of these kinases.
Distinct tissue specific functions for bone marrow regulatory T cells
Authors: Camacho Virginia , Wolock Sam , Tenen Danielle , Welner Robert
Published: January 2023

While the influence of T-regs has been explored in numerous contexts, their role within the bone marrow (BM) has not been elucidated. In this study, we establish that BM derived T-regs represent a specialized population with a tissue-specific role. Functional homing assays demonstrate that BM T-regs preferentially traffic back to this site, revealing that specific signaling cues recruit T-regs to this microenvironment. This is supported by RNA-seq data showing that the chemokine and cytokine receptor profiles of BM T-regs differ from T-regs in secondary lymphoid tissues. BM Tregs are also characterized by a higher expression of CD127/IL7Ra conventionally not expressed on peripheral T-regs. Depletion of Tregs via anti-CD25 treatment has widespread effects across several BM populations, especially the stem cell compartment. Loss of T-regs results in increased HSC cycling and proliferation, while fostering myeloid skewed differentiation. Key stem cell features including repopulation capacity and dormancy are also disrupted. Moreover, supportive niche populations are also altered by Treg depletion, as evidenced by increased cycling and proliferation of stromal cells. Non-genotoxic conditioning and transplantation, reveals that the T-reg depleted microenvironment is less supportive of HSCs, thus establishing a novel role for Tregs in maintaining niche function. Remarkably, the effects of Treg depletion are found to be longlasting. In the absence of continued treatment, cohorts sustain a myeloid bias, as well as increased numbers of HSCs and stromal cells. These observations support the conclusion that BM T-regs represent a unique subset that maintains numerous populations and have a specialized function within this tissue.
Maternal Lipid Regulation of the Development of Responsiveness to Allergen in Neonates and Infants.
Authors: Cook-Mills Joan M. , Walker Matthew
Published: January 2023

In animals and humans, offspring of allergic mothers have increased responsiveness to allergen. A major question is what are the maternal factors of allergic mothers that alter offspring responsiveness to allergen? We focused on maternal lipids, because lipids are altered during allergic inflammation and because lipids are transported to the fetal liver and are in the mother’s milk. In our studies, allergic pregnant mice had increased glucosylceramides (GlcCer) in the mother plasma, placenta, and fetal liver as compared to non-allergic mothers. In allergen-challenged pups of allergic mothers, GlcCer was elevated in the pup lung and liver. Maternal GlcCer is directly transported to the placenta, the fetal liver, mother breast tissue and the milk of the pup stomach. Administration of a pharmacolgical inhibitor of GlcCer synthase to allergic mothers reduced GlcCer to the levels of GlcCer in non-allergic mothers and blocked the development of allergic lung inflammation and generation of allergen-specific IgE in allergen-challenged pups of allergic mothers. We also demonstrated that the fetal liver and offspring of allergic mothers have increased numbers of resident dendritic cells (DCs) and that the administration of the GlcCer synthase inhibitor to the allergic mothers blocked the increase in these DCs in the offspring. In summary, allergic mothers had increased GlcCer that is transferred to offspring and mediates changes in offspring DCs and responsiveness to allergen. These data have a significant impact on our understanding of mechanisms of maternal lipid regulation of offspring development of allergies and has the potential to lead to novel interventions that significantly impact risk for allergic disease early in life.
IL-12 induced STAT4 activation plays a role in pro-inflammatory neutrophil functions
Authors: Keeter William Coles , Moriarty Alina , Butcher Matthew J. , Ma Kaiwen W. , Nadler Jerry L. , Galkina Elena
Published: January 2023

Signal transducer and activator of transcription 4 (STAT4) becomes activated via Jak/Tyk kinases in response to IL-12 and other cytokines, leading to expression of genes involved in pro-inflammatory pathways. STAT4 has been well characterized in T lymphocytes and NK cells, yet less is known of its potential role in neutrophils. Neutrophils have recently been shown to play a contributing role in atherogenesis. Our published data demonstrate that global deficiency of STAT4 results in decreased atherogenesis in atherosclerotic ApoE−/− mice. However, the cell specific role of STAT4 in atherosclerosis is unknown. Western blot analysis revealed STAT4 expression and IL-12 induced phospho-STAT4 in isolated neutrophils with no distinguishable difference between blood and bone marrow neutrophils. IL-12 dependent activation of wild-type (WT) neutrophils induced an increase in reactive oxygen species (ROS) production when compared to non-stimulated WT neutrophils in both blood and bone marrow. Similarly, IL-12 stimulated WT neutrophils showed an increase in neutrophil extracellular trap (NET) elastase activity versus unstimulated controls. IL-12 stimulated neutrophils from Stat4−/− mice displayed no changes in NET elastase activity when compared to non-stimulated STAT4 deficient neutrophils suggesting STAT4-dependent NETosis. Furthermore, IL-12 stimulated Stat4−/− neutrophils showed decreases in both ROS production and NET elastase activity when compared to IL-12 stimulated WT neutrophils. Our data suggest a novel pro-inflammatory, IL-12 dependent role of STAT4 in neutrophils. Inhibition of these processes by targeting the IL-12/STAT4 pathway may serve as a possible therapeutic approach to treating atherosclerosis.
A novel multicolor stimulated emission depletion (STED) microscopy strategy to visualize mitochondrial morphology in lymphocytes
Authors: Roesler Alexander Scott , Miozzo Pietro , Smelkinson Margery , Akkaya Billur , Brzostowski Joseph , Kabat Juraj , Theall Brandon P. , Traba Javier , Pierce Susan K. , Akkaya Munir
Published: January 2023

Immunometabolism is an emerging field that studies the activation-induced reprogramming required to alter immune cell function and meet new energetic demands. In-depth investigations of the metabolism-related cellular changes necessitate high-resolution visualization of mitochondria. However, current strategies for imaging mitochondria are optimized for model cells with high cytoplasm-to-nucleus ratios and are not readily adaptable for many immune cells. Here, we devised a multicolor STED microscopy method to assess B lymphocyte mitochondria in resting and activated states. By staining specimens with TOM-20 and MitoTracker dyes, as well as DAPI and a viability dye, our strategy allowed us to assess the morphology and volumes of mitochondria while efficiently excluding dead cells. Our analyses showed that stimulation of B lymphocytes via toll-like receptor 9 (TLR9) and/or B cell receptor (BCR) led to notable mass and volume increases in individual mitochondria within 24 hours. Furthermore, TLR9-stimulated cells demonstrated more tubular and connected mitochondrial networks consistent with their higher metabolic demands. In addition, collecting images of the same specimens side-by-side on AiryScan and standard confocal laser microscopes showed that STED microscopy performed significantly better in resolving changes in 3D mitochondrial morphology. However, none of the light microscopy imaging strategies could resolve activation-induced structural alterations in cristae or mitochondrial matrix. In conclusion, our novel STED-based multicolor imaging approach offers a powerful super-resolution tool to study immunometabolism and mitochondrial reorganization in lymphocytes at the whole cell level.
T lymphocyte immunophenotyping: 13-color flow cytometry panel design using the NovoCyte flow cytometer
Authors: Jachimowicz Lauren A. , Ye Peifang , Guenther Garret , Abassi Yama
Published: January 2023

The human immune system is highly complex and immune status is associated with disease status, treatment efficiency, and response to external stimuli such as vaccines. For example, the immunophenotyping of leukemia has become indispensable for diagnosis of hematological malignancies. Monitoring T lymphocytes has gained diagnostic importance in the prognosis and prediction of therapies. In this study, a series of T-cell markers previously used in the HIPC (Human Immunology Phenotyping Consortium) project were assessed. Through comprehensive consideration and analysis of the expression of each marker, fluorescence intensity, spectral overlap, and NovoCyte configuration, a 13-color T-cell phenotyping panel was designed. Next, peripheral blood mononuclear cells (PBMCs) were tested on a NovoCyte. Live T cells (CD3+/Aqua−) were identified as either T helper cells (Th) or cytotoxic T cells (Tc) cells by expression of CD4 and CD8 antigens. Next, further T cell subtype was determined by CCR7 and CD45RA: naïve (CD45RA+/CCR7+), effector (TEF, CD45RA+/CCR7−), effector memory (TEM, CD45RA−/CCR7−) and central memory (TCM, CD45RA−/CCR7+). Activation of T cells was assessed by the activation marker HLA-DR. Identification of Th subtypes (Th1, Th2 and Th17) was determined by CXCR3 and CCR6. Regulatory T cells (Treg) were identified as CD4+/CD25+/CD127lo/CCR4+, and memory and naïve Treg cells were further differentiated by CD45RO. With this panel, the phenotype and activation of T cell subtypes can be determined in one run which allows quickly and in-depth analysis of human T lymphocyte subsets. NovoCyte flow cytometer features high resolution of dim signals and flexible configuration and is particularly suitable for multicolor analysis.
RTA 1701 is an oral RORγt inhibitor that suppresses the IL-17A response in non-human primates
Authors: Reisman Scott A. , Lee Chun-Yue I. , Proksch Joel W. , Sakamoto Mitsumasa , Ward Keith W.
Published: January 2023

RTA 1701 is an orally-bioavailable, selective RORγt inhibitor. RORγt orchestrates the differentiation of Th17 cells and contributes to autoimmune disease pathogenesis. Inhibition of RORγt suppresses Th17 cells and the associated secretion of the effector pro-inflammatory cytokine IL-17A. As such, RTA 1701 is highly efficacious in rodent models of rheumatoid arthritis and multiple sclerosis. This study was conducted to evaluate the pharmacokinetics of RTA 1701 and its effects on ex vivo stimulation of IL-17A production in whole blood after oral administration to naïve healthy cynomolgus monkeys. Monkeys received a single administration of RTA 1701 (0.3, 3, 30, or 300 mg/kg). After a one-week washout, monkeys received the same doses of RTA 1701 once daily for 14 consecutive days. Blood was collected after a single dose and after 14 days of dosing for analysis of plasma RTA 1701 concentrations through 72 hours post-dose and for evaluation of RTA 1701 effects on ex vivo stimulation of IL-17A at 24, 30, and 72 hours post-dose. RTA 1701 exhibited oral bioavailability in monkeys with dose-dependent increases in exposure over the wide range of doses evaluated. Further, oral administration produced systemic exposure to RTA 1701 at sufficient levels to produce significant and dose-dependent suppression of ex vivo stimulation of IL-17A secretion in whole blood at 24 and 30 hours after single and repeat dosing, with IL-17A levels returning to baseline within 72 hours after a single dose. Collectively, these data demonstrate that RTA 1701 is orally bioavailable in non-human primates and significantly suppresses the IL-17A response. These data also support the future clinical development of RTA 1701 for the potential treatment of autoimmune diseases.
Targeting Dendritic Cells with Alpha 1 Antitrypsin has Therapeutic Potentials in Lupus
Authors: Elshikha Ahmed Samir , Yuan Ye , Lu Yuanqing , Chen Mong-Jen , Akbar Mohammad A. , Plate Henrike , Zeumer Leilani , Morel Laurence , Song Sihong
Published: January 2023

Increasing evidence indicate that dendritic cells (DCs) play a critical role in the development and pathogenesis of systemic lupus erythematosus (SLE). Targeting DCs in lupus may be beneficial, but it has not been yet achieved effectively. Alpha 1 antitrypsin (AAT) is a multifunctional protein with anti-inflammatory activity and immunoregulatory functions. In this study, we tested the effects of AAT on DCs and B cells in vitro and its therapeutic potential in vivo in two spontaneous mouse models of lupus. We demonstrated that AAT treatment significantly inhibited DC maturation, induced from C57BL/6 or B6.Sle1.Sle2.Sle3 lupus-prone mice, (using MHC-II IA, CD80, CD86 and CD40 as markers) and function (IFN-I, IL-1β, TNF-α, and IL-12 secretion) in response to TLR4 and TLR9 stimulation. Interestingly, the inhibitory effect of AAT on DCs inhibited B cell proliferation and antibody production induced in DC – B cell co-cultures. Importantly, in vivo AAT treatment prevented the development of kidney disease in MRL/lpr mice and AAT treatment delivered with an AAV-gene therapy vector extended the life span of NZM2410 mice, with 70% mice that received AAT remaining alive at 72 weeks of age vs. all control animals died by 46 weeks of age. In all studies, AAT treatment significantly reduced lupus-associated autoantibody levels, including anti-dsDNA IgG and anti-nuclear autoantibodies. Considering the well-known safety profile of AAT, our results strongly suggest a therapeutic potential of AAT for the treatment of SLE in humans.

This work was supported by grants from University of Florida Research Foundation and Grifols Inc.
Induction of Haploidentical Mixed Chimerism Cures Autoimmune Type 1 Diabetes
Authors: Liu Yuqing , Zhang Mingfeng , Riggs Arthur , Zhang Xi , Zeng Defu
Published: January 2023

Type 1 diabetes is an autoimmune disease in association with particular MHC, and induction of MHC-matched mixed chimerism cannot reverse autoimmunity. Haploidentical HCT has gradually become a common practice for treating non-malignant diseases, but it remains unknown whether induction of haploidentical mixed chimerism can reverse autoimmunity.

In the current studies, we induced mixed chimerism in NOD mice (H2-Ag7) by transplanting bone marrow and CD4+ T-depleted spleen cells from haploidentical (B6 × B6g7) F1 donors (H2-Ab/g7). We found that haploidentical mixed chimerism eliminated insulitis as well as prevented in pre-diabetic and cured diabetes in new-onset WT NOD mice. Interestingly, although I-As was thought to be autoimmune susceptible, induction of haploidentical mixed chimerism with I-Ag7/s transplants also effectively eliminate insulitis. Furthermore, the mixed chimerism markedly reduced the percentage of host-type but not donor-type CD4+CD8+ thymocytes and increased CD11c+CD8−SIRPα+CD11b+ migratory DC that can mediate thymic negative selection in the chimeric thymus. In the peripheral, percentage of tetramer+ autoreactive host-type Tcon cells was markedly reduced and part of the residual T cells became PD-1hiIL-7Rαlo anergic/exhausted, while percentage of host-type CD4+Foxp3+ Treg cells significantly increased. Additionally, in the pancreas of mixed chimeric BDC2.5 NOD mice, Treg cells among tetramer+ autoreactive T cells increase 5–10 fold. Taken collectively, haploidentical mixed chimerism reverses autoimmunity and eliminating insulitis in NOD mice by enhancing DC mediated negative selection in thymus and augmenting expansion of Treg, especially antigen specific Treg cells, in the periphery.