bluuebunny/crossref_metadata_2025 huggingface dataset:
MHC class II tetramers identify apolipoprotein B-specific CD4 T cells in mice and humans
Authors: Kimura Takayuki , Tse Kevin , Miller Jacqueline , McArdle Sara , Vassallo Melanie , Wolf Dennis , Baas Livia , Kobiyama Koji , Jenkins Marc K , James Eddie A , Kwok William , Hanna David B , Kaplan Robert C , Strickler Howard , Sidney John , Sette Alessandro , Ley Klaus
Published: January 2023

Immunization with atherosclerotic plaque antigens including MHC-II-restricted peptides from apolipoprotein B (ApoB, core protein of low density lipoprotein, LDL) is known to be atheroprotective in animal models. Herein we report a human ApoB peptide whose sequence is identical to mouse ApoB and which binds to both mouse (I-Ab) and human MHC class II. Immunization with this peptide reduced atherosclerotic plaque in the aortas of Apoe−/− mice fed with a Western diet and induced IL-10 producing CD25+FoxP3+ regulatory T cells (Tregs). After peptide restimulation in vivo, responding CD4+ T cells (by Nur77-GFP) were highly enriched in CD25+FoxP3+ Tregs. Using a novel I-Ab tetramer reagent, we further showed that peptide-specific CD4+ T cells expand upon vaccination, ~50% express FoxP3, and were 10-fold enriched for IL-10 producers. In human peripheral blood mononuclear cells (PBMCs) from individuals assessed for subclinical cardiovascular disease, ApoB-specific CD4+ T cells were detected using novel HLA-DR1/ApoB peptide tetramers. Peptide-specific CD4+ T cells from donors with carotid artery plaques showed more RORγt expression and less FoxP3 expression compared to those without. This is the first demonstration of ApoB-specific CD4+ T cells by tetramer in humans and the first animal model evidence of atheroprotective vaccination with a human ApoB peptide.
Chlamydia Nanovaccine Formulated with Recombinant MOMP Encapsulated in PLGA 85/15 Nanoparticles Enhances IFN-γ Production with Activation of Effector (CD44+/CD62L−) and Memory (CD44+/CD62L+) CD4+ T Cells in Balb/c Mice
Authors: Sahu Rajnish , Dixit Saurabh , Duncan Skyla , Singh Shree R , Dennis Vida
Published: January 2023

Chlamydia trachomatis remains the most commonly reported bacterial sexually-transmitted diseases, causing considerable morbidity and socioeconomic burden worldwide. To reduce its occurrence and global economic burden, vaccine development to provide protective immunity against the pathogen has been ineffective for many years due to its complex immunological mechanisms. Modern methods to develop vaccines by combining antigens with biocompatible and biodegradable polymers have emerged as attractive therapeutics. In the present study, we formulated a nanovaccine employing PLGA [poly (D, L-lactic-co-glycolic acid) 85/15 nanoparticles encapsulated with recombinant MOMP (major outer membrane protein) of Chlamydia, and investigated its immunogenicity in Balb/c mice. Mice were immunized via the intranasal and subcutaneous routes three times at two-week intervals. Immunomagnetic purified T cells from spleens were co-cultured with mitomycin-C treated splenocytes as antigen presenting cells to assess T-cell activation and proliferation, effector and memory T cells, and cytokines production. Our flow cytometry data showed an increase in effector (CD44+/CD62L−) and memory (CD44+/CD62L+) T cells specific to the CD4+ subset. T-cells re-stimulated with rMOMP produced enhanced IFN-g, which is required for protection against Chlamydia. IL-2 production was increased and correlated with a CFSE+ proliferating CD4+ T-cell population. Our data suggests that mice immunized with our Chlamydia nanovaccine triggers T-cell activation, antigen-specific effector and memory CD4+ T cells and IFN-γ production, which are required for protection against this mucosal pathogen.
A FUNCTIONAL METAGENOMIC APPROACH OF HUMAN MICROBIOTA IDENTIFIES A BACTERIAL STRAIN THAT CONFERS PROTECTION DURING INTESTINAL INFLAMMATION VIA IL-22
Authors: Guglietta Silvia , Nepelska Malgorzata , Lapaque Nicolas , Bardeli Glenda , Cultrone Annalisa , Doré Joel , Blottière Hervé , Rescigno Maria
Published: January 2023

The gastrointestinal microbiota has been shown to play an important role in resistance to pathogen invasion and in the regulation of the host metabolism. Further, it is widely recognized that the microbiota also influences the development and homeostasis of the immune system. Alteration in the microbial flora, so called dysbiosis, has been linked to many human diseases, including autoimmune and/or autoinflammatory disorders and allergies. Despite the advances in the DNA sequencing technology, a deeper understanding of the gut microbiota is still hindered by the inability to culture most of the bacterial species.

In this study, we used a high throughput cell-based functional metagenomic approach to identify several bioactive bacterial clones in the human fecal microbiota. In subsequent co-culture experiments we selected one clone, related to the phylum of Firmicutes, which indirectly activated dendritic cells through the stimulation of intestinal epithelial cells and subsequently modulated T cell functions. This effect was associated to an ABC transporter. In ex-vivo human organ culture experiments we found that this clone prevented Salmonella-induced destruction of the intestinal mucosa by stimulating the production of IL-22 in the CD4+ T cells. These results were confirmed in vivo by using a mouse model of DSS-induced colitis. Here pre-treatment of the mice with the identified clone was able to significantly reduce intestinal inflammation. This protective effect was abrogated by the administration of anti-IL22 antibody or the use of AHR antagonist.

Altogether, our functional metagenomic approach allowed the identification of bacteria and bacteria-induced genes modulating the immune system.
PGE2 controls Th17 and regulatory T cell function during intestinal immune responses
Authors: Maseda Damian , Johnson Elizabeth M , Kirk Leslie , Aronoff David M , Crofford Leslie J
Published: January 2023

Eicosanoids, particularly prostaglandin E2 (PGE2), are critical molecules in the initiation and resolution of inflammation during immune responses. PGE2 is associated with modulating autoimmunity through altering the IL-23/IL-17 axis and regulatory T cells, whose balance is critical for controlling pathogenic T cells. The inducible microsomal prostaglandin E synthase 1 (mPGES1) is a membrane enzyme that regulates local PGE2 levels and is highly expressed at sites of inflammation. Hematopoietic and stromal cells express mPGES1, but its role in T cell function and intestinal biology remains underappreciated. Th17 polarized cells produce 10-fold more PGE2 than Th1 polarized cells, and this PGE2 production is mPGES1-dependent. Interestingly, high concentrations of PGE2 can shift the production from IL-17A to IFNg during Th17 polarization, and PGE2 promotes more cell proliferation and cytokine production in Th1 than in Th17 cells. Sensing of PGE2 at physiological concentrations through its EP4 receptor proved to be critical to inhibit the generation of iTregs but not Th1 or Th17 cells. In the colon, we observed that different inflammatory insults induce different PGE2 production levels, with concomitant modulation of T cell responses in the mesenteric lymph nodes. Furthermore, mPGES1 displayed opposite effects on pathogenic and regulatory T cells in the T-cell transfer colitis model, with mPGES1 deficiency limiting intestinal damage driven by effector T cells, but enhancing de novo generation of Tregs and increasing colonic lamina propria homing of CD4+FoxP3+ cells. Taken together, these data demonstrate the pleiotropic and context-dependent actions of mPGES1 and PGE2 in intestinal inflammation and immunity.
Microbiota-dependent IL-21 signaling regulates intestinal immune cell homeostasis and immunopathology to infection
Authors: Cho Hyeseon , Spolski Rosanne , Kang Byunghyun , Leonard Warren J , Kelsall Brian L
Published: January 2023

Despite studies indicating a role for IL-21 in intestinal inflammation, how it precisely affects intestinal homeostasis and immunity to infection is not yet clear. In this study, we report a potent effect of commensal microbiota on the phenotypic manifestations of IL-21 receptor deficiency. IL-21 is expressed highly by CD4 T cells of Peyer’s patches (PPs) and small intestine lamina propria (LP) and strongly induced by co-housing with SFB-positive mice. Mice deficient in IL-21 receptor exhibit reduced numbers of GC B cells, B cell expression of AID, and IgA+ B cell populations in PPs, consistent with the known roles for IL-21 in B cell function. Consequently, IL-21R KO mice show a significant reduction in IgA+ plasmablasts and plasma cells in the small intestine LP. Interestingly, microbiota-dependent increases in RORgt+ T cells and Treg cells are observed in the small intestine of IL21-R KO mice. Neither the Th1 nor RORgt+ ILC populations are altered in the KO mice intestine compared to WT mice. Demonstrating a critical role of IL-21 signaling in immunopathology during Citrobacter rodentium infection, IL-21 receptor deficiency leads to strikingly reduced tissue pathology without affecting bacterial clearance. This reduced immunopathology likely results from dampened production of IFNg, IL-12, and IL-1b that promote severe immunopathology/lethality. Taken together, we demonstrate the regional and pleotropic effects of IL-21 signaling that fine-tunes intestinal mucosal immunity in a microbiota-dependent manner, which has significant implication for anti-IL-21 therapy to treat inflammatory bowel disease.
Innate lymphoid cells and colitis in leukocyte adhesion deficiency
Authors: Wang Baomei , Li Xiaofei , Kajikawa Tetsuhiro , Moutsopoulos Niki M , Chavakis Triantafyllos , Hajishengallis George
Published: January 2023

Leukocyte Adhesion Deficiency type I (LAD-I) is a primary immune deficiency caused by mutations in the CD18 subunit of β2 integrins. Affected individuals suffer from recurrent mucocutaneous infections and pathologic inflammation in certain mucosal barriers, including the periodontium, skin and the colon. Our laboratory has recently dissected the mechanistic basis of LAD-I–associated periodontitis but the mechanisms underlying LAD-I– associated colitis are yet to be defined. To answer this question, we utilized as model the CD18−/− mice after demonstrated that CD18 deficiency renders mice highly susceptible to Citrobacter rodentium-induced colitis, a widely used model of human colitis caused by enteropathogenic and enterohaemorrhagic Escherichia coli. Strikingly, we found that CD18−/− mice displayed significantly reduced IL-22 production and IL-22 producing Group 3 innate lymphoid cells (ILC3s). Therapeutic delivery of recombinant IL-22 (rIL-22) protected CD18−/− mice from C. rodentium–induced mortality. Furthermore, adoptive transfer of CD18+/+ monocytes partially restored IL-22 production from ILC3s and extended the survival of CD18−/− mice post C. rodentium infection. This is the first demonstration that ILC3-dependent expression of IL-22 is under β2 integrin regulation. In conclusion, these data not only identified a novel role of β2 integrin in mediating the crosstalk between intestinal macrophage and ILC3 but also can potentially lead to novel treatment of LAD-associated colitis.
The relationship between the cytokines TNF and TL1A in rheumatoid arthritis
Authors: Farley Taylor K , Richoz Nathan , Lamberth Sarah , Gregersen Peter , Cominelli Fabio , Meylan Francoise , Siegel Richard M.
Published: January 2023

The TNF-family cytokine TL1A (TNF-like ligand 1A) signaling through its receptor DR3 (Death Receptor 3) promotes immunopathology in diverse autoimmune disease models by facilitating T cell accumulation and Innate Lymphoid Cell activation at the site of inflammation while leaving systemic immune responses unaffected. TNF, a close homolog of TL1A, is a successful therapeutic in autoimmune diseases including rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). However, a significant fraction of patients do not adequately respond to TNF blockade and are in need of alternative therapies. Previous studies have reported elevated TL1A levels in serum and synovial fluid of patients with RA. Yet, how TL1A expression may be influenced by TNF, and whether blocking TL1A/DR3 interactions would be a viable treatment for RA is unknown. We found that soluble TL1A levels are specifically elevated in synovial fluid and serum from RA patients, and that TNF blockade reduces serum TL1A levels in both responders and non-responders. Additionally, in a murine model of collagen-induced arthritis, antibody blockade of TL1A reduced both clinical disease and bone destruction. To further understand the relationship between TL1A and TNF we are now utilizing the TNFDARE mouse model of spontaneous TNF driven arthritis crossed to DR3 deficient mice. By 3D micro CT imaging and cytokine analysis of the joints we will determine if the blockade of TL1A/DR3 interactions alleviates arthritis driven by excess TNF. Taken together, these studies support an investigation into TL1A blockade as a potential therapy for patients with rheumatoid arthritis.
Inhibition of BAFF binding to its receptor, BR3, by low molecular weight compounds results in suppression of activation of monocytes and B cells in vitro and in vivo model of autoimmune diseases.
Authors: Yoshimoto Keiko , Suzuki Katsuya , Sugahara Kunio , Takeuchi Tsutomu
Published: January 2023

Background and Purpose

We have demonstrated that the elevated expression of BAFF receptor (BR3) on monocytes is involved in the pathogenesis of primary Sjogren’s syndrome and BR3 is a therapeutic target to treat pSS. In our previous study, we have discovered two pyrrolopryimidine derivatives, BIK12 and BIK13, which inhibit binding of BAFF to BR3, using our original high-throughput screening system. In this study, we investigated inhibitory effects of these compounds on BAFF signaling pathways in monocytes and B cells in vitro. In addition, we analyzed the effects of the compounds on serum level of an anti-dsDNA antibody in autoimmune disease model mice.

Methods

Peripheral monocytes were stimulated with soluble BAFF (sBAFF) and cultured in vitro with or without peripheral B cells in the presence of BIK-12 or BIK-13. IL-6 production by monocytes and IgG production by B cells were measured by ELISA. MRL/lpr and NZBWF1 mice were treated with these compounds three times a week at a dose of 1.0 or 0.2 mg/kg for 6 months. The titer of an anti-ds DNA antibody was measured by ELISA.

Results and Discussion

sBAFF-induced IL-6 production by peripheral monocytes and IgG production by B cells co-cultured with sBAFF-stimulated peripheral monocytes were significantly suppressed by BIK12 and BIK13 in a dose dependent manner. Interestingly, the titer of an anti-dsDNA antibody in both MRL/lpr and NZBWF1 mice received BIK13 was significantly lower than control mice after 16 weeks’ treatment, indicating that the compound was also efficacious in vivo.
Difference between Immature Dendritic Cells (imDCs) and Mature Dendritic Cells (mDCs) Derived from Human Monocytes
Authors: Nagai Taro
Published: January 2023

We induced imDCs and mDCs from human monocytes. Then we examined their surface markers and cytokine secretion to compare with their functions.

Surface Markers

Monocytes marker CD14 still expressed on imDCs and disappeared on mDCs. However, DC marker CD209 expressed on both imDCs and mDCs as same amount. Co-stimulatory molecules, such as CD40, CD80, CD86, and HLA Class II, expressed on mDCs more than imDCs.

So, we expected that imDCs cannot induce T cell proliferation and differentiation because

Cytokine Secretion

We stimulated imDCs and mDCs by CD40L transfected L cells with or without several kinds of cytokine for 2days and measured cytokine secretion from them by ELISA.

mDCs secreted more IL-27 than imDCs, however IL-6, IL-12p70, and IL-23 secretion from them were almost same. Effects of adding cytokines on stimulation were almost same as on imDCs and mDCs. IFN-γ enhanced IL-12p70 secretion, however almost no effects against IL-6, IL-23, and IL-27 secretion. IL-4 and IL-13, which induce Th2 differentiation, had almost no effect against IL-6 and IL-27 secretion and suppressed secretions of IL-23 which induce Th17 differentiation, however enhanced secretion of IL-12 p70 which induce Th1 differentiation.

These results suggest that even though imDC may be able to induce T cell proliferation and differentiation because they secrete enough amount of cytokines for it.

Next, we would like to co-culture DCs and naive Th cells to confirm whether imDCs can induce T cell proliferation and differentiation or not.
RUNX1 regulates KIT expression and is a novel therapeutic target in human mast-cell diseases.
Authors: TOKUMASU MAYU , KATAOKA TATSUKI R. , NOGUCHI YUKI , MORITA KEN , SUGIYAMA HIROSHI , KAMIKUBO YASUHIKO , ADACHI SOUICHI
Published: January 2023

Mast cells develop from hematopoietic progenitors under SCF-dependent activation of KIT encoding a type III receptor tyrosine kinase. In addition, KIT signaling also enhances release of chemical mediators from stimulated mast cells. KIT D816V mutation, a gain-of-function mutation which is often found in mastocytosis, causes SCF-independent activation of KIT, leading to autonomous mast cell survival and proliferation. ChIP-seq analysis of mouse bone marrow-derived mast cells showed that hematopoietic transcription factor RUNX1 bound to several genomic regions between KIT exon 1 and intron 1. We therefore investigated an association between RUNX1 and the transcriptional regulation of KIT in the human mast cell lines. The global gene expression profile revealed that KIT gene expression in the cells was positively linked to RUNX1 gene expression. Using ChIP assay coupled to PCR, we found RUNX1 binding at KIT intron 1 in the cells with KIT D816V mutation. RUNX1 knockdown reduced KIT expression in both cell lines with and without KIT D816V mutation, and led to a significant decrease in cell growth of those with D816V mutation. In the RUNX1-knockdown cells, PI3K-Akt pathway of KIT downstream signaling was suppressed, and G0/G1 cell-cycle arrest occurred, which was followed by apoptosis. The chlorambucil-conjugated compound that we developed to inhibit RUNX1 binding to its DNA targets successfully and effectively decreased the KIT expression of the mast cells with KIT D816V mutation, which resulted in the cell growth suppression. These findings suggested that RUNX1 upregulates KIT expression and may be a potential therapeutic target in mast-cell diseases.
Extensive genetic diversity of CD14 promoter gene (rs2569190) polymorphism is associated with regulation of Plasmodium falciparum parasitemia but not susceptibility to infection
Authors: Thomas Bolaji , Snyder Tara , Aziz Najihah , Falade Catherine , Ojurongbe Olusola
Published: January 2023

CD14 is a multifunctional receptor expressed on many cell types, and has been shown to mediate immune response resulting in the activation of an inflammatory cascade. Genetic polymorphisms of the CD14 promoter (rs2569190) has been found to be associated with susceptibility to several diseases. In malaria infection, CD14 gene demonstrates a pathogenic profile in regulating experimental cerebral malaria, with reports of elevated levels of soluble CD14 in serum of malaria patients, with no definitive conclusions. We present a detailed analysis of genetic diversity of CD14 promoter gene polymorphism between a malaria-infected group and uninfected controls, and its association with clinical parameters of disease. Genomic DNA samples obtained from 107 Plasmodium falciparum-infected (microscopy and PCR-confirmed) patients and 380 uninfected controls were subjected to a PCR-RFLP analysis. Our results show a significant diversity (p=3.32E-06) in the genotypic frequency (3.8% versus 22.4%) of the rs2569190 mutant variant between infected and uninfected controls respectively, with the mutant allele less frequent in infected group. Mean parasitemia (parasites/μl of blood) was significantly regulated based on CD14 genetic profile (19,855 vs 37,041 vs 49,396 for mutants, heterozygotes and wild type alleles respectively), but showed no association with other markers of disease severity (fever, age of patients or anemia). Our results show the mutant variant pushed Th1 cell differentiation resulting in interferon-γ response leading to reduced parasitemia. How this affects disease severity between sub-regional or continental malaria groups deserves elucidation, including extending this results among cerebral malaria patients.
Interleukin-6 receptor α signaling on CD4+ T cells drives RBC alloantibody generation and T follicular helper cell differentiation in a murine model of RBC alloimmunization.
Authors: Arneja Abhinav , Salazar Juan E. , Jiang Wenyu , Hendrickson Jeanne E. , Zimring James C. , Luckey Chance John
Published: January 2023

Alloimmunization to non-ABO red blood cell (RBC) antigens remains a significant clinical problem in transfusion medicine. Presence of these alloantibodies can lead to delayed hemolytic transfusion reactions resulting in significant morbidity and occasional mortality upon antigenic re-exposure. For patients that generate multiple alloantibodies, provision of compatible antigen-negative RBCs can be time and resource intensive. In rare cases, it can result in the inability to locate and provide a life-saving therapy. Despite the significant clinical consequences of RBC alloimmunization, our understanding of the fundamental molecular and cellular mechanisms regulating anti-RBC antibody generation is limited. We have combined a recently developed mouse model for transfusion mediated RBC alloimmunization with mice genetically deficient in interleukin-6 receptor (IL-6Rα) to investigate the role of IL-6Rα signaling in regulating RBC alloimmunization. We have demonstrated that IL-6Rα signaling plays a key role in the generation of anti-RBC alloantibodies in response to transfused RBCs. Additionally, we have shown that RBC alloimmunization requires IL-6Rα signaling specifically on T cells, and that these signals are required for maximal in vivo differentiation of naïve anti-RBC specific CD4+ T cells into T follicular helper (TFH) cells. We have identified for the first time a specific molecule (IL-6Rα), its downstream cellular target (CD4+ T cells) and resultant functional outcome (TFH differentiation) that regulates RBC alloimmunization. Collectively, our findings support the idea that currently available biologics targeting IL-6Rα could be a viable therapeutic option for the prevention of RBC alloimmunization.
Dynamic cytokine gene expression characterized by increased IL-1 and IL-27 but decreased IL-8 occurs during development of bovine neonatal immunity.
Authors: Kaushik Azad Kumar , Pasman Yfke , Merico Daniele
Published: January 2023

We analyzed 18 RNA-seq libraries from 3 bovine calves at day 0, 7, 14 and 28, together with their dams (day 0 and 7) for cytokine gene expression. Expression of IL7, required for B- and T-cell development, significantly increases from day 0 to day 28 post-birth (LogFC 1.98, FDR 0.0054). By contrast IL7R gene expression significantly declined from day 0 to day 7 (LogFC −1.22, FDR 6.1E-08). The IL21 was expressed at low levels but IL21R gene expression increased day 0 vs day 7 (LogFC 1.17, FDR 0.000140). IL12B expression, a subunit of IL-12, increased from day 0 vs day 7 (LogFC1.14, FDR 0.016) and then declined. In parallel, IL12RB1 expression increased from day 0 to day 7 to achieve adult levels (LogFC1.31, FDR 5.5E-10). By contrast, IL12RB2 declined significantly within 7 days post-birth (LogFC −1.46, FDR 3.1E-05). Both IL12RB1 and IL12RB2 constitute IL12 receptor complex involved in T cell differentiation. The IL27 was expressed at low levels at birth but reached adult levels by day 7 (LogFC 1,66, FDR 0.006). The IL1B expression increased post-birth by day 7 (LogFC 1.44, FDR 0.0083) whereas IL1R2 gene was highly expressed at birth but declined by day 7 (LogFC −3.22, FDR 5.9E-34). Expression of chemokine IL8 that encodes CXCL8 declined day 0 vs day 7 post-birth (LogFC −1.13, FDR 0.00015). The pro-inflammatory cytokine TNFα gene expression increased by day 14 (LogFC 1.04, FDR 0.015). While various cytokines are expressed in a dynamic manner, an increase in IL-1B, IL-7, IL-12B and a decrease in IL-8 appears to be of functional significance in the development of neonatal immunity. These observations are in contrast to other species such as humans. [Supported by NSERC Discovery Grant]
Mechanisms that diversify an otherwise intrinsically processive Trav-Traj recombination program
Authors: Carico Zachary Mark , Zhang Baojun , Choudhury Kingshuk , Zhuang Yuan , Krangel Michael S.
Published: January 2023

In DP thymocytes, the Tcra locus undergoes multiple rounds of V-J rearrangement in order to generate a functional TCRα chain. While Traj segment usage is strictly ordered from 5′ to 3′ as rearrangement proceeds, it is unclear to what degree Trav usage is also ordered. To test this, we assessed the Trav-Traj combinatorial repertoire in murine DP thymocytes using 5′ RACE followed by paired end sequencing. In 129Sv/J WT thymocytes, we found a clear bias toward proximal-proximal and distal-distal joins, but Trav-Traj pairings were highly diverse, even in primary V-J rearrangement. However, combinatorial diversity was highly constrained in HYα KI thymocytes, in which a fixed primary V-J rearrangement in the endogenous Tcra locus is the starting point for secondary rearrangements. Thus, secondary rearrangement is highly processive through the V and J arrays, but surprising diversity is introduced by primary rearrangement. Tcrd rearrangement precedes Tcra rearrangement in developing thymocytes. In INT1-2KO mice, CTCF site deletion biases Trdv usage toward the most proximal gene segments. These mice, like HYα KI, displayed a substantially constrained Tcra repertoire, with limited diversity of primary rearrangements. Thus, variable truncation of the Trav array by prior Tcrd rearrangement creates diversity in an otherwise highly processive Tcra rearrangement program by imparting combinatorial diversity to primary Tcra rearrangement. This diversification is functionally important, as Trav1-Traj33 pairing and MAIT cell abundance are substantially reduced in HYα and INT1-2KO mice.
E proteins are important for restraining the ILC2 differentiation potential in the thymus
Authors: Qian Liangyue Miranda , Wang Hong-Cheng , Zhao Ying , Mengarreli Joni , Adrianto Indra , Gray-Montgomery Courtney , Urban Joseph F. , Fung Kar-Ming , Sun Xiao-Hong
Published: January 2023

Innate lymphoid cells (ILCs) have gained recognition as important regulators in various immune responses. Current paradigm states that all ILCs originate from common lymphoid progenitors (CLP) in the bone marrow through an array of downstream ILC progenitors. Id2, a member of the Id family that inhibits E protein transcription factors, is expressed in these ILC progenitors and is indispensable for ILC differentiation. Interestingly, we found that down-regulating E protein function in the thymus by Id1 overexpression or deletion of two E protein genes resulted in massive expansion of ILC2s in the thymus as well as other organs where ILC2 normally reside. The phenotypic thymic ILC2s expressed ILC2 signature genes and down-regulated T lineage-specific genes. Moreover, Id1 transgenic mice exhibited spontaneous infiltration of eosinophils and heightened type 2 immune responses to the allergen, papain, in the lung. Furthermore, these mice had increased ability in the expulsion of helminth parasites, N. Brasiliensis. These results raise an interesting question as to whether the thymus is a natural site for ILC2 production and the phenotypes of the mutant mice simply highlight such capacity. Indeed, we found that wild type DN1 and DN3 thymocytes can differentiate into IL-5 and IL-13-producing ILC2 cells in vitro. Ablation of E proteins in DN3 cells accelerated the differentiation while gain of E protein function inhibited the process. Together, our data reveal a new ILC2 differentiation program occurring in the thymus and a critical role of E proteins in restraining such a differentiation potential while ensuring proper T cell production.
Partially Maintaining E Protein Activity During ILC Development Selectively Inhibits ILC2 Development
Authors: Berrett Hannah R , Alberola-Ila Jose
Published: January 2023

Innate lymphoid cells (ILCs) provide an early response to pathogenic damage at mucosal barrier surfaces. They have beneficial roles in infection and tissue repair, as well as pathogenic roles in chronic inflammatory diseases. ILC effector subsets are defined by transcription factor expression patterns and cytokine production profiles. Recent evidence suggests ILC cytokine profiles are not static and cells can be pushed into producing different cytokines. The possibility of manipulating ILCs toward beneficial immune responses or away from pathogenic responses increases the urgency to understand how ILC effector programs are established during ILC development and maintained during homeostasis and immune responses. All ILCs are generated from a common precursor derived from the common lymphoid progenitor, and differentiation of this precursor into ILC1s, ILC2s, and ILC3s is characterized by induction of distinct transcription factors. All ILC subsets share a developmental requirement for Id2 induction. Id2 is an inhibitor of E protein transcription factor activity, but its specific functions in ILC development have not been elucidated. We have used expression of ET-2, a chimeric protein that acts as an Id dominant negative inhibitor, to partially block Id2 function during ILC development. Our experiments show that while ILC1s and ILC3s are mostly undisturbed by ET-2, ILC2s experience a profound block in development. These results demonstrate that, while all ILCs require Id2 expression, ILC2s are uniquely sensitive to the effects of ET-2 and require a more complete inhibition of E protein activity.
NF-κB signaling pathway mediates respiratory syncytial virus-induced HMGB1 release in airway epithelial cells to promote an inflammatory response
Authors: Hosakote Yashoda Madaiah , Garofalo Roberto P , Kurosky Alexander
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in young children, the elderly and immunocompromised individuals. In addition to acute morbidity, RSV infections have been linked to both the development and severity of asthma. No effective treatment or vaccine is currently available for RSV. High mobility group box 1 protein (HMGB1) is a multifunctional protein that serves as both a DNA regulatory protein and an extracellular cytokine signaling molecule that promotes airway inflammation as a damage-associated molecular pattern. Although HMGB1 is implicated in the pathogenesis of many inflammatory diseases, its involvement in RSV-induced lung inflammation is not yet investigated. The goal of this study is to investigate the molecular mechanisms that induce the release of HMGB1 and the involvement of the NF-κB and TLR4 pathway in RSV pathogenesis. Human lung epithelial cells were infected with RSV in the presence or absence of human recombinant HMGB1 and harvested over time to analyze HMGB1, NF-κB and TLR4 expression by Western blot, immunohistochemistry and qRT-PCR. Our studies with airway epithelial cells clearly show that RSV-induces a significant release of HMGB1 from cells as a result of its translocation from the nucleus to the cytoplasm and subsequently to the extracellular space; that secreted HMGB1 triggers immune responses by activating primary human monocytes via NF-κB-TLR4 signaling pathway. Blockage of one of these molecules could represent a novel therapeutic strategy in the treatment not only for RSV infection, but also for other respiratory diseases that affect human population at any age.
Influenza A virus (IAV) infection in humans leads to expansion of highly diverse CD8 T cell repertoires cross-reactive with Epstein Barr virus (EBV)
Authors: Gil Anna , Mishra Rabinarayan , Song InYoung , Aslan Nuray , Luzuriaga Katherine , Selin Liisa K
Published: January 2023

The competence of T cell responses predominantly depends on how efficient T cell receptors (TCRs) are at recognizing antigenic epitopes. We show here that during acute severely symptomatic IAV infection there was an expansion of IAV-M1/EBV-BRLF1 and IAV-M1/EBV-BMLF1 double-tetramer+ cells directly ex-vivo in 5 HLA-A2+ patients. We questioned whether this expansion specific to these two different cross-reactive responses would lead to alterations in the IAV-M158, EBV-BRLF1109 and -BMLF1280 TCR repertoires from before and during acute IAV infection. Using staining with VB mAb we found that T cell responses generated to these epitopes became surprisingly more polyclonal, with the sharing of Vb between M1, BMLF1 and BRLF1 populations which is not seen in healthy donor. Furthermore, by using next generation sequencing and single-cell analysis of TCRα and TCRβ repertoire of tetramer sorted IAV-M1 cells we showed dramatic changes in specific clonotype usage during acute IAV infection compared to before infection. In summary, these changes in TCR repertoire during acute symptomatic IAV infection suggest that during severe infection there is a preferential expansion of highly diverse cross-reactive responses between IAV and the persistent virus, EBV, which leads to permanent changes in TCR repertoires to both of these two viruses (NIH AI049320 and NIH AI109858).
Pre-existing matched antigen exacerbates the disease of influenza virus infection by attenuating antigen-specific T cell immunity
Authors: Huang Ching-Tai , Dutta Avijit , Chen Tse-Ching , Lin Chun-Yen , Lin Yung-Chang , Chang Chia-Shiang , He Yueh-Chia , Huang Yu-Lin
Published: January 2023

Influenza virus infection causes mild and self-limiting illness in most of the healthy individuals, but some healthy people develops complications as a result of the infection and suffers from severe disease and eventually dies. Here we report that pre-existing matched antigens attenuate antigen-specific immunity to the infection. High virus load and associated increase of pathogenic innate response in the lungs exacerbates the disease. The C3-HAHigh and C3-HALow transgenic mice are with inherent expression of Hemagglutinin (HA) antigen from PR8 strain of H1N1 influenza virus. Compared to wild type mice, these mice suffered from severe disease and higher mortality upon infection of wild type PR8 virus but not upon infection of reverse genetically raised PR8 virus with replaced HA from H3N2 influenza virus. Adoptively transferred PR8 virus HA-specific 6.5 CD4+ and Clone-4 CD8+ T cells responded minimally to PR8 virus infection in C3-HA mice. There was less clonotypic expansion, T-bet induction and effector cytokines IFN-γ and TNF-α production of both 6.5 CD4+ and Clone-4 CD8+ T cells. Virus neutralizing antibodies were less as well. The attenuation was associated with increased LAG-3+ but not Foxp-3+ population of HA-specific T cells in the lungs. In contrast to the instigated weak antigen-specific immunity, PR8 virus infection caused neutrophils more efficient for inflammatory IL-6 secretion and robust bronchus infiltration in C3-HA mice. These results suggest that a host factor cross-reactive to the antigens of influenza virus may exacerbate the disease with similar attenuated antigen-specific adaptive immunity accompanied by augmented innate immunity to influenza virus infection.
Duration of antigen presentation to influenza-specific CD8+ T cells regulates the development of tissue resident memory populations.
Authors: Fuller Michael John , Botta Davide , Schultz Michael D. , Bachus Holly , Ballesteros-Tato Andre
Published: January 2023

Some memory CD8+ T cells are programmed during the contraction phase of the primary response to have robust secondary responsiveness, a phenomenon termed memory programming. However, the underlying mechanism of the memory programming is not entirely understood. Using an influenza virus infection model, we show here that memory CD8+ T cell responses to the more abundant nucleoprotein (NP) antigen more vigorously proliferate than CD8+ T cell responses to the less abundant polymerase (PA) antigen. Interestingly, in spite of their differential abilities to mount secondary responses, memory NP and PA-specific CD8+ T cells are present in comparable frequencies in the lung parenchyma and airways during the memory phase. However, whereas NP-specific CD8+ T cells express low levels of CD103 and CD69, PA-specific CD8+ T cells are predominately CD103hiCD69hi, a phenotype characteristic of tissue resident memory (Trm) populations. Importantly, our data indicate that persistent antigen presentation during the contraction phase of the primary response prevented the differentiation of Trm cells. As a result, whereas prolonged antigen presentation by CD103− CD11bhi dendritic cells (DCs) during the late phase of the primary response programmed NP-specific CD8+ T cells to potently expand after re-challenge, it prevented the differentiation of NP-specific Trm cells. Collectively, our results demonstrate that CD8+ T cell responses to less abundant antigens, such as PA, preferentially develop into tissue resident memory populations.
Autophagy and TGF-β regulates innate immune response in human respiratory syncytial virus infected macrophages
Authors: Pokharel Swechha Mainali , Shil Niraj Kanti , Thapa Bhim Bahadur , Bose Santanu
Published: January 2023

Human respiratory syncytial virus (RSV) is a leading cause of hospitalization in infants and children worldwide. RSV infection manifest into two inflammatory diseases, pneumonia and bronchiolitis. Recently, we reported an involvement of transforming growth factor-β (TGF) activated SMAD2/3 signaling pathway in interferon-β (IFN-β) production from RSV infected macrophages. These studies also demonstrated a role of autophagy in triggering TGF production from RSV infected macrophages. Although IFN-β mediated antiviral response is a critical host-defense mechanism to combat virus infection, it is important to understand mechanism(s) regulating inflammation during RSV infection since it results in development of inflammatory diseases like pneumonia and bronchiolitis. Tumor necrosis factor-α (TNF) and interleukin-6 (IL-6) are critical pro-inflammatory cytokines shaping inflammation during virus infection. Therefore, we investigated the role of TGF-SMAD2/3 pathway in regulation of TNF and IL-6 production during RSV infection of macrophages. TGF-SMAD2/3 signaling pathway play an important role in pro-inflammatory response, since blockade of this pathway by TGF inhibitor and SMAD2 knockdown (via siRNA) led to diminished TNF and IL-6 production from RSV infected macrophages. Accordingly, reduced NF-κB activation was observed in RSV infected macrophages deficient in TGF-SMAD2/3 signaling. Furthermore, inhibiting autophagy in RSV infected macrophages diminished pro-inflammatory response. Thus our study has demonstrated a role of autophagy-TGF-SMAD2/3 signaling network in positively regulating NF-κB activation and subsequent production of pro-inflammatory cytokines following RSV infection of macrophages.
The role of Indoleamine 2,3-dioxygenase (IDO) in the survival of bone marrow resident long lived Plasma cells
Authors: Maharaj Shivana M , Carlson Louise M , Lee Kelvin P
Published: January 2023

Long lived plasma cells (LLPCs) are essential for sustained antibody responses and protective humoral immunity. How these cells maintain longevity and a durable antibody response is largely dependent on the complex nature of the bone marrow microenvironment in which these cells reside, and the pro survival factors produced in this niche. Work in our lab has shown that CD28 is required for LLPC survival, and that Indoleamine 2,3-dioxygenase (IDO), an enzyme that catabolizes tryptophan, is produced as a result of back signaling from CD28:CD80/86 interactions between plasma cells and bone marrow myeloid derived dendritic cells (DC). IDO is classically known to contribute to an immunosuppressive environment through its depletion of tryptophan (specifically with respect to T cells). However, to our great surprise we found that there are fewer LLPCs present in the bone marrow of IDO knockout mice in comparison to wild type mice. Additionally, live cell numbers of purified plasma cells in vitro in tryptophan free media and IDO conditioned media are greater than in media alone. These conditions also caused autophagy to occur in these plasma cells, a process shown to be required for LLPC survival. This leads us to propose a model where CD28, through back signaling to CD80/86, induces IDO production which leads to depletion of tryptophan and causes induction of autophagy, a mechanism LLPCs use to compete and survive within the bone marrow microenvironment.
PCSK9 regulates LDL-dependent uptake of bacterial lipids by HepG2 cells through LDL receptor
Authors: Grin Peter , Dwivedi Dhruva J. , Liaw Patricia C. , Fox-Robichaud Alison E.
Published: January 2023

Introduction

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key negative regulator of lipid uptake through low-density lipoprotein (LDL) receptor (LDLR), and has recently been implicated in regulating cytokine production during sepsis. We hypothesize that PCSK9 affects cytokine production by regulating hepatic LDLR-mediated uptake of bacterial lipids, such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and that this uptake is LDL-dependent.

Methods

HepG2 cells were cultured in media containing normal serum or lipoprotein-deficient serum (LPDS), and pre-treated with recombinant human PCSK9 or control, and with control IgG, or anti-LDLR antibody. HepG2 cells were also cultured in LPDS with add-back of increasing LDL concentrations. Cells were then treated for 24 h with fluorescent LPS or LTA, or controls. Media was collected for cytokine assays, and flow cytometry was performed to quantify LPS or LTA uptake by measuring fluorescence.

Results

HepG2 cells pre-treated with recombinant PCSK9 or anti-LDLR antibody had significantly decreased fluorescent LPS and LTA uptake compared to controls when cultured in normal serum. Furthermore, cells pre-treated with both anti-LDLR antibody and PCSK9 showed similar uptake as cells treated with anti-LDLR antibody alone. Neither anti-LDLR antibody nor PCSK9 had any effect on cells cultured in LPDS, but LDL add-back dose-dependently increased uptake of both LPS and LTA. Varying uptake of LPS and LTA did not affect secretion of IL-6, IL-8, IL-10, or IL-17 by HepG2 cells.

Conclusion

Bacterial lipid uptake by HepG2 cells through LDLR requires LDL, and is negatively regulated by PCSK9, but does not affect cytokine production by these cells.
Fasciola hepatica Glutathione S-tranferase suppresses the expression of inflammatory cytokines from murine macrophages in vitro and boosts the survival rate of C57Bl/6 mice against lethal doses of lipopolysaccharide
Authors: Aguayo Vasti M. , Colón Edwin , Espino Ana
Published: January 2023

Fasciola hepatica, a world-wide parasitic helminth, is a well-known master of immunomodulation. The survival of this parasite in the mammalian host is highly dependent on its ability to avoid immune responses of the host for which the parasite employs excretory-secretory products (ESPs). Glutathione S-transferase (GST), one of the proteins in the repertoire of parasite ESPs, is an enzyme recognized for its anti-oxidant properties. GST has been studied as a vaccine candidate against animal fascioliasis, but its role in the immunomodulation has been poorly studied. In a previous study, we demonstrated that GST alone incites no activation of the nuclear factor NF-κB, yet it can suppress NF-κB when THP1-CD14 cells are exposed to GST 30 minutes before stimulation with TLR ligands. In this study, we investigate whether GST could exert a similar suppressive effect on NF-κB activation in THP1-CD14 cells when added several hours after TLR-ligand stimulation and if GST is able to suppress the expression of inflammatory cytokines in bone marrow derived macrophages (bmMØs) from naïve mice induced by LPS. Finally, we performed in vivo experiments to investigate whether one 200ug intraperitoneal (IP) injection with GST could increase the survival rate of C57BL/6 mice exposed to a lethal dose of LPS. Our results demonstrate that 10ug of GST on THP1-CD14 cells up to 3 hours after TLR-ligand stimulation significantly suppressed (p≤0.01) NF-kB activation. GST also suppressed the expression of TNF-α and IL-1β cytokines (p<0.05) in bmMØs in a dose dependent manner. Importantly, a single IP injection of GST boosted the survival rate of mice by 80% against lethal doses of LPS, suggesting GST is an anti-inflammatory molecule.
Vasoactive intestinal peptide receptor - 1 deficiency causes an increase in the frequency of antibiotic producing bacteria in mouse fecal samples
Authors: Huber Benjamin , Bains Matt , Laney Caleb , Brooks Amanda , Dorsam Glenn P
Published: January 2023

New antibiotic discovery is critical because bacteria are becoming resistant to currently prescribed antibiotics. A mammalian gut peptide, vasoactive intestinal peptide (VIP), is highly expressed in the gut and possess antimicrobial activity. VIP binds two receptors, designated VPAC1 and VPAC2, and deficiency in all three have been shown to cause significant gut microbiota dysbiosis. We hypothesized that mice deficient for VIP signaling would be good candidates for exploring the existence of novel antibiotic-producing bacteria (APB). To this end, bacteria fecal samples from C57Bl/6 wild type (WT), and heterozygous (HET) and/or homozygous mutant (KO) VIP, VPAC1 and VPAC2 strains were used to test for zones of inhibition against escape pathogens. Our results showed that heterozygous VPAC1 mice resulted in the greatest increase in APB compared to WT controls, VIP or VPAC2 genetically altered strains. Initial taxonomic identification of APB from VPAC1 Het fecal samples reveled previously characterized aerobic bacteria. We conclude that reduction in VPAC1 receptor signaling in the gut, as opposed to loss of the VIP ligand or VPAC2 signaling, elevates the frequency of aerobic APB. Future studies will now focus on utilizing VPAC1 WT, HET and KO littermates to mine for the existence of novel anaerobic APB.
MAIT cells: Shaping the microbiome, contributing to Clostridium difficile infection
Authors: Smith Ashley Dawn , Zhang Irma T , Schubert Alyxandria M , Giordano Nicole P , Hastie Jessica E , Cowley Siobhan C , Carlson Paul E
Published: January 2023

Clostridium difficile (Cd) is a leading cause of nosocomial infection. Cd infection (CDI) typically occurs following antibiotic usage, which perturbs the gut microbiota leaving the host susceptible to Cd colonization. Mucosa-associated invariant T cells (MAIT) cells recognize intermediates of riboflavin biosynthesis presented on MR1, an MHC-I like related molecule. MAIT cell development is dependent on the host microbiome. MAIT cells are found in high numbers at mucosal sites and are beneficial in combatting various pulmonary infections; however their role in gut infections is unknown. We hypothesized that MAIT cells would play a role in controlling CDI. To test this hypothesis, WT and MR1−/−(lacking MAIT cells) mice were treated with antibiotics and then infected with Cd spores. Stool was collected and plated to determine Cd colonization levels for several days post-infection. Contrary to our hypothesis, MR1−/− mice showed no signs of disease or detectable levels of Cd colonization. More pathogenic strains of Cd were also tested and MR1−/− mice remained resistant. Fecal microbiota transplantation (FMT) was conducted to determine the role of the microbiota in this resistance phenotype. Susceptible WT mice given FMT from MR1−/− mice experienced dramatically lower colonization levels by day 7 and cleared detectable Cd by day 14, while WT mice given control FMT continued to exhibit high colonization levels. 16S rRNA sequencing of fecal samples from each strain revealed inherent phylum level differences in relative abundance of Bacteroidetes, Firmicutes, and Verrucomicrobia after antibiotics and FMT. Our data suggest the MR1−/− gut microbiome is resistant to Cd colonization, and this resistance is transferrable via FMT.