bluuebunny/crossref_metadata_2025 huggingface dataset:
Histone acetyltransferase p300 acts as a potent transcriptional co-activator for Pax5 (35.29)
Authors: He Ti , Yu Zhihong , Kwon Hyoung , Toleman Clifford A , Kudlow Jeffrey , Zhang Zhixin
Published: January 2023

Pax5/BSAP is an essential regulator for B lineage cell development and function. Pax5 activates the transcription of many B lineage specific genes, such as Cd19, Mb-1 and Blnk; controls the B lineage specific IgH VH→DJH recombination and meantime, represses the expression of other lineage inappropriate genes, such as Notch1, M-csfr, G-csfr, and Ccl3, to restrict B lineage cell development. It becomes important to understand how the Pax5 activity is regulated to exert these different functions. Here, we show that co-expression of histone acetyltransferase p300 dramatically enhances Pax5-mediated activation of a Pax5 responsive luciferase reporter construct. The p300-mediated enhancement is dependent on the intrinsic histone acetyltransferase activity of p300 and the activation domain of Pax5. Pax5 interacts with p300 in vitro and forms a complex with p300 in B lineage cells. Recombinant full length p300 purified from baculovirus or 293 cells directly acetylates purified Pax5 in vitro and overexpression of p300 induces Pax5 acetylation in 293 cells. Mass spectroscopy-based analysis identified that multiple lysine residues within the Pax5 DNA binding domain were acetylated upon coexpression of p300. Mutations of lysine 67, 87, 89, 103, and 142 residues on Pax5 attenuate p300-mediated enhancement. These results suggest that p300 acts as a potent co-activator for Pax5-mediated transcription function through acetylation of the Pax5 DNA binding domain.
Epigenetic Control of Killer Immunoglobulin-like Receptor (KIR) Expression in T cells (35.40)
Authors: Li Guangjin , Weyand Cornelia M. , Goronzy Jorg J.
Published: January 2023

Killer immunoglobulin-like receptors (KIR), usually restricted to NK cells, are expressed on T-cells with increasing age and contribute to age-related diseases. We examined the epigenetic changes associated with KIR expression. In T cells with absent KIR2DL4 transcripts such as the Jurkat cell line or CD4+CD28+ T cells, CpG sites of the proximal KIR2DL4 promoter were hypermethylated. DNMT inhibition in such cells induced low but appreciable KIR2DL4 transcription. DNMT inhibition was strikingly more effective in T cells that had some constitutive KIR2DL4 transcription, such as CD4 and CD8 T cells from elderly adults or the CD4+CD28−T cell line HUT78. Such cells had evidence of partial promoter demethylation; however, although the proximal nucleosome was partially relaxed, the only histone modification was an increase in dimethylated H3-Lys 4. In contrast, NK cells had a fully demethylated KIR2DL4 promoter and the full spectrum of histone modifications indicative of active transcription with H3 and H4 acetylation, di- and trimethylated H3-Lys 4, and reduced dimethylated H3-Lys 9. These results suggest that early steps of promoter activation in T cells include selective increase in H3-Lys 4 dimethylation and limited DNA demethylation. This partially accessible promoter is sensitive to DNMT inhibition, which is sufficient to induce full transcription without further histone acetylation and methylation.

Supported by NIH grant AG 15043
Phosphorylated mRNA binding proteins associated with NE/cAMP/PKA-mediated mRNA decay in S49 T lymphocytes (35.43)
Authors: LaJevic Melissa Dawn , Koduvayur Sujatha , Graf Lloyd H , Cohen Rhonna L , Chambers Donald A
Published: January 2023

We have shown that norepinephrine (NE) mediates the β-adrenergic/cAMP/PKA-dependent mRNA destabilization of mRNAs in T lymphocytes. Our goal is to elucidate the mechanism(s) involved in NE mediated Thy-1 mRNA decay in S49 T lymphoma cells. NE signal transduction in S49 cells activates PKA which could lead to the phosphorylation of effector proteins involved in ARE mediated message decay. Previous results have identified AUF1, HuR, KSRP, TIAR, and Hsc70 as candidate effector proteins. This study analyzes phosphoproteins from S49 cells and the effect of NE treatment (150μM, 30 min) on phosphorylation. NE induced phosphorylation of PKA substrate in S49 cytoplasmic extract was identified at 47, 55, 60, and 105 kD via immunoblotting. Of the candidate proteins that correlate in size, only AUF1 and Hsc70 were detected in phosphoprotein isolated from S49 whole cell extract via affinity chromatography; however, their level of phosphorylation appears unchanged with NE treatment. To determine if other binding proteins may be PKA targets, a pull down assay using a biotinylated 170 nucleotide ARE rich Thy-1 probe and 32P metabolically labeled cytoplasmic protein (100μCi/ml, 30 min) isolated at least five phospho-binding protein, two of which, 60 kD and 105 kD, increased with NE treatment. These results characterize phosphoproteins that function in the NE-mediated decay of Thy-1 and other mRNAs.

Supported by NIH
Replicating dendritic cells transmit internalized antigen to progeny for presentation (36.19)
Authors: Diao Jun , Winter Erin , Chen Wenhao , Cattral Mark S
Published: January 2023

During steady state conditions spleen dendritic cells (DC) turnover every 2–3 days. Recent evidence indicates that local proliferation of spleen DC contributes substantially to their homeostasis. We and others have shown that the majority of these dividing DC arise from a distinct population of replication-competent immediate conventional DC precursors (cDCp). cDCp generate exclusive conventional DC that continue to replicate for several generations. The objective of this study was to determine whether DC can replicate after capturing antigen, and whether replication provides a mechanism for disseminating antigen to their progeny. Real-time confocal microscopy revealed that replicating DC internalize and directly transfer model antigens to successive generations of progeny. Soluble protein antigen (ovalbumin) inherited by DC progeny in vitro or in vivo was presented effectively to OT-1 T cells. Other mechanisms of inter-DC antigen transfer (e.g. exosomes, cell membrane exchange) did not contribute to antigen acquisition by DC progeny. Our results suggest that replication of antigen-bearing DC is a fundamental behavior and may provide a novel mechanism for increasing the frequency of antigen-bearing DC, and duration of antigen presentation.
New STAT6 inhibitors with potential therapeutic application in asthma (37.1)
Authors: Zamorano Jose , Cortes Jose R , Perez-G Moises , Fernandez Carlos , Gonzalez-Nunez Maria A
Published: January 2023

Asthma is an inflammatory disease that represents an important health problem. The origin of this disease seems to be due to an immunological anomalous response to apparently innocuous antigens. In the last years, an important number of molecular intermediaries have been found to be implicated in the progression of this disease. The importance of these mediators is determined by the fact that they are actively being investigated as potential therapeutic targets. In this regard, numerous evidences indicate that Interleuquina-4 (IL-4) and IL-13 have a central role in the development of asthma through the activation of the factor of transcription STAT6. In a previous study, our group demonstrated that antiinflammatory doses of salicylates inhibited the activation of this factor of transcription. The objective of the present study was to obtain new inhibitors of STAT6 derived from salicylates by performing sequential chemical modifications. With this approach, we obtained several compounds with a planar and rigid structure, and a specific pattern of hydroxyl groups that were able to inhibit the activation of STAT6 with great efficiency. Among the obtained compounds, the named JRC688 and JRC706 inhibited the activation of STAT6 at concentrations of 20 μM. To investigate the effect of these compounds in asthma, we chose JRC706 since it had little effect on cell viability, suggesting it could have less undesirable effects. The found results indicate that the administration of JRC706 could prevent the inflammatory response in asthma. Thus, infiltrated inflammatory cells in lung and bronchoalveolar fluids, mucus production, hyperplasia of globet cells, and levels of IgE were greatly diminished in mice treated with JRC706 with regard to untreated asthmatic controls. These findings indicate that JRC706 is a good compound to investigate in asthma.
Blocking OX40L Function Inhibits TSLP-Induced Atopic Disease In Lung And Skin (37.11)
Authors: Seshasayee Dhaya , Zhou Meijuan , Shu Jean , Suto Eric , Zhang Juan , Lee Wyne Pun , Seeber Stefan , Fuentes Maria , Hymowitz Sarah , Martin Flavius
Published: January 2023

OX40L-OX40 interactions have been shown to be important for development of Th2-mediated diseases such as asthma through the use of mouse models in vivo. Thymic Stromal LymphoPoietin (TSLP), an IL7-like cytokine, has been shown to potently induce atopic immune responses and is highly expressed at sites of allergic inflammation. While the ability of TSLP to effectively induce an atopic immune cascade has been demonstrated through the use of transgenic mice, direct downstream in vivo mediators have not been identified. In our current study, we show that OX40L is a critical mediator of TSLP-mediated Th2 responses not only in vitro but also in vivo. TSLP strongly induces OX40L expression on dendritic cells and blocking a-OX40L antibodies efficiently inhibit Th2 responses induced by TSLP in vitro, and in the lung and skin in vivo. Inhibition of OX40L function was also very efficacious in decreasing Th2 cytokines and antigen-specific IgE in a mouse model of OVA-induced asthma. The use of blocking a-OX40L antibodies thus presents an effective strategy for the treatment of allergic disease associated with dysregulated Th2 immune responses.

The source of research support is Genentech, Inc.
Global patterns of gene expression following inhalation of toluene diisocyanate suggest development of allergic rhinitis (37.4)
Authors: Johnson Victor J , Kashon Michael L , Yucesoy Berran , Luster Michael I
Published: January 2023

Rhinitis is often a comorbid and preceding disease in workers afflicted with toluene diisocyanate (TDI) asthma. We hypothesized that TDI rhinitis is driven by an allergic immune response rather than innate inflammation. Global patterns of gene expression were examined via microarray in a murine model of TDI rhinitis and compared to acute nasal exposure to lipopolysaccharide (LPS). Histological analysis showed extensive inflammation of the nasal mucosa with eosinophils and neutrophils dominating in the TDI and LPS models, respectively. Effector cell recruitment correlated with expression of chemokines directed towards eosinophils in TDI rhinitis and neutrophils in LPS rhinitis. Genes involved in antigen processing/presentation, lymphocyte activation/regulation, cytokine-receptor signaling and humoral immunity were differentially regulated in TDI rhinitis. In contrast, inflammatory, innate immune and defense responses as well as response to pathogens/biotics were over represented in LPS rhinitis. These results indicate that exposure to TDI results in an antigen-driven immune response in the nasal mucosa suggestive of allergic rhinitis.

This work was supported in part by an NIEHS IAG (Y1-ES-0001 - Immunotoxicity of Workplace Xenobiotics)

The findings and conclusions have not been formally disseminated by NIOSH and should not be construed to represent any agency determination or policy.
Novel therapy for allergic disease through gene silencing of CD40 (37.7)
Authors: Suzuki Motohiko , Zhang Xusheng , Zheng Xiufen , Li Mu , Vladau Costin , Chen Dong , Garcia Bertha , Min Wei-Ping
Published: January 2023

Gene silencing is a potent, selective, and easily-inducible method for specifically blocking expression of desired genes. Gene silencing strategies have been successfully tested in animal models of various diseases. However, the therapeutic potential of gene silencing allergic diseases has not yet been reported. CD40 is an important costimulatory molecule that plays a critical role in immune responses. We attempted to develop a new therapy for allergic diseases through gene silencing using a short hairpin siRNA-expressing vector (shRNA) specific to CD40. The allergic mouse model was made by intraperitoneal immunization with ovalbumin (OVA), followed by intranasal challenges with the same antigen. CD40 shRNA was administered before or after OVA immunization. CD40 shRNA treatment reduced CD40 expression in splenic DC, remarkably reduced nasal allergic symptoms, and decreased nasal eosinophilia. The OVA-specific T cell response was inhibited after CD40 shRNA treatment. Additionally, anti-OVA specific IgE was significantly decreased in CD40-shRNA treated mice, as detected by ELISA. The production of IL-4 and IL-5 was suppressed in the CD40-shRNA treated mice. Finally, CD40 shRNA facilitated the generation of regulatory T cells, in particular, the CD4+CD25+Foxp3+ subset of cells. This study, for the first time, has demonstrated a novel therapy for allergic disease through gene silencing.
Effects of P-glycoprotein Deficiency on Epithelial Cell Responses to Toll-like Receptor Ligands (40.2)
Authors: Staley Elizabeth M , Dimmitt Reed , Lorenz Robin G
Published: January 2023

P-glycoprotein (P-gp), the product of the multi-drug resistance gene (MDR), is an ATP-dependent transmembrane pump. In intestinal epithelial cells, loss of P-gp affects tight junction proteins, and increases membrane permeability. Deficiency of this protein results in colitis in conventionally housed mice. It is believed P-gp functions to extrude xenobiotic stimuli from the intracellular environment, preventing contact with intracellular pathogen receptors. Therefore, we hypothesized that P-gp deficiency may cause colitis due to stimulation of intracellular toll-like receptors (TLRs) by excessive accumulation of bacterial antigens. HEK cells stably transfected with TLR expression plasmids were electroporated and treated with siRNA targeted for the MDR1a gene. Cells were then stimulated with TLR ligands. Inflammation was measured as IL8 secretion, an inflammatory cytokine produced by epithelial cells, and quantified by cytokine ELISA.

P-gp deficient HEK cells expressing TLR9 produced 4.9 +/− 1.3 ng/ml IL8 when treated with stimulatory CpG-OGNs, cells wild type for P-gp expression only produced 1.6 +/−.44 ng/ml IL8. No significant difference in IL8 production was detected in cells expressing TLRs 4 or 5 when treated with LPS or flagellin respectively.

P-gp may function in intestinal epithelial cells to extrude stimulatory bacterial CpG and reduce unwarranted inflammatory response.

Supported by NIH grants T32AI07051; NIHP01DK071176; & R24 DK064400
The mast cell activator Compound 48/80 is a potent adjuvant for intradermally-administered anthrax protective antigen. (41.12)
Authors: McGowen Afton , Paraggio Nicole A. , Sobel Ashley E. , Kirwan Shaun M. , Shelburne Chris P. , Pizzo Salvatore V. , Abraham Soman N. , Staats Herman F
Published: January 2023

Recent studies have reported that mast cells play a role in the trafficking of dendritic cells and lymphocytes to lymph nodes draining sites of infection. To test the hypothesis that mast cell activators could be used as vaccine adjuvants, C3H/HeN mice were intradermally vaccinated in the ear on days 0 & 21 with 0.5 μg recombinant anthrax protective antigen (rPA) alone or combined with 30 μg compound 48/80 (C 48/80), a mast cell activator. CpG oligodeoxynucleotide (CpG ODN, 1 μg) was used as a control. Ear swelling was monitored 24 hours after immunization to measure injection site inflammation. Mice immunized with rPA alone had an average ear thickness (mm) of 0.0059 while mice immunized with rPA + C 48/80 had an ear thickness of 0.0426 (p < 0.001 vs rPA alone). Mice immunized with rPA + CpG ODN had an ear thickness of 0.1519 (p < 0.001 vs rPA), significantly greater than swelling induced by C 48/80 (p < 0.001). Day 42 serum from mice immunized with PA alone had serum anti-PA IgG titers of 1:27,238 while mice immunized with PA + C 48/80 had anti-PA titers of 1:10,568,984 (p < .001 vs PA alone). Similar titers were detected in mice vaccinated with rPA plus CpG ODN. Antibodies induced with rPA + C 48/80 neutralized anthrax lethal toxin. Our findings suggest that mast cell activators provide effective vaccine adjuvant activity when delivered intradermally while inducing less injection site inflammation than CpG ODN.
CD28 costimulation is required by effector and memory CD8+ T cells (43.2)
Authors: Boesteanu Alina C. , Dolfi Douglas V , Oliai Caspian H , Borowski Annie , Katsikis Peter D
Published: January 2023

CD28 costimulation is crucial for priming of CD8+ T cell responses to pathogens, however its role in the later phase of the primary and in the secondary immune responses is not clear. To dissect the role of CD28, C57Bl/6 mice were primed intranasally with influenza virus and then treated with a non-depleting blocking anti CD28 antibody at days 6 and 8 post infection. The frequency and absolute number of virus-specific CD8+ T cells were reduced in anti CD28 treated (3.9±0.4%; 1.9±0.5x105) vs. untreated mice (11.5±1.2%; 7.4±1.7 x105). We found that anti-CD28 treatment induces apoptosis of virus-specific CD8+ cells while it did not affect the number of actively dividing cells. To study the role of CD28 during secondary CD8+ T cell responses, C57Bl/6 mice were primed with flu virus strain PR8 and day 60 memory cells were transferred into C57Bl/6 or CD80−/−CD86−/− deficient mice. Mice were then rechallenged with flu virus strain X31 and 7 days post rechallenge CD80−/−CD86−/− deficient mice exhibited a reduction in virus-specific CD8+ T cells in the lung, compared to C57Bl/6 mice (1.2±0.7x106 vs. 5.8±1.1x106, respectively). Failure of memory CD8+ T cells to expand in the absence of CD28 costimulation is due to a failure to downregulate Bcl-2 and to cell cycle arrest. Thus, effector and memory CD8+ T cells require CD28 costimulation to generate optimal immune responses against pathogens.
Mycobacterial Infection induces the secretion of High mobility group box 1 (HMGB1) protein (43.20)
Authors: Grover Ajay , Taylor Jennifer , Troudt Jolynn , Keyser Andrew , Sommersted Kirsa
Published: January 2023

High mobility group box protein 1 (HMGB1) is a nonhistone nuclear protein that acts as a pro-inflammatory cytokine and is released by monocytes and macrophages. Necrotic cells also secrete HMGB1 at the site of tissue damage that induces a variety of cellularresponses, including expression of proinflammatory mediatorssuch as TNF- , IL-1, and NO. This study investigated the secretion of HMGB1 during mycobacterial infection of macrophages in vitro and in the lungs of infected guinea pigs. We observed that infection with mycobacterium effectively induced HMGB1 release in both macrophage and monocytic cell cultures. Culture filtrate proteins from Mycobacterium tuberculosis (including Ag85A, Ag85B, ESAT-6 and CFP-10) induced maximum release of HMGB1 among different cellular components of mycobacterium. We demonstrated that HMGB1 is released by lung cells during infection of M. tuberculosis in guinea pig and increased HMGB1 secretion in lungs of guinea pigs was delayed by vaccination with BCG. The secretion of cytokines like TNF-α and IL-1β were significantly increased when J774A.1 cell cultures were incubated with HMGB1 during infection with M. bovis BCG. We conclude that HMGB1 is secreted by macrophages during tuberculosis and it may act as a signal of tissue or cellular injury and may act as a precursor to enhance the immune response.

Funding for this research was provided by NIH AI40091 (A.A.I.).
NKT cells suppress the replication of Listeria monocytogenes in cholestatic livers (43.22)
Authors: Hoffman Beth L. , Wintermeyer Philip , Holub Martin , Brossay Laurent , Gregory Stephen H.
Published: January 2023

Biliary obstruction and cholestasis can cause hepatocellular apoptosis and necrosis. Ligation of the common bile duct (BDL) in mice provides an excellent model in which to study the underlying mechanisms. NK1.1+ TCRαβint CD1d-restricted T (NKT) cells comprise approximately 30% of the hepatic lymphoid cells in mice, decidedly fewer reside in the spleen, thymus and peripheral blood. Purportedly, NKT cells play a key role in host defenses to infection. Indeed, it has been suggested that iNKT cells evolved primarily to respond to a diverse array of microbial pathogens. The increased replication of a limited number of parasites, bacteria and viruses in the organs of CD1d−/− or Jα18−/− [invariant (i)NKT cell-deficient] mice provides support for this suggestion. In many instances, however, NKT cells are not protective and their response to infection is largely detrimental. In agreement with the latter, we report here that iNKT cell-deficient mice express increased protective immunity to L. monocytogenes. Relative to wild-type animals, significantly fewer (≤1 log10) bacteria were recovered from the livers of Jα18−/− mice on day 3 post-infection i.v. with a sublethal dose of listeriae. In marked contrast to these findings, however, listerial replication, liver injury [plasma alanine aminotransferase activity (ALT)], and death from infection were increased sharply in iNKT cell-deficient mice subjected to BDL and cholestasis. Thus, iNKT cells play a critical role in host defenses to Listeria expressed in cholestatic, but not normal, livers.
The T Cell Response to Influenza in Neonates (43.29)
Authors: Lines J. Louise , Hollifield Melissa , Cauley Linda , Garvy Beth
Published: January 2023

Influenza is a significant cause of mortality and morbidity in children. However, very little data exists on the neonatal immune response to influenza. We have found that neonatal mice are highly vulnerable to influenza and only control much reduced doses of virus.

In this study, the T cell response to intranasal influenza infection was compared between mice infected as adults or at 2 days old. In neonates, despite significant expansion of T cells in draining lymph nodes, entry of T cells into the lungs was defective. In adults, high numbers of CD4 and CD8 T cells peaked in the lung interstitium and alveolar spaces by 10 days post infection. However, in neonates, CD8 T cells entered the lungs by day 10 post-infection, but not to high levels, while CD4 T cell entry was delayed until 3 weeks of age. Interestingly, while CD4 T cells reached adult levels in the interstitia, they were unable to cross the epithelial barrier into the alveoli. The delayed entry of CD4 T cells corresponded with a delay in TNFα production and a lack of IFNγ which may consequently lead to delayed upregulation of adhesion molecules and chemokines.

Despite the poor response to influenza as neonates, mice infected at 2 days old with the PR8 strain of influenza did develop T cell memory. Mice were protected when subsequently infected as adults with a lethal dose of HKx31, a strain that shares only the internal T cell antigens with PR8. These data suggest that the acute sensitivity of neonates to influenza may be a consequence of defective T cell migration.
Characterization of lymphoid DC during the resolution of Listeria monocytogenes infection (43.38)
Authors: Burt Bryan Michael , Plitas George , Nguyen Hoang , Stableford Jennifer , DeMatteo Ronald P.
Published: January 2023

Introduction: Dendritic cells are critical to innate immune responses based on their ability to secrete inflammatory cytokines and prime naïve T-cells. The role of DC during later stages of infection is not known.

Methods: Lymphoid DC (CD11c+CD8+NK1.1−B220−) were analyzed by flow cytometry and isolated by fluorescence activated cell sorting from the spleens of C57BL/6 mice infected with 5000 CFU of LM strain 10403s by tail vein injection. Mixed leukocyte reaction (MLR) was performed with 1x105 allogeneic T cells and 3x104 lymphoid DC.

Results: Following 24 hours of infection lymphoid DC accounted for 11% of conventional DC (CD11c+B220−NK1.1−) and had high expression of MHC II, CD40 and CD86. After 7 days of infection lymphoid DC increased to 47% of conventional DC and had less MHC II, CD40 and CD86 expression than uninfected controls. Lymphoid DC also lost their ability to stimulate allogeneic T cells in the MLR (6634±466 CPM for 12 hours of infection vs 320±80 for 7 days of infection).

Conclusion: Dynamic changes occur in the lymphoid DC compartment during the resolution phase of LM infection.

This work was supported by grant DK068346.
Reconstitution of immune responses occurs very rapidly after initiation of therapy for tuberculosis (43.54)
Authors: Dabitao Djeneba , Guindo O , Diallo H , Kassambara H , Washington J , Diallo S , Dao S , Tounkara A , Sereti I , Ciccone E , Catalfamo M , Lane HC , Siddiqui S
Published: January 2023

Introduction: Immune reconstitution syndrome is a potentially fatal sequelae of HAART therapy in 20% of patients with HIV and 3–5% with tuberculosis who begin treatment. The role of CD4+T cells in its pathogenesis is poorly understood.

Methods: This study was done under an IRB approved protocol to study MTb specific immunity. Groups based on HIV status and local or disseminated MTb were evaluated for CD4+ T cell counts, Purified Protein Derivative (PPD) induced proliferation and cytokine induction using intracellular cytokine staining for IFN γ, IL2, TNFα and CFSE dilution at predefined times before and after starting therapy.

Results: In preliminary results, the first 3 HIV negative patients with MTb exhibited striking increase in MTb specific immunity in the first week of therapy. Percentage of IFN γ producing CD4+T cells increased from 1.96 and 0.48 to 4.46 and 1.34, and of TNF α producing cells increased from 0.85 and 0.45 to 3.96 and 1.06 in 2 patients. Proliferative responses also showed marked increases. Percent dividing cells increased from 4.3, 4.4 and 16.3 at baseline to 36.9, 11.9 and 39.6 at 1 week and 54.8 and 72.3 at 4 weeks of therapy. Total number of CD4+ T cells also increased.

Conclusions: Tuberculosis like HIV causes quantitative and qualitative changes in CD4+T cells . These are rapidly reversed when therapy is begun .This data provides insight into the pathogenesis of the immune reconstitution syndrome.
Robust innate immune responses of murine neonates following orogastric Yersinia enterocolitica exposure (44.13)
Authors: Echeverry Andrea , Schesser Kurt , Adkins Becky
Published: January 2023

Neonates are generally thought to be highly susceptible to gastrointestinal disease, owing in part to immaturity in immune function. To study this phenomenon experimentally, we have developed a murine pediatric model of infection with the Gram-negative bacterium Yersinia enterocolitica (Ye). This model is highly relevant to the human pediatric population since Ye is a prevalent cause of gastrointestinal disorders in young children. LD50 experiments were performed on 7 day old and adult mice infected orogastrically with Ye. Remarkably, BALB/c or C57BL/6 neonatal mice were up to 50 times more resistant than adult mice. Bacterial colonization experiments indicated that Ye rapidly disseminated to the spleen and liver in adults. In striking contrast, colonization of the neonatal spleen and liver was very limited. Instead, bacterial colonization in neonates was largely restricted to the intestine and mesenteric lymph nodes (MLN). Flow cytometric and histological studies revealed markedly increased levels of neutrophils and macrophages in the neonatal MLN after infection. Importantly, in vivo neutrophil depletion led to efficient dissemination of the bacteria to the spleen and liver of neonates. Therefore, in response to some enteric bacteria, neonates appear to be able to mount vigorous mucosal immune responses, rapidly mobilizing innate phagocytes, and confining the bacterial infection to the gut.

This work was supported by NIAID grant R01 AI44923-02 (B.A.), NIAID Public Health Service grant AI53459 (K.S.), and the Department of Microbiology and Immunology, University of Miami.
Toll-like receptor 9 participation in experimental Th type 1 (Mycobacterial) pulmonary granuloma formation (44.15)
Authors: Ito Toshihiro , Schaller Matthew , Hogaboam Cory M. , Chensue Stephen W. , Kunkel Steven L.
Published: January 2023

We have established that experimental Th type 1 (Th1) responses can be studied in mice by sensitizing with Mycobacterium bovis purified protein derivative (PPD) followed by an intravenous challenge of agarose beads covalently coupled to the respective sensitizing Ags. Mycobacterial antigens (Ags) stimulate antigen presenting cells (APCs) through distinct Toll-like receptors (TLRs) resulting in rapid activation of the innate immune system. Toll-like receptor 9 (TLR9) is known to play an important role in the regulation of Th1 responses. The role of TLR9 during infection with Mycobacterium Ags has been under investigated. We demonstrate in this study that TLR9−/− mice showed greatly increased granuloma formation as assessed by morphometric analysis, and an increase in eosinophil and stromal cells, suggesting a Th-2 skewing, as compared with wild-type. Moreover, Th1 cytokine production of IL-12 and IFN-gamma was abrogated in TLR9−/− mice. These results suggest that TLR9 plays an important role in maintaining a Th1-type pulmonary granulomatous response.

This work was supported by NIH grants HL031963, HL074024, and HL031237.
Differential virus replication, cytokine production and antigen presenting function of microglia from susceptible and resistant mice after Theiler′s virus infection (44.26)
Authors: Jin Young-Hee , Mohindru Mani , Kang Min H , Fuller Alyson C , Kang Bongsu , Gallo Daniel , Kim Byung S
Published: January 2023

Infection of TMEV in CNS causes an immune-mediated demyelinating disease similar to human multiple sclerosis in susceptible (SJL) but not resistant (B6) strains of mice. To understand the mechanisms of differential susceptibility, we analyzed expression levels of co-stimulatory molecules, cytokine production and T cell stimulatory function of microglia and macrophages in the CNS of virus-infected mice. Our results indicate that a higher level of microglia and a lower level of infiltrating macrophages are found in the CNS of susceptible SJL mice compared to resistant B6 mice during early infection. In addition, the levels of TMEV replication, TNFα, IFNβ, IL-6 and MCP-1 production were consistently higher in microglia from virus-infected SJL mice. However, the levels of co-stimulatory molecule expression, as well as the ability to stimulate allogenic T cells, were significantly lower in TMEV-infected SJL mice compared to those in B6 mice. In addition, microglia from uninfected naïve mice displayed differential viral replication, T cell stimulation and cytokine production, similar to those of microglia from infected mice. These results suggest that intrinsic susceptibility to TMEV infection and glial cell cytokine production levels affect subsequent levels of cytokine production, cellular infiltration to the CNS and consequent inflammation in susceptible mice. (NS28752 / NS33008)
Inflammation by Gr+ bacteria involves LTA and its synergistic partner. (44.32)
Authors: Seo Ho-Seong , Nahm Moon H.
Published: January 2023

Innate immunity is important in early responses to bacterial infections. Although Gr+ infections constitute about 50% of bacterial invasive diseases, the innate immune response to Gr+ bacteria is much less understood than is the comparable response to Gr- bacteria. While TLR2 is known to play an important part in innate immunity and Gr+ bacterial LTA may be its ligand, the role of LTA is unclear because ¡°purified¡± LTA often has biologically active contaminants or is degraded. To investigate the role in innate immunity of LTA without these limitations, we have studied the inflammatory properties of Gr+ bacterial culture supernatant (GPCS) after using two different methods to inactivate the LTA: alkaline hydrolysis and PAF-acetylhydrolase digestion. Both inactivation methods reduce the potency of GPCS to induce TNF-a production by about 85–100%. Also, the LTA-depleted GPCS regains its activity when highly purified LTA is added to the depleted GPCS. These findings suggest that LTA is an important inflammatory molecule. However, purified LTA is more potent when it is mixed with LTA-depleted GPCS than when LTA is used alone. This discrepancy in potency suggests that while LTA is a key molecule in Gr+ bacteria-induced inflammation, a synergistic mechanism amplifies LTA-induced inflammation. We are currently investigating the synergistic molecule present in GPCS and the synergistic mechanism induced by this molecule.
Immunostimulatory activity of the heat shock protein DnaK from Francisella tularensis (45.18)
Authors: Ashtekar Amit R , Katz Jannet , Deivanayagam Champion , Larson Matt , Michalek Suzanne M
Published: January 2023

Francisella tularensis is the causative agent of tularemia, a severe debilitating disease of humans and other mammals. Since this microorganism is also considered as a ‘category A pathogen’ and a potential biowarfare agent, there is a need for an effective vaccine. Several antigens of F. tularensis including the heat shock protein DnaK have been proposed for use in a potential subunit vaccine. In this study we investigated the immunostimulatory properties of F. tularensis DnaK on murine bone marrow derived dendritic cells (DC). Recombinant DnaK was produced using a bacterial expression system and purified using a three-step purification procedure, i.e., affinity, ion exchange and size exclusion chromatography. DnaK induced DC to produce the proinflammatory cytokines IL-6, TNF-α and IL-12p40 in a dose dependant manner. DnaK did not induce IL-23p19 production. IL-10 production was low compared to IL-12p40. DnaK also induced DC maturation as demonstrated by an up-regulation of CD40 and CD86 expression. The responses were not mediated by endotoxin contamination, since the immunostimulatory activity of DnaK was resistant to polymixin B treatment, but sensitive to boiling or proteinase K digestion. An understanding of the interactions between virulence antigens of F. tularensis and immune cells is crucial for the development of a safe and efficacious vaccine against F. tularensis.
Identification of HIV gp41 membrane proximal epitope tetramer reactive populations of B220+ B cells and B220lo/neg basophils in gp41 peptide-immunized mice (45.3)
Authors: Verkoczy Laurent , Moody Anthony , Scearce Richard , Sutherland Laura , VanLeeuwen Stacie , Plonk Kelly , Cain Derek , Kelsoe Garnett , Haynes Barton
Published: January 2023

2F5 is a rare, neutralizing human mAb specific for the HIV gp41 membrane proximal region (MPER). Recently, we showed that 2F5 is a cardiolipin-binding, polyreactive autoantibody. To determine if MPER antibodies (Ab) are controlled by tolerance mechanisms, BALB/c mice were immunized with HIV-1 gp140 envelope or gp41 peptides. Immunized mice were screened for serum MPER Ab and for 2F5 antigen-specific B cells using 2F5 epitope B cell tetramers. Immunization in Ribi adjuvant did not induce anti-MPER Ab, whereas similar immunizations in TLR9, an adjuvant designed to break tolerance, did. Interestingly, in mice immunized with gp41 peptide, but not gp140, we found two distinct populations of 2F5 tetramer+ cells in bone marrow, spleen, and blood: a polyclonally-induced B220hi B-cell population that bound 2F5 tetramers via their surface Ig and a B220lo/neg cell population induced by immunization. Histological and cell surface phenotypic analysis of the B220lo/neg 2F5 tetramer+ cells indicated these were primarily basophils that bound 2F5 tetramers via MPER Ab bound to FcR. At present, the immunoregulatory significance of these distinct anti-MPER reactive populations is unknown, but their further characterization and the types of Ab they produce/bind should help in delineating why induction of neutralizing anti-MPER Ab is not achieved, despite the generation of a robust non-neutralizing MPER B cell and Ab response.
Silencing of HALP Decreases HIV-1 Infection of Primary CD4+ T-cells (45.4)
Authors: Selliah Nithianandan , Yin Jiyi , Finkel Terri H
Published: January 2023

HIV/AIDS is an ideal candidate for a novel gene therapy approach since it is an incurable and terminal disease. We have identified a set of candidate host cell genes, associated with survival of HIV-infected T-cells. We named one of these genes, “HALP”, for “HIV-Associated Life Preserver”. Recently, we used a lentiviral vector to stably express HALP shRNA in primary human CD4 T-cells. In the absence of HIV infection, silencing of HALP appeared to have no effect on viability of these cells. As predicted, silencing of HALP markedly reduced HIV infection of primary CD4+ T-cells, as measured by the percent of p24+ cells and by supernatant p24. Published data suggest that hypoxia induces HALP expression by increasing the activity of the transcription factor, Hif-1α. HIF-1 binding sites have been identified within the HALP promoter and HALP is responsive to HIF-1 in non-immune cells. We show here that HIV-1 infection of primary CD4+ T-cells increases Hif-1α mRNA expression, as measured by real time RT-PCR. Transfection of T-cells with wild-type Hif-1α or with a mutated form of Hif that is resistant to proteolysis (Hif-DPA) also increased expression of HALP mRNA. These data are consistent with a pathway of Hif-induced HALP expression in HIV-1 infected cells, leading to a hypoxia-like stress response and, ultimately, to cell survival. Our work represents one of the first attempts to search for novel cellular genes that contribute to viral inhibition of host cell apoptosis and, thus, to viral latency. HALP represents a candidate for a host cell anti-apoptotic protein, which is triggered by HIV infection, and may provide a novel molecular target for drug use and design.
NKT cells play a role in inducing toxic shock-like syndrome in severe ehrlichiosis (46.21)
Authors: Stevenson Heather Lynne , Walker David H. , Ismail Nahed
Published: January 2023

Infection with a virulent Ehrlichia strain (IOE) results in toxic shock-like syndrome characterized by severe liver injury, a weak CD4+ Th1 response, and systemic overproduction of TNF-α by CD8+ T cells. Mice lacking functional CD8+ T cells (β2m−/−) survived infection with a lethal low-dose of IOE to 30 days post-infection. Since β2m−/− mice lack conventional CD8+ T cells and CD1d-restricted NKT cells, we examined the role of NKT cells in the pathogenesis of ehrlichiosis using CD1d−/− mice. CD1d−/−mice also succumbed to lethal infection with high dose IOE, yet did not develop signs of toxic shock-like syndrome, i.e. serum TNF-α was low, liver enzymes were normal, lymphopenia was not present, and there were few apoptotic hepatic cells. CD1d−/− mice also had less activation of CD8+ T cells in the spleen and liver as indicated by lower expression of intracellular granzyme B in lymphocytes harvested from these sites. Our data provide evidence that NKT cells play a role in initiating the pathogenic CD8+ T cell response in IOE-induced toxic shock-like syndrome.
PD-1 and PDL-1 expression in patients with chronic HIV-1 infection: relationship to CD4%, immune activation and antiretroviral therapy (ART) (46.22)
Authors: Sachdeva Meenakshi , Fischl Margaret A , Pahwa Savita
Published: January 2023

In chronic HIV-1 infection, CD8 T-cell exhaustion is linked to an increased expression of programmed death molecule (PD-1), a negative regulator of activated T cells. We investigated the relative expression of PD-1 and its ligand PD-L1 in T cells and monocytes of HIV-1 infected patients at different time points of ART. In healthy controls (n=3), <10%of CD4 or CD8 T cells expressed PD-1; in comparison, expression of PD-1 was increased (P<0.05) in treatment naïve patients (n=9) in both CD4 and CD8 T cells. In patients, PD-1 expression on CD4 T cells was correlated with activation marker, HLA-DR (P=0.0004) and inversely related to percentage of CD4 T cells (P=0.0004). Percentage of cells expressing PD-L1 was significantly higher in monocytes (CD14+) versus CD4 T cells and CD8 T cells (P<0.05). Culture of PBMC with cytokines IL-2, IL-7 and IL-15 for 6 days in vitro resulted in increased expression of PD-1 and PD-L1 in CD4 and CD8 T cells, coupled with increased expression of Ki67 and HLA-DR in 3 ART naïve patients. ART was associated with a trend towards decreasing PD-1 expression. We conclude that PD-1 and PDL-1 interaction contributes to CD4 T cell loss in a state of chronic immune activation associated with HIV-1 infection.

Supported in part by the “The International Maternal Pediatric Adolescent AIDS Clinical Trials Network of the National Institute of Allergy and Infectious Diseases” (UOI-A141089 and N01-HD-3-3345) and Institutional support.
Interleukin-15 increases vaccine efficacy through a mechanism linked to dendritic cell maturation and enhanced antibody titers (47.16)
Authors: Saikh Kamal Uddin , Kissner Teri L. , Nystrom Steven , Ruthel Gordon , Ulrich Robert G.
Published: January 2023

Interleukin-15 (IL-15) is generally considered to be a growth factor for NK cells and for sustaining T-cell memory. However, previous data from our laboratory demonstrated that IL-15 is an important factor for development of human dendritic cells. In this study, we investigated the effect of IL-15 on antibody responses to STEBVax, a recombinant staphylococcal enterotoxin B (SEB) vaccine, in a pre-clinical model of toxic-shock syndrome induced by SEB. We first observed IL-15 treated mouse spleen cells in culture gained a dendritic cell-like morphology compared to untreated controls. Further, administration of IL-15 to mice resulted in an increased number of CD11c+dendritic cells recovered from spleens. A significant, IL-15 dose-dependent increase in antigen-specific antibody was observed after co-administration with vaccine. This effect required including an aluminum-based adjuvant (Alhydrogel). The co-administration of STEBVax with IL-15 and Alhydrogel also resulted in a significant increase in protection of mice from lethal toxic shock above levels obtained without IL-15. Thus, the antibody response enhanced by IL-15 appears to be mediated by mature dendritic cells. This suggests a potential use of IL-15 as an adjuvant for antibody-dependent responses to vaccines
Diverse Subsets of Regulatory T (Treg) Cells Mediates Suppression of Arthritis by An Oral Bacterial-Based Diarrheal Vaccine (47.22)
Authors: Kochetkova Irina , Trunkle Theresa , Callis Gayle , Pascual David W.
Published: January 2023

A Salmonella vaccine expressing enterotoxigenic E. coli colonization factor antigen I (CFA/I) behaves as an anti-inflammatory oral vaccine and shown to protect against EAE and collagen-induced arthritis (CIA) in an Ag-independent fashion. Immunization with Salmonella-CFA/I caused Th2 deviation, as well as the development of Treg cells, both contributing to protection. To inquire into the specific contribution of each to protection, cell-sorted CD4+CD25+ and CD4+CD25− T cells as well as total CD4+ T cells derived from DBA/1 mice orally immunized with Salmonella-CFA/I, were adoptively transferred into DBA/1 mice already induced with CIA. Treg cells produced elevated levels of IL-4, IL-10, and TGF-β, as well as IFN-γ and IL-17, as did the CD4+CD25− T cells, but in lesser amounts. CIA was followed, and mice receiving Treg cells or CD4+CD25− T cells showed reduced incidence and very weak clinical disease; however, mice receiving total CD4+ T cells showed suppressed CIA for 2 wks, after which clinical disease was identical with disease obtained with either CD4+ T cell subset. Upon termination, LN CD4+ T cells were restimulated with collagen II (CII). No IL-4 or TGF-β were detected, but IL-10 and IFN-γ were elevated for each adoptive transfer group, more so than from untreated mice. Notably, IL-17 was markedly suppressed by the total CD4+ T cells, but elevated in the others. Thus, these studies show that the collective action of input TGF-β+, IL-4+, and IL-10+ regulatory cells modify the development of CII-specific regulatory and Teff cells, most notably, the suppression of IL-17 development.

Supported by AI-41123.
Endosomal targeting of a Mycobacterium tuberculosis protein elicits potent Th1 host responses. (47.27)
Authors: Sieling Peter A. , Niazi Kayvan R. , Dy Maria C. , Swarbrick Gwendolyn , Lewinsohn David M. , Modlin Robert L.
Published: January 2023

Antigen specific IFN-γ producing CD4 T cells are critical for the control of infection by intracellular bacteria, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Whereas traditional approaches to eliciting CD4 T cell responses utilize protein antigen, DNA vaccines are more readily prepared and at less expense. DNA vaccines have been used to elicit tumor-reactive T cell responses, but little information on DNA vaccines in microbial infection is available. In contrast to protein, a ready source for antigen processing, DNA must be transcribed and translated into protein for processing to take place. Moreover, to allow processing for CD4 T cell presentation, antigen must enter the endosomal pathway. We targeted a M. tuberculosis ESAT-6 DNA vaccine into the endosomal system using sequences from lysosome-associated membrane protein (LAMP). Human dendritic cells (DCs) transfected with ESAT-LAMP presented antigen to ESAT-6-specific CD4 T cell clones, inducing 3.8 times greater IFN-γ production in comparison to T cells exposed to DCs transfected with ESAT-6 containing no targeting sequences (ESAT-STOP). More strikingly, mice immunized with ESAT-LAMP DNA exhibited stronger (17.6-fold, p<0.05) recall responses than ESAT-STOP or animals receiving vector DNA. These data indicate that DNA vaccination enhances T cell responses to microbial antigens and suggest that such an approach might stimulate protective host responses against microbial infection, including tuberculosis.
Intranasal Vaccination with an Iron-Superoxide Dismutase (FeSOD) Deficient Mutant of Francisella tularensis LVS Protects Mice against Lethal Challenge with F. tularensis SchuS4 (47.29)
Authors: Bakshi Chandra Shekhar , Metzger Dennis W
Published: January 2023

Francisella tularensis, the causative agent of tularemia is a potential bioweapon because of ease of its dissemination, multiple routes of infection, high infectivity and lethality. We have previously reported a mutant of F. tularensis LVS deficient in iron superoxide dismutase (sodBFt) and have demonstrated that this mutant is hypersensitive to oxidative stress and attenuated for virulence in mice. Herein, we evaluated further the efficacy of this mutant as a vaccine candidate against respiratory tularemia. We observed that immunization with sodBFt mutant offered 100% protection against 10–100LD100 doses of the highly virulent F. tularensis SchuS4 strain in BALB/c mice and a consistent 40% protection in C57BL/6 mice with a significantly extended median time to death as compared to naïve or LVS vaccinated mice. The sodBFt vaccinated mice exhibited significantly lowered bacterial burden, less severe histopathological lesions in the liver and spleen and a regulated production of proinflammatory cytokines following SchuS4 challenge as compared to the LVS vaccinated mice. We observed increased levels of heat shock protein GroEL, peroxidase/catalase (KatG) and bacteroferritin (Bfr) in the cell lysates and culture filtrate of sodBFt mutant as compared to F. tularensis LVS. Studies on the role of these proteins in the generation of a strong protective immunity in the sodBFt vaccinated mice are currently underway. To conclude, our results demonstrate that sodBFt mutant is a better vaccine candidate than F. tularensis LVS and this study is a major step forward towards the development of a live attenuated vaccine for the prevention of respiratory tularemia.