bluuebunny/crossref_metadata_2025 huggingface dataset:
Galectin 3 is required for MLD-STZ induced diabetes in mice (130.38)
Authors: Lukic Miodrag L , Al-Hakim Ahmed - , Mensah-Brown Eric - , Hsu Daniel K , Shahin Allen -
Published: January 2023

Galectin-3, member of ancient lectin family characterized by specific binding of β galactosides, has an antiapoptotic function in T cells, macrophages and islet cells. It was therefore of interest to evaluate the susceptibility to multiple low dose induced diabetes (MLD-STZ) in galectin (gal-3) deficient C57BL/6 mice. Gal-3 −/− and gal-3+/+ mice were treated with 5 daily injections of 40 mg/kg STZ and diabetes development evaluated by glycemia and immunohistochemistry of the pancreas. Gal-3+/+ mice developed delayed sustained and progressive hyperglucemia and mononuclear cell infiltrates in the islets. Gal-3 −/− demonstrated only mild glycemia with minimal islet pathology as evaluated by the number of infiltrating cells and insulin content. There was higher number of apoptotic cells in the islets of galectin-3 knock out than in the control gal-3+/+ mice. RT PCR analysis of pancreatic lymph node cells 17 days after diabetes induction revealed the presence of IL-23 and IL-17 in gal-3+/+ but not in Gal-3 −/− mice. TNF-α and INF-γ INOS expression was also attenuated in Gal-3 −/− mice. We concluded that antiapoptotic effect of gal-3 in diabetogenic cells favors the induction of disease. The data are also compatible with our previous finding that IL-23-Th17 axis plays a role in diabetogenesis (Eur J Immunol, 36: 216–23, 2006).

(Supported by Sheikh Hamdan Awards for Medical Research).
Anti-CD20 Therapy in NOD Model of Type 1 Diabetes (131.26)
Authors: Hu Changyun , Rodriguez Daniel , Du Wei , Ahuja Anupama. , Wong F.Susan , Shlomchik Mark , Wen Li
Published: January 2023

B cells play an important role in the pathogenesis of T1D although T1D is primarily a T cell mediated autoimmune disease. Apart from producing antibodies, B cells are important APCs and a rich source for production of various cytokines. Nonetheless, the precise roles of B cells in promoting diabetes onset and progression have not been defined. Rituximab targets human CD20 causing rapid and specific B cell depletion. A clinical trial is currently launched in treating T1D patients with Rituximab. However, there has not been a single preclinical study to test this particular therapy in T1D. To evaluate the efficacy of anti-CD20 in treating T1D, we have generated a transgenic NOD mouse expressing human CD20 on their B cells. This allows the use of anti-hCD20 antibody (2H7), which is closely related to Rituximab. Our preliminary data showed that a single cycle of anti-hCD20 treatment (4 injections within 10 days) to temporarily deplete B cells significantly delays and/or reduces the onset of diabetes in hCD20 transgenic NOD mice. This was seen at both the disease initiation and pre-diabetic stages. The same treatment also rendered 36% (5/14) of diabetic mice euglycemic, either transiently or for a long period. Anti-hCD20 treatment appears to induce and/or expand regulatory T cells and enhance TGF-β production. Our results provide extremely valuable in vivo information for the human studies using a similar approach.
CD8+ T-cells are required for the therapeutic effects of glatiramer acetate (Copaxone) on autoimmune demyelination (131.7)
Authors: Mendoza Jason P , York Nathan R. , Benagh Andrew , Karandikar Nitin J.
Published: January 2023

Murine experimental autoimmune encephalomyelitis (EAE) is an induced, autoimmune, demyelinating disease model for human multiple sclerosis (MS). Copaxone is an approved immunomodulatory therapy for MS that was discovered through studies in EAE. It is thought that the main mode of action of Copaxone is the induction of a Th1 to Th2 shift in CD4+ T-cells. We have shown that, in human MS, Copaxone therapy induces an upregulation of cytotoxic, suppressor CD8+ T-cell responses, suggesting that these cells are integrally involved in mediating the immune effects of this drug. In this study, we show that Copaxone induces robust CD8+ T-cell responses even in mice. Whereas Copaxone immunization ameliorates the clinical and histologic severity of EAE in wild type C57/BL6 mice, such protection is not observed in CD8(−/−) mice. Moreover, adoptive transfer of CD8+ T-cells from Copaxone-immunized (but not OVA-immunized) mice results in amelioration of EAE in CD8(−/−) mice, suggesting therapeutic potential for Copaxone-specific CD8+ T-cells. In conclusion, our studies provide strong evidence for the in vivo relevance of CD8+ T-cell responses during Copaxone therapy and support the hypothesis that the induction of immune modulatory CD8+ T-cells is an essential step in mediating therapeutic protection during autoimmune demyelination.

This research was supported, in part, by grants from the National Institutes of Health and the National MS Society.
Role of ATM in V(D)J recombination and cell cycle control (35.10)
Authors: Dujka Melanie E , Puebla-Osorio Nahum , Zhu Chengming
Published: January 2023

V(D)J recombination is a process specific to developing B- and T- lymphocytes in which double strand breaks (DSBs) are introduced into germline DNA and upon repair generate rearranged gene segments. This recombination event is cell cycle regulated and is confined to the noncycling G0/G1 phase of cell cycle. The protein kinase Ataxia Telangiectasia Mutated (ATM) is a vital mediator in both the DNA damage response and cell cycle. ATM was found localized at V(D)J mediated break sites, suggesting an additional function for ATM in recombination. In the absence of ATM, mice develop thymic lymphomas with chromosomal translocations. Therefore, we hypothesize a role for ATM in the cell cycle control of V(D)J recombination. Thymic lymphomas from Atm null mice revealed cells harbored multiple chromosomal translocations near sites of recombination. Wildtype and Atm null thymocytes were separated into cell cycle fractions of G0/G1 and S/G2/M. V(D)J mediated DSBs, indicative of recombination, were observed at the T cell receptor α, δ, and β loci in both cell cycle fractions of Atm null thymocytes but were found only in the G0/G1 fraction of wildtype thymocytes. Together, these results indicate the involvement of ATM in the cell cycle control of V(D)J recombination. Loss of this control can lead to sustained DNA DSBs and translocations, which could contribute to tumorigenesis.
Analysis of transcriptional regulation of mouse FoxP3 gene expression in CD4+ CD25+ regulatory T cells (35.21)
Authors: FURUUCHI KEIJI , TONE YUKIKO , KOJIMA YOSHITSUGU , TYKOCINSKI MARK L , GREENE MARK I , TONE MASAHIDE
Published: January 2023

FoxP3 is expressed at high level in CD4+CD25+ regulatory T cells (Treg) and functions as a key transcriptional factor that determines cell phenotype. Despite of the inherent interest, little is known concerning process of transcriptional regulation of FoxP3 due to lack of experimentally feasible to analyze promoter activity using transfection-based methods in natural Treg cells. We have, therefore, identified a model cell line that expresses FoxP3 at high levels upon stimulation. To identify the promoter region of the mouse FoxP3 gene, we determined the transcription initiation site by 5′RACE, using RNAs from CD4+CD25+ Treg cells and the cell line. The major start site mapped to the same position in the both type of cells, indicating that this cell line can be used as a model system. Luciferase reporter assays were performed using a 1.7kb 5′ franking fragment of the FoxpP3 gene as promoter. Although the cell line expressed FoxP3 mRNA at high levels, we could not detect any promoter activity in this region, suggesting that enhancers might be essential for this promoter to function. Indeed, we elicited that FoxP3 promoter activity by coupling it to the SV40 enhancer. We have now identified an enhancer region in the vicinity of the FoxP3 gene, that upon analysis have revealed important and unexpected insights into the transcriptional control of the FoxP3 gene.
Negative regulation of RORgt by direct binding to Egr3 (35.41)
Authors: Kersh Gilbert J , Xi Hongkang
Published: January 2023

The orphan hormone receptor RORgt is a transcription factor that is expressed in lymphoid tissue inducer cells, double positive thymocytes, and Th17 cells. Analysis of RORg-deficient mice has revealed that this factor is critical for normal function of all three of these cell types. It is therefore of interest to understand how RORgt function is regulated. We have found that RORgt function can be negatively regulated by Egr3, a zinc finger transcription factor that is induced by a variety of stimuli, including TCR signaling. Ectopic expression of both RORgt and Egr3 in a thymocyte cell line prevents RORgt from binding to target gene promoters, and blocks target gene expression. We have employed multiple strategies to demonstrate that the inhibition of RORgt function is due to direct binding of RORgt by Egr3. Immunoprecipitation of either Egr3 or RORgt brings down the other protein, even when both proteins are obtained by in vitro translation. A mammalian two-hybrid system also indicates that the two proteins physically interact. Mutational analysis of RORgt indicates that the domain responsible for steroid receptor coactivator recruitment is required for binding to Egr3. These studies demonstrate a novel function for Egr3, and suggest a mechanism for regulation of thymocyte and Th17 cell differentiation.

This work is funded by a grant from the American Cancer Society.
Identification of RNA binding proteins involved in norepinephrine mediated regulation of Thy1 mRNA (35.45)
Authors: Koduvayur Sujatha P. , LaJaevic Melissa Dawn , Graf Lloyd H , Cohen Rhonna L , Chambers Donald A
Published: January 2023

Our laboratory has shown that norepinephrine (NE) increases Thy1 mRNA decay in S49 T lymphoma cells through a ß 2 adrenergic receptor/cAMP/protein kinase A pathway. Thy1 message, like many cytokine messages, contains an adenine and uridine rich element (ARE) in its 3′untranslated region (3′UTR) that has been associated with mRNA destabilization. In this study we identify RNA binding proteins from S49 wild type (wt) and a kinase deficient mutant (kin−) and determine if NE (150uM, 30 min) treatment of the cells alters binding. Photo affinity binding assay with cytoplasmic extracts from both cell types and 32P labeled, 170 nt long Thy1 ARE probe revealed the binding of twelve proteins of 30–105 kDa. Of these AUF1, HuR, TIAR, KSRP, and Hsc70 were identified by crosslinking followed by immunoprecipitation (IP). Crosslink IP also identified protein –protein interactions between TIAR and Hsc70. A pull down assay using a biotinylated RNA probe identified PAI-1binding protein and Hsp70, in addition to the others mentioned above. NE treatment of wt and kin− cells did not change the binding profile or affect the apparent ability of these proteins to bind. These results suggest that although the identified proteins may form a part of the mRNA decay machinery, modifications of these proteins or other factors may be involved in the NE-mediated regulation of Thy1 mRNA stability.

Supported by NIH.
Are our immunoglobulin V genes pre-adapted for recognizing pathogens: a 3-dimensional analysis (35.53)
Authors: Thomson Christy , Bryson Steve , McLean Gary , Pai Emil , Schrader John
Published: January 2023

There is strong phylogenetic evidence that the immunoglobulin V genes that make a major contribution to the generation of diversity of our primary repertoire, are under selective pressure, perhaps by pathogens themselves. Here we report the X-ray crystallographic structural analysis and the consequent kinetic and thermodynamic characteristics of a representative of a subclass of human neutralizing antibodies that originate from a particular pair of Vh and Vk genes and that target a short linear epitope on a human pathogen, HCMV. By comparison of its characteristics with its putative germline ancestors, these studies demonstrate that conserved germline Vh and Vk genes encode key residues in CDR-1 and CDR-2 that are pre-adapted to provide the foundation for the generation of a high-affinity hypermutated antibody. They show that although somatic mutation results in improvements in complimentarity of the binding site, the antibody combining site of the neutralizing antibody does not conform to a lock and key model and retains considerable mobility that is stabilized by interaction with antigen. We speculate that this flexibility may enhance its effectiveness at neutralization of infectivity.
The Effect of Vitamin C on the phenotype and function of Dendritic cells during activation with LPS (36.24)
Authors: Jeong Young Joo , Woo Ah Mi , Maeng Hyung Gun , Kang Jae Seung , Shin Dong-Hoon , Lee Wang Jae , Hwang Young-il
Published: January 2023

Activating DCs produce reactive oxygen species (ROS), which are necessary during antigen processing. In this study, we hypothesized that vitamin C (VC), as an antioxidant, may affect phenotype and function of DCs following activation. Bone marrow cells isolated from mice were cultured and differentiated into DCs in the presence of GM-CSF for 7 days. DCs were pre-treated with various concentrations of VC, and activated with LPS for 24 hrs. The supernatants were collected for measurements of cytokine secretion, and the changes of several markers of DC activation were analyzed. VC in the culture media reduced intracellular ROS levels, suggesting anti-oxidant effects on DCs. The expression of CD40 and CD80 were not influenced, but those of CD86 and MHC class II were slightly reduced. The reduction of phagocytic functions of DCs with activation was suppressed by the presence of VC, dose-dependently. In the supernatants, dose-dependent reduction of IL-6 and increased secretion of IL-12p70 and IL-10 were observed with increasing concentration of VC. IFN-¥ã was not detected at all. Because VC affected some of surface marker expression and phagocytic functions of activated DCs and, most noticeably, changed cytokine secretion profiles, we also investigated the results of T cell activation by those DCs treated with VC, with respect to T cell proliferation and cytokine secretion by activated T cells.
SIRNA KNOCKDOWN OF SPHK1 PROTECTS MICE FROM IGE-MEDIATED ANAPHYLAXIS (37.6)
Authors: Melendez Alirio J. , Jayapal Manikandan , Pushparaj Peter Natesan
Published: January 2023

Sphingosine kinases are emerging a key enzymes in the intracellular signaling cascades, that mediate inflammatory responses, triggered by various inflammatory stimuli including: anaphylatoxins (C5a); bacterial-derived peptides (fMLP); and IgG or IgE receptor stimulation (FcγRI and FcεRI), in various immune cells, including in human mast cells. Moreover, we have recently reported a study on genome-wide gene expression profile of human mast cells sensitized with IgE alone, or stimulated by FcεRI aggregation, and found that sphingosine kinase 1 (SphK1) is one of the early genes to be activated. This, coupled to our previous studies indicating that SphK1 is pivotal in the signaling pathways initiated by FcεRI in human mast cells, prompted us to investigate the functional role of SphK1 in IgE-triggered inflammation/anaphylaxis in vivo. The systemic anaphylaxis reaction in mice has been considered to be a typical immediate hypersensitivity response determined by the activation of immune cells via antigen-induced aggregation of an IgE-sensitized FcεRI. To this end, we silenced the SPHK1 gene in vivo (using a specific siRNA), and investigated whether SphK1 knockdown influenced the outcome of the acute inflammatory responses, triggered during IgE-mediated systemic anaphylaxis in mice. We show here that, the siRNA for SphK1 protected mice from the IgE-mediated allergic reactions, including inhibition on: temperature changes; vascular permeability; immune-cell infiltration into organs; cytokine production; and vascular cell adhesion molecule expression. Thus, our findings strongly support a role for SpkK1 IgE-mediated anaphylaxis in vivo.
Thymic stromal lymphopoietin (TSLP) is a potent activator of human mast cells (38.4)
Authors: Jessup Heidi K. , Allakhverdi Zoulfia , Comeau Michael R. , Park-Yoon Bo-Rin , Ziegler Steven F. , Delespesse Guy
Published: January 2023

Compelling evidence suggests that the epithelial cell-derived cytokine thymic stromal lymphopoietin (TSLP) may initiate asthma or atopic dermatitis through a dendritic cell-mediated Th2 response. Here we describe how TSLP might initiate and aggravate allergic inflammation via the innate immune system. We show that TSLP stimulates the production of Th2 cytokines by progenitor-derived human mast cells (MC). MC expressed each chain of the TSLP receptor complex, i.e. the TSLP-binding chain (TSLP-R) and the IL-7R-α chain at the mRNA and protein levels. TSLP-R was also detected on MC infiltrating the bronchial mucosa of asthmatic patients as revealed by staining biopsy specimen with TSLP-R specific mAb. TSLP dose-dependently induced the release of IL-5 which was detectable after 6 h of culture and reached plateau at 24 h. The response of MC was specifically suppressed by neutralizing mAb to TSLP or TSLP-R. TSLP did not induce MC degranulation or the release of lipid mediators. In contrast, high levels of IL-5, IL-13, IL-6 and GM-CSF were released after 24 h of MC stimulation. TSLP exerted an opposing effect on the production of anti-inflammatory cytokines, i.e. it suppressed TGF-β and enhanced IL-10. Among several chemokines examined, TSLP enhanced the production of only IL-8 and I-309. Finally, we show that primary airway and skin epithelial cells release functionally sufficient levels of TSLP to stimulate human MC.
Studies of IgE receptor (FceRI) processing in human basophils (HB) (38.7)
Authors: MacGlashan Donald
Published: January 2023

Studies HB in the 1980s suggested that FceRI was not down-regulated following aggregation of the receptor. We now show that receptor, after strong stimulation, is lost from the surface and degraded, but with a very slow time course; the T1/2 for loss from the surface or degradation was 12 hours and 24 hours, respectively. FceRI degradation is inhibited with Bafilomycin A suggesting a lysosomal route of loss. Under strong stimulation there was modest loss of un-involved receptors, 13±5% loss @ 18 hrs. Loss of receptor was only partially sensitive to inhibition with a src-family kinase inhibitor, PP1, and insensitive to presence of a third generation syk inhibitor, NVP-QAB205. Previous studies have noted that there is both immature receptor (p46) and mature receptor (p60) expressed and in the current studies we noted that p46 receptor expression increased following stimulation with both IgE-dependent and IgE-independent stimuli. Resting levels were inhibited by cycloheximide, actinomycin D, MG-132, PP1 and NVP-QAB205 and enhanced with Bafilomycin A. However, these studies found that the lysosomal processing was sensitive to apoptosis and suggested that these changes were not due to changes in synthesis of p46 but to changes in degradation rate. Studies of unoccupied receptor loss also indicated endocytosis and lysosomal degradation (and some recycling), i.e., both forms of FceRI loss depend on lysosomal processing.

Supported by NIH-AI20253
Perinatal Exposure to Environmental Tobacco Smoke and Immunological Consequences in Primate Infants (40.11)
Authors: Hood Carol , Peak Janice , Spinner Abby , Datta Seema , Fiehn Oliver , Pinkerton Kent
Published: January 2023

Mechanisms of environment tobacco smoke (ETS) to adverse health in children are unknown. To understand how ETS may alter the developing immune system of nonhuman primates, rhesus macaque infants were exposed to ETS during perinatal life at 1mg/m3 total suspended particulates for 6 hours per day, 5 days per week. Four treatment groups were involved:

ETS from gestation day 40 to 3 months,ETS exposure from gestation day 40 to 13 months of age,ETS exposure starting 6 months post natal until 13 months of age,sham-control / filtered air throughout gestation and postnatal age to 13 months.

At three months of age, all ETS infants demonstrated a significant reduction in plasma protein (P = .03). Nicotine levels were three times higher in younger infants (3 months) than infants at 13 months of age (P = .002 Fisher) suggesting age-based differences in metabolism. Morphometric methods were used to examine the spleen. There was a significant increase in the relative proportion and volume of the white pulp found in the spleen following exposure to ETS at 3 months of age as well as in animals with exposure to ETS beginning at 6 months of age, compared to their control groups (P=<.0001, P =.02, P = .01, P=.04 respectively) . These findings suggest that there are critical windows of susceptibility in infants to ETS. Changes in the proportion and volume of the white pulp of the spleen by ETS may play a key role to alter the immune status of the neonate.
Differential regulation of mucosal immunity by Bacillus anthracis edema toxin and cholera toxin as adjuvants for transcutaneous vaccines (41.3)
Authors: Duverger Alexandra , Tang Wei-Jen , Leppla Stephen H , Boyaka Prosper N
Published: January 2023

Our previous studies have shown that nasal co-administration of an anthrax edema toxin (EdTx) derivative [i.e., protective antigen (PA) plus an edema factor (EF) mutant (EF N447S)], enhances mucosal and systemic immunity to both PA and co-administered unrelated protein antigen. Unlike the ganglioside targeting adjuvant, cholera toxin (CT), nasal EdTx N447S did not target CNS tissues or induce inflammatory responses in the NALT. Here, we examined whether the difference in the cell targeting of these two adjuvants would lead to different mucosal and systemic Ab responses when used in transcutaneous vaccines. We found that both CT and EdTx N447S promoted IgG Ab responses in the serum. However, transcutaneous application of EdTx N447S, or even native EdTx, did not induce IgA Ab responses in the blood or in mucosal secretions. We next analyzed APC (i.e., B cells, macrophages and DCs) in the skin, inguinal lymph nodes, mesenteric lymph nodes (MLN) and Peyer’s patches (PP) of mice 18 hrs after transcutaneous application of OVA alone, OVA plus CT or OVA plus EdTx. Transcutaneous application of CT or EdTx affected the distribution of APCs and their expression of MHC class II and co-stimulatory molecules (i.e., CD80 and CD86) in the skin and draining lymph nodes. However, only CT had a significant effect on these parameters in the MLN and intestinal PP. Ongoing studies will determine the molecular signaling (i.e., cytokines and homing receptors) triggered by the transcutaneous application of CT vs. EdTx to affect systemic vs. mucosal tissues.

(Supported by NIH Grant: AI 43197)
Head and Neck Lymph Node (HNLN) B Cells from L-Selectin−/− (L-Sel−/−) Mice Show αE Expression Independent of Cholera Toxin (CT) Exposure (41.9)
Authors: Riccardi Carol , Pascual David W.
Published: January 2023

Oral immunization with soluble protein plus adjuvant stimulates excellent regional, but not distal Ab responses. Although low level expression of distal immune B cells is observed, these wane with time. Studies examining homing receptor expression should provide insight into understanding how distal mucosal immunity is maintained. Studies showed that B lymphocyte trafficking in the HNLN and NALT is L-Sel-dependent. However, the absence of L-Sel enhanced distal B cell maintenance following oral CT immunization of L-Sel−/− mice and was partly compensated by increased αEβ7+ B cells, as evident by the significantly increased IgA antibody-forming cells (AFC) in L-Sel−/− nasal passages (NP) when compared to B6 NP. These IgA responses were supported by elevated HNLN IgA AFC responses (10- to 100-fold greater), and these AFC endured beyond 42 days, but not in B6 mice. To discern whether αEβ7 expression was influenced by CT directly, naive HNLN, mesenteric LN (MLN), and Peyer’s patches (PP) were isolated and stimulated in vitro with LPS, LPS + CT, Con A, or media. The mitogen-stimulated L-Sel−/− HNLN, MLN, and PP B cells showed early expression of αEβ7 at 48 hrs, but by 96 hrs, the L-Sel−/− HNLN clearly showed the greatest levels (30–35%) of αEβ7+ B cells. Both unstimulated and stimulated HNLN B cells showed similar levels of αEβ7 expression, and CT did not enhance αEβ7 expression. MLN and PP showed fewer (~10%) αEβ7+ B cells, and B6 B cells from each tissue showed <10% αEβ7+ B cells. These studies show that αEβ7 compensates for the absence of L-Sel on B cells, and these B cells are mostly derived from HNLN, and not from the gut.

Supported by NIH AI-55563.
Epithelial cell-intrinsic IKKβ expression regulates innate and adaptive immunity in the gut (42.4)
Authors: Zaph Colby , Troy Amy E. , Taylor Betsy C. , Berman-Booty Lisa D. , Guild Katherine J. , Du Yurong , Yost Evan A. , Gruber Achim D. , May Michael J. , Greten Florian R. , Eckmann Lars , Karin Michael , Artis David
Published: January 2023

Intestinal epithelial cells (IECs) provide a primary physical barrier against commensal and pathogenic microorganisms in the gastrointestinal (GI) tract, but the influence of IECs on the regulation of immunity is unknown. Here we show that IEC-intrinsic IKKβ-dependent gene expression is a critical regulator of dendritic cell (DC) and CD4+ T cell responses in the gut. Following infection with the parasite Trichuris, littermate control mice develop pathogen-specific TH2 cytokine responses and eradicate infection while mice with an IEC-specific deletion of IKKβ (IkkbΔIEC mice) fail to do so. Further, IkkbΔIEC mice exhibit exacerbated production of DC-derived IL-12/23p40 and TNF-α, heightened levels of CD4+ T cell-derived IFN-γ and IL-17, and develop severe intestinal inflammation. Blockade of proinflammatory cytokines during Trichuris infection ablates the requirement for IKKβ in IECs to promote CD4+ TH2 cell-dependent immunity, identifying a critical role for IECs in limiting type 1 cytokine production in the GI tract. These results suggest that the balance of IKKβ-dependent gene expression is critical for intestinal immune homeostasis by promoting mucosal immunity and limiting chronic inflammation.

This work is funded by the NIH, CCFA and the Irvington Institute.
Role of effector B and T lymphocytes and cytokine production in the protective host response to vaccination with heat killed bacilli against Burkholderia mallei (43.14)
Authors: Whitlock Greg C. , Rowland Caroline , Lukaszewski Roman A. , Judy Barbara M. , Paessler Slobodan , Torres Alfredo G. , Estes D. Mark
Published: January 2023

Burkholderia mallei, the etiologic agent of glanders disease, is a gram-negative, intracellular bacterium for which no vaccine is available. Consequently, we are interested in defining protective host immune responses to B. mallei infection with the goal of developing candidate vaccines. Here, we report on initial cell/cytokine depletion studies to investigate the possible role these effectors may play in response to vaccination with heat killed bacilli in a susceptible BALB/c mouse model of infection. While protection with heat killed bacilli does not result in sterilizing immunity, protection is afforded against an otherwise lethal infection and provides insight into potential host protective mechanisms. Our results demonstrate that mice treated with either anti-B220, TNF-α or IFN-γ monoclonal antibodies exhibited decreased survival rates, indicating a key role for vaccine induced antibody and early cytokine production in protection from lethal challenge infection by the intraperitoneal route.
PD-1 plays a downmodulatory role in a murine immunodeficiency syndrome (43.26)
Authors: Green Kathy A , Okazaki Taku , Honjo Tasuku , Green William R.
Published: January 2023

An immunodeficiency syndrome is induced by infection of C57BL/6 (B6) with the murine leukemia virus LP-BM5. Other labs have shown that this syndrome requires CD4 T and B cell interactions. We have found it to be dependent on: CD154/CD40 and CD40 associated TRAF 6 and nondependent on CD80 and CD86. Gene chip analysis on spleen cells from LP-BM5 infected B6 mice revealed that the PD-1 and IL-10 genes are highly expressed after LP-BM5 infection. PD-1, a transmembrane cellular protein, was originally identified in a T cell line undergoing cell death and its ligation is generally considered to result in negative regulation of immune cell interactions and responses. We examined the role of PD-1 in LP-BM5 induced immunodeficiency via the use of B6.PD-1 k.o. mice and found that they develop substantially more severe disease than w.t. mice, as a function of; viral dose delivered in vivo and kinetics. PD-1 ligands, PD-L1, and PD-L2 have been identified, and flow cytometry shows that PD-L1 is preferentially upregulated during in vivo LP-BM5 infection. In in vivo antibody blocking studies, after LP-BM5 infection, anti-PD-L1, but not anti-PD-L2 treatment led to exaggerated disease. Other labs have reported that PD-L1 ligation of PD-1 leads to increased IL-10 production. We have found IL-10 k.o. mice are more susceptible to LP-BM5 induced immunodeficiency than w.t. mice. Thus, PD-1/PD-L1 and IL-10 pathways both appear to play roles in the moderation of LP-BM5 pathogenesis. Adoptive transfer experiments, using combinations of w.t., vs PD-1 k.o., CD4 T vs B cells to reconstitute LP-BM5 infected Rag 1 k.o. mice, suggest that B-cell expressed PD-1 is critical for the moderation of LP-BM5 induced pathogenesis.
Vaccinia Virus-specific CD4+ T cell Responses Target a Set of Antigens Largely Distinct from those Targeted by CD8+ T cell Responses (43.34)
Authors: Moutaftsi Magdalini , Bui Huynh-Hoa , Peters Bjoern , Sidney John , Salek-Ardakani Shahram , Oseroff Carla , Pasquetto Valerie , Crotty Shane , Croft Michael , Grey Howard , Sette Alessandro
Published: January 2023

Vaccinia virus (VACV) is a potent and effective vaccine, inducing long-lasting protective cellular immune responses. Recent studies defined the antigens recognized in mice and humans by CD8+ T cell responses. However, little is known about the specificities of CD4+ T cell responses, which makes it problematic to study and monitor cellular immune responses to VACV infections.

Herein, we report for the first time the identification of VACV-specific CD4+ T cell epitopes. By screening a large set of 15-mer peptides (2146 in total) in C57BL/6 mice, we identified 14 IAb-restricted T cell epitopes. These epitopes account for about 30% of the total anti-VACV CD4+ T cell response and are derived from 13 different structural and viral genome regulation proteins. Surprisingly, no CD4+ T cell epitopes were identified that target virulence factors. While early antigens are recognized, late antigens predominate as CD4+ T cell targets. These results are in sharp contrast to what was previously found in CD8+ T cells responses, where early antigens including virulence factors are prominently recognized.

Taken together, these results highlight fundamental differences in immunodominance of CD4+ and CD8+ T cell responses to a complex pathogen and have potential implications for the development of subset vaccines.

Funded by the NIH: Contract # HHSN266200400024C and RO1 grant # RO1-AI-56268. Kirin Publication Number: 837
Cervicovaginal epithelial cell cytokine production in response to microorganisms associated with bacterial vaginosis (43.40)
Authors: Trama Jason , Libby Erika , Mordechai Eli , Adelson Martin
Published: January 2023

Bacterial vaginosis (BV) is the most common vaginal disorder among women of reproductive age. However, the pathogenesis of BV is poorly understood. Although direct causality has not been demonstrated, local epithelial cells responding to microorganisms are proposed to be the source of elevated IL-1β, IL-6 and IL-8 in the vaginal washes of women with BV. Here, we determine the inflammatory cytokine responses of immortalized cervical and vaginal epithelial cells (CVEC) when co-cultured with lactobacilli and BV-associated microorganisms including Atopobium vaginae, an anaerobic, gram-positive bacterium common in women with heterogeneous vaginal flora and more specific for BV than Gardnerella vaginalis. Dose-dependent induction of IL-8 was seen when CVEC were co-cultured with A. vaginae, but not when CVEC were co-cultured with lactobacilli or G. vaginalis at MOIs of up to 100. The induction of IL-8 was similar in endocervical, ectocervical and vaginal epithelial cells. The release of IL-8 reached a maximum by 4 hours, was sustained for 24 hours and was abrogated by heat treatment of A. vaginae. Examination of IL-1β, IL-6, IL-8, IL-10 and TNFα secretion by bead array showed induction of IL-6 and IL-8 by A. vaginae. These results suggest that A. vaginae and CVEC are contributing factors to the cytokine production and the inflammatory sequelae associated with BV.

Supported by Medical Diagnostic Laboratories LLC.
Nippostrongylus brasiliensis infection modulates Mycobacterium tuberculosis induced Th1 response (43.45)
Authors: bhatt kamlesh , Liu Zhugong , Gause W C , Salgame P
Published: January 2023

Modulation of Th1 response generated against pathogens like M tuberculosis (Mtb) by helminths is an important, area of investigation, because of the prevalence of coinfections in TB endemic regions. In this study we have designed an approach to investigate how the Th1 response generated by Mtb is modulated by helminthic infection. We have used the ovalbumin – specific TCR transgenic adoptive transfer system, wherein the ability of Mtb-pulsed dendritic cells to initiate a Th1 response was studied on the background of a helminth infection. BALB/c mice were infected with N. brasiliensis, and groups of uninfected and infected mice were injected with CFSE labeled OVA-specific TCR transgenic T cells followed by intratracheal instillation of Mtb and ovalbumin peptide-pulsed bone marrow-derived dendritic cells. Analysis of the labeled transgenic T cells harvested from the draining lymph nodes indicated proliferation of T cells from both groups of mice. However, we observed significantly reduced level of IFNγ and a reciprocal increase in IL-4 in T cells derived form the helminth-infected mice. These findings indicate that helminth infection does not abrogate the priming ability of Mtb-pulsed DCs, but interferes with their ability to promote a Th1 response. The significance of the findings lies in the potential of helminth infections to modulate resistance to tuberculosis, and also to interfere with the efficacy of tuberculosis vaccines.
Type I interferon and IL-12 induce phenotypic variegation of human CD4+ T cells (43.7)
Authors: Davis Ann M , Matthews Loderick A , Ramos Hilario J , Farrar J David
Published: January 2023

Antigen presenting cells secrete distinct patterns of innate cytokines upon activation via Toll-like receptors (TLRs). These innate cytokines shape adaptive immune responses to pathogens. For example, interleukin 12 (IL-12) is produced in response to bacterial recognition via TLRs 2, 4, 5, and 6. IL-12 induces CD4+ T helper 1 (Th1) cell development through the activation of signal transducer and activator of transcription 4 (STAT4), leading to the secretion of interferon gamma (IFN-γ). Conversely, during viral infection, signaling through TLRs 3, 7, and 9 results in the secretion of both IL-12 and type I interferon (IFN-α/β). The effects of combined IL-12 and IFN-α/β stimulation on T helper development are not well-studied. We have demonstrated that activation of naïve CD4+ T cells in the presence of IFN-α/β alone fails to induce Th1 development. However, a combination of IL-12 and IFN-α/β induces phenotypic variegation to generate two populations of T helper cells. An effector population secretes both IFN-γ and IFN-α/β, and these cells are efficient in eliminating viral infections in vitro. A second subset displays memory properties and may be involved in long-term viral immunity. Furthermore, these two subsets can be distinguished by expression of chemokine receptors. These data have important implications for the understanding of viral infections as well as for the development of novel vaccines and therapies.
Inhibition of dendritic cell IL-12 production by Giardia lamblia (44.1)
Authors: Kamda Joel D. T. , Singer Steven M.
Published: January 2023

Unlike most other enteric infections, Giardia lamblia infection does not typically induce mucosal inflammation. We therefore studied the response of bone marrow-derived dendritic cells (BMDCs) to Giardia extracts. G. lamblia is only a weak activator of BMDCs. Compared to LPS, co-incubation of G. lamblia extract results in only moderate up-regulation of the co-stimulatory molecules CD80 and CD86. In addition, G. lamblia extract causes minimal induction of IL-6 and TNF and no detectable IL-10 or IL-12. Interestingly, pre-exposure of BMDCs to G. lamblia extract inhibits the secretion of the pro-inflammatory cytokine IL-12 by LPS activated BMDCs. IL-12 suppression by G. lamblia is apparently dependent on its activation of the phosphotidylinositol 3-Kinase (PI3-K) pathway, as it is abrogated by pretreatment of BMDCs with the specific PI3-Kinase inhibitor, wortmannin. These results suggest that Giardia actively prevents inflammation, and suggests a distinct mechanism of immune regulation by this intestinal parasite.
The pattern recognition molecule mindin promotes intranasal clearance of influenza viruses. (44.10)
Authors: Jia Wei , He You-Wen
Published: January 2023

The innate immune response is essential for host defense against microbial pathogen infections and is mediated by pattern recognition molecules recognizing pathogen associated molecular patterns (PAMPs). Our previous work has demonstrated that the extracellular matrix protein mindin functions as a pattern recognition molecule for bacterial pathogens. Here we examined the role of mindin in influenza virus infection. We found that intranasal infection of mindin-deficient mice by influenza viruse resulted in dramatically increased virus titers in the lung and intranasal cavity of mutant mice. In contrast, lungs from intratracheally infected mindin-deficient mice contained similar influenza virus titers. We showed that mindin interacted with influenza virus particles directly and that mindin-deficient macrophages exhibited impaired activation after influenza virus infection in vitro. Furthermore, intranasal administration of recombinant mindin significantly enhanced the clearance of influenza virus in wildtype mice. Together, these results demonstrate that mindin plays an essential role in host innate immune response to influenza virus infection and suggest that mindin may be used as an immune enhancing agent in influenza infection.

1. This work was supported by NIH grants AI054658 and AI061364.
Role of dectin-1 in the host defense to infection with Cryptococcus neoformans (44.17)
Authors: Miyazato Akiko , Nakamura Kiwamu , Saijo Shinobu , Iwakura Yoichiro , Kaku Mitsuo , Kawakami Kazuyoshi
Published: January 2023

pathogen-associated molecular patterns (PAMP) are recognized by Toll-like receptors (TLR) and C-type lectin recptors including Dectin-1. Previously, we indicated that neither TLR2 nor TLR4 was involved in the host defence to infection with Cryptococcus neoformans, an opportunistic fungal pathogen in AIDS patients (FEMS Immunol. Med. Microbiol. 47: 148–154, 2006). In the current study, we examined the role of Dectin-1, a receptor for β-glucan, in this response. Dectin-1-deficient mice were resistant to intratracheal and intravenous infection with C. neoformans at a comparable level to wild-type mice. IFN-γ production in lung an serum was not largely different between these mice. There was not significant difference in the synthesis of IL-12p40 and TNF-α by bone marrow-derived dendritic cells upon stimulation with this fungal pathogen. Taken together, these results demonstrated that Dectin-1 did not play a major role in the host protective responses to C. neoformans infection.

This work was supported in part by a Grant-in -Aid for Science Research (C) (KAKENHI18590413) from the Ministry of Education, Culture, Sports, Science and Technology and by Tohoku University 21st COE program ′CRESCENDO′
LPS deacylation by a host enzyme shortens endotoxin tolerance (44.20)
Authors: Lu Mingfang , Munford Robert
Published: January 2023

Prior exposure to bacterial lipopolysaccharide (LPS) alters animals’ responses to a second LPS exposure. Although this reprogramming (also called endotoxin tolerance) may prevent harmful inflammatory reactions, it also may decrease resistance to subsequent infection. How animals recover from tolerance is not known. Acyloxyacyl hydrolase (AOAH) is a host lipase that inactivates LPS by cleaving the secondary fatty acyl chains from lipid A. We found that 21 days after an initial dose of LPS, long after the responses of normal mice had returned to baseline, Aoah−/− mice had diminished serum IL-6 and TNF responses to LPS challenge. Ten days after receiving LPS i.p., macrophages explanted from Aoah−/− mice and stimulated ex vivo with LPS produced less IL-6 and TNFα than did macrophages from LPS-primed control mice. Whereas the LPS that was associated with peritoneal cells of Aoah+/+ mice was completely deacylated by 10 days, LPS remained undeacylated in Aoah−/− peritoneal cells. LPS-primed Aoah−/− mice were unable to control bacterial growth and therefore were more susceptible to lethal E. coli infection. LPS inactivation by AOAH shortens the duration of tolerance and its associated immunosuppression. A better understanding of how tolerance is terminated may help ameliorate post-infection immunosuppression in humans.

Supported by NIAID grant AI18188.
Role for platelet toll-like receptor 4 (TLR4) in the formation of Neutrophil Extracellular Traps (NETs) in sepsis (44.22)
Authors: Ma Adrienne , Clark Stephen , Tavener Samantha , Patel Kamala , Chakrabarti Subhadeep , Sinclair Gary , Keys Elizabeth , Allen-Vercoe Emma , DeVinney Rebekah , Doig Christopher , Green Francis , Kubes Paul
Published: January 2023

It has been known for many years that LPS, neutrophils and platelets all participate in the pathogenesis of severe sepsis, but the inter-relationship between these players is completely unknown. Using flow chambers in vitro we suggest a novel innate immune response leading to enhanced trapping of bacteria in blood vessels. The mechanism involved platelet TLR4 detecting LPS in blood and inducing a unique response, specifically platelet binding to adherent neutrophils, but not platelet aggregation or P-selectin expression. Subsequently, the platelets stimulated very robust neutrophil activation leading to formation of NETs. These NETs retained their integrity under flow conditions and functioned to ensnare bacteria within the vasculature. Plasma from severely septic patients also induced TLR4-dependent platelet-neutrophil interactions leading to the production of NETs. We propose that this novel bacterial trapping mechanism would only occur under extreme conditions such as severe sepsis and platelet TLR4 (not leukocyte TLR4) functioned as the threshold switch for this innate immune response to occur. With the advent of antibiotics perhaps reducing the need for NET formation, we would propose that inhibiting platelet activation with TLR4 inhibitors may inhibit NET formation and reduce inadvertent tissue injury.

This work was funded by: CIHR and AHFMR
CCL2 antisense therapy improves the resistance of burn mice to Enterococcus faecalis translocation (44.23)
Authors: Shigematsu Kenji , Tsuda Yasuhiro , Kobayashi Makiko , Herndon David N , Suzuki Fujio
Published: January 2023

M2Mϕ are inhibitors of the Mϕ conversion from resident Mϕ to M1Mϕ, an antibacterial effector cell in innate immunities. CCL2 has been described as an inducer of M2Mϕ. To manipulate the inhibitory function of M2Mϕ on the generation of M1Mϕ, in this study we performed CCL2 antisense therapy in burn mice. CCL2 antisense ODN was administered s.c. to mice 2 hrs after burn injury (3rd degree, 25% TBSA burn). One day after burn injury, Mϕ were prepared from mesenteric lymph nodes of these mice, and cultured in transwells with a 10x higher number of resident Mϕ (1 x 106 cells/ml, lower chamber) in media supplemented with CpG DNA (10 μg/ml). To detect the M1Mϕ generation, the culture fluids obtained were assayed for CCL3 (a typical biomarker for M1Mϕ). In some experiments, decontaminated burn mice were infected orally with a lethal dose of E. faecalis (107 CFU/ml), and treated s.c. with CCL2 antisense ODN once daily for 5 days. In the results, M1Mϕ were not generated from resident Mϕ transwell-cultured with M2Mϕ even when they were stimulated with CpG DNA. However, M1Mϕ were generated when the same transwell-cultures were performed with resident Mϕ and Mϕ from burn mice that were previously treated with CCL2 antisense ODN. After E. faecalis infection, 75% of burn mice died, while 80% of burn mice subjected to CCL2 antisense gene therapy survived. These results indicate that M1Mϕ are inducible in burn mice subjected to CCL2 antisense therapy, and the resistance of burn mice to E. faecalis translocation is improved after this antisense gene therapy.

This study was supported by SHC NA #8840.
Inhibitory effect of glycyrrhizin on the expression of c-fos by an immunosuppressive subset of murine neutrophils (PMN-II) (44.30)
Authors: Kobayashi Makiko , Yoshida Tsuyoshi , Herndon David N , Suzuki Fujio
Published: January 2023

The root of Glycyrrhiza glabra (licorice) and its principle component (glycyrrhizin) have revealed various immunomodulatory properties that fight off attacking pathogens. In our previous studies, glycyrrhizin was shown to prevent infectious complications developed in mice with systemic inflammatory response syndrome (SIRS) through the inhibition of CCL2 production by PMN-II. c-fos (one of the nuclear transcription factors) is known as a CCL2 gene promoter. Therefore, in the present study, c-fos mRNA expression in PMN-II treated with or without glycyrrhizin was analyzed. For these studies, SIRS was induced by a flame burn injury (3rd degree, 25% TBSA burns). PMN-II were isolated from the peripheral blood of mice 18 hrs after burn injury by Ficoll-Hypague and dextran sedimentations, followed by the depletion of contaminating monocytes using magnetic beads coated with anti-F4/80 mAb. In the presence of Staphylococcus aureus antigen (0.0075%), PMN-II (1 x 106 cells/ml) were cultured with various doses of glycyrrhizin for 12 hrs, and the culture fluids obtained were assayed for CCL2. Also, the obtained cells were analyzed for c-fos mRNA by RT-PCR. In the results, 3.7 ng/ml of CCL2 was detected in the culture fluids of PMN-II, while PMN-II treated with glycyrrhizin did not produce CCL2. c-fos mRNA was expressed in PMN-II, but not in PMN from normal mice. When PMN-II were treated with 10 μg/ml of glycyrrhizin, c-fos mRNA expression was greatly decreased. These results suggest that c-fos expression in PMN-II may be controlled by glycyrrhizin when CCL2 production by these PMN is inhibited by the compound.

This study was supported by SHC NA #8840.