bluuebunny/crossref_metadata_2025 huggingface dataset:
Single cell network profiling (SCNP) assay allows for the identification of heterogeneity in the activation of multiple signaling pathways in distinct human B cell subsets (174.12)
Authors: Longo Diane , Conroy Andy , Louie Brent , Wang Ena , Pos Zoltan , Marincola Francesco , Hawtin Rachael , Cesano Alessandra
Published: January 2023

The specific functions of phenotypically defined B cell subsets in the regulation of protective immune responses and the pathogenesis of immune-mediated diseases is incompletely understood. SCNP is a multi-parametric flow cytometry based approach that simultaneously measures intracellular signaling in multiple cell subsets in heterogeneous tissues. In this study, SCNP was applied to examine 34 signaling nodes (paired modulator/intracellular readout) in B cell subsets (CD27-IgM+IgD+ naïve B cells, total CD27+ memory B cells, CD27+IgM-IgD- switched memory B cells, CD27+IgM+IgD+ IgM memory B cells) in peripheral blood mononuclear cells from 6 healthy donors. Signaling responses in B cell subsets were masked when the total B cell population was analyzed. Specifically, a higher responsiveness was observed in the memory B cell subset relative to the naïve; 14 signaling nodes showed statistically significant differences between these subsets (p<.05, paired Wilcoxon test) of which 12 displayed stronger activation in response to CD40L, SDF1α, PMA, IFNα, IFNγ, IL10 and IL27 modulation in the memory B cells. 11 signaling nodes differed significantly in IgM memory vs. switched memory B cells (p<.05, paired Wilcoxon test); 7/11 displayed stronger activation in response to CpG, R848, thapsigargin, IFNγ and IL10 modulation in IgM memory B cells. An increased understanding of B cell subset signaling may allow for the development of more effective B cell targeted therapies.
MicroRNA-29 Stabilizes IFN-γ mRNA by inhibiting GW182 (174.5)
Authors: Savan Ram , Legiewicz Michal , McFarland Adelle , Schwerk Johannes , Bindewald Eckart , Lin Fan-ching , Orr Selinda , Yoshikawa Noriko , Hegamyer Glenn , Yalamanchili Rajesh , Ramakrishnan Karthika , Kronfli Anthony , Saleh Bahara , McVicar Daniel , Carrington Mary , Colburn Nancy , Anderson Stephen , Shapiro Bruce , Le Grice Stuart , Young Howard
Published: January 2023

Tight regulation of interferon-gamma (IFN-γ) expression is critical for an optimal immune response. We have extensively studied the mechanisms responsible for the post-transcriptional regulation of IFN-γ expression. AU-rich element (ARE)-mediated decay (AMD) via the tristetraprolin (TTP) complex is responsible for robust degradation of the IFN-γ mRNA. We found that microRNA-29 (miR-29) targets the IFN-γ 3’UTR and, unlike the conventional inhibitory miRNAs, stabilizes the mRNA. Resolution of the secondary structure showed that the ARE and miR-29 binding site are in close proximity and a series of experiments manipulating the RNA structure revealed that this proximity enables miR-29 to antagonize TTP degradation. Interestingly, miR-29 is unable to degrade the mRNA even in the absence of TTP. Recently we have found that GW182, a major degradation protein in the miRISC, is unable to be recruited to the mRNA as a result of the location of the miR-29 binding site in the secondary structure. The ability of miR-29 to stabilize the IFN-γ mRNA demonstrates a novel molecular mechanism of miRNA regulation.
Crosstalk between STAT3 and RANK signaling pathways during osteoclastogenesis (174.7)
Authors: Zhang Huiyuan , Hu Hongbo , Jin Jin , Ohashi Erika , Greeley Nathaniel , Li Haiyan , Sun Shao-cong , Watowich Stephanie
Published: January 2023

Osteoclasts are a specialized cell type in the bone marrow microenvironment with a critical role in bone resorption. Osteoclasts develop from macrophage precursors in response to RANK ligand (RANKL) stimulation. Osteopenia and pathologic fractures frequently occur in patients with Hyper IgE syndrome, which is caused by loss-of-function mutations in STAT3 . How impaired STAT3 function translates to these clinical features is not understood. We found that mice with conditional Stat3 deletion in bone marrow have increased osteoclast numbers in vivo; therefore, we examined STAT3 function in RANKL-induced osteoclast differentiation from bone marrow macrophages (BMMs). We found that STAT3 negatively regulates RANK signaling by inhibiting TRAF6 ubiquitination and activation of the downstream signaling proteins NF-κB, JNK and p38 MAPK. Stat3-deficient BMMs show enhanced osteoclast gene expression and osteoclast formation in vitro. STAT3 is induced in BMMs by M-CSF, not RANKL, suggesting that M-CSF signal transduction via STAT3 conditions osteoclast precursors for appropriate RANK-RANKL signaling. We identified the deubiquitinase Uch37 as a STAT3-dependent gene regulating osteoclastogenesis. Thus, our data suggest STAT3 restrains osteoclast development and bone resorption by mediating the amplitude of RANK signaling via Uch37 regulation.
Neuroimmune semaphorin 4D is necessary for optimal lung allergic inflammatory response (175.16)
Authors: Chapoval Svetlana , Nkyimbeng-Takwi EusebuisHenry , Shanks Kathleen , Smith Elizabeth , Iyer Apoorva , Lipsky Michael , DeTolla Louis , Keegan Achsah
Published: January 2023

Neuroimmune semaphorin 4D (Sema4D) was found to be expressed and function in the nervous and immune systems. In the immune system, Sema4D is constitutively expressed on T cells and regulates T cell priming. Currently available data suggest that Sema4D has crucial stimulatory functions in a broad range of cells including macrophages, DC, NK cells, and neutrophils. We hypothesized that Sema4D plays a critical non-redundant regulatory role in allergic airway response. To test our hypothesis, we employed Sema4D-/- and WT mice in the well-defined mouse model of OVA-induced experimental asthma. We observed a decrease in eosinophilic airway infiltration in allergen-treated Sema4D-/- mice relative to WT mice. This reduced allergic inflammatory response was associated with decreased BAL IL-5, IL-13, and TGFb1 levels. In addition, T cell proliferation in OVA323-339 - restimulated Sema4D-/- cell cultures were downregulated. However, airway hyperreactivity to methacholine challenges was not affected by Sema4D deficiency. These data provide a new insight into Sema4D biology and define Sema4D as an important regulator of Th2-driven lung pathophysiology.
Allergic lung inflammation and susceptibility to Streptococcus pneumoniae (175.2)
Authors: Sanfilippo Alan , Metzger Dennis
Published: January 2023

Streptococcus pneumoniae is the most common cause of bacterial pneumonia which results in 175,000 hospitalizations as well as 40,000 deaths in the United States annually. With the introduction of a conjugate vaccine, overall disease burden has declined. However, a link between asthma and invasive pneumococcal disease (IPD) has been identified, prompting the Advisory Committee on Immunization Practices to recommend that all adults with asthma receive the pneumococcal conjugate vaccine. To study the mechanism responsible for an increased risk of IPD for individuals with asthma, we used a model of ovalbumin (OVA)-induced allergic lung inflammation (ALI). We hypothesized that mice with ALI would be more susceptible than naïve mice to an S. pneumoniae infection. Following OVA challenge, BALB/c mice were infected intranasally (i.n.) with a lethal dose of S. pneumoniae and morbidity and mortality were monitored. Surprisingly, mice with ALI were more resistant to infection than PBS treated controls. This increased resistance was highest 10 days following the final OVA challenge, despite reduced cytokine production after infection. Mice with ALI also had significantly increased early pneumococcal clearance from the lungs across a wide range of infectious challenges. We plan to further characterize the cellular response to pulmonary S. pneumoniae infection in mice with ALI to determine the mechanisms responsible for the observed increased resistance.
Polymorphisms of the Ig heavy chain 3’ Regulatory Region associates with humoral Ig Homeostasis. (176.13)
Authors: Frezza Domenico , Serone Eliseo , Daleno Cristina , Scala Alessia , Principi Nicola , Iacoacci Valentina , Karasiota Evangelista , Magrini Andrea , Massoud Renato , D'Addabbo Pietro , Plebani Alessandro , Giambra Vincenzo , Esposito Susanna
Published: January 2023

Aim. Incomplete maturation of immune system in the first years of life could affect different individual response to vaccination. To investigate the issue we studied a polymorphic genomic cis regulator of Ig heavy chain in response to vaccination with viral antigens. Method. Two cohorts of healthy subjects, one not yet 3 years and a second between 21 and 26 years old were vaccinated against H1N1, H2N3, B and HBV antigens respectively. From time zero the two cohorts were followed up for the total peripheral blood levels of Ig and for specific antibodies against the vaccine antigens. Subjects were genotyped for Ig heavy chain 3’ RR structural polymorphisms of enhancer hs1.2. Results. The serum specific antibodies in subjects Non responder, Responder and High responder to the vaccination is not associated with the levels of circulating IgM, IgG, IgA. However, the humoral concentration of Ig in the adult subjects correlates with hs1.2 allelic frequency as observed previously (p<0.001). Children with low IgM concentration versus children with high IgM show a change of hs1.2 allele *1 frequency from 47% to 1.8% and for allele *2 from 43% to 82% (p<10-9). The number of CD19 and CD27 cells were determined. Conclusions. The hs1.2 enhancer is associated with regulation of Ig levels. Children with allele *2 could have a possible advantage with wider repertoire of Ig. We suggest different controls for the production and mantainment of total and specific Ig amount with vaccination.
Mechanisms for regulation of the human antibody response to the influenza vaccine and changes with age (176.19)
Authors: Blomberg Bonnie , Romero Maria , Diaz Alain , Mendez Nicholas , Landin Ana Marie , Frasca Daniela
Published: January 2023

Aging is associated with a decline in the ability of the individual to mount protective immune responses, e.g. antibodies (immunoglobulins, Ig). Understanding the molecular mechanisms of the age-related impairment in immune functions will help to prevent infectious diseases and improve the biological quality of life in the elderly. We hypothesize that the increased pro-inflammatory status of the elderly, called inflammaging, impairs the capacity of the individual to make protective antibodies and to respond to vaccination. Our data indicate that aged B lymphocytes make TNF-α before vaccine challenge, which impairs their function,including optimal AID and class switch recombination (CSR) of Ig. We previously showed that the transcription factor E47 and enzyme, AID (activation-induced cytidine deaminase), both crucial for class switch recombination, are decreased with age in both mice and humans. We here report that human unstimulated (ex vivo) B cells from elderly have significantly higher levels of TNF-α than young subjects and that the t0 TNF level negatively correlates with the in vitro t28 influenza vaccine response (as measured by AID) which in turn correlates with the in vivo serum HAI response. Thus, these results reveal new molecular mechanisms which contribute to reduced antibody responses in aging and should have immediate impact on crucial development of effective vaccines to protect elderly individuals from diseases typical of old age.
Regulation of Central B-cell Tolerance by Aicda and Myd88 (176.20)
Authors: Kuraoka Masayuki , Snowden Pilar , Holl T. , Liao Dongmei , Reynolds Alexander , Kelsoe Garnett
Published: January 2023

V(D)J recombination generates a highly diverse repertoire of B-cell antigen receptors crucial for effective humoral immunity. This process also frequently creates autoreactive B cells that are mostly eliminated during B-cell development. Early immature B cells that are polyreactive or bind nuclear antigens are preferentially eliminated in the bone marrow at the pre-B to immature B-cell transition. Residual autoreactive clones are further suppressed in the periphery. Despite years of study, the mechanism(s) of these tolerance checkpoints are not understood. Recently, we and others have shown that expression of activation-induced cytidine deaminase (AID), the product of Aicda gene, by immature B cells plays an unanticipated role in purging self-reactive B cells in mice and humans. We now show that co-activation of immature B cells by anti-IgM F(ab’)2 and CpG DNA synergistically increases AID expression, implying that cross-talk between BCR- and Toll-like receptor 9 (TLR9)-mediated signals may regulate AID expression in immature B cells. By extension, we hypothesized that MyD-88 would also play a crucial role in central B-cell tolerance. Consistent with this idea, mice deficient for Myd88 exhibited reduced capacity to purge autoreactive immature/transitional B cells; the clonal deletion of self-reactive 3H9 VDJ knock-in mice is significantly impaired in the absence of MyD88 expression. We are currently investigating interaction of Aicda and Myd88 in central tolerance in B cells.
Regulated release of nitric oxide by nonhematopoietic stroma controls expansion of the activated T cell pool in lymph nodes (176.23)
Authors: Lukacs-Kornek Veronika , Malhotra Deepali , Fletcher Anne , Acton Sophie , Kutlu Elpek , Turley Shannon
Published: January 2023

Fibroblastic reticular cells (FRCs) and lymphatic endothelial cells (LECs) are non-hematopoietic stromal cells of lymphoid organs. They influence the migration and homeostasis of naïve T cells; however, their influence on activated T cells remains undescribed. Here we report that FRCs and LECs inhibited T cell proliferation through regulated nitric oxide synthase-2(NOS2)-dependent mechanism. Moreover, in vivo NOS2 expression by FRCs and LECs controlled the size of the activated T cell pool. Mechanistically, the expression of NOS2 and production of nitric oxide (NO) paralleled the activation of T cells and required IFNGR1 signaling in FRCs and LECs, and was augmented by TNF and direct contact with activated T cells. Comparative transcriptomic analysis revealed that various adhesion molecules and multiple components of cytokine, complement and JAK-STAT signaling pathways are upregulated in FRCs in the presence of activated T cells. Based on these data, current efforts aim to identify the molecules involved in the direct stromal cell-T cell interaction influencing NO production by FRCs. This negative regulatory feedback mechanism is likely of great relevance to regulation of immune responses either to prevent excessive T cell expansion or to avoid structural damage within LNs caused by the expanding T cell pool.
The adipokines Leptin and Wnt5a have divergent effects on mast cell function and migration (177.11)
Authors: Kolawole Elizabeth , Falanga Yves , Ryan John
Published: January 2023

Mast cells are known for their critical role in allergy and inflammatory diseases such as asthma. Furthermore, it has been shown that mast cell numbers are increased in white adipose of the obese. Due to the epidemiological relationship between allergy and obesity, we investigated the effects of adipokines on mast cell regulation. In this work we demonstrate that the adipokine Wnt5a enhances IgE-mediated cytokine production as well as migration, with greater effect on a Th2-prone background. By contrast, the adipokine Leptin, best known for its pro-inflammatory roles, suppresses IgE-mediated cytokine production. Additionally, in vivo studies showed that Leptin receptor-deficiency magnifies IgE-mediated anaphylaxis. Interestingly, the effects of Leptin may be restricted to mast cells cultured from mice with a Th1-prone background. Characterizing the role of mast cells in obesity can increase our understanding of the links between obesity, allergy and allergic disease enabling the possibility of new therapies.
Histamine-releasing factor (HRF) in asthma and atopic dermatitis (177.16)
Authors: Ando Tomoaki , Kashiwakura Jun-ichi , Kawakami Yuko , Kawakami Toshiaki
Published: January 2023

Effects of monomeric IgE on mast cell biology exhibit vast heterogeneity, depending on IgE used. Highly cytokinergic (HC) IgEs can induce many activation events such as degranulation, cytokine production, etc, whereas poorly cytokinergic (PC) IgEs can induce only weak survival promotion. Because of the reminiscence to the heterogeneity in the ability of IgE molecules to prime basophils in response to HRF, this dichotomy has led us to find that a subset of IgEs bind to HRF via their Fab portions; the IgE-binding sites were mapped to two different sites of HRF, indicating that dimeric HRF can cross-link IgE-bound FcϵRI; N19 and H3 peptides blocked HRF-Ig interactions; administration of these inhibitors drastically reduced airway inflammation in asthma models, implying that blockade of HRF-Ig interactions may represent a potential prophylactic and therapeutic strategy for the treatment of asthma. Importantly, most HC, but no PC, IgEs exhibit polyreactivity to HRF and other autoantigens. Interestingly, sera from atopic dermatitis (AD) patients showed increased reactivity to autoantigens and increased levels of HRF. Some AD patients, but not healthy individuals, had high serum levels of HRF-reactive IgE. AD sera with high titers of autoreactive IgE could induce increased IL-8 secretion from human mast cells. Collectively, these results show support the autoimmune mechanism in the pathogenesis of atopic dermatitis.
Enhancement of IgE- and mast cell-dependent cutaneous inflammation by mast cell expression of C3aR and C5aR (177.5)
Authors: Piliponsky Adrian , Schafer Beatrix , Song Chang-Ho , Gerard Norma , Gerard Craig , Tsai Mindy , Kalesnikoff Janet , Galli Stephen
Published: January 2023

Mast cells express receptors for complement anaphylatoxins C3a and C5a (i.e., C3aR and C5aR) and can be activated by C3a or C5a in vitro, and C3a and C5a are generated during IgE-dependent immediate hypersensitivity reactions in vivo. However, it is not clear to what extent expression of C3aR or C5aR by mast cells influences either C3a- or C5a-induced cutaneous responses, or IgE-dependent passive cutaneous anaphylaxis (PCA), in vivo. In this study we assessed whether the expression of C3aR or C5aR by mouse skin mast cells influences: 1) the cells’ responsiveness to i.d. injections of exogenous C3a or C5a, or 2) the extent of IgE-dependent mast cell degranulation and IgE-dependent PCA, in vivo. We measured the magnitude of cutaneous responses to i.d. injections of C3a or C5a, and the extent of IgE-dependent mast cell degranulation and IgE-dependent PCA responses, in mice containing mast cells that did or did not express C3aR or C5aR. We found that the majority of the skin swelling induced by C3a or C5a required that mast cells expressed C3aR or C5aR, respectively, and the extent of IgE-dependent degranulation of skin mast cells and of IgE-dependent PCA were significantly reduced when mast cells lacked either C3aR or C5aR. We conclude that the expression of C3aR and C5aR by skin mast cells contributes to the ability of C3a and C5a to induce skin swelling and can enhance the extent of mast cell degranulation and inflammation during IgE-dependent PCA in vivo.
Visualization of Helios-expressing T cells in the skin (179.12)
Authors: Nomura Takashi , Kabashima Kenji , Miyachi Yoshiki
Published: January 2023

The majority of Foxp3(+)CD4(+) T cells develops in thymus as thymus-derived Tregs. On the other hand, naive T cells can express Foxp3 to become "induced" Tregs under certain conditions where TGF-b plays a key role. Helios is potentially a specific marker for thymus-derived Tregs. However characteristics of Helios(+) and Helios(-) Tregs remain elusive because it is still difficult to sort these cells alive. To solve the above problem and to delineate difference between Helios(+) and Helios(-) Tregs in the skin, we established a gene-targeted mouse in which Helios-expression can be monitored by Venus, a derivative of green fluorescent protein. Venus was expressed in a subset of double negative thymocytes, intra-thymic Tregs, and 80% of peripheral Tregs in accordance with endogenous Helios expression. Characteristics of the cutaneous Tregs are currently under investigation. Our Helios/Venus mouse is a versatile tool for studying Treg-subsets in various tissues including the skin not only with flow cytometer but also under real-time two-photon microscopy. Additionally, this system would provide a novel scope for screening factors that control Helios expression and alter epigenetic state of Tregs.
The Glucocorticoid-Induced Tumor necrosis-factor related Receptor (GITR) is a redundant costimulatory molecule for conventional T (Tconv) cells and plays a dual role on Regulatory T cells (Treg). (179.17)
Authors: Ephrem Amal , Epstein Alan , Shevach Ethan
Published: January 2023

Treg control immune homeostasis, but are also responsible for the suppression of tumor immunity; thus, reversing Treg function is a promising strategy for cancer therapy. The GITR is highly expressed on Treg, and upregulated on Tconv after activation. The GITR-L is primarily expressed on APC. Manipulation of GITR/GITR-L interactions in vivo has resulted in enhancement of immune responses, but whether the enhancement is secondary to co-stimulation of Tconv or reversal of Treg-mediated suppression remains unclear. To resolve this controversy, we used GITR-/- and GITR-L-/- mice and Fc-GITR-L in several experimental models. Treatment of mice with Fc-GITR-L induced the expansion of Treg. Polyclonal GITR-/- Treg survived less well than WT Treg following transfer to normal mice suggesting that GITR/GITR-L interactions are important for the survival of Tconv. In contrast, the expansion of both antigen-specific WT Tconv and Treg was not impaired following transfer to GITR-L-/- recipients. Although the capacity of GITR-/- Treg to suppress IBD was similar to WT, injection of GITR-L exacerbated IBD in mice that received CD45RBhigh T cells and in mice that received and CD45RBhigh cells and Treg. GITR-L treatment in this latter group resulted in a loss of Foxp3 expression. Thus, the GITR is a redundant molecule for costimulation of Tconv, but engagement of the GITR on Treg can result in different outcomes (expansion or loss of Foxp3) depending on the nature of the immune stimulus.
Hyperthermia regulates FOXP3 expression in human FOXP3+ regulatory T cells (179.8)
Authors: Tran Dat
Published: January 2023

RATIONALE: Fever is a primordial response to infection and inflammation. FOXP3+ regulatory T cells (Tregs) are central in orchestrating immune responses and preventing autoimmunity. This study hypothesizes that fever or hyperthermia regulates immunity by downregulating FOXP3 to abrogate thymic derived nTregs. METHODS: FACS analysis of FOXP3 in Tregs from peripheral blood of 5 adult cancer patients treated for 6h with thermal therapy (TT, 40 celcius). For in vitro studies, Tregs (CD4+CD127-CD25hi) were FACS-sorted from PBMC of healthy donors and stimulated with anti-CD3/CD28/IL2 for 6h at 37 (normothermia) or 40 (hyperthermia) celcius to assess their FOXP3 expression. Treg suppressive function was measured in a 4d CFSE based assay with daily 4h hyperthermia. RESULTS: Patients treated with TT had a significant (p<0.0001) decreased in geometric mean fluorescent intensity (gMFI) of FOXP3 concentration between 15-40% after 6h treatment that persisted after 18h later. There was a rise in the percentage of Tregs after 6h that fell below preheat after 18h postheat (p<0.001). In vitro studies demonstrated a decreased in the gMFI of FOXP3 in Tregs after 6h hyperthermia and an abrogation of suppressive function as compared to normothermia. CONCLUSIONS: Hyperthermia can regulate immune responses by transiently abrogating Treg suppressive function through the downregulation of FOXP3 expression. Hyperthermia has the therapeutic potential to augment vaccine response and cancer treatment.
IDO modifies TLR-ligand driven macrophage cytokine production by activation of GCN2 kinase arm of the integrated stress response and subsequent CHOP/GADD153 induction (180.3)
Authors: Liu Haiyun , Bradley Jill , Huang Lei , McGaha Tracy
Published: January 2023

Microbial sepsis is characterized by an initial cytokine storm followed by immune suppression, vascular collapse, and multiple organ failure. Indoleamine 2,3 dioxygenase (IDO) is an intracellular tryptophan-catabolizing enzyme that is induced after exposure to LPS in vivo. While generally considered to have immunosuppressive function, IDO blockade was recently demonstrated to protect mice against systemic lipopolysaccharide (LPS)-challenge, however, nothing is known about the underlying molecular mechanisms. In this study, we report that IDO was induced in the spleen of mice 6 hours after exposure to a lethal dose of endotoxin, and IDO deficiency in hematopoietic cells significantly improved survival. Moreover, we found IDO mediated tryptophan depletion in macrophages increased pro-inflammatory cytokine production in response to LPS exposure. This was due to the activation of the GCN2 kinase arm of the integrated stress response and subsequent expression of CHOP (GADD153) resulting in enhanced constitutive NF-κB activity. Lack of either GCN2 or CHOP completely abrogated macrophage production of IL-6 under stress conditions. Further, disruption of GCN2 reduced production of TNF-α and IL-12 in response to LPS stimulation under any condition. Thus, taken together our findings demonstrate that IDO functions to modulate macrophage pro-inflammatory response via a novel amino acid deprivation mechanism dependent on the integrated stress response.
Analysis of bovine natural killer cell cytotoxicity following activation by pro-inflammatory cytokines(181.2)
Authors: Dar Pervaiz , Patch Jared , Kenney Mary , Waters Ryan , Glabman Raisa , Kondabattulla Ganesh , Golde William
Published: January 2023

Bovine natural killer (NK) cells are important mediators of the innate immune response to infectious diseases of cattle. Like other mammalian species, bovine NK cells express NKp46 (CD335) and do not express CD3 or a T cell receptor. However, bovine NK cells remain poorly characterized with respect to cytokine activation, in part because the commonly used lymphoma target cell line, K562, apparently fails to induce a significant cytotoxic response by bovine NK cells. We have assessed natural cytotoxicity against a bovine-derived lymphoid cell line (BL3.1) compared to human-derived K562 cells. Lymphocytes activated in vitro with bovine cytokines (IL-2, IL-15, IL-21 and their combination) for different time periods were analyzed for killing activity using these target cells. We observed enhanced killing of BL3.1 cells, suggesting that they are more susceptible to killing by bovine NK cells than K562 cells. Killing activity was detected after 24 hrs of in vitro cytokine stimulation and maximal cytotoxicity was demonstrated following 72-96 hrs of culture with cytokines. Our data indicate that the addition of IL-21 enhances IL-2 mediated cytotoxicity, whereas IL-15 has no effect on NK cytotoxicity for these target cells. Preliminary data suggest that vaccination of cattle with an inactivated foot-and-mouth disease virus (FMDV) vaccine fails to activate NK cells isolated from peripheral blood. Future studies will investigate the ability of FMDV infection to activate bovine NK cells.
Identification of bio-active collagen type II alpha 1 protein in shark cartilage (181.3)
Authors: Merly Liza , Smith Sylvia
Published: January 2023

Previous studies have shown that extracts of shark cartilage (SC) induce a significant cytokine response in human leukocytes characteristic of a Th1 immune response. The nature of the bio-active component(s), however, is unknown. The aim of the present study was to identify the bio-active component(s) in SC. Cartilage extracts treated with proteases lost 80% of their cytokine-inducing property, suggesting that the active component(s) was likely a complex protein. Employing a protocol involving acid and solid phase extraction followed by ion exchange chromatography and electrophoretic separation, a partially purified bio-active preparation was obtained from commercial shark cartilage. At least two cytokine-inducing components were separated chromatographically and an anionic fraction has been identified as a small glycoprotein with at least 40% carbohydrate content. LC-MS analysis yielded five unique peptides with 100% molecular identity with collagen type II, alpha 1 protein from the lesser spotted catshark, Scyliorhinus canicula. The presence of collagen type II, alpha 1 protein in a dietary supplement has significant health implications given its role in the pathology associated with arthritis. Our findings suggest that consumers of SC, taken as prophylaxis particularly for the maintenance of joint health, should be cautious of the potential unanticipated immunomodulatory activity associated with peptides present in commercial preparations.
Role of Ikaros in hematopoiesis and tumor suppression: Selective functions of individual zinc fingers within the DNA-binding domain of Ikaros. (42.3)
Authors: Schjerven Hilde , Frietze Seth , McLaughlin Jami , Cheng Donghui , Farnham Peggy , Witte Owen , Smale Stephen
Published: January 2023

Ikaros, a C2H2 zinc finger transcription factor, is a critical regulator of hematopoiesis and tumor suppression in the lymphoid lineage. The C2H2 zinc finger is the most prevalent DNA-binding motif in mammals, with DNA-binding domains usually containing more tandem fingers than are needed for stable sequence-specific DNA recognition. To examine the reason for the frequent presence of multiple zinc fingers, and to investigate in greater depth the role of Ikaros in hematopoiesis and tumor suppression, we generated mice lacking finger 1 or finger 4 of the 4-finger DNA-binding domain of Ikaros. Each mutant strain exhibited a specific subset of the phenotypes observed with Ikaros null mice. Of particular relevance, fingers 1 and 4 contributed to distinct stages of B- and T-cell development and finger 4 was selectively required for tumor suppression in thymocytes and in a new model of BCR-ABL+ acute lymphoblastic leukemia. These results, combined with transcriptome profiling and DNA-binding analysis, reveal that different subsets of fingers within multi-finger transcription factors can modulate binding to different target sequences and regulate distinct target genes and biological functions. These novel mutant strains provide a powerful tool to elucidate Ikaros' role in hematopoiesis and tumor suppression. Furthermore, this study demonstrates that selective mutagenesis can facilitate efforts to elucidate the functions and mechanisms of action of this prevalent class of factors.
Helicobacter pylori infection in pigs is dominated by a Th1 and cytotoxic immune response (43.17)
Authors: Kronsteiner-Dobramysl Barbara , Bassaganya-Riera Josep , Washington Cassandra , Vento Salvador , Pedragosa Mireia , Viladomiu Monica , Carbo Adria , Hontecillas Raquel
Published: January 2023

H. pylori is the leading cause for peptic ulcer disease and gastric adenocarcinoma. While iTreg are required for long-term colonization without disease, Th1 and Th17 responses are associated with lower bacterial load at the expense of gastric pathology. We have developed computational models of H. pylori infection predicting a dominant Th1-response that results in lesion development in the gastric mucosa. In this study, pigs were infected with H. pylori strain SS1 or J99. Changes in PBMC populations were monitored over 2 months. Bacterial loads were assessed at 9 and 57 days post-infection. Both strains elicit a Th1 response with increased percentage of CD4+Tbet+ cells and elevated Tbet and IFN-γ mRNA in PBMC. A subset of CD8β+ cells expressing Tbet increased due to infection. These cells expressed low levels of CD8β, a property of cytotoxic T cells previously exposed to their cognate peptide. A significant increase of NK cells was observed in infected pigs pointing towards a predominant cytotoxic immune response. Strain J99 evoked a more dramatic acute response associated with bacterial clearance, while strain SS1 showed long-term colonization capacity. Ex vivo functional assays indicate strain-specific, weak and suppressed memory immune responses suggesting that specific virulence factors contribute to suppression of immune responses and chronic persistence. Data from this study will be used in the modeling process to refine computational models of H. pylori infection.
Th17 cells are associated with pathology in human schistosomiasis (43.24)
Authors: Larkin Bridget , Mbow Moustapha , Meurs Lynn , Wammes Linda , de Jong Sanne , Labuda Lucja , Smits Hermelijn , Dieye Tanaka , Polman Katja , Mboup Souleyman , Stadecker Miguel , Yazdanbakhsh Maria
Published: January 2023

Infection with parasitic helminths of the genus Schistosoma results in a wide range of immunopathology. Studies in murine schistosomiasis suggest that Th17 cells play a major role in the development of severe pathology; conversely, regulatory T (Treg) cells represent a mechanism to curtail excessive inflammation. We assessed by flow cytometry the profile of peripheral blood (PB) CD4 T cells in a cohort of children with urinary schistosomiasis from the village of Pakh, Department of Richard Toll, Senegal. S. hematobium-infected children with bladder pathology had a significantly higher percentage of PB IL-17+ cells with higher RORγt+/ Foxp3+ and IL-17+/ IL-10+ cell ratios than infected children without pathology. To investigate the relationship between PB values and target organs, we also examined the PB T cell response in murine S. mansoni infection (S. hematobium is non-permissive in mice) and, similar to humans, found a significantly higher percentage of CD4+ IL-17+ cells in high-pathology CBA mice than in low-pathology BL/6 mice. Moreover, a significant increase in IL-17+ cells in spleen and liver granulomas together with lower Foxp3+ cells in the spleen of CBA mice denoted a good correlation between PB and target organs. Our findings for the first time demonstrate an association between pathology and PB Th17 cells in human schistosomiasis and suggest human PB T cell subsets to faithfully reflect those mediating lesions in organs affected by the disease.
Local and systemic lymphoproliferative responses to Escherichia coli O157:H7 proteins occur after colonization and are predictive of decreased fecal shedding. (43.29)
Authors: Lahmers Kevin , Hayles Andrea , Boland Kathryn , Sheng Haiqing , Hovde Carolyn
Published: January 2023

Escherichia coli O157:H7 (O157) is a zoonotic, human, enteric pathogen with potential for life-threatening complications. Cattle asymptomatically carry the bacteria in the gastrointestinal tract with colonization occurring at the rectoanal junction mucosa. Fecal material with high numbers of O157 contaminates the environment, food and water. Among the preharvest intervention attempts to reduce bovine carriage of O157, vaccination is promising. To date, immune responses to O157 have been evaluated solely by immunoglobulin response and not T cell responses. This study evaluated the bovine antigen specific lymphoproliferative responses to three recombinant O157 virulence proteins: intimin, TIR, and EspB, which elicit immunoglobulin responses among naturally exposed and experimentally challenged cattle. Lymphoproliferative responses were detected in the peripheral blood both in cattle that were challenged twice intraruminally and in naturally exposed cattle. There were also local lymphoproliferative responses in the mesorectal lymph nodes after challenge and in naturally exposed cattle. Mesorectal cell proliferation to all three proteins was ten-fold greater than proliferation in peripheral blood cells. The strength of the proliferative response prior to challenge correlates with shorter duration of shedding and decreased total shedding of O157. The presence of consistent, strong lymphoproliferative responses may be required for bovine vaccine efficacy.
Identification of novel epitopes from Mycobacterium tuberculosis (43.32)
Authors: Lindestam Arlehamn Cecilia , Swann Justine , Henderson Ryan , Greenbaum Jason , Sidney John , McKinney Denise , Park Daniel , Taplitz Randy , Grey Howard , Peters Bjoern , Sette Alessandro
Published: January 2023

Mycobacterium tuberculosis (MTB) is the one of the worldwide leading cause of death from infectious disease. The MTB genome encodes just under 4,000 different ORFs, yet epitopes recognized by human CD4+ T cells, the key cellular effector of MTB immunity, have been described for less than 2%. Our present study addresses the discovery and validation of MTB CD4+ T cell epitopes presented by HLA Class II molecules. We have identified hundreds of MTB-specific epitopes by the combined use of epitope predictions and high-throughput ex vivo ELISPOT assays using PBMC from human donors latently infected with MTB. Specificity was demonstrated by parallel testing of non-TB infected control donors. These epitopes allow us to identify previously unknown CD4+ TB antigens. We are in the process of further validating these epitopes by examining the T cell phenotype, pattern of cytokine secretion associated with epitope responses and projected population coverage as determined by quantitative binding assays, as well as investigating T cell responses as a function of clinical outcome. This study determines the breadth of MTB responses in humans and it will establish whether there is a correlation between the antigens recognized by CD4+ T cells and serological reactivity and CD8+ T cells, antigen expression in different MTB forms and other structural and biological features associated with the antigens. In addition, these epitopes can be used as tools to understand the human immunity to TB.
Adaptive immunity in the mouse model of Clostridium difficile infection (43.4)
Authors: Johnston Pehga , Gerding Dale , Knight Katherine
Published: January 2023

Clostridium difficile rivals MRSA as the most common hospital-acquired infection. C. difficile infection (CDI) can manifest as mild diarrhea to life-threatening psuedomembranous colitis. CDI occurs following a course of antibiotics, and although most patients recover fully from CDI, ~20% undergo recurrent disease caused by toxins A and/or B. Several studies demonstrate a correlation between anti-toxin antibody (Ab) and a decrease in recurrence; however; the contribution of the systemic and mucosal Ab response remains unclear. Our goal was to use the recently described mouse model of CDI to characterize the protective immune response. Mice infected with C. difficile developed protective immunity against CDI and did not develop CDI upon re-challenge. Infected mice generated both systemic and mucosal Ab directed against toxin. We infected CD4-/- mice with C. difficile, expecting they would succumb to CDI. Instead, the mice developed immunity and we found IgA anti-toxin Ab in both serum and the mucosa. pIgR-/- mice also developed serum anti-toxin Ab and were protected from CDI, indicating that mucosal anti-toxin Ab is not required for protection from CDI. Our studies suggest that protective anti-toxin Ab can be induced in T-cell deficient individuals and that systemic anti-toxin immunity may be sufficient for protection from CDI. The results should aid in development of vaccines for CDI, especially for individuals with immunodeficiency.
Immunization with pneumococcal polysaccharide SSS-3 and lipopolysaccharide protects against deadly Streptococcus pneumoniae infection in mice. (43.8)
Authors: Restori Katherine , Kennett Mary , Ross A.
Published: January 2023

To study pneumonia disease progression, we developed a mouse model of pneumonia by intranasal inoculation with Streptococcus pneumoniae, serotype 3. In order to combat mortality, mice were immunized with pneumococcal polysaccharide specific for serotype 3 (SSS-3) or co-immunized with SSS-3 and a low dose of lipopolysaccharide (LPS) 5 days prior to infection. Pneumonia severity was assessed during acute and resolution phases of infection. Immunization was clinically protective as evidenced by decreased bacterial recovery from the lung and blood during acute infection and increased survival. As expected, primary antibody production specific for SSS-3 was upregulated in immunized mice. Histopathological severity scores for lungs from immunized mice were lower, due to little or no inflammation while lungs from non-immunized mice exhibited moderate inflammation. Furthermore, immunization attenuated proinflammatory cytokine gene expression in the lung at acute time points. Lung microarray analysis 48 hr after infection revealed higher expression of a large number of genes in non-immunized mice that were expressed at nearly normal levels after co-immunization, including a highly significant reduction in serum amyloid a (Saa1, Saa2) gene expression. Our results demonstrate that immunization with SSS-3 and LPS significantly decreased lung bacterial burden and systemic dissemination while promoting resolution of infection.
A Functional Single-Nucleotide Polymorphism in Interleukin-6 Promoter is associated with Susceptibility to Tuberculosis (44.13)
Authors: Chen Xinchun , Zhang Guangliang , Zhang Mingxia
Published: January 2023

Background. Genetic variation influences susceptibility or resistance to tuberculosis (TB). Interleukin-6 contributes to protection against TB in mice, however, its role in regulating susceptibility or resistance to TB in humans is unclear. Methods. Genotyping of polymorphisms in IL-6 and IL-6R (CD126) genes were performed in two independent cohorts, namely experimental (495 cases and 358 controls) and validation (1383 cases and 1149 controls) populations. The associations of the variants with tuberculosis were tested using two case-control association studies. In addition, the regulatory effects of SNP rs1800796 (-572C>G) on IL-6 production in plasma and CD14+ monocyte cultures stimulated with a Mycobacterium tuberculosis (Mtb) product were assessed. Results. The rs1800796 polymorphism is associated with increased resistance to TB (odds ratio, 0.771; confidential interval 0.684-0.870). The rs1800796GG genotype is strongly associated with reduced risk to TB (odds ratio, 0.621; confidential interval 0.460-0.838). Interestingly, CD14+ monocytes isolated from individuals with rs1800796GG genotype produced significantly lower IL-6 in response to Mtb 19kDa lipoprotein than those with CC or CG genotype do. Conclusions. We identified a genetic polymorphism in IL-6 promoter that regulates the cytokine production and host resistance to pulmonary TB in Chinese population.
Behavior-modulating function of the Duffy Antigen Receptor for Chemokines (DARC) in mice under homeostatic conditions (44.24)
Authors: Schneider Erich , Gao Ji-Liang , Holmes Gibran , Fowler Stephen , Peiper Stephen , Murphy Philip
Published: January 2023

DARC binds promiscuously many inflammatory chemokines without intracellular signal transduction. It is mainly expressed on endothelial cells of postcapillary venules and on erythrocytes, where it acts as a transendothelial chemokine transporter and as a chemokine sink, respectively. Surprisingly, DARC was also shown to be expressed at high density in the cerebellum of human and mouse brain, but with as yet unknown function. We addressed this question by subjecting C57Bl/6 wildtype and DARC-deficient mice to behavior experiments including Morris water maze-, elevated plus maze-, rotarod- and actometer tests. While the water maze results are ambiguous, elevated plus maze trials show a strong aversion of DARC-/- mice to walk to the end of the open arm, which is consistent with anxiety-like behavior. Moreover, DARC-/- mice show greatly reduced locomotor activity, resulting from episodes of reduced mobility that occur more frequently than in the C57Bl/6 controls (elevated plus maze, actometer). Finally, DARC-/- mice spend a significantly reduced time on the rotarod compared to C57Bl/6 controls, probably indicating impaired cerebellar function. We conclude that DARC modulates brain function. Surprisingly, this appears to be happening under homeostatic conditions, although DARC binds mainly inflammatory chemokines. Since DARC shows no signaling, this effect may be indirectly caused by altered brain chemokine concentrations or may result from as yet unknown DARC signaling pathways.
Human platelets produce and constitute the main source of IL-18 in the circulation (44.25)
Authors: Allam Ossama , Samarani Suzanne , Marzouk Rim , Ahmad Ali
Published: January 2023

IL-18 is a pro-inflammatory cytokine belonging to the IL-1 family. The cytokine is known to be produced from macrophages, dendritic cells, keratinocytes, adrenal cortex etc in the body. The cytokine is produced as an inactive precursor protein, which is cleaved into mature form by activated caspase 1. We show here for the first time that human platelets contain abundant amounts of this cytokine, and release it upon activation. The platelets also contain components of the inflammasome, which activate caspase 1 and process the IL-18 precursor into mature form during the platelet activation process. Different activating agents activate platelets and release IL-18 to variable extent, the maximum being observed with thrombin. Furthermore, the released cytokine further activates platelets and enhances platelets to produce more IL-18. In addition to the cytokine, the platelets also release its antagonist, IL-18 binding protein (IL-18BP), upon activation. Surprisingly, a part of the cytokine appears to be synthesized within platelets. We also show here for the first time that inflammasome assembly occurs in platelets and the platelet-released IL-18 constitutes the major source of this cytokine in the circulation, as we found little amount of this cytokine in platelet-free plasma. Our findings have important implications for chronic inflammatory diseases, metabolic syndrome and cardiovascular diseases in which increased IL-18 levels play a pathogenic role.
IL-21 reduces production of pro-inflammatory cytokines by activated CD8 T cells (44.26)
Authors: Parmigiani Anita , Lichtenheld Mathias , Pahwa Savita
Published: January 2023

Background: Chronic HIV infection is characterized by systemic immune activation (IA), accumulation of T cells with a skewed terminally-differentiated profile and a progressive loss of IL-21 production. Experiments described below address the hypothesis that IL-21 prevents IA in healthy individuals. Methods: Purified CD8 T cells from healthy donors were activated for 48 hrs. by antiCD3+antiCD28 in the presence of IL-21 and, respectively, its absence. Cytokine synthesis and secretion were evaluated by real time PCR and ELISA. Results: CD8 T cells TCR-stimulated in the presence of IL-21 produced less pro-inflammatory cytokines TNF alpha and IL-12p70, compared to CTL stimulated in the absence of exogenous cytokine. Moreover, IL-21 induced significantly higher amounts of the anti-inflammatory cytokine IL-10 and lower levels of IL-2. Conclusions: IL-21 could act as a favorable immunomodulatory agent in the context of chronic infections such as HIV because: i) it limits the production of pro-inflammatory mediators by CD8 T cells; and ii) it induces the anti-inflammatory cytokine IL-10.