bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific regions of the ricin toxin A subunit are involved in eliciting protective antibodies: implications for vaccine design (53.5)
Authors: O'Hara Joanne , Brey Robert , Mantis Nicholas
Published: January 2023

Efforts to develop an effective vaccine against ricin are focused on engineering attenuated and stable recombinant forms of the toxin’s enzymatic A subunit (RTA). While several candidate antigens are in development, vaccine design and efficacy studies are being undertaken in the absence of a fundamental understanding of the regions of RTA that are critical in eliciting protective immunity. To address this issue, we recently identified six distinct immunodominant linear regions on RTA. Using monoclonal antibodies (MAbs) specific to each region, we determined that protective antibodies were directed against α-helices located in RTA folding domains 1 and 2, whereas non-neutralizing antibodies recognized random coils and loops that were primarily confined to folding domain 3. This suggests that neutralizing and non-neutralizing antibodies target distinct domains on RTA. In support of this hypothesis, we now report that the vast majority of conformation-dependent ricin neutralizing MAbs bind to RTA 1-33/44-198, a recombinant RTA deletion mutant lacking folding domain 3. Furthermore, preliminary studies in mice suggest that RTA 1-33/44-198 is more effective than an attenuated RTA point mutant at eliciting neutralizing antibodies. These data offer insights into the immunodominant and structural determinants on RTA that give rise to protective immunity, and for the first time provide an immunological rationale for ricin vaccine design.
Anti-inflammatory activity of Dioscoreae rhizome in macrophages by inhibition of pro-inflammatory cytokines via NF-κB signaling pathway (54.11)
Authors: Kim Seulah , Shin Seulmee , Park Yoonhee , Lee Seungjeong , Lee Chong-Kil , Kim Kyungjae
Published: January 2023

Dioscoreae rhizome (DR) has been used in traditional medicine to treat numerous diseases and reported to have anti-diabetes and antitumor activities. The pharmacological and biochemical mechanisms of the water extracts of DR (EDR) on macrophages in immune responses have not been clearly elucidated. We examined whether EDR suppresses the production of pro-inflammatory cytokines and the expression of co-stimulatory molecules. EDR reduced the production of NO and PGE2 through the suppressed gene and protein expression of iNOS and COX-2 in the both RAW 264.7 cells and peritoneal macrophages. The production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 production were suppressed by EDR. The change of gene expression by EDR treatment was analyzed by using RT-PCR, and the associated mRNA expression was inhibited same as cytokine production. Western blot analysis showed the augmentation of cytokine related protein on EDR treatment whereas anti-inflammatory cytokines, IL-4 and IL-10, did not significantly change. The expression level of co-stimulatory molecules such as ICAM-1, B7-1, and B7-2 also was reduced with the increment of EDR concentration. The nuclear translocation of NF-κB activity in LPS-activated macrophages was suppressed by EDR. These results showed EDR decreased pro-inflammatory cytokines via the inhibition of NF-κB dependent inflammatory protein level and suggesting that how EDR could be useful immunomodulatory agent for treating immunological diseases.
The immunological ménage à trois promotes inflammation in Type 2 diabetes. (54.14)
Authors: Bogdan Madhumita , McDonnell Marie , Shin Hyunjin , Rehman Qasim Rehman , Hasturk Hatice Hasturk , Apovian Caroline , Nikolajczyk Barbara
Published: January 2023

Type 2 diabetes (T2D) is an inflammatory disease promoted by changes in immune cell function. Monocytes and T cells play roles in T2D inflammation as evidenced, in part, by our demonstration of pro-inflammatory T cell subset skewing in T2D patients. However, the role of cross-talk amongst immune system cells in T2D is poorly unstudied. PBMC depletion studies showed that T2D patient T cells, unlike non-diabetic (ND) donor cells, required monocytes to maintain IL-17 hyper-secretion. In contrast, B cell depletion did not affect T2D Th17 function, but surprisingly, B cell depletion from non-diabetic (ND) mixed cell cultures significantly elevated Th17 function. Based on the role B cell IL-10 plays in inflammation resolution, we tested the hypothesis that IL-10 regulates immune cell cross-talk to block Th17 function. IL-10 addition to B cell-depleted PBMCs decreased IL-17 to “healthy” levels in both ND and T2D samples, indicating that both cell types are physiologically capable of interpreting the anti-inflammatory signals from B cells despite the presence of pro-inflammatory monocytes in T2D samples. Critically, unlike ND B cells, T2D B cells fail to secrete IL-10, thus lack of B cell IL-10 in T2D samples compounds elevated pro-Th17 cytokine production by monocytes. We conclude that IL-10 can block monocyte-mediated increases in Th17 function, and that lack of B cell IL-10 in T2D associates with IL-17-induced inflammation thus metabolic imbalance.
Anti-inflammatory and anti-bacterial effect of polyacetylene compound from Cirsium japonicum var. ussuriense (54.19)
Authors: Lee Ki-Man , Lee Geum Seon , Yim Dongsool , Kim Kyungjae , Kang Tae Jin
Published: January 2023

Cirsium japonicum var. ussuriense is known to have a variety of biological activities, including anti-inflammatory, analgesic activity and antipyretic activity. The role of 1-Heptadecene-11, 13-diyne-8, 9, 10-triol (PA), a polyacetylene compound isolated from the root of Cirsium japonicum var. ussuriense was herein investigated as an inhibitor of some macrophage functions involved in the inflammatory process. The effect of PA on the production of pro-inflammatory cytokines, interleukin-1beta (IL-1β) and tumour necrosis factor-alpha (TNF-a), and nitric oxide (NO) were tested in mouse macrophage cell line, RAW264.7 by ELISA method and griess reaction, respectively. PA inhibited lipopolysaccharide (LPS)-induced IL-1β and TNF- a production by macrophages at a dose dependent manner. PA also supressed inhibited the NO production of macrophages by LPS. LPS-induced NF-kB activity was decreased by treatment of PA. Next, anti-bacterial activity of PA with E. coli and S. aureus was maesured by MIC (Minimum Inhibitory Concentration). PA significantly inhibited bacterial growth. Therefore, these results indicated that PA has anti-inflammatory and anti-bacterial activities.
Eph receptors are involved in the pro-inflammatory response following spinal cord injury (54.21)
Authors: Glass Stephanie , Theus Michelle , Zhuang Zhiye , Liebl Daniel
Published: January 2023

Following spinal cord injury (SCI), residential microglia and astrocytes in the spinal cord are known to activate an innate immune response. Here, we examined a role for EphB3 and EphA4 receptors in the early immune response following SCI. Our results demonstrate that EphB3 and EphA4 are present on both microglia and astrocytes in the adult spinal cord, and that they are phosphorylated within 15 min after the initiation of injury. Receptor activation by the ligand ephrinB3 is thought to enhance multiple cytokine mRNA transcripts involved in the immune response, as well as mediate the release of several pro-inflammatory cytokines in vitro. These responses are significantly attenuated in cells derived from mice deficient for EphB3 and EphA4. Interestingly, analysis of the whole spinal cord, using ELISA Assay, showed a significant attenuation in interleukin-6 (IL-6) in astrocytes but not microglia derived from EphB3/EphA4 double knockout (EphB3/A4-/-) mice. To examine cytokine levels following SCI, we collected spinal cord tissues from sham or SCI wild type (WT) and EphB3/A4-/- mice at 4 and 24 hours and analyzed cytokine levels using cytometric bead array. We observed a significant increase in IL-6 and chemokine ligand 2 (CCL-2) at 4 and 24 hours post-SCI in WT but not EphB3/A4-/- mice. These data suggest a novel role for EphB3 and EphA4 in the early activation of the acute immune response, and possibly the progression of damage to the injured spinal cord.
Predominant role of monocytes rather than neutrophils in the pathogenesis of the familial autoinflammatory disease TRAPS (TNFR-associated Periodic Syndrome) (54.3)
Authors: Pelletier Martin , Bulua Ariel , Myerowitz-Vanderhoek Samuel , Kastner Daniel , Siegel Richard
Published: January 2023

Mutations in the type 1 TNF receptor (TNFR1) cause the autoinflammatory disorder TNF receptor-associated periodic syndrome (TRAPS), a disease characterized by recurrent fevers with inflammation. TRAPS-associated mutations in TNFR1 cause misfolding, retention in the ER, and loss of function as a conventional receptor, but instead initiate an intracellular signaling cascade that results in hyper-responsiveness to innate stimuli. Using mice engineered to have TRAPS-associated mutations in TNFR1, we examined the contents of peritoneal lavage fluid after intraperitoneal LPS injection in order to identify the pathogenic cell type. TNFR1 mutant mice have increased monocyte/macrophage infiltrates when compared to wild-type mice, but a similar number of recruited neutrophils. In mixed marrow chimeras, wild-type and TNFR1 mutant neutrophils were recruited similarly into the peritoneum after LPS injection, suggesting that there is no cell-intrinsic predisposition of TNFR1 mutant neutrophils to accumulate at sites of inflammation. In support of this, neutrophils from TRAPS patients and healthy donors migrate similarly in response to CXCL8. In contrast, we found that murine peritoneal macrophages from TNFR1 mutant mice produce higher amounts of two potent chemokines, CXCL1 and CXCL2, in response to LPS in vitro. These results suggest that enhanced chemokine secretion by monocytes, rather than hyper-responsiveness of neutrophils to these stimuli, is critical to the pathogenesis of TRAPS.
A role for periostin in regulation of goblet cell metaplasia (55.1)
Authors: Sehra Sarita , Nguyen Evelyn , Snider Paige , Petrache Irina , Conway Simon , Kaplan Mark
Published: January 2023

Periostin is a 90kD member of the fasciclin-containing family and functions as part of the extracellular matrix. Periostin is expressed in a variety of tissues and expression is increased in airway epithelial cells from asthmatic patients. Recent studies have implicated a role for periostin in allergic eosinophilic esophagitis. To further define a role for periostin in Th2-mediated inflammatory diseases such as asthma, we studied the development of allergic pulmonary inflammation in periostin-deficient mice. Sensitization and challenge of periostin-deficient mice with ovalbumin resulted in increased peripheral Th2 responses compared to control mice. In the lungs, periostin-deficiency resulted in increased airway resistance and significantly enhanced mucus production by goblet cells concomitant with increased expression of Gob5 and Muc5ac, compared to wild type littermates. Importantly, the physiological significance of the function of periostin is demonstrated by its ability to inhibit the expression of Gob5, a putative calcium-activated chloride channel involved in the regulation of mucus production, in primary murine airway epithelial cells. Our studies suggest that periostin may be part of a negative-feedback loop regulating allergic inflammation that could serve as a therapeutic target in the treatment of atopic disease.
Itk negatively regulates IFNγ, influencing T helper 2 response in vivo (55.16)
Authors: Kannan Arun , Sahu Nisebita , August Avery
Published: January 2023

Asthma is a major public health concern affecting millions of people each year. Allergic asthma is a T helper 2 (Th2) cell mediated inflammatory disease characterized by airway inflammation and hyper responsiveness. The Tec family tyrosine kinase Interleukin-2 Inducible T cell kinase (Itk) is primarily expressed in T cells and is critical for development, function and differentiation of T helper cells. Itk-/- mice have a defective Th2 response and are not susceptible to allergic asthma and parasitic worm infections. We observed that naïve CD4+ T cells from Itk-/- mice have substantially increased transcript levels of prototypic Th1 genes. We hypothesized that this upregulation of Th1 genes could negatively regulate polarization to Th2 effector cells. Here, we show that Itk can negatively regulate IFNγ. More importantly, in absence of IFNγ there is a rescue in the expression of Th2 related genes and Th2 mediated allergic asthma in Itk-/- mice. In the absence of Itk, IFNγ levels increase leading to a positive feed-forward loop with T-bet. This is responsible for the inability of CD4+ T cells from Itk-/- mice to optimally induce Th2 transcriptional program and develop a Th2 response upon allergen challenge. In addition to providing mechanistic insight into the observed Th2 defects in Itk-/- mice, this work has implications for understanding Itk as a potential target for Th2 mediated inflammation, T helper differentiation programs and the molecular pathology of asthma.
Effects of electroacupucture at ST36 point on regulatory T cells in a murine model of OVA-induced allergic asthma (55.18)
Authors: Youngjoo Kwon , Kim Youngeun , Lee Gihun , Ham Gyeonggeun , Shin Minkyu , Hong Moochang , Bae Hyunsu
Published: January 2023

Allergic diseases, including asthma, are defined as chronic inflammatory disorders originating from an aberrant immune response to innocuous antigens. Current therapies are effective in crosssections of patients, but remain mostly palliative. Given the capacity of regulatory T cells (Tregs) to suppress disease-promoting immune responses, enhancing Treg function represents an attractive therapeutic strategy for the treatment of allergic disease. Among the complementary therapeutic approaches to asthma, acupuncture has been widely used and its efficacy has been well-known. The selected acupoint was ST36, as it is considered of choice to treat asthma. This study was conducted to address not only EA treatment reduce allergic inflammatory processes, but also to observe whether these beneficial effects of EA on asthma could be correlated with CD4+CD25+ regulatory T cells. EA treatment began 7 days after antigen priming and was repeated for 10 consecutive days. Cellular profiles and histopathologic analysis demonstrated that peribronchial and perivascular inflammatory cell infiltrates were significantly lower in control and EA groups compared to OVA and Treg depletion groups. Additionally, the population of CD4+CD25+Foxp3+ in lung cells increased in EA groups, compared to OVA. These results imply that EA at ST 36 efficiently diminishes the bronchial immune-mediated inflammation and that these effects might correlate to regulatory T cells.
Allergic experimental asthma can be ameliorated by allergen specific immunotherapy in the absence of T-bet. (55.6)
Authors: Böhm Livia , Übel Caroline , Maxeiner Joachim , Dehzad Nina , Heinz Anke , Martin Helen , Reuter Sebastian , Schild Hansjörg , Taube Christian , Finotto Susetta
Published: January 2023

Clinical studies have demonstrated that repeated subcutaneous (s.c.) administrations of allergen are effective in the treatment of allergic diseases. It has been suggested, that the success of this treatment, also known as allergen specific immunotherapy (IT), relates to a shift of the T helper 2 (Th2) to T helper 1 (Th1) cells, a blockade of specific antibodies and/or the induction of regulatory T cells (Treg). The T box transcription factor (T-bet) is crucial for the Th1 development and previous studies demonstrated that T-bet deficient mice (T-bet KO) develop a severe asthmatic phenotype. Thus we address the question of whether T-bet is necessary for the effectiveness of IT. T-bet KO mice were analyzed in a murine model of allergic airway disease and IT. OVA-sensitized T-bet KO or BALB/c mice were treated with 3 s.c. injections of OVA (1000µg/injection) prior to airway challenge. This treatment was effective in suppressing the allergen-induced asthma manifestations such as airway hyperresponsiveness (AHR), serum IgE, lung eosinophilia and airway inflammation in sensitized and challenged wildtype mice. Consistently Th2 cytokines were reduced in the lung of IT treated mice. IT treated T-bet KO mice showed an alleviated airway disease following IT to a comparable degree as that observed in the wildtype mice. In summary, the present study shows that IT is not dependent on T-bet and that this treatment effectively suppresses the development of AHR and airway inflammation.
Borrelia burgdorferi pathogenic potential correlates with type I and type III IFN production by human immune cells. (56.18)
Authors: Krupna Michelle , Wormser Gary , Schwartz Ira , Petzke Mary
Published: January 2023

Borrelia burgdorferi (Bb) is the extracellular bacterial agent of Lyme disease. Bb elicits type I interferon (IFN) in humans via Toll-like receptors 7 and 9. Bb clinical isolates with different pathogenic potential were tested for their ability to induce IFNs in human peripheral blood mononuclear cells (PBMCs). Disseminating Bb isolates induced significantly higher levels of both type I (IFN-α) and type III IFN (IFN-λ) than did non-disseminating Bb isolates. In contrast, there was no significant difference in type II IFN (IFN-γ) production, nor in levels of IL-2, 6, 8, 10. However, non-disseminating Bb isolates induced significantly higher levels of TNF-α and IL-1β. In addition, disseminating clinical isolates were able to directly induce multiple IFN-responsive genes; non-disseminating strains required IFN-feedback signaling. A mutant of Bb was identified that was unable to induce a robust IFN-α response when compared to wild-type (wt); IFN-γ levels were not affected. Wt and mutant Bb were GFP-tagged and assessed for their ability to be phagocytosed by human immune cell populations using flow cytometry and fluorescence microscopy. There were more wt than mutant Bb associated with (p=0.01) and internalized by (p=0.034) PBMCs. These findings suggest that disseminating and non-disseminating clinical isolates may be differentially phagocytosed by dendritic cells. Intriguingly, the differential uptake appears to be mediated by factor(s) encoded on a single linear plasmid of Bb.
CD74 deficiency is protective against P. aeruginosa-induced ocular keratitis (56.20)
Authors: Gadjeva Mihaela
Published: January 2023

Eye trauma and contact lens wear are the main factors that predispose to the development of infectious keratitis. The existing therapy often fails to control the excessive tissue damage that is induced during P. aeruginosa infection. While antibiotic treatment can reduce the bacterial burden, tissue damage occurs as a result of exaggerated local inflammation. Thus, a combined therapy including agents that control local inflammatory responses could be highly beneficial. We report that deficiency for CD74, the invariant chain for MHCII, appears protective against P.aeruginosa-induced corneal damage. This protection is exemplified with reduced bacterial burdens, and reduced pathology during infection. Since CD74 deficiency impairs MHCII-driven antigen presentation, changes in the CD4+ T cell maturation are expected. Consistently, the observed improved recovery of the CD74 KO could be due to alterations of the CD4+ T cell functions. However, about 8-10% of the CD74 is expressed on the cell surface, independently of the MHCII, where it forms complexes with CD44, and serves as s receptor for macrophage migration inhibitory factor (MIF). An alternative scenario to explain the lessened pathology would be a deficiency in the MIF-CD74 signaling axes. Consistently, MIF deficient mice are protected from P.aeruginosa-induced karatitis, suggesting that MIF-driven CD74-mediated activities exacerbate pathology during acute bacterial infection.
Divergent expression patterns of IL-4 and IL-13 define unique functions in type 2 immunity (56.22)
Authors: Reinhardt Richard , Liang Hong-Erh , Sullivan Brandon , Locksley Richard
Published: January 2023

Interleukin-4 (IL-4) and interleukin-13 (IL-13) are closely related cytokines critical to host responses against parasitic helminthes. We use reporter mice genetically engineered to mark cells producing IL-4 and IL-13 in vivo, allowing the temporal and spatial assessment of cytokine production at the single-cell level. In the lymph node, IL-4 is confined to T follicular helper (Tfh) cells. Unexpectedly, Tfh cells do not make IL-13. In contrast, tissue Th2 cells produce both cytokines. Divergent IL-4 and IL-13 production also occurs in innate immune cells. Basophils produce IL-4, while innate helper type-2 (Ih2) cells make only IL-13. Cytokine production by Th2 and Ih2 cells is intimately associated with high GATA-3 expression. Conversely, Tfh cells and basophils regulate IL-4 independently of GATA-3. Partitioning of the localization and cellular expression patterns of IL-4 and IL-13 helps explain the unique roles of these cytokines in anti-helminth and allergic immunity.
A positive feedback mechanism in the regulation of mammalian chitinase responses (56.29)
Authors: Vega Karina , Diaz-Arevalo Diana , Bagramyan Karine , Hong Teresa , Kalkum Markus
Published: January 2023

Immunosuppressed patients are highly susceptible to invasive fungal infections (IFI) such as invasive pulmonary aspergillosis, which is predominantly caused by the fungus Aspergillus fumigatus. An important component of the fungal cell wall is chitin, a polymer of N-acetyl-D-glucosamine (GlcNAc). Chitin is not produced by humans, however, the chitin degrading enzymes (chitinases) chitotriosidase (Chit-1) and acidic mammalian chitinase (AMCase) are. Chitinase is predominantly produced by activated macrophages, and may possibly aid in the defense against chitin-containing pathogens. We show that serum and bronchoalveolar lavage (BAL) chitinase levels are increased in patients with IFI, and in mice after pulmonary exposure to A. fumigatus conidia. Several different stimuli, including stimulation with chitin can lead to chitinase responses. In vitro stimulation of U937 human monocytes and RAW mouse macrophages with either chitin-particles (7-12 µm) or GlcNAc increased secreted and intracellular chitinase activity, the accumulation of intracellular Chit-1, and significantly increased the mRNA levels of Chit-1 and AMCase. Accordingly, we hypothesize that Chit-1 expression is regulated through a positive feedback mechanism involving the degradation of chitin to GlcNAc by host chitinases and GlcNAc recognition that in turn upregulates chitinase expression. This potential feedback mechanism of chitinase regulation may have utility in the diagnosis of IFIs.
IL-21 contributes to the control of infection and to the severity of gastritis during Helicobacter infection (56.3)
Authors: Algood Holly , Chaturvedi Rupesh , Wilson Keith , Washington M.
Published: January 2023

The immune response to Helicobacter pylori involves a mixed T helper-1, T helper-2, and T helper-17 response. It has been suggested that T helper cells contribute to the gastric inflammatory response during infection, and that Th1 and Th17 subsets may be required for control of H. pylori colonization in the stomach. The relative contributions of these subsets to gastritis and control of infection are still under investigation. Expression of IL-21, which may be produced by a number of T cell subsets, has been reported to increase during H. pylori infection. IL-21 plays a role in both B cell stimulation and T cell differentiation. To investigate the role of IL-21 in H. pylori, we infected IL-21 deficient mice and wild-type littermates with H. pylori strain SS1. At 3 months post-infection, we assessed colonization, gastric inflammation, cellular infiltrate in the stomach, and cytokine profiles in the gastric tissue. Specifically, H. pylori-infected IL-21-/- mice have higher levels of H. pylori in their stomachs (4.5±0.4x107 CFU v. 4.3±2.3x106 CFU, **p=0.004), significantly less gastritis (gastritis scores 3.8±0.2 v. 1.2±0.3, ***p=0.0002), and reduced expression IL-17 and IFNγ (*p<0.05) compared to H. pylori-infected wild-type mice. Our results indicate that IL-21 contributes to the control of infection and severity of gastritis, suggesting IL-21 is required for activation of the immune response and induction of gastritis in response to H. pylori.
Differential kinetics of cell surface marker modulation with T cell activating reagents (57.14)
Authors: Tran Kimvan , Clor Julie , Tyagarajan Kamala
Published: January 2023

Simultaneous assessment of cell surface phenotypic markers, activation markers such as CD69 and apoptosis related markers such as CD95/FAS can provide deeper understanding of mechanism and impacts of in vitro mitogenic stimulation on immune cell populations.. Kinetic and dose response studies of PBMC’s with different stimulants was performed and followed with novel simplified assays that could assess levels of CD69 and CD95 on T and B cells followed by plate-basedmicrocapillary flow cytometry. Information on the CD4, CD8, CD3,CD19, CD45 expression levels was also obtained. Our results demonstrate that while CD4 expression on CD4 T cells was rapidly downregulated with phorbol myristate acetate or combinations with ionomycin, but was not impacted by PHA treatment. Tracking of CD95 expression levels demonstrate the elevation of CD95 on T and B cells with PHA treatment with no significant loss in CD4 expression levels. Correlation of T celll activation markers and apoptotic markers and expression levels of phenotypic markers would facilitate further elucidation of the mechanism of T cell activation.
Amelioration of β-glycerophosphate-induced vascular calcification by the IL-10 family of cytokines (57.25)
Authors: Kang Haeng-A , Lee Ki-Mo , Kang Hyung-Sik
Published: January 2023

Vascular calcification is a hallmark of cardiovascular diseases. The IL-10 family of cytokines has been known to suppress the tumor progression in a variety of human cancers, but its role has not yet been investigated in vascular calcification. To determine whether the IL-10 family of cytokines can inhibit vascular calcification, the effect of the IL-10 family of cytokines on vascular calcification was examined in β-glycerophosphate (β-GP)-induced vascular smooth muscle cells (VSMCs) calcification model. Calcium deposition was declined in the IL-10 family of cytokines-treated calcified VSMCs. The expression of Runx2 and alkaline phosphatase was also suppressed by the IL-10 family of cytokines in the calcified VSMCs. The IL-10 family of cytokines inhibited β-GP-induced activation of the Wnt/β-catenin signaling pathway in VSMCs. These results suggested that the IL-10 family of cytokines ameliorated vascular calcification by suppressing the expression of Runx2 and alkaline phosphatase through the blockade of the Wnt/β-catenin signaling pathway. These findings collectively suggest that the IL-10 family of cytokines plays a critical role in the regulation of vascular calcification, and it may be a potential target for the therapy against cardiovascular diseases.
The ubiquitin E3 ligase Cul4B regulates TTP protein turnover and TNFα mRNA stability independent of p38 (57.6)
Authors: Brooks Seth , Pfeiffer Jason
Published: January 2023

TNFα is a central mediator of inflammation and is critical for host response to infection and injury. TNFα biosynthesis is controlled by transcriptional and post-transcriptional mechanisms allowing for rapid, transient production. Tristetraprolin (TTP) is an AU-Rich Element binding protein that regulates the stability of TNFα mRNA. Previous work has demonstrated that TTP function is regulated by activation of both the p38 and ERK kinase pathways and that TTP protein is rapidly degraded by a proteasomal mechanism. Using a novel screen for TTP interacting proteins, we identified Cullin 4B (CUL4B), a scaffolding component of the CRL family of ubiquitin E3 ligases. shRNA knockdown of Cul4B causes a significant reduction in TNFα protein and mRNA in resting and LPS stimulated (100 ng/ml 90 min) mouse macrophage RAW264.7 cells. Cul4B knockdown also reduces TTP protein levels. Moreover, the TNFα half life was reduced from t1/2=68.7 min to t1/2=38.9 min. TNF-3’UTR luciferase assays utilizing wild-type and mutant TTP, which cannot be phosphorylated by p38, indicate that Cul4B operates independent of p38 activity. Based on the data to date, we conclude that Cul4B acts to inhibit TTP protein turnover and in turn limit TTP mediated decay of the TNFα mRNA. Thus, the loss of Cul4B enhanced both TTP protein and TNFα mRNA turnover. Further studies are ongoing to establish the mechanism through which Cul4B operates.
Inhibition through PD-1 is crucial for protection against M. tuberculosis infection (58.1)
Authors: Lazar-Molnar Eszter , Chen Bing , Jacobs William , Nathenson Stanley
Published: January 2023

With over one-third of the world's population currently infected with M. tuberculosis, there is an increasing need to develop novel vaccines and immunotherapies. Better understanding of the early immune responses in the lung is crucial for this purpose. We have shown that Programmed Death-1 (PD-1), a co-inhibitory receptor that is known to negatively regulate adaptive immune responses, is crucial for the development of a protective host response against M. tuberculosis. PD-1 deficient mice are extremely sensitive to tuberculosis, show uncontrolled bacterial proliferation in the lung and develop severe necrotic pneumonia, with dramatically reduced survival. Cytokine responses in the lungs of PD-1-/- and wild-type mice were profoundly different, indicating a significantly altered inflammatory response in the absence of PD-1. PD-1 blockade in wild-type mice after M. tuberculosis infection resulted in decreased survival, suggesting that immunotherapeutic approaches targeting PD-1 blockade should be considered cautiously. By studying genetically modified mouse models that have altered PD-1 or PD-L expression, our data show differential contributions of PD-L1 and PD-L2 to the immune regulatory role of PD-1 observed in tuberculosis. By providing the mechanism on the immune regulatory role of PD-1 in tuberculosis, our data will allow for the identification of novel therapeutic targets, which can be of great value for future immunotherapy.
Chemoattractant regulation of Toxoplasma gondii-induced encephalitis (58.4)
Authors: Marino Ana Paula , Murphy Philip
Published: January 2023

Some leukocyte chemoattractants and their receptors have been reported to regulate outcome in T. gondii infection of mice, however, most have not yet been evaluated. We have begun to systematically and comprehensively survey chemoattractant receptors in T. gondii infection, using the natural peroral route of administration of the organism. Chemoattractant receptor mRNA was quantified from brain extracts from C57BL/6 mice infected with 10 cysts of the ME49 strain. Parasitism peaked in the brain on the 25th day post infection, and infection was uniformly fatal by day 100. The organism induced expression of the pro-inflammatory chemoattractant receptors C5L2, Ccr1 and Ccr5; the decoy receptors, Ccrl2 and CMKLR1; and the constitutively expressed receptor ligand Cx3cl1. Mice lacking the Cx3cl1 receptor Cx3cr1 had accelerated mortality, whereas mortality was delayed in mice lacking Fpr1, a classical chemoattractant receptor. Unexpectedly, animals infected with T. gondii also had a substantial increase of Ccr7 mRNA during the entire chronic phase, and Ccr7-/- mice exhibited dramatically increased mortality, in comparison with WT mice. The poster will focus on Ccr7, delineating the microbiologic and immunologic correlates of skewed mortality in infected Ccr7-deficient mice.
Dichotomous consequences of IL-23 signaling in chemically induced colitis in mice (59.2)
Authors: Cox Jennifer , Ghilardi Nico
Published: January 2023

The interleukin-23 (IL-23) pathway has emerged as a promising therapeutic target for inflammatory bowel disease and blockade of IL-23 signaling is beneficial in mouse models of intestinal inflammation. Although the pathogenic role of IL-23R on T lymphocytes is well established, the function of IL-23R on innate immune cells has not been thoroughly examined. Here we investigate the consequence of IL-23R deletion in dextran sodium sulfate (DSS)-induced colitis, a chemically-induced acute model of inflammatory bowel disease. In IL23R-/- and IL23p19-/- mice, we observed decreased weight loss, improved histological scores, and reduced leukocyte infiltrate following DSS exposure. Surprisingly, when the IL-23R-/- allele was crossed into Rag2-/- mice, we observed exacerbated disease characterized by delayed recovery, increased epithelial damage, and reduced pSTAT3 in the epithelium in IL23R-/-Rag2-/- mice. This defect in epithelial repair in IL23R-/-Rag2-/- mice was rescued with exogenous IL22-Fc, and epithelial pSTAT3 was restored to normal levels. Depletion of Thy1+ innate lymphoid cells eliminated the majority of IL22 production in the colon lamina propria of DSS-treated mice, suggesting that these are the major IL-23 responsive innate cells in this context. In summary, we provide evidence for a protective role of IL-23 in murine colitis with implications in the therapeutic blockade of IL-23 signaling.
Role of Smad4-deficient T cells in Gastrointestinal Inflammatory Disease (59.7)
Authors: Kim Byung-Gyu , Choi Sung Hee , Robinson Janet , Lee Hyoung-Gon , Letterio John
Published: January 2023

Selective loss of Smad4 signaling in T cells leads to spontaneous gastrointestinal inflammation and cancer. However, the mechanism underlying the pathogenesis of these diseases is not completely understood. Here, we report that Smad4 is essential for TGF-β conversion of naïve T cells into Foxp3+ regulatory T (Treg) cells and for TGF-β suppression of effector T cells activity. We show that Smad4 deficient (Smad4KO) T cells do not generate Foxp3-expressing cells in inducible Treg (iTreg) differentiation condition. In suppression assays, iTreg cells from naïve WT T cells suppress T cell proliferation but Smad4KO T cells lack suppressive activity. In Smad4KO mice at 6 months of age, FoxP3+ Treg cells among CD4+ T cells are significantly reduced in the colon, but not in the thymus or spleen compared to those of WT. Moreover, TGF-β suppresses the proliferation of WT CD4+ T cells via cell cycle arrest and reduces the production of pro-inflammatory cytokines including INF-γ and IL-6, but TGF-β fails to suppress T cell proliferation and pro-cytokine production of Smad4 KO T cell. As expected, CD4+ and CD8+ T cells in the colon of Smad4KO mice are highly activated and produce pro-inflammatory cytokines, such as INF-γ and IL-6 greater compared with those of WT. Taken together, our results suggest the important role of Smad4 dependent TGF-β signaling in T cells in the maintenance of mucosal immune homeostasis and the pathogenic mechanism underlying gastrointestinal disease.
Beraprost enhances the APC function of B cells by upregulating CD86 expression levels (61.10)
Authors: Choe Jongseon , Kim Jini
Published: January 2023

Lipid mediators are emerging as important regulators of the immune system. Based upon our previous result that shows strong expression of prostacyclin synthase in the germinal center, we investigated whether prostacyclin would regulate the APC function of B cells. Due to the very short half-life of prostacyclin in experimental conditions, we used a more stable analogue beraprost. Beraprost increased the amounts of the costimulatory molecule CD86 but not CD80 on the surface of activated B cells in time- and dose-dependent manners. However, the enhancing effect of beraprost was not observed on memory B cells, centroblasts, and centrocytes. Beraprost required BCR and CD40 signals to upregulate CD86 expression levels. Other prostanoids such as PGE2, 6-keto-PGF1α, and PGF2α failed to alter CD86 expression levels, whereas other prostacyclin analogues were as potent as beraprost. Results carried out with receptor antagonists revealed that beraprost enhanced CD86 levels by binding to prostacyclin receptor IP and by increasing intracellular cAMP concentrations. Beraprost-treated B cells potently stimulated allo-T cells, which was significantly abolished by CD86 neutralization. Our data imply an unrecognized cellular and molecular mechanism about the germinal center reactions.
B cells inhibit and CR2 promotes antigen specific cellular immunity (61.15)
Authors: Silva Ines , Balin Samuel , Platt Jeffrey , Cascalho Marilia
Published: January 2023

Although T cells and B cells can respond immunologically to some tumors, whether and how they collaborate to control tumor growth is incompletely understood. To address this question we introduced a foreign antigen (HIV envelope), as a model for a tumor antigen, linked to C3d (AgC3d) in a tumor cell line and tested the fate of that line in isogeneic mice. AgC3d-positive tumors invoked antigen-specific antibodies and T cell-mediated IFN gamma secretion. Tumors expressing AgC3d grew more slowly than non-transfected cells and mice with tumors expressing AgC3d survived longer than mice with tumor alone. Slower growth of tumors was due to immunity directed against the viral antigen since AgC3d-positive tumors grew as fast as AgC3d-negative tumors in immune-deficient RAG-2 gammac-deficient hosts. Immunity and control of tumor growth required C3d since tumors expressing the antigen without C3d grew slower than tumors expressing AgC3d. Anti-tumor immunity depended on CR2 since tumors implanted in mice treated with CR2-Ig grew more rapidly than tumors grown in the absence of CR2-Ig. Control of tumors was not the direct result of C3d interaction with B cell CR2 as AgC3d-positive tumors grew slower and in some cases regressed in B cell deficient (JH-/-) mice than in wild type mice. Our findings suggest that C3d enables antigen specific responses by T cells and this response may be regulated by B cells.
Increased activity of proteasome associated deubiquitinating enzyme, USP14, accompanies lowered proteasomal proteolysis in primary human T lymphocytes during aging. (63.18)
Authors: Ponnappan Usha , Ponnappan Subramaniam , Ovaa Huib , Cane Stefania , Sullivan Dennis , Palmieri Michela
Published: January 2023

Aging is accompanied by a significant decline in proteasomal proteolysis that underlies lowered induction of T cell functional response. As USP14, found in association with the proteasome is both necessary and important in mediating ubiquitin trimming from proteasomal substrates, we evaluated functional USP14 levels by employing active site probes for proteasome associated deubiquitinating enzymes (DUBS). Our studies for the first time demonstrate that in T lymphocytes obtained from elderly donors, USP14 activity is significantly higher than those from young donors, and additionally, increased functional activity of USP14 accompanies conditions under which 26S proteasome catalytic activities are inhibited. UCH37, another DUBS associated with 26S proteasome appears to demonstrate only a modest increase in activity. Alteration in USP14 activity appears to be reciprocally regulated by the catalytic function of the 26S proteasome. Increase in USP14 activity is accompanied by an overall increase in USP14 protein but not mRNA expression in cells from the elderly. Thus, DUB activity associated with the proteasome demonstrates compensatory increase in activity during aging, and may regulate local levels of monomeric ubiquitin.
Genomic evolution of the B7 family (63.28)
Authors: Ota Yuko , Krylov Vladimir , Tlapakova Tereza , Flajnik Martin
Published: January 2023

B7 ligands are expressed by different cell types and interact with receptors on T cells to generate signals required for immunoregulation. All B7 members have structurally distinct domains in the immunoglobulin superfamily (IgSF). Some variable (V) domains of B7 ligands contain an ancestral, so-called “VJ” IgSF domain, while some constant (C) domains are of the “C1-type,” a rare form found primarily in molecules associated with adaptive immunity. Phylogenetic analysis had suggested a relationship between the V domains of B7 and MHC-linked tapasin (TAPBP), suggesting that they share a common ancestor. To study the evolutionary origins and functional aspects of B7 ligands, we used a comparative genomics approach emphasizing the genome of the amphibian Xenopus, a high-connectivity model. All known B7 family members except CD80 were found in X. tropicalis. Some B7 genes show conserved synteny between human and frog, suggesting that they are ancient and thus vital for the immune system, while the other genes were taxon-specific, perhaps having novel functions. In a more thorough bioinformatics approach, we found un-assigned B7 family genes from different vertebrate taxa including human. One of these genes is B7-H6 and the Xenopus orthologue is linked to beta-2 microglobulin (B2M). B2M is MHC-linked in sharks, presumably in the primordial organization. Thus, linkage of a B7 precursor to the MHC seems to be ancestral as part of the pre-adaptive immune complex.
Cyclosporine-resistant, Rab27a-independent mobilization of intracellular preformed CD40L mediates antigen-specific T cell help in vitro (63.8)
Authors: Gardell Jennifer , Thauland Timothy , Parker David , Koguchi Yoshinobu
Published: January 2023

CD40L is critically important for the initiation and maintenance of adaptive immune responses. CD40L expression in CD4+ T cells is regulated transcriptionally and produced from new mRNA following antigen recognition. However, recent studies with two-photon microscopy revealed that most cognate interactions between effector CD4+ T cells and APCs are too short for de novo synthesis of CD40L. Given that effector and memory CD4+ T cells store preformed CD40L (pCD40L) in a secretory compartment that comes to the cell surface within minutes of antigenic stimulation, we and others have proposed that pCD40L might mediate T cell-dependent activation of cognate APCs during these brief encounters. However, it has not been shown that the relatively small amount of pCD40L is sufficient to activate APCs, owing to the difficulty of separating the effects of pCD40L from those of de novo CD40L and other cytokines in vitro. Here we show that pCD40L surface mobilization is resistant to cyclosporine or FK506 treatment, while this treatment fully inhibits de novo cytokine expression. This allows us to dissect the role of pCD40L in APC activation. We find that pCD40L mediates selective activation of cognate but not bystander APCs in vitro and pCD40L mobilization does not depend on Rab27a, which is required for mobilization of lytic granules. Therefore, effector CD4+ T cells deliver pCD40L specifically to APCs on the same time scale as the lethal hit of CTLs but with distinct molecular machinery.
IL-23 induces apoptosis of self-reactive thymocytes in thymic negative selection by an Rorc dependent mechanism (64.19)
Authors: Li Hao , Wu Qi , Yang PingAr , Cua Daniel , Hsu Hui-Chen , Mountz John
Published: January 2023

Thymocyte apoptosis can occur by over-induction of a specific transcription factor, RORγt (or its gene, Rorc); however, the role of RORγt in thymocyte selections and the upstream signal that drives the expression of Rorc are largely unknown. We have observed that interleukin-23 (IL-23), a cytokine which has been implicated as a key regulator of Rorc, was expressed by thymic dendritic cells. To determine if the IL-23-RORγt signaling axis is involved in thymocyte selections, we have determined the expression of Il23r and Rorc in thymocytes from the male antigen-specific Db/H-Y T-cell receptor (TCR) transgenic (Tg) and the MOG35-55 antigen-specific 2D2 TCR Tg mice. Specific antigen activation induced Il23r expression on CD4+CD8+ double positive (DP) thymocytes and then upregulated the expression of Rorc with IL-23 stimulation, leading to dramatic induction of DP thymocyte apoptosis in vitro in TCR Tg+ mice. In vivo over-expression of IL-23 in B6 mice by adenovirus (AdIL-23) efficiently depleted DP thymocytes by promoting apoptosis (from 1.5% to 10%) as determined by in situ TUNEL staining. This AdIL-23 induced thymocyte apoptosis was partially blocked in Rorc+/- mice. Consistent with these, there was an increased percent of Db/H-Y Tg+ thymocytes in Il23 p19-/- mice, compared with that in p19 intact male Db/H-Y Tg mice. Taken together, our work suggests an important role for the IL-23-RORγt axis in the induction of TCR-mediated apoptosis and negative selection in thymocytes.
Notch signaling requires GATA-3 for the survival of cells entering the T-lineage development program (64.4)
Authors: Thompson Patrycja , de Pooter Renée , Zúñiga-Pflücker Juan Carlos
Published: January 2023

GATA-3 plays a crucial role in T cell development, however its role and collaboration with the Notch signaling pathway in the induction of T cell specification and commitment have not been fully elucidated. Work in our laboratory using both, in vitro and in vivo models of T cell development, has shown that GATA-3 deficiency arrests this development at the DN2 stage but does not result in B-lymphopoiesis in the presence of Notch ligand Delta-like 1 (Dll1). These GATA-3-/- progenitors express T-lineage associated genes but are unable to survive the T-specification event as indicated by their increased rate of apoptosis. Cell cycle regulation genes, mainly p19Arf and p16Ink4a (Arf/Ink), are upregulated in these cells as indicated by gene expression microarray and qRT-PCR analyses. We have thus sought to address the possibility that GATA-3 represses Arf/Ink following T cell specification/commitment in the presence of Notch signals. We are employing ChIP assay to show whether potential GATA-3 recognition sequences present in the Arf/Ink promoter region are occupied and regulated by GATA-3. Finally, using GATA-3-/-Arf-Ink-/- bone marrow progenitors, we will address a possible rescue of the early T cell developmental block observed in the absence of GATA-3. These experiments will help to identify a potential mechanism of how GATA-3 and the Notch signaling pathway collaborate in regulating T-lineage specification, commitment and survival during T cell development.
Knock-down of IL-12 secretion in dendritic cells leads to Th2 polarization of T helper cells in vitro (65.12)
Authors: Heilmann Katja , Völker Christina , Wand Inga , Micheel Burkhard , Rohayem Jaques
Published: January 2023

Dendritic cells, T lymphocytes and B lymphocytes were co-culativated in vitro in order to induce an in vitro immunization process resulting in the activation of the involved cell populations and in the production of antigen-specific antibodies. Bone-marrow derived dendritic cells were treated with IL12-siRNA to inhibit the secretion of IL-12 which is mainly responsible to induce Th1 directed immune responses. The siRNA-treated dendritic cells were then loaded with ovalbumin (OVA) as antigen and co-cultured with naive OVA-TcR-transgenic T helper cells and naive B lymphocytes. The activation steps and the production of OVA-specific antibodies were investigated by FACS, RT-PCR and ELISA. FACS and RT-PCR analysis revealed a Th2 polarization of the involved T helper cells according to their cytokine patterns and c-MAF expression. Ovalbumin-specific antibodies could be detected in the culture supernatants after co-cultivation of naive B cells with T cells and siRNA-treated dendritic cells. These studies indicate that the silencing of IL-12 can lead to a Th2 polarization of in vitro cultivated T helper cells. This step could allow an in vitro induction of antigen-specific antibody molecules in shorter time frames than the conventional hybridoma technology and can simplify the generation of antibodies of non-murine origin.