bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective loss of dominant low affinity CD4 T cells during memory differentiation (85.6)
Authors: Baumgartner Christina , Malherbe Laurent
Published: January 2023

Antigen-specific memory T cells are crucial for conveying protection against pathogens upon vaccination. The evolution of the CD4 memory T cell repertoire is not fully understood. We analyzed CD4 memory T cell differentiation of a diverse, private and broad affinity CD4 T cell repertoire that developed in draining lymph nodes of mice 7 days after immunization with moth cytochrome c peptide (MCC88-103) in MPL-based adjuvant. 30 days after primary immunization we found a dramatic remodeling of the repertoire. The initially diverse repertoire evolved into a focused and public repertoire expressing high affinity T cell receptors. Importantly, 30 days after primary immunization we observed a selective loss of one particular low affinity clonotype that dominated the primary response at day 7. Absence of this clonotype in draining lymph nodes was not due to recirculation through the animal, since we could not detect this clonotype after re-challenge of mice with the peptide 30 days after primary immunization. Additionally, when we immunized mice with yet another peptide of high peptide-MHCII stability we observed a dramatic repertoire focusing towards high affinity clonotypes to the same extent as in MCC88-103 immunized mice. Thus, after primary immunization only public, high affinity CD4 T cells differentiate into memory CD4 T cells.
Absence of Tec family kinases Itk and Btk significantly impairs FcϵRI dependent mast cell responses (86.26)
Authors: Morales Jose , Iyer Archana , Ojo Folake , Ning Gang , August Avery
Published: January 2023

While the etiology of asthma can be multi factorial, signaling through the IgE receptor (Fcϵ receptor I) on mast cells is critical to its onset. The Tec family tyrosine kinases ITK and BTK serve important roles as signal amplifiers downstream of the FcϵRI, however, their individual contribution to FcϵRI signaling has not been fully established. We have generated Itk and Btk double deficient (DKO) mice in an effort to determine the relative role of Itk and Btk to FcϵRI signaling in mast cells. Analyses of skin mast cells from these mice revealed reduced mast cell granule content and density. However, in vitro cultured bone marrow derived mast cells (BMMCs) had normal gross morphology and granule density. Mast cell histamine release was also compromised in DKO mice, likely associated with elevated endogenous IgE expression in these mice. In vitro growth analysis indicated that Btk negatively regulates BMMC growth as previously published, while the absence of both Itk/Btk did not further contribute to this phenotype. However, there was a significant reduction in FcϵRI mediated calcium responses, accompanied by reduced phosphorylation of PLCγ2 and the mitogen activated protein kinase p38. Lastly, a reduction in the production of several cytokines was also observed in DKO BMMCs. Our results suggest that Itk and Btk have redundant, complementary, and unique functions in mast cell FcϵRI signaling that could be pharmacologically manipulated to treat asthma.
Using mast cells derived from human CD34+ bone marrow stem cells to identify drugs that may produce pseudoallergic reactions (86.3)
Authors: Haskell Keith , Whritenour Jessica , Donahue Christopher , Blake William , Wolfram Thaddeus , He Linyan , Kawabata Thomas , Thiede Mark
Published: January 2023

Drugs such as morphine, vancomycin, radio-contrast dyes and smooth muscle relaxants are known to cause pseudoallergic or anaphylactoid reactions, which are very similar to Type 1 hypersensitivity reactions. These drugs directly stimulate mast cell degranulation by an IgE independent-mechanism and in drug development, there is a critical need to identify compounds that may be pseudoallergens in humans. To this end, an in vitro culture was developed to generate a reproducible supply of human mast cells Immuno-selected CD34+ bone marrow cells (106) were cultured for 63 days with weekly media changes and sequential cytokine additions. Cultures were maintained under hypoxic conditions from days 1-28 and switched to normoxic conditions from days 29-63. On day 63, the cultures were FcER1+, CD45+, CD117+, tryptase+, chymase+ CD34-, and CD123- and released histamine and PGD2 when exposed to anti-IgE, substance P, or compound 48/80. We tested several known pseudoallergens (morphine, tubocurarine, and vancomycin) for their ability to stimulate histamine release by mast cells produced from various donors. All 3 drugs reproducibly stimulated histamine release at 100uM with Morphine = tubocurarine > vancomycin. Based on these studies, it appears that the mast cells derived from CD34+ cells do respond to known pseudoallergens, and may be used to determine if drugs in development have the potential to produce pseudoallergic reactions.
TGFb1 Suppresses the Mast Cell Response In Vitro and In Vivo (86.6)
Authors: Ryan John , Fernando Josephine , Barnstein Brian
Published: January 2023

Mast cell activation is a critical component of allergic disease, and may be regulated by cytokines present in the microenvironment. We recently described the suppressive effects of TGFb1 on IgE signaling in mouse and human mast cells. We have furthered this study, demonstrating that TGF injection or aerosolization reduces mast cell IgE receptor expression in vivo, and suppresses eosinophil recruitment in a mast cell-dependent model of airway hyperresponsiveness. We find that TGFb1 inhibits expression of the key signaling molecules Syk, Fyn, Akt, and Stat5 at the protein level without any change in mRNA expression. In contrast, expression or activation of ERK, JNK, GSK, and p38 was unaffected. Fyn transfection partially reversed TGFb1-mediated suppression of IgE-induced IL-13 secretion. Our data support the theory that TGFb1 possesses potent mast cell suppressive activities both in vitro and in vivo.
Antagonistic Control of IgG-mediated Mast Cell Activation by Lyn and Fyn Kinases (86.8)
Authors: Ryan John , Falanga Yves
Published: January 2023

Mast cells are key players of the innate immune system. In addition to their role in atopic disease, mast cell involvement is strongly suspected in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, and atherosclerosis. Once activated, they degranulate and produce cytokines, resulting in the recruitment of other immune cells. It is well-appreciated that mast cells are activated via the high-affinity immunoglobulin E (IgE) receptor FcϵRI. In this study we focus on mast cell activation via the immunoglobulin G (IgG) receptor FcγRII/RIII and downstream signaling by Fyn and Lyn kinases. Lyn-deficient mast cells displayed increased FcγR expression, correlating with enhanced IgG-mediated histamine and leukotriene C4 secretion. The mast cells did not show a change in IgG-mediated cytokine production, suggesting a restricted and negative regulatory role for Lyn kinase in mast cell IgG signaling. In contrast, we show that Fyn kinase is rapidly activated by IgG crosslinkage, and is required for both IgG-mediated histamine release and cytokine production in mast cells. We obtained similar results when studying Lyn- or Fyn-deficient basophils and macrophages. Finally, we found that Lyn-deficient mice had exacerbated responses to passive systemic anaphylactic shock elicited by IgG challenge in vivo. These data illustrate the importance of Src family kinases in IgG signaling, a means by which mast cells may be activated in chronic inflammatory diseases.
Potential of cannabinoids to modulate the inflammatory response in colitis (87.1)
Authors: Zaric Violeta , Busbee Brandon , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Recent studies have suggested the role for cannabinoid (CB) type 1 and CB2 receptors in modulating gastrointestinal inflammation in animal models of experimental colitis. However, the immunosuppressive effect of the cannabinoid system has not been fully investigated. Our preliminary studies using delta-9-tetrahydrocannabinol (THC) showed promising results in a 2,4,6-trinitrobenzene sulfonic acid TNBS-induced colitis. 50mg/kg body weight of TNBS/ethanol was administered in 10 female BALB/c mice. Each group received intraperitoneal injections of vehicle or THC (10 mg/kg) once every other day. At day 6, mice were sacrificed and cells from spleen and MLN were analyzed for the expression of surface markers for T-cells, myeloid-derived suppressor cells (MDSC) and regulatory T-cells (Treg) by flow cytometry. The body weight increased in TNBS-treated mice administered THC when compared to vehicle. THC improved the survival up to 60% of mice in TNBS-induced colitis when compared to vehicle treated mice (30%). In the spleen and MLN, the ratio of T-cells is lower in THC-treated mice compared to vehicle-treated group whereas the ratio of Treg is increased in THC-treated mice. THC also reduced the ratio of MDSCs in TNBS-treated mice. THC treatment ameliorated colitis by modulating the immune response towards Treg and altering the T-cells and their subsets. Future experiments will address the potential of other cannabinoids in modulating the inflammatory response in colitis.
H.polygyrus infection induces functionally distinct FOXP3+IL-10+ and FOXP3+IL-10- cell subsets in the Lamina propria of mice (87.15)
Authors: Stoyanoff Korynn , Hang Long , Blum Arthur , Setiawan Tommy , Leung John , Weinstock Joel
Published: January 2023

Background: Infection with the murine intestinal helminth H. polygyrus (Hp) results in the regulation of inflammation in the gut Lamina propria (Lp) of mice. Infection upregulates FOXP3 expression in Lp T cells, but the effect of Hp infection on various FOXP3+ subgroups and their function in unknown. We hypothesized that Hp infection induces one or more FOXP3+ T cell subgroups and results in enhanced regulation. Method: We distinguished several FOXP3+ subgroups using a dual reporter mouse for FOXP3 and IL-10. Spleen (Sp), mesenteric lymph nodes (MLN), ileum Lp, and colon Lp cells from mice were analyzed by FACS for percentages of FOXP3 and/or IL-10 expressing cells. Subsets were also tested for their ability to regulate Sp cell proliferation. Results: The FOXP3+IL-10- subset is dominant in the Sp and MLN, the FOXP3+IL-10+ dominates in the colon Lp, and the subsets are roughly equal in the ileum Lp of infected and uninfected mice. Hp infection increases FOXP3+IL-10+ cells in ileum and colon Lp cells vs. uninfected mice. We tested the ability of FOXP3+IL-10+ cells to regulate the proliferation of Sp T cells in 48hr cultures. FOXP3+IL-10- inhibited proliferation better than FOXP3+IL-10+ cells (67.41% vs. 23.88% inhibition, 1:1 ratio). Discussion: Hp infection increases FOXP3+IL-10+ expression in the Lp. Other regulatory roles for FOXP3+IL-10+ cells are being investigated and may be important in understanding how Hp infection regulates gut inflammation.
Role of Adenosine A2B Receptors in Regulating Inflammatory Responses of TNBS-induced Experimental Colitis (87.2)
Authors: Alam Mohammad , Wilson Jeff , Ernst Peter
Published: January 2023

Background: Adenosine, an endogenous purine nucleoside exerts anti-inflammatory activities on host cells via signaling through the G protein-coupled receptors subtypes referred as A1, A2A, A2B, and A3. Both A2A and A2B adenosine receptors regulate inflammatory responses during experimental colitis. Since intestinal epithelial cells and antigen presenting cells express the A2BAR and contribute an important role in the development of colitis, we studied the effect of A2BAR gene deletion in mice during 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Methods: TNBS pre-sensitized A2BAR-null (A2BAR-/-) & wildtype mice were intrarectally instilled with TNBS and changes in body weight, colonic tissue inflammatory responses and pro-inflammatory cytokine production were compared. Liver/spleen tissues were also cultured for evaluating bacterial translocation. Results: Studies indicated that the severity of colitis was increased in A2BAR-/- mice relative to wildtype controls, as evidenced by more dramatic weight loss and increased colonic tissue damage with histopathology revealing an extensive infiltration of immune/inflammatory cells. Levels of pro-inflammatory cytokines (TNF-a) were elevated in the colons of TNBS-treated A2BAR-/- mice. A2BAR-/- mice also showed increased systemic dissemination of intestinal bacteria in the spleen/liver cultures. Conclusion: Our data suggest A2BAR play a protective, anti-inflammatory role in TNBS-induced colitis.
Growth Detection: A Novel Role for CD4 T-cells (87.20)
Authors: Bewick Sharon
Published: January 2023

One of the central goals of immunology has been to understand the cellular and molecular mechanisms of immune regulation. Progress on the cellular front has developed rapidly with the identification of distinct CD4 T-cell populations with unique functions and patterns of cytokine production. Similarly, the discovery of TLRs in the late 1990s resulted in rapid advances towards an understanding of the molecular mechanisms of immune regulation. Despite the progress being made on both the cellular and molecular fronts, however, many aspects of immune regulation remain unexplained. Two Th-cell populations - induced regulatory T-cells (iTreg) and Th17 cells have recently been characterized. How iTreg and Th17 cells integrate with other Th populations remains an active area of research. Recently, we proposed a novel mechanism of immune regulation based on detection of pathogen growth (Bewick et. al, PLoS ONE, 2009). We then showed how growth detection can arise naturally as a result of signaling interactions and maturation kinetics of iTreg and Th17 cells. Building off of the basic premise of this growth detection model, we now extend the iTreg/Th17 system to include additional immune effector cells. This allows us to show how the immune system is organized in such a way that it can accurately and robustly classify different pathogens based on growth rate.
Immune regulatory roles of microparticles in autoimmune and allergic diseases (87.22)
Authors: Kahraman Tamer , Simsek Ismail , Gursel Mayda , Hakan Erdem , Pay Salih , Dinc Ayhan , Gay Steffen , Kocabas Can , Ozcan Celal , Civelek Ersoy , Gursel Ihsan
Published: January 2023

Microparticles (MP) are small vesicles secreted from immune and non-immune cells and their diameter ranges from 200 to 700nm. Studies demonstrated that MP number and source varies in several disease conditions. Therefore, we plan to characterize the cell source, activation state and levels of MPs in different clinical states of Behcet`s Syndrome (BS) and asthma. Isolated MPs were stained with Annexin V and cell specific CD markers (i.e. CD42a, CD69, CD105, CD31 and CD146). Number of marker specific MP/ml blood was detected by FACS. In the case of BS, blood plasma of 22 healthy, 28 inactive BS patients and 29 active BS patients were examined while blood plasma of 13 healthy, 15 atopic dermatitis (AD) and 15 asthma patients were used for asthma. The number of MPs/ml plasma is increased by >3 fold in active BS (34.1±10.9 x 104) and >2 fold in inactive BS (21.3±8.3 x 104) compared to healthy subjects (9.7±3.8 x 104). We also found that majority of secreted MPs were platelet origin, this was valid for allergic patients too. Surprisingly, MPs (number/ml plasma) were much higher in healthy donors (9.8±1.3 x 105) compared to atopic dermatitis (3.5±2.9 x 105) and asthma (2.4±0.5 x 105) patients. This data suggest that plasma MPs have differential immune regulatory roles in autoimmune and allergic conditions.
A genetically determined, serine-based and phosphorylation-dependent molecular switch regulates the anti-inflammatory potential of human IgA (87.34)
Authors: Ji Chuanyi , Wu Jianming , Gibson Andrew , Edberg Jeffrey , Kimberly Robert
Published: January 2023

Monteiro and colleagues have suggested dual roles for FcαRI in immune regulation through tyrosine-based “inhibitory/activating” mechanisms mediated by the immunoreceptor tyrosine-based activation motif (ITAM) in the Fc receptor γ-chain (FcRγ) [Pasquier et al., Immunity 2005, 22(1)]. We report a second mechanism in which the natural S248G variants of the FcαRI α-chain alter the inflammatory capacity of FcαRI and constitute a serine-based, genetically determined switch for regulation of the anti-inflammatory potential of human IgA. To elucidate the basis for this α-chain mechanism, we sought allele-specific FcαRI-associated molecules. Sab (SH3-domain binding protein that preferentially associates with Btk), a trans-inhibitor of Bruton's tyrosine kinase (Btk), is recruited by the common Ser248 allele in a phosphorylation-dependent fashion, whereas the protein tyrosine kinase Lyn is reciprocally recruited by the Gly248 variant. Compared to FcαRI Gly248, recruitment of Sab by FcαRI Ser248 results in inhibition of Btk activation and suppression of IgA effector functions independent of FcRγ-pairing. Expression of dominant negative Sab releases FcαRI-mediated inhibition in a Ser248-allele specific manner. These findings suggest an immunoreceptor serine-based inhibition motif (ISIM) which functions as a molecular switch for receptor induced activation/inhibition and advances our understanding of the divergent properties of human IgA in inflammation.
Role of the 3’ heavy chain enhancers in germline transcription and switch recombination (88.1)
Authors: Dunnick Wesley , Shi Jian , Fontaine Clint , Zerbato Jennifer , Collins John
Published: January 2023

Class switch recombination (CSR) is preceded by germline transcription and then mediated by a DNA recombination event. To study germline transcription and CSR we utilize transgenic mice with a 230 kb bacterial artificial chromosome that includes a rearranged VDJ gene, the entire heavy chain C region locus, and all known 3’ enhancers. In the intact transgene, germline transcription of and CSR to the γ, ϵ, and α heavy chain genes are regulated in parallel to the endogenous genes. We have reported that deletion of a 28 kb segment, including the 3’ enhancers HS3A, HS1,2, HS3B, and HS4, results in a reduction of CSR to all heavy chain genes to about 1% of the level of the intact transgene. We have derived four new versions of the heavy chain transgene: with 1 kb deletions of (i) HS1,2 and HS3B, (ii) HS1,2, HS3B, and HS4, (iii) HS3A, HS1,2, and HS3B, and (iv) HS3A, HS1,2, HS3B, and HS4. Comparing levels of CSR in these deletion variants to intact transgenes, we find that deletion of more enhancers results in a reduction in CSR for more heavy chain genes, and the reduction is more severe for a given heavy chain gene. Nevertheless, different enhancers have different effects on different heavy chain genes. Finally, there are enhancer deletions for which the reduction in CSR is much greater than the reduction in germline transcription. This suggests that the 3’ enhancers have a role in CSR that is distinct from the enhancement of germline transcription.
Lymphotoxin alpha gene polymorphism influences the cardiomyopathy development in chagas’ disease patients (88.16)
Authors: Rodrigues Virmondes , Pisseti Cristina , Oliveira Rafael , Correia Dalmo
Published: January 2023

Chagas’ disease, caused by Trypanosoma cruzi, is an important parasitic disease in Latin America, affecting more than 8 millions of people. The chronic phase is characterized by commitment of heart or digestive organs and the reason for the development of one clinical form or another is not well known. It is speculated that host and parasite genetic characteristics contribute for it. LT-α is an inflammatory cytokine, important in the organization and maintenance of the inflammatory process by stimulating adhesion molecules, such as VCAM, ICAM and E-selectin. Polymorphism on LT-α gene, position +252, were analyzed on subjects (n= 295) from an endemic area for Chagas’ disease grouped according to serology for T. cruzi and with clinical form (cardiac or indeterminate forms).Expression of LT-α was evaluated in culture supernatants and by flow cytometry. Presence of G allele was significantly more frequent in patients with heart commitment (cardiac form) (p=0.016, OR=2.4). Patients with cardiac form produced significantly more LT-α than individuals with the indeterminate form. Furthermore, infected individuals with G allele produced significantly higher levels of LT-α than those without G allele. CD8+ T lymphocytes were the major source of LT-α in basal condition and after stimulation with specific antigens. This study presents genetic and biological evidence for a role of LT-α in the development of cardiac form of Chagas’ disease.
Identification of a novel deubiquitinating enzyme DUBS6 that regulates Sirt6-mediated suppression of NF-κB in T cells (88.17)
Authors: Fang Deyu , Yang Heeyoung
Published: January 2023

The type III histone deacetylase Sirt6 is known as a regulator of aging in diverse species by regulating NF-kB-dependent gene expression. Here, using an LC-MS/MS approach combined with immunoprecipitation, we have identified a complex of Sirt6-interacting molecules (interactome) that contains about 40 proteins from mouse primary T cells. One of the interactome members is a novel deubiquitinating enzyme (DUB); we thus define this gene as a DUB of Sirt6 (DUBS6). Co-immunoprecipitation and western blotting analysis further validated that DUBS6 interacts with Sirt6 in transiently transfected HEK293 cells as well as in mouse primary T cells. Expression of DUBS6 inhibits Sirt6 ubiquitination and thus stabilizes Sirt6. Unexpectedly, DUBS6 expression, despite increased Sirt6 stability, reversed Sirt6-mediated suppressive function of NF-kB transcription activity. These results suggest that ubiquitination of Sirt6 appears to be required for its functions and that DUBS6 is a novel regulator in Sirt6-mediated NF-kB suppression in T cells.
HuR protein is involved in S49 thymoma cell proliferation and is regulated by cAMP/PKA signaling (88.20)
Authors: LaJevic Melissa , Cohen Rhonna , Chambers Donald
Published: January 2023

The RNA binding protein human antigen R (HuR) participates in the post transcriptional regulation of many mRNAs bearing AU rich and U rich elements by influencing stabilization and translation. Based on the types of mRNAs regulated by HuR, the cellular role of HuR can be identified as pro-survival and pro-inflammatory. Using the S49 thymoma cell line, we recently showed that cAMP treatment, which triggers apoptotic signaling events, leads to a decrease in the effectiveness of HuR function. In this work, we investigate if there is a relationship between cAMP mediated cell death and HuR activity by utilizing small interfering RNA-mediated suppression of HuR in S49 cells. We found that knockdown of HuR protein decreases normal cell growth, and increases cAMP mediated apoptosis. qPCR analysis of mRNAs important in inflammation, cell growth, and apoptosis reveal that knockdown of HuR downregulates TNF-α mRNA and upregulates IL-10 and Bim mRNAs in untreated cells, and upregulates p27, and Bim mRNAs upon treatment with cAMP. Furthermore, cAMP treatment causes a PKA dependent decrease in HuR cytoplasmic levels. These results suggest that a pro-inflammatory and pro-survival role for HuR in S49 thymoma cells exists and may be compromised upon the elevation of cAMP by decreasing cytoplasmic HuR protein and allowing the expression of apoptotic factors.
The V(D)J recombination machinery is associated with the nuclear matrix (88.3)
Authors: Lange Miles , Xie Wanqin , Hong Sang Yong , Yu Zhihong , He Ti , Huang Lin , Yu Yangsheng , Marran-Nichol Kathleen , Swanson Patrick , Lu Runqing , Su Kaihong , Zhang Zhixin
Published: January 2023

Somatic recombination of immunoglobulin or T cell antigen receptor genes occurs through recombination activating gene product (RAG)-mediated cleavage at recombination signal sequences (RSS) and subsequently, the broken DNA ends are repaired by non-homologue end joining (NHEJ) enzymes. Here, we show that RAG1, RAG2, and many NHEJ factors in B lineage cells are associated with the nuclear matrix. The core RAG1 and RAG2 proteins have their own nuclear matrix targeting regions. RAG-mediated double stranded DNA breaks at the Jκ4 RSS can be readily detected in the nuclear matrix fraction in Gleevec treated Abelson transformed murine B cells or mouse bone marrow cells, indicating that the recombination reaction is ongoing on the nuclear matrix. In the HEK 293 cell-based recombination system, artificial recombination substrates are recruited to the nuclear matrix for recombination. Moreover, nuclear matrix proteins purified from 293 cells expressing the core RAG proteins or human B lineage cells expressing endogenous RAG proteins support cleavage of RSS substrates in vitro. Based on these results, we propose that the V(D)J recombination machinery is associated with the nuclear matrix.
Regulation of chromatin remodeling during thymocyte differentiation (88.5)
Authors: Dovat Sinisa , Song Chunhua , Gurel Zafer , Payne Kimberly , Popescu Marcela
Published: January 2023

The Ikaros gene encodes a DNA-binding protein that functions as a master regulator of lymphoid development. Ikaros protein interacts with histone deacetylation complex and represses transcription of its target genes via chromatin remodeling. We have previously reported that Ikaros’ activity and protein stability is regulated by CK2 kinase (Popescu et al. J Biol Chem 2009 284:13869). We studied the effect of CK2-mediated phosphorylation on Ikaros function in primary thymocytes. Ikaros proteins with phosphomimetic mutations at CK2 phosphorylation sites 1) lose the ability to associate with Sin3a, a component of the NuRD histone deacetylase, as indicated by co-immunoprecipitation assays and 2) fail to repress genes involved in thymocyte differentiation as indicated by luciferase reporter assay. The introduction of phosphoresistant mutations at five N-terminal CK2 phosphorylation sites on the Ikaros protein restored Ikaros’ association with Sin3a, as well as wild-type levels of repressor activity. The treatment of primary thymocytes with CK2 kinase inhibitors resulted in increased levels of Ikaros protein and increased Ikaros’ DNA-binding affinity to the promoter regions of its target genes, as measured by quantitative chromatin immunoprecipitation. These results demonstrate that CK2 kinase regulates thymocyte differentiation by controlling Ikaros’ association with chromatin remodeling complexes and its ability to repress the transcription of developmentally regulated genes.
Conversion of Adenines to Inosines in RNA During Virus Infections Is an Innate Immune Stimulus (89.10)
Authors: Liao Jie-Ying , Prater Chrissy , Thakur Sheetal , Zalinger Zachary , Imani Farhad
Published: January 2023

Inflammatory responses that are generated during viral infections are critical for protective anti-viral immune responses. The exact viral-derived structures (pathogen-associated molecular patterns, PAMPs) that activate uninfected cells to induce pro-inflammatory signals are not completely characterized. To date, the viral PAMPs that are identified include double-stranded RNA (dsRNA) and 5'-triphosphate-containing single-stranded RNA (ssRNA). During viral infections, the cellular enzyme adenosine deaminase acting on RNA-1 (ADAR-1) catalyzes conversion of adenines to inosines, which is a pathway for blocking viral replication. ADAR-1 activation in the infected cells leads to the presence of inosine-containing viral and cellular RNA (Ino-RNA). Here, we report that Ino-RNA from infected cells is a stimulus for inflammatory innate immune response. Treatment of primary human bronchial epithelial (PHBE) cells, primary human alveolar macrophages or C57BL/J6 mice with in vitro synthesized Ino-RNA rapidly and potently induced several markers of innate immune response such as IFN-β, TNF-α, IL-6 and RANTES. We further identified increased secondary structures in ssRNA upon conversion of adenines to inosines. The increased secondary structures activated the dsRNA-specific binding protein PKR. In addition, Ino-RNA activated the MAP Kinase pathways, which is downstream of PKR. Our results suggest that Ino-RNA is a novel viral innate immune recognition element.
IL1 is Required for CD8 T Cell Stimulation of Endothelial Cells to Produce KC in Contact Hypersensitivity (89.13)
Authors: Kish Danielle , Gorbachev Anton , Fairchild Robert
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten sensitization and challenge of the skin. Elicitation of CHS is initiated when hapten-primed CD8 T cells producing IFN-γ and IL-17 traffic to the challenge site and stimulate endothelial cells (EC) to produce CXCL1/KC. KC is required for the neutrophil recruitment directing CD8 T cell infiltration through the EC where they are activated to induce the edema of CHS. This study investigated the role of IL1 in the CD8 T cell priming and elicitation of CHS. Both KC production and neutrophil infiltration into the skin challenge site as well as CHS responses were low-absent following challenge of sensitized IL1R-/- vs. wild-type (WT) mice. Sensitization of IL1R-/- mice induced marked decreases in migration of hapten-presenting Langerhans cells into the skin-draining lymph nodes and priming of CD8+ T cells producing IFN-γ or IL-17 vs. WT mice. Transfer of WT hapten-presenting Langerhans cells to IL1R-/- mice did not correct these defects. Transfer of primed WT CD8 T cells to naïve IL1R-/- mice also failed to induce KC production to hapten challenge. These results indicate that IL1 signaling is required for optimal migration of hapten-presenting dendritic cells from the sensitized skin to the draining lymph nodes, for hapten-primed CD8 T cell priming, and for activation of these CD8 T cells by hapten-presenting endothelial cells in the challenge site to induce CXCL1/KC production and the CHS response.
ATP induced microvesicles stimulate macrophage activation (89.18)
Authors: Thomas Louis , Salter Russell
Published: January 2023

Adenosine triphosphate (ATP) has a unique role as a damage associated molecular pattern (DAMP) as it is normally kept within cells unless there is cellular/tissue injury. As a DAMP, ATP has several known mechanisms for immune cell activation. Our lab and others have reported that exogenous ATP administration to macrophages in cell culture induces rapid shedding of microvesicles (MV). In this study, we harvested MV from ATP treated macrophages and demonstrated that MV are capable of stimulating naive macrophages to release pro-inflammatory cytokines and induce co-stimulatory surface marker upregulation. MV- induced activation is dependent on a cAMP/p38 MAPK pathway and also results in NFkappaB translocation. Activation is partially dependent on TLR4, but is not due to the TLR4 agonist HMGB1, which is present in MV. TNFalpha is also present in MV but is not required to activate macrophages since MV from TNF-deficient cells are also stimulatory. Biochemical fractionation of MV was then used to show that the stimulatory component is phospholipid in nature, and we are currently working to identify the bioactive compound. These findings suggest a novel means by which macrophages at sites of cellular damage can become sterilely activated by ATP to further propagate cellular damage via shed microvesicles.
Induction of active immune suppression by co-immunization with DNA and cytokines plasmid in SPF chickens (89.26)
Authors: He Cheng , Ling Yong , Zhang Chang , Yang Junjing
Published: January 2023

Avian chlamydiosis has been widely prevalent in Chinese intensive farms, resulted in the significant economic loss as well as the huge potential threat to human being health. It is an urgent need to develop an appropriate vaccine against avian Chlamydopila psittaci. Th1-cell immunity plays a main role in the vaccination of avian Chlamydosis, but DNA vaccine based on the main outer membrane protein is unable to induce the incomplete protection in large animals. Hence, the co-vaccination with DNA and cytokine plasmids is considered to be a promising approach for improving efficacy of DNA vaccine. In this study, the SPF chickens were administered with DNA plus IL-2 plasmid, IL-12α plasmid, DNA-IL-12β plasmid and DNA-15 plasmid, respectively on day 0, day 14 and day 28. The birds inoculated with DNA were regarded as the positive control. All chickens were detected antibody levels,cytokine titers and challenged with C.psittaci to assess the protection. Results show that DNA vaccine plus four cytokine plasmids induce the significantly lower IgG antibody levels, the titers of IFN-γ and IL-12 as compared to DNA vaccine. Moreover, the spleens were observed to have heavy loads with chlamydia post challenge in four co-vaccination groups. The interesting findings indicate that DNA vaccine plus cytokine plasmids will induce the immune suppression. Keywords: omp A; Cytokine plasmid; immunosuppression; Chlamydophila psittaci
CD34+CD31+ immature myeloid cells (IMC) as an inhibitor cell on the production of antimicrobial peptide: III. Translational studies of glycyrrhizin in humanized NOD-SCID IL-2rγ-/- mice (NOD-SCID mice) (89.30)
Authors: Yoshida Shohei , Inatsu Akihito , Kobayashi Makiko , Herndon David , Suzuki Fujio
Published: January 2023

In the accompanying paper II, glycyrrhizin (GL) is shown to be inhibitory on the CD34+CD31+ IMC-mediated suppression of HBD-1 production by normal human epidermal keratinocytes (NHEK). In this study, the inhibitory effect of GL on CD34+CD31+ IMC-mediated suppression in HBD-1 production by NHEK was tested in human immune system mice (HIS mice) (X-irradiated NOD-SCID mice inoculated i.d. with NHEK and CD34+CD31+ IMC). Two days after the cell inoculation, inoculation site skin tissues were biopsied (8-mm diameter) from HIS mice treated with or without GL (Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan) 6 and 24 hrs after the cell inoculation. Five biopsied tissue samples were homogenized together on ice in PBS. The concentrations of HBD-2 in the homogenates were measured by ELISA. In the results, 4.9 μg/ml of HBD-2 was detected in skin homogenates derived from NOD-SCID mice inoculated with NHEK, while HBD-2 was not detected in skin homogenates from HIS mice. When HIS mice were treated with 10 mg/kg of GL, HBD-2 was detected in IMC-inoculation site tissues of these mice. The amount of HBD-2 detected in this homogenate was corresponded to that detected in tissues of NOD-SCID mice inoculated with NHEK alone. These results indicate that GL improves the suppressed HBD-2 production in HIS mice. This study was supported by SHC #8610.
Plasma membrane-derived vesicles (PMVs) released from apoptotic Jurkat cells express upregulated surface Fas and induce apoptosis in recipient viable cells (89.45)
Authors: Jorfi Samireh , Ansa-Addo Ephraim , El dine Safa , Inal Jameel
Published: January 2023

Apoptosis, distinct from necrosis, is irreversible programmed cell death. Characterized by membrane blebbing, DNA fragmentation and cell shrinkage it plays an important role in infection. During viral infection, depending on the virus, apoptosis may either be enhanced or inhibited. Apoptosis can be induced by dexamethasone (DEX), cychloheximide (CHX) and H2O2 in vitro in various cell types. PMV release from apoptotic cells (isolated by differential centrifugation, expressing surface phosphatidylserine and of 0.2-1μm average diameter) could be inhibited by pretreatment with calpeptin. Such PMVs, which we distinguished from apoptotic bodies and other microvesicle populations, such as exosomes, have increased Fas expression on DEX and CHX- but not H2O2-mediated apoptotic Jurkat cells and an increased capacity to induce apoptosis in recipient healthy Jurkat cells, maximally at a 5:1 PMV-cell ratio. This effect, specific to PMVs from apoptotic cells, as opposed to those from viable cells, occurred after 30 min incubation of recipient, viable cells with PMVs and dose-dependently increased up to a maximum after 5h incubation. Inhibitors of caspase and anti-Fas blocking antibodies have been used to elucidate the importance of these factors in PMV transmission of this apoptotic signal. PMV-mediated apoptosis is currently being investigated for its role in in vitro studies of Coxsackie B3 infection in HeLa cells.
Galectin binding to and expression modulation by viruses in zebrafish (89.51)
Authors: Jackson Shawn , de las Heras Sanchez Ana , Ahmed Hafiz , Ammayappan Arun , Vakharia Vikram , Vasta Gerardo
Published: January 2023

The galectin family of β-galactoside-binding proteins is evolutionarily conserved. Galectins are known to interact with and affect the infectivity and/or pathogenicity of several viruses. We hypothesize that galectin interactions with viruses influence anti-viral immune responses. We investigate the interactions of zebrafish galectins with the glycosylated (G) protein of the rhabdovirus IHNV. We (a) characterized the carbohydrates present on IHNV proteins, and (b) identified direct interactions between zebrafish galectins (Drgal) and IHNV. We assessed the effects of immune challenge on (c) galectin and (d) cytokine expression in zebrafish. Several IHNV proteins exhibit complex glycosylation patterns. One or multiple IHNV proteins exhibit terminally linked mannose, sialic acid terminally linked to galactose, galactose-β(1-3)-GalNAc, sialic acid terminally linked to galactose or GalNAc, and galactose-β(1-4)-GlcNAc. The proto type Drgal1-L2, the chimera type Drgal3-L1 and the tandem-repeat type Drgal9-L2 all appear to bind to one or more IHNV proteins. All tested Drgal appear to bind to the IHNV G protein. ZFL cells endogenously express mRNA encoding Drgal1-L2, -L3, and -L4, Drgal3-L1 and -L2, and Drgal9-L1 mRNA. Drgal mRNA expression in ZFL cells was not induced by treatment with polyI:C. ZFL cells were found to endogenously express mRNA encoding Mx, TNFα, and IFNαβ. Treatment of these cells with polyI:C induced the expression of mRNA encoding IL-10 and IFNγ2.
Stimulation and priming of innate lymphocytes by oenothein B (89.59)
Authors: Ramstead Andrew , Schepetkin Igor , Quinn Mark , Jutila Mark
Published: January 2023

Oenothein B is an immunomodulatory tannin isolated from the Epilobium angustifolium plant. This compound is known to affect myeloid cells and these effects include enhanced chemotaxis, NF-κB activation, and production of proinflammatory cytokines, including TNFα and IL-6. Its effects on other innate immune cells have not been investigated. Previous studies have shown this compound to be beneficial in tumor models; therefore, it is of interest to determine what effect this compound may have on other innate immune cells known to be beneficial in anti-tumor responses, such as NK cells, NKT cells, and γδ T cells. Previous work from our lab has shown that certain tannins have the ability to directly stimulate γδ T cells, indicating oenothein B may also have immunomodulatory effects on this cell type. Our preliminary results show that oenothein B it is capable of stimulating γδ T cells and other lymphocytes. We also show that priming with oenothein B can enhance responses by these cells to secondary stimuli. This includes augmentation of IFNγ production, which may not be reliant upon oenothein B’s ability to stimulate both macrophages and γδ T cells. The molecular mechanisms, which facilitate these responses, are currently under investigation in both bovine and human cells. Funded by NIH Contract HHSN2662004000009/N01-AI40009, PO1 AT004986, and RR020185.
Beta-mannosylceramide represents a new class of NKT agonists that induces tumor immunity by a novel IFN-γ-independent mechanism. (89.7)
Authors: O'Konek Jessica , Illarionov Petr , Stewart Khursigara Deborah , Ambrosino Elena , Castillo II Bernard , Raju Ravinder , Khalili Maryam , Kim Hee-Yong , Howell Amy , Besra Gurdyal , Porcelli Steven , Berzofsky Jay , Terabe Masaki
Published: January 2023

Type I NKT cells are a unique lymphocyte population which recognize lipid antigens presented by the non-classical MHC-like molecule CD1d. Activation of type I NKT cells by α-galactosylceramide (α-GC) induces tumor immunity by IFN-γ dependent mechanisms. In this study, we characterized how modifications of the sugar head group of α-GC impacted tumor protection in an in vivo mouse model of lung metastasis. β-mannosylceramide (β-ManCer), but not α-mannosylceramide or α-fucosylceramide, induced strong type I NKT cell-dependent tumor immunity. Because this is the first report of a β-linked glycosylcermide capable of inducing significant type I NKT cell-mediated tumor protection, we further characterized the mechanism by which β-ManCer induced tumor elimination. Interestingly, unlike α-GC and its analogs which protected through IFN-γ dependent mechanisms, β-ManCer induced very little cytokine production both in vitro and in vivo and retained protection in the absence of IFN-γ. Rather, β-ManCer protected through a TNF-α and nitric oxide synthase-dependent mechanism novel for NKT cells. Simultaneous administration of sub-therapeutic doses of β-ManCer and α-GC resulted in synergistic reduction in tumor burden, supporting the conclusion that these glycolipids work through different mechanisms. The distinct mechanism of action of β-ManCer and its ability to synergize with α-GC suggest that β-ManCer is the first representative of a new class of type I NKT cell agonists.
Early myocardial infarction with concurrent complement response studied using a novel immunofluorescent approach (89.8)
Authors: Zhang Ming , Charlagorla Pradeepkumar , Green Danielle , Balroop Javi , Shaka-Idusuyi Oghomwen , Reza Mohammed , Patel Monaliben , Matta Sravan
Published: January 2023

Experimental myocardial infarction is typically determined using TTC. TTC, reduced to a red product in intact cells by endogenous enzymes/cofactors, remains colorless in damaged cells from which the enzymes/cofactors have been removed during reperfusion. Its use is limited to studies with prolonged reperfusion and sections cannot be used for other pathological events. Two recent studies showed that propidium iodide (PI), which enters damaged cells and intercalates with DNA to produce a red fluorescent complex, can be injected in vivo following ischemia/reperfusion to assess infarct size. We build on this in a murine model, using PI to study early infarct development and to evaluate the concurrent inflammatory response as indicated by complement C3 deposition. Occlusion of the coronary artery was followed by increasing periods of reperfusion and injection of PI to define infarction. Blue fluorescent microspheres were injected to define the area at risk (AAR). Heart slices were immediately imaged for infarction and AAR, and cryosections obtained from the slices imaged for C3 using FITC-labeled antibody. PI fluorescence revealed a significant infarction at 1 hour of reperfusion which was maintained through 24 hours. C3 deposition lagged infarction development, becoming significant at 3 hours of reperfusion. In summary, in vivo injection of PI allows study of the early stages of infarction and permits analysis, in the same tissue, of concurrently occurring inflammatory events.
Signalling pathways in the inflammatory process of a severe protrein malnutrition model (90.10)
Authors: Roux María , Calabrese Graciela
Published: January 2023

Previous work has shown that dietary antigen starts an intestinal inflammatory process, regulated by TNFα, inducing inflammatory genes transcription by NF-kB factor translocation into intestinal epitelial cells (IECs) nucleus . Therefore, we study IL-17 expression and its related transcriptional factors in our model of secondary immunodeficiency by malnutrition .Western Blot was used to analyze intracellular distribution ( microsomes ,nucleus and cytosol) obtained by ultracentrifugation of IECs from wellnourrished rats (C60) ,as well as from renourrished ones (weaning rats lost 25% of intially body weight and refed with a 20% casein diet during 21 days ( R21). Detection was performed with specific biotinylated antibodies to IL-17; ROR γt , NF-k B and STAT3 and avidin- biotin peroxidase system to detect the immunoreactive bands that were revealed with the DAB substrate. Results: IL-17 was found in the microsomal fraction accompagnied by a nuclear localization of ROR γt , NF-kB and STAT 3 only for R21 rats; while no differences in nuclear Foxp3 detection was observed between C60 compared with R21. We assume that the inflammatory process is associated with a nuclear localization of ROR γt, NF-kB and STAT3 in the renourrished animals withouth activation of Foxp3. Therefore, IL-17 expression contributes to the perpetuation of the inflammatory process, as downregulation of Foxp3 strongly correlates with upregulation of IL-17.
Dendritic cell-expressed CD103 is required for inhibition of mucosal Th2 cell responses (90.3)
Authors: Mullaly Sarah , Maltby Steven , Burrows Kyle , McNagny Kelly , Zaph Colby
Published: January 2023

Dysregulated mucosal CD4 Th2 cell responses are a feature of several inflammatory diseases including asthma, allergies and some forms of inflammatory bowel disease. The integrin CD103 is expressed primarily on a subset of dendritic cells (DCs) that promote regulatory T cell development and intestinal immune homeostasis. However, the role of CD103 in Th2-dependent immunity has not been tested. Using the helminth parasite Trichuris muris, we identify a critical role for CD103 in specifically limiting Th2 responses in vivo. We find that following infection with Trichuris, CD103-deficient (CD103-/-) mice develop an enhanced Th2 response that is associated with decreased levels of IL-10. While in vitro CD4 T cell differentiation was unaffected by CD103 deficiency, bone marrow-derived CD103-/- DCs induce heightened T cell proliferation and increased IL-13 production under Th2 promoting conditions. The inhibitory role of CD103 appears to be specific for Th2 responses, as CD103-/- mice are protected from disease development in a model of chemically-induced colitis. Taken together, these results suggest that CD103 expression on DCs is critically required to specifically regulate Th2 immune responses in vivo.
Shaping of the gut microbiota by B lymphocytes influences GATA4-dependent lipid absorption and body fat accumulation (90.8)
Authors: Shulzhenko Natalia , Morgun Andrey , Gavrilova Oksana , Battle Michele , Matzinger Polly
Published: January 2023

In order to understand the role for B lymphocytes in normal physiology of intestine, we compared global gene expression in the jejunum of wild-type (WT) and B-cell knockout (KO) mice. We found about 300 genes whose expression differed between WT and B-cell KO mice. Genes down-regulated in B- cell KO intestines, were highly enriched for lipid/energy metabolism genes. Consistent with this result, we found lower amounts of para-genital fat and serum leptin level in B-cell KO mice than in their B-cell-sufficient littermates. We revealed GATA4 as the most enriched transcription factor for the down regulated genes. Conditional KO of Gata4 in the intestinal epithelium showed a high correlation with BcKO gene signature. We also found a decrease in cholesterol absorption in the gut of B-cell KO mice (p<0.005) previously observed in Gata4 intestinal KO. In addition, Gata4 KO mice perfectly recapitulated the systemic effects observed in B-cell KO mice presenting decreased levels of body fat and leptin (p<0.01). Studying the mechanism, we found that mice that have B lymphocytes but do not produce soluble antibodies exhibit a very similar gene expression profile as the B-cell KO mice implying that this phenomenon is antibody-dependent. Under germ-free conditions, we found almost no difference between B-cell KO and control mice. Thus we conclude that B lymphocytes are involved in the control of GATA4-dependent lipid absorption and accumulation of body fat by shaping the gut microflora.