bluuebunny/crossref_metadata_2025 huggingface dataset:
Type I Interferon Regulates the Dynamics of Innate Myeloid Sentinel Cells During Persistent Viral Infection (169.7)
Authors: Nayak Debasis , Johnson Kory , Heydari Sara , Roth Theodore , Zynselmeyer Bernd , McGavern Dorian
Published: January 2023

Viral infections of brain usually trigger inflammatory responses that give rise to a wide range of pathological outcomes. To gain novel insights into how the brain responds to a virus as it establishes persistence, we utilized genomic and two-photon imaging approaches to study a pure innate immune response to a lymphocytic choriomeningitis virus (LCMV) infection. This was accomplished by monitoring temporal gene expression patterns and myeloid cells dynamics in the brain of CX3CR1-GFP mice lacking LCMV-specific T cells. Within the first two weeks of infection, brain resident myeloid cells were highly activated and responded directly by sequestering DsRed-labeled viral antigen. At the genomic level, this triggered massive changes in gene expression that were attenuated over time (likely due to inhibition by viral nucleoprotein) and were all directly linked to type I interferon (IFN-I) signaling. Surprisingly, in the absence of IFN-I signaling, no genes were differentially expressed in the nervous system despite the presence of persistent virus. In addition, two-photon imaging studies revealed that IFN-I receptor deficient innate immune sentinels were completely unresponsive to the establishment of a persistent viral infection. These data demonstrate that IFN-I drives all innate immune activity in the brain following LCMV infection, and deficiency in IFN-I signaling renders LCMV invisible in its murine host.
Regulatory T cells shape the effector and memory T cell response to virus infection in the tissues (170.15)
Authors: Graham Jessica , Lund Jennifer
Published: January 2023

Regulatory T cells (Tregs) are well known for their role in dampening the immune responses to self-antigens and thereby helping to prevent autoimmune disease. However, they must also permit immune responses to occur against foreign infectious agents. Using a mouse model of West Nile Virus (WNV) infection, we examined Treg kinetics, trafficking, and activation at both acute and memory time points. We showed that Treg numbers increase in both the secondary lymphoid organs and CNS after infection, and become activated similar to effector T cells. Using the Foxp3DTR knock-in mice, we have performed experiments to study the course of WNV infection in the presence or absence of Tregs. We found that compared to ”normal” mice, Treg deficient mice had increased antigen-driven production of IFN-g from both CD4 and CD8 T cells in both the spleen and CNS. Additionally, Treg deficient mice had an increased frequency of poly-functional T cells. In mice lacking Tregs, there were greater numbers of short lived effector CD8+ cells at acute infection time points in both the spleen and brain. At a memory time point, there were significantly fewer antigen-specific T cells present in the CNS of mice deficient in Tregs at the time of infection compared to Treg sufficient mice. Studies are currently underway to examine the role of Tregs in CD4 memory using this Treg depletion model.
Analysis of early differential immunosuppressive patterns in the MAIDS model (170.23)
Authors: Akalu Yemsratch , Kwaa Abena , Sceats Lindsay , Stranford Sharon
Published: January 2023

Murine acquired immunodeficiency syndrome (MAIDS), induced by exposure to murine leukemia virus (MuLV), serves as an animal model to study human AIDS. While infection with the LP-BM5 isolate of MuLV leads to progressive immune compromise in MAIDS-susceptible C57BL/6 mice, resistant BALB/c mice launch directed and controlled immune responses to eradicate the virus and develop protective immunity. Since modulation of immunosuppressant molecule activities after viral infection may shape disease progression, we aimed to characterize key early differences in the expression of two natural immunosuppressive proteins, indoleamine 2, 3-dioxygenease (IDO) and interleukin-10 (IL-10). Preliminary experiments showed significantly higher IDO mRNA expression levels in the secondary lymphoid organs of naïve and infected disease-susceptible C57BL/6 mice than in resistant BALB/c mice. In our follow-up studies we used commercially available ELISA kit to measure IL-10 and developed our own ELISA-based assay to measure mouse-IDO. The findings indicate that expression of IDO protein is induced early after viral infection. The IL-10 ELISA indicated significant differences in IL-10 expression between the two strains of mice during the first week of infection. These results suggest that these immune mediators may preferentially activate immunosuppression pathways in MAIDS-susceptible mice.
Regulatory T cells can be expanded from HIV-1 infected individuals with preservation of suppressive function and TCR repertoire (170.26)
Authors: Angin Mathieu , King Melanie , Klarenbeek Paulus , Moodley Eshia , Rezai Ashley , Piechocka-Trocha Alicja , Toth Ildiko , Walker Bruce , Addo Marylyn
Published: January 2023

While natural regulatory T cells (Tregs) are being explored as therapeutic modalities in the context of autoimmune diseases and transplantation, their role in HIV-1 pathogenesis is less well defined and to date it is unknown if Tregs can be isolated and expanded from HIV-1 infected individuals. Tregs and conventional T cells (Tconvs) were isolated from the peripheral blood of HIV-1 infected and healthy donors using flow-based cell sorting, stimulated and cultured. Expanded Tregs were phenotyped by flow cytometry and qPCR. TCR repertoire diversity was investigated by high-throughput sequencing. Suppressive function was assessed using flow-based proliferation and chromium release cytotoxicity assays. Infectability of expanded Tregs was investigated using VSV-G pseudotyped HIV-GFP. Tregs from HIV-1+ individuals, including infants, were successfully expanded with a mean fold change of 29 at day 7. Expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS and a demethylated TSDR (Treg-specific demethylated region). The TCR repertoire of expanded Tregs was not skewed and they maintained their suppressive function in vitro. Expanded Tregs were readily infectable with HIV-GFP. Our data show that Tregs from HIV-1 positive individuals can be successfully isolated and expanded ex vivo. This approach holds great potential for detailed studies on the role of regulatory T cells in the setting of HIV-1 infection and has implications for potential future therapeutic use of Tregs.
Tick-borne flaviviruses impair IRF-1 and type I IFN signaling to inhibit IL-12 production by dendritic cells (170.6)
Authors: Robertson Shelly , Lubick Kirk , Freedman Brett , Best Sonja
Published: January 2023

Tick-borne encephalitis virus (TBEV), family Flaviviridae, is an emerging pathogen causing ~10,000 human cases of severe CNS disease annually. Dendritic cells (DCs) are early cellular targets of TBEV infection, which may contribute to viral pathogenesis. Here, we examined the cytokine response and maturation profile of murine bone marrow-derived DCs infected with LGTV, a naturally attenuated member of the TBEV serogroup. LGTV infection failed to induce DC maturation or a robust cytokine response. Treatment with LPS or LPS/IFNγ, strong inducers of inflammatory cytokines, resulted in enhanced production of TNFα and IL-6, but suppressed IL-12 in infected DCs when compared to uninfected "bystander" cells or mock-infected controls. Although late suppression of IL-12p40 involved antagonism of type I IFN signaling, early suppression was dependent on IRF-1, a key transcription factor required for anti-flaviviral immunity. LGTV infection did not affect IRF-1 mRNA expression, but rather diminished IRF-1 protein levels and nuclear localization in DCs and reduced IRF-1 transcriptional activity in Huh7 cells. The effect on IRF-1 was also observed in DCs infected with the virulent Sofjin strain of TBEV and in DCs isolated from skin-draining lymph nodes of LGTV-infected Ifnar-/- mice. Thus, IL-12p40 inhibition by LGTV involved coordinated impairment of IRF-1 function and type I IFN signaling. This work identifies IRF-1 as a novel target of flavivirus-mediated immune evasion.
Type I IFN Signature in plasmacytoid dendritic cells from young Sle 1,2,3 lupus prone mice. (171.1)
Authors: Sriram Uma , Varghese Linda , Xu Jun , Gallucci Stefania
Published: January 2023

The importance of the Type I Interferon (IFN) Signature in systemic lupus erythmatosus is well documented in patients and mouse models. We have earlier observed a distinct IFN gene Signature in the myeloid dendritic cells (mDC) of young Sle 1,2,3 lupus prone mice. In this study, we grew bone-marrow precursors in the presence of Flt3L to generate plasmacytoid dendritic cells (pDC) and analyzed the IFN Signature. The percentage of pDCs generated in culture did not differ between the Sle and the control C57BL/6 (B6) mice. We sorted pDCs (B220+CD11c+) and mDCs (CD11c+CD11b+) from the culture and analyzed the constitutive expression of IFN responsive genes (IFNb, IRF7, ISG15, Mx-1 and CXCL10) by real-time RT-PCR as sign of IFN Signature. There was minimal IFNb expression in both B6 and Sle pDCs; while the IFNb expression in mDCs was higher and much higher in Sle vs B6 mDCs like we observe in cultures supplemented with GMCSF. IRF7 gene expression was higher in pDCs than mDCs and much higher in Sle pDCs. The expression of the other genes in pDCs was lower than in mDCs in both strains, although in Sle pDCs their expression was higher than in B6 pDCs. Our results indicate that plasmacytoid DCs from young pre-diseased Sle1,2,3 lupus prone mice express an IFN Signature similarly to lupus myeloid DCs. Moreover, these results highlight the important role for IRF7 in the activation of lupus pDCs and suggest a probable mechanism for the hyper-activation of the IFN Signature in lupus.
Neutrophil expression of FasL and perforin in hapten challenged skin induce chemoattractants directing the infiltration of hapten-primed CD8 T cells into the skin (173.3)
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. Hapten-primed CD8 T cell infiltration into the skin challenge site is observed 12-16 h post-challenge and requires prior neutrophil infiltration and activation within the site. The neutrophil activities required to direct this T cell infiltration have remained undefined. Absent CHS responses to 2, 4-dinitrofluorobenzene (DNFB) in gld/perforin-/- (dKO) mice are caused by the absence of T cells in the hapten challenged skin of sensitized dKO mice at 18 h despite the presence of neutrophils 6 h after challenge. The absence of T cell infiltration into the hapten challenged skin of sensitized dKO mice was accompanied by absent expression of the T cell chemoattractants CCL1, CCL2, and CCL5 in the site at 6 h after challenge. Gr-1+ cells purified from WT bone marrow expressed high levels of FasL and perforin mRNA. Transfer of these wild-type Gr-1+ cells to sensitized dKO at time of hapten challenge restored expression of CCL1, CCL2 and CCL5 in the challenged skin as well as T cell infiltration into the site at 18 h after challenge and the edema of the CHS response. These results indicate the requirement for neutrophil expression of FasL and perforin in hapten challenged skin to induce the chemoattractants for activated T cells and other leukocytes that direct hapten-primed T cells into the challenged skin site to mediate the edema of CHS responses.
Specific antigenic recognition shifts a balance from tolerance to effector immune responses in lupus cerebritis (173.34)
Authors: Dasgupta Subhajit , Molano Ivan
Published: January 2023

Lupus cerebritis is a distinct mid brain specific disease with unknown etiology. The MRL/MpJ (Fas lpr/Faslpr) mice are used to study systemic lupus as well as organ specific lupus cerebritis. We found a decrease in CD3+CD4+CD25+ regulatory T cells (Treg) in 22 weeks lupus prone female MRL/MpJ (Faslpr/Faslpr) than asymptomatic 6 weeks old mice. Such attenuation of tolerance specific Treg cells was inversely related with CD3+CD1d+ T cells. We found, at 22 weeks of disease, there was an increase of CD3+CD1d+ T cells than 6 weeks old asymptomatic mice. We also found that, the TLR2 ligand LTA and TH17 cytokine IL17 play a role in activation of CD19+ B cells in spleen mononuclear cells. These CD19+ B cells were also found positive for CD1dgalactosylceramide. The LTA injection in female MRL/lpr mice increased expansion of CD1d+CD19+ B220+CD5- activated B cells in spleen. However, there was a decrease in CD19+CD1d+CD5+ regulatory B (Breg) cells at the acute stage of disease. The observations suggested a possibility of switch in balance from tolerance to effector immune responses in MRL mice during progression of disease. Studies are undertaken to determine the mechanism of raise in antigen specific Breg responses in maintenance of Treg tolerance and attenuation of TH17/ IL17 responses as a way to prevent mid brain inflammation in lupus cerebritis.
CD13 regulates immune cell trafficking to solid tumors. (P641) (173.5)
Authors: Vernier Kaitlyn , Shapiro Linda
Published: January 2023

While solid tumor growth clearly triggers an inflammatory response, the tumors are often able to evade attack from the innate immune system by establishing both an immunosuppressive and tumor promoting microenvironment. Moreover, increases in infiltrating myeloid cells in tumors correlates with poor prognosis suggesting a role for these cells in the control of the tumor microenvironment. The peptidase CD13 acts as a homotypic adhesion molecule expressed on myeloid cells and activated endothelium (including tumor blood vessels) that regulates the transport of myeloid cells to sites of inflammation. Naive wild type or CD13 null mice show identical immune cell profiles but display significant differences in the number and ratio of myeloid subsets recruited to the sites of disease upon inflammatory challenge prompting our investigation into its role in myeloid cell control of the tumor microenvironment. We found that allografts grown in CD13 null mice have more infiltrating monocytes and macrophages as well as an increase in tumor growth rate. Paradoxically, although tumor growth is more rapid, tumor angiogenesis is markedly decreased suggesting tumor vessels are more patent in the absence of CD13 and can more efficiently supply the growing tumor. Therefore, CD13 has a role in immune cell trafficking, infiltration into solid tumors, and tumor angiogenesis. The knowledge gained from this study will broaden our understanding of immune cell contribution to cancer progression.
Role of microRNA in 3,3-diindolylmethane (DIM)-mediated amelioration of SEB-induced acute lung injury (173.6)
Authors: Elliott David , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Staphylococcal enterotoxin B (SEB) is a CDC select agent of bioterrorism and a public health threat during nosocomial infections. A deleterious effect of SEB exposure is acute lung injury (ALI) and acute respiratory distress syndrome characterized by pulmonary infiltration. 3,3-diindolylmethane (DIM) found in cruciferous vegetables, has been shown to have anti-inflammatory properties. We studied the mechanism underlying the ameliorative effects of DIM on SEB-induced ALI. Mice were exposed to SEB [50 mg; intranasal] and administered DIM [100 mg/kg] via oral gavage. Forty-eight hours after SEB exposure, serum, lung tissue and lung infiltrating cells were collected. DIM treatment led to a decrease in lymphocytic infiltration, specifically CD4+, CD8+ and Vβ8+ T cells as well as NKT cells. DIM also led to a reduction of fluid leak in lungs. DIM [10, 25, 50 µM] induced apoptosis in splenocytes and CD3+ T cells treated in vitro with SEB in a dose-dependent manner. DIM treatment led to downregulation in microRNA, miR-706 and miR-494 that target pro-apoptotic genes, and upregulation of miR-22 and miR-34a targeting anti-apoptotic genes in lung-infiltrating cells. Thus, studies are underway to examine whether in T cells, DIM treatment leads to modulation in the expression of genes involved in apoptosis. Together, these studies demonstrate that DIM treatment may suppress SEB-mediated ALI by induction of apoptosis in T cells via alteration in expression of miRNA.
A non human primate gamma Interferon assay for quantitation of cellular immune responses to therapeutic specific and measles antigen specific responses (174.11)
Authors: Jawa Vibha , Deshpande Meghana , Zhou Lei , Wullner Danika , Chirmule Naren
Published: January 2023

We have developed and optimized an in vitro functional assay using whole blood derived peripheral blood derived mononuclear cells (PBMC) from non-human primates. The assay was used to quantitate therapeutic specific T cell responses following dosing with a fully human antibody based therapeutic. Measles-specific response from same animals was used as a control. The drug product specific response was evaluated using 50 monkeys dosed with the therapeutic. The PBMC were tested for reactivity to the therapeutic and to a control measles peptide cocktail, using an IFN-gamma ELISPOT assay and a Luminex based cytokine secretion assay. The optimization and qualification experiments describe: i) evaluation of number of cells to be plated in culture, ii) pre-resting of cells before plating, iii) viability of cells, and iv) impact of co-stimulatory cytokines (IL2, IL7, and IL15) on elicitation of antigen-specific responses. An assay specific cut point and stimulation index was calculated to distinguish responders from non-responders. Using this qualified assay, therapeutic specific responses and measles-specific T cell responses were evaluated in 56 and 50 monkeys respectively. These assays may provide an assessment of antigen-specific T cell function in vivo, and have various applications, such as evaluation of immune competence status of animals, immunomodulatory effects of drug products, and understanding the T cell dependence of anti-drug-antibody immune responses.
Effect of delay from sample collection to umbilical cord mononuclear cell isolation on T cell phenotype and function. (174.20)
Authors: Scheible Kristin , Secor-Socha Shelley , Wightman Terry , Wang Hongyue , Mariani Thomas , Topham David , Pryhuber Gloria , Quataert Sally
Published: January 2023

Background: The unpredictable nature of deliveries makes it difficult to control timing of umbilical cord mononuclear cell (UCMC) isolation. Our study tested the effect of time delay from umbilical cord collection to UCMC isolation on T cell phenotype and function. Methods: Cord blood was drawn from 15 healthy term deliveries using a standardized collection method. Each sample was divided and processed immediately, 24 hours and 48 hours after collection. Samples were analyzed using flow cytometry for phenotype and cytokine secretion, with and without in vitro stimulation. Comparisons were made to determine differences in variation and median cell frequencies over time. Results: Viability and recovery were >92% for all time-points. There was a decrease after 48 hours for CD56+ and CD3+ frequencies. CD4+ T cells showed differences after 24 hours in 4 of 23 subpopulations, and 8 subpopulations after 48 hours. No differences were seen with cytokine positive frequencies for IFNg, TNFa, IL17, or IL4/5/13 after 24 and 48 hours, but there was a decrease in IL2 and increase in MIP1β frequencies for CD4+ T cells. IL2 alone was increased for CD8+ T cells after 24 and 48 hours. Variation analysis showed high assay reproducibility, and subject-subject variation exceeded time delay variation for most populations. Conclusion: A 24-hour delay prior to UCMC isolation does not significantly change phenotype or function of most cord T cell subsets. A 48-hour delay may affect study results.
The Alarmin Interleukin-33 is a Notch Target in Quiescent Endothelial Cells (174.6)
Authors: Haraldsen Guttorm , Jetne Reidunn , Hol Johanna , Sponheim Jon , Küchler Axel , Kasprzycka Monika , Sundlisæter Eirik
Published: January 2023

The molecular mechanisms that induce and maintain expression of the alarming interleukin-33 (IL-33) in endothelial cells are unknown. Based on the observation that IL-33 is also a marker of endothelial cell quiescence, we hypothesized that the Notch signaling pathway might be involved in regulation of IL-33 expression. Here we show that activation of Notch1 by immobilized Notch ligands was sufficient to induce nuclear IL-33 expression in human umbilical vein endothelial cells (HUVECs). Furthermore, IL-33 expression in confluent HUVECs was inhibited by the γ-secretase inhibitor DAPT or by blocking antibodies to Dll4, Jagged1 or Notch1, as well as by knockdown of the transcription factor RBP-Jκ. In vivo, expression of Dll4 but not Jagged1 was generally observed in all segments of quiescent vessels in healthy organs in both human and rat tissue, and therefore well correlated with expression of IL-33. Moreover, subcutaneous injections of DAPT markedly reduced expression of vascular IL-33, compatible with a role for Notch signaling in driving vascular IL-33 expression in vivo. Taken together, our data demonstrate that the alarmin IL-33 is a target of Notch and serve to further establish Notch signaling in innate immune defense.
Environmental exposure during pregnancy modulates fetal Treg development with consequnces for the allergy risk of the child (175.10)
Authors: Herberth Gunda , Hinz Denise , Bauer Mario , Röder Stefan , Olek Sven , Hühn Jochen , Sack Ulrich , Borte Michael , Simon Jan , Lehmann Irina
Published: January 2023

Regulatory T cells (Treg) are characterized by a specific demethylated region in the FOXP3 gene. There is evidence that prenatal exposure and lifestyle factors may have the potential to cause alterations in DNA methylation. The aim of the present study was to analyze the influence of prenatal environmental exposure on Treg numbers in cord blood due to demethylation of FOXP3 and the subsequent risk for the development of allergic disease. Analyses were performed in blood samples from pregnant women in the 34th gestational week and in cord blood (n=346 mother-child pairs) in the LINA study. DNA methylation of the Treg specific demethylated region (TSDR) of the FOXP3 gene in cord blood and total and specific IgE to food (fx5) and inhalant (sx1) allergens were determined. Exposure and disease outcomes were assessed by detailed questionnaires. Prenatal exposure to tobacco smoke and the usage of disinfectants during pregnancy were associated with decreased Treg numbers in cord blood. Children with low Treg numbers in cord blood had a significantly higher risk to develop an atopic dermatitis (adj. OR 1.55, 95% CI 1.00-2.41) and sensitization to food specific allergens (adj. OR 1.55, 95% CI 1.06-2.25) during the first year of life. These results indicate that environmental exposure during pregnancy may have the potential to influence the Treg number at birth. Low numbers of Treg in cord blood may predict the development of atopic dermatitis.
CD8+ T cells are not essential for the development of murine allergic airway disease (AAD). (175.13)
Authors: McKnight Christopher , Perkins Charles , Finkelman Fred
Published: January 2023

Murine models of asthma have shown that some cell types, including CD4+ T cells, are essential for induction of AAD. The importance of other T cell subtypes, including CD8+ T cells, is controversial. Although CD8+ T cells are reported to have little importance of AAD induction in rats, CD8+ T cells are reported to contribute substantially to AAD induction in mice. Notably, the murine experiments used transfer of T cells, ovalbumin (OVA) as antigen and peritoneal sensitization with adjuvant to induce disease and primarily studied C57BL/6 mice. We investigated this disparity with the more direct approach of antibody-mediated CD8+ T cell depletion in BALB/c and C57BL/6 mice. We find that CD8+ cells are irrelevant when inducing airway hyperresponsiveness (AHR) in BALB/c mice with house dust mite (HDM) in the absence of adjuvant or with OVA plus alum, but contribute to the development of eosinophilia in both strains of OVA-immunized mice. Our OVA immunization protocol only induced AHR in BALB/c mice. Thus, CD8+ T cells do not appear to be required for AAD induction when a potent allergen relevant to human asthma is used to immunize mice that are relatively prone to develop Th2 responses, but may promote AAD in a less Th2-biased mouse strain. These observations may have relevance to the possible role of CD8+ T cells in human allergy and asthma.
Chemokines mediate ethanol exacerbation of murine cockroach-allergen induced asthma. (175.15)
Authors: Bouchard Jacqueline , Beal Dominic , Allen Jessica , Kim Jiyoun , Vaickus Louis , Remick Daniel
Published: January 2023

Contributors to the development of atopic asthma can be multifactorial, with severe cases often requiring several stimuli, including allergen, LPS, and environmental factors. Here we show that alcohol intoxication concurrent with cockroach allergen (CRA) exposure will cause significant asthmatic exacerbations in mice pre-sensitized to CRA. CRA exposures were given on days 0 and 14, then outbred mice were gavaged with water or 32% ethanol on day 21 and challenged with CRA 30 minutes post-gavage. Kinetics of asthmatic inflammation were investigated. Ethanol intoxication significantly increased eotaxins and CXC chemokines in the bronchoalveolar lavage (BAL), peaking 90 minutes post-CRA challenge. Ethanol-gavaged mice displayed 2-fold increases in BAL eosinophils and 3-fold increases in BAL neutrophils over water-gavaged controls by 16 hours post-allergen challenge. Ethanol also resulted in significant respiratory impairment, including an acute 3-fold increase in airways mucin, increased mucus plug formation and bronchiole occlusion, and a 2-fold increase in airways hyperreactivity 14 hours post-CRA challenge. Pre-administration of a cocktail of neutralizing antibodies to eotaxins and CXC chemokines reduced BAL cellular infiltrate to baseline levels and decreased airways hyperreactivity by 50%. These data provide evidence that alcohol intoxication can exacerbate asthmatic inflammation and respiratory function, which may be mediated by ethanol-induced chemokine production.
Constitutive IL-13 promotes respiratory chlamydial infection and infection-induced chronic airway hyper-responsiveness (175.18)
Authors: Starkey Malcolm , Kim Richard , Horvat Jay , Essilfie Ama-tawiah , Beagley Ken , Mattes Joerg , Foster Paul , Hansbro Philip
Published: January 2023

We have previously shown that chlamydial lung infections in early-life drive the development of chronic airway hyper-responsiveness (AHR). To elucidate the mechanisms involved, we used a murine model of chlamydial lung infection, to investigate the influence of IL-13 on infection and infection-induced AHR. Infected IL-13-/- mice had less severe infection, reduced inflammation (neutrophils, dendritic cells, macrophages and T cells), less mucus secreting cells (MSCs) in the airways and attenuated AHR. More severe infection in wild-type (WT) mice was associated with the development of alternatively activated macrophages (increased Arg-1, FIZZ-1 and Ym-1), which were suppressed in IL-13-/- mice. Surprisingly, infection of WT mice did not induce any detectable increase in IL-13 expression. However, infection decreased the level of the IL-13 decoy receptor (IL-13Rα2), which may allow constitutive IL-13 to signal. Interestingly, reconstitution of IL-13 in IL-13-/- mice restored infection-induced inflammation and MSC formation, but not AHR. Furthermore, infection-induced inflammation and mucus were Stat-6-independent, whilst infection-induced AHR was dependent upon Stat-6. Thus, early-life respiratory chlamydial infection results in decreased decoy receptor expression, which allows constitutive IL-13 to signal and promote more severe infection, which drives the development of chronic AHR that persists into adulthood.
Allergic lung inflammation decreases the anti-viral response to vaccinia virus (175.3)
Authors: Walline Crystal , Sehra Sarita , Fisher Amanda , Guindon Lynette , Wilkes David , Brutkiewicz Randy , Kaplan Mark , Blum Janice
Published: January 2023

Despite the global eradication of smallpox following widespread vaccination with vaccinia virus (VV), other members of the poxviridae family persist and continue to infect animal and human hosts. Although the lung is the most lethal route of poxvirus infection, there is little known about the local immune response to pulmonary VV infection. Moreover, VV vaccination is contraindicated in patients with atopic dermatitis as the pro-allergic milieu diminishes anti-VV responses. Our goal was to determine the role of allergic airway disease (AAD) on the local immune response to VV infection. Our studies demonstrated increased weight loss, airway hyperreactivity, mucus gene expression, lung pathology and virus titer in mice with AAD. In contrast to observations from cells exposed to pro-allergic cytokines, expression of antimicrobial peptide genes in the lung tissue of infected mice was similar in control mice, and mice with AAD. At 4 days post infection (dpi) there was a more robust innate response in mice with AAD as evidenced by increased infiltration of lung macrophages and granulocytes. By 10 dpi, there were increased numbers of IL-10+CD8+ and IFNγ+CD8+ T cells and enhanced secretion of IL-10 and IFNγ in the bronchoalveolar lavage fluid of mice with AAD, compared to controls. Mice with AAD also had increased VV-specific IgG1 and IgM serum antibodies. These studies suggest the allergic lung microenvironment may influence the anti-viral immune response to pulmonary VV infection.
Role of NKT cell in the regulation of antigen presenting cell-function during RSV infection (175.5)
Authors: Durre Kainath , Wu Sherman , Qureshi Nagib , Beal Brooks , Qureshi Mahboob
Published: January 2023

Survivors of RSV bronchiolitis and pneumonia develop increased susceptibility to asthma. We showed that skewed polarization of dendritic cells (DC) towards plasmacytoid DC (pDC) in the pups in response to RSV is inhibited by exogenous administration of osteopontin (OPN) with an associated Th1 immune response. In the present study, we examined the expression of MHC-II, an indicator of antigen presenting function, on the antigen presenting cells (APC) in the RSV-infected lungs and its dependence on NKT cells. Wild type (WT) and NKT knock out (NKT-/-) pups were infected with RSV and were treated with or without OPN. Expression of MHCII on the lung APCs, including macrophages (MQ) and DCs (pDC; CD11blo/-CD11Clo/-CD45Rhi) and mDC; CD11blo/-CD11ChiCD45Rlo/-), was determined by flowcytometry (FACS). The effect of OPN on MHCII expression in the presence or absence of NKT cells was studied. FACS revealed that expression of MHCII was higher on the pDCs than mDCs in the RSV-infected WT lungs. In the absence of NKT cells, MHCII expression on pDCs was increased. OPN treatment augmented expression of MHCII on MQ and DCs, which was further enhanced in the absence of NKT cells. These data suggest that OPN- induced MHCII expression on the lung APCs is NKT dependent. Further experiments will explore the underlying mechanism of this phenomenon, which may help developing a therapeutic strategy to stop skewed polarization of DC in RSV infected lungs in preventing susceptibility to asthma.
A Bovine Whey Protein Extract Can Inhibit TH2 Cell Function and Show the Potential to Alleviate Asthma Symptoms in a Murine Asthma Model (175.6)
Authors: Huang Po-Han , Chen Jiunn-Horng , Chen Hui-Chen
Published: January 2023

Asthma patients have dramatically increased in the past decades and cost of care is more than 11.3 billion per year. The use of steroid is the major treatment to control symptoms; however the side effects are often devastating. Seeking new drugs or new strategies in reducing the dose of steroid taken has always been an important task. A whey protein extract (WPE) from bovine that is enriched in growth factor TGF-β has been demonstrated with potential for reducing symptoms associated with mild-to moderate TH1 mediated psoriasis in human. However whether WPE also has potential for inhibiting TH2 mediated disease is still unclear. In this paper, using a murine asthma model, we have identified that daily feeding the sensitized mice with WPE before challenge was able to reduce airway inflammation, serum OVA-specific IgE and airway hyperresponsiveness. In the presence WPE, an increased in regulatory T cell population in vitro and in vivo were observed. Moreover, the production of IL-4, IL-5 and IL-13 and the expression of TH2 master transcription factor GATA3 by stimulated TH2 cells were reduced when TH2 cells were cultured with WPE. Using TGF-β blocking antibody, we further identified that TGF-β is the main component in the WPE that exert the inhibitory effect. Thus, our data suggested that WPE has a potential to alleviate the symptoms of asthma by regulating TH2 cell functions. Therefore, taking WPE regularly might be potentially beneficial for asthma patient
Aberrant expression of MCC in TRAF3-deficient mouse B lymphoma and human multiple myeloma (176.12)
Authors: Edwards Shanique , Baron Jacqueline , Moore Carissa , Liu Yan , Hart Ronald , Xie Ping
Published: January 2023

We recently reported that specific deletion of a novel tumor suppressor gene, TRAF3, in B lymphocytes leads to B lymphoma development in mice. Interestingly, B lymphoma development in B-TRAF3-/- mice exhibits a long latency (about 9 months), indicating that additional oncogenic alterations are required for B lymphomagenesis. To delineate such oncogenic alterations in TRAF3-/- B lymphomas, we performed microarray analyses and identified mutated in colorectal cancer (MCC) as a gene consistently up-regulated in B lymphomas spontaneously developed in different individual B-TRAF3-/- mice. We verified the striking up-regulation of MCC expression in TRAF3-/- B lymphomas at both the mRNA and protein levels by quantitative real time PCR and Western blot analyses, respectively. We further found that the MCC transcript and protein were aberrantly elevated in 6 human patient-derived multiple myeloma cell lines with TRAF3 deletions or relevant mutations. In contrast, MCC expression was not detected in normal mouse splenic B cells or premalignant TRAF3-/- B cells. Furthermore, MCC expression was not induced in normal splenic B cells or premalignant TRAF3-/- B cells by a variety of B cell stimuli, including CD40, BCR, LPS, and CpG. Taken together, our findings indicate that aberrant MCC expression is associated with B cell tumorigenesis. Our ongoing study will further elucidate the roles and mechanisms of MCC in TRAF3 deficiency-initiated B cell malignant transformation.
MicroRNA-568 inhibit CD4+ T cell activation through targeting NFAT5 (176.2)
Authors: Wen Weihong , Li Wei , Yang An-Gang
Published: January 2023

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally and are also known to be involved in lymphocyte development and function. In our previous study, by using microRNA array, we tried to identify the miRNAs that may regulate T cell activation, and we have reported that miR-181c can regulate T cell activation through directly targeting IL-2 (Mol Immunol, 2011, 48: 592-599). Here we reported that during CD4+ T cell activation, the expression of miR-568 was decreased. Forced expression of miR-568 can significantly inhibit the activation of Jurkat cells and human peripheral CD4+ T cells after stimulation. NFAT5 was predicted to be a potential target of miR-568, and the expression of NFAT5 was increased during CD4+ T cell activation. NFAT5 3’UTR luciferase reporter assay showed that miR-568 can inhibit the expression of luciferase, but can’t inhibit the mutated NFAT5 3’UTR luciferase reporter expression. Upregulation of miR-568 can inhibit the expression of NFAT5 in CD4+ T cells, while miR-568 inhibitors can increase the NFAT5 level. Furthermore, we found that knockdown of NFAT5 can inhibit the activation of CD4+ T cells after stimulation. Thus, our results demonstrated that microRNA-568 can inhibit CD4 T cell activation, at least in part, through targeting NFAT5.
Endogenous ROS Regulates Vα14iNKT Cells Signaling during Agonistic Fas mAb-dependent Fulminant Liver Failure (176.3)
Authors: Ajuebor Maureen , Liu Jianfeng , Adegboyega Patrick , Aw Tak Yee , Downs Isaac
Published: January 2023

Current theories recognize uncontrolled systemic activation of the immune system as an early initiating event that leads to development of acute fulminant liver failure (FLF) in mice after treatment with agonistic Fas mAb. In the present study, we identify a novel and previously unrecognized pro-inflammatory and pro-apoptotic role for endogenous ROS in stimulating signaling in IFN-γ-expressing Vα14iNKT cells in a pSTAT-1/T-bet dependent manner to promote the development of FLF. Specifically, we show that treatment of mice with N-acetylcysteine (NAC), an ROS scavenger and glutathione (GSH) precursor, almost completely abolished IFN-γ signaling and nitrosative stress in Vα14iNKT cells. Conversely, fortification of the antioxidant, GSH, in the liver due to Vα14iNKT cells deficiency, induces anti-inflammatory responses to prevent/limit the development of FLF and hepatic apoptosis in Jα18−/− mice through suppression of IFN-γ signaling and effector functions. This study provides critical new insights into how ROS regulates IFN-γ signaling in intrahepatic Vα14iNKT cells to impact inflammatory and pathological responses.
CD47 protects naïve murine B cells from anti-CD19 mAb-mediated depletion (176.6)
Authors: Gallagher Sandra , Wang Yue , Yusuf Isharat , Herbst Ronald , Carter Laura
Published: January 2023

Studies with human CD19 transgenic mice demonstrate that the primary mechanism of anti-CD19 mAb mediated B cell depletion is antibody-dependent cellular phagocytosis (ADCP) mediated by macrophages. We hypothesize that the balance between positive and negative regulators of phagocytosis is an important mechanism regulating the efficiency of B cell depletion in vivo. One potential pathway involved in this regulation is CD47, which is ubiquitously expressed on cells, binding to CD172a on macrophages to generate a negative signal and prevent phagocytosis of self. Here, we investigated the role of CD47-CD172a in anti-CD19 mAb-mediated B cell depletion. All B cells in the blood, spleen and bone marrow express CD47 to some extent; the highest level of CD47 was detected on plasma cells, which are typically resistant to depletion. Further, CD47 KO B cells are more susceptible to in vitro depletion than WT B cells. And, anti-CD47 F(ab)2 mAb blockade of CD47-CD172a enhances ADCP of WT cells. Accordingly, in vivo treatment with anti-CD19 mAb demonstrated enhanced depletion in CD47-/- mice compared to WT mice even when a more potent, afucosylated anti-CD19 mAb is utilized. In conclusion, CD47 binding to CD172a counteracts anti-CD19 mAb-mediated phagocytosis of B cells. Future studies are warranted to investigate the regulatory role of CD47 in B cell depletion in clinical settings.
Mouse CD229 ligation costimulates T cell activation (176.7)
Authors: Wang Jinghua , Wu Guoping , Hao Ruyi
Published: January 2023

CD229 (SLAMF3) is a receptor protein primarily found on mature T cells, B cells, myeloid cells, macrophages, and thymocytes. The exact functions of this receptor remain uncertain. Data from CD229 knockout mice showed that CD229 is uniquely involved in enhancing T cell activation. However, coligation of CD229 and TCR with anti-CD229 (mAb) and anti-CD3 antibodies has been demonstrated to inhibit T cell activation. To study the functions of CD229, we expressed a soluble polyHistidine tagged protein containing the mouse CD229 extracellular domain (aa48-454) and investigated its function in purified mouse CD3+ cells in vitro. Concurrent ligation of CD229 and TCR with immobilized CD229-His protein and anti-CD3 antibody significantly enhanced cell proliferation and IFN-γ secretion in murine CD3+ splenocytes in a dose-dependent manner. Furthermore, pre-treatment of CD3+ splenocytes with polyclonal anti-CD229 antibody completely abolished co-stimulative actions of CD229-His. This suggests that CD229-His co-stimulates T cells through CD229 receptors on T cell surface. Moreover, ligation of CD229 with CD229-His in CD3+ cells led to ERK1/2 phosphorylation in response to anti-CD3 stimulation. Taken together, these results suggest that CD229 acts as a hemophilic costimulatory molecule and is able to up-regulate T-cell activation.
Effects of KOB03, a polyherbal medicine in mast cell-mediated allergic response in ovalbumin-induced allergic rhinitis in vivo model and RBL-2H3 mast cells. (177.14)
Authors: Jung Jinki , Jung Hyo Won , Kang Seok Yong , Kim Songhee , Park Yong-Ki
Published: January 2023

KOB03 is a polyherbal medicine originating from Korean Traditional Medicine, which has been clinically applied for the treatment of allergic diseases. Here, we investigated the inhibitory effects of KOB03 on mast cell-mediated allergic responses on ovalbumin-induced allergic rhinitis in guinea pigs and RBL-2H3 mast cells. To determine the effect of KOB03 on mast cell-mediated allergic reactions, we investigated the parameter changes on ovalbumin (OVA)-induced allergic rhinitis in guinea pigs, and the release of allergic inflammatory mediators such as histamine, IgE and inflammatory cytokines in DNP-IgE-stimulated RBL-2H3 mast cells. The oral administration of KOB03 at doses of 200 and 500 mg/kg in OVA-sensitized/challenged guinea pig significantly decreased the serum levels of OVA-specific IgE, histamineand TNF-alpha KOB03 also inhibited the histopathological changes of nasal mucosa with the infilteration of eosinophils and inflammatory cells. Furthermore, KOB03 significantly inhibited DNP-IgE-induced release of histamine in RBL-2H3 cells via suppressing the ERK1/2 phosphorylation. These data suggest that KOB03 has an anti-allergic activity by modulating mast cell-mediated allergic responses.
Purified Natural Ara h 6: An Important Marker for IgE Responses to Peanut (177.15)
Authors: Koid Audrey , Hamilton Robert , van Ree Ronald , Versteeg Serge , Dreskin Stephen , Chapman Martin , Wuenschmann Sabina
Published: January 2023

Ara h 6 has a reported seroprevalence similar to Ara h 2, a major peanut allergen. Both allergens have a similar molecular weight (14.5kD - Ara h 6; 17-18kD - Ara h 2) and share 55% identity in their amino acid sequences. Thus, it has been difficult to obtain natural Ara h 6 (nAra h 6) free of Ara h 2. Our study compares the IgE antibody reactivity of highly purified nAra h 6 to a natural Ara h 6 reference reagent (nAra h 6-R), recombinant Ara h 6 (rAra h 6) and natural Ara h 2 (nAra h 2). SDS-PAGE of the highly purified allergen revealed a single 14.5kD band and the identity of Ara h 6 was confirmed by tandem mass spectrometry (LC/MS-MS). The purified nAra h 6 contained <0.005% traces of nAra h 2 as assessed by ELISA. Natural Ara h 6 had a lower biological activity in basophil histamine release assays than nAra h 2. Chimeric ELISA showed that 70 and 75% of peanut allergic patients (n=57) had specific-IgE to nAra h 2 and nAra h 6 respectively. Natural Ara h 6-R and rAra h 6 displayed similar IgE antibody reactivity when compared to the nAra h 6. In conclusion, Ara h 6 is a major peanut allergen, with comparable immunoreactivity to Ara h 2. The highly purified Ara h 6, free of Ara h 2, will be useful for diagnostic IgE antibody assays, and for molecular and cellular studies to further investigate the immunological mechanisms of peanut allergy.
The nascent truncation of FcϵRIβ confers calmodulin binding which is essential for microtubule formation and degranulation in human mast cells (177.17)
Authors: Cruse Glenn , Beaven Michael , Bradding Peter , Gilfillan Alasdair , Metcalfe Dean
Published: January 2023

The gene locus containing the MS4A2 gene (encoding FcϵRIβ) has strong linkage to allergy susceptibility. MS4A2 was considered as a candidate gene, but studies on the functional consequence of mutations in FcϵRIβ have been disappointing. We have identified a truncation of FcϵRIβ (t-FcϵRIβ) in humans. In this study we sought to identify the function of this variant. We determined that t-FcϵRIβ forms a complex with Fyn kinase and Gab2 in the cytosol and that the truncation of FcϵRIβ exposes a calmodulin (CaM) binding domain. Using co-IPs we show that in the presence of Ca2+, CaM binds to the t-FcϵRIβ complex, allowing phosphorylation of Fyn and Gab2, which are involved in microtubule formation. Confocal microscopy revealed translocation of the t-FcϵRIβ complex to a tubulin-rich region after cell activation. Deconvolution and 3D modeling of the imaging data demonstrated that the translocated complex surrounds the centrosome, forming an intricate network with microtubules. Utilizing lentiviral shRNA to silence the FcϵRIβ variants, we identified that knockdown of t-FcϵRIβ attenuated microtubule formation, degranulation and cytokine production in response to thapsigargin. Ca2+ influx and PGD2 production were unaffected. These data suggest that t-FcϵRIβ propagates Ca2+ signals by binding CaM and initiating microtubule formation which is essential for mast cell degranulation and cytokine release. Thus, targeting t-FcϵRIβ may offer a novel approach to treatment of allergic diseases.
Thymol inhibits passive cutaneous anaphylaxis in mice through induction of apoptotic cell death in mast cells (177.3)
Authors: Wechsler Joshua , Hsu Chia-lin , Bryce Paul
Published: January 2023

Mast cells play a critical role in atopic dermatitis through the release of preformed and de novo synthesized pro-inflammatory mediators. In 1878, HR Crocker described the use of topical thymol in the treatment of advanced eczema. We hypothesized that the efficacy of thymol in eczema is due to a direct effect on mast cells. The effect of topical thymol was assessed on allergic murine ear swelling in the mast cell-dependent passive cutaneous anaphylaxis (PCA) model as well as on ear swelling directly and mast cells in vitro. Thymol dose-dependently inhibited PCA when administered topically prior to antigen challenge. Thymol induced a dose-dependent immediate ear swelling in mice, however lacked late phase swelling seen in PCA. This effect was associated with increased mast cell degranulation histologically and was inhibited in histamine deficient mice. Thymol induced mast cell degranulation and mRNA transcription in vitro, but prevented protein secretion. This was due to decreased mast cell viability from apoptosis. Mast cell activation from thymol occurred via sustained calcium flux. Thymol-induced calcium flux was not inhibited in TRPV3 KO mast cells, but was inhibited by HC-030031, a selective TRPA1 antagonist, and 2-APB, an IP3 receptor antagonist. However, HC-030031 and 2-APB were unable to prevent thymol-induced cell death. We are currently investigating the mechanism by which thymol leads to activation-induced cell death in mast cells.