bluuebunny/crossref_metadata_2025 huggingface dataset:
Exposure to LPS induces the generation of immunosuppressive myeloid cells (95.7)
Authors: Arora Meenakshi , Ray Anuradha , Ray Prabir
Published: January 2023

The biologic effects of lipopolysaccharide (LPS) may have different consequences on the expression of asthma dependent upon whether the exposure to LPS occurs during the establishment of the atopic status, or whether the exposure to LPS occurs after the asthma phenotype is established. It has been shown that exposure to LPS during early childhood is associated with a lower incidence of asthma later in the life. Dendritic cells (DCs) are the primary antigen presenting cells of the immune system and play critical role in the induction of peripheral tolerance and regulation of T-cell responses. The diverse functions of DCs in immunity and tolerance depend on their lineage and maturation stage. We have observed that exposure of LPS can generate a population of immature myeloid cells that have potent immunosuppressive activity on T cells. These CD11c− cells are largely MHC IIlow/MHC II- and have poor antigen presenting capacity. These cells poorly stimulate proliferation of naïve allogenic T cells and are unable to stimulate T cell differentiation along the Th1 or Th2 lineage. However, these cells are capable of suppressing the proliferation and differentiation of activated CD4+ T cells both in vitro and in vivo. Our data shows that LPS reduces DC driven T cell development through the induction of cells with suppressive activity, which could be a potential strategy to suppress harmful T-cell responses in patients with allergic diseases.

(RO1 HL60207 to P.R. and RO1 HL77430 to A.R.)
Preferential Chemotaxis and Fugetaxis of CD4−CD8− Thymocytes in Response to CXCL16 (95.8)
Authors: Manilay Jennifer O. , McLelland Bryce
Published: January 2023

Directed migration of developing thymocytes to specific anatomic regions of the thymus can influence normal T cell development. However, the mechanisms which influence migration of early CD4−CD8− (double negative: DN) thymocytes are incompletely understood. We investigated the role of chemokine CXCL16 and its receptor, CXCR6, in thymocyte migration. Gene expression analysis showed that CXCR6 is expressed only in DN thymocytes, but not at later stages of development, whereas CXCL16 is expressed on stromal cells throughout the thymus. The ability of DN thymocytes to migrate in response to CXCL16 was tested with in vitro transwell assays. At concentrations of 100 and 10ng/ml, DN thymocytes demonstrated specific migration toward CXCL16, whereas migration of CD4+CD8+ thymocytes was not significantly different from controls. We also tested the fugetactic ability of DN thymocytes from CXCL16, and observed that DN thymocytes selectively migrated away at concentrations of 10 and 1ng/ml. These data demonstrate that DN thymocytes migrate differentially to CXCL16 in a dose-dependent fashion. Thus, CXCR6 and CXCL16 may be involved in localized cell migration in the thymus. ADAM10 and Notch influence cell migration in other systems; thus experiments to test their roles in the control of CXCR6-mediated migration of DN subsets in the thymus are ongoing.

Supported by UCM Faculty Startup Funds
Characterization of chicken TNFSF15 and it’s role in coccidiosis (96.10)
Authors: Lillehoj Hyun Soon , Park Soon Soon
Published: January 2023

A full-length cDNA encoding chicken tumor necrosis factor superfamily 15 (TNFSF15) was isolated and its functional role was investigated. TNFSF15 transcripts were primarily expressed in spleen, liver, intestinal intraepithelial lymphocytes (IEL), peripheral blood lymphocytes and bursa. In vitro infection of HTC macrophages with three species of Eimeria sporozoites induced TNFSF15 gene expression. In vivo experiments revealed that TNFSF15 gene was highly increased following primary infections with E. acervulina or E. maxima. In contrast, no consistent changes in transcript levels were seen following primary infection with E. tenella, or following secondary infection with any of the three Eimeria species. Following infection with E. acervulina and E. maxima, TNFSF15 transcripts were primarily expressed in intestinal CD4+ and TCR2+ IEL, respectively. A dose-dependent cytotoxic effect of recombinant TNFSF15 protein was observed on HTC and LSCC-RP9 tumor cells. These results indicate that TNFSF15 plays an important role in the local inflammatory response to Eimeria.
Massive colonic cytokine dysregulation in IL-10−/− mice revealed by multiplex analysis (96.17)
Authors: Klein John R. , Hicks John , Montufar-Solis Dina
Published: January 2023

IL-10−/− mice develop enterocolitis resulting from a failure to control the expression of proinflammatory and other immune response analytes. To better understand changes that occur in regulatory and effector cytokines in IL-10−/− mice during the progression of intestinal inflammation, multiplex analysis of 23 analytes was evaluated in colonic intraepithelial cell culture supernatants from 25 IL-10−/− and 6 control mice ranging from 7–52 weeks or age. In IL-10−/− mice, widespread cytokine dysregulation was evident across the period of the study for 18 analytes (IL-1α, IL-1β, IL-6, IL-9, IL-12p40, IL-12p70, IL-13, eotaxin, G-CSF, GM-CSF, IFN-γ, KC/CCXCL1, MCP-1, MIP-1α, MIP-1β, CCL5/RANTES, TNFα, and IL-17), whereas no activity was detected for IL-2, IL-3, IL-4, IL-5, or IL-10. Of analytes with elevated activities, 15 had a low-to-medium correlation with the degree of colonic pathology (r = 0.2649 to r = 0.6494); IL-9 and IL-13 had very high correlations with pathology (r = 0.8026 and r = 0.8137, respectively); and IL-17 had an extremely strong correlation with pathology (r = 0.9885). These findings demonstrate that the lack of IL-10 results in a complex, massive, and early process of intestinal cytokine dysregulation with IL-17 strongly linked to disease overall.
TNF-α promotes CD4 T cell infiltration of the central nervous system (CNS) by altering the polarity of CXCL12 expression at the blood-brain barrier (BBB) (96.18)
Authors: McCandless Erin Elizabeth , Piccio Laura , Cross Anne , Russell John , Klein Robyn
Published: January 2023

CXCL12 is a secondary lymphoid chemokine that localizes lymphocytes within lymph node zones and promotes CD4 T cell help. We have previously shown that expression of CXCL12 at the BBB displays basolateral polarity and localizes leukocytes to the perivascular space, similar to its function within lymphoid tissue. Induction of experimental autoimmune encephalomyelitis (EAE) leads to redistribution of CXCL12 across the BBB and exit of myelin-specific T cells from the perivascular space. In the current study we have extended our findings to human CNS specimens and examined the molecular triggers for CXCL12 redistribution at the BBB. We observed that a similar loss in CXCL12 polarity occurs within active Multiple Sclerosis (MS) lesions, suggesting that altered BBB expression of CXCL12 plays a role in the pathogenesis of MS. Using TNFR1−/− mice, we demonstrate that while loss of TNFR1 signaling does not alter peripheral immune responses, CXCL12 expression in TNFR1−/− mice remains polarized at the BBB and is associated with the accumulation of leukocytes within the perivascular space. Comparisons of myelin-specific CD4 T cells recovered from vessel-associated versus parenchymal sites revealed that only T cells that have successfully infiltrated the CNS parenchyma express both CXCR4 and TNF-α. These results suggest that TNFR1 signaling promotes leukocyte entry via redistribution of CXCL12 at the BBB.
Potential role of CCR9 in gastrointestinal involvement of ATL cells (96.21)
Authors: Nagakubo Daisuke , Jin Zhe , Hieshima Kunio , Nakayama Takashi , Shirakawa Aiko-Konno , Yoshie Osamu
Published: January 2023

Adult T-cell leukemia (ATL) is a mature CD4+ T-cell malignancy etiologically associated with human T-lymphotropic virus type 1 (HTLV-1). Previously, we have shown that ATL cells from the majority of cases are strongly positive for CCR4, which may explain the frequent skin invasion of ATL. Although ATL is also known to frequently invade the gastrointestinal tract, the chemokine receptors possibly involved in the process have not been determined yet. In this study, we examined whether ATL cells express CCR9, which is known to be involved in T-cell homing to the gastrointestinal tract. While ATL cells freshly isolated from peripheral blood hardly expressed CCR9, ATL cells cultured for 1 day consistently upregulated CCR9 in parallel with the upregulation of Tax, the HTLV-1-encoded transcriptional activator. A luciferase reporter gene under the control of the CCR9 promoter was expressed by co-transfection of an expression vector for Tax. Immunohistochemical staining demonstrated that ATL cells infiltrating gastrointestinal tract were frequently positive for CCR9. Thus, CCR9 is inducible by Tax and may play a role in the gastrointestinal involvement of ATL.

This work was supported by grants from Ministry of Education, Culture, Sports, Science and Technology, Japan and Japan Science and Technology Corporation.
Response to SDF-1 defines a subset of CD38+/ZAP-70+ CLL patients with a common genetic signature and aggressive behavior (96.23)
Authors: Deaglio Silvia , Aydin S , Vaisitti T , Bergui L , Omede’ P , Bonello L , D’Arena G , Chiorino G , Efremov D , Malavasi F
Published: January 2023

Chronic lymphocytic leukemia (CLL), traditionally considered a disease of failed apoptosis, is now seen as characterized by a different propensity to grow according to microenvironmental signals. CD38 (surface receptor) and ZAP-70 (cytoplasmic molecule) independently predict unfavorable outcome in CLL patients. We reported that CD38 is a receptor mediating interactions with nurse-like cells (NLC) and transducing proliferation signals. The production by NLC of the attractant SDF-1α and the presence of ZAP-70 in CXCR4 pathway suggested that CD38 and ZAP-70 control CLL cell migration. We show that CD38+/ZAP-70+ patients feature enhanced ability to respond to SDF-1α. The functional link between the two molecules was confirmed by showing that i) CD38 engagement leads to ZAP-70 phosphorylation in CLL cells and in ad hoc modified B line expressing a ZAP-70-myc construct. Further, ii) CD38 signals are effective only in presence of ZAP-70, as seen in 22 molecularly characterized patients.

Lastly, a separation of the patient cohort based on migration, brought to the identification of 23 differentially expressed genes: 12 are involved in cell motility, migration and neoplastic transformation.

This work provides biological evidence explaining why the combination of CD38 and ZAP-70 in routine clinics results in a more efficient identification of CLL patients with aggressive disease.

Sponsors: CLL-GRF, AIRC, FIRMS.
The alternative complement system and obesity: using knockout mouse models to investigate fat metabolism (96.24)
Authors: Cianflone Katherine , Paglialunga Sabina , Roy Christian , Lu HuiLing , Pekna Marcela
Published: January 2023

A growing body of evidence now links immunology and metabolic functions. The alternative system plays a role in injury and tissue damage as assessed by complement 3 knockout (C3KO), factor B (FBKO) and factor D (FDKO) mice with injury and septic shock. Recently the C5L2 receptor was found to act as a decoy receptor for C5a during similar conditions. Acylation stimulating protein (C3adesArg/ASP), a by-product of the alternative complement pathway also acts via C5L2 to stimulate triglyceride (TG) synthesis in adipose tissue.

Wildtype (WT), C3KO, FBKO and C5L2KO mice on a normal chow diet were studied over 12 weeks. No difference in body weight was observed between KO and WT mice. However C3KO, FBKO and C5L2KO mice displayed increased food intake by 56%, 48% and 62% respectively vs. WT mice (p<0.05). Consistent with previous findings, C3KO mice showed delayed TG clearance during a fat load. FBKO and C5L2KO mice also display delayed postprandial lipemia. Adipose tissue from C5L2KO mice showed reduced ex vivo TG synthesis (-83%, p<0.001). Also, DGAT (TG synthesis enzyme) activity and protein were significantly reduced in C3KO mice vs. WT.

Therefore, under normal physiological, alternative complement KO mice display altered fat metabolism. We attribute this phenotype to the lack of ASP and C5L2 signalling, however, further investigation is still required to understand the association between immunity and metabolism.

Funding: CIHR and NSERC
Effect of Dermatophagoides pteronyssinus on expression of monocyte chemotactic protein 1 (MCP-1)/IL-6/IL-8 in the human monocytic cell line, THP-1 (96.26)
Authors: Lee Ji-Sook , Kim In Sik , Kim Dong Hee , Kim Joo-Hwan , Jeong Hye Gwang , Ryu Ji-Sun , Choi Hye-Jin , Yun Chi-Young
Published: January 2023

RATIONALE: The house dust mite, Dermatophagoides pteronyssinus (DP) is a major source of allergens implicated in the pathogenesis of allergic disorders; they include asthma, allergic rhinitis, and atopic dermatitis. Monocyte chemotactic protein 1 (MCP-1/CCL2)/IL-6/IL-8 plays a pivotal role in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis.

METHODS: The aim of this study was to investigate the effect of DP extract (DpE) on expression of MCP-1/IL-6/IL-8 mRNA and protein and the signal transduction leading to MCP-1/IL-6/IL-8 expression induced by DpE in the human monocytic cell line, THP-1. The mRNA and protein expression of MCP-1/IL-6/IL-8 was measured by performing RT-PCR and ELISA.

RESULTS: DpE-stimulated cells showed elevated MCP-1/IL-6/IL-8 expression in a time and dose-dependent course. MCP-1/IL-6/IL-8 expression induced by DpE is strongly inhibited by PP2, an inhibitor of Src, rottlerin, an inhibitor of protein kinase C δ (PKC δ) PD98059, an inhibitor of MEK and SB202190, an inhibitor of p38 mitogen-activated protein kinase (MAPK).

CONCLUSIONS: These results indicate that DP increases MCP-1/IL-6/IL-8 expression and transduces the signal through extracellular signal-regulated kinases (ERK1/2) in human monocytes.
STAT3 mediates the initiation and progression of Lyme carditis (96.29)
Authors: Zegarra-Moro Ofelia L , Vinasco Javier , Braga Walter U , Moro Manuel H
Published: January 2023

Lyme disease is caused by Borrelia burgdorferi and represents a worldwide public health problem. This multisystemic and chronic disease may affect several organs including the heart. However, little is known about the molecular mechanisms that control and/or facilitate the development of Lyme carditis. Signal transducer and activator of transcription 3 (STAT3) is critical in mediating cytokine signaling and it has been involved in diverse pathological processes and also implicated in a variety of immune responses. The aim of this study is to examine the role of STAT3 in the initiation and progression of Lyme carditis using a murine model. Six week-old C3H and BALB/c mice were inoculated with B. burgdorferi. Hearts were excised at defined time intervals that reflected the progression of Lyme disease in the mouse model: 7, 14, 21, 30, and 60 days post-inoculation. Infected cardiac tissues were screened for expression of STAT3 and STAT3-regulated proteins by Western blot analyses, immnunoprecipitation and immunofluorescence studies. We show that STAT3, pSTAT3 and STAT3 regulated proteins were upregulated in cardiac tissues infected with B. burgdorferi. Furthermore, we found a direct correlation between upregulation of STAT3/pSTAT3 and progression of the disease during the acute phase of Lyme carditis. Our results suggest that STAT3 plays a key role in Lyme carditis and may represent a molecular target for monitoring the initiation and progression of Lyme carditis. NIH P20RR016443-04 .
HIV-1 infection alters cytokine and chemokine networks that influence innate immunity in the lung (96.9)
Authors: Nicol Marlynne Quigg , Hayete Boris , Pereira Albertina , Rarick Matt , Mathys Jean-Marie , Collins James , Skolnik Paul
Published: January 2023

Objective: To create mathematical network models of cytokine and chemokine interactions in the lungs of HIV+ subjects, utilizing protein and mRNA measurements, to better elucidate the relative importance of elements within these complex networks.

Methods: We used U937, U1 (HIV-1-infected) cell lines, alveolar macrophages from HIV+ subjects and matched healthy controls, exposed to LPS and cytokines. We measured cytokine/chemokine protein production by Luminex, and mRNA by real-time PCR, for nine moieties. Using these perturbations and NIR algorithms, functional relationship maps were constructed.

Results: Analysis of protein or mRNA data showed differences in type and magnitude of responses in infected cells. Clear differences in connectivity between the infected and uninfected cell lines for the nine moieties were also apparent in functional relationship maps derived from the protein or mRNA data. In addition, concordance of mRNA maps with maps produced from microarray data validated our network hypothesis.

Conclusions: Preliminary network models show the feasibility of obtaining biologically relevant results that model cytokine and chemokine networks in the lung, rather than individual moieties or single agonist/antagonist pairs. These networks may indicate connections not previously considered important in HIV-1 pathogenesis in the lung.

Supported by NIH grant R01HL076114
Loss of Mac-1 expression in mice leads to more severe forms of inflammatory disease in both the MRL/MpJ-Faslpr and CIA Models (97.1)
Authors: Bullard Daniel C , He Xiaodong , Schoeb Trenton R , Raman Chander
Published: January 2023

Mac-1 plays key roles in innate immune responses, and has also been implicated in the pathogenesis of diseases such as SLE and RA. Previously, we observed that Mac-1 deficiency in MRL/MpJ-Faslprmice, a model for SLE, resulted in a more severe inflammatory phenotype. To further examine mechanisms by which this adhesion molecule regulates inflammation in this model, we investigated whether loss of Mac-1 specifically altered T cell activation. We found that both Mac-1 deficient CD4+ and CD8+ T cells showed a hyperproliferative response following stimulation with low doses of anti-CD3 compared to control cells. We also analyzed T cell proliferation kinetics in Mac-1 deficient DO11.10 TCR transgenic T cells in response to Ova-peptide challenge and observed a similar hyperproliferation phenotype, suggesting that this adhesion molecule can negatively regulate T cell proliferation, independent of Faslpr gene expression. Finally, we analyzed Mac-1 deficient mice in the CIA model of RA to determine whether loss of expression of this adhesion molecule would also lead to a more severe inflammatory phenotype. We observed that Mac-1 deficient mice showed an accelerated onset of joint inflammation compared to wild type controls. Thus, these results suggest that Mac-1 can act as a negative regulator in the development of inflammatory diseases, possibly through controlling T cell activation and proliferation.
ICAM-1 expression is required for regulatory T cell localization in the lungs of M. tuberculosis infected mice (97.2)
Authors: Plessner Hillarie L. , Flynn JoAnne L.
Published: January 2023

Mycobacterium tuberculosis (Mtb) is a widespread human pathogen that persists in the lung of latently infected individuals. Control over Mtb infection relies upon migration of leukocytes into the granuloma. We hypothesized that the adhesion molecule ICAM-1 plays a role in the migration, retention and interaction of leukocytes during Mtb infection. While previous studies in ICAM-1−/− mice have indicated that ICAM-1 is required for long-term control over Mtb infection, the mechanism remains unclear. In this study, ICAM-1−/− mice infected with Mtb survived over 10 months post-infection and controlled bacterial burden. Although granuloma formation appeared normal, higher overall cell numbers were observed by H&E staining and flow cytometry. We observed inhibited T cell contraction in the absence of ICAM-1 following primary Mtb infection. T regulatory cells were significantly reduced in ICAM-1−/− mice, which could play a role on T cell contraction. We conclude that ICAM-1 expression helps to control lung pathology by supporting T regulatory cells. Future studies will attempt to elucidate the mechanism by which ICAM-1 affects T regulatory cell recruitment to or retention in the lungs.
STAT6 is a novel regulator of CD44 expression in human B cells (97.4)
Authors: Mishra Jyoti Prasad , Mishra Sasmita , Kumar Ashok
Published: January 2023

Interactions of CD44, an adhesion molecule, with its ligand, hyaluronan (HA) play a crucial role in cell migration, inflammation and immune response. The regulation of CD44 expression particularly in human B cells is not well understood. We have previously demonstrated that CD44 expression in human monocytic cells is regulated by Egr-1 and AP-1 transcription factors (Mishra et al. 2005, JBC). IL-4, a pleotropic cytokine is known to regulate CD44-HA interactions, cell migration, and differentiation of human B cells. To understand the regulation of IL-4-induced CD44 expression in human B cells, we used an EBV-transformed Burkitt’s lymphoma cell line, BL30/B95-8 as a model system. Our results suggested that IL-4 did not induce the expression of Egr-1 and AP-1 as determined by Northern and Western blot analysis and gel shift assays. To elucidate the transcription factors involved, we demonstrated by promoter analysis and gel shift assays that STAT-6 plays a critical role in IL-4-induced CD44 regulation in human B cells. We also investigated the upstream signaling events and demonstrated that IL-4-induced STAT-6 is activated by two distinct signaling pathways namely Jak-1/3 and ERK-MAPK and is independent of the IRS2/PI3 kinase pathway. Taken together, our results suggest a novel pathway of CD44 regulation involving the activation of STAT-6 through the upstream Jak-1/3 and ERK-MAPK in IL-4-stimulated human B cells.
Immobilized SDF-1α Triggers Rapid VLA-4 Affinity Increases to Stabilize Lymphocyte Tethers on VCAM-1 and Subsequently Initiate Firm Adhesion (97.7)
Authors: Lawrence Michael Brandeau , Divietro Jeffrey A. , Larson Richard S. , Sklar Larry A.
Published: January 2023

The integrin VLA-4 (?4?1) mediates tethering and rolling events as well as firm adhesion of leukocytes to VCAM-1. Unlike selectins, VLA-4 integrin-mediated lymphocyte adhesiveness can be modulated by chemokines through intracellular signaling pathways. To investigate the effects of the chemokine Stromal cell Derived Factor-1? (SDF-1?) on VLA-4-mediated lymphocyte adhesion; human peripheral blood lymphocytes (PBL) were flowed over VCAM-1 substrates in a parallel plate flow chamber with surface immobilized SDF-1?, a potent activator of firm adhesion. The initial tethering interactions had a median lifetime of 200 ms, consistent with the half-life of low affinity VLA-4-VCAM-1 bonds. Immobilized SDF-1? acted within the lifetime of a primary tether to stabilize initial tethering interactions, increasing the likelihood a PBL would remain interacting with the surface. As expected, the immobilized SDF-1? also increased the ratio of PBL firm adhesion to rolling. An LDV peptide-based small molecule that preferentially binds high affinity VLA-4 reduced PBL firm adhesion to VCAM-1 by 90%. The reduction in firm adhesion due to blockage of high affinity VLA-4 was paralleled by a four-fold increase in the fraction of rolling PBL. SDF-1? activation of PBL firm adhesion on VCAM-1 depended on induction of high affinity VLA-4 rather than recruitment of a pre-existing pool of high affinity VLA-4 as previously thought.

Supported by NIH R01 HL54614.
Cloning and Expression of Bovine CCL28 (98.10)
Authors: Hughes Melissa , Martino Ben , Voyich Jovanka , Wilson Eric
Published: January 2023

Chemokines play a vital role in mediating leukocyte homing and accumulation to tissues. The chemokine CCL28 also known as Mucosal Epithelial Chemokine (MEC) has been shown to be essential in mediating migration of IgA antibody secreting cells to the lactating mammary gland and gastrointestinal tract. This protein has also been shown to function as an antimicrobial peptide. Cows and other ruminants do not transfer antibody transplacentally as do humans and mice. Therefore efficient transfer of immunity to the suckling newborn in ruminants is highly dependant on efficient transfer of antibodies via colostrum. Neither the role of CCL28 in the transfer of immunity from mother to nursing neonate, nor the role of CCL28 as a potential antimicrobial compound in milk has yet been elucidated in the cow. To begin addressing these basic questions we are cloning and expressing bovine CCL28. Using a putative bovine sequence of CCL28 from GenBank and known sequences from mouse and human CCL28, appropriate primers with restriction sites were created for bovine CCL28. Salivary gland mRNA was collected and RT-PCR was employed to amplify the coding region of the gene. The amplified fragment was sequenced to establish concordance with GenBank records. The coding region was then inserted into a pMAL-c2G vector and is being used to generate recombinant bovine CCL28.
The key role of a G protein coupled receptor mFPR2 in the development of inflammatory airway disease (98.4)
Authors: Wang Ji Ming , Li Yingying , Hu Jinyue , Huang Jian , Gong Wanghua , Chen Keqiang
Published: January 2023

The human G protein-coupled formylpeptide receptor-like 1 (FPRL1) and its mouse homologue mFPR2 mediate leukocyte chemotaxis in response to a variety of chemotactic peptides associated with inflammation and bacterial infection. In order to ascertain the role of this receptor in innate host defense, we generated mFPR2 knockout (mFPR2−/−) mice. mFPR2−/− mice developed normally, but with reduced number of myeloid cells in the bone marrow. In pulmonary allergic inflammation induced by ovalbumin (OVA), mFPR2−/− mice showed markedly reduced myeloid cell exudation in the broncho-alveolar lavage (BAL) and cell infiltration in the lung tissue, in association with impaired epithelial proliferative responses. In addition, the BAL from OVA-treated wild type mice contained high levels of mFPR2 specific chemotactic activity, which was absent in the BAL of mFPR2 −/− mice. Our results indicate a key role for mFPR2 in the development of inflammation in the allergic airway disease.

This project has been funded with federal funds from the National Cancer Institutes, National Institutes of Health, under Contract No. NO1-CO-12400 and was also supported in part by the Intramural Research Program of the NCI, NIH.
CX3CR1 deficiency impairs the recruitment of dendritic cells to the arterial intima and reduces atherosclerotic burden (98.7)
Authors: Liu Peng , Spencer Jessica A , Johnson Ashley E , Patel Dhavalkumar D
Published: January 2023

Dendritic cells (DCs) have recently been found in atherosclerosis-predisposed regions of arteries and have been proposed to be causal in atherosclerosis. Our previous studies demonstrated that the chemokine receptor CX3CR1 is associated with arterial injury and atherosclerosis. Here we investigated if DC arterial accumulation was correlated with atherosclerosis and the mechanisms by which CX3CR1 may play a role. Mouse aortas were isolated and subjected to en face immunofluorescence analysis. We found that DCs were located predominantly in the intimal regions of arterial branch points and curvature. Consistent with the increased accumulation of intimal DCs in aging (2196.2±154) and ApoE−/−aortas (2697.5±214.3) compared to young WT aortas (904±79.5, p=0.004 and 0.05 respectively), the incidence of atherosclerosis was 88.9% for aging and 100% for ApoE−/− mice. CX3CR1 was expressed on intimal DCs, and DC numbers were decreased in CX3CR1−/− aortas (541.1±93.6 vs. 904±79.5 for WT, p=0.0008). CX3CR1-deficiency decreased atherosclerosis in aortas of ApoE−/−mice by 71% (0.41%±0.11% of aorta surface for ApoE−/−/CX3CR1−/− vs. 1.39%±0.02% for ApoE−/−, p=0.00006). Thus, the accumulation of intimal DC increases in aging and ApoE−/− aortas and correlates with the generation of atherosclerosis. CX3CR1-deficiency impairs the recruitment of DC to the aortic wall and markedly reduces the atherosclerotic burden.
Mechanisms involved in accumulation of host cells within the CNS during adoptive transfer induced EAE (99.10)
Authors: Lees Jason R , Russell John H
Published: January 2023

Adoptive transfer of activated central nervous system (CNS) antigen specific T-cells into susceptible strains of mice results in the invasion of transferred cells into the CNS. CNS invasion by these initiating T-cells and recruited monocytes culminates in the induction of experimental autoimmune encephalomyelitis (EAE). Following this initial invasion a secondary infiltrate of host T-cells of unknown function and antigenic specificity enters the CNS. The role of these secondary-infiltrating cells in the initiation, development, and resolution of EAE remains uncharacterized. In this study we utilized an adoptive transfer model of EAE induction in mice to characterize the mechanisms involved in entry of host T-cells found within the CNS following disease initiation. These cells were further examined for both phenotypic and functional characteristics at various times after disease initiation. Finally, we examined the necessity for antigenic recognition of peptides in entry and retention of cells within the CNS. Findings from this study allow us to characterize the mechanisms involved in accumulation of host cells within the CNS during EAE pathogenesis.
Plexin-A1 on dendritic cells and its interaction with a T cell surface molecule (B11)
Authors: Eun So-Young , O’Connor Brian Patrick , Wong Athena W , Ye Zhengmao , Ting Jenny P.-Y.
Published: January 2023

Plexin-A1 was first discovered on neurons and found to cause growth cone collapse in response to semaphorin3A, a repelling cue. Interestingly, we and others have recently observed that plexin-A1 is also an important factor for the interaction between antigen-presenting cells and T cells (Wong, A. W., et al. 2003, Nat. Immunol.; Takegahara, N., et al. 2006, Nat. Cell Biol.). Plexin-A1 on dendritic cells is shown to play a significant role in fully activating antigen-specific T cells in their co-culture. Effect of plexin-A1 on T cell activation is shown to be signaled through the Rho GTPase to accumulate actin filaments toward immune synapse (Eun, S.-Y., et al., 2006, J. Immunol.). Our group and others have identified a ligand for plexin-A1 on T cells as the semaphorin6D molecule, which is upregulated on activated T cells. In the co-culture of DCs and T cells, we observed that T cell proliferation became significantly reduced by intervention of soluble sema6D fusion protein conjugated with human IgG. Signaling events in T cell activation were also blocked by reduced interaction of sema6D with plexin-A1. These findings suggest that interaction of plexin-A1 and sema6D mediates activation signals for both DCs and T cells.

(This work is supported by National Institutes of Health Grant AI29564).
Role of human skin resident T cells in wound healing (B116)
Authors: toulon antoine , Bhavsar Dhaval , Tenenhaus Mayer , Pineau Nathalie , Lionel Breton , Jameson Julie , Havran Wendy Lynn
Published: January 2023

Cutaneous wound healing is a complex process requiring constant communication between cells in the form of cytokine release, cell-to-cell contacts and cell-to matrix interactions. Skin gammadelta T cells have been shown to play unique roles in tissue homeostasis and during tissue repair through local secretion of distinct growth factors in mice. However, because the distribution of resident T cells (gamma delta vs alpha beta) is different in mice and humans we aimed to characterize the role of epithelial resident T cells in wound repair in humans. In the present study we show that the freshly isolated Vdelta1 gamma delta and alpha beta resident T cells secrete IGF-1 after stimulation by PMA and ionomycin. Interestingly both cell types are activated in acute wounds and secrete IGF-1 to promote wound healing. The characterization of the functional activity of resident T lymphocytes in acute wounds should allow the development of new therapeutic strategies against chronic wounds.

Supported by grants from L’Oreal, NIH and Fondation pour la Recherche Médicale.
Nature of Bacterial Provocation Determines Susceptibility To Colitis (B117)
Authors: Henderson Abigail Lynn , Jergens Albert , Liu Zhiping , Hostetter Jesse , Dorn Andrea , Wannemuehler Michael
Published: January 2023

Development of inflammatory bowel disease (IBD) involves predisposing and triggering events including aberrant immune responses against microbial antigens. Colonization of defined flora (DF) mice with Helicobacter bilis has been shown to induce immune responses to the resident flora.

Aim: To determine the mechanisms underpinning susceptibility to colitis after colonizing DF mice with new bacterial species.

Methods: Following colonization of DF mice with H. bilis or Escherichia coli, mice were treated with a low dose (1.5 %) of dextran sulfate sodium (DSS) (i.e., inflammatory trigger). Following euthanization, severity of colonic lesions were assessed as well as flora-specific antibody and T cell responses.

Results: Colonization of DF mice with H. bilis, unlike E. coli, significantly (p<0.05) increased the severity of DSS-induced colitis. T cells from mice colonized with H. bilis and E. coli displayed in vitro ASF-specific recall responses but only the responses from the H. bilis-infected mice correlated with severity of lesions. T cell responses from E. coli-colonized mice produced more ASF-specific IL-10 than those from H. bilis colonized mice.

Implications: These studies indicate that bacterial colonization can perturb mucosal homeostasis by impairing mucosal regulatory function(s) and exacerbate host susceptibility to subsequent colitic insults resulting in IBD.

Supported by F31DK070563-01A2.
Development of Potent Anti-HIV Chimeric Immune Receptors (B120)
Authors: Yang Wen , Lu Li , Junghans Richard P
Published: January 2023

T cells that are genetically modified to express anti-HIV chimeric immune receptors (CIR) and have the potential to eradicate the reservoirs of viral infections, may be useful to suppress or cure HIV/AIDS. CD4zeta (CD4z) can bind HIV-1gp120 antigen in an MHC nonrestricted fashion through its CD4 extracellular domain (Ec) fused to the cytoplasmic domain (Cy) of z. However, the adoptive transfer of CD4z-expressing T cells to HIV/AIDS patients failed to control the viral infection in clinical trials, with the mechanisms of failure being unknown. To date, all anti-HIV CIRs developed have been TCR Signal 1-only (e.g., via Cy of z). CIRs that also provide a costimulatory signal (Signal 2; e.g., CD28) significantly enhanced their potency in inducing T cell activation. In order to develop more potent anti-HIV CD4-based CIRs, we have constructed retroviral vectors encoding CD4-based CIR with Signal1+2 (CD4mCD28z) by fusing the Ec of CD4 to Tm and Cy of CD28 with Cy of z. This is compared with a Signal 1-CIR, CD4mz, as a control representing the former Signal 1-CIR designs. Our preliminary results showed that the two CIRs expressed equally well on Phoenix cells transfected with CIR-encoding plasmid and human Jurkat T cells retrovirally transduced individually with the two CIRs. Western blot analysis confirmed that the two CIRs were cellularly expressed intact. Human CD8 T cells will be transduced individually with the two CIRs, followed by evaluating their potencies in inducing T cell activation via the CIR as well as in inhibiting anergy of the CIR-T cells. This will demonstrate the benefits of the inclusion of Signal 2 in in vitro systems as a potential predictor of a better 2nd generation agent for subsequent human in vivo application.
Specific T lymphocyte citotoxicity induced by Cu/Zn superoxide dismutase from Brucella abortus RB51 (B124)
Authors: Moreno-Lafont Martha Cecilia , Flores-Mejia Raul , Rodriguez-Cortes Octavio , Chacon-Salinas Rommel , Cancino-Diaz Mario
Published: January 2023

Brucellosis is an infectious disease affecting animals and humans, caused mainly by B. abortus. Although several vaccines have been developed, B. abortus strain RB51 is the most used for cattle, but is not useful for humans since it causes severe granulomas. To develop a vaccine for humans, Cu-Zn superoxide dismutase (SOD Cu/Zn) has been identified as an antigen which induces T-cell mediated immunity in mice vaccinated with live Escherichia coli expressing B. abortus SOD Cu/Zn or a plasmid DNA carrying the gene sodC. The aim of this work was to demonstrate the induction by SOD Cu/Zn from B. abortus RB51 of specific cytotoxic T lymphocytes (CTL) recognizing in vitro macrophages infected with B. abortus strain RB51 or 2308. The protein was extracted from recombinant E. coli-pBAD-SOD and purified in a Ni-agarose column, and then by FPLC in a molecular weight exclusion column. Fractions corresponding to 20–22 KDa were collected and mice were immunized intramuscularly with three doses of 25 μg of SOD Cu/Zn each. T lymphocytes purified from spleen with an anti CD90 coupled to magnetic beads, were tested in an in vitro cytotoxicity assay, using J774A.1 cell line infected with B. abortus strain RB51 or 2308 as target. CTL activation was evident in both cases, since macrophages infected with any bacterial strain were killed as demonstrated by neutral red dye uptake or lactate dehydrogenase release assays. These results demonstrated that the protein by itself is able to prime pCTL to kill macrophages infected with B. abortus, no matter its virulence.

Grants from CONACyT 44837-Q and SIP 20060688 and 20070239.
T cell response induced by recombinant Cu/Zn superoxide dismutase from Brucella abortus RB51, in patients with brucellosis (B125)
Authors: Lopez-Santiago Ruben , Paredes-Cervantes Vladimir , Gonzalez-Gonzalez Edith , Goznalez-Peña Araceli , Cancino-Diaz Mario
Published: January 2023

Cu/Zn superoxide dismutase (SOD), located in the periplasmic space of the cell wall of Brucella abortus, induces protective cell mediated immunity in mice challenged with virulent Brucella strains. In vitro studies have shown that the SOD induces proliferation of splenocytes, antibody production and T cell cytotoxicity. In humans, the recombinant SOD (rSOD) is recognized by serum of patients with chronic brucellosis. In order to prove the rSOD immunogenicity in humans with brucellosis, we cloned the coding sequence of the gene sodC from B. abortus RB51 into an expression system that allowed us to obtain large quantities of recombinant protein to determine the immune response in patients suffering of brucellosis. The sodC gene was cloned from B. abortus genome by PCR. A 525 bp fragment was obtained and subcloned into the pBAD-TOPO expression vector (AmpR), used to transform competent E. coli TOP-10. Ampicillin resistant clones were selected and verified for the correct 525 bp size insert. Antigenicity of rSOD purified by a Ni-column was confirmed by Western blot with a rabbit anti B. abortus RB51 serum. Patients with acute or chronic brucellosis showed a specific T cell proliferation induced by the rSOD, as assessed by BrdU incorporation assay. Furthermore, mRNA synthesis for IL-2, IL-10 and IFNγ induced by rSOD was determined by RT-PCR. It was found mRNA for IL-2 and IFNγ in acute patients, while chronically infected patients showed a tendency to express IL-10 mRNA, confirming a type 1 cytokine pattern in acute patients, whereas a type 2 in chronic patients.

Grants by CONACyT 44837-Q and SIP 20060693
IL-4 induction in RSV-infected macrophages: examining the role of NFAT family members (B128)
Authors: Levy Nyssa A , Fach Sasha J , Sacco Randy E
Published: January 2023

Respiratory Syncytial Virus (RSV) is the leading cause of respiratory illness in infants, causing severe bronchiolitis and pneumonia. In the lung, alveolar macrophages (AMΦ) are important antigen presenting cells responsible for pathogen clearance, antigen presentation and cytokine production. The effect of RSV infection on AMΦs is not currently well understood. The objective of our studies was to characterize the AMΦ response following bovine RSV (bRSV) infection. Previously, we have shown that bRSV infects AMΦs as detected by immunofluorescence. Active bRSV replication within AMΦs was confirmed by real time PCR analysis of viral NS2 mRNA. Interestingly, we have also found by real time PCR that bRSV infection induces AMΦs to upregulate the mRNA transcription of genes encoding immunomodulatory mediators, IL-4 and IL-10. Presently, we are examining the role of NFAT family members in the induction of IL-4 transcription in RSV-infected macrophages. Production of IL-4 by AMΦs is of particular significance because of the association of RSV and Th2-associated chronic lung disease.

Supported by NIH R01 AI062787
Changes in Cellular Composition and Induction of Cell Death in the Spleen Are Correlated to the Disease Symptoms in Response to Influenza A Virus (B130)
Authors: Sato Kayoko , Fujikura Daisuke , Takada Ayato , Kida Hiroshi , Miyazaki Tadaaki
Published: January 2023

The pathogenicity of influenza A viruses varies depending on the species of the host animals and the virus strains. A/PR/8 (H1N1) strain of influenza A virus causes a more severe disease than A/Aichi/2/68 (H3N2) strain in mice. However, the plausible explanation for their difference of pathogenicity is still unknown. To identify the critical determinants for the pathogenicity of these viruses, the immunological responses to these virus infection were analyzed. Mice were infected with these viruses by inhalation and the lung, spleen, and sera from the infected mice were obtained on 3, 5, and 7 days after the infection. It is noted that the weight of the spleen and the number of splenocytes in mice infected with A/PR/8 virus was decreased, whereas those in mice with A/Aichi/2/68 was increased. Furthermore, we found that A/PR/8 virus infection strongly induced cell death in the spleen and the changes of cellular profile such as the population of macrophages or T cells. Taken together, cell death induction in the spleen and the change of the composition of splenocytes could be critical characteristics for the severe pathogenicity determined by the infection with A/PR/8, compared with A/Aichi/2/68.
Cytotoxic T lymphocytes express intracellular stores of FasL via mechanisms distinct from cytolytic granule exocytosis (B16)
Authors: He Jin-Shu , Ostergaard Hanne L.
Published: January 2023

Cytotoxic T lymphocytes (CTL) lyse target cells through the release of cytolytic granules and cell surface expression of Fas ligand (FasL). We found that there is prestored FasL pool in CTL, which underwent rapid TCR-regulated cell surface expression upon target cell engagement. Current models suggest that FasL is stored in cytolytic granules and that FasL cell surface expression would be subject to the same control as degranulation. The aim of the current study is to investigate if degranulation of cytolytic granules and FasL expression pathways of CTL are controlled by differential mechanisms. FasL cell surface expression and degranulation, as measured by CD107a cell surface expression in CTLs were compared by flow cytometry under different conditions. FasL underwent an immediate and transient cell surface delivery following either TCR stimulation by anti-CD3 antibody crosslinking, or phorbol ester treatment, conditions that do not permit degranulation. Furthermore, treatment with pharmacological reagents indicated that the preformed FasL cell surface transport is PI3-kinase (PI3K)-, calcineurin- and microtubule-independent, which are all indispensable for degranulation. Finally, stored FasL resides in vesicles distinct from cytolytic granules as assessed by confocal microscopy and subcellular fractionation. Our data clearly show that CTL degranulation and FasL lytic mechanisms are fully independent with respect to regulation and localization.

This work was funded by the Canadian Institutes of Health Research (CIHR).
Structural and functional features of cells from the sole (Solea senegalensis Kaup 1858) immune system. (B169)
Authors: Salinas Irene , Anchelin Monica , Esteban Maria Angeles , Meseguer Jose
Published: January 2023

During last years, the severe decrease of fishing resources has impulsed the growing of aquaculture with the necessary diversification of production species. The sole, Solea senegalensis Kaup 1858, is a high-value flatfish reared in intensive aquaculture production in Southern Europe, but no studies on the sole immune system cells have been conducted. The knowledge on healthy immune cells will be useful for diagnosis purposes in pathologies associated to changes in these cells. This study reports a morphological study (light and electron microscopy) of the sole mature immune cells from blood, head-kidney and spleen, the characterization of the different haemopoietic stages, and two innate immune responses of these mature cells were. Phagocytic capacity was studied by transmission electron microscopy (TEM) and the respiratory burst activity was evaluated by a chemiluminescence method. Sole blood, head-kidney and spleen leukocytes were isolated and analysed by flow cytometry showing the existence of three main cell subpopulations. Heterophilic and acidophilic granulocytes, macrophages and thrombocytes from the different studied tissues showed phagocytic capacity engulfing up to 4 yeast cells (Saccharomyces cerevisae). The head-kidney leukocytes respiratory burst response showed a slope of 4836 ± 414.
Increased T-regulatory cell number and expression of CXCR4 and mu-opioid receptors on CD4+ T-cells by methadone is associated with enhancement of HIV-1 infectivity (B17)
Authors: Rodríguez José W. , Hunter Robert , Nair Madhavan , Rios-Olivares Eddy
Published: January 2023

Previous in vitro, animal-model, and epidemiological studies have indicated that opiates have conditionally variable effects on HIV-1 susceptibility and disease progression. Immunovirological and pharmacological variability are two major factors that seem to dictate whether HIV-1 infection will progress more rapidly or more slowly in HIV-1 infected opiate-exposed subjects. According to previous studies, the immunovirological effect of the opioid drug methadone has not been elucidated yet. In the present study, we analyzed various immunovirological determinants to evaluate the effects of methadone. Human lymphocytes from healthy individuals were isolated and acutely infected with HIV-1 and then assayed to determine how in vitro exposure of methadone affects HIV-1 infectivity and alters the expression of HIV-1 co-receptor (CXCR4) and mu-opioid receptor (MOR) on HIV-infected CD4+ T-cells. Our results showed that in vitro exposure of methadone increases HIV-1 replication by 4-folds. This was also correlated with a significant increase in the expression of HIV-1 co-receptor CXCR4 and mu-opioid receptor on HIV-infected CD4+ T-cells. Interestingly, we also observed a significant increase of CD4+ T-regulatory cells following methadone in vitro exposure. These results provide a new insight on the immunovirological status of methadone-treated HIV-infected patients.

Supported by NIH/RCMI Grant # G12-RR03035.