bluuebunny/crossref_metadata_2025 huggingface dataset:
Forced expression of suppressor of cytokine signaling 3 in T cells protects mice from concanavalin A-induced hepatitis (90.16)
Authors: Fushimi Soichiro , Takahata Tomohiro , Wakabayashi Hiroshi , Ogino Tetsuya , Kubo Masato , Matsukawa Akihiro
Published: January 2023

The regulatory mechanism behind T cell-mediated liver injury is not fully elucidated. Here, we demonstrate that forced expression of SOCS3 in T cells protects mice from a T cell dependent concanavalin A (ConA)-induced hepatitis. Mice with a T cell specific overexpression of SOCS3 (SOCS3-cTg) were resistant to ConA-hepatitis, as evidenced by decreased serum ALT level, reduced hepatic damage and improved mice survival relative to the control. Hepatic expression of T-bet, a Th1-specific transcription factor, was decreased in SOCS3-cTg liver relative to the control with the reduced expressions of Th1 associated cytokines (TNFa, IL-12, IFNg) and chemokines (CXCL9/10). Flow cytometric analysis of isolated hepatic lymphocytes demonstrated that activated CD4+T cells, cytotoxic T (Tc) cells and natural killer T (NKT) cells were significantly decreased in SOCS3-cTg liver. Expression of perforin and granzyme B, injurious molecules produced from Tc and NKT cells, was reduced in SOCS3-cTg liver. These results indicate that forced expression of SOCS3 in T cells prevents ConA-induced liver injury by inhibiting Th1 responses. SOCS3 delivery in T cells may be therapeutic strategy in T cell-mediated liver injury. Financial support: Grants-in-Aid from The Ministry of Education, Science, Sports and Culture, and The Ministry of Health and Welfare, Japan,
A critical role for microRNA miR-155 in CD8+ T cell responses (90.23)
Authors: STELEKATI ERIETTA , BOESTEANU ALINA C. , GRACIAS DONALD T. , MUELLER YVONNE M. , VIGORITO ELENA , NORTON JILLIAN A. , TURNER MARTIN , KATSIKIS PETER D.
Published: January 2023

The role of microRNAs in CD8+ T cells responses is largely unexplored. We have found that miR-155 is upregulated by 100-fold in activated CD8+ T cells. To test the in vivo role of miR-155 in CD8+ T cell responses, we examined bic/miR-155 deficient (bic-/-) CD8+ T cells in Influenza type A virus or Listeria monocytogenes (L.m.) infected mice. To demonstrate that miR-155 deficiency induces an intrinsic defect in CD8+ T cells, bic-/-OT-1+ and OT-1+ transgenic CD8+ T cells were adoptively transferred into C57BL/6 hosts, and then either infected with SIINFEKL peptide expressing influenza virus strain WSN-OVA or recombinant L.m. strain expressing OVA (L.m.-OVA). At day 10 post-infection the number of pulmonary bic-/-OT-1+ CD8+ T cells was reduced 3-fold compared to OT-1+ CD8+ T cells (p=0.01). With L.m.-OVA infection, day 7 post-infection numbers of splenic bic-/-OT-1+ CD8+ T cells were 10-fold lower than OT-1+ CD8+ T cells (p=0.0005). On day 3 post-infection however, no differences were found between OT-1+ or bic-/-OT-1+ CD8+ T cells transferred animals and both groups equally controlled the bacterial burden in the spleen compared to non-transferred control animals suggesting that bic-/-OT-1+ CD8+ T cells transfer normally and that miR-155 regulates CD8+ T cell responses at the later stages of infection. These findings demonstrate that miR-155 plays a critical role in CD8+ T cell responses against pathogens.
Nrf2 activation suppresses production of T cell cytokines and IgM in C57BL/6 mice (90.26)
Authors: Rockwell Cheryl E , Fields Patrick E , Crawford Robert B , Kaminski Norbert E , Klaassen Curtis D
Published: January 2023

Nrf2 is a transcription factor that upregulates cytoprotective genes in response to cellular stress. Nrf2 gene expression is induced in CD3+ cells by tert-butylhydroquinone (tBHQ), a known Nrf2 activator. In addition, tBHQ upregulates the Nrf2 target genes, Ho-1, Nqo1, and Gclc in wild-type, but not Nrf2-null CD3+ T cells. Furthermore, tBHQ and butylated hydroxyanisole (BHA), another Nrf2 activator, markedly inhibit IFNγ transcription in wild-type activated T cells, but have little effect on IFNγ transcription in Nrf2-null T cells. Likewise, tBHQ inhibits binding to both the AP-1 and NFκB response elements in wild-type CD3+ cells, but only modestly inhibits AP-1/NFκB binding in Nrf2-null CD3+ cells. Production of TNFα and IL-2 by activated T cells is also suppressed by tBHQ. In accordance with the aforementioned findings, tBHQ-treated mice exhibited diminished antibody production to sheep RBC, a T cell-dependent immune response. Collectively, the current studies suggest Nrf2 activation in CD3+ cells markedly inhibits T cell cytokine production and consequently, T cell-dependent responses, in vivo. The present studies suggest Nrf2 may represent a novel regulatory mechanism in mature murine T cells and thus may be a useful target for the development of new anti-inflammatory therapeutics. (Supported by NIH grants ES09716, ES07079, ES013714, ES09649, and RR021940.)
MHC haplotypes modulate strength of natural killer (NK) cell licensing (91.10)
Authors: Jonsson A. Helena , Yokoyama Wayne M.
Published: January 2023

NK cells react to cells that lack self-MHC class I. Yet, since many NK cells cannot recognize self-MHC, mechanisms such as NK cell licensing protect against autoreactivity. To become licensed, i.e. functionally competent to be triggered through its activation receptors, an NK cell must engage host MHC class I via at least one of its MHC class I-specific inhibitory receptors, such as the Ly49 family of receptors in the mouse. However, the general determinants of this process remain largely unknown. Herein, we investigated the licensing impact of the b, d, f, k, q, r, and s H2 haplotypes on Ly49A+ NK cells in MHC-congenic mice. Ex vivo PK136 (anti-NK1.1) stimulation assays indicated that licensing may not be a binary phenomenon as some Ly49A-MHC class I haplotype combinations produced an intermediate licensing phenotype. Ly49A surface accessibility, a measure of cis binding with MHC class I, and Ly49A tetramer binding displayed a similar variability among the MHC haplotypes. Taken together, the data indicate a correlation between strength of licensing and strength of binding of Ly49A receptors with MHC class I, both of which are inversely correlated with surface accessibility of Ly49A receptors. These findings suggest that NK cell licensing is modulated by the strength of the interaction of inhibitory receptors with host MHC class I.

Supported by HHMI and grants from the NIH.
Human lymphoid tissue inducer (LTi) cells are CD127+RORc+ and can be expanded ex vivo (91.11)
Authors: Crellin Natasha  K , Cupedo Tom , Trifari Sara , Kaplan Charles , Spits Hergen
Published: January 2023

Long recognized as essential for proper lymph node organogenesis during fetal development, recent evidence has suggested that LTi cells also play an important role in the adult. Recent work from our laboratory has identified the previously unknown human LTi as lineagenegCD127+RORc+ cells. In vitro studies suggest a direct relationship of LTi cells with CD127+RORc+ NK cells. Both LTi and CD127+ NK cells isolated from human tonsil produce IL-22, IL-17, TNFalpha, and lymphotoxin, but not IFNgamma. LTi cells are present in a low frequency in human tonsils, thus making detailed analysis challenging. Using a rigorous selection strategy, we have successfully purified LTi and CD127+ NK cells and expanded these cells 30-100 fold using a feeder cell mixture and IL-2. These expanded LTi and CD127+ NK cell lines maintain RORc expression and production of IL-22 and IL-17. Interestingly, following ex vivo expansion both LTi and CD127+ NK cells produce IFNgamma upon stimulation. An outstanding question remains the cytotoxic potential of LTi and CD127+ NK cells, which we can now investigate. Further, we are investigating the role of stimulatory cytokines in regulating IL-22 production from LTi and CD127+ NK cells. As aberrant regulation of IL-22 can contribute to inflammatory diseases, further understanding of this novel population of IL-22 producing cells is of great relevance.
Retargeting IgM Antibodies with Different Specificities to a Common Cell Type Reveals Shared Mechanisms Regulating Cellular Activation (91.12)
Authors: Keenan Thomas D , Radhakrishnan Suresh , Bell Michael , Barry Michael , Pease Larry R
Published: January 2023

IgM antibodies, as multivalent ligands, have the capacity to activate cells differentially by cross-linking targeted cell surface signaling molecules. For example, hIgM12 (B7DC Xab) activates dendritic cells (DCs), leading to potent immunomodulation, while hIgM22 binds glial cells and causes remyelination in mouse models of multiple sclerosis. Both antibodies activate common signaling intermediates in their respective cellular targets suggesting a common mechanism of action. Elucidation of these shared mechanisms became possible by targeting both antibodies to a common cell type. Our previous studies demonstrate that B7-DC expression is required to activate downstream signaling in DCs by hIgM12. We utilized a phage display system to identify 7mer peptides bound by each IgM and expressed each peptide attached to a surrogate cell surface carrier on retrovirally tranduced bone marrow-derived DCs from B7-DC -/- mice. Transduced cells were treated with hIgM12, hIgM22 or anti-carrier IgM, and subsequent activity was compared against B7-DC +/+ DCs or B7-DC-reconstituted B7-DC -/- DCs. DC activation by each IgM was specific to expression of their conjugate peptide construct. Activation by hIgM22 was indistinguishable from activation by hIgM12, while anti-carrier IgM caused no activation. This indicates that while their target specificity differs, once bound these IgMs activate cells via common mechanisms.
Modification of DC function via blockade of CD40:TRAF interactions (91.13)
Authors: Qu Xinyan , Stout Robert D , Suttles Jill
Published: January 2023

Ligation of CD40 on dendritic cells (DC) results in rapid protein kinase and transcription factor activation leading to enhanced expression of genes encoding a variety of cytokines and costimulatory molecules. CD40 does not contain cytoplasmic sequences with catalytic activity and transmits signals relying on the use of TNFR-associated factors (TRAFs), a family of adapter proteins that are involved in signaling by the TNF receptor family and the toll/interleukin-1 receptor (TIR) family members. Cell-permeable TRAF6 and TRAF2/3/5 blocking peptides (TRAFBPs) corresponding to the TRAF6 or TRAF2/3/5 binding region of CD40 were employed to better define the roles of TRAF2 and TRAF6 in CD40 signaling in DC. Both TRAF6BP and TRAF2BP inhibited CD40-stimulated DC production of TNF and IL-6, but did not impact CCL2 and CXCL8 production. ERK1/2 phosphorylation was inhibited by both TRAF6BP and TRAF2BPs, whereas JNK, p65 and AKT phosphorylation were abrogated by TRAF6, but not by TRAF2BP. Both TRAF6BP and TRAF2BP have no effect on p38 MAP kinase activation. During antigen presentation to T cells, pre-incubation of DC with TRAF6BP and TRAF2BP effectively inhibited Th1 and Th17 differentiation, but did not impact activation of IL-10-producing T cells. Thus, targeting TRAF:CD40 interactions in DC may provide a means to selectively modulate DC function.
Expression of a2V-ATPase following addition of ATP leads to cell death (91.18)
Authors: Levine Rita , Kwong Christina , Beaman Kenneth
Published: January 2023

Surface expression of a2V-ATPase (a2V) is congruent with cell destruction and regulates the immune response. Leukocytes isolated from whole blood display a2V on their surface and are highly sensitive to cell destruction following addition of ATP. Exogenous ATP enhances and alters surface expression of a2V. This augments the cell destruction of certain white cell populations.

Like the monocytic cell line studied previously, the epithelial cell lines, TOV112D and JEG3, treated with ATP significantly increased surface expression of a2V. Leukocyte populations incubated with ATP altered surface expression of a2V. Monocytes were strongly a2V positive and sensitive to ATP. The addition of ATP for 1hr resulted in total destruction of the monocyte population. Lymphocytes treated with ATP for 2hrs increased surface expression of a2V dramatically. In vitro addition of ATP led to an increase in granulocytic cell death. These data demonstrate that a2V is an important marker of cell destruction in vivo and in vitro.
Estrogen promotes pro-inflammatory response by regulating multiple transcription factors (91.7)
Authors: Dai Rujuan , Phillips Rebecca A , Karpuzoglu Ebru , Khan Deena , Ahmed S. Ansar
Published: January 2023

In vivo 17-β estradiol (estrogen) treatment has been shown to promote inflammatory responses in different types of immune cells including splenocytes. However, the molecular mechanism of estrogen mediated pro-inflammatory responses is still not clear, which impede the therapeutic intervention of estrogen related immune disorders. In the present study, we investigated the effects of estrogen on the signaling pathways, which involved in the regulation of inflammatory molecules, especially inducible nitric oxide synthase (iNOS). We found that although activation of IFNγ-STAT-1 signaling is essential for Con-A stimulated iNOS expression in mouse splenocytes, it is not sufficient. The induction of iNOS in splenocytes required synergistic activation of STAT-1 and NF-κB. Nevertheless, the induction of Monocyte chemotactic protein-1 (MCP-1) in splenocytes from estrogen-treated mice is not dependent on STAT-1 and NF-κB. We then demonstrated that estrogen increases the expression and DNA binding activity of c/EBPβ, which is also important for inflammatory regulation. Further, our data revealed that serine protease play important role in the regulation of IFNγ-STAT-1 and NF-κB signaling in splenocytes from in vivo estrogen treated mice. Together, our data indicate that estrogen promotes pro-inflammatory responses by modulating the activity of multiple transcription factors including STAT-1, NF-κB, and c/EBPβ.
Induction of IL-6 in mixed glial cultures – potential role of CGRP in CD40-dependent neuropathic pain (91.8)
Authors: Malon Jennifer , Cao Ling
Published: January 2023

We have previously demonstrated a critical role of spinal cord microglial CD40 in the development of spinal nerve L5 transection (L5Tx)-induced neuropathic pain. Cross-talk between pro-inflammatory mediators and pro-nociceptive factors is believed to play an essential role in the development of neuropathic pain. Here, we investigated the effects of the pro-nociceptive factors calcitonin gene-related peptide (CGRP) and CX3CL1 with/without CD40 on the production of several pro-inflammatory mediators by glial cells in vitro. Anti-CD40 induced low levels of nitric oxide, a known pro-nociceptive factor, while CX3CL1 enhanced CXCL10 production by mixed glia. Interestingly, while anti-CD40 and CX3CL1 had little effect on IL-6 production, CGRP induced a significant secretion of IL-6 by glia that is unaffected by CD40 co-stimulation. In vivo data indicated that spinal cord IL-6 was increased by day 1 and spinal cord microglial CD40 expression was increased in an IL-6 dependent fashion by day 3 post-L5Tx. Together, these data lead us to hypothesize that CGRP released from primary sensory nerve terminals following L5Tx induces spinal cord glial IL-6 production, which in turn enhances microglial CD40 expression, thus facilitating the development of neuropathic pain. Future studies with microglial-enriched and depleted populations will be used to further delineate this pathway.

Support of this study: 1K01DA023503-01A1 (Cao), NIDA.
CD34 mediates dendritic cell trafficking in hypersensitivity pneumonitis (92.1)
Authors: Bennett Jami L , Blanchet Marie Renee , Girard Melissa , Cormier Yvon , McNagny Kelly M
Published: January 2023

Hypersensitivity pneumonitis (HP) is an inflammatory lung disease characterized by a chronic T cell airway infiltration. Dendritic cells (DC) have been reported to play a crucial role in trafficking of antigen to the lymph nodes and antigen presentation in this disease. However, little is know about what triggers and mediates DC trafficking in HP. CD34 is a sialomucin best known for it's expression on hematopoietic progenitors. This protein, which was recently reported as a facilitator of cell trafficking, is also expressed on a few subsets of mature cells such as DCs. We therefore explored whether CD34 expression in DCs was involved in DC trafficking to and from the lung in a mouse model of HP, using wild type and Cd34-/- mice. Lung inflammation and DC recruitment was evaluated in both strains. Results show that lack of CD34 expression leads to a lower lung inflammation (as characterized by lower total cell counts in the broncho-alveolar lavage). DC recruitment was reduced in the alveoli of Cd34-/- mice, but was increased in the lung tissue. Using bone marrow chimeric mice, this effect was attributed to the Cd34-/- environment (including potentially radio-resistant DCs). Lastly, intraveinous injection of wild type DCs reconstituted disease in Cd34-/- mice. We conclude that CD34 could mediate DC trafficking in HP and possibly other inflammatory lung diseases.

Supported by the Canadian Institutes of Health Research (CIHR), AllerGen Network Centre of Excellence (KMM), Multiple Sclerosis Society of Canada (JLB), Michael Smith Foundation for Health Research (KMM, MRB), and FRSQ (MRB)
Curcumin stimulates cyclooxygenase-2 (COX-2), prostaglandin I2 synthase (PGI2S) and PGE2 synthase (PGE2S) gene expression in human coronary artery endothelial cells (93.19)
Authors: Tan Xiaoyu , Poulose Ellen M. , Stechschulte Daniel J. , Dileepan Kottarappat N.
Published: January 2023

Curcumin is reported to decrease atherosclerosis progression in Apo-E/LDL receptor knockout mice. PGI2 and PGE2 are generated from arachidonic acid (AA) by the COX-PGI2S/PGE2S pathway and are known to have important roles in cardiovascular function. In this study, we evaluated the in vitro effect of curcumin on the expression and function of COX-2, PGI2S and PGE2S in human coronary artery endothelial cells (HCAEC). HCAEC monolayers were incubated with curcumin (1.25-10 μM), and the mRNA and protein expressions were monitored by qRT-PCR and Western blots, respectively. PGI2 and PGE2 levels in the culture supernatants were quantified by EIA. Incubation of HCAEC with curcumin led to concentration-dependent increases in COX-2 mRNA and protein expression without change in COX-1 expression. Curcumin also induced 2-fold increases in PGI2S and PGE2S gene expression. Despite the increase in COX-2, PGI2S and PGE2S gene expression, curcumin failed to increase PGI2 or PGE2 production. Furthermore, curcumin inhibited LPS-induced PGE2 and PGI2 production by HCAEC and exogenous AA could not reverse the inhibitory effect. These results show that curcumin acts both as an inducer of COX-2, PGI2S and PGE2S gene expression and as an inhibitor of stimulated PGE2 and PGI2 synthesis. The ability of curcumin to regulate prostanoid biosynthesis in HCAEC indicates its therapeutic potential for cardiovascular disease. (Supported by NIH R01-HL070101 and the Carey Arthritis Fund).
IL17 stimulates granulopoiesis via IL6 induction (93.5)
Authors: Sun Honghong , Worthen Scott
Published: January 2023

IL-6 is an important inflammatory cytokine required for Th17 cell differentiation. IL-17, a proinflammatory cytokine involved in many inflammatory diseases, is produced by Th17 cells, under the control of transforming growth factor-b and IL-6. IL17 may mediate neutrophil recruitment and granulopoiesis through G-CSF-dependent and -independent mechanisms. Using a novel in vitro culture and differentiation system and an in vivo lung inflammation/injury model, we demonstrate that addition of G-CSF and IL17 promotes functional neutrophil generation. The numbers of neutrophils released upon IL-17 stimulation is comparable to or more than that of G-CSF stimulation. In confirmation of the result published by Kolls et al, IL17 induces modest G-CSF production. However, IL-17 also induces a dramatic increase in IL6 expression that is not observed in G-CSF-treated cultures. Furthermore, both G-CSF and IL17-induced granulopoiesis is attenuated in IL6-deficient mice. Our data also show an increased population of lin-/c-kit+/Sca1- cells in IL17 and IL6-treated cultures. The Stat3 and pAKT are activated upon IL17 stimulation and contribute to activation of IL6 transcription. Taken together, IL-17 produced by T cells stimulates granulopoiesis through the release IL-6 and G-CSF and is critical for the initiation of innate immunity.
Delineation of hypoxia versus inflammatory contributions to tissue injury by attenuation of G-alpha-i signaling (93.7)
Authors: Moratz Chantal  M , Egan R , Zacharia A , Simovic M , Gist I , Dalle Lucca J , Tsokos G C
Published: January 2023

The complement dependent pathology resulting from mesenteric ischemia/reperfusion insult instigates tissue injury in mesenteric and systemic organs. In this study the contribution of G-protein receptor signaling through G[alpha]i to stages of tissue injury is delineated. Global G[alpha]i inhibition, by PTX, attenuated mesenteric injury and production of lipid modulators such as leukotriene B4. Systemic inflammation was inhibited as assessed by lung tissue injury scores and monitoring cellular infiltrates. To delineate the role of G[alpha]i signaling in mesenteric versus systemic inflammatory responses, an inhibitor of pulmonary inflammatory chemotactic G[alpha]i signaling, anti-CXCR2 was used. As expected PTX and anti-CXCR2, abrogated pulmonary inflammation. In contrast, anti-CXCR2 was not effective in mitigating mesenteric injury. The results from global versus specific G[alpha]i inhibition in local and systemic injury indicate multiple mechanisms by which G[alpha]i signaling facilitate tissue injury. The G[alpha]i linked signaling effects of complement activation and chemokine induced migratory control are distinct and can be mitigated independently. Delineating which signals govern particular components of tissue injury will be useful in developing therapeutics which can control specific deleterious aspects of systemic inflammation after trauma while maintaining the protective aspects of complement activation.
A novel analysis of single particle trajectories quantifies the dynamic interactions between LFA-1 and the actin cytoskeleton (94.13)
Authors: Das Raibatak , Cairo Christopher W , Coombs Daniel
Published: January 2023

Dynamics of cell-surface proteins are strongly influenced by their localized interactions with cytoskeletal attachments or intracellular signaling molecules. We developed a technique to analyze single particle trajectories to quantify these interactions. We modeled trajectories of a cell membrane-associated protein interacting with a homogeneously distributed binding partner, and applied this model to analyze the motion of the integrin receptor LFA-1 on the surface of T cells. Our analysis reveals that the diffusion of LFA-1 is two-state, with distinct diffusion coefficients in its free and cytoskeleton-bound forms. We also quantify the rates of transition between the two states, and we show that cellular activation leads to large changes in the LFA-1-cytoskeleton interactions.
Macrophage migration inhibitory factor and the regulation of adhesion molecule expression on the blood brain barrier during experimental autoimmune encephalomyelitis (94.15)
Authors: Cox Gina Mavrikis , Kithcart Aaron P , Williams Jessica , Smith Kristen , Shawler Todd , Whitacre Caroline C
Published: January 2023

Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system (CNS). Cerebrospinal fluid (CSF) isolated from patients during relapse was found to contain greater levels of macrophage migration inhibitory factor (MIF) relative to samples collected during remission, thus suggesting a role for MIF in the pathogenesis of MS. We have demonstrated that upon induction of experimental autoimmune encephalomyelitis (EAE), mice deficient in MIF exhibit reduced clinical signs as well as fewer inflammatory infiltrates in the brain and spinal cord. MIF deficient mice, however, do not differ from wild type controls in the function of autoreactive lymphocytes in the periphery. These findings suggest that MIF is necessary for progression of EAE in the CNS, particularly at the level of the blood brain barrier (BBB). Mice treated with anti-MIF antibody show impaired migration of lymphocytes into the CNS and down-regulation of adhesion molecules on the BBB. Thus, we hypothesize that MIF is acting at the level of the BBB, regulating adhesion molecule expression and therefore access to the CNS. We are analyzing the expression of adhesion molecules, such as VCAM-1 and ICAM-1 in the brain and spinal cord of MIF deficient and small molecule inhibitor-treated mice. (Supported by NIH grant AI 064320 and National MS Society grant RG3272)
Effect of the sphingolipid synthesis inhibitor, myriocin, in a murine pulmonary inflammation model (94.19)
Authors: Edling Andrea E , Mahan Amy , Leonard John , Williams John , Kaplan Johanne , Perricone Michael
Published: January 2023

Effect of the sphingolipid synthesis inhibitor, myriocin, in a murine pulmonary inflammation model Sphingolipids are signaling molecules involved in many aspects of cell function. Therapeutic modulation of the sphingolipid pathway is a potential approach to manage inflammation. Myriocin is a naturally occurring fungal metabolite that inhibits serine palmitoyltransferase (SPT), a vital enzyme in sphingolipid biosynthesis, and has been shown to have immunosuppressive properties. Using a murine acute pulmonary inflammation model as a surrogate inflammatory disease model, we evaluated the immune-modulatory effects of myriocin. Myriocin was administered at 0.3mg/kg, i.p., shortly after LPS challenge. Cellular infiltration into the lungs 24hrs and 48hrs after LPS challenge was significantly inhibited by myriocin. Macrophage, neutrophil, and lymphocyte numbers in the bronchoalveolar lavage fluid (BALf) were all significantly reduced by myriocin treatment as compared to the vehicle control group. Plasma ceramide levels were reduced 24hrs after myriocin treatment. There was no effect of myriocin on the pro-inflammatory cytokine TNFα, or chemokines KC and MIP-2 in the BALf. However, myriocin inhibited MCP-1 induced migration activity of BALf macrophages. These data provide support for the therapeutic modulation of the sphingolipid pathway as a strategy to suppress inflammation.
CD73-generated Adenosine Regulates Thrombomodulin Expression (94.29)
Authors: Qu Dongfeng , Esmon Naomi , Esmon Charles , Thompson Linda
Published: January 2023

Thrombomodulin (TM), as a thrombin modulator, plays a role in anti-coagulation. TM also plays an important role in regulating inflammation and vascular permeability. As TM expression levels are regulated by cAMP, and adenosine receptors (AR) modulate intracellular cAMP levels, we investigated whether TM expression can be regulated by AR signaling triggered by CD73-generated adenosine. Mouse endothelial cells were treated with either the general AR agonist NECA, or agonists specific for different AR subtypes, and TM expression levels were measured. Protein levels increased 2-fold in response to NECA; mRNA levels increased 4-fold. TM protein and mRNA levels also increased in response to A2AAR and A2BAR agonists, but not to A1AR or A3AR agonists. TM protein and mRNA levels also increased after AMP treatment and these increases were abrogated by a CD73 inhibitor. TM mRNA levels increased 4- to 6-fold in lung, heart, and liver of mice treated with NECA; protein levels increased 2-fold. Wild type and CD73-/- mice were treated with LPS and TM expression levels were measured in lung tissues. TM mRNA levels decreased further in lung tissue of CD73-/- mice than in wild-type mice after LPS treatment. These data suggest that AR signaling can modulate TM expression and that CD73-generated adenosine can be very important in regulating TM mRNA levels in vivo during an inflammatory response.

This work is funded by NIH grants AI18220 and P20RR016904.
Type-I interferon modulates monocyte recruitment and maturation in chronic inflammation (94.4)
Authors: Lee Pui Y , Li Yi , Kumagai Yutaro , Weinstein Jason , Nacionales Dina , Butfiloski Edward , Kellner Erinn , Akira Shizuo , Sobel Eric , Satoh Minoru , Reeves Westley
Published: January 2023

Chronic inflammation is characterized by continuous recruitment and activation of immune cells such as monocytes in response to a persistent stimulus. Production of pro-inflammatory mediators by monocytes leads to tissue damage and perpetuates the inflammatory response. However, the mechanism(s) responsible for the sustained influx of monocytes in chronic inflammation are not well defined. In chronic peritonitis induced by 2,6,10,14-tetramethylpentadecane (TMPD; pristane), the persistent recruitment of Ly6Chi inflammatory monocytes into the peritoneum was abolished in type-I interferon (IFN-I) receptor deficient mice but was unaffected by the absence of IFN-γ, TNF-α, IL-6 or IL-1. IFN-I signaling stimulated the production of chemokines (CCL2, CCL7, and CCL12) that recruited Ly6Chi monocytes via interactions with the chemokine receptor CCR2. Interestingly, after TMPD treatment, the rapid turnover of inflammatory monocytes in the inflamed peritoneum was associated with a lack of differentiation into Ly6Clo monocytes/macrophages, a more mature subset with enhanced phagocytic capacity. In contrast, Ly6Chi monocytes/macrophages differentiated normally into Ly6Clo cells in IFN-I receptor deficient mice. The effects of IFN-I were specific for monocytes as granulocyte migration was unaffected in the absence of IFN-I signaling. Taken together, our findings reveal a novel role of IFN-I in promoting the recruitment of inflammatory monocytes via the chemokine receptor CCR2. Continuous monocyte recruitment and the lack of terminal differentiation induced by IFN-I may help sustain the chronic peritoneal inflammatory response.
Differentiation of blood monocytes to dendritic cells following reverse transmigration across the blood-brain barrier (94.6)
Authors: Talreja Jaya , Prameya Rukmini , Dorovini-Zis Katerina
Published: January 2023

Dendritic cells (DC) are absent in the normal human central nervous system (CNS) but their presence has been documented in neuroinflammation. The origin of CNS DC is still controversial. The objective of this study was to determine whether monocytes that cross the blood-brain barrier (BBB) differentiate into DC. Primary human brain microvessel endothelial cells (HBMEC) were cultured on collagen gels. Purified human CD14+ monocytes were incubated with confluent HBMEC for 48h and the reverse transmigrated (RT) cells were characterized by FACS analysis. Approximately 50% of the monocytes reverse transmigrated across HBMEC monolayers and 70% of the RT cells expressed DC-SIGN. Monocytes incubated on gels in the absence of HBMEC retained their phenotype. The percentage of RT cells expressing CD83 increased from 25% to 70% when human CNS myelin was incorporated into the gels, and there was significant increase in CCR7 expression. Analysis of culture supernatants showed high levels of IL-6, TNF-α, PGE2 and IL-1β when HBMEC were cultured on myelin gels suggesting that these cytokines may contribute to the differentiation of monocytes to mature DC. There was an associated increase of MIP-3β, MCP-1, MIP-1α, and MIP-1β in the supernatants. Mixed lymphocyte reactions by co-culturing CFSE-labeled T cells and RT cells showed that RT cells were capable of stimulating allogeneic T cell proliferation. In conclusion, the results suggest that monocytes which reverse-transmigrate across HBMEC differentiate into mostly mature DC capable of inducing T cell proliferative responses. (Supported by MSS, Canada)
ICOS regulates CD4 T cell exit from the lymph nodes (95.15)
Authors: Moore Tamson V. , Clay Bryan S. , Histed Alex , Shilling Rebecca A. , Sperling Anne I.
Published: January 2023

Inducible costimulator (ICOS) belongs to the CD28 family of costimulatory receptors. Although ICOS has been found to augment CD4 T cell differentiation and cytokine production, its effects on proliferation and survival are controversial. To dissect the effects of ICOS on T cell survival and proliferation, ICOS-/- or wild-type TCR-transgenic T cells were adoptively transferred and followed after immunization. ICOS-/- cells divided significantly less than wild-type CD4 T cells by CFSE dilution. Unexpectedly, when the total number of cells was examined, more ICOS-/- cells were found in the lymph nodes and fewer in the spleen. We then shifted our focus to the effects of ICOS on CCR7 and CD62L, receptors known to retain cells in the lymph nodes. Interestingly, ICOS-/- TCR-transgenic cells had a defect in down-regulation of CD62L and CCR7 compared to wild-type cells. In a model of Th2-mediated airway inflammation, ICOS-/- mice had significantly fewer CD4 T cells in their airways than wild-type mice, and the CD4 T cells in the draining lymph node expressed higher levels of CCR7. Finally, ICOS-/- cells migrated more to the draining lymph nodes than the spleen compared to wild-type cells in inflamed mice. Together, these data suggest that ICOS-/- CD4 T cells have defective down-regulation of CCR7 and CD62L, leading to a defect in cell egress from lymph nodes and migration to sites of inflammation.
Targeting regulatory T cells in vaccination: In silico identified small molecule CCR4 antagonists exert adjuvant activity (96.12)
Authors: BAYRY Jagadeesh , Tchilian Elma Z , Davies Matthew N , Beverley Peter C.L. , Flower Darren R , Tough David F
Published: January 2023

The efficacy of vaccines can be greatly improved by the addition of adjuvants, which enhance and modify immune responses. At present, few adjuvants are available for human use, and the most common, alum, stimulates immunity that is suboptimal for protection against many infections. We have used computer modeling and virtual screening to identify potential small molecule adjuvants that target specific cell receptors. The chemokine receptor CCR4 was posited as an adjuvant target, based on its expression on CD4+CD25+ regulatory T cells, which normally act to down-regulate immune responses. A high proportion (16%) of small molecules that were predicted by virtual screening to be CCR4 antagonists were able to inhibit CCR4-mediated cellular migration in vitro. Importantly, CCR4 antagonists enhanced human T cell proliferation in an in vitro immune response model and increased T cell and antibody responses in vivo when used in combination with vaccines. The results suggest that in silico screening will prove an aid to the rational design of molecular vaccine adjuvants targetting specific receptors.
Inhibition of VEGF reduces antigen specific CD8 T cell mediated CNS vascular permeability (96.16)
Authors: Johnson Aaron J. , Suidan Georgette L. , Chen Yi , Pirko Istvan
Published: January 2023

CD8 T cells and vascular endothelial growth factor (VEGF) have been implicated in promoting vascular permeability in neurological disease. We have created an in vivo system to study BBB disruption using the Theilers murine encephalomyelitus virus (TMEV) model of multiple sclerosis. During acute TMEV infection, 70% of CNS infiltrating CD8 T cells are specific for a TMEV peptide, VP2 121-130, presented in the context of the Db Class I molecule. Intravenous injection of the VP2 121-130 peptide during the peak of CD8 T cell expansion in the CNS results in fatal vascular permeability characterized by: 1.) Astrocyte activation as detected by GFAP expression, 2.) Alteration of blood brain barrier tight junction proteins ZO-2, Occludin and Claudin 5, 3.) Gadolinium enhancing lesions on MRI, and 4.) Severe motor deficit. Prior to these overt changes in CNS vascular permeability we observe dramatic increases in neuronal VEGF expression. Inhibition of VEGF results in a titratable reduction of CNS vascular permeability, demonstrating the importance of this cytokine in BBB disruption. Inhibition of VEGF also resulted in greatly reduced phosphorylation of CNS VEGF receptor Flk-1, demonstrating the appropriate blockade of VEGF was occurring in the CNS. These experiments demonstrate the therapeutic potential of VEGF inhibition in neurologic diseases characterized by CNS vascular permeability.
Production of a cytokine inducing IL-2 production from T cell by dendiritic cells stimulated with a sesquiterpene compound TG651 (96.19)
Authors: Kang Hyun-Jung , Im Sun-A , Lee Young-Hee , Lee Young-Ran , Song Sukgil , Lee Chong-Kil
Published: January 2023

Dendritic cells (DCs) have been known to produce cytokines such as IL-1, IL-6, IL-10, IL-12, IFN-á and IFN-ã. None of those cytokines has been shown to activate T cells to produce IL-2 by itself. Here, we report that DCs stimulated with TG651, a sesquiterpene compound, produce a cytokine that induces IL-2 production from T cells. The cytokine, termed as IL-2SF, was produced from DCs as a membrane bound form as well as a soluble form. IL-2SF was produced from DCs within 5 min after TG651 stimulation, and did not require new protein synthesis, although presence of approximately 10 % fetal bovine serum was required for optimal production. IL-2SF activated both CD4 and CD8 T cells to produce IL-2. Soluble form of IL-2SF appeared to be stable at neutral - acidic condition, but destroyed readily at alkaline condition when treated at 56oC for 30 min. Ultrafiltration experiments showed that the MW of soluble form of IL-2SF is larger than 50 KD. Although the molecular entity of IL-2SF remains to be elucidated, the present study may lead to discover a novel cytokine that induces IL-2 production from T cells.
The crucial role for the first gata-1 site in the exon1 of CCR3 in its transcription. (96.20)
Authors: Uhm Tae Gi , Kim Byung Soo , Kang Jin Hyun , Park Choon-Sik , Chung Il Yup
Published: January 2023

GATA-1 is shown to regulate development/differentiation of myeloid lineages including eosinophils. CCR3 is almost exclusively expressed on eosinophils and has been extensively studied for a molecular target to interfere eosinophils-associated diseases such as asthma. There are several putative gata-1 sites in exon1 of CCR3 and the regions flanking the exon1. To know whether GATA-1 is involved in CCR3 expression and further which gata-1 site is responsible for GATA-1 binding, we performed chromatin immunoprecipitation assay and mutation analysis using peripheral blood eosinophils, developing eosinophils, and cell lines that express varying degrees of CCR3 and GATA-1. We found that GATA-1 strongly bound to the regions in the exon1 and the intron1 downstream the exon1. K562 cells, which endogenously express the highest level of GATA-1, exhibit much higher CCR3 reporter activity than cells expressing low or intermediate levels of GATA-1. Introduction of DN-GATA-1 resulted in a decrease in CCR3 reporter activity. Among eight point mutants for the gata-1 sites, the first gata-1 sequence of exon1 is solely responsible for CCR3 transcription, which was subsequently confirmed by EMSA.
CD8 T CELLS PRODUCING IFN-G AND IL-17 INDUCE THE INNATE IMMUNE RESPONSE REQUIRED FOR T CELL MEDIATED RESPONSES IN THE SKIN (96.3)
Authors: Kish Danielle D.
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to skin sensitization and challenge with hapten. Hapten-primed CD8 T cell recruitment into the challenged skin to mediate CHS requires prior CXCL1/KC and CXCL2/MIP-2 directed neutrophil infiltration. Factors inducing KC and MIP-2 production in the skin challenge site during CHS to dinitrofluorobenzene (DNFB) were investigated. KC and MIP-2 were produced 3-6 h after challenge of DNFB sensitized, but not unsensitized, mice and required hapten-specific CD8 T cells. Hapten-primed CD8 T cells stimulated hapten-labeled endothelial cell (EC) cultures to produce KC that was blocked by either anti-IFN-g or anti-IL-17 mAb. Addition of rIFN-g plus rIL-17 stimulated cultured EC to produce KC. DNFB sensitization induced hapten-specific CD8 T cell populations producing IFN-g; and IL-17. Induction of KC and MIP-2 in the skin challenge site of sensitized wild type mice was accompanied by expression of IFN-g, IL-17, and IL-21 mRNA and all were absent following challenge of sensitized IFN-g-/- and IL-17-/- mice. Co-transfer of CD8 T cells from sensitized IFN-g-/- and IL-17-/- mice, but not each population alone, to naïve mice restored hapten challenge induced KC production. Activation of hapten-primed CD8 T cell populations producing IFN-g and IL-17 is required to induce production of the KC and MIP-2 required to initiate elicitation of CHS.
Fibrotic footprint of Oncostatin M-induced pulmonary disease (97.10)
Authors: Mozaffarian Afsaneh , Anders Penny M , Trueblood Esther S , Luzina Irina G , Todd Nevins W , Atamas Sergei P , Arnett Heather A
Published: January 2023

Oncostatin M (OSM), an IL-6 family cytokine, has been implicated in a number of biological processes including the induction of inflammation and modulation of extracellular matrix (ECM). Recently, we observed that OSM is elevated in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis as well as scleroderma. In mice, delivery of OSM to the lungs results in recruitment of inflammatory cells and an increase in collagen deposition in the lungs, with pathological correlates to characteristic human interstitial lung disease. We used genetically modified mice to show that the fibrotic response is largely independent of B and T lymphocytes, eosinophils and mast cells. To investigate the relationship between OSM-induced inflammation and OSM-induced fibrosis, we used both protein and genomic array approaches to generate a "fibrotic footprint" for OSM. While the IL-4/IL-13 and TGF-β pathways are generally intertwined in fibrosis, we show that OSM is capable of driving lung fibrosis independent of these pathways. For comparison, we show the expression patterns of OSM versus bleomycin-induced lung fibrosis to highlight the unique mechanisms underlying disease pathogenesis. The demonstration that OSM is a potent mediator of lung inflammation and ECM accumulation, and upregulated in patients, provides a rationale for targeting OSM in human disease.
IL-6 modulates TGF-β expression in skin from IL-6 deficient mice (97.11)
Authors: Luckett-Chastain Lerin R , Smith Mackenzie L , Gallucci Randle M
Published: January 2023

IL-6 and Tgf-β have been shown to play a role in skin development and maintenance. However, a link between these two cytokines has yet to be identified therefore; therefore, in this study we investigated the modulation of Tgf-β1 and Tgf-βR2 by IL-6 in skin. Recombinant IL-6 treatment of IL-6KO lattices and intradermal injections of IL-6 into unwounded mice showed a significant induction of Tgf-β1 mRNA and protein, where Tgf-β1 expression localized in the dermis, while Tgf-βR2 expression resided primarily in the epidermis in IL-6KO mice. IL-6 also increased Tgf-βR2 mRNA and protein expression, and potentiates TGFb mediated decreases in proliferation in cultured keratinocytes. Wounded IL-6KO and C57BL/6 control mice showed Tgf-β1 and βR2 mRNA expression was significantly greater in unwounded and seven day-old wounds from WT mice, however; protein expression was not different. Use of inhibitors indicated that IL-6 modulates Tgf-β through a Mapk/Erk dependent mechanism. These studies indicate that IL-6 has the ability to modulate the expression of Tgf-β and Tgf-βR2 mRNA in dermal fibroblasts and unwounded skin through a Mapk/Erk dependent mechanism. Protein expression levels indicated additional regulation by factors other than IL-6 within the wound. IL-6 treatment also potentiates the differentiative effects of Tgf-β on cultured keratinocytes. These results may provide a possible mechanism defining the role of IL-6 in skin maintenance and may indicate a new association of IL-6 with Tgf-β in pathologies associated fibrosis.

This work was funded by NIH/NIGMS grant R01 GM067745.
An accessory role for B cells in the IL-12 induced activation of resting murine NK lymphocytes (97.2)
Authors: Haddad Evette Adam , Senger Laura , Takei Fumio
Published: January 2023

IL-12 is produced during early innate immune responses. Via their potent production of IFN-γ, NK cells are important mediators of the anti-microbial and anti-cancer effects of IL-12. Since the IL-12 receptor is expressed on activated but not resting NK cells, it is still unclear how IL-12 stimulates resting NK cells. Here we reveal that the activation of resting murine NK cells by IL-12 is dependent upon B cells. Upon stimulation of bulk spleen cells with IL-12, NK cells are the main source of IFN-γ. The activation of NK cells with IL-12 is dependent on endogenous IL-18 as spleen cells from IL-18 knock out (KO) mice are poorly stimulated, and purified NK cells are not stimulated with IL-12 unless IL-18 is also added. To stimulate purified NK cells with IL-12 alone, B cells, but not NKT, macrophages or dendritic cells, are absolutely required. Co-culturing B cells with NK cells induces IFN-γ production by NK cells in a contact-dependent manner. Purified B cells from IL-18 KO mice are not able to stimulate NK cells in the presence of IL-12, but IL-18 KO NK cells are still stimulated with IL-12 as long as normal B cells are present. This suggests that B cells may be a source of endogenous IL-18 during IL-12 stimulation. Intraperitoneal injection of IL-12 induces higher levels of IFN-γ and IL-18 in the sera of wild type mice than those of B cell deficient mice. Thus, we propose that B cells are important accessory cells to NK cell stimulation by IL-12.

(Supported by a grant from the National Cancer Institute of Canada)
Anti-inflammatory effects of cannabinoid receptor-2-selective agonists in immune cells (98.12)
Authors: Adhikary Sabina , Ganea Doina
Published: January 2023

A few studies have been published showing effects of cannabinoids in modulating immune response from T helper cell type 1 (Th1) to T helper cell type 2 (Th2) in a model of Legionella pneumophila infection. Cannabinoids act through two cloned receptors, CB1 and CB2. The CB1 receptor is expressed primarily in the CNS, whereas the CB2 receptor is highly expressed in immune cells in the periphery. Previous results from our laboratories indicated that the CB2 agonist had an anti-inflammatory effect on dendritic cells (DCs) and T cells. DCs differentiated in the presence of CB2 agonist produced less IL-12 upon LPS stimulation and T cells produced less IFN-gamma when co-cultured with these DCs in syngeneic culture in the presence of specific antigen. Here, we report that CB2 agonist treatment of DCs resulted in reduced STAT-1 phosphorylation. Also, treatment of polyclonally activated CD4+ T cells resulted in reduced expression of IL-12 receptor beta 2 chain, which correlated with decreased IFN-gamma production. Our data lead us to believe that CB2 agonists can modulate immune responses by acting directly on CD4+ T cells or through their effects on DCs.

This work is supported by NIH/NIAID R01AI05230, and training grant T32 DA07237 (SA)