bluuebunny/crossref_metadata_2025 huggingface dataset:
The naturally arising FoxP3+ CD4+ regulatory T cells preferentially respond to weak TCR stimulation due to constitutive nuclear translocation of NFAT (89.20)
Authors: Guo Xiaohua , Jensen Peter , Chen Xinjian
Published: January 2023

Constituting a small fraction of CD4 T cells, the naturally arising FoxP3+ CD4+ Tregs are potent suppressors required for maintenance of self-tolerance. Despite their high potency in suppressing autoimmunity, Tregs appear not to suppress protective immunity against common microbial infections, as a Treg-intact host is fully immunocompetent. How Tregs suppress certain but not all immunity is incompletely understood since the signaling pathway selectively activating Tregs has not yet been determined. We recently reported that weak allo-stimulation preferentially activates allo-reactive Tregs. Here we report that Tregs preferentially respond to all weak TCR stimulation including Ag-specific or anti-CD3-mediated TCR stimuli. In search for the underlying mechanism, we found that in freshly isolated Tregs a major fraction of NFAT molecules but not of the other two major transcription factors NF-κB and AP-1 was localized in the nucleus. Resting or treating Tregs ex vivo with the calcineurin inhibitor cyclosporine A (CsA) did not alter NFAT nuclear localization. Consistently, CsA failed to inhibit anti-CD3/CD28-induced proliferation of Tregs in cultures supplemented with IL-2. Similarly, PMA that activates NF-κB and AP-1 but not NFAT selectively induced proliferation of Tregs but not Tcons when used alone without ionomycin. Our results together with the data of others demonstrate that NFAT, through interacting with FoxP3, is constitutively located in the nucleus of Tregs, and its constitutive nuclear localization not only endows the Tregs with an anergic phenotype and capacity to suppress, but also the ability to preferentially respond to weak TCR stimulation or stimuli that activate NF-κB and AP-1 but not NFAT.
PPARgamma drives conversion of inducible Tregs by stimulating dendritic cell production of retinoic acid (89.23)
Authors: Housley William J , O'Conor Catherine A , Clark Robert B
Published: January 2023

Regulatory T cells are critical in preventing autoimmunity, and peripherally-generated inducible regulatory T cells (iTregs) may also play an important regulatory role. Retinoic acid (RA) has been shown to enhance iTreg generation, and specific subsets of gastrointestinal (GI) dendritic cells (DCs) enhance iTreg generation through production of RA. The factors that regulate DC secretion of RA are unknown. We postulated that the nuclear hormone receptor PPARγ may regulate murine DC production of RA and DC-mediated generation of iTregs. PPARγ ligands are expressed in the GI tract and both down-regulate immune responses and ameliorate murine models of autoimmunity. Using splenic DCs and the synthetic PPARγ ligand Ciglitizone (Cig), we show that PPARγ activation in vitro increases iTreg generation. This increase can be blocked by LE 540, an RA inhibitor, or Citral, an RA synthesis inhibitor, and is associated with an upregulation of mRNA for RA-synthesis enzymes. In vivo administration of Cig also increases splenic DC expression of RA-synthesis genes, and, ex-vivo, these DCs enhance iTreg generation. Finally, the PPARγ-inhibitor GW9662 blocks both the baseline iTreg generation and the Cig-induced increase in iTreg generation. Our results show that PPARγ activation can increase DC RA production and DC-mediated iTreg generation, suggesting a new role for PPARγ in immunoregulation (NIH: 1R56AI072533-01A1).
“Induction” of Regulatory T cell Function is IL-35 and IL-10 dependent (89.35)
Authors: Collison Lauren W , Pillai Meenu R , Chaturvedi Vandana , Vignali Dario AA
Published: January 2023

Regulatory T cells (TR) are believed to suppress naïve effector T cell (TN) proliferation in vitro in a contact-dependent, cytokine-independent manner, based in part on experiments in which TR and TN are separated by a permeable membrane. We show that the production of interleukin-35 (IL-35), a novel inhibitory cytokine expressed by natural TR, increases substantially following contact with TN. Surprisingly, TR were able to mediate potent suppression of TN across a permeable membrane when placed in direct contact with TN in the upper chamber of a Transwell plate. Suppression was IL-35 and IL-10 dependent, as shown by the use of IL-35 or IL-10 deficient TR and IL-10 addition or neutralization. In addition, by utilizing TN that were unable to be activated due to fixation or inappropriate Vβ stimulation, we show that TN activation was required for maximal potentiation of TR suppression. Not only was activation required for maximum potentiation of TR suppression, but TN-derived autocrine IL-10 was also required for maximal suppression. Interestingly, both TN and polarized effector T cells (Th1 or Th2) are capable of potentiating TR suppression in a cell contact independent manner. These data suggest that it is the 'induction' of suppression, rather than the 'function', of TR that is obligatorily contact-dependent. Moreover, they provide evidence that IL-10 is key for maximal regulatory activity, as seen across a Transwell membrane, providing a potential explanation for the previously reported conflicting in vitro and in vivo data.
FOXP3 activation in Treg cells, - atomic level aspects of FOXP3 structure and biochemistry that govern function (89.4)
Authors: Li Bin , Song Xiaomin , Zhou Zhaocai , Wang Qiang , Shen Yuan , Greene Mark I
Published: January 2023

The path by which signaling transduction from the cell surface to the nucleus leads to FOXP3 activation and function is undefined. The additional complexity of creation of an ordered FOXP3 ensemble of transcriptional cofactors on chromatin is not understood. FOXP3 activation in Treg cells may be regulated by extra-cellular stimuli that occur through innate, toll-like and cytokine receptors. Our studies found that TCR receptor and pro-inflammatory cytokine signals including TGF-beta and IL-6 regulate FOXP3 binding to DNA in human CD4+CD25+ Treg cells. Here we report our recent progress in understanding the role of cell surface receptor signals and pro-inflammatory cytokines in regulating biochemical modifications of the FOXP3 molecule altering its activity in Treg cells. We have now determined the biochemical features of how pro-inflammatory cytokines and pharmaceutical agents affect FOXP3 activity, and consequently how they may affect immune responses in human inflammatory disease. Moreover, we also have deduced the atomic structure of large parts of the FOXP3 molecule including the zinc finger and leucine zipper domains. These regions are important for dimerization and oligomerization of FOXP3 as well as interactions with other molecules. Our structural and biochemical studies establish the atomic and molecular basis for understanding human XLAAD/IPEX autoimmune diseases.

Research grants include NIH AI073489-01A1 (M.I.G.) and Nidus Laboratories (B.L., and Z.Z.).
Suppression of the Immune Response to Human FVIII with FVIII Transgene Modified Tolerogenic Dendritic Cells in Hemophilia A Mice (89.42)
Authors: Su Rui-Jun , Epp Angela , Wu Xiaoping , Josephson Neil
Published: January 2023

The development of inhibitory antibodies to factor VIII (FVIII) is the most serious complication in hemophilia A patients. In this study we evaluated the ability of tDCs transduced with a human FVIII transgene foamy virus (FV) vector to modulate the immune response to FVIII in hemophilia A mice. The tDCs were generated by culturing bone marrow cells in RPMI1640/10%FBS supplemented with IL-10 and the neural peptides VIP and PACAP38. Na ?ve hemophilia A mice were treated with 2 weekly infusions of tDC-F8, control tDCs (tDCs-Ctrl), or with no cells (Neg-Ctrl) prior to challenge with four weekly intravenous doses of 0.2 µg rhFVIII. Following immunization the inhibitor titers in tDC-F8 mice were 60-61% lower than the controls. The regulatory T cell related markers such as FOXP3 were up-regulated on splenic CD4+ T cells from tDC-F8 mice and the CD4+ T cell proliferation response to FVIII was suppressed by 80 - 90%. In pre-immunized mice, treatment with 4 weekly infusions of FVIII vector transduced tDCs lowered inhibitor titers by 54%. In contrast, treatment with untransduced tDCs had no significant effect. Adoptive transfer of CD4+ T cells from tDC-F8 mice suppressed the immune response to FVIII in subsequently immunized naïve secondary recipients, but not to OVA, an unrelated antigen. We also observed that infusion of tDCs transduced with FV vectors co-expressing FVIII and IL-10 into naïve mice resulted in a 83 - 85% reduction in inhibitor titers after subsequent immunization. The CD4+ T cell proliferation response to FVIII was suppressed over 90%. In summary, these data indicate that FVIII vector transduced tDCs are useful in suppressing the immune response to FVIII in hemophilia A mice and that co- expression of IL-10 by the vector modified tDCs further enhances their effectiveness.
The neuropeptide npT1 is specifically expressed in human CD25+ regulatory T cells (89.5)
Authors: Prots Iryna , Skapenko Alla , Lipsky Peter E. , Schulze-Koops Hendrik
Published: January 2023

The mechanisms that regulate the generation of peripheral Tregs are largely unknown. To gain insights into Treg differentiation on a molecular level, we performed gene expression profiling at different stages of peripheral Treg development. Peripheral Tregs were generated from human naive CD4+CD25- T cells in the presence of interleukin (IL)-4. Gene expression of CD25+ Treg cells was assessed at days 3, 5, 7, and 10 of culture by microarrays and was compared to that of CD25- T cells and of CD25+ effector T cells generated in the absence of IL-4. At early stages of Treg development, the majority of identified genes were involved in cell cycle control, whereas at later time points, Treg-specific genes predominantly encoded for transcription factors and signal transduction molecules. Of interest, the neuropeptide, npT1 was strongly upregulated during late Treg development and was virtually absent in CD25- T cells. Upregulation of npT1 in CD25+ effector T cells was below 5% compared to CD25+ Tregs. Expression of npT1 in in vitro generated Tregs was confirmed by RT-PCR in independent experiments. Of note, npT1 was also expressed by resting and activated natural occurring CD25+ Tregs purified from the peripheral blood of healthy donors. Thus, npT1 is a novel Treg specific protein that is expressed in late developing and functionally mature peripheral and natural occurring Tregs.
Antigen-specific tolerance to the fetus in murine pregnancy (89.9)
Authors: Perchellet Antoine , Petroff Margaret
Published: January 2023

Tolerance of the maternal immune system is believed to be important for successful pregnancy as the fetus is semi-allogeneic. We examined maternal T cell tolerance in mice using a system in which a model antigen, ovalbumin (OVA), is expressed exclusively in the fetus. This is achieved by breeding females that lack OVA to male mice that transgenically express membrane-bound OVA under the control of the β-actin promoter. By employing T cell receptor (TCR) transgenic mice specific for a MHC class II-restricted epitope of OVA (OT-II) as mothers, we investigated the fate of fetus-specific CD4+ T cells during gestation. OVA-specific CD4+ T cells displayed an activated phenotype in the spleen and lymph nodes of OVA-bred OT-II mice, consistent with their encounter of fetal antigen in peripheral lymphoid tissues. A small percentage of OVA-specific CD4+ T cells were deleted in the periphery of OVA-bred OT-II mice, with evidence of TCR downregulation in a portion of the remaining T cells. Interestingly, deletion and TCR downregulation were also observed in the thymus of OVA-bred OT-II mice, suggesting that fetal antigen may affect central tolerance. These preliminary data indicate that fetal antigen-specific maternal CD4+ T cells are tolerized during gestation. This project is supported by NIH grants HD045611 and HD049480, and a Lied Endowed Basic Science Pilot grant. A. Perchellet is supported by NIH training grant T32HD007455.
Plasmacytoid dendritic cells are crucial for Th17 differentiation (90.14)
Authors: Ishifune Chieko , Maekawa Yoichi , Yoshimoto Takayuki , Yasutomo Koji
Published: January 2023

Plasmacytoid DCs (pDCs) produces type I interferon by CpG stimuli, and controls both innate and adaptive immune responses. However, it is largely unknown that pDCs are involved in Th17 differentiation together with controlling other types of helper T cell functions. Therefore, we here examined if pDCs control Th17 differentiation.

To investigate T cell differentiation ability of pDCs, CpG-activated BM-derived pDCs (CD11c+mPDCA-1+B220+) were co-cultured with CD4+ T cells in the presence of anti-CD3 mAb. We found that pDCs have stronger ability to induce Th17 than conventional DCs (cDCs)(CD11c+mPDCA-1-B220-). We also observed stronger Th17 inducing ability of pDCs than cDCs even when OT-II T cells were stimulated by pDCs or cDCs cultured with OVA protein. Th17 differentiation by pDCs was inhibited by addition of IL-6 neutralizing antibody and pDCs could not induce Th17 from STAT3 deficient T cells. Furthermore, CpG-activated BM-derived pDCs could produce more Th17-related cytokines than cDCs. Experimental autoimmune encephalomyelitis (EAE) is a Th17-mediated disease. To investigate if pDCs are involved in progressing EAE, pDCs were depleted by mPDCA-1 mAb in EAE mice. The clinical score of EAE and the production of IL-17 from CNS-infiltrated lymphocyte were much lower in pDCs-depleted mice than control Ig-treated mice. Taken together, those results suggest that pDCs are crucial DCs subset for the differentiation of Th17.
Interleukin-27: A Modulator of Th1 Differentiation in human newborns (90.18)
Authors: Doganci Aysefa , Zepp Fred , Meyer Claudius U
Published: January 2023

Objective: The initial description of IL-27 indicated that it plays a role in the early regulation of T helper type 1 (Th1) initiation, induces proliferation of naïve CD4 T cells and synergizes with IL-12 in IFN-γ production. Furthermore it has broad inhibitory effects on Th2 and Th17 subsets of T cells as well as the expansion of inducible regulatory T cells. As an IL-12-related cytokine IL-27 is frequently present at sites of inflammation that can promote both anti- and pro-inflammatory immune responses. To highlight the effects of IL-27 in the specific context of the neonatal immune response, we compared its role in CD4+ T cells in both human cord and adult blood.

Methodology/Principal Findings: Umbilical cord blood samples were collected from healthy full-term infants born by cesarean sections. Leukocytes from adults were enriched from leucapheresis products. After density centrifugation, the mononuclear cell fraction (PBMC) was washed and cells were magnetically labelled for CD4+ T cells and enriched in a magnetic field, following stimulation with IL-27 for ascertainment of T cell response in vitro.

Our data indicate that addition of IL-27 induced the production of IFNγ (n= 9-11; p=0,047) in neonatal CD4+ T cells, whereby IL-17 is decreased considerably. Furthermore we noticed enhanced phosphorylation of Stat-1 (n=6-9; p=0,008) in cord blood compared to adult blood. These data suggest that binding of IL-27 to its receptor initiates Th1 responses in naïve T cells of human newborns.

Conclusions/Significance: Taken together, our results suggest that IL-27 compensate the restricted Th1 immune response in human newborns to a higher extend as compared to adult T cells.
Notch ligands jagged-1 and delta-like 4 differentially regulate effector and memory CD4+ T cell expansion during Th2 inflammation (90.21)
Authors: Schaller Matthew A , Coelho Ana Lucia , Lindell Dennis M , Lincoln Pamela , Carson William F , Ito Toshihiro , Cavassani Karen A , Chensue Stephen W , Hogaboam Cory M , Lukacs Nicholas W , Kunkel Steven L
Published: January 2023

Notch ligand jagged-1 is constitutively expressed on dendritic cells, whereas Notch ligand delta-4 is induced by TLRs. It has been hypothesized that jagged-1 is important for directing Th2 responses, while delta-4 plays a role in suppressing Th2 cytokines. To determine the role of jagged-1 in Th2 responses, we administered anti jagged-1 antibody to mice during the formation of S. Mansoni induced lung granulomas in sensitized mice. Our results showed a higher amount of IL-4 in lymph nodes and an increase in the CD4 effector cell population (CD44hi CD62lo). To determine if this increase in effector cells arose from an increase in effector or memory cell proliferation, we transferred either memory (CD44hiCD62L+CCR7+) or effector cells into congenic S. Mansoni sensitized mice, induced granuloma formation, and administered anti jagged-1 antibody. Our results indicate that effector cell expansion is significantly reduced in the absence of jagged-1, and central memory cell expansion is increased under the same conditions. This occurred in an antigen specific manner and was true regardless of if the inflammation was Th1 or Th2 in nature. Additional experiments revealed that delta-4 had the opposite effect as jagged-1. A blockade of delta-4 during Th2 inflammation caused an increase in effector cell proliferation, thus defining opposing roles for these two molecules in directing memory and effector cell expansion.

Funding provided by NIH: N0105330-397736
Nitric oxide regulates TLR sensitivity and cytokine production by PDCA1+/Ly6C+ “inflammatory” dendritic cells and dendritic cell programming of Th1 immune responses. (90.28)
Authors: Giordano Daniela , Li Chang , Clark Edward E
Published: January 2023

Studies using Nitric Oxide Synthase 2 knockout (NOS2 KO) mice have implicated nitric oxide (NO) in the control of Th1 T cells, autoreactive T cell and prevention of autoimmunity. We investigated whether the effect of NO on DCs might contribute to the NO-mediated control of Th1 immune responses. We found that GM-CSF and IL4-derived bone marrow DCs (GM/IL4-DCs) from NOS2 KO mice have enhanced survival and responsiveness to TLR signals compared to WT DCs. TLR stimuli induce a substantial up-regulation in NOS2 KO GM/IL4-DCs of a Ly6Ch DC subset characterized by: 1) high expression of co-stimulatory molecules and other maturation markers (CD86h/MHCIIh/PDCA1+); 2) a substantial increase in inflammatory cytokine production; and 3) decreased Mac3 expression compared to WT DCs. The DC subset dysregulated in the absence of NO resembles the recently described "inflammatory" DC. Furthermore, TLR-stimulated OVA-loaded, NOS2 KO GM/IL4-DCs compared to WT DCs induced increased IFNγ production of OTII CD4+ T cells either in vitro or in vivo. Our findings suggest that autocrine NO drives DC differentiation toward an "inflammatory" DC with a more macrophage phenotype and with lower co-stimulator/antigen-presenting capability and cytokine production, and thus toward a DC with less potential to activate Th1 immune responses. The [EC1]NO effects on DC programming of T cells may be crucial for the protective role of NO in autoimmune diseases. (Supported by NIH grants AI44257 and AI52203).

[EC1]less potential for adaptive immunity, why would this be a good thing?
Dissection of the reciprocal interaction between T helper cells and B cells during an immune response in vivo (90.4)
Authors: Baumjohann Dirk , Lanzavecchia Antonio , Sallusto Federica
Published: January 2023

Cognate interaction of B cells with antigen-specific T helper cells in secondary lymphoid organs is required for production of high-affinity antibodies and for generation of memory B cells and long-lived plasma cells. To gain insight into the nature of signals regulating these different aspects of B cell responses, we set up an experimental system in which CD3ε-/- mice - which lack T cells but have normal B cell development - are reconstituted with small numbers of TCR-transgenic T cells. Serum antibody levels, germinal center reaction, as well as generation of antibody-secreting cells and long-lived plasma cells were monitored at different time points after primary and secondary immunization. We found that adoptively transferred antigen-specific CD4+ T cells induced effective primary antibody responses; however, serum antibody levels were not sustained. Further analysis revealed that upon immunization T cells proliferated extensively but became exhausted, did not respond to secondary antigenic stimulation, and also failed to provide B cell help upon booster immunization. Although immunoglobulin class switch recombination, initiation of germinal center reaction, and differentiation of B cells to antibody-secreting short-lived plasma blasts were comparable in reconstituted CD3ε-/- mice, affinity-maturation was impaired and long-lived plasma cells were not generated or did not migrate to the bone marrow. Data will be presented on the mechanistic link between B cell-T cell interaction and the exhausted state of T helper cells in our experimental system. The results of the study suggest the existence of distinct early and late T cell-dependent events in initiation and maintenance of humoral immune responses.

This work was supported by a Boehringer Ingelheim Fonds PhD Scholarship to D.B.
Jak2 deficiency prevents mice from LPS-induced sepsis by modulating innate immunity without affecting adaptive immunity through STAT5 and STAT6 pathways (91.17)
Authors: Zhong Jixin
Published: January 2023

Dendritic cells (DCs) serve as a key component of innate immunity and bridge innate and adaptive immune responses against to bacteria and other pathogens. Many immune disorders are thought to be a consequence of out-of-control of inflammatory response to bacterial toxins, such as sepsis is a result of excessive inflammation with a suppression of adaptive immunity post-sepsis. Therefore, manipulating the function of DC in innate immune response without affecting its capability to initiate adaptive immunity could be a novel therapeutic approach for treatment of sepsis. Using an inducible Jak2 deficiency model, here we have demonstrated that Jak2 could be a perfect target to solely suppress DC's capacity in innate immune response. Other than its functionality in DC development, Jak2 also plays a pivotal role in modulation of innate immune response. DCs deficient for Jak2 display an immature phenotype characterized by low expression of MHC-II and costimulatory molecules and a reduced secretion of pro-inflammatory cytokines upon stimulation. However, their capability to initiate adaptive immune response was not impaired. As a result, mice deficient for Jak2 are resistant to LPS-induced septic shock. Our studies further revealed that Jak2/SATA5 signaling is implicated in DC development, while Jak2/STAT6 signaling is responsible for the regulation of pro-inflammatory cytokine secretion in DCs.
MyD88-dependent Inhibition of TLR4/TRIF Associated Signaling after Monophosphoryl Lipid A Stimulation (91.20)
Authors: Cekic Caglar , Casella Carolyn R. , Mitchell Thomas C.
Published: January 2023

TLR4 stimulation leads to the activation of both MAL/MyD88 and TRAM/TRIF pathways. Recently we identified synthetic monophosphoryl Lipid A (sMLA) as a Trif-biased agonist of TLR4. However, not all TRIF dependent genes were induced optimally after sMLA stimulation. In this study we show that there is a MyD88-dependent inhibition of IFNβ expression after sMLA but not after synthetic diphosphoryl Lipid A (sDLA) stimulation in monocytic dendritic cells (DCs). The IFNβ promoter has binding sites for NF-κB, AP-1 and IRF3, transcription factors activated by IKKα/β, MAPKs (JNK, p38 and ERK) and TBK1, respectively. Functional studies with specific inhibitors showed that JNK and p38 but not ERK1/2 played a major role in IFNβ expression both in MyD88KO and Wild type (WT) DCs. However, phospho-activation of these MAPKs as well as of IKKα/β were decreased in MyD88KO vs. WT DCs after sMLA or sDLA stimulation. However, sMLA but not sDLA induced stronger IRF3 phospho-activation in MyD88KO cells when compared to WT suggesting a MyD88 dependent regulation of TBK1 activation. Preliminary studies indicate differential kinetics in MyD88 recruitment after sMLA stimulation, which may be responsible for this inhibitory effect via an unknown mechanism. These findings identify a novel role for MyD88 in regulation of IFNβ expression based on ligand structure.

This work was supported by the NIH/NIAID and the AHA.
SHIP deficient macrophages promote inflammation by supporting the generation of Th17 cells (91.5)
Authors: Tarasenko Tatiana N , Bolland Silvia
Published: January 2023

SHIP is an SH2-containing inositol phosphatase expressed in all hematopoietic cells and proposed as negative regulator of the immune response. SHIP-null mice show increased mortality due to a myeloproliferative-like syndrome. To better understand the function of SHIP we used a conditional disruption of SHIP in the macrophage-monocyte lineage (LysMcreSHIPfl/fl), which partially recapitulates the myeloproliferative phenotype of the null deletion. Macrophages from LysMcre SHIPfl/fl mice display an M2 phenotype likely due to enhanced signaling through the GM-CSF receptor. Deletion of SHIP in macrophages not only leads to myeloid hyperproliferation, but also to lymphocyte activation as a secondary effect. LysMcreSHIPfl/fl conditional mice develop splenomegaly with a large number of activated lymphocytes. We determined that T cells in these mice are biased towards the Th17 phenotype and expand the regulatory population. LysMcreSHIPfl/fl mice show higher mortality and greater induction of Th17 cells in the autoimmune model of EAE. Taken together, we have uncovered a specific regulatory role of SHIP signaling in macrophages that has important consequences in negatively regulating macrophage function directly and T cell development indirectly.
Targeting the Activated Macrophage with Folate-Conjugated Compounds in Experimental Atherosclerosis (92.11)
Authors: Ayala-López Wilfredo , Xia Wei , Henne Walter A , Low Philip S
Published: January 2023

Macrophages are central to the development of atherosclerosis. Folate-receptor positive (FR+) macrophages have been reported to orchestrate inflammatory processes in multiple inflammatory diseases, but the role of FR+ macrophages in atherosclerosis is unknown. Here, we report the use of folate to target radioactive and fluorescent imaging agents to macrophages present within aortas of atherosclerotic ApoE-/- mice. Folate-targeted 99m-Tc and folate-DyLight680 were synthesized and injected into ApoE-/- mice that were fed either normal or Western chow for 25 weeks. Mice and their excised aortas were then imaged to assess accumulation of both folate conjugates. Flow cytometric analysis of single cell suspensions of collagenase-digested aortas was also performed. Folate-targeted compounds, but not nontargeted compounds, were found to accumulate in atherosclerotic lesions of ApoE-/- mice, with greater uptake in mice fed Western chow than normal chow. The targeted cells were also confirmed to be FR+ macrophages. These data demonstrate that FR+ macrophages are present in atherosclerotic lesions and that they can be targeted with folate-conjugated compounds. Folate targeting could conceivably be developed for the staging of atherogenesis and treating of the associated disease.
TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation from monocyte/macrophage lineage precursor cells via TRAF-6 dependent pathway (92.14)
Authors: Hsu Ping-Ning , Huang Shih-Chia
Published: January 2023

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. Recent evidence indicates that the TNF-related apoptosis-inducing ligand (TRAIL) of the TNF ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated T cells. We show in this work that TRAIL can induce osteoclast formation from human monocytes and murine RAW264.7 macrophages. We demonstrated that both cell models differentiate into osteoclast-like cells in the presence of TRAIL in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. The TRAIL-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. However, TRAIL-induced osteoclastogenesis is dependent on activation of NF-£eB, ERK, and p38 MAP kinase. The TRAIL-induced osteoclastogenesis was significantly inhibited by treatment with TRAF-6 siRNA and TRAF-6 decoy peptide, indicating this pathway is TRAF-6 dependent. Thus, our data demonstrate that TRAIL induces osteoclast differentiation via direct engagement with the TRAIL death receptor through a signaling pathway distinct from apoptosis. Our results indicate that in addition to triggering apoptosis, TRAIL induces osteoclast differentiation. It provides a novel role for TRAIL in regulating osteoclast differentiation and in osteoimmunology.
A CCR2/CCR5-dual antagonist, BMS-A, offers a potential novel oral therapy for the treatment of autoimmune disease (92.6)
Authors: Zhao Qihong , Pang Jian , McIntyre Kim , Gilloolly Kathleen , Townsend Robert , Postelnek Jennifer , Grafstrom Robert , He Bokang , Ford Gregory , Conarello Stacey , Xie Jenny , Davies Paul , Barrish Joel , Trzaskos James , Salter-Cid Luisa , Mckinnon Murray , Mandlekar Sandhya , Carter Percy
Published: January 2023

CCR2 and CCR5 are two chemokine receptors that are important in the pathogenesis of animal models of autoimmune diseases. Since the two receptors play complementary roles in the immune system, targeting both receptors simultaneously might afford greater efficacy than targeting either alone. The present study describes the identification and characterization of BMS-A, an orally bioavailable small-molecule CCR2/5-dual antagonist. BMS-A was identified as active against CCR2 and CCR5 in radioligand binding assays. Functional assays and biochemical studies demonstrate that it is a potent, selective, competitive, and reversible antagonist against both receptors. It is also a pan-antagonist in that it inhibits functional responses triggered by all ligands of both receptors. When studied in vivo in CCR2-dependent homeostatic and inflammatory models in mouse as well as monkey, BMS-A potently inhibits monocyte trafficking. Its in vivo activity against CCR5-mediated inflammatory processes was established in a CCR2-null mouse model of inflammation, wherein it further reduces macrophage infiltration into tissue. In mouse models of autoimmune disease, such as collagen-induced arthritis, BMS-A robustly reduces the clinical score and tissue destruction, as well as local and systemic inflammation. Thus, BMS-A is potentially a novel oral therapy for the treatment of autoimmune disease.
5-Lipoxygenase mediates IL-4-induced ROS generation and MCP-1 expression in human aortic endothelial cells (93.12)
Authors: Kim Paul H. , Lee Won Hee , Lee Yong Woo
Published: January 2023

Pro-oxidative and pro-inflammatory pathways in vascular endothelium have been implicated in the critical early pathological events in atherosclerosis. The present study was designed to investigate the role of 5-lipoxygenase (5-LOX) in IL-4-induced reactive oxygen species (ROS) generation and MCP-1 expression in vascular endothelium. Real-time RT-PCR and ELISA showed that IL-4 significantly up-regulated the mRNA and protein expression of MCP-1 in primary human aortic endothelial cells (HAEC) and mouse aortas. To determine whether 5-LOX is involved in IL-4-induced MCP-1 expression, HAEC were pre-treated with nordihydroguaiaretic acid (NDGA), a selective 5-LOX inhibitor, for 30 min and then exposed to 10 ng/ml of IL-4. Inhibition of 5-LOX by NDGA significantly attenuated IL-4-induced overexpression of MCP-1 mRNA and protein in HAEC. Additionally, dihydroethidium (DHE) and dichlorodihydrofluorescein (DCF) fluorescence staining showed that pre-treatment of HAEC with NDGA significantly suppressed IL-4-induced ROS generation. Our results suggest that IL-4 induces ROS generation and MCP-1 expression in vascular endothelium through 5-LOX-dependent pathway. These data may contribute to understanding the cellular and molecular mechanisms involved in IL-4-mediated progression of atherosclerosis. (This work was supported by NIH/NHLBI [Grant#R15HL085229] and NSF MILES IGERT).
Therapeutic use of proteasome inhibitors in bacterial superantigen- and LPS-induced acute systemic inflammatory response syndromes (93.13)
Authors: rajagopalan govindarajan , Tilahun Ashenafi Y , David Chella S
Published: January 2023

Bacterial superantigen-induced toxic shock syndrome (TSS) and sepsis are characterized by a profound systemic inflammatory response. Given the important role of NF¿B pathway in inflammation, we investigated whether a clinically approved proteasome inhibitor, Bortezomib, capable of inhibiting NF¿B pathway, be of therapeutic use in such conditions using HLA class II transgenic mice. Bortezomib significantly blocked staphylococcal enterotoxin B (SEB)-induced T cell activation and cytokine production in vitro. In SEB-induced TSS, in vivo administration of Bortezomib significantly reduced serum levels of several pro-inflammatory cytokines and chemokines. Nevertheless, 100% of SEB challenged mice treated with Bortezomib succumbed to TSS, and Bortezomib sensitized mice to TSS at concentrations of SEB which are otherwise non-lethal (i.e., as low as 2 μg/mouse), even when administered 24 hours after exposure to SEB. Sensitization to TSS by Bortezomib was not due to the pro-apoptotic effects of Bortezomib. Serum biochemical parameters and histopathological findings suggested acute liver failure as the possible cause of mortality. In sepsis model, whereas LPS alone (10 μg/mouse) was seldom lethal, it was uniformly lethal when administered along with Bortezomib. In conclusion, Bortezomib sensitizes mice to certain acute systemic inflammatory diseases.

Supported by R01AI068741 and AI 72211
Systemic Inflammation in Murine Choroidal Neovascularization (CNV) (93.14)
Authors: Raikwar Himanshu P , Coughlin Beth , Rohrer Baerbel M
Published: January 2023

Laser-induced photocoagulation of RPE-Bruch's membrane is a conventional method of inducing CNV in mice, which resembles in many aspects the pathology of human exudative age-related macular degeneration (AMD). The presence of complement components and inflammatory cells in the CNV and AMD lesions support an "inflammation-related theory of AMD". Here we further examined the inflammatory processes in experimentally-induced CNV in mice. The spleens of CNV mice were enlarged on day 6, with a corresponding increase in weight and cellularity of spleenic mass. In particular the CD11b (monocyte/macropgage) population was increased. At the protein level, T-bet, transcription factor for IFNγa proinflammatory/Th1 cytokine, was upregulated in the spleen. Furthermore serum levels of IFNγand anaphylotoxin were elevated in CNV mice. Taken together, CNV seems to involve both the innate (complement system) and adaptive (Th1) immune response, acting both locally within the eye as well as systemically. Distinguishing the different contributions of local and systemic effects to CNV might aid in the design of treatment strategies.

This work was supported in part by the Foundation Fighting Blindness, and an unrestricted grant to MUSC from Research to Prevent Blindness, Inc., New York, NY. BR is a Research to Prevent Blindness Olga Keith Weiss Scholar.
Anti-inflammatory Adjuvants in Resuscitation Fluids Improves Survival in Experimental Hemorrhage (93.15)
Authors: Dong Weihong , Cai Bolin , Pena Geber , Pisarenko Vadim , Lee Marlon , Ramos Laura , Deitch Edwin , Ulloa Luis
Published: January 2023

Conventional resuscitation fluids reestablish tissue perfusion, but they fail to prevent lethal inflammatory responses. We reasoned that ethyl pyruvate (EP) can provide a therapeutic anti-inflammatory potential to improve survival during resuscitation. Survival, organ damage, inflammatory responses and characteristic pathways were analyzed after resuscitation of hemorrhagic rodents and swine. All control rodents and swine without resuscitation died within the first 6 hrs after hemorrhage. Resuscitation with Hextend reestablished arterial blood pressure and tissue perfusion, yet 90% of the animals died within 6 hrs after hemorrhage. All the animals treated with 50 mM EP survived hemorrhage. EP prevented systemic inflammation and improved survival in a concentration dependent manner. Unlike Hextend, resuscitation with EP ablated TNF and HMGB1 production, preserved the intestinal epithelium, and prevented bacterial endotoxin in the serum. From a molecular perspective, EP inhibited NF-kB and PARP activation without affecting IKBa activation in major organs and culture of macrophages. USAMRMC#05308004, AHA06352230N, NIH-GM084125.
Visualizing the rapid and localized keratinocyte expression of dendritic epidermal T cell antigens in response to wounding (93.3)
Authors: Komori Heather Kiyomi , Witherden Deborah A , Teyton Luc , Havran Wendy L
Published: January 2023

Within the skin, dendritic epidermal T cells (DETC) function at the interface of innate and adaptive immunity, playing important roles in homeostasis, tumor surveillance and wound repair. Central to their role in wound repair is stimulation of DETC through their monoclonal Vγ3Vδ1 T cell receptor (TCR) by an uncharacterized antigen expressed on stressed keratinocytes. While only DETC proximal to the wound site become activated, expression patterns and kinetics of antigens that stimulate DETC activation are uncharacterized. Using soluble DETC TCR tetramers, we show that the DETC TCR binds to a glycosylated, cell surface expressed, protein that is constitutively expressed by transformed epithelial cell lines in vitro. Additionally, in situ studies show that DETC TCR ligands are rapidly upregulated by keratinocytes proximal, but not distal to, wound edges. Following antigen expression, DETC become activated and antigen expression is subsequently down-modulated. Furthermore, inhibition of TCR-ligand interactions using DETC TCR tetramers delays wound repair in vivo, highlighting the importance of rapid, TCR mediated, DETC activation by keratinocytes for proper wound healing. This study represents the first visualization of DETC TCR ligand expression by keratinocytes in murine skin and provides a mechanism for the rapid and localized activation of DETC in response to wounding. Supported by the National Institutes of Health (AI064811 to W.L.H, A142267 to L.T.), and the National Science Foundation Graduate Research Fellowship to H.K.K.
Beneficial effects of fullerene derivative on lipopolysccharide-induced pro-inflammatory pathways in human aortic endothelial cells (93.9)
Authors: Lee Won Hee , Kim Paul H. , Lee Yong Woo
Published: January 2023

Inflammation in vascular endothelium has been implicated in the critical early pathological events in atherosclerosis. The present study was designed to examine the hypothesis that fullerene derivative, polyhydroxylated fullerene [C60(OH)18±22], may attenuate pro-inflammatory pathways in human aortic endothelial cells (HAEC). The quantitative real-time RT-PCR, enzyme-linked immunosorbent assay (ELISA), and cell-based enzyme-linked immunosorbent assay (CELISA) showed that pre-treatment of HAEC with increasing concentrations of polyhydroxylated fullerene (5, 25, and 50 μM) significantly and dose-dependently attenuated lipopolysaccharide (LPS)-induced up-regulation of mRNA and protein expression of pro-inflammatory mediators such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Our results suggest that polyhydroxylated fullerene may play a potential protective role in the progression of atherosclerosis via its anti-inflammatory action. Additionally, it will provide insights to novel preventive and therapeutic approaches for atherosclerosis specifically targeted against pro-inflammatory pathways in vascular endothelium (This work was supported by the Institute for Critical Technology and Applied Science).
CXCR3-dependent mechanisms are required for plasma cell recruitment into the CNS (94.18)
Authors: Marques Cristina P. , Sedlack Stefanie N. , Stohlman Stephen A. , Bergmann Cornelia C.
Published: January 2023

CXCR3 and its ligands, CXCL-9, -10, and -11 regulate T cell trafficking to sites of infection, but their role in recruiting antibody secreting cells (ASC) is unknown. Neurotropic coronavirus induces CXCR3 ligands early during infection, and CXCL10 remains elevated throughout CNS persistence. In addition, lymph nodes reveal increased CXCR3 expression on ASC prior to their accumulation in the CNS. CXCR3-dependent ASC recruitment was thus studied in CXCR3-/- mice, as well as following CXCL10 neutralization. Infected CXCR3-/- mice exhibited increased mortality and clinical disease. Uncontrolled CNS virus replication compared to wt mice coincided with a decrease in lymphocyte recruitment and increased T and B cell accumulation in lymph nodes. Both total and virus-specific ASC in the brain were reduced by ~80%. In contrast, CXCL10 inhibition in wt mice did not significantly alter CNS inflammation or morbidity. Furthermore, Ab treatment increased the relative frequency of B cells, CD138+ plasma blasts, and both total and virus-specific ASC in the CNS. Nevertheless, CD4 T cell percentages remained reduced. Thus, although the absence of CXCR3 reduced ASC recruitment to the CNS, CXCL10 blockade did not inhibit protective ASC accumulation. CXCL10-independent ASC recruitment to the CNS suggests CXCL9-mediated support of local CNS humoral immunity.

Supported by NIH AI 47249
Role of inducible costimulator and inducible costimulator ligand in bleomycin-induced lung and skin fibrosis model (94.22)
Authors: tanaka chichiro , Fujimoto Manabu , Sato Shinichi , Takehara Kazuhiko , Hasegawa Minoru
Published: January 2023

Inducible costimulator (ICOS) is a CD28 homolog with an expression restricted to T cells, and its only interaction partner, ICOS ligand (ICOSL), is highly expressed on B cells, macrophage, and dendritic cells. To assess the role of ICOS-ICOSL signaling pathway in tissue fibrosis, bleomycin-induced lung and skin fibrosis model was examined in mice deficient for ICOS and/or ICOSL. Daily subcutaneous injection of bleomycin induced fibrosis in the lungs and skin. The loss of ICOS improved the survival rate as a result of ameliorated inflammatory cell infiltration, collagen deposition, and proinflammatory cytokine expression. By contrast, the deficiency of ICOSL or both ICOS and ICOSL reduced the survival rate in consequence of aggravated inflammation and tissue fibrosis. Interestingly, ICOSL expression on macrophage and B cell was ~300% elevated in ICOS-deficient mice compared with wild type mice during this process. Thus, ICOSL expression levels inversely associated with the severity of this mouse model. These findings suggest that ICOSL expression on antigen presenting cells has regulatory roles independent of ICOS-ICOSL pathway for the development of bleomycin-induced tissue fibrosis.
Monocyte chemoattractant protein-1 (MCP-1), not MCP-3, is the primary chemokine required for monocyte recruitment in thioglycollate-induced peritonitis (94.5)
Authors: Yoshimura Teizo , Takahashi Munehisa , Galligan Carole , Tessarollo Lino
Published: January 2023

In this study, we generated a novel MCP-1-deficient (designated as MCP-1Ä/Ä) mouse model using the Cre/loxP system. As expected, MCP-1 was not produced by LPS-activated MCP-1Ä/Ä macrophages; however, the production of MCP-3 was unexpectedly increased. In contrast, macrophages from another mouse line with a neo gene cassette in intron 2 produced a significantly lower level of MCP-1 and MCP-3. Decreased MCP-3 production was also detected in previously generated MCP-1-deficient mice by the insertion of a neo gene cassette in exon 2 (designated as MCP-1 KO). Altered MCP-1 and/or MCP-3 production was observed in vivo in each mouse model in response to intraperitoneal injection of thioglycollate (TG). The up- and down-regulation of MCP-3 production in MCP-1Ä/Ä and MCP-1 KO mice, respectively, provided us with a unique opportunity to evaluate the role for MCP-3 in the recruitment of monocytes. Despite the increased MCP-3 production in MCP-1Ä/Ä mice, TG-induced monocyte accumulation was still reduced by about 50% compared to WT mice, which was similar to the reduction detected in MCP-1 KO mice. Thus, up-regulated MCP-3 production did not compensate for the loss of MCP-1, and MCP-3 appears to be a less effective mediator of monocyte recruitment than MCP-1 in this model. Our results also suggest the participation of other mediators regulating the recruitment of monocytes in this model.
CCL20 and beta-defensin induce arrest of human Th17 cells on inflamed endothelium under flow conditions (95.1)
Authors: Yssel Hans , Ghannam Soufiane , Giot Jean-Philippe , Jorgensen Christian , Pène Jérôme
Published: January 2023

CCR6 is a chemokine receptor that is specifically expressed at the cell surface of Th17 cells, a population of cytokine producing CD4+ T cells with strong inflammatory properties. Ligand-mediated triggering of chemokine receptors is involved in T lymphocyte migration towards and diapedesis into inflamed tissue. Here, we have determined the involvement of CCR6 in this process by analyzing the capacity of CCR6-specific ligands to induce arrest of Th17 lymphocytes onto endothelial cells in vitro under flow conditions. We show that under inflammatory conditions both CCL20 and beta-defensin-2 are capable to induce arrest of tissue-derived Th17, but neither Th1 nor Th2, cells on HUVEC, stimulated with IL-1beta and TNF-alpha 48 h prior to the adhesion assay. Chemokine-induced arrest of Th17 cells was found to be dependent, at least in part, on the expression of CD54. Although specific for Th17 cells, CCR6 expression is not a stable trait, as antigen-specific stimulation resulted in the inhibition of cell surface expression of CCR6 with a complete loss of expression observed after 76h of stimulation. CCR6 expression was restored following subsequent culture in vitro which was intrinsic to Th17 cells and which was independent of the addition of cytokines to the culture medium. As CCL20 is not only secreted by inflamed endothelium, but is also produced rapidly by activated Th17 cells, these results show that CCR6 plays an important role in the migratory capacity of human Th17 cells towards inflamed tissue and, furthermore, that antigen-specific activation induces a transient state of non-responsiveness to CCR6-specific ligands.