bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo opposing regulation of vasoactive intestinal peptide receptor-1 and -2 throughout the T cell immune response (90.25)
Authors: Vomhof-DeKrey Emilie E , Haring Jodie , Dorsam Glenn P
Published: January 2023

This study investigates vasoactive intestinal peptide receptor 1 (VPAC1) and VPAC2 regulation during the immune response of murine T cells. VPAC1 is an anti-proliferative, G-protein coupled receptor that has been shown to be downregulated upon ex vivo T cell activation, whereas VPAC2 was shown to be upregulated. To date, regulation of VPAC1 and 2 have not been elucidated in primary, murine T cells throughout the phases of a T cell immune response following infection and whether they have a role in memory T cell evolution. Splenic CD4 T cells were isolated from C57Bl/6J mice 24hr post in vivo activation with anti-CD3. Flow cytometry (Accuri) analysis confirmed 90% activation of cells (CD69+/CD25+). QPCR analysis of total RNA showed VPAC1 levels decreased 93% in activated CD4 T cells, while VPAC2 levels increased 66%, possibly suggesting that these receptors regulate early components of the CD4 T cell response. These results prompted us to pursue an in vivo activation model utilizing OT-I and OT-II T cells. We hypothesize in CD4 T cells, VPAC1 levels will decrease upon activation and gradually increase to naïve levels throughout contraction into memory phase, while VPAC2 levels will increase upon activation and gradually decrease to lower naïve levels. The reciprocal expression pattern of these receptors may indicate they have opposing functions in regulating the kinetics of the T cell response and memory cell evolution.

1KO1 DK064828, COBRE- 2P20RR015566
CD70 drives the proliferation of memory CD8 T cells during secondary responses (90.5)
Authors: Al-Shamkhani Aymen , Taraban Vadim Y , Buchan Sarah L
Published: January 2023

Recent studies have either showed CD27 to be necessary or unnecessary for memory CD8 T cell responses. Heterogeneity in memory CD8 T cells in their ability to respond to CD27 signaling and/or differential use of CD27 ligand (CD70) by different dendritic cell subsets may explain the variable dependence on CD27 in different models. To distinguish between these two possibilities, we used soluble recombinant CD70 in vivo in order to overcome the requirement for endogenous CD70 expression. We show that secondary expansion and function of central and effector memory CD8 T cells are boosted by administration of soluble CD70. In vivo BrdU incorporation studies demonstrated increased proliferation of CD8 memory T cells in mice that had received soluble CD70, compared with those that received antigen alone. Furthermore, activation of CD27-low effector memory CD8 T cells led to CD27 upregulation to a level similar to that expressed by central memory T cells. We conclude that optimal proliferation of memory CD8 T cells requires a costimulatory signal via CD27 and that both central and effector memory CD8 T cells are equally competent in responding to CD27 ligation by CD70. Therefore, discrepancies in the requirement for CD70 during secondary responses reported in previous studies are likely to be due to differential use of CD70 by alternative antigen presenting cells. This work is funded by Cancer Research UK.
A novel monoclonal antibody that identified a transmembrane isoform of CD160 (CD160-TM) selectively expressed on human NK cell upon activation (90.9)
Authors: Bensussan Armand , Giustiniani Jerome , Marie-Cardine Anne
Published: January 2023

CD160 has been initially identified as a GPI-anchored MHC-class I activating receptor mainly expressed on peripheral blood NK cells. Here, we report the identification of an additional CD160-related mRNA generated through alternative splicing of CD160 gene, which encoded a putative CD160 transmembrane isoform (CD160-TM). We first establish that CD160-TM surface expression is highly restricted to NK lymphocytes and is activation-dependent. In addition, we provide evidences that CD160-TM represent a novel activating receptor, as assessed by the increased CD107a NK cell surface mobilization observed upon its engagement with a specific monoclonal antibody. Finally, we study the activating signaling obtained following CD160-TM engagement. The identification of CD160-TM therefore provides new possibilities regarding the role of CD160 isoforms in the regulation of NK cell functions.
DAP12 renders liver dendritic cells resistant to maturation (91.14)
Authors: Sumpter Tina L , Thomson Angus W
Published: January 2023

Liver APC are constitutively exposed to gut-derived LPS, yet are refractory to LPS-induced maturation. We and others have hypothesized that resistance to LPS in liver DC reflects molecular inhibition of NF-κB activation. DNAX-activating protein of 12kDa (DAP12), a transmembrane adaptor protein, dampens co-stimulatory molecule and cytokine expression associated with inhibition of NF-κB in myeloid (m)DC. In this study, we evaluated the function of DAP12 in mDC (CD11c+CD11b+NK1.1-B220-) purified from livers or spleens of Fms-like tyrosine kinase 3 ligand (10 μg/day/10d)-mobilized C57BL/10 mice (in which DC populations were enriched), then cultured overnight with LPS. To test the hypothesis that DAP12 impairs LPS-induced maturation of liver mDC, DAP12 was silenced with siRNA (400nM) 2h prior to LPS stimulation. Liver mDC did not upregulate CD80, CD86, B7-H1 (PD-L1), B7RP or IL-12 to the extent of splenic mDC in response to LPS. Silencing DAP12 enhanced expression of CD80, CD86, IL-12 and the Th1-polarizing molecule, Delta4, but not B7-H1 or B7RP in liver and spleen mDC in response to LPS. These effects were more pronounced in liver mDC. We conclude that the comparatively immature state of liver mDC may be regulated, in part, by DAP12. Supported by American Liver Foundation and American Transplantation Society Fellowships and NIH RO1AI60994.
Estrogen receptors promote langerin+ dendritic cell differentiation and migration from skin during inflammation in vivo (91.3)
Authors: Bajana Sandra , Agrawal Hemant , Carreras Esther , Kovats Susan
Published: January 2023

CD207/langerin+ dendritic cells in the epidermis (eLC) and dermis (dLC) initiate immune responses in the skin. We previously published that estrogen receptor alpha (ERα) signaling promotes the differentiation of langerin+ DC in a GM-CSF-driven culture model. To compare the efficiency of dLC differentiation from ERα+ and ERα-/- bone marrow (BM) cells in vivo, we generated mixed competitive ERα+/ERα-/- BM chimeric mice. In the radiation-induced inflammatory environment early after reconstitution, dLC in cutaneous lymph nodes (cLN) were derived preferentially from ERα+ BM cells, indicating that during systemic inflammation ERα signaling promotes dLC differentiation from short term repopulating progenitors. To determine the relative ability of ERα+ and ERα-/- LC to become activated during local skin inflammation, green fluorescent dye in acetone/dibutylphthalate was applied to the skin of ERα+ and ERα-/- mice. Compared to ERα-/- mice, after 24 hr, ERα+ mice contained significantly greater numbers of langerin+ CD11b+ eLC that had acquired the green dye and migrated to cLN. Moreover, ERα+ mice contained higher numbers of new inflammatory CD11bhi CD11clo F4/80+ myeloid cells in the spleen. In sum, ERα signaling in response to endogenous estrogens promotes the development, activation and migration of skin-derived langerin+ DC during inflammation in vivo. Supported by OCAST HR06-157 & NIH AI079616, RR020143RR020143.
SHP-2 constitutively suppresses natural killer cell polarization and granule release in response to tumor target cells (91.4)
Authors: Purdy Amanda K , Campbell Kerry S
Published: January 2023

The tyrosine phosphatase, SHP-2, promotes the full activation of Ras/ERK in many cytokine and growth factor receptor pathways. In contrast, SHP-2 inhibits activation of natural killer (NK) cells by binding to inhibitory receptors, known as killer cell Ig-like receptors (KIRs). To determine how SHP-2 impacts NK cell activation pathways, we employed retroviral shRNA knockdown and over-expression strategies in human NK cell lines. In response to stimulation with tumor cells, SHP-2 shRNA expressing NK cells had elevated cytolytic activity and IFN-γ production, whereas cells over-expressing wild type or gain-of-function mutants of SHP-2 exhibited dampened activities. SHP-2 suppressed microtubule organizing center (MTOC) polarization and granzyme B release in a dose-dependent manner, but NK-tumor cell conjugation was not significantly affected. The suppressed NK cell function was independent of inhibitory KIR. In summary, our data demonstrate that SHP-2 constitutively suppresses NK cell activation in response to tumor cells. This suppression occurs at a stage after NK-tumor cell adhesion, but upstream of MTOC-based cytoskeletal rearrangement and granule release. These studies have identified a new, KIR-independent suppressive role for SHP-2 in regulating NK cell function and provide mechanistic insight for this role.

This work was supported by NIH grants CA083859 (K.S.C) and CA009035-32 (A.K.P).
TIM-3 is involved in NO imbalance of Atherosclerosis through the IkappaB/NF-kappaB Pathway (91.6)
Authors: Hou Nan , Liu Ying , Ju Ying , Zhang Xiaoning , Zhao Di , Zhang Zhenyu
Published: January 2023

TIM-3 (T cell immunoglobulin and mucin domain-3) is proved to regulate the immune system in many inflammaroty diseases. However, the role of TIM-3 on atherosclerosis has not been studied. We found TIM-3 was highly expressed on monocytes/macrophages of human peripheral blood,but low on T cells, either CD4+ or CD8+ T cells. And the level of Tim-3 expression on monocytes/macropahges from patients with atherosclerotic disease was higher than that from healthy subjects (93.35¡À12.14% vs 81.04¡À 24.07%,t=4.968,P<0.0001). Immunochemistry staining demonstrated the upregualtion of Tim-3 on RAW264.7, a macrophage like cell line, incubated with oxidized low-density lipoprotein (oxLDL) or LPS. Moreover, Tim-3 blocking antibody acted as an adjuvant during Nitic oxide (NO) production of RAW 264.7.Consistent to that,overexpresstion of Tim-3 by transfection inhibited NO production of RAW264.7.Futher study showed Tim-3 blocking antibody enhanced phosphorylation of NF-kappaB p65 and degradation of IkappaB.Thus TIM-3 inhibits NO production of macrophages mainly through the IkappaB/NF-kappaB pathway and is involved in NO imbalance in Atherosclerosis.

correspondence to: Professor Chunhong Ma, Institute of Immunology, Shandong University School of Medicine.E-mail address: machunhong@sdu.edu.cn.
Characterization of folate receptor positive macrophages and monocytes and folate receptor-mediated specific targeting in inflammation (92.10)
Authors: Xia Wei , Hilgenbrink Andrew R , Matteson Eric L , Lockwood Michael B , Morgenstern David E , Cheng Ji-Xin , Low Philip S
Published: January 2023

Folate receptor (FR) expressing macrophages have been shown to play an integral role in a number of inflammatory diseases. (1) In this report, we characterize these FR+ macrophages and further investigate whether monocytes, the precursors to these macrophages, also express FR. We demonstrate that FR expression is observed on a subset of highly activated peritoneal and synovial macrophages, but not on other infiltrating immune cells or resident macrophages. We further find that a subset of human peripheral blood monocytes also expresses FR, albeit at a lower level than activated macrophages. Surface antigen characterization of the FR+ monocytes suggests markers characteristic of an inflammatory phenotype (CD14hiCD16), while the resident monocytes (CD14loCD16+) express few or no FR. Preliminary clinical data also suggest that the numbers of peripheral FR+ monocytes can be reduced by a folate-targeted immunotherapy. These results suggest that activated macrophages and inflammatory monocytes express a FR that can be targeted with folate-linked drugs without promoting drug uptake by non-activated macrophages and resident monocytes.

1. Xia W, Hilgenbrink AR, et al. Blood. 2008 Oct 24. [Epub ahead of print]

This work was supported in part by a grant from Endocyte, Inc.
ESM inhibits the production of proinflammatory cytokines in macrophages (92.13)
Authors: Moon Sunhee , Lee Sungwon , Shin Seulmee , Kown Jeonghak , Gong Hyunseok , Lee Seungjeong , Kim Kyungjae
Published: January 2023

Salvia miltiorrhiza (SM) has been commonly used in traditional medicine to treat vascular diseases such as hypertension, arteriosclerosis, and inflammatory disease. It is little kwon that extract of SM alters the immune response of inflammatory reaction. Macrophage, essential cellular components of the body's host defense system, has critical functions in both innate and adoptive system. Macrophages exhibit a particularly vigorous response to endotoxin, which induces production of a variety of inflammatory mediators, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), prostagrandinE2 (PGE2), and nitric oxide (NO) via the inducible nitric oxide synthase (iNOS). In this study, we examined that inhibition effect of ethanol extract of SM (ESM) on pro-inflammatory mediators in LPS-induced murine macrophage cell line RAW 264.7 cells as well as in primary macrophages. ESM deceased the production NO and pro-inflammatory cytokine such as IL-1β, IL-6, TNF-α, and COX-2 in LPS-stimulated macrophage. The variation of gene expression level by ESM treatment was determined by RT-PCR and the relation of mRNA expression of pro-inflammatory cytokine in activated macrophage was suppressed by ESM. In western blot analysis, the level of cytokine protein was decreased in activated macrophage by ESM dose-dependently manner. In addition, the expression of co-stimulatory molecules such as ICAM-1, B7-1, and B7-2 was decreased by ESM dose-dependently. These results showed that ESM has immunomodulatory activities in both innate and adoptive immunity, and they provide the critical information for the understanding of the mechanism of inflammation studies.

Key words: Salvia miltiorrhiza, anti-inflammatory, immunomodulator, cytokines
Oxidative stress-induced protein radical formation precedes follicular dendritic cell apoptosis in murine sepsis (92.3)
Authors: Chatterjee Saurabh , Deterding Leesa , Tucker Jeff , Corbett Jean , Mason Ronald P
Published: January 2023

New studies indicate that most deaths from sepsis are actually the result of a substantially impaired immune response that is due to extensive death of immune effector cells including follicular dendritic cells (FDCs). However studies by Hotchkiss et al, and others have shown that FDC's undergo caspase 3-mediated apoptosis at 36 h post sepsis with profound depletion of these cells by 48 hours, but the underlying causes of apoptosis have been unknown. This study hypothesizes the role of oxidative stress-induced protein radical formation in FDCs as the principle cause of apoptosis in murine sepsis. This study utilizes immuno-spin trapping to detect DMPO (spin trap) nitrone adducts in FDCs isolated from Lipopolysaccharide (LPS)-primed mice after flow sorting. The isolated FDCs were also screened by LC/MS/MS analysis to detect possible proteins that contained DMPO nitrone adducts. Results indicated that mice administered LPS had DMPO nitrone adduct formation at 24 hours post sepsis as detected by western blot analysis and confocal microscopy. Mice administered allopurinol mice showed significant decrease in adduct formation, annexin V binding, thus indicating the role of xanthine oxidase in oxidative stress-induced protein radical formation and apoptosis. This clearly suggested a role of xanthine oxidase-mediated oxidative stress in FDC apoptosis in the germinal center of septic mice.
An evaluation of fetal/neonatal and chronic adult ethanol exposure (FNEE and CAEE) on murine cutaneous dendritic cell (CDC) maturation and migration using an organ culture system (92.5)
Authors: Parlet Corey  Patrick , Schlueter Annette J.
Published: January 2023

Alcoholism and fetal alcohol syndrome are associated with increased rates of infection. CDCs, consisting of Langerhans cells (LCs), langerin+ dermal dendritic cells (LdDCs) and langerin - dermal dendritic cells, initiate adaptive immune responses by transporting antigen from the skin to the regional lymph node (RLN). We previously reported a migratory delay of langerin+ CDCs (LCDCs) into the RLN due to CAEE. Migration of CDCs into the RLN is driven by chemokine receptors (CCRs) and adhesion molecules (AMs). To determine if either CAEE or FNEE impair the CCR/AM expression needed for timely transit into the RLN, skin explants were cultured (+/-) CPG & CCL21, and the phenotypes of emigrated CDCs (eCDCs) from control and EtOH exposed mice were evaluated by flow cytometry. CAEE reduced the numbers of emigrated LCs (eLCs). It also reduced CCR7 expression on eLCs and emigrated LdDCs (eLdDCs) and LFA-1 expression on eLdDCs. In contrast, FNEE induced a trend towards increased numbers of eLCs without affecting CCR7 and LFA-1 expression. These findings suggest that diminished CDC migratory capacity is a result of CAEE and not FNEE. The decreased expression of CCR7 and LFA-1 provides a mechanism for the migratory delay exhibited by LCDCs as a result of CAEE and ultimately, a mechanism to explain increased skin infections in alcoholics. Evaluation of additional AMs/CCRs are underway for both models. NIH RO1 AA014405 and AA014406 supported this work.
Synergy of TRIF-dependent TLR3 and MyD88-dependent TLR7 in up-regulating expression of mouse FPR2, a promiscuous G-protein-coupled receptor, in microglial cells (92.9)
Authors: Chen Keqiang , Huang Jian , Gong Wanghua , Dunlop Nancy M. , Cui Youhong , Wang Ji Ming
Published: January 2023

Human G-protein-coupled formyl peptide receptor-like 1 (FPRL1) and its mouse homologue mFPR2 mediate leukocyte chemotaxis to a variety of pathogen and host-derived peptides, including amyloid â42, a key causative factor in Alzheimer's disease. In mouse microglial cells, mFPR2 is up-regulated by several TLR ligands, resulting in increased cell response to and endocytosis of A â42 peptide. Since microglial cells may be exposed to multiple TLR agonists under pathological conditions, we investigated the consequence of activating TLR3, whose signaling is dependent on the adaptor TRIF, and TLR7, which signals through MyD88. The TLR3 agonist Poly(I:C) and the TLR7 agonist R837, when used alone, each dose-dependently increased the expression of functional mFPR2 in microglial cells. Combination of suboptional concentrations of Poly(I:C) and R837 showed a marked synergistic effect on induction of mFPR2. We further observed that Poly(I:C) enhanced I êB-á phosphorylation induced by R837. Our results indicate that the TRIF-dependent TLR3 and MyD88-dependent TLR7 cooperate to regulate microglial responses which are known to be involved in brain inflammation and neurodegenerative disease.
Age-related changes in inflammatory gene expression in response to the brain irradiation (93.10)
Authors: Lee Won Hee , Sonntag William E. , Lee Yong Woo
Published: January 2023

The aim of the present study was to determine the effect of aging on the levels of expression of pro-inflammatory mediators in response to irradiation in rat brain. Male F344×BN rats (4, 16, and 24 months old) received either whole brain irradiation with a single dose of 10 Gy γ-rays or sham-irradiation, and were maintained for 4, 8, and 24h post-irradiation. The expression levels of various pro-inflammatory mediators such as cytokines, chemokines, adhesion molecules, and matrix metalloproteinases were analyzed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). A significant and marked up-regulation of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and matrix metalloproteinase-9 (MMP-9) was observed in hippocampal regions isolated from irradiated animals at all ages compared with sham-irradiated controls. On the contrary, the expression levels of pro-inflammatory mediators up-regulated by irradiation were significantly attenuated with age. These results demonstrate that radiation-induced pro-inflammatory responses in the brain are significantly influenced by age. (This work was supported by NINDS R01NS056218)
The sympathetic nervous system regulates the release of anti-inflammatory peptides from salivary glands (93.18)
Authors: Morris Katherine E , St. Laurent Chris D , Hoeve Ryan S , Wickware Jim , Forsythe Paul , Mathison Ron , Befus A Dean
Published: January 2023

Chronic and acute stress have profound effects on inflammation. In rats, allergic inflammation is regulated by the sympathetic nervous system acting on salivary glands. Human asthma is frequently accompanied by salivary gland inflammation. Salivary gland dysfunction in stressed individuals could enhance asthma severity. Salivary gland prohormone SMR1 (submandibular rat-1) is cleaved into two peptides that are anti-inflammatory in rats, mice, dogs, sheep, cats, and human cells in pulmonary inflammation, food allergy, septic shock, pancreatitis, and spinal cord injury. We hypothesized that modulation of the autonomic nervous system would change the expression, processing, and secretion of SMR1 and its peptides.

Rats were injected with saline, isoproterenol, or pilocarpine, or the superior cervical ganglion was excised. Saliva, blood, and tissues were collected and analyzed for SMR1. Adrenergic stimulation caused the majority of SMR1 into be secreted into saliva in 60 min. Removal of the superior cervical ganglion that innervates the salivary glands changed SMR1 protein levels in the salivary glands. SMR1 secretion into saliva in response to acute stress may provide a large pool of SMR1-derived peptide products that mediate anti-inflammatory responses locally and systemically. This research is funded by AllerGen NCE.
Identification and characterization of a potent and selective antagonist of the prostaglandin D2 receptor (94.23)
Authors: Eberhardt Christine , Wright Dale , Gomez Anna , Cook Adam , Doherty George , Corrette Christopher , Hunt Kevin , Clark Christopher , Kim Ganghyeok , Romoff Todd , Delisle Kirk , Hayter Laura , Neale Jason , Dean Brian , Chantry David , Carter Laura , Burgess Laurence E
Published: January 2023

Prostaglandin D2 (PGD2) is a potent lipid mediator released from activated mast cells during allergic responses which exerts its pro-inflammatory effects through the GPCR DP2 also referred to as CRTH2. DP2 is expressed on cells associated with allergic inflammation: TH2 cells, eosinophils, mast cells, and basophils.

We have developed orally bioavailable, potent and selective small molecule antagonists of DP2. One of these compounds, ARRY-060, inhibits binding of PGD2 to DP2 (IC50 = 3 nM) and blocks PGD2-stimulated calcium flux with similar potency. Inhibition was fully surmountable by increasing concentrations of PGD2, consistent with a competitive mode of binding. In primary human basophils treatment with PGD2 stimulated a robust chemotactic response that could be blocked by ARRY-060. Selectivity for DP2 was examined by profiling against related prostanoid receptors and no inhibition of these targets was observed, including DP1. In a guinea pig asthma model, ARRY-060 (50 mg/kg, po) decreased total cellular influx in bronchial alveolar lavage. Together, these results support the development of DP2 inhibitors for the treatment of allergic disease.
Loss of Fcgamma receptors ameliorates initiation and progression of atherosclerosis in hyperlipidemic mouse model (94.24)
Authors: Nagarajan Shanmugam
Published: January 2023

OxLDL generated during hyperlipidemia is known to induce an autoimmune response resulting in the production of anti-oxLDL IgG. In humans and in the hyperlipidemic mouse model, the titer of autoantibodies against oxLDL correlates with the progression of atherosclerosis. Although the presence of anti-oxLDL IgG is well documented, the contribution of FcγRs to the initiation and/or progression of atherosclerosis has not been studied in detail. First, to address the presence of oxLDL-IC in atherosclerotic lesions, descending aorta from wild type (WT) and apoE-/- mice fed chow diet for 18 weeks and oxLDL-IC in the lysates were determined. OxLDL-IC was detected in the aortic lysates of both WT and apoE-/- mice. However, the levels were elevated about 8 fold in apoE-/- mice. The presence of anti-oxLDL IgG in atherosclerotic lesions in patients and animal models, suggests the possibility of oxLDL-IC deposition in the lesions. To determine the role of FcγRs in the initiation and/or progression of atherosclerosis, we have generated apoE-/-γ-chain-/- mice by mating γ-chain-/- mice with apoE-/- mice. ApoE-/- and apoE-/-γ-chain-/- mice (age 4 wk) were fed chow diet for 20 weeks. Plasma cholesterol levels were similar in apoE-/- and apoE-/-γ-chain-/- mice. Atherosclerotic lesions in aortic sinus were determined by staining cryosections with Oil Red O. Enface analysis of the descending aorta was performed by staining descending aorta from the aortic arch to the iliac bifurcation with Sudan IV. Both analyses showed about a 50% reduction in lesions in apoE-/-γ-chain-/- mice compared to apoE-/- mice. These novel findings suggest activating FcγRs contribute to the progression of atherosclerosis. (Supported by NIH HL086674).
Sphingosine kinase-1 mediates TNF activation of the integrin VLA-5 for leukocyte adhesion (94.26)
Authors: Bonder Claudine S , Sun Wai Y
Published: January 2023

For leukocyte recruitment to sites of inflammation the classical paradigm states that selectins (eg P- and E-selectin) are important for initial tethering and rolling with subsequent interaction via integrins and cellular adhesion molecules (eg α4β1, VCAM-1 and ICAM-1) enabling their adhesion and emigration from the microvasculature. Previous studies have shown that the lipid enzyme sphingosine kinase-1 (SK-1) is essential for TNFα-induced E-selectin, VCAM-1 and ICAM-1 expression on endothelial cells (EC). However, its role in regulating the expression of other key adhesion molecules is still unclear.

Herein we demonstrate that TNFα activates the integrin VLA-5 (α5β1) without altering total expression levels on human umbilical vein ECs. Moreover, inhibition studies with an SK-1 mutant suggest that TNFα-induced β1 activation is SK-1 dependent. Finally, a parallel plate flow chamber assay suggests that leukocyte adhesion to the TNFα-treated endothelium under shear stress can be attenuated by blocking VLA-5 or its ligand angiopoitein-2 on the ECs.

Taken together, these results suggest that TNFα-induced activation of VLA-5 is SK-1 dependent and mediates, in part, leukocyte adhesion to the endothelium. Given that excessive leukocyte recruitment during inflammation results in chronic and fatal diseases, the regulation of integrins such as VLA-5 is key for developing successful therapeutics to alleviate disease.

This work was supported by an NHMRC Project Grant, Florey Fellowship and Arthritis Australia.
Increased death receptor-induced apoptosis in ezrin-deficient type I T cells (94.28)
Authors: Lai Ming-Zong , Kuo Wen-Chih
Published: January 2023

Ezrin is reported to be essential for Fas-induced apoptosis. Here we showed that ezrin interacts with Fas, yet knockdown of ezrin increased Fas- and TRAIL-induced apoptosis in type I T cells like H9. Expression of the dominant negative form of ezrin also led to increased Fas- and TRAIL-triggered cell death in H9 cells. Similarly, downregulation of ezrin in normal human T lymphocytes resulted in elevated Fas-stimulated cell death. In contrast, ezrin knockdown in type II cells, including Jurkat and CEM cells, did not affect Fas- and TRAIL-induced apoptosis. Ezrin knockdown led to an accelerated caspase-8 activation and enhanced activation of caspase-3 in H9 cells. Ezrin deficiency did not affect the internalization of Fas after Fas engagement in H9 cells. Instead, an enhanced formation of DISC was observed in H9 cells with ezrin knockdown. Together, our results suggest that ezrin plays a negative role in the recruitment of Fas into signaling complex in type I T cells, and deficiency in ezrin removes the constraint imposed by ezrin and facilitates the assembly of death receptor.
Mechanism of platelet adhesion to neutrophils in sepsis (94.3)
Authors: Urrutia Rossana , Ma Adrienne C. , Kubes Paul
Published: January 2023

Sepsis is a systemic inflammatory response to infection which has a high mortality rate in the intensive care unit (ICU) resulting in 215000 deaths a year in United States. Neutrophils and platelets play an important role in the induction of inflammation and defense against infection in this pathology. Recent studies by our group have revealed that in severe sepsis TLR4 stimulated platelets interact with already adherent neutrophils. This interaction leads to the formation of Neutrophil Extracellular Traps (NETs) in the circulation, which helps to enhance bacterial trapping and clearance from the body; however, this may also enhance host injury in the liver sinusoids and pulmonary capillaries. To further elucidate the mechanisms by which activated platelets can adhere to immobilized neutrophils in the setting of sepsis, we used an in vitro flow chamber system using LPS and septic plasma as platelet stimulants. Antibodies to several adhesion molecules on platelets and neutrophils were tested. TLR4 activated platelets did not express P-Selectin on their surface and their interaction with immobilized neutrophils was not P-selectin-dependent. Findings show that LFA-1 and CD11c/CD18 on neutrophils, and JAM-A and ICAM-2 on platelets are involved in LPS-induced platelet adhesion to immobilized neutrophils. In addition, results from septic plasma stimulated platelets show that LFA-1 and CD11c/CD18, but not JAM-A, are involved in platelet adhesion to immobilized neutrophils. Blocking the adhesion molecules that take place in platelet adhesion to immobilized neutrophil could prevent NET formation and tissue damage specifically in lungs and liver capillaries providing a target for new therapeutics in this pathology.
Thromboxane A2 receptor regulates the severity of experimental murine colitis (94.30)
Authors: Dann Sara , Ditto David , Spehlmann Martina , Kagnoff Martin , Le Dzung , Eckmann Lars
Published: January 2023

Background: Prostanoids, lipid mediators of the COX pathway, are elevated in inflammatory bowel disease and are regulators of intestinal inflammation, yet the roles of individual prostanoids are poorly understood. We evaluated the role of the prostanoid thromboxane A2 (TXA2) in a murine model of colitis. Methods: Colitis was induced by feeding DSS to mice lacking the receptor for TXA2 and their normal littermate controls. Disease severity was analyzed by clinical and histological parameters. Production of TXA2 and expression of its synthases were determined by ELISA and real-time RT-PCR, respectively. Platelet aggregation was assessed using collagen and TXA2 receptor antagonist, and activation was measured by the release of platelet factor 4 (PF4/CXCL4). Results: Our results show that expression of COX-2 and TXA2 were increased in colitis. Mice lacking the TXA2 receptor showed attenuated intestinal inflammation and ulceration, and were protected against colitis-associated weight loss and mortality. Platelet aggregation responses were enhanced in colitic animals. Activation led to increased levels of PF4, which enhanced CXCL1 (KC) induced migration of neutrophils. Conclusions: These results indicate that TXA2 promotes colitis through activation of platelets and augmentation of neutrophil recruitment. Therefore, blockade of the TXA2 system maybe of therapeutic value in intestinal inflammation.
The interaction of α9 integrin and its ligands provides microenvironments critical for the development of autoimmune arthritis (94.9)
Authors: Kanayama Masashi , Kon Shigeyuki , Kurotaki Daisuke , Nakayama Yosuke , Morimoto Junko , Uede Toshimitsu
Published: January 2023

It has been shown that osteopontin (OPN) is critically involved in various Th1 type immune-mediated disorders such as rheumatoid arthritis (RA) and multiple sclerosis. In this study, we demonstrated that the expression of both OPN and tenascin-C (TN-C), and its common receptor, α9 integrin are up-regulated at the pathological foci of autoimmune arthritis. The local production of OPN and TN-C in arthritic joints is due to mainly synovial fibroblsts and to the lesser extent synovial macrophages. Majority of synovial fibroblasts and a subpopulation of synovial macrophages express α9 integrin. Importantly, autocrine and paracrine interactions of α9 integrin on synovial macrophages and fibroblasts and its ligands differently contribute to the production of pro-inflammatory cytokines and chemokines. α9 integrin is also involved in the recruitment and accumulation of inflammatory cells, through interaction with vascular cell adhesion molecule-1 (VCAM-1). Inhibition of α9 integrin function with an inhibitory anti-α9 integrin antibody significantly reduces production of arthrogenic cytokines and chemokines and ameliorates on-going arthritis. Thus, we identified α9 integrin as a critical intrinsic regulator that control the development of autoimmune arthritis. These results suggest that α9 integrin serves as a novel therapeutic target for the treatment of autoimmune disorders including RA.
Do the lungs deliver naïve CD4 T cells to draining nodes? (95.13)
Authors: Caucheteux Stephane M , Paul William E
Published: January 2023

~80% of lung CD4 T cells express low levels of CD44, high levels of CD62L, CD45RB, CCR7 and CD127 consistent with their being naïve. When Rag2-/- TCR transgenic cells are transferred to syngeneic mice, naïve phenotype transgenic cells are found in the lung. These cells disappear rapidly upon treatment with FTY-720 but are still found in mice treated with anti-CD62L, implying they are part of the recirculating pool but do not require L-selectin to enter the lung. Upon intranasal immunization with low (1 μg) antigen dose, the response is limited to the nodes draining the airways. When these low antigen doses are used, responses are not efficient as shown by the persistence of naïve antigen-specific CD4 T cells in the non-draining nodes. Nonetheless, "naïve" antigen-specific cells are lost from the lung; they reappear as activated cells at about 2.5 days after immunization, a response that is inhibited by FTY720. These results indicate that primary responses occur in the organized draining lymphoid tissue, as would be expected, but imply that the afferent lymphatics may be an important source for the delivery of naive CD4 T cells from tissues to the responding lymph nodes.
The role of the Septin cytoskeleton in primary T cell migration (95.3)
Authors: Gilden Julia K , Krummel Matthew F
Published: January 2023

The highly-conserved Septin family of GTP-binding proteins form filamentous networks in many cell types and have been implicated in a variety cell-biological processes, including membrane partitioning, molecular scaffolding, cytoskeletal regulation, and vesicle trafficking. We have previously reported that Septins are required for the regulation of shape, rigidity, motility, and membrane dynamics of a T cell line. Here, we extend our findings to primary cells. We found that during transmigration of a T cell through an endothelial monolayer under laminar flow, T cell Septins formed 'ribs' coplanar with the endothelial cells, a structure that may play a stabilizing role at this cell-cell contact. Biochemical and microscopic evidence showed that septin localization was altered following stimulation through CCR7 and through the TCR. Septins also played a role in the proper trafficking of T cells in vivo. Taken together, these results support a role for Septins in shape changes throughout the life of a T cell.
Both CD62L (L-selectin) and β7 integrin are Required for the Induction of Chronic Colitis (95.4)
Authors: Ostanin Dmitry V. , Bao Richard , Grisham Matthew B
Published: January 2023

L-selectin and beta7 integrin are T-cell associated molecules that are important for cell trafficking. The objective of this study was to assess the role of T-cell-associated CD62L and β7 in the induction of chronic colitis. Naïve (CD45RBhigh) CD4+ T cells were flow purified from the spleens of wild type (WT) or CD62L-/-xβ7-/- double deficient (DKO) mice and transferred into immunodeficient RAG-1-/- recipients. Transfer of WT T-cells induced robust colitis at 8 wks (mean histological score±SEM: 13.2±0.6) whereas transfer of DKO T-cells induced little or no disease at 8 wks (2.9±0.7) and only mild colitis at 25 wks (8.2±2.1) post-transfer. Total CD4+ T-cell numbers were reduced in the colonic LP of DKO→RAG-/- vs. WT→RAG-1-/- mice at both time points; however intracellular staining revealed that the few DKO LP CD4+ T-cells at 8 and 25 wks still produced significant amounts of IFN-γ and IL-17 comparable to that produced by WT LP CD4+ T cells. Analysis of CFSE-labeled WT or DKO T-cells transferred into RAG-1-/- mice revealed a defect in short-term homing to the MLN and colonic LP, but not in the in vivo activation/proliferation. These data suggest that DKO T-cells fail to induce colitis because of defects in T-cell trafficking rather than defects in T-cell activation and production of pro-inflammatory cytokines (supported by PO143785 and CCFA1787).
Lymphocyte subset recruitment during an immune response differs between peripheral and mucosal lymphoid tissues (95.7)
Authors: Grailer Jamison J. , Conway Rochelle M. , Steeber Douglas A.
Published: January 2023

Lymphocyte recirculation through peripheral lymphoid tissue depends primarily on the adhesion molecules L-selectin and α4β7 integrin. While most naïve lymphocytes express both molecules, subset-specific differences exist. These differences, along with tissue-specific expression of vascular ligands, results in preferential migration patterns among subsets. In the present studies, lymphocyte subset migration into peripheral (PLN) and mesenteric lymph nodes (MLN) was examined following s.c. and oral immunization, respectively. Naïve CD4+ T cell migration into PLNs increased after s.c. immunization with KLH-alum, peaking at 72 h. Naïve CD8+ T cell migration started lower than CD4+ cells, but increased steadily following immunization, reaching equivalent levels after 7 days. By comparison, B cell migration increased little and plateaued after 24 h. Among memory/effector cells, CD8+ T cell migration increased gradually up to 5 days post-immunization, and decreased thereafter, while CD4+ T cells increased modestly and plateaued after 24 h. In contrast to activated PLNs, oral immunization with cholera toxin + KLH resulted in peak increases in migration of all T cell subsets into the MLN at 24 h post-immunization. Interestingly, no changes in migration into the Peyer's patches were observed. These results indicate that activated PLN and MLN have different recruitment kinetics for lymphocyte subsets.
Role of GIT2 in T cell migration and development (95.8)
Authors: Phee Hyewon , Mollenauer Marianne N. , Weiss Arthur
Published: January 2023

The G-protein-coupled receptor (GPCR)-kinase-interacting proteins (GIT) 1 and 2 are multi-domain proteins involved in diverse cellular processes. In fibroblasts, GIT is thought to regulate focal adhesion (FA) turnover, cell spreading, and motility through interaction with various proteins. In T lymphocytes, GIT interacts with PIX (Pak-interacting Rac/Cdc42 guanine exchange factor) and PAK (p21-activated kinase) and forms a trimolecular complex. Our understanding of the physiological roles of GIT in T cells is very limited.

To determine the role of GIT1 and GIT2 in vivo, we generated GIT1- and GIT2- deficient mice. GIT1 is highly expressed in the brain, heart and testes. GIT2 is the major isoform expressed in hematopoietic cells. We found that GIT1 deficiency increased lethality, leading to perinatal death within 1-2 days after birth with a defect in brain development. GIT2-deficient mice were born healthy at normal Mendelian ratios. Interestingly, GIT2-deficient splenic T cells showed increased basal and chemokine-dependent migration. Furthermore, immature double positive thymocytes from GIT2-deficient mice showed increased basal and chemokine-induced migration. Since migration of thymocytes in the thymus has been tightly linked to their development, we crossed these mice to TCR transgenic mouse lines to better define the alterations in T-cell development and function by limiting compensation by repertoire selection. We found that the transition from the CD4 and CD8 double positive stage to CD4 single positive stage was greatly inhibited in the thymus of TCR transgenic GIT2-deficient mice. We are currently testing the hypothesis that GIT2 deficient-thymocytes have an altered migratory behavior leading to a defect in CD4 single positive thymocyte development.

*H.P. is supported by the Leukemia and Lymphoma Society Special Fellow Award.
Type I IFN-induced chemokine production and the regulation of immune effector cell trafficking from the bone marrow during acute MCMV infection (96.4)
Authors: Crane Meredith J , Hokeness-Antonelli Kirsten L , Salazar-Mather Thais P
Published: January 2023

Monocytes/macrophages are key innate effector cells in the response against murine cytomegalovirus (MCMV) infection. Activation of the chemokine receptor CCR2 on the monocyte/macrophage surface directs cell trafficking. In CCR2-deficient C57BL/6 mice, fewer monocytes/macrophages enter MCMV-infected livers. Consequently, downstream antiviral responses, such as NK cell recruitment, are impaired. Experiments were performed to better understand the origin of this defect. We hypothesized that CCR2 activation on bone marrow monocytes is required for release into the circulation, thus regulating their availability to the liver. We have demonstrated that monocytes accumulate in the bone marrow and are diminished in the circulation of CCR2-deficient mice. NK cells also accumulate in the bone marrow of CCR2-deficient mice, suggesting that impaired monocyte responses inhibit NK cell release. Monocyte mobilization from the bone marrow is likely coordinated by expression of the CCR2 ligands CCL2, CCL7 and CCL12. These chemokines are produced by F4/80+ bone marrow cells and detected in the serum in a type I IFN-dependent manner. These results further support the central role for type I IFN and chemokine-chemokine receptor interactions in establishing effective antiviral immune responses during MCMV infection.

Supported by NIH R01CA-102708 and the Nicole Rosenthal Hartnett '91 Graduate Fellowship.
Unique immune regulation by alternatively spliced IL-4 (97.3)
Authors: Atamas Sergei P , Lockatell Virginia , Todd Nevins W , Luzina Irina G
Published: January 2023

We have previously shown that IL-4δ2, a naturally occurring in humans and other species splice variant of IL-4, competes with IL-4 for receptor binding and inhibits some of IL-4 effects on lymphocytes and monocytes. Numerous groups reported that the levels and the ratio of IL-4δ2 and IL-4 mRNAs change in immune, inflammatory, and infectious diseases. It has been suggested that recombinant human (rh) IL-4δ2 can be used therapeutically to neutralize the harmful effects of excess IL-4 in vivo, although it was not clear whether IL-4δ2 mRNA expressing cells secrete IL-4δ2 protein. Transfection of HEK293 or Jurkat cells with IL-4δ2-encoding constructs resulted in IL-4δ2 protein secretion confirmed by LC/MS, ELISA, and Western blotting. To investigate whether IL-4δ2 has no independent activity and acts only as an inhibitor of IL-4, we overexpressed human IL-4δ2 in mouse lungs. Adenovirus-mediated gene delivery of IL-4δ2 caused lymphocytic infiltration similar to that in IL-4-overexpressing mice, as well as proinflammatory/Th1 changes in pulmonary milieu, with the induced levels of TNF-α, IL-1, IFN-γ, IL-12p40, and MCP-1 significantly exceeding those induced by gene delivery of IL-4. Germline deficiency of STAT6 had no effect, whereas deficiency of IL-4Rα significantly attenuated IL-4δ2-induced or IL-4-induced changes in the lungs. Stimulation of primary human T cells or PBMC, but not fibroblasts or A549 cells, with rhIL-4δ2 induced phosphorylation of Jak1, Jak3, and Tyk2, but not of STAT6. IL-4δ2 potently induced production of MCP-1 in human T cells and upregulated IL-4δ2 mRNA production in an autocrine fashion by several hundred fold. Thus, IL-4δ2 is secreted as a protein, acts as a potent autocrine regulator of inflammation and immunity in an IL-4Rα-dependent STAT6-independent fashion, and is a potential novel target for future therapies.
PARP-14 mediates IL-4-enhanced metabolic function and survival of B cells (97.4)
Authors: Cho Sung Hoon , Boothby Mark
Published: January 2023

IL-4 is survival factor for lymphocytes and other hematopoietic cells. Whether there are mechanisms of pro-survival signaling induced by IL-4 apart from PI3K-Akt activation is not fully clear. Our laboratory identified PARP-14, a poly-ADP-ribose polymerase subfamily member, as a Stat6-interacting protein. PARP-14 is highly expressed in lymphoid organs, influences B cell subset ratios as well as the IgA response to antigen, and has intrinsic ADP-ribosyltransferase activity. ADP-ribosyltransferases and PARPs catalyze mono- and poly-ADP-ribosylation, transferring ADP from NAD+ to target proteins. ADP-ribosylation is a post-translational modification used by bacterial exotoxins to impact signal transduction, or, in the case of the mammalian PARP-1, to influence gene transcription and DNA repair or trigger apoptosis. Almost nothing is known about biological roles or mechanisms of action of other mammalian PARPs. We now show that PARP-14 is essential for full survival signaling despite normal Akt activation in B cells. Investigation of the mechanism(s) revealed that PARP-14 mediates IL-4 induction of gene products known to promote cell survival, and also metabolic functions of B cells. Moreover, intervention that restored glycolytic activity in IL-4-treated B cells also rescued pro-survival signaling. These results reveal a novel link to metabolic regulation via a mammalian ADP-ribosyltransferase.
Helicobacter pylori Induces Gastric Epithelial Cell Production of TGF-β that Suppresses CD4+ T Cells (97.7)
Authors: Beswick Ellen J , Pinchuk Irina V , Schmitt David A , Reyes Victor E
Published: January 2023

CD4+ T cells are recruited to the gastric mucosa during H. pylori infection, but are generally hyporesponsive. CD4+CD25+FoxP3+ regulatory T cells (Treg) are present during infection and impact the CD4+ T cell response. We have previously shown that epithelial cell responses to H. pylori play an important role in inhibiting CD4+ T cell responses via the local induction of Treg cells from naïve CD4+ T cells. As TGF-β plays a role in the development of Tregs, we hypothesized that gastric epithelial cells (GECs) produce TGF-β during H. pylori infection that contributes to Treg development and suppresses local CD4+ T cell effector function. In order to investigate this, we examined GEC production of TGF-β after exposure to H. pylori and found them to produce significant levels of TGF-β that were partially dependant on the cag PAI virulence factor of H. pylori. TGF-β produced by GEC in response to H. pylori increased the local development of CD4+CD25+FoxP3+ Treg cells from naïve T cells in co-culture with GEC and decreased proliferation of activated CD4+ T effector cells in co-culture with GEC. These events were decreased when TGF-β neutralizing antibodies were present. The observations presented here introduce a novel mechanism used by H. pylori via gastric epithelial cells leading to inhibition of CD4+ T cells that may contribute to chronicity of infection.