bluuebunny/crossref_metadata_2025 huggingface dataset:
Delivery of signal 0 by dendritic cell-derived IL-6 promotes naïve T cell trafficking across high endothelial venules (P5098)
Authors: Fisher Daniel , Muhitch Jason , Lu Theresa , Evans Sharon
Published: January 2023

Dendritic cells (DC) are master regulators of adaptive immunity, providing cognate antigen (signal 1) and costimulation (signal 2) required for T cell activation in addition to cytokines (IL-12, TNF, IL-1 and IL-6; signal 3) that drive T cell expansion. Here we show that DC play an unexpected role in shaping the size of the naive T cell pool within draining nodes by promoting trafficking across vascular checkpoints termed high endothelial venules (HEV). Immunization with bone marrow-derived mouse DCs matured ex vivo by TLR4 or TLR9 agonists (LPS and ODN-CpG, respectively), but not a TLR2 agonist (Pam3CSK4), increased the density of two HEV homing molecules, peripheral lymph node addressin (PNAd) and intercellular adhesion molecule-1 (ICAM-1). Elevated PNAd and ICAM-1 supported increased trafficking of naïve T cells early in the adaptive immune response (12-48 h), preceding the peak phase of proliferative expansion of endothelial cells lining HEV in draining nodes. Loss of a single cytokine, IL-6, from CD11c+CD11b+CD8α- immunizing DC abrogated the early effects on HEV adhesion. Induction of HEV adhesion further required CCR7 chemokine receptor-dependent access of IL-6-producing DCs to afferent lymphatics at the immunization site. We propose that DC-derived IL-6 functions as a previously unrecognized signal 0 to fine-tune HEV adhesion, thereby increasing the probability that rare Ag-specific T cells receive activating signals during the initial phase of adaptive immunity.
CRK/CRKL proteins regulate T cell migration and activation (P5104)
Authors: Huang Yanping , Clark Fiona , Park Tae-ju , Curran Tom , Burkhardt Janis
Published: January 2023

Crk adaptor proteins include three family members: CrkI and CrkII are from the alternatively spliced crk gene, and CrkL is from the crk-like gene. These ubiquitously expressed proteins play important roles in multiple signaling pathways. However, their functions in T cells are poorly characterized, partly because germline-deficiency for Crk or CrkL leads to lethality in mice. By breeding Crkflox/flox:CrkLflox/flox mice to CD4Cre mice, we generated mice conditionally deficient for both Crk and CrkL in mature T cells. These mice are viable and fertile. We find that T cells lacking both Crk and CrkL develop normally, but are defective in chemotactic responses to multiple chemokines. Further analysis revealed that while Rac1 activation is intact, Rap1 activation is perturbed, as is tyrosine phosphorylation of the Rap1 GEF C3G. Consistent with this, these cells exhibit reduced chemokine-stimulated adhesion to ICAM-1 and fibronectin, and defective diapedesis. Using a cutaneous DTH model, we show that Crk and CrkL are required for efficient T cell migration to sites of inflammation. In addition to regulating T cell migration, Crk proteins also control T cell activation. Double deficient T cells showed significant proliferative defects upon allogeneic antigen stimulation. These results demonstrate a key role for Crk proteins in T cell function. We are currently working on defining Crk/CrkL-dependent pathways and analyzing functions of Crk proteins in singly deficient T cells.
Expression of Leukotriene B4 receptor 1 on CD8+ T cells is essential for their migration to tumors, immune-surveillance and cancer immunity (P5106)
Authors: Sharma Rajesh , Chheda Zinal , Jala Venkatakrishna , Haribabu Bodduluri
Published: January 2023

The role of Leukotriene B4 (LTB4) receptor 1, BLT1 as a pro-inflammatory mediator in various inflammatory diseases is well known. However, its role in regulating immune responses to tumor is yet unknown. Here, we studied the role of BLT1 in CD8+ T cell mediated anti-tumor response using implantable TC-1 cervical cancer and B16F10 melanoma mice models. BLT1−/− mice had significantly accelerated tumor growth, reduced survival, and decreased CD8+ T cells as well as their effector molecules such as IFN-γ, granzyme-B and IL-2 within tumors as shown by flow cytometry and confocal microscopy, compared to the WT controls. Moreover, the tumor growth patterns in BLT1−/− mice was similar to WT mice that were depleted of their CD8+ T cells. Evaluation of CTL responses by in vivo killing assay revealed no differences in the cytolytic ability between WT and BLT1-/- CD8+ T cells. Adoptive transfer of tumor primed CD8+ T cells from WT but not BLT1-/- mice reduced tumor growth in Rag2-/- mice. In Rag2-/- mice, while the numbers of WT or BLT1-/- CD8+ T cells in dLN remain similar, the recruitment of BLT1-/- CD8+ T cells into tumor was dramatically reduced as compared to WT CD8+ T cells. Analysis of CD8+ T cells from the dLN revealed no difference in the expression of other T cell chemokine receptors. These results demonstrate a previously unidentified, yet crucial role of BLT1 on CD8+ T cell in their effective migration into the tumor and hence in immune-surveillance and anti-tumor immunity.
Mast cells and the Stat5 activation pathways are required for allergen-induced and spontaneous skin inflammation (P6026)
Authors: Ando Tomoaki , Xiao Wenbin , Tomimori Yoshiaki , Hong Hong , Yamashita Hirotaka , Kimura Miho , Kawakami Yuko , Kawakami Toshiaki
Published: January 2023

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Repeated epicutaneous applications of allergen (Dermatophagoides farinae extract) and staphylococcal enterotoxin B induced AD-like skin lesions on mice. We showed that this dermatitis requires mast cells and T cells, but not B cells or eosinophils, for full expression of dermatitis. The clinical severity of dermatitis was correlated with numbers of mast cells. GM-CSF, TSLPR and FcϵRI were required to attain maximal clinical scores. Moreover, high similarity was found in gene expression patterns in allergen-induced lesional skin and human AD skin, supporting the clinical relevance of our mouse model. We have also been studying spontaneously occurring AD-like skin lesions in phospholipase C (PLC)-β3-deficient mice. Mast cells, but not T or B cells, were indispensable for the development of skin lesions. Remarkable abundance of mast cells in the skin of PLC-β3-/- mice was due to the loss of an adaptor function of PLC-β3 to inhibit IL-3-mediated proliferation of mast cells via SHP-1-mediated suppression of Stat5 activity. Consistent with this, increased numbers of mast cells with phosphorylated Stat5 were found in lesional skin of PLC-β3-/- mice. Therefore, this study demonstrates a critical importance of mast cells in AD and suggests that AD development is controlled by elements of the Stat5-activating pathways, as well as other components (PLC-β3 and SHP-1) of the recently discovered multi-molecular complex SPS.
Frequent ingestion of the curry spice curcumin inhibits mastocytosis and suppresses intestinal anaphylaxis in a murine model of food allergy (P6034)
Authors: Mathias Clinton , Shah Sagar , Statham Rebecca , Gambrah Amos , Henchey Elizabeth , Schneider Sallie
Published: January 2023

IgE antibodies and mast cells play critical roles in the establishment of allergic responses to food antigens. Curcumin, the active ingredient of the curry spice turmeric, has anti-allergic properties, and thus may have the capacity to modulate mucosal mast cell activation and function during allergic responses. Here we show that frequent ingestion of curcumin inhibits mast cell expansion and suppresses intestinal anaphylaxis in a murine model of ovalbumin (OVA)-induced food allergy. Intragastric exposure to OVA in i.p. sensitized BALB/c mice induced a robust IgE-mediated response accompanied by enhanced OVA-IgE levels, intestinal mastocytosis, elevated serum mMCP-1 and acute diarrhea. In contrast, mice exposed to oral curcumin throughout the experimental regimen appeared to be normal and did not exhibit intense allergic diarrhea or a significant enhancement of OVA-IgE and intestinal mast cell expansion and activation. Furthermore, allergic diarrhea, and mast cell activation and expansion were also suppressed in mice exposed to curcumin during the challenge phase alone, despite the presence of normal levels of OVA-IgE. This suggested that curcumin may have a direct suppressive effect on intestinal mast cell homeostasis and function during food allergy. In summary, our data demonstrate a protective role for curcumin during allergic responses to food antigens, suggesting that frequent ingestion of this spice may modulate the outcome of disease in susceptible individuals.
Expression of ADAM10 on mast cells: effects on IgE and stem cell factor activation (P6010)
Authors: Faber Travis , Pullen Nicholas , Fernando Josephine , Kolawole Motunrayo , Barnstein Brian , Conrad Daniel , Ryan John
Published: January 2023

Mast cells are innate immune cells best characterized for their role in allergy. In response to cross-linking of the high-affinity IgE receptor, Fc{epsilon}RI, mast cells release mediators such as histamine, leukotrienes, IL-6, and TNF-α. Additionally, mast cells are widely noted for their secreted proteases like chymases and cathepsins. We have decided to investigate the role that the protease A Disintegrin and Metalloproteinase 10 (ADAM10) plays on murine mast cells. To date, we have discovered mast cells express ADAM10 in vitro and in vivo, and that Treg cytokines TGF-β1 and IL-10 down regulate mast cell ADAM10. IL-10 effects depend on STAT3. ADAM10 deletion results in abrogated mast cell migration through Collagen IV to stem cell factor, but not to antigen. Also, ADAM10 deletion decreases IL-6 release post cross-linking, but does not alter other commonly released cytokines such as TNF-α, IL-13, and MIP-1α. Because of these findings, we hypothesize that ADAM10 plays a pro-inflammatory role in mast cell migration and also in allergic disease.
Early growth-response 2 is critical for mast cell-directed CD4+ T cell migration via specific regulation of CCL1 production. (P6012)
Authors: Lin Tong-Jun , Wu Zhengli , MacNeil Adam , Junkins Robert , Berman Jason
Published: January 2023

Mast cells are well positioned in host tissue for detecting environmental signals, including allergens, leading to activation of the high affinity IgE receptor FcϵRI, and initiating a signaling cascade that perpetuates the production of biologically potent mediators including chemokines. We have identified a novel target of mast cell FcϵRI activity in the transcription factor early growth-response 2 (Egr2) and sought to characterize its function therein. Egr2 was transiently activated following FcϵRI-mediated signaling, targeted the promoter of the chemokine CCL1, and was critical for allergen-induced mast cell CCL1 production. Egr2-deficient mast cells were incapable of directing CD4+ T cell migration via the CCL1-CCR8 axis. In a model of allergic asthma, reconstitution of mast cell-deficient mice with Egr2-deficient mast cells demonstrated that mast cell Egr2 was essential for migration of CD4+ T cells to the inflamed lung. These findings position Egr2 as a critical regulator of mast cell-directed CD4+ T cell migration.
Uracil nucleotides enhance the degranulation of human basophils induced by anti-IgE antibody via a purinergic receptor (P6016)
Authors: Nakano Manabu , Kudo Fujimi , Nakamura Toshiya , Kasai Kosuke , Ito Kyoko , Takami Hideki , Ito Koichi
Published: January 2023

The P2Y receptors (P2YRs) are a family of G protein-coupled purinergic receptors that are stimulated by ATP, ADP, UTP, UDP, and UDP-G, and their interaction induces a wide range of biological effects in various cells through phospholipase C activation, inositol triphosphate generation, and Ca2+ release from intracellular stores. In our previous study, we demonstrated that in neutrophils, ATP regulates chemotaxis and phagocytosis via P2YR. In the present study, we examined the distribution and function of P2YR in another innate immune cell type, basophils, the degranulation of which is associated with type I allergy. To date, P2YR have been subdivided into 8 different subclasses: P2Y1, 2, 4, 6, 11, 12, 13, and 14. Of these 8 subclasses, our RT-PCR results showed the predominant expression of P2Y2, P2Y4, and particularly P2Y6, which are known as uracil nucleotide-sensitive P2YRs. Interestingly, HPLC analysis indicated that basophils spontaneously secrete UTP, the ligand for P2Y2 and 4, whereas UDP, the ligand for P2Y6, is probably generated from UTP in a metabolic pathway. Although the addition of UTP and UDP did not directly induce basophil degranulation, these nucleotides clearly enhanced the degranulation induced by anti-IgE cross-linking. These results suggest that uracil nucleotides act as potent inducers of histamine release in basophils.
Programming of immunological memory in NK cells (P1419)
Authors: Sun Joseph
Published: January 2023

Immunological memory is a hallmark of the adaptive immune system. Although natural killer (NK) cells have traditionally been classified as a component of the innate immune system, they share many developmental and functional similarities with B and T cells. NK cells have recently been shown in mice and humans to experience a selective education process during development, undergo a clonal-like expansion during virus infection, generate long-lived progeny (i.e. memory cells), and mediate recall responses against previously encountered pathogens - all characteristics previously ascribed only to B and T cells in mammals. The molecular events that control NK cell responses are not well understood. Using a mouse model of cytomegalovirus infection, we demonstrate that both pro-inflammatory IL-12 and type I-IFN signaling are indispensible and play non-redundant roles in the generation of virus-specific NK cell memory. Furthermore, we discovered that antigen-specific proliferation and protection by NK cells (100-1000 fold) is mediated by the transcription factor Zbtb32, which is induced by pro-inflammatory cytokines and promotes a cell cycle programme in NK cells by repressing the anti-proliferative factor Blimp-1. A greater understanding of the molecular mechanisms controlling NK cell responses will provide novel strategies for tailoring vaccines to target infectious disease.
Maternal high fat diet alters Th17 responses and bacterial colonization in newborn mice. (P3156)
Authors: Mirpuri Julie , Raetz Megan , Wilhelm Cara , Sturge Carolyn , Yarovinsky Felix
Published: January 2023

The American diet is dominated by high-fat foods and 50% of women of childbearing age are either overweight or obese. Studies in adult mice show that the Th17 response is important in regulating the microbiota and modulating development of diet-induced obesity. The effect of maternal high fat diet on the acquisition of the microbiota and on the Th17 cytokine responses are unknown. Objective: To define the effect of maternal high fat diet on acquisition of the microbiota and Th17 responses in offspring. Methods: 454 pyrosequencing of bacterial 16S rRNA gene in colonic fecal contents from offspring of mothers on a high fat diet and regular diet at 1-, 3-, and 6-weeks of age. Analysis of Th17 and inflammatory cytokines in the colon of offspring by qRT-PCR and ELISA. Results: 1-week-old pups from mothers on high fat diet had a unique microbiota that was dominant in Firmicutes and segmented filamentous bacteria compared to control newborn mice. Offspring of mothers on high fat diet showed a persistent change with age in their colonic microbiota even when maintained on a regular diet. There was increased production of inflammatory cytokines in pups born to mothers on high fat diet, with markedly increased expression of IL-17 and IL-22. Conclusions: Acquisition of the microbiota was unique in pups of mothers on a high fat diet with increased baseline inflammation and an altered Th17 response. Long-term consequences are under investigation and may contribute to programming obesity.
Evaluation of a mouse model of food allergy for determination of threshold oral elicitation doses (P6224)
Authors: Gangur Venugopal , Gonipeta Babu , Para Radhakrishna , Kim Eunjung , Parvataneni Sitaram
Published: January 2023

Food allergies are a significant public health problem because they trigger life-threatening systemic allergic reactions. We have previously reported a novel mouse model that uses transdermal exposure (TDE) to nut protein for sensitization followed by oral challenge (OC) to elicit systemic reactions. Here we evaluated the utility of this model to determine the oral threshold doses for hazelnut (HN). Groups of mice (n=8-10/group) were sensitized with 2, 3, 4 or 5 TDE to HN or saline, followed by OC with HN to induce systemic anaphylaxis. The data demonstrated that a minimum of 4 TDE to HN and a definition of >/=1 o C drop in rectal temperature (RT) at 30 minutes post OC, ensures 100% positive responses and <10% false positive responses. We then evaluated threshold oral elicitation doses in three scenarios: (1) homogeneous 4 TDE to allergen, followed by repeated OC with different doses of HN at weekly intervals; 2) homogeneous 4 TDE to allergen, followed by single OC per dose per group; and 3) heterogeneous (i.e., 4, 5 or 6) TDE with HN followed by single/repeated OC to simulate the human exposure conditions that are generally heterogeneous. The percent responder curves were established for each case and the NOAEL and LOAEL (25 and 50% responses) were estimated. The NOAELs were </=125 mg/Kg; the 25% response LOAELs ranged from 270-312 mg/Kg; and the 50% response LOAELs ranged from 362-412 mg/Kg.
The effect of simulated gastric and intestinal digestion on temporal stability and immunoreactivity of peanut, almond and pine nut protein allergens (P6206)
Authors: Toomer Ondulla , Do Andrew , Pereira Marion , Williams Kristina
Published: January 2023

Current models of digestibility utilize pepsin stability to assess the safety of allergenic versus non-allergenic food proteins. Dietary protein digestion in vivo, however, requires acid denaturation and protease cleavage by pepsin, trypsin, and/or chymotrypsin. The ability of this approach to identify food protein stability in the mammalian gut may be limited. We determined the temporal stability and immunoreactivity of almond, pine nut, and peanut allergenic proteins using simulated physiologic gastric and intestinal digestive conditions in vitro. Gel electrophoresis and immunoblot analyses were used to determine protein stability and immunoreactivity, respectively. Peanut, almond, and pine nut proteins were pepsin- and pancreatin-stable, and immunoreactive for up to 1 h after initiating digestion. Moreover, successive acid denaturation, and pepsin and pancreatin cleavage were necessary to hydrolyze these allergenic proteins and reduce their IgG and IgE-binding capacity, which suggests that digestibility models must be improved for more accurate safety assessment of food allergens.
C1q and adiponectin trigger efferocytosis via a shared pathway (P1287)
Authors: Bohlson Suzanne , Galvan Manuel
Published: January 2023

Failure to efficiently clear apoptotic cells (efferocytosis) is associated with autoimmunity. Complement component C1q is required for efferocytosis, and deficiency in C1q leads to development of autoimmunity. We recently identified a novel molecular mechanism for C1q-dependent efferocytosis in mouse macrophages. We found that C1q elicited the expression and function of Mer tyrosine kinase and the Mer ligand, Ga6: a receptor-ligand pair that mediates efferocytosis. To define the signal transduction pathway downstream of C1q, pathway analysis was performed using the transcriptome from C1q-treated macrophages. This analysis revealed that the adiponectin signaling pathway was significantly upregulated with C1q. Adiponectin is a well characterized adipokine with critical roles in glucose and fatty acid metabolism, and it is structurally homologous to C1q. Similar to C1q, adiponectin triggered expression of Mer that correlated with enhanced engulfment of apoptotic cells, and a soluble Mer-Fc fusion protein inhibited adiponectin-dependent efferocytosis. Furthermore, C1q triggered activation of AMP kinase, a central regulator of adiponectin signaling and cellular metabolism. Inhibition of AMPK signaling resulted in inability to upregulate C1q-dependent Mer expression in macrophages. Our results suggest that C1q and adiponectin activate macrophages via a common signaling pathway, and this pathway may contribute to the molecular crosstalk between inflammation and metabolism.
Innate immunity in pancreatic cancer patients and changes after immuno-enhancing nutrition (P1308)
Authors: Slotwinski Robert , Dabrowska Aleksandra , Lech Gustaw , Slodkowski Maciej
Published: January 2023

The purpose of our study was to investigate the changes of innate immunity after immunonutrition in pancreatic cancer patients. Sixty-six patients operated on for pancreatic cancer were included. Before and after preoperative nutrition TLR4,NOD1,MyD88 and TRAF6 gene expression in peripheral blood leukocytes were measured using RT Real-Time PCR. The randomized studies were performed in patients who received the pre-operative enteral nutrition (immunonutrition with glutamine or PAFAs and standard supplementation) and in patients without pre-operative nutrition. Results: TLR4, NOD1 and TRAF6 gene expressions were significantly higher (before surgery and nutrition respectively p= 0.008; 0.0008 ; 0.01) and MyD88 was significantly lower (p= 0.0002) as compared to the healthy controls. Treatment with glutamine was associated with decreased TLR4 gene expression (before versus after immunonutrition p=0.01). After PUFAs and standard nutritional support no changes in gene expressions were detected. Conclusions: Overexpression of TLR4, NOD1 and TRAF6 gene of pancreatic cancer patients may reveal the up-regulation of the innate antibacterial response. This disorders may contribute to cancer progression and increased susceptibility to postoperative septic complications. Preoperative enteral immunonutrition containing glutamine by reducing the overexpression of TLR4 gene may reduce the susceptibility of malnourished patients to post-operative septic complications.
The anti-inflammatory effects of vitamin D in mammary epithelial cells through sCD14 (P1329)
Authors: Simmons Katrina , Sellati Timothy , Welsh JoEllen
Published: January 2023

Many studies have shown vitamin D to regulate immune cell responses, yet few have investigated whether vitamin D alters the immune responses of epithelial cells in barrier tissues, such as the mammary gland, to alter immune signaling within tissue microenvironments. Microarray screening and qPCR of human mammary epithelial cells treated with 1α,25-dihydroxyvitamin D (the active form of vitamin D) identified CD14 as a 1,25D induced gene. To follow up these findings, we examined whether 1,25D induced the expression of membrane CD14 or soluble CD14 and found that 1,25D increased the accumulation of sCD14 in a dose- and time- dependent manner but did not alter mCD14. To determine the effects of 1,25D-induced sCD14 from HME cells on Toll-like receptor 4 activation and cytokine secretion from monocyte-derived macrophages, we incubated HEK-Blue TLR4 reporter cells and differentiated THP1 macrophages in conditioned media from 1,25D-treated HME cells ± purified E.coli lipopolysaccharide. We found that conditioned media from 1,25D-treated cells had no significant effects on TLR4 activation in HEK Blue cells yet did promote the secretion of IL-10 from differentiated THP-1 macrophages in the presence of E.coli lipopolysaccharide. These results are the first to demonstrate vitamin D’s effects on the expression and secretion of CD14 in mammary epithelial cells and support the hypothesis that vitamin D promotes an anti-inflammatory environment via secretion of sCD14 in mammary tissue.
Enhanced ICOS-L expression on DC in IL-10 deficiency and its impact on T cell subsets in respiratory tract infection (P1332)
Authors: Gao Xiaoling , Yang Jie , Yang Xi
Published: January 2023

Association of ICOS-L expression and IL-10 production by dendritic cells (DCs) has been commonly found in infectious disease models. The DCs with higher ICOSL expression and IL-10 production are reportedly more efficient in inducing regulatory T cells (Treg). To test whether IL-10 production and ICOS-L expression has a causal relationship, we examined ICOS-L expression by DC in IL-10 knockout (KO) mice in Chlamydia muridarum (Cm) lung infection. Moreover, we examined the development of T cell subsets, including Treg, Th17 and Th1 cell, in the condition of IL-10 deficiency and its relationship with ICOSL/ICOS signaling following infection. Surprisingly, we found that the IL-10 KO mice exhibited significantly higher ICOS-L expression by DCs. And the IL-10 KO mice showed lower Treg but higher Th17 and Th1 responses. Further analysis of Th17 and Th1 cells showed that only the Th17 response was dependent on ICOS signaling. Consistently, most of the Th17 cells were ICOS+, while most of the Th1 cells were ICOS- in the infected mice. The data suggest that ICOSL expression is independent of IL-10 production and that ICOS-L expression on DC in the presence or absence of IL-10 co-stimulation may respectively promoting Treg or Th17 response, without significant impact on Th1. The data also suggest that the enhanced protection in IL-10 KO mice may be due to the synergy of Th17 and Th1 responses.
MKK3/P38 is essential for cHSP60-induced inflammatory response (P1341)
Authors: Shi Liyun , Kang Yanhua , Wang Fang , Lu Zhe , Wang Cuili
Published: January 2023

Heat shock protein (HSP) 60 is the major component of Chlapidia pneumonia (C. pneumonia), and has been recognized to be related with inflammatory disorders clinically. Nevertheless, much remains to be learned about the immunopathologic property of C. pneumonia HSP60 (cHSP60). Here we report that the MAPK kinase 3 (Mkk3) is required for cHSP60-induced lung inflammation in mice, as mice with deletion of Mkk3 displayed profoundly reduced infiltration of neutrphils, production of proinflammaoty mediators, and inflammatory symptoms in lung tissue. P38 is selectively activated by Mkk3, and plays a critical role in activating production of inflammatory cytokines through transcriptional and post-transcriptional mechanisms. We also identified MSK1 to act downstream of p38 and contribute to the inflammatory response upon cHSP60 stimulation. Specific knockdown of MSK1 led to a defective phosphorylation of NF-κB/RelA at Ser276, causing the alleviated inflammatory reaction. We thus reveal the biological function of cHSP60 in vivo as well as in in vitro, and establish that MKK3/p38/MSK1 axis is essential for its pathological role, and maybe C. pneumonia-associated inflammatory diseases.
The novel cytokine IL-17D at the intersection of cellular stress and immune activation (P1364)
Authors: Saddawi-Konefka Robert , O'Sullivan Timothy , Gross Emilie , Tran Miller , Bui Jack
Published: January 2023

Our laboratory has recently identified a novel role for the cytokine IL-17D as an important mediator of the antitumor immune response, and the loss of IL-17D as a key component of tumor progression. Our data show for the first time that overexpression of the cytokine IL-17D is sufficient to induce tumor rejection or growth delay. Tumors expressing IL-17D displayed an increased infiltration of natural killer cells, which gave way to a commensurate increase of M1-type macrophages. While examining the regulation of IL-17D, we came across the surprising finding that IL-17D can be induced by oxidative stress signals via the transcription factor nrf2. Nrf2, responsible for regulating the expression of the anti-oxidant response element genes, is known as the primary cellular responder to oxidative stress. Although the role of nrf2 in cancer biology has been well studied, the role of nrf2 in the regulation of immune responses (especially via IL-17D) has yet to be defined. We find that nrf2 is necessary to induce IL-17D expression in a variety of tumor and non-tumor cells, and that this regulation is active in both tumor progression and viral infection. These studies constitute an intriguing and novel connection between oxidative cellular stress and immune activation.
The development and effector state of innate type-2 lymphocytes is orchestrated by a Gfi1-dependent regulatory network (P1367)
Authors: Lesch Justin , Yan Donghong , Zhou Meijuan , Eastham-Anderson Jeffrey , Diehl Lauri , Lee Wyne , Xu Min , DeVoss Jason , Spoonder Chauncey , Singh Harinder
Published: January 2023

Innate type-2 lymphoid cells (ILC2s) have been shown to play a vital role in protective immune responses against helminthic parasites. The regulatory networks that orchestrate the expansion and effector state of ILC2s are largely unknown. Here we report that the transcriptional repressor Growth factor independence-1 (Gfi1) functions to control the development of ILC2s and the proper specification of their effector state in response to Nippostrongylus brasiliensis infection, papain challenge, and IL33- or IL25 instigated inflammation. Consistent with these findings, IL5 and IL13 levels are reduced and eosinophilia, goblet cell hyperplasia and worm clearance are severely impaired in the peritoneal cavity and lung of Gfi1-/- mice. Analysis of ILC2s reveals a stringent requirement for Gfi1 in their response to IL33 and suggests a prominent role for this signaling pathway in the lung. Strikingly, conditional inactivation of Gfi1 in ILC2s results in reduced expression of IL5 and the mis-expression of IL17. Given that Gfi1 is implicated in reciprocally regulating Th2 and Th17 cell fates, our results reveal it to be a conserved regulatory component which functions in both innate and adaptive immune cells.
A critical role for neddylation in regulating dendritic cell function. (P1369)
Authors: Mathewson Nathan , Toubai Tomomi , Sun Yaping , Tamaki Hiroya , Wang Ying , Oravecz-Wilson Katherine , Kapeles Steven , Hou Guoqing , Reddy Pavan
Published: January 2023

Post-translational modifications (PTMs) of proteins modulate cellular processes. However, the role, specifically that of neddylation (a type of PTM), in regulating immune cells is not known. Neddylation of Cullin-RING ligase (CRL) protein modulates the process of ubiquitination. Thus, CRL activity is critical for the quotidian functions of cells. We explored the role of neddylation in dendritic cells (DCs) utilizing the small molecule inhibitor of neddylation (MLN4924) and the knockdown of SAG, a critical adapter element of CRL. Inhibition of neddylation in the DC significantly reduced the expression and release of several proinflammatory cytokines (TNFα, IL-6) and significantly reduced the ability of DCs to stimulate T cells (P<0.001) following stimulation by several TLR and NLR ligands. This suppression of DC functions was more than that caused by the known anti-inflammatory drug dexamethasone. Mechanistic studies showed that reduction of IκB degradation was critical for the suppression of DCs by the inhibitors of neddylation. This was further confirmed by the absence of NF-κB translocation and function. However, MAPK/ERK pathway was unperturbed by the inhibition of neddylation. Our results demonstrate a novel biological role of inhibition of the PTM, neddylation, in regulating DC functions.
Programmed cell death 4 is an inhibitor of autophagy via targeting ATG5 (P1371)
Authors: Zhang Lining
Published: January 2023

PDCD4 (programmed cell death protein 4), an inhibitor of protein transcription and translation, is involved in tumor and inflammation diseases. However, its underlining mechanisms remain unclear. Autophagy, an evolutionarily conserved catabolic process, is reported to regulate survival and death of cells. Here, we demonstrated that PDCD4 inhibits autophagy in multiple cells including immune cells and tumor cells, by which it controls cell fate. Mechanically, PDCD4 suppressed translation of an essential autophagy related gene ATG5 by its MA-3 domain. Our data indicate that PDCD4 is endogenous inhibitor of Atg5.
Phospholipid scramblase 1 activity mediates phospholipid alterations leading to an endothelial response following hypoxia and reoxygenation (P1295)
Authors: Archer Slone Emily , Fleming Sherry
Published: January 2023

Ischemia, lack of blood flow, and reperfusion, return of blood flow, is a common phenomenon affecting millions of Americans each year. Roughly 30,000 Americans per year experience intestinal ischemia-reperfusion (IR), which is associated with a 70-80% mortality rate. Previous studies of the intestine established a role for innate immune cells and naturally occurring antibodies (Ab) in IR-induced pathology. Furthermore, administration of two monoclonal Ab that recognize phospholipids (PL) or the PL binding protein β2-glycoprotein I (β2-GPI) restores tissue damage in IR-resistant Rag-1-/- mice to wildtype levels. These data indicate involvement of a lipid or lipid-like moiety in mediating IR-induced damage. We hypothesized that PL scramblase 1 (PLSCR1), a protein regulating bilayer asymmetry, is involved in altering the PL of endothelial cells during hypoxia leading to β2-GPI binding and subsequent inflammation. To test our hypothesis, an endothelial cell line, MS1, was subjected to hypoxia (1% O2) followed by reoxygenation as an in vitro model of IR. PLSCR1 transcription, protein expression and activity were assessed. Mass spectrometry detected PL alterations and β2-GPI binding was confirmed by immunohistochemistry. Inflammatory markers including prostaglandins were also evaluated. Our data demonstrate hypoxia and reoxygenation-induced PLSCR1 activity in PL scrambling of endothelial cells. The altered PL arrangement enhances β2-GPI binding and leads to intestinal IR pathology.
Regulation of lytic granules for normal cytotoxic T lymphocyte function (P1140)
Authors: Pattu Varsha , Halimani Mahantappa , Peuschel Monika , Krause Elmar , Rettig Jens
Published: January 2023

Cytotoxic T lymphocytes (CTLs) function to kill bacterial and viral infected target cells by releasing cytotoxic components such as perforin and granzymes that are contained within lytic granules, into the infected cell. Release occurs via the fusion of lytic granules at the contact zone between infected cell and CTL, the immunological synapse (IS). Defect in the fusion of lytic granules results in the dysfunction of CTLs, the underlying cause of the fatal disease Familial Hemophagocytic Lymphohistiocytosis (FHL). In order for fusion to occur, lytic granules must first arrive, dock and prime at the IS. Mutations within Munc 13-4, a priming factor, for lytic granules, results in FHL subtype-3. We have identified the presence of additional priming factors in CTLs. Targeted gene knockouts of these priming factors resulted in defects in lytic granule fusion at the immunological synapse. These findings demonstrate the presence of more regulatory mechanisms for the most critical event in CTLs, lytic granule fusion. Using high-resolution microscopy and evanescent wave imaging we aim to dissect the precise regulation of lytic granule fusion, which in turn would give us more insight into the regulation and function of CTLs.
Context specific regulation of microRNA targets in the immune system (P1142)
Authors: Loeb Gabriel , Canner David , Khan Aly , Darnell Robert , Leslie Christina , Rudensky Alexander
Published: January 2023

MicroRNAs (miRNAs) are key gene expression regulators—their misexpression can cause both immune dysfunction and cancer. However, most miRNA targets remain unknown, limiting mechanistic insight. Combining genetic and biochemical approaches we generated a transcriptome-wide map of miR-155 targets in T cells. High throughput sequencing of cross-linked immunoprecipitated (HITS-CLIP) Argonaute bound transcript fragments in miR-155 sufficient and deficient cells identified hundreds of direct targets for this miRNA in T cells. This map shed insight on known and novel miRNA phenotypes and demonstrated that miRNAs directly regulate transcripts without canonical target motifs. In addition to T cells, miR-155 is expressed in many innate and adaptive immune cells following activation and its deletion leads to varied cell-type specific phenotypes. We hypothesized that miRNAs have diverse functions in different contexts through regulation of different targets. To reveal the extent and mechanism of context specific miRNA targeting, we employed RNA-seq and HITS-CLIP in wild type and miR-155 deficient T cells, B cells, and dendritic cells. Furthermore, we employed polyA-seq to determine how alternative polyadenylation affects context specific targeting. Immune system complexity is generated from a limited set of genes; by revealing how regulatory factors are repurposed in different cell types, our studies will provide insight into fundamental mechanisms of immunological gene regulation.
NBR1 in the regulation of T cell development and differentiation (P1000)
Authors: Yang Jun-Qi , Liu Hongzhu , Diaz-Meco Maria , Moscat Jorge
Published: January 2023

NBR1 (neighbor of BRCA1 gene 1) is a newly discovered signaling molecule containing a Phox and Bem1 (PB1) domain. PB1-domains are protein interaction modules. PB1 domain-containing molecules participate in diverse biological processes, which also include p62, Par-6, MEK5a, MEKK3, PKCζ and PKC λ/ι besides NBR1. We have recently reported that NBR1 regulates Th2 function and allergic airway inflammation. However, the role of NBR1 in the T cell development and in the regulation of other CD4+ T cell subsets remains to be elucidated. With the use of CD4+ T cell specific conditional NBR1-deficient mice, we found that NBR1 did not affect T cell development. Thymocyte subpopulations (double negative, double positive, CD4 or CD8 single positive cells) were similar between conditional NBR1-deficient mice and their WT littermates. CD4+ and CD8+ cells in the spleen and blood also did not differ significantly between these two groups. Furthermore, NBR1-deficiency did not affect the development of natural Treg in vivo and the differentiation of induced Treg in vitro. However, NBR1 was found selectively regulate Th2 function without affect Th1 cells. Interestingly, our preliminary data indicated that NBR1-deficiency might affect Th17 differentiation in vitro. These data suggest that NBR1 has no effect on T cell development, whereas it may selectively affect some CD4+ T cell subsets, especially Th2 and Th17. Further study is needed to elucidate the underlying mechanisms.
Insulin impairs regulatory T cell function: implications for obesity (P1020)
Authors: Han Jonathan , Patterson Scott , Ehses Jan , Levings Megan
Published: January 2023

Chronic inflammation is known to drive metabolic dysregulation in obesity and type 2 diabetes. Although the precise origin of the unchecked inflammatory responses in obesity is unclear, it is known that over-production of pro-inflammatory cytokines such as TNF-α by innate immune cells has a key role in the development of metabolic dysfunction. One key hallmark of obesity is high levels of the pancreatic hormone insulin, and we hypothesized that there may be an unknown link between hyperinsulinemia and chronic inflammation. Here we show that high levels of insulin impair the ability of regulatory T cells to suppress inflammatory responses via effects on the AKT/mTOR signaling pathway. Insulin strongly activates AKT/mTOR signalling in regulatory T cells, leading to specific inhibition of the production of the anti-inflammatory cytokine IL-10. As a result, insulin hinders the ability of regulatory T cells to suppress the production of TNF-α by macrophages. Regulatory T cells from the visceral adipose tissue of hyperinsulinemic, obese mice also have a decrease in IL-10 production and a parallel increase in production of IFN-γ. These data suggest that the hyperinsulinemia associated with obesity may contribute to the development of obesity-associated inflammation via a previously unknown effect on regulatory T cells function.
PD-1, B7-H1 and IL-10 in protection from kidney ischemia reperfusion injury (P1022)
Authors: Kinsey Gilbert , Jaworska Katarzyna , Ratajczak Joanna , Stevens Brain , Huang Liping , Okusa Mark
Published: January 2023

Regulatory T cells (Tregs) protect the kidney from inflammation, tissue damage and loss of function induced by ischemia-reperfusion (IR) in an IL-10 and PD-1 dependent manner, but the mechanisms of action for Tregs in IR are not completely understood. We hypothesized that Treg PD-1 is engaged by its ligand B7-H1, leading to increased IL-10 production by Tregs, which suppresses inflammation and protects from kidney IR. Similar to our previous observation that WT Tregs lose their ability to protect mice from kidney IR if treated with a PD-1 blocking antibody (ex vivo, prior to adoptive transfer), PD-1 KO Tregs did not offer any protection from kidney IR. In a model of mild renal IR that does not result in significant renal dysfunction in isotype control antibody-treated mice (plasma creatinine (PCr) level: 0.5±0.1, n=5 vs. sham PCr: 0.3±0.1, n=3) blockade of B7-H1 prior to IR causes a marked reduction in renal function (PCr: 1.3±0.3, n=6) accompanied by increased acute tubular necrosis and accumulation of neutrophils in the kidney. Stimulation of isolated WT Tregs with plate-bound anti-CD3, in the presence of IL-2, resulted in significant production of IL-10. In contrast to our hypothesis, addition of plate-bound B7-H1 Ig fusion protein dose-dependently inhibited IL-10 production by WT Tregs. Taken together these results suggest PD-1 and its ligand B7-H1 interact to protect from ischemic kidney injury, but argue against enhanced IL-10 production by Tregs as the mechanism.
Farnesyltransferase inhibitors differentially affect cytokine secretion from CD4+ T cell subsets in mice (P1004)
Authors: Field Kenneth , McMichael James , Dieck Chelsea , Hayes Michael , Gaylo Alison , Crosby Erika
Published: January 2023

Farnesyltransferase inhibitors (FTIs) have been shown to modulate alloreactive immune responses in mouse models of transplantation (A.E. Gaylo, et al., Transplant Immunology 20 (2009) 163-170) and graft versus host disease (A.-K. Hechinger, et al. Haematoligica 98 (2013) 31-40). FTIs can block cytokine secretion from T cells and this activity may explain the drug’s immunomodulatory mechanism. FTIs could modulate immune responses by blocking T cell cytokine secretion thereby affecting the differentiation of stimulated CD4+ T cells into Th1, Th2, or other effector subtypes. To test this hypothesis, we have examined the ability of FTIs to affect cytokine secreation from various T cell subsets and T cell polarization in mice during an allorejection response. Using qPCR we quantified cytokine gene expression in graft-draining lymph nodes (GDLNs) isolated from mice during the rejection of an MHC class II-mismatched skin allograft (bm12 donors to B6 recipients). Using ELISpot assays, we also measured the number of alloreactive Th1 (IFN-γ producing) and Th2 (IL-4-producing) cells in GDLNs. In vitro T cell polarization of polyclonally-stimulated CD4+ T cells isolated from spleen was measured using IFN-γ, IL-4, IL-17, and FoxP3 intracellular staining. We found that FTI treatment differentially affects cytokine secretion from the CD4+ T cell subsets studied. These effects lead to differences in Th1/Th2 cell polarization that could explain the immunomodulatory activity of FTIs.
Intravenous transfer of MOG-pulsed, bone marrow-derived dendritic cells modulates expression of co-stimulatory molecules and cytokines by CD4+ T cells (P5217)
Authors: Zhang Guang-Xian , Zhou Joseph , Ciric Bogolijub , Rostami Abdolmohamad
Published: January 2023

Dendritic cells (DCs) can induce immune tolerance in vivo; however, the mechanisms of DC-mediated tolerance have not been fully elucidated. Here we demonstrate that intravenous (i.v.) transfer of bone marrow-derived DCs pulsed with MOG peptide blocks the development of experimental autoimmune encephalomyelitis (EAE). Transfer of MOG-pulsed DCs leads to down-regulation of IL-17A and IFN-γ production, while the production of IL-10 is up-regulated. The numbers of CD4+IL-10+IFN-γ+ T cells and CD4+FoxP3+IL-10+ regulatory T cells are also increased after MOG-pulsed DC transfer. Intravenous transfer of MOG-pulsed DCs down-regulates protein expression of OX40, CD154 and CD28 on CD4+ T cells, whereas expression of CD152, CD80, PD-1, ICOS and BTLA is up-regulated. Our results indicate that DCs pulsed with MOG inhibit CD4+ T cell activity via modulation of co-stimulatory signaling.
Characterization of a small molecule compound VPR-66 as an inverse agonist for ROR proteins (P5167)
Authors: Lee Hyun-Ku , McAlpine Jennifer , Chitta Sriram , Bouzida Djamal , Singh Gita , Rosenberg Jonathan , Alton Gordon , Singh Sujay
Published: January 2023

Retinoic Acid Receptor (RAR)-Related Orphan Receptors RORα, RORγ and RORγt are key transcription factors required for differentiation of naive CD4+ helper cells into Th17 cells that play an important role in driving autoimmune and inflammatory diseases by secretion of IL-17 and other pro-inflammatory cytokines. Recently development of the Th17 immunomodulators as potential therapeutics for autoimmune diseases has been focused on ROR proteins to regulate differentiation and IL-17 production of Th17 cells. We designed a small molecule compound VPR-66 via the molecular modeling study, which is hypothesized to bind to the ligand-binding domain (LBD) of ROR proteins. VPR-66 was characterized by two different cell-based reporter assays, in which the LBD or full-length of ROR proteins were utilized. In the LBD-based assay, VPR-66 selectively suppressed the RORα and RORγt LBD-mediated transcriptional activity but showed little or no effects on VP16 control protein- or RARα LBD-mediated transcriptional activity. Similarly, in the full-length ROR-based assay, VPR-66 significantly inhibited RORγ- or RORγt-induced IL-17A promoter activity while VPR-66 did not affect the promoter activity in the absence of ROR protein expression. Taken together, it is proposed that VPR-66 acts as an inverse agonist specific for ROR proteins and its binding to ROR proteins interferes with transcriptional complex formation for IL-17 induction.
GABA exerts anti-inflammatory and immunosuppressive effects (P5175)
Authors: Prud'homme Gerald , Glinka Yelena , Wang Qinghua
Published: January 2023

Gamma amino-butyric acid (GABA) is an inhibitory neurotransmitter in the CNS, but it also exerts important functions in the immune system and the islets of Langerhans. Thus, functional GABA receptors have been identified on immune cells and islet cells. Previously, we found that in both autoimmune (NOD) and streptozotocin (STZ)-induced diabetes GABA increases islet-cell mass, while exerting anti-apoptotic effects. GABA induced the regeneration of islet beta cells. It also suppressed inflammatory cytokine production, which is likely important in allowing survival of new islet beta cells. In this study, we examine how GABA exerts immunoprotective effects. We report that GABA suppresses both T cells and macrophages. It increases TGF-beta production and regulatory T cells (Tr or Treg). Notably, GABA inhibits NF-kB activation in both lymphocytes and pancreatic islet beta cells. It also protects beta cells from apoptosis induced by various mechanisms, and this may be related to NF-kB inhibition. The mechanisms by which GABA exerts these effects are largely unknown. However, in immune cells it stimulates GABA type A receptors, which act as ligand-gated chloride channels. This may open voltage-dependent calcium channels in the membrane and decrease intra-cellular calcium levels. Conclusion: GABA acts on both T cells and macrophages and exerts potent anti-inflammatory effects, which are protective in models of type 1 diabetes.