bluuebunny/crossref_metadata_2025 huggingface dataset:
Aberrant glycogen synthase kinase 3β phosphorylation contributes to abnormal cytokine secretion in obese subjects. (116.29)
Authors: Yen Chia-Liang , Shieh Chi-Chang K.
Published: January 2023

To clarify the role of obesity in the immune responses, we investigated the relationship between insulin-induced signaling and Toll-like Receptor (TLR)-induced leukocyte cytokine secretion in obese subjects. Peripheral blood samples were collected from metabolically normal (MNO) and metabolically abnormal (MAO) obese subjects and stimulated with TLR agonists. We found elevated IL-1β, IL-10 and decreased IL-6, IFN-γ secretion in both MNO and MAO leukocytes with TLR stimulation when compared with normal leukocytes. Changes in TLR-induced cytokine secretion correlated with biochemical markers including fasting glucose, insulin, homeostasis model assessment (HOMA) index, and body mass index (BMI). Moreover, peripheral blood mononuclear cells (PBMCs) collected from MAO subjects showed enhanced basal Akt / Glycogen Synthase Kinase 3β (GSK3β) phosphorylation which was not further increased by insulin and lipopolysaccharide (LPS). We also found IκB accumulation in PBMCs isolated from normal subjects treated with GSK3β inhibitor lithium chloride. These findings indicate that TLR-induced cytokine secretion is affected by obesity even in subjects who have not reached the criteria of metabolic syndrome. Enhanced leukocyte GSK3β phosphorylation may contribute to aberrant immune response in obese subjects.
Characterization and functional study of CPAP-associated protein-1(CPAPAP-1) in NF-κB-mediated transcriptional activation (116.3)
Authors: Fu Ru-Huei , Liu Shih-Ping , Huang Chin-Mao , Lin Ya-Hsien , Shyu Woei-Cherng , Lin Shinn-Zong
Published: January 2023

Nuclear factor-kappaB (NF-κB) is a Rel transcription factor that controls the gene expression involved in immune response, inflammation, apoptosis, and proliferation. Centrosomal P4.1-associated protein (CPAP) is an associating partner of the p65 subunit (RelA) of NF-κB. Molecular biology assays and cell biology experiments showed that the role of CPAP is a coactivator of NF-κB-mediated transcription. The C-terminal region of CPAP appears to contribute NF-κB-mediated transcriptional activation. However, knowledge about the factors that interact with the C-terminal region of CPAP is still little. In a yeast two-hybrid screen using the C-terminal region of CPAP as bait, we isolated a novel associating partner named CPAP-associated protein-1 (CPAPAP-1, GenBank: BC114945). CPAPAP-1 has the effector domain (cAMP) of the CAP family of transcription factors. We confirmed that CPAPAP-1 associates with C-terminal region of CPAP by using Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments. After treatment with TNFα, a portion of CPAPAP-1 was observed to accumulate in the nucleus. We also confirmed that CPAPAP-1 controlled activation of TNFα-induced NF-κB by using NF-κB-dependent reporter gene expression assay and RNA interference technique. These results suggest that CPAPAP-1 functions as a coactivator of NF-κB-mediated transcription.
Low dose ionizing radiation exposure can alter unmotivated mouse behavior and brain-based inflammatory mediators (116.34)
Authors: York Jason , Blevins Neil , Peterlin Molly , Cengel Keith , Freund Gregory
Published: January 2023

Individuals exposed to ionizing radiation (IR) experience acute radiation syndrome, the prodromal phase of which includes symptoms such as malaise and fatigue. These symptoms and related adverse biobehaviors have mental, physical and motivational elements that can be difficult to experimentally isolate. We’ve developed an experimental system to test IR-induced fatigue in mice using gamma rays to deliver total body IR in both motivated and unmotivated settings. 6h after a 50cGy dose of 137Cs whole body radiation, mice had a 34% reduction in spontaneous distance moved and average velocity of movement compared to non-irradiated mice. In contrast, the motivated behavior social exploration of a novel juvenile showed non-statistically significant trends toward reduced activity up to 6h post-IR. Examination of brain-based immunity showed whole brain TNF-α mRNA was significantly increased at 4 and 6h post-IR and this increase was coupled to 1) an up-regulation of IL-1RA and down-regulation of IL-6 mRNAs 4h post-IR and 2) a down-regulation of IL-1β, IFN-γ and F4/80 mRNAs at 6h post-IR. Together these data show that shortly after low-dose IR, mouse activity is reduced but can be overcome via a motivator. This altered locomotion is tied to a brain-based alteration in pro/anti-inflammatory balance. These data suggest anti-inflammatory therapies such as corticosteroids or anti-TNF/IL-1 agents may be effective countermeasures for prodromal symptoms due to IR exposure.
The role of IL-1β on IKKβ inhibitors induced neutrophilia (116.4)
Authors: Lee Chih-Yuan , Lai Ting-Yu , Hsu Li-Chung , Yeh Tzu-Wen , Enzler Thomas , Karin Michael
Published: January 2023

Transcription factor NF-κB and its activating kinase IκB Kinase β (IKKβ) are critical for inflammation and immunity. Pharmacological inhibitors of IKKβ-NF-κB could be used for the treatment of inflammation. It was unexpected that IKKβ inhibitors (MLN120B) cause neutrophilia in mice but the mechanism remained unknown. IL-1β acted as a growth factor for myeloid progenitors and a survival factor for mature neutrophils. We tried to investigate the role of IL-1β on IKKβ inhibitors induced neutrophilia. Using conditional deletion of loxP-flanked Ikbkb alleles in myeloid cells in mice, we found severe neutrophilia after IKKβ deletion. Without any stimulus, Ikkβ-deleted mice produced more circulating IL-1β than age-matched wild-type mice. Isolated Ikkβ-deleted monocytes and macrophages secreted higher amounts of IL-1β than wild-type controls without stimulation. Treatment of wild-type mice with IKKβ inhibitors also led to neutrophilia within 8 days, which would be prevented by concomitant treatment with IL-1 receptor antagonis. Neutrophilia in mice after deletion of IKKβ seems IL-1β dependent. Excessive IL-1β signaling is responsible for the uncontrolled neutrophilia and inflammation in IKKβ-deleted mice. Enhanced IL-1β production represents a compensatory mechanism for maintaining immunity when NF-κB is inhibited and compensates for loss of NF-κB-dependent antibacterial immunity.
Role of novel microRNAs in immunological dysfunction associated with PTSD (117.13)
Authors: Zhou Juhua , Nagarkatti Prakash , Zhong Yin , Ginsberg Jay , Nagarkatti Mitzi
Published: January 2023

The immunological dysfunction in combat veterans with post-traumatic stress disorder (PTSD) has not been understood. The current study aimed to evaluate the role of novel microRNAs (miRNAs) in immunological dysfunction associated with PTSD. Our studies demonstrated that both PBMC and CD4+ T cells in peripheral blood significantly increased in PTSD patients as compared with controls. Further studies showed that the percentage of IFN-γ producing Th1 cells was much higher in PTSD patients than normal donors. Multiple cytokine assays indicated that PBMC from PTSD patients produced more IL-1β, IL-2, IL-17, G-CSF and IFN-γ than those from controls. Our results demonstrate that PTSD is associated with proinflammatory response. High-throughput miRNA array hybridization analysis showed that several novel miRNAs such as miR-664 and miR-1291were significantly up-regulated, whereas miR-125a and miR-181c were dramatically down-regulated in PTSD patients. Real-time PCR confirmed that several of these miRNAs such as miR-125a and miR-181c significantly decreased in PTSD patients. Our further studies demonstrated that down-regulation of miR-125a may be responsible for increased IFN-γ production in PTSD patients. The results suggest that some novel miRNAs may play a role in immunological dysfunction associated with PTSD and such novel miRNAs may be used as biomarkers for diagnosis or as a therapeutic remedy for PTSD.
In a murine model of sepsis, IL-6 increases early and remains high until week 4, when its decrease may be related to late-phase immunodeficiency. (117.27)
Authors: Valdes-Ferrer Sergio , Rosas-Ballina Mauricio , Olofsson Peder , Pavlov Valentin , Ochani Mahendar , Chavan Sangeeta , Tracey Kevin
Published: January 2023

Chronic sepsis survival is still an unchartered territory, where system-wide, sometimes perennial dysfunction occurs, usually with dire consequences. In the present study, polimicrobial abdominal sepsis -or sham surgery- was induced using the CLP model. Long-term survivors (>7 days) were sacrificed at weekly intervals, and blood and spleen were collected. Splenic and circulating leukocyte populations were analyzed by flow cytometry. Cytokines were measured by bead-array assay. We found an early increase in splenic size and weight, followed by massive, significant influx of granulocytes, monocytes, and B-cells. Splenic monocytes and granulocytes remained high until week 4. In contrast, splenic T-cell populations remained stable after sepsis, and B-cells returned to baseline by week 3. In peripheral circulation, we found early increases in IL-6 (25-fold; p=0.01), and TNF (7-fold; p=0.01). Other cytokines, including IL-2, IFN-γ, and IL-17A were not different between groups. Serum IL-6 concentration dropped back to levels similar to those of sham mice by week 4. Strikingly, around this time all surviving mice developed spontaneous abdominal abscesses. This is interesting since IL-6 levels have been shown to be a good predictor of early mortality. The fact that a late-phase decrease may be related to a the development of a state of imunosuppression opens new avenues to understanding CARS syndrome/immunoparalysis state known to be devastating in long term septic patients.
INCB38579 is a novel, potent and selective histamine H4 receptor antagonist with activity in rodent models of inflammatory pain (117.28)
Authors: Shin Niu , Covington Maryanne , Bian Di , Bowman Kevin , Li Yanlong , Soloviev Maxim , Yao Wenqing , Newton Robert , Sherle Peggy
Published: January 2023

The histamine H4 receptor (H4) has been considered as an attractive therapeutic target for a number of diseases, such as allergy, asthma, atopic dermatitis and inflammatory pain. INCB38579 is a novel human and rodent active H4 receptor antagonist, with at least 50-fold selectivity over the human H1, H2 and H3 receptors and good pharmacokinetic properties in rats and mice. The compound potently inhibits histamine binding to and signaling through recombinant human and mouse H4 and blocks histamine-induced migration of human and mouse dendritic cells, as well as cell shape change and migration of human eosinophils. This novel H4 inhibitor is efficacious in blocking histamine-induced pruritus in mice and carrageenan-induced acute inflammatory pain in rats. Interestingly, when examined in a formalin pain model, INCB38579 could significantly reduce the relatively long lasting inflammation associated pain. A good correlation between the predicted protein binding adjusted potency from in vitro studies and the analgesic effect in vivo was observed. These results suggest that INCB38579 can serve as a useful tool for pharmacologic characterization of H4 and further supports the hypothesis that the H4 receptor is a target for the development of new therapeutics for various inflammatory diseases.
IL-33 expression by mast cells is regulated by a Sphk-calcium-NFAT dependent pathway (117.5)
Authors: Hsu Chia-Lin , Bryce Paul
Published: January 2023

IL-33 is an IL-1 family cytokine that can act extracellularly via its receptor, T1/ST2 or intracellularly. Functionally, it promotes Th2-associated immunity by enhancing several cell type activation and survival. However, the regulation of IL-33 expression is still unclear. Previously, we showed that basal expression of IL-33 in mast cells was upregulated upon IgE activation. Ca2+ mobilization was sufficient and necessary to enhance IL-33 expression in mast cells. We wanted to further investigate the pathway that drives IL-33 expression. Two major Ca2+-dependent pathways induced in mast cells are inositol trisphosphate (IP3) bound to its receptor, IP3R, and sphingosine kinase 1/2 (Sphk1/2) generated sphingosine-1-phosphate (S1P). PI3K is the upstream regulator for Sphk1. Here, we demonstrate that inhibition of PI3K and Sphk activity decreases IL-33 expression via IgE activation while ionomycin stimulation, which bypasses the inhibition, restores IL-33 expression. Interruption of NFAT and calcineurin interaction, which is downstream of Ca2+ mobilization, also decreases IL-33 expression and ionomycin can not restore it. Blocking of IP3R or NF-kB does not influence IL-33 expression but inhibits another IL-1 family cytokine, IL-1b. In summary, IL-33 is upregulated upon cross-linking of IgE receptors on mast cells and expression is dependent on Ca2+ mobilization and regulated by a PI3K-Sphk1-NFAT pathway.
IL-27 promotes IL-10 production and B7-H1 expression by plasmacytoid DC and augments their expansion of allogeneic Foxp3+ CD4+ T cells (147.2)
Authors: Matta Benjamin , Raimondi Giorgio , Thomson Angus
Published: January 2023

IL-27 is an immunoregulatory cytokine that influences T cell responses and can promote Treg function. Plasmacytoid dendritic cells (pDC) possess immunoregulatory properties that can favor induction of immune tolerance. We investigated the influence of IL-27 on pDC phenotype, function, and interaction with allogeneic T cells. pDC (mPDCA-1+) were immunobead-purified from livers and spleens of Flt3L-treated C57BL/6 mice. Cells were cultured with CpG B, IL-27, or both, and their phenotype and cytokine production analyzed by flow cytometry. T cell allostimulatory capacity of pDC was determined in CFSE MLR, including analysis of Foxp3 expression. CpG B upregulated MHC class II and CD86 on pDC, however, liver pDC expressed greater levels of the coregulator B7-H1, IL-27p28, IL-10, and the IL-27 receptor (R)/WSX-1 compared to spleen pDC. Exposure to exogenous IL-27 augmented B7-H1 and IL-10 but decreased CD86 expression only on liver pDC. Inhibition of STAT3 blunted upregulation of B7-H1. Liver pDC exposed to exogenous IL-27 selectively expanded CD4+Foxp3+ T cells, despite no change in overall CD4+ T cell proliferation. In conclusion, IL-27 enhances the immunoregulatory potential of pDC through upregulation of B7-H1 and IL-10, but not CD86. IL-27-conditioned liver pDC exhibit enhanced ability to expand allogeneic CD4+Foxp3+ T cells compared to spleen pDC. IL-27 may facilitate immunoregulation through the modulation of pDC function and subsequent control of allogeneic T cell responses.
Comparison of in vitro immunomodulatory effects of rhesus bone marrow-derived MSC and MAPC, and dermal fibroblasts (147.22)
Authors: Sindberg Greg , Lindborg Beth , Bansal-Pakala Pratima , O'Brien Timothy
Published: January 2023

In the transplant community, natural suppressive cells derived from animals offer vast advantages over traditional chemical immunosuppressive agents that have very harsh side effects on both the transplant and the patient. The bulk of this research has focused on T regulatory cells (Tregs), given their known role of quelling and suppressing immune responses to natural infection. Other cells, such as mesenchymal stem cells (MSC), have also been observed to exert suppressive capabilities. As therapeutic immunomodulatory cells, MSCs have an advantage over Tregs in that they are much less immunogenic and may potentially be utilized from allogeneic derivations whereas Tregs require patient specific development in the form of autogenic isolations from blood, which greatly limits the available supply. In addition to MSCs, fibroblasts and multipotent adult progenitor cells (MAPCs), which are phenotypically very similar to MSCs, have each been considered to have immunomodulatory potential but mechanisms are poorly understood. Using Rhesus bone marrow-derived MSC and MAPC and dermal fibroblasts we showed that all 3 types of cells strongly (80-90%) inhibited proliferation of (αCD3 stimulated) CD4 and CD8 T-lymphocytes in an in vitro assay. Suppression was maximal with 1:1 cell ratios and effects diminished with serial dilution of test cells. The findings suggest that all 3 lineages have potential as an alternative to Tregs for immunosuppression in organ transplantation.
Anti-DR5 antibody eliminates inflammatory macrophages rebalancing IL-23/Th17 axis and regulatory T cells in arthritis (147.26)
Authors: Li Jun , Hsu Hui-Chen , Yang PingAr , Wu Qi , Li Hao , Mountz John
Published: January 2023

Dysregulated macrophages (MΦ), IL-23/Th17 axis, and regulatory T cells (Tregs) have been implicated in the pathogenesis of rheumatoid arthritis (RA). Therapeutic strategies that can restore the homeostasis of these factors in RA are unknown. Death receptor 5 (DR5) can mediate apoptosis upon binding to an anti-human DR5 antibody, TRA-8. To investigate the immunomodulatory effects of TRA-8, a 3kb mouse promoter/Floxed-STOP/humanized mouse DR5 transgenic (Tg+) mouse was generated and crossed with lysozyme M.Cre mice to achieve MΦ specific expression. Collagen II (CII)-induced arthritis was induced by chicken CII. TRA-8 (0.2 mg/mouse) was I.V. weekly initiated on day 30 until mice were sacrificed 3-mo post CII. TRA-8 treatment resulted in a 60% reduction of CD11b+ MΦ in the draining lymph nodes (LN) of Tg+ mice. Within the CD11b+ cells, ~60% of the Gr1+Ly6G+ activated MΦ and ~90% of the IL-23+ MΦ were eliminated by TRA-8. The depletion also leads to elevation of CD4+Foxp3+ Tregs (3.6% to 5.9%, P<0.05) and reduction of the CD4+IL-17+ Th17 cells (1.4% to 0.6%, P<0.05). After TRA-8, joint histopathology showed a significantly decreased MAC3+ MΦ infiltration, synovial hyperplasia, and destruction (arthritis score: 9.8 versus 1.5, P<0.01). Neither systematic toxicities nor increased infection were found. Our study suggested that TRA-8 can be developed into a novel biologic which depletes the IL-23 secreting inflammatory macrophages (MΦ) to restore the balance between Th17 and Tregs.
Role of Th1, Th2 and Th17 cytokines on the expression of various inflammatory cytokines, defensive proteins and chemokines by primary human epithelial keratinocytes (148.10)
Authors: Rahman Saifur , Collins Mary , Williams Cara , Ma Hak
Published: January 2023

Psoriasis and Atopic Dermatitis are common autoimmune/inflammatory diseases of the skin. Although the exact etiology is not clear, recent data suggest a dysregulated interplay between skin infiltrates and keratinocytes may play a role in disease progression. To better understand this relationship primary human fetal epithelial keratinocytes were cultured in the absence or presence of various Th1 (IFNg), Th17 (IL-17, IL-21 and IL-22) and Th2 cytokines (IL-4, IL-13) that are known to play a role in either psoriasis or AD. Our results demonstrate that individual treatment with either Th1 or Th17 cytokines (IFN-γ, IL-17 or IL-21) induced the proinflammatory cytokine gene expression of IL-1a and IL-6. However, the Th2 cytokines IL-4 and IL-13 had no effect on expression of the proinflammatory molecules. Additionally, IFN-γ or IL-22 alone or in combination increased the expression of ICAM-1, highlighting the potential for these cells to facilitate leukocyte recruitment to the skin epidermis. Interestingly, only treatment with IL-22 individually or in combination was found to markedly increase Defensin b, Filaggrin, S100A7 and S100A8, genes important in maintaining and protecting the integrity of the skin whereas treatment with only IL-21 or in combination markedly increased the expression of Twist-1, a signature gene for epithelial mesenchymal transition. Taken together, these results suggest various cytokines have unique and redundant roles in regulating keratinocyte activity.
The role of T cells in the development of arthritis in Inherited Inflamed Joints mice. (148.12)
Authors: Shaum Katherine , Combs Matt , McMahon Eileen
Published: January 2023

The IIJ (Inherited-Inflamed-Joints) mouse strain spontaneously develops an inflammatory, possibly autoimmune, arthritis at an early age. While only a third develop disease, incidence displays a distinct female-bias and mice have elevated Ig, autoantibodies, and an increased percent of double negative (DN; CD4-CD8-CD3+) T cells in their secondary lymphoid organs. The goal of our research was to further examine the T cell compartment. The thymi of AR IIJ mice were significantly decreased in size compared to non-arthritic (NAR) IIJ and SJL controls. Flow cytometry revealed significant disregulation in thymocyte subpopulations and immunohistochemistry indicated disrupted cortical/medullary organization. AR IIJ mice also had increased thymic apoptosis based on Annexin V/PI staining and TUNEL assays were performed to investigate the spatial localization of apoptosis. While it remains unclear whether the DN T cells in AR IIJ mice contributes directly to arthritis development, we used flow cytometry to show that they were present in the blood before clinical symptoms began. Finally, since the IIJ strain was originally derived from a transgene-negative mouse from a breeding colony of 5B6 TCR transgenics, we used flow cytometry and PCR to verify the absence of the transgenes. Overall, our studies confirm that IIJ mice have significant disruption in the T cell compartment and that this disruption occurs before onset of clinical disease suggesting a possible causal role in pathogenesis.
Enhanced T cell responses to modified and unmodified myelin epitopes in subjects with multiple sclerosis (148.19)
Authors: James Eddie , Kwok William
Published: January 2023

MS is an autoimmune disease characterized by infiltration of CD4+ T cells and other immune cells into the central neural system, leading to demyelination and neuronal damage. Myelin antigens are known to undergo post-translational modifications such as citrullnation and phosphorylation. However, responses to the modified forms of these antigens have been minimally studied. We used a systematic approach to investigate CD4+ T-cell responses to modified and unmodified myelin epitopes. We first characterized the ability of DR1501 to accommodate citrulline or arginine containing sequences, demonstrating that citrulline is accepted within three of its binding pockets. Based on previously published methods, we developed an algorithm to predict DR1501 restricted epitopes within modified and unmodified peptides from MBP, PLP, and MOG. Predicted epitopes that were able to bind to DR1501 protein were used to stimulate PBMCs from MS and healthy control subjects with DR1501 haplotypes. A subset of these sequences elicited tetramer positive T cell responses in the majority of subjects with MS, but not healthy controls. In some cases citrullination or phosphorylation altered the antigenicity of these peptides. These findings highlight the potential importance of post-translationally modified sequences in multiple sclerosis and suggest specificities that could be used for immune monitoring or antigen specific therapies.
Chemokine-directed B cell trafficking in rabbit GALT during diversification of the primary antibody repertoire (149.4)
Authors: Lanning Dennis , Knight Katherine
Published: January 2023

Rabbits preferentially rearrange the 3’-most VH gene segment during VDJ gene rearrangement. Early in life, rabbit B cells diversify these VDJ genes in gut-associated lymphoid tissue (GALT) in response to signals acquired from intestinal commensal bacteria, and thus generate a diversified primary antibody repertoire. To gain insight into the cell-cell and cell-microbe interactions required for VDJ gene diversification, we examined B cell surface expression levels of four chemokine receptors that mediate trafficking to important sites within GALT during repertoire diversification. We stained B cells from two- through seven-day-old appendix with fluorescence-labeled Ig-fusion proteins of the chemokines CXCL12, CCL20, CXCL13 and CCL21 and analyzed them by flow cytometry. The changes we observed in relative chemokine receptor expression levels during the first week of life suggest a model in which B cells, upon entering appendix follicles, first traffic to the follicle-associated epithelium, where they likely encounter commensal bacteria sampled from the intestinal microbiota. B cells subsequently migrate to, and likely receive additional signals from, the follicular dendritic cell network. Upon becoming activated, B cells then traffic to the basal region of the follicle, where they proliferate and diversify their VDJ genes, thereby generating the primary antibody repertoire.
Characterization of monoclonal antibodies to human CXCR7 (149.9)
Authors: Bonnevier Jody , Loges Naomi , Bostrom Kerri , Stump Julie , Brumbaugh Kathy , Munshi Cyrus , Ni Jessie
Published: January 2023

The G protein-coupled chemokine receptor CXCR7 binds with high-affinity to CXCL12/SDF-1 and CXCL11/I-TAC. It is also a co-receptor for several HIV and SIV strains. The expression of CXCR7 in human cell types has been studied extensively and analyzed at the transcription and protein levels. Depending on the antibody used, the reported expression profiles of CXCR7 on human cells differ significantly. This calls into question the specificity of some of the antibodies used in the studies. In this poster, we described how our monoclonal antibody panel to CXCR7 was developed, and present vigorous testing results using flow cytometry, Western blot and molecular biology approaches on multiple CXCR7 transfectants, primary cells and cell line, to establish the specificity of our antibodies. Using different specific monoclonal antibodies within our panel, we showed that the profiles of human CXCR7 expression on hematopoietic cells differ. Although the basis of these differences is not known, it likely can be attributed to cell context and the specific epitopes recognized by the different antibodies. Our panel of anti-CXCR7 should be useful for future studies to define interaction partners that interact with CXCR7 on different cell types.
Thymic stromal lymphopoietin can restore functional T cell-independent antibody responses in young mice (150.5)
Authors: Dickinson Gregory , Liu Hongqi , Shriner Anne , Leonard Warren , Alugupalli Kishore
Published: January 2023

Unlike adults, young children cannot mount an antibody response to T cell-independent (TI) antigens such as Pneumococcal polysaccharides (PPS) and the basis for this impairment remains elusive. The ontogeny of B cells early in life is IL-7-independent, whereas in adults it is IL-7-dependent. Despite having TI B cell subsets, young wildtype or adult IL-7-/- mice are impaired in anti-PPS response and do not survive Pneumococcal challenge. In contrast, enforced expression of IL-7 restores protective anti-PPS responses in young mice, indicating that IL-7-dependent B cells are critical for TI responses. Thymic stromal lymphopoietin (TSLP) is structurally similar to IL-7 and utilizes IL-7Rα for signaling, therefore it may also contribute to the development of TI B cell responses. Indeed, we found that young TSLP transgenic mice generate an anti-PPS response and protective immunity comparable to that of adult mice. TSLP can be induced upon activation of TLR2 and TLR9. Borrelia hermsii, an agent of relapsing fever, activates TLR2 and TLR9 signaling and induces a protective TI antibody response even in young mice, suggesting that TSLP induced during this infection could play a role in anti-B. hermsii responses. Indeed, IL-7Rα-/- mice (deficient in both IL-7 and TSLP signaling) have a more severe impairment in anti-B.hermsii responses and suffer more prolonged bacteremia than IL-7-/- or TSLPR-/- mice. These data indicate that TSLP can restore functional TI responses in the young.
IL-21R is critical for the development of memory responses (150.9)
Authors: Lowe Leslie , Rankin Andrew , MacLeod Heather , Keegan Sean , Andreyeva Tatyana , Bloom Laird , Collins Mary , Nickerson-Nutter Cheryl , Guay Heath , Young Deborah
Published: January 2023

The development of long-term humoral immunity, characterized by the formation of high affinity memory B cells and long-lived plasma cells in the bone marrow, is a major goal of vaccination, but the mechanisms involved in the generation of long-term humoral immunity are not well understood. IL-21 can induce B cell differentiation into plasma cells and influence isotype switching to some subclasses of IgG. Using IL-21R KO mice, we show for the first time a role for the IL-21 pathway in the development of long-term humoral immunity. Antigen specific IgG serum responses to the T cell-dependent antigen, NP-CGG, were delayed in IL-21R KOs, but reached comparable titers to those measured in WT within 3 weeks. The formation of germinal centers, which give rise to long-lived plasma cells and memory B cells, was also delayed in IL-21R KO, but reached similar magnitudes as in WT within 2 weeks. IL-21R KO generated normal numbers of NP-specific IgG plasma cells in their bone marrow at ~1.5 months after immunization. NP-specific serum IgG antibody responses were of similar relative affinity in IL-21R KO and WT at this time, indicating that NP-specific B cells affinity matured in IL-21R KO. However, IL-21R KO failed to generate NP-specific secondary IgG antibody responses following rechallenge with NP-CGG. These results indicate that the IL-21 pathway is necessary for the formation of memory B cell responses but not high affinity long-lived plasma cells.
Insight into the mechanism of STAT5 tyrosine phosphorylation in IgE sensitized, antigen-stimulated mast cells (151.13)
Authors: Pullen Nicholas , Barnstein Brian , Wilson Bridget , Falanga Yves , Mizuno Kazuya , Ryan John
Published: January 2023

Previously we established that STAT5 expression is required for IgE-mediated mast cell function. STAT5 tyrosine phosphorylation is swiftly and transiently enhanced by FcϵRI aggregation in an IL-3- and MAPK-independent manner. However, a more detailed understanding of the signaling apparatus responsible in this situation is wanting. Recently, we have observed that STAT5 co-localizes with FcϵRI during mast cell activation and that lipid raft dissipation interrupts STAT5 activation. Downstream of FcϵRI we find that this mode of STAT5 activity is also independent of BTK, PI3K, Akt, Syk, and Lyn, whereas removal of Fyn kinase ablates the effect. Immunoprecipitation demonstrates that STAT5 is associated with Fyn, and this co-IP decreases concomitant with the induction of IgE-mediated STAT5 tyrosine phosphorylation. Examination of other signaling-related factors reveals that SHP-1 phosphatase deficiency enhances STAT5 tyrosine phosphorylation, even prior to stimulation. Loss of GAB2, an adaptor protein that associates with Lyn and SHP-1, also augments STAT5 activation. Targeting of STAT5A or B with knock-outs and specific siRNA pools shows that IgE-mediated mast cell cytokine production is selectively dependent upon the STAT5B isoform. Altogether these data implicate Fyn as the major positive mediator of STAT5 after FcϵRI engagement, and demonstrate importantly distinct roles for STAT5A and STAT5B in mast cell function.
Adipose eosinophils promote alternatively activated macrophages that maintain metabolic homeostasis (151.21)
Authors: Molofsky Ari , Wu Davina , Liang Hong-Erh , Bando Jennifer , Jouihan Hani , Ricardo-Gonzalez Robert , Chawla Ajay , Locksley Richard
Published: January 2023

Eosinophils are innate Th2-related granulocytes that are elevated in asthma, allergy, and helminth infection, and are frequency associated with alternatively activated macrophages (AAMs). Whereas adipose classically activated macrophages are associated with obesity and type-2 diabetes, AAM help maintain ‘lean physiology’. Adipose AAM can be induced by the cytokine interleukin-4 (IL-4), although a definitive cellular source of IL-4 within adipose tissue is not known. Surprisingly, we find eosinophils are resident within mouse white adipose tissues, where they are the major interleukin-4 (IL-4) expressing cells. Elevated adipose eosinophils promote Arginase-1+ AAM in an IL-4/13 dependent manner, and, in the absence of eosinophils or IL-4/IL-13, adipose AAM are diminished. In mouse models of obesity, adipose eosinophils inversely correlate with weight, and a genetic absence of eosinophils results in increased body fat and insulin resistance. Conversely, infection with the helminth Nippostrongylus brasiliensis leads to prolonged adipose eosinophilia and decreased insulin resistance. Eosinophils are evolutionarily conserved cells whose function has remained elusive, present in high levels in many naturally parasitized human populations. We speculate that eosinophils participate in integrating metabolic and immune signals to maintain homeostasis during chronic parasitism.
Increased activities of cysteine cathepsins in mouse models of asthma (151.23)
Authors: Yin Fangfang , Yu Mang , Liu Chen , Paulick Margot , Verdoes Martijn , Stein Paul , Galli Stephen , D'Andrea Annalisa , Bogyo Matthew
Published: January 2023

Asthma is a severe disorder characterized by chronic inflammation of the airways and increased responsiveness to nonspecific stimuli. During an asthma attack, a number of immune cells are recruited to the lung. In addition to cytokines, these cells secrete a number of proteolytic enzymes or proteases to mediate the inflammation and airway remodeling associated with asthma. Cysteine cathepsins (CCs), a family of lysosomal proteases, have been found to be present in asthma patients, but little is known about their role in asthma pathology. In our study, we have used small molecule activity-based probes that specifically target CCs to investigate the in vivo profiles of CCs in two mouse models of asthma. The two models differ in their mechanism of initiating allergic inflammation by sensitization either in presence of the adjuvant alum (mast cell independent, Alum+) or in its absence (mast cell dependent, Alum-). In the Alum- model, we have found that the activities of CCs, especially Cathepsin S, were elevated in asthmatic lungs. Using immunostaining and flow cytometry, we have discovered that macrophage is a major cell type that produces elevated CCs in asthmatic lungs, while in the fluids of bronchoalveolar lavage, both macrophage and eosinophils are the main cell types that are associated with CC activities. We are now profiling CC activities in Alum+ model. These studies will allow us to gain a complete understanding of CC activities during asthmatic responses.
Low level stimulation of FcϵRI on human skin mast cells modulates expression of adenosine receptors A2aAR and A3AR. (151.5)
Authors: Gomez Gregorio , Schwartz Lawrence
Published: January 2023

Adenosine is a purine nucleoside that can potentiate and inhibit IgE-induced degranulation of mast cells. Extracellular adenosine acts through four G protein-coupled receptors (A1AR, A2aAR, A2bAR and A3AR). This study shows that low level stimulation of IgE receptor (FcϵRI), but not complement factor C5a receptor (C5aR), on human skin mast cells (SMCs) differentially modulates the expression of A2aAR and A3AR. Stimulation of SMCs with the anti-FcϵRIα mAb 22E7 at a concentration that does not induce degranulation temporally induced a 5-fold increase in A2aAR and a 5-fold decrease in A3AR. A2bAR was not affected and A1AR, which is not expressed in resting SMCs, was not induced. Mechanistically, the FcϵRI-induced increase in A2aAR was dependent on the NF-κB transcription factor since it was mostly blocked by the NF-κB inhibitor BAY-11. In contrast, A3AR levels were further reduced when NF-κB was inhibited. Since A2aAR couple predominantly with Gs proteins (adenylate cyclase activation) and A3AR with Gi proteins (adenylate cyclase inhibition), cAMP levels were measured. Exposure to adenosine of SMCs incubated ±22E7 resulted in a significant increase in cAMP levels in 22E7-stimulated SMCs relative to those cultured without 22E7. These data suggest that altered expression of A2aAR and A3AR by low-level stimulation of FcϵRI which promotes increased cAMP production upon adenosine exposure might act to raise the threshold for mast cell degranulation under certain conditions.
MKK3 regulates mast cell IL-4 production through Egr1. (151.9)
Authors: Lin Tong-Jun , MacNeil Adam
Published: January 2023

Mast cells play a central role in allergic inflammation and are activated through crosslinking of IgE receptors, initiating a signaling cascade resulting in production of biologically potent mediators. Signaling pathways in the regulation of specific mediators remain incompletely defined. Here, we examined the role of mitogen-activated protein kinase (MAPK) kinase 3 (MKK3) in IgE-dependent mast cell activation. In an in vivo model of passive cutaneous anaphylaxis, MKK3-deficient mice showed a deficit in late phase IgE-dependent inflammation. To characterize the mechanism of this deficiency, we cultured bone marrow-derived mast cells (BMMCs) from wild-type and MKK3 knockout (KO) mice. We found that IgE-mediated mast cell activation induced rapid MKK3 phosphorylation by 5 minutes, diminishing slowly after 6 hours. In MKK3-deficient BMMCs, phosphorylation of p38 was reduced at early and later time points. Among 40 cytokines tested using a protein array, IL-4 was the only cytokine specifically down-regulated in MKK3-deficient BMMCs. Furthermore, early growth response-1 (Egr1) bound to the promoter of IL-4 in FcϵRI-activated mast cells and Egr1 transcription factor activity was diminished from 1-6 hours in MKK3-deficient BMMCs. Thus, MKK3 is a specific regulator of IgE-induced IL-4 production likely through regulating Egr1 activity in mast cells.
Activation of Nrf2 skews CD4+ cells toward Th2 differentiation (152.15)
Authors: Rockwell Cheryl , Zhang Mingcai , Fields Patrick , Klaassen Curtis
Published: January 2023

Nuclear erythroid 2 related factor 2 (Nrf2) is a transcription factor activated by cell stress, including oxidative and electrophilic stimuli, resulting in the upregulation of a battery of cytoprotective genes. Although best known for its role in detoxication, Nrf2 has also been shown to have a number of anti-inflammatory effects. The role of Nrf2 in T cells is largely unknown, however. Nrf2 can be activated experimentally by a number of different pharmacological agents, including tert-butylhydroquinone (tBHQ), which is also used commercially as a food preservative. The present studies found that the Nrf2 activator, tBHQ, suppresses production of IFNγ in activated CD4+ cells, while concurrently inducing production of IL-4, IL-5, and IL-13. These effects were also observed in CD4+ cells that were stimulated with anti-CD3/anti-CD28 in the presence tBHQ for 5 days and then restimulated without further addition of Nrf2 activators, suggesting that the effects are due to CD4+ cell differentiation. Furthermore, the current studies suggest that activation of Nrf2 diminishes T-bet binding activity, while concurrently enhancing GATA-3 binding activity. Collectively, the present studies suggest that activation of Nrf2 has a suppressive effect on Th1 differentiation, while promoting Th2 differentiation, which likely occurs through inhibition of T-bet activity and potentiation of GATA-3 activity.
Immunoproteasome activity controls the balance of inflammatory and regulatory gene expression in T helper cells (152.21)
Authors: Suzuki Erika , Muchamuel Tony , Arastu-Kapur Shirin , Kirk Christopher
Published: January 2023

The immunoproteasome (iPr), a unique form of the proteasome found in cells of the immune system, regulates cytokine production and differentiation of Th1 and Th17 cells in vitro in an NF-kB independent manner. However, the role of the iPr in other T-cell subsets and the specific molecular signaling events regulated by the iPr are not well understood. We found that FoxP3 and TGF-β expression increased in Treg cells exposed to a selective iPr inhibitor (ONX 0914, formerly PR-957) but that IL-4 production in Th2 cells was unaffected by iPr inhibition. Interestingly, increased FoxP3 expression was noted in Th0 and Th1 cells exposed to ONX 0914. Inhibition of the iPr resulted in reduced phosphorylation of STAT1 and STAT3 in Th1 and Th17 cells, respectively. Decreased basal and IL-6 stimulated STAT3 phosphorylation was noted immediately following iPr inhibition suggesting a receptor proximal event but at later timepoints coincided with reduced gene expression. After a brief exposure to ONX 0914, Th17 and Treg cells show a similar pattern of protein accumulation as measured by 2D Diffference Gel Electrophoresis and included the regulatory subunit of PP2A, a protein phosphatase known to regulate STAT3 and other transcription factors. Collectively, these results suggest that the iPr regulates Th cell plasticity via PP2A activity.
Altered T cell differentiation mediated by heterozygous SOCS1 expression: implications for autoimmunity (152.23)
Authors: Wilson Tenisha , Larkin III Joseph
Published: January 2023

Suppressor of cytokine signaling-1 (SOCS1) is an intracellular protein that negatively regulates cytokine signaling. Heterozygous expression of SOCS1 leads to T lymphocyte mediated autoimmunity, suggesting that reduced expression of SOCS1 can disrupt immune system tolerance through aberrant cytokine signaling in lymphocytes. Immune system tolerance to self-tissues is dependent upon proper differentiation of naïve T lymphocytes (Tresp) subsequent to activation. Although it is known that cytokines are essential for Tresp differentiation, it is poorly understood how heterozygous expression of SOCS1 affects this process. We demonstrate that heterozygous expression of SOCS1 does not affect Treg induction from naïve Tresp. However, it does disrupt Th17 differentiation and results in a Th1 bias, as SOCS1 heterozygous Tresp that have been placed in Th17 inducing conditions have decreased IL-17 and increased IFN-gamma message expression. These results suggest that the systemic autoimmunity experienced by SOCS1 heterozygous mice may not be due to decreased Treg induction, but rather a predominant Th1 bias caused by dysregulated IFN-gamma signaling as a result of reduced expression of SOCS1.
TCR-CARMA1-NF-κB controls Th17 differentiation (152.4)
Authors: Molinero Luciana , Alegre Maria-Luisa
Published: January 2023

IL-17 producing-CD4 T cells play a key role in immune responses against extracellular bacteria and autoimmunity. The transcription factor NF-kB is required for T cell activation and effector functions. Here, we investigated the role of T cell receptor (TCR)-mediated NF-kB in Th17 differentiation. In vitro, cells deficient in the adaptor molecule CARMA1, necessary for TCR-NF-kB signaling, did not differentiate towards a Th17 program, as shown by absence of IL-17A or IL-17F, IL-22, IL-21, IL-23R and CCR6. Consistently, CARMA1-deficient mice did not develop Th17-driven experimental autoimmune encephalomyelitis. CARMA1 selectively affected Th17 but not Th1 or Th2 differentiation in a cell intrinsic manner. While CARMA1 controls both NF-kB and JNK2 TCR-mediated activation, only NF-kB-impaired T cells (IkBaDN-Tg) had defective Th17 differentiation, confirming that Th17 differentiation requires intact TCR-CARMA1-NF-κB signaling. Despite impaired Th17 differentiation, CARMA1-deficient cells had intact IL-6 or TGFb signaling, as assessed by p-STAT3 and p-Smad2/3. Moreover, induction of transcription factors essential for Th17 differentiation such as RORgt, proceeded normally in CARMA1-deficient T cells. Notably, accessibility to the IL-17 locus was impaired in CARMA1-deficient cells. Our results demonstrate that TCR-CARMA1-NF-κB controls Th17 differentiation by enabling IL-17 locus accessibility. Targeting T cell NF-kB could be potentially used as therapy for autoimmune diseases.
The differentiation of pathogenic Th17 cells is negatively regulated by the transcription factor ELF4 (152.6)
Authors: Lee Ping-Hsien , Lacorazza H. Daniel
Published: January 2023

T helper 17 (Th17) cells are potent inducers of tissue inflammation involved in several autoimmune diseases such as multiple sclerosis (MS). Although the importance of environmental factors such as IL-6, IL-21, and IL-23 has been well established, the transcriptional network regulating lineage specification and acquisition of effector function in Th17 cells is not entirely understood. We found that loss of the transcription factor ELF4 led to increased in vitro Th17 differentiation without significant alteration in Th1, Th2, and Treg polarization. The increased differentiation of Elf4-null Th17 cells was also observed in the absence of TGFβ signaling, known to abrogate pathogenicity of Th17 cells, suggesting that polarized Elf4-null CD4+ T cells may be more pathogenic. By using experimental allergic encephalomyelitis (EAE), a mouse model for human MS, we found that Elf4-null mice exhibited an earlier onset and increased severity of disease compared to wild-type mice, supporting a negative role of ELF4 in pathogenicity of Th17 cells in vivo. To understand how ELF4 modulates Th17 differentiation, we investigated cytokine signaling in Elf4-null CD4+ T cells. Increased phosphorylation of STAT3 was observed in Elf4-null CD4+ T cells after 5-min and 30-min IL-6 stimulation. Collectively, our study identified a novel intrinsic regulator that may inhibit Th17 differentiation by dampening cytokine signaling through STAT3 inhibition.
Characterization of different subsets of human follicular helper T (TFH) cells (152.9)
Authors: Choi Jinyoung , Kim Sang Taek , Lainez Begona , Craft Joseph
Published: January 2023

Follicular helper T (TFH) cells are specialized CD4+ T cells that are localized in B cell follicles and help B cells produce antigen-specific antibodies via contact and secretion of IL-21 and IL-4. In mice, they are characterized by high expression of CXCR5, PD-1, ICOS, and Bcl-6, and low expression of P-selectin glycoprotein ligand-1 (PSGL-1); however, the study of TFH cells in human is more limited. Here we identified three subsets of human tonsillar TFH cells (CXCR5hiPD-1hiICOShiPSGL-1lo) based upon the expression of CD127 and CD57 and characterized them using flow cytometry, tissue distribution, microarray analysis, and B cell help, and compared them to traditional Th effector subsets and naïve cells. TFH cells, residing in the B cell follicle and germinal center (GC) by confocal microscopy, were CD45RAloCXCR5hiPD-1hiICOShiPSGL-1lo. Following sorting, the three subsets of TFH cells produced comparable levels of IL-21 and IL-4, and expressed transcripts of Bcl6. In addition, these subsets efficiently induced GC B cells to produce switched immunoglobulin. By contrast, Th1 and Th17 cells, as defined by IFNγ and IL-17 secretion and by Tbx21 and Rorc expression, respectively, were CD45RAloCXCR5loPD-1loPSGL-1hi, with T cell zone residence. These studies define characteristics of TFH subsets in human tonsils and potentially pave the way for identifying therapeutic targets in SLE. Work to investigate their unique function and differential gene expression is underway.
Early Growth Response Gene-2 regulates the development of B and T cells (153.15)
Authors: Li Suling , Symonds Alistair L.J. , Zhu Bo , Liu Mengya , Raymond Meera V. , Miao Tizong , Wang Ping
Published: January 2023

Understanding of how transcription factors are involved in lymphocyte development still remains a challenge. It has been showed that Egr-2 deficiency results in impaired NKT cell development and defective positive selection of T cells. Here we investigated the development of T, B and NKT cells in Egr-2 transgenic mice and the roles in the regulation of distinct stages of B and T cell development. The expression of Egr1, 2 and 3 were analysed at different stages of T and B cell development by RT-PCT and results showed that the expression was strictly regulated at different stages. Forced expression of Egr-2 in CD2+ lymphocytes resulted in a severe reduction of CD4+CD8+ (DP) cells in thymus and pro-B cells in bone marrow, which was associated with reduced expression of Notch1 in ISP thymocytes and Pax5 in pro-B cells, suggesting that retraction of Egr-2 at the ISP and pro-B cell stages is important for the activation of lineage differentiation programs. In contrast to reduction of DP and pro-B cells, Egr-2 enhanced the maturation of DP cells into single positive (SP) T and NKT cells in thymus, and immature B cells into mature B cells in bone marrow. Our results demonstrate that Egr-2 expressed in restricted stages of lymphocyte development plays a dynamic, but similar role for the development of T, NKT and B cells