bluuebunny/crossref_metadata_2025 huggingface dataset:
Ubiquitination system defects and abnormal tolerance in CD4+ T cells from systemic lupus erythematosus patients. (135.16)
Authors: Gomez-Martin Diana , Galindo-Campos Miguel , Díaz-Zamudio Mariana , Crispín José , Alcocer-Varela Jorge
Published: January 2023

Ubiquitin ligases, such as Cbl-b, GRAIL and Itch are included in the anergy genetic program, and their deficiency in murine models has been related to a resistant to anergy phenotype in T cells and lupus like disease. The aim was to analyze Cbl-b, GRAIL and Itch expression in T cells from SLE patients upon anergy induction. We included 14 SLE patients in clinical remission and 5 active untreated SLE patients. Cbl-b RNA expression was measured by RT-PCR in peripheral CD4+ T cells from SLE patients and controls upon anergy induction (ionomycin). Cell proliferation was measured by CFSE. Cytokine production was analyzed by luminometry and activation markers surface expression by flow cytometry We show that CD4+ T cells from SLE patients display decreased expression of Cbl-b RNA upon anergy induction vs resting (p=0.020) and ex vivo (p=0.030) conditions. Also, a resistant to anergy phenotype (increased proliferation and IL-2 production upon ionomycin) was evidenced. Decreased IL-17 and IFN-γ production as well as increased CD69 expression were found in CD4+ T cells from SLE patients after anergy induction. The expression of GRAIL and Itch was not significantly altered. Our data suggest that CD4+ T cells from SLE patients in clinical remission display abnormalities in the ubiquitination system, specifically, deficient Cbl-b expression which is related to the resistant to anergy phenotype, and should be considered as part of the intrinsic T cell defects in SLE.
Naïve T cells found in the islets of NOD mice do not cause Type I diabetes (135.17)
Authors: Young Ellen , Talmage Garrick , Larry Fatima , Buntzman Adam , Hess Paul , Tisch Roland , Frelinger Jeffrey
Published: January 2023

The islets of non obese diabetes-prone (NOD) mice become chronically inflamed during progression of autoimmune Type I diabetes. In a previous study we found that there are substantial numbers of naïve T cells in the islets of NOD mice. Up to 30% of CD4+ T cells and 40% of CD8+ T cells have a naïve phenotype of CD62Lhigh and CD44low in the islets of 12 to 14 week old female NOD mice. Since mice with higher levels of inflammation had higher levels of naïve T cells we wondered if the inflammation levels of individual islets would also affect the accumulation of naïve T cells. We examined the phenotype of T cells emerging from individual islets. We found an inflammation threshold of 100 to 200 CD4+ and 20 to 70 CD8+ T cells in individual islets. Above this threshold we found significant numbers of naïve T cells. To determine if these naïve T cells were contributing to diabetogenesis, we separated naïve and effector T cells from the islets of NOD mice and transferred either naïve or effector T cells to NOD scid mice. Islet effector T cells could transfer diabetes to NOD scid mice while islet naïve T cells could not. We conclude that individual islets progress as unique sites of inflammation that evolve individually to form environments of tertiary lymphoid structure that support the migration of naïve T cells into the islets. Given the large number of B cells also in the islet, the presence of naïve T cells may allow for epitope spreading to also occur in the islets.
A hematopoetic factor contributes to brain microhemorrhage in CD8 T cell induced blood brain barrier disruption (135.19)
Authors: Johnson Aaron , Chen Yi , Suidan Georgette , McDole Jeremiah , Lohrey Anne , Pirko Istvan
Published: January 2023

Blood brain barrier (BBB) disruption is a common feature of neurological disease. We developed a novel model of CD8 T cell mediated BBB disruption using the Theilers murine encephalomyelitis virus (TMEV) model of multiple sclerosis. At 7 days post TMEV infection, 50-70% of CNS infiltrating CD8 T cells are specific for an immunodominant virus peptide VP2 121-130 presented in the context of the Db class I molecule. Intravenous injection of VP2 121-130 peptide at 7 days post TMEV infection results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. Despite having an identical CD8 T cell response, 129 SvIm mice survive administration of the VP2 121-130 peptide. We performed bone marrow transplants to determine if a hematopoetic factor was responsible for this fatal syndrome. 129 SvIm mice were reconstituted with C57BL/6 or autologous 129 SvIm bone marrow. Similar brain infiltration and purity of transplanted CD8 T cells was observed during acute TMEV infection in reconstituted mice. However, 129 SvIm mice reconstituted with C57BL/6 bone marrow had increased microhemorrhage formation as measured by T2* weighted MRI and became moribund. Meanwhile, 129 SvIm mice receiving an autologous bone marrow transplant had significantly less microhemorrhage formation and remained resistant to becoming moribund. These studies demonstrate that a hematopoetic factor is responsible for the onset of brain microhemorrhage in immune mediated fatal BBB disruption.
Liver Th1 cells drive the accumulation of Myeloid Derived Suppressor Cells during acute hepatitis in an IFN-γ-dependent manner. (135.2)
Authors: Cripps James , Wang Jing , Maria Ann , Blumenthal Ian , Gorham James
Published: January 2023

Th1 responses in the liver are the basis for parenchymal damage in autoimmune hepatitis (AIH). The cellular pathways that inhibit liver Th1 responses are not well understood. BALB/c mice lacking TGF-β1 spontaneously and rapidly develop acute Th1 cell-mediated necroinflammatory hepatitis. Liver damage requires interferon gamma (IFN-γ) production by CD4+ T cells. We hypothesized that the Th1 response would also activate pathways capable of down-regulating T cell responses, specifically, a myeloid derived suppressor cell (MDSC) population. Myeloid cells were indeed abundant in liver parenchyma in histological sections of Tgfb1KO livers. Characterization of this cell type showed that they were CD11b+Gr-1+ cells that strongly suppressed T cell proliferation in vitro, thus MDSCs. By contrast, Tgfb1WT liver CD11b+Gr-1+ cells were much less abundant and exhibited no T cell suppression capabilities. Suppression required nitric oxide (NO) and cell-cell contact, but not arginase, PDL1, or H2O2. Tgfb1KO MDSCs suppressed T cell proliferation by inducing apoptosis. Further studies using Tgfb1KO/IfngKO mice showed that IFN-γ was required both for MDSC accumulation in liver as well as for MDSC suppressor activity. These results demonstrate that, during hepatitis, Th1 cells activate through IFN-γ production an MDSC negative feedback pathway that can inhibit T cell responses through NO. Why this negative feedback pathway ultimately fails in AIH remains critical to elucidate.
Targeted depletion of macrophages using an anti-human DR5 antibody prevented arthritis in a novel human/mouse chimeric DR5 Tg mouse (135.29)
Authors: Li Jun , Yang Ping-Ar , Wu Qi , Hsu Hui-Chen , Mountz John
Published: January 2023

The severity of rheumatoid arthritis (RA) correlates with the activation and abundance of macrophages in the inflamed tissues and circulation. The purpose of this study is to generate a humanized mouse (hu/mo) DR5 transgenic (Tg) mouse in which the hu/mo DR5 is targeted to the monocytes/macrophages and promote apoptosis by an anti-human DR5 antibody, TRA-8. The DR5 Tg consists of a human extracellular domain, a mouse transmembrane and intracellular domain under the regulation of the 3kb mo DR5 promoter. A Floxed STOP was inserted between the promoter and hu/mo DR5. Targeted expression of the DR5 Tg in monocytes/macrophages was generated by crossing the DR5 Tg with the lysozyme-Cre (LysMCre) mice. Arthritis was induced by intradermal injection of chicken type II collagen (CII) in CFA/IFA on day 0 and boost on day 21. There was a high percentage of CD11b+ macrophage in the spleen of CII immunized non-Tg mice (10.7%) compared to naïve mice (2.5%). TRA-8 (0.2 mg) was administrated I.V. every 3 days for 3 doses starting at day 14 after boost. After TRA-8 treatment, there was a significant decrease (55.6%) of CD11bhighGr-1+ macrophages. TRA-8 treatment also suppressed the development of arthritis in LysMCre+ hu/mo DR5+ Tg mice. This study demonstrated that selectively reducing the numbers of activated macrophage by using an anti-human DR5 antibody is a novel therapeutic strategy applicable to human RA, which might lead to prevention of joint inflammation and arthritis.
CD11b+Ly6C+ cells, myeloid derived suppressor cells, affect T cell responses during demyelinating disease (135.38)
Authors: Olson Julie , Glazer Alex
Published: January 2023

Theiler’s murine encephalomyelitis virus (TMEV) is a mouse model of multiple sclerosis in which mice develop a chronic progressive demyelinating disease associated with a myelin- specific CD4+ T cell response. Our previous studies have demonstrated that the innate immune response directly affects the initiation and progression of demyelinating disease. We recently determined that the predominant innate immune cells infiltrating into the central nervous system (CNS) following TMEV infection are CD11b+Ly6C+ cells. CD11b+Ly6C+ cells are immature myeloid cells that exit the bone marrow without maturing. Thus, we wanted to determine the role of these cells in the immune response during demyelinating disease. CD11b+Ly6C+ cells isolated from the CNS during TMEV infection expressed high levels of IL-10 and iNOS and low levels of IL-12, and expressed low levels of costimulatory molecules and MHC class II. Most interestingly, CD11b+Ly6C+ cells isolated during TMEV- induced demyelinating disease suppressed CD4+ T cell responses against virus and myelin antigens. Furthermore, depletion of Ly6C+ cells during TMEV infection lead to increased expression of IFNγ, IL-12, and IL-17 in the CNS associated with increased T cell responses. These results suggest that these CD11b+Ly6C+ cells are myeloid derived suppressor cells that suppress T cell responses and affect the progression of demyelinating disease. This work was supported by the National Multiple Sclerosis Society Grant RG3625.
Lipoteichoic acid from Staphylococcus aureus attenuates differentiation of pre-osteoclast into mature osteoclast (136.26)
Authors: Yang Jihyun , Kim Jiseon , Ryu Young Hee , Yun Cheol-Heui , Han Seung Hyun
Published: January 2023

Bacterial virulence factors regulate differentiation and functions of osteoclasts, which mediate the bone-resorptive process. Here, we investigated the effect of a major cell wall virulence factor of Gram-positive bacteria, lipoteichoic acid (LTA), on the osteoclast differentiation. Highly-pure and structurally-intact LTA was prepared from Staphylococcus aureus through sequential application of organic solvent extraction and hydrophobic-interaction column chromatography followed by ion-exchange column chromatography. Osteoclast precursors were prepared from C57BL/6 mouse bone marrow cells using M-CSF and RANKL. Staphylococcal LTA suppressed the differentiation of osteoclast precursors to mature osteoclasts concordantly with an inhibition of the bone-resorptive capacity. Meanwhile, LTA augmented the phagocytic and inflammatory potential of the pre-osteoclasts. The LTA inhibition of osteoclast differentiation appears to be mediated through Toll-like receptor 2 (TLR2), and interestingly both MyD88-dependent and -independent signaling pathways. Furthermore, LTA reduced phosphorylation of ERK and JNK during the osteoclast differentiation process followed by a decreased DNA-binding activity of AP-1. Conclusively, our results suggest that LTA from S. aureus attenuates the differentiation of pre-osteoclast to mature osteoclast through TLR2 with partial involvement of MyD88 signaling pathways.
RIG-I mediates rhabdovirus-induced inflammatory immune responses of primary human astrocytes (136.3)
Authors: Furr Samantha , Moerdyk-Schauwecker Megan , Grdzelishvili Valery , Marriott Ian
Published: January 2023

Astrocytes are ideally positioned to respond to invading pathogens of the central nervous system (CNS) and produce key inflammatory mediators following exposure to neurotropic rhabdoviruses such as rabies virus and vesicular stomatitis virus (VSV). However, the mechanisms by which resident CNS cells perceive such viral challenges have not been defined. Recently, several cytosolic DExD/H box RNA helicases including retinoic acid-inducible gene I (RIG-I) have been described that function as intracellular sensors of replicative RNA viruses. Here, we demonstrate that primary human astrocytes constitutively express RIG-I and show that such expression is elevated following exposure to VSV. Evidence for the functional nature of RIG-I expression in these cells comes from the observation that this molecule associates with its downstream effector molecule, interferon promoter stimulator-1, following VSV infection and from the finding that a specific ligand for RIG-I elicits astrocyte immune responses. Importantly, RIG-I knockdown significantly reduces inflammatory cytokine production by VSV-infected astrocytes and inhibits the production of soluble neurotoxic mediator(s) by these cells. These findings directly implicate RIG-I in the initiation of inflammatory immune responses by human glial cells and provide a potential mechanism underlying the inflammation and neuronal cell damage associated with acute rhabdoviral infections of the CNS.
Accelerated wound healing in mice by local administration of sustained-release CpG oligodeoxynucleotide (136.4)
Authors: Sato Takashi , Shimosato Takeshi , Yamamoto Masaki , Kobayashi Nobuaki , Miyazawa Naoki , Ishigatsubo Yoshiaki , Klinman Dennis
Published: January 2023

Recent reports show that wounds in mice lacking MyD88 heal at a delayed rate, suggesting that the innate immune pathway can affect the healing process. Preliminary studies revealed that the kinetics of wound healing in TLR9 KO mice were also delayed when compared to congenic controls, suggesting that this activation pathway may contribute physiologically to wound healing. Synthetic oligodeoxynucleotides (ODN) expressing unmethylated CpG motifs mimic the ability of bacterial DNA to stimulate an innate immune response via TLR9. We incorporated CpG ODN into a vehicle (basement membrane extract) that slowly released bioactive ODN to wound sites. The effect of the ODN was then examined using a murine excisional skin biopsy model. Results show that CpG ODN treatment significantly accelerates wound repair in normal adult and aged mice (p < 0.0001 and p < 0.01, respectively). Consistent with these effects being mediated by CpG signaling via TLR9, no effect was observed in TLR9 KO mice. Histologic studies indicate that CpG treatment promotes local accumulation of macrophages, increases production of VEGF, and accelerates neovascularization, resulting in accelerated granulation and wound closure in wildtype (but not TLR9 KO mice, p < 0.01). Current findings suggest that immunostimulatory oligonucleotides may represent a novel, inexpensive, safe and effective means of improving wound repair.
Mindbomb proteins are E3 ubiquitin ligases essential for TBK1-mediated antiviral activity (136.6)
Authors: Wang Lingyan , Li Shitao , Kong Young-Yun , Dorf Martin
Published: January 2023

First line host defense against virus attack includes the innate immune system which recognizes and mounts rapid antiviral responses. An important aspect in defense against viral infections is the production of type 1 interferon which provides a protective state to surrounding cells. TBK1 is a critical kinase involved in cellular transcriptional responses to infection and production of type I interferon. We found that TBK1 undergoes Lys63 ubiquitination following viral infection. The E3 ligases Mindbomb 1 (MIB1) and Mindbomb 2 (MIB2) associate with the TBK1 complex and confer K63-linked ubiquitin to TBK1, resulting in activation of downstream IRF signaling. Two lysine residues on TBK1 are ubiquitin acceptor sites and critical for innate antiviral defense. Ectopic expression of MIB1 or MIB2 augments TBK1-induced antiviral responses. Deficiency of MIB1 and MIB2 demonstrates that these genes are critical for TBK1 ubiquitination, type 1 interferon production, and TBK1-mediated antiviral activity. These results identify MIB1 and MIB2 as positive regulators of antiviral responses through polyubiquitination of TBK1.
Molecular mechanism underlying lipopolysaccharide-mediated expression of LCN2 in mesangial cells (136.9)
Authors: Glaros Trevor , Li Liwu
Published: January 2023

The neutrophil gelatinase-associated lipocalin 2 (LCN2) is a critical inflammatory mediator highly expressed in both acute and chronic inflammatory conditions. In particular, LCN2 produced in kidney mesangial cells contributes to tubular damage and kidney failure. In this report, we demonstrated that the interleukin 1 receptor associated kinase 1 (IRAK-1) is essential for lipopolysaccharide (LPS)-induced expression of LCN2 in kidney mesangial cells in vtro and kidney tissues in vivo. Mechanistically, we demonstrated that C/EBPδ is critically involved in the expression of LCN2. Through chromatin immunoprecipitation assays, we showed that LPS recruits C/EBPδ to the LCN2 promoter from WT, but not IRAK-1-/- cells. Furthermore, we revealed that both de novo synthesis and activation of C/EBPδ mediated by IRAK-1 are required for LCN2 expression. Blockage of new protein synthesis using cyclohexamide significantly reduces C/EBPδ and LCN2 expression in WT, but not IRAK-1-/- cells. The expression of C/EBPδ and LCN2 can also be effectively blocked by an IKKϵ inhibitor in WT, but not IRAK-1-/- cells, suggesting the IRAK-1 contributes to C/EBPδ expression though an IKKϵ-dependent mechanism. Taken together, this study has identified an integrated intracellular network involved in LPS-induced expression of LCN2. In addition, this research provides evidence that IRAK-1 is an important target with therapeutic potential in treating acute and chronic inflammatory diseases.
An Imbalance in sVEGFR1 and VEGF-A mediates corneal neovascularization after HSV-1 ocular infection (137.23)
Authors: Mulik Sachin , Suryawanshi Amol , Sharma Shalini , Rouse Barry
Published: January 2023

Ocular transparency is essential for optimal vision. The cornea maintains avascularity by expressesing soluble VEGFR1 (sVR) which binds resident VEGFA and constrain its pro-angiogenic activity. Here we report that early on, herpes simplex virus 1 (HSV-1) infection leads to an increase in VEGFA expression while low sVR levels in vitro as well as in vivo in infected mice corneas. In addition, we found degradation of sVR protein which was subsequently attributed to matrix metallopeptidase 7 and 9 as observed by in-vitro and ex-vivo degradation assays. Furthermore, mice receiving an inhibitor of MMPs showed decreased degradation of sVR and diminished HSV induced corneal angiogenesis. The second peak of VEGFA observed during clinical phase of herpes stromal keratitis (HSK) was attributed to infiltrating neutrophils and their depletion during this stage resulted in low VEGFA load and diminished corneal angiogenesis. Finally, the provision of recombinant sVR to HSV infected mice diminished angiogenic response supporting the notion that the activity of sVR has a constraining effect on the angiogenic activity of VEGFA. Taken together our results support the notion that the binding of sVR to VEGFA acts to limit the extent of ocular angiogenesis in pathological situations such as observed in HSK. Thus therapies designed to increase the presence, or diminish the breakdown, of sVR may be a useful therapeutic approach to manage the severity of HSK, an important cause of human blindness.
Dual roles of CD4+CD25+Foxp3+ Tregs in regulating Th17 cells and promoting host resistance to Candida albicans (137.7)
Authors: Pandiyan Pushpa , Conti Heather , Gaffen Sarah , Lenardo Michael
Published: January 2023

Th17 cells and CD4+CD25+Foxp3+ regulatory T cells (Tregs) are thought to promote and suppress inflammatory responses, respectively. However, their direct interaction in the context of an inflammation is unclear. Here we show that under Th17 conditions, Tregs suppress late but not early Th17 responses. Moreover, Tregs boost IL-17A, IL-17F and IL-22 in Th17 cells at early time-points. Transient up-regulation of Th17 cytokines by Tregs plays a protective role in clearing Candida albicans infection in mice. However, Tregs regain their suppressive functions over time and suppress Th17 cell mediated inflammation in Candida infected mice. Thus, Tregs play dual roles by initially promoting protective Th17 cells, but subsequently reducing the pathology of Th17 driven inflammation.
Control of RSV-induced lung injury by alternatively activated macrophages is IL-4Rα-, TLR4-, and IFN-β-dependent (137.8)
Authors: Shirey Kari Ann , Pletneva Lioubov , Puche Adam , Keegan Achsah , Prince Gregory , Blanco Jorge , Vogel Stefanie
Published: January 2023

Severe RSV-induced bronchiolitis has been associated with a mixed “Th1” and “Th2” cytokine storm. We hypothesized that differentiation of “alternatively activated” macrophages (AA-Mϕ) would mediate resolution of RSV-induced lung injury. RSV infection of murine lung and peritoneal macrophages resulted in IL-4 and IL-13 production, IL-4Rα/STAT6-dependent AA-Mϕ differentiation, and led to significantly enhanced inflammation in lungs of IL-4Rα-/- mice. Adoptive transfer of wild-type macrophages to IL-4Rα-/- mice restored RSV-inducible AA-Mϕ phenotype and diminished lung pathology. RSV-infected TLR4-/- and IFN-β-/- macrophages and mice also failed to express AA-Mϕ markers, but exhibited sustained proinflammatory cytokine production (e.g., IL-12) in vitro and in vivo and epithelial damage in vivo. TLR4 signaling was required for PPARγ expression, a DNA-binding protein that induces AA-Mϕ genes, while IFN-β regulates IL-4, IL-13, IL-4Rα, and IL-10 expression in response to RSV. RSV-infected cotton rats treated with a COX-2 inhibitor increased expression of lung AA-Mϕ. These data suggest novel treatment approaches for RSV that promote AA-Mϕ differentiation.
ITAM-containing adaptors regulate NKG2D-mediated cytotoxicity in human NK cells (138.15)
Authors: Neff Jadee , Bida Anya , Schoon Renee , Dick Christopher , Leibson Paul , Billadeau Daniel
Published: January 2023

NKG2D is expressed on cytotoxic lymphocytes and is an important immunosurveillance receptor that recognizes stress ligands such as MICA on tumor cells or virally infected cells. Unlike traditional immune activating receptors, NKG2D couples to the non-ITAM-containing transmembrane adaptor DAP10, which recruits PI3K and Grb2 to initiate target cell lysis. Current understanding predicts an ITAM-independent mechanism of NKG2D-mediated cytotoxicity. However, we show that in primary human NK cells, the ITAM-containing adaptors DAP12, FcϵR1γ, and CD3ζ selectively regulate the Grb2-Vav1 branch of NKG2D-mediated signal transduction and cytotoxicity. Furthermore, this ITAM-adaptor regulation of NKG2D is independent of ITAM activation. Thus our data reveal a more complex interplay between NKG2D and other activating receptors than was previously imagined, and we have uncovered a novel mechanism of ITAM-adaptor regulation of cell-mediated cytotoxicity. A more thorough understanding of immunoreceptor signaling networks will lead to more precise therapies when fighting diseases such as cancer, autoimmunity, and infection.
Signaling through kainate receptors enhances murine B cell proliferation and Ig production. (138.17)
Authors: Chaimowitz Natalia , Sturgill Jamie , Conrad Daniel
Published: January 2023

Abundant evidence indicates an interplay between the immune and nervous systems. Altered plasma glutamate levels, for example, have been strongly correlated with immunodeficiency states, such as AIDS and malignancies, suggesting that glutamate is capable of acting as an immunomodulator. While glutamate receptor signaling has been examined in some immune cells (e.g. T cells), the effects of glutamatergic stimuli on B lymphocytes remain unknown. To determine if glutamate is capable of modulating B cell function, we first examined mouse B cells for glutamate receptor expression. Furthermore, mouse splenocytes were cultured in glutamate-enriched media; proliferation and antibody production were determined. FACS analysis shows that splenic B2 B cells express the receptor subunits that comprise the kainate receptor (KAR), a specific subtype of ionotropic glutamate receptor. KAR surface expression was found to be upregulated upon B cell stimulation. Moreover, when splenocytes and B cells were stimulated with IL-4 and CD40 ligand in glutamate enriched media, there was a significant increased in cellular proliferation and antibody (IgG1, IgE) production. The KAR specific antagonist NS-102 is being used to confirm that these effects are mediated by KARs. The data also suggests that ADAM10 is required for the proliferative enhancing activity of glutamate. These results suggest that glutamate is capable of enhancing B cell function.
Human peripheral CD4+ T-cells expressing complement receptor 1 (CR1, CD35) (138.3)
Authors: Reilly Brian , Pfeiffer Anne , Petney Katrina , Atkinson John
Published: January 2023

CD4+ T-cells display an array of functions that can be traced to distinct lineages or to a variety of effector fates open to a naïve T cell. The path taken is dependent not only on the antigen-specific signal received through the TCR, but also on individual signals received through other cellular receptors. Earlier studies have shown the existence of CD35 on human CD4+ T-cells. To date, no further characterization of CD35 expression on other CD4+ subsets has been described. Here, using purified CD4+ T-cells, the molecular basis of CD35 expression is further explained. Known subsets were characterized by flow cytometry as naïve, central memory (CM), effector memory (EM), or T follicular helper cells (Tfh). All subsets contained small numbers of CD35+ cells with preferential expression on naïve and CM cells. If CFSE-loaded T-cells were activated in vitro by TCR cross-linking, the division index of CD35+ cells was 2-fold greater than that of CD35neg cells. This suggests that expression of CD35 defines a functional subset of CD4+ T-cells. Further analysis using cytokine microarrays demonstrated several cytokines to be elevated following co-engagement of CD3 and CD35 compared to TCR triggering alone. Employing cytokine secretion assays at the single-cell level, a bystander effect was observed with strong enhancement of IFN-γ production in CD35neg T-cells following co-engagement of CD3 and CD35. These results point to a regulatory role for CD35+ cells in T-cell activation.
Specific deletion of TRAF3 in B lymphocytes leads to B lymphoma development in mice (138.6)
Authors: Moore Carissa , Liu Yan , Shao Changshun , Bishop Gail , Covey Lori , Morse Herbert , Xie Ping
Published: January 2023

TRAF3 is a cytoplasmic adaptor protein utilized by the tumor necrosis factor receptor superfamily and Toll-like receptors for signaling. To explore the in vivo functions of TRAF3 in B cells, we recently generated B cell-specific TRAF3-deficient (B-TRAF3-/-) mice, and reported that TRAF3 is a critical regulator of peripheral B cell survival. In this study, we examined the hypothesis that the prolonged survival of TRAF3-/- B cells may predispose them to transformation. We found that approximately 50% of B-TRAF3-/- mice spontaneously developed B lymphomas by the age of 18 months. Our histopathological and immunophenotyping data revealed that B1 lymphomas, marginal zone B lymphomas, and immunoblastic B lymphomas developed in different individual B-TRAF3-/- mice. TRAF3-/- B lymphoma cells were malignant, as demonstrated by the identification of clonal B lymphoma cells in multiple organs in diseased mice, including spleen, ascites, bone marrow, cervical and mesenteric lymph nodes, and occasionally, kidney and lung. Corroborating our findings, recent evidence demonstrates that deletions and mutations of the TRAF3 gene frequently occur in human patients with B-cell lymphomas and multiple myeloma. Thus, our results, together with the evidence from human patients, indicate that TRAF3 is a tumor suppressor gene in B lymphocytes. Our findings suggest that TRAF3 downstream signaling components may serve as important therapeutic targets for the treatment of B lymphomas.
PKC-θ mediates negative feedback on regulatory T cell function (139.1)
Authors: Zanin-Zhorov Alexandra , Ding Yi , Kumari Sudha , Attur Mukundan , Hippen Keli , Brown Maryanne , Blazar Bruce , Abramson Steven , Lafaille Juan , Dustin Michael
Published: January 2023

T cell receptor (TCR) dependent regulatory T cell (Treg) activity controls effector T cell (Teff) function and is acutely inhibited by tumor necrosis factor (TNF). Protein kinase C-θ (PKC-θ) recruitment to the immunological synapse is required in effector cells to recruit Carma-1 in the pathway to NF-κB activation. Surprisingly, PKC-θ was sequestered away from the Treg immunological synapse. Furthermore, PKC-θ inhibition or suppression by RNAi enhanced Treg function, demonstrating PKC-θ mediated negative feedback. Treg integrate TCR and TNF signals through control of PKC-θ location such that inhibition of PKC-θ protected Treg from inactivation by TNF, restored activity of defective Treg from rheumatoid arthritis patients, and enhanced protection of mice from inflammatory colitis. Treg freed of PKC-θ mediated negative feedback can function in the presence of TNF and thus have therapeutic potential in control of inflammatory diseases.
Interleukin-2 inhibits programmed death-1 receptor expression on CD4+Foxp3+ regulatory T cells (139.11)
Authors: Channappanavar Rudragouda , Twardy Brandon , Suvas Susmit
Published: January 2023

Programmed death-1 (PD-1) is induced in response to TCR stimulation on T cells. In this report, we demonstrate that stimulation of splenocytes (SPCs) obtained from unmanipulated C57BL/6 mice with high (1.0 µg/ml) and low (0.1 µg/ml) dose of plate-bound anti-CD3 mAb resulted in an increased proportion of PD-1+ Treg when measured at different time-points (24h, 48h, 72h and day 4) post-stimulation. Interestingly, addition of recombinant IL-2 in anti-CD3 (0.1 µg/ml) stimulated SPCs culture significantly reduced proportion of PD-1+ Treg in comparison to control cultures with no exogenous IL-2. No significant difference in the frequency of PD-1+ Treg was evident in cultures stimulated with high dose of anti-CD3, irrespective to the addition of IL-2. Since Treg homeostasis in the periphery is largely regulated by IL-2 cytokine, we next determined whether in vivo neutralization of IL-2 can augment PD-1 expression on Treg. Our results showed that 48 h post intra-peritoneal administration of anti-IL-2 mAb in C57BL/6 mice, frequency of PD-1+ Treg significantly increased both in spleen and peripheral lymph nodes when compared to PBS treated control mice. We also determined an age related increase in PD-1 expression on Treg in C57BL/6 that correlates with an age-dependent reduction in IL-2 production in the periphery. Taken together, our results suggest that IL-2 signaling impede PD-1 expression on Treg both under homeostatic condition and in response to weak but not strong TCR stimulus.
Role of Krüppel-like factor 4 (Klf4) in regulation of T cell number and TH-17 differentiation (139.12)
Authors: An Jie , Golech Susanne , Klaewsongkram Jettanong , Huston Gail , Wood William , Zhang Yongqing , Wersto Robert , Becker Kevin , Swain Susan , Weng Nan-ping
Published: January 2023

Krüppel-like factor 4 (Klf4), a zinc finger-containing transcription factor of the Krüppel-like factor family, plays critical role in cell differentiation, growth, and cycle. We report here a role of Klf4 in T cell development and differentiation. The expression of Klf4 was high in thymocytes and resting mature T cells, and was down-regulated in activated mature T cells. In the absence of Klf4, modest but significant reductions of T cell numbers in thymus (~27%) and in spleen (~25%), and a similar degree reduction of spleen CD4 T cells and a less degree loss of spleen CD8 T cells were found in Klf4 deficient mice. Gene expression analysis revealed an alteration of genes involved in cell cycling and proliferation in the absence of Klf4 in thymocytes, implying their role in control of T cell number. Furthermore, we found that significant reduction of IL-17 expressing CD4 T cells (TH-17) during in vitro differentiation in mice absence of Klf4 compared to the wild-type mice. We then demonstrated that Klf4 directly binds the promoter of IL17a but not to the promoter of ROR-γt gene, suggesting that Klf4 directly regulates Il17a in TH-17 differentiation. Together, these findings demonstrate that Klf4 regulates T cell development and TH-17 differentiation.
SHP-1 negatively regulates Th17 cell development. (139.6)
Authors: Mauldin Ileana , Lorenz Ulrike
Published: January 2023

T helper 17 cells (Th17) are a recently identified T cell subset characterized by the production of the inflammatory cytokine IL-17. Activation of the transcription factor STAT3 downstream of IL-6 or IL-21-mediated signaling has been shown to be essential for Th17 differentiation; however how STAT3 is regulated is still poorly understood. We investigated whether the protein tyrosine phosphatase SHP-1, which is recognized for its regulatory role in JAK/STAT signaling, influences Th17 development. Using a genetic mouse model of SHP-1 deficiency, we show that mice homozygous (me/me) or heterozygous (me/+) for the motheaten mutation possess increased numbers of Th17 cells in their Peyer’s patches and intestinal lamina propria compared to wild type (+/+) mice. To gain a better mechanistic understanding of how SHP-1 regulates Th17 development, we performed ex vivo Th17 skewing assays comparing SHP-1-deficient and wild type mice. These studies demonstrate that T cells from me/+ mice are hyper-responsive to IL-6 or IL-21 stimulation compared to +/+ T cells, indicating that SHP-1 negatively regulates IL-6 and/or IL-21-driven Th17 development. Finally, biochemical analyses of primary CD4+ T cells from +/+, me/+ and me/me mice demonstrate that SHP-1 decreases the overall cytokine-induced phosphorylation of STAT3. Collectively, these data suggest that SHP-1 negatively regulates STAT3 signaling downstream of IL-6 and IL-21 and thereby limits Th17 generation.
IL-2 induces perforin mediated cytotoxicity in CD4 cells via STAT5 signaling (139.9)
Authors: Brown Deborah , Canfield Jenna , Lee Sarah , Condon Shirley
Published: January 2023

Cytolytic CD4 T cells have been documented in various chronic infections; however, the factors influencing development of these cells have not been fully elucidated. We have compelling data indicating that IL-2 has profound effects on the differentiation of perforin-mediated cytotoxicity in CD4 effectors. To further dissect the role of IL-2 in CD4 CTL generation, TCR Tg mice deficient in IL-2 or CD25 were used. Low concentrations of IL-2 are necessary in the first 24 h of culture to drive cytolytic CD4 cell differentiation. Cytotoxicity, diminished in the absence of CD25, suggests that signaling through the high affinity IL-2R complex is critical for full cytolytic potential. Phosphorylation of STAT5 peaked at 18-24 h, consistent with duration of exposure to IL-2. IL-7, a common γ chain cytokine that activates STAT5 also induces moderate cytolytic activity. STAT5 binds to the promoter region of perforin and granzyme B and studies are underway to determine whether blocking STAT5 activation can inhibit the development of perforin mediated cytotoxicity in CD4 CTL. Thus, a threshold of IL-2 signaling within the first 48 h is responsible for driving cytolytic activity. Whether this signaling involves STAT5 exclusively, or synergizes with other signaling pathways is a topic of further investigation. These data have implications in the design of vaccines against chronic infections such as HIV, CMV and EBV and in adoptive immunotherapy against certain tumors.
In vivo imaging of monocyte mediated neutrophil extravasation during acute arthritis (140.6)
Authors: Wang Baomei , Runnels Herb , Zinselmeyer Bern , Allen Paul , Miller Mark
Published: January 2023

Rheumatoid arthritis (RA) is an autoimmune disease characterized by progressive inflammation with massive infiltration of leukocytes into the synovium of multiple joints. Although monocyte/macrophage infiltration is a prominent feature and correlated with disease severity, the exactly role of monocytes in the induction phase of joint inflammation is unknown. Using two-photon microscopy, we tested whether monocytes regulate the recruitment of neutrophils during K/B×N serum induced arthritis. We examined neutrophil trafficking dynamics in vivo in LysM-GFP mice using the established i.v. transfer model and a modified in which serum was injected subcutaneously (s.c.) into the footpad. The s.c. model had several advantages including: more synchronized cell trafficking behaviors, shorter disease course and focused inflammation in the affected limb. We found that clodronate-liposome depletion of monocytes impaired neutrophil recruitment in our model at two separate steps. First, transendothelial cell migration was inhibited significantly, leaving many neutrophils stranded along the endothelial surface. Moreover, the cells that managed to extravasate showed defective chemotaxis and localization to the joint. Our results suggest that monocytes serve as key mediators of neutrophil extravasation and may play a second role in controlling the local tissue distribution of neutrophils during arthritis. Key words: Arthritis, Monocyte, Neutrophil, two photon microscopy
Cross-talk between chemoattractant and integrin signaling required for robust calcium flux in neutrophils (140.7)
Authors: Zhang Hong , Ley Klaus
Published: January 2023

Neutrophils are recruited to sites of inflammation in response to chemoattractants including fMLP through a sequence of events involving integrin activation, adhesion and migration into the tissue. Chemoattractant couples through GPCRs to PLCβ, leading to production of DAG and IP3, which in turn induces Ca2+ release from ER. Integrin engagement also elicits a rise in [Ca2+]i. Here, we show that [Ca2+]i in response to fMLP is severely defective in neutrophils lacking β2 integrin (Itgb2-/-). This defect in Ca2+signaling leads to downstream defects in neutrophil spreading, migration and respiratory burst. The Ca2+ signaling defect in neutrophils from Itgb2-/- mice was phenocopied in Plcγ2-/- mice that lack PLCγ2, an enzyme downstream of integrin signaling. Plcγ2-/- neutrophils were still able to arrest under flow, but the adhesion was not productive in that the cells failed to spread. Similar phenotypes were seen in Fgr/Hck, Syk or Tyrob/Fcrγ KO mice lacking molecules known to be involved in integrin signaling. These findings suggest a model in which a small Ca2+signal triggered by PLCβ is followed by an integrin-dependent boost of Ca2+release, resulting in a sufficient level of [Ca2+]i to trigger Ca2+ influx and downstream effector functions. This unexpected cross-talk between chemoattractant receptor and integrin signaling provides a mechanism for robust neutrophil recruitment and explains some of the severe functional defects in mice and patients lacking β2 integrins.
Maternal exposure to diesel exhaust particles alters neonatal dendritic cell DNA methylation profiles and skews function towards pro-Th2 responses. (141.13)
Authors: Fedulov Alexey , Yang Zhiping , Kobzik Lester
Published: January 2023

Neonates of mother mice exposed to allergen are born with increased risk of allergy; their dendritic cells (DC) carry the increased risk phenotype when transferred into normal recipients. These ‘pro-allergic’ DCs have vastly altered DNA methylation profiles. We investigated if epigenetic changes and ‘pro-allergic’ skewing also exist in DCs from offspring of mothers exposed during pregnancy to a pollutant linked to Th2 polarization, diesel exhaust particles (DEP). METHODS: Purified splenic CD11c+ DC of 14-d.o. allergen-naïve offspring from DEP-exposed or vehicle control mothers (n=6/group), as well as from positive control neonates born to OVA-sensitized and challenged females (n=3) were adoptively transferred to 3-d.o. recipients (n=12/group), followed by a low-dose OVA protocol which does not induce responses in normal controls. DCs were also screened for genome-wide DNA methylation changes. RESULTS: Recipients of DCs from neonate donors born to DEP-exposed mothers, but not to vehicle controls, revealed increased airway responsiveness and increased lung eosinophilia, which was similar in magnitude to recipients of DC from neonates born to asthma mothers. DNA methylation testing revealed shared patterns of epigenetic alterations in the DCs from DEP and asthma groups not seen in controls. CONCLUSION: Pregnancy exposure to DEP leads to ‘pro-allergic’ skew in neonatal DC; shared DNA methylation alterations suggest epigenetic involvement in increased allergy risk.
PU.1 expression in T cells is required for the development of IL-9 producing T cells in allergic inflammation (141.14)
Authors: Sehra Sarita , Chang Hua-Chen , Goswami Ritobrata , Han Ling , Nguyen Evelyn , Nutt Stephen , Kaplan Mark
Published: January 2023

The well documented Ets family transcription factor, PU.1 is an important regulator of mast cell, granulocyte and B cell development. Previous studies from our laboratory have identified a novel role for PU.1 in regulating Th2 cell cytokine expression. To further define a role for PU.1 in the development of allergic inflammation in vivo, we studied the development of allergic airway inflammation in mice with a conditional deletion of PU.1 in T cells. Despite the development of normal Th2-responses in the periphery, PU.1 deficient T cells have attenuated allergic inflammation in the lung as compared to wild type mice. To elucidate the mechanism for decreased inflammation in the lung, analysis of cytokines and chemokines from mice that had T cells lacking PU.1, demonstrated significantly decreased levels of IL-9 in the bronchoalveolar lavage fluid concomitant with a significant reduction in the pro-allergic chemokines, CCL17 & CCL22 in lung tissue. We identified PU.1 as a factor that promotes the Th9 phenotype by both repressing Th2 cytokine production and increasing IL-9 production. PU.1 was required for normal generation of IL-9 secreting T cells in vitro and ectopic expression of PU.1 increased IL-9 and chemokine production from Th9 cultures. Together, these data suggest a critical role for PU.1 in generating the Th9 phenotype and in the development of allergic inflammation.
IL-13 is required and sufficient for airway acidification in allergic airway inflammation (141.16)
Authors: Zimmermann Nives , Rothenberg Marc , Kottyan Leah
Published: January 2023

Clinical studies have shown acidification of airways in asthma. Importantly, studies have suggested that acidification contributes to the pathophysiological process. However, the mechanism of acidification is unclear. We developed a novel method for measuring the acidity of mouse airways and demonstrated that mouse airways are acidified during models of allergic airway inflammation. Our studies determined that airway acidification does not develop in IL-13-deficient mice and that IL-13 delivery alone is sufficient to induce airway acidification. There are multiple ways IL-13 could lead to acidification, including direct effects on epithelial cells or through recruitment of inflammatory cells. We demonstrated a partial role for eosinophils in airway acidification as CCR3 and IL-5-deficient mice had decreased extent of airway acidification in allergen-challenged mice. Furthermore, using dimethyl amiloride, a specific inhibitor of the Na+/H+ exchanger, we demonstrated significant inhibition of airway acidification in allergic airway inflammation, suggesting a role for ion (proton) channels. In summary, our studies demonstrate that mouse airways are acidified during allergic airway inflammation. We also showed that the mechanism of airway acidification in asthma involves IL-13-mediated pathways including eosinophils and proton channels. These results have considerable implications for the development of therapies that target airway acidification.
CD23 dependent transcytosis of IgE and immune complex across the polarized human respiratory epithelial cells (141.20)
Authors: Zhu Xiaoping , Palaniyandi Senthilkumar , Li Zili , Conrad Daniel
Published: January 2023

IgE is frequently present in airway secretions and its level can be enhanced in human patients with allergic rhinitis and bronchial asthma. However, it remains completely unknown how IgE appears in the airway secretions. In this study, we showed that CD23 (FceRII) was expressed in the established or primary human airway epithelial cells; its expression was significantly up-regulated when airway epithelial cells were subjected to IL-4 stimulation. In a transcytosis assay, CD23 was capable of transporting IgE across the polarized human Calu-3 monolayer in a bidirectional manner. Furthermore, CD23 was able to transport IgE derived immune complexes. CD23 specific antibody or soluble CD23 significantly reduced the efficiency of IgE or immune complex transcytosis, suggesting a specific receptor-mediated transport by CD23. Exposure of Calu-3 monolayer to IL-4 also enhanced the transcytosis of either human IgE or the immune complex. Transcytosis of both IgE and the immune complex was further verified in primary human airway epithelial cell monolayers. Furthermore, the transcytosed immune complexes were competent in inducing degranulation of the cultured human mast cells. Since airway epithelial cells are the first cell layer to come into contact with inhaled allergens, our study implies CD23-mediated IgE transcytosis in human airway epithelial cells may play a critical role in initiating and contributing to the perpetuation of airway allergic inflammation.
The role of Tyrosine kinase 2 on Th17 development in a murine model of allergic asthma (141.7)
Authors: Übel Caroline , Lehr Hans-Anton , Müller Mathias , Finotto Susetta
Published: January 2023

Mutations of the Janus Kinase Tyrosine Kinase 2 (Tyk2) in humans are associated with hypereosinophilia, Hyper-IgE and susceptibility to viral and mycobacterial infections. This syndrome was found to be the result of defective signaling of both IL 12 and IFNalpha as well as abnormal responses to IL 6, IL-10 and IL-23. Here we describe asthmatic Tyk2 deficient mice suffered from extreme airway eosinophilic inflammation and hyper IgE production compared to wild type mice. As these symptoms are associated with an increase in Th2 cytokines IL-4 and IL-5 in the airways, the major goal of the project is to investigate the role of Tyk2 in CD4+ T cell subset differentiation in the lung in a murine model of acute asthma. Th2 and early Th17 effector T cells are important in establishing the inflammation seen in the airways in this disease. In contrast, regulatory T cells and late developed Th17 T cells have a protective function on it. Under Th17 skewing conditions naïve CD4+ T cells from Tyk2 deficient mice showed an IL-6-dependent defect on Th17 cell development. Consistently, we found hyper-proliferation of Th2 cells associated with a constitutive inhibition of the Th1 and Th17 pathway in the lung of asthmatic Tyk2 deficient mice compared to wild type mice. In summary these data suggest that overexpression of Tyk2 could lead to amelioration of the asthmatic symptoms because this would suppress the pathogenic disease-specific CD4+ T effector cells in the lung.