bluuebunny/crossref_metadata_2025 huggingface dataset:
Tetracyclines tigecycline and doxycycline inhibit LPS-induced nitric oxide production by RAW 264.7 murine macrophages. (101.3)
Authors: Kaushik Vidhu , Beduya Dino , Kalampokis Ioannis , Kohlhoff Stephan , Joks Rauno O , Durkin Helen G , Nowakowski Maja
Published: January 2023

We previously reported that minocycline and doxycycline strongly suppress serum IgE and decrease IgE production by PBMC from patients with asthma (Smith-Norowitz et al, Ann Allergy, Asthma, & Immunol 2002; 89:172–9; Durkin et al, AAAAI Annual Meeting San Diego, Feb 23–27, 2007). We have now studied the effects of doxycycline and tigecycline on LPS-induced nitric oxide (NO) production by murine macrophages (RAW 264.7). Cell cultures were incubated for 1 – 5 days with or without LPS (200ng/mL, phenol extracted from Salmonella typhimurium) and doxycycline or tigecycline (1 – 10 μg/ml). Accumulation of nitrite, stable metabolite of NO, was determined using a modified colorimetric Griess reaction. Both doxycycline, and to a lesser extent tigecycline, consistently suppressed LPS-induced nitric oxide production in dose-dependent manner (doxycycline: 23–30%, tigecycline: 10–23%). The tetracycline mediated suppression of NO production by macrophages in culture is independent of antimicrobial activities, but the underlying mechanism of this effect remains to be elucidated. Since nitric oxide is an important mediator of inflammatory responses, our results suggest that the anti-inflammatory effects of tetracyclines may be utilized in the treatment of IgE mediated allergic disorders and may decrease exhaled breath nitric oxide, an emerging biomarker of allergic airway disease.
Oxylipins Derived from Docosapentaenoic Acid (DPAn-6) Possess Potent Anti-inflammatory Activity (101.4)
Authors: Arterburn Linda M , Dangi Bindi , Nauroth Julie , Teymourlouei Mah , Obeng Marcus
Published: January 2023

Background: The resolution of inflammation is an active process mediated by endogenous lipids formed by lipoxygenases and cyclo-oxygenases at the site of inflammation. Enzymatically oxygenated derivatives (oxylipins) of the omega-3 fatty acids DHA and EPA (resolvins) have potent resolution activity.

Objectives: To determine if oxylipins are enzymatically synthesized from the omega-6 fatty acid DPAn-6 and whether these molecules possess pro-resolution, anti-inflammatory properties.

Results: Two major metabolites, 17-hydroxy-DPAn-6 and 10,17-dihydroxy-DPAn-6, were formed from DPAn-6 following reaction with soybean 15-lipoxygenase. The identity of these metabolites was confirmed by LC/MS/MS, NMR, and UV analysis. 17-hydroxy-DPAn-6 was also formed following incubation of DPAn-6 with whole human blood. In a mouse air pouch model of acute inflammation, local administration of 100 ng (0.005 mg/kg) of either 17-hydroxy-DPAn-6 or 10,17-dihydroxy DPAn-6 resulted in 70–80% reduction in leukocyte trafficking, comparable to that observed following indomethacin (5 mg/kg ip) administration. The anti-inflammatory activity of both compounds was confirmed in a rat hind paw edema model of acute inflammation. Intravenous administration of 5 μg (~0.025 mg/ml) of either compound resulted in ~25% reduction in paw edema post carrageenan challenge. Both DPAn-6 oxylipins were also active in the hind paw model after oral administration.

Conclusions: Oxylipins derived from DPAn-6 are natural compounds possessing potent anti-inflammatory activity. These compounds or analogs thereof are candidates for new drugs that act uniquely to resolve inflammation.
A potential role for Citalopram as an immunoenhancer on morphine-induced immunosuppression (101.9)
Authors: Nguyen Thao , Vallejo Ricardo , Benyamin Ramsin , Kramer Jeff , Vogel Laura A.
Published: January 2023

Chronic pain patients are often challenged with depression stemming from the long-term physiopsychological effects of their condition. Consequently, chronic pain patients are often treated with morphine, which can induce immunosuppression, along with an antidepressant. The antidepressant, Citalopram, is a serotonin reuptake inhibitor that may have immunomodulatory effects. We investigated whether Citalopram has immunoenhancing effects on morphine-induced immune suppression. Adult mice were pretreated for 7 days with either saline or Citalopram (10 or 30 mg/kg, twice daily intraperitoneal injections), followed by immunosuppression by subcutaneous implantation of a 25 mg morphine pellet for 48 h. Spleen, thymus, and lymph nodes were harvested to analyze total cell numbers. Total splenocyte numbers in all Citalopram-treated animals were significantly higher than saline-treated animals, while no differences were found in the thymus or lymph nodes. More specifically, a high dose of Citalopram (30 mg/kg) resulted in significantly higher B cell numbers in the lymph nodes, where T cell and NK cell numbers were not different. Interestingly, splenic lymphocyte populations did not differ between treatments. In summary, Citalopram may have a specific potential in the attenuation of morphine’s immunosuppressive effect by enhancing splenocyte numbers and lymph node B cell populations.
DONOR SPLENOCYTES DECORATED WITH A NOVEL FORM OF FASL INDUCES TOLERANCE TO CARDIAC ALLOGRAFTS (102.11)
Authors: Yolcu Esma Shirwan , Zhao Hong , Gu Xiao , Lacelle Chantale , Bandura-Morgan Laura , Askenasy Nadir , Shirwan Haval
Published: January 2023

AICD is critical to tolerance to self antigens and apoptosis induced by Fas/FasL interaction plays a critical role in this process. We recently developed a novel approach designated as ProtEx™ that allows for the generation of chimeric immunological ligands with core streptavidin (SA) and their display on biotinylated cells in a rapid and effective manner. Importantly, the chimeric proteins have potent immunological activities possibly due to their ability to form tetramers and higher structures. We generated a chimeric form of FasL (SA-FasL) with potent apoptotic activity and previously demonstrated that the treatment of ACI rats with WF splenocytes decorated with SA-FasL pre- and post-transplantation of WF hearts resulted in tolerance. We herein tested if pre- or post-treatment alone results in tolerance to cardiac allografts. Pre-treatment of ACI rats with unmodified donor WF splenocytes (13x106) on days -6 to mobilize alloreactive T cells and prime them for apoptosis followed by two doses of SA-FasL-decorated cells administered on days -3 and -1 resulted in tolerance to >80% of the WF hearts transplanted on day 0. Posttreatment with SA-FasL-decorated donor cells on days +1, +3, and +5 resulted in tolerance to >60% of the WF hearts without the use of any adjuvant immunosuppression. In marked contrast, all the animals treated with donor splenocytes decorated with control SA protein rejected their grafts in a normal tempo. Immunomodulation with donor cells decorated with SA-FasL represents a practical, effective, and safe alternative approach to gene therapy for the induction of transplantation tolerance. NIH (R21 DK61333, R01 AI47864, R21 AI057903, R21 HL080108), ADA (1-05-JF-56).
IL-10-mediated Induction of regulatory Foxp3+CD4+ T cells by F4/80+ macrophages in mixed allogeneic chimeras (102.12)
Authors: Zhao Yong , Liu Guangwei , Ma Haixia
Published: January 2023

Antigen-presenting cells including macrophages play a crucial role in shaping immune response to pathogens or allografts. The immune regulatory function of macrophages has not been fully understood. In the present study, the ability of donor F4/80+ splenic macrophages (SPMs) to induce recipient regulatory T cells (Treg) was studied using a B6-->Balb/c mixed chimeric mouse model, in which transplantation tolerance has been achieved. B6 F4/80+ SPMs in mixed chimeras induced significantly lower cell proliferation, more IL-10 and TGF-â products, and less IL-2 and IFN-ã products of recipient CD4+ T cells than B6 F4/80+ SPMs in non-chimeric B6 mice did, whereas they showed similar immunogenicity to the third part C3H CD4+ T cells. Importantly, donor B6 F4/80+ SPMs in mixed chimeras could induce recipient Foxp3+CD4+ Tregs from Foxp3-CD25-CD4+ T cells. Furthermore, donor B6 F4/80+ SPMs in mixed chimeras produced more IL-10 and less IFN-ã than those in non-chimeric mice when cultured with recipient Balb/c but not the third party CD4+ T cells. The induction of CD4+ Tregs by donor F4/80+ SPMs could be significantly blocked by anti-IL-10, but not TGF-â mAb. Therefore, donor F4/80+ SPMs may have the ability to induce recipient Foxp3+CD4+ Tregs partially via IL-10-dependent pathway. This study identified an additional mechanism for macrophages to induce and/or maintain transplant tolerance in mixed allogeneic chimeras.
Dendritic cells supply IL-2 for gain of Treg function in vitro (102.14)
Authors: Nasr Mohamed Elmahadi
Published: January 2023

The CD4+CD25+ regulatory T cell plays an important role in controlling immunity to self and foreign antigens. CD4+CD25+ require T cell receptor and IL-2 receptor activation for in vivo development/ expansion, in vitro expansion and for in vitro function, but the source of IL-2 remains unknown.

we used the DO11.10 transgenic mouse model, whose T cell receptor shows allogeneic cross-reactivity to B6. Purified dendritic cells from WT or IL-2 −/− B6 were labeled with cell tracker green and cultured overnight with natural CD4+CD25+ Tregs from DO11.10 transgenic mice, the RD6 inhibitory hybridoma, or the control RD6N non-inhibitory hybridoma. DCs were FACS sorted then used to stimulate fresh CD4+CD25− DO11.10 effectors T cells in an IL-2 ELISPOT assay. CD4+CD25+ cells suppressed CD4+CD25− effectors cells up to 90% when stimulated with WT dendritic cells, but only 29% when stimulated with IL-2 −/− DCs. Similar results were obtained using the RD6 inhibitory hybridoma> RD6 suppressed a mean of 97% with WT dendritic cells and only 25% with IL-2 −/− dendritic cells. The result supports a model that Tregs require signal 3 activation for the APC to gain inhibitory function.
Natural Killer (NK) T cells subset predominance after irradiation due to Bcl-2 protection against p53 dependent apoptosis (102.15)
Authors: Yao Zhenyu , Liu Yinping , Strober Samuel
Published: January 2023

Previously, we found the percentage of regulatory NK T cells among all T cells in the spleen of mice treated with total lymphoid irradiation (TLI) increased markedly. In the current study, we gave total body irradiation (TBI) to wild type C57BL/6, p53−/− and Bcl-2 transgenic mice, and measured the number of surviving NK T and non NK T cells and their intracellular Bcl-2 levels. In some studies, the turnover rate of cells was examined by BrdU staining. 24 hours after irradiation, the percentage of NK T cells among all T cells had increased from 3% in the untreated controls to 65% in mice given 3,000 cGy. Whereas the number of non NK T cells decreased at least 1000 fold, the number of NK T cells decreased approximately 50 fold. The BrdU incorporation of NK T cells from irradiated mice was markedly reduced as compared to untreated mice, and was similar to that of non NK T cells. A subset of NK T cells (8–13%) and non NK T cells (1–6%) in untreated mice expressed a high level of Bcl-2. After 3,000 cGy, most surviving T cells were Bcl-2hi cells. The number of Bcl-2hi NK T cells decreased by about two fold while the number of Bcl-2hi non NK T cells decreased by about 100 fold. In p53−/− and Bcl-2 transgenic mice, both NKT and non NKT cells showed similar increased resistance to apoptosis after TBI. In addition, in p53−/− mice there was no significant increase in the percentage or decrease in the number of Bcl-2hi cells between NK T and non NK T cells after TBI. Our data indicate that the increased percentage of NK T cells amongst all T cells after irradiation is due to greater radioresistance of NK T cells as compared to non NK T cells. The loss of non-NK T cells is mainly due to the p53-dependent pathway of apoptosis. Almost all surviving NK T cells express the Bcl-2hi phenotype, but constitutive high expression of Bcl-2 alone cannot explain the unique radioresistance of NK T cells.
Exposure to NIMA-like alloantigens induces vigorous in vivo T cell responses in murine neonates (102.17)
Authors: Opiela Shannon Jacqueline , Adkins Becky
Published: January 2023

During early life, fetuses and neonates are exposed to allogeneic non-inherited maternal antigens (NIMA), through the passage of maternal cells and/or molecules across the placenta and in the breastmilk. Currently, it is thought that this early exposure results in the development of tolerance to NIMA. This is manifest in later life as an increased acceptance of grafts expressing the NIMA antigens. However, there also is evidence that exposure to NIMA during early life can lead to priming instead. In a murine model, we have shown that priming of neonates occurs when they are exposed to low doses of NIMA-like alloantigens. As these two possibilities, tolerance or priming, are extremely important for the outcome of transplantation, our lab has been engaged in studies to understand the in vivo responses of early life exposure to low doses of NIMA-like alloantigens. Following exposure, neonates developed both primary and memory cytotoxic responses to alloantigen. These responses were accompanied by vigorous Th1 and Th2 responses that exceeded the responses of adults similarly exposed. Overall, we conclude that exposure to low doses of NIMA-like alloantigens induces the development of robust cytotoxic and cytokine responses in neonates. This suggests that under specific conditions, early exposure to NIMA may lead to highly efficient immunological priming of all arms of T cell adaptive immunity, rather than tolerance.

This work was supported by NIAID grant number R01 AI44923-02 (B.A.)
The activating receptor, NKp30, is important in the interactions between rat natural killer cells and dendritic cells (102.20)
Authors: Wai Lu-En , Su Wei-Wei , Krams Sheri M
Published: January 2023

There is considerable interest in using dendritic cells (DC) to induce transplant tolerance. DC and natural killer (NK) cells have been found to interact in a bi-directional manner. DCs can activate human NK cells to more effectively kill, while NK cells can mediate both maturation and killing of DCs via an NK cell activation receptor, NKp30. NK cells expressing this receptor are elevated post-transplantation prompting us to examine NK cell interactions with DC in a rodent experimental system.

Wildtype and rNKp30-transfected RNK-16 cells (rat NK cell line) were co-cultured with bone marrow-derived DCs (BMDC). There was a significant increase in IFNγ production when rNKp30+, but not rNKp30−, NK cells were co-cultured with both immature and mature BMDCs. This NK-DC interaction required cell contact. Flow cytometric analysis of BMDCs with the rNKp30-Fc fusion protein (as compared to a control Fc fusion protein) indicated the presence of rNKp30 ligand(s) on the surface of DCs. Several tumor cell lines also specifically bound rNKp30-Fc. rNKp30-Fc binding was abolished by pretreatment of the tumor cells with trypsin suggesting the ligand is a protein. Furthermore, we established that the expression level of putative rNKp30 ligands correlated with the ability to activate NK cells, as indicated by the levels of IFNγ. Understanding NK cell interactions with DC may suggest potential new targets for tolerance induction.
NFAT1 in Cord Blood-Derived Human CD4+ T-Cells: Regulatory Implications for Graft vs. Host Disease (102.27)
Authors: Weitzel Richard Patrick , Lesnewski M , Fanning L , Laughlin Mary J
Published: January 2023

Clinical benefits of umbilical cord blood (UCB) grafts include the lower incidence of acute GVHD following allogeneic transplantation. We have previously demonstrated dramatically lower expression of the NFAT1 transcription factor and its associated transcripts and known binding partners in UCB CD4+ cells following primary stimulation when compared to T-cells obtained from adult blood (AB). We hypothesize that NFAT1 and NFAT1-dependent factors play a critical role in the increased proliferation and decreased cytokine production seen in UCB T-cells. We have now demonstrated in vitro via RNAi-mediated knockdown in AB-derived primary T-cells with RT-PCR and Cytometric Bead Assay that expression of a GVHD-promoting, Th1-type cytokine profile is dependent on expression of NFAT1, and that knockdown of NFAT1 results in a transcriptional and cytokine profile similar to that seen in UCB. However, microarray and RT-PCR time point analyses indicate NFAT1 transcripts are not changed in UCB versus AB CD4+ cells. This may suggest a crucial regulatory role for post-translational events which may preferentially occur in UCB-derived T-cells in vivo. Indeed, we have observed ubiquitination of NFAT1 and rapid rescue of NFAT1 expression following treatment with proteasome inhibitor in UCB-derived T-cells in vitro. NFAT1 in UCB CD4+ cells is shown by co-IP to be significantly more ubiquitinated than in AB-derived cells. Overall, our data indicates that regulation of NFAT1 through ubiquitination and proteasomal degradation, and its subsequent downstream effects may constitute a key difference between T-cells of adult and neonatal origin; specifically that this post-translational modification may serve as a method by which UCB-derived T-cells may prevent NFAT1 activity, impacting their allogeneic response.
In Vitro Methotrexate as a Practical Approach to Selective Allodepletion (102.7)
Authors: Mundy Dorothy I. , Sathe Atul , Ortega Sterling , Collins Robert H. , Karandikar Nitin J.
Published: January 2023

Graft-versus-Host Disease (GvHD) is a major cause of transplant-related morbidity and mortality in recipients of allogeneic hematopoietic stem cell transplantation. GvHD is mediated predominantly by alloreactive donor T-cells, therefore selective depletion of the alloreactive cells from the graft may alleviate GvHD, while maintaining other advantages conferred by donor T-cells, such as graft survival, antiviral immunity and graft-versus-leukemia effect. In this study, we evaluated the ability of methotrexate, a clinically approved anti-metabolite drug, to deplete alloreactive T-cells in mixed lymphocyte reactions (MLR). We show that methotrexate potently inhibits the proliferation of alloreactive T-cells in primary in vitro MLR. In addition, re-exposure of methotrexate-treated cells to the same allostimulus, results in a selective inhibition of the alloreactive immune response while the responses to third party allostimuli and viral antigens are preserved. In conclusion, our results provide strong pre-clinical evidence that in vitro treatment with MTX, a practical FDA-approved agent, results in specific allodepletion and may have excellent potential as an effective approach for preventing or minimizing GvHD.
EFFECT OF AGING ON HOST DENDRITIC CELL (DC) NUMBERS AND FUNCTION FOLLOWING HEMATOPOIETIC STEM CELL CONDITIONING REGIMEN (102.8)
Authors: Abu-Arja Rolla F , Park Joon Seong , Wilke Werner W , FAN JI , Schlueter Annette J
Published: January 2023

Graft versus host disease (GVH) incidence and severity increases with older patient age. Host DC are responsible for initiating GVH, likely from activation during transplant conditioning. In this study, young (8–14 wk) and older (12–16 mo) mice were treated with conditioning of lethal irradiation (950–1100 cGy) and assessed 6 h and 18 h later for changes in DC costimulatory molecule expression and cytokine secretion. Older mice had slightly higher numbers of splenic classical DC (cDC; CD11chi Cl II+ B220-) and plasmacytoid DC (pDC; CD11clo Cl IIlo B220+) both before and after irradiation. No differences were observed in CD40, CD80, or CD86 expression between young and older mice, either before or 6 h after irradiation. IL-12, IFN?, and TNF? producing cDC were more numerous in young than in older untreated mice. These cells decreased in young mice and increased in older mice at 6 and 18 h after irradiation. IL-12, IFN?, and TNF? producing pDC were more frequent in older than in young mice prior to irradiation, and both older and young mice had increased frequency of cells producing these cytokines after irradiation. Older mice had a higher frequency of IL-10+ cDC and pDC prior to irradiation. After irradiation, young mice had a higher frequency of IL-10+ cells. These findings indicate the balance of cytokine production in older mice after conditioning with irradiation is shifted to stimulatory rather than suppressive cytokines
Role of Hepatic Kupffer Cells in Inducing T cell Tolerance (128.12)
Authors: You Qiang , Cheng Linling , Ju Cynthia
Published: January 2023

The liver is known to favor the induction of immunological tolerance rather than immunity. Although Kupffer cells (KC) have been indicated to play a role in liver tolerance to allografts and soluble antigens, the mechanisms involved remain unclear. We hypothesized that KC could induce T cell tolerance by acting as ¡°poor¡± antigen presenting cells, as well as an active suppressor of T cell activation. The phenotypes of KC were characterized by flow cytometry. The abilities of KC to activate T cells and to suppress T cell activation induced by spleen dendritic cells (DC) were examined by in vitro proliferation assays using OTII cells. We found that, comparing with DC, KC expressed significantly lower levels of MHCII, B7.1, B7.2, and CD40. This is consistent with our observation that KC were not as potent as DC in eliciting OVA-specific T cell proliferation. More importantly, we found that KC could inhibit DC-induced OVA-specific T cell response. Further investigation of the mechanism involved in such suppression revealed that KC could induce apoptosis of the activated T cells. In summary, our data suggest that KC are ¡°poor¡± stimulators of T cells, and that they can cause tolerance through induction of T cell apoptosis. These findings indicate that KC play a critical role in regulating immune reactions within the liver and contributing to liver-mediated systemic immune tolerance.
Intravenous Administration of MOG35-55 Peptide Inhibits Experimental Autoimmune Encephalomyelitis by Suppressing IL-17/STAT4 Signaling Pathway (128.16)
Authors: Jiang Zhilong , Zhang Guang-Xian , Rostami A.M.
Published: January 2023

Intravenous (i.v.) administration of encephalitogenic peptide can effectively prevent experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. The mechanism underlying the induction of immune tolerance is not fully understood. In this study, we induced i.v. tolerance against EAE by administration of MOG35-55 peptide and determined the role of IL-17/STAT4 signaling pathways in this process. In EAE mice, IL-17 and tyrosine phosphorylated STAT1, STAT3, STAT4, ERK1/2 and NF-κBp65 were highly elevated in spleen cells and spinal cords, while they were significantly inhibited in tolerized mice. Administration of exogenous IL-17 effectively reversed MOG-induced immune tolerance, accompanied by an elevated level of phosphorylated STAT4. IL-17 activated STAT4 in a dose-dependent manner in vitro. CD11b+ cells in tolerant mice were tolerogenic, characterized by suppressed STAT4 activation; adoptive transfer of CD11b+ cells from the tolerized mice effectively delayed onset and reduced the severity of EAE. We conclude that the IL-17/STAT4 signaling pathway is closely related to EAE induction, which is negatively modulated in MOG-induced immune tolerance. These findings have important implications for the development of novel strategies to treat EAE/MS.
Suppressive Role of CD72 in Experimental Autoimmune Encephalomyelitis (128.22)
Authors: Li Daniel H , Creusot Remi , Ousman Shalina S , Fontoura Paulo , Sobel Raymond A , Cao Thai M , Ho Peggy , Youssef Sawsan , Robinson William H , Hong Anita , Steinman Lawrence , Parnes Jane R
Published: January 2023

CD72, a type II transmembrane protein expressed predominantly on B cells, acts as a negative regulator of B cell signaling and activation, and promotes B cell apoptosis through the B-cell receptor. However, whether CD72 plays a role in autoimmunity in vivo is unknown. We found that aged CD72-deficient mice naturally developed lupus-like autoimmune disease. To examine whether CD72 might play a role in multiple sclerosis [MS], we induced experimental autoimmune encephalomyelitis (EAE), a useful animal model for MS, in both wild-type (WT) and CD72-deficient (CD72KO) mice. We found that immunization with MOG (35–55) peptide induced an earlier onset and more severe clinical disease in CD72KO mice than in WT mice. CD72KO B cells had greater capacity for antigen presentation to CD4+ T cells as determined by T cell proliferation. MOG (35–55) immunized CD72KO mice also had higher anti-MOG IgM antibody response in the late phase (Day 35) of disease compared to WT mice. The present results indicate that CD72 may play an important role in the modulation of human autoimmune diseases such as lupus and MS.
Protection against autoimmune arthritis induced by hsp65-mediated tolerance involves enhanced production of both IFN-γ and antigen-specific antibodies (128.23)
Authors: Satpute Shailesh R. , Moudgil Kamal D.
Published: January 2023

Induction of antigen-specific immune tolerance is a desired goal of many therapeutic approaches against autoimmune diseases. Adjuvant arthritis (AA), an animal model of human rheumatoid arthritis (RA), is a Th1-mediated disease and it can be induced in Lewis (LEW) (RT.1l) rats by s.c. injection of heat-killed M. tuberculosis. Arthritic LEW rats raise T cell and antibody responses to mycobacterial heat shock protein 65 (Bhsp65). We employed a novel tolerogenic approach by introducing a Bhsp65-IgG heavy chain gene construct retrovirally ex vivo into B cells and then injecting these cells i.p. into syngeneic recipients. In another approach, we injected LEW rats i.p. with soluble Bhsp65. The former approach was successful in preventing as well as treating ongoing AA, whereas the latter was effective in the prevention. We observed significantly reduced T cell proliferative response to Bhsp65 in rats injected with either Bhsp65-IgG-expressing B cells or soluble Bhsp65 compared to the respective controls. This reduced T cell proliferation was restored by IL-2. Interestingly, there was increased production of IFN-γ by Bhsp65-primed cells in the Bhsp65-treated LEW rats, whereas not much change was observed in IL-10 secretion. The frequency of CD4+FoxP3+ T cells in peripheral blood did not change following tolerization. However, a high level of serum anti-Bhsp65 antibodies was observed in tolerized rats much earlier during the course of AA than that in controls. Regulation of a Th1-mediated autoimmune disease by a pro-inflammatory cytokine (IFN-γ) and the disease-protective attribute of antibodies represent novel aspects of this study. B cell-mediated suppression of established autoimmunity offers a promising approach for the treatment of RA.

(Support: Arthritis Foundation and NIH)
T cell tolerance to AChR, the autoantigen in myasthenia gravis, is mediated primarily by peripheral mechanisms (128.25)
Authors: Williams Earlanda , Stacy Sue , Carlisle Patricia , Infante Anthony , Lenardo Michael , Kraig Ellen
Published: January 2023

In myasthenia gravis (MG), a T-cell dependent antibody response is directed against acetylcholine receptor (AChR) at the neuromuscular junction. To study T cell tolerance, we generated a transgenic mouse in which the TAChRα chain is expressed as a self protein. This transgenic was bred to nude mice and thymus transplants were performed to produce progeny expressing TAChRα in the thymus and/or the periphery (muscle). These mice were immunized with the TAChRα dominant epitope, p146–162, and in vitro proliferation of responding T cells was measured. Since AChR is expressed at low levels in the thymus, it was presumed that central tolerance would be important. However, this was not the case; only mice with peripheral expression of TAChRα showed tolerance (a lack of proliferation). Furthermore, CFSE-labeled TAChR specific T cells from a TCR transgenic were adoptively transferred into TAChR transgenic (tolerizing) and non-transgenic recipients. Following immunization, proliferation of the transferred T cells was lower in the TAChR transgenic host. Moreover, when the TAChR transgene was bred to scurfy mice, progeny lacking Tregs (foxp3−/Y) exhibited an increase in serum anti-TAChR Ig. Autoantibodies were not seen in the foxp3+/Y mice or in mice lacking Aire function (which have defective central tolerance). These results are consistent with a primary role for peripheral tolerance in regulating the response to AChR.

Supported by an APC Subcontract, a UTHSCSA ERC grant and NIH (T32AG021890, T32DE11438).
Mechanism of B-cell mediated tolerance in LPS versus CpG treated cells (128.33)
Authors: Carey Indira , Su Yan , Li Xin , Scott David
Published: January 2023

Our laboratory has developed a gene therapy protocol for B-cell mediated tolerance induction, whose efficacy has been demonstrated in multiple mouse models for human disease, including diabetes, MS, uveitis and hemophilia. We recently found that LPS activated B cells were tolerogenic, whereas those activated with CpG were not. These observations were made in B cells retrovirally transduced to present various experimental antigens, as well as in specific antigen-presenting transgenic B cells. To further elucidate the mechanism of LPS vs CpG action in B-cell mediated tolerance, we examined differential cytokine expression in mouse primary B cells by Real Time PCR and multiplex protein profiling. Since different migration patterns might be associated with B cell activation by LPS versus CpG, we also tracked B cell migration to lymph nodes after activation. While Real Time PCR data suggests that there is no dramatic difference in IL10 mRNA levels between LPS and CpG treated samples, preliminary multiplex protein profiling data indicate that IL10 may be important for tolerance. Interestingly, our results also show that CpG treatment may affect B cell proliferation and migration, compared to LPS. Thus, LPS and CpG treated B cells mediate differential effects on B cell function/fate. These data may explain the activity of B cells exposed to naked DNA vs those that are retrovirally transduced.

(Supported by NIH AI35622)
Monitoring FoxP3 expression in Tolerance to Factor VIII (128.4)
Authors: Skupsky Jonathan , Scott David W.
Published: January 2023

The FoxP3 transcription factor has emerged as a marker for regulatory T cells in the mouse. Our lab has identified a gene therapy based approach that allows us to abrogate the immune response in models for autoimmune diseases and the deleterious immune response in hemophilic patients being treated with factor VIII. Utilizing the FoxP3-GFP knock-in mouse that has been mated to our hemophilic fVIII−/− mouse, we are able to monitor fluctuations in the FoxP3 by flow cytometry. We have observed a relative increase in FoxP3 when tolerance is induced and a relative decrease when an immune response is initiated towards fVIII. We have also developed an in vitro model to follow FoxP3 as it changes in different populations with certain stimuli. Thus, we found that ~75% of CD4+CD25+ cells in a resting population are FoxP3 positive and 12% of all CD4+ cells are FoxP3+. After a single day of stimulation of splenocytes with soluble anti-CD3 antibody, nearly all the CD4+ cells become CD4+CD25+ while less than 6% remain FoxP3+. When presentation of anti-CD3 via Fc receptors is blocked, activation (monitored by CD25 levels) is abrogated, but FoxP3 levels also decrease. While this drop in the frequency of FoxP3 expression in CD4 T cells can be explained in part by the activation of the CD4+CD25− effector cells, we propose that anti-CD3 antibody is also actively diminishing FoxP3 levels in the regulatory T cell population.

Supported by HL061883.
B cells and T regulatory cells regulate spontaneous autoimmune thyroiditis (SAT) in NOD.H-2h4 mice (128.7)
Authors: Yu Shiguang , Braley-Mullen Helen
Published: January 2023

SAT is characterized by infiltration of thyroids by T and B cells. Although B cell-deficient NOD.H-2h4 mice are resistant to SAT, they develop SAT following transient depletion of T reg by anti-CD25. Depletion of T reg does not increase SAT severity in WT mice. WT and B cell deficient mice have similar numbers of CD4+CD25+Foxp3+ T cells, suggesting that T reg in WT and B cell deficient mice may differ in function, or effector T cells of WT mice may be resistant to suppression, i.e. B cells might modulate T reg activity. T reg from WT and B cell-deficient mice did not differ in their expression of surface TGF-β, or in their ability to suppress anti-CD3 induced proliferation, and effector cells from WT and B cell-deficient mice are equally suppressed by T reg. Studies are in progress to determine if T reg from WT and B cell deficient mice differ in their ability to suppress SAT in vivo. To determine if T reg might inhibit SAT in B cell-depleted WT mice, newborn WT mice were given anti-IgM to deplete B cells; half also got anti-CD25 to deplete T reg. B cell-depleted WT mice developed minimal SAT, but after T reg depletion, they develop SAT comparable to unmanipulated WT mice, suggesting that T reg can inhibit SAT in B cell depleted WT mice. Depletion of B cells in adult WT mice using anti-CD20 reduces SAT severity and anti-thyroglobulin antibody. Studies are in progress to determine if T reg depletion can reverse the effects of anti-CD20 on SAT development.

(Supported by Arthritis National Research Foundation, Children′s Miracle Network, and VA Merit Review).
Myelin specific regulatory T cells expand from naturally occurring regulatory T cells and accumulate in the CNS during EAE (128.8)
Authors: Oukka Mohamed , Bettelli Estelle , Korn Thomas , Gao Wenda , Sobel Ray , Strom Terry , Wucherpfennig Kai , Kuchroo Vijay
Published: January 2023

FoxP3 is a lineage specific marker for regulatory T cells (T-reg). We have generated FoxP3 knock-in mice by introducing a bicistronic GFP reporter into the endogenous FoxP3 locus, allowing us to faithfully track T-reg in vivo. We have also generated a MOG 35-55/IAb-tetramer. The combination of these two technologies enables us to study the in vivo behavior of myelin specific T-reg and effector T cells (T-eff) during EAE. Upon immunization with MOG 35-55, we identified a population of MOG-tetramer-reactive T-reg in the peripheral lymphoid compartment. T-reg trafficked to the CNS where they were readily detected as early as day 10 following immunization. At this stage and till the peak of the disease, an overwhelming number of T-eff also targeted the CNS. Thus, the ratio of T-reg and T-eff at the initiation of disease was 1:15. However at the onset of recovery, this ratio changed in favor of T-reg (T-reg/T-eff close to 1:1. During recovery, 50% of the CNS-derived T-reg were IL-10 producers compared to only 10% in the peripheral immune compartment. FOXP3+ T cells isolated from the CNS are effective in suppressing naive MOG-specific T cells, but fail to control CNS-derived T-eff that secrete IL-6 and TNF. Our data suggest that in order for CD4+FOXP3+ T-reg to effectively control autoimmune reactions in the target organ, it may also be necessary to control tissue inflammation
Phenotypes of TLR7-overexpressing B cells suggest a TLR7-mediated increase in antigen presentation (129.11)
Authors: Pisitkun Prapaporn , Deane Jonathan A. , Bolland Silvia
Published: January 2023

Autoantibodies against nuclear antigens secreted by autoreactive B cells are a hallmark of systemic autoimmunity. Failure to eliminate autoreactive B cells in the germinal center reaction has been reported in lupus disease. We have recently shown that TLR7 duplication in Yaa mice accelerates the onset of lupus disease. To better understand TLR7 roles in the pathogenesis of lupus disease, we created transgenic TLR7 (B6.Tg. TLR7) mice. We found an increase of spontaneous germinal center formation in both B6.Tg. TLR7 mice and B6. Yaa which is TLR7 dependent. We have carried out microarray analysis of gene expression profiles of follicular B cells (CD9-CD43-) from B6.Tg. TLR7 compared to B6. The upregulated genes in B6.Tg. TLR7 consist of interferon inducible genes, cell cycle regulators, MHC, and immunoglobulin genes. We have confirmed increased protein expression of phosphorylated stat1 (Ser727) by western blot and of H2-Ab1 by flow cytometry in B6.Tg. TLR7. These results suggest the possibility of increased antigen presentation by TLR7-overexpressing B cells in the high interferon environment.
Distinct subsets of dendritic cells drive tolerance in two models of CD4 T cell mediated autoimmunity (129.14)
Authors: Jain Renu , Divekar Rohit D , Nast Danielle M , Lee Hyun-Hee , Ellis Jason S , Hardaway John C , Haymaker Cara L , Hoeman Christine M , Zaghouani Habib
Published: January 2023

Dendritic cells (DCs) are professional antigen presenting cells that are capable of presenting both self and foreign antigens to T cells thereby inducing either tolerance or immunity respectively. What is still not clear is whether T cell tolerance requires a specific subset or any population of DCs can drive tolerance. To address this question different subsets of DCs were tested for suppression of autoimmunity in two major animal models- type I diabetes (T1D) and experimental allergic encephalomyelitis (EAE).

There are 3 major subsets of DCs in spleen- CD8α+, CD8α−CD4+ and CD8α−CD4−. In this study we investigated the ability of these subsets to induce tolerance in TID and EAE. Indeed the CD8α−CD4− DC subset mediated protection against diabetes in non-obese diabetic (NOD) mice. In fact, mice had reduced destructive infiltration within the islets and the antigen-specific lymphocytes showed reduced proliferation and an increase in IFNγ and IL-10 response. Surprisingly, in EAE it is the CD8α−CD4+ DC subset that supported protection against the disease. The lymphocytes from treated mice displayed more of a Th1 biased response. Both the protective subsets were mature and expressed significant levels of class II MHC and costimulatory molecules. However, the subsets trafficked differently to spleen and draining lymph nodes. Thus, distinct DC subsets drive tolerance in the two models of autoimmunity despite the fact that both diseases are mediated by CD4+ T cells.

Research supported by grants DK65748 and NS37406 from NIH.
Susceptibility to autoimmune myocarditis in mice: analysis of the Eam1 susceptibility locus (129.18)
Authors: Ligons Davinna LaChelle , Guler Mehmet L. , Li Haiyan S. , Rose Noel R.
Published: January 2023

H-2sA.SW and B10.S mice are susceptible and resistant to experimental autoimmune myocarditis (EAM), respectively. Our previous studies suggested that Eam1 on chromosome 1 is a non-MHC locus conferring susceptibility to cardiac myosin-induced (EAM) in A.SW mice. B10.A Eam1 congenic mice, carrying the Eam1 locus on the EAM-resistant B10.S background, develop EAM comparable to A.SW mice, suggesting that Eam1 plays a key role in the development of disease. We have previously reported that lymph node cells (LNC) of A.SW mice are more resistant to cyslophosphamide (Cy)-induced apoptosis in comparison to B10.S mice, and a similar apoptosis-resistant phenotype is found in NOD mice has been linked to the Idd5 locus overlapping with Eam1. Therefore, we hypothesized that the mechanisms by which Eam1 mediates the susceptibility to EAM could be due to its regulatory role in apoptosis. In the current study, we found that LNC from B10.A Eam1 congenic mice present an intermediate susceptibility to Cy-induced apoptosis, i.e. the percentage of apoptotic LNC with activated caspase 3, 8, 9 in congenic mice falls in between A.SW and B10.S mice. Interestingly, data suggest that A.SW mice have significantly lower levels of apoptotic LNC following immunization with cardiac myosin in CFA than B10.S mice. Our results therefore suggest that Eam1 partially down regulates LNC apoptosis and delays the removal of self-activated T cells, thereby providing an explanation for the role of Eam1 in the susceptibility to EAM.

Supported in part by NIH grant HL077611
Production of CCL2 by CNS resident cells regulates development of EAE through the recruitment of TNF-α and iNOS expressing macrophages (129.19)
Authors: Dogan Rukiye Nazan , Elhofy Adam , Karpus William J
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) is demyelinating disease of the central nervous system (CNS), which serves as an animal model for Multiple Sclerosis (MS). Increased expression of CCL2, monocyte chemoattractant protein-1 has been detected in CNS inflammatory infiltrates, which consist of T cells, macrophages, and B cells. The critical role for CCL2 in MS-like diseases was identified in mice that lack this molecule and develop less severe EAE and impaired macrophages recruitment into the CNS. While CCL2 production is essential for macrophage infiltration into the CNS, it remains unclear which cell source is critical in disease induction. We constructed radiation bone marrow chimera to investigate the cell-specific source of CCL2 production in EAE. Mice lacking CCL2 production by resident CNS cells showed a delayed and diminished clinical disease as result of decreased macrophage infiltration into the CNS. Moreover, TNF-α and iNOS producing TipDC recruitment to CNS was reduced in mice devoid of CCL2 production by CNS resident cells. Mice lacking CCL2 production by infiltrating mononuclear cells developed EAE similar to controls. Our study demonstrates that CCL2 production by CNS resident cells is the major contributors to trafficking of macrophages into the CNS and subsequent disease progressions.

Supported by NIH NS034510
Possible mechanism of shedding of BAFF in human T cells (129.2)
Authors: Yoshimoto Keiko , Takahashi Yasue , Setoyama Yumiko , Suzuki Katsuya , Tsuzaka Kensei , Abe Tohru , Takeuchi Tsutomu
Published: January 2023

B cell activating factor of the TNF family, or BAFF, is a type II membrane-bound protein and the extracellular C-terminal fragment is released from the cells as soluble BAFF (sBAFF). BAFF is mainly produced in monocytes and dendritic cells, and regulates proliferation, differentiation, and survival of B cells. Our recent study has revealed that the expression of BAFF is abnormally elevated in peripheral T cells of SLE patients, suggesting that BAFF plays an important role in the pathogenesis of autoimmune diseases. In the present study, we investigated the expression of BAFF in T cells of Sjögren’s syndrome (SS), and found that the expression of BAFF was enhanced in peripheral T cells of SS patients upon stimulation. We further analyzed the mechanism of release of sBAFF from T cells, since this step may provide a therapeutic possibility to treat the diseases. Particularly, we focused on furin and matrix metalloproteinases, which are membrane bound proteases and responsible for shedding of several proteins. Investigation with specific inhibitors against these proteases revealed that at least furin and MMP9 was involved in the release of sBAFF.
Differential involvement of Th1 or Th17 cells in pathogenic autoimmune processes triggered by different TLR ligands (129.22)
Authors: Shi G. , Vistica B. P. , Yu C-R. , Cox C. A. , Montalvo V. , Wawrousek E. F. , Egwuagu C. E. , Gery I.
Published: January 2023

We have previously shown that various TLR ligands are capable of converting naïve CD4 cells specific to hen egg lysozyme (HEL) to become effector cells that initiate inflammation in eyes of recipient mice expressing HEL in their lens. In addition, our data revealed that pertussis toxin (PTX) is superior to all other tested TLR ligands in this activity. Here, we examined the involvement of Th1 or Th17 cells in the pathogenic processes triggered by different TLR ligands. Using qPCR, we found that eyes of PTX-treated recipients expressed both IL-17 and IL-22 transcripts ~15, ~10 and ~6 fold higher than eyes from recipients treated with poly (I:C) (PIC), CpG oligodeoxynucleotide (CpG), or LPS, respectively. Although inflamed eyes from PTX-treated mice also expressed IFN-γ more than eyes of the other groups, the differences were markedly lower: ~5, ~ 4 and ~ 4 fold, respectively. Furthermore, intracellular staining of infiltrating cells collected from the inflamed eyes revealed that cells stained for IL-17 were ~5 fold higher than cells stained for IFN-γ in eyes of PTX-treated mice, ~2 fold higher in eyes of the LPS group, approximately equal in the PIC group, but ~2 fold lower in the CPG group.
IL13 induces IL17 by regulating dendritic cell maturation in autoimmune myocarditis (129.25)
Authors: Cihakova Daniela , Barin Jobert , Baldeviano G Christian , Kimura Miho , Talor Monica V , Rose Noel R
Published: January 2023

IL13 KO BALB/c mice developed severe myosin-induced experimental autoimmune myocarditis (EAM). This severe disease is characterized by increased inflammation, increased total intracardiac CD45+ leukocytes, and progression to dilated cardiomyopathy. Hearts of IL13 KO mice had increased levels of the proinflammatory and profibrotic cytokines IL1β, IL18, IFNγ, TGFβ1, IL4 and histamine. However, another proinflammatory cytokine, IL17 is significantly decreased in both hearts (p= 0.0005) and spleens of IL13 KO mice on day 21 of EAM. The decrease of IL17 is significantly correlated with decrease of another Th17 cytokine – TNF α in the hearts and spleens of IL13 KO mice. The substantial decrease of the Th17 population in the absence of IL13 is not due to the absence of cytokines required to induce Th17, since IL13 KO mice have increased levels of IL1β and TGFβ. This Th17 defect is also not caused by increased IL27, which is known to impair Th17 development, since IL27 is actually significantly decreased in IL13 KO mice. IL23, which is necessary for sustaining the Th17 population, is significantly decreased in IL13 KO mice. It has been shown that IL13 induces production of IL23 by dendritic cells. We have observed severely impaired DC maturation both in vivo as well as in vitro in IL13 KO mice. Thus, we hypothesize that IL13 is required for maturation and IL23 production by DCs; therefore, IL13 is required for Th17 differentiation.

(Supported by 2R01 HL67290).
IL-23 and IL-17 in pathogenesis of experimental ocular autoimmunity: requirement for IL–23 may extend beyond its role in sustaining the IL-17 effector response (129.26)
Authors: Caspi Rachel , Silver Phyllis , Cua Daniel , Chen Zoe , Chan Chi-Chao , Luger Dror
Published: January 2023

Experimental autoimmune uveitis (EAU) represents autoimmune uveitis in humans. Here we examined the role of the IL-23/IL-17 pathway in EAU. We immunized IL-23p19, IL-12p35, IL-12p40, IFN-γ and IL-17 KO mice with the uveitogenic Ag IRBP. Alternatively, we treated wild type mice immunized for EAU with Abs to IL-23p19 or to IL-17 throughout the disease course (prevention), or only during the effector phase (reversal). IL-23p19 KO were resistant to EAU, showing reduced Ag-specific DTH, IL-2, IFN-γ, IL-17, IL-1β, IL-6 and IL-5. In contrast, IL-12p35 and IFN-γ KO mice developed exacerbated EAU, DTH and IL-17 responses. Treatment with anti-IL-23p19 protected from EAU and reduced immunological responses, but was unable to reverse disease, indicating that EAU is dependent on early availability of IL-23. In contrast, anti-IL-17 prevented as well as reversed EAU. Interestingly, severe EAU could be induced with a Th1 cell line that does not produce IL-17. In vivo cytokine neutralization revealed requirement for IFN-γ and TNF-α, but not for IL-17, suggesting that under some conditions an antigen-specific Th1 effector is sufficient to induce EAU. In keeping with this, IL-17 KO mice were still susceptible to disease. These data raise the possibility that the nonredundant need for IL-23 in EAU may extend beyond its role in promoting the Th17 effector response and point to IL-23 and IL-17 as new therapeutic targets for uveitis.