[Show abstract][Hide abstract] ABSTRACT: Follicular T helper (Tfh) cells are a critical population of CD4 T helper cells that are primarily localized in the germinal centers (GCs) to help B cell maturation and antibody production. Tfh cells can be identified in tissue sections based on the expression of a panel of classical Tfh surface makers, transcription marker(s), and effector-function cytokines, as well as by their unique anatomic proximity to other GC cells, including follicular dendritic cells (FDC) and GC B cells. Here, we describe an immunofluorescence staining method for visualization of GC Tfh cells in frozen spleen tissue sections of the autoimmune BXD2 mouse using a confocal imaging strategy. Tfh cells were characterized based on the expression of CD4, CXCR5, Bcl6, IL-21, and IL-17.
[Show abstract][Hide abstract] ABSTRACT: The breakdown in tolerance of autoreactive B cells in the lupus-prone NZM2410-derived B6.Sle1.Sle2.Sle3 (TC) mice results in the secretion of autoantibodies. TC dendritic cells (DCs) enhance B cell proliferation and antibody secretion in a cytokine-dependent manner. However, the specific cytokine milieu by which TC DCs activate B cells was not known. In this study, we compared TC and C57BL/6 (B6) control for the distribution of DC subsets and for their production of cytokines affecting B cell responses. We show that TC DCs enhanced B cell proliferation through the production of IL-6 and IFN-γ, while antibody secretion was only dependent on IL-6. Pre-disease TC mice showed an expanded PDCA1+ cells prior to disease onset that was localized to the marginal zone and further expanded with age. The presence of PDCA1+ cells in the marginal zone correlated with a Type I Interferon (IFN) signature in marginal zone B cells, and this response was higher in TC than B6 mice. In vivo administration of anti-chromatin immune complexes upregulated IL-6 and IFN-γ production by splenic DCs from TC but not B6 mice. The production of BAFF and APRIL was decreased upon TC DC stimulation both in vitro and in vivo, indicating that these B cell survival factors do not play a role in B cell modulation by TC DCs. Finally, TC B cells were defective at downregulating IL-6 expression in response to anti-inflammatory apoptotic cell exposure. Overall, these results show that the TC autoimmune genetic background induces the production of B cell-modulating inflammatory cytokines by DCs, which are regulated by the microenvironment as well as the interplay between DC.
PLoS ONE 08/2014; 9(8):e102151. DOI:10.1371/journal.pone.0102151 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transient thymic involution is frequently found during inflammation, yet the mode of action of inflammatory cytokines is not well defined. Here we report that interleukin-23 (IL-23) production by the thymic dendritic cells (DCs) promotes apoptosis of the CD4(hi)CD8(hi) double-positive (DP) thymocytes. A deficiency in IL-23 signalling interferes with negative selection in the male D(b)/H-Y T-cell receptor (TCR) transgenic mice. IL-23 plus TCR signalling results in significant upregulation of IL-23 receptor (IL-23R) expressed predominantly on CD4(hi)CD8(hi)CD3(+)αβTCR(+) DP thymocytes, and leads to RORγt-dependent apoptosis. These results extend the action of IL-23 beyond its peripheral effects to a unique role in TCR-mediated negative selection including elimination of natural T regulatory cells in the thymus.
[Show abstract][Hide abstract] ABSTRACT: Germinal centers (GCs) provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. In this study, we show that there are significantly higher frequencies of CXCR5(+)ICOS(+) TFH cells in autoimmune BXD2 mice, and these cells express both IL-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra(-/-) mice, the majority of CXCR5(+) TFH cells from BXD2-Il17ra(-/-) mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra(-/-), disrupted TFH-B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling 16 (RGS16) to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16(-/-) mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on postdifferentiated IL-17RA(+) TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.
The Journal of Immunology 07/2013; 191(4). DOI:10.4049/jimmunol.1300479 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bidirectional interactions between granulocyte–macrophage colony-stimulating factor–positive (GM-CSF+) T cells and interferon regulatory factor 5–positive (IRF-5+) macrophages play a major role in autoimmunity. In the absence of SH2 domain–containing phosphatase 1 (SHP-1), GM-CSF–stimulated cells are resistant to death receptor (DR)–mediated apoptosis. The objective of this study was to determine whether TRA-8, an anti–DR5 agonistic antibody, can eliminate inflammatory macrophages and CD4 T cells in the SHP-1–deficient condition.
Ubiquitous Cre (Ubc.Cre) human/mouse-chimeric DR5–transgenic mice were crossed with viable SHP-1–defective motheaten (mev/mev) mice. TRA-8 was administered weekly for up to 4 weeks. The clinical scores, histopathologic severity, and macrophage and CD4 T cell phenotypes were evaluated. The role of TRA-8 in depleting inflammatory macrophages and CD4 T cells was also evaluated, using synovial fluid obtained from patients with rheumatoid arthritis (RA).
The levels of inflammatory macrophages (interleukin-23–positive [IL-23+] IRF-5+) and CD4 T cells (IL-17+ GM-CSF+) were elevated in mev/mev mice. In DR5-transgenic mev/mev mice, DR5 expression was up-regulated in these 2 cell populations. TRA-8 treatment depleted these cell populations and resulted in a significant reduction in inflammation and in the titers of autoantibodies. In synovial cells from patients with RA, the expression of IRF5 and DR5 was negatively correlated with the expression of PTPN6. TRA-8, but not TRAIL, suppressed RA inflammatory macrophages and Th17 cells under conditions in which the expression of SHP-1 is low.
In contrast to TRAIL, which lacks the capability to counteract the survival signal in the absence of SHP-1, TRA-8 eliminated both IRF-5+ IL-23+ M1 macrophages and pathogenic GM-CSF+ IL-17+ CD4 T cells in a SHP-1–independent manner. The results of the current study suggest that TRA-8 can deplete inflammatory cell populations that result from a hyperactive GM-CSF/IRF-5 axis.
[Show abstract][Hide abstract] ABSTRACT: Given the protective roles of 25-hydroxyvitamin D (25[OH]D or vitamin D) in musculoskeletal health and the potential beneficial effects of vitamin D supplementation in reducing the risk of various chronic diseases, intensive repletion of vitamin D has been widely advocated. Of note, CD8 T cells have the highest levels of the vitamin D receptor compared with other major immune cells. The effects of vitamin D on CD8 T cells during aging, however, remain unclear. This study determined the relationship between vitamin D levels and CD8 T-cell status in 34 healthy female subjects (all >60 years old). The CD8 T cell phenotype was defined by the surface expression of CD28 and CD95. The low-25(OH)D serum groups (≤30 ng/ml) had higher percentages of CD28(+)CD95(-)CD8(+) (naïve) T cells and lower percentages of CD28(+)CD95(+)CD8(+) (effector) T cells. By contrast, subjects with high levels of 25(OH)D had very low percentages of naïve CD8 T cells but very high percentages of effector CD8 T cells. There was a significant inverse correlation between 25(OH)D levels and the frequency of naïve CD8 T cells. The results show that higher levels of vitamin D are correlated with decreased frequencies of naïve CD8 T cells during early aging, suggesting that higher levels of 25(OH)D accelerate CD8 T-cell senescence. These results warrant the further evaluation of the effects of vitamin D supplementation in immune aging.
Advances in Aging Research 05/2013; 2(2):72-80. DOI:10.4236/aar.2013.22010
[Show abstract][Hide abstract] ABSTRACT: Marginal zone macrophages (MZMs) act as a barrier to entry of circulating apoptotic debris into the follicles of secondary lymphoid organs. In autoimmune BXD2 mice, there is a progressive reduction in the function and numbers of MZMs. Absence of MZMs results in retention of apoptotic cell (AC) debris within the marginal zone (MZ) and increased loading of AC Ags on MZ B cells and MZ-precursor (MZ-P) B cells. The MZ-P B cells are capable of translocating the AC Ags to the follicular zone and stimulating T cells. Both MZMs and MZ-P B cells from BXD2 mice express low levels of tolerogenic signals and high levels of inflammatory signals. Thus, the current study suggests a multifaceted mechanism in which MZMs maintain tolerance to apoptotic autoantigens and suppress their translocation to follicles. Lack of clearance of apoptotic debris by MZMs drives follicular Ag-transportation by MZ-P B cells to stimulate an autoimmune response.
The Journal of Immunology 03/2013; 190(9). DOI:10.4049/jimmunol.1300041 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Macrophages play a central role in the pathogenesis of rheumatoid arthritis (RA). There is an imbalance of inflammatory and antiinflammatory macrophages in RA synovium. Although the polarization and heterogeneity of macrophages in RA have not been fully uncovered, the identity of macrophages in RA can potentially be defined by their products, including the co-stimulatory molecules, scavenger receptors, different cytokines/chemokines and receptors, and transcription factors. In the last decade, efforts to understand the polarization, apoptosis regulation, and novel signaling pathways in macrophages, as well as how distinct activated macrophages influence disease progression, have led to strategies that target macrophages with varied specificity and selectivity. Major targets that are related to macrophage development and apoptosis include TNF-α, IL-1, IL-6, GM-CSF, M-CSF, death receptor 5 (DR5), Fas, and others, as listed in Table 1. Combined data from clinical, preclinical, and animal studies of inhibitors of these targets have provided valuable insights into their roles in the disease progression and, subsequently, have led to the evolving therapeutic paradigms in RA. In this review, we propose that reestablishment of macrophage equilibrium by inhibiting the development of, and/or eliminating, the proinflammatory macrophages will be an effective therapeutic approach for RA and other autoimmune diseases.
Current Rheumatology Reports 08/2012; 14(5):445-54. DOI:10.1007/s11926-012-0272-4 · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine the therapeutic efficacy and immunomodulatory effect of an anti-human death receptor 5 (DR5) antibody, TRA-8, in eliminating macrophage subsets in a mouse model of type II collagen-induced arthritis (CIA).
A human/mouse-chimeric DR5-transgenic mouse, under the regulation of a mouse 3-kb promoter and a loxP-flanked STOP cassette, was generated and crossed with an ubiquitous Cre (Ubc.Cre) mouse and a lysozyme M-Cre (LysM.Cre)-transgenic mouse to achieve inducible or macrophage-specific expression. Chicken type II collagen was used to induce CIA in mice, which were then treated with an anti-human DR5 antibody, TRA-8. Clinical scores, histopathologic severity, macrophage apoptosis and depletion, and T cell subset development were evaluated.
In human/mouse DR5-transgenic Ubc.Cre mice with CIA, transgenic DR5 was most highly expressed on CD11b+ macrophages, with lower expression on CD4+ T cells. In human/mouse DR5-transgenic LysM.Cre mice, transgenic DR5 was restrictively expressed on macrophages. Both in vivo near-infrared imaging of caspase activity and TUNEL staining demonstrated that TRA-8 rapidly induced apoptosis of macrophages in inflamed synovium. Depletion of pathogenic macrophages by TRA-8 led to significantly reduced clinical scores for arthritis; decreased macrophage infiltration, synovial hyperplasia, osteoclast formation, joint destruction, cathepsin activity, and inflammatory cytokine expression in joints; reduced numbers of Th17 cells; and an increased number of Treg cells in draining lymph nodes.
The anti-human DR5 antibody TRA-8 was efficacious in reducing the severity of arthritis via targeted depletion of macrophages and immunomodulation. Our data provide preclinical evidence that TRA-8 is a potential novel biologic agent for rheumatoid arthritis therapy.
[Show abstract][Hide abstract] ABSTRACT: BE-3-3-3-3 (1,15-(ethylamino)4,8,12-triazapentadecane) is a bis(ethyl)polyamine analogue under investigation as a therapeutic agent for breast cancer. Since estradiol (E(2)) is a critical regulatory molecule in the growth of breast cancer, we examined the effect of BE-3-3-3-3 on estrogen receptor α (ERα) positive MCF-7 cells in the presence and absence of E(2). In the presence of E(2), a concentration-dependent decrease in DNA synthesis was observed using [(3)H]-thymidine incorporation assay. In the absence of E(2), low concentrations (2.5-10 μM) of BE-3-3-3-3 increased [(3)H]-thymidine incorporation at 24 and 48 h. BE-3-3-3-3 induced the expression of early response genes, c-myc and c-fos, in the absence of E(2), but not in its presence, as determined by real-time quantitative polymerase chain reaction (qPCR). BE-3-3-3-3 had no significant effect on these genes in an ERα-negative cell line, MDA-MB-231. Chromatin immunoprecipitation assay demonstrated enhanced promoter occupation by either E(2) or BE-3-3-3-3 of an estrogen-responsive gene pS2/Tff1 by ERα and its co-activator, steroid receptor co-activator 3 (SRC-3). Confocal microscopy of BE-3-3-3-3-treated cells revealed membrane localization of ERα, similar to that induced by E(2). The failure of BE-3-3-3-3 to inhibit cell proliferation was associated with autophagic vacuole formation, and the induction of Beclin 1 and MAP LC3 II. These results indicate a differential effect of BE-3-3-3-3 on MCF-7 cells in the absence and presence of E(2), and suggest that pre-clinical and clinical development of polyamine analogues might require special precautions and selection of sensitive subpopulation of patients.
[Show abstract][Hide abstract] ABSTRACT: The pathogenesis of systemic lupus erythematosus (SLE) is multifactorial and multi-genetic. Chronic inflammation associated
with lupus is thought to be due to loss of self-tolerance due to molecular mimicry, environment trigger, hormonal factors
or apoptosis defects. Defects in apoptosis will be the focus of this chapter. Defects in apoptosis can lead to abnormal clonal
deletion of autoreactive cells or failure to downmodulate an inflammatory response. Although the Fas death domain family of
molecules are the primary pathway for elimination of inflammatory cells, defects in these death domain molecules are rarely
observed in lupus and lupus-like syndromes. Patients with autoimmune-lymphoproliferative (ALPS) syndrome disease have defects
in Fas, and we have reported one patient with SLE that exhibits a mutation of Fas ligand. Other death domain family molecules
such as death receptor 3 (DR3), DR4, DRS, Fas ligand 2 (FasL2) have not been studied in SLE. Also, there are signaling pathways
for apoptosis including Fas-associated protein with death domain (FADD), tumor necrosis factor receptor-1 associated death
domain (TRADD), FADD-like interleukin-113 converting enzyme (FLICE) which are important in apoptosis signaling. The bc1-2
family modulate apoptosis, and have been reported to be abnormal in human autoimmune disease. Soluble inhibitors of Fas apoptosis
including a soluble form of Fas which lacks the transmembrane exon are elevated in SLE patients. Different genetic and environmental
factors are proposed to interfere with apoptosis and clearance of inflammatory cells at several levels leading to the cellular
defects of T cell dysfunction and B cell hyperactivity observed in patients with SLE.
[Show abstract][Hide abstract] ABSTRACT: To determine whether functional suppression of the catalytic domain of activation-induced cytidine deaminase (AID) can suppress the hyperreactive germinal center (GC) responses in BXD2 mice.
We generated transgenic BXD2 mice expressing a dominant-negative (DN) form of Aicda at the somatic hypermutation site (BXD2-Aicda-DN-transgenic mice). Real-time quantitative reverse transcriptase-polymerase chain reaction was used to determine the expression of Aicda and DNA damage/repair genes. Enzyme-linked immunosorbent assay was used to measure serum levels of autoantibodies and immune complexes (ICs). Development of GCs and antibody-containing ICs as well as numbers of proliferative and apoptotic cells were determined using flow cytometry and/or immunohistochemical analyses. Development of arthritis and kidney disease was evaluated histologically in 6-8-month-old mice.
Suppression of the somatic hypermutation function of AID resulted in a significant decrease in autoantibody production without affecting the expression of DNA damage-related genes in GC B cells of BXD2-Aicda-DN-transgenic mice. There was decreased proliferation, increased apoptosis, increased expression of caspase 9 messenger RNA in GC B cells, and lower numbers of GCs in the spleens of BXD2-Aicda-DN-transgenic mice. Decreased GC response was associated with lower levels of IgG-containing ICs. Anti-IgM- and anti-CD40 plus anti-Ig-induced B cell proliferative responses were decreased in BXD2-Aicda-DN-transgenic mice.
Inhibition of the AID somatic hypermutation function in BXD2 mice suppressed development of spontaneous GCs, generation of autoantibody-producing B cells, and autoimmunity in BXD2 mice. Suppression of AID catalytic function to limit selection-based survival of GC B cells could become a novel therapy for the treatment of autoimmune disease.