Basic Science Highlights

Stuart M. Levine, MD

Abstract # 139: Vitamin D Limits TLR9/IFN-Alpha Induced Antigen Presenting Cell Maturation

Abstract # 580 : Vitamin D Deficiency Is Associated with B Cell Hyperreactivity in Systemic Lupus Erythematosus

Authors:

Melissa  A. Lerman, Homaira Rahimi, Edward M. Behrens and Jon M. Burnham, Children's Hospital of Philadelphia, Philadelphia, PA

Lauren R. Cole1, Susan Macwana2, Rufei Lu2, Wendy Klein2, Virginia Roberts2, Amy Dedeke1, Sherry R. Crowe2, Gillian M. Air1, Linda F. Thompson3, Joel M. Guthridge2 and Judith A. James3, 1University of Oklahoma Health Sciences Center, Oklahoma City, OK, 2Oklahoma Medical Research Foundation, Oklahoma City, OK, 3OMRF & OUHSC, Oklahoma City, OK

Background: 

It has recently been recognized that vitamin D, in addition to its primary role in bone metabolism, plays an important part in regulating, and perhaps suppressing, human immune responses. Though the mechanisms accounting for this regulation are not yet clear, several exciting new studies suggest that vitamin D might be directly involved in modulating the initiation of an immune response.  Vitamin D deficiency is quite commonly observed in patients with SLE; the studies presented here examine how this might be playing a role in the disease process itself.

Methods: 

In the first study, bone marrow cells from mice were differentiated into antigen presenting cells, and stimulated with TLR9 agonists in the presence and absence of 1,25-Vitamin D.  Cell surface expression of activation markers was monitored via flow cytometry (FACS).  Normal human monocytes were incubated in the presence of both i) normal and SLE sera, and ii) IFN, with or without vitamin D, and activation markers were similarly measured by FACS.

In the second study, 25-vitamin D levels were measured in both SLE patients and healthy controls, and a variety of measures of humoral immune activation were measured.  Isolated peripheral blood mononuclear cells (PBMCs) from SLE patients were evaluated for markers of activation.

Results:  

Vitamin D significantly inhibited TLR9-induced dendritic cell activation in the murine cells, as evidenced by a marked reduction in cell surface activation markers (MHC II, CD40, CD86).  When human monocytes were incubated in the presence of IFN or SLE serum, these same activation markers were highly expressed as well. Notably, co-incubation with vitamin D significantly reduced this activation (p<0.05 for all markers).  Nearly 75% of the SLE patients were found to be vitamin D deficient.  These patients were more likely to have higher antibody levels, exaggerated humoral immune responses, and upregulation of B cell activity markers such as pLYN and pERK1/2.

Significance: 

These studies are exciting, in that they demonstrate a mechanistic link between vitamin D and immune activation that is clinically relevant in patients with SLE.  By showing that vitamin D can suppress TLR9-mediated dendritic cell activation, and that patients with SLE and co-incident vitamin D deficiency have exaggerated humoral immune responses, these studies pave the way for clinical therapeutic trials of vitamin D treatment for patients with both SLE and other immune mediated disorders.

Abstract # 581: Gadd45a Overexpression Contributes to Autoimmunity by Demethylating DNA in Lupus T Cells

Authors:  

Yaping Li1, Ming Zhao1, Yongqi Luo1, Sha Zhao1, Xiujuan Zhang1, Heng Yin1, Hai Long1, Yuwen Su1, Bruce C. Richardson2 and Qianjin Lu1 The Second Xiangya Hospital, Central South University, Changsha, China, 2University of Michigan, Ann Arbor, MI

Background: 

Epigenetic modifications have been shown in recent years to play a prominent role in modulating gene expression in a variety of human cancers.  The role of methylation and other modifications in regulating immune responses is becoming increasingly better understood.  For instance, demethylation of certain regulatory regions (CD11a and CD70) in CD4+ T cells can lead to autoreactivity and the promotion of uncontrolled humoral immune responses.  One demethylating enzyme, Gadd45a, is known to be induced by UV light, and as such might be particularly important in mediating epigenetic modifications in patients with SLE.

Methods: 

CD4+ T cells were isolated from both healthy controls and patients with SLE.  Gadd45a mRNA and protein levels in these cells were measured by RT-PCR and Western blotting respectively. Methylation status of genomic DNA was analyzed using a commercially available kit.  Gadd45a was over-expressed in normal CD4+ T cells cells by expression plasmid transfection, or knocked down by transfection with Gadd45a siRNA. CD11a and CD70 mRNA levels were assessed by PCR and FACS, and T cell proliferation and B cell IgG production were measured for each transfection state.

Results: 

SLE patients had significantly higher Gadd45a at both the RNA (p=0.006) and protein (p=0.026) levels than normal controls, and significantly reduced global methylation levels.  These reduced levels correlated with disease activity.  Further, UVB irradiation normal T cells led to increased Gadd45a levels within 6 hours, with persistent demethylation and increased CD11a/CD70 levels noted at 48 hours.  Control CD4+ T cells transfected with Gadd45a had decreased methylation and increased activation; conversely, cells in which Gadd45a was knocked down by siRNA were more hypermethylated, had reduced activation, and were less efficient at simulating B cell IgG production.

Significance: 

This study shows a convincing link between methylation status at T cell activation regulatory sites and the generation of both T cell help and B cell reactivity. The authors have shown that an enzyme induced by UV light (a well-known SLE flare stimulus), Gadd45a, can mediate these effects, and that this enzyme is indeed upregulated in SLE patients.  These data identify Gadd45a as an attractive therapeutic target in SLE.

Abstract # 610:  The Surface Expression Level of DC-STAMP Defines the Fusogenic Potential of Osteoclast Precursors (OCP): RANKL-Induced DC-STAMPlo OCP Are the Master-Fusogens

Authors:

Kofi A. Mensah1, Yahui Grace Chiu1, L. Xing1, Christopher Ritchlin2 and E.M. Schwarz1, 1University of Rochester, Rochester, NY, 2University of Rochester Medical Center, Rochester, NY

Background: 

Important studies by this group in the past several years have revealed an increased number of circulating osteoclast precursors (OCPs) in the peripheral blood of patients with psoriatic arthritis, a condition associated with significant bone destruction.  These cells have been classically identified by their ability to differentiate into osteoclasts in culture after the addition of the cytokines MCSF and RANK ligand (RANKL).  Though powerful, this methodology is time consuming; finding a method for identifying and enumerating OCPs before and after treatment by a more direct method is thus urgently needed. One recently described molecule, DC-STAMP, has been shown to be crucial to osteoclast fusion, and is an attractive putative disease activity biomarker in psoriatic arthritis (PsA).

Methods: 

A murine macrophage cell line was incubated with RANKL to induce osteoclast formation, and resultant cells were evaluated for DC-STAMP expression by FACS.  Expression of macrophage fusion genes was examined by PCR in both high and low DC-STAMP-expressing cells.  PBMCS from both healthy controls and patients with psoriatic arthritis were incubated with MCSF and RANKL to induce osteoclast formation, and then stained with a DC-STAMP monoclonal antibody.  

Results: 

When murine macrophages (which all express DC-STAMP) were incubated with RANKL, two distinct populations of DC-STAMP expressing cells were noted after 3 days (DCSTAMPhi and DCSTAMPlo).  Curiously, only the DC-STAMPlo cells expressed high levels of fusogenic genes (cd9, cd47 and others), indicating that these cells are the primary fusing OCPs.  Similarly, PBMC culture resulted in the generation of high and low DC-STAMP- expressing cells; only the DC-STAMPlo cells showed the characteristic morphology of fusing cells (pseudopods). The authors suggest that the persistent DC-STAMPhi cells become the feeding cells for ongoing fusion, and that as they fuse they lose their DC-STAMP from the cell surface.  Psoriatic arthritis patients had higher levels of DC-STAMP on their mononuclear cells than did healthy controls.

Significance: 

These data demonstrate that RANKL mediates the down-regulation of DC-STAMP in a subset of OCPs, and by doing so induces the expression of fusogenic genes and the characteristic morphologic changes seen during osteoclast formation.  These same populations could be seen in PsA patients, suggesting that DC-STAMP expression, as well as the proportion of DC-STAMP hi and lo populations, might serve as real-time biomarkers of osteoclast differentiation and dynamic bone destruction in PsA.

Abstract # 624: The Role of Uric Acid in Inflammation to Sterile Cell Death

Authors: 

Hajime Kono1, Chun-Jen Chen2, Fernando Ontiveros1 and Kenneth L. Rock1, 1UMass Med School, Worcester, MA, 2National Taiwan Univ., Taipei, Taiwan

Background: 

Antibodies recognizing uric acid (UA) released from dying cells have recently been identified, thus suggesting a role for natural immune responses to this nucleic acid breakdown product in the homeostatic clearance of cellular debris.  The role of uric acid in mediating immune responses to necrotic cells in vivo has not yet been elucidated.

Methods: 

Two novel transgenic mice expressing high levels of either intra- or extra-cellular uricase (which deplete intracellular and extracellular UA respectively) were employed for these studies.  Systemic inflammation and cell death was induced by i) peritoneal instillation of sterile necrotic cells, and ii) high dose oral acetaminophen (Tylenol) to induce hepatic necrosis.  UA levels were further depleted from the serum with recombinant uricase or allopurinol. Neutrophil recruitment into the peritoneum, and liver myeloperoxidase (MPO) activity (a measure of neutrophil activation) were measured in mice with varying UA levels in the different compartments.

Results: 

Interestingly, mice depleted of either intra-or extra-cellular uric acid displayed a marked reduction neutrophilic influx to areas of cellular necrosis.  For instance, liver MPO activity was reduced 5-fold in mice transgenic for intracellular uricase, indicating a role for xanthine oxidase in the UA-mediated clearance of necrotic cells.  These results were recapitulated in normal mice depleted of UA by more traditional means (with allopurinol or recombinant uricase).    

Significance:

This interesting study follows a study published earlier this year demonstrating novel, immune mediated homeostatic functions of uric acid.  It suggests that intracellular uric acid, produced both following cell-death mediated nucleic acid breakdown, serves as a “danger” signal to the immune system that sterile cell death has occurred and needs to be cleared.  It also raises the possibility that overly aggressive uric acid lowering with allopurinol or other UA lowering drugs might be deleterious.

Abstract #1212:  FLIP: The Master Regulator of Myeloid Differentiation and Survival, a Novel Therapeutic Target in Chronic Inflammation

Authors:

Qi Quan Huang1, Zan Huang1, Robert Birkett1, Lixin Kan1, Sandeep Gurbuxani2, John Crispino1, Harris R. Perlman1 and Richard M. Pope1, 1Northwestern University Feinberg School of Medicine, Chicago, IL, 2University of Chicago, Chicago, IL

Background:

It is well recognized that macrophages and neutrophils are key cells in generating the inflammatory response in rheumatoid arthritis and other inflammatory syndromes.  Whether their inappropriate activation in these conditions is the result of aberrant differentiation is unknown.  One potential regulator of myeloid differentiation is FLIP, which is induced by inflammatory cytokines and leads to resistance to cell death.  Whether FLIP activation plays a role in keeping these cells in the inflammatory milieu is the question addressed by this study.

Methods: 

In an elegant series of genetic manipulations in mice, FLIP was selectively knocked out in cells of the myeloid lineage alone.  The resultant animals were assessed for growth and developmental defects, ability to form myeloid cells, and secrete cytokines.  Their tissues were evaluated for the presence of myeloid cells.  Inflammatory arthritis was induced in the FLIP knock out animals with anti-GPI sera, and the effects of FLIP under-expression was assessed.   

Results: 

FLIP knock out animals were smaller, and almost half died before 7 months of age.  Neutrophils were increased on the peripheral blood and infiltrated the major organs.  Conversely, macrophages were reduced both in the spleen and peritoneal cavity.  Interestingly, the neutrophils were mature and functional, showing that FLIP is not required for normal neutrophil development.  The knock out mice had markedly elevated serum levels of inflammatory cytokines including TNF-a, and IL6, although they developed a less severe arthritis than wild type animals. 

Significance:

These elegant studies reveal the importance of FLIP in mediating macrophage development and neutrophil apoptosis.  Amazingly, though the animals had markedly elevated neutrophil numbers both in the tissues and in the periphery, and had increased levels of inflammatory cytokines, they were protected from serum-induced arthritis.  The authors postulate that this is due to the selective reduction of FLIP in tissue macrophages- if this is true, then macrophage-expressed FLIP might be a novel therapeutic target for inflammatory arthritis.  However, the deleterious systemic consequences of absent FLIP are a daunting hurdle to this hypothesis.

Abstract #1896: All-Trans Retinoic Acid Promotes the Differentiation of Itreg Cells Via Smad and Non-Smad Signaling Pathways

Abstract #1969: All-Trans Retinoic Acid Restores the Stability and Functionality of Ntregs in the Inflammatory Milieu

Authors:

Xiao H. Zhou1, Ling Lu1, Julie Wang1, Hejian Zou2, David A. Horwitz1, David Brand3, Huimin Fan4, Zhongmin Liu4 and Song Guo Zheng1, 1University of Southern California, Los Angeles, CA, 2Huashan Hospital, Fudan University, Shanghai, China, 3VA Medical Center, Memphis, Memphis, TN, 4East Hospital, Tongji University, Shanghai, China

Xiao H. Zhou1, Ning Kong2, Julie Wang1, Hejian Zou3, Huimin Fan4, David Brand5, Zhongmin Liu4 and  Song Guo Zheng1, 1University of Southern California, Los Angeles, CA, 2University of Southern California, Los Angeles, CA ; Huashan Hospital, Fudan University, Shanghai, China, 3Huashan Hospital, Fudan University, Shanghai, China, 4East Hospital, Tongji University, Shanghai, China, 5VA Medical Center, Memphis, Memphis, TN

Background:

This year’s highlights begin and end with a discussion of vitamins! We have already seen the important functions that Vitamin D plays in the initiation and propagation of immune responses. We now turn our attention to a form of Vitamin A, all-trans retinoic acid (ATRA).  Most physicians are aware of ATRA’s role as an anti-leukemic agent that can induce the differentiation of immature promyelocytic leukemia cells into mature, non-leukemic neutrophils.  However, recent studies reveal that ATRA can also promote the development of regulatory T cells (tregs), which have been shown to be depleted or dysfunctional in a variety of autoimmune disease, from SLE to Wegener’s.

Methods:

Since it is know that TGF-b can induce CD4+/CD25+ T-cells to form tregs, these cells from both wild type and smad3 knock out mice (which are deficient in TGF-b signaling) were incubated in the presence and absence of ATRA. A variety of methods were used to assess resultant treg function and modulation by ATRA.  In the second study, both wild type and foxp3 transgenic mice (which have increased numbers of tregs) were treated with IL6 to induce inflammation (a treatment known to drive inflammatory Th17 cell formation from tregs).  The effects of ATRA on IL6-mediated treg: Th17 differentiation were assessed by several different assays.

Results: 

In the first study, ATRA treatment resulted in an increased number of tregs, and worked via both TGF-b dependent and independent pathways.  ATRA treatment resulted in changes in the expression of foxp3, the main treg transcription factor, at the transcriptional and protein level, demonstrating a direct effect on gene expression and cellular differentiation. In the second study, ATRA blocked the IL-6 mediated conversion of tregs to inflammatory Th17 cells.  ATRA treatment preserved the treg phenotype of IL-6 treated cells; when these cells were transferred to wild type mice immunized with Type 2 collagen, arthritis severity was markedly reduced. 

Significance:

These 2 abstracts reveal that ATRA can both drive the differentiation of induced tregs and can restore the function of inflammation-differentiated tregs, revealing an additional exciting possible therapeutic application for this vitamin A derivative in the treatment of autoimmune disease.

AddThis Social Bookmark Button