Lawrence Steinman - US grants

Local, systemic and neurological complications have been observed following pertussis (whooping cough) vaccination in children. These often occur soon after primary or secondary immunization. The neurological syndrome ranges from minor irritability to convulsions, coma, and on rare occasions death. Children who recover show varying degrees of permanent neurological sequelae. The pathogenesis of this encephalopathy is unknown, and the lack of an animal model has retarded our understanding of this disease. We have described an animal model for pertussis immunization encephalopathy. A clinical syndrome with pathological features closely resembling post-immunization encephalopathy can be reproduced in the mouse after immunization with heat-killed Bordetella pertussis vaccine and exposure to a foreign antigen, if the inoculated animal has a susceptible H-2 genotype. We will analyze the mechanisms whereby B. pertussis immunization produces encephalopathy in four major areas. Genetic mapping studies will localize the locus of susceptibility to encephalopathy within H-2. Immunologic experiments will a) study the role of antibody isotype using switch variants of monoclonal antibodies, b) ascertain whether transposon induced B. pertussis mutants are adequate vaccines, and c) analyze the characteristics of pertussis triggered lymphocytosis. Therapy of B. pertussis encephalopathy will be tried with anti-I-A and anti-L3T4 antibody. Physiologic studies on B. pertussis dependent T cell homing to the central nervous systmem will be carried out. These experiments may help to characterize the rare encephalopathic reactions to B. pertussis and may lead to the development of a safer B. pertussis vaccine.

DESCRIPTION (Adapted from the Applicant's Abstract): In this competing renewal application, the Principal Investigator wishes to expand his studies on immunotherapy for autoimmune disease by comparing selective anti-TCR therapy with less selective approaches targeting either class II MHC, adhesion molecules or cytokines. Three different models for the induction of relapsing EAE will be utilized; these include 1) spinal cord homogenate, 2) T-cell clones specific for MBP 1-11 and 3) superantigen. Five specific aims are proposed. Aim 1 is to determine whether highly selective anti-TCR therapy will succeed in clinical paradigms where "epitope spreading" might occur. Aim 2 is to determine whether non-selective anti-cytokine therapy or anti- adhesion molecule therapy will be beneficial in clinical paradigms where "epitope spreading" does occur. Aim 3 is to assess the role of the cytokines IL-4 and IL-10 in suppression of EAE. In Aim 4, the role of a variety of adhesion molecules, cytokines, class II and CD4 in selective and non- selective homing to the CNS will be assessed. Aim 5 is to assess the possibility of using DNA vaccination against TCR Vb for treatment of EAE. It is hoped that these studies will provide the basis for future clinical trials in MS patients.

Three areas of study are covered in the final year of this grant. We are continuing investigations of the mechanism of reaction of anti-I-A antibody in reversing experimental allergic encephalomyelitis (EAE). T cell clones which induce EAE are central to this aspect of this study. These T cell clones are restricted to I-A molecules and recognize specific peptides on myelin basic protein. The second part of this investigation involves monoclonal antibodies directed against T cell subset markers. An antibody to the L3T4 antigen on helper/inducer T cells has been shown to reverse EAE as effectively as anti I-A antibody. The mechanism underlying this observation will be studied. Finally, we are using transposon induced mutations in Bordetella pertussis to design a safer pertussis vaccine. We have shown that transposon induced mutations which markedly reduce the amount of pertussis toxin available in these organisms leads to a greatly reduced encephalopathic potential.

This grant has funded work on altered peptide ligands (APL) for therapy of autoimmune disease, specifically multiple sclerosis (MS). Work undertaken during the previous five years has provided a framework for the first clinical trials on patients with relapsing remitting MS. In the next grant period we propose to carry out pre-clinical experiments that will optimize APL therapy in animal models of MS, and to develop the next generation of APL therapy utilizing techniques involving vaccination with naked DNA. We will also demonstrate how microbes have the capacity to modulate autoimmune disease via structural mimicry with self-molecules. The specific aims of this grant are to 1) subvert epitope spreading with APL. In this aim we will see whether after intermolecular epitope spreading has occurred, if an APL which targets a single epitope on a single myelin molecule, would be able to suppress ongoing EAE, and reverse paralysis. This is a stringent demand, but will be a useful paradigm to optimize therapeutic regimens in Phase II clinical trials with relapsing remitting MS patients. In Aim 2 we will analyse how APL pulsed dendritic cells will serve to reverse EAE. In Aim 3 we will attempt APL therapy via vaccination with DNA minigenes encoding myelin epitopes. We have succeeded in preventing EAE with DNA vaccination using a minigene encoding a myelin PLP epitope. We will optimize this approach utilizing myelin minigenes paired in tandem with cytokine and chemokine constructs. Finally in Aim 4 we will study APL as microbial mimics that modulate EAE. We have succeeded with preliminary studies, in demonstrating subversion of epitope spreading, with microbial sequences that mimic myelin epitopes. The optimization of such APL derived from microbes will be studied here. This work with APL may be applicable not only to MS, but to other autoimmune diseases including juvenile diabetes, rheumatoid arthritis, and inflammatory bowel disease.

We have discovered VDJ beta and VJ alpha gene rearrangements in MS brain plaques that encode amino acid sequences identical to those seen in T cell clones from humans, mice and rats that have specificity for myelin basic protein (MBP) peptide 87-99. Using a high-efficiency expression system for TCR alpha and beta chains, we will rigorously prove that a major T cell response in the MS lesion is directed to MBPp87-99. We have developed potent T cell receptor antagonists for p87-99 that suppress ongoing paralysis in rodent models of EAE, and suppress proliferative and cytotoxic responses to p87-99 in humans. We will determine the putative contacts for amino acids in the TCR CDR3 regions with residues of p87-99. A core motif containing MBP87-91 (VHFFK) is found in this epitope. A number of microbes share sequence homology with this core motif-VHFFK, and stimulate MBPp87-99 specific T cells. We will determine whether microbial epitopes can trigger the transfected construct, as efficiently as the native MBP epitope. This would indicate how these T cells might become activated during infection, supporting the notion that self- reactivity arises from the molecular mimicry of self-antigens by microbes. Since T cells reactive to MBP p87-99 trigger EAE, we will test whether these microbial sequences either trigger EAE, or alternatively whether they serve as TCR or MHC antagonists and can block disease induction. Thus we will consider whether molecular mimics of MBP epitopes can induce disease or protect from pathology. We have demonstrated that a dominant antibody response found in the MS brain plaque and in MS cerebrospinal fluid is directed to an overlapping region of the MBP molecule from p86-99. This epitope is identical to the epitope restricted by HLA DR2 beta (HLA DRB1*1501), and overlaps with the epitope restricted by HLA DR2 alpha (HLA DRB5*0101). We will analyze immunoglobulin gene rearrangements in MS brain using RT-PCR, and in CSF using single cell PCR. We will see whether there are restricted Ig CDR3 motifs in MS. We will compare these CDR3 motifs to those found in murine monoclonal antibodies raised against the identical epitope p86-99. Our persistent goal is to develop selective immunotherapy for multiple sclerosis, a chronic disease of the central nervous system affecting approximately 250,000 Americans.

Our goal is to design of T cell receptor and MHC blocking peptides with predictable properties that might be used therapeutically to treat multiple sclerosis. Originally we formulated a non-immunogenic peptide based on the based on the sequence of myelin basic protein (MBP) that binds to I-Au with much greater affinity relative to the encephalitogenic peptide Ac1-11 of MBP. This peptide reverses EAE, and even prevents subsequent relapses, if given at the time that signs of EAE first begin to appear. We shall extend these findings to develop inhibitors of MHC and of TCR specific for MBP peptide 87-99 in the Lewis rat. The reason for pursuing inhibitors of the TCR and of MHC-binding of peptide 87-99, follows from some serendipitous finding regarding a major set of TCR rearrangements in MS brain lesions. We analyzed T cell receptor (TCR) gene rearrangements directly from MS brain plaques. Rearrange Vbeta 5.2 genes were detected in the brains of all patients who were HLA DR2. A common Vbeta t.2-Dbeta-Jbeta sequence in these MS brain plaques was identical to that described for the VDJ region of a Vbeta 5.2 T cell clone. This clone from an MS patient, who was HLA DR2, was cytotoxic for targets with MBP peptide 89-106. The deduced amino acid sequence of this VDJ rearrangement, LRG, was also described previously in T Cells, cloned from EAE lesions, which were specific for MBP peptide 87-99. VDJ sequences with specificity for this MBP epitope constitute a large fraction (40%) of the TRC Vbeta 5.2N(D)N rearrangements in MS lesions. The capacity of T cells with these VDJ sequences to cause EAE, and the prevalence of such sequences in demyelinated lesions, indicated that T cells with this rearranged TCR, may be critical in MS. We have analyzed the MBP peptide 87-99 to determine the putative interaction sites with MHC and with TCR. Based on these studies we have designed peptide analogues of MBP 87-99 that interfere with either MHC or TCR binding, including the LRG motif found in the CDR3 of TCR commonly transcribed in MS lesions. Some of these TCR antagonists prevent EAE. We thus plan to develop MBP analogues that interfere with TCR and MHC recognition of MBP peptide 87-99. We will test these antagonists to determine if they can reverse EAE after the first signs of disease have appeared. We shall also develop CD4+ T cell clones specific for human MBP peptide 87-99 and restricted to HLA DRB1*1501(DR2), and test whether the blockers from the Lewis rat system also block their human counterparts. Ultimately we would hope to apply these results to clinical trials in MS.

The long term objective of this proposal is to use short, cationic peptides as a efficient method of delivering biopolymers, such as intact autoantigens, immunodominant peptides, and antisense oligonucleotides into the cytoplasm of antigen presenting cells and T lymphocytes to induce antigen specific anergy and/or affect the profile of secreted cytokines as a therapy for experimental allergic encephalomyelitis (EAE). We have demonstrated that conjugation of a peptide, corresponding to nine amino acids from the HIV tat protein, to protein antigens results in their rapid uptake by a variety of cells and permits the molecules to enter both the MHC class I and ll biosynthetic pathways, which results in a dramatic increase in their antigenicity and immunogenicity. In addition, to delivering antigens to the MHC class I and ll molecules, we propose to use the tat peptide to transport PNA antisense reagents into cells to modulate cytokine production characteristic of inflammation. The studies outlined in this project seek to explore the therapeutic potential of specifically suppressing proinflammatory cytokine expression by introducing antisense PNA for the transcriptional activator NF-kappaB into murine T cell clones. If successful, these methods could be used as therapeutic strategies in mice in vivo to reduce inflammation in the CNS of mice. This project has three specific aims, 1. demonstrate that conjugation of the tat peptide to intact myelin basic protein and proteolipid protein as well as their immunodominant determinants and altered peptide ligands dramatically increases their ability to induce antigen specific tolerance and/or modify the cytokine profile of the autoantigen specific clones, 2. demonstrate that tat conjugated proteins and peptides enter antigen presenting cells, such as dendritic cells and small resting B cells, and explore whether adoptive transfer of the antigen loaded cells more efficiently induces either immune stimulation or tolerance than immunization with the peptides or proteins, and 3. demonstrate that the expression of a variety of proinflammatory proteins can be dramatically reduced with HIV-1 tat conjugated antisense PNA against the 65 kilodalton subunit of NF-kappaB in vitro and that the antisense PNA constructs can reduce inflammation when directly injected into rodents with EAE.

Vaccination was initiated empirically 200 years ago by Jenner and consolidated conceptually 100 years ago by Pasteur, and the procedure still provides surprises. Among the latest is vaccination with DNA. A particular variable region gene of the T cell receptor, Vbeta8.2, is rearranged and its product is expressed on pathogenic T cells that induce experimental autoimmune encephalomyelitis (EAE) and its product is expressed on pathogenic T cells that induce experimental autoimmune encephalomyelitis (EAE) in H-2/u mice following immunization with myelin basic protein (MBP). Vaccination of H-2/u mice with naked DNA pathogenic portion of the MBP molecule, indicated in the vaccinated mice there was a reversal of the autoimmune response from Th2 to Th2. This shift may make this approach attractive for treatment of recently of Th1 mediated diseases like multiple sclerosis, juvenile diabetes, and rheumatoid arthritis. We have recently extended this approach to the treatment of autoimmune diseases with altered peptide ligands. In this portion of the program project proposal, we aim to: 1. Extend studies on the mechanism whereby DNA vaccination with TCR Vb constructs induces Th2 responses and suppresses EAE. 2. Test vaccination with DNA minigens encoding myelin epitopes for peptide- and APL-based treatment of EAE. 3. Investigate bacterial CpG sequences inducing gamma interferon and IL- 12, and their application for suppression of EAE. 4. Utilize DNA vaccination to chemokines for treatment of EAE. 5. Develop tandem cytokine and chemokine constructs for treatment of EAE.

DESCRIPTION (provided by applicant): EAE has served as a useful model for MS, yet many therapies which succeed in preventing or reversing paralysis in the animal model, do not succeed when applied to MS. However, approved drugs for MS like beta interferon and Copaxone have been successful in both EAE and MS. There are several convenient models of EAE in rodents with both acute and relapsing features along with demyelination. We shall examine transcription profiles in MS lesions using gene microarray technologies, comparing the transcriptional profiles of these lesions from six MS brains. Genes of interest found in microarray analysis will be corroborated with real time PCR RNAse protection, and Western blotting in selective cases. Lesions, where mRNA was isolated, will also be characterized histopathologically and immunohistochemically. We shall continue comparison of active and chronic lesions, where we have already identified key differences in transcription of immunoglobulin genes p38kinase and alpha-1-antichymotrypsin. Profiles from MS brain will be compared to those obtained from the CNS of rodents with relapse, remission, or acute attacks of EAE. We shall also study transcriptional profiles of pathogenic T cell clones that cause EAE, either stimulated with native myelin peptide or altered peptide ligands. APL's have been successful in treating EAE, and have now been taken into phase II trials in MS. Finally we will study the role of osteopontin a gene identified in large scale transcriptional profiling of EAE and MS. We demonstrate that osteopontin is expressed in MS and EAE lesions, and that in animals with the osteopontin gene deleted there is a profound change in the regulation of disease relapses. The discovery of the role of osteopontin, first identified in large scale profiles, exemplifies how this approach may help us to understand MS and to develop new therapies.

The Immunology Program at Stanford seeks to provide the best training possible for our pre- and postdoctoral students. The Program is interdepartmental in organization, with 46 faculty from 11 departments and 4 divisions (in the Department of Medicine), in the Schools of Medicine and Humanities and Sciences. The faculty's research is at the forefront of the key areas in immunology today - molecular, cellular, and clinical -- and they are leaders in the development of new technologies and in the application of current knowledge of basic immune mechanisms to the investigation of human immunological diseases and their treatment. The research and training activities in immunology benefit from this multidisciplinary approach and from our tradition of interactions and collaborations among the labs. Predoctoral trainees are required to develop strong background in basic biomedical sciences through coursework and they receive extensive and broad-based research training through laboratory rotations and thesis research. Both pre- and postdoctoral trainees develop professional skills and perspectives through participation in program activities including the weekly Immunology Seminar Series (bringing in over 30 scientists from around the world each year), 1 graduate student and 2 postdoctoral fellow journal clubs in immunology, the annual Stanford Immunology Scientific Conference at Asilomar, and the course in the Responsible Conduct of Research. Trainees are encouraged to present their research at local and national conferences. Trainees have access to modern laboratories, many in new or newly-renovated buildings, and to state-of-the-art specialized research facilities such as the cell-sorting and analysis (FACS) facility, the Protein and Nucleic Acid Facility, the Electron Microscope and Cell Imaging Facilities, the Molecular Modeling Facility, and the transgenic/gene knockout mouse facilities.

Bacterial DNA and immunostimulatory CpG oligonucleotides (CpG-ODN) activate the innate immune system to produce proinflammatory cytokines. Shown to be potent Th1-like adjuvants, stimulatory CpG-motifs are currently utilized as effective therapeutic vaccines for various animal models of infectious diseases, tumors, allergies, and autoimmune diseases. We have shown that it is possible to induce antigen specific Th2 immunity to myelin, using a co-vaccination strategy with DNA encoding IL-4 and myelin proteins. This approach ameliorated and actually reversed ongoing EAE. We recently discovered that the application of an immunoinhibitory GpG-ODN, with a single base switch from CpG to GpG, can effectively inhibit the immunostimulatory response of its CpG-ODN counterpart. Moreover, this inhibitory GpG-ODN is not only capable of counteracting the stimulatory effect of CpG-ODN in vitro, it is also capable of suppressing the disease severity of experimental autoimmune encephalomyelitis (EAE) in mice, a Th1-mediated animal disease model for multiple sclerosis, and inducing a Th2 shift, much as DNA co-vaccination with genes encoding myelin and IL-4 [Garren et al, 2001]. We will explore the utility of the GpG motif for therapy of autoimmune disease, and examine the underlying mechanism whereby it exerts its effects. We will extend pre-clinical studies on the mechanism whereby GpG-ODN induces reduced expression of MHC class II, promotes a Th2 shift, and enhances antigen specific Th2 responses. We will test a co-vaccination strategy using GpG-ODN plus genes encoding myelin to prevent and reverse acute EAE, and to block further relapses, if given after the initial acute attack in chronic relapsing EAE. We will use our recently developed proteomic myelin array to monitor epitope spreading and the nature of the T cell response, when DNA vaccines encoding myelin proteins are used to treat relapsing remitting EAE after the acute attack.

Bacterial DNA and immunostimulatory CpG oligonucleotides (CpG-ODN) activate the innate immune system to produce proinflammatory cytokines. Shown to be potent Th1-like adjuvants, stimulatory CpG-motifs are currently utilized as effective therapeutic vaccines for various animal models of infectious diseases, tumors, allergies, and autoimmune diseases. We have shown that it is possible to induce antigen specific Th2 immunity to myelin, using a co-vaccination strategy with DNA encoding IL-4 and myelin proteins. This approach ameliorated and actually reversed ongoing EAE. We recently discovered that the application of an immunoinhibitory GpG-ODN, with a single base switch from CpG to GpG, can effectively inhibit the immunostimulatory response of its CpG-ODN counterpart. Moreover, this inhibitory GpG-ODN is not only capable of counteracting the stimulatory effect of CpG-ODN in vitro, it is also capable of suppressing the disease severity of experimental autoimmune encephalomyelitis (EAE) in mice, a Th1-mediated animal disease model for multiple sclerosis, and inducing a Th2 shift, much as DNA co-vaccination with genes encoding myelin and IL-4 [Garren et al, 2001]. We will explore the utility of the GpG motif for therapy of autoimmune disease, and examine the underlying mechanism whereby it exerts its effects. We will extend pre-clinical studies on the mechanism whereby GpG-ODN induces reduced expression of MHC class II, promotes a Th2 shift, and enhances antigen specific Th2 responses. We will test a co-vaccination strategy using GpG-ODN plus genes encoding myelin to prevent and reverse acute EAE, and to block further relapses, if given after the initial acute attack in chronic relapsing EAE. We will use our recently developed proteomic myelin array to monitor epitope spreading and the nature of the T cell response, when DNA vaccines encoding myelin proteins are used to treat relapsing remitting EAE after the acute attack.

DESCRIPTION (provided by applicant): These studies bring together laboratories who have published innovative papers on large scale microarrays to detect antibodies to proteins, peptides, lipids and carbohydrates. "Epitope spreading" is an immunological hallmark of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. It is defined as the expansion of antigen-specific immune responses beyond those targeted in the initial immunization. The complexity of the process includes spread not only to other peptide epitopes of the same protein molecule, defined as intramolecular spreading, but to other molecules, defined as intermolecular spreading. Spreading of the immune response is not confined to peptide epitopes, but also includes immune responses to lipids and carbohydrates. If tolerization to antigen specific autoimmune responses is desirable for treatment of autoimmune disease, then one must devise practical measures to tolerize the immune system across a wide front including multiple proteins/peptides, carbohydrates and lipids. We shall tolerize animals with ongoing EAE, using key proteins/peptides, lipids and carbohydrates that are targeted by autoantibodies detected on the arrays used to study both MS and EAE. We have already shown promising clinical and pre- clinical results with tolerization to these components of the myelin sheath. Both tolerization to proteins and to lipids ameliorates paralysis in EAE. We hypothesize that in order to reduce epitope spreading it will be necessary to tolerize to potentially pathogenic autoantigenic peptides/proteins, lipids AND carbohydrates, and not simply to one of these types of chemical constituents. We will now see if tolerizing individually to each of these distinct chemical components of myelin is optimal, or if tolerization in concert to proteins/peptides AND lipids AND carbohydrates, achieves even more beneficial results. In New Aim 1 we will undertake a) structure function studies on promising lipids that are the target of the immune response in MS and EAE. b) We will analyze how these tolerogens influence epitope spreading. c) We will investigate the mechanisms of action for induction of tolerance in depth on each promising candidate that suppresses ongoing EAE in three different models of EAE. In New Aim 2 we will undertake studies on tolerization to various mannose clusters that are the target of the immune response in MS and EAE, comparing for example, (Man9)n and [(Man9)4]n in reducing disease in three models of relapsing and progressive EAE. In New Aim 3, we shall tolerize animals with ongoing EAE, using SIMULTANEOUS administration of key proteins/peptides, lipids and carbohydrates shown to be targeted by autoantibodies detectable with the arrays used to study MS and EAE. We will now see if tolerizing in concert to proteins/peptides AND lipids AND carbohydrates achieves even more beneficial results in terms of reducing relapses, improving clinical function, reducing epitope spreading in three models of EAE, than tolerizing INIDIVIDUALLY to each of these three chemical types.Antigen specific tolerance is a long sought after goal for treatment of autoimmune disease. We shall develop strategies for tolerizing to proteins, lipids and carbohydrates of the myelin sheath. This approach may lead to better therapies to treat multiple sclerosis.

DESCRIPTION (provided by applicant): Our preliminary results have shown a direct suppression of IL-17 by 1,25(OH)2D3 as well as reversal of paralysis and inhibition of progression of EAE [a murine model of multiple sclerosis (MS)] by 1,25(OH)2D3 which is associated with an inhibition of IL-17. We propose to examine the mechanisms involved. We hypothesize that understanding the mechanisms involved will result in new concepts in our understanding of the interaction between the vitamin D endocrine system and the immune system that may suggest therapeutic targets for the control of MS and other TH17 dependent inflammatory diseases including inflammation induced bone loss. In Specific Aim 1 we will examine the effect of in vivo treatment of EAE mice with 1,25(OH)2D3 on the production of IL-17 and other cytokines (brains and spinal cords as well as splenocytes and lymph nodes will be isolated from EAE mice and the effect of 1,25(OH)2D3 on the production of various cytokines including those produced by TH17 cells (IL-17A, IL-17F, IL-21, IL-22), TH2 cells (IL-4, IL-5), regulatory T cells (IL-10 and TGF?) and innate immune cells (IL-23, IL-12, IL6 and the anti-inflammatory cytokine, IL-27) will be assessed. In preliminary results we noted for the first time that 1,25(OH)2D3 has a direct inhibitory effect on activated IL-17 expression and transcription. The mechanisms involved will be examined (1,25(OH)2D3 may mediate this repression by inhibiting activation mediated by NFAT, Runx1 and ROR gamma transcription factors). Genome-wide analysis of NFAT, Runx1 and vitamin D receptor (VDR) binding sites in CD4+T cell DNA isolated from EAE mice treated with vehicle or 1,25(OH)2D3 using ChIP-seq will also be done. These studies will enable us to identify new target genes and to assess how functional relationship among genes involved in immune function may be altered after 1,25(OH)2D3 treatment. These studies would provide mechanisms for the reversal of paralysis by 1,25(OH)2D3. It is possible that the mechanisms we identify may reflect more general mechanisms involved in a therapeutic role of 1,25(OH)2D3 in the control of pathological immune responses. In Specific Aim 2, since clinical studies are being done treating MS patients with high dose vitamin D, we also propose to examine the effect of high dose dietary vitamin D on paralysis, on the progression of EAE and the production of IL-17 and other cytokines. These studies provide a unique opportunity to combine the expertise of the Steinman lab in multiple sclerosis and immunology and the Christakos lab in vitamin D to increase our understanding of the interaction between the vitamin D endocrine system and the immune system. Findings from these studies may suggest new therapeutic targets and treatment strategies for MS and other TH17 dependent inflammatory diseases. This application is appropriate for the R21 mechanism which supports projects that "involve considerable risk but may lead to a breakthrough in a particular area that could have a major impact on a field of biomedical or clinical research." PUBLIC HEALTH RELEVANCE: An increased understanding of the interaction between the vitamin D endocrine system and the immune system, with a focus on IL-17, a central player in the mammalian immune system, may suggest possible therapeutic targets and treatment strategies for the control of MS and other TH17 dependent inflammatory diseases, including inflammation induced bone loss.