Mining the Human Gut Microbiota for Immunomodulatory Organisms.

Within the human gut reside diverse microbes coexisting with the host in a mutually advantageous relationship. Evidence has revealed the pivotal role of the gut microbiota in shaping the immune system. To date, only a few of these microbes have been shown to modulate specific immune parameters. Herein, we broadly identify the immunomodulatory effects of phylogenetically diverse human gut microbes. We monocolonized mice with each of 53 individual bacterial species and systematically analyzed host immunologic adaptation to colonization. Most microbes exerted several specialized, complementary, and redundant transcriptional and immunomodulatory effects. Surprisingly, these were independent of microbial phylogeny. Microbial diversity in the gut ensures robustness of the microbiota's ability to generate a consistent immunomodulatory impact, serving as a highly important epigenetic system. This study provides a foundation for investigation of gut microbiota-host mutualism, highlighting key players that could identify important therapeutics.

Treg cells require Izumo1R to regulate γδT cell-driven inflammation in the skin.

Izumo1R is a pseudo-folate receptor with an essential role in mediating tight oocyte/spermatozoa contacts during fertilization. Intriguingly, it is also expressed in CD4+ T lymphocytes, in particular Treg cells under the control of Foxp3. To understand Izumo1R function in Treg cells, we analyzed mice with Treg-specific Izumo1r deficiency (Iz1rTrKO). Treg differentiation and homeostasis were largely normal, with no overt autoimmunity and only marginal increases in PD1+ and CD44hi Treg phenotypes. pTreg differentiation was also unaffected. Iz1rTrKO mice proved uniquely susceptible to imiquimod-induced, γδT cell-dependent, skin disease, contrasting with normal responses to several inflammatory or tumor challenges, including other models of skin inflammation. Analysis of Iz1rTrKO skin revealed a subclinical inflammation that presaged IMQ-induced changes, with an imbalance of Rorγ+ γδT cells. Immunostaining of normal mouse skin revealed the expression of Izumo1, the ligand for Izumo1R, electively in dermal γδT cells. We propose that Izumo1R on Tregs enables tight contacts with γδT cells, thereby controlling a particular path of skin inflammation.

The transcriptional landscape of αß T cell differentiation.

The differentiation of αßT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4(+)CD8(+) stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4(+) or CD8(+) lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation.

Identification of transcriptional regulators in the mouse immune system.

The differentiation of hematopoietic stem cells into cells of the immune system has been studied extensively in mammals, but the transcriptional circuitry that controls it is still only partially understood. Here, the Immunological Genome Project gene-expression profiles across mouse immune lineages allowed us to systematically analyze these circuits. To analyze this data set we developed Ontogenet, an algorithm for reconstructing lineage-specific regulation from gene-expression profiles across lineages. Using Ontogenet, we found differentiation stage-specific regulators of mouse hematopoiesis and identified many known hematopoietic regulators and 175 previously unknown candidate regulators, as well as their target genes and the cell types in which they act. Among the previously unknown regulators, we emphasize the role of ETV5 in the differentiation of γδ T cells. As the transcriptional programs of human and mouse cells are highly conserved, it is likely that many lessons learned from the mouse model apply to humans.

Sequestration of gut pathobionts in intraluminal casts, a mechanism to avoid dysregulated T cell activation by pathobionts.

T cells that express the transcription factor RORγ, regulatory (Treg), or conventional (Th17) are strongly influenced by intestinal symbionts. In a genetic approach to identify mechanisms underlying this influence, we performed a screen for microbial genes implicated, in germfree mice monocolonized with Escherichia coli Nissle. The loss of capsule-synthesis genes impaired clonal expansion and differentiation of intestinal RORγ+ T cells. Mechanistic exploration revealed that the capsule-less mutants remained able to induce species-specific immunoglobulin A (IgA) and were highly IgA-coated. They could still trigger myeloid cells, and more effectively damaged epithelial cells in vitro. Unlike wild-type microbes, capsule-less mutants were mostly engulfed in intraluminal casts, large agglomerates composed of myeloid cells extravasated into the gut lumen. We speculate that sequestration in luminal casts of potentially harmful microbes, favored by IgA binding, reduces the immune system's actual exposure, preserving host-microbe equilibrium. The variable immunostimulation by microbes that has been charted in recent years may not solely be conditioned by triggering molecules or metabolites but also by physical limits to immune system exposure.

Identification and validation of a tumor-infiltrating Treg transcriptional signature conserved across species and tumor types.

FoxP3+ T regulatory (Treg) cells are central elements of immunologic tolerance. They are abundant in many tumors, where they restrict potentially favorable antitumor responses. We used a three-pronged strategy to identify genes related to the presence and function of Tregs in the tumor microenvironment. Gene expression profiles were generated from tumor-infiltrating Tregs (TITRs) of both human and mouse tumors and were compared with those of Tregs of lymphoid organs or normal tissues from the same individuals. A computational deconvolution of whole-tumor datasets from the Cancer Genome Atlas (TCGA) was performed to identify transcripts specifically associated with Tregs across thousands of tumors from different stages and locations. We identified a set of TITR-differential transcripts with striking reproducibility between tumor types in mice, between mice and humans, and between different human patients spanning tumor stages. Many of the TITR-preferential transcripts were shared with "tissue Tregs" residing in nonlymphoid tissues, but a tumor-preferential segment could be identified. Many of these TITR signature transcripts were confirmed by mining of TCGA datasets, which also brought forth transcript modules likely representing the parenchymal attraction of, or response to, tumor Tregs. Importantly, the TITR signature included several genes encoding effective targets of tumor immunotherapy. A number of other targets were validated by CRISPR-based gene inactivation in mouse Tregs. These results confirm the validity of the signature, generating a wealth of leads for understanding the role of Tregs in tumor progression and identifying potential targets for cancer immunotherapy.

Network pharmacology of JAK inhibitors.

Small-molecule inhibitors of the Janus kinase family (JAKis) are clinically efficacious in multiple autoimmune diseases, albeit with increased risk of certain infections. Their precise mechanism of action is unclear, with JAKs being signaling hubs for several cytokines. We assessed the in vivo impact of pan- and isoform-specific JAKi in mice by immunologic and genomic profiling. Effects were broad across the immunogenomic network, with overlap between inhibitors. Natural killer (NK) cell and macrophage homeostasis were most immediately perturbed, with network-level analysis revealing a rewiring of coregulated modules of NK cell transcripts. The repression of IFN signature genes after repeated JAKi treatment continued even after drug clearance, with persistent changes in chromatin accessibility and phospho-STAT responsiveness to IFN. Thus, clinical use and future development of JAKi might need to balance effects on immunological networks, rather than expect that JAKis affect a particular cytokine response and be cued to long-lasting epigenomic modifications rather than by short-term pharmacokinetics.

Identifying species of symbiont bacteria from the human gut that, alone, can induce intestinal Th17 cells in mice.

Th17 cells accrue in the intestine in response to particular microbes. In rodents, segmented filamentous bacteria (SFB) induce intestinal Th17 cells, but analogously functioning microbes in humans remain undefined. Here, we identified human symbiont bacterial species, in particular Bifidobacterium adolescentis, that could, alone, induce Th17 cells in the murine intestine. Similar to SFB, B. adolescentis was closely associated with the gut epithelium and engendered cognate Th17 cells without attendant inflammation. However, B. adolescentis elicited a transcriptional program clearly distinct from that of SFB, suggesting an alternative mechanism of promoting Th17 cell accumulation. Inoculation of mice with B. adolescentis exacerbated autoimmune arthritis in the K/BxN mouse model. Several off-the-shelf probiotic preparations that include Bifidobacterium strains also drove intestinal Th17 cell accumulation.

Variation and genetic control of gene expression in primary immunocytes across inbred mouse strains.

To determine the breadth and underpinning of changes in immunocyte gene expression due to genetic variation in mice, we performed, as part of the Immunological Genome Project, gene expression profiling for CD4(+) T cells and neutrophils purified from 39 inbred strains of the Mouse Phenome Database. Considering both cell types, a large number of transcripts showed significant variation across the inbred strains, with 22% of the transcriptome varying by 2-fold or more. These included 119 loci with apparent complete loss of function, where the corresponding transcript was not expressed in some of the strains, representing a useful resource of "natural knockouts." We identified 1222 cis-expression quantitative trait loci (cis-eQTL) that control some of this variation. Most (60%) cis-eQTLs were shared between T cells and neutrophils, but a significant portion uniquely impacted one of the cell types, suggesting cell type-specific regulatory mechanisms. Using a conditional regression algorithm, we predicted regulatory interactions between transcription factors and potential targets, and we demonstrated that these predictions overlap with regulatory interactions inferred from transcriptional changes during immunocyte differentiation. Finally, comparison of these and parallel data from CD4(+) T cells of healthy humans demonstrated intriguing similarities in variability of a gene's expression: the most variable genes tended to be the same in both species, and there was an overlap in genes subject to strong cis-acting genetic variants. We speculate that this "conservation of variation" reflects a differential constraint on intraspecies variation in expression levels of different genes, either through lower pressure for some genes, or by favoring variability for others.

Genome-wide and species-wide dissection of the genetics of arthritis severity in heterogeneous stock mice.

OBJECTIVE: Susceptibility to inflammatory arthritis is determined by a complex set of environmental and genetic factors, but only a portion of the genetic effect can be explained. Conventional genome-wide screens of arthritis models using crosses between inbred mice have been hampered by the low resolution of results and by the restricted range of natural genetic variation sampled. The aim of this study was to address these limitations by performing a genome-wide screen for determinants of arthritis severity using a genetically heterogeneous cohort of mice. METHODS: Heterogeneous stock (HS) mice derive from 8 founder inbred strains by serial intercrossing (n>60), resulting in fine-grained genetic variation. With a cohort of 570 HS mice, we performed a genome-wide screen for determinants of arthritis severity in the K/BxN serum-transfer model. RESULTS: We mapped regions on chromosomes 1, 2, 4, 6, 7, and 15 that contain quantitative trait loci influencing arthritis severity at a resolution of a few megabases. In several instances, these regions proved to contain 2 quantitative trait loci: the region on chromosome 2 included the C5 fraction of complement known to be required for K/BxN serum-transfer arthritis but also contained a second adjacent quantitative trait locus, for which an intriguing candidate is Ptgs1 (Cox1). Interesting candidates on chromosome 4 included the Padi family, encoding the peptidyl arginine deiminases responsible for citrulline protein modification; suggestively, Padi2 and Padi4 RNA expression was correlated with arthritis severity in HS mice. CONCLUSION: These results provide a broad overview of the genetic variation that controls the severity of K/BxN serum-transfer arthritis and suggest intriguing candidate genes for further study.

The same systemic autoimmune disease provokes arthritis and endocarditis via distinct mechanisms.

The immune mechanisms that provoke concomitant inflammation of synovial joints and cardiac valves in disorders such as rheumatic fever and systemic lupus erythematosus remain poorly defined. Here, we report the discovery of spontaneous endocarditis-in addition to their well-studied autoimmune arthritis-in K/BxN T cell receptor (TCR) transgenic mice. The same adaptive immune system elements were required for initiation of arthritis and endocarditis, and both diseases were dependent on autoantibodies. In contrast, the participation of key innate immune system molecules and perhaps T cells as effectors of inflammation differed between the 2 target tissues. Arthritis in K/BxN TCR transgenic mice depended primarily on complement C5 and not FcRgamma-using receptors; conversely, endocarditis depended essentially on FcRgamma receptors and not C5. Elucidating how a single systemic autoimmune disease engages distinct immune effector pathways to damage different target tissues is essential for optimizing the treatment of such disorders.

Deficiency of CXCR2, but not other chemokine receptors, attenuates autoantibody-mediated arthritis in a murine model.

OBJECTIVE: Chemokines coordinate leukocyte trafficking in homeostasis and during immune responses. Prior studies of their role in arthritis have used animal models with both an initial adaptive immune response and an inflammatory effector phase. We undertook analysis of chemokines and their receptors in the effector phase of arthritis using the K/BxN mouse serum-transfer model. METHODS: A time-course microarray analysis of serum-transferred arthritis was performed, examining ankle tissue, synovial fluid, and peripheral blood leukocytes. Up-regulation of chemokines was confirmed by quantitative reverse transcriptase-polymerase chain reaction. The functional relevance of chemokine induction was assessed by transferring serum into mice deficient in CCR1-7, CCR9, CXCR2, CXCR3, CXCR5, CX(3)CR1, CCL2, or CCL3. Further mechanistic analysis of CXCR2 involved treatment of arthritic mice with a CXCR2 antagonist, bone marrow (BM) cell transfers with CXCR2(+/-) and CXCR2(-/-) donors and recipients, flow cytometry of synovial cells, and competition experiments measuring enrichment of CXCR2-expressing neutrophils in arthritic joints of mice with mixed CXCR2(+/+) and CXCR2(-/-) BM cells. RESULTS: Gene expression profiling revealed up-regulation of the CXCR2 ligands CXCL1, CXCL2, and CXCL5 in the joint in parallel with disease activity. CXCR2(-/-) mice had attenuated disease relative to CXCR2(+/-) littermates, as did mice receiving the CXCR2 inhibitor, while deficiency of other chemokine receptors did not affect arthritis severity. CXCR2 was required only on hematopoietic cells and was widely expressed on synovial neutrophils. CXCR2-expressing neutrophils were preferentially recruited to arthritic joints in the presence of CXCR2-deficient neutrophils. CONCLUSION: CXCR2 (but not other chemokine receptors) is critical for the development of autoantibody-mediated arthritis, exhibiting a cell-autonomous role in neutrophil recruitment to inflamed joints.

Critical roles for interleukin 1 and tumor necrosis factor alpha in antibody-induced arthritis.

In spontaneous inflammatory arthritis of K/BxN T cell receptor transgenic mice, the effector phase of the disease is provoked by binding of immunoglobulins (Igs) to joint surfaces. Inflammatory cytokines are known to be involved in human inflammatory arthritis, in particular rheumatoid arthritis, although, overall, the pathogenetic mechanisms of the human affliction remain unclear. To explore the analogy between the K/BxN model and human patients, we assessed the role and relative importance of inflammatory cytokines in K/BxN joint inflammation by transferring arthritogenic serum into a panel of genetically deficient recipients. Interleukin (IL)-1 proved absolutely necessary. Tumor necrosis factor (TNF)-alpha was also required, although seemingly less critically than IL-1, because a proportion of TNF-alpha-deficient mice developed robust disease. There was no evidence for an important role for IL-6. Bone destruction and reconstruction were also examined. We found that all mice with strong inflammation exhibited the bone erosion and reconstruction phenomena typical of K/BxN arthritis, with no evidence of any particular requirement for TNFalpha for bone destruction. The variability in the requirement for TNF-alpha, reminiscent of that observed in treated rheumatoid arthritis patients, did not appear genetically programmed but related instead to subtle environmental changes.

The K/BxN mouse model of inflammatory arthritis: theory and practice.

Mice expressing the KRN T cell receptor transgene and the MHC class II molecule A(g7) (K/BxN mice) develop severe inflammatory arthritis, and serum from these mice causes similar arthritis in a wide range of mouse strains, owing to pathogenic autoantibodies to glucose-6-phosphate isomerase (GPI). This model has been useful for the investigation of the development of autoimmunity (K/BxN transgenic mice) and particularly of the mechanisms by which anti-GPI autoantibodies induce joint-specific imflammation (serum transfer model). In this chaper, after a summary of findings from this model system, we describe detailed methods for the maintenance of a K/BxN colony, crossing of the relevant TCR and MHC genes to other strain backgrounds, evaluation of KRN transgenic T cells, measurement of anti-GPI antibodies, induction of arthritis by serum transfer, and clinical and histological evaluation of arthritis.

MUCOSAL IMMUNOLOGY. Individual intestinal symbionts induce a distinct population of RORγ⁺ regulatory T cells.

T regulatory cells that express the transcription factor Foxp3 (Foxp3(+) T(regs)) promote tissue homeostasis in several settings. We now report that symbiotic members of the human gut microbiota induce a distinct T(reg) population in the mouse colon, which constrains immuno-inflammatory responses. This induction­which we find to map to a broad, but specific, array of individual bacterial species­requires the transcription factor Rorγ, paradoxically, in that Rorγ is thought to antagonize FoxP3 and to promote T helper 17 (T(H)17) cell differentiation. Rorγ's transcriptional footprint differs in colonic T(regs) and T(H)17 cells and controls important effector molecules. Rorγ, and the T(regs) that express it, contribute substantially to regulating colonic T(H)1/T(H)17 inflammation. Thus, the marked context-specificity of Rorγ results in very different outcomes even in closely related cell types.

Auxin stimulates S6 ribosomal protein phosphorylation in maize thereby affecting protein synthesis regulation.

Auxin is known to stimulate protein synthesis in many plant tissues, but the mechanisms involved in this process are unknown. The present research inquires whether auxin might regulate selective translation of mRNAs by inducing S6 ribosomal protein phosphorylation on the 40S ribosomal subunit in maize (Zea mays L.). Maize embryonic axes auxin-stimulated by natural (IAA) or synthetic (Dicamba or 1-NAA) auxins, selectively increased ribosomal protein synthesis. This effect was not reproduced by auxin inactive analogue 2-NAA. Enhanced S6 ribosomal protein phosphorylation on the 40S ribosomal subunit was also observed after auxin stimulation, as measured by [32P] incorporation into this protein. This increment did not occur when stimulation was performed with the inactive auxin analogue. Further, increased recruitment into polysomes of two 5'TOP-like mRNAs, encoding for the initiation translation factor eIF-iso4E and the S6 ribosomal protein, was also found after auxin stimulation of maize axes. A positive correlation was established between the levels of S6 ribosomal protein phosphorylation and the S6 ribosomal protein transcript recruitment into polysomes by means of okadaic acid or heat shock application to maize axes. These data indicate that auxin stimulates S6 ribosomal protein phosphorylation on maize ribosomes, concomitant to the recruitment of specific mRNAs (5'TOP-like mRNAs) into polysomes for translation. It is proposed that by this mechanism auxin regulate the synthesis of specific proteins in maize tissues.

Type 1 diabetes in NOD mice unaffected by mast cell deficiency.

Mast cells have been invoked as important players in immune responses associated with autoimmune diseases. Based on in vitro studies, or in vivo through the use of Kit mutant mice, mast cells have been suggested to play immunological roles in direct antigen presentation to both CD4(+) and CD8(+) T cells, in the regulation of T-cell and dendritic cell migration to lymph nodes, and in Th1 versus Th2 polarization, all of which could significantly impact the immune response against self-antigens in autoimmune disease, including type 1 diabetes (T1D). Until now, the role of mast cells in the onset and incidence of T1D has only been indirectly tested through the use of low-specificity mast cell inhibitors and activators, and published studies reported contrasting results. Our three laboratories have generated independently two strains of mast cell-deficient nonobese diabetic (NOD) mice, NOD.Cpa3(Cre/+) (Heidelberg) and NOD.Kit(W-sh/W-sh) (Leuven and Boston), to address the effects of mast cell deficiency on the development of T1D in the NOD strain. Our collective data demonstrate that both incidence and progression of T1D in NOD mice are independent of mast cells. Moreover, analysis of pancreatic lymph node cells indicated that lack of mast cells has no discernible effect on the autoimmune response, which involves both innate and adaptive immune components. Our results demonstrate that mast cells are not involved in T1D in the NOD strain, making their role in this process nonessential and excluding them as potential therapeutic targets.

Gene expression during the generation and activation of mouse neutrophils: implication of novel functional and regulatory pathways.

As part of the Immunological Genome Project (ImmGen), gene expression was determined in unstimulated (circulating) mouse neutrophils and three populations of neutrophils activated in vivo, with comparison among these populations and to other leukocytes. Activation conditions included serum-transfer arthritis (mediated by immune complexes), thioglycollate-induced peritonitis, and uric acid-induced peritonitis. Neutrophils expressed fewer genes than any other leukocyte population studied in ImmGen, and down-regulation of genes related to translation was particularly striking. However, genes with expression relatively specific to neutrophils were also identified, particularly three genes of unknown function: Stfa2l1, Mrgpr2a and Mrgpr2b. Comparison of genes up-regulated in activated neutrophils led to several novel findings: increased expression of genes related to synthesis and use of glutathione and of genes related to uptake and metabolism of modified lipoproteins, particularly in neutrophils elicited by thioglycollate; increased expression of genes for transcription factors in the Nr4a family, only in neutrophils elicited by serum-transfer arthritis; and increased expression of genes important in synthesis of prostaglandins and response to leukotrienes, particularly in neutrophils elicited by uric acid. Up-regulation of genes related to apoptosis, response to microbial products, NFkB family members and their regulators, and MHC class II expression was also seen, in agreement with previous studies. A regulatory model developed from the ImmGen data was used to infer regulatory genes involved in the changes in gene expression during neutrophil activation. Among 64, mostly novel, regulatory genes predicted to influence these changes in gene expression, Irf5 was shown to be important for optimal secretion of IL-10, IP-10, MIP-1α, MIP-1ß, and TNF-α by mouse neutrophils in vitro after stimulation through TLR9. This data-set and its analysis using the ImmGen regulatory model provide a basis for additional hypothesis-based research on the importance of changes in gene expression in neutrophils in different conditions.

Protective effects of C-type natriuretic peptide on linear growth and articular cartilage integrity in a mouse model of inflammatory arthritis.

OBJECTIVE: The C-type natriuretic peptide (CNP) signaling pathway is a major contributor to postnatal skeletal growth in humans. This study was undertaken to investigate whether CNP signaling could prevent growth delay and cartilage damage in an animal model of inflammatory arthritis. METHODS: We generated transgenic mice that overexpress CNP (B6.SJL-Col2a1-NPPC) in chondrocytes. We introduced the CNP transgene into mice with experimental systemic inflammatory arthritis (K/BxN T cell receptor [TCR]) and determined the effect of CNP overexpression in chondrocytes on the severity of arthritis, cartilage damage, and linear growth. We also examined primary chondrocyte cultures for changes in gene and protein expression resulting from CNP overexpression. RESULTS: K/BxN TCR mice exhibited linear growth delay (P < 0.01) compared to controls, and this growth delay was correlated with the severity of arthritis. Diminished chondrocyte proliferation and matrix production was also seen in K/BxN TCR mice. Compared to non-CNP-transgenic mice, K/BxN TCR mice with overexpressed CNP had milder arthritis, no growth delay, and less cartilage damage. Primary chondrocytes from mice overexpressing CNP were less sensitive to inflammatory cytokines than wild-type mouse chondrocytes. CONCLUSION: CNP overexpression in chondrocytes can prevent endochondral growth delay and protect against cartilage damage in a mouse model of inflammatory arthritis. Pharmacologic or biologic modulation of the CNP signaling pathway may prevent growth retardation and protect cartilage in patients with inflammatory joint diseases, such as juvenile idiopathic arthritis.

Abrogation of antibody-induced arthritis in mice by a self-activating viridin prodrug and association with impaired neutrophil and endothelial cell function.

OBJECTIVE: To test a novel self-activating viridin (SAV) prodrug that slowly releases wortmannin, a potent phosphoinositide 3-kinase inhibitor, in a model of antibody-mediated inflammatory arthritis. METHODS: The SAV prodrug was administered to K/BxN mice or to C57BL/6 (B6) mice that had been injected with K/BxN serum. Ankle thickness was measured, and histologic changes were scored after a 10-day disease course (serum-transfer arthritis). Protease activity was measured by a near-infrared imaging approach using a cleavable cathepsin-selective probe. Further near-infrared imaging techniques were used to analyze early changes in vascular permeability after serum injection, as well as neutrophil-endothelial cell interactions. Neutrophil functions were assessed using an oxidative burst assay as well as a degranulation assay. RESULTS: SAV prevented ankle swelling in mice with serum-transfer arthritis in a dose-dependent manner. It also markedly reduced the extent of other features of arthritis, such as protease activity and histology scores for inflammation and joint erosion. Moreover, SAV was an effective therapeutic agent. The underlying mechanisms for the antiinflammatory activity were manifold. Endothelial permeability after serum injection was reduced, as was firm neutrophil attachment to endothelial cells. Endothelial cell activation by tumor necrosis factor alpha was impeded by SAV, as measured by the expression of vascular cell adhesion molecule. Crucial neutrophil functions, such as generation of reactive oxygen species and degranulation of protease-laden vesicles, were decreased by SAV administration. CONCLUSION: A novel SAV prodrug proved strongly antiinflammatory in a murine model of antibody-induced inflammatory arthritis. Its activity could be attributed, at least in part, to the inhibition of neutrophil and endothelial cell functions.