Adriana Ortiz-Lopez

Critical Roles for Interleukin 1 and Tumor Necrosis Factor α 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)–α was also required, although seemingly less critically than IL-1, because a proportion of TNF-α–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 TNFα for bone destruction. The variability in the requirement for TNF-α, reminiscent of that observed in treated rheumatoid arthritis patients, did not appear genetically programmed but related instead to subtle environmental changes.

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.

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 microbiotas 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.

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

Significance Th17 cells accumulate in the gut, where they mediate barrier defenses and repair but can also provoke inflammatory disease. In mice, segmented filamentous bacteria (SFB) is sufficient to induce Th17 cells in the gut, but functionally analogous microbes in humans have not been defined. Here, we identified Bifidobacterium adolescentis as one of several human symbiont bacterial species that could, alone, induce Th17 cells in the small intestine of mice. B. adolescentis and SFB exhibited overlapping but also distinct activities, suggesting multiple routes to intestinal Th17 induction. Like SFB, B. adolescentis exacerbated autoimmune arthritis, arguing for its pathological relevance. Our results help to inform the search for therapeutic targets in diseases associated with Th17 responses and mucosal dysfunction. 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.

The K/BxN Mouse Model of Inflammatory Arthritis

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.

A Point Mutation in atpC1 Raises the Redox Potential of the Arabidopsis Chloroplast ATP Synthase γ-Subunit Regulatory Disulfide above the Range of Thioredoxin Modulation

The light-dependent regulation of chloroplast ATP synthase activity depends on an intricate but ill defined interplay between the proton electrochemical potential across the thylakoid membrane and thioredoxin-mediated redox modulation of a cysteine bridge located on the ATP synthase γ-subunit. The abnormal light-dependent regulation of the chloroplast ATP synthase in the Arabidopsis thaliana cfq (coupling factor quick recovery) mutant was caused by a point mutation (G to A) in the atpC1 gene, which caused an amino acid substitution (E244K) in the vicinity of the redox modulation domain in the γ-subunit of ATP synthase. Equilibrium redox titration revealed that this mutation made the regulatory sulfhydryl group energetically much more difficult to reduce relative to the wild type (i.e. raised the Em,7.9 by 39 mV). Enzymatic studies using isolated chloroplasts showed significantly lower light-induced ATPase and ATP synthase activity in the mutant compared with the wild type. The lower ATP synthesis capacity in turn restricted overall rates of leaf photosynthesis in the cfq mutant under low light. This work provides in situ validation of the concept that thioredoxin-dependent reduction of the γ-subunit regulatory disulfide modulates the proton electrochemical potential energy requirement for activation of the chloroplast ATP synthase and that the activation state of the ATP synthase can limit leaf level photosynthesis.

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 FcRγ-using receptors; conversely, endocarditis depended essentially on FcRγ 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.

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-1β, 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.

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.Cpa3Cre/+ (Heidelberg) and NOD.KitW-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.

Network pharmacology of JAK inhibitors

Significance JAK kinase inhibitors (JAKis) have advanced options for treatment of autoimmune diseases. Because JAKs are signaling hubs for several cytokine receptors, JAKis’ overall impact on the immune system and how they actually improve diseases like rheumatoid arthritis remain poorly understood. Combined immunophenotyping and genomic profiling revealed broad JAKi effects on the immunogenomic network, irrespective of inhibitor fine specificity, with effects on population homeostasis and coregulated gene-expression networks, particularly in innate immunocytes. Persistent repression by JAKis of IFN signature genes lasted beyond drug clearance and correlated with changes in the structure of the underlying chromatin, with direct implications for practical use of the drugs. Further JAKi development may need to take into account their broad network and epigenomic effects. 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.