Modulation of plasmacytoid dendritic cells response in inflammation and autoimmunity.

The discovery of toll-like receptors (TLRs) and the subsequent recognition that endogenous nucleic acids (NAs) could serve as TLR ligands have led to essential insights into mechanisms of healthy immune responses as well as pathogenic mechanisms relevant to systemic autoimmune and inflammatory diseases. In systemic lupus erythematosus, systemic sclerosis, and rheumatoid arthritis, NA-containing immune complexes serve as TLR ligands, with distinct implications depending on the additional immune stimuli available. Plasmacytoid dendritic cells (pDCs), the robust producers of type I interferon (IFN-I), are providing critical insights relevant to TLR-mediated healthy immune responses and tissue repair, as well as generation of inflammation, autoimmunity and fibrosis, processes central to the pathogenesis of many autoimmune diseases. In this review, we describe recent data characterizing the role of platelets and NA-binding chemokines in modulation of TLR signaling in pDCs, as well as implications for how the IFN-I products of pDCs contribute to the generation of inflammation and wound healing responses by monocyte/macrophages. Chemokine modulators of TLR-mediated B cell tolerance mechanisms and interactions between TLR signaling and metabolic pathways are also considered. The modulators of TLR signaling and their contribution to the pathogenesis of systemic autoimmune diseases suggest new opportunities for identification of novel therapeutic targets.

TLR9 ligand sequestration by chemokine CXCL4 negatively affects central B cell tolerance.

Central B cell tolerance is believed to be regulated by B cell receptor signaling induced by the recognition of self-antigens in immature B cells. Using humanized mice with defective MyD88, TLR7, or TLR9 expression, we demonstrate that TLR9/MYD88 are required for central B cell tolerance and the removal of developing autoreactive clones. We also show that CXCL4, a chemokine involved in systemic sclerosis (SSc), abrogates TLR9 function in B cells by sequestering TLR9 ligands away from the endosomal compartments where this receptor resides. The in vivo production of CXCL4 thereby impedes both TLR9 responses in B cells and the establishment of central B cell tolerance. We conclude that TLR9 plays an essential early tolerogenic function required for the establishment of central B cell tolerance and that correcting defective TLR9 function in B cells from SSc patients may represent a novel therapeutic strategy to restore B cell tolerance.

Dichotomous roles of RIPK3 in regulating the IFN response and NLRP3 inflammasome in human monocytes.

Regulation of the profile and magnitude of toll-like receptor (TLR) responses is important for effective host defense against infections while minimizing inflammatory toxicity. The chemokine CXCL4 regulates the TLR8 response to amplify inflammatory gene and inflammasome activation while attenuating the interferon (IFN) response in primary monocytes. In this study, we describe an unexpected role for the kinase RIPK3 in suppressing the CXCL4 + TLR8-induced IFN response and providing signal 2 to activate the NLRP3 inflammasome and interleukin (IL)-1 production in primary human monocytes. RIPK3 also amplifies induction of inflammatory genes such as TNF, IL6, and IL1B while suppressing IL12B. Mechanistically, RIPK3 inhibits STAT1 activation and activates PI3K-Akt-dependent and XBP1- and NRF2-mediated stress responses to regulate downstream genes in a dichotomous manner. These findings identify new functions for RIPK3 in modulating TLR responses and provide potential mechanisms by which RIPK3 plays roles in inflammatory diseases and suggest targeting RIPK3 and XBP1- and NRF2-mediated stress responses as therapeutic strategies to suppress inflammation while preserving the IFN response for host defense.

Chronic activation of pDCs in autoimmunity is linked to dysregulated ER stress and metabolic responses.

Plasmacytoid dendritic cells (pDCs) chronically produce type I interferon (IFN-I) in autoimmune diseases, including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). We report that the IRE1α-XBP1 branch of the unfolded protein response (UPR) inhibits IFN-α production by TLR7- or TLR9-activated pDCs. In SSc patients, UPR gene expression was reduced in pDCs, which inversely correlated with IFN-I-stimulated gene expression. CXCL4, a chemokine highly secreted in SSc patients, downregulated IRE1α-XBP1-controlled genes and promoted IFN-α production by pDCs. Mechanistically, IRE1α-XBP1 activation rewired glycolysis to serine biosynthesis by inducing phosphoglycerate dehydrogenase (PHGDH) expression. This process reduced pyruvate access to the tricarboxylic acid (TCA) cycle and blunted mitochondrial ATP generation, which are essential for pDC IFN-I responses. Notably, PHGDH expression was reduced in pDCs from patients with SSc and SLE, and pharmacological blockade of TCA cycle reactions inhibited IFN-I responses in pDCs from these patients. Hence, modulating the IRE1α-XBP1-PHGDH axis may represent a hitherto unexplored strategy for alleviating chronic pDC activation in autoimmune disorders.

CXCL4 synergizes with TLR8 for TBK1-IRF5 activation, epigenomic remodeling and inflammatory response in human monocytes.

Regulation of endosomal Toll-like receptor (TLR) responses by the chemokine CXCL4 is implicated in inflammatory and fibrotic diseases, with CXCL4 proposed to potentiate TLR responses by binding to nucleic acid TLR ligands and facilitating their endosomal delivery. Here we report that in human monocytes/macrophages, CXCL4 initiates signaling cascades and downstream epigenomic reprogramming that change the profile of the TLR8 response by selectively amplifying inflammatory gene transcription and interleukin (IL)-1ß production, while partially attenuating the interferon response. Mechanistically, costimulation by CXCL4 and TLR8 synergistically activates TBK1 and IKKε, repurposes these kinases towards an inflammatory response via coupling with IRF5, and activates the NLRP3 inflammasome. CXCL4 signaling, in a cooperative and synergistic manner with TLR8, induces chromatin remodeling and activates de novo enhancers associated with inflammatory genes. Our findings thus identify new regulatory mechanisms of TLR responses relevant for cytokine storm, and suggest targeting the TBK1-IKKε-IRF5 axis may be beneficial in inflammatory diseases.

Chemokines form nanoparticles with DNA and can superinduce TLR-driven immune inflammation.

Chemokines control the migratory patterns and positioning of immune cells to organize immune responses to pathogens. However, many chemokines have been associated with systemic autoimmune diseases that have chronic IFN signatures. We report that a series of chemokines, including CXCL4, CXCL10, CXCL12, and CCL5, can superinduce type I IFN (IFN-I) by TLR9-activated plasmacytoid DCs (pDCs), independently of their respective known chemokine receptors. Mechanistically, we show that chemokines such as CXCL4 mediate transcriptional and epigenetic changes in pDCs, mostly targeted to the IFN-I pathways. We describe that chemokines physically interact with DNA to form nanoparticles that promote clathrin-mediated cellular uptake and delivery of DNA in the early endosomes of pDCs. Using two separate mouse models of skin inflammation, we observed the presence of CXCL4 associated with DNA in vivo. These data reveal a noncanonical role for chemokines to serve as nucleic acid delivery vectors to modulate TLR signaling, with implications for the chronic presence of IFN-I by pDCs in autoimmune diseases.

Noncytotoxic Inhibition of the Immunoproteasome Regulates Human Immune Cells In Vitro and Suppresses Cutaneous Inflammation in the Mouse.

Inhibitors of the immunoproteasome (i-20S) have shown promise in mouse models of autoimmune diseases and allograft rejection. In this study, we used a novel inhibitor of the immunoproteasome, PKS3053, that is reversible, noncovalent, tight-binding, and highly selective for the ß5i subunit of the i-20S to evaluate the role that i-20S plays in regulating immune responses in vitro and in vivo. In contrast to irreversible, less-selective inhibitors, PKS3053 did not kill any of the primary human cell types tested, including plasmacytoid dendritic cells, conventional dendritic cells, macrophages, and T cells, all of which expressed genes encoding both the constitutive proteasome (c-20S) and i-20S. PKS3053 reduced TLR-dependent activation of plasmacytoid dendritic cells, decreasing their maturation and IFN-α response and reducing their ability to activate allogenic T cells. In addition, PKS3053 reduced T cell proliferation directly and inhibited TLR-mediated activation of conventional dendritic cells and macrophages. In a mouse model of skin injury that shares some features of cutaneous lupus erythematosus, blocking i-20S decreased inflammation, cellular infiltration, and tissue damage. We conclude that the immunoproteasome is involved in the activation of innate and adaptive immune cells, that their activation can be suppressed with an i-20S inhibitor without killing them, and that selective inhibition of ß5i holds promise as a potential therapy for inflammatory skin diseases such as psoriasis, cutaneous lupus erythematosus, and systemic sclerosis.

Adrenomedullin and truncated peptide adrenomedullin(22-52) affect chondrocyte response to apoptotis in vitro: downregulation of FAS protects chondrocyte from cell death.

Chondrocyte apoptosis may have a pivotal role in the development of osteoarthritis. Interest has increased in the use of anti-apoptotic compounds to protect against osteoarthritis development. In this work, we investigated the effect of adrenomedullin (AM), a 52 amino-acid hormone peptide, and a 31 amino-acid truncated form, AM(22-52), on chondrocyte apoptosis. Bovine articular chondrocytes (BACs) were cultured under hypoxic conditions to mimic cartilage environment and then treated with Fas ligand (Fas-L) to induce apoptosis. The expression of AM and its calcitonin receptor-like receptor (CLR)/receptor activity-modifying protein (RAMP) (receptor/co-receptor) was assessed by immunostaining. We evaluated the effect of AM and AM(22-52) on Fas-L-induced chondrocyte apoptosis. FAS expression was appreciated by RT-qPCR and immunostainings. The expression of hypoxia-inducible factor 1α (HIF-1α), CLR and one co-receptor (RAMP2) was evidenced. With BACs under hypoxia, cyclic adenosine monophosphate production increased dose-dependently with AM stimulation. AM significantly decreased caspase-3 activity (mean 35% decrease; p = 0.03) as a marker of Fas-L-induced apoptosis. Articular chondrocytes treated with AM showed significantly reduced cell death, along with downregulated Fas expression and production, as compared with AM(22-52). AM decreased articular chondrocyte apoptosis by downregulating a Fas receptor. These findings may pave the way for novel therapeutic approaches in osteoarthritis.

Structure-Activity Relationships of Noncovalent Immunoproteasome ß5i-Selective Dipeptides.

The immunoproteasome (i-20S) has emerged as a therapeutic target for autoimmune and inflammatory disorders and hematological malignancies. Inhibition of the chymotryptic ß5i subunit of i-20S inhibits T cell activation, B cell proliferation, and dendritic cell differentiation in vitro and suppresses immune responses in animal models of autoimmune disorders and allograft rejection. However, cytotoxicity to immune cells has accompanied the use of covalently reactive ß5i inhibitors, whose activity against the constitutive proteasome (c-20S) is cumulative with the time of exposure. Herein, we report a structure-activity relationship study of a class of noncovalent proteasome inhibitors with picomolar potencies and 1000-fold selectivity for i-20S over c-20S. Furthermore, these inhibitors are specific for ß5i over the other five active subunits of i-20S and c-20S, providing useful tools to study the functions of ß5i in immune responses. The potency of these compounds in inhibiting human T cell activation suggests that they may have therapeutic potential.

Plasmacytoid dendritic cells promote systemic sclerosis with a key role for TLR8.

Systemic sclerosis (SSc) is a multisystem life-threatening fibrosing disorder that lacks effective treatment. The link between the inflammation observed in organs such as the skin and profibrotic mechanisms is not well understood. The plasmacytoid dendritic cell (pDC) is a key cell type mediating Toll-like receptor (TLR)-induced inflammation in autoimmune disease patients, including lupus and skin diseases with interface dermatitis. However, the role of pDCs in fibrosis is less clear. We show that pDCs infiltrate the skin of SSc patients and are chronically activated, leading to secretion of interferon-α (IFN-α) and CXCL4, which are both hallmarks of the disease. We demonstrate that the secretion of CXCL4 is under the control of phosphatidylinositol 3-kinase δ and is due to the aberrant presence of TLR8 on pDCs of SSc patients, which is not seen in healthy donors or in lupus pDCs, and that CXCL4 primarily acts by potentiating TLR8- but also TLR9-induced IFN production by pDCs. Depleting pDCs prevented disease in a mouse model of scleroderma and could revert fibrosis in mice with established disease. In contrast, the disease was exacerbated in mice transgenic for TLR8 with recruitment of pDCs to the fibrotic skin, whereas TLR7 only partially contributed to the inflammatory response, indicating that TLR8 is the key RNA-sensing TLR involved in the establishment of fibrosis. We conclude that the pDC is an essential cell type involved in the pathogenesis of SSc and its removal using depleting antibodies or attenuating pDC function could be a novel approach to treat SSc patients.

Musculoskeletal Involvement in SSc Is Associated with Worse Scores on Short Form-36 and Scleroderma Health Assessment Questionnaire and Lower Tumor Necrosis Factor-α Gene Expression in Peripheral Blood Mononuclear Cells.

BACKGROUND: Systemic sclerosis (SSc) is characterized by a wide variety of symptoms and disease manifestations including joint pain, gastrointestinal dysfunction, interstitial lung disease, and cardiomyopathy. QUESTIONS/PURPOSES: Using the Scleroderma Health Assessment Questionnaire (SHAQ) and Short Form-36 (SF-36) we explored how patient-reported physical health, mental health, and functional status related to these clinical characteristics and to cytokine levels utilizing the Hospital for Special Surgery Scleroderma Registry. METHODS: In a cross-sectional study of 185 patients meeting the 2013 ACR/EULAR criteria for SSc, we compared disease features and patient-reported outcomes (PROs). Interleukin-6 (IL-6), interleukin-1ß (IL1ß), and tumor necrosis factor-α (TNFα) levels were assessed by luminex and ELISA assays in a subset of 32 patients. The Pearson correlation coefficient, Spearman correlation coefficient, two-sample t test or Wilcoxon rank sum test, ANOVA or Kuskal-Wallis test, and Pearson chi-squared or Fisher's exact test were performed as applicable to detect the association between disease manifestations, PROs, and blood biomarkers. RESULTS: The modified Rodnan skin score (MRSS) was positively correlated with SHAQ scores. Patients who had musculoskeletal involvement scored worse on both the SHAQ and SF-36. Lower levels of TNFα expression in PBMCs were also correlated with musculoskeletal involvement. No other significant correlations were found between clinical factors, PROs, and cytokine data. CONCLUSION: Musculoskeletal outcomes are a major determinant of quality of life and function in patients with SSc. These results emphasize the importance of musculoskeletal outcomes in clinical studies of SSc.

Protective role of systemic furin in immune response-induced arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is an autoimmune joint disease associated with chronic inflammation of the synovium that causes profound damage of joints. Inflammation results in part from the influx of immune cells secreting inflammatory cytokines and the reduction in the number of Treg cells. We undertook this study to assess the effect of furin, a proteinase implicated in the proteolytic activity of various precursor proteins and involved in the regulation of both proteinase maturation and immune cells, in an experimental model of RA. METHODS: The effect of furin and its inhibitor α1-PDX was tested in mice with collagen-induced arthritis (CIA). Joints were processed for histology and protein expression. Levels of cytokines were measured in joint tissue, and Treg cell numbers were measured in spleens. RESULTS: Furin expression and activity were high in the synovial pannus in RA patients and mice with CIA. Systemic administration of furin prevented increases in the arthritis score, joint destruction, and bone loss, in contrast to systemic administration of the furin inhibitor α1-PDX, which enhanced these parameters. By preventing the development of synovial pannus, furin reduced the expression of metalloproteinases in the joints. In contrast, α1-PDX enhanced synovial proliferation and the expression and activity of matrix metalloproteinases. Furthermore, furin reversed the local Th1/Th2 balance and restored the number of Treg cells in the spleen, indicating mediation by immune cells. CONCLUSION: These findings show the protective role of exogenous furin against RA, mediated by an immune response. The data suggest the potential therapeutic use of furin or its derivatives in autoimmune diseases including RA.

Targeting bone alleviates osteoarthritis in osteopenic mice and modulates cartilage catabolism.

OBJECTIVE: Subchondral bone modifications occur early in the development of osteoarthritis (OA). The level of bone resorption might impact cartilage remodeling. We therefore assessed the in vivo and in vitro effects of targeting bone resorption in OA and cartilage metabolism. METHODS: OA was induced by meniscectomy (MNX) in ovariectomized osteopenic mice (OP) treated with estradiol (E2), pamidronate (PAM), or phosphate buffered saline (PBS) for 6 weeks. We assessed the subchondral bone and cartilage structure and the expression of cartilage matrix proteases. To assess the involvement of bone soluble factors in cartilage metabolism, supernatant of human bone explants pre-treated with E2 or PAM were transferred to cartilage explants to assess proteoglycan release and aggrecan cleavage. OPG/RANKL mRNA expression was assessed in bone explants by real-time quantitative PCR. The role of osteoprotegerin (OPG) in the bone-cartilage crosstalk was tested using an OPG neutralizing antibody. RESULTS: Bone mineral density of OP mice and osteoclast number were restored by E2 and PAM (p<0.05). In OP mice, E2 and PAM decreased ADAMTS-4 and -5 expression, while only PAM markedly reduced OA compared to PBS (2.0±0.63 vs 5.2±0.95; p<0.05). OPG/RANKL mRNA was increased in human bone explants treated with both drugs (2.2-3.7-fold). Moreover, supernatants from bone explants cultured with E2 or PAM reduced aggrecan cleavage and cartilage proteoglycan release (73±8.0% and 80±22% of control, respectively, p<0.05). This effect was reversed with osteoprotegerin blockade. CONCLUSION: The inhibition of bone resorption by pamidronate in osteopenic mice alleviates the histological OA score with a reduction in the expression of aggrecanases. Bone soluble factors, such as osteoprotegerin, impact the cartilage response to catabolic factors. This study further highlights the importance of subchondral bone in the regulation of joint cartilage damage in OA.

Adrenomedullin, a neuropeptide with immunoregulatory properties induces semi-mature tolerogenic dendritic cells.

Dendritic cells (DC) play a pivotal role in tolerance. Adrenomedullin (AM), a neuropeptide with anti-apoptotic and anti-inflammatory effects, may decrease T helper type 1 effector cells and induce regulatory T (Treg) cells. The aim of this study was to evaluate AM effects on murine dendritic cell (DC) maturation and functions. Bone marrow-derived DC were produced and stimulated with CpG motifs, lipopolysaccharide or AM for 24 hr. Then, DC maturation and expression of AM and AM receptors were evaluated. Compared with lipopolysaccharide-stimulated or CpG-stimulated DC, AM-stimulated DC had lower levels of co-stimulatory molecule expression and pro-inflammatory cytokine release. The AM induced high levels of interferon-γ but not of interleukin-10. Importantly, AM inhibited lipopolysaccharide-induced maturation of DC. However, allogeneic T-cell stimulation and endocytic capacity of AM-stimulated DC were comparable to those of semi-mature and mature DC. Moreover, DC expressed AM and its receptors at a basal level, and AM receptor expression increased with DC maturation. The AM stimulation induced indoleamine 2,3-dioxygenase (IDO) expression, promoting Treg cell expansion. For the first time, we describe the DC maturation phenotype by a neuropeptide (AM). We have demonstrated that AM and its receptors are expressed in DC and that exogenous AM can modify the DC phenotype and functions and can induce a semi-mature DC phenotype with IDO expression. These results indicate close interactions among immune system regulation mechanisms and calcitonin-like peptides.

Adrenomedullin(22-52) combats inflammation and prevents systemic bone loss in murine collagen-induced arthritis.

OBJECTIVE: Adrenomedullin(22-52) is a truncated peptide derived from adrenomedullin, a growth factor with antiapoptotic and immunoregulatory properties. It can act as an agonist or an antagonist depending on cell type. Its in vivo effects are unknown, but adrenomedullin(22-52) could possess immunomodulatory properties. This study was undertaken to evaluate the effect of adrenomedullin(22-52) in a mouse model of arthritis. METHODS: DBA/1 mice with collagen-induced arthritis (CIA) were treated with 1.2 µg/gm adrenomedullin(22-52) , adrenomedullin, or saline at arthritis onset. Bone mineral density was measured at the beginning of the experiment and when mice were killed. Mouse joints were processed for histologic analysis and protein studies, and spleens were examined for Treg cell expression. Cytokine expression was studied in mouse joint tissue and serum. RESULTS: In mice with CIA, adrenomedullin and adrenomedullin(22-52) reduced clinical and histologic arthritis scores and shifted the pattern of articular and systemic cytokine expression from Th1 to Th2, as compared to untreated mice with CIA (controls). Tumor necrosis factor α, interleukin-6 (IL-6), and IL-17A levels were significantly decreased in the joints of mice with CIA treated with adrenomedullin or adrenomedullin(22-52) as compared to controls, whereas IL-4 and IL-10 levels were increased. Adrenomedullin(22-52) was more effective than adrenomedullin in modulating cytokine content and enhanced Treg cell function without changing Treg cell expression compared to controls. Adrenomedullin receptor binding and transcriptional adrenomedullin receptor expression were markedly increased in joints from controls, whereas adrenomedullin receptor binding was considerably decreased in treated animals. Mice with CIA treated with adrenomedullin or adrenomedullin(22-52) had considerably fewer apoptotic chondrocytes and diminished cartilage degradation. Adrenomedullin(22-52) completely prevented systemic bone loss by preserving osteoblastic activity, but without changes in osteoclastic activity. CONCLUSION: Our findings indicate that adrenomedullin(22-52) , which has no vasoactive or tumor-inducing effects, is a potent antiinflammatory and bone-protective agent in this arthritis model.

Adrenomedullin increases fibroblast-like synoviocyte adhesion to extracellular matrix proteins by upregulating integrin activation.

INTRODUCTION: Rheumatoid arthritis (RA) is characterized by bone and cartilage invasion by fibroblast-like synoviocytes (FLSs). Adrenomedullin, a peptide with anabolic and antiapoptotic properties, is secreted by rheumatoid FLSs. Adrenomedullin also increases the expression of adhesion molecules in endothelial cells and keratinocytes. Here, we investigated whether adrenomedullin mediated FLS adhesion to extracellular matrix (ECM) proteins. METHODS: FLSs were isolated from synovial tissues from RA and osteoarthritis (OA) patients. Plates were coated overnight with the ECM proteins vitronectin, fibronectin, and type I collagen (Coll.I). Adrenomedullin was used as a soluble FLS ligand before plating. We tested interactions with the adrenomedullin receptor antagonist (22-52)adrenomedullin and with the protein kinase A (PKA) inhibitor H-89, and inhibition of co-receptor RAMP-2 by siRNA. Cell adhesion was measured by using color densitometry. Activation of α2 and ß1 integrins was evaluated by fluorescent microscopy; integrin inhibition, by RGD peptides; and the talin-integrin interaction, by immunoprecipitation (IP). RESULTS: Adrenomedullin specifically increased RA-FLS adhesion to vitronectin, fibronectin, and Coll.I; no such effect was found for OA-FLS adhesion. Basal or adrenomedullin-stimulated RA-FLS adhesion was inhibited by (22-52)adrenomedullin, H-89, and RAMP-2 siRNA. Adrenomedullin-stimulated adhesion was inhibited by RGD peptides, and associated with α2 and ß1 integrin activation. This activation was shown with IP to be related to an integrin-talin interaction and was significantly decreased by (22-52)adrenomedullin. CONCLUSIONS: Adrenomedullin-stimulated RA-FLS adhesion was specific for ECM proteins and mediated by α2 and ß1 integrins. This effect of adrenomedullin was dependent on adrenomedullin receptors. These results support a new role for adrenomedullin in rheumatoid synovial fibroblast pathobiology.