Induction of S100A9 homodimer formation in vivo.

We show here that increased S100A8 and S100A9 protein expression is induced in spleen of animals with active inflammation or with inoculated tumors. In tumor bearing animals an increased expression was also detected in the lung. To further analyze the induced proteins, we performed chemical cross-linking followed by Western blotting. We observed in protein extracts from spleen that both S100A8/S100A9 heterodimers as well as S100A9 homodimers were formed, both after tumor and inflammatory challenge. The cellular source for S100A9 homodimers were CD11b+GR1+ cells. S100A9 homodimers were also secreted into the extracellular space. Lastly, in the spleen from normal and tumor bearing animals cells expressing relatively higher levels of S100A9 compared to S100A8 could be observed by immunohistochemistry. Taken together, these data show that the biologically potent dimeric form of S100A9 is induced in vivo in situations of tumor burden or inflammatory challenge.

IgA Structure Variations Associate with Immune Stimulations and IgA Mesangial Deposition.

IgA1 mesangial deposition is the hallmark of IgA nephropathy and Henoch-Schönlein purpura, the onset of which often follows infections. Deposited IgA has been reported as polymeric, J chain associated, and often, hypogalactosylated but with no information concerning the influence of the IgA repertoire or the link between immune stimuli and IgA structure. We explored these issues in the α1KI mouse model, which produces polyclonal human IgA1 prone to mesangial deposition. Compared with mice challenged by a conventional environment, mice in a specific pathogen-free environment had less IgA deposition. However, serum IgA of specific pathogen-free mice showed more galactosylation and much lower polymerization. Notably, wild-type, α1KI, and even J chain-deficient mice showed increased polymeric serum IgA on exposure to pathogens. Strict germfree conditions delayed but did not completely prevent deposition; mice housed in these conditions had very low serum IgA levels and produced essentially monomeric IgA. Finally, comparing monoclonal IgA1 that had different variable regions and mesangial deposition patterns indicated that, independently of glycosylation and polymerization, deposition might also depend on IgA carrying specific variable domains. Together with IgA quantities and constant region post-translational modifications, repertoire changes during immune responses might, thus, modulate IgA propensity to deposition. These IgA features are not associated with circulating immune complexes and C3 deposition and are more pertinent to an initial IgA deposition step preceding overt clinical symptoms in patients.

The anti-tumor effect of the quinoline-3-carboxamide tasquinimod: blockade of recruitment of CD11b(+) Ly6C(hi) cells to tumor tissue reduces tumor growth.

BACKGROUND: Previous work has demonstrated immunomodulatory, anti-tumor, anti-metastatic and anti-angiogenic effects of the small molecule quinoline-3-carboxamide tasquinimod in pre-clinical cancer models. To better understand the anti-tumor effects of tasquinimod in transplantable tumor models, we have evaluated the impact of the compound both on recruitment of myeloid cells to tumor tissue and on tumor-induced myeloid cell expansion as these cells are known to promote tumor development. METHODS: Mice bearing subcutaneous 4 T1 mammary carcinoma tumors were treated with tasquinimod in the drinking water. A BrdU-based flow cytometry assay was utilized to assess the impact of short-term tasquinimod treatment on myeloid cell recruitment to tumors. Additionally, long-term treatment was performed to study the anti-tumor effect of tasquinimod as well as its effects on splenic myeloid cells and their progenitors. Myeloid cell populations were also immune-depleted by in vivo antibody treatment. RESULTS: Short-term tasquinimod treatment did not influence the proliferation of splenic Ly6C(hi) and Ly6G(hi) cells, but instead reduced the influx of Ly6C(hi) cells to the tumor. Treatment with tasquinimod for various periods of time after tumor inoculation revealed that the anti-tumor effect of this compound mainly operated during the first few days of tumor growth. Similar to tasquinimod treatment, antibody-mediated depletion of Ly6C(hi) cells within that same time frame, caused reduced tumor growth, thereby confirming a significant role for these cells in tumor development. Additionally, long-term tasquinimod treatment reduced the splenomegaly and expansion of splenic myeloid cells during a later phase of tumor development. In this phase, tasquinimod normalized the tumor-induced alterations in myeloerythroid progenitor cells in the spleen but had only limited impact on the same populations in the bone marrow. CONCLUSIONS: Our results indicate that tasquinimod treatment reduces tumor growth by operating early after tumor inoculation and that this effect is at least partially caused by reduced recruitment of Ly6C(hi) cells to tumor tissue. Long-term treatment also reduces the number of splenic myeloid cells and myeloerythroid progenitors, but these effects did not influence established rapidly growing tumors.

Myeloid-related protein 14 promotes inflammation and injury in meningitis.

BACKGROUND: Neutrophilic inflammation often persists for days despite effective antibiotic treatment and contributes to brain damage in bacterial meningitis. We propose here that myeloid-related protein 14 (MRP14), an abundant cytosolic protein in myeloid cells, acts as an endogenous danger signal, driving inflammation and aggravating tissue injury. METHODS: The release pattern of MRP14 was analyzed in human and murine cerebrospinal fluid (CSF), as well as in isolated neutrophils. Its functional role was assessed in a mouse meningitis model, using MRP14-deficient mice. RESULTS: We detected large quantities of MRP14 in CSF specimens from patients and mice with pneumococcal meningitis. Immunohistochemical analyses and a cell-depletion approach indicated neutrophils as the major source of MRP14. In a meningitis model, MRP14-deficient mice showed a better resolution of inflammation during antibiotic therapy, which was accompanied by reduced disease severity. Intrathecal administration of MRP14 before infection reverted the phenotype of MRP14-deficient mice back to wild type. Moreover, intrathecal injection of MRP14 alone was sufficient to induce meningitis in a Toll-like receptor 4 (TLR4)-CXCL2-dependent manner. Finally, treatment with the MRP14 antagonist paquinimod reduced inflammation and disease severity significantly, reaching levels comparable to those achieved after genetic depletion of MRP14. CONCLUSIONS: The present study implicates MRP14 as an essential propagator of inflammation and potential therapeutic target in pneumococcal meningitis.

S100A9 expressed in ER(-)PgR(-) breast cancers induces inflammatory cytokines and is associated with an impaired overall survival.

BACKGROUND: Breast cancer is the most common cancer form among women today. Depending on hormone receptor status, breast cancers are divided into different subtypes with vastly varying prognosis. S100A9 is a calcium-binding protein that is associated with inflammation and expressed not only in myeloid cells but also in some tumours. The role for S100A9 in the malignant cells is not well characterised; however, previous studies have shown that the protein could have important immune-modulating properties. METHODS: Using a human breast cancer cohort consisting of 144 tumour samples and in vitro analysis of human breast cancer cell lines, we investigated the expression and function of S100A9 in human breast cancer. RESULTS: We show that S100A9 expression in breast cancer correlated with the ER(-)PgR(-) breast tumour subtype (P<0.001) and with Ki67 (P=0.024) and was expressed both in the malignant cells and in the tumour-infiltrating anti-inflammatory CD163(+) myeloid cells (P<0.001). Stromal expression of S100A9 also correlated to nodal stage, tumour size and Her2 positivity. Within the ER(-)PgR(-) subgroup, all Her2(+) and EGFR(+) tumours expressed S100A9 in the cytoplasm. Both cytoplasmic staining in the malignant cells as well as stromal S100A9 expression in myeloid cells correlated with a decreased overall survival in breast cancer patients. Furthermore, rS100A9 homodimers induced expression of pro-inflammatory cytokines (IL-6, IL-8 and IL-1ß) in a TLR4- and EGFR-dependent manner in human breast cancer cells in vitro. CONCLUSION: We suggest that S100A9 could be viewed as a novel therapeutic target for patients with ER(-)PgR(-) breast cancers.

Amelioration of experimental autoimmune encephalomyelitis by the quinoline-3-carboxamide paquinimod: reduced priming of proinflammatory effector CD4(+) T cells.

Quinoline-3-carboxamide compounds (Q compounds) have demonstrated efficacy in treating autoimmune disease in both humans and mice. However, the mode of action of these compounds is poorly understood. Here, we show that preventive treatment with the Q compound paquinimod (ABR-215757) during the first 5 days after induction of experimental autoimmune encephalomyelitis is sufficient to significantly ameliorate disease symptoms. Parallel cell-depletion experiments demonstrated that Ly6C(hi) inflammatory monocytes play an essential role in this phase. The paquinimod-induced amelioration correlated with reduced priming of antigen-specific CD4(+) T cells and reduced frequency of IFN-γ- and IL-17-producing cells in draining lymph nodes. Importantly, the treatment did not inhibit T-cell division per se. In mice with established experimental autoimmune encephalomyelitis, the numbers of Ly6C(hi) CD115(+) inflammatory monocytes and CD11b(+)CD11c(+) dendritic cells (DCs) were reduced in spleen, but not in bone marrow or draining lymph nodes of treated mice. Inflammatory monocyte-derived DCs and CD4(+) T cells were also reduced in the brain. In contrast, there was no decrease in DC subsets previously shown to be critical for effector CD4(+) T-cell development in lymph nodes. Taken together, these data indicate that preventive treatment with paquinimod ameliorates experimental autoimmune encephalomyelitis by reducing effector T-cell priming and, on prolonged treatment, displays a selective effect by decreasing distinct subpopulations of splenic CD11b(+) myeloid cells.

Increased serum levels of S100A8/A9 and S100A12 are associated with cardiovascular disease in patients with inactive systemic lupus erythematosus.

OBJECTIVES: Patients with SLE have an increased morbidity and mortality from cardiovascular disease (CVD). The reason for this is not entirely understood, but is believed to be partly related to the long-lasting inflammatory process seen in SLE. The aim of the present study was to investigate whether there is an association between CVD and serum levels of the proinflammatory proteins S100A8/A9 and S100A12 in SLE. METHODS: Serum levels of S100A8/A9 and S100A12 were measured with ELISA in 237 SLE patients with clinically inactive disease and without infections, as well as in 100 healthy individuals. Cardiovascular manifestations were defined according to the SLICC/ACR Damage Index (SLICC/ACR-DI). RESULTS: Serum levels of S100A8/A9 were elevated in our inactive SLE patients as compared with healthy individuals (P < 0.0001), which was not seen for S100A12 (P = 0.12). SLE patients with a history of CVD had increased serum levels of both S100A8/A9 and S100A12 compared with patients with no CVD or venous thromboembolism (P = 0.003 and P = 0.006, respectively). The presence of organ damage according to SLICC/ACR-DI was associated with an increase in both S100A8/A9 and S100A12 serum levels (P = 0.001 and P = 0.006, respectively). CONCLUSION: Elevated serum levels of S100A8/A9 and S100A12 may be used as an indicator of severe disease and CVD in SLE, suggesting that SLE patients with elevated serum S100A8/A9 and S100A12 concentrations may benefit from more intense cardiovascular primary preventive strategies and possibly also from more intense and early immunosuppressive treatment.

Pharmacokinetics, tolerability, and preliminary efficacy of paquinimod (ABR-215757), a new quinoline-3-carboxamide derivative: studies in lupus-prone mice and a multicenter, randomized, double-blind, placebo-controlled, repeat-dose, dose-ranging study in patients with systemic lupus erythematosus.

OBJECTIVE: To assess the efficacy of paquinimod, a new immunomodulatory small molecule, in a murine lupus model, and to evaluate its pharmacokinetics and tolerability in systemic lupus erythematosus (SLE) patients at doses predicted to be efficacious and safe and determine the maximum tolerated dose. METHODS: The efficacy of paquinimod was studied in lupus-prone MRL-lpr/lpr mice and compared with that of established SLE treatments. Dose-response data and pharmacokinetic data were used to calculate effective and safe clinical doses of paquinimod. The pharmacokinetics and tolerability of paquinimod were evaluated in a phase Ib double-blind, placebo controlled, dose-ranging study in which cohorts of SLE patients received daily oral treatment for 12 weeks. RESULTS: Paquinimod treatment resulted in disease inhibition in MRL-lpr/lpr mice, comparable to that obtained with prednisolone and mycophenolate mofetil; prominent effects on disease manifestations and serologic markers and a steroid-sparing effect were observed. In patients with SLE, the pharmacokinetic properties of paquinimod were linear and well suitable for once-daily oral treatment. The majority of the adverse events (AEs) were mild or moderate, and transient. The most frequent AEs were arthralgia and myalgia, reported with the highest dose levels of paquinimod (4.5 mg/day and 6.0 mg/day). At the 4.5 mg/day dose level and higher, some AEs of severe intensity and serious adverse events were reported. CONCLUSION: Paquinimod effectively inhibited disease and had a steroid-sparing effect in experimental lupus. Results from preclinical models together with pharmacokinetic data were successfully translated into a safe clinical dose range, and doses of up to 3.0 mg/day were well tolerated in the SLE patients. Taken together, the promising combined data from a murine model and human SLE support the future clinical development of paquinimod.

Induction of nuclear factor-κB responses by the S100A9 protein is Toll-like receptor-4-dependent.

Interactions between danger-associated molecular patterns (DAMP) and pathogen-associated molecular patterns (PAMP) and pattern recognition receptors such as Toll-like receptors (TLRs) are critical for the regulation of the inflammatory process via activation of nuclear factor-κB (NF-κB) and cytokine secretion. In this report, we investigated the capacity of lipopolysaccharide (LPS) -free S100A9 (DAMP) protein to activate human and mouse cells compared with lipoprotein-free LPS (PAMP). First, we showed that LPS and S100A9 were able to increase NF-κB activity followed by increased cytokine and nitric oxide (NO) secretion both in human THP-1 cells and in mouse bone marrow-derived dendritic cells. Surprisingly, although S100A9 triggered a weaker cytokine response than LPS, we found that S100A9 more potently induced IκBα degradation and hence NF-κB activation. Both the S100A9-induced response and the LPS-induced response were completely absent in TLR4 knockout mice, whereas it was only slightly affected in RAGE knockout mice. Also, we showed that LPS and S100A9 NF-κB induction were strongly reduced in the presence of specific inhibitors of TLR-signalling. Chloroquine reduced S100A9 but not LPS signalling, indicating that S100A9 may need to be internalized to be fully active as a TLR4 inducer. This was confirmed using A488-labelled S100A9 that was internalized in THP-1 cells, showing a raise in fluorescence after 30 min at 37°. Chloroquine treatment significantly reduced the fluorescence. In summary, our data indicate that both human and mouse S100A9 are TLR4 agonists. Importantly, S100A9 induced stronger NF-κB activation albeit weaker cytokine secretion than LPS, suggesting that S100A9 and LPS activated NF-κB in a qualitatively distinct manner.

Inhibition of metastasis in a castration resistant prostate cancer model by the quinoline-3-carboxamide tasquinimod (ABR-215050).

BACKGROUND: Tasquinimod (ABR-215050) is an orally active quinoline-3-carboxamide analog that has completed phase II clinical trial in patients with castration resistant prostate cancer, showing promising inhibiting effects on the occurrence of metastasis and delayed disease progression. Its mechanism of action is not fully elucidated, but previous studies show anti-angiogenic effects and strong interaction with the S100A9 protein. METHODS: This study was performed to evaluate if tasquinimod inhibits prostate cancer metastasis, by using both orthotopic and intratibial xenograft models. Animals were treated with tasquinimod, and tumor growth characteristics as well as molecular markers for metastasis and angiogenesis were analyzed. RESULTS: The results show that formation of lung and lymph node metastases from orthotopic castration resistant prostate tumors was inhibited by tasquinimod treatment. Importantly, establishment of tumors in the bone after intratibial implantation was suppressed by tasquinimod. In addition, establishment and growth of subcutaneous tumors were affected. Both in primary tumors and serum from treated mice an upregulation of thrombospondin 1 was observed. Further, downregulation of the hypoxia driven genes VEGF, CXCR4, and LOX was detected in the primary tasquinimod-treated tumors and decreased expression of chemotactic ligand SDF-1 was demonstrated in the lungs. Thus, these molecular changes could contribute to the anti-angiogenic and anti-metastatic effects of tasquinimod. CONCLUSIONS: In conclusion, this study and clinical data show that tasquinimod interferes with the metastatic process, presumably by inhibition of tumor establishment. Therefore, tasquinimod is an interesting treatment option for patients with prostate cancer prone to metastasis.

CD11b+Ly6C++Ly6G- cells show distinct function in mice with chronic inflammation or tumor burden.

BACKGROUND: S100A9 has been shown to be important for the function of so called Myeloid Derived Suppressor Cells (MDSC). Cells with a similar phenotype are also involved in pro-inflammatory processes, and we therefore wanted to investigate the gene expression and function of these cells in animals that were either subjected to chronic inflammation, or inoculated with tumors. METHODS: CD11b(+)Ly6C(++) and Ly6G(+) cells were isolated from spleen, tumor tissue or inflammatory granulomas. S100A9, Arginase 1 and iNOS gene expression in the various CD11b(+) cell populations was analyzed using Q-PCR. The suppressive activity of the CD11b(+) cell populations from different donors was studied in co-culture experiments. RESULTS: S100A9 was shown to be expressed mainly in splenic CD11b(+)Ly6C(+)G(+) cells both at the RNA and protein level. Arginase I and iNOS expression could be detected in both CD11b(+)Ly6C(+)Ly6G(+) and CD11b(+)Ly6C(+)G(-)/C(++)G(-) derived from tumors or a site of chronic inflammation, but was very low in the same cell populations isolated from the spleen. CD11b(+) cells isolated from mice with peritoneal chronic inflammation were able to stimulate T lymphocytes, while CD11b+ cells from mice with peritoneal tumors suppressed T cell growth. CONCLUSION: An identical CD11b(+)Ly6C(++)G(-) cell population appears to have the ability to adopt immune stimulatory or immune suppressive functions dependent on the presence of a local inflammatory or tumor microenvironment. Thus, there is a functional plasticity in the CD11b(+)Ly6C(++)G(-) cell population that cannot be distinguished with the current molecular markers.

Identification of human S100A9 as a novel target for treatment of autoimmune disease via binding to quinoline-3-carboxamides.

Despite more than 25 years of research, the molecular targets of quinoline-3-carboxamides have been elusive although these compounds are currently in Phase II and III development for treatment of autoimmune/inflammatory diseases in humans. Using photoaffinity cross-linking of a radioactively labelled quinoline-3-carboxamide compound, we could determine a direct association between human S100A9 and quinoline-3-carboxamides. This interaction was strictly dependent on both Zn++ and Ca++. We also show that S100A9 in the presence of Zn++ and Ca++ is an efficient ligand of receptor for advanced glycation end products (RAGE) and also an endogenous Toll ligand in that it shows a highly specific interaction with TLR4/MD2. Both these interactions are inhibited by quinoline-3-carboxamides. A clear structure-activity relationship (SAR) emerged with regard to the binding of quinoline-3-carboxamides to S100A9, as well as these compounds potency to inhibit interactions with RAGE or TLR4/MD2. The same SAR was observed when the compound's ability to inhibit acute experimental autoimmune encephalomyelitis in mice in vivo was analysed. Quinoline-3-carboxamides would also inhibit TNFalpha release in a S100A9-dependent model in vivo, as would antibodies raised against the quinoline-3-carboxamide-binding domain of S100A9. Thus, S100A9 appears to be a focal molecule in the control of autoimmune disease via its interactions with proinflammatory mediators. The specific binding of quinoline-3-carboxamides to S100A9 explains the immunomodulatory activity of this class of compounds and defines S100A9 as a novel target for treatment of human autoimmune diseases.

Tasquinimod (ABR-215050), a quinoline-3-carboxamide anti-angiogenic agent, modulates the expression of thrombospondin-1 in human prostate tumors.

BACKGROUND: The orally active quinoline-3-carboxamide tasquinimod [ABR-215050; CAS number 254964-60-8), which currently is in a phase II-clinical trial in patients against metastatic prostate cancer, exhibits anti-tumor activity via inhibition of tumor angiogenesis in human and rodent tumors. To further explore the mode of action of tasquinimod, in vitro and in vivo experiments with gene microarray analysis were performed using LNCaP prostate tumor cells. The array data were validated by real-time semiquantitative reversed transcriptase polymerase chain reaction (sqRT-PCR) and protein expression techniques. RESULTS: One of the most significant differentially expressed genes both in vitro and in vivo after exposure to tasquinimod, was thrombospondin-1 (TSP1). The up-regulation of TSP1 mRNA in LNCaP tumor cells both in vitro and in vivo correlated with an increased expression and extra cellular secretion of TSP1 protein. When nude mice bearing CWR-22RH human prostate tumors were treated with oral tasquinimod, there was a profound growth inhibition, associated with an up-regulation of TSP1 and a down- regulation of HIF-1 alpha protein, androgen receptor protein (AR) and glucose transporter-1 protein within the tumor tissue. Changes in TSP1 expression were paralleled by an anti-angiogenic response, as documented by decreased or unchanged tumor tissue levels of VEGF (a HIF-1 alpha down stream target) in the tumors from tasquinimod treated mice. CONCLUSIONS: We conclude that tasquinimod-induced up-regulation of TSP1 is part of a mechanism involving down-regulation of HIF1alpha and VEGF, which in turn leads to reduced angiogenesis via inhibition of the "angiogenic switch", that could explain tasquinimods therapeutic potential.

Indoleamine 2,3-dioxygenase (IDO) activity influence tumor growth in the TRAMP prostate cancer model.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) activity has been shown to be expressed in local lymph nodes and induce immune suppression of tumor immunity. Here we analyze the effect of IDO expression on prostate tumor growth using the transgenic adenocarcinoma of mouse prostate (TRAMP) animal model. METHODS: Mice deficient in IDO expression were crossed to TRAMP mice and the time to the appearance of palpable tumors were measured. Immune histology was used to analyze the IDO expressing cells in tumors and in local lymph nodes. The levels of the substrate for IDO (tryptophane) and its product (kynurenine) was measured by HPLC. RESULTS: We found that systemic IDO activity, determined as the kynurenine/tryptophan ratio in serum, correlated with the presence of palpable tumor. Immunohistological analysis showed increased numbers of IDO expressing cells in local lymph nodes. In tumors, IDO expression could be detected in the tumor stroma by both CD31+ and CD31(-) cells. Essentially no CD45+, IDO expressing cells could be detected in the tumors. The influence of IDO activity on tumor progression was analyzed by back-crossing TRAMP mice with IDO(-/-) animals and J-chain negative (J(-/-)) mice that have perturbed IDO activity. In both crosses a delayed tumor incidence was observed. CONCLUSION: Our results argue for a role for IDO mediated immune suppression in the early stages of prostate cancer progression. However, since the intra-tumor IDO expression in J(-/-) mice was indistinguishable from that of C57BL/6 animals the IDO expression in the tumor tissue appears to be irrelevant for TRAMP tumor incidence.

Antioxidant metabolism induced by quinic acid. Increased urinary excretion of tryptophan and nicotinamide.

For over 50 years, hippuric/quinic acids were believed to have no biological efficacy. Here data are presented to support the hypothesis that quinic acid is not responsible for any efficacy, but rather that quinic acid nutritionally supports the synthesis of tryptophan and nicotinamide in the gastrointestinal (GI) tract, and that this in turn leads to DNA repair enhancement and NF-kB inhibition via increased nicotinamide and tryptophan production.Moreover, it is shown that quinic acid is a normal constituent of our diet, capable of conversion to tryptophan and nicotinamide via the GI tract microflora, thus providing an in situ physiological source of these essential metabolic ingredients to humans. The concentrations of quinic and hippuric acids in the diet were dependent on each other when analysed in urine, as was evidenced by a significant linear regression analysis that included unsupplemented control subjects (n = 45, p < 0.001). Thus, these ingredients were identified as major dietary components, and not simply originating from environmental pollution as previously had been thought.

A subset of dendritic cells express joining chain (J-chain) protein.

Joining chain (J-chain) is well known as an integrated component of dimeric immunoglobulin A (IgA) and pentameric IgM. We show here that the J-chain protein is also expressed in a subset of CD11c+ dendritic cells (DC) in C57BL/6 mice. J-chain knockout mice (J-/- mice) had a reduced fraction of CD4-/CD8alpha+ and mPDCA-1+ DC in the spleen. J-/- mice also had reduced levels of RNA for the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in the spleen. Furthermore, in lymph nodes from C57BL/6 mice the majority of J-chain-expressing CD11c+ cells also expressed IDO, while the number of IDO-expressing cells in lymph nodes and the amount of IDO protein in splenic CD11c+ cells were reduced in J-/- mice. Also, J-/- mice had a lower ratio of kynurenine/tryptophan in serum compared to C57BL/6 mice, indicating a lower overall IDO activity in J-/- mice. We also show that J-/- mice are less susceptible to tolerance induction than C57BL/6 mice. In conclusion, our data show that J-chain protein is expressed outside the B-cell compartment in a subset of immunoregulatory DC that are compromised in animals that cannot express J-chain.

Paquinimod prevents development of diabetes in the non-obese diabetic (NOD) mouse.

Quinoline-3-carboxamides (Q compounds) are immunomodulatory compounds that have shown efficacy both in autoimmune disease and cancer. We have in here investigated the impact of one such compound, paquinimod, on the development of diabetes in the NOD mouse model for type I diabetes (T1D). In cohorts of NOD mice treated with paquinimod between weeks 10 to 20 of age and followed up until 40 weeks of age, we observed dose-dependent reduction in incidence of disease as well as delayed onset of disease. Further, in contrast to untreated controls, the majority of NOD mice treated from 15 weeks of age did not develop diabetes at 30 weeks of age. Importantly, these mice displayed significantly less insulitis, which correlated with selectively reduced number of splenic macrophages and splenic Ly6Chi inflammatory monocytes at end point as compared to untreated controls. Collectively, these results demonstrate that paquinimod treatment can significantly inhibit progression of insulitis to T1D in the NOD mouse. We propose that the effect of paquinimod on disease progression may be related to the reduced number of these myeloid cell populations. Our finding also indicates that this compound could be a candidate for clinical development towards diabetes therapy in humans.

Autoinhibitory regulation of S100A8/S100A9 alarmin activity locally restricts sterile inflammation.

Autoimmune diseases, such as psoriasis and arthritis, show a patchy distribution of inflammation despite systemic dysregulation of adaptive immunity. Thus, additional tissue-derived signals, such as danger-associated molecular patterns (DAMPs), are indispensable for manifestation of local inflammation. S100A8/S100A9 complexes are the most abundant DAMPs in many autoimmune diseases. However, regulatory mechanisms locally restricting DAMP activities are barely understood. We now unravel for the first time, to our knowledge, a mechanism of autoinhibition in mice and humans restricting S100-DAMP activity to local sites of inflammation. Combining protease degradation, pull-down assays, mass spectrometry, and targeted mutations, we identified specific peptide sequences within the second calcium-binding EF-hands triggering TLR4/MD2-dependent inflammation. These binding sites are free when S100A8/S100A9 heterodimers are released at sites of inflammation. Subsequently, S100A8/S100A9 activities are locally restricted by calcium-induced (S100A8/S100A9)2 tetramer formation hiding the TLR4/MD2-binding site within the tetramer interphase, thus preventing undesirable systemic effects. Loss of this autoinhibitory mechanism in vivo results in TNF-α-driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9-/- mice with 2 independent TNF-α-transgene mouse strains. Since S100A8/S100A9 is the most abundant DAMP in many inflammatory diseases, specifically blocking the TLR4-binding site of active S100 dimers may represent a promising approach for local suppression of inflammatory diseases, avoiding systemic side effects.