SMAC mimetics promote NIK-dependent inhibition of CD4+ TH17 cell differentiation.

Second mitochondria-derived activator of caspase (SMAC) mimetics (SMs) are selective antagonists of the inhibitor of apoptosis proteins (IAPs), which activate noncanonical NF-κB signaling and promote tumor cell death. Through gene expression analysis, we found that treatment of CD4+ T cells with SMs during T helper 17 (TH17) cell differentiation disrupted the balance between two antagonistic transcription factor modules. Moreover, proteomics analysis revealed that SMs altered the abundance of proteins associated with cell cycle, mitochondrial activity, and the balance between canonical and noncanonical NF-κB signaling. Whereas SMs inhibited interleukin-17 (IL-17) production and ameliorated TH17 cell-driven inflammation, they stimulated IL-22 secretion. Mechanistically, SM-mediated activation of NF-κB-inducing kinase (NIK) and the transcription factors RelB and p52 directly suppressed Il17a expression and IL-17A protein production, as well as the expression of a number of other immune genes. Induction of IL-22 production correlated with the NIK-dependent reduction in cMAF protein abundance and the enhanced activity of the aryl hydrocarbon receptor. Last, SMs also increased IL-9 and IL-13 production and, under competing conditions, favored the differentiation of naïve CD4+ T cells into TH2 cells rather than TH17 cells. These results demonstrate that SMs shape the gene expression and protein profiles of TH17 cells and inhibit TH17 cell-driven autoimmunity.

Wnt5a is a TLR2/4-ligand that induces tolerance in human myeloid cells.

Innate immune responses are rapid, dynamic and highly regulated to avoid overt reactions. This regulation is executed by innate immune tolerance mechanisms that remain obscure. Wnt5a is a signalling protein mainly involved in developmental processes and cancer. The effect of Wnt5a on inflammatory myeloid cells is controversial. Here, we combine primary cell cultures, in vitro binding studies, mass spectrometry and Drosophila protein modelling to show that Wnt5a is a direct ligand of toll-like receptor (TLR) 2 and 4. The binding promotes a MyD88-non-canonical nuclear factor of kappa B (NFκB) and AP-1 signalling cascade, with contradictory profiles in mouse (pro-inflammatory) and human (anti-inflammatory) myeloid immune cells. These data reveal that the true nature of Wnt5a in inflammatory cells, is to regulate TLR signals, and in human myeloid cells it acts as an endogenous, tolerance-associated molecular pattern (TAMP), inducing IL-10 and innate immune tolerance.

The immunomodulatory quinoline-3-carboxamide paquinimod reverses established fibrosis in a novel mouse model for liver fibrosis.

Quinoline-3-carboxamides (Q substances) are small molecule compounds with anti-inflammatory properties. In this study, we used one of these substances, Paquinimod, to treat a novel model for chronic liver inflammation and liver fibrosis, the NOD-Inflammation Fibrosis (N-IF) mouse. We show that treatment of N-IF mice significantly reduced inflammation and resulted in the regression of fibrosis, even when the treatment was initiated after onset of disease. The reduced disease phenotype was associated with a systemic decrease in the number and reduced activation of disease-promoting transgenic natural killer T (NKT)-II cells and their type 2-cytokine expression profile. Paquinimod treatment also led to a reduction of CD115+ Ly6Chi monocytes and CD11b+ F4/80+ CD206+ macrophages.

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.

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.

A New Mouse Model That Spontaneously Develops Chronic Liver Inflammation and Fibrosis.

Here we characterize a new animal model that spontaneously develops chronic inflammation and fibrosis in multiple organs, the non-obese diabetic inflammation and fibrosis (N-IF) mouse. In the liver, the N-IF mouse displays inflammation and fibrosis particularly evident around portal tracts and central veins and accompanied with evidence of abnormal intrahepatic bile ducts. The extensive cellular infiltration consists mainly of macrophages, granulocytes, particularly eosinophils, and mast cells. This inflammatory syndrome is mediated by a transgenic population of natural killer T cells (NKT) induced in an immunodeficient NOD genetic background. The disease is transferrable to immunodeficient recipients, while polyclonal T cells from unaffected syngeneic donors can inhibit the disease phenotype. Because of the fibrotic component, early on-set, spontaneous nature and reproducibility, this novel mouse model provides a unique tool to gain further insight into the underlying mechanisms mediating transformation of chronic inflammation into fibrosis and to evaluate intervention protocols for treating conditions of fibrotic disorders.

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.

CD14 Is a Co-Receptor for TLR4 in the S100A9-Induced Pro-Inflammatory Response in Monocytes.

The cytosolic Ca2+-binding S100A9 and S100A8 proteins form heterodimers that are primarily expressed in human neutrophils and monocytes. We have recently shown that S100A9 binds to TLR4 in vitro and induces TLR4-dependent NF-κB activation and a pro-inflammatory cytokine response in monocytes. In the present report we have further investigated the S100A9-mediated stimulation of TLR4 in monocytes. Using transmission immunoelectron microscopy, we detected focal binding of S100A9 to monocyte membrane subdomains containing the caveolin-1 protein and TLR4. Furthermore, the S100A9 protein was detected in early endosomes of the stimulated cells, indicating that the protein could be internalized by endocytosis. Although stimulation of monocytes with S100A9 was strictly TLR4-dependent, binding of S100A9 to the plasma membrane and endocytosis of S100A9 was still detectable and coincided with CD14 expression in TLR4-deficient cells. We therefore investigated whether CD14 would be involved in the TLR4-dependent stimulation and could show that the S100A9-induced cytokine response was inhibited both in CD14-deficient cells and in cells exposed to CD14 blocking antibodies. Further, S100A9 was not internalized into CD14-deficient cells suggesting a direct role of CD14 in endocytosis of S100A9. Finally, we could detect satiable binding of S100A9 to CD14 in surface plasmon resonance experiments. Taken together, these results indicate that CD14 is a co-receptor of TLR4 in the S100A9-induced cytokine response.

Platelet-Derived S100A8/A9 and Cardiovascular Disease in Systemic Lupus Erythematosus.

OBJECTIVE: Levels of S100A8/A9, a proinflammatory and prothrombotic protein complex, are increased in several diseases, and high levels predispose to cardiovascular disease (CVD). Recently, platelet S100A8/A9 synthesis was described in mice and humans in relation to CVD. The aim of this study was to investigate the role of platelet S100A8/A9 in systemic lupus erythematosus (SLE), a disease with markedly increased cardiovascular morbidity, as well as the exact platelet distribution of the S100A8/A9 proteins. METHODS: The occurrence and distribution of platelet S100A8/A9 protein were detected by enzyme-linked immunosorbent assay, electron microscopy, Western blotting, and flow cytometry in healthy controls (n = 79) and in 2 individual cohorts of SLE patients (n = 148 and n = 318, respectively) and related to cardiovascular morbidity. RESULTS: We observed that human platelets expressed S100A8/A9 proteins, and that these were localized in close proximity to intracellular membranes and granules as well as on the cell surface upon activation with physiologic and pathophysiologic stimuli. Interestingly, S100A8/A9 was enriched at sites of membrane interactions, indicating a role of S100A8/A9 in cell-cell communication. S100A8/A9 levels were highly regulated by interferon-α, both in vivo and in vitro. Patients with SLE had increased platelet S100A8/A9 content compared with healthy individuals. Increased levels of platelet S100A8/A9 were associated with CVD, particularly myocardial infarction (odds ratio 4.8, 95% confidence interval 1.5-14.9, P = 0.032 [adjusted for age, sex, and smoking]). CONCLUSION: Platelets contain S100A8/A9 in membrane-enclosed vesicles, enabling rapid cell surface deposition upon activation. Furthermore, platelet S100A8/A9 protein levels were increased in SLE patients, particularly in those with CVD, and may be a future therapeutic target.

Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells.

We show here, by using surface biotinylation, followed by Western blotting or surface plasmon resonance analysis, that very low levels of S100A8 and/or S100A9 can be detected on the surface of THP-1 cells or freshly isolated human monocytes. This was supported by immune-electron microscopy where we observed membrane-associated expression of the proteins restricted to small patches. By using confocal microscopy we could determine that S100A8 and S100A9 protein in THP-1 cells or freshly isolated human monocytes was mostly present in vesicular structures. This finding was confirmed using immune-electron microscopy. Subcellular fractionation and confocal microscopy showed that these vesicular structures are mainly early endosomes and endolysosomes. Our subsequent studies showed that accumulation of S100A8 and S100A9 in the endolysosomal compartment is associated with induction of their release from the cells. Furthermore, an inhibitor of lysosomal activity could modulate the release of S100A8 and S100A9 in the extracellular milieu. Our current results suggest that the S100A8 and S100A9 proteins are primarily associated with certain kinds of cytosolic vesicles and may be secreted via an endolysosomal pathway.

S100A9 as a Pharmacological Target Molecule in Inflammation and Cancer.

Upon tissue injury and infection both stressed and dying cells can release proteins that normally reside inside the cells. Some of the released proteins become ligands of various cell surface receptors expressed by local cells and such proteins are denoted as damage associated molecular patterns (DAMPs). Binding of some DAMPs to certain cell surface receptors induces signals emanating in the production of pro-inflammatory cytokines, ultimately leading to an inflammatory response. Our laboratory is interested in the S100A9 protein, a bona fide DAMP protein. This protein normally resides inside monocytes and neutrophils and in these cells it forms heterodimers with the S100A8 protein. The S100A8/A9 heterodimer is released in large amounts during several types of inflammatory disease and is currently used clinically as a biomarker in some diseases. The fact that several different proinflammatory functions have been ascribed to this protein makes it a potential target for the development of small molecule inhibitors. We have developed several such inhibitors, some of which are already in phase III clinical development. This review describes our efforts to investigate the biological functions of the S100A9 protein as well as our ongoing efforts of developing second-generation, more specific, small molecule inhibitors of its pro-inflammatory functions.

The quinoline-3-carboxamide paquinimod (ABR-215757) reduces leukocyte recruitment during sterile inflammation: leukocyte- and context-specific effects.

Quinoline-3-carboxamides (Q-compounds) are currently in clinical development for both autoimmune disease and cancer. We have previously shown that the Q-compound paquinimod (ABR-215757) significantly ameliorates disease symptoms in several mouse models of human inflammatory disease. Considering that recruitment of inflammatory cells into tissue is a common denominator of these models, we have in this report investigated whether paquinimod would interfere with cell accumulation during sterile peritoneal inflammation. To mimic the cell recruitment elicited by tissue injury, we used necrotic cells to induce the acute inflammatory response. We show that per oral treatment with paquinimod significantly reduced the accumulation of Ly6C(hi) inflammatory monocytes and eosinophils, but not neutrophils, in this model, and that this correlated with reduced number of such cells also in the omentum. Treatment also reduced the accumulation of these cell populations at a subcutaneous site of inflammation. In alum-induced inflammation, however, neutrophils were the dominant cell population and paquinimod failed to reduce the accumulation of inflammatory cells. Taken together, our results indicate that paquinimod selectively inhibits cell recruitment during acute sterile inflammation, but that this effect is context-dependent. These data have important implications for the understanding of the mechanism of action of Q-compounds in both pre-clinical and clinical settings.

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.

Common interactions between S100A4 and S100A9 defined by a novel chemical probe.

S100A4 and S100A9 proteins have been described as playing roles in the control of tumor growth and metastasis. We show here that a chemical probe, oxyclozanide (OX), selected for inhibiting the interaction between S100A9 and the receptor for advanced glycation end-products (RAGE) interacts with both S100A9 and S100A4. Furthermore, we show that S100A9 and S100A4 interact with RAGE and TLR4; interactions that can be inhibited by OX. Hence, S100A4 and S100A9 display similar functional elements despite their primary sequence diversity. This was further confirmed by showing that S100A4 and S100A9 dimerize both in vitro and in vivo. All of these interactions required levels of Zn++ that are found in the extracellular space but not intracellularly. Interestingly, S100A4 and S100A9 are expressed by distinct CD11b+ subpopulations both in healthy animals and in animals with either inflammatory disease or tumor burden. The functions of S100A9 and S100A4 described in this paper, including heterodimerization, may therefore reflect S100A9 and S100A4 that are released into the extra-cellular milieu.

Human S100A9 protein is stabilized by inflammatory stimuli via the formation of proteolytically-resistant homodimers.

S100A8 and S100A9 are Ca(2+)-binding proteins that are associated with acute and chronic inflammation and cancer. They form predominantly heterodimers even if there are data supporting homodimer formation. We investigated the stability of the heterodimer in myeloid and S100A8/S100A9 over-expressing COS cells. In both cases, S100A8 and S100A9 proteins were not completely degraded even 48 hrs after blocking protein synthesis. In contrast, in single transfected cells, S100A8 protein was completely degraded after 24 h, while S100A9 was completely unstable. However, S100A9 protein expression was rescued upon S100A8 co-expression or inhibition of proteasomal activity. Furthermore, S100A9, but not S100A8, could be stabilized by LPS, IL-1ß and TNFα treatment. Interestingly, stimulation of S100A9-transfected COS cells with proteasomal inhibitor or IL-1ß lead to the formation of protease resistant S100A9 homodimers. In summary, our data indicated that S100A9 protein is extremely unstable but can be rescued upon co-expression with S100A8 protein or inflammatory stimuli, via proteolytically resistant homodimer formation. The formation of S100A9 homodimers by this mechanism may constitute an amplification step during an inflammatory reaction.

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.

S100A9 and tumor growth.

We have investigated the role of the Ca(2+)-binding protein S100A9 on tumor growth in prostate cancer and T-cell lymphoma models. We found that the expression of, S100A9 and its interaction with Toll-like receptor 4 (TLR4) is critical for tumor growth in these settings.

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.

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.