Toni Weinhage

Proinflammatory S100A12 Can Activate Human Monocytes via Toll-like Receptor 4

RATIONALE S100A12 is overexpressed during inflammation and is a marker of inflammatory disease. Furthermore, it has been ascribed to the group of damage-associated molecular pattern molecules that promote inflammation. However, the exact role of human S100A12 during early steps of immune activation and sepsis is only partially described thus far. OBJECTIVES We analyzed the activation of human monocytes by granulocyte-derived S100A12 as a key function of early inflammatory processes and the development of sepsis. METHODS Circulating S100A12 was determined in patients with sepsis and in healthy subjects with experimental endotoxemia. The release of human S100A12 from granulocytes as well as the promotion of inflammation by activation of human monocytes after specific receptor interaction was investigated by a series of in vitro experiments. MEASUREMENTS AND MAIN RESULTS S100A12 rises during sepsis, and its expression and release from granulocytes is rapidly induced in vitro and in vivo by inflammatory challenge. A global gene expression analysis of S100A12-activated monocytes revealed that human S100A12 induces inflammatory gene expression. These effects are triggered by an interaction of S100A12 with Toll-like receptor 4 (TLR4). Blocking S100A12 binding to TLR4 on monocytes or TLR4 expressing cell lines (HEK-TCM) abrogates the respective inflammatory signal. On the contrary, blocking S100A12 binding to its second proposed receptor (receptor for advanced glycation end products [RAGE]) has no significant effect on inflammatory signaling in monocytes and RAGE-expressing HEK293 cells. CONCLUSIONS Human S100A12 is an endogenous TLR4 ligand that induces monocyte activation, thereby acting as an amplifier of innate immunity during early inflammation and the development of sepsis.

The functional-374T/A polymorphism of the receptor for advanced glycation end products may modulate Crohn's disease

The receptor for advanced glycation end products (RAGE) is involved in innate immune mechanisms. Polymorphisms of the RAGE gene have been described as a factor amplifying inflammation in susceptible patients, but the association with Crohns disease (CD) is not known. The coding RAGE polymorphism G82S (rs2070600) and two promoter polymorphisms, -374T/A (rs1800624) and -429T/C (rs1800625), were studied in two samples from Germany and the United States consisting of 421 and 317 CD patients and 549 and 218 controls, respectively. To test the functional relevance, additional data on serum soluble RAGE (sRAGE), tissue RNA, and protein levels were collected and immunohistochemical stainings of bowel tissue of CD patients and healthy controls as well as models of experimental (dextran sodium sulfate-induced) colitis in RAGE knockout and wild-type mice were performed. The -374T/A RAGE promotor single nucleotide polymorphism (SNP) was negatively associated with CD (odds ratio = 0.708, 95% confidence interval = 0.535-0.938, P = 0.016) and with stenosis (OR = 0.627, P = 0.04) in the German sample. Transmission disequilibrium testing confirmed an undertransmission of the -374A allele. Serum sRAGE levels were higher in patients in complete remission of the -374AA/TA group (1,975 ± 299 pg/ml; -374TT group: 1,310 ± 153 pg/ml SE, P < 0.05) and showed a trend toward decreased levels in CD patients with active disease compared with CD patients in remission. Further in vitro and in vivo studies indicated that an increase of sRAGE ameliorates inflammation. The -429T/C and the G82S polymorphism were not associated with CD. The -374T/A RAGE polymorphism leading to facilitated RAGE gene transcription may to some degree protect from developing a stricturing subphenotype of CD, most likely by increasing levels of sRAGE, which neutralizes proinflammatory mediators.

Myeloid-Related Proteins 8 and 14 Contribute to Monosodium Urate Monohydrate Crystal-Induced Inflammation in Gout

Monosodium urate monohydrate (MSU) crystal–induced interleukin‐1β (IL‐1β) secretion is a critical factor in the pathogenesis of gout. However, without costimulation by a proIL‐1β–inducing factor, MSU crystals alone are insufficient to induce IL‐1β secretion. The responsible costimulatory factors that act as a priming endogenous signal in vivo are not yet known. We undertook this study to analyze the costimulatory properties of myeloid‐related protein 8 (MRP‐8) and MRP‐14 (endogenous Toll‐like receptor 4 [TLR‐4] agonists) in MSU crystal–induced IL‐1β secretion and their relevance in gout.

Reprogramming of Monocytes by GM-CSF Contributes to Regulatory Immune Functions during Intestinal Inflammation

Human and murine studies showed that GM-CSF exerts beneficial effects in intestinal inflammation. To explore whether GM-CSF mediates its effects via monocytes, we analyzed effects of GM-CSF on monocytes in vitro and assessed the immunomodulatory potential of GM-CSF–activated monocytes (GMaMs) in vivo. We used microarray technology and functional assays to characterize GMaMs in vitro and used a mouse model of colitis to study GMaM functions in vivo. GM-CSF activates monocytes to increase adherence, migration, chemotaxis, and oxidative burst in vitro, and primes monocyte response to secondary microbial stimuli. In addition, GMaMs accelerate epithelial healing in vitro. Most important, in a mouse model of experimental T cell–induced colitis, GMaMs show therapeutic activity and protect mice from colitis. This is accompanied by increased production of IL-4, IL-10, and IL-13, and decreased production of IFN-γ in lamina propria mononuclear cells in vivo. Confirming this finding, GMaMs attract T cells and shape their differentiation toward Th2 by upregulating IL-4, IL-10, and IL-13 in T cells in vitro. Beneficial effects of GM-CSF in Crohn’s disease may possibly be mediated through reprogramming of monocytes to simultaneously improved bacterial clearance and induction of wound healing, as well as regulation of adaptive immunity to limit excessive inflammation.

Immune Suppression via Glucocorticoid-Stimulated Monocytes: A Novel Mechanism To Cope with Inflammation

Glucocorticoids (GCs) are used as first-line therapies for generalized suppression of inflammation (e.g., allergies or autoimmune diseases), but their long-term use is limited by severe side effects. Our previous work revealed that GCs induced a stable anti-inflammatory phenotype in monocytes, the GC-stimulated monocytes (GCsMs) that we exploited for targeted GC-mediated therapeutic effects. We demonstrate that GCsMs interact with T cells in suppressing proliferation, as well as cytokine release of CD8+ and, especially, CD4+ T cells in vitro, and that they support generation of Foxp3+ cells. Therefore, we tested their immunosuppressive potential in CD4+ T cell–induced colitis in vivo. We found that injection of GCsMs into mice with severe colitis abolished the inflammation and resulted in significant clinical improvement within a few days. T cells recovered from GCsM-treated mice exhibited reduced secretion of proinflammatory cytokines IFN-γ and IL-17. Furthermore, clusters of Foxp3+ CD4+ T cells were detectable at local sites of inflammation in the colon. Thus, GCsMs are able to modify T cell responses in vitro and in vivo, as well as to downregulate and clinically cure severe T cell–mediated colitis.

Peripheral monocyte functions and activation in patients with quiescent Crohn's disease.

Recent developments suggest a causal link between inflammation and impaired bacterial clearance in Crohn’s disease (CD) due to alterations of intestinal macrophages. Studies suggest that excessive inflammation is the consequence of an underlying immunodeficiency rather than the primary cause of CD pathogenesis. We characterized phenotypic and functional features of peripheral blood monocytes of patients with quiescent CD (n = 18) and healthy controls (n = 19) by analyses of cell surface molecule expression, cell adherence, migration, chemotaxis, phagocytosis, oxidative burst, and cytokine expression and secretion with or without lipopolysaccharide (LPS) priming. Peripheral blood monocytes of patients with inactive CD showed normal expression of cell surface molecules (CD14, CD16, CD116), adherence to plastic surfaces, spontaneous migration, chemotaxis towards LTB4, phagocytosis of E. coli, and production of reactive oxygen species. Interestingly, peripheral blood monocytes of CD patients secreted higher levels of IL1β (p<.05). Upon LPS priming we found a decreased release of IL10 (p<.05) and higher levels of CCL2 (p<.001) and CCL5 (p<.05). The expression and release of TNFα, IFNγ, IL4, IL6, IL8, IL13, IL17, CXCL9, and CXCL10 were not altered compared to healthy controls. Based on our phenotypic and functional studies, peripheral blood monocytes from CD patients in clinical remission were not impaired compared to healthy controls. Our results highlight that defective innate immune mechanisms in CD seems to play a role in the (inflamed) intestinal mucosa rather than in peripheral blood.

Pro-inflammatory cytokine environments can drive IL-17 over-expression by γδT cells in systemic juvenile idiopathic arthritis

Systemic‐onset juvenile idiopathic arthritis (JIA) is speculated to follow a biphasic course, with an initial systemic disease phase driven by innate immune mechanisms and interleukin‐1β (IL‐1β) as a key cytokine and a second chronic arthritic phase that may be dominated by adaptive immunity and cytokines such as IL‐17A. Although a recent mouse model points to a critical role of IL‐17–expressing γ/δ T cells in disease pathology, in humans, both the prevalence of IL‐17 and the role of IL‐17–producing cells are still unclear.

Correlation of Secretory Activity of Neutrophils With Genotype in Patients With Familial Mediterranean Fever

Familial Mediterranean fever (FMF) is an autoinflammatory disorder caused by pyrin‐encoding MEFV mutations. Patients present with recurrent but self‐limiting episodes of acute inflammation and often have persistent subclinical inflammation. The pathophysiology is only partially understood, but neutrophil overactivation is a hallmark of the disease. S100A12 is a neutrophil‐derived proinflammatory danger signal that is strongly elevated in active FMF. This study was undertaken to characterize the secretory activity of neutrophils in vitro and investigate the association of S100A12 with disease activity and genotype in patients with FMF.

Secretory Activity of Neutrophils Correlates With Genotype in Familial Mediterranean Fever

Familial Mediterranean fever (FMF) is an autoinflammatory disorder caused by pyrin‐encoding MEFV mutations. Patients present with recurrent but self‐limiting episodes of acute inflammation and often have persistent subclinical inflammation. The pathophysiology is only partially understood, but neutrophil overactivation is a hallmark of the disease. S100A12 is a neutrophil‐derived proinflammatory danger signal that is strongly elevated in active FMF. This study was undertaken to characterize the secretory activity of neutrophils in vitro and investigate the association of S100A12 with disease activity and genotype in patients with FMF.

Granulocyte Macrophage Colony-Stimulating Factor–Activated CD39+/CD73+ Murine Monocytes Modulate Intestinal Inflammation via Induction of Regulatory T Cells

Background & Aims Granulocyte macrophage colony-stimulating factor (GM-CSF) treatment induces clinical response in patients with active Crohn’s disease. To explore whether monocytes mediate GM-CSF effects in vivo, we used a mouse model of chronic colitis induced by dextran sulfate sodium (DSS). Methods Murine bone marrow-derived monocytes were activated with GM-CSF in vitro, and gene expression, phenotype, and function of GM-CSF-activated monocytes (GMaM) were analyzed. Therapeutic effects of GMaM were assessed in a model of chronic colitis induced by repeated cycles of DSS. Monocytes were administered intravenously and their immunomodulatory functions were evaluated in vivo by clinical monitoring, histology, endoscopy, immunohistochemistry, and expression of inflammatory markers in the colon. The distribution of injected monocytes in the intestine was measured by in vivo imaging. Results GMaM expressed significantly higher levels of anti-inflammatory molecules. Production of reactive oxygen species was also increased while phagocytosis and adherence were decreased. GMaM up-regulated CD39 and CD73, which allows the conversion of adenosine triphosphate into adenosine and coincided with the induction of Foxp3+ (forkhead-box-protein P3 positive) regulatory T cells (Treg) in cocultures of GMaM and naive T cells. In chronic DSS-induced colitis, adoptive transfer of GMaM led to significant clinical improvement, as demonstrated by reduced weight loss, inflammatory infiltration, ulceration, and colon shrinkage. As GMaM migrated faster and persisted longer in the inflamed intestine compared with control monocytes, their presence induced Treg generation in vivo. Conclusions GM-CSF leads to specific monocyte activation that modulates experimental colitis via mechanisms that include the induction of Treg. We demonstrate a possible mechanism of Treg induction through CD39 and CD73 expression on monocytes.