During malaria infection, high levels of proinflammatory molecules (e.g., cytokines, chemokines) correlate with disease severity. Even if their role as activators of the host immune response has been ...studied, the direct contribution of hemozoin (HZ), a parasite metabolite, to such a strong induction is not fully understood. Previous in vitro studies demonstrated that both Plasmodium falciparum HZ and synthetic HZ (sHZ), beta-hematin, induce macrophage/monocyte chemokine and proinflammatory cytokine secretion. In the present study, we investigated the proinflammatory properties of sHZ in vivo. To this end, increasing doses of sHZ were injected either i.v. or into an air pouch generated on the dorsum of BALB/c mice over a 24-h period. Our results showed that sHZ is a strong modulator of leukocyte recruitment and more specifically of neutrophil and monocyte populations. In addition, evaluation of chemokine and cytokine mRNA and protein expression revealed that sHZ induces the expression of chemokines, macrophage-inflammatory protein (MIP)-1alpha/CCL3, MIP-1beta/CCL4, MIP-2/CXCL2, and monocyte chemoattractant protein-1/CCL2; chemokine receptors, CCR1, CCR2, CCR5, CXCR2, and CXCR4; cytokines, IL-1beta and IL-6; and myeloid-related proteins, S100A8, S100A9, and S100A8/A9, in the air pouch exudates. Of interest, chemokine and cytokine mRNA up-regulation were also detected in the liver of i.v. sHZ-injected mice. In conclusion, our study demonstrates that sHZ is a potent proinflammatory agent in vivo, which could contribute to the immunopathology related to malaria.
Following tissue injury after trauma, the activation of innate immune pathways results in systemic inflammation, organ failure and an increased risk of infections. The objective of this study was to ...characterize the kinetics of the S100A8/S100A9 complex, a new-recognized alarmin, as well as its soluble receptor sRAGE, over time after trauma as potential early biomarkers of the risk of organ damage.
We collected comprehensive data from consenting patients admitted to an ICU following severe trauma. The blood samples were taken at Day 0 (admission), Day1, 3 and 5 S100A8/A9 and sRAGE were measured by ELISA. Biomarkers levels were reported as median (IQR).
Thirty-eight patients sustaining in majority a blunt trauma (89%) with a median ISS of 39 were included. In this cohort, the S100A8/A9 complex increased significantly over time (p = 0.001), but its levels increment over time (D0 to D5) was significantly smaller in patients developing infection (7.6 vs 40.1 mcg/mL, p = 0.011). The circulating level of sRAGE circulating levels decreased over time (p < 0.0001) and was higher in patients who remained in shock on day 3 (550 vs 918 pg/mL; p = 0.02) or 5 (498 vs 644 pg/mL; p = 0.045). Admission sRAGE levels were significantly higher in non-survivors (1694 vs 745 pg/mL; p = 0.015) and was higher in patients developing renal failure (1143 vs 696 pg/mL, p = 0.011).
Our findings reveal an interesting association between the biomarker S100A8/9 least increase over time and the presence of infectious complication after trauma. We describe that the sRAGE decline over time is in relation with shock and markers of ischemic injury. We also confirm the association of sRAGE levels measured at admission with mortality and the development of renal failure.
This work illustrates the importance of following the circulating level of biomarker overtime. The utilization of S1008/9 as a tool to stratify infection risk and trigger early interventions need to be validated prospectively.
Summary Calgranulins are a family of powerful chemoattractants, which have been implicated as biomarkers in inflammatory diseases. To determine how different respiratory diseases affect the ...expression of calgranulins, we measured the expression of S100A8/A9 and S100A12 in bronchoalveolar lavage fluid (BALF) of acute respiratory distress syndrome (ARDS) patients and healthy volunteers by ELISA. Analysis of calgranulin expression revealed a high level of S100A12 in the lavages of patients suffering from ARDS compared to controls ( p <0.001). Based on the hypothesis that the increased expression of S100A12 relative to the S100A8/A9 heterodimer was a characteristic of respiratory diseases with neutrophilic inflammation, we measured calgranulin expression in BALF of cystic fibrosis (CF) patients. Despite similarly elevated levels of S100A8/A9, S100A12 was significantly higher in ARDS compared to CF BALF ( p <0.001). The differential expression of calgranulins was unique for inflammatory markers, as an array of cytokines did not differ between CF and ARDS patients. Since ARDS is an acute event and CF a chronic inflammation with acute exacerbations, we compared calgranulin expression in sputum obtained from CF and patients with chronic obstructive lung disease (COPD). Levels of S100A12 and S100A8/9 were elevated in CF sputum compared to COPD sputum, but the ratio of S100A12 to S100A8/A9 was similar in COPD and CF and reflected more closely than seen in healthy controls. The results indicate that the regulation of human calgranulin expression and the ratio of S100A8/A9 to S100A12 may provide important insights in the mechanism of respiratory inflammation.
The neutrophil cytoplasmic protein S100A8/A9 (along with S100A8 and S100A9) is chemotactic and stimulates neutrophil adhesion by activating the β2‐integrin CD11b/CD18. It is also essential to ...neutrophil migration in vivo in response to monosodium urate monohydrate (MSUM) crystals, the principal etiologic agent of gout. S100A8/A9 is present in the synovial fluid of patients with gout and arthritis and is secreted by activated monocytes; however, its mechanism of release by neutrophils remains unknown. The aim of this study was to identify the mechanism of stimulation of the release of S100A8/A9 by MSUM‐activated neutrophils. Here, we show that S100A8/A9 is released by neutrophils stimulated with MSUM crystals and that this release could be enhanced by preincubating neutrophils with granulocyte macrophage‐colony stimulating factor. Antibodies directed against CD11b and CD16 blocked the release induced by MSUM crystals, suggesting that Fc receptor for immunoglobulin G (FcγR)IIIB (CD16) and CD11b/CD18 were involved in the stimulation by MSUM crystals. Neutrophil preincubation with the Src kinase inhibitor 4‐amino‐5‐(4‐chlorophenyl)‐7‐(t‐butyl) pyrazolo3,4‐dpyrimidine and the Syk tyrosine kinase inhibitor trans‐3,3′,4,5′‐tetrahydrozystilbene significantly reduced the release of S100A8/A9, suggesting that the Src tyrosine kinase family and Syk were involved. In addition, wortmannin reduced neutrophil release of S100A8/A9, indicating a potential involvement of phosphatidylinolitol‐3 kinase in this release. Preincubation of neutrophils with the tubulin depolymerization promoters nocodazole and vincristine reduced MSUM‐induced release, suggesting a tubulin‐associated pathway of release. These results indicate that S100A8/A9 is released by MSUM crystal‐stimulated neutrophils following activation of CD11b, CD16, Src kinases, Syk, and tubulin polymerization.
Th17 cells play a critical role in the pathogenesis of rheumatoid arthritis (RA), but the mechanisms by which these cells regulate the development of RA are not fully understood. We have recently ...shown that α2β1 integrin, the receptor of type I collagen, is the major collagen-binding integrin expressed by human Th17 cells. In this study, we examined the role of α2β1 integrin in Th17-mediated destructive arthritis in the murine model of collagen-induced arthritis (CIA). We found that α2β1 integrin is expressed on synovial Th17 cells from CIA mice and its neutralization with a specific mAb significantly reduced inflammation and cartilage degradation, and protected the mice from bone erosion. Blockade of α2β1 integrin led to a decrease in the number of Th17 cells in the joints and to a reduction of IL-17 levels in CIA mice. This was associated with an inhibition of receptor activator of NF-κB ligand levels and osteoclast numbers, and reduction of bone loss. We further show that α2β1 integrin is expressed on synovial Th17 cells from RA patients, and that its ligation with collagen costimulated the production of IL-17 by polarized human Th17 cells by enhancing the expression of retinoic acid receptor-related orphan receptor C through ERK and PI3K/AKT. Our findings provide the first evidence, to our knowledge, that α2β1 integrin is an important pathway in Th17 cell activation in the pathogenesis of CIA, suggesting that its blockade can be beneficial for the treatment of RA and other Th17-associated autoimmune diseases.
S100A8 and S100A9 are intracellular calcium-binding proteins produced by myeloid cells that promote neutrophil/monocyte recruitment at inflamed tissues by enhancing attachment to endothelial cells. ...Although the intracellular functions of these proteins, i.e., myeloid-related proteins (MRP)-8 and MRP-14, are not completely understood, these proteins exhibit prominent extracellular cytokine-like functions and are considered reliable markers of inflammation in diverse diseases. As S100A8 and S100A9 have been reported to be rapidly released in response to components derived from infectious agents, we hypothesized that they play an important role in the modulation of key microbicidal phagocyte functions. In this study, we report for the first time that MRPs are powerful inducers of NO production by murine macrophages (Mphi). This increase in NO production was linked to an increased inducible NO synthase expression both at gene and protein level. This induction was concomitant with an important phosphorylation of SAPK/JNK, but also of MEK and ERK kinases. Upon stimulation with MRPs, NF-kappaB was rapidly translocated to the nucleus (30 min). When Mphi were treated concomitantly with IFN-gamma, another activator of Mphi functions, we observed a strong synergy in NO production, synergy that resulted from the engagement of exclusive signaling pathways: SAPK/JNK, ERK and NF-kappaB were involved in signaling of MRPs, whereas IFN-gamma uses the JAK/STAT pathway. This suggests that the synergy results from interactions of transcription factors in the promoter region. Finally, we observed this effect to be dependent on TLR4. Collectively, our study unravels the importance of MRPs as potent new inducers of Mphi NO production.
Th17 cells play a critical role in the pathogenesis of rheumatoid arthritis (RA), but the mechanisms by which these cells regulate the development of RA are not fully understood. We have recently ...shown that alpha 2 beta 1 integrin, the receptor of type I collagen, is the major collagen-binding integrin expressed by human Th17 cells. In this study, we examined the role of alpha 2 beta 1 integrin in Th17-mediated destructive arthritis in the murine model of collagen-induced arthritis (CIA). We found that alpha 2 beta 1 integrin is expressed on synovial Th17 cells from CIA mice and its neutralization with a specific mAb significantly reduced inflammation and cartilage degradation, and protected the mice from bone erosion. Blockade of alpha 2 beta 1 integrin led to a decrease in the number of Th17 cells in the joints and to a reduction of IL-17 levels in CIA mice. This was associated with an inhibition of receptor activator of NF- Kappa B ligand levels and osteoclast numbers, and reduction of bone loss. We further show that alpha 2 beta 1 integrin is expressed on synovial Th17 cells from RA patients, and that its ligation with collagen costimulated the production of IL-17 by polarized human Th17 cells by enhancing the expression of retinoic acid receptor-related orphan receptor C through ERK and PI3K/AKT. Our findings provide the first evidence, to our knowledge, that alpha 2 beta 1 integrin is an important pathway in Th17 cell activation in the pathogenesis of CIA, suggesting that its blockade can be beneficial for the treatment of RA and other Th17-associated autoimmune diseases.
Extracellular vesicles (EVs) and their contents (proteins, lipids, messenger RNA, microRNA, and DNA) are viewed as intercellular signals, cell-transforming agents, and shelters for viruses that allow ...both diagnostic and therapeutic interventions. EVs circulating in the blood of individuals infected with human immunodeficiency virus (HIV-1) may provide insights into pathogenesis, inflammation, and disease progression. However, distinguishing plasma membrane EVs from exosomes, exomeres, apoptotic bodies, virions, and contaminating proteins remains challenging. We aimed at comparing sucrose and iodixanol density and velocity gradients along with commercial kits as a means of separating EVs from HIV particles and contaminating protein like calprotectin; and thereby evaluating the suitability of current plasma EVs analysis techniques for identifying new biomarkers of HIV-1 immune activation. Multiple analysis have been performed on HIV-1 infected cell lines, plasma from HIV-1 patients, or plasma from HIV-negative individuals spiked with HIV-1. Commercial kits, the differential centrifugation and density or velocity gradients to precipitate and separate HIV, EVs, and proteins such as calprotectin, have been used. EVs, virions, and contaminating proteins were characterized using Western blot, ELISA, RT-PCR, hydrodynamic size measurement, and enzymatic assay. Conversely to iodixanol density or velocity gradient, protein and virions co-sedimented in the same fractions of the sucrose density gradient than AChE-positive EVs. Iodixanol velocity gradient provided the optimal separation of EVs from viruses and free proteins in culture supernatants and plasma samples from a person living with HIV (PLWH) or a control and revealed a new population of large EVs enriched in microRNA miR-155 and mitochondrial DNA. Although EVs and their contents provide helpful information about several key events in HIV-1 pathogenesis, their purification and extensive characterization by velocity gradient must be investigated thoroughly before further use as biomarkers. By revealing a new population of EVs enriched in miR-155 and mitochondrial DNA, this study paves a way to increase our understanding of HIV-1 pathogenesis.
S100A8 and S100A9 are cytoplasmic proteins expressed by phagocytes. High concentrations of these proteins have been correlated with various inflammatory conditions, including autoimmune diseases such ...as rheumatoid arthritis and Crohn’s disease, as well as autoinflammatory diseases. In the present study, we examined the effects of S100A8 and S100A9 on the secretion of cytokines and chemokines from PBMCs. S100A8 and S100A9 induced the secretion of cytokines such as IL-6, IL-8, and IL-1β. This secretion was associated with the activation and translocation of the transcription factor NF-κB. Inhibition studies using antisense RNA and the pharmacological agent BAY-117082 confirmed the involvement of NF-κB in IL-6, IL-8, and IL-1β secretion. S100A8- and S100A9-mediated activation of NF-κB, the NLR family, pyrin domain-containing 3 (NLRP3) protein, and pro-IL-1β expression was dependent on the generation of reactive oxygen species. This effect was synergistically enhanced by ATP, a known inflammasome activator. These results suggest that S100A8 and S100A9 enhance the inflammatory response by inducing cytokine secretion of PBMCs.
Background
Biological agents have allowed remarkable improvement in controlling autoimmune arthropathies, although none of the numerous biologics readily available represent a universal treatment ...standard. Moreover, classical and genetic predictors are currently unsatisfactory to predict individual response to a biologic, and the best treatment selection is still based on a trial-and-error approach. Here, we report a clinical case demonstrating the usefulness of examining the leukocytes’ secretome of patients. We set up and standardized a protocol that examines a patient’s immune responses to establish the secretome of the blood mononuclear leukocytes and personalize the biotherapy.
Case presentation
A 24-year-old woman was diagnosed with active early rheumatoid arthritis. The initial treatment regimen (prednisone, methotrexate, hydroxychloroquine, naproxen) was inefficient, as well as the anti-TNF adalimumab. The diagnosis was revised as possible rheumatoid arthritis-like psoriatic arthritis and adalimumab was replaced by abatacept (IgG1 Fc-CTLA-4) to no avail. Five years later, abatacept was replaced by the anti-IL-12/IL-23 ustekinumab with no objective control over the symptoms. The patient was thus enrolled in a prospective study based on the quantification of cytokines secreted by peripheral blood leukocytes stimulated with well-known immune activators of pattern recognition receptors and cytokine signalling. The results of this study revealed that plasma concentrations of cytokines were similar between the patient and healthy donors. In comparison to leukocytes from healthy donors, the patient’s secretome showed a unique overproduction of IL-6. The anti-IL-6 receptor tocilizumab was, therefore, administered with a rapid improvement of her active psoriatic arthritis that remained dependent on low prednisone dosage. Clinical parameters progressively returned to normal levels and her quality of life was greatly improved, despite the major delay to begin the present personalized treatment.
Conclusions
An efficient way to effectively treat patients with complex autoimmune arthropathies, and avoid irreversible disability, is to know their leukocytes’ secretome to identify abnormally secreted cytokines and personalize their biotherapy, as exemplified by this case report.
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