Specific targeting of the IL-23 receptor, using a novel small peptide noncompetitive antagonist, decreases the inflammatory response.

IL-23 is part of the IL-12 family of cytokines and is composed of the p19 subunit specific to IL-23 and the p40 subunit shared with IL-12. IL-23 specifically contributes to the inflammatory process of multiple chronic inflammatory autoimmune disorders, including psoriasis, multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis. So far, one antibody targeting the shared p40 subunit of IL-12 and IL-23, Ustekinumab, is approved clinically to treat psoriasis. However, there are no treatments inhibiting specifically the IL-23 proinflammatory response. We have developed small IL-23R-specific antagonists by designing all D-peptides arising from flexible regions of IL-23R. Of these peptides, we selected 2305 (teeeqqly), since in addition to its soluble properties, it inhibited IL-23-induced STAT3 phosphorylation in spleen cells. Peptide 2305 specifically binds to IL-23R/IL-12Rß1-expressing HEK-293 cells and not to cells devoid of the receptor. Peptide 2305 showed functional selectivity by modulating IL-23-induced gene expression in IL-23R/IL-12Rß1-expressing cells and in Jurkat cells; 2305 does not inhibit IL-12-induced cytokine expression in IL-12Rß-IL-12Rß2-HEK-293 cells. Finally, compared with anti-p40 treatment, 2305 effectively and selectively inhibits IL-23-induced inflammation in three in vivo mouse models: IL-23-induced ear inflammation, anti-CD40-induced systemic inflammatory response, and collagen-induced arthritis. We, hereby, describe the discovery and characterization of a potent IL-23R small-peptide modulator, 2305 (teeeqqly), that is effective in vivo. 2305 may be more convenient, less cumbersome, less costly, and most importantly, more specific than current biologics for the treatment of inflammatory conditions, and conceivably complement the actual therapies for these chronic and debilitating inflammatory diseases.

Syk is a novel target of arsenic trioxide (ATO) and is involved in the toxic effect of ATO in human neutrophils.

The anticancer dug arsenic trioxide (ATO) is known to be toxic for human mononuclear and neutrophil cell populations by a mechanism that needs to be further investigated. Due to the well-characterized role of Syk kinase in phagocytosis and because activation and involvement of Syk in response to ATO treatment has never been reported in any cells, we decided to determine whether or not Syk is involved in the mode of action of ATO. Human neutrophils were freshly isolated and incubated in vitro with ATO and the role of Syk was evaluated in different neutrophil functions. We found that ATO increased phosphorylation of Syk and this was reversed by piceatannol, a Syk-specific pharmacological inhibitor. In addition, ATO increased the phagocytic ability of neutrophils and degranulation via Syk activation. Finally, using both pharmacological inhibition and cellular depletion of Syk via an antisense approach, we found that this kinase is involved in apoptosis. We conclude that Syk activation is an important step in the mode of action of ATO in mature immune cells such as neutrophils and is involved in rapid, intermediate and lengthy biological processes. This is the first study to report that Syk is activated by ATO.

IL-4R(alpha), a new member that associates with Syk kinase: implication in IL-4-induced human neutrophil functions.

Although Syk has been reported to be associated with IL-2R beta [corrected] and IL-15R alpha in some hematopoietic cells, its association has never been investigated in the IL-4/IL-4R system. In this study, we demonstrate for the first time that Syk is constitutively associated with IL-4R(alpha)in human polymorphonuclear neutrophils (PMNs) and that IL-4 stimulation increases the amount of Syk associated with IL-4R(alpha). Moreover, upon IL-4 treatment, a pool of Syk associated with IL-4R(alpha) is phosphorylated. We also report that such association is not unique to PMNs because Syk associates with IL-4R(alpha) in Raji and in PBMC cells. Stimulation of PMNs by IL-4 increased the amount of Syk associated with PLC-gamma2, pAkt, and alpha-tubulin. Pretreatment of cells with the Syk-selective inhibitor piceatannol or Syk inhibitor II, significantly inhibited the ability of IL-4 to enhance phagocytosis and cell adhesion and to delay apoptosis, and these results correlate with the ability of piceatannol to reduce Syk activation and its association with IL-4R(alpha). Down-regulation of Syk by antisense techniques demonstrates the importance of Syk in the antiapoptotic effect of IL-4. We conclude that association of Syk to IL-4R(alpha) is of biological significance and that IL-4R(alpha) is a new candidate to be added to the few cytokine receptor components which associate with Syk.

Interleukin (IL)-4 induces leukocyte infiltration in vivo by an indirect mechanism.

Interleukin (IL)-4 is a cytokine known mainly for its anti-inflammatory activity. Using the in vivo murine air pouch model, we found that IL-4 significantly increased the number of leukocytes after 9 hours of treatment, consisting mainly of neutrophil (60%) and monocytic (40%) cell populations. Using an antibody array, we found that the expression of several analytes (predominantly CCL2) was increased by IL-4 before the arrival of leukocytes. The IL-4-induced expression of CCL-2 was confirmed by ELISA. Air pouch resident lining cells were harvested and were found to express IL-4Ralpha. CCL2 mRNA expression was monitored in lining cells, cells isolated from the air pouch skin, in RAW264.7 macrophage and in epithelial Mode-K cells and its expression was increased in response to IL-4 in all conditions. We conclude that IL-4 can attract leukocytes in vivo by an indirect mechanism involving the production of several analytes by, at least, resident cells.

Immune system: maturation of myeloid cells.

Human and animal cell lines are important laboratory tools that can be used for studying a variety of cell functions. Some cell lines are blocked at a certain step of maturation and can be used in order to study the mechanisms involved in cell maturation. Because such cell lines can be differentiated toward a more mature-like phenotype after the addition of agents, they can replace primary cells in large screening experiments preventing daily isolation. The human promyelocytic HL-60 leukemia cell line is an attractive model for studying the events which occur in myeloid cells. Interestingly, these cells can be differentiated toward monocytes, macrophages, or granulocytes as described in the present chapter.

Herpes simplex virus-1 up-regulates IL-15 gene expression in monocytic cells through the activation of protein tyrosine kinase and PKC zeta/lambda signaling pathways.

IL-15 plays a seminal role in innate immunity through enhancing the cytotoxic function as well as cytokine production by NK and T cells. We have previously shown that exposure of PBMC as well as monocytic cells to different viruses results in immediate up-regulation of IL-15 gene expression and subsequent NK cell activation as an innate immune response of those cells to these viruses. However, no signaling pathway involved in this up-regulation has been identified. Here we show for the first time that HSV-1-induced up-regulation of IL-15 gene expression is independent of viral infectivity/replication. IL-15 gene is up-regulated by HSV-1 in human monocytes, but not in CD3+ T cells. HSV-1 induces the phosphorylation of protein tyrosine kinases (PTKs) and protein kinase C (PKC) for inducing IL-15 expression in monocytic cells. Inhibitors for PTKs reduced HSV-1-induced PTK activity, DNA binding activity of NF-kB as well as IL-15 gene expression. In contrast, an inhibitor for membrane-bound tyrosine kinases had no effect on these events. Experiments using PKC inhibitors revealed that phosphorylation of PKC zeta/lambda (PKC zeta/lambda), DNA binding activity of NF-kB and HSV-1-induced up-regulation of IL-15 were all decreased. Furthermore, we found that HSV-1-induced IL-15 up-regulation was also dependent on PTKs regulation of PKC phosphorylation. Thus, we conclude that IL-15 up-regulation in HSV-1-treated monocytic cells is dependent on the activity of both PTKs and PKC zeta/lambda.

Interaction of monocytic cells with respiratory syncytial virus results in activation of NF-kappaB and PKC-alpha/beta leading to up-regulation of IL-15 gene expression.

Respiratory syncytial virus (RSV) is a major human respiratory pathogen, particularly for infants. RSV is also a powerful inducer of cytokines, one of which is IL-15, an important immunoregulatory cytokine. IL-15 plays a key role in NK and T cell development and differentiation and also regulates NK cell/macrophage interaction, as well as monocyte/macrophage and granulocyte function. We have shown previously that different viruses up-regulate IL-15 gene expression in human PBMCs. Recently, we found that RSV induces the expression of IL-15 mRNA in the monocytic line THP-1. The signaling pathway involved in such virus-induced up-regulation of IL-15 has not yet been identified. We report here a study describing this mechanism. Because of the involvement of the protein kinase C (PKC) and the transcription factor NF-kappaB in the regulation of others cytokines by RSV as well as the involvement of NF-kappaB in the transactivation of IL-15, our hypothesis was that RSV induced the expression of IL-15 in THP-1 cells through the PKC and NF-kappaB activation. We demonstrate here that RSV-induced up-regulation of IL-15 expression in THP-1 cells involves the phosphorylation of PKC-alpha/beta. Further, inhibition of PKC by different specific inhibitors blocks this up-regulation. Using the electromobility shift assay, we show that the activated form of NF-kappaB binds to the IL-15 promoter sequence. We further confirm, using an ELISA assay, the involvement of p65 in the transcription of IL-15. This study, demonstrating the ability of RSV to induce IL-15 expression, might explain, at least in part, the exacerbated, inflammatory response triggered by RSV infection.

Induction of apoptosis by herpes simplex virus-1 in neonatal, but not adult, neutrophils.

We report a study on the effect of herpes simplex virus 1 (HSV-1) infection on apoptosis of neutrophils from both adults and neonates and present evidence showing that HSV-1 enhances apoptosis in neonatal, but not adult, neutrophils. HSV-1 enhanced the expression of both Fas and Fas ligand on the surface of neonatal neutrophils. Treatments with anti-Fas antibody and a Fas ligand inhibitor significantly reduced the induction of apoptosis by HSV-1. Using an ELISA assay, it was found that HSV-1 infection also leads to increased release of soluble FasL from HSV-1-infected neonatal neutrophils. Increased neonatal neutrophil apoptosis following HSV-1 infection may represent an important mechanism by which HSV-1 may diminish the antiviral response of neonatal neutrophils and might explain, at least in part, the severity of infections that are caused in newborns by this herpesvirus.