Induction of insulin‐like growth factor‐I by interleukin‐17F in bronchial epithelial cells

Cite this as: M. Kawaguchi, J. Fujita, F. Kokubu, G. Ohara, S‐K Huang, S. Matsukura, Y. Ishii, M. Adachi, H. Satoh and N. Hizawa, Clinical & Experimental Allergy, 2010 (40) 1036–1043. Background Increased expression of IL‐17F has been noted in the airway of asthmatic patients, but its role in asthma has not been fully elucidated. Insulin‐like growth factor‐I (IGF‐I) is known to be involved in airway remodelling and inflammation, while its regulatory mechanisms remain to be defined. Objective To further clarify the biological function of IL‐17F, we investigated whether IL‐17F is able to regulate the expression of IGF‐I in bronchial epithelial cells. Methods Bronchial epithelial cells were stimulated with IL‐17F in the presence or absence of T‐helper type 2 cytokines. Various kinase inhibitors were added to the culture to identify the key signalling events leading to the expression of IGF‐I, in conjunction with the use of short interfering RNAs (siRNAs) targeting mitogen‐ and stress‐activated protein kinase (MSK) 1, p90 ribosomal S6 kinase (p90RSK), and cyclic AMP response element‐binding protein (CREB). Results IL‐17F significantly induced IGF‐I gene and protein expression, and co‐stimulation with IL‐4 and IL‐13 augmented its production. MAP kinase kinase (MEK) inhibitors and the Raf1 kinase inhibitor significantly inhibited IGF‐I production, and the combination of PD98059 and Raf1 kinase inhibitor showed further inhibition. Overexpression of Raf1 and Ras dominant‐negative mutants inhibited its expression. MSK1 inhibitors significantly blocked IL17F‐induced IGF‐I expression. Moreover, transfection of the siRNAs targeting MSK1, p90RSK, and CREB blocked its expression. Conclusions In bronchial epithelial cells, IL‐17F is able to induce the expression of IGF‐I via the Raf1–MEK1/2–ERK1/2–MSK1/p90RSK–CREB pathway in vitro.     

Pannexin‐1‐dependent caspase‐1 activation and secretion of IL‐1β is regulated by zinc

Inflammatory processes induced by IL‐1β are critical for host defence responses, but are also implicated in disease. Zinc deficiency is a common consequence of, or contributor to, human inflammatory disease. However, the molecular mechanisms through which zinc contributes to inflammatory disease remain largely unknown. We report here that zinc metabolism regulates caspase‐1 activation and IL‐1β secretion. One of the endogenous mediators of IL‐1β secretion is adenosine triphosphate, acting via the P2X7‐receptor and caspase‐1 activation in cells primed with an inflammatory stimulus such as LPS. We show that this process is selectively abolished by a brief pre‐treatment with the zinc chelator N,N,N′,N′‐tetrakis‐(2‐pyridylmethyl) ethylene diamine (TPEN). These effects on IL‐1β secretion were independent of rapid changes in free zinc within the cell, not a direct effect on caspase‐1 activity, and upstream of caspase‐1 activation. TPEN did however inhibit the activity of pannexin‐1, a hemi‐channel critical for adenosine triphosphate and nigericin‐induced IL‐1β release. These data provide new insights into the mechanisms of caspase‐1 activation and how zinc metabolism contributes to inflammatory mechanisms.     

OK‐432‐stimulated chemokine secretion from human monocytes depends on MEK1/2, and involves p38 MAPK and NF‐κB phosphorylation,in vitro

Interaction between the immune system and cancer cells allows for the use of biological response modifiers, like OK‐432, in cancer therapy. We have studied the involvement of monocytes (MOs) in the immune response to OK‐432 by examining MCP‐1, MIP‐1α and MIP‐1β secretion, in vitro. OK‐432‐induced IL‐6/TNF‐α secretion has previously been shown to depend on mitogen‐activated protein kinases (MAPKs) ERK1/2 and p38, and we therefore investigated the role of these MAPKs in OK‐432‐induced chemokine secretion. Here we demonstrate that pharmacological MEK1/2 kinase inhibition generally impaired chemokine secretion from MOs, whereas p38 MAPK inhibition in particular reduced MIP‐1α production. Furthermore, simultaneous inhibition of MEK1/2 and Syk kinase was seen to have an additive impact on reduced MCP‐1, MIP‐1α and MIP‐1β secretion. Based on single cell flow cytometry analyses, OK‐432, lipoteichoic acid (LTA) and lipopolysaccharide (LPS) were seen to induce p38 MAPK and NF‐κB phosphorylation in MOs with different time kinetics. LTA and LPS have been shown to induce ERK1/2 phosphorylation, whereas the levels of phosphorylated ERK1/2 remained constant following OK‐432 treatment at the time points tested. Toll‐like receptors (TLRs) recognize pathogen‐associated molecular patterns, and we demonstrate increased TLR2 cell surface levels on the MO population, most profoundly following stimulation with LTA and OK‐432. Together these results indicate that modulation of MEK1/2 and p38 MAPK signalling could affect the response to OK‐432 treatment, having the potential to improve its therapeutic potential within cancer and lymphangioma treatment.     

The protein kinase PKR is critical for LPS‐induced iNOS production but dispensable for inflammasome activation in macrophages

Inflammasomes are multi‐protein platforms that drive the activation of caspase‐1 leading to the processing and secretion of biologically active IL‐1β and IL‐18. Different inflammasomes including NOD‐like receptor (NLR) family pyrin domain‐containing 3 (NLRP3), NLR caspase‐recruitment domain‐containing 4 (NLRC4) and absent in melanoma 2 (AIM2) are activated and assembled in response to distinct microbial or endogenous stimuli. However, the mechanisms by which upstream stimuli trigger inflammasome activation remain poorly understood. Double‐stranded RNA‐activated protein kinase (PKR), a protein kinase activated by viral infection, has been recently shown to be required for the activation of the inflammasomes. Using macrophages from two different mouse strains deficient in PKR, we found that PKR is important for the induction of the inducible nitric oxide synthase (iNOS). However, PKR was dispensable for caspase‐1 activation, processing of pro‐IL‐1β/IL‐18 and secretion of IL‐1β induced by stimuli that trigger the activation of NLRP3, NLRC4 and AIM2. These results indicate that PKR is not required for inflammasome activation in macrophages.     

The differential effects of 1,25‐dihydroxyvitamin D3 on Salmonella‐induced interleukin‐8 and human beta‐defensin‐2 in intestinal epithelial cells

Salmonellosis or Salmonella, one of the most common food‐borne diseases, remains a major public health problem worldwide. Intestinal epithelial cells (IECs) play an essential role in the mucosal innate immunity of the host to defend against the invasion of Salmonella by interleukin (IL)?8 and human β‐defensin‐2 (hBD‐2). Accumulated research has unravelled important roles of vitamin D in the regulation of innate immunity. Therefore, we investigated the effects of 1,25‐dihydroxyvitamin D3 (1,25D3) on Salmonella‐induced innate immunity in IECs. We demonstrate that pretreatment of 1,25D3 results in suppression of Salmonella‐induced IL‐8 but enhancement of hBD‐2, either protein secretion and mRNA expression, in IECs. Furthermore, 1,25D3 enhanced Salmonella‐induced membranous recruitment of nucleotide oligomerization domain (NOD2) and its mRNA expression and activation of protein kinase B (Akt), a downstream effector of phosphoinositide 3‐kinase (PI3K). Inhibition of the PI3K/Akt signal counteracted the suppressive effect of 1,25D3 on Salmonella‐induced IL‐8 expression, while knock‐down of NOD2 by siRNA diminished the enhanced hBD‐2 expression. These data suggest differential regulation of 1,25D3 on Salmonella‐induced IL‐8 and hBD‐2 expression in IECs via PI3K/Akt signal and NOD2 protein expression, respectively. Active vitamin D‐enhanced anti‐microbial peptide in Salmonella‐infected IECs protected the host against infection, while modulation of proinflammatory responses by active vitamin D prevented the host from the detrimental effects of overwhelming inflammation. Thus, active vitamin D‐induced innate immunity in IECs enhances the host's protective mechanism, which may provide an alternative therapy for invasive Salmonella infection.     

Isoniazid‐induced control of Mycobacterium tuberculosis by primary human cells requires interleukin‐1 receptor and tumor necrosis factor

Proinflammatory cytokines are critical mediators that control Mycobacterium tuberculosis (Mtb) growth during active tuberculosis (ATB). To further inhibit bacterial proliferation in diseased individuals, drug inhibitors of cell wall synthesis such as isoniazid (INH) are employed. However, whether INH presents an indirect effect on bacterial growth by regulating host cytokines during ATB is not well known. To examine this hypothesis, we used an in vitro human granuloma system generated with primary leukocytes from healthy donors adapted to model ATB. Intense Mtb proliferation in cell cultures was associated with monocyte/macrophage activation and secretion of IL‐1β and TNF. Treatment with INH significantly reduced Mtb survival, but altered neither T‐cell‐mediated Mtb killing, nor production of IL‐1β and TNF. However, blockade of both IL‐1R1 and TNF signaling rescued INH‐induced killing, suggesting synergistic roles of these cytokines in mediating control of Mtb proliferation. Additionally, mycobacterial killing by INH was highly dependent upon drug activation by the pathogen catalase‐peroxidase KatG and involved a host PI3K‐dependent pathway. Finally, experiments using coinfected (KatG‐mutated and H37Rv strains) cells suggested that active INH does not directly enhance host‐mediated killing of Mtb. Our results thus indicate that Mtb‐stimulated host IL‐1 and TNF have potential roles in TB chemotherapy.     

Brucella abortus nitric oxide metabolite regulates inflammasome activation and IL‐1β secretion in murine macrophages

NLRP3 inflammasome is a protein complex crucial to caspase‐1 activation and IL‐1β and IL‐18 maturation. This receptor participates in innate immune responses to different pathogens, including the bacteria of genus Brucella. Our group recently demonstrated that Brucella abortus‐induced IL‐1β secretion involves NLRP3 inflammasome and it is partially dependent on mitochondrial ROS production. However, other factors could be involved, such as P2X7‐dependent potassium efflux, membrane destabilization, and cathepsin release. Moreover, there is increasing evidence that nitric oxide acts as a modulator of NLRP3 inflammasome. The aim of this study was to unravel the mechanism of NLRP3 inflammasome activation induced by B. abortus, as well as the involvement of bacterial nitric oxide (NO) as a modulator of this inflammasome pathway. We demonstrated that NO produced by B. abortus can be used by the bacteria to modulate IL‐1β secretion in infected murine macrophages. Additionally, our results suggest that B. abortus‐induced IL‐1β secretion depends on a P2X7‐independent potassium efflux, lysosomal acidification, cathepsin release, mechanisms clearly associated to NLRP3 inflammasome. In summary, our results help to elucidate the molecular mechanisms of NLRP3 activation and regulation during an intracellular bacterial infection.     

NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase‐4 and caspase‐5

Humans encode two inflammatory caspases that detect cytoplasmic LPS, caspase‐4 and caspase‐5. When activated, these trigger pyroptotic cell death and caspase‐1‐dependent IL‐1β production; however the mechanism underlying this process is not yet confirmed. We now show that a specific NLRP3 inhibitor, MCC950, prevents caspase‐4/5‐dependent IL‐1β production elicited by transfected LPS. Given that both caspase‐4 and caspase‐5 can detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, we generated human monocytic cell lines in which caspase‐4 and caspase‐5 were genetically deleted either individually or together. We found that the deletion of caspase‐4 suppressed cell death and IL‐1β production following transfection of LPS into the cytoplasm, or in response to infection with Salmonella typhimurium. Although deletion of caspase‐5 did not confer protection against transfected LPS, cell death and IL‐1β production were reduced after infection with Salmonella. Furthermore, double deletion of caspase‐4 and caspase‐5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL‐1β maturation, downstream of cytoplasmic LPS detection by caspase‐4/5. We also show that both caspase‐4 and caspase‐5 are functionally important for appropriate responses to intracellular Gram‐negative bacteria.     

Galectin‐3 is an amplifier of the interleukin‐1β‐mediated inflammatory response in corneal keratinocytes

Interleukin‐1β (IL‐1β) is a potent mediator of innate immunity commonly up‐regulated in a broad spectrum of inflammatory diseases. When bound to its cell surface receptor, IL‐1β initiates a signalling cascade that cooperatively induces the expression of canonical IL‐1 target genes such as IL‐8 and IL‐6. Here, we present galectin‐3 as a novel regulator of IL‐1β responses in corneal keratinocytes. Using the SNAP‐tag system and digitonin semi‐permeabilization, we show that recombinant exogenous galectin‐3 binds to the plasma membrane of keratinocytes and is internalized into cytoplasmic compartments. We find that exogenous galectin‐3, but not a dominant negative inhibitor of galectin‐3 polymerization lacking the N‐terminal domain, exacerbates the response to IL‐1β by stimulating the secretion of inflammatory cytokines. The activity of galectin‐3 could be reduced by a novel d ‐galactopyranoside derivative targeting the conserved galactoside‐binding site of galectins and did not involve interaction with IL‐1 receptor 1 or the induction of endogenous IL‐1β. Consistent with these observations, we demonstrate that small interfering RNA‐mediated suppression of endogenous galectin‐3 expression is sufficient to impair the IL‐1β‐induced secretion of IL‐8 and IL‐6 in a p38 mitogen‐activated protein kinase‐independent manner. Collectively, our findings provide a novel role for galectin‐3 as an amplifier of IL‐1β responses during epithelial inflammation through an as yet unidentified mechanism.     

PI3Kγ kinase activity is required for optimal T‐cell activation and differentiation

Phosphatidylinositol‐3‐kinase gamma (PI3Kγ) is a leukocyte‐specific lipid kinase with signaling function downstream of G protein‐coupled receptors to regulate cell trafficking, but its role in T cells remains unclear. To investigate the requirement of PI3Kγ kinase activity in T‐cell function, we studied T cells from PI3Kγ kinase‐dead knock‐in (PI3Kγ^KD/KD) mice expressing the kinase‐inactive PI3Kγ protein. We show that CD4⁺ and CD8⁺ T cells from PI3Kγ^KD/KD mice exhibit impaired TCR/CD28‐mediated activation that could not be rescued by exogenous IL‐2. The defects in proliferation and cytokine production were also evident in naïve and memory T cells. Analysis of signaling events in activated PI3Kγ^KD/KD T cells revealed a reduction in phosphorylation of protein kinase B (AKT) and ERK1/2, a decrease in lipid raft formation, and a delay in cell cycle progression. Furthermore, PI3Kγ^KD/KD CD4⁺ T cells displayed compromised differentiation toward Th1, Th2, Th17, and induced Treg cells. PI3Kγ^KD/KD mice also exhibited an impaired response to immunization and a reduced delayed‐type hypersensitivity to Ag challenge. These findings indicate that PI3Kγ kinase activity is required for optimal T‐cell activation and differentiation, as well as for mounting an efficient T cell‐mediated immune response. The results suggest that PI3Kγ kinase inhibitors could be beneficial in reducing the undesirable immune response in autoimmune diseases.     

PKC,ERK/p38 MAP kinases and NF‐κB targeted signalling play a role in the expression and release of IL‐1β and CXCL8 in Porphyromonas gingivalis‐infected THP1 cells

Porphyromonas gingivalis is a keystone pathogen in periodontitis and is gaining importance in cardiovascular pathogenesis. Protease‐activated receptors (PARs), toll‐like receptors (TLRs) and nucleotide‐binding oligomerization domain (NOD) on monocytes recognize the structural components on P. gingivalis, inducing inflammatory intermediates. Here, we elucidate the modulation of PARs, TLRs, NODs, and the role of MAPK and NF‐κB in IL‐1β and CXCL8 release. THP1 cells were stimulated with P. gingivalis wild‐type W50 and its isogenic gingipain mutants: Rgp mutant E8 and Kgp mutant K1A. We observed modulation of PARs, TLRs, NOD, IL‐1β and CXCL8 expression by P. gingivalis. Gingipains hydrolyse IL‐1β and CXCL8, which is more evident for IL‐1β accumulation at 24 h. Inhibition of PKC (protein kinase C), p38 and ERK (extracellular signal‐regulated kinases) partially reduced P. gingivalis‐induced IL‐1β at 6 h, whereas PKC and ERK reduced CXCL8 at both 6 and 24 h. Following NF‐κB inhibition, P. gingivalis‐induced IL‐1β and CXCL8 were completely suppressed to basal levels. Overall, TLRs, PARs and NOD possibly act in synergy with PKC, MAPK ERK/p38 and NF‐κB in P. gingivalis‐induced IL‐1β and CXCL8 release from THP1 cells. These pro‐inflammatory cytokines could affect leucocytes in circulation and exacerbate other vascular inflammatory conditions such as atherosclerosis.     

Mechanism of interleukin‐1β‐induced proliferation in rat hepatic stellate cells from different levels of signal transduction

Hepatic stellate cells (HSCs) are the major producers of collagen in the liver. Their conversion from resting cells to proliferative, contractile, and activated cells is a critical step leading to liver fibrosis that is characterized by the deposition of excessive extracellular matrix. Interleukin‐1 (IL‐1) may play a role in maintaining HSC in a proliferative state that is responsible for hepatic fibrogenesis. The aim of this study was to study the roles of the IL‐1 type I receptor (IL‐1R1), c‐Jun N‐terminal kinase (JNK), and activation protein‐1 (AP‐1) in IL‐1β–mediated proliferation in rat HSCs. We showed that IL‐1β can upregulate proliferation in rat HSCs; however, inhibition of the JNK pathway could inhibit HSCs proliferation. Furthermore, IL‐1β activated IL‐1R1 expression, the JNK signaling pathway, and AP‐1 activity in a time‐dependent manner in rat HSCs. These data demonstrate that IL‐1β could promote the proliferation of rat HSCs and that the IL‐1R1, JNK, and AP‐1 pathways were involved in this process. In summary, IL‐1β‐induced proliferation is possibly mediated by the IL‐1R1, JNK, and AP‐1 pathways in rat HSCs. Therefore, drugs that block these pathways may inhibit the proliferation of HSCs and suppress liver fibrosis.     

Interleukin‐17A participates in podocyte injury by inducing IL‐1β secretion through ROS‐NLRP3 inflammasome‐caspase‐1 pathway

Studies show that the Th17/IL ‐17A axis plays an important role in the pathogenesis of kidney diseases. Previously, we also showed that IL ‐17A may play a role in the pathogenesis of primary nephrotic syndrome; however, the underlying mechanism(s) is unclear. The aim of this study was to explore the molecular mechanism of IL ‐17A‐inducing podocyte injury in vitro. In this study, the NLRP 3 inflammasome activation and the morphology of podocytes were detected by Western blot and immunofluorescence. The results showed that podocytes persistently expressed IL ‐17A receptor and that NLRP 3 inflammasome in these cells was activated upon exposure to IL ‐17A. Also, activity of caspase‐1 and secretion of IL ‐1β increased in the presence of IL ‐17A. In addition, IL ‐17A disrupted podocyte morphology by decreasing expression of podocin and increasing expression of desmin. Blockade of intracellular ROS or inhibition of caspase‐1 prevented activation of the NLRP 3 inflammasome, thereby restoring podocyte morphology. Taken together, the results suggest that IL ‐17A induces podocyte injury by activating the NLRP 3 inflammasome and IL ‐1β secretion and contributes to disruption of the kidney's filtration system.     

Transforming growth factor‐β/Smad ‐ signalling pathway and conjunctival remodelling in vernal keratoconjunctivitis

Background Vernal keratoconjunctivitis (VKC) is a chronic ocular allergic inflammation characterized by corneal complications and the formation of giant papillae. Sma‐ and Mad‐related proteins (Smad) modulate extracellular matrix gene expression during wound healing, inflammation and tissue remodelling. Objective To investigate the relationship between allergic inflammation and TGF‐β/Smad signalling pathway, expression in VKC patients and in primary cultured conjunctival fibroblasts exposed to mediators found previously over‐expressed in VKC. Methods Smad‐2, ‐3, ‐7, phospho‐(p)Smads, TGF‐β1 and ‐β2 were evaluated in the conjunctiva of normal subjects (CT) and VKC patients by immunohistochemistry. The expression of Smads, pro‐collagen I (PIP), TGF‐β1, ‐β2, mitogen‐activated protein kinase (p38/MAPK), c‐Jun N‐terminal kinase (JNK) and extracellular signal‐regulated kinase (ERK1/2) were also determined in conjunctival fibroblast cultures exposed to histamine, IL‐4, ‐13, TGF‐β1, IFN‐γ and TNF‐α using immunostaining or RT‐PCR. Results Immunostaining for Smad‐2, ‐3, pSmad‐2, ‐3, TGF‐β1, ‐β2 and PIP was significantly increased in VKC stroma compared with CT. In conjunctival fibroblast cultures, Smad‐3 and PIP were stimulated by histamine, IL‐4, ‐13 and TGF‐β1 exposure, while PIP was reduced by IFN‐γ, and TNF‐α mRNA expression of Smad‐3 was increased by histamine, while Smad‐7 was reduced by IL‐4. In addition, histamine, IL‐4 and TNF‐α increased JNK and ERK1/2 expression. Conclusion and Clinical Relevance The TGF‐β/Smad signalling pathway is over‐expressed in VKC tissues and modulated in conjunctival fibroblasts by histamine, IL‐4, TGF‐β1 and TNF‐α. These mechanisms may be involved in fibrillar collagen production, giant papillae formation and tissue remodelling typical of VKC and might provide new therapeutic targets for its treatment. Cite this as: A. Leonardi, A. Di Stefano, L. Motterle, B. Zavan, G. Abatangelo and P. Brun, Clinical & Experimental Allergy, 2011 (41) 52–60.