Exploring the Role of IL-32 in HIV-Related Kaposi Sarcoma.

The intracellular proinflammatory mediator IL-32 is associated with tumor progression; however, the mechanisms remain unknown. We studied IL-32 mRNA expression as well as expression of other proinflammatory cytokines and mediators, including IL-1α, IL-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α, the proangiogenic and antiapoptotic enzyme cyclooxygenase-2, the IL-8 receptor C-X-C chemokine receptor (CXCR) 1, and the intracellular kinase focal adhesion kinase-1. The interaction of IL-32 expression with expression of IL-6, TNF-α, IL-8, and cyclooxygenase-2 was also investigated. Biopsy specimens of 11 HIV-related, 7 non-HIV-related Kaposi sarcoma (KS), and 7 normal skin tissues (NSTs) of Dutch origin were analyzed. RNA was isolated from the paraffin material, and gene expression levels of IL-32 α, ß, and γ isoforms, IL1a, IL1b, IL6, IL8, TNFA, PTGS2, CXCR1, and PTK2 were determined using real-time quantitative PCR. Significantly higher expression of IL-32ß and IL-32γ isoforms was observed in HIV-related KS biopsy specimens compared with non-HIV-related KS and NST. The splicing ratio of the IL-32 isoforms showed IL-32γ as the highest expressed isoform, followed by IL-32ß, in HIV-related KS cases compared with non-HIV-related KS and NST. Our data suggest a possible survival mechanism by the splicing of IL-32γ to IL-32ß and also IL-6, IL-8, and CXCR1 signaling pathways to reverse the proapoptotic effect of the IL-32γ isoform, leading to tumor cell survival and thus favoring tumor progression.

Abnormal epigenetic changes during differentiation of human skeletal muscle stem cells from obese subjects.

BACKGROUND: Human skeletal muscle stem cells are important for muscle regeneration. However, the combined genome-wide DNA methylation and expression changes taking place during adult myogenesis have not been described in detail and novel myogenic factors may be discovered. Additionally, obesity is associated with low relative muscle mass and diminished metabolism. Epigenetic alterations taking place during myogenesis might contribute to these defects. METHODS: We used Infinium HumanMethylation450 BeadChip Kit (Illumina) and HumanHT-12 Expression BeadChip (Illumina) to analyze genome-wide DNA methylation and transcription before versus after differentiation of primary human myoblasts from 14 non-obese and 14 obese individuals. Functional follow-up experiments were performed using siRNA mediated gene silencing in primary human myoblasts and a transgenic mouse model. RESULTS: We observed genome-wide changes in DNA methylation and expression patterns during differentiation of primary human muscle stem cells (myoblasts). We identified epigenetic and transcriptional changes of myogenic transcription factors (MYOD1, MYOG, MYF5, MYF6, PAX7, MEF2A, MEF2C, and MEF2D), cell cycle regulators, metabolic enzymes and genes previously not linked to myogenesis, including IL32, metallothioneins, and pregnancy-specific beta-1-glycoproteins. Functional studies demonstrated IL-32 as a novel target that regulates human myogenesis, insulin sensitivity and ATP levels in muscle cells. Furthermore, IL32 transgenic mice had reduced insulin response and muscle weight. Remarkably, approximately 3.7 times more methylation changes (147,161 versus 39,572) were observed during differentiation of myoblasts from obese versus non-obese subjects. In accordance, DNMT1 expression increased during myogenesis only in obese subjects. Interestingly, numerous genes implicated in metabolic diseases and epigenetic regulation showed differential methylation and expression during differentiation only in obese subjects. CONCLUSIONS: Our study identifies IL-32 as a novel myogenic regulator, provides a comprehensive map of the dynamic epigenome during differentiation of human muscle stem cells and reveals abnormal epigenetic changes in obesity.

Alternatively spliced isoforms of IL-32 differentially influence cell death pathways in cancer cell lines.

Alternative splicing is a biological mechanism that enables the synthesis of several isoforms with different or even opposite functions. This process must be tightly regulated to prevent unwanted isoform expression favoring pathological processes. Some isoforms of interleukin 32 (IL-32) are reported to be more potent in inducing inflammation, however the role in cell death remains to be investigated. This study demonstrates that IL-32γ and IL-32ß can induce caspase-8-dependent cell death whereas this was not observed for IL-32α. Overexpression of IL-32ß or IL-32γ but not IL-32α, resulted in enhanced expression of the survival cytokine IL-8. Furthermore, restoring the IL-8 signaling pathway by overexpressing CXCR1 in HEK293 cells, rescued IL-32ß but not IL-32γ-induced cell death. Interestingly, IL-32γ was able to downregulate CXCR1 and thereby induce cell death. Subsequent studies into the role of IL-32 in thyroid cancer (TC) revealed that several IL-32 isoforms, IL-8, and CXCR1 are expressed in TC cell lines and specimens. Remarkably, TC cell lines were found to produce high concentrations of IL-8, indicating an important role for IL-8 in the survival-signaling pathway in these cells. Intriguingly, a significant correlation between the IL-8 receptor CXCR1 and IL-32γ was observed in TC specimens, while this was not observed for the other IL-32 splice variants. Blocking IL-32 alternative splicing by Isoginkgetin resulted in predominant expression of IL-32γ splice variants and cell death in TC cell lines. All together, modulation of IL-32 alternative splicing could represent a novel strategy for the treatment of malignancies, in particular thyroid cancer.

IL-32 promotes angiogenesis.

IL-32 is a multifaceted cytokine with a role in infections, autoimmune diseases, and cancer, and it exerts diverse functions, including aggravation of inflammation and inhibition of virus propagation. We previously identified IL-32 as a critical regulator of endothelial cell (EC) functions, and we now reveal that IL-32 also possesses angiogenic properties. The hyperproliferative ECs of human pulmonary arterial hypertension and glioblastoma multiforme exhibited a markedly increased abundance of IL-32, and, significantly, the cytokine colocalized with integrin αVß3. Vascular endothelial growth factor (VEGF) receptor blockade, which resulted in EC hyperproliferation, increased IL-32 three-fold. Small interfering RNA-mediated silencing of IL-32 negated the 58% proliferation of ECs that occurred within 24 h in scrambled-transfected controls. Reduction of IL-32 neither affected apoptosis (insignificant changes in Bak-1, Bcl-2, Bcl-xL, lactate dehydrogenase, annexin V, and propidium iodide) nor VEGF or TGF-ß levels, but siIL-32-transfected adult and neonatal ECs produced up to 61% less NO, IL-8, and matrix metalloproteinase-9, and up to 3-fold more activin A and endostatin. In coculture-based angiogenesis assays, IL-32γ dose-dependently increased tube formation up to 3-fold; an αVß3 inhibitor prevented this activity and reduced IL-32γ-induced IL-8 by 85%. In matrigel plugs loaded with IL-32γ, VEGF, or vehicle and injected into live mice, we observed the anticipated VEGF-induced increase in neocapillarization (8-fold versus vehicle), but unexpectedly, IL-32γ was equally angiogenic. A second signal such as IFN-γ was required to render cells responsive to exogenous IL-32γ; importantly, this was confirmed using a completely synthetic preparation of IL-32γ. In summary, we add angiogenic properties that are mediated by integrin αVß3 but VEGF-independent to the portfolio of IL-32, implicating a role for this versatile cytokine in pulmonary arterial hypertension and neoplastic diseases.

Novel insights into the biology of interleukin-32.

Interleukin (IL)-32 is known as a proinflammatory cytokine that is likely involved in several diseases, including infections, chronic inflammation, and cancer. Since the first report in 2005, IL-32 has been the subject of numerous studies to unravel the biological function of this molecule. For example, silencing of endogenous IL-32 in primary or cell lines of human origin consistently suppressed responses to Toll-like receptors. The protein folding structure of the six isoforms of IL-32 does not resemble that of any classical cytokine and as of this writing, a specific IL-32 receptor has not been identified. Instead, we propose a mechanism by which exposure to extracellular IL-32 or overexpression of the molecule results in binding to intracellular partners that influences functions such as gene expression, cell death, or survival. As such, this review offers insights into the role of IL-32 in several diseases, host defense, inflammation, immune function, and cancer. Finally, possibilities to target IL-32 in several diseases are proposed.

Inflammation-dependent secretion and splicing of IL-32{gamma} in rheumatoid arthritis.

Different splice variants of the proinflammatory cytokine IL-32 are found in various tissues; their putative differences in biological function remain unknown. In the present study, we report that IL-32γ is the most active isoform of the cytokine. Splicing to one less active IL-32ß appears to be a salvage mechanism to reduce inflammation. Adenoviral overexpression of IL-32γ (AdIL-32γ) resulted in exclusion of the IL-32γ-specific exon in vitro as well as in vivo, primarily leading to expression of IL-32ß mRNA and protein. Splicing of the IL-32γ-specific exon was prevented by single-nucleotide mutation, which blocked recognition of the splice site by the spliceosome. Overexpression of splice-resistant IL-32γ in THP1 cells or rheumatoid arthritis (RA) synovial fibroblasts resulted in a greater induction of proinflammatory cytokines such as IL-1ß, compared with IL-32ß. Intraarticular introduction of IL-32γ in mice resulted in joint inflammation and induction of several mediators associated with joint destruction. In RA synovial fibroblasts, overexpression of primarily IL-32ß showed minimal secretion and reduced cytokine production. In contrast, overexpression of splice-resistant IL-32γ in RA synovial fibroblasts exhibited marked secretion of IL-32γ. In RA, we observed increased IL-32γ expression compared with osteoarthritis synovial tissue. Furthermore, expression of TNFα and IL-6 correlated significantly with IL-32γ expression in RA, whereas this was not observed for IL-32ß. These data reveal that naturally occurring IL-32γ can be spliced into IL-32ß, which is a less potent proinflammatory mediator. Splicing of IL-32γ into IL-32ß is a safety switch in controlling the effects of IL-32γ and thereby reduces chronic inflammation.

A promoter polymorphism in human interleukin-32 modulates its expression and influences the risk and the outcome of epithelial cell-derived thyroid carcinoma.

Interleukin (IL)-32 is an intracellular proinflammatory mediator that strongly modulates the inflammatory reaction. Recent studies have suggested the involvement of IL-32 in the pathogenesis of malignancies. We aimed to assess whether a known germ-line polymorphism in the IL32 promoter modulates IL-32 expression, and whether it influences susceptibility and/or outcome of epithelial cell-derived thyroid carcinoma (TC). In this study, IL32 genotype was assessed in 139 TC patients and 138 healthy controls and was correlated with TC susceptibility and clinical outcome. Furthermore, IL-32 messenger RNA expression and protein were assessed in TC tissues and functional consequences of genetic variants of IL32 were studied in a model of human primary immune cells. Results demonstrate substantial IL-32 expression in TC tumor tissue. Lipopolysaccharide (LPS) stimulation of primary immune cells revealed 2-fold higher expression of IL-32γ, but not IL-32ß, in cells homozygous for the ancient T allele. Furthermore, production of LPS-induced cytokines was increased in cells bearing this T allele. Genetic analysis revealed that the ancient T allele was overrepresented in TC patients with odds ratio (95% confidence interval) = 1.71 (1.06-2.75). In addition, the cumulative radioactive iodine (RAI) dose received after total thyroidectomy was significantly higher in TC patients bearing the ancient T allele. In conclusion, individuals bearing genetic variants of IL32 that lead to an increased IL-32γ gene expression and higher production of proinflammatory cytokines have higher risk for developing epithelial cell-derived TC. Subsequently, they require higher dosages of RAI to achieve successful tumor remission. These data suggest an important role of IL-32 in the pathogenesis of TC.

Interleukin 32 (IL-32) contains a typical α-helix bundle structure that resembles focal adhesion targeting region of focal adhesion kinase-1.

IL-32 can be expressed in several isoforms. The amino acid sequences of the major IL-32 isoforms were used to predict the secondary and tertiary protein structure by I-TASSER software. The secondary protein structure revealed coils and α-helixes, but no ß sheets. Furthermore, IL-32 contains an RGD motif, which potentially activates procaspase-3 intracellular and or binds to integrins. Mutation of the RGD motif did not result in inhibition of the IL-32ß- or IL-32γ-induced cytotoxicity mediated through caspase-3. Although IL-32α interacted with the extracellular part of αVß3 and αVß6 integrins, only the αVß3 binding was inhibited by small RGD peptides. Additionally, IL-32ß was able to bind to αVß3 integrins, whereas this binding was not inhibited by small RGD peptides. In addition to the IL-32/integrin interactions, we observed that IL-32 is also able to interact with intracellular proteins that are involved in integrin and focal adhesion signaling. Modeling of IL-32 revealed a distinct α-helix protein resembling the focal adhesion targeting region of focal adhesion kinase (FAK). Inhibition of FAK resulted in modulation of the IL-32ß- or IL-32γ-induced cytotoxicity. Interestingly, IL-32α binds to paxillin without the RGD motif being involved. Finally, FAK inhibited IL-32α/paxillin binding, whereas FAK also could interact with IL-32α, demonstrating that IL-32 is a member of the focal adhesion protein complex. This study demonstrates for the first time that IL-32 binds to the extracellular domain of integrins and to intracellular proteins like paxillin and FAK, suggesting a dual role for IL-32 in integrin signaling.

Towards a role of interleukin-32 in atherosclerosis.

BACKGROUND: IL-32 has been previously shown to promote inflammation in rheumatoid arthritis patients and to contribute to IL-1ß-induced ICAM-1 as well as other proinflammatory cytokines synthesis in human umbilical endothelial cells (HUVECs). Given the high rate of atherosclerosis in RA, these observations suggest that IL-32 may be involved in the inflammatory pathways of atherosclerosis. METHODS: mRNA and protein levels of IL-32 were determined in human atherosclerotic arterial vessel wall tissue by quantitative real-time PCR and immunohistochemistry. HUVEC and M1/M2 macrophages were stimulated with proinflammatory cytokines and TLR ligands to assess IL-32 mRNA induction. Human THP1 macrophages were transduced with AdIL-32γ, to investigate induction of several proatherosclerotic mediators. Finally, aortas from IL-32γ transgenic mice were studied and compared with aortas from age-matched wild-type mice. RESULTS: IL-32 expression was detectable in human atherosclerotic arterial vessel wall, with the expression of IL-32ß and IL-32γ mRNA significantly enhanced. TLR3-ligand Poly I:C in combination with IFNγ were the most potent inducers of IL-32 mRNA expression in both HUVEC and M1/M2 macrophages. Adenoviral overexpression of IL-32γ in human THP1 macrophages resulted in increased production of CCL2, sVCAM-1, MMP1, MMP9, and MMP13. The IL-32γ transgenic mice chow a normal fat diet exhibited vascular abnormalities resembling atherosclerosis. CONCLUSIONS: IL-32 acts as a proinflammatory factor and may be implicated in the inflammatory cascade contributing to atherosclerosis. By promoting the synthesis of matrix metalloproteinases, it may further contribute to plaque instability. Further studies are warranted to investigate whether IL-32 may serve as a potential therapeutic target in fighting atherosclerosis.

Interleukin-32: a predominantly intracellular proinflammatory mediator that controls cell activation and cell death.

In this review, we will discuss the current knowledge on IL-32 and provide new insights regarding the biological function of IL-32. IL-32 is seen as a cytokine that can induce a range of proinflammatory mediators and contribute to autoimmune diseases, such as rheumatoid arthritis, however present knowledge demonstrates that IL-32 is not a classical cytokine. We present the history of this cytokine, the role of IL-32 in several diseases and discuss a possible novel role of intracellular IL-32 in cell homeostasis. Taken into account the observed biological functions of IL-32, it may belong to a class of cytokines, like IL-1α, IL-33, and IL-37, with both intracellular and extracellular functions.

Tumour necrosis factor alpha-driven IL-32 expression in rheumatoid arthritis synovial tissue amplifies an inflammatory cascade.

OBJECTIVE: To investigate the interplay between IL-32 and tumour necrosis factor alpha (TNFα) during the chronic inflammation of rheumatoid arthritis (RA) and to assess whether anti-TNFα treatment of RA patients modulates synovial IL-32 expression. METHODS: Induction of IL-32γ by Pam3Cys, lipopolysaccharide, IL-1ß or TNFα was investigated in human fibroblast-like synoviocytes (FLS). Stimulation of TNFα production by IL-32γ was studied by adenoviral overexpression of IL-32γ (AdIL-32γ) and lipopolysaccharide stimulation of THP1 cells. Silencing of endogenous IL-32 was employed to study cytokine regulation in FLS. AdIL-32γ followed by TNFα stimulation was performed in FLS to investigate cytokine induction. Immunohistochemistry was applied to study IL-32 expression in synovial biopsies from RA patients. RESULTS: TNFα potently induced IL-32γ expression in FLS. Increased TNFα, IL-1ß, IL-6 and CXCL8 production was observed after IL-32γ overexpression and lipopolysaccharide stimulation of THP1 cells. TNFα stimulation of FLS after silencing IL-32γ resulted in diminished IL-6 and CXCL8 production, whereas IL-32γ overexpression resulted in enhanced IL-6 and CXCL8 levels. Remarkably, the mechanism through which IL-32γ overexpression induced TNFα, IL-1ß and CXCL8 was by counteracting messenger RNA decay. Importantly, treatment of RA patients with anti-TNFα resulted in significant reduction of IL-32 protein in synovial tissue. CONCLUSIONS: TNFα is a potent inducer of endogenous IL-32 expression and IL-32 itself contributes to prolonged TNFα production, thus inducing an important auto-inflammatory loop. Treatment of RA patients with anti-TNFα antibodies diminished IL-32 expression in synovial tissue. The potent anti-inflammatory effect of TNFα blockade in RA patients may be partly due to the reduction of synovial IL-32 expression.

Differential requirement for the activation of the inflammasome for processing and release of IL-1beta in monocytes and macrophages.

The processing of pro-interleukin-1beta depends on activation of caspase-1. Controversy has arisen whether Toll-like receptor (TLR) ligands alone can activate caspase-1 for release of interleukin-1beta (IL-1beta). Here we demonstrate that human blood monocytes release processed IL-1beta after a one-time stimulation with either TLR2 or TLR4 ligands, resulting from constitutively activated caspase-1 and release of endogenous adenosine triphosphate. The constitutive activation of caspase-1 depends on the inflammasome components, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and NALP3, but in monocytes caspase-1 activation is uncoupled from pathogen-associated molecular pattern recognition. In contrast, macrophages are unable to process and release IL-1beta solely by TLR ligands and require a second adenosine triphosphate stimulation. We conclude that IL-1beta production is differentially regulated in monocytes and macrophages, and this reflects their separate functions in host defense and inflammation.

Differential function of the NACHT-LRR (NLR) members Nod1 and Nod2 in arthritis.

The pathogenesis of chronic joint inflammation remains unclear, although the involvement of pathogen recognition receptors has been suggested recently. In the present article, we describe the role of two members of the NACHT-LRR (NLR) family, Nod1 (nucleotide-binding oligomerization domain) and Nod2 in a model of acute joint inflammation induced by intraarticular injection of Streptococcus pyogenes cell wall fragments. Here, we show that Nod2 deficiency resulted in reduced joint inflammation and protection against early cartilage damage. In contrast, Nod1 gene-deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage, consistent with a model in which Nod1 controls the inflammatory reaction. To explore whether the different function of Nod1 and Nod2 occurs also in humans, we exposed peripheral blood mononuclear cells (PBMCs) carrying either Nod1ins/del or Nod2fs mutation with SCW fragments in vitro. Production of both TNFalpha and IL-1beta was clearly impaired in PBMCs carrying the Nod2fs compared with PBMCs isolated from healthy controls. In line with results in Nod1 gene-deficient mice, PBMCs from individuals bearing a newly described Nod1 mutation produced enhanced levels of proinflammatory cytokines after 24-h stimulation with SCW fragments. These data indicate that the NLR family members Nod1 and Nod2 have different functions in controlling inflammation, and that intracellular Nod1-Nod2 interactions may determine the severity of arthritis in this experimental model. Whether a distorted balance between the function of Nod1 and/or Nod2 is involved in the pathogenesis of human autoinflammatory or autoimmune disease, such as rheumatoid arthritis, remains to be elucidated.

IL-32gamma and Streptococcus pyogenes cell wall fragments synergise for IL-1-dependent destructive arthritis via upregulation of TLR-2 and NOD2.

OBJECTIVE: To investigate the potential synergism between interleukin (IL) 32γ and Streptococcus pyogenes cell wall (SCW) fragments in the development of destructive arthritis. METHODS: An adenoviral vector encoding human IL-32γ (AdIL-32γ) was constructed and validated in HeLa cells. Fibroblast-like synoviocytes (FLS) were transduced with AdIL-32γ and stimulated with Toll-like receptor 2 (TLR-2) and nucleotide oligomerisation domain (NOD) 2 ligands. Expression levels of several proinflammatory cytokines, chemokines, matrix degrading enzymes, TLR-2 and NOD2 were measured by quantitative real-time PCR. Furthermore, IL-6 and CXCL8 protein levels were determined. In-vivo synergy between IL-32γ and SCW was studied by intra-articular injection of AdIL-32γ in C57Bl/6 mice followed by SCW injection. The contribution of endogenous IL-1 was assessed in mice deficient for both IL-1α and IL-1ß. RESULTS: IL-32γ synergise with TLR-2/NOD2 ligands to induce proinflammatory cytokines, chemokines and matrix degrading enzymes in AdIL-32γ-transduced FLS. In mice, AdIL-32γ transduction followed by the injection of SCW displayed aggravated joint inflammation and cartilage destruction. However, IL-1-deficient mice were protected against IL-32γ/SCW-induced joint changes, indicating a requirement for IL-1 in downstream events triggered by IL-32γ plus SCW. To elucidate the synergistic mechanism, the authors investigated the expression of two pattern recognition receptors involved in sensing SCW fragments. TLR-2 and NOD2 receptor expression was enhanced by IL-32γ and Pam3Cys/muramyl dipeptide stimulation in FLS. CONCLUSIONS: Here the authors show that IL-32γ aggravates SCW-induced arthritis by the upregulation of TLR-2/NOD2 expression and promotes severe joint erosion in an IL-1-dependent fashion. Targeting of IL-32γ may provide a novel therapy to prevent destructive arthritis.

Differential effects of human and plant N-acetylglucosaminyltransferase I (GnTI) in plants.

In plants and animals, the first step in complex type N-glycan formation on glycoproteins is catalyzed by N-acetylglucosaminyltransferase I (GnTI). We show that the cgl1-1 mutant of Arabidopsis, which lacks GnTI activity, is fully complemented by YFP-labeled plant AtGnTI, but only partially complemented by YFP-labeled human HuGnTI and that this is due to post-transcriptional events. In contrast to AtGnTI-YFP, only low levels of HuGnTI-YFP protein was detected in transgenic plants. In protoplast co-transfection experiments all GnTI-YFP fusion proteins co-localized with a Golgi marker protein, but only limited co-localization of AtGnTI and HuGnTI in the same plant protoplast. The partial alternative targeting of HuGnTI in plant protoplasts was alleviated by exchanging the membrane-anchor domain with that of AtGnTI, but in stably transformed cgl1-1 plants this chimeric GnTI still did not lead to full complementation of the cgl1-1 phenotype. Combined, the results indicate that activity of HuGnTI in plants is limited by a combination of reduced protein stability, alternative protein targeting and possibly to some extend to lower enzymatic performance of the catalytic domain in the plant biochemical environment.

Interplay between thyroid cancer cells and macrophages: effects on IL-32 mediated cell death and thyroid cancer cell migration.

PURPOSE: Interleukin 32 (IL-32) is a pro-inflammatory cytokine of which different isoforms have been identified. Recently, IL-32 has been shown to act as a potent inducer of cell migration in several types of cancer. Although previous research showed that IL-32 is expressed in differentiated thyroid cancer (TC) cells, the role of IL-32 in TC cell migration has not been investigated. Furthermore, tumour-associated macrophages (TAMs) may play a facilitating role in cancer cell migration. The aim of this study was to explore whether the interaction between TC cells and TAMs results in increased expression of IL-32 in TC cells and to investigate whether this affects TC cell migration. METHODS: TPC-1 cells were co-culture with TC-induced or naive macrophages. Next, transcriptome analysis on TPC-1 cells was performed and supernatants were used for stimulation of TPC-1 cells. IL-32ß and IL-32γ were exogenously overexpressed in TPC-1 cells using transient transfection, after which an in vitro gap closure assay was performed to assess cell migration, and the expression of migratory factors was assessed using RT-qPCR. RESULTS: We found that TC-induced macrophages induced IL-32 expression in TC cells and that TAM-derived TNFα was the main inducer of IL-32ß expression in TC cells. Overexpression of IL-32ß and IL-32γ did not affect TC cell migration, but increased cell death. Finally, we found that IL-32ß overexpression led to increased mRNA expression of the pro-survival cytokine IL-8, while the expression of other migratory factors was not affected. CONCLUSIONS: From our data, we conclude that TAM-derived TNFα induces IL-32ß in TC cells. Although IL-32ß does not affect TC cell migration, alternative splicing of IL-32 towards the IL-32ß isoform may be beneficial for TC cell survival through induction of the pro-survival cytokine IL-8.

Interleukin-32 upregulates the expression of ABCA1 and ABCG1 resulting in reduced intracellular lipid concentrations in primary human hepatocytes.

BACKGROUND AND AIMS: The role of interleukin (IL-)32 in inflammatory conditions is well-established, however, the mechanism behind its role in atherosclerosis remains unexplained. Our group reported a promoter single nucleotide polymorphism in IL-32 associated with higher high-density lipoprotein (HDL) concentrations. We hypothesize that endogenous IL-32 in liver cells, a human monocytic cell line and carotid plaque tissue, can affect atherosclerosis by regulating (HDL) cholesterol homeostasis via expression of cholesterol transporters/mediators. METHODS: Human primary liver cells were stimulated with recombinant human (rh)TNFα and poly I:C to study the expression of IL-32 and mediators in cholesterol pathways. Additionally, IL-32 was overexpressed in HepG2 cells and overexpressed and silenced in THP-1 cells to study the direct effect of IL-32 on cholesterol transporters expression and function. RESULTS: Stimulation of human primary liver cells resulted in induction of IL-32α, IL-32ß and IL-32γ mRNA expression (p < 0.01). A strong correlation between the expression of IL-32γ and ABCA1, ABCG1, LXRα and apoA1 was observed (p < 0.01), and intracellular lipid concentrations were reduced in the presence of endogenous IL-32 (p < 0.05). Finally, IL32γ and ABCA1 mRNA expression was upregulated in carotid plaque tissue and when IL-32 was silenced in THP-1 cells, mRNA expression of ABCA1 was strongly reduced. CONCLUSIONS: Regulation of IL-32 in human primary liver cells, HepG2 and THP-1 cells strongly influences the mRNA expression of ABCA1, ABCG1, LXRα and apoA1 and affects intracellular lipid concentrations in the presence of endogenous IL-32. These data, for the first time, show an important role for IL32 in cholesterol homeostasis.

Cytokines and microbicidal molecules regulated by IL-32 in THP-1-derived human macrophages infected with New World Leishmania species.

BACKGROUND: Interleukin-32 (IL-32) is expressed in lesions of patients with American Tegumentary Leishmaniasis (ATL), but its precise role in the disease remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, silencing and overexpression of IL-32 was performed in THP-1-derived macrophages infected with Leishmania (Viannia) braziliensis or L. (Leishmania) amazonensis to investigate the role of IL-32 in infection. We report that Leishmania species induces IL-32γ, and show that intracellular IL-32γ protein production is dependent on endogenous TNFα. Silencing or overexpression of IL-32 demonstrated that this cytokine is closely related to TNFα and IL-8. Remarkably, the infection index was augmented in the absence of IL-32 and decreased in cells overexpressing this cytokine. Mechanistically, these effects can be explained by nitric oxide cathelicidin and ß-defensin 2 production regulated by IL-32. CONCLUSIONS: Thus, endogenous IL-32 is a crucial cytokine involved in the host defense against Leishmania parasites.

The NOD2 receptor is crucial for immune responses towards New World Leishmania species.

American Tegumentary Leishmaniasis is a chronic infection caused by Leishmania protozoan. It is not known whether genetic variances in NOD-like receptor (NLR) family members influence the immune response towards Leishmania parasites and modulate intracellular killing. Using functional genomics, we investigated whether genetic variants in NOD1 or NOD2 influence the production of cytokines by human PBMCs exposed to Leishmania. In addition, we examined whether recognition of Leishmania by NOD2 contributes to intracellular killing. Polymorphisms in the NOD2 gene decreased monocyte- and lymphocyte-derived cytokine production after stimulation with L. amazonensis or L. braziliensis compared to individuals with a functional NOD2 receptor. The phagolysosome formation is important for Leishmania-induced cytokine production and upregulation of NOD2 mRNA expression. NOD2 is crucial to control intracellular infection caused by Leishmania spp. NOD2 receptor is important for Leishmania recognition, the control of intracellular killing, and the induction of innate and adaptive immune responses.

IL-32 promoter SNP rs4786370 predisposes to modified lipoprotein profiles in patients with rheumatoid arthritis.

Patients with rheumatoid arthritis (RA) are at higher risk of developing cardiovascular diseases (CVD). Interleukin (IL)-32 has previously been shown to be involved in the pathogenesis of RA and might be linked to the development of atherosclerosis. However, the exact mechanism linking IL-32 to CVD still needs to be elucidated. The influence of a functional genetic variant of IL-32 on lipid profiles and CVD risk was therefore studied in whole blood from individuals from the NBS cohort and RA patients from 2 independent cohorts. Lipid profiles were matched to the specific IL-32 genotypes. Allelic distribution was similar in all three groups. Interestingly, significantly higher levels of high density lipoprotein cholesterol (HDLc) were observed in individuals from the NBS cohort and RA patients from the Nijmegen cohort homozygous for the C allele (p = 0.0141 and p = 0.0314 respectively). In contrast, the CC-genotype was associated with elevated low density lipoprotein cholesterol (LDLc) and total cholesterol (TC) in individuals at higher risk for CVD (plaque positive) (p = 0.0396; p = 0.0363 respectively). Our study shows a functional effect of a promoter single-nucleotide polymorphism (SNP) in IL32 on lipid profiles in RA patients and individuals, suggesting a possible protective role of this SNP against CVD.