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M-CSF inhibits anti-HIV-1 activity of IL-32, but they enhance M2-like phenotypes of macrophages
Abstract and Figures
M-CSF promotes the differentiation and survival of macrophages, and preferentially induces anti-inflammatory M2, rather than proinflammatory M1 macrophages. Recently, another cytokine, IL-32, was also shown to promote macrophage differentiation. In this article, we provide the first evidence, to our knowledge, that M-CSF has both additive and inhibitory effects on the macrophage-related activities of IL-32. When added to M-CSF-derived macrophages, M-CSF and IL-32 promoted macrophage survival, which was further enhanced by their combination. However, they had different effects on HIV-1 replication; that is, it was stimulated by M-CSF and inhibited by IL-32. Interestingly, the anti-HIV-1 activity of IL-32 was counteracted by M-CSF. Such inhibitory effect of M-CSF was not observed with IL-32-induced M1-like features including high cytokine/chemokine production and strong expression of the costimulatory molecule CD80. However, IL-32-treated macrophages unexpectedly showed also M2-like features including increased phagocytic activity, and high expression of CD14 and the scavenger receptor CD163, and the expression of CD14 and CD163 was further upregulated by cotreatment with M-CSF. The findings of this study regarding the unique functional interplay between M-CSF and IL-32 increase our understanding of the mechanisms that regulate the survival and M1/M2 ratio of macrophages, as well as HIV-1 replication in macrophages.
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... A possible explanation for this is the inflammatory activation of CD4 1 T cells by IL-32, which results in an increased susceptibility to HIV-1 (6,15). However, the concentrations of IL-32 used in studies using CD4 1 T cells were higher than those in studies using macrophages (250500 ng/ml versus 10100 ng/ml) (6,9,10,15). Moreover, Mesquita et al. reported that when added at 100 ng/ml, rhIL-32g reduced but did not increase viral production induced by PHA in CD4 1 T cells of HIV-1infected individuals (16). ...
... Human peripheral blood MDMs were prepared as previously described (10,18). Briefly, PBMCs were seeded onto multiwell plates or dishes, and monocytes were enriched by allowing them to adhere. ...
... The HIV-1 infection assay was performed as previously described (10,18). MDMs were incubated with the viruses (p24 Gag concentration 10 ng/ml) at 37 • C for 2 h. ...
The proinflammatory cytokine IL-32 is elevated in the plasma and tissues of HIV-1-infected individuals. However, its significance in HIV-1 infection remains unclear because IL-32 inhibits and stimulates viral production in monocyte-derived macrophages (MDMs) and CD4+ T cells, respectively. In this study, we initially found that the inhibitory effect on human MDMs depends on SAMHD1, a dNTP triphosphohydrolase that inhibits viral reverse transcription. IL-32 increased the unphosphorylated active form of SAMHD1, which was consistent with the reduced expression of the upstream cyclin-dependent kinases. Indeed, IL-32 lost its anti-HIV-1 activity in MDMs when SAMHD1 was depleted. These results explain why IL-32 inhibits HIV-1 in MDMs but not CD4+ T cells, because SAMHD1 restricts HIV-1 in noncycling MDMs but not in cycling CD4+ T cells. Another unique feature of IL-32 is the induction of the immunosuppressive molecule IDO1, which is beneficial for HIV-1 infection. In this study, we found that IL-32 also upregulates other immunosuppressive molecules, including PD-L1, in MDMs. Moreover, IL-32 promoted the motility of MDMs, which potentially facilitates intercellular HIV-1 transmission. Our findings indicate that IL-32 has both the direct inhibitory effect on HIV-1 production in MDMs and the indirect stimulatory effects through phenotypic modulation of MDMs, and they suggest that the stimulatory effects may outweigh the inhibitory effect because the window for IL-32 to inhibit HIV-1 is relatively confined to SAMHD1-mediated reverse transcription suppression in the viral life cycle.
... Macropinocytic and phagocytic activities were also quantified using flow cytometry. 35,36 Briefly, prior to macropinocytosis and phagocytosis assays, cells were incubated with lucifer yellow (Sigma) and fluorescent microspheres (fluoresbrite carboxylate microspheres with a 0.7-μm diameter; Polysciences), respectively. ...
... The expression of phosphorylated SAMHD1 and IFITIM3 was analyzed by Western blot as described previously. 26,36 The antibodies used were as follows: anti-SAMHD1 (12586-1-AP; Proteintech), antiphosphorylated (pThr592) SAMHD1 (D7O2M; Cell Signaling Technology), anti-IFITM3 (11714-1-AP; Proteintech), and antiactin (EPR16769; Abcam). ...
Despite effective antiretroviral therapy (ART), HIV-1 persists in cells, including macrophages, which is an obstacle to cure. However, the precise role of macrophages in HIV-1 infection remains unclear because they reside in tissues that are not easily accessible. Monocyte-derived macrophages (MDMs) are widely used as a model in which peripheral blood monocytes are cultured and differentiated into macrophages. However, another model is needed because recent studies revealed that most macrophages in adult tissues originate from the yolk sac and fetal liver precursors rather than monocytes, and the embryonic macrophages possess a self-renewal (proliferating) capacity that MDMs lack. Here, we show that human induced pluripotent stem (iPS) cell-derived immortalized macrophage-like cells (iPS-ML) are a useful self-renewing macrophage model. They proliferate in a cytokine-dependent manner, retain macrophage functions, support HIV-1 replication, and exhibit infected MDM-like phenotypes, such as enhanced tunneling nanotube formation and cell motility, and resistance to viral cytopathic effect. However, several differences are also observed between MDMs and iPS-ML, most of which can be explained by the proliferation of iPS-ML. For instance, proviruses with large internal deletions, which increased over time in individuals receiving ART, are enriched more rapidly in iPS-ML. Interestingly, inhibition of viral transcription by HIV-1-suppressing agents is more obvious in iPS-ML. Collectively, our present study proposes that iPS-ML model is suitable for mimicking the interplay between HIV-1 and self-renewing tissue macrophages, the newly recognized major population in most tissues that cannot be fully modeled by MDMs alone.
... Macrophages can be polarized to the immune suppressive M2 macrophages by some cytokines, such as IL-8, which promoted the metastasis of cancers [19,20]. Previous studies showed that recombinant IL-32 promoted M2 polarization in Mycosis fungoides and macrophage infected with HIV in vitro [21,22]. However, the relationship between tumor cell-derived IL-32 and macrophage in ESCC remain unclear and require further research, and the pathway involved in this process also remain unclear. ...
... The results implied us that EV-IL-32 was phagocytosed by macrophage and mediated the communication between tumor cells and macrophages. Existing published studies reported that recombinant IL-32 promoted M2 polarization [21,22], but the relationship between EV-IL-32 and macrophage has not been studied before. Studies have reported that tumor cell-derived EV could prepare the distant tumor microenvironment for accelerated metastasis [49,50]. ...
Background
Metastasis is the leading cause of mortality in human cancers, including esophageal squamous cell carcinoma (ESCC). As a pro-inflammatory cytokine, IL-32 was reported to be a poor prognostic factor in many cancers. However, the role of IL-32 in ESCC metastasis remains unknown.
Methods
ESCC cells with ectopic expression or knockdown of IL-32 were established and their effects on cell motility were detected. Ultracentrifugation, Transmission electron microscopy and Western blot were used to verify the existence of extracellular vesicle IL-32 (EV-IL-32). Coculture assay, immunofluorescence, flow cytometry, and in vivo lung metastasis model were performed to identify how EV-IL-32 regulated the crosstalk between ESCC cells and macrophages.
Results
Here, we found that IL-32 was overexpressed and positively correlated to lymph node metastasis of ESCC. IL-32 was significantly higher in the tumor nest compared with the non-cancerous tissue. We found that IL-32β was the main isoform and loaded in EV derived from ESCC cells. The shuttling of EV-IL-32 derived from ESCC cells into macrophages could promote the polarization of M2 macrophages via FAK-STAT3 pathway. IL-32 overexpression facilitated lung metastasis and was positively correlated with the proportion of M2 macrophages in tumor microenvironment.
Conclusions
Taken together, our results indicated that EV-IL-32 derived from ESCC cell line could be internalized by macrophages and lead to M2 macrophage polarization via FAK-STAT3 pathway, thus promoting the metastasis of ESCC. These findings indicated that IL-32 could serve as a potential therapeutic target in patients with ESCC.
... M2 macrophages contribute to tumor cell migration by secreting enzymes, which destroy the stromal membrane of endothelial cells, and also secrete some cytokines to promote tumor angiogenesis, consistent with their immunosuppressive and procancer properties [41]. It has been clearly demonstrated that IL34 and M-CSF can promote the differentiation of TAMs to M2 macrophages [42][43][44]. In this study, we found that P4HA3 promoted IL34 and M-CSF expression in colon cancer cells, which also suggests that P4HA3 might promote the differentiation of TAMs to M2 types, thereby promoting tumor progression. ...
Cancer immunotherapies have greatly changed the prospects for the therapy of many malignancies, including colon cancer. Macrophages as the effectors of cancer immunotherapy provide considerable promise for cancer treatment. Prolyl 4-hydroxylase subunit alpha 3 (P4HA3) plays a cancer-promoting role in a variety of cancers, including colon cancer. In the present work, we provided evidence for the first time that P4HA3 promoted colon cancer cell escape from macrophage phagocytosis, and preliminarily explored its possible molecular mechanism. Immunohistochemistry was used to detect the expression of P4HA3 in tissues. Bioinformatics methods were used to analyze the tumor public databases (including TCGA database and GEO database). Macrophage phagocytosis assay and flow cytometric analysis were used to detect the phagocytic capacity of macrophages. Western blot and qRT-PCR were used to detect the expression of related markers (such as P4HA3, CD47, CD24, IL-34, and M-CSF). First, we found that P4HA3 was significantly and highly expressed in both colon cancer tissues and cells, and that P4HA3 had a positive correlation with lymph node metastasis, Dukes stage and also strongly correlated with poorer survival. Subsequently, we found that P4HA3 was strongly associated with the macrophage infiltration level in colon cancer. Immediately we also found that decreasing P4HA3 expression promoted macrophage phagocytosis in colon cancer cells, whereas P4HA3 overexpression produced the opposite effect. Finally, we demonstrated that P4HA3 promoted the expression of cluster of differentiation 47 (CD47) in colon cancer cells. Moreover, P4HA3 caused colon cancer cells to secrete Interleukin 34 (IL34) and Macrophage colony stimulating factor (M-CSF), which further induced macrophages to differentiate to M2 type and thereby contributed to the progression of colon cancer. We have demonstrated that P4HA3-driven CD47 overexpression may act as an escape mechanism, causing colon cancer cells to evade phagocytosis from macrophages.
... But also, CSF-1-differentiated macrophages could undergo a later polarization to the M1-like phenotype, and still higher expressions of M2 markers, i.e., IL-10, CCL14, and CD206, are retained [30]. Furthermore, it has been explored that another cytokine IL-32γ which could induce both M1 and M2 phenotypes if given as co-treatment with CSF-1 preferentially fast-tracks the M2 polarization in differentiating BM HSCs [160]. ...
Macrophages are one of the first innate immune cells to reach the site of infection or injury. Diverse functions from the uptake of pathogen or antigen, its killing, and presentation, the release of pro- or anti-inflammatory cytokines, activation of adaptive immune cells, clearing off tissue debris, tissue repair, and maintenance of tissue homeostasis have been attributed to macrophages. Besides tissue-resident macrophages, the circulating macrophages are recruited to different tissues to get activated. These are highly plastic cells, showing a spectrum of phenotypes depending on the stimulus received from their immediate environment. The macrophage differentiation requires colony-stimulating factor-1 (CSF-1) or macrophage colony-stimulating factor (M-CSF), colony-stimulating factor-2 (CSF-2), or granulocyte–macrophage colony-stimulating factor (GM-CSF) and different stimuli activate them to different phenotypes. The richness of tissue macrophages is precisely controlled via the CSF-1 and CSF-1R axis. In this review, we have given an overview of macrophage origin via hematopoiesis/myelopoiesis, different phenotypes associated with macrophages, their clinical significance, and how they are altered in various diseases. We have specifically focused on the function of CSF-1/CSF-1R signaling in deciding macrophage fate and the outcome of aberrant CSF-1R signaling in relation to macrophage phenotype in different diseases. We further extend the review to briefly discuss the possible strategies to manipulate CSF-1R and its signaling with the recent updates.
Graphical Abstract
... Aberrant expression of some genes such as CSF-1 and FGF-1 has been reported to promote malignant differentiation of TAMs. 28,35 In MMP-21-associated KEGG functional enrichment results, we found that CSF-1 and FGF-1 were involved in macrophage differentiation, so we speculated that MMP-21 might affect the polarization of macrophages via these cytokines ( Figure 5A). Analysis of TCGA data sets displayed the potential connections between MMP-21 and these two molecules (R = 0.343, p < 0.001 for CSF-1 and R = 0.614, p < 0.001 for FGF-1) ( Figure 5B). ...
MMP-21 is a newly-identified member of Matrix metalloproteinases family and has been reported to regulate both embryonic development and tumor progression. However, roles of MMP-21 in HCC remains largely unclear. In this study, we used western blot, qPCR and IHC to determine the upregulation of MMP-21 in HCC tissues, and showed that the increase of MMP-21 was associated with vascular invasion and poor prognosis. Although changing levels of MMP-21 in HCC cell lines had no significant effects on cell migration or invasion abilities in in vitro transwell tests, both IHC analysis and in vivo mouse model proved that upregulated MMP-21 promoted metastasis. Functional enrichments of MMP-21 using TCGA data suggested that MMP-21 might regulate metastasis via macrophages. Further experiments proved that MMP-21 enhanced macrophage recruitment via increasing CCL-14 levels and promoted M2-type polarization of macrophage via elevating the expression of CSF-1 and FGF-1. Taken together, this study revealed that MMP-21 controlled the tumor microenvironment remodeling and functional regulation of macrophages to regulate HCC metastasis.
... The expression of cell surface molecules on macrophages, which were detached from dishes using the enzyme-free cell dissociation buffer, was determined by flow cytometry on a FACSVerse (BD Biosciences) using G2; BioLegend). The phagocytic activity of cells was determined by measuring the uptake of fluorescent microspheres (Fluoresbrite carboxylate microspheres, 0.7 μm in diameter; Polysciences), as described previously 33 . The macropinocytic activity was determined by measuring the uptake of Lucifer yellow (Sigma), as described previously 34 . ...
Recent studies have revealed that tissue macrophages are derived from yolk sac precursors or fetal liver monocytes, in addition to bone marrow monocytes. The relative contribution of these cells to the tissue macrophage pool is not fully understood, but embryo-derived cells are supposed to be more important because of their capacity to self-renew. Here, we show the presence of adult bone marrow-derived macrophages that retain self-renewing capacity. The self-renewing macrophages were readily obtained by long-term culture of mouse bone marrow cells with macrophage colony-stimulating factor (M-CSF), a key cytokine for macrophage development. They were non-tumorigenic and proliferated in the presence of M-CSF in unlimited numbers. Despite several differences from non-proliferating macrophages, they retained many features of cells of the monocytic lineage, including the differentiation into dendritic cells or osteoclasts. Among the transcription factors involved in the self-renewal of embryonic stem cells, Krüppel-like factor 2 (KLF2) was strongly upregulated upon M-CSF stimulation in the self-renewing macrophages, which was accompanied by the downregulation of MafB, a transcription factor that suppresses KLF2 expression. Indeed, knockdown of KLF2 led to cell cycle arrest and diminished cell proliferation in the self-renewing macrophages. Our new cell model would be useful to unravel differences in phenotype, function, and molecular mechanism of proliferation among self-renewing macrophages with different origins.
Interleukin 32 (IL-32) is a proinflammatory cytokine secreted from several kinds of cancer cells. In the present study, we investigated the significance of IL-32 in lung adenocarcinoma by immunohistochemistry and bioinformatics analysis. IL-32 was positive in cancer cells of 21 cases (9.2%) of total 228 cases. Increased IL-32 gene expression was linked to worse clinical course in TCGA analysis, however, IL-32 expression in immunohistochemistry was not associated to clinical course in our cohort. It was also found that high IL-32 expression was seen in cases with increased lymphocyte infiltration. In vitro studies indicated that IFN-γ induced gene expression of IL-32 and PD1-ligands in lung adenocarcinoma cell lines. IL-32, especially IL-32β, also induced overexpression of PD1-ligands in human monocyte-derived macrophages. Additionally, Cancer-cell-derived IL-32 was elevated by stimulation with anticancer agents. In conclusion, IL-32 potentially induced by inflammatory conditions and anticancer therapy and contribute to immune escape of cancer cells via development the immunosuppressive microenvironment. IL-32 might be a target molecule for anti-cancer therapy.
Silk fibroin (SF) and sericin (SS), the two major proteins of silk, are attractive biomaterials with great potential in tissue engineering and regenerative medicine. However, their biochemical interactions with stem cells remain unclear. In this study, we employed multiomics to obtain a global view of the cellular processes and pathways of mesenchymal stem cells (MSCs) triggered by SF and SS to discern cell-biomaterial interactions at an in-depth, high-throughput molecular level. Integrated RNA sequencing and proteomic analysis confirmed that SF and SS initiated widespread but distinct cellular responses and potentiated the paracrine functions of MSCs that regulate extracellular matrix deposition, angiogenesis, and immunomodulation through differentially activating the integrin/PI3K/Akt and glycolysis signaling pathways. These paracrine signals of MSCs stimulated by SF and SS effectively improved skin regeneration by regulating the behavior of multiple resident cells (fibroblasts, endothelial cells, and macrophages) in the skin wound microenvironment. Compared to SS, SF exhibited better immunomodulatory effects in vitro and in vivo, indicating its greater potential as a carrier material of MSCs for skin regeneration. This study provides comprehensive and reliable insights into the cellular interactions with SF and SS, enabling the future development of silk-based therapeutics for tissue engineering and stem cell therapy. This article is protected by copyright. All rights reserved.
Die Tumormikroumgebung (TME) spielt eine wichtige Rolle in Bezug auf das Ansprechen von Therapien, Tumorwachstum und die Bildung von Metastasen. In den letzten Jahren konnte belegt werden, dass Tyrosinkinaseinhibitoren (TKIs) Einfluss auf Zellen des TME haben und vor allem die dort vorherrschenden Zellen, Tumor-assoziierte Makrophagen (TAM), durch die TKIs moduliert werden können. Sie entsprechen meist dem M2-Phänotyp, sezernieren antiinflammatorische Zytokine und sind protumoral, indem sie u.a. die Metastasierung und das Tumorwachstum unterstützen. Zentrale Targets für die Reprogrammierung von Makrophagen stellen sowohl der NF-κB als auch die Inhibition des CSF1-Rezeptors dar. An diesen beiden Schlüsselstellen wirken u.a. TKIs. In den durchgeführten Versuchen wurden drei TKIs verwendet – Dasatinib, Src-Kinase-Inhibitor-I, Bosutinib – um die Ergebnisse von Vorarbeiten zu verifizieren und um zu untersuchen, ob ein Klasseneffekt in Bezug auf eine gesteigerte IL-12-Produktion vorliegen könnte. Ein wichtiger Ansatzpunkt in der Bekämpfung von Tumoren ist die Aktivierung von Immunzellen gegen Tumorzellen, in unserem Fall eine Modulation von TAM in Richtung M1-Makrophagen. Eine signifikante Änderung des Phänotyps konnte nicht festgestellt werden. Allerdings wurde eine gesteigerte IL-12-Produktion aller Makrophagensubtypen durch die Inkubation mit Dasatinib- bzw. Src-kinase-inhibitor-I oder Bosutinib gezeigt. IL-12 ist ein wichtiges proinflammatorisches Schlüsselzytokin des Immunsystems, indem es u.a. NK-Zellen und T-Zellen aktiviert. Die funktionellen Auswirkungen der verstärkten IL-12-Produktion in Hinblick auf NK-Zellen haben wir untersucht. Eine deutlich verstärkte Aktivierung anhand Aktvierungsmarker von NK-Zellen konnten wir nicht beweisen. Allerdings wurde eine erhöhte Zytotoxizität durch Ko-Kultivierung der NK-Zellen mit den unterschiedlichen Makrophagensubtypen und gleichzeitiger Inkubation mit Dasatinib demonstriert. Die erhöhte IL-12-Produktion von APCs sowie verringerte IL-10-Produktion und der Einfluss auf andere Immunzellen, hier am Beispiel der NK-Zellen, zeigen u.a. das therapeutische Potential der TKIs als antineoplastisch wirksame Substanz. Als alleinige Therapie ist deren Wirkungsbereich nach den hier vorliegenden Ergebnissen jedoch noch zu gering. In Kombination mit anderen Therapieoptionen stellen die TKIs allerdings ein mögliches Therapieregime dar. Der genaue Wirkmechanismus und die dadurch entstehenden Veränderungen sind noch genauer zu untersuchen. Ein weiteres Ziel ist in vitro etablierte Ergebnisse auch in die klinische Anwendung einfließen zu lassen. Der zweite Teil der Arbeit befasste sich mit einem proinflammatorischen Zytokin IL-32γ und dessen Wirkung auf Makrophagen. Wie bereits auch bei den TKIs wurde der Einfluss des Interleukins auf das Tumormikromilieu und die entsprechenden Auswirkungen untersucht. IL-32γ wirkt nicht nur selbst als proinflammatorisches Zytokin, sondern reguliert eine Vielzahl an weiteren Zytokinen. Der Einfluss von IL-32 auf das Tumormikromilieu und dessen Zellen stellt einen der zentralen Interessenspunkte dar. In unseren Versuchen konnte unter IL-32γ eine effektivere Antigenpräsentation der Makrophagen – gemessen an einer verstärkten Expression von CD80 und CD86 – gezeigt werden. Auf der anderen Seite wurde das antiinflammatorische Zytokin, IL-10, von IL-32γ-stimulierten Makrophagen ebenfalls verstärkt sezerniert. Eine Ko-Kultivierung von Makrophagen und NK-Zellen und gleichzeitige Inkubation mit IL-32γ führte bei NK-Zellen zu einer verstärkten Aktivierung sowie zu einer erhöhten Zytotoxizität. Die Auswirkungen auf NK-Zellen deuten eine antitumorale Wirkung von IL-32γ an. Das breite Wirkspektrum des Interleukins ist vielversprechend und könnte neue Therapiestrategien eröffnen, wofür allerdings weitere Versuche sowohl in vitro als auch in vivo notwendig sind, um das Interleukin und seine Wirkungen genauestens zu verstehen.
The effects of macrophage colony-stimulating factor (M-CSF) on CD4 receptor expression, susceptibility to human immunodeficiency virus type 1 (HIV) infection, and anti-HIV activity of dextran sulfate and soluble-CD4 were studied in cultured, human primary macrophages. M-CSF stimulated macrophage cells to express the CD4 receptor, and this resulted in an increase of both the number of CD4+ cells and the density of the receptor on the cell surface. M-CSF also significantly enhanced the susceptibility of macrophage cells to HIV infection. Interestingly, the anti-HIV activity of dextran sulfate and soluble-CD4 (two compounds that interfere with HIV-CD4 binding with different mechanisms) was reduced 100-fold and fivefold, respectively, in M-CSF- treated macrophages. Human blood concentrations of M-CSF are reported to be similar to those used in this work (1,000 U/mL); thus, it is conceivable that also in vivo this cytokine may modify the susceptibility of macrophages to HIV and the ability of dextran sulfate and soluble CD4 to inhibit HIV replication. These results suggest that the in vitro study in M-CSF-treated macrophages of promising drugs inhibitors of HIV-CD4 binding could provide further insights into the potential efficacy of these compounds in patients.
Polymorphisms in the gene encoding the transcription factor IRF5 that lead to higher mRNA expression are associated with many autoimmune diseases. Here we show that IRF5 expression in macrophages was reversibly induced by inflammatory stimuli and contributed to the plasticity of macrophage polarization. High expression of IRF5 was characteristic of M1 macrophages, in which it directly activated transcription of the genes encoding interleukin 12 subunit p40 (IL-12p40), IL-12p35 and IL-23p19 and repressed the gene encoding IL-10. Consequently, those macrophages set up the environment for a potent T helper type 1 (T(H)1)-T(H)17 response. Global gene expression analysis demonstrated that exogenous IRF5 upregulated or downregulated expression of established phenotypic markers of M1 or M2 macrophages, respectively. Our data suggest a critical role for IRF5 in M1 macrophage polarization and define a previously unknown function for IRF5 as a transcriptional repressor.
Interleukin-32 was recently identified as a pro-inflammatory cytokine produced by T-lymphocytes, natural killer cells, epithelial cells, and blood monocytes. IL-32 is induced by IFN-γ in a time-dependent manner suggesting a role for IL-32 in innate and adaptive immune responses. In this study we present evidence that Human immunodeficiency virus promotes interleukin-32 production at both mRNA and protein levels. Our results showed that there is a 74% increase in the serum levels of IL-32 among HIV patients as compared to healthy individuals. There was a three-fold increase in the promoter activity of the IL-32 in the present infections HIV clone. This increase in IL-32 promoter activity was substantiated by increased IL-32 mRNA and protein levels. We have also demonstrated that IL-32 suppresses HIV replication. Our results show that HIV LTR activity was increased by more than six-folds when endogenous IL-32 was knocked down by IL-32-specific siRNA whereas it decreased by one-fold when IL-32 was over expressed in the cells. Similarly a more than two-fold increase and a 50% decrease in HIV p24 values were noted when IL-32 was knocked down and when IL-32 was over expressed in the cells, respectively. Our present work shows that raised IL-32 levels in HIV infection may in turn hamper HIV replication; one of the protective mechanisms of nature.
We have isolated a cDNA clone from a human activated NK cell-derived cDNA library that identifies a transcript (NK4) that is selectively expressed in lymphocytes. The expression of this transcript is increased after activation of T cells by mitogens or activation of NK cells by IL-2 (lymphokine-activated killer cells). The transcript levels demonstrated by Northern blot analysis increase by 12 h after activation, remain high for at least 48 h, and require protein synthesis for expression. Southern blot analysis of B lymphoblastoid lines derived from 18 unrelated individuals reveal variable banding patterns suggestive of polymorphism within the NK4 gene. No homology was found between the sequence of the coding region of this transcript and any sequences in the GenBank data base. Sequence homology to the U1 small nuclear RNA was found within the 3' untranslated region immediately upstream of the site of polyadenylation, suggesting a possible role for U1 in the polyadenylation process. Sequence analysis indicates the transcript would encode a protein having a mass of 27 kDa. The presence of a signal sequence and lack of a transmembrane region suggests that the protein is secreted. In addition, the protein contains an RGD sequence that may be involved in cellular adhesion. This transcript appears to encode a novel product common to the activation pathways of both NK cells and T cells.
We characterized the molecular species of human macrophage colony- stimulating factor (hM-CSF) found in serum and urine, using immunoblot analysis after partial purification on an antibody-bound affinity column. Although antibodies were prepared using the recombinant product of the large form of hM-CSF with a molecular weight (MW) of 85 Kd as the antigen, this immunoblot system was also capable of detecting the small form of hM-CSF with a MW of 40 to 60 Kd. A single band with a MW of 43 Kd, which reacted with anti-recombinant hM-CSF IgG but not with control IgG, was found when serum and urine from normal adults underwent electrophoresis on reduced sodium dodecyl sulfate- polyacrylamide gel and subsequent immunoblotting. This band represented a subunit of the large form of hM-CSF, because the large form of hM-CSF is a homodimer of a subunit with a MW of 43 Kd and the small form of hM- CSF is a homodimer of a subunit with a MW of 20 to 30 Kd. Analysis of serum and urine from leukemic patients and pregnant women, who had higher serum levels of hM-CSF than normal adults, showed only a single band with a MW of 43 Kd as a hM-CSF-specific molecule. These results suggest that the large form of hM-CSF is the major species in human body fluids.
Diversity and plasticity are hallmarks of cells of the monocyte-macrophage lineage. In response to IFNs, Toll-like receptor engagement, or IL-4/IL-13 signaling, macrophages undergo M1 (classical) or M2 (alternative) activation, which represent extremes of a continuum in a universe of activation states. Progress has now been made in defining the signaling pathways, transcriptional networks, and epigenetic mechanisms underlying M1-M2 or M2-like polarized activation. Functional skewing of mononuclear phagocytes occurs in vivo under physiological conditions (e.g., ontogenesis and pregnancy) and in pathology (allergic and chronic inflammation, tissue repair, infection, and cancer). However, in selected preclinical and clinical conditions, coexistence of cells in different activation states and unique or mixed phenotypes have been observed, a reflection of dynamic changes and complex tissue-derived signals. The identification of mechanisms and molecules associated with macrophage plasticity and polarized activation provides a basis for macrophage-centered diagnostic and therapeutic strategies.
We describe the gene structure, regulation, signal transduction. and functions of a cytokine, interleukin (IL)-32. An IL-18 unresponsive cell was converted to a responsive cell by transfection of the IL-18 receptor β chain, and IL-18-induced microarray revealed high expression of a cytokine-like gene. Although IL-32 does not share sequence homology with known cytokine families, IL-32 induces various cytokines, human TNFα, and IL-8 in THP-1 monocytic cells as well as mouse TNFα and MIP-2 in Raw macrophage cells. IL-32 activates typical cytokine signal pathways of nuclear factor-kappa B (NF-κB) and p38 mitogen-activated protein kinase. IL-32 mRNA is highly expressed in immune tissue rather than other tissues. Human IL-32 exists as four splice variants, and IL-32 from other species were found as expressed sequence tag clones in the databank. Induced in human peripheral lymphocyte cells after mitogen stimulation, in human epithelial cells by IFNγ, and in NK cells after exposure to the combination of IL-12 plus IL-18, IL-32 may play a role in inflammatory/autoimmune diseases.
Infection of CD4(+) chemokine coreceptor(+) targets by HIV is aided and abetted by the proficiency of HIV in eliminating or neutralizing host cell-derived defensive molecules. Among these innate protective molecules, a family of intracellular apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases, is constitutively expressed but inactivated by HIV viral infectivity factor. The ability of interferon-alpha (IFN-alpha) to augment cytidine deaminases offered the possibility that the balance between virus and target cell might be altered in favor of the host. Further characterization of transcriptional profiles induced by IFN-alpha using microarrays, with the intention to identify and dissociate retroviral countermaneuvers from associated toxicities, revealed multiple molecules with suspected antiviral activity, including IL-27. To establish whether IFN-alpha toxicity might be sidestepped through the use of downstream IL-27 against HIV, we examined whether IL-27 directly regulated cytidine deaminases. Although IL-27 induces APOBECs, it does so in a delayed fashion. Dissecting the underlying regulatory events uncovered an initial IL-27-dependent induction of IFN-alpha and/or IFN-beta, which in turn, induces APOBEC3, inhibited by IFN-alpha/beta receptor blockade. In addition to macrophages, the IL-27-IFN-alpha connection is operative in CD4(+) T cells, consistent with an IFN-alpha-dependent pathway underlying host cell defense to HIV.
Polarized activation of cells of the monocyte-macrophage lineage into M1 and M2 cells is an operationally useful, simplified descriptor of the functional plasticity of these cells. Ghassabeh and colleagues now put to the test the actual in vivo validity and significance of the M1/M2 paradigm.
